Distilled water (ml) 1 0.8 0.6 0.4 0.2 – 0.8
20% TCA (ml) – 111111
Mix and incubate all the tubes at 37C for 30 min. Centrifuge all the test tubes at 5000 rpm for
10 – 15 min and discard the supernatant completely.
0.5 N NaOH (ml) 0.5 0.5 0.5 0.5 0.5 0.5 0.5
Distilled water (ml) 1.5 1.5 1.5 1.5 1.5 1.5 1.5
Biuret reagent (ml) 2 2 2 2 2 2 2aAmount present in volume of BSA standard added in test tubes
9.5 Calculations
Plot a graph between amount of BSA at x-axis and absorbance at y-axis. Then
extrapolate quantity of proteins present in given urine sample from the graph. The
extrapolated amount will give amount of protein in 0.2 ml of urine sample. Express
protein amount per liter of urine sample or 24 h urine sample. The protein concen-
tration in urine sample can also be calculated by using equation:
Amount of protein¼OD of testamount of standardðmgÞ 1000
OD of standardvolume of sampleðmlÞ 1000
¼xg=L of urineNote For calculation, take BSA protein amount and OD of same standard, i.e., if
amount of S 2 standard is taken, then take OD of S 2 only. In formula, the value
1000 at denominator is taken to convert protein amount to per gram and at numerator
is used to convert value to per liter of urine.
Precautions
- Filter or centrifuge the urine, if urine sample is turbid.
- Don’t dissolve CuSO 4 in sodium-potassium tartrate solution directly in the
preparation of Biuret reagent.
9.6 Clinical Significance
The clinical significance of proteinuria has been discussed in the previous chapter.
9.6 Clinical Significance 41