effects of physical exercise and hydration on homocysteine

UNIVERSIDAD POLITÉCNICA DE MADRID FACULTAD DE CIENCIAS DE LA ACTIVIDAD FÍSICA Y DEL DEPORTE (INEF)

Effects of physical exercise and hydration on

homocysteine concentrations in physically active male adults

Efectos del ejercicio físico y la hidratación sobre las

concentraciones de homocisteína en varones físicamente activos

Tesis Doctoral Internacional International PhD Thesis

Beatriz Maroto Sánchez Licenciada en Ciencias de la Actividad Física y del Deporte

2015

Madrid 2015 Todos los derechos reservados.

ISBN: 978-84-608-4357-3

Edita: Fundación General de la Universidad Politécnica de Madrid C/ Pastor, 3 – 28003 Madrid

Imprime: Llar Digital C/ Caballeros, 13-Bajo. – 12001 Castellón

Maroto Sánchez B, 2015

II

International PhD Thesis

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DEPARTAMENTO DE SALUD Y RENDIMIENTO HUMANO

FACULTAD DE CIENCIAS DE LA ACTIVIDAD FÍSICA Y DEL DEPORTE

Effects of physical exercise and hydration on homocysteine concentrations in physically active male adults

Efectos del ejercicio físico y la hidratación sobre las

concentraciones de homocisteína en varones físicamente activos

Beatriz Maroto Sánchez Licenciada en Ciencias de la Actividad Física y del Deporte

2015

DIRECTORES DE TESIS

Marcela González-Gross Ms Sc, PhD.

Catedrática de Universidad Universidad Politécnica de Madrid

Pedro J. Benito Peinado

Ms Sc, PhD. Prof. Titular de Universidad

Universidad Politécnica de Madrid


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MIEMBROS DEL TRIBUNAL

MIEMBROS DEL TRIBUNAL SUPLENTES

Ricardo Mora Rodriguez

PhD Catedrático de Universidad

Universidad de Castilla-La Mancha Spain

Gonzalo Palacios Le Blé

PhD Investigador

Universidad Politécnica de Madrid Spain

Alejandro González de Agüero Lafuente PhD

Profesor Ayudante Doctor Universidad de Zaragoza

Spain

Christina Breidenassel PhD

Associated Professor University of Bonn

Germany

Francisco José Sánchez Muniz PhD

Catedrático de Universidad Universidad Complutense de Madrid

Spain

Gabriel Rodríguez Romo PhD

Profesor Titular de Universidad Universidad Politécnica de Madrid

Spain

Margarita Perez Ruiz PhD

Profesora Titular de Universidad Universidad Europea de Madrid

Spain


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TRIBUNAL DE LA TESIS

Tribunal nombrado por el Mgfco. y Excmo. Sr. Rector de la Universidad Politécnica de

Madrid, el día ____________________________________________________de 2015.

Presidente D. ____________________________________________________________

Vocal D. _______________________________________________________________

Vocal D. _______________________________________________________________

Vocal D. _______________________________________________________________

Secretario D. ____________________________________________________________

Realizado el acto de defensa y lectura de Tesis el día, ___________________________

en ____________________________________________________________________

Calificación: ____________________________________________________________

EL PRESIDENTE LOS VOCALES

EL SECRETARIO


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A mi maravillosa familia,

en especial a mi padre por su apoyo incondicional.

A Jorge, por ayudarme a mantener siempre el equilibrio.


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List of Contents

List of Tables ........................................................................................................... XIIIList of Figures ........................................................................................................... XVList of Abbreviations and Symbols ....................................................................... XVIIList of publications from the thesis .......................................................................... XIXGranted Research Projects and Funding .................................................................. XXIABSTRACT .................................................................................................................. 1RESUMEN ................................................................................................................... 31 CHAPTER 1. INTRODUCTION ........................................................................... 51.1 Research Background ............................................................................................ 51.2 Statement of the Research Problems ................................................................... 301.3 Structure of the Thesis ......................................................................................... 312 CHAPTER 2. OBJECTIVES AND HYPOTHESIS ............................................. 333 CHAPTER 3. GENERAL MATERIAL AND METHODS ................................. 353.1 Sample of the study ............................................................................................. 353.2 Ethical issues ....................................................................................................... 353.3 Experimental Design ........................................................................................... 363.4 Materials .............................................................................................................. 433.5 Statistical Analysis ............................................................................................... 444 CHAPTER 4. STUDY 1: El ejercicio agudo aumenta las concentraciones de homocisteína en varones físicamente activos. Acute exercise increases homocysteine concentrations in physically active males. .................................................................. 454.1 Resumen .............................................................................................................. 454.2 Abstract ................................................................................................................ 454.3 Introducción ......................................................................................................... 464.4 Material y métodos .............................................................................................. 474.5 Resultados ............................................................................................................ 504.6 Discusión ............................................................................................................. 554.7 Conclusiones ........................................................................................................ 575 CHAPTER 5. STUDY 2: Effect of rehydration after acute exercise on homocysteine concentrations and related parameters. ................................................ 595.1 Abstract ................................................................................................................ 595.2 Introduction .......................................................................................................... 595.3 Material and Methods .......................................................................................... 605.4 Results .................................................................................................................. 655.5 Discussion ............................................................................................................ 695.6 Conclusion ........................................................................................................... 716 CHAPTER 6. STUDY 3: Hydration during exercise prevents the increase of homocysteine concentrations ...................................................................................... 736.1 Abstract ................................................................................................................ 736.2 Introduction .......................................................................................................... 736.3 Material and Methods .......................................................................................... 756.4 Results .................................................................................................................. 806.5 Discussion ............................................................................................................ 896.6 Conclusion ........................................................................................................... 927 CHAPTER 7. GENERAL DISCUSSION ............................................................ 93


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8 CHAPTER 8. CONCLUSIONS ......................................................................... 101REFERENCES ......................................................................................................... 103

APPENDIX ............................................................................................................... 113ACKNOWLEDGMENTS ........................................................................................ 159SUMMARIZED CV/CURRÍCULUM VITAE ABREVIADO ................................ 163


XIII

List of Tables

Table 1. The effect of acute exercise on tHcy concentrations ....................................... 15

Table 2. The effect of chronic exercise on tHcy concentrations .................................... 19

Table 3. Relation of physical activity (PA) levels and cardiorespiratory fitness with

tHcy concentrations ........................................................................................................ 22

Table 4. Implicated biomarkers related to tHcy concentrations and exercise ............... 25

Table 5. List of methods and devices used for the different biochemical parameters ... 40Table 6. Laboratory material .......................................................................................... 43

Table 7. Características generales de los sujetos ........................................................... 51

Table 8. Parámetros físicos recogidos durante la prueba máxima y la prueba submáxima

........................................................................................................................................ 51

Table 9. Concentraciones de tHcy, Folato, Vitamina B12 y Creatinina antes y después

del ejercicio en prueba máxima y prueba submáxima .................................................... 52

Table 10. Correlaciones de Pearson entre las variables tHcy, folato, Vitamina B12 y

creatinina antes y después en pruebas máxima y submáxima ........................................ 54

Table 11. Drink composition ......................................................................................... 63

Table 12. General characteristics of the participants at baseline ................................... 65

Table 13. Total homocysteine, folate, vitamin B12 and creatinine concentrations before,

after exercise and 2 hours after rehydration protocol ..................................................... 66

Table 14. Pearson correlation coeficients between tHcy, vitamin B12, folate and

creatinine ........................................................................................................................ 67

Table 15. Anthropometric characteristics and genotype of the studied sample ............ 80

Table 16. Heart rate and blood pressure before and after the exercise tests .................. 81

Table 17. Weight lost and urine osmolarity before and after exercise .......................... 81

Table 18. Change of Plasma Volume (%) after all four tests ........................................ 82

Table 19. Total homocysteine concentrations corrected (C) and uncorrected (U) by

haemoconcentration ........................................................................................................ 84

Table 20. Folate and vitamin B12 concentrations corrected and uncorrected by


Table 21. Creatine and Creatinine concentrations corrected and uncorrected by


Table 22. Sodium, Potassium, Chloride and Magnesium values ................................... 88


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List of Figures

Figure 1. Factors related to tHcy concentrations ............................................................. 6

Figure 2. Methionine-Homocysteine cycle ...................................................................... 8

Figure 3. Mechanisms of heat dissipation .................................................................... 27

Figure 4. Factors affecting heat gain and heat loss during exercise ............................. 28

Figure 5. Experimental protocol of the study ................................................................ 36

Figure 6. Niveles de tHcy antes y después de la prueba máxima .................................. 53

Figure 7. Niveles de tHcy antes y después de la prueba submáxima ............................ 53

Figure 8. Percentage of change (%) in total homocysteine between “before” and “after

exercise” in exercise tests. Sample splitting by tertiles .................................................. 67

Figure 9. Percentage of change (%) in total homocysteine between “after exercise” and

“2 hours after rehydration” with water and sport drink. Sample splitting by tertiles ..... 68

Figure 10. Experimental protocol .................................................................................. 76

Figure 11. Corrected total homocysteine concentrations (µmol/L) in all 4 tests .......... 83

Figure 12. Uncorrected total homocysteine concentrations (µmol/L) in all 4 tests ...... 83

Figure 13. Percentage of change (%) of corrected total homocysteine concentrations . 85


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List of Abbreviations and Symbols

ACE I/D Angiotensin-Converting Enzyme ACSM American College of Sports Medicine AGAT Glycine Amidinotransferase ANOVA Analysis of variance ATP Adenosine Three Phosphate BHTM Betaine Homocysteine Methyltransferase BIA Bioelectrical impedance analysis BMI Body Mass Index bp base pairs BP Blood Pressure C Corrected CBS Cystathionine synthase cm Centimetre(s) cm2 Square centimetre(s) Cl Chloride CV Coefficient of variation CVD Cardiovascular disease CVR Cardiovascular risk CYS Cystathionine D Deletion dL Decilitre(s) DXA Dual energy X-ray absorptiometer EDTA Ethylene-Diamineteraacetic Acid et al. et alii (= and others) g Gram(s) GAA Guaninoacetic acid GAMT Glycine amidinotransferase h Hour(s) Hb Hemoglobin Hcy Homocysteine Hct Hematocrit HR Heart Rate I Insertion K Potassium kg Kilogram(s) km Kilometre(s) L Litre(s) mg Milligram(s) min Minute(s) mL Millilitre(s) mmEq Milliequivalent(s) mmHg Millimetre(s) of mercury mmol Millimole(s) Mg Magnessium MTHFR Methyl-Tetrahydrofolate Reductase MS Methyonine synthase n Number of


XVIII

ng Nanograms Na Sodium NS Non significant PA Physical activity pg Pictogram(s) PCR Polimerase Chain Reaction r Correlation coefficient R² Coefficient of determination RFLP Restriction Fragment Length Polymorphism s seconds SAH S-AdenosylhomocysteineSAM S-AdenosylhomocysteineSD Standard deviationSPSS Statistical Package for Social SciencestHcy Total HomocysteineTHF TetrahydrofolateU UncorrectedUK United KingdomUSA United States of AmericaVE VentilationVO2 Oxigen ConsumptionVO2max Maximal oxygen UptakeWHO World Health Organizationyr Years5-MTHFR 5-MethylΔPV Change of Plasma Volume α Alpha % Percentage ® Registered Trademark °C Degrees Celsius µL Microlitre(s) µmol Micromole(s)


XIX

List of publications from the thesis

Mielgo-Ayuso J, Maroto-Sánchez B, Luzardo-Socorro R, Palacios G, Palacios Gil-

Antuñano N, González-Gross M; EXERNET Study Group. Evaluation of nutritional

status and energy expenditure in athletes. Nutr Hosp. 2015 Feb 26;31 Suppl 3:227-36.

doi: 10.3305/nh.2015.31.sup3.8770. (JCR: 1.04).

Maroto-Sánchez Beatriz, Lopez-Torres Olga, Palacios Gonzalo, González-Gross

Marcela. What do we know about Homocysteine and exercise? A review from the

literature. CCLM. (In Press). (JCR: 2.70).

Maroto-Sánchez B, Valtueña J, Albers U, Benito PJ, González-Gross M. Acute physical

exercise increases homocysteine concentrations in young trained male subjects. Nutr

Hosp. 2013 Mar-Apr;28(2):325-32. doi: 10.3305/nh.2013.28.2.6300. (JCR: 1.04).

Maroto-Sánchez Beatriz, Lopez-Torres Olga, Valtueña Jara, Benito Pedro J, Palacios

Gonzalo, Díaz Martínez Ángel Enrique, González-Lamuño Domingo, Carru Ciriaco,

Zinellu Angelo, González-Gross Marcela. Hydration effect on increased homocysteine

concentrations after exercise. (Submitted).

Maroto-Sánchez Beatriz, Lopez-Torres Olga, Valtueña Jara, Benito Pedro J, Palacios

Gonzalo, Díaz Martínez Ángel Enrique, González-Lamuño Domingo, Carru Ciriaco,

Zinellu Angelo, González-Gross Marcela. Hydration during exercise prevents the

increase of homocysteine concentrations. JPAH. (Submitted). (JCR: 2.09).


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XXI

Granted Research Projects and Funding

This PhD Thesis has been financially supported by the following fundings and grants:

- The author has been contracted with funds from the ImFine research group of

the Universidad Politécnica de Madrid.

- Research Funds from the Universidad Politécnica de Madrid.

- Grant for attending the ECSS (European College of Sport Science) annual

congress. (Liverpool2011). Powerade Grant (SE08110001).

- Grant from the Social Council of the Univeridad Politécnica de Madrid for

internships abroad (year 2011): Internship at the Health and Wellness Center,

Colorado School of Medicine at the University of Colorado, Denver, Colorado.

U.S.A, June-September (2012).

- Grant from the European Hydration Institute (EHI) for the project “Fluid intake

in elderly. “Differences in hydration habits between an active and a non-active

Spanish population”. Project number: E131115081 (2013).

- Grant to participate at the 10th Annual Obesity Summer Boot Camp for experts

and new proffesionals in obesity research. Alberta, Canadá, July 18th to July

26th (2015).

- Aditional support from Instituto de Salud Carlos III (Centro de Investigación

Biomédica en Red. Fisiopatología de la Obesidad y Nutrición) CIBERobn

CB12/03/30038.


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1

ABSTRACT

The current thesis analyzes the effect of exercise and hydration on total homocysteine

(tHcy) concentrations and the relationship with the implicated parameters, like folate,

vitamin B12, and creatine in physically active male adults. The work is based on the

results of the study conducted at the Faculty of Physical Activity and Sport

Sciences of the Technical University of Madrid. A total of 29 physically active

voluntary healthy males from the Region of Madrid were recruited. The main

outcomes of this thesis are: a) tHcy concentrations increased after acute exercise

with both, maximal (VO2max) and submaximal (65 % of VO2max) tests in

physically active male subjects independently of their baseline tHcy status. b) After 2 h

of rehydration with a sport drink, tHcy concentrations, which had previously increased

during an acute exercise, decreased significantly, although they didn´t recover to

baseline values. c) An adequate hydration protocol during acute aerobic submaximal

exercise prevents the increase of tHcy concentrations and maintains these

concentrations at baseline up to 2 h post-exercise. d) Serum tHcy concentrations

increased after submaximal exercise when the hydration protocol during exercise was

not applied. Furthermore, tHcy concentrations reached maximal values 6 h after the end

of exercise. e) At 24 h, tHcy concentrations recovered baseline values independently

whether or not there was a hydration protocol during exercise. f) There is a need to

clarify the underlying mechanisms related to cardiovascular risk due to the transient

increase of tHcy concentrations induced by acute exercise. Further research

analayzing the relationship between tHcy concentrations after acute exercise and

the implication of creatine, vitamin B12 and folate as related parameters in the

homocysteine metabolism is needed. Finally, tHcy concentrations increased above the

recommended values after an acute aerobic submaximal exercise; nevertheless, a good

hydration protocol maintains tHcy concentrations at baseline and prevents the

further increase in a sample of physically active male adults.

Key words: Homocysteine, Exercise physiology, Metabolism, Nutrition


2

What is already known on this topic? What does this PhD Thesis add? There is no consensus about the relationship

between physical exercise in its different

modalities, and total homocysteine (tHcy) serum

concentrations.

Moreover, the exact mechanism by which these

amino acid concentrations may be affected by

exercise is still unknown.

In the introduction of the present thesis, there is a

unified and differentiate classification of the

studies that analyzed until now the relationship

between tHcy serum concentrations and exercise

in its different modalities: effect of acute exercise,

effect of chronic exercise, physical activity level,

and cardiorespiratory fitness. Moreover, the

possible mechanisms proposed until now from the

different investigations are discussed.

Some studies have observed an increase in serum

tHcy concentrations after acute exercise. However,

others have not observed changes. Furthermore,

these studies use different methodologies and study

samples. It is necessary to get deeper knowledge

on the effect of acute exercise in physically active

subjects with a strict laboratory protocol and

methodologies.

This thesis provides conclusive results about the

effect of acute exercise both, maximal and

submaximal, increasing tHcy concentrations after

applying a strict methodological laboratory

protocol in a group of physically active adult

males. Moreover, it shows the behaviour of these

concentrations up to 24 h after exercise. Thus, it

can contribute to the knowledge of the effects of

acute exercise to apply to athletes of the same

characteristics.

Dehydration during exercise affects all the

physiologic systems in the human body. Moreover,

the importance of hydration from the health

perspective on biomarkers affected during exercise

is less studied. There are no data on the effect of an

adequate hydration protocol on tHcy serum

concentrations during and after acute aerobic

submaximal exercise.

To offer, for the first time, data that show that an

adequate hydration protocol during aerobic

submaximal exercise maintains tHcy

concentrations at baseline levels up to 2 h after the

exercise and prevents the further increase.

There is a consensus about the inverse relationship

between folate, and vitamin B12 concentrations and

tHcy concentrations at baseline. However, there are

not conclusive data in the scenario of

physical exercise. It is necessary to get

deeper knowledge on the understanding of the

behaviour of folate and vitamin B12 and other

parameters like creatine with tHcy concentrations

after acute exercise.

This thesis analyzes the relationship between

folate, vitamin B12, and creatine concentrations

with those of tHcy, before and after acute aerobic

submaximal exercise. The effect of acute exercise

showed an increase in vitamin B12 and creatinine

in line with those observed for the homocysteine

concentrations.


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RESUMEN

La presente tesis analiza el efecto del ejercicio físico agudo y la hidratación sobre las

concentraciones de homocisteína total (tHcy) y su relación con los parámetros

implicados en el metabolismo de la homocisteína como el folato, la vitamina B12, y la

creatina en una muestra de varones jóvenes físicamente activos.

El trabajo se basa en los resultados del estudio realizado en la Facultad de Ciencias de la

Actividad Física y del Deporte de la Universidad Politécnica de Madrid. Para el cual se

contó con un total de 29 voluntarios sanos físicamente activos de la Comunidad de

Madrid. Los principales resultados de esta tesis son: a) Las concentraciones de tHcy

aumentaron después del ejercicio agudo tanto tras una prueba de intensidad máxima

(VO2max) como una submáxima (65 % of VO2max) en varones físicamente activos

independientemente de las sus concentraciones basales de tHcy. b) Las concentraciones

de tHcy disminuyeron 2 h después del ejercicio físico aeróbico submáximo tras aplicar

un protocolo de hidratación con una bebida para deportistas. c) Un adecuado protocolo

de hidratación durante el ejercicio físico agudo previno el aumento de las

concentraciones de tHcy hasta 2 h después del ejercicio. d) Las concentraciones de tHcy

aumentaron a las 6 h tras la finalización del ejercicio únicamente en los test en los que

no se siguió un protocolo de hidratación durante el ejercicio físico. e) A las 24 h tras el

ejercicio, las concentraciones de tHcy volvieron a los niveles basales

independientemente de si se aplicó un protocolo de hidratación durante el ejercicio o no.

f) Es necesario aclarar si existen mecanismos subyacentes relacionados con el riesgo

cardiovascular debido al aumento transitorio de las concentraciones de tHcy inducidas

por el ejercicio agudo. Se necesitan más estudios que analicen la relación entre las

concentraciones de tHcy después del ejercicio físico agudo y la implicación de la

creatina, vitamina B12 y folato como parámetros relacionados en el metabolismo de la

homocisteína. El efecto agudo del ejercicio físico aumenta las concentraciones de tHcy

por encima de los valores recomendados; sin embargo, un adecuado protocolo de

hidratación mantiene las concentraciones a niveles basales y previene el posterior

aumento en una muestra de varones adultos físicamente activos.

Palabras Clave: Homocisteina, Fisiología del ejercicio, Metabolismo, Nutrición


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¿Qué se sabe en este ámbito? ¿Qué añade esta Tesis Doctoral? Existe falta de consenso acerca de la relación

entre el ejercicio físico en sus diferentes

modalidades con las concentraciones de

homocisteína total (tHcy) séricas.

Además, no está claro el mecanismo exacto por el

cual las concentraciones de este aminoácido

pueden verse afectadas por el ejercicio.

En la introducción de la presente tesis se resumen y

analizan las investigaciones que hasta la fecha han

estudiado la relación entre las concentraciones de

tHcy séricas y el ejercicio físico en sus diferentes

modalidades: efecto agudo del ejercicio, el efecto

crónico del ejercicio, el nivel de actividad física, y

el fitness cardiorespiratorio. Además, se discuten

los posibles mecanismos que hasta la fecha se han

propuesto en las diferentes investigaciones.

Existen algunos estudios que han observado un

aumento en las concentraciones séricas de tHcy

tras el ejercicio físico agudo. Sin embargo, otros

no han observado cambios. Estos estudios,

además, utilizan metodologías y muestras de

estudio muy variadas. Es necesario profundizar en

el efecto del ejercicio físico agudo en sujetos

físicamente activos con un protocolo y

metodologías estrictas de laboratorio.

Esta tesis ofrece resultados concluyentes que

muestran que el efecto del ejercicio físico agudo

tanto de intensidad máxima como submáxima,

aumenta las concentraciones de tHcy séricas tras

aplicar un estricto protocolo metodológico en

laboratorio en un grupo de varones adultos

físicamente activos. Además, muestra el

comportamiento de estas concentraciones hasta 24

h después del ejercicio. Así, se puede contribuir al

conocimiento del efecto del ejercicio agudo y

aplicarlo a deportistas de las mismas características.

La deshidratación durante el ejercicio físico afecta

todos los sistemas fisiológicos en el cuerpo

humano. Sin embargo, el estudio de la

hidratación sobre algunos parámetros bioquímicos

desde el punto de viste de la salud es escaso. No

existen datos del efecto que puede tener un

adecuado protocolo de hidratación sobre las

concentraciones de tHcy séricas tanto durante

como después del ejercicio físico agudo.

Se presentan, por primera vez, datos que muestran

que un adecuado protocolo de hidratación durante

el ejercicio mantiene los niveles de tHcy en niveles

basales después del ejercicio aeróbico submáximo

hasta las 2 h y previene el posterior aumento.

Existe consenso entre la relación inversa de las

concentraciones de vitamina B12 y folato con las

de tHcy en reposo. Sin embargo, en el escenario

del ejercicio no hay datos concluyentes. Es

necesario profundizar en el conocimiento del

comportamiento de la vitamina B12 y el folato y

otros parámetros relacionados como la creatina

con las concentraciones de tHcy tras el ejercicio

físico agudo.

Esta tesis analiza la relación entre las

concentraciones de vitamina B12, folato y creatinina

con las de tHcy antes y después del ejercicio físico

agudo. Tras el ejercicio físico todos los parámetros

aumentan en línea con las concentraciones de tHcy.

En cuanto a la relación entre la tHcy y la creatina,

no se han obtenido resultados concluyentes.


5

1 CHAPTER 1. INTRODUCTION

1.1 Research Background

1.1.1 Homocysteine

Homoysteine (Hcy) is an endogenous sulfur-containing, non protein-forming amino

acid synthesized from the essential amino acid methionine. In recent years, numerous

studies have shown that elevated Hcy concentrations (also called

hyperhomocysteinemia) have a strong relationship with cardiovascular diseases (CVD)

(39). There is evidence supporting Hcy concentrations as a powerful independent

predictor of coronary heart disease, cerebrovascular disease and venous thrombosis

(70). Boushey et al. (12) conducted the first meta-analysis with a total of 27 studies.

Results concluded that Hcy was an independent risk factor for atherosclerotic disease, in

the coronary, cerebral and peripheral vessels. Nevertheless, the debate about if high Hcy

concentrations represent a risk factor or at least as a marker is open (133). Moreover,

this meta-analysis showed that an increment of 5 µmol/L in total homocysteine (tHcy)

concentrations was associated with 60 % and 80 % increased risk for coronary heart

disease in men and women, respectively.

Following research has demonstrated that by lowering tHcy 3 µmol/L the risk of

ischemic heart disease is reduced by 16 % in participants with the gene-

mutation Methyl-tetrahydrofolate reductase (MTHFR) (128), which increases Hcy

concentrations and will be explained in depth below. Studies strongly suggest

that elevated Hcy concentrations in blood increase the risk of CVD independently of

the other CVD risk factors (70), but how does elevated Hcy increase the risk for CVD?

1.1.2 Homocysteine as a risk factor

The first mechanism is the endothelial dysfunction. Hcy inhibits endothelium-dependent

anticoagulant reactions (61, 78, 105), induces the expression of procoagulants (41, 70),

decreases interactions between endothelial cells and plasminogen activators (59, 70) and

impairs the bioavailability of endothelium-derived nitric oxide that inhibits blood vessel

dilation (70, 116). On the other hand, the platelet aggregation and thrombosis are the

other mechanisms by which Hcy contributes to a risk for CVD. During the oxidation of

Hcy, the formation of the hydrogen peroxide occurs causing oxidative damage by


6

increasing platelet activity. In turn, this reaction causes endothelial dysfunction,

decreased nitric oxide production and therefore, accelerate atherosclerosis (55).

Cardiovascular risk is gradual and proportional to the concentration of tHcy in blood

(48). Thus, people with tHcy concentrations at the upper limit from those that could be

considered normal, have a higher increase of cardiovascular risk respect to those with

lower concentrations. The following figure shows the known factors that are related to

tHcy concentrations.

Figure 1. Factors related to tHcy concentrations

Plasma Hcy concentrations in fasting conditions considered within the reference range

in adults are from 5 to 15 µmol/L, levels greater than this values are considered

Hyperhomocysteinemia (19). Hcy levels are usually higher in men than women and also

increase with age in both sexes. Furthermore, it has been suggested that the desirable

concentrations must not exceed 10 µmol/L (8, 88, 106). Omenn et al. (96), based on


7

data from case-control, observational and meta-analysis studies, have also postulated a

plasma tHcy > 10 µmol/L as a cut-off point risk for ischaemic heart disease.

1.1.3 Homocysteine metabolism

Hcy concentrations are influenced by nutritional, clinical and lifestyle factors

such as smoking, coffee consumption, excessive alcohol intake, lack of exercise,

obesity, stress, malabsorption or suboptimal intake of vitamin B12 and folate, reduced

kidney function, or intake of medications that can reduce the absorption of vitamins

(110).

Methionine is the only known precursor of Hcy in humans. Three enzymes are directly

involved in the Hcy metabolism: methionine synthase (MS), betaine homocysteine

methyltransferase (BHMT), and cystathionine B-synthase (CBS). Several other

enzymes are indirectly involved. Vitamins B6, and B12 are cofactors to these enzymes

and folate is a substrate in de MS-mediated reaction. Disturbances on the methionine-

homocysteine metabolism either caused by genetic enzyme defect or owing to

deficiency of cofactors, normally result in a cellular accumulation of Hcy and

subsequently increased levels in the blood stream (7). In plasma, only about 1 % of Hcy

exists in the free reduced form. About 70 % of plasma Hcy is bound to albumin. The

rest forms disulphides, predominantly with cysteine or as the homocysteine dimer. The

sum of all the forms is termed total homocysteine. The liver and the kidney are

supposed to be the most important organs for uptake and metabolism of Hcy (6, 7).

Moreover, renal excretion is not an important route of elimination, being only a 1 % of

the Hcy filtered by the glomeruli and normally found in urine (7, 57). The rest of the

Hcy is reabsorbed and metabolized. Thus, the kidneys are Hcy-metabolising rather than

Hcy-excreting. The excess of methionine turns into Hcy by enzymatic transmethylation

reactions (103).

Hcy is formed from the essential amino acid methionine, provided by food proteins.

Methionine is converted in the presence of adenosine three phosphate (ATP)

into S-adenosylmethionine (SAM). SAM is an important methyl donor in many

biological reactions. After donating its methyl group, SAM is converted

into S-adenosylhomocysteine (SAH), which can be hydrolyzed into Hcy. There

are two physiologic pathways to metabolize Hcy: remethylation, and

transsulfuration. The remethylation pathway allows the recovery of methionine,

while the transsulfuration pathway, converts Hcy into cystathionine (CYS).


8

The remethylation of Hcy into methionine is catalyzed by the enzyme MS and its

cofactor methylcobalamin. A methyl group provided by 5-methyltetrahydrofolate (5-

MTHF) is transferred to MS, which then transfers it to Hcy producing

methionine. Homocysteine catabolism via the transsulfuration pathway is

mediated by the enzyme CBS, which requires vitamin B6 (pyridoxal 5- phosphate)

as a cofactor. CYS is then produced, and converted into cysteine and alpha-

ketobutyrate in presence of cystathioninase, another vitamin B6 dependent enzyme.

Cysteine can be further transformed into glutathione, the major constituent of the

antioxidant defense in humans (7). Additionally, Hcy has a key role in the one-carbon

metabolism. This role is shared with 5-MTHF. Both Hcy and 5-MTHF are substrates

for the production of tetrahydrofolate (THF) as well as methionine. THF is the form of

folate that can be used to synthesize purins (for DNA synthesis).

Figure 2. Methionine-Homocysteine cycle

THF: TetraHydroFolate CBS: Cystathionine b-Synthase BHMT: Beatine – Homocysteine MethylTransferase MTHFR: 5, 10-MethyleneTetraHydroFolate Reductase 5, 10 MTHF: 5, 10- MethylTetraHydroFolate 5 MTHF: 5-MethylTetraHydroFolate B6: Vitamin B6 B12: Vitamin B12 B2: Vitamin B2


9

Hcy catabolism via the transsulfuration pathway is favored under methionine overload

situation (after meals). On the other hand, Hcy-remethylation to methionine is

favored during the relative methionine shortage within the cells (fasting

conditions). The integrity of Hcy metabolism depends on the availability of vitamin

B12, vitamin B6 and folate in addition to several key enzymes. Important steps in the

metabolism of Hcy are illustrated in figure 2.

Hcy metabolism is tightly regulated under normal physiological situations. SAM can

enhance the transsulfuration pathway and inhibit the remethylation pathway. When

folate in the form of 5-MTHF is available, the production of SAM will be enhanced and

SAM can inhibit the formation of 5-MTHF from 5, 10-methylenetetrahydrofolate by

inhibiting the enzyme MTHFR (7). Unlike in folate deficiency, Vitamin B12 deficiency

is accompanied by a slight elevation of Hcy because the role of SAM in enhancing

the transsulfuration pathway is not affected by Vitamin B12 deficiency. SAM is the

main methyl donor in many biochemical reactions in humans like in the

synthesis of methylated phospholipids (phosphatidylcholine), nucleic acids,

amino acids, and neurotransmitters. Above all, the ratio SAM/SAH indicates the

methylation potential of the cell and is more important than the absolute

concentrations of each of these compounds. For example, a low SAM/SAH ratio

causes DNA-hypomethylation thus affecting gene expression (7).

1.1.4 Genetics and homocysteine

The MTHFR C677T gene polymorphism is the single most important genetic

determinant of blood Hcy values in the general population. MTHFR catalyzes the

irreversible conversion of 5, 10 methylenetetrahydrofolate to 5-MTHF in the

methionine cycle (114, 132), impairing their ability to fully activate (methylate) folic

acid to 5-MTHFR, the bioactive form of the B vitamin. Inheritance of the recessive T

allele results in reduced enzyme activity and increased Hcy concentrations; this is

especially true under low-folate conditions (23). The prevalence of the 677TT genotype

varies across regions and ethnic groups (24), being most common in Mexican (32 %),

Chinese (26 %) and southern Italian populations (20 %) and least common in those of

African origin (0.3–0.8 %) (11). The frequency of the 677TT genotype in Caucasians

ranges from 8 % to 14 % in North America, to 6 % to 14 % in northern Europe and 15


10

% to 20 % in southern Europe (134). Individuals who inherit this gene variant from both

parents have a significantly higher (14-21 %) risk of vascular disease than those who do

not. It should be noted that genetic factors have a much lower influence on

homocysteinemia than nutritional or lifestyle factors, and that a diet rich in folate and

vitamin B12 is an effective measure to prevent excessive Hcy and related conditions

(132).

1.1.5 Folate

Folate also called vitamin B9 is an essential water-soluble B-vitamin naturally found in

foods such as leafy green vegetables, legumes, egg yolks, liver and some citrus fruits

(69). Folate plays an important role in mental and emotional health. The main functions

are the normal cell growth and replication as well as synthesize, repair and methylate

DNA. Folate acts as a cofactor in certain biological reactions. The term folic acid refers

to the synthetic compound that is used in supplements and fortified foods (85). The term

folate will refer in this thesis to both, folic acid and natural food folate (132). Decreased

folate availability may occur when there is impaired folate absorption (e.g,

inflammation and infection of the gastrointestinal tract, specific gastrointestinal

diseases, and reduced dietary intake (135). As mentioned before, folate has a tight

relationship with vitamin B12 status, and Hcy concentrations sharing multiple pathways

interacting with one another (114). All three parameters together with inflammation or

infection are the biological predictors of folate status and have important roles in

influencing tHcy concentrations, although there are multiple factors that influence folate

status, including the physiological status (age, pregnancy/lactation), MTHFR

C677T gene polymorphism, or contextual factors such as comorbidity and low

socioeconomic status.

1.1.6 Vitamin B12

Vitamin B12, also called cobalamin, is an essential water-soluble B vitamin that plays a

key role in the normal functioning of the brain and central nervous system and for the

formation of red blood cells. It is normally involved in the metabolism of every cell in

the human body, especially in the DNA synthesis and regulation, and also in the fatty

acid amino acid metabolisms. Vitamin B12 is found in most animal derived foods,

including meat (especially liver), fish and shellfish, poultry, eggs, milk, and milk


11

products (131). Vitamin B12 is a co-substrate of various cell reactions involved in

methylation synthesis of nucleic acid and neurotransmitters. Vitamin B12 deficiencies

are commonly caused by low intakes of this vitamin, but can also result from wrong

absorption, certain intestinal disorders, low presence of binding proteins, and intake of

certain medications. Its deficiency could cause potential, severe and irreversible

damage, especially to the brain and nervous system. Slightly lower levels than

normal could have an effect on a variety of symptoms such as fatigue, depression, and

affect memory (3). Concentrations of vitamin B12 are closely related to

concentrations of blood Hcy, because as explained before, the active metabolite

of vitamin B12 is required for the methylation of Hcy in the production of methionine,

which is involved in a number of biochemical processes related to neurotransmitters

(132).

1.1.7 Creatine

Creatine is a compound endogenously synthesized by humans in the liver, kidney and

pancreas from arginine, glycine and methionine. It is also naturally founded in meat and

fish. Creatine from the diet is metabolized and then excreted by urine in the

form of creatinine. Muscles constitute a great source of creatine, about a 90-95 % of

creatine is located in the skeletal muscle; one third corresponds to free-creatine,

while the other two thirds are in the form of phosphocreatine. Muscles are a dynamic

storage and a rapid source of high-energy phosphate for high intensity performances

and short duration physical activities, where creatine is involved in a series of

phosphorylation/dephosphorylating reactions (115).

The first step in creatine synthesis is the reversible transfer of the amino group of

arginine to glycine to form guanidinoacetic acid (GAA) and ornithine in a reaction

catalyzed by the enzyme arginine: glycine amidinotransferase (AGAT), which is very

active in kidneys. Next, the irreversible transfer of a methyl group from SAM to GAA is

catalyzed by the enzyme guanidinoacetate N-methyltransferase (GAMT) (13, 28). The

products of this reaction are creatine and SAH. Therefore, creatine metabolism is

tightly conected to that of Hcy (119).

Creatine synthesis is responsible for a considerable consumption of SAM in the liver

and Hcy formation (28, 117). In short duration high intensity exercises, creatine

phosphate is required as an immediate energy source for muscle contraction. Thus, in

high intensity exercises, the increase in creatine synthesis demand can be a key factor in


12

the methyl balance modulation and one of the most important factors related

to increased Hcy in blood (29). However, the studies performed until now only analyzed

the effect of creatine supplementation before exercise in rats and humans, and

had contradictory results (27). Moreover, the relationship between tHcy and blood

creatine before and after acute exercise needs further investigation.

1.1.8 The beneficial effects of physical activity and exercise

Regular physical activity and exercise are associated with numerous physical and

mental health benefits in men and women. There is a long list of benefits

regarding physical activity and exercise for human health. Among many others,

exercise and physical activity decrease the risk of developing CVD, stroke, type 2

diabetes, and some forms of cancer (e.g., colon and breast cancers) (21).

Furthermore, regular physical activity lowers blood pressure; improves lipoprotein

profile, C-reactive protein, and other CVD biomarkers; enhances insulin sensitivity,

and plays an important role in weight management (21). Regarding mental

benefits, physical activity prevents and improves mild to moderate depressive

disorders and anxiety (44).

The physiologic responses of the body due to an aerobic or endurance exercise take

place in the musculoskeletal, cardiovascular, respiratory, endocrine, and immune

systems (62). Moreover, the physiological response to exercise is dependent on the

intensity, duration and frequency of the exercise as well as the environmental

conditions. During physical exercise, requirements for oxygen and substrate in skeletal

muscle are increased, as are the removal of metabolites and carbon dioxide. Chemical,

mechanical and thermal stimuli affect alterations in metabolic, cardiovascular and

ventilatory function in order to support these increased demands (15).

For the understanding of some results from this thesis it will be important to

pay attention to the difference between two terms of exercise physiology: the responses

and the adaptations to exercise. A response is an acute or short-term change

(adjustment) in the body that is associated with exercise; these responses refer to

the acute effect of exercise. In contrast, an adaptation to exercise involves a long-term

change in the body due to exercise training; the adaptations refer to the chronic effect

of exercise. The study of these types of responses and adaptations provides the

scientific basis for the field of exercise physiology (62).


1.1.9 The relation between exercise and homocysteine concentrations

The beneficial effect of physical exercise on cardiovascular health has been strongly

demonstrated (4, 44). However, it is unclear if exercise or physical activity can modify

or have an effect on tHcy concentrations. In the last few years, some research has

been focused on the role of exercise on tHcy concentrations; however, results obtained

from several studies are contradictory and sometimes inconclusive (33, 64, 70, 74). The

studies have been focused on the impact of lifestyle factors, physical activity level, and

cardiorespiratory fitness, chronic effect of exercise or acute effect of exercise on tHcy

concentrations. There is a variety of study populations in the different investigations

such as sedentary, elderly, athletes, obese, women or children as well as a

variety in the methodology applied among all the studies, hence, it is difficult

to reach an agreement (70). Some researchers have demonstrated reduced tHcy

concentrations after a training period (74, 101); others have related high physical

activity levels and cardiorespiratory fitness with lower Hcy concentrations (76,

109). In contrast, some studies have shown higher tHcy concentrations after acute

exercise, training period or after a specific sport competition (32, 64, 74). The

controversial results may be due, in part, to the lack of standardization among exercise

protocols, study populations, type of exercise intervention, training programs, or

timing and methodology of blood samples collection. In addition, one important

aspect is the wrongly generalized concept of “exercise” as a sole term for

different exercise responses involved in the physiological effects of exercise. All

in all, it could lead to a possible misunderstanding in the extrapolation of the

conclusions from the results. Altogether this makes it necessary to interpret

carefully previous results and to clarify the different research lines in the context

of “exercise effect” on tHcy concentrations.

