Clinical Biochemistry 46 (2013) 1447–1452

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Clinical Biochemistry

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Obesity and diabetes related plasma amino acid alterations

Yong Zhou a,1, Ling Qiu b,1, Qian Xiao a, Yi Wang a, Xiangying Meng a, Rong Xu b, Siyang Wang b, Risu Na b,⁎a Department of Endocrinology, Dahua Hospital, No.901 Old Hu Min Road, Shanghai 200237, Chinab Department of Endocrinology, Xuhui District Central Hospital, No.966 Huaihai Road, Shanghai 200031, China

⁎ Corresponding author at: Department of Endocrinolopital, No.966 Huaihai Road, Shanghai 200031, China. Fax:

E-mail addresses: shzhouyy@163.com (Y. Zhou), qiulxiao0813@hotmail.com (Q. Xiao),wangyi9093@126.com ((X. Meng), xx.rong@hotmail.com (R. Xu), misswsy@hotmnarisuBM@163.com (R. Na).

1 These authors contributed equally to this work.

0009-9120/$ – see front matter © 2013 The Canadian Shttp://dx.doi.org/10.1016/j.clinbiochem.2013.05.045

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 13 March 2013Received in revised form 4 May 2013Accepted 6 May 2013Available online 19 May 2013

Keywords:Amino acid concentration profilesDiabetesInsulin resistanceObesity

Objectives: The objective of the study is to evaluate whether plasma amino acid (AA) differences are re-lated with obesity or diabetes.

Design and methods: In 126 diabetes and 100 non-diabetes participants, the plasma concentrations of 42(AAs) were analyzed with a liquid chromatography tandem mass spectrometry technology (LC–MS/MS). Bothgroups were divided into obese and lean individuals and we compared intra- and inter-group differences be-tween the groups.

Results: In obese non-diabetic participants, 19 AA plasma concentrations were different compared tolean non-diabetic individuals, from which 15 were essential AAs, whereas in the diabetic group onlythree AAs differed in the obese compared to the lean patients. When comparing the overall AA differencesbetween diabetics and non-diabetics, 16 AA concentrations were enhanced and 11 AA concentrations were

reduced in the diabetic patients. A multivariate linear regression analysis revealed correlations between:FBG and Cystathionine, Proline and Citrulline; HbA1c and Glycine, Proline and Sarcosine; Cholesterol andSerine, β-alanine, Proline and Cystathionine; HDL-C and β-alanine, 1-methylhistidine and Proline; andLDL-C and α-Amino n-butyric acid and Hydroxyproline. Triglycerides were related with γ-aminobutyricacid, Serine and Alanine. Fasting insulin was related with 3-methylhistidine, Asparagine, Alanine,γ-aminobutyric acid and Cystathionine.

Conclusions: The concentrations of 19 plasma AAs differed between non-diabetic obese and lean individ-uals, which were mostly superimposed by diabetes. Between diabetic and non-diabetic participants plasma AAconcentration differences were obvious and some of these alterations were correlated to other factors likeblood glucose, lipids, insulin and hemoglobin status.

© 2013 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Introduction

While people in the present developed cities of China become in-creasingly less active andmore obese, the incidence of type 2 diabetesis growing at an alarming rate. Altered AA concentrations in sera aredescribed for kidney diseases and cardiovascular or stroke related pa-thologies [1–5] and prevailing theories for insulin resistance not onlyfocus on lipid mediated mechanisms but also include insulin resis-tance mediated by elevated concentrations of AAs and inflammatorysignaling molecules as well as combinations of these [6]. On theother hand since Floyd et al. started their pioneer work [7], manyprevious research reported that some AAs are promoting insulin

gy, Xuhui District Central Hos-+86 21 51980039.in52172@hotmail.com (L. Qiu),Y.Wang), lingzhi771@126.comail.com (S. Wang),

ociety of Clinical Chemists. Publishe

secretion both in vitro and in vivo [8–11]. Dunger et al. suggestedthat high serum concentrations of AAs (AA) can induce residual anddamaged B cells to secrete insulin as well as promote proliferationof islet B cell precursors in a streptozocin (STZ) diabetes animalmodel [12]. Despite many years of research, the role of obesity inthe development of these disorders and diseases is still not fully elu-cidated. However, in recent years, the application of ‘omic’ technolo-gies for comparisons of lean and obese subjects has enhanced ourunderstanding in this research area. Recently, compelling supportfor the idea, that branched chain amino acid (BCAA) diets might berelated to pathophysiologies was provided by findings of elevatedserum BCAA, several related AAs and metabolite concentrations inobese, insulin-resistant subjects compared with lean control subjects,and these concentrations were linearly related to a homeostasismodel assessment index [13]. Wang et al. demonstrated that fastingplasma concentrations of five AAs including BCAA and aromatic AAwere elevated up to 12 yr prior to the onset of diabetes in high-risksubjects [14]. This study focused on altered serum AA concentrationprofiles between obese and lean people as well as diabetes andnon-diabetes patients in order to find correlations between serumAA pattern and other metabolic factors.

d by Elsevier Inc. All rights reserved.

