CLIN. CHEM.

36/11, 1906-1910 (1990)

Evaluation of Dialysis Treatment in Uremic Patients by Gel Filtration of Serum

Jesus Osada,1 Teodoslo Gsa,2 Carmen Sanz,4 Isabel Mlllan,3 and JulIo Botella4

A group of substances of molecular masses between 300 MaterIals and Methods

and 1500 Da have been found to be toxic metabolites in Patients and Sera
patients with uremia. We determined the concentration in
serum of these molecules in the following groups of patients: Pre-dialysis blood samples were collected from five
two hemodialyzed groups (one with cuprophane and the groups: Group A, six patients hemodialyzed with a cu-

other with polyacrylonitnle dialyzers), one group treated with prophane dialyzer; Group B, six patients hemodialyzed
continuous ambulatory peritoneal dialysis, one group of with polyacrylonitrile; Group C, six patients dialyzed with
nondialyzed azotemic patients, and one control group of chronic ambulatory peritoneal dialysis (CAPD); Group D,
six nondialyzed azotemic patients (NDAP), whose condi-
healthy persons. Ultrafiltrates of the subjects’ sera were
tions did not require any dialysis; and Group E, healthy
fractionated on Sephadex G-15 followed by ion-exchange
individuals with normal kidney function. The procedures
chromatography. Eluates were monitored by absorbance at
followed were in agreement with the Helsinki Declaration.
254 and 206 nm. Partially characterized peaks P1 and P2,
The blood samples were taken and serum was obtained
obtained by gel filtration, correlated with the concentration of
by centrifugation within 60 mm after collection. In every
creatinine in serum; their concentrations were significantly (P case, samples were analyzed in duplicate runs.
<0.01) larger in hemodialyzed groups than in peritoneal We diluted 2 mL of each serum sample with 2 mL of
dialyzed or in nondlalyzed azotemic patients. After ion- distilled water, then passed this through a Centriflo CF-50
exchange chromatography, two peaks (P’5 and P’6) corre- membrane (Amicon, Danvers, MA) for 20 mm at 900 x gin
lated with serum creatinine and also were larger in hemodi- a Beckman refrigerated centrifuge (Beckman Instruments,
alyzed patients than in the other groups. Apparently, ade- Brea, CA). The nonfiltered residue was resuspended in 2
quate discrimination is obtained by gel-filtration analysis and mL of distilled water and the ultrafiltration step was
further analysis by ion-exchange chromatography does not repeated. Both ultrafiltrates were pooled and lyophilized.
provide additional information in most of the affected pa- Before assay, we dissolved the lyophilized material in 2 mL
tients. of distilled water.

AddItional Keyphrases: hemodialysis . peritoneal dialysis Methods

chromatography, ion-exchange creatinine
. - azotemia
Each subject was monitored for serum creatinine and
creatinine clearance. Serum creatinine was estimated ac-
A group of poorly characterized compounds with molec- cording to the Jaff#{233} kinetic colorimetric method, with an
ular masses ranging from 300 to 1500 Da are important Astra-45 analyzer (Beckman Instruments Inc.). Creatiine
toxic metabolites in uremia even in low concentrations (1, clearance was determined as described by Henry et al. (11).
2). Called “middle molecules” (1, 3), this group of sub- Analytical chromatography by gel filtration. Gel ifitration
stances may be implicated in the development of neuropa- was performed according to the method of Furst et al. (12),
thy and anemia in some uremic patients (2-5). After with use of a 75 x 0.5 cm column packed with Sephadex
successful renaltransplantation, uremic middle molecules G-15 (Pharmacia, Uppsala, Sweden). After equilibrating
disappear from serum, earlier than the normalization of the column with 0.01 mollL Tris HC1 buffer,
- pH 8.6, we
the concentrations of creatinine (6). applied 0.5 mL of the ultraiiltrate. The column was eluted
Most of the uremic middle molecules belong to a poorly with the same buffer at a flow rate of 10 mL/h. The eluates
characterized group of peptides with limited diffusibility were monitored at 254 nm and 206 nm with a dual-channel
through hemodialysis membranes. Some are incompletely ultraviolet spectrometer (Uvicord Ill; LKB, Bromma, Swe-
removed from uremic serum by hemodialysis (5, 7). den). At both wavelengths the range of absorbance was 0 to
Chronic dialysis therapy has been successful in the 1 A. Peak separation was complete in 2 h. Effluents were
treatment of end-stage renal patients because it reverses collected with a fraction collector (7000 Ultrarac; LKB).
most uremic symptoms. However, objective criteria as to Fractions with molecular masses between 1000 and 1500
which solutes should be preferentially eliminated by dial- Da (determined by comparison with the oxytocin elution
ysis remain controversial (8-10). peak) were collected and lyophilized. The dry powder was
In this paper, we have studied the concentrations of resuspended in 0.4 mL of 0.01 mol/L Tris HCI buffer,
- pH
middle molecules in sera of several patients treated with 8.6.
different systems of dialysis to determine whether analysis Analytical ion-exchange chromatography. Two 35 X 0.5
of middle molecules would help identify the best dialysis cm columns were used, one as a comparison column (to
method. These results are the basis for the present report. control background) and the other as a sample column.
Both were packed with diethylaminoethyl-Sephadex A-25
(Pharmacia), equilibrated with 0.01 mol/L Tris 11C1 -

