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Technical Briefs |
1
Laboratory Medicine,
2
Nephrology, and
3
Urology, University Hospital Charité, Humboldt University Berlin, 10098 Berlin, Germany;
4
Dade Behring Marburg GmbH, 35001 Marburg, Germany;
5
Gesellschaft für Biotechnologische Forschung, Department of Protein Glycosylation, 38124 Braunschweig, Germany;
a address correspondence to this author at: Department of Urology, Research Division, University Hospital Charité, Humboldt University Berlin, Schumannstrasse 20/21, 10098 Berlin, Germany
Recent studies have shown that low-molecular weight proteins in
serum seem to be better markers for detecting reduced glomerular
filtration rate (GFR) than the conventional measurement of serum
creatinine (1)(2). Several proteins, such as ribonuclease,
1-microglobulin,
ß2-microglobulin (B2M), and cystatin C have
been compared with serum creatinine (2)(3). Whereas serum
creatinine is increased only after a reduction of ~50% in GFR, the
above proteins, especially cystatin C, are already increased within
that so-called creatinine-blind range (1)(4)(5)(6). Recently,
another low-molecular weight protein, the ß-trace protein (BTP),
isolated primarily from cerebrospinal fluid (7), was shown
to be increased in patients with renal diseases (8)(9).
However, there is no information on the relationship of BTP and a
standard measure of GFR such as inulin clearance. Therefore, to
investigate the potential clinical usefulness of BTP for early
detection of reduced GFR, we have performed corresponding measurements
of BTP and inulin clearance.
The study included 115 diabetic patients (44 women; mean age, 53.4 years; 71 men, mean age, 52.9 years); 57 had type I diabetes and 58 had type II. GFR was determined by measuring the inulin clearance. A priming dose of 2.5 g of inulin (Inutest; Laevosan) was administered intravenously within 30 s, followed by a constant infusion (15 mL/h) of inulin at a rate dependent on serum creatinine (infusion rate, 1.5 g/h for serum creatinine concentrations <130 µmol/L; 0.75 g/h for serum creatinine concentrations between 130 and 265 µmol/L; and 0.38 g/h for serum creatinine concentrations between 265 and 450 µmol/L).
After 30 min for equilibration and after the patients were instructed to empty their bladders, two spontaneously voided urine samples were collected at 60-min intervals. Blood samples were taken before bolus injection as well as at the beginning and at the end of each urine collection period. After centrifugation (1600g for 15 min), samples of serum and urine were stored at -80 °C until analysis. A fully enzymatic assay for the quantification of inulin was used (10). GFR was calculated by taking the mean of the two clearances and correcting that value to 1.73 m2 of body surface area. Serum creatinine concentrations were determined by an enzymatic method, and B2M was determined by a turbidimetric method; both methods were performed on a Hitachi 717 analyzer with reagents supplied by Boehringer Mannheim (Creatinine plus and Tina-quant ß2-microglobulin). Controls with assigned values showed interassay imprecision (CV) of <5%.
For determination of BTP, a newly developed nephelometric research assay was performed on a BNA II analyzer (Dade Behring Marburg). This assay is based on the principle of latex particle-enhanced immunonephelometry using rabbit polyclonal antibodies against BTP. Calibration of the assay is based on highly purified BTP from cerebrospinal fluid characterized by amino acid sequencing and quantitative amino acid analysis. For the default sample dilution of 1:100, the basic measuring range is ~0.25–15.8 mg/L. The total analytical imprecision (intraassay plus interassay; n = 40) of the assay, calculated from two control materials and three serum samples with concentrations of 1.51–7.89 mg/L, was between 2.33% and 6.5%.
Statistical calculations were performed with SPSS 7.5 for Windows (SPSS Software). The diagnostic validity was evaluated by the ROC curve analysis. GraphROC for Windows, Ver. 2.1, was used for calculations of areas under the curves (11). Regression analysis was performed with the EVAPAK for Windows software, Ver. 3.01 (12). P <0.05 was considered statistically significant. The study was performed in accordance with ethics standards of the Helsinki Declaration of 1975 (revised in 1985).
The GFR was <80 mL/min in 41 patients and >80 mL/min in 74 patients,
which was considered as the lower cutoff limit of GFR. BTP showed
curvilinear behavior in relation to GFR, as did creatinine and B2M
(Fig. 1
, A-C). There were significant correlations between GFR and the
reciprocal concentrations of creatinine, B2M, and BTP
(r = 0.666, 0.514, and 0.672, respectively;
P <0.05); the differences between the correlation
coefficients, however, were not statistically significant (P
>0.05). The median values (and ranges) were 88 µmol/L
(49–331 µmol/L) vs 69 µmol/L (33–137 µmol/L) for creatinine,
2.17 mg/L (0.51–12.1 mg/L) vs 1.44 mg/L (0.85–3.59 mg/L) for B2M, and
0.82 mg/L (0.44–6.44 mg/L) vs 0.52 mg/L (0.32–1.09 mg/L) for BTP and
differed significantly between the two groups (Mann–Whitney
U-test, P <0.0001). For BTP, an upper 97.5%
reference limit of 0.79 mg/L was calculated by both parametric and
nonparametric approaches (13) considering patients with GFR
values >80 mL/min as individuals with normal GFR. Fig. 1D
shows ROC
curves of the three analytes to discriminate between patients with
normal (>80 mL/min) and reduced (<80 mL/min) GFR values. The curve of
BTP is above the curves of creatinine and B2M. The area under the BTP
curve is significantly higher than the areas under the two other
curves, demonstrating a higher power of discrimination
(P = 0.042). When the point with the highest diagnostic
efficiency (0.79 for creatinine and B2M, 0.81 for BTP) was selected as
the optimal decision limit, the threshold was 91 µmol/L for
creatinine, 2.34 mg/L for B2M, and 0.64 mg/L for BTP. The corresponding
diagnostic sensitivities and specificities were 49% and 96% for both
creatinine and B2M and 76% and 84% for BTP.
