Development of a modified copper-cadmium reduction method for rapid assay of total nitric oxide

Abstract

The biomolecule, nitric oxide (NO) is short lived and is converted to two stable products—nitrite (NO₂) and nitrate (NO₃). NO₃ can be measured by colorimetric procedure involving enzymatic conversion to NO₂ and by HPLC. But these methods, though sensitive and specific, require expensive reagents or equipment. Reduction of NO₃ to NO₂ by copper-coated cadmium granules is a popular method devised by Cortas and Wakid (Clin. Chem., 1990, 36, 1440), but it takes 120 min. Our aim of this study is to modify the Cortas and Wakid method for rapid reduction of NO₃ to NO₂ for total NO estimation in serum. After reduction, NO₂ is estimated by Griess reaction. As per our results, complete reduction of NO₃ could be achieved by both the methods, but it took only 5 min by our method. Standard graphs were linear by both of the methods. Factors were found to be 143.4 and 196.7 by the Cortas and Wakid method and by our method, respectively. Recovery of NO₂ from NO₃ was found to be 105% and 106%, respectively. We measured total NO₂ in sixteen sera by both the methods and our method's coefficient of correlation was found to be 0.9924. Our method is comparable to that of Cortas and Wakid for complete reduction of NO₃ with the greatest advantage of decrease in the time span from 120 to 5 min. This is due to our development of an increased copper coating method on cadmium granules. Our rapid method saves much time for total NO estimation.

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Introduction

Nitric Oxide (NO) is a short lived product of a five-electron oxidation of amino acid L-arginine catalysed by nitric oxide synthase. The transient and volatile nature of NO makes it unsuitable for estimation by convenient methods; however, two stable oxidation products, nitrate (NO₃) and nitrite (NO₂), are formed. In biological fluids, nitrate can be measured by colorimetric procedure involving enzymatic conversion to NO₂ by nitrate reductase and by ion-exchange chromatography.^1,2 Though the methods are more sensitive and specific, but these require costly reagents or equipments. Reduction of nitrate to nitrite by a metal and subsequent estimation of NO₂ by diazotization (Griess reaction) has become a common procedure as it is sensitive, specific and inexpensive.^3,4 But this method is not free of drawbacks. Some metals reduce nitrite further to NO and some biological constituents (e.g. ascorbic acid and phosphate) interfere.⁵

Reduction of nitrate to nitrite by copper-coated cadmium granules is a popular method.⁵ The method has been refined by use of standard addition and by precipitation with zinc hydroxide. But the drawback of this method is that a long time (120 min) is required for reduction of NO₃.⁵

Aims and objectives

Development of a modified Copper-Cadmium Reduction method for rapid assay of total Nitric Oxide in serum

Materials and methods

Materials

All the chemicals used were of analytical grade.

Cadmium granules. Using a wire cutter we made small pieces (60–100 mg) of cadmium granules and stored them in H₂SO₄ (0.1 mol L⁻¹).

Glycine buffer. 1.5 gm of glycine was dissolved in about 90 ml of water, pH was adjusted to 9.7 with 2 mol L⁻¹ of NaOH solution and the final volume was made up to 100 ml (200 mmol L⁻¹). This is stable for one month at 4–8 °C.

CuSO₄ solution. A) 5 mmol L⁻¹ in glycine buffer for Cortas and Wakid method.

 B) 200 mmol L⁻¹ in glycine buffer for our Rapid Cadmium-copper reduction method.

ZnSO₄ solution. 75 mmol L⁻¹ in analytical water.

NaOH solution. 55 mmol L⁻¹ in analytical water.

Sulfanilamide. 1000 mg was dissolved in 100 ml of 3 mol L⁻¹ HCl solution (58 mmol L⁻¹). This is stable for one year at room temperature.

N-Naphthylethylene diamine dihydrochloride. 20 mg was dissolved in 100 ml of water (0.8 mmol L⁻¹). Stored at 4–8 °C.

Standards. 10, 25, 50, 75 and 100 μmol L⁻¹ working standards were prepared by diluting stock of 1.0 mmol L⁻¹ solution of NaNO₂ or KNO₃ in 10 mmol L⁻¹ of Na₂B₄O₇ solution on the day of use. Stock standards are stable for at least nine months at room temperature, while the working standard are stable for three days.⁵

Specimens. Left out sera from Clinical Biochemistry Laboratory was used for this study.

