1. HEAT AND ACETIC ACID TEST (BOILING TEST)
This test is based on the principle that proteins
get precipitated when boiled in an acidic solution.
Urine should be clear; if not, filter or use supernatant from a centrifuged sample.
Urine should be just acidic (check with litmus paper); if not, add 10% acetic acid drop by drop until blue litmus paper turns red.
A test tube is filled 2/3rds with urine. The tube is inclined at an angle and the upper portion is boiled over the flame. (Only the upper portion is heated so that convection currents generated by heat do not disturb the precipitate and the upper portion can be compared with the lower clear portion). Compare the heated part with the lower part. Cloudiness or turbidity indicates presence of either phosphates or proteins (Figure 821.1). A few drops of 10% acetic acid are added and the upper portion is boiled again. Turbidity due to phosphates disappears while that due to proteins does not.
Figure 821.1 Principle of heat test for proteins
test occurs with tolbutamide and large doses of penicillins.
The reagent area of the strip is coated with an indicator and buffered to an acid pH
which changes color in the presence of proteins (Figures 821.2 and 821.3). The principle is known as “protein error of indicators”.
Figure 821.2 Principle of reagent strip test for proteins. The principle is called as ‘protein error of indicators’ meaning that one color appears if protein is present and another color if protein is absent. Sensitivity is 5-10 mg/dl. The test does not detect Bence Jones proteins, hemoglobin, and myoglobin
The reagent area is impregnated with bromophenol blue indicator buffered to pH 3.0 with citrate. When the dye gets adsorbed to protein, there is change in ionization (and hence pH) of the indicator that leads to change in color of the indicator. The intensity of the color produced is proportional to the concentration of protein. The test is semi-quantitative.
Figure 821.3 Grading of proteinuria with reagent strip test (above) and sulphosalicylic acid test (below)
Reagent strip test is mainly reactive to albumin. It is false-negative
in the presence of Bence Jones proteins, myoglobin, and hemoglobin. Overload (Bence Jones) proteinuria and tubular proteinuria may be missed entirely if only reagent strip method is used. This test should be followed by sulphosalicylic acid test, which is a confirmatory test. Highly alkaline urine, gross hematuria, and contamination with vaginal secretions can give false-positive reactions. Also read: URINE STRIP TEST — UNDERSTANDING ITS LIMITATIONS.
3. SULPHOSALICYLIC ACID TEST
Addition of sulphosalicylic acid to the urine causes formation of a white precipitate if proteins are present (Proteins are denatured by organic acids and precipitate out of solution).
Take 2 ml of clear urine in a test tube. If reaction of urine is neutral or alkaline, a drop of glacial acetic acid is added. Add 2-3 drops of sulphosalicylic acid (3 to 5%), and examine for turbidity against a dark background (Figure 821.3).
This test is more sensitive and reliable than boiling test.
False-positive test may occur due to gross hematuria, highly concentrated urine, radiographic contrast media, excess uric acid, tolbutamide, sulphonamides, salicylates, and penicillins.
False-negative test can occur with very dilute urine.
The test can detect albumin, hemoglobin, myoglobin, and Bence Jones proteins.
Comparison of reagent strip test and sulphosalicylic acid test is shown in Table 821.1.
Table 821.1 Comparison of two tests for proteinuria
||Reagent strip test
||Sulphosalicylic acid test
|2. Proteins detected
||All (albumin, Bence Jones proteins, hemoglobin, myoglobin)
|5. Type of test
QUANTITATIVE ESTIMATION OF PROTEINS
Indications for quantitative estimation of proteins in urine are:
- Diagnosis of nephrotic syndrome
- Detection of microalbuminuria or early diabetic nephropathy
- To follow response to therapy in renal disease
Proteinuria >1500 mg/ 24 hours indicates glomerular disease; proteinuria >3500 mg/24 hours is called as nephrotic range proteinuria; in tubular, hemodynamic and post renal diseases, proteinuria is usually < 1500 mg/24 hours.
Grading of albuminuria is shown in Table 821.2. There are two methods for quantitation of proteins:
- Estimation of proteins in a 24-hour urine sample, and
- Estimation of protein/creatinine ratio in a random urine sample.
Table 821.2 Grading of albuminuria
1. Quantitative estimation of proteins in a 24-hour urine sample: Collection of a 24-hour sample is given earlier. Adequacy of sample is confirmed by calculating expected 24-hour urine creatinine excretion. Daily urinary creatinine excretion depends on muscle mass and remains relatively constant in an individual patient. In adult males creatinine excretion is 14-26 mg/kg/24 hours, while in women it is 11-20 mg/kg/24 hours. Various methods are available for quantitative estimation of proteins: Esbach’s albuminometer method, turbidimetric methods, biuret reaction, and immunologic methods.
2. Estimation of protein/creatinine ratio in a random urine sample: Because of the problem of incomplete collection of a 24-hour urine sample, many laboratories measure protein/creatinine ratio in a random urine sample. Normal protein/creatinine ratio is < 0.2. In low-grade proteinuria it is 0.2-1.0; in moderate, it is 1.0-3.5; and in nephrotic- range proteinuria it is > 3.5.
This is defined as urinary excretion of 30 to 300 mg/24 hours (or 2-20 mg/dl) of albumin in urine.
Significance of Microalbuminuria
- Microalbuminuria is considered as the earliest sign of renal damage in diabetes mellitus (diabetic nephropathy). It indicates increase in capillary permeability to albumin and denotes microvascular disease. Microalbuminuria precedes the development of diabetic nephropathy by a few years. If blood glucose level and hypertension are tightly controlled at this stage by aggressive treatment then progression to irreversible renal disease and subsequent renal failure can be delayed or prevented.
- Microalbuminuria is an independent risk factor for cardiovascular disease in diabetes mellitus.
Detection of Microalbuminuria: Microalbuminuria cannot be detected by routine tests for proteinuria. Methods for detection include:
- Measurement of albumin-creatinine ratio in a random urine sample
- Measurement of albumin in an early morning or random urine sample
- Measurement of albumin in a 24 hr sample
Test strips that screen for microalbuminuria are available commercially. Exact quantitation can be done by immunologic assays
like radioimmunoassay or enzyme
linked immunosorbent assay.
BENCE JONES PROTEINURIA
Bence Jones proteins are monoclonal immunoglobulin light chains (either κ or λ) that are synthesized by neoplastic plasma
cells. Excess production of these light chains occurs in plasma cell
dyscrasias like multiple myeloma and primary amyloidosis. Because of their low molecular weight and high concentration they are excreted in urine (overflow proteinuria).
Bence Jones proteins have a characteristic thermal behaviour. When heated, Bence Jones proteins precipitate at temperatures between 40°C to 60°C (other proteins precipitate between 60-70°C), and precipitate disappears on further heating at 85-100°C (while precipitate of other proteins does not). When cooled (60-85°C), there is reappearance of precipitate of Bence Jones proteins. This test, however, is not specific for Bence Jones proteins and both false-positive and -negative results can occur. This test has been replaced by protein electrophoresis of concentrated urine sample (Figure 821.4).
Figure 821.4 Urine protein electrophoresis showing heavy Bence Jones proteinuria (red arrow) along with loss of albumin and other low molecular weight proteins in urine
Further evaluation of persistent overt proteinuria is shown in Figure 821.5.
Figure 821.5 Evaluation of proteinuria.
Note: Quantitation of proteins and creatinine clearance are done in all patients with persistent proteinuria