3.7.1.5 Urea Test Lab Report
3.7.2.1.5 Urea test
PRINCIPLE
Urea is hydrolyzed in the presence of urease to produce ammonia and CO2. The ammonia produced combines with 2 – oxoglutarate and NADH in the presence of GLDH to yield glutamate and NAD.
Urea + H2O + 2H+ 2NH4+ + CO2
NH4+ + 2-Oxoglutarate +NADH H2O +NAD+ + Glutamate
The decrease in absorbance due to the decrease of NADH concentration in unit time is proportional to the urea concentration. Ammonia which is produced by different decomposition processes is also determined by this method.
REAGENTS COMPOSITION
5 volume of Reagent 1 contains buffer and was mixed with 1volume of enzyme reagent (R2). Working reagent were prepared fresh prior to use. …show more content…
REAGENTS:
R1: - 0.9% sodium chloride solution.
R2:- Diazo reagent
Diazo reagent: - 7ml of sulphanilic acid + 1ml of sodium nitrite PROCEDURE
The test tubes were labelled blank, sample and standard. 250µl of R2 and 2ml of R1 was pipetted into the test tubes labelled sample and standard while only the R1 and 200µl of sample was pipetted into the test tube labelled blank. 200µl of sample and standard was also pipetted into the tubes labelled samples and standard respectively. The mixtures were mixed and allowed to stand for 7minutes at room temperature, after 7minutes, the absorbance were read at 546nm against the blank.
The concentration of bilirubin was calculated by the formula:
Total Bilirubin Conc. = [Sample Abs./Standard Abs.] × standard Conc.
3.6.2.2.5 Estimation of albumin content
PRINCIPLE
The test is based on the amount of albumin that binds Bromocresol green indicator in an acid medium to form a green BCG complex, the amount which is directly proportional to the albumin concentration present in the sample.
REAGENTS
Reagent I: Bromocresol green (0.24mM/L) + Succinate
PRINCIPLE
Urea is hydrolyzed in the presence of urease to produce ammonia and CO2. The ammonia produced combines with 2 – oxoglutarate and NADH in the presence of GLDH to yield glutamate and NAD.
Urea + H2O + 2H+ 2NH4+ + CO2
NH4+ + 2-Oxoglutarate +NADH H2O +NAD+ + Glutamate
The decrease in absorbance due to the decrease of NADH concentration in unit time is proportional to the urea concentration. Ammonia which is produced by different decomposition processes is also determined by this method.
REAGENTS COMPOSITION
5 volume of Reagent 1 contains buffer and was mixed with 1volume of enzyme reagent (R2). Working reagent were prepared fresh prior to use. …show more content…
REAGENTS:
R1: - 0.9% sodium chloride solution.
R2:- Diazo reagent
Diazo reagent: - 7ml of sulphanilic acid + 1ml of sodium nitrite PROCEDURE
The test tubes were labelled blank, sample and standard. 250µl of R2 and 2ml of R1 was pipetted into the test tubes labelled sample and standard while only the R1 and 200µl of sample was pipetted into the test tube labelled blank. 200µl of sample and standard was also pipetted into the tubes labelled samples and standard respectively. The mixtures were mixed and allowed to stand for 7minutes at room temperature, after 7minutes, the absorbance were read at 546nm against the blank.
The concentration of bilirubin was calculated by the formula:
Total Bilirubin Conc. = [Sample Abs./Standard Abs.] × standard Conc.
3.6.2.2.5 Estimation of albumin content
PRINCIPLE
The test is based on the amount of albumin that binds Bromocresol green indicator in an acid medium to form a green BCG complex, the amount which is directly proportional to the albumin concentration present in the sample.
REAGENTS
Reagent I: Bromocresol green (0.24mM/L) + Succinate