Methods

Deproteinization⁶. 250 μL of water/standard/serum was mixed with 1000 μL of ZnSO₄ solution and then, 1250 μL of NaOH solution was dispensed. This solution was kept at room temperature for 10 min and centrifuged to get transparent a colourless supernatant. The final pH was around pH 7.0

Activation of cadmium granules

Cortas and Wakid procedure. The cadmium granules were swirled for 1–2 min in 5 mmol L⁻¹ CuSO₄ solution. CuSO₄ solution was drained completely, then rinsed three times with CuSO₄ solution and lastly by water. Copper-coated granules were used within 10 min.

Our rapid procedure. This is a crucial step in our procedure. Acid from the granules was rinsed with water with three times. In a 100 ml beaker approximately 20 g of granules and 30 ml of 200 mmol L⁻¹ CuSO₄ solution were added. This was shaken gently on a shaker until the deep blue colour of CuSO₄ solution fades. This took about 10 min. CuSO₄ solution was drained off and fresh CuSO₄ solution was poured to submerge the granules. This was hand shaken gently for 30 s and the liquid was drained off. Granules were dried well with tissue paper. These were used within 20 min. Prolonged exposure of the granules to air diminishes their reductive ability and turns to blackish colour (Fig. 1).

Fig. 1 Changes in colour of cadmium granule from native to activated and then after prolong air exposure.

Reduction procedures

Cortas and Wakid procedure. 1000 μL of water/standards/serum filtrates were placed in glass tubes (12 mm × 75 mm) and then 1000 μL of glycine buffer were added. The reaction was started by adding about 1.5 g of activated cadmium granules in each tube. The tubes were shaken gently for 120 min on a shaker.

Our rapid procedure. 600 μL of water/standards/serum filtrates were placed in glass tubes. The reaction was started by adding two granules of Cu-coated cadmium. These were put on a shaker for 5 min. We omitted addition of equal volume of glycine buffer.

Nitrite assay. Nitrite was estimated by Griess reaction. From the above tubes 500 μL of sample were placed into fresh glass tubes. To it 250 μL sulfanilamide solution were mixed in, followed by 250 μL NED solution. We then waited for 10 min at room temperature for a pink colour development and absorbance was read at 545 nm within 60 min.

Results

We compared the effectivity of our method with that of the Cortas and Wakid Cd reduction method for reduction of nitrate to nitrite. The efficiency of our method to coat cadmium granules by copper was much greater (Fig. 2).

Fig. 2 Colours of cadmium granules.

Reduction of standard solutions of NO₃ to NO₂ (recovery test) were found to be 105% by the Cortas and Wakid method and 106% by our method (Tables 1 and 2). Standard graphs were also linear by both of the methods (Fig. 3a and b). We have checked linearity from 10–100 μmol L⁻¹. The factor (concentration of standard divided by absorbance of standard at 545 nm) was found to be 143.4 by the Cortas and Wakid method and 196.7 by our method. The blank absorbances were 0.036 and 0.024, respectively. Total NO₂ was measured in 16 serum samples by both of the methods (Table 2). Mean values were found to be 35.53 ± 33.82 μmol L⁻¹ by the Cortas and Wakid method and 39.03 ± 35.86 μmol L⁻¹ by our method, but the difference was insignificant (P > 0.40). Linear regression analysis gave a co-efficient of correlation (R²) 0.9924 with an X-axis intercept of 1.4884 (Fig. 4). Intra-assay coefficient of variation (C.V.) of samples (n = 8) were 5.2% by both the methods.

Table 1 Recovery of NO₂ from NO₃ by the Cortas and Wakid Cd-Cu reduction method (A₅₄₅) Factor: 143.4 and Recovery: 105%

μmol/L	NO2	NO3 → NO2
10	0.068	0.070
25	0.160	0.179
50	0.332	0.343
75	0.492	0.522
100	0.696	0.709

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Table 2 Recovery of NO₂ from NO₃ by our Rapid Cd-Cu reduction method (A₅₄₅) Factor: 196.7 and Recovery: 106%

μmol/L	NO2	NO3 → NO2
10	0.048	0.051
25	0.122	0.127
50	0.238	0.265
75	0.360	0.392
100	0.489	0.487

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Fig. 3 (a) Standard graph of total NO assay by the Cortas and Wakid Cd–Cu reduction method. (b) Standard graph of total NO assay by our Rapid Cd-Cu reduction method.

