CLIN.CHEM.38/8, 1429-1436 (1992)

CLINICALCHEMISTRY,Vol.38, No.8, 1992 1429

Assessmentof Urea and Other Uremic Markersfor Quantificationof DialysisEfficacyRaymond C. Vanholder, Rfta V. De Smet, and Severin M. Ringoir

To validate azotemic markers as an index for intradialyticchanges in solute concentration, we compared eightsolutes (pseudouridine, xanthine, hypoxanthine, peak 4,peak 5, p-hydroxyhippuric acid, indoxyl sulfate, and hip-punc acid) with five classical azotemic markers (urea,creatinine, uric acid, phosphate, and potassium). Wedetermined concentrations by reversed-phase HPLC cou-pled to ultraviolet absorption or photometrically. Sevencompounds showed significant intercorrelation (P<1 0): urea, pseudouridine, uric acid, peaks 4 and 5,p-hydroxyhippuricacid, and creatinine. The hippuric acidconcentration change after dialysis correlated with thechange for these seven compounds and also with indoxylsulfate, hypoxanthine, potassium, and the group of un-identified ultraviolet-absorbing HPLC peaks accumulatingin uremia. We conclude that urea only partially representsthe concentration changes of other retention compoundsafter dialysis; alternative markers, e.g., hippurate, shouldbe considered.

ddft1onal Keyphra.es: azotemlc markers pseudYjridineurate p-hydroxyhlppuric acid cxeatinine . hippuratechromatography, reversed-phase

The uremic syndrome is a complex of clinical andmetabolic disturbances caused by retention of azotemicwaste products (1-5). Its correction by dialysis treat-cnent is effective, but this is an unselective process forremoving uremic toxins. The definition of adequacy ofdialysis has long been controversial (6-9). The mostreliable index for adequate dialysis resulted from exten-3ive studies by the National Cooperative Dialysis StudyNCDS) Group, which produced an outline for dialysisiose prescription following the ratio of total urea clear-ance over distribution volume (Ks/V) and kinetic pm-iples (10-13).

Kinetic modeling of urea clearance allows a quicknbiased estimation of dialysis efficacy, although math-matical or methodological factors may introduce incon-istencies (14-18). Urea is a water-soluble non-protein-ound small molecule (M 60) with almost single-com-artment kinetics, and it may not be representative ofle other compounds retained as toxins in uremia andliminated by dialysis.In addition, synthetic dialyzer membranes with large

ores may have a completely different removal patternian that of cellulosic membranes, which were the onlyiembranes investigated in the NCDS study (9, 13).andsay and Spanner (17) indicated that syntheticiembranes required a lower urea Ks/V than did cellu-

Nephrology Department, University Hospital, 185 De Pin-lsan, B-9000 Ghent,Belgium.Received October 31, 1991; acceptedFebruary 27, 1992.

losic membranes to obtain the same rate of proteincatabolism; they attributed this to a better removal ofhigher-Me molecules through the synthetic membranes.

To further investigate the indexes for adequate bloodpurification and to circumvent the objections formulatedagainst urea in these days of more-sophisticated dialysisstrategies and membranes, we investigated 12 azotemicsolutes besides urea and compared their behavior dur-ing dialysis.

MaterIals and Methods

Reagents and StandardsAll chemicals for standard solutions were analytical

grade from Sigma Chemical Co. (St. Louis, MO), excepturea and phosphate (Boehringer Mannheim, Mann-helm, FRG). Methanol and water were HPLC-grade(Ailtech, Deerfield, IL). p-Hydroxyhippuric acid wasobtained courtesy of A. Schoots, Eindhoven, The Neth-erlands.

HPLC AnalysisSerum samples were centrifuged at 1800 x g through

a membrane with a cutoff of 30 kDa (Centrifreetm;Amicon Co., Danvers, MA), leaving non-protein-boundmaterial in the ultrafiltrate. We then analyzed thesesamples with an automated HPLC system comprisingan LKB 2153 autoinjector (Pharmacia LKB Biotechnol-ogy, Uppsala, Sweden) coupled to an ultraviolet absor-bance detector (LKB 2238 Uvicord Sil, at 254 nm) (19).The chroinatograms were recorded with a Model 3390Aintegrator (Hewlett-Packard, Avondale, PA). Ultravio-let spectra were registered with a photodiode arraydetector (1040A HPLC detection system; Hewlett-Pack-ard, Waldbmonn,FRG).