1.1.10 Homocysteine and exercise. Mechanisms

The exact mechanism by which exercise affects tHcy concentrations continues to be

unknown. During prolonged exercise, skeletal muscle increases protein and amino acid

catabolism (13), a cortisol-dependent regulation that results in simultaneous liver amino

acid uptake to induce glucose synthesis (99). Moreover, mechanical contraction favors

the pool of methionine in the blood stream. The possible responses have focused on the

protein turnover during exercise, which could alter tHcy concentrations by increasing

methionine metabolism, or by decreasing vitamin B12 or folate availability. On the other

13


14

hand, during high intensities the increase of the methyl group turnover (70), which

implicates the creatine synthesis, appears to be another important pathway in the

relation between exercise and tHcy concentrations.

In the review conducted by Lanae & Manore, (70) the authors concluded that no

consistent relationship has been found between Hcy concentrations and exercise in

the last 10 years. The authors evidenced the necessity of conducting further

studies controlling nutritional status, the methodology of exercise

intervention, and the study population among many other factors.

Moreover a recent systematic review published in 2014 highlights some of the

results regarding the effects of training programs and physical activity on tHcy

concentrations, but studies with a period less than 6 weeks were excluded,

and consecuently, some of the studies analyzing the acute effect of exercise.

1.1.11 What do we know about homocysteine and exercise?

As mentioned before, it is important to discern the type of exercise, training programs of

physical activity levels that affect tHcy concentrations. In order to understand the state

of the art, a review of the literature was conducted. The studies analyzing tHcy

concentrations related to exercise in the last 15 years are included below (Tables 1-4).

A total of 30 articles are divided in four different groups: 1) effect of acute exercise on

tHcy concentrations, 2) chronic effect of exercise on tHcy concentrations, 3) relation of

physical activity level and cardiorespiratory fitness with tHcy concentrations and 4)

biomarkers related to tHcy and exercise.

The effect of acute exercise on tHcy concentrations

Table 1 summarizes the intervention studies containing the effect of acute exercise on

tHcy. From 11 articles, 7 studies were performed in athletes, recreational athletes or

well-trained participants, 3 in healthy sedentary or inactive participants for at least 6

months and 1 study in rats.

Nine studies consistently reported a significant increase of tHcy immediately after a

single bout of aerobic or resistance exercise (p < 0.05), independently of the type of

exercise, duration, intensity, intervention protocol or training level of the subjects. By

contrast, two studies showed no significant differences for tHcy with or without

exercise intervention.


15

Tab

le 1

. The

eff

ect o

f acu

te e

xerc

ise

on tH

cy c

once

ntra

tions

Ref

eren

ce

Titl

e A

im

Typ

e of

Exe

rcis

e Su

bjec

ts

Var

iabl

es

Res

ults

and

con

clus

ions

T

ype

Inte

nsity

D

urat

ion

Her

rman

n et

al.

(200

3)

Hom

ocys

tein

e in

crea

ses

durin

g en

dura

nce

exer

cise

.

To

asse

ss

plas

ma

hom

ocys

tein

e af

ter

mar

atho

n, 1

00km

run,

and

12

0km

bik

e ra

ce.

Aer

obic

: -M

arat

hon

race

-100

km

run

-120

km

mou

ntai

n bi

kera

ce

Vig

orou

s >

than

1hou

r10

0 re

crea

tiona

l at

hlet

es (♀

and

♂)

(Mar

atho

n n=

46;

100

km ru

n n=

12;

Mou

ntai

n bi

ke ra

ce

n=42

).

-Hcy

-Fol

ate

-B12

-↑ H

cy o

vera

l sam

ple

afte

r exe

rcis

e.

-↑ H

cy 6

4 %

afte

r mar

atho

n gr

oup.

-N

S H

cy: A

fter 1

00 k

m ru

n an

d 12

0km

bik

e ra

ce.

Kon

ig e

t al.

(200

3)

Influ

ence

of t

rain

ing

volu

me

and

acut

e ph

ysic

al e

xerc

ise

on th

e ho

moc

yste

ine

leve

ls in

en

dura

nce-

train

ed m

en:

Inte

ract

ions

with

pl

asm

a fo

late

and

vi

tam

in B

12.

To a

sses

s th

e in

fluen

ce o

f ex

tens

ive

endu

ranc

e tra

inin

g an

d ac

ute

exer

cise

on

pl

asm

a co

ncen

tratio

ns

of

Hcy

, vi

tam

in B

12, a

nd fo

lic a

cid

in

42

wel

l-tra

ined

m

ale

triat

hlet

es.

Aer

obic

: -S

prin

ttri

athl

on(S

wim

min

g40

0 m

,bi

cycl

e 25

000

m, r

un 4

000

m)

Vig

orou

s >

than

1hou

r39

trai

ned

(27.

1 yr

). -H

cy-F

olat

e-B

12

-↑ H

cy a

fter 1

h an

d 24

h o

fco

mpe

titio

n.-N

S in

B12

afte

r com

petit

ion.

-↑ fo

late

1 h

afte

r com

petit

ion.

-↑ fo

late

in L

ow T

rain

ing

grou

paf

ter c

ompe

titio

n.

Rea

l et a

l. (2

004)

Ef

fect

s of m

arat

hon

runn

ing

on p

lasm

a to

tal

hom

ocys

tein

e co

ncen

tratio

ns.

To

inve

stig

ated

th

e ch

ange

s in

pla

sma

tHcy

co

ncen

tratio

ns 2

4h b

efor

e an

d af

ter a

mar

atho

n ra

ce.

Aer

obic

: M

arat

hon

race

: 42.

195

m

Vig

orou

s >

than

1hou

r22

non

-pro

fess

iona

l ♂

athl

etes

(35.

6 yr

).-H

cy-B

12-F

olat

e-M

THFR

677T

T ge

noty

pe

-↑ H

cy (1

9 %

) 24

h po

st-r

ace.

-NS

fola

te a

nd B

12 p

ost-r

ace

(24

h).

-Cor

rela

tion

Hcy

-Fol

ate

Post

race

.

Gel

ecek

et

al. (

2007

) In

fluen

ces o

f acu

te a

nd

chro

nic

aero

bic

exer

cise

on

the

plas

ma

hom

ocys

tein

e le

vel.

To

inve

stig

ate

the

influ

ence

of

subm

axim

al

acut

e ae

robi

c ex

erci

se a

nd

aero

bic

train

ing

on H

cy

leve

ls.

Trea

dmill

ae

robi

c ex

erci

se

Vig

orou

s (7

0-80

%

HR

max

)

-30

min

69 ♂

and

♀

(21.

12yr

) (3

gro

ups:

Acu

te

n=22

; crh

onic

n=

29; c

ontro

l n=2

8).

-Hcy

-Lip

id p

rofil

e-↑

Hcy

acu

te su

bmax

imal

exe

rcis

e.

Sotg

ia e

t al.

(200

7).

Acu

te v

aria

tions

in

hom

ocys

tein

e le

vels

are

re

late

d to

cre

atin

e ch

ange

s ind

uced

by

phys

ical

act

ivity

.

To

inve

stig

ate

whe

ther

th

e m

odifi

catio

n in

Hcy

le

vel

afte

r a

mod

erat

e ph

ysic

al

activ

ity

was

ex

plai

nabl

e in

the

light

of

the

com

mon

co

nnec

tion

of

phys

ical

ac

tivity

an

d H

cy to

cre

atin

e.

Incr

emen

tal

cycl

oerg

omet

er

max

imal

test

Incr

emen

tal

to

exha

ustio

n

16 y

oung

subj

ects

(2

1-37

yr).

-Sed

enta

ry (6

)-A

thle

tes (

10)

-tH

cy-r

Hcy

-Cre

atin

ine

-↑ C

reat

inin

e in

crea

sed

afte

rex

erci

se in

bot

h gr

oups

.-N

S tH

cy a

fter e

xerc

ise.


16

Ven

ta e

t al.

(200

9).

Plas

ma

vita

min

s, am

ino

aci

ds,a

nd re

nal f

unct

ioni

n po

st-e

xerc

ise

hy

perh

omoc

yste

inem

ia.

To st

udy

the

effe

ct o

f di

ffer

ent a

cute

aer

obic

ex

erci

ses o

n pl

asm

a,

redu

ced,

and

tota

l Hcy

(r

Hcy

, tH

cy) a

nd c

yste

ine

(rC

ys, t

Cys

) and

on

its

met

abol

ical

ly re

late

d vi

tam

ins a

nd a

min

o ac

ids.

Incr

emen

tal

spec

ific

to

exha

ustio

n te

st

(Cyc

le

ergo

met

er

and

kay

ak

ergo

met

er)

Incr

emen

tal

to

exha

ustio

n

-28

min

-21

min

-15

cycl

ists

-14

kaya

kers

(14-

22yr

)

-Hcy

-rH

cy-F

olat

e-B

12-C

reat

inin

e

-↑ H

cy a

fter a

cute

exe

rcis

e re

late

d to

the ↑i

n rH

cy.

-↑ B

12 a

nd c

reat

inin

e af

ter a

cute

exer

cise

s.-N

S in

Fol

ate

inde

pend

ently

of t

heex

erci

se a

nd v

itam

in st

atus

.

Dem

inic

e et

al

. (20

11)

Cre

atin

e su

pple

men

tatio

n re

duce

s inc

reas

ed

hom

ocys

tein

e co

ncen

tratio

n in

duce

d by

acu

te e

xerc

ise

in

rats

.

To e

valu

ate

the

effe

ct o

f cr

eatin

e su

pple

men

tatio

n on

hom

ocys

tein

e m

etab

olis

m a

fter a

cute

ae

robi

c an

d an

aero

bic

exer

cise

.

-Sw

imm

ing

4%

Bod

yw

eigh

t loa

d-6

x30

verti

cal

jum

ps 5

0 %

body

wei

ght

load

-Mod

erat

eae

robi

cex

erci

se.

-Mod

erat

ean

aero

bic

exer

cise

-1 h

our

112

wis

tar r

ats

(4 g

roup

s):

-Aer

obic

exe

rcis

e-A

erob

ic e

xerc

ise

+cr

eatin

esu

pple

men

tatio

n.-A

naer

obic

exer

cise

-Ana

erob

icex

erci

se +

supp

lem

enta

tion

-Hcy

-Cre

atin

e-↑

Hcy

leve

ls a

fter a

naer

obic

exer

cise

.-↑

cre

atin

e af

ter e

xerc

ise

in th

e 4

grou

ps.

-↓ H

cy w

hen

crea

tine

supp

lem

enta

tion

inde

pend

ently

of

the

type

exe

rcis

e.

Biz

heh

and

Jaaf

ari

(201

1)

The

effe

ct o

f a si

ngle

bo

ut c

ircui

t res

ista

nce

exer

cise

on

hom

ocys

tein

e, h

s-C

RP

and

fibrin

ogen

in

sede

ntar

y m

iddl

e-ag

ed

men

.

Exam

ine

the

effe

ct o

f a

sing

le b

out o

f circ

uit

resi

stan

ce e

xerc

ise

on

Hcy

.

Res

ista

nce

train

ing

prog

ram

: C

ircui

t of 1

0 re

sist

ance

ex

erci

ses

-35

% o

fR

M-1

2 s x

3tim

es-2

3 he

alth

y in

activ

e♂ -1

4 tra

inin

g-9

con

trol

-Hcy

-hs-

CR

P-↑

Hcy

afte

r exe

rcis

e.

-↑ h

s-C

RP.

Igle

sias

- G

utie

rrez

et

al.

(201

2)

Tran

sien

t inc

reas

e in

ho

moc

yste

ine

but n

ot

hype

rhom

ocys

tein

emia

du

ring

acut

e ex

erci

se a

t di

ffer

ent i

nten

sitie

s in

sede

ntar

y in

divi

dual

s.

To d

eter

min

e th

e ki

netic

s of

seru

m h

omoc

yste

ine

at

diff

eren

t int

ensi

ties.

2 cy

cle

exer

cise

is

ocal

oric

tri

als

(400

kcal

): H

igh

inte

nsity

(H

i) an

d Lo

w

inte

nsity

(Li).

-40

% V

O2

peak

-80

% V

O2

peak

-40

min

-40

min

-8 se

dent

ary ♂

(18-

27 y

r)-H

cy-B

12

-B6

-Fol

ate

-C67

7TM

THFR

geno

type

-↑ H

cy d

urin

g ex

erci

se in

bot

h H

i(h

igh

inte

nsity

) and

Li (

Low

inte

nsity

).-H

i max

val

ue o

f Hcy

: 25

min

afte

rex

erci

se.

-Li m

ax v

alue

of H

cy 3

7.5

min

afte

rex

erci

se.

-Hcy

reco

vere

d at

24

h.↑

Fola

te, B

12, B

6 du

ring

exer

cise

inbo

th H

i and

Li.

-Lar

ge v

aria

bilit

y in

cor

rela

tions

.

Con

tinua

tion

1 of

tabl

e 1


17

Mal

es: ♂

; Fe

mal

es: ♀

; ↑: I

ncre

ase;

Dec

reas

e: ↓

; NS:

No

stat

istic

al d

iffer

ence

s; y

r: y

ears

; B12

: Vita

min

B12

; B6:

Vita

min

B6;

Hcy

: Hom

ocys

tein

e; tH

cy: t

otal

Hom

ocys

tein

e.

Ham

mou

da

et a

l. (2

012)

Effe

ct o

f sho

rt-Te

rm

max

imal

exe

rcis

e on

bi

oche

mic

al m

arke

rs o

f m

uscl

e da

mag

e, to

tal

antio

xida

nts s

tatu

s, an

d ho

moc

yste

ine

leve

ls in

fo

otba

ll pl

ayer

s.

To a

sses

s the

eff

ect o

f sh

ort-t

erm

max

imal

ex

erci

se o

n m

arke

rs o

f m

uscl

e da

mag

e,

hom

ocys

tein

e an

d to

tal

antio

xida

nts s

tatu

s in

train

ed su

bjec

ts.

Win

gate

test

s -M

axim

alsp

rint

-30

s18

♂ fo

otba

ll pl

ayer

s (17

.5yr

) -H

cy-C

reat

inin

e-C

K

-NS

Hcy

afte

r exe

rcis

e.-↑

Cre

atin

ine.

Dem

inic

e et

al

. (20

13)

Shor

t-ter

m c

reat

ine

supp

lem

enta

tion

does

no

t red

uce

incr

ease

d ho

moc

yste

ine

conc

entra

tion

indu

ced

by a

cute

exe

rcis

e in

hu

man

s.

To e

valu

ate

the

effe

cts o

f cr

eatin

e su

pple

men

tatio

n on

hom

ocys

tein

e (H

cy)

plas

ma

leve

ls a

fter a

cute

ex

erci

se in

hum

ans.

Acu

te

anae

robi

c hi

gh in

tens

ity

-Spr

int

exer

cise

6x35

m te

st

23 y

oung

socc

er

play

ers d

ivid

ed in

2

grou

ps:

-Cre

atin

esu

pple

men

tatio

n-P

lace

bo

-Hcy

-Cre

atin

e-F

olat

e-B

12

-↑ H

cy le

vels

afte

r exe

rcis

e (1

8 %

).-↑

cre

atin

e le

vels

afte

r 7 d

ays o

fcr

eatin

e su

pple

men

tatio

n.-N

S Fo

late

.-↑

B12

afte

r exe

rcis

e.

Con

tinua

tion

2 of

tabl

e 1

18

The effect of chronic exercise on tHcy concentrations

The chronic effects of exercise and training programs on tHcy concentrations are shown

in table 2. From 10 articles, six studies analyzed aerobic training programs andother 4

studies, resistance-training programs. Regarding the cronic effect of aerobic training

on tHcy, 2 articles reported a decrease in tHcy, 4 showed no tHcy changes, one of

them only in blacks and 2 studies observed a tHcy increase after an aerobic training

program. On the other hand, from those 4 studies performing resistance-training

programs, 2 of them showed a decrease in tHcy and the other two showed an

increase of tHcy after exercise interventions.



19

Tab

le 2

. The

eff

ect o

f chr

onic

exe

rcis

e on

tHcy

con

cent

ratio

ns

Ref

eren

ces

Titl

e A

im

Typ

e of

exe

rcis

e

Subj

ects

V

aria

bles

Res

ults

T

ype

Freq

uenc

y In

tens

ity a

nd

dura

tion

Peri

od

trai

ning

Ran

deva

, et

al.

(200

2)

Exer

cise

Dec

reas

es

Plas

ma

Tota

l H

omoc

yste

ine

in

Ove

rwei

ght Y

oung

W

omen

with

Pol

ycys

tic

Ova

ry S

yndr

ome.

To e

xam

ine

the

effe

cts o

f ex

erci

se o

n pl

asm

a to

tal

hom

ocys

tein

e co

ncen

tratio

ns in

you

ng

over

wei

ght o

r obe

se

Poly

cyst

ic O

vary

Syn

drom

e (P

CO

S) w

omen

.

Aer

obic

bris

k w

alki

ng

At l

east

3

wal

ks p

er

wee

k 20

-60

min

dur

atio

n

6 mon

ths

exer

cise

21 o

bese

♀

(30.

6 yr

) di

vide

d in

2

grou

ps:

-Exe

rcis

e(1

2)-N

on e

xerc

ise

(9)

-Hcy

-Fol

ate

-Cre

atin

ine

-↓ H

cy e

xerc

ise

grou

p.-P

atie

nts w

ith h

ighe

r Hcy

leve

ls

in e

xerc

ise

g rou

p al

so h

ave

the

high

er ↓

afte

r exe

rcis

e tra

inin

g.-N

S in

B12

, Fo

late

Cre

atin

ine

betw

een

base

line

and

6 m

onth

s af

ter i

n bo

th g

roup

s.-↓

Hcy

by

regu

lar e

xerc

ise.

Vin

cent

et

al.

(200

3)

Hom

ocys

tein

e an

d Li

popr

otei

n Le

vels

Fo

llow

ing

Res

ista

nce

Trai

ning

in O

lder

Adu

lts.

To e

xam

ine

the

effe

ct o

f 6

mon

ths o

f hig

h- o

r low

-in

tens

ity re

sist

ance

exe

rcis

e on

seru

m h

omoc

yste

ine

and

lipop

rote

in (a

) lev

els i

n ad

ults

age

d 60

–80

year

s.

6 m

onth

s re

sist

ance

tra

inin

g

13 re

p 50

%

of 1

RM

or 8

re

p 80

% 1

R

M

3 tim

es

per

wee

k fo

r 24

w

eeks

43 ♂

and

♀

(60-

80 y

r)

-Con

trol (

10)

-Low

inte

nsity

(18)

-Hig

hin

tens

ity (1

5)

-Hcy

-↓ H

cy in

bot

h tra

inin

g gr

oups

.

Kon

ig e

t al.

(200

3)

Influ

ence

of t

rain

ing

volu

me

and

acut

e ph

ysic

al e

xerc

ise

on th

e ho

moc

yste

ine

leve

ls in

en

dura

nce-

train

ed m

en:

Inte

ract

ions

with

pla

sma

fola

te a

nd v

itam

in B

12.

To a

sses

s the

influ

ence

of

exte

nsiv

e en

dura

nce

train

ing

and

acut

e ex

erci

se

on p

asm

a co

ncen

tratio

ns o

f H

cy, v

itam

in B

12, a

nd fo

lic

acid

in 4

2 w

ell-t

rain

ed m

ale

triat

hlet

es.

Aer

obic

(S

prin

t tri

athl

on:

Swim

min

g 40

0 m

, bi

cycl

e 25

000

m, r

un

4000

m).

Mea

n du

ratio

n: 6

7.1

min

39 tr

ainn

ed

27.1

yr

-Hcy

-Fol

ate

-B12

-NS

Hcy

afte

r tra

inin

g.-N

S Fo

late

afte

r tra

inin

g.

Bor

eham

et

al. (

2005

) Tr

aini

ng e

ffec

ts o

f sho

rt bo

uts o

f sta

ir cl

imbi

ng o

n ca

rdio

resp

irato

ry fi

tnes

s, bl

ood

lipid

s, an

d ho

moc

yste

ine

in

sede

ntar

y yo

ung

wom

en.

To st

udy

the

train

ing

effe

cts

of e

ight

wee

ks o

f sta

ir cl

imbi

ng o

n V

O2 m

ax,

bloo

d lip

ids,

and

hom

ocys

tein

e in

sede

ntar

y,

but o

ther

wis

e he

alth

y yo

ung

wom

en.

Aer

obic

: Sta

ir cl

imbi

ng

Prog

ress

ive

8 w

eeks

15

♀ (1

8.8

yr)

-Sta

ir cl

ibin

g(n

=8)

-Con

trol

(n=7

)

-Hcy

-N

S H

cy.


20

Mal

es: ♂

; Fe

mal

es: ♀

; ↑: I

ncre

ase;

Dec

reas

e: ↓

; NS:

No

stat

istic

al d

iffer

ence

s; y

r: y

ears

; B12

: Vita

min

B12

; B6:

Vita

min

B6;

Hcy

: Hom

ocys

tein

e; tH

cy: t

otal

Hom

ocys

tein

e.

Oku

ra e

t al.

(200

6)

Effe

ct o

f re

gula

r ex

erci

se

on

hom

ocys

tein

e co

ncen

tratio

ns:

the

HER

ITA

GE

Fam

ily

Stud

y.

Whe

ther

re

gula

r ae

robi

c ex

erci

se c

ould

aff

ect p

lasm

a to

tal h

omoc

yste

ine

(tHcy

), an

d w

heth

er t

here

w

ere

sex-

rela

ted

or

raci

al

diff

eren

ces i

n tH

cy c

hang

es.

Aer

obic

ex

erci

se

train

ing.

C

yclo

ergo

met

er

55 %

VO

2max

30

min

La

st 6

wee

ks:

75 %

of V

O2

max

50

min

3

times

per

w

eek

20

wee

ks

730

subj

ects

bl

ack

and

with

m

en a

nd ♀

(1

7-65

yr)

.

-Hcy

-B6

-B12

-NS

tHcy

in B

lack

s with

train

ing.

-↑ H

cy si

gnifi

cant

ly in

whi

tes.

-Hyp

erho

moc

yste

inem

ia le

vels

decr

ease

d si

gnifi

cant

ly w

ithre

gula

r aer

obic

exe

rcis

e.-N

S B

6.-↓

B12

in a

ll gr

oups

.-↑

Fol

ate

only

in b

lack

s.V

ince

nt e

t al

. (20

06)

Res

ista

nce

Trai

ning

Lo

wer

s Ex

erci

se-I

nduc

ed

Oxi

dativ

e St

ress

an

d H

omoc

yste

ine

Leve

ls in

O

verw

eigh

t an

d O

bese

Old

er A

dults

.

To c

ompa

re o

xida

tive

stre

ss

and

leve

ls o

f ho

moc

yste

ine

and

chol

este

rol

in n

orm

al-

wei

ght

and

over

wei

ght

olde

r ad

ults

afte

r re

sist

ance

ex

erci

se.

Res

ista

nce

exer

cise

s. 8-

13 re

p50

-80

% R

M6 m

onth

s 49

old

er (6

0-72

yr)

. (N

orm

al

wei

ght,

over

wei

ght

and

obes

e)

-Hcy

-↓ H

cy in

bot

hov

erw

eigh

t/obe

se a

nd n

orm

al-

wei

ght r

esis

tanc

e tra

inin

ggr

oups

com

pare

d w

ith c

ontro

lgr

oups

.

Gel

ecek

et

al. (

2007

) In

fluen

ces

of

acut

e an

d ch

roni

c ae

robi

c ex

erci

se

on

the

plas

ma

hom

ocys

tein

e le

vel.

To in

vest

igat

e th

e in

fluen

ce

of

subm

axim

al

acut

e ae

robi

c ex

erci

se a

nd a

erob

ic

train

ing

on H

cy le

vels

.

Trea

dmill

ae

robi

c ex

erci

se

Bris

k w

alki

ng

Aer

obic

6

wee

ks

69 ♂

and

♀

(21.

12 y

r)

(Acu

te n

=22;

cr

honi

c n=

29;

cont

rol n

=28)

.

-Hcy

-Lip

idpr

ofile

-NS

Hcy

chr

onic

exe

rcis

e.

Guz

el e

t al.

(201

2)

Long

-Ter

m

Cal

listh

enic

Ex

erci

se–R

elat

ed

Cha

nges

in

Blo

od L

ipid

s, H

cy,

Nitr

ic O

xide

Lev

els

and

Bod

y C

ompo

sitio

n in

M

iddl

e-A

ged

Hea

lthy

Sede

ntar

y W

omen

.

To in

vest

igat

e th

e ef

fect

s of

ca

llist

heni

c ex

erci

ses

on

plas

ma

lipid

s, H

cy,

tota

l ni

tric

oxid

e (N

O).

Cal

liste

nic

exer

cise

s in

term

edia

te

inte

nsity

.

50 m

in 3

tim

es/w

eek

12

wee

ks

42 m

iddl

e-ag

ed h

ealth

y se

dent

ary ♀

(4

1,40

yr)

.

-Hcy

-NO

-↑H

cy le

vels

by

long

-term

calli

sthe

nic

exer

cise

s.

Mol

ina-

Lópe

z et

al.

(201

3)

Effe

ct

of

folic

ac

id

supp

lem

enta

tion

on

Hom

ocys

tein

e co

ncen

tratio

n an

d as

soci

atio

n w

ith t

rain

ing

in h

andb

all p

laye

rs.

To

eval

uate

nu

tritio

nal

stat

us

for

mac

ronu

trien

ts

and

folic

aci

d in

mem

bers

of

a

high

-per

form

ance

ha

ndba

ll te

am,

with

a f

olic

ac

id su

pple

men

tatio

n.

Trai

ning

pe

riod

(han

dbal

l pl

ayer

s).

4 da

y/w

eek

Gro

ups:

<

60 %

60

-80

%<

80 %

of 1

RM

4 mon

ths

14 H

andb

all

play

ers h

igh

perf

orm

ance

(2

2.9

yr).

-Hcy

-↑ H

cy a

t wee

k 8

and

16re

spec

t bas

elin

e.-

Cor

rela

tion

inve

rse

betw

een

Hcy

and

folic

aci

d at

16

wee

k in

< 60

% in

tens

ity g

roup

.

Cho

i et a

l. (2

014)

R

egul

ar E

xerc

ise

Trai

ning

In

crea

ses

the

Num

ber

of

Endo

thel

ial

Prog

enito

r C

ells

and

Dec

reas

es H

cy

Leve

ls in

Hea

lthy

Perip

hera

l Blo

od.

To

dete

rmin

e w

heth

er

endo

thel

ial

prog

enito

r ce

ll co

lony

-for

min

g as

say

EPC

nu

mbe

rs c

ould

be

incr

ease

d th

roug

h re

gula

r ex

erci

se

train

ing.

Aer

obic

tre

adm

ill

exer

cise

.

Ana

erob

ic

exer

cise

.

30 m

inut

es a

t 60

% o

f HR

. 28

day

re

gula

r ex

erci

se

5 (2

5-30

yr)

. -H

cy-A

fter 2

8 da

ys o

f tra

inin

g:↓

Hom

ocys

tein

e le

vels

.-I

nver

se C

orre

latio

n be

twee

nEP

C-C

FU a

nd H

cy in

Hea

lthy

men

.

Con

tinua

tion

1 of

tabl

e 2


21

Relation of Physical Activity (PA) levels and cardiorespiratory fitness with tHcy

concentration

Table 3 shows the studies analyzing the association between PA levels and/or

cardiorespiratory fitness with tHcy concentrations. A total of 9 studies were categorized

into this group. Three of the 5 studies focused on cardiorespiratory fitness found an

inverse association with tHcy concentrations in women. On the other hand, six articles

described and analyzed the correlation between PA levels and tHcy. Three of them

found lower tHcy concentrations in athletes or in subjects with higher levels of PA

compared to sedentary ones. Otherwise, neither intensity, nor duration or frequency

showed significant associations with tHcy across the nine studies.


22

Tab

le 3

. Rel

atio

n of

phy

sica

l act

ivity

(PA

) lev

els a

nd c

ardi

ores

pira

tory

fitn

ess w

ith tH

cy c

once

ntra

tions

Ref

eren

ces

Titl

e A

im

Subj

ects

V

aria

bles

R

esul

ts

Ku

et a

l. (2

005)

Le

vels

of h

omoc

yste

ine

are

inve

rsel

y as

soci

ated

with

ca

rdio

vasc

ular

fitn

ess i

n w

omen

, but

no

t in

men

: dat

a fr

om th

e N

atio

nal

Hea

lth a

nd N

utrit

ion

Exam

inat

ion

Surv

ey 1

999–

2002

.

To a

sses

s the

ass

ocia

tion

betw

een

elev

ated

hom

ocys

tein

e an

d ca

rdio

vasc

ular

fitn

ess.

1444

non

-in

stitu

tiona

lized

ad

ults

(20-

49 y

r).

-Car

dior

espi

rato

ryfit

ness

by

Subm

axim

alte

st

-Hcy

leve

ls w

ere

inve

rsel

y as

soci

ated

toca

rdio

resp

irato

ry fi

tnes

s in

wom

en, b

ut n

otin

men

.

Rou

ssea

u et

al

. (2

005)

Pl

asm

a ho

moc

yste

ine

is r

elat

ed t

o fo

late

inta

ke b

ut n

ot tr

aini

ng st

atus

To

de

term

ine,

w

heth

er

diet

ary

fact

ors

such

as

inta

kes

of f

olat

e,

vita

min

B

6 an

d B

12

wer

e as

soci

ated

w

ith

low

er

plas

ma

tHcy

in a

thle

tes.

74

wel

l tra

ined

at

hlet

es

4 gr

oups

: Se

dent

ary,

EE1

, EE

2, E

E3 a

nd

grou

ped

by ty

pe

of e

xerc

ise.

-Hcy

-Die

tary

in

take

of

vita

min

s B

6, B

12

and

fola

te

(7-d

ay

diet

ary

and

activ

ity re

cord

s)

-H

cy w

as ↓

in

athl

etes

with

hig

h EE

(ene

rgy

expe

nditu

re)

com

pare

d to

ath

lete

sw

ith lo

wer

EE.

-Hcy

was

↓ in

aer

obic

ath

lete

s co

mpa

red

toin

term

itten

t at

hlet

es a

nd s

eden

tary

sub

ject

sbu

t not

with

ana

erob

ic g

roup

.

Unt

et a

l. (2

007)

H

omoc

yste

ine

stat

us in

form

er to

p-le

vel m

ale

athl

etes

: pos

sibl

e ef

fect

of

phy

sica

l act

ivity

and

phy

sica

l fit

ness

.

To st

udy

the

efec

t of p

hysi

cal

activ

ity a

nd p

hysi

cal f

itnes

s on

Hcy

stat

us in

top

athl

ets.

118

mid

dle

aged

77

form

er ♂

at

hlet

es a

nd 3

3 se

dent

ary

cont

rols

(3

5-78

yr)

.

-Hcy

-Phy

sica

l act

ivity

-Phy

sica

lly a

ctiv

e ex

ath

lete

s sho

wed

low

er H

cy c

ompa

red

to se

dent

ary

ones

.-N

o re

latio

n am

ong

Hcy

and

exe

rcis

e,fr

eque

ncy

dura

tion

and

inte

nsity

.-C

urre

nt p

hysi

cal a

ctiv

ity a

ndca

rdio

resp

irato

ry fi

tnes

s are

inve

rsel

yas

soci

ated

with

↑ H

cy le

vel i

n m

iddl

e-ag

edfo

rmer

ath

lete

s.D

ankn

er e

t al.

(200

7)

Phys

ical

act

ivity

is in

vers

ely

asso

ciat

ed w

ith to

tal h

omoc

yste

ine

leve

ls, i

ndep

ende

nt o

f C67

7T

MTH

FR g

enot

ype

and

plas

ma

B

vita

min

s

To fu

rther

elu

cida

te th

e ob

serv

ed

asso

ciat

ion

betw

een

hom

ocys

tein

e an

d ph

ysic

al

activ

ity

620 ♂

and

♀,

(70.

5 yr

). -P

A-M

THFR

C67

7Tge

noty

pe-F

olat

e-B

12

-Phy

sica

lly a

ctiv

e su

bjec

ts h

ad ↓

Hcy

leve

ls.

-Inv

erse

cor

rela

tions

bet

wee

n bo

dy m

ass

inde

x, p

las m

a fo

late

, B12

and

Hcy

leve

ls.

-Fol

ate,

B12

, and

C67

7T g

enot

ype,

asso

ciat

ed w

ith H

cy le

vels

.R

uiz

et a

l. (2

007)

H

omoc

yste

ine

leve

ls in

chi

ldre

n an

d ad

oles

cent

s are

ass

ocia

ted

with

the

met

hyle

nete

trahy

drof

olat

e re

duct

ase

677C

> T

gen

otyp

e, b

ut n

ot w

ith

phys

ical

act

ivity

, fitn

ess o

r fat

ness

: Th

e Eu

rope

an Y

outh

Hea

rt St

udy

To e

xam

ine

the

asso

ciat

ions

tHcy

w

ith p

hysi

cal a

ctiv

ity,

card

iore

spira

tory

fitn

ess a

nd

fatn

ess i

n ch

ildre

n an

d ad

oles

cent

s.

301

child

rens

and

37

9 ad

oles

cent

s. -P

A (a

ccel

erom

eter

)-C

677T

MTH

FRge

noty

pe-C

ardi

ores

pira

tory

fitne

ss

-PA

, fitn

ess a

nd b

ody

fat a

re n

ot a

ssoc

iate

dw

ith tH

cy le

vels

in c

hild

ren

and

adol

esce

nts,

even

afte

r con

trolli

ng fo

rpr

esen

ce o

f the

MTH

FR C

677T

.-T

gen

otyp

e is

the

mai

n in

fluen

ce o

n ↑

tHcy

leve

ls in

thes

e su

bjec

ts.


23

Mal

es: ♂

; Fe

mal

es: ♀

; ↑: I

ncre

ase;

Dec

reas

e: ↓

; NS:

No

stat

istic

al d

iffer

ence

s; y

r: y

ears

; B12

: Vita

min

B12

; Hcy

: Hom

ocys

tein

e; tH

cy: t

otal

Hom

ocys

tein

e.

Rui

z et

al.

(200

7)

Car

diov

ascu

lar F

itnes

s Is

Neg

ativ

ely

Ass

ocia

ted

With

Hom

ocys

tein

e Le

vels

in

Fem

ale

Ado

lesc

ents

.

To e

xam

ine

the

asso

ciat

ion

betw

een

card

iova

scul

ar fi

tnes

s an

d ho

moc

yste

ine

leve

ls in

ad

oles

cent

s.

156

adol

esce

nts

(14.

8 yr

). 20

m sh

uttle

run

test

.

-Fol

ic a

cid

-B12

-Hcy

-C

677T

MTH

FRG

enot

ype

-Hcy

hig

her i

n 67

7CT

AN

D T

T ge

noty

pes

com

pare

d to

CC

in a

dole

scen

ts.

-Car

diov

ascu

lar f

itnes

s is n

egat

ivel

yas

soci

ated

with

Hcy

leve

ls in

fem

ale

adol

esce

nts.

-No

asso

ciat

ion

betw

een

Car

diov

ascu

lar

fitne

ss a

nd H

cy in

mal

es.

-Hcy

and

B12

inve

rsel

y as

soci

ated

to H

cy.

Di S

anto

lo, e

t al.

(200

8)

Ass

ocia

tion

of re

crea

tiona

l phy

sica

l ac

tivity

with

hom

ocys

tein

e, fo

late

an

d lip

id m

arke

rs in

you

ng w

omen

.

To a

sses

s the

influ

ence

of

recr

eatio

nal p

hysi

cal a

ctiv

ity in

yo

ung

heal

thy

wom

en o

n ho

moc

yste

ine.

-124

You

nghe

alth

yre

crea

tiona

l ♀at

hlet

es.

-116

con

trols

(23

yr).

-Hcy

-Fol

ate

-Lip

id m

arke

rs-C

reat

inin

e-R

ecre

atio

nal P

A

-Hcy

inve

rsel

y to

fola

te a

nd p

ositi

ve to

crea

tinin

e.-R

ecre

atio

nal P

A n

o as

soci

atio

n to

Hcy

leve

ls a

mon

g yo

ung

wom

en.

Lana

e M

. Jou

bert

and

Mel

inda

M.

Man

ore

(200

8)

The

Rol

e of

Phy

sica

l Act

ivity

Lev

el

and

B-V

itam

in S

tatu

s on

Blo

od

Hom

ocys

tein

e Le

vels

.

To d

eter

min

e w

heth

er p

lasm

a H

cy v

alue

s, in

depe

nden

t of

plas

ma

B-v

itam

in c

once

ntra

tions

, ar

e hi

gher

in a

ctiv

e th

an le

ss

activ

e m

asle

s and

fem

ales

.

Hea

lthy

youn

g (2

6 yr

) phy

sica

lly

activ

e ♂

div

ided

in

gro

ups:

M

oder

ate

to h

igh

inte

nsity

.

-PA

(7 d

ay P

hysi

cal

activ

ity re

cord

)-H

cy-V

itam

ins B

-Hcy

, ind

epen

dent

of p

lasm

a B

-vita

min

leve

ls, w

as n

ot d

iffer

ent b

etw

een

PA le

vels

in n

on-s

upp l

emen

ting

youn

g ad

ults

.-F

olat

e in

vers

ely

asso

ciat

ed to

Hcy

.

Dan

kner

et a

l. (2

009)

C

ardi

ores

pira

tory

Fitn

ess a

nd

Plas

ma

Hom

ocys

tein

e Le

vels

in A

dult

Mal

es a

nd F

emal

es

To fu

rther

exp

lore

the

rela

tions

hip

betw

een

card

iore

spira

tory

fitn

ess a

nd

plas

ma

tota

l hom

ocys

tein

e le

vel.

2576

adu

lts (6

2 %

♂

) (30

-59

yr).

-tH

cy-N

o as

soci

atio

n w

as fo

und

betw

een

leve

lof

car

dior

espi

rato

ry fi

tnes

s and

pla

sma

tHcy

in m

en o

r wom

en.

Con

tinua

tion

1 of

tabl

e 3


24

Implicated biomarkers related to tHcy concentrations and exercise

Table 4 summarizes those studies that analyze the relation of implicated biomarkers on

the tHcy and exercise. A total of 12 studies were selected into this group. Associations

of tHcy with folate and vitamin B12 before exercise were observed in 2 studies. Two

studies also showed an inverse correlation after exercise between tHcy and folate. One

of these investigations was different than those that found the correlation pre-exercise.

Furthermore, most of the studies showed higher values of folate, vitamin B12 or B6 after

acute exercise. Regarding creatinine, 4 of the studies found high creatinine

concentrations after the acute effect of exercise.


25

Tab

le 4

. Im

plic

ated

bio

mar

kers

rel

ated

to tH

cy c

once

ntra

tions

and

exe

rcis

e

Ref

eren

ces

Titl

e A

im

Subj

ects

V

aria

bles

R

esul

ts

Her

rman

n et

al

. (20

03)

Hom

ocys

tein

e in

crea

ses d

urin

g en

dura

nce

exer

cise

. To

ass

ess p

lasm

a ho

moc

yste

ine

afte

r m

arat

hon,

100

km ru

n, a

nd 1

20km

bik

e ra

ce.

100

recr

eatio

nal

athl

etes

♀ a

nd ♂

. -M

arat

hon

(n=4

6)-1

00 k

m ru

n (n

=12)

-Mou

ntai

n bi

ke ra

ce(n

=42)

-Hcy

-Fol

ate

-B12

Cha

nges

in H

cy c

orre

late

s with

tim

e of

exe

rcis

e at

rest

.

Hig

h H

cy p

rera

ce w

ere

asso

ciat

ed

with

rela

tivel

y lo

w fo

late

and

B12

.

Kon

ig e

t al.

(200

3)

Influ

ence

of t

rain

ing

volu

me

and

acut

e ph

ysic

al e

xerc

ise

on th

e ho

moc

yste

ine

leve

ls in

end

uran

ce-tr

aine

d m

en:

Inte

ract

ions

with

pla

sma

fola

te a

nd

vita

min

B12

.

