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NDT Advance Access originally published online on August 23, 2007
Nephrology Dialysis Transplantation 2008 23(1):309-314; doi:10.1093/ndt/gfm510
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© The Author [2007].
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oxfordjournals.org


Serum levels of beta-trace protein and its association to diuresis in haemodialysis patients

Thomas Gerhardt1, Uwe Pöge1, Birgit Stoffel-Wagner2, Burkhard Klein1, Hans-Ulrich Klehr1, Tilman Sauerbruch1 and Rainer P. Woitas1

1Department of Internal Medicine I and 2Institute of Clinical Biochemistry, University of Bonn, Germany

Correspondence and to: Dr Thomas Gerhardt, Department of Internal Medicine I, University Hospital, Sigmund-Freud-Str. 25, D-53105 Bonn, Germany. Email: thomas.gerhardt{at}ukb.uni-bonn.de



   Abstract
Top
 Abstract
Introduction
 Patients and methods
 Results
 Discussion
 References
 
Background. Beta-trace protein (BTP) has been proposed as an alternative endogenous marker of the glomerular filtration rate. However, possible determinants of BTP in ESRD patients undergoing regular renal replacement therapy have not been evaluated.

Methods. Serum levels of BTP, beta-2-microglobulin, creatinine and urea were analysed before and after dialysis treatment in 73 patients [haemodialysis (HD) n = 52; haemodiafiltration (HDF) n = 21]. Patients were categorized into four groups with residual diuresis (RD) < 0.5 l/day (group 1; n = 24), 0.5–1 l/day (group 2; n = 18), 1.1–1.5 l/day (group 3; n = 12) and >1.5 l/day (group 4; n = 19). Subsequently RD was compared to pre-treatment levels of BTP.

Results. HD treatment did not affect BTP serum levels [pre-treatment 8.1 ± 4.1 mg/l (mean+SD) vs post-treatment 7.7 ± 4.1 mg/l; –0.6 ± 16.1%; ns]. However, in 6 out of 21 patients undergoing HDF BTP levels were reduced by more than 20%. Overall, the resulting decrease in serum concentration was minuscule (9.6 ± 6.2 vs 8.3 ± 4.9 mg/l; –14 ± 21.9%; P = 0.03). BTP serum levels were tightly associated to RD of the four groups. Comparison of BTP levels showed significant differences between patients of groups 1 vs 3 and 4 as well as 2 vs 4.

Conclusions. BTP serum levels may serve as a surrogate marker for residual renal function since HD and HDF do not exert clinical relevant alterations on them. Furthermore, BTP serum concentrations appear strongly associated to RD.

Keywords: beta-trace-protein; beta-2-microglobulin; haemodiafiltration; haemodialysis; residual renal function



   Introduction
Top
 Abstract
 Introduction
Patients and methods
 Results
 Discussion
 References
 
Beta-trace-protein (BTP) (MW 25.2 kDa) was primarily isolated as prostaglandin D2 synthase from cerebrospinal fluid and found to be increased in the serum of patients with renal failure [1]. The half-life of BTP is approximately 1.2 h and it is virtually exclusive excreted via the kidneys [2]. Therefore, it has been proposed as a new endogenous marker of glomerular filtration rate (GFR) [3–5].

Markedly increased serum levels of BTP have been reported in patients undergoing haemo- and peritoneal dialysis [6]. Although the mode of renal replacement therapy (RRT) importantly affects removal of middle molecules and small-molecular weight proteins [7], the impact of major determinants of RRT on BTP has not been investigated to date. The aim of this study was to evaluate the serum levels of BTP in patients on chronic RRT, with special emphasis on patient and treatment characteristics as well as residual diuresis (RD).



   Patients and methods
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 Abstract
 Introduction
 Patients and methods
Results
 Discussion
 References
 
Seventy-three patients (24 female, 49 male) on continuous intermittent RRT aged 59 ± 15.2 years (mean + SD) were included in the study.