1448 Y. Zhou et al. / Clinical Biochemistry 46 (2013) 1447–1452

Subjects and methods

Subjects

100 non-diabetic volunteers and 126 patients with non-insulindependent diabetes mellitus (NIDDM) were selected for this study.The non-diabetic group included 80 obese subjects (mean age,33 ± 6.3 yr; BMI 27–35 kg/m2) and was compared with 20 lean sub-jects (mean age, 32.7 ± 7.3 yr; BMI, 17–21 kg/m2) [15]. In the volun-teer group, nobody had any evidence of renal, hepatic, cardiovascular,or other major organ system diseases evaluated by routine history,physical examination, and laboratory screening test. There was nofamily history of diabetes, and no subject was taking any medication,including oral contraceptives. Menstrual cycles were normal, andthere was no clinical evidence of any polycystic ovary syndrome. Nosubjects were participating in any strenuous exercise or were exces-sively sedentary. The other diabetic group of 126 patients included31 obese patients (mean age, 60.1 ± 5.9 yr; BMI, 27–35 kg/m2),which were compared with 95 lean patients (mean age, 60.5 ±5.8 yr; BMI, 17–21 kg/m2). All diabetic patients were diagnosedwith fasting blood glucose ≥7.0 mmol/L (126 mg/dL) or postprandialblood glucose ≥11.1 mmol/L (200 mg/dL). Exclusion criteria wereknown cardiovascular diseases, liver or kidney disease, diabetes withserious complications and musculoskeletal conditions prohibitingexercise training. All subjects' written informed consent was obtainedbefore their participation. This study was conducted according to theguidelines laid down in the Declaration of Helsinki and all proceduresinvolving human patients were approved by the Ethical committee ofthe Dahua Hospital and Xuhui District Central Hospital.

Methods

Fasting EDTA plasma was collected from each participant in themorning after 10 h fasting and stored at −80 °C. The fasting plasmaglucose concentration was determined by the glucose oxidase meth-od. For the determination of glucose specific activity, the plasma wasde-proteinized according to the Somogyi procedure [16]. Convention-al glucose was measured with the hexokinase method (Hitachi 911analyzer). The reagents for the Hitachi analyses were obtainedfrom Roche (Indianapolis, IN; glucose, lactate, and uric acid) andWako USA (Richmond, VA; total ketones, -hydroxybutyrate, andnon-esterified free fatty acids). The plasma insulin concentrationwas determined by radioimmunoassays (RIAs) [17]. HbA1c was mea-sured by ion-exchange high-performance liquid chromatography(HPLC; Bio-Rad, Hercules, CA, USA). Total cholesterol, high densitylipoprotein cholesterol (HDL-C) and triglyceride levels were mea-sured via enzymatic procedures using an auto analyzer (Bayer,

Table 1Demographic data and plasma substrate concentrations in the basal state of the obese and

Diabetes mellitus

Obesity(BMI 27–35 kg/m2)

Lean(BMI 17–21 kg/m2)

Total P

Age (yr) 60.1 ± 5.9 60.5 ± 5.8 60.4 ± 5.8 .721Gender

Male 15(24.6%)

46(75.4%)

61 .997

Female 16(24.6%)

49(75.4%)

65

FBG (mmol/L) 6.5 ± 1.1 5.6 ± 0.9 5.8 ± 1.1 .000PBG (mmol/L) 11.1 ± 2.3 8.0 ± 1.6 8.8 ± 2.2 .000HbA1c (mmol/L) 8.6 ± 1.3 6.9 ± 1.1 7.3 ± 1.4 .000Cholesterol (mmol/L) 6.5 ± 1.9 4.3 ± 1.0 4.9 ± 1.6 .000HDL-C (mmol/L) 0.9 ± 0.3 1.1 ± 0.3 1.1 ± 0.3 .002LDL-C (mmol/L) 2.7 ± 0.9 2.1 ± 0.5 2.3 ± 0.7 .001Triglyceride (mmol/L) 2.4 ± 1.0 1.2 ± 0.6 1.5 ± 0.9 .000Fasting insulin (mU/L) 27.2 ± 12.9 12.8 ± 5.9 16.4 ± 10.2 .000

USA). Low density lipoprotein cholesterol (LDL-C) was calculated bythe Friedewald equation [18]. 42 AAs were measured using a liquidchromatography tandem mass spectrometry technology (LC–MS/MS)as described previously [19].

Statistical analyses

Because the most distribution of amino acid was non-normal, thus,the non-parametric test was used to compare the difference of variableswith non-normal distribution, and t-test to compare the difference of var-iables with normal distribution. Simple (Pearson's) correlation coeffi-cients were calculated with standard formulas. A multivariate linearregression analysis was performed to evaluate independent associationsamong variables.