‘Human Nutrition Research Center on Aging, Tufts University, buffer, pH 8.6, and eluted with a linear gradient of NaCl
711 Washington St. Boston, MA 02111. (0-1 mol/L), performed with an LKB 11300 Ultrograd
2Seyicio de Bioqulmica Clfnica, 3Servicio de Bioestadfstica,
Gradient mixer. The flow rate was 10 mL/h. Detection at
and Servicio de Nefrologf a, Hospital “Puerta de Hierro,” San
Martin de Porres 4, 28035 Madrid, Spain. 206 nm was monitored in the range of 0-1 A; however, at
Received January 16, 1990; accepted September 12, 1990. 254 mu we used a 0-0.2 A range. Separation was complete

1906 CLINICAL CHEMISTRY, Vol.36, No. 11, 1990

 A A
nm
in 3 h (12). Concentrations were expressed as millimeter of I,.

- In nm
peak height per milliliter of serum.
Statistical analysis. The Shapiro-Wilk test was applied
to establish the behavior of distributions. Whenever the
Shapiro-Wilk test rejected the hypothesis of normal distri- P.akb I’

bution, or when the Bartlett test for homogeneity of vari-

ances was significantly different, we calculated the overall
significance of differences with the Kruskal-Wallis (one-
way analysis of variance) test. If the differences were
significant, we then tested the differences between the flm.
Fig. 2. Chromatographic profile obtained by ion-exchange chroma-
groups pair-wise, using the Mann-Whitney U-test (13).
tography of a uremic serum
Differences were considered nonsignificant when P >0.05.
We used dlethylamlnoethyl-Sephadex A-25, equilibrated with 0.01 moL/L
Association between variables was assessed by Spear- Tns KCI buffer, pH 8.6, and eluted with the same buffer but with a continuous
man’s rank-order correlation coefficient (r.) (14). Precision ionic strength gradient to NaCI, 1 mol/L Flow rate was 10 mI/h
studies were carried out by duplicate analysis of samples in
different days and results were expressed as CV (%).
Table 1. ConcentratIons of Peaks ObtaIned by Gel
Results FiltratIon MonItored at 254 nm
Peek P1 Peak P2
Figures 1 and 2 show the chromatographic profiles ob-
tamed by gel ifitration and ion-exchange chromatography, Patient group n Mean (and range), mm/mL of serum
respectively. Gel filtration of uremic serum yielded five or
Cuprophane 6 15 (5-20)’#{176} 7 (4...1#{216})a.b.c
six peaks at 254 mu. In contrast, no peak appeared in
Polyacrylonitnie 6 12 (5-17)a.b.c 6 (4...8)a.b.c
healthy controls (data not shown). At 206 mu, the chro-
CAPD 6 4 (1...9)a.d.e 3 (1-4)’
matogram showed peaks in both groups. Peaks P1 and P2,
NDAP 6 3 (l....5)a.d.e I (o.7-.2.5)
with retention times of 39 and 50 miii, respectively, were
Healthy controls 7 0 (0-0) 0 (0-0)
collected and further processed by ion-exchange chroma-
tography. The profile of uremic serum at 254 urn showed 11 P <0.01 vs healthy controls; b P <0.01 vs NDAP; C P <0.01 vs CAPD; d p
<0.01 vs polyacnj$onitrile; and P
#{149}<0.01 vs cuprophane dialysis (Mann-
peaks by ion-exchange chromatography, whereas no peaks
Whitney’s U-test to compare the different groups par-wise).
could be detected at this wavelength in sera from healthy
subjects. The reproducibility (between-run CV) of retention