Treatment consisted of haemodialysis (HD) in 52 and of haemodiafiltration (HDF) using the post-dilution method in 21 patients. Further treatment characteristics are given in Table 1.


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  		Table 1. Treatment characteristics and pre-treatment levels of crea, urea, BTP and B2MG

 
Five different high flux dialyzers were used throughout the study: polysulfone membranes: Braun HI-PS 18 (effective surface 1.8 m2, UFC 55 ml/h/mmHg; Braun, Germany) (HDF n = 6; HD n = 2), FX60 (effective surface 1.4 m2, UFC 46 ml/h/mmHg; Fresenius, Germany) (HDF n = 6; HD n = 15), F60S (effective surface 1.35 m2, UFC 40 ml/h ml/h/mmHg; Fresenius, Germany) (HDF n = 1; HD n = 20) and polyamide membranes: Arylane H 6 (effective surface 1.57 m2: UFC 69 ml/h/mmHg; Hospal, Switzerland) (HDF n = 2; HD n = 5), Polyflux 14 S (effective surface 1.4 m2, UFC 62 ml/h/mmHg; Gambro, Germany) (HDF n = 6; HD n = 10). Single pool Kt/V was calculated using the Efficacy© software (Riegel, Kuhlmann et al., Homburg, Germany).

The study was approved by the local ethics committee and informed consent was obtained from all patients enrolled in the study.

Parameters and assays
Before and after the dialysis treatment, 5 ml serum was drawn for determination of the analytes as described in detail elsewhere [8].

Serum creatinine (crea) was measured with a modified Jaffe-method using the DimensionTM Clinical Chemistry System (Dade Behring, Marburg, Germany). Serum beta-2-microglobulin (B2MG) and BTP were analysed by fully automated, latex-enhanced immunonephelometry (N latex B2 mg, N latex BTP, respectively, on a Nephelometer II; Dade-Behring). Total 95% reference intervals established from a representative cohort of 100 female and 100 male healthy blood donors (median age, 31 years; 2.5–97.5 percentiles, 19.0–60.5 years) were 0.402–0.738 mg/l for BTP, 1.085–2.015 mg/l for B2MG and 0.6–1.2 mg/dl for crea as described elsewhere [4]. The reduction of the analytes by treatment was corrected for body weight loss as described by Bergström and Wehle [9] to counteract ultrafiltration effects on serum levels of the analytes.

Data on RD were documented at study inclusion. In out-patients attending the study, volumes of RD were determined by a 24 h urine collection during a ‘long interval’ as routinely performed at home quarterly, whereas in in-patients the urine was collected by the nurse at the ward.

Statistical analysis
Results were expressed as the arithmetic mean and standard deviation (mean ± SD). Statistical analysis was performed using the StatView 5.0TM Software (Version for Windows; SAS Institute Inc., Cary, NC, USA).

Differences between two groups (HD vs HDF) were analysed by the Mann–Whitney–U-test or t-test for unpaired data and pre- vs post-session data by the Wilcoxon Sum Rank test or paired t-test, where applicable. Differences between BTP serum levels in the four groups categorized by RD and the groups with different dialysers were analysed by ANOVA (+ Bonferroni/Dunn post-hoc test). P-values <0.05 were considered significant.



   Results
Top
 Abstract
 Introduction
 Patients and methods
 Results
Discussion
 References
 
Age, body weight, duration of the dialysis sessions, the solution-flow rate, the fluid removal rate and kt/V did not differ between HD and HDF groups (Table 1). On average, the blood flow rate was some 24 ml/min higher in the HDF group. The mean volume of replacement fluid in the HDF group was 17.5 ± 5 l per session.

Pre-treatment levels of BTP
Pre-treatment levels of BTP were markedly elevated in all patients studied. The mean serum level was 8.5 ± 4.8 mg/l being 11.5-fold the upper normal range derived from a control group of 200 healthy blood donors (0.402–0.738 mg/l). Pre-treatment levels of all analytes are given in Table 1.

