212 D. H. Krieter et al.

in chronic renal failure. Biochem Biophys Res Commun 1995; 215:
98105
13. Moser B, Roth G, Brunner M et al. Aberrant T cell activation and
heightened apoptotic turnover in end-stage renal failure patients:
a comparative evaluation between non-dialysis, haemodialysis, and
peritoneal dialysis. Biochem Biophys Res Commun 2003; 308: 581
585
14. Joon J, Gollapudi S, Pahl M et al. Nave and central memory T-cell
lymphopenia in end-stage renal disease. Kidney Int 2006; 70: 371376
15. Sarantopoulos S, Stevenson KE, Kim HT et al. High levels of B-
cell activating factor in patients with active chronic graft-versus-host
disease. Clin Cancer Res 2007; 13: 61076114
16. Mackay F, Schneider P, Rennert R et al. BAFF and April: a tutorial
on B cell survival. Annu Rev Immunol 2003; 21: 231264
17. Mayne C, Amanna I, Nashold F et al. Systemic autoimmunity in
26. Wardemann H, Yurasov S, Schaefer A et al. Predominant autoantibody
production by early human B cell precursors. Science 2003; 301:
13741377
27. Waldschmidt T, Noelle R. Long live the mature B cell, a BAFFling
mystery resolved. Science 2001; 293: 20122013
28. Kalled L. Impact of the BAFF/BR3 axis on B cell survival, germinal
center maintenance and antibody production. Semin Immunol 2006;
18: 290296
29. Woodland R, Schmidt M, Thompson C. BLyS and B cell homeostasis.
Semin Immunol 2006; 18: 318326
30. Kipps T. The CD5 B cell. Adv Immunol 1989; 47: 117185
31. Herzenberg L, Haughton G, Rajewsky K. CD5 B cells in development
and disease. Ann NY Acad Sci 1992; 651: 591601
32. Martin F, Kearney JF. B1 cells: similarities and differences with other
B cell subsets. Curr Opin Immunol 2001; 13: 195201

BAFF-R-mutant A/WySnJ strain mice. Eur J Immunol 2008; 38: 587
598
18. Raskova J, Ghobrial I, Czerwinski DK et al. B-cell activation
and immunoregulation in end-stage renal disease patients receiving
hemodialysis. Arch Intern Med 1987; 147: 8993
19. Balin SJ, Platt JL, Cascalho M. Noncognate function of B cells in
transplantation. Transplantation 2009; 22: 593598
20. Pistoia V. Production of cytokines by human B cells in health and
disease. Immunol Today 1997; 18: 343350
21. Hornung V, Rothenfusser S, Britsch S et al. Quantitative expression
of Toll-Like receptor 110 mRNA in cellular subsets of human periph-
eral blood mononuclear cells and sensitivity to CpG oligodeoxynu-
cleotides J Immunol 2002; 168: 45314537
22. Bernasconi NL, Onai N, Lanzavecchia A. A role for Toll-like receptors
in acquired immunity: up-regulation of TLR9 by BCR triggering in
naive B cells and constitutive expression in memory B cells. Blood
2003; 101: 4500450
23. Johnson S, Shah N, Panoskaltsis-Mortari A et al. Murine and human
IL-7 activate STAT5 and induce proliferation of normal human pro-B
cells. J Immunol 2005; 175: 73257331
24. Milne CD, Paige CJ. IL-7: a key regulator of B lymphopoiesis. Semin
Immunol 2006; 18: 2030
25. Sims G, Ettinger R, Shirota Y et al. Identification and characterization
of circulating human transitional B cells. Blood 2005; 105: 43904398
Downloaded from http://ndt.oxfordjournals.org/ at Pontificia Universidad Catlica de Chile on November 25, 2016
33. Uckun F. Regulation of human B-cell ontogeny. Blood 1990; 76:
19081923
34. Gray D. Immunological memory: a function of antigen persistence.
Trends Microbiol 1993; 1: 3941
35. Bouts A, Davin J, Krediet R et al. Children with chronic renal failure
have reduced numbers of memory B cells. Clin Exp Immunol 2004;
137: 589594
36. Descamps-Latscha B, Chatenoud L. T cells and B cells in chronic
renal failure. Semin Nephrol 1996; 16: 183191
37. Raska K Jr, Raskova J, Shea SM et al. T cell subsets and cellular
immunity in end-stage renal disease. Am J Med 1983; 75: 734740
38. Degiannis D, Mowat AM, Galloway E et al. In-vitro analysis of B
lymphocyte function in uraemia. Clin Exp Immunol 1987; 70: 463
470
39. Fernandez-Fresnedo G, Ramos MA, Gonzalez-Pardo MC et al. B
lymphopenia in uremia is related to an accelerated in vitro apoptosis
and dysregulation of Bcl-2. Nephrol Dial Transplant 2000; 15: 502
510
40. Hoy WE, Cestero RV, Freeman RB. Deficiency of T and B lympho-
cytes in uremic subjects and partial improvement with maintenance
hemodialysis. Nephron 1978; 20: 182188
41. Deenitchina SS, Ando T, Okuda S et al. Cellular immunity in
hemodialysis patients: a quatitative analysis of immune cell subsets
by flow cytometry. Am J Nephrol 1995; 15: 5765