To a

sses

s the

influ

ence

of e

xten

sive

en

dura

nce

train

ing

and

acut

e ex

erci

se o

n pa

sma

conc

entra

tions

of H

cy, v

itam

in B

12,

and

folic

aci

d in

42

wel

l-tra

ined

mal

e tri

athl

etes

.

39 tr

ainn

ed (2

7 yr

). -H

cy-F

olat

e-B

12

-NS

in B

12 b

oth,

trai

ning

and

com

petit

ion.

-Tra

i nin

g vo

lum

e an

d fo

late

prec

ompe

titio

n, n

egat

ivel

y co

rrel

ated

to H

cy 2

4 h

afte

r com

petit

ion.

Rea

l et a

l. (2

004)

Ef

fect

s of m

arat

hon

runn

ing

on p

lasm

a to

tal h

omoc

yste

ine

conc

entra

tions

. To

inve

stig

ated

the

chan

ges i

n pl

asm

a tH

cy

conc

entra

tions

24

h be

fore

and

afte

r a

mar

atho

n ra

ce.

22 n

on p

rofe

ssio

nal ♂

at

hlet

es (3

5.6

yr).

-Hcy

-B12

-Fol

ate

-MTH

FR67

7TT

geno

type

-C

orre

latio

n H

cy -

fola

te a

nd B

12

pre-

race.

-C

orre

latio

n H

cy-F

olat

e po

st-r

ace.

-N

S in

Fol

ate

and

B12

pos

t-rac

e (2

4 h)

.

Sotg

ia e

t al.

(200

7).

Acu

te v

aria

tions

in h

omoc

yste

ine

leve

ls

are

rela

ted

to c

reat

ine

chan

ges i

nduc

ed b

y ph

ysic

al a

ctiv

ity.

To

inve

stig

ate

whe

ther

the

mod

ifica

tion

in

Hcy

leve

l afte

r a m

oder

ate

phys

ical

act

ivity

w

as e

xpla

inab

le in

the

light

of t

he c

omm

on

conn

ectio

n of

phy

sica

l act

ivity

and

Hcy

to

crea

tine.

16 y

oung

subj

ects

(2

1-37

yr)

. -S

eden

tary

(6)

-Ath

lete

s (10

)

-tH

cy-r

Hcy

-Cre

atin

ine

-↑ C

reat

inin

e af

ter e

xerc

ise

in b

oth

grou

ps.

Ven

ta e

t al.

(200

9).

Plas

ma

vita

min

s, am

ino

acid

s, an

d re

nal f

unct

ion

in p

oste

xerc

ise

hype

rhom

ocys

tein

emia

.

To st

udie

d th

e ef

fect

of d

iffer

ent a

cute

ae

robi

c ex

erci

ses o

n pl

asm

a, re

duce

d, a

nd

tota

l Hcy

(rH

cy, t

Hcy

) and

cys

tein

e (r

Cys

, tC

ys) a

nd o

n its

met

abol

ical

ly re

late

d vi

tam

ins a

nd a

min

o ac

ids.

-15

cycl

ists

-14

kaya

kers

(14-

22 y

r)

-Hcy

-rH

cy-F

olat

e-B

12-C

reat

inin

e

-↑ B

12 a

nd c

reat

inin

e af

ter a

cute

exer

cise

s.-N

S in

Fol

ate

inde

pend

ently

of t

heex

erci

se a

nd v

itam

in st

atus

.

Dem

inic

e et

al

. (20

11)

Cre

atin

e su

pple

men

tatio

n re

duce

s in

crea

sed

hom

ocys

tein

e co

ncen

tratio

n in

duce

d by

acu

te e

xerc

ise

in ra

ts.

To e

valu

ate

the

effe

ct o

f cre

atin

e su

pple

men

tatio

n on

hom

ocys

tein

e m

etab

olis

m a

fter a

cute

aer

obic

and

an

aero

bic

exer

cise

.

112

wis

tar r

ats:

-A

erob

ic e

xerc

ise

-Aer

obic

exe

rcis

e +

crea

tine

sup.

-Ana

erob

ic e

xerc

ise

-Ana

erob

ic e

xerc

ise

+su

p

-Hcy

-Cre

atin

e-C

reat

ine

supp

lem

enta

tion

Low

erH

cy b

oth

anae

robi

c an

d ae

robi

cgr

oups

.-C

reat

ine

plas

ma

and

mus

cle ↑

afte

rex

erci

se in

bot

h su

pple

men

tati o

ngr

oups

aer

obic

and

ana

erob

ic.


26

Mal

es: ♂

; Fe

mal

es: ♀

; ↑: I

ncre

ase;

Dec

reas

e: ↓

; NS:

No

stat

istic

al d

iffer

ence

s; y

r: y

ears

; B12

: Vita

min

B12

; B6:

Vita

min

B6 H

cy: H

omoc

yste

ine;

tHcy

: tot

al

Hom

ocys

tein

e.

Igle

sias

- G

utie

rrez

et

al. (

2012

)

Tran

sien

t inc

reas

e in

hom

ocys

tein

e bu

t no

t hyp

erho

moc

yste

inem

ia d

urin

g ac

ute

exer

cise

at d

iffer

ent i

nten

sitie

s in

sede

ntar

y in

divi

dual

s.

To d

eter

min

e th

e ki

netic

s of s

erum

ho

moc

yste

ine

at d

iffer

ent i

nten

sitie

s. 8

sede

ntar

y ♂

(18-

27

yr) i

nact

ive

for a

t le

ast 6

mon

ths.

-Hcy

-B12

-B

6 -F

olat

e-C

677T

MTH

FRge

noty

pe

-↑ F

olat

e, B

12, B

6 , d

urin

g ex

erci

se

in b

oth

Hi a

nd L

i.-L

arge

var

iabi

lity

in c

orre

latio

ns.

-Fol

ate

and

B6 r

ecov

ered

at 1

9 h.

Ham

mou

da e

t al

. (20

12)

Effe

ct o

f sho

rt-Te

rm m

axim

al e

xerc

ise

on

bioc

hem

ical

mar

kers

of m

uscl

e da

mag

e,

tota

l ant

ioxi

dant

s sta

tus,

and

hom

ocys

tein

e le

vels

in fo

otba

ll pl

ayer

s.

To a

sses

s the

eff

ect o

f sho

rt-te

rm m

axim

al

exer

cise

on

mar

kers

of m

uscl

e da

mag

e,

hom

ocys

tein

e an

d to

tal a

ntio

xida

nts s

tatu

s in

trai

ned

subj

ects

.

18 ♂

foot

ball

play

ers

(17.

5 yr

). -H

cy-C

reat

inin

e-C

K

-↑C

reat

inin

e af

ter e

xerc

ise

test

.

Dem

inic

e et

al

. (20

13)

Shor

t-ter

m c

reat

ine

supp

lem

enta

tion

does

no

t red

uce

incr

ease

d ho

moc

yste

ine

conc

entra

tion

indu

ced

by a

cute

exe

rcis

e in

hum

ans.

To e

valu

ate

the

effe

cts o

f cre

atin

e su

pple

men

tatio

n on

hom

ocys

tein

e (H

cy)

plas

ma

leve

ls a

fter a

cute

exe

rcis

e in

hu

man

s.

23 y

oung

socc

er

play

ers d

ivid

ed in

2

grou

ps.

-Cre

atin

e su

p.-P

lace

bo

-Hcy

-Cre

atin

e-F

olat

e-B

12

-NS

Fola

te a

fter e

xerc

ise

-↑ B

12 af

ter e

xerc

ise

-Cre

atin

e su

pple

men

tatio

n do

n´t

decr

ease

Hcy

afte

r sup

plem

enta

tion

or a

fter e

xerc

ise

cre a

tine

don´

tin

fluen

ce B

12 o

r fol

ate

leve

ls.

Ran

deva

et a

l. (2

002)

Ex

erci

se D

ecre

ases

Pla

sma

Tota

l H

omoc

yste

ine

in O

verw

eigh

t You

ng

Wom

en w

ith P

olyc

ystic

Ova

ry

Synd

rom

e.

To e

xam

ine

the

effe

cts o

f exe

rcis

e on

pl

asm

a to

tal h

omoc

yste

ine

conc

entra

tions

in y

oung

ove

rwei

ght o

r ob

ese

PCO

S w

omen

.

21 o

bese

♀ (3

0.6

yr)

divi

ded

in 2

gro

ups:

Ex

erci

se (1

2) a

nd

non-

exe

rcis

e (9

).

-Fol

ate

-B12

-Cre

atin

ine

-NS

afte

r tra

inin

g pe

riod

in B

12,

fola

te c

reat

inin

e be

twee

n ba

selin

ean

d 6

mon

ths a

fter i

n bo

th g

roup

s.

Oku

ra e

t al.

(200

6)

Efec

t of r

egul

ar e

xerc

ise

on h

omoc

yste

ine

conc

entra

tions

: th

e H

ERIT

AG

E Fa

mily

Stu

dy.

Whe

ther

regu

lar a

erob

ic e

xerc

ise

coul

d af

fect

pla

sma

tota

l hom

ocys

tein

e (tH

cy),

and

whe

ther

ther

e w

ere

sex-

rela

ted

or ra

cial

diff

eren

ces i

n tH

cy c

hang

es.

730

subj

ects

bla

ck

and

wite

s ♂ a

nd ♀

(1

7-65

yr)

.

-Hcy

-B6

-B

12

-NS

B6.

-↓ B

12 in

all

grou

ps a

fter t

rain

ing.

-↑ F

olat

e on

ly in

Bla

cks a

fter

train

ing.

M

olin

a-Ló

pez

et a

l. (2

013)

Effe

ct o

f fol

ic a

cid

supp

lem

enta

tion

on

hom

ocys

tein

e co

ncen

tratio

n an

d as

soci

atio

n w

ith tr

aini

ng in

han

dbal

l pl

ayer

s.

To e

valu

ate

nutri

tiona

l sta

tus f

or

mac

ronu

trien

ts a

nd fo

lic a

cid

in m

embe

rs

of a

hig

h-pe

rfor

man

ce h

andb

all t

eam

, and

de

term

ine

the

effe

ct o

f a n

utrit

iona

l in

terv

entio

n w

ith fo

lic a

cid.

14 H

andb

all p

laye

rs

high

per

form

ance

(2

2.9

yr).

-Hcy

-Fol

ate

-Int

ensi

tytra

inin

g

-Neg

ativ

e co

rrel

atio

n of

Hcy

and

fola

te w

ith tr

aini

ng in

tens

ity o

f 60

%.

-Neg

ativ

e co

rrel

atio

n be

twee

n H

cyan

d fo

late

at w

eek

8.

Con

tinua

tion

2 of

tabl

e 4


27

1.1.12 Hydration and exercise

Normal hydration, often called euhydration, is important for health and wellbeing. Even

small losses of body water can have a negative effect on muscle strength, endurance and

Maximal oxigen uptake (VO2max). Normal hydration status is the condition of healthy

individuals to maintain water balance that depends on the difference between water gain

and water loss (86). Under normal conditions, water entry into the organism proceeds

from fluid intake (around 2300 mL/day) as well as the production of water from

reactions of cellular metabolism (200 mL/day). Concerning water output sources, the

main outputs are in the form of urine (1500 mL/day), followed by cutaneous

perspiration (350 mL/day), pulmonary ventilation (350 mL/day), sweat (150 mL/

day) and faeces (150 mL/day) (86).

During prolonged exercise there is a progressive increase in body temperature that is

determined primarily by the balance between the rate of metabolic heat production and

heat dissipation. An increased rate of metabolic heat production and body temperature

will rise if heat loss is not increased accordingly (111). Increased sweating leads to a

condition called cardiac drift, which includes symptoms such as peripheral displacement

of blood, a reduction in central blood volume, and reduction in stroke volume causing a

compensatory increase in heart rate (16).

Figure 3. Mechanisms of heat dissipation (73)


28

During exercise, as fluid losses by sweat increase, fluid intake should also increase. Due

to that a slight state of dehydration (a water loss of only 1 % - 2 % of body weight),

will negatively affect both physical and mental performance (86).

Furthermore, dehydration produces a negative effect on the cardiovascular system,

thermoregulation, besides compromising metabolic, endocrine and excretory systems,

resulting in decreased physical performance and also cognitive function.

Figure 4. Factors affecting heat gain and heat loss during exercise (66)

The consequences of dehydration include reduced training capacity, reduced sports

performance, and compromised thermoregulation and cardiovascular functions.

Dehydration and hyperthermia not only impair physiological function and exercise

performance but also represent a major threat to the health and well being of

individuals exercising in the heat (100).

Therefore, athletes must be concerned to drink accordingly in order to reduce this

performance loss.

In the last decades, the knowledge that dehydration impairs performance in sports has

been controversial and there are two polarised supporting lines of debate (54, 100). A

number of studies stated that dehydration over 2 % of body mass loss in a hot

enviroment impairs aerobic, mental and physical performance (17, 34, 100, 112). On the

other hand, recent studies suggest that the level of dehydration up to 4 % body mass

does not alter physical performance (54, 100, 129). However those results had been

studied in well-trained male cyclist well aclimatized to exercising in a hot


29

enviroment. Thus, it is important to interpret performance results carefully in order

to extrapolate to the general population and health recommendations contexts in order

to not underestimate the detrimental effect of dehydration (20, 100).

A proper protocol hydration during exercise will influence cardiovascular function,

thermoregulatory function, muscle performance, plasma and fluid volume status, and

exercise performance (16). The main goal of drinking during exercise is to prevent

excessive dehydration (2 % weight loss from water deficit) and excessive changes in

electrolyte balance to avoid compromised exercise performance (111).

Fluid replacement should approximate sweat and urine losses and at least maintain

hydration at less than 2 percent body weight reduction. Hydration before and during

exercise is essential for a good performance during exercising, but hydration after

exercise is equally as important. A high rate of fluid consumption during the first two

hours of post-exercise rehydration is known to increase plasma volume significantly and

to result in substantial urine production (75). The recommendations to ensure a rapid re-

hydration are to replace 1.5 L of fluid for each Kg of body mass loss (111).

The main goal of rehydration is the return of physiologic function, after dehydration

induced by exercise, the rehydration solution should include water to restore hydration

status, carbohydrates to replenish glycogen stores, and electrolytes for faster

rehydration and ability to maintain blood volume (111).

Composition and characteristics of the sport drinks

- Carbohydrate concentration: 5-8 %.

- Beverage temperature: 10 °C to 15 °C.

- Osmolarity: 80-400 mEq/L.

- Mineral content (especially Na +): 20 to 30 mEq/L.- Taste: must have a pleasant taste to encourage voluntary hydration and rehydration. It

is important to achieve an appropriate balance between fluid intake and fluid losses in

athletes or, what is the same, an optimal state of hydration before, during and after

exercise (86).

1.1.13 Dehydration and haemoconcentration

Hemoglobin, hematocrit, and red blood cell count have an important relationship to the

transport of oxygen and therefore may influence performance in endurance and aerobic


30

exercise. During an extended aerobic exercise, plasma volume is reduced producing

haemoconcentration, reflected by an increase in the hematocrit (Htc) value (2).

The basic mechanism of haemoconcentration consists in the passage of liquid from the

blood to the interstitial and intracellular spaces. Furthermore, an increase in sweating

produces an increased loss of body water that will contribute to a greater

haemoconcentration, being important to balance with an adequate intake of water.

Finally, there is evidence that the increase in cell metabolism, can contribute to the

osmotic absorption of the interstitial fluid compartments and vascular cells (36). It has

been shown that exercise at 75 % VO2max produces a reduction in plasma volume of 5 %

to 10 % (22). Therefore, it has been recommended to make corrections of plasma

volume when biochemical parameters are analyzed in high-intensity exercise (72). In

this regard, Dill and Costill (31) proposed the estimate of changes in blood plasma

based on the hematocrit and hemoglobin concentration, since these two parameters are

directly related (127).

1.2 Statement of the Research Problems

There are a limited number of studies analyzing the effects of exercise on tHcy

concentrations and the results are sometimes inconclusive, but most of the

investigations analysing the acute effect of exercise on tHcy concentrations reported

increased tHcy concentrations after exercise. Due to the detrimental effect of high tHcy

concentrations related to cardiovascular and cerebrovascular health, there is a necessity

of further studies analyzing the effect of acute exercise, the underliyng mechanisms and

the possible prevention of tHcy increase induced by acute exercise. Furthermore, the

intensity and duration of exercise have been reported as a factor that could be related to

the increase of tHcy concentrations (64). The first study of the present thesis assesses

the effect of acute exercise by two different intensities (maximal and submaximal) on

tHcy concentrations in active male subjects (study 1).

Second, rehydration as an important factor restoring all the physiologic systems in the

human body, as well as its implication in restoring plasma volume should be taken

into account as a possible factor not only for restoring water, glycogen and

electrolyte losses or recovering plasma volume, but also its possible implication in

affected blood parameters by exercise and dehydration as is the case of Hcy.

However, to the best of our knowledge, there are not previous studies analysing the


31

effect of rehydration on increased tHcy concentrations after an acute aerobic

submaximal exercise. For this reason and due to the results obtained in study 1,

further analyses will be focused on the effect of a 2 h of rehydration protocol with

two different drinks after acute aerobic submaximal exercise on tHcy concentrations

and related parameters (study 2).

And finally, there are no existing studies analysing hydration during exercise from the

perspective of health as a preventive factor and analysing the behaviour of Hcy after

acute exercise when a hydration protocol is implemented. Therefore, study 3 will

analyze the effect of a hydration protocol during exercise on tHcy concentrations

with two different drinks, in order to find out if a proper hydration during a

single bout of exercise could prevent the increase of tHcy concentrations induced by

exercise.

This thesis will contribute to a better understanding of the current knowledge of tHcy

responses after acute exercise and the effect of hydration as a possible important

component restoring altered biomarkers related to health as is Hcy in this case, that has

not been measured until now.

1.3 Structure of the Thesis

This thesis consists of 8 chapters. Chapter 1 is a general introduction of the whole

thesis. Chapter 2 corresponded to the objective and hypothesys. Chapter 3, presents the

general material and methods explanation of the thesis, chapters 4 to 6 are the core of

the thesis that are the experimental studies presented in manuscript format according to

the requirements of the scientific journals to which they were submitted,

with corresponding parts of introduction, material and methods, results,

discussion and conclussions. Chapter 7 contains a general discussion of the

entire thesis and summarises the main outcomes of the three studies. Lastly,

chapter 8 includes the specific and general conclusions of the thesis.

Therefore, there are some repetitions among chapters in the thesis. For the reader’s

benefit, references of each chapter are removed and placed at the end of the thesis.


32


33

2 CHAPTER 2. OBJECTIVES AND HYPOTHESIS

General objective

To analyze the effects of acute aerobic submaximal exercise and hydration on tHcy

concentrations and related parameters in a sample of physically active males.

Specific objectives

Study 1:

- To assess the effect of maximal and submaximal acute exercise on tHcy

concentrations and related parameters as vitamin B12, folate and creatinine in physically

active adult males.

Study 2:

- To analyze the effect of a 2 h rehydration protocol with water and with a sport drink on

tHcy concentrations and related parameters after acute submaximal exercise in a hot

environment in physically active adult males.

Study 3:

- To analyze the effect of a hydration protocol during acute submaximal exercise on

tHcy concentrations and related parameters and the subsequent behaviour immediately

after, at 2 h, 6 h and 24 h in physically active adult males.

- To assess the correlation between tHcy concentrations and related parameters such as

folate, vitamin B12, creatine and creatinine immediately after acute exercise, at 2 h, 6 h

and 24 h.

- To study the implementation of a hydration protocol from the health perspective as an

important factor in relation to altered tHcy concentrations after acute exercise.

Hypothesis

An adequate hydration protocol during exercise prevents the increase of tHcy

concentrations after acute aerobic submaximal exercise.


34

Alternative Hypothesis

An adequate hydration protocol during exercise increases tHcy concentrations after

acute aerobic submaximal exercise.

Null Hypothesis

An adequate hydration protocol doesn´t affect tHcy concentrations after acute aerobic

submaximal exercise.


35

3 CHAPTER 3. GENERAL MATERIAL AND METHODS

This doctoral thesis is an experimental study funded by the ImFINE Research Group

and coordinated by Prof. Marcela González Gross from the Technical University of

Madrid.

3.1 Sample of the study

3.1.1 Subject recruitment

Twenty nine males (mean age 27.55±7.16 yr) without known pathology, healthy and

physically active were recruited by means of advertisements published at the Faculty of

Physical Activity and Sport Sciences–INEF of the Technical University of Madrid

(Spain) inviting them to voluntarily participate in the study. In this study, only

males were included to avoid any distortion in the response to exercise mediated by

ovarian cycle. Criteria for participants’ selection included males aged between 18

to 42 yr, being physically active at the moment of the presentation to the study, (At

least 3 days per week of aerobic exercise), non-smokers and not having any of the

exclusion criteria listed below.

Exclusion criteria: To have any central or peripheral cardiovascular risk factor, diabetes,

kidney or liver problems, known asthmatic complications, total cholesterol > 200 mg/dl,

systolic blood pressure > 160 mmHg or diastolic blood pressure > 100 mmHg, history

of toxic abuse, history of inflammation or cancer, orthopedic limitations, medications

that may affect metabolic and cardiovascular function, following a vegetarian diet,

intake of B-vitamins supplement or vitamin-fortified food or creatine supplementation

during the last two months and being a smoker.

First data collection took place during May 2010 and the second data collection took

place from October 2011 to March 2012. After finishing the fieldwork, 29 subjects from

34 were elegible for the study.

3.2 Ethical issues

Participants were informed of the nature and purpose of the study and signed an

informed consent prior to conducting the tests (Appendix). The sample selection and

study protocol were performed following the ethical guidelines of the Declaration of


36

Helsinki (1964) as revised in Edinburgh (2000) and other national regulations for

research projects involving human subjects: Protection of personal data, Law 15/1999 of

13 December on the Protection of Personal Data provided in the current legislation

(Royal Decree 1720/2007 of 21 December). The protocol was approved by the Ethics

Review Board of the Technical University of Madrid.

3.3 Experimental Design

The present study is a randomized and counterbalanced crossover design, where

each participant acts as his own control. The study consisted of two intervention

periods. Specific details will be described in each study. Figure 5 summarizes a general

view of the protocol of the present thesis.

Figure 5. Experimental protocol of the study

3.3.1 Medical examination

Subjects were required to complete a medical examination in order to ensure there was

no medical contraindication for participation in the study. In their first visit a basal

blood pressure and a fasting blood routine analysis took place between 8 to10 a.m.

Approximately, 30 mL of blood were collected from an antecubital vein in serum and

ethylene-diamineteraacetic acid (EDTA) tubes (Sarstedt AG & Co., Nümbrecht,

Germany). After blood sampling, a breakfast was offered for all participants.


37

In their 2nd visit a basal electrocardiogram was performed with the Jaeger®

electrocardiograph (Erich Jaeger, Germany). In addition, body mass (kg) with a

Detecto® scale (Lafayette Instruments Company, Lafayette, Indiana, USA), and height

(cm) with a conventional rack stadiometer (Holtain Limited, Crymych, UK) were

registered. Body composition (Body mass, water content, Fat mass, Lean body mass

and Body Mass Index) was analyzed by Bioelectrical Impedance Analysis (BIA) with a

TANITA BC 418 MA (Tanita Corp., Tokyo, Japan). Moreover, body composition (% of

fat, % of lean mass and Bone Mineral Density (BMD) (g/cm2) by the X-ray

absorptiometry dual-energy (DXA) was registered in ten participants from the

whole sample with the Lunar Prodigy TM scanner (General Electric, Madison,

Wisconsin, USA).

3.3.2 Exercise protocols

All the exercise tests were performed on a treadmill (H/P/COSMOS® 3P 4.0, H/P/

Cosmos Sports & Medical, Nussdorf-Traunstein, Germany) at the Laboratory

of Exercise Physiology, at the Faculty of Physical Activity and Sport Sciences-

INEF, Technical University of Madrid (Laboratory number 214, Laboratory Network

of the Region of Madrid, Spain).

Maximal exercise test

All subjects completed an incremental maximal test according to the protocol described

bellow (92). Individual maximal oxygen uptake VO2max was determined to establish the

individual load at 65 % of oxygen consumption (VO2) of each subject for the further

tests of the study.

The protocol was as follows: Starting at 1 initial resting minute at 0% of slope, followed

by 3 minutes at 6 km per hour (km/h) (1 % of slope), with a speed increase of 0.2 km/h

every 12 s until exhaustion, followed by an active recovery of 2-minutes walking at

6 km/h followed by a 3-minute of sitting passive recovery.

Submaximal exercise tests

The protocol of the submaximal treadmill tests were as follows:

Tests were performed at constant load at an intensity of 65 % of the individual VO2max.

This test consisted of an initial 1-minute resting (0 % of slope), 3-minutes warm up at a


38

rate of 6 km/h and 1 % of slope, then 40 minutes running at constant intensity (65 % of

de individual VO2max) and finally, an active recovery of 2 minutes walking and 3

minutes sitting of passive recovery. Tests were performed in a hot environment in order

to get the subjects dehydrated (mean temperature of 30 °C and 60 % of mean

relative humidity). There was a one-week washout period between tests.

3.3.3 Hydration protocols

Hydration protocols are explained in chapters 5 and 6.

3.3.4 Standardization of previous diet and exercise

After enrolment and in order to standardize the results, subjects were instructed not to

perform strenuous exercise between the 24 h prior to testing and until the last

blood sample collection, as well as not to eat any food, drink coffee or caffeinated

beverages within 2 h prior to performing the tests. Furthermore, to ensure

euhydration status before each trial, subjects followed a standardized hydration

protocol by ingesting an average of 350 mL of water 2 h before testing, according

to American College of Sports Medicine (ACSM) recommendations (111). The diet

and hydration instructions for the participants are included in the appendix of the

present thesis.

3.3.5 Physiological measures

During exercise tests, heart rate (HR) was controlled using a Polar S810® (Polar

Electro, Kempele, Finland); the VO2 and ventilation (VE) were measured with the gas

analyzer Jaeger Oxycon Pro (Erich Jaeger, Viasys Healthcare, Germany). Blood

pressure (BP) was measured before and after each exercise test.

3.3.6 Temperature and Humidity conditions

Submaximal tests were performed in a hot environment in order to get the subjects

dehydrated (mean temperature of 30 °C and 60 % of mean relative humidity), controlled

by plastics, heaters and a weather station.

3.3.7 Anthropometric measurements and body composition

Weight was measured in light and dry clothes and without shoes with an electronic scale

(Type TANITA BC-418, Tokio, Japan). Height was measured barefoot positioning the

subject´s head in the Frankfort plane with a rack stadiometer (Holtain Limited,


39

Crymych, UK). Body composition were measured with a portable BIA, TANITA BC

418 (Tanita Corp., Tokio Japan) with a 200 kg maximum capacity and a +/- 100 g error

margin was used to measure the body mass, percentage of body fat and muscle mass.

Moreover in a subsample of 10 subjects, body composition was also measured by DXA

(Lunar Prodigy TM scanner, General Electric, Madison, Wisconsin, USA).

3.3.8 Blood Samples processing

Blood samples (10 mL) were collected from an antecubital vein. Extraction was

performed by standard venipuncture with vacuum moth Vacutainer® tubes containing

EDTA as anticoagulants or gel for serum (Sarstedt AG & Co., Nümbrecht, Germany)

for assessing the different biomarkers and blood parameters. The tubes were

immediately placed on ice and after clot formation was centrifuged for 10 minutes at

3000 rpm. The serum was separated in 1 mL eppendorf sample and was stored at -80 °C

until processing. For hematological parameters, a complete hematological analysis was

performed within the first hour after extraction. Routine biochemistry analysis was

carried out using standard methodologies. The methods and devices used for the

analysis of specific biochemical parameters are presented in table 5.

The analysis of all biochemical parameters was carried out at the Biochemistry

Laboratory of the Faculty of Physical Activity and Sport Sciences-INEF, of the

Technical University of Madrid (Laboratory number 242, Laboratory Network of the

Region of Madrid) and at the Clinical laboratory of the Sports Medicine Center of the

High Sports Council (HSC, Spain).

Biochemical parameters

Serum tHcy concentrations were determined by immunoassay technology for detecting

fluorescence polarization (FPIA); Abbott AxSYM, Abbott Park, USA, Total CV ≤ 6 %)

and by enzymatic assay (AU400 analyzer, Beckman Instruments, Ltd., Bucks, UK; CV

≤ 6 %).

Serum vitamin B12 was determined by technology enzyme immunoassay microparticles

(MEIA), Abbott AxSYM, Abbott Park, USA, Total CV ≤ 11 %) and by

electrocheluminescence immnoassay (Elecsys 2010 analyzer, Roche Diagnostics, IN,

USA; CV ≤ 10 %).


40

Serum folate was determined by the immunoassay ion capture technology (ICIA),

Abbott AxSYM, Abbott Park, USA, ≤ 19 % total CV) and by electrocheluminescence

immunoassay (Elecsys 2010, Roche Diagnostics, IN, USA, CV ≤ 11 %).

Serum creatinine was analyzed by the colorimetric-kinetic method (JAFFÉ) by

autoanalyzer spectrophotometer Clima MC-15 (RAL, SA, Spain Total Cv ≤ 3 %) and

creatinine by colorimetric analyzer (Beckman AU400, Beckman Instruments, Ltd.,

Bucks, UK). Creatine was assessed by P/ACE Beckman capillary electrophoresis diode

array detector (Beckman instruments, Fullerton, CA, USA) as described by Zinellu et al.

(136).

Table 5. List of methods and devices used for the different biochemical parameters

Parameter Sample Method Analyzer

Lactate Serum Enzimatic colorimetric Clima MC-15 (RAL) RAL, SA, Spain

CK-M Serum Kinetic U.V Test Clima MC-15 (RAL) RAL, SA, Spain

Total protein Serum Colorimetric (BIURET) Clima MC-15 (RAL) RAL, SA, Spain

Potassium Serum Flame Photometer Ion3 Flame Photometer (RAL) RAL, SA, Spain

Sodium Serum Flame Photometer Ion3 Flame Photometer (RAL) RAL, SA, Spain

Chloride Serum Flame Photometer Ion3 Flame Photometer (RAL) RAL, SA, Spain

Magnesium Serum Enzimatic colorimetric Clima MC-15 (RAL) RAL, SA, Spain

Total Homocysteine Serum

Immunoassay technology fluorescence polarization (FPIA)

Abbott AxSYM, Abbott Park, USA

Enzymatic assay AU400 analyzer, Beckman Instruments, Ltd, Bucks

Vitamin B12 Serum

Enzymmunoassay microparticles (MEIA)

Abbott AxSYM, Abbott Park, USA,

Electrocheluminescence immnoassay

Elecsys 2010 analyzer, Roche Diagnostics, IN, USA

Folate Serum

Immunoassay ion capture technology (ICIA)

Abbott AxSYM, Abbott Park, USA

Electrocheluminescence immnoassay

Elecsys 2010 analyzer, Roche Diagnostics, IN, USA

Creatinine Serum

Colorimetric-kinetic (JAFFÉ) Clima MC-15

Clima MC-15 (RAL) RAL, SA, Spain

Colorimetric analyzer (Beckman AU400)

Beckman Instruments, Ltd, Bucks, UK

Creatine Serum Capillary electrophoresis diode array detector P/ACE Beckman

Beckman instruments, Fullerton, CA, USA


41

Blood samples collection

Timing of blood sample collection and the specific biochemistry analysis will be

described in each study.

Sample pre-treatment and transport

After sample fieldwork, 5 mL of blood from each participant was collected in EDTA,

shipped on dry ice and sent to the Laboratory of Pediatrics, Faculty of Medicine,

University of Cantabria and preserved at -20°C until genetic analysis. Moreover an

eppendorph of 5 mL with serum was collected on dry ice and send to the Clinical

laboratory of the Faculty of Medicine, Dept. of Biomedical Sciences of University of

Sassari (Sardinia, Italy) for the creatine analysis.

3.3.9 Genetic analysis

Whole blood (5 mL) from each participant was collected in EDTA and sent to the

Laboratory of Pediatrics, Faculty of Medicine, University of Cantabria. DNA was

extracted from each sample using the "QIAamp® DNA Blood Mini Kit" from QIAGEN

(Hilden, Germany) and the genotyping was performed afterwards. The DNA samples

were preserved at -20 °C.

The analysis of the MTHFR C677T (rs1801133) polymorphism was done based on the

polymerase chain reaction (PCR) and Restriction Fragment Length Polymorphism

(RFLP) techniques described by Frosst et al. (40). The primers used for amplification

were as follows: sense primer 5’- TGA AGG AGA AGG TGT CTG CGG GA -3’

(exonic); antisense primer 5’- AGG ACG GTG CGG TGA GAG TG -3’ (intronic).

PCR reaction was made in a total volume of 50 µL containing: 3 µL genomic DNA, 1.5

mM MgCl2, 0.2 mM dNTP mix, 0.5 µM each primer, 10 % dimethyl sulfoxide

(SIGMA, Sant Louis, MO, USA) and 2U Taq polymerase (BioTaq Polimerase, Bio-

Line, London, UK), using a GeneAmp® PCR System 2400 thermal cycler (Perkin

Elmer, Applied Biosystems Division, Foster City, CA, USA). The amplification

consisted of initial denaturation (94 ºC, 5 min); 38 cycles consisting of denaturation (94

ºC, 1 min), annealing (55 ºC, 45 sg), and extension (72 ºC, 1 min); and final extension

(72 ºC, 10 min). PCR products were electrophoresed in 1.5 % agarose gel to verify

successful amplification of the 198 bp fragments.


42

The amplified product was digested with 0.4 U the restriction enzyme Hinf I (GE

Healthcare Life Sciences, Uppsala, Sweden) at 37 ºC for 4 h. The fragments were

resolved on a 3 % agarose MS-12 (Laboratorios Conda, Madrid, Spain) gel with Tris-

borate EDTA (89 mM Tris-borate, 2 mM EDTA) (Serva, Heidelberg, Germany) buffer

and visualized under ultraviolet illumination after staining with ethidium bromide (10

mg/mL) (Serva, Heidelberg, Germany). This digestion produced fragments of the

following sizes: 198 bp in 677C homozygotes; 198, 175 and 23 bp in 677CT

heterozygotes; and 175 and 23 bp in 677TT homozygotes. The 23-bp fragment was too

small to be resolved on the gel.

PCR amplification to detect Angiotensin Converting Enzime (ACE) I/D polymorphism

(rs4340) was carried out using the previously published primers by Hohenfellner et al.

(67): Ace Id Up 5’-CTG GAG ACC ACT CCC ATC CTT TCT-’3 and Ace Id Down

5’-GAT GTG GCC ATC ACA TTC GTC AGA T-’3. PCR reaction was made in a total

volume of 50 µL containing: 3 µL genomic DNA, 1.5 mM MgCl2, 0.2 mM dNTP mix,

0.5 µM each primer, 10% dimethyl sulfoxide (SIGMA, Sant Louis, MO, USA) and 2U

Taq polymerase (BioTaq Polimerase, Bio-Line, London, UK), using a GeneAmp® PCR

System 2400 thermal cycler (Perkin Elmer, Applied Biosystems Division, Foster City,

CA, USA). Thermocycling consisted of denaturation at 94°C for 30 sg, annealing at 58

°C for 45 s, and extension at 72 °C for 2 min for 38 cycles, followed by a final

extension at 72°C for 7 min. Then, electrophoresis of the amplified products was

performed on an agarose gel low electroendosmosis D-1 (Conda Laboratories, Madrid,

Spain) to 1.5%, for 30 min at 100 volts, allowing the samples genotyping based on

fragments sizes. The presence of a fragment of 288 base pairs (bp) in the gene, resulting

PCR fragments of 490 bp for allele I (insertion), or 190 bp for the D allele (deletion)

respectively.

Because the D allele in heterozygous samples is preferentially amplified, all samples

genotyped as DD, were re-amplified with an insertion-specific primer pair, previously

described by Lindpaintner et al. (80): PCR II Up 5’-TGG GAC CAC AGC GCC CGC

CAC TAC-’3 and PCR II Down 5’- TCG CCA GCC CTC CCA TGC CCA TAA’, with

identical PCR conditions except for an annealing temperature of 67 ºC. The reaction

yields a 335-bp amplicon only in the presence of an I allele, and no product in samples

homozygous for DD.


43

3.3.10 Plasma Volume and correction for haemoconcentration

In order to control the haemoconcentration by changes in plasma volume (ΔPV) during

exercise and during rehydration, study 3 will present data with the corrections of the

concentrations calculated by using the Dill & Costill (31) equation by using Hematocrit

(Hct) in g/dL and Hemoglobin (Hb) in % as follows:

ΔPV (%) =100 x ((Hb pre / Hb post) x (100-Htc post) / (100 - Htc pre) -1),

The concentrations of biochemical parameters at post-exercise and 2 h, 6 h and 24

h recovery were corrected for hemoconcentration as follows:

Parameter C = Parameter U / (1 - ∆VP (%) / 100),

where “c” and “u” sub-indices denote “Corrected” and “Uncorrected”

concentrations, respectively.

3.4 Materials

The following parts summarize all materials used for the physiological and clinical

analysis. More precisely, Table 6 shows the used laboratory materials and facilities.

Table 6. Laboratory material

Product Specification Manufacturer

Treadmil HP COSMOS 3P 4.0 Cosmos Sports & Medical, Nussdorf-Traunstein, Germany

Gas analyzer Jaeger Oxycon Pro Erich Jaeger, Viasys Healthcare, Germany

Electrocardiogram Electrocardiograph Jaeger Erich Jaeger, Germany

Heart Rate monitor Polar S810® Polar Electro, Kempele, Finland

Scale Scale Detecto Lafayette Instruments Company, Lafayette, Indiana, USA

Stadiometer Rack stadiometer Holtain Limited, Crymych, UK

Bioelectrical Impedance Analysis (BIA) analyzer

TANITA BC 418 MA Tanita Corp., Tokyo, Japan

Densitometry X- ray (DXA) analyzer

Lunar Prodigy TM scanner

General Electric, Madison,

Wisconsin, USA


44

Chemical reagents

The chemical reagents used were of the highest purity available, and stored as the

optimum conditions indicated by the merchant. Then, they are classified with regard to

the method in which they were used, indicating manufacturers where they were

acquired and catalog number.

3.5 Statistical Analysis

The analisys of the data were performed with the Statistical Package for Social Sciences

(SPSS) versions from 15.0 to 20.0 for Windows (SPSS Inc., Chicago, Illinois, USA).

Descriptive statistics are shown as mean ± standard deviation (SD) unless

otherwise stated. P-values < 0.05 were considered as statistically significant. The

detailed description of statistic procedure is presented in each study.


45

4 CHAPTER 4. STUDY 1: El ejercicio agudo aumenta las

concentraciones de homocisteína en varones físicamente activos. Acute

exercise increases homocysteine concentrations in physically active

males.

4.1 Resumen

Introducción: Niveles altos de Homocisteína (Hcy) se han identificado como un factor

de riesgo cardiovascular. En relación con la práctica de ejercicio físico los resultados

son contradictorios. Objetivos: El objetivo del presente estudio fue determinar la

influencia del ejercicio físico agudo tanto máximo como submáximo sobre las

concentraciones de homocisteína total (tHcy) y parámetros relacionados. Material y

métodos: Diez varones (23,5 ± 1,8 años) físicamente activos realizaron un test

incremental máximo hasta el agotamiento y un test submáximo a una intensidad del 65

% del consumo máximo de oxígeno (VO2max) en tapiz rodante. Se extrajeron muestras

sanguineas antes e inmediatamente después del ejercicio y se analizaron las

concentraciones de tHcy, folato, vitamina B12 y creatinina séricas. Resultados: Las

concentraciones de tHcy séricas aumentaron significativamente inmediatamente

después del ejercicio en ambos tests, máximo (p < 0,05) y submáximo (p < 0,01). El

folato y la vitamina B12 también aumentaron de manera significativa tras los dos tests de

ejercicio (p < 0,05). Los niveles de creatinina aumentaron únicamente de manera

significativa después del test máximo (p < 0,001). Las concentraciones de folato y de

tHcy mostraron una relación significativamente inversa en todos los puntos analizados

en ambos tests (p < 0,05). Conclusión: El ejercicio agudo tanto máximo como

submáximo aumenta las concentraciones de homocisteína séricas en varones jóvenes

físicamente activos.