We used an Ultrasphere octadecylsilyc (ODS) column(4.6 mm x 25 cm; Beckman Inc., Mervue, Ireland) with5-ian particles and a 4.6 mm x 5 cm guard columnpacked with the same material. The solvent gradientwas 100% aqueous ammomum formate buffer (50mmol/L, pH 4.0) to 60% methanol at 45 mm and 67%methanol at 50 mm. Flow rate was 0.9 mL/min, and 1001L of sample was injected. We estimated peak heightand expressed it as relative to the height of an internalnaphthalene sulfonic acid standard (10 mg/L) to obtainresults independent of variations in detector sensitivity,sample size, and other components. The naphthalenestandard was eluted in a zone of the chromatogram thatwas free of other compounds. Studies with variousstandard solutions revealed correlation between peakheight and concentration for the identified solutes in theobserved concentration range (r >0.95). Peak heightwas preferred to peak area as an index of concentrationbecause of the complex nature of the solutions and the

I II II 31 II

Study Protocols

-ISI Nil

Fig. 1. Representativechromatogramsof azotemicserum samplesbefore and afterdialysisThe peaksevaluatedas single compounds are darkened; the naphthalenesulfonicstandard is marked by an asterisk

presence of multiple peaks. Peak area measurementdoes not always correctly define each peak nor permitcalculation of the exact area (20). For well-definedpeaks, however, peak height and area were highlysignificantly correlated.

Figure 1 illustrates representative chromatograxns,before and after dialysis; pseudouridine, uric acid, hy-poxanthine, xanthine, p-hydroxyhippuric acid, indoxylsulfate, hippuric acid, and two unidentified peaks (peaks4 and 5) were evaluated. All identified compounds havea molecular mass between 136 (hypoxanthine) and 251(indoxyl sulfate) Da. Protein binding, determined byheat deproteinization (21), was <10% for all compoundsexcept p-hydroxyhippuric acid (12%, SD 2%), hippuricacid (41%, SD 2%), and indoxyl sulfate (93%, SD 1%).We characterized substances by their retention times. Astandard solution was analyzed by HPLC, both aloneand after being added to serum ultraflltrate; tentativelyidentified peaks in the ultrafiltrate increased in heightwithout new peaks appearing. We confirmed the iden-tity of the peaks by superimposing the sample spectraobtained at 200-400 nm on spectra for standards havingthe same retention times. Intra- and interassay varia-tions of individual peak heights obtained in a standardsolution and in an ultraflltrate of uremic serum pro-cessed by HPLC (10 measurements) resulted in CVs<5%, except for the interassay CV of unidentified peaks4 and 5(8.5% and 8.3%, respectively), which was attrib-uted to insufficient resolution between these peaks.

To study the effects of these retention products onprotein binding of theophylline, we used semiprepara-tive HPLC (19) with 2-mL samples. HPLC conditionswere the same as for the analytical method, but we usedan RSil reversed-phase C18 preparative column, 10 mm(i.d.) x 25 cm, with 10-pun particles (Bio-Rad, Eke,Belgium). The flow rate was 3 mL/min; 6-mL fractionswere collected, lyophilized, and added to normal serumto study their impact on protein binding of theophylline.

1430 CLINICALCHEMISTRY,Vol.38, No.8, 1992

Concentration changes: comparison of azotemic aolutes. We used serum samples collected during 65 treatment sessions in 11 equilibrated patients who had beeion maintenance hemodialysis for �1 year. Serum samples were taken for photometric and HPLC analysiifrom the arteriovenous fistula before and immediatelafter dialysis. Samples were collected on crushed icecentrifuged, and deep-frozen at -80 #{176}Cuntil processingWe calculated the percentage concentration changes foieach solute during dialysis.

Dialyses were performed three times per week atblood flow rate of 300 mL/min and a dialysis-fluid flosirate of 500 mL/inin (22), with bicarbonate dialysatcontaining no glucoseand 1 mmol of potassium per liteiwith individual delivery-monitoring systems (BelIco BI760, Mirandola, Italy, and Fresenius 2008 C, OberurselFRG). There were 32 conventional 4-h hemodialysetthrough polysulfone P8400 membrane (F6; Freseniusand 33 hemodiafiltration sessions of 3 (n = 15) or 4 h (r= 18) through polysulfone P8600 membrane (F60; Fresenius) with 10 L of postdilutional fluid substitutior(23). Blood was collected after an equilibration period o�1 wk on the same treatment.