Results

Demographic data and plasma substrate concentrations in the basalstate between two groups

Body mass index and plasma glucose/insulin in the basal state ofdiabetic and non-diabetic subjects are shown in Table 1. Plasma glu-cose, FBG, PBG, HbA1c, LDL-C, HDL-C, Triglyceride and Fasting insulinconcentrations were higher in diabetic than in non-diabetic subjectsexcept Cholesterol. In the basal state of diabetic patients, the FBG,PBG, HbA1c, Cholesterol, HDL-C, LDL-C, Triglyceride and Fasting insu-lin of obese patients were significantly increased compared to thelean patients. In the healthy group, the FBG, PBG, HbA1c, Cholesterol,and HDL-C values were also higher in obese than in lean subjects ex-cept LDL-C, Triglyceride and Fasting insulin. In addition, for the dia-betic group, there was no difference in lean and obesity proportionsbetween females and males, whereas in the healthy group, 94.8%obese participants were male and 69.5% of the lean participantswere female.

Plasma AA concentration comparison of obese and lean subjects in thenon-diabetic group

In 100 non-diabetic subjects, from 42 measured AAs 19 serum con-centrations differed in obese subjects (Table 2), from which Asparticacid, Histidine, β-alanine, Alanine, Glutamic acid, 3-methyl histidine,Argininosuccinic acid,α-Amino adipic acid, Proline, Ornithine, Cysteine,Lysine, Valine, Tyrosine, Isoleucine, Leucine, Phenylalanine and Trypto-phan were increased, whereas the Glycine serum concentration waslower. Interestingly, 15 of the 19 differing AAs were essential AAs,suggesting that they are not metabolized in obese people and thereforeaccumulate in the blood.

lean diabetes mellitus and non-diabetic participants.

Non-diabetes mellitus (DM vs. non-DM)P-value

Obesity(BMI 27–35 kg/m2)

Lean(BMI 17–21 kg/m2)

Total P

33.1 ± 6.1 32.7 ± 7.3 33 ± 6.3 .808 .000.000

73(94.8%)

4(5.2%)

77 .000

7(30.4%)

16(69.6%)

23

4.6 ± 0.5 4.3 ± 0.3 4.6 ± 0.4 .004 .0006.1 ± 0.6 5.5 ± 0.4 6.0 ± 0.6 .000 .0005.7 ± 0.4 5.1 ± 0.3 5.6 ± 0.4 .000 .0005.1 ± 0.8 4.2 ± 0.7 4.9 ± 0.8 .000 1.0001.2 ± 0.3 1.5 ± 0.3 1.3 ± 0.3 .000 .0002.6 ± 0.7 2.4 ± 0.6 2.6 ± 0.7 .207 .0021.2 ± 0.6 1.0 ± 0.5 1.2 ± 0.6 .134 .0037.5 ± 3.1 6.2 ± 2.8 7.2 ± 3.0 .079 .000

Table 2Intra- and inter-comparisons of plasma AA concentrations in obese and lean subjects with and without diabetes.

Diabetes mellitus (mean ± SD) Non-diabetes mellitus (mean ± SD) (DM vs. non-DM)P-value

