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Conventionally, Lowry and Bradford assays have been used to estimate the level of protein in human biological fluids. It is known that intact proteins would be predominantly reabsorbed and/or not filtered and hence, would be expected to be present at low levels in human urine in healthy individuals. However, it has been documented that urinary peptides are also detectable in this fluid by the Lowry's method along with intact proteins. In contrast, the Bradford assay has been shown to predominantly detect intact proteins. Hence, the quantification and validation of the levels of the urinary peptides, by these widely used and accepted assays, can pave the way for analytic work to characterize them and evaluate their possible role in health and disease.
The principal barrier to the passage of blood protein that has been thought to reside in the glomerular capillary wall. The restricted permeability of the glomerulus to filter the macromolecules is based on size, configuration and electrical charge. It is previously considered as the most of the proteins which get filtered get degradaded and reabsorbed by renal tubules into the blood stream. But now 95% of albumins in humans get reabsorbed from the proximal tubules and get degraded to small peptides (<10 kDa) which is excreted in urine.
Determining albumin (normal 24 hour normal values are approximately <150 mg/day) in the urine is helpful for early detection and treatment of patients at risk for renal (kidney) disease. The major conditions in which elevated levels of albumin in urine may be present include Type 1 and Type 2 Diabetes, hypertension and renal disease found in pregnancy. There have also been several reports in the literature of the association between the presence of specific peptides and diseases. For e.g., urine C-peptide is considered to be the gold standard for endogenous insulin secretion in Type I Diabetics. Characterization of the proteins/peptides, while extremely important, would perforce require their quantification and obtain comparable values consistent with those in the peer-reviewed literature. Hence, it is our
endeavor to estimate reproducibly the peptides present in human urine in health individuals after normalization to Creatinine levels.
MATERIALS AND METHODS :
The following instructions are therefore followed for the collection of 24hr urine specimen.
1. Collect the urine during each 24 hr period. It does not depends upon how much urine is urinated.
2. Collection of urine should be time dependent e.g. first urine sample to be collected will be at 6:10 am and second exactly after 6 hrs that is 12:10 pm.
3. Similarly collect the urine sample for evening and night.
4. The storage of each bottle should be made at room temperature if we have to perform the experiment within two days. Otherwise kept it in refrigerator for longer period experiment.
The spot and 6 hours and 24 hours urine sample would be collected using accepted procedures and normalized to Creatinine levels.
Protein/peptide levels will be assayed for by the Lowry and Bradford methods.
1.Stock standard of BSA :
Weigh accurately 50 mg of bovine serum albumin (fraction volume) and dissolve in distilled water and make up to 50 ml in a standard flask.
2. Working Standard : Dilute 10 ml of the stock BSA solution to 50 ml with distilled water in standard flask. One ml of this solution contains 200microgram of protein.
It is similar for both the assays.
FOR BRADFORD'S ASSAY :
Protein stock solution was dissolved in PBS (Phosphate Buffered Saline).
Composition of PBS: 8 gm of NaCl, 0.2 gm of KCl, 0.4 gm of Disodium hydrogen phosphate, 0.24 gm of Potassium dihydrogen phosphate. Maintain a pH 7.4, volume at 1 litres, autoclave for 30 minutes.
Bradford Reagent : The assay reagent is made by dissolving 100 mg of Coomassie Brillient Blue G250 in 50 ml of 95% ethanol. The solution is then mixed with 100 ml of 85% phosphoric acid and make up to 1 L with distilled water. The reagent should be filtered through Whatmann no.1 filter paper and then stored in an amber bottle at room temperature.
The absorbance is measured at 595 nm.
FOR LOWRY'S ASSAY :
We used a modified assay for the determination of urinary peptide. The reagent was prepared as follows.
Solution 1 : Alkaline sodium carbonate solution.
Take 2 gm of sodium hydroxide in 400 ml of double distilled water. Mix well and add 10 gm of anhydrous sodium carbonate. Shake well and make up to 500 ml using distilled water.
Solution 2 : Copper sulphate solution.