There was no significant correlation between pre-dialysis serum levels of BTP and age, body weight or time since start of RRT. BTP serum concentrations measured in male patients were not statistically different from those measured in female patients (data not shown).

Influence of haemodialysis and haemodiafiltration on serum concentration of analytes
As shown in Figure 1 and Table 2 HD and HDF treatment reduced crea, urea and B2MG significantly. In contrast, BTP levels were not altered significantly by HD treatment (pre-treatment 8.1 ± 4.1 mg/l vs post-treatment 7.7 ± 4.1 mg/l, P = ns), whereas a small reduction vs baseline was found for BTP levels after HDF treatment (9.6 ± 6.3 vs 8.3 ± 4.9 mg/l, P = 0.03). Figure 2 shows in detail the pattern of BTP serum-level reduction delineating a distinct range that was more pronounced in HDF patients (–0.6 ± 16 vs –14 ± 22%; P = 0.027).


Figure 1
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  		Fig. 1. Percent reduction of the studied analytes in comparison to pre-treatment levels shown as the dotted baseline. Data are given as box plots indicating 25th and 75th percentile and the median; the whiskers designate 10th and 90th percentile.

 

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  		Table 2. Pre- and post-treatment levels of crea, urea, BTP and B2MG divided in HD and HDF

 

Figure 2
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  		Fig. 2. Post-treatment BTP reduction (%) depicted according to the dialyse membrane and mode of treatment applied.

 
Influence of treatment determinants on reduction of analytes
Reduction of BTP was analysed in accordance to the characteristics of dialysers used (Figure 2).

UF-corrected reduction of BTP correlated weakly but significantly to the dialysers surface (r = 0.283; P = 0.023) and the membranes ultrafiltration-coefficient (r = 0.254; P = 0.042). Applying ANOVA analysis the UF corrected percental BTP reduction in patients treated with Braun HI-PS dialysers (22.9 ± 26.5%) was significantly larger compared to treatments performed with F 60 S (–1.5 ± 13.1%; P = 0.0016) dialysers. No differences were observed between all other filters. Although blood flow- (r = 0.103; P = 0.9) and solution-flow rate (r = 0.108; P = 0.4) were not correlated to BTP reduction, there was a weak but significant correlation to the duration of the dialysis session (r = 0.264; P = 0.027). Single-pool Kt/V as a measure of dialysis dose was significantly correlated to the reductions of crea, urea and B2MG, but not to that of BTP (Table 3).


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  		Table 3. Correlation between body weight loss corrected, relative reduction of analytes to single-pool Kt/V (spKt/V)

 
Pre-treatment levels of analytes and RD
Patients were categorized into four groups with RD <0.5 l/day (group 1; n = 24), 0.5–1 l/day (group 2; n = 18), 1.1–1.5 l/day (group 3; n = 12) and >1.5 l/(group 4; n = 19).

As shown in Figure 3A, BTP levels were significantly different between patients of groups 1 vs 3 and 4 as well as 2 vs 4. In contrast, no differences were found for pre-dialysis B2MG levels between the groups (Figure 3B). Pre-treatment crea and urea levels were significantly different between patients of groups 1 and 4 only (Figure 3C and 3D).


Figure 3
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  		Fig. 3A–D. Pre-treatment levels of the analytes in four groups of patients categorized by RD (l/24 h). Significance levels are indicated by horizontal brackets. RD = residual diuresis (l/24 h).

 
By applying ROC analysis, a serum BTP concentration of 8.2 mg/l was identified as criterion [sensitivity of 75.5% (95% CI: 61.1–86.6), specificity of 70.8% (48.9–87.3)] to distinguish between no relevant RD and more than 0.5 l diuresis per day. The positive predictive value was calculated as 84.1%, the negative predictive value tested as 59.3%.



