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Urinary beta-trace protein as a tubular marker of renal dysfunction in patients with chronic kidney disease
Clinica Chimica Acta 411 (2010) 1154–1155
Contents lists available at ScienceDirect
Clinica Chimica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l i n c h i m
Letter to the Editor Urinary beta-trace protein as a tubular marker of renal dysfunction in patients with chronic kidney disease
Urinary protein analysis is of particular value in early diagnosis, prevention, screening, monitoring and treatment of chronic kidney disease (CKD). Beta-trace protein (BTP) or prostaglandin D2 synthase (EC 5.3.99.2.) is a low-molecular-weight protein (23–29 kDa) which has been detected in cerebrospinal fluid, serum, urine, amniotic fluid, and seminal plasma. It is almost completely excreted via the kidneys, has a constant production rate and is stable in urine pH [1]. BTP has been introduced as a potential marker in detecting impaired renal function [2]. Proteinuria is a well-known biomarker of kidney damage. Proteins with low molecular weight, such as α1-microglobulin (A1M), β2microglobulin (B2M) and cystatin C are biomarkers of proximal tubular injury because they are freely filtered at the glomerulus and then reabsorbed and metabolized in the proximal tubule [3–5]. Whereas BTP has the same properties like these proteins, it has been recognized as a tubular marker. The aim of this study was to investigate the clinical usefulness of the urinary levels of beta-trace protein for detection of renal impairment in patients with chronic kidney disease and to compare them with the levels of other tubular proteins. The study enrolled 134 patients with chronic kidney disease (72 females and 62 males), aged 20–80 years and 50 healthy volunteers with similar age and sex distribution. For all participants, 24-hour urine samples were collected and stored at 4 °C during collection. The study was conducted in accordance with the Helsinki Declaration. All study participants gave written informed consent. Creatinine, total protein and B2M were determined in urine samples on the day of collection. B2M was measured after the addition of stabilization buffer. Cystatin C, α1-microglobulin and BTP were measured later in urine samples stored at −70 °C. Results for urinary proteins were expressed in mg per day, and for urinary creatinine in mmol per day. BTP, A1M, cystatin C and B2M in urine samples were measured by a latex particle-enhanced immunonephelometric assay on a Behring Nephelometer II analyzer (Dade Behring, Marburg, Germany). The detection limits, specified by the manufacturer, for cystatin C, B2M and A1M were: 0.05 mg/L; 0.21 mg/L and 5.64 mg/L, respectively. Urinary creatinine was determined by a kinetic alkaline picrate method and urinary total protein by a turbidimetric method using benzethonium chloride, both on an ARCHITECT ci8200 analyzer (Abbott Diagnostics, Wiesbaden, Germany). Median, range, Mann–Whitney U-test and Spearman's test were used in statistical analysis. A P b 0.05 was considered statistically significant. Statistical analysis was performed using MedCalc for Windows, version 9.5.2.0 (MedCalc software, Mariakerke, Belgium). The median value in healthy males was 2.04 mg/day, and ranged from 0.50 to 4.68 mg/day (0.188 mg/mmol creatinine; 0.043– 0.635 mg/mmol creatinine). The median value in healthy females was 2.02 mg/day, and ranged from 0.32 to 3.80 mg/day (0.143 mg/mmol 0009-8981/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2010.04.013
creatinine; 0.028–0.516 mg/mmol creatinine). There was no statistically significant difference between genders considering the values of urinary BTP expressed in mg/day (P = 0.2793) and expressed in mg per mmol of creatinine (P = 0.3527), although the last ones were slightly higher in males. The healthy individuals had a median concentration of 2.03 mg/day, range: 0.32–4.68 mg/day (0.177 mg/mmol creatinine; 0.028–0.635 mg/mmol creatinine). Urinary values of BTP in patients with CKD (median, 3.93 mg/day; range, 0.14–63.52 mg/day) were significantly higher than in healthy individuals (P b 0.0001). Median (range) values for creatinine, total protein, cystatin