The assessment of ADMA as a The assessment of ADMA as a prognostic marker for prognostic marker for

haemodynamic instability in patients haemodynamic instability in patients with sepsiswith sepsis

Felicity Dempsey Student No. 30478703

Assignment Date September 30th 2005

MSc in Biomedical Science

Introduction to ADMA

Asymmetric Di Methyl Arginine is an endogenous inhibitor of Nitric Oxide Synthase ( NOS) which is found in human blood and urine.See Fig. 1. (Boger R.et al. 1998)

It is derived from the catabolism of methylated arginine residues by proteins designated Protein Arginine Methyl Transferase Type I and II (PRMT I, II) (Leiper J. Vallance P. 1999)

It is further metabolised to to form Citrulline and Methylamines via the enzyme Dimethylarginine Dimethylaminohydrolase.(DDAH) (Maxwell A. 2002)

Fig 1.

ADMA inhibits NOS by competing with L-arginine for the amino acid transporter system across cell membranes.(Leiper J. Vallance P. 1999)

ADMA therefore interferes with all the biological functions associated with NOS (Boger R. Vallance P. 2003)

Pathophysiological Role of ADMA Derangements of the ADMA/NOS pathway lead to endothelial

dysfunction and the initiation of vascular disorders.(Cooke J. 2004)

ADMA appears to be an independent predictor of acute coronary events and associated morbidity and mortality.( Dayoub H. Achan V. et al 2003)

The NOS system becomes uncoupled by the competitive inhibition of ADMA which results in the production of damaging superoxides instead of endogenous NO. (Sydow K. Munzel T. 2003)

The production of these superoxides results in oxidative damage to proteins,lipids and DNA .(Sydow K. Munzel T 2003)

Increased levels of ADMA are therefore associated with diseases such as athereosclerosis, diabetes mellitus, hypertension and renal failure.

ADMA inhibition of NOS •Platelet Aggregation

causes •Monocyte Adhesion

an increase in •Release of Super Oxide Radicals

these biological • Proliferation of Vascular Smooth Muscle Cells

functions •Oxidation of LDL

(Adapted from Boger R et al 1998)

Aims of Study

This study will investigate whether ADMA levels are altered in patients with severe sepsis.

This study will explore the possibility of a relationship between ADMA levels and(a) the occurrence of shock

(b) the severity of organ failure

(c) survival in a cohort of patients with severe sepsis.

The study will also explore changes in ADMA levels over the first 7 days of intensive care stay.

The study will explore the relationship between ADMA and CRP levels which has previously been reported (Zoccali et al. 2002).

Serum samples will be taken from a cohort of 46 patients admitted to ICU with sepsis.

Control samples will be taken at one time point from a group of colleagues.

Methods (1)

Previous methods for the measurement of ADMA included paper chromatography, electrophoresis, thin layer chromatography, and ion exchange chromatography.

This study will use the ELISA method developed by Schulze et al. in 2004(Schulze F. Wesemann R. at al 2004)

The ADMA ELISA kit uses a microtitre plate format. ADMA is bound to the solid phase of the plate.

ADMA in the samples competes with solid phase ADMA for a fixed number of rabbit anti-ADMA binding sites. When the system is in equilibrium, free antigen and free antigen/antibody complexes are removed by washing.

The antibody bound to the solid phase ADMA is detected by anti-rabbit peroxidase.The substrate TMB/peroxidase (3,3’,5,5’,tetramethyl benzidine/peroxidase) reaction is monitored at 450nm.

The amount of antibody bound to the solid phase of the plate is inversely proportional to the amount of ADMA in the sample.

Methods (2)

CRP will be measured on the Roche Modular DIIP using a particle enhanced immunoturbidimetric method.(Roche Tina Quant a C Reactive protein)

All data will be stored on an excel spreadsheet, in an anonymous format and will be reported as pooled data.

The outcome measurements will be

1. requirement for inotropic support on day 1 and day 7.

2. survival or mortality.

3. Organ Failure Score

Statistics will be analysed using the JMP programme ( SAS, Cary, NC). Between group comparisons for continuous variables will be analysed by paired t test. Spearman rank correlation coefficient will be used to analyse the relation between continuous variables.