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In summary, our data support the view that BTP may be suitable as an indicator of reduced GFR even in the creatinine-blind range. Additional detailed studies are required, but are worthwhile because the search for improved markers of GFR continues (14).
Acknowledgments
This work was supported in part by the Fund of the German Chemical Industry (Grant 400770 to K.J.) and includes parts of the doctoral thesis of F.P.
Footnotes
fax 4930-2802-1402, e-mail klaus.jung{at}charite.de
References
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The following articles in journals at HighWire Press have cited this article:
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  S. M. Rombach, M. C. Baas, I. J. M. ten Berge, R. T. Krediet, F. J. Bemelman, and C. E. M. Hollak The value of estimated GFR in comparison to measured GFR for the assessment of renal function in adult patients with Fabry disease Nephrol. Dial. Transplant., March 9, 2010; (2010) gfq108v1. [Abstract] [Full Text] [PDF]
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T. Gerhardt, U. Poge, B. Stoffel-Wagner, B. Klein, H.-U. Klehr, T. Sauerbruch, and R. P. Woitas Serum levels of beta-trace protein and its association to diuresis in haemodialysis patients Nephrol. Dial. Transplant., January 1, 2008; 23(1): 309 - 314. [Abstract] [Full Text] [PDF]
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 C. A. White, A. Akbari, S. Doucette, D. Fergusson, N. Hussain, L. Dinh, G. Filler, N. Lepage, and G. A. Knoll A Novel Equation to Estimate Glomerular Filtration Rate Using Beta-Trace Protein Clin. Chem., November 1, 2007; 53(11): 1965 - 1968. [Abstract] [Full Text] [PDF]
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 A. Zhang, Z. Dong, and T. Yang Prostaglandin D2 inhibits TGF-beta1-induced epithelial-to-mesenchymal transition in MDCK cells Am J Physiol Renal Physiol, December 1, 2006; 291(6): F1332 - F1342. [Abstract] [Full Text] [PDF]
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A. R. Huber and L. Risch Recent Developments in the Evaluation of Glomerular Filtration Rate: Is There a Place for {beta}-Trace? Clin. Chem., August 1, 2005; 51(8): 1329 - 1330. [Full Text] [PDF]
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U. Poge, T. M. Gerhardt, B. Stoffel-Wagner, H. Palmedo, H.-U. Klehr, T. Sauerbruch, and R. P. Woitas {beta}-Trace Protein Is an Alternative Marker for Glomerular Filtration Rate in Renal Transplantation Patients Clin. Chem., August 1, 2005; 51(8): 1531 - 1533. [Full Text] [PDF]
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H. Oda, Y. Shiina, K. Seiki, N. Sato, N. Eguchi, and Y. Urade Development and Evaluation of a Practical ELISA for Human Urinary Lipocalin-Type Prostaglandin D Synthase Clin. Chem., September 1, 2002; 48(9): 1445 - 1453. [Abstract] [Full Text] [PDF]
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E. Arrer, C. Meco, G. Oberascher, W. Piotrowski, K. Albegger, and W. Patsch {beta}-Trace Protein as a Marker for Cerebrospinal Fluid Rhinorrhea Clin. Chem., June 1, 2002; 48(6): 939 - 941. [Full Text] [PDF]
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G. Filler, F. Priem, N. Lepage, P. Sinha, I. Vollmer, H. Clark, E. Keely, M. Matzinger, A. Akbari, H. Althaus, et al. {beta}-Trace Protein, Cystatin C, {beta}2-Microglobulin, and Creatinine Compared for Detecting Impaired Glomerular Filtration Rates in Children Clin. Chem., May 1, 2002; 48(5): 729 - 736. [Abstract] [Full Text] [PDF]
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 N. Hirawa, Y. Uehara, M. Yamakado, Y. Toya, T. Gomi, T. Ikeda, Y. Eguchi, M. Takagi, H. Oda, K. Seiki, et al. Lipocalin-Type Prostaglandin D Synthase in Essential Hypertension Hypertension, February 1, 2002; 39(2): 449 - 454. [Abstract] [Full Text] [PDF]
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R. P. Woitas, B. Stoffel-Wagner, U. Poege, P. Schiedermaier, U. Spengler, and T. Sauerbruch Low-Molecular Weight Proteins as Markers for Glomerular Filtration Rate Clin. Chem., December 1, 2001; 47(12): 2179 - 2180. [Full Text] [PDF]
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F. Priem, H. Althaus, K. Jung, and P. Sinha {beta}-Trace Protein Is Not Better than Cystatin C as an Indicator of Reduced Glomerular Filtration Rate Clin. Chem., December 1, 2001; 47(12): 2181 - 2181. [Full Text] [PDF]
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