C, A1M and B2M in patients were: 11.18 (2.38– 22.39) mmol/day, 0.98 (0.04–17.94) g/day, 0.27 (0.07–14.40) mg/day, 39.91 (10.85–450.0) mg/day and 0.75 (0.22–172.3) mg/day, respectively. Urinary concentrations of A1M, cystatin C and B2M in the healthy individuals were below the method detection limit. The highest correlation was observed between BTP and A1M urinary concentrations (r = 0.871). Likewise, beta-trace protein correlated significantly with cystatin C and total protein in 24-hour samples. Slightly weaker correlation was obtained with B2M concentrations. The negative correlation between urinary BTP and urinary creatinine was very poor, but significant (Table 1). Beta-trace protein belongs to the lipocalin family of proteins with carrier and enzymatic functions. It is a very stable enzyme and is highly resistant against heat treatment and protease digestion [1]. Serum and urinary levels of BTP have been shown to be increased in patients with various renal diseases [2]. In this study, we also found markedly increased BTP urinary concentrations in patients with CKD (range 0.14–63.52 mg/day). Reported data showed the upper reference limit of 0.45 mg/mmol creatinine for BTP measured by immunonephelometry [6]. In our findings, BTP concentrations ranged from 0.028 to 0.635 mg/mmol creatinine in healthy individuals. There was no statistically significant difference between genders, although the males had slightly higher concentrations. Slightly higher urinary BTP values in healthy males were also found in other studies and they can be explained with the presence of BTP in seminal plasma [7]. In this study, BTP was in significant correlation with all the measured urinary proteins (Table 1). Only correlation between urinary BTP and urinary creatinine was very poor, but significant. These results are in accordance with the recent findings where similar significant correlations between urinary BTP and urinary A1M and total protein, and no correlation with urinary creatinine were obtained [6]. BTP had the strongest correlation with A1M (r = 0.871). Urinary A1M is a well established early marker of proximal tubular damage and its excretion has been successfully used in the diagnosis of various types of renal tubular disease [5]. It has been considered as the best marker of tubular damage, because it is stable, easily measurable, sensitive in detecting tubular dysfunction and it is not significantly affected by inflammation and nonrenal pathological conditions [3,5]. Urinary concentrations of BTP also highly correlated with cystatin C concentrations (r = 0.759). Because of its small size and high isoelectric point (pI = 9.2), cystatin C is more freely filtered at the glomerulus than other low-molecular-weight proteins. However,
Letter to the Editor Table 1 The correlations between beta-trace protein and α1-microglobulin, cystatin C, total protein, β2-microglobulin and creatinine in 24-hour urine samples from patients with chronic kidney disease. Correlation beta-trace protein with urinary markers Urinary marker
rSpearman
95% CI
P
α1-Microglobulin Cystatin C Total protein β2-Microglobulin Creatinine
0.871 0.759 0.684 0.497 −0.175
0.805–0.915 0.632–0.846 0.582–0.765 0.309–0.648 − 0.334 to − 0.0052
b0.0001 b0.0001 b0.0001 b0.0001 0.0440
cystatin C may have lower diagnostic utility, because of very low levels in urine of patients with normal renal function [8,9]. Likewise, good correlation between BTP and B2M (r = 0.497) was found. The usefulness of B2M is limited because of its instability in urine at physiological pH and increased production in other diseases such as autoimmune diseases, infections and tumors [9]. BTP seems to be useful as a diagnostic marker for the early detection of renal tubular damage, because it correlates highly with A1M and other tubular proteins, has a constant production rate, and its concentrations do not vary in situations with an acute-phase reaction. Unlike B2M, it is stable over a wide pH range and, unlike A1M, does not bind other proteins [6,10]. Therefore, the results of our study support other reported data in stating that BTP may be a useful and alternative marker for tubular damage in patients with chronic kidney disease.
