**2.1 Subjects**

198 Chronic Kidney Disease

and plasma synthesized from endothelial cells (Figure 1). It is shown that high ADMA level increases the cardiovascular incident risk by 34% and mortality risk by 52% (4-8). Increased ADMA concentration has a high prevalence in hyperhomocysteinemia, coronary artery diseases, hypercholesterolemia, diabetes mellitus, hypertension, preeclampsia, peripheral arterial occlusive disease, impaired renal function and other diseases (7,9,10). Reduced nitric oxide (NO)-dependent vasodilation is regarded as an early indicator of atherosclerotic diseases (7,14). It is documented that adult patients with renal failure have 2-6 times higher ADMA than healthy subjects due to reduced renal excretion and reduced enzymatic degradation (15). NO is synthesized from L-arginine via NO synthase enzyme. NO inhibition decreases endothelial derived vasodilation and increases vascular resistance. Reduced NO availability can occur in patients with CKD. Moreover CKD can contribute to the accelaration of hypertension and cardiovascular complications. It appears that the increase in endogenic NO inhibitors like ADMA plays a major role in this process (11, 15- 17). It has been shown that Hcy stimulates ADMA formation and plasma ADMA levels elevate in humans and animals by hyperhomocysteinemia (18-20). Increased serum Hcy level in adult CKD patients is an independent risk factor for cardiovascular system mortality. Elevated ADMA and hyperhomocysteinemia may be due to decreased renal excretion (18-22). It is reported that ADMA formation may be related with Hcy metabolism (18,19). It was found that there is a significant interaction of serum fibrinogen and CKD with

respect to risk of both fatal/nonfatal coronary events and death (20–24).

Fig. 1. Biochemical pathway for generation and degradation of ADMA and homocysteine.

This prospective study was carried out in 65 untreated mild chronic kidney disease (35 men and 30 women; mean age 55.2 ± 9.6 years) and 65 healthy control subjects with matched age, sex and body mass index (BMI). The creatinine clearance was calculated by the Cockcroft-Gault Formula (25). Patients having creatinine clearance less than 75 ml/min were considered to have mild CKD. Body mass index was determined as weight divided by the square of height (kg/m2). The underlying causes of CKD were glomerulonephritis (n=17), interstitial nephropathy (n=12), autosomal dominant polycystic kidney disease (n=13), chronic pyelonephritis (n=7) and urological problems (n=5). No cause was identified in 11 cases. The exclusion criteria were diabetes mellitus, active hepatitis, malignancy, smoking and infectious disease. Patients using vitamin supplements were also excluded.

The study protocol was approved by the Ethics Committee of the Dicle University School of Medicine (Diyarbakir, Turkey) and written informed consent was obtained from each participant.

#### **2.2 Methods**

In all patients, venous blood samples were drawn between 7:00 AM after a 12-h fastened, and the serum was frozen at -70 C in aliquots until biochemical analysis were performed.

*ADMA Measurement:* ADMA was measured by HPLC according to the method described by Chen et al. (26). Mobile phases consisting of 50 mM sodium acetate (pH 6.8), methanol and tetrahydrofuran (THF) (A, 82:17:1; B, 22:77:1) were used. All separations were performed at 270C and at a flow-rate of 1.0 ml/min. The wavelengths of fluorescence detector were set at 338 nm and 425 nm for excitation and emission, respectively. 20 mg of 5-sulfosalicylic acid (5-SSA) was added to 1 ml plasma, and the mixture was left in an ice bath for 10 min. The precipitated protein was removed by centrifugation at 2000 g for 10 min. o-Phthaldialdehyde (OPA) (10 mg) was dissolved in 0.5 ml of methanol, and 2 ml of 0.4 M borate buffer (0.4 M boric acid adjusted to pH 10.0 with potassium hydroxide) and 30 μl of mercaptoethanol were added. The derivatization was performed by mixing 10 μl of sample or working standard solution and 100 μl of OPA reagent and reacting for 3 min before autoinjecting onto the column.

*NO Measurement:* The serum level of NO was measured using a colorimetric method based on the Griess reaction (27), in which nitrite is reacted with sulphanilamide and N-(1 naphthyl) ethylenediamine to produce an azo dye that can be detected at 540 nm. This was carried out after enzymatic reduction of nitrate to nitrite with nitrate reductase.

*Hcy Measurement:* Serum level of Hcy was measured using HPLC with fluorescence detection (Shimadzu RF-10A fluorescence detector; Shimadzu Co., Kyoto, Japan).

The Effects of Asymmetric Dimethylarginine (ADMA), Nitric Oxide (NO) and Homocysteine (Hcy) on Progression of Mild Chronic Kidney Disease (CKD): Relationship Between Clinical and... 201

Age (years) 54.9 ± 10.1 55.2 ± 9.6

Number of patients (M/F) 35/30 35/30

Body mass index (kg/m2) 24.90 ± 2.1 24.70 ± 2.6

Systolic BP (mmHg) 110.20 ± 10.4 \*128.40 ± 22.4

Diastolic BP (mmHg) 72.20 ± 11.6 \*84.40 ± 16.3

Creatinine clearance (ml/min) 90.20 ± 15.1 \*52.50 ± 15.3

Urea (mg/dl) 31.50 ± 6.2 \*61.30 ± 14.6

Creatinine (mg/dl) 1.20 ± 0.42 \*1.61 ± 0.73

Calcium (mg/dl) 8.73 ± 1.2 8.91 ±1.08

Phosphate (mg/dl) 4.10 ± 1.09 4.09 ± 1.2

Albumin (g/dl) 3.82 ± 0.9 3.94 ± 0.5

Protein (g/dl) 6.40 ± 1.1 6.01 ± 0.3

Glucose (mg/dl) 87.90 ± 16.2 90.10 ± 15.4

Insulin (µu/ml) 11.60 ± 2.9 12.04 ± 2.83

Triglyceride(mg/dl) 118.30 ± 20.2 120.10 ± 18.5

Total cholesterol (mg/dl) 184.20 ± 22.1 185.60 ± 19.4

HDL-C (mg/dl) 45.80 ± 12.4 42.02 ±14.3

LDL-C (mg/dl) 113.20 ± 12.8 \*142.12 ± 18.6

hsCRP (mg/dl) 1.914 ± 0.667 \*7.048 ±2.249

Fibrinogen (g/L) 2.835 ± 0.646 \*4.574±0.521

ADMA (µmol/L) 0.512 ± 0.116 \*0.837±0.189

Nitric oxide (µmol/L) 75.67 ± 8.626 \*44.31±7.811

Homocystein (µmol/L) 6.256 ± 1.629 \*18.37 ± 3.192

Table 1. Clinical and laboratory data of patients with CKD and healthy subjects.

BP: Blood Pressure; HDL-C: High Density Lipoprotein Cholesterol; LDL-C: Low Density Lipoprotein Cholesterol; hsCRP: High sensitive C Reactive Protein; ADMA: Asymmetric dimethylarginine

\*P < 0.001; Data are reported as means ± SD.

Healthy Subjects (n=65)

Chronic Kidney Disease (n=65)

Urea, creatinine, calcium, phosphate, albumin, protein, high sensitive CRP (hsCRP), insulin, glucose, total cholesterol, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and triglyceride assays were determined by standard laboratory methods according to the established methodology. The serum level of fibrinogen was measured by the Clauss method using a commercial kit. All routine laboratory measurements were carried out using certified assay methods.

Statistical analysis of the differences between groups of subjects was performed using the Kolmogorov-Smirnov and unpaired student's t-test or by the Mann-Whitney nonparametric test as appropriate. Pearson's correlation analyses were performed.