4.2 Abstract

Introduction: High homocysteine concentrations (Hcy) have been identified as a

cardiovascular risk factor. Regarding physical exercise, the results are contradictory.

Objectives: The aim of this study was to determine the influence of maximal and

submaximal acute exercise on total serum homocysteine concentrations (tHcy) and

related parameters. Material and methods: Ten physically active male subjects (mean

age: 23.51 ± 1.84), performed two treadmill tests, a maximal to exhaustion test and a


46

submaximal constant test at an intensity of 65 % of maximal oxygen uptake (VO2max).

Serum tHcy concentrations, folate, vitamin B12 and creatinine were analyzed before and

immediately after each test. Results: serum tHcy concentrations increase significantly

after both, maximal (p < 0.05) and submaximal (p < 0.01) tests. Folate and vitamin B12

concentrations also increased significantly after both tests (p < 0.05). Creatinine levels

only increased after the maximal test (p < 0.001). Folate and tHcy concentrations had an

inverse significant correlation (p < 0.05) in all the measurement points in both tests.

Conclusion: acute exercise, both maximal and submaximal increases serum

tHcy concentrations in young physically active males.

4.3 Introducción

La homocisteína (Hcy) es un aminoácido sulfurado que se forma como producto

intermedio en el ciclo metabólico de la metionina, cuyas concentraciones elevadas en

sangre (> 100 µmol/L) dan lugar a homocistinuria y enfermedad aterosclerótica precoz

(12). Desde los años 90 se vino observando que también niveles moderadamente

elevados (> 10-12 µmol/L) se correlacionan con un mayor riesgo de enfermedad cardio

y cerebrovascular (12, 53). Las concentraciones de Hcy total (tHcy) se ven afectadas

por factores no modificables como la edad, el sexo y las afecciones metabólicas

hereditarias (46) y por factores modificables como los hábitos nutricionales o el

tratamiento con fármacos (52). Sin embargo, menos conocido es el efecto que ejerce la

práctica de ejercicio físico sobre las concentraciones de tHcy (47, 64, 76, 124). Se ha

comprobado que estas concentraciones son mayores en varones que en mujeres y parece

que la respuesta al ejercicio es diferente dependiendo del sexo (109).

Los resultados en relación a la práctica de ejercicio físico con los niveles de tHcy

encontrados hasta ahora en las diferentes investigaciones son contradictorios, sin estar

bien definido el tipo de ejercicio e intensidades que provocan cambios en la tHcy (10,

65). En relación al efecto del ejercicio crónico o entrenamiento prolongado, algunos

estudios han demostrado una disminución de las concentraciones de tHcy tras el

entrenamiento a largo plazo (74, 101); otras investigaciones, sin embargo, no han

observado cambios (9).

En relación al efecto agudo del ejercicio físico algunos estudios han mostrado un

aumento en las concentraciones de tHcy inmediatamente después del ejercicio físico


47

intenso (10, 32, 47, 64, 95), aunque otros han observado que estas concentraciones

disminuyen (101), y otros no han encontrado ningún efecto (60).

Como posible explicación a estas divergencias, se han propuesto la variación en la

intensidad, el tipo de ejercicio y del estado vitamínico en especial de folato y de

vitamina B12 (29). A su vez se conoce que el folato es la vitamina que de forma

individual presenta un mayor grado de asociación inversa con los niveles de tHcy (12,

30). En el caso de la vitamina B12, los resultados en los diferentes estudios también

apuntan hacia una correlación inversa con los niveles de tHcy (51), si bien es más débil

que la observada para el folato. El objetivo del presente trabajo fue analizar el efecto del

ejercicio físico agudo máximo y submáximo sobre las concentraciones de tHcy y

parámetros relacionados en varones jóvenes físicamente activos.

4.4 Material y métodos

Sujetos

Diez sujetos varones, sanos sin patología conocida de 18 a 28 años (edad media: 23,5 ±

1,8 años) estudiantes de la Facultad de Ciencias de la Actividad Física y del Deporte

(INEF) de la Universidad Politécnica de Madrid participaron en el estudio.

Se realizó de un muestreo incidental de voluntarios, con una población muy homogénea

para comprobar el efecto de las pruebas.

La selección de la muestra tras el muestreo incidental se realizó mediante presentación

voluntaria por parte de los sujetos al estudio, difundido y publicado en la Facultad de

Ciencias de la Actividad Física y del Deporte-INEF. Siguiendo con las directrices éticas

de la Declaración de Helsinki para la investigación con seres humanos (World Medical

Association, 2004), los participantes fueron informados de la naturaleza y finalidad del

estudio y firmaron un consentimiento informado previo a la realización de las pruebas.

Los criterios de inclusión fueron los siguientes: Varones con edad comprendida entre 18

y 28 años, físicamente activos (realización de actividad física regular, mínimo 2 o 3 días

por semana), no fumadores y sanos, es decir, no padecer ninguna de las patologías

indicadas en los criterios de exclusión del presente estudio.

Los criterios de exclusión fueron presentar algunas de las siguientes patologías: riesgo

cardiovascular, central o periférico; diabetes, problemas renales o hepáticos conocidos,

complicaciones asmáticas, colesterol plasmático > 8 milimoles por litro (mmol/L),


48

presión arterial sistólica > 160 milímetros de mercurio (mmHg) o diastólica > a 100

mmHg, historial de abuso de alcohol o drogas, historial previo de inflamación o cáncer,

limitaciones ortopédicas, medicaciones que puedan afectar a la función cardiovascular

metabólica y seguir una dieta vegetariana.

Procedimiento experimental

Examen médico previo

Los sujetos se sometieron a un examen médico previo con el fin de asegurar que no

existía contraindicación médica para realizar el estudio. Se les realizó un

electrocardiograma con un electrocardiógrafo Jaeger® (Erich Jaeger, Alemania).

Además, se registró el peso en Kg con una báscula Detecto® (Lafayette Instruments

Company, Lafayette, Indiana, USA), la talla en cm con un estadiómetro convencional

de cremallera (Holtain Limited, Crymych, Reino Unido) y la composición corporal: %

de masa grasa, % de masa magra y DMO (Densidad Mineral Ósea) (g/cm2); mediante

Absorciometría por rayos X de energía dual (DXA), con el escáner Lunar ProdigyTM

(General Electric, Madison, Wisconsin, USA).

Protocolo de los test físicos

Tras ser seleccionados para el estudio y para la estandarización de los resultados, los

sujetos fueron instruidos en no realizar ejercicio físico intenso las 24 horas previas a las

pruebas, ni de ingerir alimentos ni café o bebidas con cafeína en las 2 horas previas a la

realización de las pruebas.

Los sujetos realizaron dos pruebas en tapiz rodante (H/P/COSMOS 3P 4.0®,

H/P/Cosmos Sports & Medical, Nussdorf-Traunstein, Alemania), una prueba de

esfuerzo incremental máxima y una prueba submáxima de carga constante a una

intensidad del 65 % del VO2max de cada sujeto. Se estipuló un periodo de dos días como

mínimo entre la realización de las dos pruebas.

Prueba incremental máxima: se realizó una prueba incremental en rampa hasta el

agotamiento siguiendo el protocolo descrito por Myers y Bellin (92), el cual se describe

a continuación: 1 minuto inicial de reposo, un calentamiento de 3 minutos a una

velocidad de 6 kilómetros por hora (km/h) y a continuación un incremento de la

velocidad de 0,2 km/h cada 12 segundos hasta el agotamiento del sujeto. Tras la


49

finalización, se siguió una recuperación activa de 2 minutos a 6 km/h y una

recuperación pasiva de 3 minutos sentado.

Prueba submáxima: A partir de los resultados de VO2max obtenidos en la prueba máxima

se estableció la intensidad al 65 % para la realización de la prueba submáxima para cada

sujeto. Esta prueba constaba de 1 minuto inicial de reposo, un calentamiento de 3

minutos a una velocidad de 6 km/h y a continuación 40 minutos a velocidad constante, y

recuperación activa de 2 minutos a 6 km/h y una recuperación pasiva de 3 minutos

sentado.

La prueba se realizó a una temperatura ambiental media de 30 ºC, una humedad relativa

del 60 % que se controló durante toda la prueba mediante la colocación de un plástico

aislante, calefactores y una estación meteorológica, tratando de reproducir una de las

situaciones meteorológicas habituales en España durante gran parte del año. Asimismo,

durante ambas pruebas se controlaron parámetros fisiológicos como la frecuencia

cardiaca (FC), mediante un monitor Polar S810, el Consumo de Oxígeno (VO2) y la

Ventilación (VE) con el analizador de gases Jaeger Oxycon Pro (Erich Jaeger, Viasys

Healthcare, Alemania).

Las pruebas se realizaron en el Laboratorio de Fisiología del Esfuerzo de la Facultad de

Ciencias de la Actividad Física y del Deporte (INEF-UPM) (Laboratorio número 214 de

la Red de Laboratorios de la Comunidad de Madrid).

Muestras sanguíneas y procesamiento

Se extrajeron muestras de sangre (10 mL) antes e inmediatamente después de cada una

de las pruebas, y se analizaron los siguientes parámetros bioquímicos: tHcy, folato,

vitamina B12 y creatinina. La extracción se realizó mediante punción venosa estándar

con palomilla en tubos al vacío Vacutainer®. Los tubos se colocaron inmediatamente

sobre hielo y una vez formado el coagulo, se centrifugó la muestra durante 10 minutos a

3.000 r.p.m. Se separó el suero en eppendorfs de 1 mL y se conservó la muestra a -80 ºC

hasta su procesamiento.

Las concentraciones totales de tHcy, fueron determinadas por la tecnología de

inmunoensayo por detección de fluorescencia polarizada, (FPIA; Abbott AxSYM,


50

Abbott Park, USA, CV total ≤ 6 %). Las concentraciones de vitamina B12 fueron

determinadas por la tecnología de enzimoinomunensayo de micropartículas (MEIA;

Abbott AxSYM, Abbott Park, USA, CV total ≤ 11 %). El folato sérico fue determinado

por la tecnología inomunensayo de captura de ion (ICIA; Abbott AxSYM, Abbott Park,

USA, CV total ≤ 19 %). La creatinina fue analizada por el método colorimétrico-

cinético (JAFFÉ), mediante espectrofotómetro autoanalizador CLIMA MC-15, (RAL,

S.A, España Cv total ≤ 3 %).

El análisis de todos los parámetros bioquímicos se llevó a cabo en el Laboratorio de

Bioquímica de la Facultad de Ciencias de la Actividad Física y del Deporte (INEF-

UPM) (Laboratorio número 242 de la Red de Laboratorios de la Comunidad de

Madrid).

Análisis estadístico

Todas las variables fueron promediadas en el paquete estadístico SPSS v.15.0 para

Windows (SPSS Worldwide Headquarters, Chicago, IL), donde se tomó la media y la

desviación estándar (DE) como estadísticos descriptivos. Se analizó la normalidad a

través de la prueba de Kolmogorov-Smirnov, además de analizar la asimetría y curtosis

de las variables, obteniéndose que todas las variables tenían un comportamiento normal

y era procedente utilizar estadística paramétrica. Para todas las variables estudiadas se

realizó el test t para muestras pareadas. Por tratarse de un reducido número de sujetos y

para verificar que no eran dependientes de la distribución, además, se realizó la prueba

no paramétrica de muestras relacionadas mediante el test de rangos y signos de

Wilcoxon. Para el análisis de las correlaciones entre las variables se realizó la prueba

del coeficiente de correlación de Pearson.

Se estableció para todos los análisis un valor de significación alpha < 0,05.

4.5 Resultados

Las características generales de los sujetos se muestran en la Tabla 7.


51

Table 7. Características generales de los sujetos

Características N Media DE Mínimo Máximo

Edad (años) 10 23,5 1,8 21,7 28,1

Talla (cm) 10 178,2 6,4 164,2 186,4

Peso (kg) 10 78,8 8,8 61,1 89,4

% Masa Grasa 10 16,0 4,7 7,4 24,9

% Masa Magra 10 84,0 4,7 75,1 92,6

DMO (g/cm2) 10 1,3 0,1 1,2 1,4

DE: Desviación estándar; DMO: Densidad Mineral Ósea

Los datos de la capacidad física de trabajo durante las pruebas máxima y submáxima se

muestran en la Tabla 8.

Table 8. Parámetros físicos recogidos durante la prueba máxima y la prueba submáxima

Variables Prueba Máxima

Media ±DE (min-máx)

Prueba Submáxima Media ±DE (min-máx)

VO2 max (mL/min) 4704,1 ± 575,2 (3937-5970)

4037,8 ± 341 (3556 - 4547)

VO2 peso (mL/min/Kg) 60 ± 5,3 (51,8 - 69,9)

51,9 ± 5,7 (41,7 - 59,1)

FC final (ppm) 193,4 ± 7,2 (180 - 202) -

FC max (ppm) - 188,4 ± 9,6 (175 - 202)

FC media (ppm) - 163,3 ± 12,1 (145,9 - 177,9)

V Aeróbica máx. (Km/h) 17,8 ± 1,1 (15,9 - 19,4) -

W/Peso (W/Kg) 4,0 ± 0,3 (3,6 - 4,4)

2,5 ± 0,3 (2 - 2,8)

VE (L/min) 161,6 ± 24,1 (130 - 214)

111,4 ± 9,6 (98 - 126)

VEL media (Km/h) - 11 ± 1,3 (8,6 - 12,4)

FC: Frecuencia Cardiaca; VO2: Consumo de Oxígeno; V: Velocidad; VE: Ventilación por minuto; W: Carga; VEL: Velocidad.


52

En la Tabla 9 se muestran los resultados en relación a todos los parámetros analizados

antes y después de realizar las dor pruebas. Las concentraciones séricas de tHcy

aumentaron de manera significativa (p < 0,05) tras la prueba máxima, superando el

valor esperado, al igual que en la prueba submáxima, en la que el efecto del ejercicio

fue más potente desde el punto de vista estadístico (p < 0,01).

Table 9. Concentraciones de tHcy, Folato, Vitamina B12 y Creatinina antes y después del ejercicio en prueba máxima y prueba submáxima

tHcy: Homocisteína Total; D.E: Desviación Estandard * Diferencias significativas entre momento antes y después (p<0,05) ** Diferencias significativas entre momento antes y después (p<0,01) a Diferencias significativas entre pruebas en el momento antes (p<0,05) b Diferencias significativas entre pruebas en el momento después (p<0,05)

En las figuras 6 y 7 se puede observar el efecto de ambas pruebas sobre los niveles de

tHcy en todos los sujetos estudiados, observando que las concentraciones de tHcy

aumentaron por encima del valor esperado tanto en la prueba máxima como en la

submáxima. Además, en la prueba submáxima, el aumento de las concentraciones de

tHcy se dio en todos los sujetos estudiados.

Prueba Máxima Prueba Submáxima

Media ±D.E (mín-máx)

Media ±DE (mín-máx)



Variables N Antes Después Antes Después

tHcy (µmol/L) 10 13,3±5,4

(6,3-25,7) 14,6±6*

(6,9-27,9) 12,3±4,5 (6,9-22,7)

14,4±6,3** (7,4-29,9)

Folato (ng/L) 10 8,9±2,3

(5,6-13,2) 10,3±2,6** (6,4-14,4)

8.8±10.5 (4,6-13,3)

10,5±3,0** (5,9-15,8)

Vitamina B12 (pg/mL)

10 504,6±135 (283,3-680,5)

548,9±135,6* (320,8-723)

466,8±129,3 (227-654)

507,7±147,9**b (236,4-766,2)

Creatinina (mg/dL) 10 0,8±0,1

(0,6-1) 2±0,3** (1,7-2,5)

1,4±0,4a (0,8-1,8)

1,5±0,2b (1,2-1,9)


53

Figure 6. Niveles de tHcy antes y después de la prueba máxima

Figure 7. Niveles de tHcy antes y después de la prueba submáxima

tHcy Antes

tHcy

Des

pués

µmol/L

tHcy

Des

pués

tHcy Antes

Línea de tendencia

µmol/L

Línea de tendencia


54

Las concentraciones séricas de folato y vitamina B12 aumentaron de manera

significativa después del ejercicio, tanto en la prueba máxima como en la prueba

submáxima. Sin embargo, la creatinina sólo aumentó de forma significativa después de

la prueba submáxima.

En la Tabla 10 se muestran las correlaciones entre las variables estudiadas. En cuanto a

la tHcy y el folato, se observó que existía una correlación negativa antes del ejercicio en

ambas pruebas en la prueba máxima (r = -0,69; p < 0,05) y en la prueba submáxima (r =

-0,87; p < 0,01). Al finalizar las pruebas esta relación se siguió manteniendo e incluso

aumentó, en la prueba máxima (r = -0,87; p < 0,01) y en la prueba submáxima (r = 0,94;

p < 0,001).

Para el resto de parámetros analizados no se encontraron correlaciones significativas en

ninguno de las dos pruebas.

Table 10. Correlaciones de Pearson entre las variables tHcy, folato, Vitamina B12 y creatinina antes y después en pruebas máxima y submáxima

Prueba Máxima Prueba Submáxima

Variables tHcy antes

tHcy después

tHcy antes

tHcy después

Folato Antes -0,69* -0,86** -0,87** -0,91**

Folato Después -0,82** -0,87** -0,88** -0,94**

Vitamina B12 Antes -0,13 0,01 -0,20 -0,26

Vitamina B12 Después -0,24 0,07 0,02 0,07

Creatinina Antes 0,21 0,14 -0,30 -0,41

Creatinina Después 0,34 0,34 0,19 0,37

tHcy: Homocisteína total * correlación significativa (p<0,05)** correlación significativa (p<0,01)


55

4.6 Discusión

El resultado principal del presente estudio indicó que el efecto agudo del ejercicio, tanto

tras una prueba incremental de intensidad máxima (VO2max) y 10 minutos de duración,

como tras una prueba submáxima al 65 % del VO2max y una duraciónd de 40 minutos

aumentó significativamente las concentraciones séricas de tHcy en sujetos jóvenes

varones físicamente activos. Esto indica que la elevación de las concentraciones de tHcy

post-esfuerzo es independiente de la duración y la intensidad de la prueba, al menos en

sujetos varones entrenados. En un estudio similar, pero realizado en varones más

jóvenes y en pruebas realizadas en cicloergómetro y kayak-ergómetro, Venta y col.

(124) también observaron una hiperhomocisteinemia post-esfuerzo en todos los casos.

Estos resultados arrojan algo de luz frente a los datos discrepantes publicados en la

bibliografía. Después de un esfuerzo en agudo realizado en cicloergómetro, Sotgia y col

(115) no observaron diferencias en las concentraciones de tHcy. En un estudio similar,

Gaume y col (45), incluso observaron una reducción de las concentraciones de tHcy

post-esfuerzo. En cambio, y de forma similar a nuestros resultados, varios autores han

encontrado concentraciones aumentadas tras el ejercicio (64, 74), aunque existen

variaciones en cuanto a duración y método utilizado (64, 124). En este sentido, la

diferencia en los protocolos utilizados puede ser una de las razones de la discrepancia de

los datos publicados.

En cuanto a la prueba de esfuerzo submáxima, las concentraciones de tHcy aumentaron

en todos los participantes del estudio. De acuerdo con nuestros resultados, se han

encontrado respuestas parecidas en la bibliografía, en la que se muestra un aumento de

tHcy tras este tipo de ejercicios (32, 47). En el estudio de Gelecek y col. (47) se observó

un aumento de tHcy tras la realización de un ejercicio aeróbico agudo en tapiz rodante

durante 30 minutos con una intensidad de 70-80 % de la FC máxima, apoyando los

resultados del presente estudio.

El mecanismo exacto por el que la concentración sérica de tHcy aumenta tras el

ejercicio físico agudo es desconocido. Algunos estudios apoyan la teoría de que la

demanda metabólica inducida por el ejercicio hace que el metabolismo del folato y de la

vitamina B12 y B6 sea mayor, resultando como consecuencia un aumento de los niveles

de tHcy (101, 115). Entre todas las vitaminas del grupo B, existe amplio consenso de


56

que el folato es la vitamina que más influencia tiene sobre las concentraciones de tHcy

(30, 107) a diferencia de la vitamina B12, cuyo efecto es menos evidente (1). De hecho,

en nuestros resultados se aprecia una correlación inversa significativa entre las

concentraciones de folato y tHcy, que no sólo se mantiene sino que aumenta en ambas

pruebas después del esfuerzo. Sin embargo, no se encontraron correlaciones

significativas en ninguno de los puntos entre tHcy y vitamina B12. Cabe destacar que la

correlación negativa entre folato y tHcy se mantiene aun aumentando también las

concentraciones de folato después del esfuerzo

Las concentraciones de folato del presente estudio mostraron un aumento significativo

después de ambas pruebas, lo que podría apoyar esta hipótesis, justificando la necesidad

de determinar si las personas que realizan una alta actividad física tienen mayores

necesidades de folato y vitaminas B para mantener los niveles de tHcy lo más bajo

posibles (71). En cambio, Venta y col. (124) no observaron aumentos significativos de

las concentraciones de folato post-esfuerzo, y sí de vitamina B12.

Por otro lado, se ha hipotetizado que la síntesis de creatina puede afectar a los niveles

circulantes de tHcy (47). Durante la práctica de ejercicio físico intenso, el aumento del

consumo de oxígeno y de la producción de radicales libres puede incrementar el

catabolismo de la metionina, con un consecuente aumento de la formación de tHcy

provocando la regeneración de muchas de las moléculas que contienen metilo,

particularmente la creatina durante altas intensidades de ejercicio (71, 124). En el

presente estudio se han analizado las concentraciones de creatinina como producto de

desecho de la creatina, las cuales aumentaron significativamente tras la prueba máxima

por encima del valor esperado, afectando de manera muy regular a todos los casos. Sin

embargo, en la prueba submáxima no se encontraron diferencias significativas. En

cuanto a la relación entre creatinina y tHcy no se encontró ninguna correlación

significativa, por lo que no se ha podido relacionar la influencia del aumento de la

creatinina tras el ejercicio físico con el aumento de los niveles de tHcy. En este sentido,

un estudio realizado en ratas analizó la influencia de una suplementación previa de

creatina durante 28 días sobre los niveles de tHcy inducidos por el ejercicio aeróbico y

anaeróbico. Los resultados mostraron que la suplementación de creatina disminuyó los


57

niveles de tHcy inducidos por el ejercicio en todas las fases medidas

independientemente del ejercicio realizado (29).

4.7 Conclusiones

Nuestros datos indican que en varones jóvenes entrenados, esfuerzos aeróbicos de alta

intensidad tanto máximos de corta duración como submáximos de duración media se

produce una elevación de las concentraciones de tHcy post-esfuerzo. Son necesarios

estudios que analicen el comportamiento de este incremento y sus repercusiones sobre

la salud a corto, medio y largo plazo.


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5 CHAPTER 5. STUDY 2: Effect of rehydration after acute exercise on homocysteine concentrations and related parameters.

5.1 Abstract

Current research has demonstrated an increase in total homocysteine (tHcy)

concentrations after acute exercise. Considering the importance of hydration as a control

mechanism on the body´s physiological responses, the aim of the present study was to

assess the effect of a post-exercise hydration protocol on tHcy concentrations and related

parameters after an acute aerobic submaximal exercise. Methods: Nineteen young

trained male participants (23.5 ± 1.8 yr) completed 2 submaximal 40-minutes treadmill

tests (65% VO2max) followed by a rehydration protocol, after one test subjects drunk

water (W) and after the other one a sport drink (SP). Results: Serum tHcy, vitamin B12,

folate and creatinine were analyzed before, after exercise and 2 h after the rehydration

protocol. Concentrations of tHcy significantly increased after both tests (p < 0.001).

Furthermore, tHcy concentrations decreased only significantly with the SP (p < 0.05).

Significant correlations were found between tHcy and folate before exercise (r = -0.553, p

< 0.05) and 2 h after rehydration only in one of the submaximal tests (r = -0.708, p <

0.01). The correlation analysis showed a high variability. Conclusions: After 2 h of

rehydration with water and with a sport drink, tHcy concentrations continued being above

the recommended values. Furthermore, an adequate reydration protocol after exercise

with a sport drink could be better than water in reducing the elevated tHcy induced

by acute aerobic submaximal exercise. Further research analyzing the effect of an

acute exercise and the role of hydration protocol on tHcy concentrations up to 24 h is

needed.

5.2 Introduction

Hyperhomocysteinemia results from altered methyl group metabolism and is considered

as an independent risk factor for cardiovascular disease (CVD), including atherosclerosis,

coronary artery disease, cerebrovascular disease, and myocardial infarction (81, 87, 94,

123). High blood concentrations of Hcy (Hcy) (> 100 µmol/L) induce endothelial

dysfunction, oxidative stress mechanisms and inflammatory vascular processes (12, 122),

but even concentrations around 12-15 µmol/L are considered a cardiovascular risk factor

(12) or at least a marker (133).

Hcy balance and hyperhomocysteinemia prevention depends on a number of

substrates, cofactors and coenzymes (93). Both nutritional (including B vitamins) and


60

hormonal factors have been demonstrated to have an influence on total homocysteine

(tHcy) blood concentrations (51, 70, 83, 95, 115).

Moreover, some studies have attempted to study the effect of exercise on tHcy

concentrations, but results are not consistent. Lack of standardization, different protocols

used, type of exercises, intensities and duration, make it difficult to reach an agreement.

Some available data demonstrated an increase in tHcy levels immediately after moderate

or high intensity exercise (32, 64, 83, 95, 115), but no consistent data are available

regarding the responsible mechanisms and the health impact. It has also been speculated

that the synthesis of creatine during exercise could affect circulating tHcy levels (124).

Exhausting exercise increases the synthesis of creatine and plasma protein regeneration

(46). Given that physical exercise induces changes in protein and amino acid metabolism

it is important to understand whether Hcy concentrations are affected by exercise and to

determine possible mechanisms especially in active populations. On the other hand,

all physiological systems in the human body are influenced by dehydration (16, 91).

This particularly important role of hydration on physical exercise contributes to an

adequate homeostatic balance. Previous efforts have been made regarding the

relationship between hydration and performance but less to health-related aspects.

Considering the importance of hydration as a control mechanism on the body´s

physiological responses it is important to study its possible implication on tHcy

concentrations after exercise. Since tHcy concentrations increase after moderate and

high-intensity acute exercise (83), the aim of the present study was to assess the

effect of controlled rehydration on tHcy concentrations and related parameters after

acute aerobic submaximal exercise.

5.3 Material and Methods

5.3.1 Participants

Nineteen apparently healthy active young males (mean age 23.5 ± 1.8 yr) participated in

the study. Participants were recruited by means of advertisements published at the

Faculty of Physical Activity and Sport Sciences–INEF of the Technical University of

Madrid (Spain) inviting them to voluntarily participate in the study.

The study has been performed following the ethical guidelines of the Declaration of

Helsinki for research involving human subjects (World Medical Association, 2004).

Participants were informed of the nature and purpose of the study and signed an informed


61

consent prior to conducting the tests. The protocol was approved by the Ethics Review

Board of the Technical University of Madrid.

Inclusion and exclusion criteria

Criteria for participants’ selection included: being physically active (at least 3 days of

physical activity per week), a non-smoker and healthy (not having any of the diseases

listed in the exclusion criteria listed below).

Exclusion criteria were: Having any central or peripheral cardiovascular risk factor

including diabetes, kidney or liver problems, known asthmatic complications, plasma

cholesterol > 8 mmol per liter (mmol/L), systolic blood pressure > 160 mmHg or

diastolic blood pressure > 100 mmHg, history of alcohol or drug abuse, history of

inflammation or cancer, orthopedic limitations, medications that may affect metabolic

and cardiovascular function, following a vegetarian diet, intake of B-vitamins

supplement, vitamin-fortified food or creatine supplementation during the last two

months.

5.3.2 Design

Medical examination

On their first visit, participants were required to complete a medical examination in order

to ensure there was not any medical contraindication to participate in the study.

Participants underwent an electrocardiogram using an electrocardiogram Jaeger ® (Erich

Jaeger, Germany). Weight was recorded with a scale Detecto ® (Lafayette Instruments

Company, Lafayette, Indiana, USA), height was measured with a conventional zipper

stadiometer (Holtain Limited, Crymych, UK). Body composition was analyzed by

Bioelectrical Impedance Analysis (BIA) with a TANITA BC 418 MA (Tanita Corp.,

Tokyo, Japan).

Participants completed an incremental maximal test on a treadmill (H/P/COSMOS ®

3P 4.0, H/P/Cosmos Sports & Medical, Nussdorf-Traunstein, Germany) to determine

their individual maximal oxygen uptake (VO2max), following the protocol described by

Myers & Bellin in 2000 (92); starting at 1 initial resting minute at 0 % of slope,

followed by 3 minutes at 6 km per hour (km/h) (1 % of slope), with a speed increase

of 0.2 km/h every 12 seconds until exhaustion, followed by an active recovery of 2-

minutes walking at 6 km/h followed by a 3-minute of sitting passive recovery.


62

VO2max was used to establish the individual load at 65 % of VO2max of each participant

for further tests.

Experimental protocol

Participants completed two treadmill tests, performed in a hot environment in order to get

the subjects dehydrated (mean temperature of 30 °C and 60 % of mean relative humidity),

controlled by plastics, heaters and a weather station. The load was constant with an

intensity of 65 % of their VO2max on a treadmill (H/P/COSMOS ® 3P 4.0, H/P/Cosmos

Sports & Medical, Nussdorf-Traunstein, Germany). Each test consisted of an initial 1-

minute resting, 3-minutes warm up at a rate of 6 km/h, then 40 minutes running at

constant intensity (65 % of VO2max) and finally, an active recovery of 2 minutes walking

and 3 minutes sitting of passive recovery. After both tests participants followed a

rehydration protocol during 2 hours, one of the tests with water and the other one with a

sport drink, randomly assigned, (see rehydration protocol). There was a one-week

washout period between tests.

Tests were performed at the Laboratory of Exercise Physiology, at the Faculty of

Physical Activity and Sport Sciences-INEF, Technical University of Madrid (Laboratory

number 214, Laboratory Network of the Region of Madrid, Spain).

Randomization

Participants were randomly assigned to complete the 2 tests through counterbalanced

drawing. Each volunteer was randomly assigned to drink water or the sport beverage in

the first or the second treadmill test. The type of beverage after each test was randomized

and counterbalanced. It was not a blind randomized trial as the taste of the drink was

not hidden.

Standardization of the diet and exercise

In order to standardize the results, participants were instructed not to perform high or

moderate exercise 24 hours prior to testing, and not to consume any food, drink,

coffee or caffeinated beverages within 2 hours prior to tests. To ensure a euhydration

status before each trial, subjects were instructed to follow a standard hydration protocol 2

hours before the test, with a water average intake of 350 mL, according to

ACSM 2007 recommendations (111).


63

Furthermore, there was a diet control during all the participation in the study.

Rehydration protocol

Participants were weight with the same light and dry clothes before and after each of the

submaximal exercise tests, in order to calculate water loss through sweat during exercise.

It was obtained by subtracting the initial body mass from the final body mass.

After the end of the tests, participants followed a 2 h post-exercise controlled

rehydration. The total volume intake during the post-exercise controlled rehydration

phase was the same amount (in mL) as the body mass loss (in g) in each test, calculated

individually for each participant.

Half of the total amount was drunk during the first hour and the other half during the second hour distributed in regular intervals.

Drink composition

The types of drink used for the rehydration protocol were water and a sport drink. Table

11 shows the composition of the drinks.

Table 11. Drink composition

Bottled Water Sport drink Dry residue 265 mg/L, bicarbonates 276 mg/L, sulfates 6.9 mg/L, chlorides 4.3 mg/L, calcium 90.4 mg/L, magnesium 2.7 mg/L and sodium 2.1 mg/L.

Ingredients: Water, sucrose, acidulant citric acid, mineral salts: sodium citrate, magnesium chloride, calcium chloride and potassium citrate. Flavourings and stabilizers E-414 and E-445 dye E-133Energy value (Values for each 100 mL): 31 kcal; proteins 0 g; carbohydrates 7.5 g, which sugar added: 7.5 g; fat 0 g, which saturated fatty acids: 0 g; dietary fiber 0 g; sodium 50 mg; added minerals: calcium 1.3 mg; potassium 12.5 mg; magnesium 0.6 mg.

Physiologic measures

During exercise, heart rate (HR) was controlled using a Polar S810® (Polar Electro,

Kempele, Finland); the oxygen uptake (VO2) and ventilation (VE) were measured with

the gas analyzer Jaeger Oxycon Pro (Erich Jaeger, Viasys Healthcare, Germany).

Blood sample processing

Blood samples (10 mL) were collected immediately before, immediately after and 2

hours after each of the treadmill tests.


64

The extraction was performed by standard venipuncture in vacuum Vacutainer ® tubes.

Tubes were placed on ice immediately and after clot formation samples were centrifuged

during 10 minutes at 3000 rpm. Serum was distributed and conserved on eppendorf of 1

mL at -80 ºC until processing.

For hematological parameters, a complete hematological analysis was performed within

the first hour after extraction and was obtained by an automated hematology analyzer

(Celltac E MEK-7222J/K, Nihon Kohden Corporation, Tokyo, Japan) at the Laboratory

of the Faculty of Physical Activity and Sport Sciences-INEF, of the Technical University

of Madrid (Laboratory number 242, Laboratory Network of the Region of Madrid).

Routine biochemistry analysis was carried out with the Clima MC-15 (RAL) RAL, SA,

Spain, using standard methodologies.

Serum tHcy was determined by immunoassay technique for detection of fluorescence

polarization (FPIA; Abbott AxSYM, Abbott Park, USA, CV ≤ 6 %) and by enzymatic

assay (Beckman AU400, Beckman Instruments, Ltd., Bucks, UK, CV ≤ 6 %). Serum B12

concentrations were determined by microparticle enzyme immunoassay technique

(MEIA; Abbott AxSYM, Abbott Park, USA, CV ≤ 11 %) and by electrocheluminescence

immnoassay on Elecsys 2010 (Roche Diagnostics, IND, USA, CV ≤ 10 %). Serum Folate

was determined by immunoassay ion capture technique (ICIA; Abbott AxSYM, Abbott

Park, USA, CV ≤ 19 %) and by electrocheluminescence immnoassay on Elecsys 2010

(Roche Diagnostics, IND, USA, CV ≤ 11 %). Creatinine was analyzed by kinetic

colorimetric method (Jaffe) with an autoanalyzer CLIMATE spectrophotometer MC-15,

(RAL, SA, Spain, CV ≤ 3 %) and by colorimetric analyzer (Beckman AU400, Beckman

Instruments, Ltd., Bucks, UK, CV ≤ 3%).

The analysis of all the specific biochemical parameters was carried out at the

Biochemistry Laboratory of the Faculty of Physical Activity and Sport Sciences-INEF, of

Technical University of Madrid (Laboratory number 242, Laboratory Network of the

Region of Madrid) and at the Clinical laboratory of the Sports Medicine Center of the

High Sports Council (CSD, Spain).

5.3.3 Statistical Analysis

SPSS v.20.0 for Windows (SPSS Worldwide Headquarters, Chicago, IL) was used for the

statistical analysis.


65

As all the variables followed a normal distribution, parametric statistics were

used. Standard statistical methods were used for the calculation of the means and

standard deviation (± SD).

Two-way analysis of variance for repeated measures (ANOVA) was used to determine

any differences in each variable between points (before, after and two hours after

hydration) and among tests (water and sport drink), multiple evaluations were made using

the Bonferroni post-hoc test.

Pearson correlation coefficient was used for analysis of correlations between variables.

Percentage (%) of change was calculated within each drink between points before-after

(1-2) and between after-two hours after hydration (2-3) on the variable homocysteine.

Non-parametric tests were performed (median and Related Samples Wilcoxon signed

Rank test) to compare the hydration protocols.

Tertiles were also calculated according to baseline tHcy levels. Changes of tHcy from

basal levels were observed graphically in a scatter plot. A non-parametric test of Kruskal-

Wallis was performed to study the changes after exercise in tHcy in respect of

baseline concentrations.

Significance was set at p < 0.05 for all analysis.

5.4 Results

Table 12 shows the descriptive characteristics of the studied sample regarding height,

weight, VO2max, percentage of fat mass and lean mass.

Table 12. General characteristics of the participants at baseline

VO2max:maximaloxygenuptake

Characteristics N Mean SD Min Max

Age (years) 19 22.8 1.78 21 28 Height (cm) 19 176.9 7.17 160.8 188 Weight (Kg) 19 75.4 8.93 54.6 89.4 Lean mass (Kg) 19 64.9 7.12 50.9 74.0 Fat mass (Kg) 19 9.06 4.04 2.10 17.0 % Mass Fat 19 12.1 4.91 3.40 19.9 VO2max (mL/min) 19 4586 554 3798 5970 VO2max Relative to weight (mL/min/kg) 19 61.15 5.08 51.80 69.90


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Table 13 shows the analyzed blood parameters according to both tests, before,

immediately after and 2 hours after exercise. A significant increase of tHcy levels (p < 0.001) was observed after exercise in both tests (11.32 % and 12.37 % of increase with

respect to pre-exercise values).

Table 13. Total homocysteine, folate, vitamin B12 and creatinine concentrations before, after exercise and 2 hours after rehydration protocol

*p<0.05betweenmeasuredpointswithintest.**p<0.01betweenmeasuredpointswithintest.tHcy:TotalHomocysteine;Before:beforeexercise;After:afterexercise;2h:2hoursafterrehydration.

Concentrations of tHcy decreased after 2 hours of rehydration, only significantly for the

SP drink (p < 0.05). No differences were found between tests at any of the measured

points.

Concentrations of folate and vitamin B12 also increased significantly after exercise in

both tests (p < 0.01). After the rehydration protocol, folate and vitamin B12 decreased

significantly again with both drinks (p < 0.05).

Creatinine levels showed an increased tendency after exercise tests, and in line with the

other parameters analyzed, decreased significantly after both hydration protocols (p <

0.05).

Pearson partial correlations are shown in table 14. Significant inverse correlations were

found between tHcy and folate before exercise and 2 hours after hydration with W while

no significant correlation was found between tHcy and B12 concentrations at any

measured point. A significant correlation was found between tHcy and creatinine before

exercise only before SP test (p < 0.05).

Variables W test Before

Mean±SD (Min-Max)

W test After Mean±SD (Min-Max)

W test 2h Mean±SD (Min-Max)

SP test Before Mean±SD (Min-Max)

SP test After Mean±SD (Min-Max)

SP test 2h Mean±SD (Min-Max)

tHcy (µmol/L)

10.82±2.153 (6.91-16.07)

12.51±2.640** (7.41-18.92)

12.08±2.901 (6.82-18.72)

10.77±1.878 (7.97-14.42)

12.62±2.605** (8.90-18.58)

11.98±2.798* (8.34-17.76)

Vitamin B12 (pg/mL)

492.2 ± 161.5 (184.90-958.40)

547.9±176.2** (212.7-1036)

533.96±152.9** (202.9-923.2)

502.58±169.8 (199.8-954.7)

552.2±172.3** (232.3-1010)

504.9±156.8** (238.7-925.2)

Folate (ng/L)

8.677±2.61 (4.08-13.3)

10.50±2.806** (6.58-15.89)

9.568±2.701* (5.60-14.81)

8.231±2.201 (5.23-14.2)

10.05±2.718** (6.55-15.40)

8.384±2.268** (5.60-13.39)

Creatinine (mg/dL)

1.27±0.286 (0.80-1.80)

1.411±0.210 (1.10-1.90)

1.232±0.297** (0.90-2.10)

1.22±0.288 (0.70-2.00)

1.46±0.314 (1.11-2.20)

1.22±0.24** (0.90-1.80)


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Table 14. Pearson correlation coeficients between tHcy, vitamin B12, folate and creatinine

Vitamin B12 (pg/mL)

Folate (ng/L)

Creatinine (mg/dL)

W test

tHcy before (µmol/L) r = -0.194 r = -0.553* r = -0.135

tHcy after (µmol/L) r = -0.22 r = -0.399 r = 0.316

tHcy 2h (µmol/L) r = -0.184 r = -0.708** r = 0.284

SP test

tHcy before (µmol/L) r = -0.086 r =-0.354 r = -0.549*

tHcy after (µmol/L) r = -0.101 r =-0.265 r = -0.132

tHcy 2h (µmol/L) r = -0.033 r =-0.276 r = -0.297

r:Pearsoncorrelationcoefficient;*p<0.05;**p<0.01.tHcy:TotalHomocysteine;Before:beforeexercise;Afterafterexercise;2h:2hoursafterrehydration;W:watertest;SP:sportdrinktest.