1155
[3] Herget-Rosenthal S, Poppen D, Husing J, et al. Prognostic value of tubular proteinuria and enzymuria in nonoliguric acute tubular necrosis. Clin Chem 2004;50: 552–8. [4] Mijusković Z, Maksić DJ, Hrvačević R, et al. Urinary cystatin C as a marker of tubular dysfunction. J Med Biochem 2007;26:98–102. [5] Penders J, Delanghe JR. Alpha 1-microglobulin: clinical laboratory aspects and applications. Clin Chim Acta 2004;346:107–18. [6] Vynckier LL, Flore KMJ, Delanghe SE, Delanghe JR. Urinary β-trace protein as a new renal tubular marker. Clin Chem 2009;55:1241–3. [7] Melegos DN, Diamandis EP, Oda H, Urade Y, Hayaishi O. Immunofluorometric assay of prostaglandin D synthase in human tissue extracts and fluids. Clin Chem 1996;42:1984–91. [8] Guder WG. Clinical biochemistry of renal function. Riv Med Lab - JLM 2002;3:28–31. [9] Lamb E, Newman DJ, Price CP. Kidney function tests. In: Burtis CA, Ashwood ER, Bruns DE, editors. Tietz textbook of clinical chemistry and molecular diagnostics. 4th Ed. St. Louis: Elsevier Saunders; 2006. p. 797–835. [10] Filler G, Priem F, Lepage N, et al. β-Trace protein, cystatin C, β2-microglobulin, and creatinine compared for detecting impaired glomerular filtration rates in children. Clin Chem 2002;48:729–36.
Marijana Dajak⁎ Institute of Medical Biochemistry, Clinical Center of Serbia Višegradska 26, 11000 Belgrade, Serbia ⁎ Corresponding author. Tel./fax: + 381 11 3615631. E-mail address: [email protected] (M. Dajak). Svetlana Ignjatović Institute of Medical Biochemistry, Clinical Center of Serbia and School of Pharmacy, Belgrade, Serbia Biljana Stojimirović Institute of Urology and Nephrology, Clinical Center of Serbia and School of Medicine, Belgrade, Serbia
Acknowledgements This study was conducted as a part of the project No. 145010B, financially supported by the Ministry of Science, Technology and Development of the Republic of Serbia. References [1] Urade Y, Hayaishi O. Biochemical, structural, genetic, physiological, and pathophysiological features of lipocalin-type prostaglandin D synthase. Biochim Biophys Acta 2000;1482:259–71. [2] Melegos DN, Grass L, Pierratos A, Diamandis EP. Highly elevated levels of prostaglandin D synthase in the serum of patients with renal failure. Urology 1999;53:32–7.
Snežana Gajić Institute of Urology and Nephrology Department, Dr Dragiša Mišović Clinic, Belgrade, Serbia Nada Majkić-Singh Institute of Medical Biochemistry, Clinical Center of Serbia and School of Pharmacy, Belgrade, Serbia
8 April 2010
Contents lists available at ScienceDirect
Clinica Chimica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l i n c h i m
Letter to the Editor Urinary beta-trace protein as a tubular marker of renal dysfunction in patients with chronic kidney disease
Urinary protein analysis is of particular value in early diagnosis, prevention, screening, monitoring and treatment of chronic kidney disease (CKD). Beta-trace protein (BTP) or prostaglandin D2 synthase (EC 5.3.99.2.) is a low-molecular-weight protein (23–29 kDa) which has been detected in cerebrospinal fluid, serum, urine, amniotic fluid, and seminal plasma. It is almost completely excreted via the kidneys, has a constant production rate and is stable in urine pH [1]. BTP has been introduced as a potential marker in detecting impaired renal function [2]. Proteinuria is a well-known biomarker of kidney damage. Proteins with low molecular weight, such as α1-microglobulin (A1M), β2microglobulin (B2M) and cystatin C are biomarkers of proximal tubular injury because they are freely filtered at the glomerulus and then reabsorbed and metabolized in the proximal tubule [3–5]. Whereas BTP has the same properties like these proteins, it has been recognized as a tubular marker. The aim of this study was to investigate the clinical usefulness of the urinary levels of beta-trace protein for detection of renal impairment in patients with chronic kidney disease and to compare them with the levels of other tubular proteins. The study enrolled 134 patients with chronic kidney disease (72 females and 62 males), aged 20–80 years and 50 healthy volunteers with similar age and sex distribution. For all participants, 24-hour urine samples were collected and stored at 4 °C during collection. The study was conducted in accordance with the Helsinki Declaration. All study participants gave written informed consent. Creatinine, total protein and B2M were