CHAPTER 5 MEASUREMENT OF UREA AND URIC ACID IN BLOOD SERUM

Transcription

1 79 CHAPTER 5 MEASUREMENT OF UREA AND URIC ACID IN BLOOD SERUM 5.1 INTRODUCTION Urea and Uric acid are the metabolic nitrogenous wastes present in the body that can be measured in blood and urine. Serum uric acid reflects the interactions of four major processes: dietary purine intake, endogenous purine metabolism, urinary urate excretion, and intestinal uricolysis. Uric acid is a metabolite of purines, nucleic acids and nucleoproteins. Consequently, abnormal levels may be indicative of a disorder in the metabolism of these substances. Hyperuricaemia may be observed in renal dysfunction, gout, leukemia, polycythaemia, atherosclerosis, diabetes, hypothyroidism, or in some genetic diseases. Decreased levels are present in patients with Wilson s disease and Fanconis syndrome (Peters and Van Slyke 1946). The existing methods could be conveniently divided into two groups: reductive and enzymatic. The reductive methods are non-specific and involve the oxidation of uric acid with phosphotungstate reagent to allantoin with resultant blue coloring of tungstate solution. The enzymatic methods are specific. They involve the catalytic oxidation of uric acid with the enzyme uricase to allantoin with the formation of hydrogen peroxide (Tietz 1986). The peroxide concentration which is directly proportional to the concentration of uric acid could then be determined by a number of methods (Fossati et al 1980, Matsubara et al 1994, Miland et al 1996, Nakaminami et al 1999, Galbán et al 2001).

2 80 The amount of urea nitrogen is a breakdown product of protein metabolism. Urea formed in the liver as the end product of protein metabolism enters into the blood and is ultimately eliminated in the urine by the kidneys. Most kidney diseases affect urea excretion so that blood urea nitrogen (BUN) levels increase in the blood. It may also increase the dehydration or bleeding in the stomach and/or intestines or result in any side effect of some medications. Raised levels may also be seen in any state causing hypovolemia heart failure, starvation and urinary tract obstruction, etc. Urea is one of the first substances to be determined in biological fluids as one of the parameters of liver function tests. In routine procedures urea determination in biological fluids is carried out with chemical reagents and enzymatic methods (Orsonneaue et al 1992, Naslund et al 1998). 5.2 METHODOLGY Serum Separation The serum separation procedure is followed as per section (3.2.1) Urea Sample Preparation For sample preparation (Urea by Berthelot method a kit supplied by Aspen Laboratories, Baddi, Himachal Pradesh, India) has been used. To 10 l of the serum first 50 l of urease enzyme is added and incubated for 5 minutes at 37 o C. Then 1.5ml of phenol reagent and 1.5 ml of hypochlorite reagent are added and incubated for 20 minutes at 37 o C. The principles involved for this reaction are as follows: Urea +H 2 O Urease Ammonia + CO 2 Ammonia + Phenol+ Hypochlorite Nitropruside Indophenol

3 81 Urease hydrolyses urea into ammonia and carbon dioxide. In alkaline conditions, ammonia reacts with hypochlorite and phenol in the presence of nitropruside to form Indophenol colored complex. The intensity of the color is directly proportional to the concentration of urea in the sample Uric acid Sample Preparation 1 ml of uricase enzyme has been added to 20 l of serum (Uricase based on POD - a kit supplied by Merck, Mumbai, India). The solution is mixed well and incubated at 37 o C for 10 minutes. The principle involved in this reaction is represented as Uric acid + H 2 O + O 2 Uricase Allantoin + C O 2 +H 2 O 2 4-Aminoantipyrine + TBHBA + 2H 2 O 2 POD Quinoneimine dye + 3H 2 O Uric acid is hydrolyzed by uricase to allontoin and hydrogen peroxide. Hydrogen peroxide so formed reacts with 4-aminoantipyrine and 2,4,6-tribromo-3- hydroxy benzoic acid (TBHBA) in the presence of enzyme peroxidase (POD) to produce Quinoneimine dye compound. The intensity of the color is directly proportional to the concentration of uric acid in the sample Measurements The nonlinear refractive index (n 2 ) is calculated using the standard relations as briefed in section (3.2.3) 5.3 RESULTS AND DISCUSSION Measurement of Absorbance Spectra The absorption spectra are measured using UV-Vis spectrophotometer (SHIMADZU- UV-2401PC), and the spectra for both urea

4 82 and uric acid are found to be broad banded as depicted in Figures 5.1 and 5.2 respectively. Both have exhibited good absorption at 532 nm. Hence for further study 532 nm Nd:YAG laser is used Absorbance (a.u) Wavelength (nm) Figure 5.1 UV-Vis Spectra of standard urea with reagent Absorbance (a.u) Wavlength (nm) Figure 5.2 UV-Vis Spectra of standard uric acid with reagent