Received for publication: 18.3.09; Accepted in revised form: 14.7.09

Nephrol Dial Transplant (2010) 25: 212218

doi: 10.1093/ndt/gfp437
Advance Access publication 15 September 2009

Protein-bound uraemic toxin removal in haemodialysis

and post-dilution haemodiafiltration

Detlef H. Krieter1 , Andrea Hackl1 , Annie Rodriguez2 , Lela Chenine2 , Helene Leray Moragues2 ,
Horst-Dieter Lemke3 , Christoph Wanner1 and Bernard Canaud2

1
Division of Nephrology, Department of Medicine, University of Wurzburg, Wurzburg, Germany, 2 Department of Nephrology,
Hopital Lapeyronie, University of Montpellier, Montpellier, France and 3 EXcorLab GmbH, Obernburg, Germany
Correspondence and offprint requests to: Detlef H. Krieter; E-mail: krieter_d@medizin.uni-wuerzburg.de

Abstract elimination by dialysis therapy remains difficult. In the

Background. The accumulation of larger and protein- present study, the impact of the albumin permeability of re-
bound toxins is involved in the uraemic syndrome but their cently introduced advanced high-flux dialysis membranes


C The Author 2009. Published by Oxford University Press [on behalf of ERA-EDTA]. All rights reserved.
For Permissions, please e-mail: journals.permissions@oxfordjournals.org
Protein-bound toxins in HD and HDF
on the removal of such substances was tested in haemodial-
ysis and online post-dilution haemodiafiltration.
Methods. Two types of polyethersulfone membranes only
differing in albumin permeability (referred as PU and
PU+) were compared in eight patients on maintenance
dialysis in a prospective cross-over manner. Treatment set-
tings were identical for individual patients: time 229
22 min; blood flow rate 378 33 mL/min; dialysate flow
rate 500 mL/min; substitution flow rate in haemodiafil-
tration 94 9 mL/min. Removal of the protein-bound
compounds p-cresyl sulfate (pCS) and indoxyl sulfate (IS)
was determined by reduction ratios (RRs), dialytic clear-
ances and mass in continuously collected dialysate. In ad-
dition, the elimination of the low-molecular weight (LMW)
proteins beta2 -microglobulin, cystatin c, myoglobin (myo),
free retinol-binding protein (rbp) and albumin was
measured.
Results. Plasma levels of the protein-bound toxins were
significantly decreased by all treatment forms. However,
the decreases were comparable between dialysis mem-
branes and between haemodialysis and haemodiafiltration.
The RRs of total pCS ranged between 40.4 25.3 and
47.8 10.3% and of total IS between 50.4 2.6 and
54.6 8.7%. Elimination of free protein-bound toxins as
assessed by their mass in dialysate closely correlated pos-
itively with the pre-treatment plasma concentrations being
r = 0.920 (P < 0.001) for total pCS and r = 0.906 (P <
0.001) for total IS, respectively. Compared to haemodialy-
sis, much higher removal of all LMW proteins was found
in haemodiafiltration. Dialysis membrane differences were
only obvious in haemodialysis for the larger LMW proteins
myo and rbp yielding significantly higher RRs for PU+
(myo 46 9 versus 37 9%; rbp 18 5 versus 15 5%; of high-performing dialysis membranes only differing in
P < 0.05). Additionally, the albumin loss varied between
membranes and treatment modes being undetectable with
PU in haemodialysis and highest with PU+ in haemodi-
afiltration (1430 566 mg).
Conclusions. The elimination of protein-bound compounds
into dialysate is predicted by the level of pre-treatment
plasma concentrations and depends particularly on diffu-
sion. Lacking enhanced removal in online post-dilution
haemodiafiltration emphasizes the minor significance of
convection for the clearance of these solutes. Compared to
LMW proteins, the highly protein-bound toxins pCS and
IS are less effectively eliminated with all treatment forms.
For a sustained decrease of pCS and IS plasma levels, al-
ternative strategies promise to be more efficient therapy
forms.
Keywords: dialysis membrane; end-stage renal disease;
haemodiafiltration; haemodialysis; protein-bound toxins
Introduction
A large number of retained compounds are involved in
uraemic toxicity. Among them are larger solutes and
protein-bound compounds, which seem to be related to
deleterious biological and clinical effects but are difficult
to remove by dialysis [1]. Particularly, protein-bound toxins
represent a challenge for extracorporeal renal replacement
213
strategies because only the unbound, mostly low-molecular
solute can pass current dialysis membranes while the bound
fraction is retained.
P-cresol is one of the most frequently studied protein-
bound toxins in end-stage renal disease to date. The free p-
cresol serum concentration is significantly associated with
cardiovascular disease in non-diabetics on haemodialysis
and is suited to predict overall mortality in this patient
group [2,3]. The interrelation of p-cresyl sulfate (pCS), the
main in vivo metabolite of p-cresol, with vascular disease
in uraemia may be derived from its pro-inflammatory effect
on unstimulated leucocytes leading to oxidative stress and,
consequently, atherosclerosis [4].
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Similar to other protein-bound compounds, pCS is poorly
removed by haemodialysis because of its protein bind-
ing and its high ratio of distribution volume to clearance
[5]. Compared with low-flux, high-flux dialysis membranes
generally do not enhance the elimination of protein-bound
toxins, while albumin-leaking super-flux membranes are
superior especially in removing indoxyl sulfate (IS) [6,7].
Convection seems to positively impact protein-bound toxin
removal because haemodiafiltration is able to increase p-
cresol clearance without leading to excessive albumin loss
[8].
Recently, advanced synthetic high-flux dialysis
membranes were introduced for extracorporeal renal
replacement therapy. These membranes are characterized
by optimized, i.e. steeper sieving profiles with an improved
selective permeability for low-molecular weight (LMW)
proteins and an efficiency in conventional haemodialysis
similar to online haemodiafiltration with standard high-
flux membranes [9,10]. In the present study, two such types
albumin permeability were compared in haemodialysis and
online high-efficiency post-dilution haemodiafiltration
regarding the removal of protein-bound toxins and LMW
proteins.
Patients and methods
Study design
Study approval was given by the local ethics committee, and the competent
authorities were notified (registration no. 0007565). The study design was
prospective, randomized and cross-over.
Eight stable chronic kidney disease stage 5 patients on regular three
times weekly maintenance dialysis were enrolled into the study after they
had given written informed consent. The patients concomitant medica-
tions were continued in an unchanged manner.
Each patient underwent randomly one study week of three con-
secutive haemodialysis treatments with the PUREMA
R
H dialysis
membrane (referred as PU; synthetic, high-flux polyethersulfone,
1.9 m2 , -sterilized; Xenium 190 dialyser, Baxter Healthcare, McGaw
Park, IL, USA) and one week with the PUREMA
R
H+ dialysis mem-
brane (referred as PU+; 1.9 m2 ; Pureflux
R
190 H+ dialyser, Nipro Corp.,
Osaka, Japan). The KO A for urea of PU and PU+ measured in vitro in
aqueous conditions at a blood flow rate (QB ) of 300 mL/min and dialysate
flow rate (QD ) of 800 mL/min were 1598 140 and 1667 165 mL/min,
respectively. The two dialysis membranes only differed in the sieving co-
efficient for albumin being 0.002 for PU and 0.003 for PU+. After the
haemodialysis period, the patients were randomly subjected to one week of
three consecutive online post-dilution haemodiafiltration treatments with
each of the dialysis membranes.
Haemodialysis and haemodiafiltration were performed using Fresenius