determined in urine samples on the day of collection. B2M was measured after the addition of stabilization buffer. Cystatin C, α1-microglobulin and BTP were measured later in urine samples stored at −70 °C. Results for urinary proteins were expressed in mg per day, and for urinary creatinine in mmol per day. BTP, A1M, cystatin C and B2M in urine samples were measured by a latex particle-enhanced immunonephelometric assay on a Behring Nephelometer II analyzer (Dade Behring, Marburg, Germany). The detection limits, specified by the manufacturer, for cystatin C, B2M and A1M were: 0.05 mg/L; 0.21 mg/L and 5.64 mg/L, respectively. Urinary creatinine was determined by a kinetic alkaline picrate method and urinary total protein by a turbidimetric method using benzethonium chloride, both on an ARCHITECT ci8200 analyzer (Abbott Diagnostics, Wiesbaden, Germany). Median, range, Mann–Whitney U-test and Spearman's test were used in statistical analysis. A P b 0.05 was considered statistically significant. Statistical analysis was performed using MedCalc for Windows, version 9.5.2.0 (MedCalc software, Mariakerke, Belgium). The median value in healthy males was 2.04 mg/day, and ranged from 0.50 to 4.68 mg/day (0.188 mg/mmol creatinine; 0.043– 0.635 mg/mmol creatinine). The median value in healthy females was 2.02 mg/day, and ranged from 0.32 to 3.80 mg/day (0.143 mg/mmol 0009-8981/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2010.04.013
creatinine; 0.028–0.516 mg/mmol creatinine). There was no statistically significant difference between genders considering the values of urinary BTP expressed in mg/day (P = 0.2793) and expressed in mg per mmol of creatinine (P = 0.3527), although the last ones were slightly higher in males. The healthy individuals had a median concentration of 2.03 mg/day, range: 0.32–4.68 mg/day (0.177 mg/mmol creatinine; 0.028–0.635 mg/mmol creatinine). Urinary values of BTP in patients with CKD (median, 3.93 mg/day; range, 0.14–63.52 mg/day) were significantly higher than in healthy individuals (P b 0.0001). Median (range) values for creatinine, total protein, cystatin C, A1M and B2M in patients were: 11.18 (2.38– 22.39) mmol/day, 0.98 (0.04–17.94) g/day, 0.27 (0.07–14.40) mg/day, 39.91 (10.85–450.0) mg/day and 0.75 (0.22–172.3) mg/day, respectively. Urinary concentrations of A1M, cystatin C and B2M in the healthy individuals were below the method detection limit. The highest correlation was observed between BTP and A1M urinary concentrations (r = 0.871). Likewise, beta-trace protein correlated significantly with cystatin C and total protein in 24-hour samples. Slightly weaker correlation was obtained with B2M concentrations. The negative correlation between urinary BTP and urinary creatinine was very poor, but significant (Table 1). Beta-trace protein belongs to the lipocalin family of proteins with carrier and enzymatic functions. It is a very stable enzyme and is highly resistant against heat treatment and protease digestion [1]. Serum and urinary levels of BTP have been shown to be increased in patients with various renal diseases [2]. In this study, we also found markedly increased BTP urinary concentrations in patients with CKD (range 0.14–63.52 mg/day). Reported data showed the upper reference limit of 0.45 mg/mmol creatinine for BTP measured by immunonephelometry [6]. In our findings, BTP concentrations ranged from 0.028 to 0.635 mg/mmol creatinine in healthy individuals. There was no statistically significant difference between genders, although the males had slightly higher concentrations. Slightly higher urinary BTP values in healthy males were also found in other studies and they can be explained with the presence of BTP in seminal plasma [7]. In this study, BTP was in significant correlation with all the measured urinary proteins (Table 1). Only correlation between urinary BTP and urinary creatinine was very poor, but significant. These results are in accordance with the recent findings where similar significant correlations between urinary BTP and urinary A1M and total protein, and no correlation with urinary creatinine were obtained [6]. BTP had the strongest correlation with A1M (r = 0.871). Urinary A1M is a well established early marker of proximal tubular damage and its excretion has been successfully used in the diagnosis of various types of renal tubular disease [5]. It has been considered as the best marker of tubular damage, because it is stable, easily measurable, sensitive in detecting tubular dysfunction and it is not significantly affected by inflammation and nonrenal pathological conditions [3,5]. Urinary concentrations of BTP also highly correlated with cystatin C concentrations (r = 0.759). Because of its small size and high isoelectric point (pI = 9.2), cystatin C is more freely filtered at the glomerulus than other low-molecular-weight proteins. However,