Figure 8 shows the % of change of tHcy concentrations “before exercise” and “after

exercise” splitting the sample by tertiles. THcy increased in all cases, independently of

the tHcy basal levels.

tHcy:TotalHomocysteine;W:Watertest;SP:Sportdrinktest

Figure 8. Percentage of change (%) in total homocysteine between “before” and “after exercise” in exercise tests. Sample splitting by tertiles


68

Figure 9 shows the % of change of tHcy concentrations between “after exercise” and “2 h

after rehydration”. There were no statically significant differences, but a steady better

tHcy recovery was observed with SP than with W in those subjects with higher

levels of tHcy (3º tertile).

tHcy:TotalHomocysteine;W:Watertest;SP:Sportdrinktest

Figure 9. Percentage of change (%) in total homocysteine between “after exercise” and “2 hours after rehydration” with water and sport drink. Sample splitting by tertiles


69

5.5 Discussion

This study contributes to a better understanding of the response of tHcy concentrations

after exercise and the implication of a controlled rehydration protocol on increased tHcy

concentrations after an acute aerobic submaximal exercise (65 % of VO2max). There were

a variety of studies aiming to investigate the effect of exercise on tHcy concentrations

after different types of exercices and intensities, but, to the best of our knowledge, this

is the first one that shows the effect of fluid intake with a standardized rehydration

protocol on tHcy concentrations after a single bout of acute aerobic exercise.

Our results showed an increase in tHcy concentrations after aerobic submaximal exercise

in young trained male participants. Moreover after 2 h of rehydration, tHcy

concentrations showed a significant decrease after the rehydration protocol only with the

sport drink. Comparison with previous studies is difficult because there are no studies

analyzing the recovery of tHcy after exercise with the implementation of a hydration

protocol.

In the last years, different mechanisms have been proposed to explain the increase in

tHcy post-exercise. Some explanations are focused on renal blood flow and filtration

reduction during exercise (124), others sustain the theory that increased metabolic rate

during exercise induces higher folate uptake as a consequence of an increase in Hcy

levels (76). But on the contrary, recently, Iglesias-Gutierrez et al. (68), concluded that

neither of these mechanisms explain the lack of linear relationship between pre and post-

exercise tHcy concentrations.

A quite new hypothesis proposed that changes on tHcy could be related to energy

expenditure and substrate utilization, and dependent on duration and intensity of exercise

(71, 130), but in our previous study (83), we found a similar significant increase of tHcy

concentrations after exercise at two different intensities and duration tests, maximal

intensity (VO2max) and submaximal intensity (65 %) of VO2max. In agreement, Iglesias-

Gutierrez et al. (68) did not find any relationship between tHcy concentrations and the

different substrate utilization at low and high intensities. One of the most interesting

findings of the present study is that the increase of tHcy concentration is subject

independent, increasing in all of them independently of their basal tHcy concentrations

before exercise.

Moreover, we observed a post-exercise increase of creatinine. It has been suggested that

during high intensity or exhaustive exercise, which relies on anaerobic or protein-derived


70

energy sources, tHcy production could increase due to the metabolism of protein turnover

(70, 74). But, in contrast our results did not find a relationship between creatinine as end-

product of creatine and tHcy in any of the measured points.

Our results showed an inverse correlation between folate and tHcy at basal status,

coinciding with a variety of results in different studies (51, 64, 83, 93). Nevertheless,

there was a tendency of losing the strong negative correlation between tHcy and

folate after exercise that is contradictory to our previous results (83). Even if after

rehydration serum folate concentrations decreased significantly with both

rehydration protocols, the previous relation with tHcy after exercise was not recovered.

This could be probably due to the high demand of folic acid as methyl donor for the

remethylation of methione from homocysteine in the post-exercise phase (63). On

the contrary, vitamin B12, also implicated in the methionine-tHcy metabolism, did not

show any correlation with tHcy at any of the measured points. Previous studies stated

that correlations of vitamin B12 are usually weaker than those of folate (52).

In relation to the rehydration effect on tHcy concentrations after exercise, our results

showed a slightly higher tHcy decrease with the SP than with W although there were no

significant differences among tests in this point and the higher reduction of SP could be

explained also by the slightly higher increase of tHcy concentrations in this test.

However, tHcy concentrations did not return to basal values with any of the beverages.

Beverage composition probably plays an important role in this context. It seems to be due

to the effect of the carbohydrate content of the sport drink. Previous research has shown a

better effect of carbohydrate-electrolyte solutions than water when rapid rehydration is

required (38). The rate of gastric emptying of ingested fluids is determined primarily by

the volume, osmolarity, and energy density of the gastric contents. Ingestions of drinks

that contain carbohydrates and electrolytes may offer a gradual return to pre-dehydration

levels and tend to prevent any decrease in circulating sodium concentration, maintaining

better the plasma volume and resulting in a smaller urine fluid loss. Some investigations

highlight the importance of avoiding rapid increase in plasma volume and corresponding

reduction in sodium concentration and osmolarity during post-exercise rehydration to

ensure that diuresis does not occur and that retention of ingested fluid is maximized

(38). In this way, as hydration with a sport beverage (carbohydrate and electrolyte

containing) helps to maintain plasma volume better than water, we could hypothesize that


71

sport drinks will do better than water to control elevated tHcy and other blood parameters

that could be altered during exercise.

Regarding dehydration, sweat rates and sweat composition depends on external

temperature, humidity and exercise intensity, but also on individual differences (84). Our

results showed a mean lost of 1.27 kg in the W test and 1.45 kg in the SP test after

exercise. That corresponds to a 1.7 % and 1.92 % of weight lost, respectively. As the

guidelines of ACSM in 2007 indicated, “If proper controls are made, body water changes

can provide a sensitive estimate of acute total body water changes to access hydration

changes during exercise” (111). The body mass changes in the present study show an

important dehydration status, that is consistent with exercise in a warm environment

(ambient temperature > 30 ºC). Dehydration between 1 to 2 % of body weight

begins to compromise physiologic function and increases an athlete´s risk of

developing an exertional illness (16). This level of dehydration is common in many

sports and better established on long duration exercise, but there is a need of well-

defined hydration protocols for sports with duration of less than one hour. Usually, in

exercises less than 1 hour, only water is recommended, but those studies are based

on getting deeper into performance aspects, and less into health-related aspects.

Regarding our results, recommendations on hydration and beverages must be

reviewed, in relationship with some risk parameters like tHcy, beyond thermoregulatory

effects.

It is important to emphasize that during the time of exercise without hydration tHcy

increases, but its possible health consequences are still unknown. These higher

concentrations are maintained during 1 hour or more plus recovery time, and could also

affect subsequent B vitamin values and other parameters. Therefore, it could be

interesting to study if a controlled hydration during exercise could help to maintain

tHcy concentrations at baseline levels and the relationship of the other implicated

parameters.

5.6 Conclusion

Concentrations of tHcy increase after acute aerobic submaximal exercise in young trained

males independently of the initial concentrations at baseline. Furthermore, 2 h of

a rehydration protocol with a sport drink, decreased tHcy concentrations significantly,

but concentrations continued being above the recommended values. More research


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analyzing tHcy behaviour and the effect of hydration on this parameter up to 24 hours

after acute aerobic exercise is needed.


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6 CHAPTER 6. STUDY 3: Hydration during exercise prevents the increase of homocysteine concentrations

6.1 Abstract

Background: Several studies have demonstrated an increase in total serum homocysteine

(tHcy) after acute exercise. Objective: To assess the effect of hydration on tHcy and

related parameters after acute aerobic submaximal exercise. Methods: Twenty trained

males (29.4±7.9 yr) completed 4 treadmill tests at constant intensity (65 % of VO2max): 2

non-hydration tests (NH1 and NH2) and 2 tests with hydration during exercise with 2

different beverages, water (H1) and a sport drink (H2). After all 4 tests, subjects followed

a 2 h rehydration protocol, with water after NH1 and H1, and with a sport drink after

NH2 and H2. Serum tHcy, vitamin B12, folate, creatine and creatinine were analyzed

before (pre0), after (post0), at 2 h, 6 h and 24 h. Methylenetetrahydrofolate reductase

(MTHFR) C677T and Angiotensin Converting Enzyme (ACE) Insertion/Deletion (I/D)

polymorphisms were controlled. Results: tHcy concentrations increased after exercise in

NH1 and NH2 reaching significant differences at 6 h (p < 0.05). Concentrations of tHcy

were maintained at baseline up to 2 h after exercise in H1 and H2. At 24 h tHcy

concentrations recovered to baseline in all tests. Vitamin B12 increased at 6 h in NH1,

NH2 and H2 (p < 0.05). Serum creatine concentrations increased at post0 in the 4 tests

(p< 0.05). Creatine and creatinine concentrations reached maximum at 6 h in the 4 tests

(p < 0.05). No differences were found in MTHFR C677T or ACE I/D genetic

groups. Conclusions: Hydration during acute aerobic submaximal exercise

maintains tHcy concentrations at baseline up to 2 hours and prevents the further

increase. The increase of tHcy induced by acute exercise could be related to the

demand of creatine and vitamin B12. The underlying mechanisms need further

investigation. 6.2 Introduction Elevated total serum homocysteine (tHcy) concentration, even a mild increase, has been

consistently considered in humans as a risk factor for cardiovascular diseases and stroke,

for neurodegenerative disorders and for the development of atherosclerosis (35, 39).

However, more recently, the debate is open as to whether homocysteine is a marker,

or a causative agent (97). Homocysteine synthesis occurs in the liver in the

remethylation pathway of the methionine metabolism, after conversion to S-

adenosylmethionine (SAM), the most important methyl group donor in the body

(14).


74

Physical exercise induces changes in protein and amino acid metabolism, and

therefore the effect of exercise on homocysteine response is gaining importance. Over

the past few years there has been controversial data regarding the effect of exercise on

tHcy concentrations, but lately, several studies have consistently demonstrated a tHcy

increase immediately after acute vigorous exercise (27, 47, 64, 68). The exact

mechanism by which exercise affects tHcy continues to be unknown. Some authors

have suggested that exercise accelerates protein catabolism and the pool of amino acids

in the muscle (121). As a result it may lead to an increase in homocysteine synthesis

(70, 104). Moreover, as described in several studies, tHcy is strongly correlated to

folate and vitamin B12, but in exercise different responses have been observed. An

explanation would be that the synthesis of tHcy during exercise and the increase of

folate and vitamin B12 demand lead to a depletion losing the strong correlation after

exercise (82). In addition, homocysteine metabolism is affected by several enzyme

mutations, the methylenetetrahydrofolate reductase (MTHFR) C677T

polymorphismbeing the most prevalent. In those with the MTHFR 677TT genotype,

enzyme activity is lowered. Therefore, these individuals might require an increased intake

of folate to maintain or control blood levels of folate or tHcy (43).

Furthermore, prolonged exercise induces marked dehydration and hyperthermia if the

fluid lost is not replaced during exercise. The detrimental effects of dehydration on

cardiovascular, thermoregulatory and metabolic function are well documented (50, 77,

113). Previous studies have provided compelling evidence that dehydration during

exercise in the heat results in significant perturbations in cardiovascular function in

healthy humans compared with euhydrated normothermic and euhydrated heat-stressed

individuals (50). The combination of exercise in the heat and dehydration leads the

human body to a stress situation inducing elevations in tHcy concentrations (118). These

stressors increase catecholamine secretions leading to blood pressure elevation. The

Angiotensin Converting Enzyme (ACE) is involved in all this process (118). As the rise

in tHcy concentrations may have been sympathetically-mediated and is closely related

with the stressor stimuli, the relation between ACE insertion/deletion (I/D) polymorphism

on increased tHcy concentrations during exercise should be studied.

Dehydrated cells could have a catabolic effect, promoting glycogen and possible protein

breakdown. Our previous results (82) have shown that hydration after exercise helps to

reduce tHcy concentrations with both, water and a sport drink, the tHcy recovery rate

being higher with a sport drink than with water after 2 hours of a rehydration protocol,


75

but no data exist regarding the effect of hydration during exercise. Because hydration

could have an important effect on the physiological changes induced by exercise,

the main objective of this study was to assess the response of tHcy and its related

parameters such as folate, vitamin B12, creatine, creatinine, MTHFR C677T and ACE

I/D polymorphisms, with and without a hydration protocol during exercise, and the

different effects on these parameters regarding water and sport drink hydration. 6.3 Material and Methods

Participants

Twenty males (mean age 29.4±7.9 yr) without known pathology, healthy and physically

active were recruited at the Faculty of Physical Activity and Sport Sciences–INEF of the

Technical University of Madrid (Spain).

The sample selection and study protocol were performed following the ethical guidelines

of the Declaration of Helsinki for research involving human subjects (World Medical

Association, 2004). Participants were informed of the nature and purpose of the study and

signed an informed consent prior to conducting the tests. The protocol was approved by

the Ethics Review Board of the Technical University of Madrid.

Inclusion and exclusion criteria

Inclusion criteria were to be male, physically active (at least 3 days per week of aerobic

exercise), a non-smoker and healthy (not having any of the diseases listed below).

Exclusion criteria were to have any central or peripheral cardiovascular risk factor,

diabetes, kidney or liver problems, known asthmatic complications, total cholesterol >

200 mg/dl, systolic blood pressure > 160 mmHg or diastolic blood pressure > 100 mmHg,

history of toxic abuse, history of inflammation or cancer, orthopaedic limitations,

medications that may affect metabolic and cardiovascular function, vegetarian diets,

intake of B-vitamins supplement or fortified food during the last two months.

Medical examination

On their first visit, subjects were required to complete a medical examination in order to

ensure there was no medical contraindication to participate in the study. Participants

underwent an electrocardiogram and were weighed and measured. Body composition was

analyzed by Bioelectrical Impedance Analysis (BIA) with a TANITA BC 418 MA

(Tanita Corp., Tokyo, Japan).


76

During this session, subjects completed an incremental maximal test on a treadmill

(H/P/COSMOS® 3P 4.0, H/P/Cosmos Sports & Medical, Nussdorf-Traunstein,

Germany), according to the protocol described by (92). Individual maximal oxygen

uptake (VO2max) was determined to establish the individual load at 65 % of oxygen

consumption (VO2) of each subject for the further tests of the study.

Exercise protocol

All subjects completed 4 tests at constant load with an intensity of 65 % of their VO2max

on a treadmill as described in a previous study, (83). Tests were performed in a hot

environment in order to get the subjects dehydrated (mean temperature of 30 °C and 60 %

of mean relative humidity), controlled by heaters and a weather station. Two of the

exercise tests were performed without hydration during exercise (NH1 and NH2) and the

other 2 tests with hydration during exercise (H1 and H2) (Figure 10). After all of the tests

subjects followed a rehydration protocol (See below hydration protocol).

During exercise, heart rate (HR), VO2 and ventilation (VE) were controlled. Blood

pressure (BP) was measured before and after each exercise test.

Figure 10. Experimental protocol


77

Hydration protocol

Subjects were weighed in light clothes before and after each of the tests, in order to

calculate water loss through sweat during exercise. The hydration protocols were as

follows:

Subjects followed a 2 hour post-exercise controlled rehydration with water in NH1 and

with a sport drink in NH2. In the hydration tests, subjects drank 250 mL during exercise

and followed a 2 hours post-exercise controlled rehydration with water (H1) and with a

sport drink (H2). The composition of the drinks is the same as described previously in

study 2 (Table 11).

The drinking volume during the post-exercise controlled rehydration phase was the same

as the weight lost during exercise, individually measured for each participant in each of

the 4 tests. It was obtained by subtracting the initial body weight from final body weight.

The difference in grams (g) was considered in volume in millilitres (mL). Half of the total

amount was drunk during the first hour and the other half during the second hour

distributed in regular intervals.

Hydration during exercise

The hydration protocol during exercise was a follows: Participants followed a controlled

hydration during the exercise test. The volume intake was 250 mL distributed in 2 doses

of 125 mL in minute 15 and minute 30.

Randomization

Subjects were randomly assigned through counterbalanced drawing to complete the 2

non-hydration tests: NH1 and NH2; and the 2 hydration tests, H1 and H2.

The type of beverage after each test was randomized and counterbalanced. Although it

was a randomized trial, it was not blind, because the taste of the drink was not hidden.

Standardization of previous diet and exercise

After enrolment and in order to standardize the results, subjects were instructed not to

perform strenuous exercise 24 hours prior to testing, and not to consume any food, drink

coffee or caffeinated beverages within 2 hours prior to performing the tests. To ensure

euhydration status before each trial, subjects had to follow a standardized hydration


78

protocol by ingesting an average of 350 mL of water 2 hours before performing the

tests, according to ACSM recommendations (111).

Blood sample processing

Blood samples (10 mL) were collected at pre0, post0, 2 h, 6 h, and 24 h after each of the

treadmill tests.

The extraction was performed by standard venipuncture in vacuum Vacutainer ® tubes.

Tubes were placed on ice immediately and after clot formation samples were centrifuged

during 10 minutes at 3000 rpm. Serum was distributed and stored at -80 ºC until

processing.

Serum tHcy was determined using an enzymatic assay (AU400 analyzer, Beckman

Instruments, Ltd., Bucks, UK; CV ≤ 6 %). Vitamin B12 was analyzed using an

electrocheluminescence immnoassay (Elecsys 2010 analyzer, Roche Diagnostics, IN,

USA; CV ≤ 10 %). Serum Folate was determined using an electrocheluminescence

immunoassay (Elecsys 2010, Roche Diagnostics, IN, USA, CV ≤ 11 %). Creatinine was

analyzed using a colorimetric analyzer (Beckman AU400, Beckman Instruments, Ltd.,

Bucks, UK; CV≤ 2 %) and creatine was determined by capillary electrophoresis using a

diode array detector (P/ACE Beckman, Fullerton, CA, USA). Serum soudium, chloride

and magnesium were measured by indirect potentiometry technique (Beckman AU400,

Beckman Instruments, Ltd., Bucks, UK) .The rest of the routine biochemistry analysis

was carried out with the colorimetric analyzer (Beckman AU400, Beckman Instruments,

Ltd., Bucks, UK CV≤ 6 %) using standard methodologies. Urine osmolarity was

determined by freezing point depression with an osmometer Osmo Station OM-6050

(Menarini Diagnostics, Florence, Italy, CV ≤ 1 %)

For hematological parameters, a complete hematological analysis was performed within

the first hour after extraction and was obtained by an automated hematology analyzer

(Celltac E MEK-7222J/K, Nihon Kohden Corporation, Tokyo, Japan) at the Laboratory

of the Faculty of Physical Activity and Sport Sciences-INEF, of the Technical University

of Madrid (Laboratory number 242, Laboratory Network of the Region of Madrid).

The analysis of all the biochemical parameters was carried out at the Clinical laboratory

of the Sports Medicine Center of the High Sports Council (HSC, Spain). Creatine was


79

measured at the Clinical laboratory of the Faculty of Medicine, Dept. of Biomedical

Sciences of University of Sassari (Sardinia, Italy).

Genetic analysis

Whole blood (5 mL) from each participant was collected in ethylene-diamineteraacetic

acid, EDTA and sent to the Laboratory of Pediatrics, Faculty of Medicine, University of

Cantabria. DNA was extracted from each sample using the "QIAamp® DNA Blood Mini

Kit" from QIAGEN (Hilden, Germany) and the genotyping was performed afterwards.

The DNA samples were preserved at -20 °C.

The analysis of the MTHFR C677T (rs1801133) polymorphism was done based on the

PCR and RFLP techniques (40).

Polymerase chain reaction (PCR) amplification to detect ACE I/D polymorphism

(rs4340) was carried out using the previously published primers (67).

6.3.1 Statistical Analysis

As all the variables followed a normal distribution, parametric statistics were

used. Standard statistical methods were used for the calculation of the means and

standard deviation (±SD).

Two-way analysis of variance for repeated measures (ANOVA) was used to determine

any differences in each variable between points (Pre0, Post0, at 2 h, 6 h and 24 h) and

among tests (NH1, NH2, H1, H2), multiple evaluations were made using the Bonferroni

post-hoc test. Percentage of change was calculated within each drink between points

Pre0, Post0, 2 h, 6 h and 24 h on the variable tHcy. Correlation analysis was made by

using Pearson correlation coefficient in order to check the relationship among the

analyzed variables.

All the parameters were corrected by haemoconcentration following the calculations

described by Dill & Costill (1974) (31).

Differences in Systolic blood pressure, weight and urine osmolarity at pre0 and post0

were analyzed using Student t test.

For the interaction of ACE I/D and MTHFR polymorphisms on tHcy, HR and Systolic

BP a univariate model followed by a Kruskal-Wallis tests were used due to the small

sample size in each genotype group.

For all tests used, a value of p < 0.05 was considered statistically significant.


80

SPSS v.20.0 for Windows (SPSS Worldwide Headquarters, Chicago, IL) was used for the

statistical analysis.

6.4 Results

Table 15 shows the general characteristics and genotype of the studied sample. The

distributions of C677T MTHFR and ACE I/D genotypes revealed that 5 % of the sample

(n=1) was TT homozygous, 45 % (n = 9) heterozygous (CT) while 50 % (n = 10) CC

homozygous. Concentrations of tHcy did not differ between C677T MTHFR or ACE I/D

genotype groups at any sampling point. Moreover, ACE genotype was not related with

heart rate neither with blood pressure during exercise in any of the 4 tests.

Table 15. Anthropometric characteristics and genotype of the studied sample

ACE I/D: Angiotensin Converting Enzyme gene Insertion (I) and Deletion (D) polymorphism; MTHFR: Methylene tetrahydrofolate reductase polymorphism; CC genotype: without mutation; CT genotype: Heterozygous; TT genotype: MTHFR C677T common mutation.

Physiological parameters registered during tests including heart rate and blood pressure

stratified by the four exercise tests are shown in table 16. Systolic BP was higher after

tests without hydration compared to hydration tests (NS).

Characteristics SD

Age (yr) 29.4 7.903 Height (cm) 176.0 7.163 Weight (kg) 76.1 7.854 Body Mass index (BMI) 24.4 1.909 Basal Metabolism (Kcal) 1960 167.7 Fat Mass (kg) 11.3 4.184 %Fat 8.8 3.644 Lean mass (kg) 67.3 5.842 Total Body Water (kg) 49.3 4.281

Polymorphism Genotype % (n)

MTHFR C677T CC 50 % (10) CT 45 % (9) TT 5 % (1)

ACE I/D DD 20 % (4) ID 65 % (13) II 15 % (3)


81

Table 16. Heart rate and blood pressure before and after the exercise tests

** Significant differences between previous point within each test (p<0.001). Pre0: before exercise; Post0: immediately after exercise; NH1: non-hydration during exercise and water hydration after exercise; NH2: non-hydration during exercise and sport drink hydration after exercise; H1: water hydration during and after exercise; H2: sport drink hydration during and after exercise.

Urine osmolarity before and after tests and weight lost are shown in table 17. There were

no statistical differences between osmolarity before and after exercise in any of the 4

exercise tests. Weight lost was significant in the four exercise tests (p < 0.001).

Table 17. Weight lost and urine osmolarity before and after exercise

NH1 (±SD)

NH2 (±SD)

H1 (±SD)

H2 (±SD)

Weight lost after tests (kg) 1.31±0.37** 1.39±0.29** 1.20±0.32** 1.08±0.34**

Osmolarity Pre0 (mosm/L) 596.2±310.3 666.5±309 581.4±329.1 747.2±240.5

Osmolarity Post0 (mosm/L) 583.9±303.9 670.6±294.4 516.9±285 674.1±269.1

** Significant differences between previous point within each test (p<0.001). Pre0: before exercise; Post0: immediately after exercise; NH1: non-hydration during exercise and water hydration after exercise; NH2: non-hydration during exercise and sport drink hydration after exercise; H1: water hydration during and after exercise; H2: sport drink hydration during and after exercise.

NH1 ±SD

NH2 ±SD

H1 ±SD

H2 ±SD

Systolic Blood pressure pre0 (mmHg)

121.11±6.46 124.30±5.75 116.50±9.52 118.708.90

Systolic Blood pressure post0 (mmHg)

139.60±12.21** 131.11±16.53 127.22±10.60** 125.83±10.67

Diastolic Blood pressure pre0 (mmHg)

66.83±7.22 69.15±4.68 67.70±7.18 70.35±7.54

Diastolic Blood pressure post0 (mmHg)

68.30±7.79 65.05±7.22 64.44±5.60 65.88±8.32

HR max (bpm) 174±23 174±16 162±13 164±14

HR mean (bpm) 153±14 151±15 140±12 141±13


82

Table 18 shows the descptive data as mean and SD of plasma volume changes (%) after

tests.

Table 18. Change of Plasma Volume (%) after all four tests

ΔVP (%) ±SD

NH1 NH2 H1 H2

Post0 -7.82±5.28 -10.77±3.36 -8.45±8.79 -9.69±5.57

2h 1.57±8.17 2.86±6.15 -4.98±9.27 3.53±5.82

6h 4.92±10.71 4.51±8.15 1.02±7.17 7.25±8.37

24h 3.87±9.93 0.49±12.31 0.60±8.47 2.75±7.45

Post0: immediately after exercise; 2h: 2 hours after exercise; 6h: 6 hours after exercise; 24h: 24 hours after exercise; NH1: non-hydration during exercise and water hydration after exercise; NH2: non-hydration during exercise and sport drink hydration after exercise; H1: water hydration during and after exercise; H2: sport drink hydration during and after exercise.

Results of tHcy concentrations, corrected and uncorrected by haemoconcentration, in all

measured points of the 4 tests are shown in table 19. Mean values of homocysteine > 10

µmol/L were observed after tests at all measured points. Serum tHcy concentrations

showed a significant increase (p < 0.05) after acute aerobic submaximal exercise only in

the uncorrected data for NH1 and NH2 tests. Moreover, tHcy concentrations still

increased over the time reaching their maximum values at 6 h (p < 0.05) that represents

an increase of 1.93 µmol/L (18.90 %) and 2.11 µmol/L (21.21 %) from pre0, for NH1 and

NH2, respectively. However, tHcy did not show any change after exercise in either H1 or

H2 tests. These concentrations were maintained from baseline up to 2 h. Furthermore,

after the 2 hours of rehydration phase, tHcy concentrations started an increasing tendency

reaching their maximum values at 6 h, that means 0.64 µmol/L (6.20 %) and 1.45 µmol/L

(14 %) of increase from pre0 for H1 and H2, respectively. At 24 h, tHcy concentrations

recovered baseline values in all four tests. Corrected an uncorretcted tHcy concentrations

are represented in figures 11 and 12.


83

* Significant differences from baseline (p<0.05); Pre0: before exercise; Post0: immediately afterexercise; 2h: 2 hours after exercise; 6h: 6 hours after exercise; 24h: 24 hours after exercise; NH1: non-hydration during exercise and water hydration after exercise; NH2: non-hydration during exercise andsport drink hydration after exercise; H1: water hydration during and after exercise; H2: sport drinkhydration during and after exercise.

Figure 11. Corrected total homocysteine concentrations (µmol/L) in all 4 tests

** Significant differences from baseline (p<0.01); * Significant differences from baseline (p<0.05); Pre0: before exercise; Post0: immediately after exercise; 2h: 2 hours after exercise; 6h: 6 hours after exercise; 24h: 24 hours after exercise; NH1: non-hydration during exercise and water hydration after exercise; NH2: non-hydration during exercise and sport drink hydration after exercise; H1: water hydration during and after exercise; H2: sport drink hydration during and after exercise.

Figure 12. Uncorrected total homocysteine concentrations (µmol/L) in all 4 tests

**

**

** **

*


84

Tab

le 1

9. T

otal

hom

ocys

tein

e co

ncen

trat

ions

cor

rect

ed (C

) and

unc

orre

cted

(U) b

y ha

emoc

once

ntra

tion

.

*Si

gnifi

cant

diff

eren

ces

from

pre

viou

s po

int w

ithin

eac

h te

st (p

<0.

05);

**

from

pre

viou

s po

int w

ithin

eac

h te

st (p

<0.

001)

; a fr

om b

asel

ine

with

in e

ach

test

(p<

0.05

); b di

ffere

nces

bet

wee

n sa

me

poin

t C a

nd U

.tH

cy: t

otal

ser

um h

omoc

yste

ine;

Pre

0: b

efor

e ex

erci

se; P

ost0

: im

med

iate

ly a

fter

exer

cise

; 2h:

2 h

ours

afte

r ex

erci

se; 6

h: 6

hou

rs a

fter

exer

cise

; 24h

: 24

hour

s af

ter

exer

cise

; N

H1:

non

-hyd

ratio

n du

ring

exe

rcis

e an

d w

ater

hyd

ratio

n af

ter

exer

cise

; N

H2:

non

-hyd

ratio

n du

ring

exe

rcis

e an

d sp

ort

drin

k hy

drat

ion

afte

r ex

erci

se;

H1:

wat

er h

ydra

tion

duri

ng a

nd a

fter

exer

cise

; H

2: s

port

dri

nk h

ydra

tion

duri

ng a

nd a

fter

exer

cise

. C

: co

rrec

ted

by

haem

ocon

cent

ratio

n; U

: unc

orre

cted

by

haem

ocon

cent

ratio

n.

NH

1 N

H2

H1

H2

(µm

ol/L

) tH

cy U

±SD

tH

cy C

±SD

tH

cy U

±SD

tH

cy C

±SD

tH

cy U

±SD

tH

cy C

±SD

tH

cy U

±SD

tH

cy C

±SD

Pre0

10

.21±

1.44

10

.21±

1.45

9.

95±1

.65

9.95

±1.6

5 10

.33±

1.94

10

.33±

1.94

10

.36±

1.96

10

.36±

1.96

Po

st0

11.7

6±2.

33**

10

.90±

2.01

b11

.71±

1.69

**

10.6

5±1.

47 b

10.9

8±2.

26

10.1

2±1.

89 b

11.2

6±1.

72

10.4

1±1.

60 b

2h

11.2

9±2.

14

11.5

4±2.

27 a

10.6

3±1.

95*

10.9

6±2.

00 b

10.5

9±2.

09

10.1

9±2.

32 b

9.81

±1.7

6 10

.30±

1.94

b

6h

11.3

6±1.

89 a

12.1

4 ±2

.60*

,a,b

11.4

3±1.

95

12.0

6±2.

26 a,

b10

.80±

1.89

10

.97±

2.03

10

.78±

2.37

11

.81±

2.24

b

24h

10.1

4±1.

51**

10

.68±

2.10

10

.72±

1.28

11

.11±

2.43

10

.29±

2.15

10

.49±

2.34

10

.40±

1.65

10

.81±

1.45


85

The percentage of change (%) of tHcy among the measured points in the 4 exercise tests

is shown in figure 13.

tHcy: total serum homocysteine Pre0: before exercise; Post0: immediately after exercise; 2h: 2 hours after exercise; 6h: 6 hours after exercise; 24h: 24 hours after exercise; NH1: non-hydration during exercise and water hydration after exercise; NH2: non-hydration during exercise and sport drink hydration after exercise; H1: water hydration during and after exercise; H2: sport drink hydration during and after exercise.

Figure 13. Percentage of change (%) of corrected total homocysteine concentrations

Tables 20 and 21 present concentrations of folate and vitamin B12, and creatinine and

creatine respectively, corrected and uncorrected by haemoconcentration in the exercise

tests. Comparing with baseline levels, vitamin B12 values showed a significant increase

at 6 h in NH1, NH2 and H2 tests. On the contrary, no significant differences were

observed in serum folate concentrations. Serum creatine concentrations significantly

increased at post0 in the 4 tests (NH1, NH2 and H2: p < 0.01; and H1: p < 0.05),

decreasing at 2 h, in the 4 tests, being significant for NH1, NH2, and H2 (p < 0.05). The

highest creatine concentrations were reached at 6 h in the 4 tests (p < 0.05). No

significant differences were observed in serum creatinine at Post0 in any of the tests.

Moreover, at 6 h, creatinine also significantly increased to maximum values in all four 4

tests (p < 0.05). Values of Na, K, Cl and Mg as mean and SD are presented in table 22.

Singnificant changes have been observed in serum concentrations of Na, K, Cl, and Mg,

although mean levels were within the reference values in all measured points.


86

Tab

le 2

0. F

olat

e an

d vi

tam

in B

12 c

once

ntra

tions

cor

rect

ed a

nd u

ncor

rect

ed b

y ha

emoc

once

ntra

tion

NH

1 N

H2

H1

H2

(ng/

mL

) Fo

late

U

±S

D

Fola

te C

±SD

Fo

late

U

±S

D

Fola

te C

±SD

Fo

late

U

±S

D

Fola

te C

±SD

Fo

late

U

±S

D

Fola

te C

±SD

Pre0

8.

89±3

.34

8.89

±3.3

4 9.

04±3

.37

9.04

±3.3

7 8.

85±1

.84

8.85

±1.8

4 8.

88±2

.78

8.88

±2.7

8 Po

st0

10.4

0±2.

59

9.67

b±2.

46

10.8

7±3.

32 *

9.

94±3

.08

b10

.03±

3.11

9.

22±2

.69

b10

.37±

3.44

**

9.24

±3.1

1 b

2h

9.67

±3.2

1 9.

85±3

.08

8.92

±2.5

0 **

9.

24±2

.74 b

10.1

0 ±2

.65

a,c

9.70

±2.8

4 9.

69±4

.26

9.92

±4.5

3 b

6h

9.05

±2.4

3 9.

62±2

.42

b9.

20±2

.65

9.63

±2.6

2 b

9.45

±2.1

3 9.

55±2

.14

9.58

±2.9

2 10

.13±

3.16

b

24h

9.00

±2.5

9 9.

33±2

.30

9.17

±3.3

8 9.

33±3

.56

8.69

±2.3

3 8.

70±2

.53

8.96

±2.7

6 9.

26±3

.01

(pg/

mL

) V

itam

in B

12 U

±SD

V

itam

in B

12 C

±SD

V

itam

in B

12 U

±SD

V

itam

in B

12 C

±SD

V

itam

in B

12 U

±SD

V

itam

in B

12 C

±SD

V

itam

in B

12 U

±SD

V

itam

in B

12 C

±SD

Pre0

45

8.22

±154

.82

458.

22±1

54.8

2 47

3.65

±167

.91

473.

65±1

67.9

1 46

5.60

±165

.39

465.

60±1

65.3

9 47

9.62

±180

.93

479.

62±1

80.9

3 Po

st0

522.

58±1

73.4

7 *

484.

46±1

57.0

4 b

513.

75,b

±174

.19*

* 46

9.20

±159

.95

510.

69±1

62.6

7 b

472.

64±1

52.3

9 b

561.

75±2

30.4

2 50

9.63

±219

.32

2h

505.

07±1

60.3

2 51

3.86

±149

.34

475.

77 ±

153.

63 *

,b49

5.30

±179

.97

504.

43±1

81.4

5 48

6.55

±193

.27

b49

8.04

±199

.13

512.

47±2

21.1

3 6h

51

4.51

±207

.65

543.

03 ±

184.

75 *

,a51

0.53

±17

9.61

*,a53

6.02

±18

1.22

a49

4.59

±173

.12

498.

97±1

66.7

4 b

514.

58±1

83.3

0 55

8.51

±24

6.12

a

24h

483.

55±1

85.2

0 *

503.

95±1

79.4

0 50

0.04

±198

.52

506.

55±1

87.4

2 *

487.

01±1

65.4

1 50

7.15

±198

.25

525.

10±1

91.2

8 54

7.30

±23

6.93

a

*Si

gnifi

cant

diff

eren

ces

from

pre

viou

s po

int w

ithin

eac

h te

st (p

<0.

05);

**

from

pre

viou

s po

int w

ithin

eac

h te

st (p

<0.

001)

; a fr

om b

asel

ine

with

in e

ach

test

(p<

0.05

); b di

ffere

nces

bet

wee

nsa

me

poin

t C a

nd U

; c di

ffere

nces

with

test

NH

2 at

sam

e po

int (

p<0.

05).

tHcy

: tot

al s

erum

hom

ocys

tein

e; P

re0:

bef

ore

exer

cise

; Pos

t0: i

mm

edia

tely

afte

r ex

erci

se; 2

h: 2

hou

rs a

fter

exer

cise

; 6h:

6 h

ours

afte

r ex

erci

se; 2

4h: 2

4 ho

urs

afte

r ex

erci

se; N

H1:

non

-hy

drat

ion

duri

ng e

xerc

ise

and

wat

er h

ydra

tion

afte

r ex

erci

se;

NH

2: n

on-h

ydra

tion

duri

ng e

xerc

ise

and

spor

t dr

ink

hydr

atio

n af

ter

exer

cise

; H

1: w

ater

hyd

ratio

n du

ring

and

afte

r ex

erci

se; H

2: sp

ort d

rink

hyd

ratio

n du

ring

and

afte

r exe

rcis

e; C

: cor

rect

ed b

y ha

emoc

once

ntra

tion;

U: u

ncor

rect

ed b

y ha

emoc

once

ntra

tion.


87

Tab

le 2

1. C

reat

ine

and

Cre

atin

ine

conc

entr

atio

ns c

orre

cted

and

unc

orre

cted

by

haem

ocon

cent

ratio

n

NH

1 N

H2

H1

H2

(mg/

dL)

Cre

atin

e U

±SD

C

reat

ine

C

±S

D

Cre

atin

e U

±SD

C

reat

ine

C

±S

D

Cre

atin

e U

±SD

C

reat

ine

C

±S

D

Cre

atin

e U

±SD

C

reat

ine

C

±S

D

Pre0

2.

68±1

.19

2.68

±1.1

9 2.

53±0

.91

2.53

±.91

2.

73±1

.13

2.73

±1.1

3 2.

59±1

.15

2.59

±1.1

5 Po

st0

3.66

±1.3

5 **

3.

41 ±

1.30

**,b

3.67

±1.6

5 **

3.

41 ±

1.43

*,a,

b3.

37±1

.17

* 3.

13 ±

1.12

*,b

3.87

±1.7

0 **

3.

56 ±

1.63

*,b

2h

2.42

±0.9

4 **

2.

47*±

.97

2.61

±1.0

2 *

2.70

±1.

12 *

,b2.

32±0

.83

* 2.

23±.

82*

2.54

±1.3

4 **

2.

70 ±

1.59

*,b

6h

4.50

±2.

99 *

,a4.

76 ±

3.14

*,a,b

3.91

±2.3

0 a

4.12

±2.

37 a,

b3.

90±2

.38

3.93

±2.

39 *

4.

59±3

.58

* 5.

12 ±

4.03

*,a,

b

24h

2.49

±1.0

0 *

2.59

±1.0

1*

2.73

±1.1

2 2.

92±1

.76

2.81

±1.4

4 2.

86±1

.46

2.66

±1.1

8 2.

78±1

.23

(mg/

dL)

Cre

atin

ine

U

±S

D

Cre

atin

ine

C

±S

D

Cre

atin

ine

U

±S

D

Cre

atin

ine

C

±S

D

Cre

atin

ine

U

±S

D

Cre

atin

ine

C

±S

D

Cre

atin

ine

U

±S

D

Cre

atin

ine

C

±S

D

Pre0

1.

13±0

.13

1.13

±0.1

3 1.

13±0

.08

1.13

±0.0

8 1.

08±0

.10

1.08

±0.1

0 1.

14±0

.10

1.14

±0.1

0 Po

st0

1.27

±0.1

3 **

1.