4008 H monitors (Fresenius Medical Care, Bad Homburg, Germany).
Treatment duration, QB and QD , as well as the infusion flow rate (QI )
214
in post-dilution haemodiafiltration were kept constant for each patient.
The ultrafiltration flow rate (QUF ) of each session was set according
to the individual patients interdialytic weight gain. Anticoagulation was
performed by unchanged adoption of the previous routine heparinization.
Quantitation of treatment efficacy
Treatment efficacy was determined during the third (mid-week) consecu-
tive session of each respective period by measuring instantaneous plasma
clearances (K), dialytic clearances and reduction ratios (RRs). The total
mass (MTD ) of protein-bound toxins and albumin was detected in contin-
uously collected dialysate.
K was measured after 30 min and 180 min of the treatment for the
small solutes urea (60 Da) and phosphate (PO4 ; 96 Da) and for the LMW
proteins beta2 -microglobulin (b2m; 11,800 Da), cystatin C (cysc; 13 400
Da), myoglobin (myo; 17 800 Da), and free retinol-binding protein (rbp;
21.200 Da). Plasma concentrations were determined in blood samples
obtained from the arterial (Cart ) and the venous (Cven ) blood line of the
extracorporeal circuit. During sampling, QUF was maintained. QUF was
taken into account for calculation of K [11]. Plasma water clearances
were obtained by calculation according to equation 1 using the patients
haematocrit level (Hct) at the time of the respective clearance sampling
and the pre-dialysis total protein level (TP, g/L) [12]. To account for solute
shift from the blood cell, solute partition coefficients (SPC) were assumed
as 0.86 (urea), 0.5 (phosphate) and 0 (LMW proteins), respectively [12].
K plasma = Q B (1 0.0107 TP) (SPC Hct + (1 Hct))
(1)
((Cart Cven )/Cart ) + (Q UF Cven /Cart ) (mL/min)
RRs were determined for the LMW proteins b2m, cysc, myo and rbp,
and the protein-bound substances for pCS, p-cresyl glucuronide (pGC)
and IS. Plasma concentrations were measured in blood samples drawn
from the arterial blood line before (Cpre ) and at the end (Cpost ) of each
treatment after reduction of QB to 50 mL/min for 30 s, and QD was turned
off. RR was calculated according to equation 2 using corrected Cpost
(Cpost-corr ) [13]. Cpost was corrected for extracellular volume changes
based on differences in the patients pre- (BWpre ) and post-dialysis body
weight (BWpost ) (equation 3) [14].
RR = (1 Cpost-cor r/Cpre ) 100 (%) (2)
Cpost-corr = Cpost /(1 + ((BWpre BWpost )/0.2 BWpost )) (mg/L) (3)
MTD was determined by continuously collecting a spent dialysate frac-
tion of 10 mL/min during the whole dialysis duration (t) via a T-connector
inserted into the dialysate drainage line. The concentrations (CD ) of pCS,
IS and albumin were determined in an aliquot taken from the collected
spent dialysate. MTD was calculated according to equation 4.
MTD = CD (Q D + Q UF ) t (mg) (4)
Obtaining reliable clearances for protein-bound solutes from simulta-
neous arterial and venous concentrations is very difficult because of only
small differences across the dialyser [5]. Therefore, despite producing
rather a rough estimate, dialytic clearances of the protein-bound toxins
were calculated by dividing MTD by the log mean of the pre- and post-
treatment plasma levels and the treatment duration [8].
Based on the distribution volume of pCS, which is about 205 mL/kg
body weight and much larger than the plasma volume [5], the mass transfer
rate and the mass balance error were estimated for the protein-bound toxins
pCS and IS as an equivalent for adsorption onto the dialysis membranes.
The whole body mass of protein-bound solutes was calculated by multi-
plying the plasma concentration with the total volume of distribution. The
mass transfer rate was derived from the difference between whole body
mass before and after treatment. The mass balance error was calculated
as the difference between the mass transfer rate and the mass recovered in
dialysate.
Analytical methods
The total and unbound (free) portions of the protein-bound substances
pCS and IS were determined by high-performance liquid chromatography
(HPLC) according to Meert et al. [15]. Blood samples were collected in
tubes containing 1 mg tetrahydrolipstatin to avoid the activation of the
lipoprotein lipase in plasma by heparin and the generation of free fatty
D. H. Krieter et al.
acids that would compete with the protein-bound solutes for their binding
sites. Different to methods described earlier [6], deproteinization of the
plasma samples was performed with heat (95 C for 30 min) instead of
acidification to avoid hydrolysis of the p-cresol conjugates allowing the
detection of conjugated pCS.
As standards, pCS was kindly provided by Prof. Raymond Vanholder,
University of Ghent, Belgium, while IS was purchased from Sigma,
Taufkirchen, Germany. The standards were further purified by reversed
phase HPLC. They equalled coeluting peaks derived from plasma of
uraemic individuals by mass spectroscopy [HCT IonTrap LC/MS-system
(Bruker Daltronic GmbH, Bremen, Germany)].
Small solutes were determined with a Cobas c 111 analyser (Roche
Instrument Center, Rotkreuz, Switzerland). LMW protein concentrations
were measured by laser immunonephelometry (BN ProSpec Analyzer,
Dade Behring GmbH, Marburg, Germany).
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Data analysis
Descriptive analysis of the results was performed by calculating mean
values standard deviations (SD). After testing for normal distribution of
the samples, within-subject between-treatment differences were analysed
by ANOVA and a Tukey post hoc test. Comparative statistical analyses
of within-subject within-treatment changes from baseline were assessed
using the two-sided paired t-test. Correlation coefficients were determined
for pre-treatment plasma concentrations and MTD of protein-bound com-
pounds according to Pearson. A P-value of <0.05 was considered sta-
tistically significant. The statistical analysis was performed by means of
the Minitab Release 4 statistical software package (Minitab, Inc., State
College, PA, USA).
Results
Patient data
One female and seven male patients had a mean age of 63
12 years. Underlying renal diseases were cast nephropathy
(n = 2), diabetic nephropathy (n = 1), glomerulonephritis
(n = 1), hypertensive nephropathy (n = 1), primary amy-
loidosis (n = 1), Alport syndrome (n = 1) and unknown
(n = 1). The mean duration on dialysis treatment was 81.8
144.0 months (range 3411 months). The mean post-
dialysis body weight was 70.8 18.6 kg. All patients were
anuric. Four patients had efficient native arteriovenous
fistulas and four patients had patent bi-flow dialysis
catheters for blood access.
Treatment data
The treatment duration was 229 22 min. QB and QD
averaged at 378 33 mL/min and 500 0 mL/min, re-
spectively. QI in post-dilution haemodiafiltration was 94
9 mL/min. QUF was not different for the four treatment pe-
riods (PU in HD 2673 510 mL; PU+ in HD 2726
816 mL; PU in HDF 3013 651 mL; PU+ in HDF
2838 1208 mL). Six patients received standard heparin,
one patient fractionated heparin and another patient did not
receive any heparin due to severe thrombopenia associated
with lupus erythematodes.
Laboratory data
The relative binding of protein-bound toxins was not dif-
ferent before and after treatment being 96.0 2.4 and
96.3 3.1% for pCS and 95.0 4.6 and 94.9 3.7% for
IS. Except for the free portion of pCS, which was not sig-
nificantly decreased by all treatment modes, haemodialysis
Protein-bound toxins in HD and HDF 215
Table 1. Pre- and post-treatment plasma concentrations and mass in dialysate of the total and free portion of the protein-bound uraemic toxins pCS
and IS

