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ORIGINAL ARTICLE
Year : 2007  |  Volume : 17  |  Issue : 1  |  Page : 7-9
 

Urine protein thiols in chronic renal failure patients


Department of Biochemistry, Kasturba Medical College, Manipal, India

Correspondence Address:
M Prakash
Department of Biochemistry, Kasturba Medical College, Manipal - 576 104
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0971-4065.35013

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 ~ Abstract 

Proteinuria is the most common finding in chronic renal failure (CRF). The thiol groups on proteins contribute most to the antioxidant status of the body. Serum protein thiols were found to be decreased in CRF. The current study was designed to find out the levels of such protein-bound thiols in the urine of patients with CRF. The study was conducted on urine and serum samples of 20 healthy controls and 40 CRF cases. Serum and urine protein thiol levels were determined by spectrophotometric method using dithionitrobenzoic acid (DTNB). Serum albumin, urine protein, and urine creatinine levels were estimated in 24-h urine samples by an automated analyzer. There was significant decrease in serum albumin ( P <0.01) and serum protein thiols ( P <0.01) in CRF patients compared to healthy controls. In the urine samples, there was significant increase in protein ( P <0.01) and decrease in protein-bound thiols ( P <0.01) in CRF cases compared to healthy controls. Serum albumin correlated positively with serum protein thiols (r = 0.561, P <0.01), and urinary protein correlated negatively with urinary protein-bound thiols (r = -0.410, P <0.009). Protein-bound thiols, the major antioxidants in the body, are decreased in CRF cases. Due to increased consumption of protein-bound thiols in such an oxidative environment, there was significant decrease in protein-bound thiols in urine.


Keywords: Thiols, chronic renal failure, proteinuria, serum protein thiols, urine protein


How to cite this article:
Mallikarjunappa S, Prakash M. Urine protein thiols in chronic renal failure patients. Indian J Nephrol 2007;17:7-9

How to cite this URL:
Mallikarjunappa S, Prakash M. Urine protein thiols in chronic renal failure patients. Indian J Nephrol [serial online] 2007 [cited 2019 Nov 21];17:7-9. Available from: http://www.indianjnephrol.org/text.asp?2007/17/1/7/35013



 ~ Introduction Top


Albumin, a major plasma protein which maintains the oncotic pressure, is also an important extracellular antioxidant. Albumin contains an exposed cysteine -SH (thiol) group and provides the bulk of total plasma thiol, a well-known antioxidant. [1]

Proteinuria is the most common finding in chronic renal failure (CRF) patients. CRF is an inflammatory state, with increased reactive oxygen species (ROS) and imbalance of ROS and the antioxidant defence mechanism. [2] Several previous studies have indicated an increased production of ROS [3] and the occurrence of lipid peroxidation [4],[5] in the plasma and the red blood cells of CRF patients.

Serum protein thiols were found to be decreased in CRF patients who were on conservative management or on hemodialysis. Such decreased protein thiols were correlated positively with the decrease in albumin levels. [6] The current study was designed to establish the relationship between serum albumin and protein thiols with protein-bound thiols in the urine of CRF patients.


 ~ Materials and Methods Top


The study was carried out on 40 CRF patients on conservative management and 20 healthy controls. Patients having GFR <30 ml/min and serum creatinine >1.6 mg/dl for more than 3 months, along with clinical and sonological findings were considered as having CRF. All of them were on a renal diet (50 gm protein and 5 gm salt/day). The healthy controls were not on any kind of prescribed medication or dietary restrictions. This study was approved by the institutional review board and informed consent was obtained from all the subjects involved in the study.

Under aseptic conditions, blood samples (5 ml) were drawn into plain vacutainers from the antecubital veins. The collected blood was allowed to clot for 30 min and then centrifuged at 2000 g for 15 min for clear separation of the serum. All assays were performed immediately after the serum was separated. From the same cases and controls, 24-h urine samples were collected in bottles containing toluene and was then assayed for urinary protein, creatinine, and protein thiols.

5 5´ dithio- bis (2-nitrobenzoic acid) (DTNB), was obtained from Sigma chemicals, St. Louis, M, USA. All other reagents were of analytical grade.

Serum and urine protein thiols were measured by a spectrophotometric method using DTNB. [7],[8] For this, 900 ml of 0.2 M Na 2 HPO 4 containing 2 mM of Na 2 EDTA, 100 ml serum, and 20 ml of 10 mM DTNB in 0.2 M Na 2 HPO 4 were taken in an Eppendorf tube and warmed to 37°C. The solution was mixed in a vortex mixer and transferred to a cuvette and the absorbance was measured at the end of 5 min at 412 nm. Both sample and reagent blanks were prepared and absorbances were noted at 412 nm. The absorbance of the sample and reagent blanks were subtracted from the serum absorbance values to obtain the corrected values. The calibration curve was produced using glutathione dissolved in phosphate-buffered saline (PBS). The protein thiol concentration in serum was determined from the standard curve, using the corrected absorbance values for serum. The protein thiol groups in plasma were calculated from the total thiol. However, there is little difference between total thiols and protein thiols, as the concentration of glutathione in plasma is very low. [6],[8] Values were expressed in µmoles/l for serum protein and in µmoles/g for urine protein. Serum creatinine and albumin and urinary total proteins and creatinine levels were estimated by spectrophotometric methods using an automated analyzer (Hitachi 911).