No patients received erythropoietin during the stud3or the 3 mo preceding the study. Beverages and foodwere withheld during the treatment sessions.

Concentration changes: comparison of hemodiafihtra.tion and hemodialysis. We used a similar approach inscrossover study. We consecutively treated 10 patients bhemodiaflltration (P8600; 10-L substitution, 3 or 3.5 hand conventional hemodialysis (P8400,4 h) in a randoixorder. These patients differed from those evaluatedduring the first protocol (1). Each treatment lasted for Iwk, and we collected blood before and after dialysis althe last session for photometric and HPLC analysis, asin the first protocol.

Hemodiaflltration and hemodialysis (4 h) with thsame P8600 membrane were also compared for 1patients. For hemodialysis with large-pore P8600 membrane, we reduced the transmembrane pressure byautomated decrease in the blood-compartment pressand an increase in the dialysate pressure, which rduced the ultrafiltration rate to the required minim[technical setup: Fresenius 2008 C coupled to arius (Oberursel, FRG) SM 40017 ultrafiltration module

Medication was unchanged during the evaluatioperiod.

Drug-protein binding studies. To test the toxic infience of the solutes evaluated in the first two protocolby using a biochemical function with pathophysiologiconsequences, we studied the effect of the lyophilisateHPLC fractions of deproteinized uremic serum on ptein binding of ‘4C-labeled theophylline (Amemshhit., Amersham, Bucks., UK; concentration 5.8 ‘imospecific radioactivity 58 Ci/mol) to normal serum. Dproteinized serum samples were obtained by heat deturation (n =3) or by acetonitrile extraction (n =4) (224).

Main ± SO

Concentration

Table 1. Percentage Concentration Changes and Concentrations Before and After Treatment

CLINICALCHEMISTRY,Vol. 38, No. 8, 1992 1431

Uric acid, zmoVLp-Hydroxyhippuricacid, pmot/LHippuric acid, zmoVLPeak 4Urea,mmol/LPeak5Creatlnine,imoVLPseudourldine, unoVLPhosphate, mmol/LPotassium,mmol/LHypoxanthine, izmoI/LXanthine, zmoI/LIndoxyl sulfate,pmol/L

66± 966± 964 ± 1161 ± 1160 ± 1058 ± 1255± 748 ± 1042 ± 1332± 929±3621 ± 27

2±30

VM

410 ± 7424 ± 21

272 ±1541.7± 0.8’

12 ± 41.4± 0.6’

1052 ±35638±8

1.8± 0.44.9 ± 0.8

15 ± 1210 ± 534±35

After

128 ± 428±6

97 ± 550.6 ± 0.3’5±20.6 ± 0.4’

482 ±21720±61.0± 0.23.4 ± 0.6

10 ± 37±3

30 ± 16

‘Relative peak height vs Internalstandard (peaks 4 and 5 are unidentified).

We measured drug-protein binding after adding 50pL of drug solution to 250 pL of normal serum (19). Tothis mixture we added the lyophilisate of deproteinizedserum samples or of HPLC fractions from deproteinizeduremic serum. The results were compared with those fora control sample with no lyophilisate added. Afterincubation at 37#{176}Cfor 30 mm, portions were ultrafil-trated through an anisotropic hydrophilic ultrafiltrationmembrane (Centrifree), to obtain the free fraction of thedrug. Samples (100 1zL)were processed for scintillationcounting (Packard Tri-.carb B 2450), after addition of 5mL of scintillation liquid (Ultima Gold; Canberra Pack-ard, Benelux, Belgium). Disintegrations per minutewere calculated by the external standard ratio method.Protein binding was calculated as the percentage boundfraction (PB) vs. total (pre-ultraflltration) concentrationby the formula

PB = [1 - (RAU/RA+)] x 100where RA is the radioactivity in the samples afterultrafiltration through the Centrifree membrane, andRA is the radioactivity in the total sample beforesubmission to ultrafiltration.

In addition, we dissolved known azotemic solutes in250 L of distilled water and, after lyophilization, addedthese to 250 zL of normal serum, to study their effect ontheophylline-protein binding: urea (71 mmol/L), creat-inine (1768 unol/L), uric acid (1196 imol/L), pseudo-

dine (205 zmoI/L), indoxyl sulfate (1194 .unol/L),-hydroxyhippuric acid (204 xno1/L), phosphorus (2260

oJ/L), and hippuric acid (1116 zmol/L) (n = 7 each).ese concentrations were chosen to be in the upper

of those currently encountered in dialyzed ure-.cs.

hemical and StatisticalProceduresUrea and phosphate were determined enzymatically

ccording to standard methods, and creatinine accord-g to the Jaffe reaction. Potassium was measured by

e photometry.