p-Cresyl sulfate Indoxyl sulfate

Pre Post Dialysate Pre Post Dialysate

(mg/L) (mg/L) (mg) (mg/L) (mg/L) (mg)

PU HD Total 19.6 17.8 10.5 9.3 88 84 22.1 7.9 11.0 4.0# 114 50
Free 0.9 0.8 0.5 0.4 88 84 1.0 0.5 0.4 0.2 114 50
PU+ HD Total 19.8 15.6 11.5 10.5 78 62 18.4 9.8 9.2 6.1# 89 50
Free 0.9 0.8 0.5 0.5 78 62 0.8 0.4 0.5 0.4 89 50
PU HDF Total 21.4 18.2 11.2 8.8 69 78 16.8 8.8 7.8 4.8# 75 41
Free 0.9 0.9 0.5 0.5 69 78 0.6 0.3 0.3 0.2 75 41
PU+ HDF Total 20.4 20.5 10.4 9.6 86 80 17.7 11.5 8.3 6.1 85 57
Free 0.9 1.0 0.4 0.4 86 80 0.6 0.4 0.3 0.2 85 57

Downloaded from http://ndt.oxfordjournals.org/ at Pontificia Universidad Catlica de Chile on November 25, 2016
P < 0.05 versus pre-treatment concentration.
P < 0.01 versus pre-treatment concentration.
# P < 0.001 versus pre-treatment concentration.
Since bound toxins cannot pass the dialysis membrane, the free and bound portion in dialysate are equal. Results of haemodialysis and online post-
dilution haemodiafiltration with the PU and the more permeable PU+ dialysis membrane are given. No differences were found between PU and
PU+ in the same treatment mode and between haemodialysis and haemodiafiltration.

