Citation: Cell Death and Disease (2012) 3, e377; doi:10.1038/cddis.2012.112
& 2012 Macmillan Publishers Limited All rights reserved 2041-4889/12 http://www.nature.com/CDDIS
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A Noce1,7, M Ferrannini2,7, R Fabrini3,7, A Bocedi3, M Dess4, F Galli5, G Federici6, R Palumbo2, N Di Daniele1 and G Ricci*,3
Kt/Vurea ratio is commonly used to assess the delivered dose of dialysis in maintenance hemodialysis (MHD) patients. This parameter only reects the efcacy of dialytic treatments in removing small toxins, but not middle and protein-bound toxins. Erythrocyte glutathione transferase (e-GST), an enzyme devoted to cell depuration against a lot of large and small toxins, is overexpressed in uremic patients. Aim of the present study is to verify whether e-GST may represent a novel biomarker to assess the adequacy of different dialytic techniques complementary to Kt/Vurea parameter. Furthermore, it will be investigated whether e-GST could reect the average adequacy of multiple dialytic sessions and not of a single one treatment as it occurs for Kt/Vurea.
One hundred and three MHD patients and 82 healthy subjects were tested. Fourty four patients were treated with standard bicarbonate hemodialysis (HD) and 59 patients were on online hemodialtration (HDF). In all MHD patients e-GST activity was 60% higher than in healthy controls. In HDF, e-GST activity was lower than in HD subgroup (8.20.4 versus 10.00.4 U/gHb,
respectively). Single-pool Kt/Vurea and total weekly Kt/Vurea were higher in HDF than in HD, but no correlation was found between e-GST activity and Kt/Vurea data. e-GST, whose level is stable during the erythrocyte life-span, provides information on the long-term depurative efcacy of dialysis treatments.
Cell Death and Disease (2012) 3, e377; doi:http://dx.doi.org/10.1038/cddis.2012.112
Web End =10.1038/cddis.2012.112 ; published online 23 August 2012
Subject Category: Internal Medicine
Uremic syndrome is characterized by the accumulation of uremic toxins due to inadequate kidney function. In literature more than 90 compounds are identied as uremic toxins. The European Uremic Toxin (EUTox) Work Group proposed a practical classication based on physicalchemical characteristics that inuence their dialytic removal:1 small solutes (o500 Da), with urea as a prototypal compound, middle molecules (4500 Da) such as beta-2-microglobulin, and large solutes, which include an heterogeneous class of molecules such as small and middle protein-bound molecules that are bound to plasma proteins (these include for instance p-cresol, homocysteine and a series of reactive carbonyls such as 4-hydroxynonenal, malondialdehyde, methylglyoxal and so on), tissue proteins released by cell damage and products of protein damage by the reaction with reactive oxygen species and reducing sugars, and the products of their metabolism by proteolytic events, cross-linking and so on.24
An ideal dialytic therapy should remove all of these compounds. However, only small toxins are easily removed
Keywords: chronic kidney disease; erythrocyte glutathione S-transferase; hemodialysis adequacy; Kt/Vurea; oxidative stress; uremic toxinsAbbreviations: ADPKD, autosomal dominant polycystic kidney disease; BUN, blood urea nitrogen; CDNB, 1-chloro-2,4-dinitrobenzene; hs-CRP, high-sensitivity C-reactive protein; DNDGIC, dinitrosyl-diglutathionyliron complex; e-GST, erythrocyte glutathione transferase; EPO, erythropoietin; ERI, EPO resistance index; ESRD, end-stage renal disease; GSH, glutathione; Hb, hemoglobin; HD, standard bicarbonate hemodialysis; HDF, hemodialtration; K-DOQI, kidney disease outcomes quality initiative; MHD, maintenance hemodialysis; spKt/Vurea, single-pool Kt/Vurea
Received 17.5.12; revised 26.6.12; accepted 28.6.12; Edited by A Finazzi-Agro0
Erythrocyte glutathione transferase: a new biomarker for hemodialysis adequacy, overcoming the Kt/Vurea
dogma?
