DETERMINATION OF NITRATE CONCENTRATION IN SERUM

DETERMINATION OF NITRATE CONCENTRATION IN SERUM AS AN INDEX OF NITRIC OXIDE (NO)SYNTHETASE ACTIVITY IN HEALTHYINDIAN MALE SUBJECTS

K N Maruthy, Rani Gupta
Department of Physiology, St. John’s Medical College, Bangalore 560 034.

The present study was taken up to find out the serum nitrate levels in healthy human volunteers by a single step enzymatic assay using nitrate reductase from Aspergillus species.1 Serum nitrate of 27 male subjects was measured and was found to be 0.8 - 26.4 m mol/L (mean 9.24 m mol/L, median - 8 m mol/L).
INTRODUCTION

Nitric oxide is now recognised as a gas of biological importance. It is produced endogenously from L-Arginine by action of Nitric oxide synthatases, in mamallian tissue almost ubiquitously in constitutive and as well as inducible forms.

Vascular endothelium, neuronal synapses, blood vessels, leucocytes, macrophages, platelets are few of the well known sites of nitric oxide (NO) generation in physiological and pathological states. Physiologically it is responsible for basal vasomotor tone, which is vasodilator in nature. This is imitated by compounds such as nitroglycerine and sodium nitroprusside which have well established clinical efficiency as vasodilators. The endothelium derived relaxation factor for vascular smooth muscle is identical to the nitric oxide radical.[2] Nitric oxide inhibits the platelet aggregation and thereby involved in homeostasis of vasculature.

Many biological processes such as neurotransmission, tumour cell killing, immunity, inflammatory response are controlled by nitric oxide. Thus nitric oxide has both cytotoxic and cytoprotective actions.[3] Nitric oxide has a very short plasma half life of 6-60 sec.[4] and within 10-20 minutes NO gets converted into stable nitrate compound and is excreted through kidney. As per the previous studies the serum nitrate values range between 4.0 - 45.3 m mol/L (mean 19.7 m mol/L).[5]

The present study is aimed to estimate normal serum nitrate levels in healthy Indian adult males using a single step enzymatic assay.[1]

MATERIAL AND METHODS

Samples : Serum samples were obtained from 27 healthy male volunteers in the age group of 18-37 years after an overnight fast. These subjects were either medical students or laboratory workers and they were advised not to consume protein rich diet the previous day. The subjects were rested for at least twenty minutes before collection of sample in order to avoid the changes in serum nitrate levels secondary to physical activity. Samples were collected in accordance with the Medical Ethical Committee regulations of our institute and were stored at -20°C.

Chemicals : Nitrate reductase - from Aspergillus species and FAD were obtained from Sigma-Aldrich, B-NADPH from Spectrochem, and Sodium nitrate Potassium dihydrogen phosphate and dipotassium hydrogen phosphate were obtained from Nice chemicals.

Assay : 500 micro liter eppendoff tubes containing 250 micro liter of 100 micro mol/L potassium phosphate buffer was equilibrated to room temperature (pH 7.5). To this 50 micro liter of distilled water, 50 micro liter 0.2 micro mol/L FAD and 10 micro liter of 12 milli mol/L Beta-NADPH with 100 micro liter of nitrate calibrator solution were added in varying concentration for calibration (Sodium nitrate 50,100,150 and 200 micro mol/L).

To start the enzymatic assay reaction 40 micro liter of 500 U/L nitrate reductase was mixed and immediately kept in dark for 45 minutes as FAD is photo labile. After the incubation period the absorbance at 340 nm was recorded by means of UV spectro photometer (Pye Unicam - SP8-400). Each assay was performed in triplicates.

Reagent blank : 250 micro liter of potassium phosphate buffer was taken and to this 50 micro liter 0.2 micro mol per liter FAD and 10 micro litre of 12 milli mole per litre of B-NADPH and 150 micro litre of distilled water (instead of nitrate calibrator or serum) in presence of 40 micro litre of 500 U/L nitrate reductase incubated in dark for 45 minutes and absorbance value noted as reagent blank.