Table 2. Reduction ratios (RRs) corrected for weight loss, dialytic clearances and mass balance errors of the total and free portion of the protein-bound
uraemic toxins pCS and IS

p-Cresyl sulfate Indoxyl sulfate

Dialytic Dialytic
clearance Mass transfer Mass balance clearance Mass transfer Mass balance
RR (%) (mL/min) (mg) error (mg) RR (%) (mL/min) (mg) error (mg)

PU HD Total 45.6 2.0 31.6 18.2 131 112 43 44 50.4 2.6 31.7 12.6 168 65 53 30
Free 47.5 18.7 49.9 26.3 54.5 9.9 51.7 18.1
PU+ HD Total 47.3 14.8 24.5 14.7 123 83 45 53 52.2 12.2 25.5 12.7 141 49 51 52
Free 29.0 33.4 40.0 24.4 43.3 27.2 42.5 21.5
PU HDF Total 47.8 10.3 21.1 19.9 150 130 81 62 54.6 8.7 21.4 10.8 134 78 60 41
Free 34.8 44.2 33.8 29.6 45.0 33.3 36.0 17.4
PU+ HDF Total 40.4 25.3 26.6 19.5 142 142 56 65 53.3 8.9 24.1 14.3 139 103 54 46
Free 42.9 28.3 49.0 26.0 56.1 8.4 40.7 23.2

Results of haemodialysis and online post-dilution haemodiafiltration with the PU and the more permeable PU+ dialysis membrane are given. No
differences were found between PU and PU+ in the same treatment mode and between haemodialysis and haemodiafiltration.

and haemodiafiltration with both PU and PU+ led to a
significant reduction of the plasma concentrations of free
and total IS and total pCS (Table 1). The RRs for total pCS
(between 40.4 25.3% in PU+ HDF and 47.8 10.3% in
PU HDF) and total IS (between 50.4 2.6% in PU HD
and 54.6 8.7% in PU HDF) were all on a similar level
(Table 2). There was no significant difference between any
of the four treatment forms regarding pre-treatment plasma
concentrations, RRs and MTD of protein-bound toxins.
Compared with the total fraction, the dialytic clearances
of the free fractions of pCS and IS were considerably
higher (Table 2). No significant difference between treat-
ment modes was noted.
The mass balance error for total pCS (between 43
44 and 81 62 mg) and total IS (between 51 52 and
60 41 mg) was not different between dialysis membranes
(Table 2). Even after the dialysis forms were combined ir-
respective of the type of membrane, no difference between
haemodialysis and haemodiafiltration was determined
(Table 2).
While a significant effect of any treatment form on the
removal of the protein-bound compounds was absent, the
MTD as one measure for solute elimination closely corre-
lated positively with the pre-treatment plasma concentra-
tions. For the total and free portions, this linear relationship
was r = 0.906 and r = 0.800 for IS (P < 0.001) and r =
0.920 and r = 0.873 for pCS (P < 0.001), respectively.
The instantaneous plasma clearances of PU and PU+
for urea at 30 min and PO4 at 30 and 180 min were
significantly lower in haemodialysis compared with
haemodiafiltration (Table 3). For all LMW proteins,
K was lower (P < 0.001) in haemodialysis than in
haemodiafiltration without differences between the two
dialysis membranes (Table 3). Similarly, the LMW protein
RRs were throughout lower (P < 0.001) in haemodialysis
mode (Table 4). Compared to PU+, the RRs obtained in
haemodialysis with PU were lower for the larger LMW
proteins myo and rbp (Table 4). For the smaller middle
molecules b2m and cysc, the RRs were almost identical in
haemodialysis with both dialysis membranes.
Significant differences were found between the dial-
ysis membranes with respect to the albumin loss into
dialysate during haemodialysis and haemodiafiltration. In
haemodialysis with PU, the albumin loss was always
216 D. H. Krieter et al.
Table 3. Instantaneous plasma clearances (K) of haemodialysis and online post-dilution haemodiafiltration with the PU and the more permeable
PU+ dialysis membrane

Instantaneous plasma clearances (mL/min)

urea PO4 b2m cysc myo rbp

PU HD 30 min 266 12 223 16# 63 10 69 8 24 10 2 9

180 min 264 18 226 27## 59 9 58 9 19 9 4 8
PU+ HD 30 min 257 19 214 24 60 9 64 8 25 8 8 9
180 min 256 18 219 24## 62 7 62 9 25 7 7 9
PU HDF 30 min 286 27 242 25 96 6 100 6 55 15 13 13
180 min 277 57 242 37 102 10 94 4 52 8 6 12
PU+ HDF 30 min 289 21 251 25 97 9 100 11 52 9 11 10
180 min 286 23 258 26 94 11 88 7 54 14 69