by all dialytic techniques, even if some improvements have been introduced by the use of more efcient dialyser membranes such as advanced high-ux and protein-leaking hemodialyser membranes, and treatment methods that are based for instance on hemodialtration (HDF) techniques.57
Despite this, the hemodialysis adequacy and dosing are usually discussed only in terms of Kt/Vurea, a mathematical
model that takes into account the urea clearance in a single hemodialysis session.8,9 Recent studies showed that Kt/Vurea in dialysis cannot represent correctly the removal of other solutes and uid, indicating that this parameter alone should not be used as the sole indicator of dialysis adequacy.10,11
Thus, other dialysis biomarkers have been proposed (i.e., p-cresol,12 beta-2-microglobulin,13 guanidine compounds,14 high-throughput molecular ngerprinting assays15), but their
quantications require complex and expensive procedures and again they only measure the efciency of a single dialytic session. Thus, the identication of new clinical indicators able to reveal the degree of blood purication from small as well as
1Department of Internal Medicine, Nephrology and Hypertension Unit, Tor Vergata University, Viale Oxford 81, 00133 Rome, Italy; 2Nephrology and Dialysis Unit, S. Eugenio Hospital, Piazzale dellUmanesimo 10, 00144 Rome, Italy; 3Department of Chemical Sciences and Technologies, University of Rome Tor Vergata, Via della Ricerca Scientica, 00133 Rome, Italy; 4Department of Laboratory Medicine, Tor Vergata University, Via Montpellier 1, 00133 Rome, Italy; 5Department of Internal Medicine, University of Perugia, Via del Giochetto, 06126 Perugia, Italy and 6Childrens Hospital IRCCS Bambin Ges, Piazza SantOnofrio 4, 00165 Rome, Italy *Corresponding author: G Ricci, Department of Chemical Sciences and Technologies, University of Rome Tor Vergata, Via della Ricerca Scientica 1, 00133 Rome, Italy. Tel: +39 6 72594379; Fax: +39 6 72594328; E-mail: mailto:[email protected]
Web End [email protected]
7These authors contributed equally to this work.
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A Noce et al
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Table 1 Epidemiological parameters of hemodialytic patients and healthy controls
Total uremic patients
Control group
Total uremic patients
large toxins in a wide range of dialytic sessions will be of
medical interest.
Erythrocyte glutathione transferase (e-GST), an enzyme compartmentalized in the red cells and then non-dialyzable, could be ideal for this role. GSTs represent a super-family of ubiquitous enzymes devoted to cell protection16 by promoting the conjugation of glutathione with toxins of very different shapes.17,18 Alternatively, GSTs may act as ligandins by binding and sequestering a variety of small or large toxic compounds and peptides. An example of this ligandin role is the specic binding of GSTP1-1 to Jun-kinase, a pro-apoptotic enzyme that becomes inactive when bound to GST.19 Human cytosolic GSTs are dimeric proteins grouped into seven different gene-independent classes termed Alpha, Mu, Pi, Theta, Omega, Sigma and Zeta. Human glutathione transfer-ase P1-1 (hGSTP1-1) is a homodimeric intracellular protein of about 46 kDa expressed in different organs and cell types. The GSTP1-1 is the most abundant form of intra-erythrocyte transferase representing 95% of entire GST pool.20
In healthy subjects, the intra-cellular level of e-GST remains virtually constant during childhood and adult life,21 increasing only in two pathological conditions, that is hyperbilirubinemia and uremia.22,23 No other pathologies have been reported to induce e-GST hyper-activity. We recently conrmed these results observing that the activity of e-GST increases from5.80.4 U/gHb in healthy subjects to 10.20.5 U/gHb in maintenance hemodialysis (MHD) patients.24 This hyper-activity represents a defense reply against systemic toxicity of the uremic condition. For the rst time a signicant increase in e-GST activity has been also found in chronic kidney disease patients under conservative therapy with a positive correlation with disease severity weighted according to the ve stages of chronic renal failure of kidney disease outcomes quality initiative (K-DOQI) classication.24 Interestingly, patients belonging to the fourth stage display e-GST activity (121 U/gHb) higher than those under dialysis(10.20.4 U/gHb), conrming that e-GST may be a reasonable natural biomarker of blood toxicity,24 that is an endogenous probe whose concentration reects the level of circulating toxins. The aim of the present study is to verify whether e-GST may represent a novel biomarker able to assess the adequacy of different dialytic techniques (i.e., standard bicarbonate hemodialysis (HD) and HDF) complementary to the Kt/Vurea
parameter. Furthermore, it will be investigated whether e-GST could reect the adequacy not of a single treatment but of many dialytic sessions accomplished during a few weeks. The possible correlation between e-GST activity and single-pool Kt/Vurea (spKt/Vurea) and total weekly Kt/Vurea will be also investigated. Results obtained open the way to further clinical studies on the use of e-GST as biosensor for long-time exposure to uremic toxicity and dialysis efciency.
