Chemical Examination of Urine Protein
Introduction
IN the normal kidney only a small amount of low molecular weight protein is filtered at the glomerulus. Glomerular membrane prevents the passage of high molecular weight proteins such as albumin (mol. wt. 69000) and gamma globulin (mol. wt. 1,80000). After filtration most of the protein is reabsorbed in the tubules. About < 150 mg protein /24 hrs is generally excreted which is not detected by chemical methods.
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Renal tubules secrete a mucoproteins called as Tamm-Horsfall protein. This protein is normally excreted in urine. There are two main mechanisms by which proteinuria can occur
- Glomerular damage
- Defect in the reabsorption process of the tubules.
In glomerular damage the capillary walls become more permeable. Large molecular weight protein, mainly albumin, is passed in urine. The presence of protein may be the first sign of a serious problem. However, it is necessary to find out the related clinical condition whether it is pr renal, renal or postrenal.
Some of the conditions associated with glumeular proteinuria (renal conditions) are glomerular nephrites, hypertension and lipoid nephrosis. In diminished tubular reabsorption low molecular weight protein are not completely reabsorbed and hence they are present in urine. The related conditions are
- Pyelonephritis.
- Renal tubular acidosis.
- Cystinosis.
- Interstitial nephritis.
- Rejection of kidney allografts.
Proteinuria is also found in the wide range of perenal conditions which produces it because of secondary effects on the kidneys. The proteinuria generally disappear when the primary disease is cured.
Examples are
- Dehydration,
- Intestinal obstruction,
- Myocardial infraction,
- Intra abdominal tumours, (since there is intra abdominal pressure on ascites)
- Fevers.
In postrenal condition’s, proteins may be added in urine as it passes along the urinary tract. Lesions of the renal pelvis, bladder, prostate and urethra can all lead to such a condition.
The urine should be clear if it is a turbid, it is necessary to centrifuges it. In that case, superintendent is tested for protein and sediment for the microscopic examination. Even after centrifugation, if the urine is turbid then filter it. If urine is alkaline, add a few drops of glacial acetic acid and make a slightly acidic.
Requirements
- Test Tube
- Pasteur pipettes
- 3 g/dl sulfosalicylic acid
- Glacial acetic acid
- Bunsen burner
Methods
- Sulfosalicylic Acid test method
- Heat test or Heat and acetic acid method
- Strip Test
- Eshbach’s method (Quantitative method)
Procedure (Sulfosalicylic Acid test method)
Principle:- If proteins are present in urine, these are present in soluble form. However, when Sulfosalicylic acid reagent is added, these proteins appear as white precipitate due to denaturation by the acidic reagent. Presence of turbidity at the top of the reaction mixture indicate presence of protein in urine.
- Take a clean dry life and test.
- Transfer 3 to 4ml of centrefuged Urine to a small test tube.
- Add two to three drop of Sulfosalicylic acid on the top of the specimen.
- Observed for turbidity after five minutes.
Observations
- No formation of diabetes at the upper portion of urine : Protein absent
- Formation of turbidity : Protein present
- If proteins are present, grade the result according to the degree of turbidity as trace, +, ++, +++, ++++.
Procedure (Heat test or Heat and acetic acid method)
Principle:- If proteins are present in urine due to heating, these are denatured and their specific structure get disrupted. Denatured proteins appear as white precipitate. Presence of turbidity which persist after the addition of glycerol acidic acid indicates presence of protein in urine.
- Place 5 to 10ML of clear urine in a test tube.
- Boil the upper portion over a flame.
- If turbidity develops, add one to two drops of glacial acetic acid.
- If the turbidity is due to phosphate precipitation it will clear.
- Reboiled the specimen.
Observation after reboiling the specimen
- No turbidity : protein absent
- Presence of turbidity : proteins present
- Grade the degree of observability as mentioned above.
Bence Jones Protein
It consists of dimers of either kappa or lambda, light chain from immunoglobulins. The molecular weight is very small about 44000, hence it is easily filtered through the normal glomerulus. It was first detected by Henry Bence Jones in 1847.
Bence Jones protein has unusual solubility properties. It precipitates when heated to 40 to 60 degree Celsius but becomes soluble again when boiled. It reappears after cooling. There is malignant proliferation of plasma cells in multiple myeloma, usually in the bone marrow. Nearly 50 to 80% patient with multiple myeloma will have Bence Jones protein in their urine. The remaining cases can be diagnosed by serum electrophoresis.