Obesity Lean Total P Obesity Lean Total P

Phosphoserine 0.4 ± 0.2 0.4 ± 0.3 0.4 ± 0.3 .758 0.6 ± 0.3 0.5 ± 0.2 0.6 ± 0.3 .335 .000Phosphoric acid ethanolamine 0.1 ± 0.1 0.1 ± 0.2 0.1 ± 0.2 .598 2.6 ± 2.7 2.1 ± 2.1 2.5 ± 2.6 .464 .000Taurine 119.5 ± 47.2 117.2 ± 53.2 117.8 ± 51.6 .834 136.1 ± 79.7 145.4 ± 54.1 137.9 ± 75.1 .622 .019Asparagine 93.7 ± 16.3 96.6 ± 23.3 95.9 ± 21.8 .517 92.3 ± 16.8 90.3 ± 19.4 91.9 ± 17.2 .652 .124Serine 172.6 ± 37.7 182.8 ± 50.9 180.3 ± 48.1 .303 200.7 ± 50.9 215.5 ± 27 203.7 ± 47.3 .213 .000Hydroxyproline 23.6 ± 9.5 22.4 ± 12.1 22.7 ± 11.5 .609 20.4 ± 8.4 18.4 ± 7.4 20 ± 8.2 .340 .049Glycine 325.4 ± 98.1 343.1 ± 115.7 338.8 ± 111.5 .445 328.5 ± 48.6 384.2 ± 82 339.6 ± 60.6 .008 .940Glutamine 991.1 ± 225.8 991.5 ± 254.4 991.4 ± 246.8 .994 858.3 ± 97.4 830.1 ± 165.8 852.7 ± 113.9 .473 .000Aspartic acid 20.3 ± 13.5 22.7 ± 18.1 22.1 ± 17.1 .490 29.1 ± 8.8 23.4 ± 5.4 28 ± 8.5 .007 .001Ethanolamine 181.8 ± 13.4 182.4 ± 16.4 182.2 ± 15.7 .860 178.2 ± 9.6 176.4 ± 8 177.8 ± 9.3 .425 .009Histidinea 98.6 ± 17.5 98.6 ± 19.4 98.6 ± 18.9 .986 111.4 ± 11.9 102.5 ± 13.3 109.6 ± 12.6 .004 .000Threoninea 160.6 ± 41.4 162.4 ± 47.4 161.9 ± 45.9 .853 167.7 ± 25.4 181.5 ± 39.7 170.5 ± 29.1 .151 .091Citrulline 45.8 ± 18.8 51.1 ± 18.5 49.8 ± 18.7 .166 35.6 ± 8.2 36 ± 7 35.7 ± 7.9 .850 .000Sarcosine 3.7 ± 4.4 4.2 ± 5.1 4.1 ± 4.9 .588 2.4 ± 0.7 2.3 ± 0.6 2.4 ± 0.7 .544 .000β-Alanine 24.8 ± 5.1 25.1 ± 6 25 ± 5.7 .808 22.2 ± 8.9 16.9 ± 9.2 21.1 ± 9.1 .021 .000Alanine 719.8 ± 248.2 613.4 ± 203.6 639.6 ± 219.3 .018 653.3 ± 127.4 527.6 ± 125.3 628.2 ± 136.1 .000 .632Glutamic acid 150.4 ± 82.8 162.4 ± 138.4 159.5 ± 126.8 .649 108 ± 29.6 61.6 ± 18.8 98.8 ± 33.4 .000 .0001-Methylhistidine 10.2 ± 7.7 8.8 ± 7.5 9.2 ± 7.6 .381 12.9 ± 5.2 10.3 ± 7.8 12.3 ± 5.9 .083 .0013-Methyl histidine 8.8 ± 7.2 6.8 ± 3.7 7.3 ± 4.9 .161 4.9 ± 1.2 3.8 ± 1.2 4.7 ± 1.3 .000 .000Argininosuccinic acid 260.8 ± 48 257.4 ± 56.2 258.3 ± 54.1 .765 296 ± 46.1 248.7 ± 58.3 286.5 ± 52 .000 .000Carnosine 0.3 ± 0.2 0.3 ± 0.4 0.3 ± 0.4 .740 0.4 ± 0.4 0.4 ± 0.4 0.4 ± 0.4 .973 .002Anserine 0.4 ± 0.2 0.3 ± 0.2 0.3 ± 0.2 .403 0.6 ± 0.5 0.7 ± 0.5 0.6 ± 0.5 .471 .000Citrulline 1.1 ± 1.2 0.8 ± 0.6 0.9 ± 0.8 .157 18.9 ± 161.1 0.6 ± 0.4 15.2 ± 144.1 .614 .321Arginine 128.8 ± 32.8 136.9 ± 35.3 134.9 ± 34.8 .257 138.5 ± 23.9 134.4 ± 19.4 137.7 ± 23 .476 .474α-Amino adipic acid 2.1 ± 0.8 1.8 ± 0.7 1.9 ± 0.7 .071 2.8 ± 0.7 1.7 ± 0.4 2.6 ± 0.7 .000 .000γ-Aminobutyric acid 1.2 ± 1.3 1.6 ± 1.5 1.5 ± 1.4 .191 n/a n/a n/a n/a .000β-Amino isobutyric acid 4.3 ± 4.3 4.2 ± 3.8 4.2 ± 3.9 .945 2.2 ± 1.5 2.5 ± 1.5 2.2 ± 1.5 .434 .000α-Amino n-butyric acid 26.6 ± 8.6 30.3 ± 9.1 29.4 ± 9.1 .051 28.4 ± 8 26.9 ± 11 28.1 ± 8.6 .495 .276δ-Hydroxylysine 0.6 ± 0.6 0.5 ± 0.4 0.5 ± 0.5 .556 1.3 ± 0.8 1.2 ± 0.7 1.3 ± 0.8 .904 .000Proline 298 ± 102.4 239.9 ± 72.6 254.2 ± 84.3 .006 231.5 ± 58.5 191.6 ± 71.5 223.5 ± 63 .011 .002Ornithine 116.3 ± 50.3 140.5 ± 114.8 134.6 ± 103.1 .257 134.3 ± 40.1 112.2 ± 42.4 129.9 ± 41.3 .032 .644Cystathionine 2.7 ± 2.8 2.8 ± 4.0 2.8 ± 3.7 .876 2.9 ± 2.2 3.4 ± 1.5 3 ± 2.1 .248 .572Cysteine 45.4 ± 34.2 35.2 ± 30.4 37.8 ± 31.6 .119 37.3 ± 10.8 24.2 ± 7.8 34.7 ± 11.5 .000 .314Lysine 261.5 ± 50.4 260.5 ± 60.7 260.7 ± 58.1 .930 292.5 ± 42.1 244.2 ± 52.3 282.9 ± 48.1 .000 .002Methioninea 48.5 ± 12.2 43.3 ± 8.5 44.6 ± 9.8 .011 42.5 ± 10.6 42.1 ± 7 42.4 ± 10 .860 .100Valinea 357.6 ± 78.9 342.5 ± 74.7 346.2 ± 75.7 .338 366.2 ± 60.3 280.5 ± 51.5 349 ± 67.8 .000 .769Tyrosine 88.1 ± 18.2 88.7 ± 25.4 88.5 ± 23.8 .910 95.4 ± 15.2 77.1 ± 12.6 91.7 ± 16.4 .000 .257Homocysteine 0.8 ± 0.2 0.8 ± 0.3 0.8 ± 0.2 .262 4.2 ± 1.7 4.7 ± 1.6 4.3 ± 1.7 .289 .000Isoleucinea 107.1 ± 31.2 98.8 ± 27.1 100.9 ± 28.2 .156 106.3 ± 19.1 74.8 ± 14.4 100 ± 22.2 .000 .798Leucinea 188.1 ± 49.8 179.6 ± 46.2 181.7 ± 47 .389 207.8 ± 34.4 148.1 ± 22.1 195.9 ± 40.2 .000 .017Phenylalaninea 96 ± 26.4 96.5 ± 27.4 96.4 ± 27.1 .939 99.8 ± 14 82.9 ± 10 96.4 ± 14.9 .000 .986Tryptophana 50.9 ± 14.2 50.7 ± 12.7 50.8 ± 13.0 .929 64.6 ± 9.3 59.5 ± 10.5 63.6 ± 9.7 .035 .000