Results
GST hyper-activity in dialyzed patients. One hundred and eighty ve healthy subjects and uremic patients have been involved in this study and some epidemiological parameters are shown in Table 1. Furthermore, the number of patients grouped for primary causes of end-stage renal disease (ESRD) (Table 1) did not differ between HD and HDF
subgroups. On 82 healthy subjects (control group), e-GST activity was 5.60.4 U/gHb, a value very close to that reported in a previous investigation.24 For the 103 uremic patients their e-GST activity was 9.00.3 U/gHb (Figure 1a and Table 2).
On comparing the e-GST activities of the control group versus all uremic patients, we observed a signicant statistically difference (Po0.0001) (Figure 1a). Moreover, we investigated whether the e-GST activity was related to different hemodialysis techniques (convective versus diffusive) and/or to dialytic dose.
First, we matched HDF and HD groups for e-GST and several parameters (Table 2). In the 59 HDF-group patients the mean spKt/Vurea and total weekly Kt/Vurea were
1.500.03 and 4.60.1 respectively; the mean e-GST activity was 8.20.4 U/gHb. For the 44 HD-group, the mean spKt/Vurea value was 1.300.05, the total weekly Kt/Vurea3.90.1, whereas the mean e-GST activity of this group was10.00.4 U/gHb. The difference in e-GST activity between the HD-group and HDF-group is better highlighted in Figure 1b, where the relative increase of e-GST (from the mean value of healthy subjects) is reported.
From statistical analysis, we found no differences for age, HD vintage, albumin, EPO dose, EPO resistance index (ERI) and high-sensitivity C-reactive protein (hs-CRP), between the two groups (Table 2). However, we observed statistical signicant differences for e-GST activity (P 0.003), blood
urea nitrogen (BUN)-predialysis (P 0.0001), spKt/Vurea
(P 0.0002) and total weekly Kt/Vurea (P 0.0006) (Table 2).
In a second experimental step, we evaluated whether e-GST activity is related to the dialytic dose. For this purpose, all uremic patients were divided in two subgroups, using a cutoffX1.3 for Kt/Vurea, according to NFK-DOQI guidelines.25
In patients with spKt/Vureao1.3, e-GST was 9.70.7
(S.D. 3.2), whereas in patients with spKt/VureaX1.3 was
8.70.4 (S.D. 2.9), without any statistically signicant
HDF group
HD group
Number 82 103 59 44 Male/female 36/46 65/38 37/22 28/16 Average age (years) 442a 6116a 6118a 6114a HD vintage (months) 8783a 7666a 10195a
Cause of ESRD(a) Chronic glomerulonephritis
18 10 8
(b) Nephroangiosclerosis 38 21 17(c) Pyelonephritis and Interstitial Nephritis
8 5 3
(d) Diabetic nephropathy 27 16 11(e) ADPKD 7 4 3(f) Other disease or unknown cause
5 3 2
Abbreviations: ESRD, end-stage renal disease; ADPKD, autosomal dominant polycystic kidney disease
aData are expressed as meanS.D.