Downloaded from http://ndt.oxfordjournals.org/ at Pontificia Universidad Catlica de Chile on November 25, 2016
P < 0.001 versus PU HDF and PU+ HDF.
P < 0.01 versus PU HDF and PU+ HDF.
# P = 0.01 versus PU HDF and P < 0.001 versus PU+ HDF.
## P < 0.01 versus PU+ HDF.
Urea (60 Da); PO4, phosphate (96 Da); b2m, beta2 -microglobulin (molecular weight 11,800 Da); cysc, cystatin c (13,400 Da); myo, myoglobin (17,600
Da); rbp, retinol-binding protein (21.200 Da)

Table 4. Reduction ratios of low-molecular weight proteins corrected for

weight loss of haemodialysis and online post-dilution haemodiafiltration
with the PU and the more permeable PU+ dialysis membrane

Reduction ratios [%]

b2m cysc myo rbp

PU HD 68 6 68 6 37 9 15 5
PU+ HD 68 6 67 6 46 9 18 5
PU HDF 78 5 77 5 65 6 19 8
PU+ HDF 78 6 76 5 66 7 26 16

P < 0.001 versus PU HDF and PU+ HDF.

P < 0.05 versus PU+ HD and P < 0.001 versus PU HDF and PU+
HDF.
b2m, beta2 -microglobulin (molecular weight 11 800 Da); cysc, cystatin
c (13 400 Da); myo, myoglobin (17 600 Da); rbp, retinol-binding protein
(21 200 Da)
below the detection limit of 200 mg, while with PU+,
it averaged at 482 154 mg per treatment. In haemodi-
afiltration, an albumin loss of 809 206 and 1430
566 mg was determined with PU and PU+, respectively
(Figure 1).
Discussion
This small-scale study was performed to compare the im-
pact of two differently permeable innovative dialysis mem-
branes applied in haemodialysis and post-dilution haemodi-
afiltration on the removal of protein-bound toxins and
LMW proteins. Both haemodialysis and haemodiafiltra-
tion decreased the plasma concentrations of all substances
significantly during a single treatment. Despite the fact that
the infusion volume met the criteria for high efficiency
haemodiafiltration and in contrast to the effect on LMW
proteins, no differences in the removal of the protein-bound
toxins was found neither between treatment modes nor be-
tween the two different dialysis membranes.
This finding is inconsistent with the results from a pre-
vious trial, which reported slightly superior total p-cresol
removal by highly convective renal replacement therapy
Fig. 1. Albumin loss in dialysate of a single session of haemodialysis
and online post-dilution haemodiafiltration with the PU and the more
permeable PU+ dialysis membrane. Mean values and standard deviations
are depicted. P < 0.05 versus PU+ HD; P < 0.001 versus PU HDF
and PU+ HDF; # P = 0.002 versus PU+ HDF.
including post-dilution haemodiafiltration [8]. This dis-
crepancy may be explained by several factors: (1) the ef-
fect of convection on protein-bound toxin removal is poor
compared with diffusion. Although the transferability of
data from recent in vitro studies on continuous venove-
nous haemofiltration and haemodiafiltration to our results
is difficult, they suggest that an increase of protein-bound
toxin removal by higher ultrafiltration rates (i.e. convec-
tion) is little effective [16]. Alterations of the dialysate
flow rate and a larger dialysis membrane surface area are
more important measures to enhance protein-bound toxin
elimination [17]. In this respect, it must be noted that no
difference in the removal of most protein-bound toxins, in-
cluding p-cresol and IS, was found between low- and high-
flux dialysis [6]. In contrast to high-flux dialysis, in which
convection plays a considerable role, low-flux dialysis is a
purely diffusive therapy form; (2) less efficacy differences
between haemodialysis and haemodiafiltration, particularly
with regard to convection. The dialysis membranes tested in
our study represent recently introduced advanced high-flux
Protein-bound toxins in HD and HDF
membranes. With regard to LMW protein removal, even the
less permeable PU membrane has proven to be superior in
haemodialysis compared to the Helixone
R
membrane used
in the referenced trial [8,9]. This difference was attributed
to enhanced internal filtration, i.e. more convection, allow-
ing the elimination of a larger quantity within a wider range
of LMW proteins [9]. Accordingly, our results for dialytic
clearances and mass in dialysate of pCS were rather high al-
ready in haemodialysis; (3) differences in the measurement
of p-cresol. Bammens et al. applied gas chromatographic
mass spectrometry for the determination of total p-cresol
[8]. We used an HPLC method to differentiate the p-cresol
conjugate pCS. pGC, the quantitatively less important con-
jugate accounting for total p-cresol, may behave completely
different from pCS if its protein binding is different. The
strength of binding is of particular importance for the dia-
lytic elimination of protein-bound substances. The higher
the degree of the protein binding the more difficult is
the removal of a substance by dialysis therapy. Therefore,
total p-cresol, i.e. pCS and pGC, may have slightly different
kinetics during dialysis as pCS alone.
The two dialysis membranes under investigation only dif-
fered in albumin permeability but only marginally to avoid
potential hazards of excessive albumin loss in haemodi-
afiltration. Although significant differences in albumin
loss per session were detected between PU and PU+ in
haemodialysis and haemodiafiltration, the maximum loss,