5 Measurement of Nonlinear Refractive Index The results of typical Z-scan normalized transmittance measurement for urea and uric acid are shown in Figures 5.3 and 5.4 respectively. As the concentration of the standard urea and uric acid increases, the normalized transmittance peak increases whereas the valley decreases respectively. It is found that ΔT p-v as well as refractive index value increase linearly with concentration of standard urea and uric acid as seen in Figures 5.5(a), (b) and 5.6(a), (b). Figure 5.7 (a) and (b) shows the linear variation of optical density with concentration of urea and uric acid respectively as measured with conventional colorimetric method. The experiments are repeated five times and the mean value of the nonlinear refractive index (n 2 ) is calculated from the normalized transmittance values. This calculated value is assumed to be the standard for measurement of unknown urea and uric acid content present in blood sample. This is arrived by plotting a linear graph of urea and uric acid concentration Vs nonlinear refractive index. The nonlinear refractive index value is first measured against the reagent blank solution mg/dl 50 mg/dl 60 mg/dl Normalised Transmittance Z (mm) Figure 5.3 Z-scan data of the standard urea

6 mg/dl 6 mg/dl 8 mg/dl Normalised Transmittance Z (mm) Figure 5.4 Z-scan data of the standard uric acid T p-v 0.3 R 2 =0.98 Y= x Concentration of urea (mg/dl) Figure 5.5(a) Linear variation of ΔT p-v with urea concentration

7 T p-v 0.3 R 2 =0.99 Y=0.0467x Concentration of uric acid (mg/dl) Figure 5.5(b) Linear variation of ΔT p-v with uric acid concentration n 2 x10-8 cm 2 /W R 2 =0.99 Y= Concentration of urea (mg/dl) Figure 5.6(a) Linear variation of n 2 with urea concentration

8 n 2 x10-8 cm 2 /W R 2 =0.99 Y= x Concentration of uric acid (mg/dl) Figure 5.6(b) Linear variation of n 2 with uric acid concentration 0.25 Optical Density (a.u) R 2 =0.99 Y= x Concentration of urea (mg/dl) Figure 5.7(a) Linear variation of optical density with urea concentration

9 87 Optical Density (a.u) R 2 =0.99 Y= x Concentration of uric acid (mg/dl) Figure 5.7(b) Linear variation of optical density with uric acid concentration The calibration has been made with the conventional colorimetric method and the results are tabulated in Table 5.1 for urea and Table 5.2 for uric acid. The common urea level in blood serum is mg/dl. The common uric acid level in blood serum is mg/dl range for Males and mg/dl range for Females. Table 5.1 Nonlinear refractive index (n 2 ) values for standard urea Standard Urea Concentration (mg/dl) Nonlinear refractive index n (cm 2 /W) ± ± ± ± ± ± 0.68

10 88 Table 5.2 Nonlinear refractive index (n 2 ) values for standard uric acid Standard Uric acid concentration (mg/dl) Nonlinear refractive index n (cm 2 /W) ± ± ± ± ± 0.38 From the Z-scan results we infer that, the nonlinear refractive index n 2 values for the common level of urea in blood serum (10-50 mg/dl) are ± 0.31 and ± cm 2 /W respectively. Likewise, for common level of uric acid in blood serum for males mg/dl and their corresponding n 2 values are 6.45 and cm 2 /W respectively. For females mg/dl and their corresponding n 2 values are 5.15 and ± cm 2 /W respectively Evaluation with Conventional Method Many trials have been performed to measure the urea and uric acid level with our proposed method. The blood samples are collected from six volunteers (Three males and three female). We could see that the results arrived are in good agreement with those of the conventional colorimetric method for urea Table 5.3 and for uric acid Table 5.4. This ascertains that the proposed method is on equality with the conventional colorimetric method.

11 89 Table 5.3 Serum urea measurement using colorimetric method and Z-scan method-a Comparison Sample collection Urea level Concentration of urea (mg/dl) Colorimetric method Z-scan method Male Normal Female Normal Male Normal Female Normal Female Normal Male Normal Each value is the mean of 5 individual observations. The P value (t-test value) is less than 0.01 at 1% significance level. Table 5.4 Serum uric acid measurement using colorimetric method and Z-scan method-a Comparison Sample collection Uric acid level Concentration of uric acid (mg/dl) Colorimetric method Z-scan method Female Normal Male Normal Male Normal Female Normal Male Normal Female Normal Each value is the mean of 5 individual observations. The P value (t-test value) is less than 0.01 at 1% significance level.

12 CONCLUSION We have measured the nonlinear refractive index values for urea and uric acid present in the serum sample by Z scan method with 532 nm Nd:YAG CW laser. The Z-scan measurements indicate that urea and uric acid in standard sample and blood sample exhibit nonlinear optical properties. Comparative analysis of these values with the one obtained by conventional colorimetric method shows that they are in good agreement. Thus Z-scan technique is found to be suitable for measurement of bioanalytes.