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Table 2 Correlations of various parameters between HD- and HDF groups
Parameters
Total uremic patients (103)
HDFgroup (59) P (HD versus HDF)
Albumin (g/l) 3.920.04 4.020.08 3.900.05 0.352 BUN predialysis (mg/dl) 1124 914 1287 0.0001 EPO (IU)a 11383698 120341266 10898776 0.979 EPO resistance indexa 171 172 162 0.468 Age (years) 612 612 612 0.952 HD vintage (months)a 878 10115 769 0.420 spKt/Vurea 1.440.03 1.300.05 1.500.03 0.0002
Total weekly Kt/Vurea 4.30.1 3.90.1 4.60.1 0.0006 hs-CRP (mg/dl)a 0.640.09 0.550.08 0.710.09 0.711 e-GST activity (U/gHb) 9.00.3 10.00.4 8.20.4 0.003
Data are as meanS.E.M., Po0.05 is considered statistically signicant
aMannWhitney test
HD group (44)
Figure 1 e-GST activity in all the uremic patients (HD- and HDF group) and in the Control group. (a) aPo0.0001 e-GST activity in total uremic patients versus
Control group. bPo0.0001 e-GST activity in HD group versus HDF group. (b) Relative increase in e-GST activity from the mean value of healthy subjects observed in HD group and in HDF group
Figure 2 e-GST activity in uremic patients subdivided basis on Kt/Vurea values. The cut-off for Kt/Vurea is Z1.3
difference (P 0.156) (Figure 2). In addition, we did not nd
any linear correlation between e-GST activity and sp Kt/Vurea
values in all uremic patients (r2 0.0378; P 0.049). Other
established markers for malnutrition, inammation and dialysis efciency showed slight or no correlation with e-GST, that is albumin (r2 0.0202, P 0.153), hs-CRP
(r2 0.0336; P 0.064), BUN predialysis (r2 0.1906;
Po0.001), BUN postdialysis (r2 0.0822; P 0.003).
Furthermore, to assess whether the administration of EPO may inuence the e-GST activity, we evaluated the possible correlation between e-GST and variables like EPO dose and ERI. We did not nd any linear correlation between e-GST and EPO dose (r2 0.0199, P 0.139) or e-GST and ERI
(r2 0.0396, P 0.0797).
Hyper-activity of e-GST is due to hyper-expression of the enzyme. Various factors may cause the hyper-activity of e-GST observed in uremic patients. For example, it is well known that the life-span of RBC is shorter in hemodialysis patients26 and that young RBC display somewhat higher GST activity than old RBC.27 Previous observations, however, showed that the proportion of young and old cells
are the same, but e-GST was higher in hemodialysis patients than in healthy controls.23 This difference was particularly pronounced in young cells and decreased as cell age increased. Again, the supplementation with EPO, which might increase the number of young cells in circulation, was not found to interfere with the mean concentrations of e-GST in EPO-responsive patients.23 Thus, it may be concluded that the observed increased activity in uremic patients is not due to an increased proportion of young erythroid elements in the circulation.
Other factors may cause e-GST hyper-activity, that is the presence of intra-erythrocyte GST activators, post-translational enzyme modications, overexpression or slower turnover break-down of this enzyme. Clarifying the origin of this hyper-activity will be essential to establish whether e-GST could be used as a biomarker for the adequacy of a single dialytic session or it reects the dialytic efciency of multiple dialytic sessions performed during several weeks. In fact,
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GST as dialysis biosensor A Noce et al
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Figure 3 (a) Comparison between specic activities of e-GST puried by afnitychromatography from healthy controls (5.6 U/gHb) and hemodialysis patients(15 U/gHb), see Materials and Methods section. (b) The same e-GST samples usedfor experiment in (a) were mixed with variable amounts of DNDGIC and after 1 minincubation the residual activity was measured. Each point is the mean of threedistinct measurementsS.E.M. Figure 4 e-GST activity of two patients in distinct dialytic sessions during a
week before (lled symbols) and after (open symbols) the hemodialysis treatment. () diffusive dialysis; (K) convective dialysis. In the day 2, no dialysis has been performed. Each point represent the mean of three distinct activity measurementsS.E.M.