Letter to the Editor Table 1 The correlations between beta-trace protein and α1-microglobulin, cystatin C, total protein, β2-microglobulin and creatinine in 24-hour urine samples from patients with chronic kidney disease. Correlation beta-trace protein with urinary markers Urinary marker
rSpearman
95% CI
P
α1-Microglobulin Cystatin C Total protein β2-Microglobulin Creatinine
0.871 0.759 0.684 0.497 −0.175
0.805–0.915 0.632–0.846 0.582–0.765 0.309–0.648 − 0.334 to − 0.0052
b0.0001 b0.0001 b0.0001 b0.0001 0.0440
cystatin C may have lower diagnostic utility, because of very low levels in urine of patients with normal renal function [8,9]. Likewise, good correlation between BTP and B2M (r = 0.497) was found. The usefulness of B2M is limited because of its instability in urine at physiological pH and increased production in other diseases such as autoimmune diseases, infections and tumors [9]. BTP seems to be useful as a diagnostic marker for the early detection of renal tubular damage, because it correlates highly with A1M and other tubular proteins, has a constant production rate, and its concentrations do not vary in situations with an acute-phase reaction. Unlike B2M, it is stable over a wide pH range and, unlike A1M, does not bind other proteins [6,10]. Therefore, the results of our study support other reported data in stating that BTP may be a useful and alternative marker for tubular damage in patients with chronic kidney disease.
1155
[3] Herget-Rosenthal S, Poppen D, Husing J, et al. Prognostic value of tubular proteinuria and enzymuria in nonoliguric acute tubular necrosis. Clin Chem 2004;50: 552–8. [4] Mijusković Z, Maksić DJ, Hrvačević R, et al. Urinary cystatin C as a marker of tubular dysfunction. J Med Biochem 2007;26:98–102. [5] Penders J, Delanghe JR. Alpha 1-microglobulin: clinical laboratory aspects and applications. Clin Chim Acta 2004;346:107–18. [6] Vynckier LL, Flore KMJ, Delanghe SE, Delanghe JR. Urinary β-trace protein as a new renal tubular marker. Clin Chem 2009;55:1241–3. [7] Melegos DN, Diamandis EP, Oda H, Urade Y, Hayaishi O. Immunofluorometric assay of prostaglandin D synthase in human tissue extracts and fluids. Clin Chem 1996;42:1984–91. [8] Guder WG. Clinical biochemistry of renal function. Riv Med Lab - JLM 2002;3:28–31. [9] Lamb E, Newman DJ, Price CP. Kidney function tests. In: Burtis CA, Ashwood ER, Bruns DE, editors. Tietz textbook of clinical chemistry and molecular diagnostics. 4th Ed. St. Louis: Elsevier Saunders; 2006. p. 797–835. [10] Filler G, Priem F, Lepage N, et al. β-Trace protein, cystatin C, β2-microglobulin, and creatinine compared for detecting impaired glomerular filtration rates in children. Clin Chem 2002;48:729–36.
Marijana Dajak⁎ Institute of Medical Biochemistry, Clinical Center of Serbia Višegradska 26, 11000 Belgrade, Serbia ⁎ Corresponding author. Tel./fax: + 381 11 3615631. E-mail address: [email protected] (M. Dajak). Svetlana Ignjatović Institute of Medical Biochemistry, Clinical Center of Serbia and School of Pharmacy, Belgrade, Serbia Biljana Stojimirović Institute of Urology and Nephrology, Clinical Center of Serbia and School of Medicine, Belgrade, Serbia
Acknowledgements This study was conducted as a part of the project No. 145010B, financially supported by the Ministry of Science, Technology and Development of the Republic of Serbia. References [1] Urade Y, Hayaishi O. Biochemical, structural, genetic, physiological, and pathophysiological features of lipocalin-type prostaglandin D synthase. Biochim Biophys Acta 2000;1482:259–71. [2] Melegos DN, Grass L, Pierratos A, Diamandis EP. Highly elevated levels of prostaglandin D synthase in the serum of patients with renal failure. Urology 1999;53:32–7.
Snežana Gajić Institute of Urology and Nephrology Department, Dr Dragiša Mišović Clinic, Belgrade, Serbia Nada Majkić-Singh Institute of Medical Biochemistry, Clinical Center of Serbia and School of Pharmacy, Belgrade, Serbia
8 April 2010