which was observed with PU+ in haemodiafiltration, aver-
aged at only about 1.4 g. Such a value is regarded as to be by
far within accepted ranges and is much lower compared with
albumin-leaking super-flux dialysers used in haemodialy-
sis mode [18,7]. Compared with low-flux dialysis, removal
of protein-bound toxins, especially IS, by super-flux dial-
ysis is superior and is not only attributed to compounds
bound to albumin passing or adsorbing to the membrane
[7]. With respect to albumin loss and convection, the differ-
ences between low- and super-flux dialysers are much larger
than those between PU and PU+ in either haemodialy-
sis or haemodiafiltration. The performance characteristics
of PU and PU+ were very similar and differences in
haemodialysis were not observed for solutes smaller than
myoglobin, a molecule of 17 600 Da. In haemodiafiltra-
tion, the differences in LMW protein removal between the
membranes disappeared and averaged on very high lev-
els comparable to those reported in previous trials [9,10].
Therefore, it is conclusive to find similar pCS and also
IS removal with both types of the innovative PUREMA
R
high-flux membrane.
According to Ficks law, the solute flux in haemodialy-
sis is proportional to the concentration difference between
plasma water and dialysate, provided that the distance and
area of the diffusion front, i.e. the dialysis membrane, as
well as the diffusivity, which is a property of a specific
solute, are constant [19]. The MTD is essentially the resul-
tant of solute flux. For small solutes, such as unbound pCS
and IS, the concentration gradient is the driving force for
diffusion [19]. The free and bound compounds in plasma
are equilibrated. Depending on the solute protein binding
coefficient, unbinding of solute occurs as free solute is re-
moved [20], as also indicated by constant relative bind-
ing despite decreased concentrations from pre- to post-
217
treatment. In this study, MTD must be regarded as a par-
ticularly sensitive parameter for the removal of pCS and IS
because the tested dialysis membranes were almost iden-
tical and, therefore, adsorption and other physicochemical
differences between the membranes can be neglected. Ac-
cordingly, the mass balance errors for total pCS and IS as
a measure of adsorption was not different between dial-
ysis membranes and also treatment modes despite con-
siderable amounts were estimated. We further analysed
the data by correlating the pooled MTD and pre-treatment
plasma concentrations of protein-bound compounds. In-
terestingly, an extremely close correlation was found for
all free and total protein-bound toxins suggesting that the
Downloaded from http://ndt.oxfordjournals.org/ at Pontificia Universidad Catlica de Chile on November 25, 2016
removal of protein-bound solutes is predicted by the pre-
treatment plasma concentration. The pre-treatment plasma
concentration in steady state at a constant generation rate
is determined by the intra- and extracorporeal clearance.
Since no difference in protein-bound solute removal be-
tween treatment forms was found, the impact of convec-
tion must be of minor significance. In fact, it must be
governed primarily by diffusion. This conclusion is in ac-
cordance with the findings from a previous in vitro trial
studying the effects of ultrafiltration and dialysate flow
in continuous haemofiltration and haemodiafiltration [16].
Moreover, the close correlations of MTD and pre-treatment
plasma concentrations underline the validity of our
measurements.
Conclusions
The elimination of protein-bound compounds into dialysate
is governed particularly by diffusion and is predicted by the
level of pre-treatment plasma concentration. RRs for free
and total pCS and IS, which are involved in oxidative stress,
endothelial dysfunction and potentially cardiovascular dis-
ease [4,2125], hardly reached 50% in both haemodialysis
and online post-dilution haemodiafiltration. Although our
knowledge about kinetics and distribution of these com-
pounds is limited, such values must be regarded as inad-
equate. For a sustained decrease of pCS and IS plasma
levels, alternative strategies promise to be more efficient
therapy forms. These could comprise adsorptive measures,
either applied orally [25,26] or during extracorporeal ther-
apy [27,28], which interact with protein-bound compounds
or their precursors, and techniques, which alter the strength
of the protein binding in plasma.
Acknowledgements. Part of this study has been published in abstract
form and presented at the XLV Congress of the ERA-EDTA, 2008, Stock-
holm, Sweden, and at the XXX Congress of the American Society of
Nephrology, 2008, PA, USA. The study was supported by a grant from
Baxter Healthcare, McGaw Park, IL, USA.
Conflict of interest statement. D.H.K. has received lecturer fees from Bax-
ter Healthcare and Membrana GmbH. H.-D.L. is a full-time employee
of Membrana GmbH, Wuppertal, Germany, manufacturer of PUREMA
R
H. C.W. serves on the BAXTER European Renal Advisory Board for peri-
toneal dialysis and received lecturer fees from Fresenius Medical Care.
B.C. has received fees for lectures from Fresenius Medical Care, Amgen,
Roche and Janssen-Cilag.