a Indicates essential AAs, all amino acid concentrations are shown as μmol/L.

1449Y. Zhou et al. / Clinical Biochemistry 46 (2013) 1447–1452

Plasma AA concentration comparison of obese and lean subjects in thediabetic group

In the diabetic patient group, we found that only three AA concen-trations were increased in the obese compared to the lean diabetespatients, which were Alanine, Proline and Methionine (Table 2).Thus, we suggest that there is no big difference in the AA metabolismpathways between obese and lean diabetic patients. Because theirmetabolism seems to be profoundly altered independent from theirbody weight, we further investigated whether there are AA serumconcentration differences between the diabetic and non-diabeticgroups and we compared the AA concentrations in all subjects' blood.

Overall comparison of plasma AA concentrations in the diabetic andnon-diabetic groups

We found that Phosphoserine, Phosphoric acid ethanolamine, Tau-rine, Serine, Aspartic acid, Histidine, 1-methylhistidine, Argininosuccinicacid, Carnosine, Anserine, α-Amino adipic acid, δ-Hydroxylysine, Lysine,Homocysteine, Leucine and Tryptophan were apparently reduced tolower concentrations in diabetic patients compared to non-diabetic sub-jects, and the AA Hydroxyproline, Glutamine, Ethanolamine, Citrulline,Sarcosine, β-alanine, Glutamic acid, 3-methyl histidine, γ-aminobutyric

acid, β-Amino isobutyric acid and Proline occurred in higher concentra-tions in the blood of diabetic patients (Table 2). Next, we analyzedwhether therewas a correlation between theseAAalterations and insulinresistance in the diabetic group.

Correlation of AA and insulin/glucose level concentrations in diabetespatients

To further examine the AA difference between diabetic andnon-diabetic patients, we analyzed the AA alterations for correlationswith the blood lipid index in the blood with a single factor regressionanalysis (Table 3) and a deep multiple factor regression analysis(Table 4) and found, that FBG was related with Cystathionine, Prolineand Citrulline, HbA1c was related with Glycine, Proline and Sarcosine,Cholesterol was related with Serine, β-alanine, Proline andCystathionine, HDL-C was related with β-alanine,1-methylhistidineand Proline, LDL-C was related with α-Amino n-butyric acid and Hy-droxyproline, Triglyceridewas relatedwith γ-aminobutyric acid, Serineand Alanine, and Fasting insulin was related with 3-methylhistidine,Asparagine, Alanine, γ-aminobutyric acid and Cystathionine. Prolinewas particularly related with FBG, HbA1c, Cholesterol and HDL-C,whereas γ-aminobutyric acid and Alanine were related with Triglycer-ides and Fasting insulin.

Table 3Correlation of plasma amino acid and insulin/glucose levels in diabetic patients.

FBG PBG HbA1c Cholesterol HDL-C LDL-C Triglyceride Fasting insulin

Phosphoserine Pearson correlation (r) .053 .031 − .020 .149 − .056 .106 − .036 − .087Significance (two-tail) P .552 .727 .820 .095 .530 .236 .687 .332

Phosphoric acid ethanolamine Pearson correlation (r) − .080 − .064 − .061 − .183 − .040 − .022 .023 − .056Significance (two-tail) P .374 .474 .495 .040 .657 .804 .798 .533

Taurine Pearson correlation (r) .012 .014 − .062 − .066 − .091 .076 .085 − .013Significance (two-tail) P .892 .876 .496 .471 .315 .404 .347 .889

Asparagine Pearson correlation (r) − .107 − .093 − .127 − .098 .026 − .069 − .131 − .144Significance (two-tail) P .232 .300 .155 .273 .775 .441 .143 .106

Serine Pearson correlation (r) − .075 − .109 − .184 − .315 − .069 − .078 − .173 − .087Significance (two-tail) P .405 .224 .039 .000 .445 .387 .053 .335

Hydroxyproline Pearson correlation (r) .024 − .030 − .060 .072 − .086 − .141 − .013 .041Significance (two-tail) P .790 .742 .508 .424 .337 .114 .885 .645