   Discussion
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
References
 
In this study, we show that serum levels of BTP are not altered significantly by HD. Although HDF treatment on average results in a small diminution of BTP this appears to be without clinical relevance in comparison to the other analytes studied. Furthermore, we found a striking association of BTP serum concentration with the amount of RD, suggesting BTP as a surrogate marker of residual renal function (RRF).

On average, we found an 11.5-fold increase of pre-treatment BTP serum levels in our cohort compared to the upper normal range of healthy blood donors. In accordance with previous findings in non-ESRD patients [10], clinical characteristics like gender, age and body weight were not significantly connected to pre-dialysis BTP levels.

Melegos and colleagues reported increased serum levels of BTP in patients undergoing HD and PD [6], however, little is known about the exact impact of renal replacement on this protein. In contrast, the impact of HD on serum levels of B2MG, a representative of the LMWP-class, has previously been well studied by Kabanda and coworkers [11]. In accordance with this study, both high-flux HD and HDF, reduced B2MG levels significantly in our cohort. However, we could demonstrate that in contrast to B2MG, BTP is not significantly eliminated by high-flux HD. During HDF treatment we exclusively used the post-dilution mode, which has been shown to be superior to the predilution mode with respect to the removal of LMWP in the 12–33 kD range [12–14]. Post-dilution HDF treatment leads to the expected plunge in serum concentrations of B2MG, crea and urea but only to a far smaller reduction of BTP serum concentration which seems to be not clinically relevant in everyday practice.

ANOVA analysis showed considerable differences in BTP reduction ability by different filters. These results suggest that the observed drop in serum BTP levels in patients undergoing HDF treatment are solely attributable to the BRAUN HI-PS membrane, preferably used in this cohort. Since we found only a poor correlation to UFC and dialyser surface, this effect must be attributed to other factors currently not known. It may be conceivable that our observed results for HDF treatment are confounded by the small number of patients undergoing HDF and the heterogeneity of membranes used. Increase of BTP serum levels after treatment in some patients may be attributed to concentration effects not completely counterbalanced by the Bergström formula applied in our study.

Since BTP is not substantially cleared by RRT and to a large extent independent from important determinants of RRT and dialysis dose—calculated as single-pool Kt/V—it was tempting to analysed its relation to RRF. Unfortunately, when we designed our study the measurement of RRF by urea-clearance was not implemented, which adds a drawback to our investigation. We therefore analysed BTP pre-treatment levels in sight of RD, the latter serving as a surrogate marker of RRF. After categorization of patients into groups according to their RD, BTP levels showed an inverse association with the RD. Furthermore, BTP allowed for a far better differentiation of patients with different RRF than pre-treatment creatinine and urea. Despite the recommendation to measure RRF in regular intervals in patients on chronic haemodialysis [8], the best clearance measure is still under debate. Both, creatinine and urea clearance have little accuracy estimating GFR at very low levels, especially of less than 1 ml/min [15]. Therefore, an established relation between BTP serum levels and RRF could ease patient management.

Our data may provide an explanation as to why lipoprotein-type prostaglandin D synthase, which has been shown to be the same protein as BTP [16], has previously been linked to cardiovascular injury [17]. This might only reflect the relation of impaired renal function and cardiovascular risk which is well-established [18]. Moreover, BTP is also under suspicion to contribute to the progression of renal failure [19], while underlying mechanisms have not been reported. Thus, one might speculate that the observed increase of BTP serum levels in patients with increasing impairment of renal function reflects only accumulation.

This is the first study to show that BTP serum levels are associated with RD of HD patients. Furthermore, we found that BTP, an endogenous marker of GFR, is virtually not cleared by RRT to a clinically important extent. This property of BTP may favour its use in the surveillance of patients on chronic RRT, but also in patients resolving acute renal failure while being on RRT. Due to limitations of our study, namely small sample size and heterogeneity of filters and treatment modes, additional systematic investigations are warranted to further elucidate the value of BTP as a diagnostic tool in patients on RRT.