218
References
1. Vanholder R, Van Laecke S, Glorieux G. What is new in uremic
toxicity? Pediatr Nephrol 2008; 23: 12111221
2. Meijers BK, Bammens B, De Moor B et al. Free p-cresol is associated
with cardiovascular disease in hemodialysis patients. Kidney Int 2008;
73: 11741180
3. Bammens B, Evenepoel P, Keuleers H et al. Free serum concentrations
of the protein-bound retention solute p-cresol predict mortality in
hemodialysis patients. Kidney Int 2006; 69: 10811087
4. Schepers E, Meert N, Glorieux G et al. P-cresylsulphate, the main in
vivo metabolite of p-cresol, activates leucocyte free radical production.
Nephrol Dial Transplant 2007; 22: 592596
5. Martinez AW, Recht NS, Hostetter TH et al. Removal of p-cresol
sulfate by hemodialysis. J Am Soc Nephrol 2005; 16: 34303436
6. Lesaffer G, De Smet R, Lameire F et al. Intradialytic removal of
protein-bound uraemic toxins: role of solute characteristics and of
dialyser membrane. Nephrol Dial Transplant 2000; 15: 5057
7. De Smet R, Dhondt A, Eloot S et al. Effect of the super-flux cellulose
triacetate dialyser membrane on the removal of non-protein-bound
and protein-bound uraemic solutes. Nephrol Dial Transplant 2007;
22: 20062012
8. Bammens B, Evenepoel P, Verbeke K et al. Removal of the protein-
bound solute p-cresol by convective transport: a randomized crossover
study. Am J Kidney Dis 2004; 44: 278285
9. Krieter DH, Morgenroth A, Barasinski A et al. Effects of a polyelec-
trolyte additive on the selective dialysis membrane permeability for
low-molecular-weight proteins. Nephrol Dial Transplant 2007; 22:
491499
10. Krieter DH, Hunn E, Morgenroth A et al. Matching efficacy of online
hemodiafiltration in simple hemodialysis mode. Artif Organs 2008;
32: 903909
11. Hoenich NA, Frost TH, Kerr DNS. Dialysers. In: Drukker F,
Parsons FM, Maher JF (eds) Replacement of Renal Function by Dial-
ysis. Hague/Boston/London: Martinus Nijhoff Publishers, 1979, 80
124
12. Henderson LW. Biophysics of ultrafiltration and hemofiltration.
In: Jacobs C, Kjellstrand CM, Koch KM, Winchester JF (eds).
Replacement of Renal Function by Dialysis. 4th edn, Dor-
drecht/Boston/London: Kluwer, 1996, 114145
13. Basile G, Casino F, Lopez T. Percent reduction in blood urea concen-
tration during dialysis estimates Kt/V in a simple and accurate way.
Am J Kidney Dis 1990; 15: 4045
14. Bergstrom J, Wehle B. No change in corrected 2-microglobulin con-
centration after cuprophane haemodialysis. Lancet 1987; 1: 628629
D. H. Krieter et al.
15. Meert N, Eloot S, Waterloos MA et al. Effective removal of protein-
bound uraemic solutes by different convective strategies: a prospective
trial. Nephrol Dial Transplant 2009; 24: 562570
16. Meyer TW, Walther JL, Pagtalunan ME et al. The clearance of protein-
bound solutes by hemofiltration and hemodiafiltration. Kidney Int
2005; 68: 867877
17. Meyer TW, Leeper EC, Bartlett DW et al. Increasing dialysate flow
and dialyzer mass transfer area coefficient to increase the clearance
of protein-bound solutes. J Am Soc Nephrol 2004; 15: 19271935
18. Krieter DH, Canaud B. High permeability of dialysis membranes:
what is the limit of albumin loss? Nephrol Dial Transplant 2003; 18:
651654
19. Sargent JA, Gotch FA. Principles and biophysics of dialysis. In: Jacobs
C, Kjellstrand CM, Koch KM, Winchester JF (eds). Replacement
of Renal Function by Dialysis. 4th edn, Dordrecht/Boston/London:
Downloaded from http://ndt.oxfordjournals.org/ at Pontificia Universidad Catlica de Chile on November 25, 2016
Kluwer, 1996, 34102
20. Farrell BC, Grib NL, Fry DL et al. A comparison of in vitro and in vivo
solutes-protein binding interactions in normal and uremic subjects.
Trans Am Soc Artif Intern Organs 1972; 18: 268276
21. Cerini C, Dou L, Anfosso F et al. P-cresol, an uremic retention solute,
alters the endothelial barrier function in vitro. Thromb Haemost 2004;
92: 140150
22. Dou L, Bertrand E, Cerini C et al. The uremic solutes p-cresol and
indoxyl sulfate inhibit endothelial proliferation and wound repair.
Kidney Int 2004; 65: 442451
23. Dou L, Jourde-Chiche N, Faure V et al. The uremic solute indoxyl
sulfate induces oxidative stress in endothelial cells. J Thromb Haemost
2007; 5: 13021308
24. Tumur Z, Niwa T. Indoxyl sulfate inhibits nitric oxide production and
cell viability by inducing oxidative stress in vascular endothelial cells.
Am J Nephrol 2009; 29: 551557
25. Shimoishi K, Anraku M, Kitamura K et al. An oral adsorbent, AST-
120 protects against the progression of oxidative stress by reducing
the accumulation of indoxyl sulfate in the systemic circulation in renal
failure. Pharm Res 2007; 24: 12831289
26. Evenepoel P, Bammens B, Verbeke K et al. Acarbose treat-
ment lowers generation and serum concentrations of the protein-
bound solute p-cresol: a pilot study. Kidney Int 2006; 70: 192
198
27. Meyer TW, Peattie JWT, Miller JD et al. Increasing the clearance of
protein-bound solutes by addition of a sorbent to the dialysate. J Am
Soc Nephrol 2007; 18: 868874
28. Meijers BK, Weber V, Bammens B et al. Removal of the uremic
retention solute p-cresol using fractionated plasma separation and
adsorption. Artif Organs 2007; 32: 214219
Received for publication: 28.5.09; Accepted in revised form: 4.8.09