Results were expressed as mean ± SD, and weresubmitted to statistical comparison by FriedniRn’s anal-ysis of variance, followed by Wilcoxon’s sign test forpaired analysis, and were correlated by linear correla-tion-regression analysis. Differences were consideredstatistically significant when P <0.05.

ResultsConcentration Changes In Azotemic Solutes

Table 1 summarizes the percentage concentrationchanges and concentrations before and after treatmentfor the 13 compounds in 65 experiments. The highestpercentage concentration changes were for uric acid andp-hydroxyhippuric acid (66%, SD 9%). Changes ex-ceeded 60% for only 4 of the 13 compounds-uric acid,p-hydroxyhippuric acid, hippuric acid, and peak4-whereas the concentrations of hypoxanthine, xan-thine, and indoxyl sulfate changed by <30%. Moreover,the concentration changes of the latter three compoundsdisplayed substantial scatter. Urea concentrationchanges averaged 60% (SD 10%). The percentage con-centration changes for 6 of the 13 compounds differedfrom this value by >10%.

Comparing concentration changes with one anotherby the Friednsn analysis of variance revealed highlysignificant differences (P <10-s). Paired comparisonsrevealed for virtually all data significant intercompari-son differences (Table 2). The concentration change forurea was significantly less than that for hippurate andp-hydroxyhippurate (P <0.001 for each).

Correlation-regression analysis of the concentrationchanges of single retention compounds in these 65experiments revealed statistical significance for 41 of 78regression analyses (52.6%). The best-fitting correla-tions (r >0.70) are illustrated in Figure 2 and involveseven compounds: uric acid, pseudouridine, p-hydroxy-hippuric acid, urea, creatinine, and unidentified peaks 4and 5 (Table 3). Consequently, the determination coef-ficient (r2) rarely exceeded 0.50 (only in seven regression

PS UA HX XA P4 P5 PH IS HA UR

PS - 7.Oa 3.5 6.4 6.6 6.3 7.0 3.7 6.8 6.7UA - 6.6 7.0 4.8 5.5 22b

64b 20b 59HX - NS 6.2 5.7 6.6 2.1 6.6 5.9XA - 6.8 6.7 6.9 NS 6.9 6.8p4 - 3.2

-

4.85.2

-

6.05.16.4

-

22b

3.9NS

6.3

21b

NS5.05.8

K6.87.0NS

6.97.07.0NS7.07.0

- 4.0 5.9

80

- 6.5 7.0- 4.6

65

50

35

20

HIPPURIC ACID RI%) CREATININE R(%i PEAK 4 R(%) URIC ACID R’%

650

C,

C)50

35

20

,,

,,

/

I

20 35 50 b5 50

URICACID R% UREA R(%

FIg. 2. The best-fittingcorrelations(r >0.70) for intercomparingpatterns ofconcentrationchanges

20

p#{149}OHHIPPURICACID R’%

PS - 0.73 0.72UA - 0.61P4P5URCRPH

Table 2. Intercomparlson of the percentage concentration changes of different solutes

1432 CLINICAL CHEMISTRY, Vol. 38, No.8, 1992

P5

PH

HA

CR

CR P5.8 30b

6.6 6.9536.6 5.14.7 6.227b 577.0 6.949

6.8- 5.2 6.7

#{149}Results shown are zvaiues of statistical comparison. M are P <0.001exceptwhere otherwise noted, b P <0.05.PS:pseudouridlne, UA: uric acid, HX:hypoxanthine,XA: xanthine, P4: unidentIfied peak 4, P5: unidentifiedpeak 5, PH: p.hydroxyhlppurlcacid, IS: Indoxylsulfate, HA: hippuric acid, UR: urea, CR:creatinine,P: phosphate, K: potassium; NS: nonsignificant.

0

U

U

0.0.

II9

analyses), which suggests that, in moat cases, the vari-ation in percentage concentration change for onemarker is only partially explained by the variation foranother marker.

