| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Technical Briefs |
1
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia 3800
2
Biochemistry Unit, SHCN Pathology Service, Monash Medical Centre, Clayton, Victoria, Australia 3168
aauthor for correspondence: fax 61-3-9876-5851, e-mail wayne.comper{at}med.monash.edu.au
It has recently been discovered that filtered proteins in the kidney are not excreted in their intact forms (1)(2). During renal passage, high-molecular mass proteins, including albumin, transferrin, and IgG, undergo degradation. The products of this degradation are then excreted in urine as heavily degraded fragments. In rats, 
90% of the excreted albumin is heavily degraded to fragments with molecular masses <10 000 Da, and in humans this percentage is even higher (1)(2).
The excretion of filtered protein in the form of protein fragments that are not detected by conventional protein assays has not been recognized in the clinical chemistry literature. Previous studies have shown that several techniques used routinely to measure urinary total protein and more specific techniques used to measure urinary albumin concentrations are insensitive to albumin fragments (3). Such techniques include immunochemical albumin assays, the benzethonium chloride method, the sulfosalicylate assay, the pyrogallol red assay, and the Coomassie blue assay for proteins (1)(3). This means that analysis of the total amount of a particular protein (intact plus fragments) excreted has never been accomplished and that total urinary protein/peptide excretion has been severely underestimated. The problem is particularly evident for normal and microalbuminuric urine: Whereas normal human excretion of intact albumin (as measured by RIA) is <25 mg/day, the excretion of accompanying derived fragments can be >1300 mg/day (1).
The aim of the present study was to analyze urine samples from 20 diabetic patients with varying degrees of microalbuminuria for albumin by immunochemical nephelometry (Beckman Array Protein Analysis System) and for total urinary protein by the Biuret assay (for peptide bonds) (4) and by the benzethonium chloride method (5). Analysis of samples from 20 diabetic subjects (Table 1
) demonstrated that the Biuret total protein value was 40- to 760-fold higher than the value obtained by nephelometry. This is in accord with the large quantity of fragments present in such urine samples that are not measured by nephelometry. The calibration curve for the Biuret assay, made with human albumin concentrations of 0–10 g/L, gave an absorbance range (550 nm) of 0.079–0.650 absorbance units (AU). The absorbance values for urine plus distilled water blanks were 
0.032 AU, and for urine plus alkaline tartrate (without copper), blanks were 0.040 AU. All urine Biuret results were corrected using sample blanks of urine plus alkaline tartrate.
|
The benzethonium assay results were higher than those for nephelometry (urinary albumin) but still far lower than the Biuret values. The total urinary protein measurements of 11 control urines exhibited similar ratios, with the Biuret method giving 1538 ± 1048 mg/L (mean ± SD), the benzethonium method giving 53 ± 26 mg/L, and urinary albumin by nephelometry giving 5.1 ± 2.4 mg/L. The Lowry assay, which measures certain amino acids (6), and the Biuret assay on six control urines gave protein concentrations of 2734 ± 886 and 1915 ± 1132 mg/L, respectively. For six diabetic urines, the results were 2677 ± 1254 mg/L (Lowry) and 2023 ± 687 mg/L (Biuret). The results from either the Biuret or the Lowry method were not significantly different for either the controls (P = 0.164, not significant) or the diabetic patients (P = 0.367, not significant). The results for the Bradford assay on the same samples were 24.7 ± 19.8 and 55.6 ± 38.7 mg/L, respectively.
Both nephelometry and the benzethonium assay, along with others in routine clinical use, do not detect all protein fragments (3). The excretion rates of protein based on the Biuret values are extremely high (Table 1
) and in the same range as those measured previously when the peptide fragments were taken into account (1).
The absorbance spectra of the Biuret chromogen in a urine sample and an albumin solution are shown in Fig. 1A
. The profiles are directly comparable, and there were no anomalous peaks at 550 nm, the wavelength used in the assay.
|
The Biuret assay showed quantitative recovery (99.3% ± 0.7%; n = 6) of albumin added to human diabetic urine (for controls, the recovery was 98.7% ± 0.5%; n = 5), demonstrating that the urine matrix does not affect the chromogen and thus validating the use of the Biuret assay to measure total protein concentration of urine samples.