Table 3 Serum total NO assay (in μmol/L)

Sr. No.	Cortas and Wakid Cd reduction method	Our rapid Cd reduction method
1	7.74	9.83
2	23.51	28.71
3	19.19	13.96
4	38.00	35.2
5	13.62	16.32
6	30.10	34.80
7	124.16	132.94
8	18.92	23.40
9	28.10	34.02
10	23.08	28.12
11	27.96	34.61
12	83.30	88.10
13	92.32	100.29
14	13.48	14.16
15	11.32	14.16
16	13.76	15.93
Mean ± S.D.	35.535 ± 33.82	39.034 ± 35.86
	t = 0.284, P > 0.40

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Fig. 4 Correlation of serum total NO assay (μmol L⁻¹).

Reduction of NO₃ to NO₂ with time had been studied. We found complete reduction by 4 min and increasing the time up to 8 min did not change the reduction process (Table 3).

Table 4 Time course for reduction of NO₃ to NO₂ for 100 μmol L⁻¹ of KNO₃

Time/min	A 545
0	0.001
1	0.112
2	0.251
3	0.364
4	0.470
5	0.470
6	0.473
7	0.471
8	0.473

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Discussion

Estimation of the total NO is of importance for understanding of many biological processes. The enzymatic procedure using nitrate reductase enzyme is accurate, but it takes 15 min of time and is also expensive. The NO assay by Cortas and Wakid is also accurate, but very time consuming. Copper-cadmium alloy used by Sastry et al. for their NO assay also takes 60 min of time for reduction.⁷ So, we were compelled to do an experiment to shorten the time. The greatest advantage of our method is the time reduction from 120 min to only 5 min for reduction of NO₃ to NO₂. This was possible due to the ability of our method for increased copper coating on cadmium granules (Fig. 2). This increased copper coating increased the efficiency of conversion of NO₃ to NO₂. We checked the linearity of standard graph up to 100 μmol L⁻¹ of NO₂. However, upper limit of linearity was 250 μmol L⁻¹ as found by Cortas and Wakid.⁵ ¦The sensitivity of assay by our method is less as compared to the Cortas and Wakid method (0.197 vs. 0.143 μmol L⁻¹).

Values of serum NO₂ estimation by both the methods (Table 2) showed very good correlation (Fig. 4). Intra-assay C.V. were found to be same by both the methods (5.2%).

We did not add glycine buffer during the reduction of NO₃ as we found that the increased copper coating on Cd granules used to get dissolved in the buffer. In our experimental conditions, the time required for complete reduction of NO₃ was 4 min (Table 3). After 4 min there were no changes in absorbance up to 8 min which indicates that NO₂ was not further reduced to NO. Interference by other metal ions were not studied as it is a small modification of the widely used Cortas and Wakid method⁵ with the exceptions that

i) increased copper coating on cadmium where chemicals were the same and

ii) during reduction procedure we omitted addition of the glycine buffer.

Sastry et al. used copper-cadmium alloy in acidic media,⁷ Cortas and Wakid used alkaline media,⁵ whereas we used neutral media for reduction of nitrate to nitrite to measure NO in biological samples.

Our copper coated cadmium granules effectivity is not stable. The granules begin loosing reduction capacity after about 20 min and turns to a black colour on prolonged standing (Fig. 1). Thus we have developed a rapid method that anyone can adopt for total NO assay. It is a fast and reliable method and will provide the choice of a cheaper alternative.

Conclusion

Our rapid method saves much time for total NO assay.

References

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2. D. P. Jong and M. Burgraff, Clin. Chim. Acta, 1983, 132, 63–71.
3. E. Hegesh and J. Shiloah, Clin. Chim. Acta, 1982, 125, 107–115.
4. C. D. Usher and T. M. Telling, J. Sci. Food Agric., 1975, 26, 138–144.
5. N. K. Cortas and N. W. Wakid, Clin. Chem., 1990, 36, 1440–1443.
6. M. Somogyi, J. Biol. Chem., 1930, 86, 655–663.
7. K. V. H. Sastry, R. P. Moudgal, J. Mohan, J. S. Tyagi and G. S. Rao, Anal. Biochem., 2002, 306, 79–82.

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