18±0

.13

b1.

26±0

.14*

* 1.

13±0

.13

b1.

20±0

.15*

1.

11±0

.13

b1.

24±0

.14

* 1.

14±0

.13

b

2h

1.16

±0.1

1**

1.19

±0.1

5 1.

15±0

.09

**

1.19

±0.1

4 b

1.13

±0.1

3 a

1.09

±0.1

7 b

1.14

±0.1

5 *

1.19

±0.1

7 b

6h

1.21

±0.2

0 1.

28 ±

0.25

a,b

1.18

±0.1

4 1.

24 ±

0.17

a,b

1.19

±0.1

5 a

1.21

±0.

16*,a

1.18

±0.1

0 1.

29 ±

0.19

a,b

24h

1.11

±0.0

9 1.

16±0

.15

1.11

±0.1

0 1.

13 ±

0.18

*

1.13

±0.1

0 a

1.14

±0.1

5 1.

13±0

.12

1.16

±0.1

8 *

* Si

gnifi

cant

diff

eren

ces

from

pre

viou

s po

int w

ithin

eac

h te

st (p

<0.

05);

**

from

pre

viou

s po

int w

ithin

eac

h te

st (p

<0.

001)

; a fr

om b

asel

ine

with

in e

ach

test

(p

<0.

05);

b diffe

renc

es b

etw

een

sam

e po

int C

and

U. c

diffe

renc

es b

etw

een

sam

e po

int w

ith H

1 te

st.

tHcy

: to

tal s

erum

hom

ocys

tein

e; P

re0:

bef

ore

exer

cise

; Po

st0:

imm

edia

tely

afte

r ex

erci

se;

2h:

2 ho

urs

afte

r ex

erci

se;

6h:

6 ho

urs

afte

r ex

erci

se;

24h:

24

hour

s afte

r exe

rcis

e; N

H1:

non

-hyd

ratio

n du

ring

exe

rcis

e an

d w

ater

hyd

ratio

n af

ter e

xerc

ise;

NH

2: n

on-h

ydra

tion

duri

ng e

xerc

ise

and

spor

t dri

nk h

ydra

tion

afte

r ex

erci

se; H

1: w

ater

hyd

ratio

n du

ring

and

afte

r ex

erci

se; H

2: s

port

dri

nk h

ydra

tion

duri

ng a

nd a

fter

exer

cise

; C: c

orre

cted

by

haem

ocon

cent

ratio

n; U

: un

corr

ecte

d by

hae

moc

once

ntra

tion.


88

Tab

le 2

2. S

odiu

m, P

otas

sium

, Chl

orid

e an

d M

agne

sium

val

ues

*Si

gnifi

cant

diff

eren

ces

from

pre

viou

s po

int w

ithin

eac

h te

st (

p<0.

05);

**

from

pre

viou

s po

int w

ithin

eac

h te

st (

p<0.

001)

; a fr

om b

asel

ine

with

in e

ach

test

(p<

0.05

).Pr

e0: b

efor

e ex

erci

se; P

ost0

: im

med

iate

ly a

fter

exer

cise

; 2h:

2 h

ours

afte

r ex

erci

se; 6

h: 6

hou

rs a

fter

exer

cise

; 24h

: 24

hour

s af

ter

exer

cise

;N

H1:

non

-hyd

ratio

n du

ring

exe

rcis

e an

d w

ater

hyd

ratio

n af

ter

exer

cise

; NH

2: n

on-h

ydra

tion

duri

ng e

xerc

ise

and

spor

t dri

nk h

ydra

tion

afte

rex

erci

se; H

1: w

ater

hyd

ratio

n du

ring

and

afte

r exe

rcis

e; H

2: sp

ort d

rink

hyd

ratio

n du

ring

and

afte

r exe

rcis

e.

Sodi

um (m

Eq/

L)

±S

D

Pota

ssiu

m (m

Eq/

L)

±S

D

NH

1 N

H2

H1

H2

NH

1 N

H2

H1

H2

Pre0

13

9.02

±3.3

3 13

9.96

±2.2

7 13

9.26

±3.0

8 13

8.48

±5.6

7 4.

61±0

.69

4.55

±0.3

9 4.

53±0

.41

4.51

±0.3

4

Post

0 14

0.64

±1.7

0 14

0.62

±1.9

5 13

8.67

±10.

77

140.

41±3

.00

4.85

±0.3

5 4.

87±0

.28

4.66

±0.4

3 4.

68±0

.30

2h

137.

63±1

.45

* 13

8.30

±4.4

9 13

7.96

±5.2

8 13

4.25

±13.

49

4.71

±0.4

0 4.

35±0

.40

**

4.60

±0.3

8 4.

60±1

.44

6h

140.

18±1

.58

* 14

0.29

±1.8

2 14

0.92

±1.8

1 a

139.

88±4

.23

4.38

±0.3

4 *

4.18

±0.3

6 a

4.20

±0.2

9 *

4.13

±0.3

2 a

24h

139.

27±1

.94

139.

20±2

.54

139.

93±2

.65

139.

01±4

.79

4.56

±0.2

6 4.

68±0

.56*

4.

56 ±

0.44

*,a

4.44

±0.3

3*

Chl

orid

e (m

mol

/L)

±S

D

Mag

nesi

um (m

mol

/L)

±S

D

NH

1 N

H2

NH

1 N

H2

NH

1 N

H2

NH

1 N

H2

Pre0

10

3.42

±2.9

7 10

3.82

±2.5

2 10

4.69

±3.3

0 10

3.12

±4.3

4 1.

96±0

.13

1.92

±0.1

4 1.

96±0

.14

1.91

±0.1

0

Post

0 10

5.75

±1.7

2 10

5.88

±2.4

6 *

104.

34±8

.68

105.

82±2

.77

* 1.

92±0

.12

1.92

±0.1

3 1.

94±0

.17

1.91

±0.1

3

2h

101.

73±1

.63

* 10

1.94

±3.2

2 **

10

2.41

±4.6

9 99

.46±

9.56

*

1.96

±0.1

3 1.

92±0

.18

1.92

±0.1

6 1.

94±0

.17

6h

103.

50±2

.32

**

102.

79±2

.02

104.

10±2

.20

102.

56±3

.27

2.01

±0.1

7 1.

97±0

.20

1.98

±0.1

5 2.

02±0

.19

24h

103.

67±1

.73

103.

96±2

.29

* 10

4.71

±2.6

4 10

3.50

±3.8

5 1.

94±0

.13

1.96

±0.1

4 1.

94±0

.13

1.95

±0.1

0


89

6.5 Discussion

The present investigation revealed important data about the effect of acute exercise and

hydration on tHcy concentrations and its post-recovery behaviour. To the best of the

author´s knowledge, there are no previous data regarding hydration effect on tHcy

related to exercise. Results showed that tHcy concentrations were almost one point

higher immediately after an acute submaximal exercise at 65 % of VO2max without

hydration than pre-exercise. Moreover, our data demonstrated that tHcy concentrations

progressively increased in the following hours reaching their maximum values at 6 h

after exercise. These results are in acccordance with those reported by Real et al.

(102), who found a mean of 2 µmol/L of increase after 24 h of acute exercise. They

hypothesized that this increase could be relevant for cardiovascular risk in non well-

trained athletes but further investigations are needed. Hydration during exercise

(independently of water or sport drink) maintains tHcy baseline concentrations until

2 h, and the increase of tHcy concentrations after 6 h was lower than in the non-

hydration protocol during exercise. These results could be explained by the effect of

hydration on the maintenance of volemia (38), owing to the fact that all physiological

systems in the human body are influenced by dehydration (16, 91). This finding

presents a new perspective beyond performance in hydration research, with its effects

in the CVD context. It is important to take into account that the fluid intake of the

subjects was only controlled until 2 h after the exercise tests. From 2 h to 24 h the

ingestion of fluid and food was ad libitum.

In the last few years, a variety of studies have consistently shown an increase in tHcy

concentrations after acute exercise (29, 46, 83, 124). Controversial data still exist

regarding if this increment is or not produced immediately after exercise and how long

this rise is maintained. Our results could be in line with those found by Konig et al. (74)

and Iglesias-Gutierrez et al. (68) who observed the highest tHcy increase 1h and 38

minutes, respectively, after acute exercise.

Previous results analyzing the effect of post-exercise rehydration showed that the

decrease of tHcy concentrations after 2 h of rehydration was higher with a sport drink

than with water. However, tHcy did not return to basal values with any of the beverages

after 2 hours (82). On the other hand, body weight changes produced during exercise

showed an important dehydration, on the contrary, urine osmolarity that is considered

as a hydration marker, did not reflect dehydration status after exercise.


90

This result could be explained because fluid ingestion can temporarily produce a urine

sample that does not reflect hydration status (98), since kidneys can filter fluid

consumed close to the test and produce a urine sample that indicates a wrong “well

hydrated” status.

Furthermore, haemoconcentration has been proposed as a possible cause of the

increase of tHcy during exercise (9). The controversial data regarding the immediate

effects of exercise on tHcy concentrations in the literature could be due to a lack of

methodology in correcting results by haemoconcentration. In order to compare both,

corrected and uncorrected concentrations, tHcy concentrations were adjusted for

hemoglobin and hematocrit values following the method proposed by (31). Different

results were obtained without correcting for haemoconcentration comparing to

corrected values. This investigation demonstrated that although

controlling for haemoconcentration, tHcy concentrations continue increasing after

exercise up to 6 h. Moreover the main differences among the responses are at 2 h,

where uncorrected concentrations of tHcy showed a decrease due to the volume plasma

changes, and in contrast, corrected concentrations showed they were still high in

respect to baseline values.

Involuntary dehydration occurs primarily in humans when they are exposed to various

stressors including exercise. Greenleaf (56) explains that involuntary dehydration is

controlled by more several factors including the rate of fluid absorption from the

gastrointestinal system, the level of cellular hydration involving the osmotic-

vasopressin interaction with sensitive cells or structures in the central nervous system,

and the hypovolemic-angiotensin II stimuli. Otherwise, as the patterns of findings for

blood pressure and heart rate are similar to changes in tHcy concentrations, we could

suggest that the rise in tHcy concentrations may have been sympathetically mediated

and is closely related with the stressor stimuli. Systolic BP was higher after tests

without hydration compared to hydration tests. But although subjects with DD

genotype had higher Systolic BP after tests, ACE I/D genotype were not related

to any of the cardiovascular measures nor to tHcy concentrations after exercise.

The exact mechanism by which exercise modulates blood tHcy concentrations continues

to be poorly understood (28, 74). Konig et al. (74) argued that since increased tHcy

depends on methionine availability, if a single bout of physical exercise increases

methionine availability, then increased tHcy during exercise may be partly explained by

increased transsulfuration activity.


91

The role of folate and vitamin B12 has been studied in our study as important

cofactors for the proper function of the methionine cycle. The inverse correlation

between folateand vitamin B12 with homocysteine has been demonstrated in several

studies (51, 65, 83, 93). But controversial data were found in the exercise context.

Previous results showed a tendency to lose the strong negative correlation between

tHcy and folate after moderate exercise (82). Authors have been arguing that the

synthesis of tHcy during exercise could explain the increase of vitamin B12 and folate,

both implicated in methionine-homocysteine metabolism. The high demand of folic

acid as a methyl donor for the remethylation of methione from homocysteine in the

post-exercise phase could explain this response. Previous studies stated that

correlations of vitamin B12 are usually weaker than those of folate (52); on the

contrary, this study showed a large variability of correlations of folate and vitamin

B12 with tHcy in all phases of the tests, probably due to the increase of both

parameters from post0 to 6 h. Another important parameter implicated in the

homocysteine synthesis is creatine. Its synthesis has been previously discussed as an

important factor related to increased tHcy after high-intensity exercise (29). Because

creatine synthesis is responsible for a considerable consumption of S-

adenosylmethionine (SAM) in the liver and homocysteine formation, previous

studies have hypothesized that creatine supplementation may down-regulate

endogenous formation of creatine and reduce homocysteine concentration in humans

after acute exercise (29). Lately, this author observed this response in an animal model

but not in humans (27). But regarding the endogenous formation of creatine, results of

the present investigation showed an increase of creatine and creatinine after exercise,

reaching the highest values also at 6 h after exercise. These data support the idea

reported by Zinellu et al. (115) in which creatine demand is increased after high

intensity exercise as substrate utilization, and consequently increases the formation of

tHcy. This would mean that the implication of vitamins such as folate and vitamin

B12 and the methionine cycle operates differently at rest than with exercise, and

we could hypothesize that the demand of creatine after acute exercise increases

muscle anabolism and the methionine requirement for protein synthesis, thereby its

availability for SAM-dependent transmethylation reactions and subsequently

increasing homocysteine production resulting in an increased tHcy and related

parameters after few hours of acute exercise.


92

The MTHFR polymorphism impairs the ability to process folate and is associated with

various diseases (vascular disease, cancer, neurology disorders, diabetes, psoriasis, etc).

MTHFR is important for folate metabolism, which is an integral process for cell

metabolism in DNA, RNA and protein methylation (79). This defective gene leadsto

elevated levels of tHcy in some MTHFR recessive homozygous genotypes. Only 5 % of

the studied sample was homozygous (TT) and no correlation has been observed

between MTHFR genotype and tHcy concentrations or related parameters, probably due

to the small sample size of each genotype group.

6.6 Conclusion

An adequate hydration protocol during aerobic submaximal exercise with both, water

and sport drink, maintains tHcy concentrations at baseline up to 2 h after finishing the

exercise in physically active male adults. Furthermore hydration during exercise

prevents the further increase of tHcy concentrations at 6 h. Vitamin B12 and creatine

concentrations had the same increase response after 6 h of exercise as tHcy

concentrations. Finally, at 24 h, tHcy concentrations returned to basleine values. The

rise of tHcy concentrations after acute exercise needs further investigation.


93

7 CHAPTER 7. GENERAL DISCUSSION

This thesis aims at analyzing the effect of acute exercise on tHcy concentrations and the

associations with related parameters. Additionally, in order to study the effect of

hydration on tHcy concentrations after exercise, different hydration protocols have been

analyzed. This part will summarize the results of the different studies and discuss

them in common with those described in the literature.

Effect of acute exercise on tHcy concentrations with and without hydration during

exercise

One of the main findings of this thesis is that tHcy concentrations have a different

behaviour depending on the hydration protocols. Results showed that hydration during

acute exercise at 65 % of VO2max, independently of the type of beverage, water or a

sport drink, maintained tHcy concentrations at baseline up to 2 h after exercise. In

contrast, when exercise tests were performed without, tHcy concentration increased

significantly (p < 0.05) reaching values beyond the limit of the recommended cut-off

point (> 10 µmol/L). Both corrected and uncorrected data, showed an increase

behaviour immediately after exercise, but significant differences were only found when

correction by hemoconcentration was not used. On the other hand, 6 h after exercise,

tHcy concentrations continued being higher than at baseline, but this increase was only

significant when there was not hydration during exercise.

To the best of our knowledge, this is the first investigation analyzing the

implementation of a controlled hydration protocol on tHcy concentrations during and

after acute exercise. This data corroborates the hypothesis of the present thesis, and

demonstrates that hydration during aerobic submaximal exercise has a protective effect

on the increase of tHcy concentrations during and after exercise.

Some experimental studies have found an increase of tHcy after acute exercise (5, 28,

29, 47, 64, 68, 74, 102, 124). Our results found higher tHcy concentrations immediately

after acute aerobic submaximal exercise with a continuous increase reaching

maximum values at 6 h. Twenty-four hours after exercise tHcy concentrations

recovered to baseline values. In line with our results, Herrmann et al. (64) found a 64

% increase after a marathon race. On the other hand, Konig et al. (74) found

higher tHcy concentrations 1h after acute exercise, but after 24 h of a triathlon

competition also observed high tHcy concentrations. The study by Herrmann et al.

(64) speculates that the intensity and duration of exercise could determine the tHcy


94

response, because only marathon runners showed significant tHcy increase in

both, from baseline and comparing by groups. These authors suggested that the

high intensity sustained by the runners compared to the 100 km run and mountain

bike race, can have more rest periods during trials, being a possible answer for this

response (64). Results from study 1 could determinate that independently of the

intensity (maximal or at 65 % of VO2max), and the duration (around 10 min and 40

min respectively) tHcy concentrations increased in both exercise tests. In line with

our results, other studies that included different intensities showed that the tHcy

increase was independent of the intensity of the exercise (68) or type of exercise

(124). On the other hand, Sotgia et al. (115) who compared athletes and non-athletes

found no changes in tHcy after acute exercise but a decrease in the homocysteine

reduced form. Hammouda et al. (60) did not find any change in tHcy concentrations

after acute exercise. This probably is due to the fact that the intervention was

performed only by a 30 s Wingate test, not long enough to stimulate the

methionine synthesis.

Omenn et al. (96) have postulated tHcy levels > 10 µmol/L as a cut-off point risk for

ischemic heart disease. Our data revealed a mean of the overall sample higher than 10

µmol/L after acute exercise in all tests. We could hypothesize that this effect indicates a

relevant issue in increasing acute myocardial event risks during this special time, in this

specific sample. But this observation should be studied carefully, because there are not

available data about the exact effects on health of this specific response (102).

Another interesting observation from this study is that the mean baseline values of the

overall sample were 10.60 µmol/L. This means that our sample reached the limit of the

recommended tHcy levels for the normal adult population (106). Moreover, there is not

available data demonstrating if these values in a specific physically active sample could

be considered a risk or not. It could be necessary to study, if the trained people and

athletes have different reference values of tHcy concentrations than the normal

population without constituting an adverse effect for health or, on the contrary, if it

could constitute a risk for cardiovascular events during acute exercise in sport

or competitive events in some specific subjects.

It is important to remember that this effect is different from the effects found by

chronic exercise. Some researchers highlighted an exercise-induced-fall in tHcy after

training (18, 101, 125, 126). On the other hand, some studies provided data that

training does not contribute to decreasing tHcy concentrations (9).


95

Interestingly, Okura et al. (95) found different responses depending on the

baseline tHcy status. An increased tHcy was observed after training in those

within normal tHcy concentrations at baseline; in contrast, the contrary effect was

observed in subjects with hyperhomocysteinemia at baseline, where tHcy decreases

after training. The investigations of Molina-López et al. (89) and Guzel et al. (58) also

found increased tHcy after training exercise programs Konig et al. (74) concluded

that although acute exercise significantly increases tHcy, chronic endurance exercise

was not associated with higher plasma tHcy concentrations. In summary, it could be

concluded that acute exercise induces an increase in tHcy; in contrast, no consensus

exists regarding training effects because all the analyzed investigations used a

variety of exercise interventions, with different intensities, duration and mode of

exercise.

Regarding cardiorespiratory fitness and physical activity level, Kuo et al. (76) found

that tHcy concentrations were inversely associated with cardiorespiratory fitness in

adult women but not in men, independently of body mass index (BMI), age, race,

vitamins B, creatinine levels and physical activity, among other factors. The study

conducted by Ruiz et al. (109) reported no association of tHcy with any measure of

physical activity such as cardiorespiratory fitness or physical activity level when

controlling for MTHFR genotype in children. In contrast, a further study by Ruiz

et al. (108) showed that cardiorespiratory fitness was negatively associated with

Hcy levels in young women when controlling for MTHFR genotype. These results are

in accordance with those found by a previous study (76) and suggest that this

negative association could be due to sex hormone modulation.

Differences in rates of homocysteine remethylation (42) and estrogen levels (90) may

contribute to the homocysteine sex dimorphism. In another investigation conducted by

Dankner et al. (25), results didn’t show association between tHcy and cardiorespiratory

fitness in adult males. Joubert & Manore (70), concluded that tHcy could be dependent

on the individual fitness level of participants. Dankner et al. (24) found a negative

correlation between PA levels and tHcy in the elderly, independently of the

vitamin B status and MTHFR genotype. Nygard et al. (94) found that physical

inactivity was associated to higher tHcy. These authors suggest that exercise exerts its

most favorable effect in subjects with hyperhomocysteinemia, as shown by Unt et al.

(120) who found higher tHcy concentrations in ex-athletes returning to a sedentary

lifestyle comparing to those who continued being active.


96

In contrast, another investigation, didn´t find any association between PA levels and

tHcy concentrations (71). Furtheremore, they reported that individuals who had higher

levels of PA had also higher tHcyconcentrations and may need a vitamin B

supplementation to keep blood tHcy concentrations as low as possible. The

intervention studies that analyze the relationship of tHcy and PA levels have some

limitations due the low accuracy of questionnaires used to assess PA like the 7-diary

Physical Activity record. Additionally, the population and their characteristics differ

among studies and which makes it difficult to reach a consistent conclusion. Low

cardiorespiratory fitness seems to be associated with high tHcy concentrations; in

contrast, results analyzing physical activity and its relation with tHcy concentration

need further research.

The exact mechanisms by which serum tHcy increases are still unknown (26).

Some authors have speculated that haemoconcentration could be some of the

explanation of the increased tHcy after acute exercise (64). In order to get an

approximation of whether haemoconcentration is the reason for the increase of tHcy

after acute exercise, both corrected and uncorrected tHcy concentration data were

analyzed. Uncorrected tHcy data were significantly high immediately after exercise

when there was not hydration during exercise (p < 0.05). On the other hand,

corrected tHcy concentrations by haemoconcentration at the same point, showed the

same behaviour, although we didn´t found significant differences from before

exercise. As tHcy corrected concentrations were higher than at baseline, this

demonstrated that a part of the increase of tHcy is due to the haemoconcentration,

and another part is due to mechanisms involved in the effect of exercise. Since

high serum tHcy concentrations represent a cardiovascular risk, the effects of acute

exercise on these concentrations should be studied without haemoconcentration

corrections, in order to study the “real concentration in blood” during that specific

moment. Thus, it is important to differentiate two aspects: Research aiming to

analyze the underlying effect of acute exercise on increased tHcy related to CVD

should not include the haemoconcentration corrections; in contrast, research aiming

to analyze the exact mechanisms involved in the effect of exercise on tHcy after

acute exercise, should necessarily take into account the corrections for

haemoconcentration.

Effect of rehydration after acute exercise on tHcy concentrations

Rehydration after exercise is a common and a necessary routine for all athletes. Since


97

hydration has an effect in restoring plasma volume, fluid losses and physiological

systems altered by acute exercise (17), our objective was to compare the effect of two

different drinks commonly used by trained people and athletes during sports or

competitive events. First at all, to discuss the effect of high tHcy concentrations as a

risk, we will discuss the effect of hydration without the corrections

for haemoconcentration tHcy data. Our uncorrected results showed that 2 h of a

rehydration protocol after aerobic submaximal exercise reduces tHcy concentrations

significantly with a sport drink. Moreover, the same tendency response was

observed for water. Otherwise, after the post-exercise rehydration protocol phase,

tHcy concentrations continued being higher than baseline with both, water and a sport

drink. The differences between drinks seem to be due to the hydration effect of a

carbohydrate sport beverage. Ingestion of drinks that contain carbohydrates and

electrolytes may offer a gradual return to pre-dehydration levels and tend to prevent

any decrease in circulating sodium concentration, better maintaining the plasma

volume and resulting in a smaller urine fluid loss. Some investigations highlight the

importance of avoiding rapid increase in plasma volume and corresponding

reduction in sodium concentration and osmolarity during post-exercise rehydration

to ensure that diuresis does not occur and that retention of ingested fluid is

maximized (38). In this way, as hydration with a sport beverage (containing

carbohydrates and electrolytes) helps to maintain plasma volume better than water,

we could hypothesize that sport drinks will do better than water to control elevated

tHcy and other blood parameters that could be altered during exercise. Secondly,

regarding the mechanisms involved if we correct this data by

haemoconcentration, it can be observed that the response is different. Concentrations of

tHcy showed values still higher at 2 h than after exercise, being this increase significant

respect to baseline (p < 0.05) with water but not with the sport drink. This result could

be explained by the hypothesis that after the increase of tHcy due to the acute

exercise there is an increase of the synthesis of methyl compounds, such as

creatine, hours after finishing the activity leading to a high tHcy formation (29, 64).

Regarding the analyzed related parameters, our results showed an inverse

correlation between folate and tHcy before exercise, but in contrast, this correlation

was lost after exercise and hours later. There was an exception for the first study,

where the inverse correlation was maintained after maximal and submaximal exercise.


98

This result could be due in part to the low number of subjects and on the other

hand, due to the same rate of change among tHcy and folate in the maximal and

submaximal tests. According to our results, the literature shows a great variability in

the correlations with folate and vitamin B12 with tHcy concentrations. The relations

of the known factors that influence tHcy and its implication with exercise have

attracted considerable attention. These factors include B vitamins, such as folate,

vitamin B12 and vitamin B6 blood levels as cofactors of several enzymes involved

in homocysteine metabolism. Proper intake of vitamin B6, vitamin B12 and folate can

help to maintain low Hcy concentrations and support the increased demand on

metabolism during high intensity exercise. The inverse correlations among serum

vitamin B12, and folate with tHcy are well established in the literature (52), but this

interaction may be modulated by exercise or training. The correlation between

blood vitamin B12 and folate with tHcy before exercise has been observed in various

studies. However, results are less clear and sometime controversial regarding exercise

or training program.

Regarding the effect of exercise on these vitamins, our data showed an increase in folate

and vitamin B12 after acute exercise, the same as found previously in another study (68).

It seems that there is a consensus about the increase of folate and vitamin B12 after acute

exercise, competition or training programs. In addition, some authors have speculated

that tHcy increases because vitamin B6 levels are too low for reducing homocysteine

and converting it to cysteine via the transsulfuration pathway. It should be remembered

that vitamin B6 is required as a coenzyme of transaminases, decarboxylases and

glycogen phosphorylate in metabolic pathways of energy production. In contrast,

results from the study of Iglesias Gutierrez didn´t report any relationship between

vitamin B6 and substrate utilization during different intensities throughout the trials

(68). On the other hand, Herrmann et al. (65) suggest that endurance athletes had a

higher prevalence of vitamin B deficiency due to the high necessity of vitamin B6 and

folate not only during exercise but also during training because vitamin B6 is

necessary to fuel working muscles and to repair damaged tissues (70).

The C677T polymorphism of the MTHFR gene has been established as an important

genetic determinant of elevated tHcy (37). No correlation has been observed between

C677T polymorphisms and tHcy concentrations or related parameters, probably due to

the small sample size of each genotype group.


99

Moreover, the combination of exercise in the heat and dehydration leads the human

body to a stress situation inducing elevations in tHcy concentrations (118). These

stressors increase catecholamine secretions leading to blood pressure elevation. The

ACE is involved in all these processes (118), and the rise in tHcy concentrations is

closely related with the stressor stimuli. But our results didn´t find any relationship with

the ACE insertion/deletion (I/D) polymorphism and it was not related with heart rate

neither with blood pressure during exercise in any of the 4 tests.

Creatine is responsible for a considerable consumption of S-adenosylmethionine in the

liver for homocysteine formation. There is evidence that physical activity may also alter

homocysteine metabolism by increasing protein and/or methyl group turnover (49).

During high intensity exercise, creatine-phosphate is required as an immediate

energy source for muscle contraction. The increase in creatine synthesis demand can

be a key factor in the methyl balance modulation and one of the most important factors

related to increased tHcy (115). Results of the present investigation showed an

increase of creatine and creatinine after exercise, reaching the highest values also

at 6 h after exercise. These data support the idea reported by Sotgia et al. (115) in

which creatine demand is increased after high intensity exercise as substrate

utilization, and consequently increases the formation of tHcy.

Some authors have studied the effects of creatine supplementation followed by

exercise interventions like Deminice et al. (27) in 2014, who reported that 0.3 g/kg of

creatine supplementation during 7 days were unable to lower tHcy concentrations

either at rest or after acute exercise. Surprisingly, some animal research showed the

opposite results, finding a decrease in plasma tHcy after creatine supplementation

(29, 117). These contradictory data suggest that inhibition of the endogenous

methylation demand by creatine supplementation can reduce tHcy levels in animals,

but not in humans (27). However, more studies are necessary to examine activities of

key enzymes on creatine synthesis after acute exercise and its relation to tHcy

concentrations.

A few limitations of the present thesis deserve comment. Because of the small sample

size, the genetic profile can only be taken as a control measure. Moreover, the lack of a

strict control of the diet from 2 h to 24 h after the tests makes it difficult to interpret

some results at 6 h.


100

Some strengths of the present thesis should also be stated here, which have been previously

mentioned. This Study is the first that analyzes the effect of a controlled hydration protocol

on total homocysteine concentrations after acute aerobic submaximal exercise. This is also

the first study analyzing the effect of acute exercise with and without hemoconcentration

corrrections. MTHFR and ACE polymorphisms have been included as control variables.

Moreover, among the strengths the inclusion of a standardized protocol, the strict following

of the fieldwork, the homogeneity of the study sample and the control of all parameters

during the experimental fieldwork at the laboratory must be mentioned.


101

8 CHAPTER 8. CONCLUSIONS

Study 1

- Acute exercise increases tHcy concentrations after both maximal intensity and

submaximal intensity tests (100% and 65 % of VO2max, respectively) in physically

active adult males.

- Folate, and vitamin B12, increased after both, maximal and submaximal tests while

creatinine only increased after the maximal test. On the other hand, only folate

showed a significant correlation with tHcy before and after exercise tests.

Study 2

- Concentrations of tHcy increased after 40 minutes of submaximal aerobic exercise in

a hot environment.

- Two hours of an adequate rehydration protocol after submaximal aerobic exercise

with a sport drink decreased tHcy concentrations. Nevertheless, at 2 h, tHcy

concentrations didn´t recover baseline levels.

- Folate and vitamin B12 as related parameters involved on Hcy metabolism also

increased significantly after submaximal aerobic tests. But the correlation analyses

showed a high variability.

Study 3 - A proper hydration protocol during submaximal aerobic exercise, with both water

and a sport drink maintains tHcy concentrations at baseline up to 2 h after exercise

and prevents the further increase at 6 h.

- After the increase of tHcy concentrations induced by acute exercise, tHcy

concentrations recovered baseline levels at 24 h.

- Vitamin B12 and creatinine concentrations as related parameters involved in the tHcy

metabolism, also increased after exercise tests in line with the behaviour response of

tHcy concentrations.

- The correlations of folate, vitamin B12, creatinine and creatine with tHcy showed a

high variability in all measured points.


102

General Conclusion

Total Homocysteine concentrations increased significantly after acute aerobic

submaximal exercise in physically active male subjects. An adequate hydration protocol

during aerobic submaximal exercise, with both, water or a sport drink, maintained tHcy

concentrations at baseline up to 2 h, and prevented the further increase in a sample of

physically active male subjects.


103

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APPENDIX


114


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CONSENTIMIENTO INFORMADO

PROYECTO EFECTO DE LA HIDRATACIÓN SOBRE LOS NIVELES DE

HOMOCISTEÍNA TRAS EL EJERCICIO FÍSICO EN VARONES FÍSICAMENTE ACTIVOS

El objetivo de este estudio es medir los cambios en los niveles de diferentes parámetros

tras el ejercicio y la influencia que tiene la rehidratación sobre éstos.

El estudio se realizará en la Facultad de Ciencias de la Actividad Física y del Deporte

(INEF). El estudio consiste en la realización de un análisis de la composición corporal

mediante impedancia bioeléctrica (BIA), una prueba de esfuerzo máxima, y cuatro

pruebas submáximas al 65 % de su VO2 máximo con una duración de 50 minutos cada

una, todas ellas realizadas en tapiz rodante, y la extracción de unos 10 mL de sangre

venosa en diferentes momentos de las pruebas. Las muestras sanguíneas se extraerán

por punción venosa estándar con palomilla en tubos al vacío Vacutainer®. La persona

encargada de las extracciones sanguíneas será la Técnico de laboratorio de bioquímica

de la Facultad de Ciencias de la Actividad Física y del Deporte-INEF. Universidad

Politécnica de Madrid, Técnico en diagnóstico clínico.

Durante las pruebas máximas estará presente en todo momento un médico especialista

en Medicina de la Educación Física y de la Facultad de Ciencias de la Actividad Física

y del Deporte-INEF. Universidad Politécnica de Madrid, quien se hará cargo del

Examen médico, realización de espirometría, electrocardiograma, supervisión y control

durante las pruebas máximas de esfuerzo e interpretación de los resultados recogidos en

estas pruebas.

En sangre se medirán parámetros hematológicos y bioquímicos de rutina y también se

realizará un análisis genético de un polimorfismo relacionado con el metabolismo de la

homocisteína. Metil Tetrahidrofolato Reductasa (MTHFR) es el nombre de un gen que

produce cierta enzima, también conocido como metilentetrahidrofolato reductasa.


116

El polimorfismo genético hace referencia a la existencia en una población de múltiples

alelos de un gen. Es decir, un polimorfismo es una variación en la secuencia de un lugar

determinado del ADN entre los individuos de una población. En el caso de la Metil

Tetrahidrofolato Reductasa (MTHFR), el polimorfismo más frecuente es el de la

sustitución de una base nitrogenada por otra, sustitución de una C (citosina) por una T

(timina) en la posición 677. Esta variante, que es relativamente frecuente en la

población, da lugar a una MTHFR de actividad enzimática reducida a temperaturas más

elevadas (es termolábil). Si una persona padece esta mutación genética tiene elevados

los niveles de homocisteina y podría llegar a padecer hiperhomocisteinemia, hecho

reconocido como factor de riesgo cardiovascular asociado con mayor frecuencia de

infartos de miocardio. Por ello, en el presente estudio se comprobará la presencia de este

polimorfismo en los sujetos, en las muestras sanguíneas tomadas.

Las pruebas se llevarán a cabo del 30 de Enero al 1 de marzo de 2012. Las pruebas se

realizarán dejando al menos una semana entre cada prueba submáxima. Se realizarán

dos pruebas submáximas rehidratando solo, una vez finalizada la prueba, en una de ellas

con agua y en la otra con una bebida para deportistas. Se realizarán otras dos pruebas

submáximas hidratando durante la prueba y al finalizar ésta, en una de ellas con agua y

en la otra con bebida para deportistas.

Su participación en este estudio es totalmente voluntaria.

Si actualmente sufre alguno de los siguientes casos, usted no debería tomar parte en los

test físicos a menos que un facultativo le autorizara por escrito a hacerlo:

- Riesgo cardiovascular, central o periférico. - Diabetes. - Problemas renales o hepáticos conocidos. - Complicaciones asmáticas. - Colesterol plasmático mayor de 8 mmol/litro. - Presión arterial sistólica mayor de 160 mm Hg o diastólica mayor de 100 mmHg. - Historial de abuso de alcohol o drogas. - Historial previo de inflamación o cáncer. - Limitaciones ortopédicas. - Medicaciones que puedan afectar a la función cardiovascular o metabólica. - Seguir una dieta vegetariana - Toma de cualquier suplemento vitamínico - Toma de cualquier suplementos protéico. - Tabaco


117

Con la finalización del estudio usted recibirá, un informe detallado con los resultados

más relevantes de las pruebas que haya realizado.

El riesgo de llevar a cabo estas actividades es similar al riesgo de desarrollar ejercicios

moderados y por tanto podría llegar a provocar fatiga, agujetas, esguinces, lesión

muscular, mareos o desvanecimientos. Así mismo, existe el riesgo de sufrir una parada

cardiaca, infarto o muerte súbita.

La información y datos recogidos en los diferentes cuestionarios realizados durante este

estudio respetarán siempre lo establecido por la Ley Orgánica 15/1999 de Protección de

Datos de Carácter Personal.

Otra información que usted debe saber

Seguro

De acuerdo con la Legislación Española vigente, este tipo de estudio no requiere de

ningún seguro que le proporcione cobertura frente a eventuales adversidades ya que se

trata de una práctica física común en la sociedad.

Información adicional

Ante cualquier eventualidad que pudiera surgir mientras que está participando en este

estudio o para cualquier pregunta sobre el mismo que desee realizar tras leer este

documento, por favor diríjase a los responsables del estudio.

Recuerde que siempre puede dejar de realizar las pruebas en el momento que usted lo

desee y así lo solicite.

El abajo firmante declara haber sido informado de los riesgos e implicaciones que

conlleva la participación en el presente estudio, y autoriza la realización de las pruebas

detalladas sobre su persona. Así mismo, autoriza a las estudiantes de Doctorado de la

Universidad Politécnica de Madrid, Beatriz Maroto Sánchez y Olga López Torres, y a la

directora de Tesis, Marcela González Gross, de la Universidad Politécnica de Madrid, a

que utilicen los datos derivados de las pruebas en estudios, comunicaciones y

publicaciones de carácter científico, siempre garantizando la confidencialidad de los

datos y su uso anónimo.


118

Doy mi consentimiento expreso para la determinación del polimorfismo genético

C677T de la Metiltetrahidrofolato Reductasa.

Nombre del informado: Nombre del investigador:

DNI: DNI:

Firma del informado Firma del investigador

Madrid, a ……… de Enero de 2012


119

CASE REPORT

PROJECT: Efecto de la hidratación sobre los niveles de homocisteína tras el ejercicio físico en varones físicamente activos

Fecha Teléfono Correo electrónico

Nombre y Apellidos Código

Fecha de nacimiento Edad Sexo

Dirección Profesión

COMIENZO DEL ESTUDIO:

A B Consentimiento informado Sí No Sí No El sujeto ha firmado el consentimiento informado Se entrega hoja de instrucciones del estudio

Fecha y firma del investigador (A): Fecha y firma del investigador (B):


120

SELECCIÓN DE SUJETOS A B

Criterios de Inclusión Sí No Sí No 01 Varón con edad comprendida entre los 18 y 28 años 02 Físicamente activos: Realización de actividad física aeróbica regular

(mínimo 3 días por semana).