The results were expressed as mean ± standard deviation (SD). A P value of <0.05 was considered statistically significant. Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS-10, Chicago, USA). An independent t-test was used to compare mean values. Pearson correlation was applied to find out the correlation between the parameters.


 ~ Results Top


There was significant decrease in serum albumin ( P <0.0001) and serum protein thiols ( P <0.0001) in CRF patients as compared to healthy controls. In the urine samples, there were significant increase in protein ( P <0.0001) and a decrease in protein-bound thiols ( P <0.0001) in CRF cases as compared to healthy controls [Table - 1]. Serum albumin correlated positively with serum protein thiols (r = 0.561, P <0.001) [Figure - 1], and urine protein correlated negatively with urine protein thiols (r = -0.410, P <0.009) [Figure - 2].


 ~ Discussion Top


The results presented in this study demonstrate that the concentration of protein thiol groups (P-SH) in serum were markedly reduced in CRF patients compared to healthy controls. Decreased plasma P-SH levels may be due to enhanced free-radical generation in CRF, which is an inflammatory condition. [6] Albumin is an important chain-breaking extracellular antioxidant; it contains an exposed cysteine -SH group and provides the bulk of the total 'serum thiols.' These reduced thiol groups are oxidized by electron-deficient free radicals and, in the process, there occurs oxidation of the albumin molecule itself. [6],[9] This type of antioxidant property makes albumin a 'sacrificial' antioxidant. [1]

Since there is loss of albumin in the urine of CRF patients, logically, albumin-bound thiol groups should increase in urine. Contrary to this expectation, CRF patients showed significantly low levels of urinary protein thiols; there was also significant decrease in serum albumin and protein-bound thiol groups in CRF patients. These findings suggests that the albumin excreted in the urine is deficient in thiol groups. In that case, what is the fate of the albumin-bound thiol groups? We speculate that the decrease in protein thiols in the urine of CRF patients could be because of increased oxidation of albumin-bound thiol groups in the serum due to the existing oxidative stress. [6],[9] Increased oxidation of reduced -SH groups present on albumin decreased levels of serum protein thiols. [6],[9] Excretion of such albumin, deficient in the reduced form of thiol groups, in the urine decreased the levels of protein-bound thiols in urine.


 ~ Conclusion Top


Our data suggests that increased consumption of serum protein-bound thiols in CRF patients may be the possible biochemical basis for decrease in protein-bound urinary thiols.

 
 ~ References Top

1.Himmelfarb J, McMonagle E. Albumin is the major plasma protein target of oxidant stress in uremia. Kidney Int 2001;60:358-63.  Back to cited text no. 1  [PUBMED]  [FULLTEXT]
2.Galle J, Seibold S. Has time come to use antioxidant therapy in uremic patients? Nephrol Dial Transplant 2003;18:1452-5.  Back to cited text no. 2  [PUBMED]  [FULLTEXT]
3.Luciak M, Trznadel K. Free oxygen species metabolism during hemodialysis with different membranes. Nephrol Dial Transplant 1991;6:66-70.  Back to cited text no. 3  [PUBMED]  
4.Dasgupta A, Hussain S, Ahmad S. Increased lipid peroxidation in patients on maintenance hemodialysis. Nephron 1992;60:56-9.  Back to cited text no. 4  [PUBMED]  
5.Lucchi I, Banni S, Botti B, Cappelli G, Medici G, Melis MP, et al . Conjugated diene fatty acids in patients with chronic renal failure: Evidence of increased lipid peroxidation. Nephron 1993;65:401-9.  Back to cited text no. 5    
6.Prakash M, Upadhya S, Prabhu R. Protein thiol oxidation and lipid peroxidation in patients with uremia. Scand J Clin Lab Invest 2004;64:599-604.  Back to cited text no. 6  [PUBMED]  [FULLTEXT]
7.Ellman GL. Tissue sulfhydryl groups. Arch Biochem Biophys 1959;82:70-7.  Back to cited text no. 7  [PUBMED]  
8.Hu ML. Measurement of protein thiol groups and glutathione in plasma. In : Parker L, editor. Methods of enzymology. vol 233. Academic Press: California; 1994. p. 380-5.  Back to cited text no. 8    
9.Himmelfarb J, McMonagle E, McManamin E. Plasma protein thiol oxidation and carbonyl formation in chronic renal failure. Kidney Int 2000;58:2571-8.  Back to cited text no. 9    


    Figures

  [Figure - 1], [Figure - 2]
 
 
    Tables

  [Table - 1]

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