For hippuric acid (Table 3), we also found a significantcorrelation with the group of seven compounds. Thehighest correlation was with p-hydroxyhippuric acid (r= 0.72). The correlation of hippuric acid with urea wassignificantly weaker (r = 0.46, P <0.01 by FisherZ-transformation). The correlation regression of hippu-ric acid vs. urea indicates that when urea concentrationchanges as a result of dialysis were 50-60% (correspond-ing to a KIV of ± 1.0), the hippuric acid concentrationschanged in a markedly broader range, from 40% to 80%.

Table 3. Results of CorrelatIon-Regression Analyses

HA

PS UA P4 P5 UR CR PH HA0.69 0.62 0.69 0.79 0.60.63 0.73 0.78 0.69 0.5

- 0.55 0.54 0.49 0.74 0.5- 0.46 0.66 0.62 0.4

- 0.66 0.47 0.4- 0.62 0.4

- 0.7

#{149}Results shown are best.fltting correlations In a cluster of seven conpounds,and the correlationofhippuric acid to this cluster.All correlationsesignificant. Values >0.44 correspond to P <0.001, those >0.55 to<0.00001. AbbrevIationsas In Table 2.

Clustercompound

PSUAP4P5URCRPHHA

HX

NSNS

0.25NSNSNSNS

0.32

XA IS

NS NSNS NSNS NSNS NSNS NSNS NSNS 0.30NS 0.55

P

NSNSNSNSNS

0.43NSNS

H.niodlalysls HemodlafiltratlonK

0.330.41NSNS

0.290.520.400.38

Abbreviationsas In Table 2. NS:nonsignificant,correspondsto a correlationIndex <0.24. Correlation indicesof0.24, 0.44, and 0.54 or more, correspondto P <0.05,0.001, and 0.00001, respectIvely.

Uricacidp.Hydroxyhippurlc acidHippuric acidPeak 4UreaPeak 5CreatininePseudouridinePhosphatePotassiumHypoxanthineXanthineIndoxylsulfate

69± 564± 561 ± 865± 762± 861 ± 959± 550± 543± 933± 932 ± 2130 ± 21

-45 ± 35

70± 972± 8b

72± 7b

70± 70

63± 868 ± 12058± 856 ± 100

44 ± 1531 ± 1045±2622±2213 ± 35

In addition, concentration changes of hippuric acidcorrelated specifically with those of indoxyl sulfate (r =

0.55, P <10) (Table 4), which was not the casefor anyof the 12 other solutes, including urea. Hippuric acidfurther was correlated (r = 0.88, P <10) with theaverage concentration changes of the unidentified ultra-violet-absorbing peaks eluted by HPLC (these were notconsidered as single compounds in the present study).Five substances-hypoxanthine, xanthine, indoxyl sul-fate, phosphate, and potassium-correlated with theother compoundsor with one another only weakly or notat all (Table 4). Hippuric acid, however, correlatedsignificantly with three of these five compounds, incontrast to urea, which showed no correlation withthem.

Hemodiafiltrationand HemodialysisCompared

a Mean ± SD, before vs after treatment. b.c Significantly different from

hemodlalyslsresult: b P <0.01; #{176}P <0.05.

7’ i

60

=

50

a-

311

CLINICALCHEMISTRY,Vol.38, No.8, 1992 1433

Table 4. Results of the Correlation-RegressionAnalyses of Five Compounds with Weak to No

Correlations with the Cluster and with Hippuric Acid

We also compared the concentration changes of these13 compounds by a crossover study involving differentdialysis strategies. Conventional hemodialysis withsmall-pore P5400, compared with hemodiafiltrationwith large-pore P5600, gave statistically significantdifferent results for uric acid, p-hydroxyhippuric acid,hippuric acid, peaks 4 and 5, and pseudouridine, but notfor urea and creatinine (Table 5).

When hemodialysis and hemodiaflltration were bothperformed with polysulfone PS 600 dialyzers, the differ-ences were still significant for p-hydroxyhippuric acid[concentration changes from 66% (SD 9%) to 70% (SD6%); P <0.051 and hippuric acid (changes from 61% (SD15%) to 69% (SD 11%); P <0.01), but not for the othercompounds (data not shown).