Glycine Pearson correlation (r) − .141 − .126 − .196 − .204 .020 − .066 − .108 − .045Significance (two-tail) P .116 .159 .028 .022 .825 .464 .228 .616

Glutamine Pearson correlation (r) − .055 .060 .000 .136 − .016 .011 .020 − .033Significance (two-tail) P .539 .501 1.000 .129 .861 .903 .823 .715

Aspartic acid Pearson correlation (r) − .135 − .076 − .119 − .295 − .091 .070 − .012 .034Significance (two-tail) P .130 .399 .185 .001 .312 .434 .895 .706

Ethanolamine Pearson correlation (r) .033 − .013 .049 − .066 − .275 − .011 .057 .075Significance (two-tail) P .710 .889 .583 .460 .002 .898 .524 .406

Histidine Pearson correlation (r) .044 .076 .098 − .062 − .069 .056 .067 .115Significance (two-tail) P .625 .396 .275 .492 .444 .532 .456 .201

Threonine Pearson correlation (r) − .028 − .025 − .076 − .055 − .026 − .019 − .020 − .062Significance (two-tail) P .753 .780 .401 .544 .774 .833 .828 .494

Citrulline Pearson correlation (r) − .165 − .044 − .060 − .071 .142 .052 − .057 .007Significance (two-tail) P .065 .628 .506 .432 .113 .560 .529 .936

Sarcosine Pearson correlation (r) − .158 − .016 − .192 .088 .235 − .076 − .113 − .121Significance (two-tail) P .077 .859 .031 .329 .008 .400 .210 .179

β-Alanine Pearson correlation (r) .046 − .111 .068 − .257 − .268 − .034 .041 .106Significance (two-tail) P .613 .216 .451 .004 .002 .708 .648 .236

Alanine Pearson correlation (r) .117 .063 .111 .107 − .026 .057 .163 .223Significance (two-tail) P .193 .482 .218 .234 .770 .525 .068 .012

Glutamic acid Pearson correlation (r) − .040 − .110 − .123 − .140 − .082 − .082 .028 .025Significance (two-tail) P .655 .221 .169 .119 .359 .364 .755 .781

1-Methylhistidine Pearson correlation (r) − .058 − .075 − .034 − .005 .157 .018 .010 .091Significance (two-tail) P .520 .405 .705 .952 .079 .839 .916 .309

3-Methylhistidine Pearson correlation (r) − .136 − .011 − .023 − .003 .144 .093 .062 .240Significance (two-tail) P .129 .902 .798 .975 .108 .299 .490 .007

Argininosuccinic acid Pearson correlation (r) .181 .052 .093 − .042 − .086 − .073 − .017 .107Significance (two-tail) P .043 .562 .301 .644 .341 .415 .848 .232

Carnosine Pearson correlation (r) − .103 .032 − .087 .035 .118 − .080 − .073 − .088Significance (two-tail) P .252 .725 .331 .695 .189 .372 .415 .325

Anserine Pearson correlation (r) .007 .010 .047 .087 − .066 .163 .054 .013Significance (two-tail) P .939 .915 .602 .331 .460 .069 .546 .884

High Citrulline Pearson correlation (r) − .122 − .028 − .071 .026 .050 − .003 .106 .192Significance (two-tail) P .173 .752 .427 .773 .577 .970 .239 .032

Arginine Pearson correlation (r) .082 .020 .052 .002 − .096 .057 .004 .058Significance (two-tail) P .361 .828 .562 .979 .284 .525 .966 .522

α-Amino adipic acid Pearson correlation (r) .033 .107 .021 .155 − .113 .003 .135 .138Significance (two-tail) P .713 .235 .816 .083 .208 .974 .133 .122

γ-Aminobutyric acid Pearson correlation (r) .007 − .043 − .119 .143 .206 − .104 − .162 − .192Significance (two-tail) P .939 .633 .186 .110 .020 .248 .070 .031

β-Amino isobutyric acid Pearson correlation (r) − .046 .019 − .125 .047 − .022 .018 − .040 − .115Significance (two-tail) P .606 .834 .163 .599 .809 .845 .657 .200

α-Amino n-butyric acid Pearson correlation (r) .008 − .064 − .122 − .113 .031 − .171 − .158 − .116Significance (two-tail) P .926 .473 .173 .208 .733 .056 .078 .197

δ-Hydroxylysine Pearson correlation (r) − .025 .035 − .133 − .168 .041 − .100 − .018 − .045Significance (two-tail) P .783 .698 .137 .060 .645 .265 .838 .614

Proline Pearson correlation (r) .156 .085 .187 .133 − .160 .012 .149 .225Significance (two-tail) P .081 .345 .036 .139 .073 .891 .095 .011

Ornithine Pearson correlation (r) − .196 − .085 − .081 − .219 − .027 − .013 − .064 − .031Significance (two-tail) P .028 .343 .369 .014 .763 .885 .478 .729

Cystathionine Pearson correlation (r) − .214 − .070 − .109 − .208 .069 − .052 − .066 − .063Significance (two-tail) P .016 .439 .225 .019 .444 .564 .465 .483