times for the peaks was 6% and 8% in gel ifitration and
ion-exchange chromatography, respectively. dures of hemodialysis. CAPD patients presented signifi-
Analytical precision (CV) was <16%. To calculate this cantly (P <0.01) lower values than the hemodialyzed
CV, we used results of duplicate analyses on different days, group; their values were similar to those of uremic patients
31 pairs of data for each peak. The means and ranges for without treatment. Peaks P1 and P2, evaluated at 254 nm,
each peak (mmlmL) were as follows: P1 at 254 am, 6.8 are not present in healthy subjects. They can be detected in
(0-20); P1 at 206 mu, 28(0-67); P2 at 254 am, 3.4(0-10); P2 healthy subjects at 206 mu, although their values are
at 206 mu, 30(5.5-56); P’5 at 206 mu, 9.5(0-23); and P’6 at significantly lower than in any uremic group (Figure 3).
254 am, 5.3 (0-18.5). The values obtained for peaks P1 and P2 at 254 mu behave
Table 1 shows the values for peaks 1 and 2 obtained by similarly between the different groups (Table 1) as well as
gel filtration, as monitored at 254 mu. The highest values for P1 at 206 mu (Figure 3). In contrast, peak P2 measured
were observed in the hemodialyzed patients (cuprophane at 206 mu (Figure 3) does not discriminate between healthy
and polyacrylonitrile), with no statistical variation be- controls and peritoneal-dialysis patients, or between
tween the values obtained by these two different proce- healthy controls and nondialyzed azotemic patients.
Figure 4 and Table 2 show the values obtained by
ion-exchange chromatography for peaks P’6 (206 mu) and
P’6 (254 mu). These are the only two peaks that are
206 nm
significantly associated with serum creatinine or creati-
- 2o4 nm
nine clearance. The concentration of middle molecules in
oxitocin [Mr lose Da] these two peaks is higher in hemodialyzed patients (with-
out any difference between type of dialyzer) than in perito-
neal dialysis and in nondialyzed azotemic patients. This is
particularly evident for P’6, which is absent in healthy
subjects (Table 2) but shows the same concentration in
peritoneal-dialysis patients as in nondialyzed azotemic
patients. Therefore, P’6 does not discriminate between
these groups (Table 2). However, the concentration of P’6
discriminates well between hemodialyzed patients and the
SO SO 70 so
Time
other groups studied. P’5 is highly variable in CAPD
patients (Figure 4) and does not discriminate well between
Fig. 1. Chromatographic profile obtained by gel filtration from a this group and polyacrylonitrile-hemodialyzed patients nor
uremic serum between CAPD and nondialyzed azotemic patients. The
We applied 0.5 mL of ultraflltrate to Sephadex G-15 and eluted it with 0.01 values for CAPD also overlap with those obtained in
mol/L TrIs- HCI buffer, pH 8.6, at a flow rate of 10 mL/h. The eluates were
monitored at 254 and 206 nm. Fractions between 1000 and 1500 Da healthy subjects.
(according to the oxytocin elutlon peak) were collected Figure 5 shows the serum concentrations of creatinine in

CLINICAL CHEMISTRY, Vol. 36, No. 11, 1990 1907

 70
50 -

-j

E 60 -J
E 40 -

30 -

20 -.