   References
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 

  1. Hoffmann A, Nimtz M, Conradt HS. Molecular characterization of beta-trace protein in human serum and urine: a potential diagnostic marker for renal diseases. Glycobiology (1997) 7:499–506.[Abstract/Free Full Text]
  2. Olsson JE, Link H, Nosslin B. Metabolic studies on 125I-labelled beta-trace protein, with special reference to synthesis within the central nervous system. J Neurochem (1973) 21:1153–1159.[CrossRef][ISI][Medline]
  3. Priem F, Althaus H, Birnbaum M, Sinha P, Conradt HS, Jung K. Beta-trace protein in serum: a new marker of glomerular filtration rate in the creatinine-blind range. Clin Chem (1999) 45:567–568.[Free Full Text]
  4. Woitas RP, Stoffel-Wagner B, Poege U, Schiedermaier P, Spengler U, Sauerbruch T. Low-molecular weight proteins as markers for glomerular filtration rate. Clin Chem (2001) 47:2179–2180.[Free Full Text]
  5. Poge U, Gerhardt TM, Stoffel-Wagner B, et al. beta-Trace protein is an alternative marker for glomerular filtration rate in renal transplantation patients. Clin Chem (2005) 51:1531–1533. Epub 2005 Jun 10.[Free Full Text]
  6. 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) 53:32–37.[CrossRef][ISI][Medline]
  7. Clark WR, Winchester JF. Middle molecules and small-molecular-weight proteins in ESRD: properties and strategies for their removal. Adv Ren Replace Ther (2003) 10:270–278.[CrossRef][ISI][Medline]
  8. I. NKF-K/DOQI Clinical Practice Guidelines for Hemodialysis Adequacy: update 2000. Am J Kidney Dis (2001) 37:S7–S64.[ISI][Medline]
  9. Bergstrom J, Wehle B. No change in corrected beta 2-microglobulin concentration after cuprophane haemodialysis. Lancet (1987) 1:628–629.[Medline]
  10. Filler G, Priem F, Lepage N, et al. Beta-trace protein, cystatin C, beta(2)-microglobulin, and creatinine compared for detecting impaired glomerular filtration rates in children. Clin Chem (2002) 48:729–736.[Abstract/Free Full Text]
  11. Kabanda A, Jadoul M, Pochet JM, Lauwerys R, van Ypersele de Strihou C, Bernard A. Determinants of the serum concentrations of low molecular weight proteins in patients on maintenance hemodialysis. Kidney Int (1994) 45:1689–1696.[ISI][Medline]
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  14. Ahrenholz P, Winkler RE, Ramlow W, Tiess M, Thews O. On-line hemodiafiltration with pre- and postdilution: impact on the acid-base status. Int J Artif Organs (1998) 21:321–327.[ISI][Medline]
  15. Chandna SM, Farrington K. Residual renal function: considerations on its importance and preservation in dialysis patients. Semin Dial (2004) 17:196–201.[CrossRef][ISI][Medline]
  16. Hoffmann A, Conradt HS, Gross G, Nimtz M, Lottspeich F, Wurster U. Purification and chemical characterization of beta-trace protein from human cerebrospinal fluid: its identification as prostaglandin D synthase. J Neurochem (1993) 61:451–456.[ISI][Medline]
  17. Eguchi Y, Eguchi N, Oda H, et al. Expression of lipocalin-type prostaglandin D synthase (beta-trace) in human heart and its accumulation in the coronary circulation of angina patients. Proc Natl Acad Sci USA (1997) 94:14689–14694.[Abstract/Free Full Text]
  18. Wang AY, Wang M, Woo J, et al. Inflammation, residual kidney function, and cardiac hypertrophy are interrelated and combine adversely to enhance mortality and cardiovascular death risk of peritoneal dialysis patients. J Am Soc Nephrol (2004) 15:2186–2194.[Abstract/Free Full Text]
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Received for publication: 25.10.06
Accepted in revised form: 2. 7.07


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