erythrocytes are enucleated cells and their enzyme biosynthesis does not occur in mature cells. Thus, although a post-translational activation or the presence of a specic enzyme activator could be a short-time responsiveness to an increased level of toxins, an overexpression of e-GST likely reects a long-time-mediated exposition to toxins, which may be estimated on the basis of the erythrocytes survival (E58.9 days for dialyzed patients).28 To solve this enigma, we compared the specic activity of e-GST puried with afnity chromatography from dialyzed patients with the one coming from healthy subjects. The similarity of the two values found in the fully puried samples (9410 U/mg and 836 U/mg, respectively) suggests the increase of activity found in dialyzed patient to be due to a real increased level of this enzyme and not to enzyme activation (Figure 3a), conrming previous suggestions obtained by western blotting analysis.23
A further convincing evidence has been obtained using a very specic and selective inhibitor of e-GST, that is the dinitrosyldiglutathionyl-iron complex. This compound binds with extraordinary afnity to all mammalian GSTs, with a KD of about 10 9 M29,30 and causing a complete enzyme inhibition. This phenomenon is almost stoichiometric and allows a precise quantication of enzyme present in a given solution. As shown in Figure 3b the amount of DNDGIC necessary to inhibit 50% of e-GST puried from dialyzed patients (15 U/gHb) is about
three times higher than that necessary for that from healthy subjects (5.6 U/gHb), indicating the presence of about three times higher concentration of e-GST in the dialyzed patients. Thus, e-GST hyper-activity reects a true higher level of this protein in the erythrocyte. The occurrence of possible e-GST break-down during the erythrocyte life-span is never been described; on the contrary, an extraordinary stability of e-GST activity in intact erythrocytes has been reported.24 Thus, a reasonable (even if not denitive) explanation for the increased e-GST activity in dialyzed patients is that an overexpression of e-GST occurred in the maturation phase of the red cells. In this scenario e-GST may be considered a biosensor of average blood toxicity in a span of a few weeks.
A convincing evidence that e-GST activity is not affected by a short-time variation of circulating toxins is reported in Figure 4 showing pre- and post-dialysis levels of e-GST of two patients (the rst one under HD and the second one under HDF) measured during one week of therapy. In each patient, the e-GST activity is almost identical either in the pre and post dialysis as well as in the distinct dialytic sessions with a maximum variation of about 5% (average variation 4%) for
the patient under HD and about 6% (average variation 3%)
for the patient under HDF.
Discussion
All along nephrologists dose only few toxins to quantify the degree of renal dysfunction as well as to determine dialytic dose and adequacy in MHD patients using Kt/Vurea, as
recommended by International guidelines. However, Kt/Vurea
is an urea-centric mathematical model commonly adopted to quantify the detoxication from a single compound in a single dialytic session. Curiously, doubts have arisen by considering urea as a true marker of the uremic disease as the EUTox group dened it as a not necessarily toxic solute.10 Indeed, uremic syndrome is more than urea accumulation and the toxic solutes that contribute to uremic illness are different from urea and probably most of them remain to be identied. As a consequence, adequate dose of dialysis is difcult to dene because theoretically we should calculate the fractional removal of each single toxin and their intrinsic toxicity. Otherwise, even if nowadays Kt/Vurea provides a useful tool to avoid grossly inadequate dialysis and Kt/Vurea 41.3 is
indicated by international guidelines as the dose of dialysis to achieve, in the future the urea removal standard will be fundamentally awed. Relatively recent studies on dialysis adequacy failed to demonstrate that increasing Kt/Vurea over
1.3 could decrease patient morbidity and improve
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survival.31,32 Moreover, clinical studies have shown that a further limit of Kt/Vurea as a predictor of mortality, is that both terms of the ratio are predictors.33 Data presented in this paper suggest that e-GST may be an innovative tool able to measure the efciency of a dialytic process complementary or alternative to the Kt/Vurea. In fact e-GST fullls remarkable