Cysteine Pearson correlation (r) − .081 .025 − .018 .117 .053 .026 .031 .083Significance (two-tail) P .370 .779 .842 .191 .556 .770 .726 .353

Lysine Pearson correlation (r) .157 .095 .087 − .036 − .103 − .110 − .006 .067Significance (two-tail) P .080 .289 .331 .689 .252 .222 .948 .454

Methionine Pearson correlation (r) .165 .100 .107 .062 − .057 .128 .154 .116Significance (two-tail) P .065 .267 .232 .493 .523 .153 .085 .195

Valine Pearson correlation (r) .117 .100 .114 .045 − .134 .007 .139 .156Significance (two-tail) P .192 .263 .203 .614 .134 .936 .119 .082

Tyrosine Pearson correlation (r) − .009 − .038 − .061 − .112 − .019 − .147 − .067 .036Significance (two-tail) P .917 .675 .499 .211 .829 .101 .456 .691

1450 Y. Zhou et al. / Clinical Biochemistry 46 (2013) 1447–1452

Table 3 (continued)

FBG PBG HbA1c Cholesterol HDL-C LDL-C Triglyceride Fasting insulin

Homocysteine Pearson correlation (r) .063 .046 .031 .079 .118 − .009 − .001 − .068Significance (two-tail) P .486 .606 .734 .380 .187 .925 .988 .449

Isoleucine Pearson correlation (r) .166 .130 .080 .037 − .094 − .036 .082 .120Significance (two-tail) P .063 .146 .371 .681 .295 .693 .362 .182

Leucine Pearson correlation (r) .146 .117 .093 − .012 − .142 − .007 .101 .136Significance (two-tail) P .102 .193 .300 .892 .114 .941 .259 .130

Phenylalanine Pearson correlation (r) − .066 − .035 − .139 − .261 − .104 .031 − .004 .058Significance (two-tail) P .461 .694 .120 .003 .249 .728 .963 .522

Tryptophan Pearson correlation (r) .129 .017 .097 − .001 − .121 − .124 .076 .113Significance (two-tail) P .151 .846 .282 .987 .176 .165 .396 .207

1451Y. Zhou et al. / Clinical Biochemistry 46 (2013) 1447–1452

Discussion

Metabolomics is the comprehensive study ofmetabolites in biofluids,tissues or cellular extracts and the metabolic profile of a sample maybe assessedusing a variety of techniques including ProtonNMRspectros-copy, LC–MS and GC–MS. The role of metabolomics in the field ofnutrition is growing and its utility in a number of studies has beendemonstrated [20,21]. In the present study, we have applied a compre-hensive set of analytical tools to gain a deeper understanding of AAdifferences between obese and lean humans and diabetic andnon-diabetic patients. Branched-chain amino acids (BCAAs) in blood se-rums are reported to be elevated from the obesity-related low-gradeinsulin-resistant state to pronounced diabetes [22]. Newgard et al. pro-posed, that increased BCAA levels activate the mammalian Target ofRapamycin/protein 6 kinase 1 (mTOR/S6K1) pathway and phosphoryla-tion of Insulin receptor substrate 1 (IRS1) onmultiple serines, contribut-ing to insulin resistance. In addition increased BCAA catabolic flux might

Table 4Correlation of plasma amino acid and serum lipid, FBG, PBG and HbA1c levels in diabeticpatients.

Dependentvariable

Regressioncoefficient

Standarderror

SignificanceP value

FBG (mmol/L) Constant term 5.764 .326 .000Cystathionine − .083 .025 .001Proline .004 .001 .001Citrulline − .014 .005 .006

PBG (mmol/L) No significant impactfactors

HbA1c (mmol/L) Constant term 7.450 .483 .000Glycine − .003 .001 .012Proline .004 .001 .007Sarcosine − .054 .024 .024

Cholesterol(mmol/L)

Constant term 7.081 .781 .000Serine − .009 .003 .001β-Alanine − .054 .022 .017Proline .004 .002 .008Cystathionine − .093 .037 .012

HDL-C (mmol/L) Constant term 1.588 .149 .000β-Alanine − .016 .005 .0011-Methylhistidine .011 .004 .007Proline − .001 .000 .024

LDL-C (mmol/L) Constant term 3.052 .260 .000α-Amino n-butyric acid − .018 .007 .009Hydroxyproline − .011 .005 .045

Triglyceride(mmol/L)

Constant term 1.951 .318 .000γ-Aminobutyric acid − .134 .050 .008Serine − .004 .002 .006Alanine .001 .000 .016

Fasting insulin(mU/L)

Constant term 19.972 3.971 .0003-Methylhistidine .606 .173 .001Asparagine − .147 .042 .001Alanine .015 .004 .000γ-Aminobutyric acid −1.541 .584 .009Cystathionine − .540 .235 .023

Note: Amultivariate linear regression analysiswas donewith FBG, PBG, HbA1c, Cholesterol,HDL-C, LDL-C, Triglycerides and Fasting insulin as dependent and AA as the independentvariable.