03 Sanos: No padecen ninguna de las patologías descritas en los criterios de exclusión.


A

B

Cristerios de exclusión Sí No Sí No

01 Riesgo cardiovascular, central o periférico.

02 Diabetes.

03 Problemas renales o hepáticos conocidos.

04 Complicaciones asmáticas.

05 Colesterol plasmático mayor de 8 mmol/litro.

06 Presión arterial sistólica mayor de 160 mm Hg o diastólica mayor de 100mmHg.

07 Historial de abuso de alcohol o drogas.

08 Historial previo de inflamación o cáncer.

09 Limitaciones ortopédicas

10 Medicaciones que puedan afectar a la función cardiovascular o metabólica.

11 Seguir una dieta vegetariana

12 Suplemento vitamínico o proteico

13 Tabaco 14 Consumo de alcohol por encima del consumo moderado:


121

EXÁMEN FÍSICO

A B El sujeto ha participado en esta parte del estudio Sí No Sí No

A B

Sí No Sí No 01 Impedancia Bioeléctrica 02 Espirometría 03 Electrocardiograma 04 Peso 05 Altura 06 Tensión Arterial

Incidencias: Fecha y firma del investigador (A): Fecha y firma del investigador (B): DATOS DE BIOIMPEDANCIA

A B El sujeto ha participado en esta parte del estudio Sí No Sí No

A B

Sí No Sí No

01 Porcentaje grasa

02 Masa total de grasa (g)

03 % masa magra

04 Cantidad total de masa magra (g)

05 Densidad mineral ósea Incidencias: Fecha y firma del investigador (A): Fecha y firma del investigador (B):


122

PRUEBA MÁXIMA

A B Antes Después

01 Tensión Arterial 02 Frecuencia Cardiaca Máxima (FCmáx) 03 VO2máx 04 Velocidad Cinta/VO2max Incidencias: Fecha y firma del investigador (A): Fecha y firma del investigador (B):

Prueba 1 Rehidratación

Prueba 2 Rehidratación

Prueba 3 Hidratación

Prueba 4 Hidratación

Sorteo Aleatorio Hidratación

PRUEBA SUBMÁXIMA 1 REHIDRATACIÓN

A B BEBIDA Antes Después

01 Tensión Arterial 02 Peso Liquido

perdido 03 VO2 65%

04 Velocidad Cinta 05 FC Máx

06 FC media

07 Temperatura Media

08 Humedad Relativa Media

Incidencias: Fecha y firma del investigador (A): Fecha y firma del investigador (B):


123

PRUEBA SUBMÁXIMA 2 REHIDRATACIÓN



perdido 03 VO 2 65%

04 Velocidad Cinta 05 FCMáx

06 FC media



Incidencias: Fecha y firma del investigador (A): Fecha y firma del investigador (B): PRUEBA SUBMÁXIMA 3 HIDRATACIÓN



perdido 03 VO 2 65%


06 FC media





124

PRUEBA SUBMÁXIMA 4 HIDRATACIÓN



perdido 03 VO 2 65%


06 FC media





125

RECOGIDA DE MUESTRAS SANGUÍNEAS

A/B Prueba máx

Prueba submax 1 Prueba submax 2 Prueba submax 3

Prueba submax 4

Muestras sangre

1 2 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5

FECHA

HORA



126

CONTROL DE INGESTA DE ALIMENTOS

A/B Café o similares Alcohol

Suplementos

A/B Prueba máx Prueba submax 1 Prueba submax 2 Prueba submax 3

Prueba submax 4

1-2 1-2 3 4 5 1-2 3 4 5 1-2 3 4 5 1-2 3 4 5

Ultima comida Fecha Hora

¿Qué comida? Antes 2 horas 6 horas 24 horas

MAXIMA

SUB 1

SUB 2

SUB 3

SUB 4

Incidencias:

Fecha y firma del investigador (A):

Fecha y firma del investigador (B):

Comentarios:


127

INFORME DE EFECTOS ADVERSOS Fecha:

01 Muerte * 02 Hosptalización ** 03 Riesgo vital 04 Incapacidad persitente 05 Otros

* Si muere:

Fecha de la muerte Probebla causa de la muerte: Autopsia realizada? Sí No

** Si es hospitalizado:

Fecha de hospitalización:

Descripción del efecto adverso 01 Síntomas

02 Desarrollo

03 Diagnóstico

04 IInvestigaciones

05 Resultados

06 Tratamiento

07 Otros comentarios



128

FIN DEL ESTUDIO

Ha completado el sujeto todas las pruebas del estudio? Sí No

Si no, por favor, especifique: Fecha de retirada del sujeto:

Principal razón para interrumpir la participación: 01 Petición formulada por el personal 02 Criterio de exclusión 03 Efecto adverso 04 Otros:

Comentarios: Fecha y firma del investigador (A): Fecha y firma del investigador (B):


129

FORMULARIO PARA PARTICIPACIÓN EN EL PROYECTO

EFECTO DE LA HIDRATACIÓN SOBRE LOS NIVELES DE HOMOCISTEÍNA TRAS EL EJERCICIO FÍSICO EN VARONES FÍSICAMENTE ACTIVOS

Rellene el siguiente formulario marcando con una x.

Nombre y Apellidos:

SI NO

Edad comprendida entre 18-45 años

Ejercicio físico aeróbico mínimo 3 días por semana Horas/Tipo de ejercicio.

Riesgo cardiovascular, central o periférico

Diabetes

Problemas renales o hepáticos conocidos

Complicaciones asmáticas

Colesterol plasmático mayor de 8 mmol/litro

Historial de abuso de alcohol o drogas

Historial previo de inflamación o cáncer

Limitaciones ortopédicas

Medicaciones que puedan afectar a la función cardiovascular o metabólica

Seguir una dieta vegetariana

Suplemento vitamínico Tipo y Cantidad.

Suplementación protéico Cantidad.

Consumo habitual de tabaco Cigarros diarios.

Ingesta de alcohol superior a 20 g de alcohol/día: Cerveza: 0,5 l Vino: 0,5 l Bebidas destiladas: 0,06 l

Especificar cantidad semanal (vasos, copas, latas).

Madrid, a ……… de Enero de 2012 Firma del investigador:


130

HOJA DE INSTRUCCIONES PARA EL PARTICIPANTE:

INFORMACION PREVIA A LA REALIZACION DEL ESTUDIO

EFECTO DE LA HIDRATACIÓN SOBRE LOS NIVELES DE HOMOCISTEÍNA

TRAS EL EJERCICIO FÍSICO EN VARONES FÍSICAMENTE ACTIVOS

Los participantes del estudio realizarán las pruebas en 5 sesiones:

Antes de comenzar el estudio los participantes deberán firmar el consentimiento

informado y recoger la hoja de instrucciones.

SESIÓN 1.

Reconocimiento médico, Tanita y prueba máxima.

Esta sesión será desarrollada durante la semana del 30 de Enero en horario de tarde:

• RECONOCIMIENTO MÉDICO: Se les tomará la tensión arterial, se les

realizará una historia clínica y se les pesará y medirá. Se realizará una

espirometría y un electrocardiograma.

• BIA: Se realizará un examen de la composición corporal: Porcentaje de masa

grasa y masa magra corporal, así como la cantidad total de ambas.

• PRUEBA MÁXIMA:

Esta prueba se realizará en tapiz rodante.

Se compone de una fase de toma de contacto (1 minuto en reposo), otra fase de

calentamiento (3 minutos) y una última fase principal de desarrollo de la prueba,

en la que la velocidad del tapiz será incrementada gradualmente de manera

continua (0,2 km/h cada 12 segundos). La finalización de la prueba será

determinada por la fatiga del sujeto y vendrá seguida de 2 minutos de

recuperación activa (6 km/h) y 3 minutos de reposo (sentado).

El desarrollo de esta prueba permitirá la obtención de datos sobre los umbrales

de los sujetos, los cuales serán necesarios para el desarrollo de las posteriores

pruebas submáximas que componen el estudio.


131

SESIÓN 2.

Prueba submáxima 1. Rehidratación al finalizar la prueba.

Esta sesión se desarrollará durante los días 6 de febrero al 1 de Marzo de 2011, en

horario de mañana.

La prueba se realizará en tapiz rodante.

- 5 minutos de calentamiento, a una velocidad de 6 km/h,

- 40 minutos a una intensidad correspondiente al 65 % del VO2max del sujeto

- 5 minutos de recuperación: 4 minutos de recuperación activa a una velocidad de 6

km/h y 1 minuto recuperación pasiva sentado.

La prueba se desarrolla a una temperatura media de 30 ºC y una humedad relativa de

entre 60-70 %.

Toma de muestras y mediciones

Evaluación del peso corporal, antes y después de la prueba.

Recogida de muestra de orina antes y después de la prueba.

Muestras sanguíneas:

Las muestras de sangre se tomarán antes e inmediatamente después de la prueba y a las

2 h, 6 h y 24 h tas su finalización.

Protocolo de Rehidratación

Tras finalizar la prueba, se comenzará con el protocolo de rehidratación con una de las

bebidas (agua mineral embotellada o una bebida para deportistas, según sorteo

aleatorio). Los participantes deberán beber en las 2 h siguientes a la finalización de la

prueba la misma cantidad de bebida (en mL) que el peso corporal perdido durante la

prueba. Además no podrán ingerir ningún otro alimento ni líquido durante estas 2 h.

SESIÓN 3.

Prueba submáxima 2. Rehidratación al finalizar la prueba.

El protocolo a seguir en esta prueba es exactamente igual al de la prueba anterior,

variando únicamente el protocolo de rehidratación, que en esta ocasión consistirá en la

bebida que no se tomó en la primera prueba.





Las muestras de sangre se tomarán antes, inmediatamente después de la prueba, a las 2

h, 6 h y 24 h tas su finalización.


132

SESIÓN 4.

Prueba submáxima 3. Hidratación durante la prueba.

El protocolo a seguir en esta prueba es exactamente igual al de las pruebas anteriores,

variando, únicamente, el protocolo de hidratación a seguir durante la realización de la

prueba, que en esta ocasión consistirá en administrar 250 mL de una de las bebidas

(agua o una bebida para deportistas) durante el desarrollo de la prueba, divididos en dos

tomas de 125 mL cada una, la primera a los 15 minutos y la segunda a los 30 minutos

del inicio de la prueba.

Tras la finalización de esta prueba se seguirá un protocolo de rehidratación durante 2 h

que se le indicará tras la prueba.







SESIÓN 5.

Prueba submáxima 4. Hidratación durante la prueba.

El protocolo a seguir en esta prueba es exactamente igual al de la prueba anterior,

variando únicamente la hidratación, que en esta ocasión consistirá en administrar la

bebida que no se tomó en la prueba anterior.


Evaluación del peso corporal, Antes y después de la prueba.





DIRECTRICES A SEGUIR DURANTE LA PARTICIPACIÓN EN EL ESTUDIO.

• Ejercicio Físico Antes de la prueba: Los participantes no deberán realizar

ejercicio físico en las 24 horas previas a la prueba, ni podrán realizar ejercicio

físico el mismo día de la prueba hasta las 24 horas tras la finalización de las

extracciones sanguíneas.


133

• Alimentos antes de la prueba: No deberán ingerir alimentos sólidos durante las

2 horas previas a la prueba ni ingerir líquidos en los 20 minutos previos.

• Alcohol y otras sustancias: No se deberá consumir alcohol entre las 24 horas

previas a la prueba y la última extracción sanguínea de la prueba.

• Ropa: Todos los participantes acudirán con dos pantalones deportivos similares.

Deberán acudir a la prueba con ropa deportiva cómoda y ligera, así como un

calzado adecuado para correr.

• Protocolo de alimentación y de hidratación: Los participantes deberán seguir

estrictamente las pautas de alimentación que se les entregará en la reunión

informativa. Además, deberán seguir estrictamente el protocolo de hidratación

establecido en cada prueba.

• Puntualidad: Imprescindible ser puntual a la hora de la realización de las

pruebas ya que un pequeño retraso hará que todos los participantes tengan que

modificar su horario de pruebas. El laboratorio está disponible para el estudio en

esas horas estrictas.

• Muy importante asistir puntualmente a la extracción de tomas sanguíneas.

• Habrá una semana de descanso entre la realización de todas las pruebas

submáximas.

• Los participantes deberán estar 10 minutos antes de la realización de la prueba

en la puerta del laboratorio de fisiología del esfuerzo. En la plata 7º de la

Facultad de Ciencias de la Actividad Fïsica y del Deporte-INEF.


Investigador:

Investigador Responsable:


134

INSTRUCCIONES PARA EL PARTICIPANTE:

PROTOCOLO DE ALIMENTACIÓN



Directrices a seguir antes de la prueba:

Desayunar mínimo dos horas antes de la prueba submáxima.

- NO tomar CAFÉ, NI TE

- NO tomar leche, mantequillas o alimentos enriquecidos (Vitaminas D,

Vitaminas B2, B6 B12, Acido Fólico o B9 ni Omega 3)

No comer nada en las dos horas antes de la prueba submáxima.

- Importante venir hidratado, comenzar la prueba sin sensación de sed: Beber

únicamente agua de manera normal según las pautas establecidas en la reunión

informativa (ingerir 350 mL de agua 2 h antes del comienzo de la prueba):

Directrices a seguir en el mismo día tras la realización de la prueba:

• Ingesta total de Hidratos de Carbono: 8 g x kg de peso. (patata asada o

cocida, pasta, arroz, panes integrales, legumbres…)

• Ingesta total de Proteínas: 1,5 g x kg de peso (pollo, pavo o ternera magra,

huevo, leche…)

• Grasas: Evitar todo tipo de alimento graso (bollería industrial, fritos, quesos

grasos…)

• Lácteos: Consumo moderado, no enriquecidos, a poder ser desnatados o bajo en

grasa.

• Líquidos: Agua, consumir en las 24 horas siguientes a la finalización de la

prueba mínimo el 50 % del total del líquido perdido durante la prueba.

Directrices Generales a seguir durante la duración del estudio.

Suplementos

Dejar de tomar inmediatamente cualquier tipo de suplemento vitamínico y protéico así

como alimentos enriquecidos, hasta la finalización del estudio. (Suplemento de

proteínas, suplemento de creatina, cualquier suplemento vitamínico).


135

Además se deberá dejar de tomar cualquier aporte extra en alimentos enriquecidos con

vitaminas, u omega 3, ácido fólico o vitamina B12 (cereales enriquecidos, leche

enriquecida, yogures enriquecidos, margarinas, etc).

Cerveza

Limitar el consumo de cerveza a 2 cervezas de 33 mL a la semana (*Debido al alto

contenido en ácido fólico y vitamina B12).

No consumir cerveza las 48 h antes de la prueba ni hasta la última extracción sanguínea

del día de la prueba.

Alcohol

Se podrá tomar una copa de vino al día, excepto las 24 horas anteriores a la prueba y

hasta la finalización de la última extracción sanguínea.

Se podrá tomar en total 4 copas a la semana de alcohol destilado (whisky, ron,

vodka…), excepto desde las 24 horas antes a la prueba hasta la finalización de la última

extracción sanguínea.

Café y bebidas estimulantes: Café, Te, bebidas energéticas y refrescos de Cola.

Día de antes: un café o té como máximo.

El día de la prueba: No tomar ni café ni té.

Refrescos de Cola: Máximo una lata al día 33cl.

(Red bull, Monster, Burn…): No tomar ningún tipo de bebida energética durante ningún

momento del estudio.

Vegetales

Consumo normal de vegetales a la semana (150 g al día). No tomar el día anterior ni el

día de la prueba vegetales de hoja verde.

Carne

Consumo normal de carne a la semana (3 días)

Ingesta normal del resto de alimentos (atención a que no estén enriquecidos en

Vitaminas tipo D, tipo B, fólico y Omega3)


Investigador:

Investigador Responsable:


136

PROTOCOLO DE HIDRATACIÓN



Prueba Submáxima 1 :

Tras finalizar la prueba, se comenzará con el protocolo de rehidratación con una de las

bebidas (agua mineral embotellada o bebida para deportistas, según sorteo aleatorio).

Los participantes deberán beber en las 2 h siguientes a la finalización de la prueba la

misma cantidad de bebida (en mL) que el peso corporal perdido durante la prueba, dato

que se obtendrá del peso perdido de los sujetos durante la prueba.

Se beberá la mitad de la bebida durante la 1º hora y la otra mitad durante la 2º hora.

*Durante las 2 h del protocolo de rehidratación no se podrá ingerir ningún alimento ni

ningún otro líquido.

Prueba Submáxima 2:

El protocolo a seguir será igual que el de la prueba submáxima 1, variando únicamente

el tipo de bebida que será la contraria a la que se tomó en la prueba submáxima 1.


Se administrará un total de 250 mL de una de las bebidas (agua o bebida para

deportistas según sorteo aleatorio) durante el desarrollo de la prueba, divididos en dos

tomas de 125 mL cada una. La primera toma seá administrada a los 15 minutos y la

segunda a los 30 minutos tras el inicio de la prueba.

Para poder administrar la bebida se le separará el tubo del analizador de gases de la

boquilla y con un vaso y una pajita se le facilitará la ingesta de la bebida sin paralizar la

prueba de ejercicio.

Tras la finalización del ejercicio se seguirá el mismo protocolo de rehidratación

explicado en las pruebas 1 y 2. La bebida a ingerir durante las 2 h posteriores será la

misma bebida que ha sido administrada durante el ejercicio


El protocolo de hidratación a seguir en esta prueba es exactamente igual al de la prueba

submáxima 3, variando únicamente la bebida a ingerir, en esta ocasión consistirá en

administrar la bebida contraria a la que se tomó en la prueba submáxima 3.


137

PROTOCOLO EN EL TRATAMIENTO DE LAS MUESTRAS DE SANGRE EN

EL PROYECTO DE HIDRATACIÓN



MATERIAL

Por sujeto se necesitarán:

• 6 Tubos de EDTA K3 (5/4 mL): (Acido Etilen Diamino Tetracético),

generalmentetripotásico o dipotásico, para determinaciones de hematología en sangre

completa. Inhibe el proceso de coagulación eliminando el calcio de la sangre. Reduce

la activación plaquetaria protegiendo a las plaquetas durante el contacto de la sangre

con la superficie interna de vidrio del tubo. Es el aditivo idóneo para la realización

del recuento de leucocitos, plaquetas, y hematíes y también para la determinación de

la fórmula leucocitaria, citometría de flujo y determinación de plomo, ya que se

conserva la morfología de las células de la sangre.

• 6 Tubos separador GEL 7/5 mL: Recomendado para pruebas en las que se analice

el suero. Su gel separador inerte en el fondo del tubo proporciona una barrera entre el

coágulo y el suero de la muestra. El gel, por su densidad, se mueve durante la

centrifugación hacia la parte superior del tubo, formando una barrera entre el

sobrenadante (suero) y el sedimento (coágulo de fibrina y células). El interior del

tubo está recubierto de silicona y de partículas de sílice micronizadas para acelerar el

proceso de coagulación y prevenir la adherencia de los hematíes a la pared del tubo.


138

• 2 Contenedores de orina de 100 mL• Palomilla seguridad 21G. ¾ (0.8x19)• Portatubos Vacutainer• Guantes látex• Alcohol 96º• Gasas• Eppendorf• Puntas de pipetas• Esparadrapo

Procedimiento experimental:

Antes de comenzar las pruebas físicas del estudio los sujetos acudirán en ayunas al

laboratorio de bioquímica para realizar un análisis de sangre que se obtendrá mediante

punción venosa.

Genética: Envío a la Universidad de Cantabria POLIMORFISMO C667T DE Mthfr ; POLIMORFISMO ACE I/D

Centrifuga a 3000 rpm 10-12minutos

Tubo de EDTA K3 Tubos separador GEL

BIOQUÍMICAHEMOGRAMA


139

Recogida de muestras en las sesiones de pruebas de ejercicio: RECOGIDA DE MUESTRA1 (INMEDIATAMENTE ANTES DEL EJERCICIO):

RECOGIDA DE MUESTRA 2: INMEDIATAMENTE DESPUÉS DEL EJERCICIO (40 MINUTOS):

RECOGIDA DE MUESTRA 3, 4 Y 5 . (2 h, 6 h Y 24 h RESPECTIVAMENTE) DESPUÉS DEL

EJERCICIO:



140

Departamento de Salud y Rendimiento Humano. Facultad de Ciencias de la Actividad Física y del Deporte-INEF. Universidad Politécnica de Madrid c/ Martín Fierro 7 E-28040 Madrid

INFORME DE LA PARTICIPACIÓN EN EL PROYECTO:

EFECTO DE LA HIDRATACIÓN SOBRE LOS NIVELES DE HOMOCISTEÍNA TRAS EL EJERCICIO

FÍSICO EN VARONES FÍSICAMENTE ACTIVOS

Facultad de Ciencias de la Actividad Física y del Deporte

Universidad Politécnica de Madrid

ImFINE Research Group

141

El día 1/31/2012, ____________ comenzó su participación en el proyecto

“Efecto de la hidratación sobre los niveles de homocisteína tras el ejercicio físico en

varones físicamente activos” realizado en la Facultad de Ciencias de la Actividad Física y

del Deporte – INEF.

Se realizó una prueba de valoración funcional completa con los siguientes

resultados:

La exploración clínica, el electrocardiograma de reposo y en ejercicio, y la

espirometría son normales y se concluyen las siguientes observaciones particulares

a :

Prueba de esfuerzo: directa, de carácter máximo, realizada en tapiz rodante con

protocolo incremental, detenida debido a alcanzar criterios de maximidad. Llega a una

frecuencia cardiaca de 205 lpm y con un consumo máximo de oxígeno de 73,5 ml/min/kg.

• Respuesta clínica: asintomática

• Respuesta eléctrica: negativa para cambios isquémicos, no alteraciones del ritmo

ni de la conducción con respecto a la basal.

• Respuesta hemodinámica: adecuada al esfuerzo.

• Recuperación: normal.

Recomendaciones: Se recomienda realizar un reconocimiento médico-deportivo anual.

Los rangos de entrenamiento según los umbrales son: para el método C.E1 de 142-192

latidos, C.V: 142-205 latidos, C.I: 142-202 latidos, recomendando entrenamiento con

pulsómetro en estos rangos.

1 C.E Continuo Extensivo, C.V. Continuo Variable, C.I Continuo Intensivo.

142

Propietario

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tras

Propietario

Textbox

,

Propietario

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142-180

Propietario

Textbox

142-195

Propietario

Textbox

182-195

En el momento de la prueba máxima, _________ ha obtenido los siguientes valores:

Análisis de la composición corporal mediante impedancia bioeléctrica (BIA):

BIOIMPEDANCIA ELÉCTRICA

Peso 76,6kg % grasa 14,3 %

BMI

Talla 173,6cm Masa magra

(Kg) 11,2 Kg

26,

Rango de peso normal

SEEDO

Sobrepeso I

Metabolismo Basal (estimado)

1949 Kcal/día

Masa grasa (Kg) 67,4 Kg

Agua Total corporal

49,3 kg

VARIABLES ERGOESPIROMÉTRICAS* Consumo de Oxígeno

Máximo 5753mL/min Frecuencia Cardiaca Máxima

205 lpm

Consumo de Oxígeno al Umbral Aeróbico 3471mL/min

Umbral Ventilarorio 1 (Aeróbico) 147 lpm

Consumo de Oxígeno al Umbral Anaeróbico 5211mL/min

Umbral Ventilarorio 2 (Anaeróbico) 197 lpm

Consumo de Oxígeno Máximo Relativo

(VO2max/kg) 73,5mL/min/kg Índice de Recuperación

Cardiaca** 46 %

(Normal)

* La información sobre las variables ergoespirométricas se completa con el propio informe de la prueba deesfuerzo.** Calderón FJ, Brita JL, Gonzalez C, Machota V. Estudio de la recuperación de la frecuencia cardíaca endeportistas de élite. Selección1997;6(3):101-5.

143

Propietario

Textbox

190

Informe del líquido ingerido y perdido durante las pruebas submáximas.


Fecha: 2/13/2012

Líquido Ingerido: Agua

(Rehidratación de 2 horas después de la prueba)

Condiciones ambientales

Temperatura media: 30ºC

Humedad Relativa media: 65%

Peso Antes de la prueba 78,6 kg

Peso Después de la prueba 76,4 kg

Peso perdido durante la prueba: 2,2 kg

Líquido ingerido 2,2 L


Fecha: 2/6/2012

Líquido Ingerido: Sport drink

(Rehidratación de 2 horas después de la prueba)




Peso Antes de la prueba 78, kg





Fecha: 2/27/2012

Líquido Ingerido: Agua

Hidratación durante la prueba + rehidratación 2 horas después de la prueba






Peso perdido durante la prueba: 2 kg

Líquido ingerido 2 L

144


Fecha: 3/8/2012

Líquido Ingerido: Sport drink

Hidratación durante la prueba + rehidratación 2 horas después de la prueba





Peso Después de la prueba 77, kg



145

de 1

Fecha informe: 1/03/2012

Nombre:

Fecha análisis: 30/1/2012

BIOQUIMICA Valores normales

• GLUCOSA: 81 mg/dL 60-115 mg/dL

• COLESTEROL: 165 mg/dL 100-220 mg/dL

• TRIGLICÉRIDOS: 71 mg/dL 40-160 mg/dL

• GOT: 20 U/L 10-40 U/L

• GPT: 17 U/L 10-55 U/L

• ACIDO ÚRICO: 4,3 mg/dL 3,6-7,7 mg/dL

• UREA: 39,6 mg/dL 15-45 mg/dL

• PROTEÍNAS TOTALES: 7,6 mg/dL 6,6-8,3 mg/dL

• CREATININA: 0,9 mg/dL 0,7-1,4 mg /dL

• CREATINA 1,29 1,28-5,21 mgl/L

• CREATINA KINASA: 274*U/L 20-240 U/L

• SODIO: 128,4 mEq/L 135-148 mEq/L

• POTASIO: 7,1* mEq/L 3,5-5 mEq/L

• CLORURO: 96,9 mmol/L 95-115 mmol/L

• MAGNESIO: 2,1* mmol/L 0,66-1,03 mmol/L

• HOMOCISTEÍNA: 6,9 mmol/L 5-10 µmol/L

• VITAMINA B12 200-900 pg/mL: 487,4 pg/mL

• FOLATO: 19,24 ng/mL 6-20 ng/mL

146

Informe Ergoespirométrico

Identificación: Apellidos:

Nombre: F. Nacimiento:

Sexo: Altura:

Peso: Edad:

Doctor:

S26

male

76,6 kg

Operador:

173,6 cm

30 Years

Protocolo: PJB_1CINTA Ergómetro: TapízHora: 18:42:52 Fecha: 31/01/2012

Parámetros máximos de la prueba

Departamento de Rendimiento HumanoU.P.M

Sumario VT1

Nº 8

VT2

Manual

MaxVO2 Teor Max

Vatios

MaxVO2

%pred

Recup

120 sec

Promediado 15 SegundosTime min 08:30 13:45 14:45 16:15 18:30

Load 211 300 318 241 348 132 84

Speed km/h 12.2 17.4 18.4 20.1 6.0

V'CO2 ml/min 2936 5384 6048 6087 0

V'O2 3471 5211 5753 3025 5634 190 0

V'E 70 130 153 118 175 129 0

VO2/kg ml/min/kg 45.3 68.0 75.1 73.5 0.0

O2/HR 23.6 26.5 28.5 15.9 27.5 179 0.0

HRR % % 23 -4 -6 -8 25

HR 147 197 202 190 205 106 142

VO2%m % 60 91 100 98 0

W/kg W/kg 2.8 3.9 4.2 4.5 1.1

11/04/2013 12:45

Comentarios:

Prueba nº. 2 11/04/2013 12:45:06El umbral aeróbico se encuentra en el minuto 8:30 a una frecuencia cardíaca de 147 latidos/min y un% del VO2 max del 60 %. La velocidad para este umbral ha sido de 12.2 km/h a un ritmo de 4 min55 seg en 1000 m.El umbral anaeróbico se encuentra en el minuto 13:45 a una frecuencia cardíaca de 197 latidos/miny un % del VO2 max del 91 %. La velocidad para este umbral ha sido de 17.4 km/h a un ritmo de 3min 25 seg en 1000 m.La frecuencia cardíaca máxima alcanzada durante la prueba ha sido de 205 latidos/min.

147

Propietario

Textbox

190

Propietario

Textbox

190

Propietario

Textbox

Identificación: S26

11/04/2013 12:45

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Apellidos:

Gráficas de la prueba

148

Time min

Load W

Speed km/h

HR 1/min

V'O2 ml/min

V'CO2 ml/min

V'E L/min

RER EqO2 EqCO2 PETO2 kPa

PETCO2 kPa

00:05 0 0.0 83 1587 1308 40 0.82 23.9 29.0 13.00 4.71 00:10 0 0.0 85 641 574 20 0.89 30.0 33.5 13.77 4.31 00:15 0 0.0 82 428 386 16 0.90 33.5 37.1 13.90 4.25 00:20 0 0.0 80 465 410 16 0.88 31.0 35.2 13.66 4.40 00:25 0 0.0 88 613 541 19 0.88 29.6 33.5 13.45 4.53 00:30 0 0.0 90 360 311 12 0.86 31.4 36.3 13.24 4.67 00:35 0 0.0 88 353 288 11 0.82 29.2 35.8 13.00 4.75 00:40 0 0.0 89 385 299 13 0.78 29.6 38.1 12.99 4.62 00:45 0 0.0 81 564 447 17 0.79 27.0 34.0 12.88 4.66 00:50 0 0.0 83 467 366 15 0.78 28.8 36.7 13.09 4.55 00:55 0 0.0 80 416 325 14 0.78 29.5 37.8 13.05 4.55 00:59 0 0.0 86 594 475 18 0.80 28.6 35.8 13.10 4.54 01:00 0 0.0 83 378 300 14 0.79 31.9 40.3 13.20 4.44 Fase referencia: 01:05 0 0.0 77 613 493 19 0.80 28.7 35.7 13.40 4.39 01:10 29 2.8 88 830 685 23 0.82 26.5 32.1 12.70 4.82 01:15 84 6.0 89 1048 842 27 0.80 23.8 29.6 13.05 4.71 01:20 84 6.0 110 1122 864 25 0.77 20.8 27.0 13.58 3.86 01:25 84 6.0 112 1349 1021 28 0.76 19.7 26.0 11.80 5.23 01:30 84 6.0 111 1419 1077 31 0.76 21.0 27.6 12.30 4.90 01:35 84 6.0 116 1419 1130 34 0.80 22.5 28.3 12.87 4.71 01:40 84 6.0 142 1201 1013 30 0.84 23.8 28.2 13.04 4.74 01:45 84 6.0 109 1288 1076 32 0.84 23.4 28.0 12.85 4.86 01:50 84 6.0 107 1348 1093 32 0.81 22.9 28.3 12.64 4.87 01:55 84 6.0 106 1354 1068 31 0.79 21.9 27.7 12.49 4.99 02:00 84 6.0 112 1436 1174 34 0.82 22.9 28.0 12.55 5.02 02:05 84 6.0 112 1526 1149 32 0.75 19.9 26.4 11.84 5.21 02:10 84 6.0 112 1636 1212 35 0.74 20.2 27.2 12.30 4.91 02:15 84 6.0 111 1777 1308 38 0.74 20.3 27.5 12.24 4.91 02:20 84 6.0 110 1540 1141 33 0.74 20.4 27.6 11.90 5.16 02:25 84 6.0 111 961 702 23 0.73 21.5 29.5 14.90 2.70 02:30 84 6.0 111 2151 1570 42 0.73 19.0 26.0 12.01 4.95 02:35 84 6.0 102 1660 1241 36 0.75 20.6 27.6 12.25 4.91 02:40 84 6.0 105 1632 1230 36 0.75 20.8 27.6 12.25 5.00 02:45 85 6.1 100 1077 807 26 0.75 22.2 29.7 12.79 4.54 02:50 83 6.0 110 2099 1545 40 0.74 18.6 25.3 11.91 5.05 02:55 84 6.0 106 1752 1333 38 0.76 20.8 27.3 12.39 4.89 03:00 84 6.0 106 1687 1322 37 0.78 21.0 26.7 12.30 4.98 03:05 84 6.0 108 1608 1261 36 0.78 21.2 27.0 12.39 5.02 03:10 84 6.0 99 1543 1222 35 0.79 21.7 27.4 12.47 4.96 03:15 84 6.0 103 370 279 13 0.76 31.3 41.5 13.75 3.99 03:20 84 6.0 105 2069 1606 42 0.78 19.4 25.0 12.11 5.12 03:25 84 6.0 106 1644 1280 36 0.78 21.1 27.0 12.35 5.00 03:30 84 6.0 93 1654 1303 37 0.79 21.3 27.1 12.25 5.15 03:35 84 6.0 105 1562 1239 35 0.79 21.3 26.8 12.34 5.15 03:40 84 6.0 106 1690 1336 37 0.79 20.9 26.5 12.17 5.21 03:45 84 6.0 103 1656 1315 37 0.79 21.1 26.6 12.31 5.17 03:50 84 6.0 104 1621 1293 36 0.80 21.3 26.8 12.28 5.15 03:55 84 6.0 102 890 713 23 0.80 23.7 29.5 14.75 3.08 03:59 84 6.0 104 1988 1550 40 0.78 19.4 24.8 11.87 5.34 04:00 84 6.0 105 1671 1301 36 0.78 20.3 26.1 12.13 5.12 Fase de esfuerzo: 04:05 98 6.8 105 1531 1193 34 0.78 21.0 27.0 12.41 4.95 04:10 115 7.8 104 1669 1311 36 0.79 20.3 25.9 12.23 5.18 04:15 117 8.0 113 1815 1383 37 0.76 19.4 25.5 11.93 5.27 04:20 142 8.2 114 1965 1510 40 0.77 19.6 25.5 12.00 5.23 04:25 141 8.1 116 1839 1447 39 0.79 20.3 25.8 12.14 5.19 04:30 142 8.1 112 1761 1492 42 0.85 23.0 27.2 12.91 4.83 04:35 147 8.4 116 2857 2341 56 0.82 19.2 23.4 11.99 5.31 04:40 152 8.7 122 2312 1773 46 0.77 19.0 24.8 11.81 5.34 04:45 150 8.6 126 2528 1893 50 0.75 19.0 25.3 11.70 5.29 04:50 150 8.6 124 2709 2103 57 0.78 20.4 26.3 12.05 5.16 04:55 151 8.7 126 2550 2055 55 0.81 21.0 26.0 12.16 5.22 05:00 152 8.7 126 2385 1864 46 0.78 18.8 24.0 13.64 3.92 Almacenar ECG 1 05:05 152 8.7 125 2779 2215 58 0.80 20.3 25.4 12.02 5.24 05:10 159 9.1 125 2578 2092 56 0.81 21.0 25.9 12.17 5.28 05:15 157 9.0 126 1640 1316 37 0.80 21.5 26.8 13.54 4.12 05:20 160 9.2 129 3457 2757 67 0.80 19.0 23.9 12.06 5.28 05:25 160 9.2 128 2737 2258 60 0.82 21.4 26.0 12.38 5.10 05:30 160 9.2 126 2764 2331 61 0.84 21.4 25.3 12.22 5.39 05:35 163 9.4 125 2594 2189 56 0.84 21.0 24.9 12.07 5.44 05:40 163 9.4 128 1709 1364 39 0.80 21.5 26.9 13.45 4.21 05:45 168 9.6 128 3385 2687 64 0.79 18.5 23.3 11.78 5.50 05:50 168 9.7 129 3041 2443 64 0.80 20.4 25.3 12.12 5.25

11/04/2013 12:45149

Time min

Load W

Speed km/h

HR 1/min

V'O2 ml/min

V'CO2 ml/min

V'E L/min


PETCO2 kPa

05:55 168 9.7 125 2739 2249 58 0.82 20.5 24.9 12.17 5.25 06:00 171 9.8 128 2907 2362 61 0.81 20.2 24.9 12.07 5.39 Almacenar ECG 2 06:05 177 10.2 131 3019 2492 64 0.83 20.5 24.9 12.19 5.30 06:10 175 10.0 134 2764 2262 56 0.82 19.5 23.8 13.01 4.67 06:15 174 10.0 134 3127 2597 67 0.83 20.8 25.1 12.25 5.30 06:20 174 10.0 135 3034 2550 65 0.84 20.9 24.9 12.30 5.28 06:25 176 10.2 135 2916 2468 64 0.85 21.2 25.0 12.18 5.41 06:30 182 10.5 136 3041 2597 66 0.85 21.0 24.6 12.13 5.53 06:35 181 10.4 136 2997 2526 65 0.84 20.9 24.8 12.08 5.50 06:40 181 10.4 136 3196 2672 68 0.84 20.7 24.8 12.20 5.37 06:45 181 10.4 138 3086 2597 67 0.84 20.9 24.9 12.22 5.38 06:50 183 10.5 136 3101 2670 67 0.86 21.1 24.5 12.10 5.51 06:55 183 10.5 141 3103 2582 66 0.83 20.5 24.6 11.79 5.58 07:00 186 10.7 139 3068 2509 60 0.82 19.1 23.4 12.90 4.76 Almacenar ECG 3 07:05 188 10.8 144 3373 2853 73 0.85 20.9 24.7 12.27 5.38 07:10 188 10.8 142 3176 2711 69 0.85 21.0 24.6 12.23 5.43 07:15 191 11.0 142 3221 2731 69 0.85 20.7 24.4 12.16 5.39 07:20 191 11.0 141 3237 2786 71 0.86 21.3 24.7 12.20 5.46 07:25 196 11.3 144 3288 2812 72 0.86 21.2 24.8 12.33 5.37 07:30 194 11.2 145 3295 2794 69 0.85 20.4 24.0 11.93 5.59 07:35 200 11.5 142 3491 2984 76 0.85 21.1 24.7 12.30 5.44 07:40 198 11.4 143 1892 1625 45 0.86 22.6 26.3 13.96 3.91 07:45 199 11.4 146 4179 3534 83 0.85 19.6 23.1 12.03 5.54 07:50 201 11.6 146 3396 2925 75 0.86 21.4 24.8 12.28 5.45 07:55 201 11.6 146 3402 3004 71 0.88 20.6 23.3 11.97 5.74 08:00 206 11.9 148 3578 3065 77 0.86 20.8 24.3 12.14 5.54 Almacenar ECG 4 08:05 207 11.9 148 3571 3097 78 0.87 21.3 24.6 12.26 5.47 08:10 206 11.8 151 3534 3085 77 0.87 21.1 24.2 12.14 5.56 08:15 208 12.0 151 3537 3082 77 0.87 21.2 24.3 12.02 5.68 08:20 213 12.3 151 3325 2806 64 0.84 18.8 22.3 11.58 5.93 08:25 212 12.2 148 3720 3137 76 0.84 19.8 23.5 11.88 5.71 08:30 211 12.2 147 3304 2814 67 0.85 19.8 23.2 11.51 5.98 08:35 211 12.2 148 3778 3170 77 0.84 19.9 23.7 11.80 5.68 08:40 214 12.3 150 3741 3274 81 0.88 21.1 24.2 12.14 5.58 08:45 214 12.3 153 3699 3234 79 0.87 20.8 23.7 12.01 5.69 08:50 219 12.6 156 3850 3403 85 0.88 21.4 24.3 12.30 5.51 08:55 219 12.6 154 3620 3302 78 0.91 21.2 23.2 12.18 5.67 09:00 222 12.8 153 3883 3445 85 0.89 21.4 24.1 12.28 5.57 09:05 222 12.8 158 3748 3332 82 0.89 21.4 24.0 12.36 5.48 Almacenar ECG 5 09:10 222 12.8 156 3809 3370 82 0.88 20.9 23.6 12.04 5.75 09:15 227 13.1 158 2209 1947 53 0.88 23.0 26.1 13.91 4.05 09:20 225 13.0 160 4562 4014 92 0.88 19.7 22.4 11.99 5.76 09:25 224 12.9 160 4103 3676 93 0.90 22.1 24.6 12.43 5.40 09:30 229 13.2 160 3780 3388 82 0.90 21.0 23.4 12.20 5.69 09:35 233 13.4 160 3880 3410 82 0.88 20.5 23.3 11.94 5.74 09:40 233 13.4 160 4141 3682 89 0.89 21.0 23.6 12.14 5.66 09:45 232 13.4 163 3996 3592 87 0.90 21.2 23.5 12.13 5.70 09:50 235 13.6 165 4234 3788 93 0.89 21.5 24.0 12.35 5.56 09:55 236 13.6 165 4037 3676 89 0.91 21.6 23.8 12.18 5.76 10:00 240 13.8 167 4261 3889 91 0.91 20.9 22.9 12.02 5.89 10:05 240 13.8 167 4112 3680 89 0.89 21.2 23.7 11.91 5.85 Almacenar ECG 6 10:10 240 13.8 165 4293 3895 93 0.91 21.2 23.4 12.06 5.84 10:15 243 14.0 167 4231 3841 92 0.91 21.2 23.4 12.14 5.72 10:20 243 14.0 167 4081 3815 91 0.93 21.8 23.3 12.25 5.76 10:25 244 14.1 167 4346 3938 96 0.91 21.4 23.7 12.31 5.64 10:30 246 14.2 171 3907 3490 78 0.89 19.4 21.7 14.05 3.91 10:35 248 14.3 173 4350 3932 94 0.90 21.1 23.4 12.15 5.73 10:40 248 14.3 173 4447 4090 100 0.92 22.0 23.9 12.40 5.59 10:45 248 14.3 175 4432 4084 97 0.92 21.3 23.2 12.23 5.73 10:50 255 14.7 171 4500 4132 98 0.92 21.4 23.3 12.17 5.79 10:55 253 14.6 173 4466 4135 100 0.93 21.9 23.7 12.37 5.67 11:00 253 14.6 173 4397 4076 95 0.93 21.2 22.8 12.02 5.94 11:05 256 14.8 173 4551 4172 99 0.92 21.2 23.2 12.09 5.85 Almacenar ECG 7 11:10 256 14.8 171 4497 4168 98 0.93 21.3 23.0 12.23 5.82 11:15 258 14.9 173 4315 4036 91 0.94 20.7 22.2 11.91 6.13 11:20 263 15.2 173 4768 4380 105 0.92 21.6 23.5 12.16 5.82 11:25 261 15.1 177 4692 4375 106 0.93 22.1 23.7 12.42 5.68 11:30 259 14.9 178 4741 4436 106 0.94 21.8 23.3 12.22 5.87