Drug-Protein Binding Studies

The influence of several different deproteinized serumfractions, obtained by semipreparative HPLC, on pro-tein binding of theophylline is illustrated in Figure 3.Binding in fractions 3-4, 6, and 9-12 was significantlydecreased. Further characterization of the peaks bycomparison with standard solutions revealed the pres-ence of pseudouridine and uric acid in fraction 6, p-hy-droxyhippuric acid in fraction 9, and hippuric acid and

Table 5. ComparIson of Concentration Changes withVarious Dialysis Strategies

concn change

I 5 II 15 28FRACTION(d

Fig.3. Effectof Iyophilisate of HPLCfractionsonproteinbindingoftheophylline(%of totaldrug)The left-most poInt (triangle) represents normal serum. Each fraction wascollectedover 2 mm. : P <0.05; : P <0.01 vs normalserum value. Fraction3 contains NaCI; hippuric acid and Indoxyl sulfate are eluted In fraction 12

indoxyl sulfate in fraction 12. In fractions 3 and 4, NaC1is eluted, which inhibits the protein binding of theophyl-line in a dose-dependent way (19).

Lyophilisate of solutions of hippuric acid and indoxylsulfate inhibited the protein binding of theophylline by15.6% and 12.0%, respectively (P <0.01 vs normalserum); there was no inhibition by the remaining sol-utes under study, including urea, creatinine, and uric

acid (Table 6).These data indicate that the current dialysis markers

urea and creatinine have no effect on theophyllinebinding. In contrast, binding inhibition was proven forhippuric acid and indoxyl sulfate, two compoundsshow-ing a distinct pattern of concentration changes in bothdialysis study protocols.

DiscussionSeven substances-the prevailing azotemia markers

urea, creatinine, and uric acid together with peaks 4

Salute

UreaCreatinineUricacidPseudouridineIndoxyl sulfatep.HydroxyhippuncacidPhosphorusHippuric acid

a P <0.01 vs normal serum.

Percent change

-2.6 ± 3.9-3.8 ± 3.3+1.4 ± 1.5-3.5 ± 1.5

-12.0 ± 1.sa-4.1 ± 2.3-0.2 ± 1.3

-15.6 ± 3.2a

1434 CLINICALCHEMISTRY, Vol. 38, No.8, 1992

Table 6. Effect of Individual Solute. on Protein BindIngof Theophylline

and 5, p-hydroxyhippuric acid, and pseudouridine-show highly significant mutual correlations in dialysis-induced concentration changes. However, the concen-tration changes for xanthine, hypoxanthine, indoxylsulfate, potassium, and phosphorus are not correlatedwith the data obtained with these seven compounds.

Hippuric acid correlates with the latter group, butalso has a unique additional correlation with indoxylsulfate (Table 4) and with a group of unidentified minorpeaks. This suggests that the concentration changes ofthe latter compoundsmight be controlled by a commonprocess, which is of interest because hippurate andindoxyl sulfate are the only organic acids we evaluatedthat have substantial protein binding. It would beuseful if the mechanistic behavior of hippurate wererepresentative for the concentration changes of thelarge group of remaining retention solutes, many ofwhich are organic acids. This possibility is suggested bythe remarkable correlation of hippuric acid concentra-tion changes with the average concentration changes ofminor ultraviolet-absorbing peaks eluted by HPLC. Wesuggest that hippurate may reflect the concentrationchanges not only of small water-soluble compounds,represented by urea, but also of a group of compoundswith different behavior, for which urea is not represen-tative. Nevertheless, we are aware that differencesamong the various compoundsremain marked: even forthis small group of compounds of similar molecularmasses, the validity of using each solute as a marker topredict the concentration changes of the other com-pounds is limited.

Different concentration changes may depend on thedialysis strategy used. Although concentration changesof urea and creatinine during conventional hemodialy-sis and hemodiaflltration were similar, the changes forhippuric acid were strikingly different (Table 5). Thus, asimilar concentration change in urea or creatinine is notnecessarily paralleled by a similar concentrationchange in other compounds.

The percentage removal values for hemodialysis re-ported in Table 5 are sometimes greater than the overallpercentage removals (hemodialysis + hemodiaflltra-tion) reported in Table 1. The percentage removal dur-ing dialysis depends not only on dialysis strategy anddialyzer characteristics, but also on the quality of vas-cular access, the flow rate of blood and dialysate, and the

patient’s distribution volumes for each solute. Theseinterindividual, uncontrollable differences may explainwhy the results in Table 5 in general seem somewhatmore favorable than those reported in Table 1, becausethe protocols were performed with different patientpopulations.