contribute to increased gluconeogenesis and glucose intolerance via glu-tamate transamination to alanine [13]. However, besides dietary obser-vations in animals, whether enhanced BCAA concentrations in bloodsera are the reason or the cause of insulin insensitivity is not clear [23].A recent article reported a strong association of insulin resistance traitswith Glutamine, Glutamate and the Glutamine-to-Glutamate ratios insera [24]. For obesity, results showed abnormal metabolism of twobranched-chain amino acids, two aromatic AAs, and fatty acid synthesisin obese men [25] and plasma concentrations of Phenylalanine, Histi-dine, Glutamic acid and Lysine in cows with Fat Cow Syndrome (FCS)were significantly enhanced, while concentrations of Threonine, Gluta-mine, Asparagine and Citrulline were significantly lowered [26]. In ourstudy we found that there were significant differences of serum AA con-centrations between healthy adipose and lean individuals. The obesepeople had higher serum concentrations of 19 AAs, 15 of them essentialAAs compared to lean people. Interestingly the most obvious concentra-tion enhancement was visible for Citrulline, which has previously beendescribed to be elevated in sera of obese people due to changes of theirsmall intestine [27]. Notably among the enhancedAAswere Valine, Tyro-sine, Isoleucine, Leucine and Phenylalanine, which have been suggestedto be prognostic indicators for developing diabetes II, with Isoleucine,Phenylalanine and Tyrosine predicting future diabetes with a morethan fivefold higher risk [14]. In our analyses we also found, that 11 AAconcentration increases were similar between young adipose peopleand old diabetics (data not shown). On the other hand the differentserum AA patterns almost completely disappeared in diabetes patients,in which the differences between obese and lean people seemed to beequalized by insulin resistance. Besides the smaller differences betweenadipose and lean diabetics, their overall plasmaAA concentrationswereessentially different compared to healthy individuals, with enhance-ment of 16 AA and reduction of 11 AA concentrations. Enhancedconcentrations of especially BCAA are reported to be elevated ininsulin-resistant states and high protein dietary is suggested to impairglucose metabolism [28]. Also other authors suggested, that Leucine,Isoleucine or Threonine decreases glucose uptake, because they mightserve as alternative energy source [29]. On the other hand some authorsreported enhanced glucose uptake of diabetes II patients, when theycombined carbohydrate ingestion with a free AA mixture, particularlycontaining Leucine and Phenylalanine [30] and oxidation of Glutamate,which was also enhanced in our diabetic patients, mediatesAA-stimulated insulin secretion in pancreatic b-cells [8]. Taken togetherthere are divergent opinions about the role of AAs in glucose uptakeranging from enhancement to inhibition and even no function at all atleast in non-diabetic obese individuals [31]. The partly enhanced serumAA levels and particularly that of Glutamic acid in our diabetes patientsmight be explained as a body measure to enhance insulin secretion ofpancreatic beta cells [8,30] and reduced serum AA concentrationsmight indicate, that AA is an alternative energy source [29]. Enhancedserum lipid levels in diabetes patients is well known as a high risk factorfor developing cardiovascular diseases [32] and also elevated triglycerideconcentrations occur frequently in diabetes II patients. A recent publica-tion suggested that differences in total triglycerides and HDL cholesterolare predictive for diabetes development [33], but less is known about

1452 Y. Zhou et al. / Clinical Biochemistry 46 (2013) 1447–1452

correlations of lipids and AA levels in diabetics. In our analyses we foundthat serine serum concentrations in diabetic patients were lower than innon-diabetics, which was related with Cholesterol and Triglycerides, in-versely. In contrast Proline concentrations were higher in diabetics,which correlated with FBG, HbA1c, Cholesterol, and HDL-C (Table 4).Thus, it might be possible to add or reduce Serine or Proline as supple-ments in diabetic diets to adjust the blood glucose and blood lipids. In ad-dition a longer term monitoring of specific serum AA concentrationpattern, which we derived in our study, may be of clinical significanceas prognostic value for the development of diabetes particularly inobese people. A limitation of this study was the small sample size andfurther investigations with a higher number of patients are necessaryto further elucidate the role of serum AA concentrations in diabetes de-velopment. In conclusion, there is strong evidence supporting the hy-pothesis that altered AA levels in obese young subjects are associatedwith developing insulin resistance and diabetes. Once the diabetes ismanifest, the obesity related different patterns of particular AA concen-tration enhancements are not detectable anymore. Betweenhealthy sub-jects and diabetics huge differences of serum AA concentrations areoccurring, which partly correlate with other factors like FBG, HAb1c,Cholesterol, HDL-C, triglycerates and Fasting insulin. However, the mo-lecular mechanism leading to the changed concentrations of circulatingAA remains to be identified. Progress in this field is required to enhanceour understanding of the role of AA in the development of insulin resis-tance and to support their usage as markers of diabetes risk prognosesand treatments.

Conflict of interest

None declared.

Acknowledgments

We specially thank Feng Wang and Ting Lu for collecting thematerials.

This work was supported by a grant from the Project ofvildagliptin in the phase IV clinical trial of Beijing Novartis co., LTD(MK-0431-313-00) and vildagliptin intervention study of BeijingNovartis co., LTD.

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