11/04/2013 12:45150

Time min

Load W

Speed km/h

HR 1/min

V'O2 ml/min

V'CO2 ml/min

V'E L/min


PETCO2 kPa

11:35 262 15.1 180 4771 4497 106 0.94 21.8 23.1 12.18 5.90 11:40 266 15.3 180 4799 4531 109 0.94 22.2 23.5 12.37 5.74 11:45 266 15.4 182 4604 4332 103 0.94 21.8 23.2 12.25 5.81 11:50 267 15.4 182 4714 4428 104 0.94 21.5 22.9 12.08 5.99 11:55 268 15.4 182 4779 4523 107 0.95 21.9 23.1 12.20 5.93 12:00 276 15.9 182 4877 4595 107 0.94 21.5 22.8 12.17 5.92 12:05 274 15.8 184 4854 4657 107 0.96 21.6 22.5 12.07 6.04 Almacenar ECG 8 12:10 274 15.8 184 3729 3609 85 0.97 22.3 23.0 12.28 6.05 12:15 275 15.9 184 5533 5139 112 0.93 19.9 21.4 11.92 6.09 12:20 277 16.0 186 4941 4715 111 0.95 21.9 23.0 12.25 5.89 12:25 281 16.2 186 4888 4712 113 0.96 22.5 23.4 12.37 5.85 12:30 281 16.2 184 4982 4863 115 0.98 22.6 23.2 12.44 5.84 12:35 281 16.2 186 4767 4669 108 0.98 22.2 22.6 12.28 5.95 12:40 284 16.4 186 4843 4713 109 0.97 22.0 22.6 12.13 6.09 12:45 284 16.4 186 4844 4723 110 0.98 22.2 22.7 12.26 5.95 12:50 287 16.6 186 4993 4899 118 0.98 23.0 23.5 12.52 5.78 12:55 287 16.6 191 4972 4918 118 0.99 23.2 23.4 12.46 5.90 13:00 287 16.6 189 5211 5154 119 0.99 22.4 22.7 12.35 5.98 13:05 293 16.9 189 4968 4935 116 0.99 22.8 22.9 12.55 5.81 13:10 291 16.8 189 4956 4967 118 1.00 23.4 23.3 12.45 5.89 Almacenar ECG 9 13:15 291 16.8 191 4779 4662 106 0.98 21.8 22.3 12.32 6.09 13:20 294 17.0 192 5402 5355 130 0.99 23.5 23.7 12.64 5.76 13:25 294 17.0 196 5267 5393 131 1.02 24.3 23.7 12.73 5.80 13:30 292 16.9 192 5138 5237 126 1.02 24.0 23.6 12.75 5.73 13:35 297 17.2 194 5363 5496 133 1.02 24.3 23.7 12.83 5.69 13:40 297 17.1 194 5136 5337 131 1.04 25.1 24.1 12.91 5.65 13:45 300 17.4 197 5106 5299 124 1.04 23.9 23.0 12.59 5.95 13:50 304 17.6 194 5000 5023 115 1.00 22.5 22.4 12.57 5.91 13:55 305 17.6 197 5484 5555 133 1.01 23.6 23.3 12.64 5.86 14:00 306 17.7 197 5393 5559 136 1.03 24.6 23.9 12.91 5.65 14:05 306 17.7 200 5387 5562 136 1.03 24.6 23.8 12.83 5.73 14:10 307 17.7 197 5372 5604 139 1.04 25.3 24.3 13.03 5.57 Almacenar ECG 10 14:15 312 18.0 200 5410 5683 139 1.05 25.1 23.9 12.92 5.73 14:20 312 18.0 197 5423 5669 141 1.05 25.4 24.3 12.91 5.69 14:25 313 18.1 194 5155 5467 130 1.06 24.8 23.4 12.93 5.72 14:30 314 18.1 200 4977 5139 117 1.03 22.9 22.2 12.66 5.95 14:35 315 18.2 197 6041 6234 155 1.03 25.0 24.3 13.05 5.53 14:40 318 18.4 200 5606 5949 151 1.06 26.4 24.8 13.15 5.54 14:45 318 18.4 202 5614 5962 154 1.06 26.8 25.2 13.26 5.46 14:50 318 18.4 202 5601 6009 155 1.07 27.1 25.3 13.30 5.46 14:55 322 18.6 202 5638 6075 154 1.08 26.7 24.8 13.32 5.41 15:00 322 18.6 202 5570 5977 156 1.07 27.5 25.6 13.39 5.34 15:05 325 18.8 202 5641 6081 159 1.08 27.5 25.5 13.41 5.34 15:10 325 18.8 202 5526 5923 158 1.07 28.0 26.1 13.38 5.34 Almacenar ECG 11 15:15 325 18.8 202 5577 5970 161 1.07 28.2 26.3 13.52 5.23 15:20 329 19.0 202 5685 6095 158 1.07 27.1 25.3 13.30 5.45 15:25 329 19.0 200 5609 5997 156 1.07 27.3 25.5 13.36 5.37 15:30 330 19.1 205 5586 5969 158 1.07 27.7 25.9 13.45 5.28 15:35 331 19.2 202 5605 6012 162 1.07 28.3 26.4 13.49 5.27 15:40 333 19.3 208 5528 5929 160 1.07 28.3 26.4 13.49 5.29 15:45 335 19.3 205 5707 6091 164 1.07 28.1 26.4 13.46 5.30 15:50 335 19.3 202 5554 5985 164 1.08 28.8 26.7 13.56 5.20 15:55 339 19.6 202 5529 5966 160 1.08 28.3 26.3 13.54 5.26 16:00 338 19.5 206 5349 5624 143 1.05 26.1 24.8 13.28 5.46 16:05 341 19.7 202 5704 6131 175 1.07 30.0 27.9 13.72 5.10 16:10 341 19.7 203 5540 6014 172 1.09 30.3 27.9 13.76 5.07 Almacenar ECG 12 16:15 348 20.1 205 5652 6110 178 1.08 30.7 28.4 13.86 4.98 16:20 346 20.0 202 5522 6040 172 1.09 30.3 27.7 13.76 5.12 16:22 345 19.9 208 5715 6277 178 1.10 30.4 27.7 13.77 5.13 Fase recuperación:Almacenar ECG 13 16:25 345 19.9 206 5395 5899 175 1.09 31.6 28.9 14.03 4.83 16:30 281 16.2 203 5389 5974 170 1.11 30.7 27.7 13.87 5.04 16:35 230 13.3 208 5491 6102 167 1.11 29.8 26.8 13.70 5.21 16:40 192 11.1 206 5082 5644 149 1.11 28.7 25.8 13.47 5.44 16:45 115 7.8 206 5347 5800 158 1.08 28.9 26.6 13.55 5.32 16:50 83 6.0 200 4053 4533 114 1.12 27.6 24.7 13.31 5.58 16:55 84 6.0 197 4992 5451 138 1.09 27.0 24.7 12.96 5.87 17:00 84 6.0 194 4646 5044 128 1.09 26.9 24.8 13.15 5.67

11/04/2013 12:45151

Time min

Load W

Speed km/h

HR 1/min

V'O2 ml/min

V'CO2 ml/min

V'E L/min


PETCO2 kPa

17:05 84 6.0 189 4707 5189 136 1.10 28.3 25.7 13.34 5.55 17:10 84 6.0 189 4107 4569 107 1.11 25.6 23.0 12.80 6.12 17:15 83 6.0 182 4184 4753 119 1.14 27.8 24.4 13.30 5.68 17:20 83 6.0 178 4290 4939 132 1.15 30.0 26.1 13.57 5.47 Almacenar ECG 14 17:25 84 6.0 177 2619 3171 86 1.21 32.0 26.4 14.04 5.07 17:30 84 6.0 171 2725 3354 112 1.23 40.0 32.5 14.65 4.47 17:35 84 6.0 160 2241 2757 86 1.23 37.4 30.4 14.34 4.83 17:40 84 6.0 146 2492 2962 96 1.19 37.2 31.3 14.26 4.82 17:45 84 6.0 138 2386 2793 85 1.17 34.6 29.6 13.96 5.07 17:50 84 6.0 128 1676 1956 60 1.17 34.3 29.4 13.84 5.25 17:55 84 6.0 128 337 399 10 1.19 21.1 17.8 13.53 5.47 18:10 84 6.0 134 0 0 0 0.00 0.0 0.0 18.38 - 18:25 84 6.0 142 0 0 0 0.00 0.0 0.0 18.38 - Almacenar ECG 15 18:40 56 5.0 138 0 0 0 0.00 0.0 0.0 18.38 - 18:55 56 5.0 138 0 0 0 0.00 0.0 0.0 18.38 - 19:15 57 5.1 135 0 0 0 0.00 0.0 0.0 18.38 - Almacenar ECG 16 19:30 57 5.1 131 0 0 0 0.00 0.0 0.0 18.46 - 19:45 57 5.1 128 0 0 0 0.00 0.0 0.0 18.46 - 20:00 57 5.1 127 0 0 0 0.00 0.0 0.0 18.46 - 20:20 57 5.1 126 0 0 0 0.00 0.0 0.0 18.46 - Almacenar ECG 17 20:35 3 1.5 122 0 0 0 0.00 0.0 0.0 18.46 - 20:50 0 0.0 120 0 0 0 0.00 0.0 0.0 18.46 - 21:05 0 0.0 122 0 0 0 0.00 0.0 0.0 18.46 - 21:25 0 0.0 125 0 0 0 0.00 0.0 0.0 18.46 -

11/04/2013 12:45152

Valores de Espirometría en reposo

Informe ECG y Espirometría Basal

Identificación: S26Nombre:Sexo:Peso:Doctor:

male76,6 kg

Apellidos:F. Nacimiento: Altura: 173,6 cm Edad:Operador:

Teor Med1 %M1/T[L]FVC 4.88 6.05 124.0[L]FEV 1 4.10 4.82 117.4[%]FEV 1 % VC MAX 81.81 79.66 97.4

[L/s]MMEF 75/25 4.78 4.51 94.4[L/min]MVV 144.72 154.11 106.5[L/min]FEV 1*30 144.72 144.60 99.9

[L]ERV 1.54[L]VT 0.55 1.16 211.5

LABORATORIO DE FISIOLOGÍA DEL ESFUERZO

Facultad de Ciencias de la Actividad Física y del Deporte 01/04/2013 15:18

Prueba nº. 1 01/04/2013 15:17:51Electrocardiograma normal, sin ninguna alteración evidente y compatible con la normalidad.Frecuencia cardíaca en reposo de 69 latidos/min.

Comentarios

Facultad de Ciencias de la Actividad Física y delDeporte-INEF Madrid

UNIVERSIDAD POLITÉCNICA DE MADRID

1 2 3 4 5 6 7 8

Vol [L]

10

5

0

5

10

Flow [L/s]

F/V in

F/V es

1

153

Propietario

Textbox

A continuación se incluyen los registros de ECG recogidos en reposo: en decúbito supino, en bipedestación y en bipedestación tras hiperventilar.

Ritmo ECG basal 25 mm/s 10.0 mm/mV MF Todos los segmentos almacenadosTime - Load - HR 69PR 167QT 383QTc B 411

I

II

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ACKNOWLEDGMENTS

Han sido 5 años de mucho trabajo, esfuerzo y dedicación, 5 años llenos de momentos

intensos, de subidas y bajadas, de épocas maravillosas y de otras no tan buenas. Pero

miro atrás, veo todo lo vivido y me doy cuenta de que la “Tesis” ha significado un

crecimiento y conocimiento personal mucho más grande de lo que había imaginado.

Llegados al final de esta etapa me gustaría agradecer a todas las personas que de una

manera u otra me han acompañado en este duro camino y de antemano pido disculpas si

me olvido de alguien.

En primer lugar quiero dar mi más sincero agradecimiento a la persona que ha hecho

posible que empezara mi carrera en el mundo de la investigación y que haya llegado

hasta aquí, mi directora de tesis, Marcela González Gross, gracias por abrirme las

puertas, por enseñarme tanto y por tu incondicional apoyo a lo largo de todos estos

años, por haber confiado siempre en mi y por tu constante dedicación y disposición para

mi desarrollo profesional.

A mi director de Tesis, Pedro J Benito, gracias por toda tu ayuda, por tus consejos, por

estar ahí siempre que lo he necesitado y por darme un empujón cuando no tenía fuerzas.

Marcela, Pedro, ha sido un placer y estoy profundamente agradecida de haber tenido la

suerte de contar con vosotros como directores de tesis y de recorrer este camino con

vosotros. Gracias.

A mi familia, gracias a todos por haberme apoyado, aguantado, y acompañado todos

estos años. Por haber vivido conmigo todas las alegrías y sufimientos como si fueran

vuestros, por ser una familia unida a pesar de estar a veces lejos, y por haberme hecho

tan fácil siempre compaginar la vida familiar con mi carrera. A mi padre, por ser la

persona más influyente en mi vida, por estar ahí siempre, por apoyarme en mi carera

profesional como si fuera tuya, por todo tu apoyo emocional. Sin ti nada de esto hubiera

sido posible. GRACIAS PAPA. A mi madre, por todo tu cariño, por tu apoyo, por tu

increíble modo de ver la vida, hacerme ver que las cosas son mucho más fáciles y por

estar a mi lado siempre. A mi hermana Cristina, mi gemela, mi mitad… gracias por

hacerme sentir acompañada durante todos estos años incluso desde la otra punta del

mundo, gracias por tu apoyo y comprensión aun sin saber lo que estoy haciendo, ni los

motivos por los que no tengo todo el tiempo que me gustaría para hablar de vez en

cuando un rato largo por skipe. Gracias por hacerme comprender que nuestra unión está

por encima de la distancia y el tiempo. Te quiero. A mi hermana Marta, por darme

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ánimo y apoyo durante todos estos años, por comprenderme y escucharme. Por haberme

dedicado tiempo siempre que lo he necesitado, por tus consejos y porque siempre me

has demostrado que estés donde estés y pase lo que pase estás ahí siempre que lo

necesite.

A mi abuela, por ser una de las personas que más admiro, por tu fortaleza, porque con

95 años no dejas de sorprenderme. Por todo el apoyo que me has dado durante todos

estos años, por todo lo que me ayudaste durante mi estancia en Colorado. Por hacerme

saber que no tengo que preocuparme por nada y que siempre estás ahí para que pueda

seguir tranquila con mi carrera profesional. Gracias Abuela.

A Jorge, porque apareciste en mi vida a mitad de este duro camino y desde entonces

todo se ha vuelto más fácil. Gracias por darme fuerzas para seguir cuando pensaba que

no me quedaban. Por ayudarme a levantarme y a mantener siempre el equilibrio. Por

hacerme feliz y por darme paz. Por ser la persona más atenta que he conocido y tener la

suerte de tenerte a mi lado, por hacerme cada día la vida más fácil hasta en las cosas

más insignificantes para que no tenga que preocuparme nada más que de mi carrera

profesional. Por tu infinita comprensión, gracias gracias gracias por ser mi compañero

de vida. Te amo.

A mi mejor amiga María, porque desde que nos conocimos no nos hemos separado. Por

todos los momentos que me has dado, por nuestras risas. Por formar parte de mi vida y

por hacerme sentir durante estos años que daba igual la hora y el lugar, siempre era

buen momento para vernos. A Vir, por tantos momentos y por tus visitas al INEF a la

hora de comer para poder vernos un rato. A mis queridas amigas, en primer lugar a

Giorgia, por tu manera emocionante de ver la vida y tu constante cariño; a Irene, porque

nos encontramos en uno de los momentos más importantes de mi Tesis y no has dejado

de estar ahí. A Vera e Itziar, por la música que nos une, por vuestras sonrisas, por que

os admiro a cada una por su forma de ser. Porque quiero teneros cerca, agradezco haber

tenido la suerte de conoceros y pasar tan buenos momentos estos últimos años.

A Jara, a mi querida compañera de investigación, a quien he tenido la suerte de conocer

mejor cada año. Te admiro en lo personal y lo profesional, porque eres una persona

llena de positivismo y sinceridad. Gracias por haberme ayudado y enseñado tanto

durante estos años y que gracias a la investigación hayas acabado siendo una gran

amiga. Gracias de corazón a Olga, quien ha estado mano a mano conmigo durante la

elaboración de todo este proyecto y sin su ayuda y colaboración, este trabajo no hubiera

sido posible.

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Gracias a todos mis compañeros de trabajo del grupo ImFine, que tanto me han

ayudado, con los que he trabajado durante todos estos años y han sido una parte

imprescindible para que esta tesis fuera posible, a Raquel Pedrero, Raquel Aparicio,

Jorge Marín, Raquel Luzardo, David Cañada, Juan Mielgo, Sergio Calonge, Gonzalo

Palacios, Rosa Mª Torres, Alejandro Urzanqui, Juan José Gómez, Ulrike Albers,

Francisco Fuentes, Juan Carlos Ortiz, Agustín Meléndez, Vicente Ortega, Mª Jesus

Morón, Fernando Novella, Javier Jimenez y a aquellos que han trabajado con el grupo

durante estos años, a Rebecca Scherer, Claudia Rumi y Marta García.

A Enrique Díaz, por su colaboración en el proyecto y su ayuda en el análisis de las

muestras en el laboratorio. A Ciriaco Carru por su colaboración en el análisis de

laboratorio. A Teresa Amigo y Domingo Gonzalez Lamuño por su colaboración en el

análisis genético y toda la ayuda que me han prestado.

A Laura Barrios del Consejo Superior de Investigaciones Científicas (CSIC) por su

asesoramiento en el tratamiento estadístico de los datos; a Paloma Navarro, por las

gestiones administrativas necesarias para la realización de esta tesis.

A todos los compañeros del laboratorio de fisiología del esfuerzo, Miguel, Blanca,

Esther y en especial a Rocío, por ayudarme tanto en mir primeros pasos. A mis amigos

y compañeros de doctorado y del INEF que han hecho que estos años se llenen de

buenos momentos y risas, Peter, Isma, Miguel, Sergio, Yaiza, y en especial a Jabo y

Cesar, quienes han sido un apoyo constante durante estos años y han sido “mi familia”.

Gracias por estar ahí siempre, por los buenos momentos que hemos pasado juntos y por

haberme cuidado tanto. No tengo suficientes palabras de agradecimiento para vosotros.

A Mercedes Galindo, a Javier Rojo y a Javier Calderón por su profesionalidad y

participación en el estudio como médicos supervisores durante las pruebas de esfuerzo.

A mi director de la estancia, el profesor James O Hill, por haberme dado la posibilidad

de pasar 3 meses en el Health and Wellness center de la Universidad de Colorado, de

rodearme de profesionales y de haber aprendido tanto allí. A mis amigos de Colorado

Audrey M, Audrey y Nico, quienes fueron mis compañeros durante mi estancia en

Colorado y me abrieron sus brazos como una más. Merci.

Quiero agradecer especialmente al profesor, Josep A. Tur, y a todos los miembros del

grupo de investigación NUCOX de la Universidad de Illes Baleares que han estado

involucrados en el proyecto de investigación de hidratación en personas mayores, y sin

los cuales no hubiera sido posible el desarrollo del trabajo de la Beca de Hidratación del

EHI.

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Gracias en especial al Dr. Rafael Urrialde, por su apoyo en la difusión de los resultados

de esta tesis.

Gracias al Departamento de Salud y Rendimiento Humano de la Facultad de Ciencias

de la Actividad Física y del Deporte de la Universidad Politécnica de Madrid por

haberme dado la oportunidad de realizar la tesis doctoral.

Y finalmente quiero agradecer a cada uno de los participantes que se presentaron

voluntarios para la realización de este proyecto, por su entera disposición y por su

colaboración. Gracias, porque si ellos este trabajo no hubiera sido posible.

Sin más, daros las gracias a todas las personas que habéis formado parte de mi vida

durante estos últimos años. A las que siguen y con las que he perdido el contacto,

Gracias a todas las personas que habéis confiado en mí y que me habéis dado fuerzas.

Gracias a todos y cada uno de vosotros, sin los que hubiera sido imposible llegar a este

momento, al final de esta etapa de profundo aprendizaje en todos los sentidos. Espero

seguir contando con vosotros para siempre.

GRACIAS.

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SUMMARIZED CV/CURRÍCULUM VITAE ABREVIADO

Academic education and postgraduate training

• M.Sc in Physical Activity and Sport Sciences. University of Alcalá de Henares. Madrid. 2004-2009.

• Master Degree in Physical Activity and Sport Sciences. Technical University of Madrid. 2009-2010.

• Master Thesis: Effects of physical exercise on plasma homocysteine levels in young trainedmales.

• B. Sc Physical Education for Primary School. Camilo José Cela University. 2009-2010. Madrid.• Collegiate number 54 481 by the Illustrious Association of Graduates in Sport and

Physical.Activity Sciences of the Community of Madrid (COPLEF).• Member of the Spanish Nutrition Society (SEN).• Researcher member of consolidated Research Group: ImFINE. Improvement (of health) by

fitness, nutrition and exercise. Faculty of Physical Activity and Sport Sciences (INEF).Technical University of Madrid Spain. Date: 2010-Ongoing.

Stays abroad • Internship at the Health and Wellness Center at the University of Colorado, Denver, Colorado.

U.S.A, June-September 2012, duration: 3 months.

Other achievements • Member of the recognized Research Group of Technical University of Madrid: Improvement (of

health) by fitness, nutrition and exercise. (ImFINE research group).• European Hydration Institute scholarship for the project: “Fluid intake in elderly. Differences in

hydration habits between an active and a non-active Spanish population. 2013.• Competitive grant from the Social Council of the UPM for stays abroad. 2011. Stay of three

months in Colorado University at the research center Health and wellness Center, Denver,Colorado. USA.

• Grant to participate at the 10th Annual Obesity Summer Boot Camp for experts and newproffesionals in obesity research. Alberta, Canadá, July 18th to July 26th (2015).

Research projects • Fluid intake in elderly. Differences in hydration habits between an active and a non-active

Spanish population. (E131115081). Funding organization: European Hydration Institute.Duration: 2013-2014.

• ACTIVEAGE – Capacity Building for Physical Activity Programs for Aging People.(EAC/S06/2012). Funding Organization: European Comission/DG Education and Culture.Duration: 2012-2015.

• Determinantes de riesgo de primeros eventos cardiovasculares. Un estudio coordinado de casos ycontroles anidado de la cohorte PREDIMED. Antioxidantes y estrés oxidativo (PI11/01791).Funding Organization Instituto de Salud Carlos III, Ministerio de Sanidad. Duration: 2012-2014.

• Study of influence of rehydration on homocysteine levels after exercise. Funding: Own funds ofthe ImFine Reseacrh Group. Duration: 2010-ongoing.

• EXERNET Longitudinal Study: Influence of Lifestyle, in deteriorating physical condition, bodycomposition and quality of life in people over 65 do not institutionalized. (147/11). Ministryof Health, Social Affairs and Equal-Institute of Aging and Social Services. 2012-2014.

• HELLP. Health as a lifelong learning process (III) (DE-2010-ERA-MOBIP-ZuV-29975-1-28).Funding Organization: Erasmus Program. European Community. Duration: 2010-2011.

• Mission X Train like an astronaut. (SE10111501). Funding Organization: NASA, (NationalAeronautic and Space Administration), ESA (European Space Agency). Duration 2010-Ongoing

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Teaching and invited lectures • Teaching support: Nutrition and dietetics, ETSI Agronomists. Technical University of Madrid.

2012-2013.• Oficial teaching: Summer Course teacher at the Technical University of Madrid. "Physical

activity and healthy lifestyle." July 2011.• Invited lecture: Hydration and Exercise effects on homocysteine levels. Faculty of Physical

Activity and Sport Sciences. Technical University of Madrid. October 2011.

Scientific Publications • Mielgo-Ayuso J, Maroto-Sánchez B, Luzardo-Socorro R, Palacios G, Palacios Gil-Antuñano N,

González-Gross M; EXERNET Study Group. Evaluation of nutritional status and energyexpenditure in athletes. Nutr Hosp. 2015 Feb 26;31 Suppl 3:227-36. doi:10.3305/nh.2015.31.sup3.8770. (JCR: 1.04)

• Maroto-Sánchez Beatriz, Lopez-Torres Olga, Palacios Gonzalo, González-Gross Marcela.What do we know about Homocysteine and exercise? A review from the literature. CCLM (InPress). (JCR: 2.70)

• Maroto-Sánchez Beatriz, Lopez-Torres Olga, Valtueña Jara, Benito Pedro J, Palacios Gonzalo,Díaz Martínez Ángel Enrique, González-Lamuño Domingo, Carru Ciriaco, Zinellu Angelo,González-Gross Marcela. Hydration during exercise prevents the increase of homocysteineconcentrations. JPAH. (Submitted) (JCR: 2.09)

• Palacios G, Pedrero-Chamizo R, Palacios N, Maroto-Sánchez B, Aznar S, González-Gross M.Biomarkers of physical activity and exercise. Nutr Hosp. 2015 Feb 26;31(s03):237-244.

• Maroto-Sánchez B, Valtueña J, Albers U, Benito PJ, González-Gross M. Acute physicalexercise increases homocysteine concentrations in young trained male subjects. Nutr Hosp. 2013Mar-Apr;28(2):325-32. doi: 10.3305/nh.2013.28.2.6300. (JCR: 1.04)

• Maroto-Sánchez Beatriz, Lopez-Torres Olga, Valtueña Jara, Benito Pedro J, Palacios Gonzalo,Díaz Martínez Ángel Enrique, González-Lamuño Domingo, Carru Ciriaco, Zinellu Angelo,González-Gross Marcela. Hydration effect on increased homocysteine concentrations afterexercise. (Submitted)

Other scientific publications and abstracts • Association between homocysteine, folate and vitamin B12 with fitness in people over 55 years.

Aparicio-Ugarriza R, J Mielgo-Ayuso, Luzardo-Socorro R, Maroto-Sánchez B, Palacios G, DryR, Argelich E, M Bibiloni, Tur P, González-Gross M. III Congress of the Spanish Federation ofNutrition Food and Dietetics Societies. 2015.

• Exercise and hydration effect on homocysteine Concentrations, cardiovascular adjustment andgenotype influence. Maroto-Sanchez, B. 3rd Meeting for Young Researchers - SpanishNutrition Society (SEN). SEVILLE. Spain. 2015.

• Assessment of muscle strength in relation to consumption of drinks in Spanish elderly Luzardo-Socorro R, Aparicio-Ugarriza R, Maroto-Sánchez B, Marín-Puyalto J, Palacios G, González-Gross M. Libro de abstracts VII Simposio Internacional de actualizaciones en entrenamiento dela fuerza, 2014: 82. ISSN 978-84-697-1880-3.

• Fluid intake, biomarkers and body composition differences between physically active and non-active elderly people. Maroto-Sánchez B, Luzardo-Socorro R, Aparicio-Ugarriza R, Palacios G,Diaz AE, González-Gross M. International Journal of Community Nutrition 2014, 0 (suppl);2014; 123. ISSN 2386-673X.

• Adequacy of muscular mass estimations provided by bioimpedance analysis for the assessmentof body composition in subjects over 55. Aparicio-Ugarriza R, Marín-Puyalto J, Luzardo-Socorro R, Maroto-Sánchez B, Tur JA, Palacios G. International Journal of CommunityNutrition 2014, 0 (suppl); 2014; 122. ISSN 2386-673X.

• Comparison between 2-compartment and 3-compartment bioimpedance analysis estimations ofbody composition in a population over 55. Marín-Puyalto J, Aparicio-Ugarriza R, Luzardo-Socorro R, Maroto-Sánchez B, Palacios G, Tur JA, González-Gross M. Arch Med Deporte2014; 31 (3):170-199.

• Fluid intake in elderly people. Differences between active and non-active elderly people.Maroto-Sánchez B, Luzardo-Socorro R, Parylack SJ, Aparicio-Ugarriza R, Marín-Puyalto J,Palacios G, Tur JA, González-Gross M. Nutr Hosp. 2014; (Supl.1) 30:1-64.

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• Daily beverages consumed by Spanish elderly. Maroto-Sánchez B, Scherer R, López-Torres O,Luzardo R, Tur JA, Palacios G, González-Gross M. Nutr Hosp. 2013;(Supl. 6)28:31-32, ISSN(Versión papel): 0212-1611 • ISSN (Versión electrónica): 1699-5198.

• Water intake decreases with age in Spanish elderly. Scherer R, Maroto-Sánchez B, López-Torres O, Luzardo R, Tur JA, Palacios G, González-Gross M. Nutr Hosp. 2013;(Supl. 6)28:62-63, ISSN (Versión papel): 0212-1611 • ISSN (Versión electrónica): 1699-5198.

• Hydration and non-hydration during exercise: effects on homocysteine concentrations andrelated parameters. Maroto-Sánchez B, López-Torres O, Diaz AE, Carru C, Benito PJ,González-Gross M. Ann Nutr Metab 2013;63(suppl 1): 463. ISSN:0250-6807

• Evolution of physical fitness in a 4 year-period in non-institutionalized elderly: Madrid-EXERNET longitudinal study. O.López-Torres, R. Pedrero-Chamizo, G. Palacios, B. Maroto,D. Cañada, A. Melendez, M. Gonzalez-Gross. Ann Nutr Metab 2013; 63(suppl 1):472-473.ISSN:0250-6807

• Niveles de fuerza prensil en población mayor de vida independiente: Pedrero-Chamizo, R;López-Torres O, Maroto B, Palacios G, Meléndez A, González-Gross M. Madrid-EXERNETestudio multicéntrico. 6º Simposio de actualización en entrenamiento de la fuerza. 2013. ISBN.978-84-695-9116-1. pp. 67-68.

• Recovery post-effort up to 24 hours of homocysteine concentrations and related parameters byrehydration controlled. Maroto B, Lopez-Torres O, Palacios G, Zinellu A, Benito PJ, González-Gross M. Archivos de Medicina del Deporte. 2012. 5, 151. ISSN: 0212-8799.

• Relationship of creatine and creatinine with increased tHcy after intense exercise. Maroto-Sánchez B, López-Torres O, Diaz AE, Carru C, Benito PJ, González-Gross M. III InternacionalSimposium of exercise and health in special populations. EXERNET and II INEF PostgraduateConvention. Abstract Book. 2012. ISBN: 978-84-96398-68-9.

• Exercise and hydration: effects on homocysteine levels. Beatriz Maroto, Jara valtueña, Pedro J.Benito, Agustín Meléndez, Marcela González-Gross. ImFine Research Group. II NationalHydration Congress. Revista Española de Nutrición Comunitaria. 2012; 18 (1): 34. ISSN:1135-3074.

• Influence of exercise on plasma homocysteine levels. Maroto-Sánchez B, García-González C,Pedrero R, Benito PJ, Meléndez A, González-Gross M. ImFINE research group. EuropeanCollege of Sport Science: Book of Abstracts of the 16th Annual Congress of the EuropeanCollege of Sport Science.United Kingdom. Edited by N. Tim Cable, Keith Georg. ISBN 978-09568903-0-6. 391.

• Influence of rehydration on Plasma Homocisteine Levels after exercise. Maroto-Sánchez B,García-González C, Pedrero R, Benito PJ, Meléndez A, González-Gross M. ImFINE researchgroup. European College of Sport Science: Book of Abstracts of the 16th Annual Congress ofthe European College of Sport Science.United Kingdom. Edited by N. Tim Cable, Keith Georg.ISBN 978-09568903-0-6. 391.

• Eating disorders, detection and prevention of bulimia in school. Maroto-Sánchez, B. Facets ofHealth Literacy. Educational Guidelines for Schools, Universitties, Teacher Training Collegesand Sport. Ed. Konrad Kleiner. March, 2011.114-115. ISBN: 978-3-85199-326-1.

• Influence of a maximal and submaximal test on plasma homocysteine levels. García-GonzálezC, Maroto-Sánchez B, Pedrero-Chamizo R, Meléndez A, Benito PJ, González-Gross M.Archivos de Medicina del Deporte. 2010; 5,139. ISSN:0212-8799.

• Influence of hydration on plasma homocysteine levels after physical exercise. Maroto-SánchezB, García-González C, Pedrero-Chamizo R, Meléndez A, Benito PJ, González-Gross M.Archivos de Medicina del Deporte. 2010; 5,139.ISSN:0212-8799.

Book Chapters • Specific nutritional requirements in some sports. Food and Nutrition in working life: Exercise

and sports. López-Torres O, Maroto-Sánchez B, González-Gross M. En: Alimentación yNutrición en la vida activa: ejercicio físico y deporte. Calvo C, Gómez-Candela C, Benito PJ,Iglesias C. UNED. 2013. ISBN 978-84-362-6706-8.

• Exercise and cognitive function. Gonzalez-Gross. M, Valtueña. J, Fuentes. F, Maroto B.Editores: Casajús, JA, Vicente Rodriguez G, Physical Activity and Health in SpecialPopulations. EXERNET. Editorial CSD. ICD Collection. 2011. P 413-430. ISBN 978-84-7949-216-8.

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Participation in conferences, courses and seminars: • 3rd Meeting for Young Researchers - Spanish Nutrition Society (SEN). SEVILLE. Spain. 2015.

Scientific communication: Exercise and hydration effect on homocysteine Concentrations,cardiovascular adjustment and genotype influence. Maroto-Sanchez, B.

• III Congress of the Spanish Federation of Nutrition Food and Dietetics Societies. 2015. Scientificposter presentation: Association between homocysteine, folate and vitamin B12 with fitness inpeople over 55 years. Aparicio-Ugarriza R, J Mielgo-Ayuso, Luzardo-Socorro R, Maroto-Sánchez B, Palacios G, Dry R, Argelich E, M Bibiloni, Tur P, González-Gross M.

• VII International Symposium updates in Strength Training. VII International Symposium inStrength Training. Technical University of Madrid. Spain December 2014. Oral communication:Rating muscle strength in relation to consumption of drinks in Spanish elderly. Luzardo-SocorroR, Aparicio-Ugarriza R, Sánchez Maroto-B, Marin-Puyalto J, Palacios G, González-Gross M.

• III World Congress of Public Health Nutrition. Las Palmas de Gran Canaria, Spain. November2014. Scientific poster presentation: Fluid intake, body composition and biomarkers Differencesbetween physically active and non-active elderly people. Maroto-Sánchez B, Luzardo-SocorroR, Aparicio-Ugarriza R, Palacios G, Diaz AE, M. González-Gross; Adequacy of muscle massestimations provided by bioimpedance analysis for the assessment of body composition insubjects over 55. Aparicio-Ugarriza R, Marin-Puyalto J, Luzardo-Socorro R, Maroto- SánchezB, Tur JA, G. Palacios.

• World Conference on Kinanthropometry. Murcia, Spain. July 2014. Oral communication:Comparison between 2-compartment and three-compartment bioimpedance analysis estimationsof body composition in a population over 55. Marin-Puyalto J, Aparicio-Ugarriza R, R-SocorroLuzardo, Maroto-Sánchez B, Palacios G, Tur JA, González-Gross M.

• XVI Meeting of the Spanish Society of Nutrition. Days octaves UNAV update. Pamplona, Spain.2014. Scientific poster presentation: Fluid intake in older people. Differences between physicallyactive and inactive people. Maroto-Sánchez B, R-Socorro Luzardo, Parylack SJ, Aparicio-Ugarriza R, Marin-Puyalto J, Palacios G, Tur JA, González-Gross M.

• IV International Symposium Updates in Strength Training. Technical University of Madrid.Madrid, Spain. 2013. Scientific poster presentation: Strenght levels in a population ofindependent living: Madrid-EXERNET multicenter study. Pederoro-Chamizo, R; Lopez-Torres,O; Maroto, B; Palacios, G; Melendez, A; González-Gross, M.

• III National Hydration Congress and I International Hydration Congress. Madrid, Spain.December 2013. Scientific poster presentation: Daily beverages consumed by Spanish elderly.Maroto-Sánchez B, Scherer R, Lopez-Torres O, R Luzardo, Tur JA, Palacios G, González-Gross M. Department of Health and Human Performance. Faculty of Physical Activity and SportSciences (INEF). Polytechnic University of Madrid. Spain; Water intake decreases With Age inSpanish elderly. Scherer R, B Maroto-Sanchez, Lopez-Torres O, R Luzardo, Tur JA, PalaciosG, González-Gross M. ImFINE Research Group. Department of Health and HumanPerformance. Faculty of Physical Activity and Sport Sciences (INEF). Polytechnic University ofMadrid. Spain.

• 20TH International Congress of Nutrition. Granada, Spain. November 2013. Scientific posterpresentation: Hydration and non-hydration during exercise: Effects on homocysteineconcentrations and related parameters. Maroto- Sánchez B, Lopez-Torres O; Diaz AE, Carru C,PJ Benito Gonzalez-Gross M1. ImFINE Research Group. Faculty of Physical Activity and SportINEF. Polytechnic University of Madrid.

• 1st meeting for Young Researchers. Spanish Nutrition Society (SEN). CSIC. Madrid. Spain.February 7, 2013. Scientific communication: Effect of physical activity and hydration onhomocysteine levels and related parameters. B. Maroto-Sánchez ImFINE Research Group.Faculty of Physical Activity and Sport INEF. Polytechnic University of Madrid.

• XIV National Congress of Sports Medicine of the Spanish Federation of Sports MedicineSantander, November 2012. Scientific Communication: Recovery post-effort up to 24 hours ofhomocysteine concentrations and related parameters by rehydration controlled. Maroto B,Lopez-Torres O, Palacios G, Zinellu A, Benito PJ, González-Gross M. ImFine Research Group.

• III Internacional Simposium of exercise and health in special populations. EXERNET and IIINEF Postgraduate Convention. Madrid, October 2012. Scientific Poster Presentation:Relationship of creatine and creatinine with increased tHcy after intense exercise. Maroto B,López-Torres O, Diaz AE, Carru C, Benito PJ, González-Gross M.

• II National Hydration Congress. Madrid, Spain. November 2011. Scientific Communications:Exercise and hydration: effects on homocysteine levels. Beatriz Maroto, Jara Valtueña, Pedro J.

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Benito, Agustín Meléndez, Marcela González-Gross. ImFine Research Group. (Selected among the top 5 communications).

• 16th ECSS Congress. 2011. European College of Sport Science. Liverpool, UK. July 2011.Scientific Communications: Influence of rehydration on Plasma Homocysteine Levels afterexercise. Maroto B, García-González C, Pedrero R, Benito PJ, Meléndez A, González-Gross M.Oral presentation: Influence of Exercise on Plasma Homocysteine Levels. Maroto B, García-González C, Pedrero-Chamizo R, Benito PJ, Meléndez A, González-Gross M. ImFINE researchgroup. Poster Presentation.

• International HELLP- Symposium: Facets of Health Literacy: Educational Guidelines forSchools, Universities, Teacher Training Colleges and Sport. Centre of Sport Science andUniversity Sports. University of Vienna. Vienna, Austria. March 2011. ScientificCommunication: Eating disorders, detection and prevention of bulimia in school. Maroto B.

• IV International Congress of Sports and Physical Activity for Elderly. (Universidad de Málaga,Junta de Andalucía, Ayuntamiento de Málaga, Diputación de Málaga y el Consejo Superior deDeportes). Málaga, March 2011.

• 13 th National Congress of Sports Medicine of the Spanish Federation of Sports Medicine-1International Congress of Basque Society of Sports Medicine. Bilbao, October 2010. FEMEDE.Scientific Communications: Influence of maximal and submaximal tests on PlasmaHomocysteine levels. Maroto B, García-González C, Pedrero R, Benito PJ, Meléndez A,González-Gross M.; Influence of rehydration on Plasma Homocisteine Levels after exercise.Maroto B, García-González C, Pedrero-Chamizo R, Meléndez A, Benito PJ, González-Gross M.

Organization of scientific events • CONEFTADOS: 1st State meeting of exchange of experiences in promoting physical activity

and health at school. May 2015. Madrid, Spain. Technical Secretariat of the Organization.• III International Symposium of exercise and health in special populations. EXERNET and II

INEF Postgraduate Convention. October 2012. Madrid, Spain. Member of the OrganizingCommittee.

• International Congress PRONAF (for overweight and Obesity treatment: Nutrition and PhysicalActivity Programs). December 2011. Madrid, Spain. Member of the Organizing Committee.

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