Serum blank : Some procedure as above except for 100 micro litre of serum instead of distilled water and 40 micro litres water of distilled water to replace nitrate reductase. Incubated in dark for 45 minutes and absorbance value noted as serum blank.

Sample : Same as serum blank except that 40 ml of nitrate reductase was added instead of distilled water, incubated in dark for 45 min and absorbance value noted.

Calculation : The nitrate concentration in the given sample was calculated by

C = Delta A X factor

C = Nitrate concentrations

Delta A = Absorbance of serum blank - absorbance of sample - change in the absorbance of reagent blank

Factor = V_T ₁ X1 X1

V_s X I X E _340mm

V_s = Sample volume

V_T = Total volume of reaction mixture

E_340nm = millimolar absorptivity of Beta NADPH at 340 nm

I = light path 1 centimeter

Factor = 0.833

RESULTS

Linearity : As per the method described above the reaction was linear upto 200 m mol/L using sodium nitrate standard solution as calibrator. (Fig. 1).

In 27 male subjects serum nitrate levels were measured. Nitrate levels were 0.8 - 26.4 m mol/L (mean 9.24 m mol/L). The serum nitrate levels were found to be similar when compared to the past studies.[1],[3],[5] Better assessment would be obtained if kidney function would be studied simultaneously, because the nitrate are primarily excreted through kidney.

Fig. 1 Nitrate conc. vs. Absorbance change

Precision studies

Intra assay precision was determined by 6 replicate of pooled serum from healthy subjects, the cv,% was 4.39 with mean nitrate level of 9.33 m mol/L SD æ 0.41 Inter assay precision was also determined by the same pooled sample, the cv,% was 4.67 with mean nitrate level of 9.2 m mol/L SD æ 0.43. The pooled sample was stored at -20 degree Celsius.

DISCUSSION

The enzymatic method used1 here for determining the values of Indian subjects is a specific single step assay where nitrate reductase enzyme extracted from aspergillus species is used for conversion of nitrate to nitrite in presence of B-NADPH, FAD is used to complete the reduction within 45 minutes. Deproteinization of serum is a must in methods using copper/cadmium metal catalyst or Escherichia coli derived nitrate reductase, but the present method requires no deproteinization. The only disadvantage of the method used is it requires a UV spectrophotometer which may not be easily available.

There are other methods like high performance liquid chromotography (HPLC), capillary electrophoresis and gas chromatography with mass spectrometer. All these methods require expensive equipments.

Determination of serum nitrate is a useful indicator for NO radical formation. The values we have reported are in accordance with the previous studies.

ACKNOWLEDGEMENT
Financial support received from Rameshwardasji Birla Smarak Kosh, Medical Research Centre, Bombay Hospital Avenue, Mumbai. We thankfully acknowledge for the support given.
REFERENCES

Phuong Nhi Boris and Christian Bories. Nitrate determination in biological fluids by an enzymatic one-step assay with Nitrate reductase. Clin Chem 1995; 4 (16) : 904-7.

Moncada S, Palmer RMJ, Higgs EA. Nitric oxide : Physiology, Pathophysiology and Pharmocological reviews. 1991; 43 (2) : 109-34.

Giovannoi G, Land JM, Keir G, Thompson EJ, Heales SJR. Adaptation of the nitrate reductase and Griess reaction method for the measurement of serum nitrate plus nitrite levels. Ann Clin Biochem 1997; 34 : 193-98.

Steven S Gross. Nitric oxide : Pathophysiological mechanisms. Annu Rev Physiol 1995; 57 : 737-69.

Han Moshage, Bart Kok, Hohannes R, Hviozenga, Peter LM Jansen. Nitrite and Nitrate determinations in plasma; A critical Evaluation. Clin Chem 1995; 41 (6) : 892-96.

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