10 10-i

0 0
I-C NDAP CAPD AN CU I-C NDAP CAPD AN (U
Fig. 3. Mean (and SD) concentration of P1 (left) and P2 (right) by gel filtration and measured at 206 nm (cf., Table 1, at 254 nm)
AN, polyacrylonltille; CAPO, continuous ambulatory peritoneal dialysis; CU, cuprophane; HC, healthy controls; and NDAP, nondialyzed azotemic patients.
Statistical analysis was by Mann-Whftney’s U-test: <0.01 vs HC; b P <0.01 vs NDAP; C P <0.01 vs CAPD; (I p <0.01 vs AN; #{149}
P <0.01 vs Cu

20 - -J

1500

a,b,d,e ,

10 -
1000 a,c,d,e T

500

0- 0
I-C NDAP CAPD AN I-C NDAP CAPD AN
FIg. 4. Mean (SD) concentration of peak P’5 obtained by ion- Fig. 5. Mean (SD) concentration of creatinine in serum
exchange chromatography and measured at 206 nm Abbreviations and statistical significance as in Fig. 3
Abbreviations and statistical significance as in Fig. 3

molecules with peripheral neuropathy and uremic anemia

Table 2. COflCefltratlOfls of Peak P’8 ObtaIned by (16). In vitro studies have shown that middle molecules
Ion-Exchange Chromatography have a depressive effect on lymphoblast proliferation and
Peak P’1 (254 nm), mmlmL of on mixed lymphocyte reaction (17). All these toxic actions
serum of middle molecules point out the need of monitorizing
concentrations of middle molecules to assess therapy in
Patient group Mean Rang.
uremic diseases. Achieving this general use will require a
Cuprophane 10.3 5-I 85&b.c
simplified procedure.
Polyacrylonitrile 11.6 8.5-1 6,c In this work, we have used the separation method of
0_6d.e
CAPD 2.4 F#{252}rstet al. (12), modified by increasing the flow rate to 10
NDAP 2.2 0-$#{176} mLfh, which yields a different profile from the one they
Healthy controls 0 0-0 reported. In addition, material for peak 7c was no longer
Footnotes as In Table 1. available to be cochromatographed in our conditions, which
obliged us to rename our peaks. Dr. Cueille kindly sent us
material that produces the b42 peak but, when cochromato-
the different groups. Table 3 shows significant Spearman’s graphed this did not coincide with any of our observed
coefficient of rank correlation values between serum crea- peaks, a discrepancy referred to by Dr. Cueille (18).
tunune or creatinine clearance and P1, P2, P’5, and P’6. All The reproducibility of the procedure adapted from FOrst’s
of these peaks are positively correlated with the concentra- method is similar to that of other studies (19), which allows
tion of serum creatinine and negatively with creatinine us to study middle molecules with reasonable confidence in
clearance. various stages of renal failure. From the results (Tables 1
and 2 and Figures 3 and 4), we can conclude that CAPD is
DIscussion the best dialysis procedure for eliminating these com-
Plasma middle molecules appear more associated with pounds; a similar result was obtained by Bergstrom et al.
complications of uremia such as edema, pericarditis, and (20). The effectiveness of this procedure could be due to
intercurrent infections than is any other serum analyte steady-state conditions in this type of dialysis, or to the
(15). Other clinical studies have also associated middle continued lavage of the peritoneum with hypertonic solu-

1908 CLINICAL CHEMISTRY. Vol.36, No. 11, 1990

 Table 3. Sign Iflcant (P < 0.01 ) Rank-Orde r Correlatlo n C oefflclents

Creetinlne
‘ 254 nm
P1

206 nm
Spearman’.

254 nm
r,

P2

206 nm
P5,,
206 nm
‘
P1,
254 nm
Creatinine - 0.71 0.63 0.62 0.57 0.76 0.61
Creatinine clearance -0.74 -0.85 -0.85 -0.75 -0.7 -0.76 -0.77

tions, which would result in increased porosity of the for the b42 peak and Drs. Ordovas and Kam for their critical
membrane (21), or to a minor metabolic alteration (20). For revision of this manuscript. We are also grateful to Miss Aurora
whatever reason, CAPD appears to remove neurotoxic Osada for her help in drawing graphics and to Miss Roselle Obrien
for her help in preparing this manuscript. This investigation was
middle molecules more efficiently than any other type of supported by a grant awarded by FISS (INSALUD).
dialysis (3, 7,21-24).
The different types of hemodialysis systems studied References
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1910 CLINICAL CHEMISTRY, Vol.36, No. 11, 1990