To test whether the matrix of the urine (or urinary pigment) contributes to the Biuret chromogen, diabetic urine samples were filtered through an Amicon/Millipore membrane with a molecular mass cutoff of 500 (cat. no. 13022). This filtration indicated that most of the proteinaceous material (85.8% ± 9.4%; n = 5) was retained. Filtration of urine through an Amicon/Millipore membrane with a molecular mass cutoff of 10 000 (cat. no. 13622) gave a recovery of 96.5% ± 0.9% (n = 5) of the Biuret-reactive material in the filtrate. These experiments demonstrate that the protein fragments in diabetic human urine for the samples studied have molecular masses mainly between 500 and 10 000 Da. Similar results were obtained for control urines, where the membrane with a molecular mass cutoff of 500 retained 81.8% ± 6.4% (n = 6) of the material, whereas filtration through the membrane with a cutoff of 10 000 recovered 97.7% ± 0.8% (n = 6) in the filtrate.
The HPLC profile of a control urine sample is shown in Fig. 1B
(peaks measured by absorbance at 214 nm were also apparent when measured at 278 nm; not shown). Urine retained by the filter after filtration through a membrane with a molecular mass cutoff of 500 gave a similar profile (not shown). The whole profile was altered substantially by proteolytic digestion of the urine sample by trypsin (Fig. 1C
) or Glu C, another endoproteinase (not shown). These studies demonstrate that the HPLC peaks in human urine are proteinaceous materials.
Overall, these studies demonstrate that large quantities of low-molecular mass protein-derived material exists in urine, which had not been recognized previously.
Acknowledgments
We would like to acknowledge the kind assistance of Shane Reeve and Dr. Ian Smith of the Baker Institute, Melbourne, Australia, in performing the proteolytic digestions.
References
The following articles in journals at HighWire Press have cited this article:
|
|
 |
|
  B. L. West, M. M. Picken, and D. J. Leehey Albuminuria in acute tubular necrosis Nephrol. Dial. Transplant., October 1, 2006; 21(10): 2953 - 2956. [Full Text] [PDF]
|
|
|
|
 |
|
 H. Rincon-Choles, T. L. Vasylyeva, P. E. Pergola, B. Bhandari, K. Bhandari, J.-H. Zhang, W. Wang, Y. Gorin, J. L. Barnes, and H. E. Abboud ZO-1 Expression and Phosphorylation in Diabetic Nephropathy. Diabetes, April 1, 2006; 55(4): 894 - 900. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. L. Hortin and B. Meilinger Cross-Reactivity of Amino Acids and Other Compounds in the Biuret Reaction: Interference with Urinary Peptide Measurements Clin. Chem., August 1, 2005; 51(8): 1411 - 1419. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Agarwal On the nature of proteinuria with acute renal injury in patients with chronic kidney disease Am J Physiol Renal Physiol, February 1, 2005; 288(2): F265 - F271. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Gai, V. Cantaluppi, C. Fenocchio, D. Motta, S. Masini, A. Pacitti, and G. Lanfranco Presence of Protein Fragments in Urine of Critically Ill Patients with Acute Renal Failure: A Nephrologic Enigma Clin. Chem., October 1, 2004; 50(10): 1822 - 1824. [Full Text] [PDF]
|
|
|
|
 |
|
 G. C. Hard and Kanwar Nasir Khan Invited Review: A Contemporary Overview of Chronic Progressive Nephropathy in the Laboratory Rat, and Its Significance for Human Risk Assessment Toxicol Pathol, February 1, 2004; 32(2): 171 - 180. [Abstract] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) and undiluted urine (•) with the Biuret method (A) and HPLC analyses of a human control urine before (B) and after (C) trypsin digestion.