Applying convection in hemodiaflltration presumablypromotes the removal of larger molecules (25). Thedifferent behavior of hippuric acid (compared with urea)may in part be attributed to the protein binding ofhippurate, which will produce an elimination patternsimilar to that of larger molecules (26); this makes thestudy of hippuric acid as a marker all the more inter-esting. Consequently, hippurate concentration changesmay be slightly but significantly more important thanurea concentration changes (Table 1).

On the other hand, hippurate, which is -40% proteinbound, was removed as well as urea was, whereasindoxyl sulfate, which is 93% bound, was removedpoorly, if at all. The virtually complete protein bindingof indoxyl sulfate greatly hampers its elimination.Other, partly undefined additional factors, e.g., multi-compartment distribution, acid-base shifts, and intra-dialytic increases in protein binding, may also play arole.

Our data emphasize how the behavior of azotemiccompounds during dialysis might differ, depending onthe dialysis strategy, and show the weakness of urea asa marker in new dialysis strategies and membranes. Foridentical changes in urea concentration, concentrationchanges of other compounds may behave differently,which goes unrecognized by conventional kinetic anal-ysis of urea disposition.

A marker of azotemic retention and concentrationchanges becomes more interesting if it displays sometoxicity by itself. Urea and the like (e.g., creatinine,guanidines) are currently believed to display no toxic-ity, or to be toxic only at concentrations greatly exceed-ing those observed today in maintenance dialysis (27-29). In contrast, hippuric acid interferes with drug-protein binding, glucose metabolism, erythropoiesis,and transport of organic acids acrosscell membranes inrenal tubules, chorioid plexus, brain, cerebrospinalfluid, ciliary bodies, thyroid, liver, and erythrocytes, atconcentrations currently registered in uremia (24, 30-37). As an organic anion compound, hippuric acid mayalter the accumulation of this group of substances inspecific organs, e.g., the brain, and in the entire body; inaddition, an increase in organic anions may widen the“anion gap,” which might add to their toxic impact.Inhibition of protein binding and the failure of organicanion transport are probably related to each other (38).

To appreciate directly the biochemical impact of the I

substances under study, we evaluated the influence ofHPLC-eluted fractions from deproteinized and fraction-ated uremic serum on the protein binding of theophyl-line. Hippuric acid and indoxyl sulfate were among theinhibitors of this binding, whereas urea, creatinine,pseudouridine, and uric acid had no effect (Figure 3,Table 4). Thus, urea and similar compounds have no

CLINICALCHEMISTRY,Vol.38, No.8, 1992 1435

“toxic” impact on drug-protein binding, whereas othersolutes that are not currently used as dialysis markersinterfere with this function.

Xanthine, hypoxanthine, phosphorus, potassium, andindoxyl sulfate only weakly or nonsignificantly corre-lated with most other compounds, including urea; thissupports our assumption that the concentration changesof this group of solutes will not be predicted by currentmarkers (Table 4). These results parallel what is foundin the predialysis stage of chronic renal failure; i.e.,different biochemical analytes change at different rates(39). However, concentration changes in three of theabove-mentioned compounds (hypoxanthine, indoxylsulfate, and potassium) correlate with those of hippuricacid.

The lack of correlation between urea and phosphateagrees with earlier studies, which demonstrated a sta-bilization or even an increase in phosphate concentra-tion during the last hour(s) of dialysis (40, 41), incontrast to the progressive and almost linear decrease ofurea. Conceivably, therefore, the postdialysis phosphateconcentration will not be appropriately predictable fromthe predialysis phosphate values nor from the kineticdata for urea.

In conclusion, there is no consistent behavior ofsmall-Me azotemic solutes upon dialysis. Changes inseven compounds, containing urea, creatinine, and uricacid, show a high intercorrelation. Hippuric acid, mea-sured by HPLC or photometrically (42), correlates wellwith these seven compounds, but further correlatesspecifically with other compounds such as indoxyl sul-fate, differs from urea in its response to dialysis, anddisplays toxicity in relation to protein binding of theo-phylline. The use of alternative marker molecules in-stead of urea, e.g., hippuric acid, to study and evaluatesolute concentration changes should be considered. Oneshould take into account the possibility that hippuricacid concentration depends on the dialysis strategy.

Portionsof this work have been published in abstract form(Nephrol Dial Transplant 1991;5:701and IntJ Artif Organs 1991;14: 578). This project was funded by theBelgian National Fundsfor Medical Research (NFWGO).

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