Dissolve 1 gm of copper sulphate in 50 ml of distilled water.
Solution 3 : Sodium potassium tartarate solution.
Dissolve 1 gm of sodium potassium tartarate in 50 ml water.
Solution 4 :Mixed reagent
Add 0.5 ml of solution 2 with 0.5 ml of solution 3.To this mixture add 99 ml of solution 1.
Solution 5 : Folin Ciocalteau reagent.
The absorbance is measured at 670 nm.
CREATININE TEST (Modified Jaffe's method) : For in vitro quantitative determination of Creatinine in human urine. Creatinine reacts with Picric Acid in an alkaline medium to form an orange coloured complex.
WORKING REAGENT PREPARATION : Preparing working reagent by mixing equal volume of Reagent 1(picrate reagent) with reagent 2(sodium hydroxide) to make up the desired volume. Mix gently for 3 minutes.
Dilution factor for Lowry's and Bradford's methods for urine sample according to micrograms proteins/ microlitres :
Dilution factor for Lowry's method
Dilution factor for Bradford's method
STATISTICAL ANALYSIS :
Statistical analysis was done by using the Microsoft Excel. Analysis of variance (ANOVA) followed by multiple comparison was done to compare the mean values.
The determination of urinary peptides has historically been debatable issue for many decades. Numerous methodologies have been applied to detect the urinary peptide most of the commonest and available method is based on colorimetric determination of urinary peptide that is by Lowry dye binding methods using dye like methyl orange, bromocresol green, pyrogallol red, Biorad based on the Bradford reaction, sulfosalicylic acid, turbidimetric or nephalometric methods. In our study, we estimated urinary peptides level based on differences and protein measured based on Lowry and Bradford assay. In other words the difference is substraction of Lowry values from the Bradford values to enable us to estimate urinary peptides level. This approach was based on the work reported by Dr. M. Prakash.
Since Creatinine levels are considered to be a quantitative indicator for renal function, spot test was done and the albumin to Creatinine ratio is determined. And the total protein to the Creatinine ratio was measured. The following values for the same is reported.Using Creatinine level correlate positively with peptide level which indicates that as urine Creatinine level get decreases due to renal injury or renal failure. The rate of formation of this complex is measured by reading the change in absorbance at 505nm ia a selected interval of time and is proportional to the concentration of Creatinine.
STANDARD 1(after 30 secs)
STANDARD 2(after 120 secs)
TEST 1(after 30 secs)
TEST 2(after 120 secs)
Note : It was observed similar Creatinine values were obtained for both the 24hrs and the random sample. This is consistent with the aspect information in the peer reviewed literature.
Correlation between 6 hrs urine sample by Lowry's method.
Correlation of 12 hrs urine sample by Lowry's method.
Correlation of 18 hrs urine sample by Lowry's method.
Correlation of 24 hrs urine sample by Lowry's method.
Correlation of 6 hrs urine sample by Barford's method.
Correlation of 12 hrs urine sample by Bradfod's method.
Correlation of 18 hrs urine sample by Bradfod's method
Correlation of 24 hrs urine sample by Barford's method.
Comparison between the 24 hrs urine sample by Lowry's and Barford's methods.
Urine Creatinine (mg/day)= AT2-AT1 / AS2-AS1 x 2 x Dilution factor x 24 hr Urine Sample.
STANDARD 1(after 30 seconds)
STANDARD 2(after 120 seconds)
TEST 1(after 30 seconds)
TEST 2(after 120 seconds)
Estimation of Creatinine in sample 1, sample 2 and random sample.
With the help of this Creatinine formula we calculated the result and the amount was less than 150 mg/day which is a normal level of Creatinine.
1.It is expected that we will be able to further validate this approach for the estimation of urinary peptides.
2. This approach will provide an opportunity evaluate the extent of intra and inter-individual variability in the levels of proteins/peptides using the two methods.
3. The standardized protocols can be further evaluated by concomitant measurements in urine samples from diseased and healthy individuals subsequent to the evaluation of albumin levels in these fluids in comparison with human serum albumin standards.