Results

ADMA and CRP Day 1 and Day 7 in patients with Sepsis

Group

n

Control

10

Sepsis Day 1

46

p

ADMA umol/L

0.63 (0.71-0.57) 0.88 (1.09-0.59 0.05

Comparison of ADMA levels in Sepsis and Control Groups

Day 1

n= 46

Day 7

n = 32

p

ADMA umol/L

0.88 (1.09-0.59) 1.06 (1.34-0.73) =0.01

CRP mg/dL

18.32(26.1-10.9) 7.39 (13.6-3.89) <0.0001

*All values are quoted as median and interquartile ranges*

ADMA levels in relation to Outcome Measures

Day 1 Inotrope group No Inotrope

p

ADMA

umol/L

0.91 (1.25-0.69)

N =29

0.56 (0.98-0.49)

n =15

<0.0001

Day 7 Death Discharge

ADMA

umol/L

1.53 (1.59-1.14)

n =5

1.04 (1.15-0.67)

n =24

0.03

All values are quoted as median and interquartile range

Results

Spearman’s Rho p

SOFA Day 1 0.49 0.0007

SOFA Day 7 0.44 0.02

CRP Day 1 -0.53 0.0008

CRP Day 7 0.012 0.95

Correlation coefficients between ADMA levels, SOFA scores and CRP values

There was no relationship between CRP and SOFA score on Day 1 or Day 7

Discussion

• ADMA has already been shown to be involved in disease states such as cardiovacular disease, liver failure, atherosclerosis, renal failure.

• This study shows that ADMA is strongly involved in sepsis in ICU patients also.• It would therefore be possible to risk stratify patients according to ADMA levels,

and thus initiate novel therapeutic interventions in this cohort of patients.• The study also showed that ADMA was strongly related to SOFA scores and

therefore to the prediction of multi organ failure.

• The ELISA method for ADMA used in this study was labour intensive and expensive.

Conclusions

• ADMA levels are elevated in patients with severe sepsis, and increase over time.

• ADMA levels were greater in patients with severe sepsis who required inotropic support on Day 1.

• ADMA levels in patients with severe sepsis were greater on Day 7 in patients who died than in survivors.

• ADMA correlated with SOFA score on Day 1 and 7.

• There was an inverse relation between ADMA and CRP on day1 in patients with severe sepsis.

• ADMA may be a better index of disease severity than CRP

References

•Boger R. H. Bode-Boger S. M. et al (1998) Asymmetric Dimethylarginine: A Novel Risk Factor for Endothelial Dysfunction, Circulation ,98 , (18) 1842-1847

•Boger R.H. Vallance P. Cooke J.P.( 2003) Asymmetric Dimethylarginine(ADMA) ; a key regulator of nitric oxide synthase. Atherosclerosis Supplement ,4. 1-3

•Cooke J.P. (2004) Asymmetrical Dimethylarginine, The Űber Marker ? Circulation 109, 1813-1819•Dayoub H. Vinod A. et al. (2003) Dimethylarginine dimethylaminohydrolase regulates Nitric Oxide

Synthase. Circulation. 108, 3042-3053•JMP Statistics Software, SAS Institute, Cary , North Carolina, USA.•Leiper J. Vallance P. (1999) Biological Significance of Endogenous Methylarginines that inhibit Nitric

Oxide Synthases. Cardiovascular Research, 43: 542-548•Maxwell A. (2002) Mechanisms of dysfunction of the Nitric Oxide Pathway in vascular disease. Nitric

Oxide; Biology and Chemistry, 6, (2) 101-124•Sydow K. Munzel T. (2003) ADMA and oxidative stress Atherosclerosis Supplements. 4 , 41-51•Zoccali C. Bendetto F.A. (2002) Asymmetric Dimethylarginine, C-Reactive Protein, and Carotid Intima-Media Thickness in end stage renal disease ; Journal American Society Nephrology: 13, 490-496 •Roche Tina Quant a C-Reactive Protein CRPLX . Cat No. 03002012 •Schulze F. Wesemann R et al. (2004) Determination of asymmetric dimethyl arginine (ADMA) using a

novel ELISA assay. Clin. Chem. Laboratory Medicine. 42 (12) 1377-1383•SOFA; Sequential Organ Assessment Score (American College of Chest Physicians 1992)

Discussion•Increased ADMA levels are a result of increased production or decreased metabolism of the protein.

•Accelerated protein breakdown is characteristic of critical illness and sepsis and may contribute to increased ADMA levels.

•Renal failure as part of MOF also contributes by reduced excretion of ADMA .

•Increased ADMA contributes to organ failure by blocking NOS leading to reduced NO activity.No has essential anti microbial and anti viral properties.

•The reduced NO activity may result in persistent infection and perpetuation of the inflammatory response.

•Result; failure to clear the initial sepsis and development of organ failure

Metabolism of ADMA in Critical Illness