improvements and novelty elements: (a) it can be considered not as a classical biomarker, but more appropriately an endogenous biosensor of blood toxicity being naturally over-expressed when the toxin level increases; (b) its expression is reasonably linked not to the quantitative level of blood toxins nor to their size, but probably to their own specic toxicity; (c) contrary to the Kt/Vurea parameter, e-GST reects the adequacy of multiple dialytic sessions within 12 months of life span of circulating erythrocytes; (d) spectrophotometric assay of e-GST activity is fast, requires only 0.05 ml of blood, and it is not expensive. As shown in Figure 3 data from the present study demonstrate that patients under convective hemodialysis express e-GST (8.2 U/gHb) signicantly lower than that observed in patients under traditional diffusive hemodialysis (10.0 U/gHb), suggesting that the former group is exposed to a lower level of circulating toxins and thus to a more efcient dialytic therapy. This idea fully agree with recent observations, indicating an advantage of convective over pure diffusive strategies for patient survival.34 This e-GST variation observed in the two different hemodialytic treatments is not negligible; with e-GST activity in healthy subjects set as reference value, i.e., 5.6 U/gHb, the convective hemodialysis lowers the hyper-activity of e-GST observed in patients under diffusive hemodialysis by 443% (Figure 1b).
e-GST activity of patients under convective treatment(8.2 U/gHb) is, however, far from that expected from an ideal dialysis that may be characterized by a GST value near that of the healthy group (5.6 U/gHb). Tentatively, a value o6 U/gHb could be the goal to be realized by means of future and more advanced dialysis procedures. In conclusion, e-GST seems to reveal toxicological aspects of uremic syndrome (Figure 1) wider than those of the urea-centric Kt/Vurea (Figure 2)
explaining the absence of correlation between these two parameters. In this context it is not surprising that e-GST does not or poorly correlate with other established markers (albumin, hs-CRP, BUN predialysis and postdialysis), which reect a short-term toxicity or the adequacy of a single dialytic session. As the erythrocyte is an enucleated cell, its enzymatic content is exclusively determined before or during its maturation and remains unchanged during the cell lifespan, which is about 60 days in dialyzed patients.28 Data
presented in this paper demonstrate that the increased e-GST activity found in dialyzed patients is likely due to an hyper-expression of the enzyme and not due to an enzymatic activation (Figure 3). Thus, e-GST activity must reect reasonably a blood exposure to toxins during about 2 months and then the average adequacy of multiple dialytic sessions. This property, also conrmed by the invariance of e-GST activity during distinct dialytic sessions within a week or before/after a single dialysis (Figure 4), displays analogy with HbA1c used as long-term marker for blood exposure to glucose35 and make e-GST a marker of uremic toxicity and dialytic adequacy very different from Kt/Vurea. A larger trial
involving several hundreds of dialyzed patients is now in
progress in a number of Italian dialysis centers to further conrm the relevance of e-GST in the clinical monitoring of ESRD patients.
Materials and MethodsPatients and study design. The study protocol complied with the declaration of Helsinki and a written fully informed consent was provided by all patients and healthy subjects before enrollment into the study. The present research is approved by the Ethical Committee of our Institution (Comitato Etico Indipendente dellAzienda Ospedaliera Universitaria Policlinico Tor Vergata).
The study is a cross-sectional investigation of healthy subjects and MHD patients. Eighty-two healthy controls with normal renal function and no history of diabetes mellitus served as healthy controls. Blood samples were collected from the anticubital vein and stored into K-3-EDTA tubes at 4 1C for no more than 2 days.
One hundred and three uremic patients were enrolled. Inclusion criteria were: age between 18 and 80 years, hemodialytic therapy since 6 months at least and the same hemodialysis technique since 3 months. Blood samples were collected from the arterial site of the vascular access before the dialysis at the end of the long interdialytic interval. Samples were stored with the same modality as for the control group.
Exclusion criteria in both healthy controls and MHD patients were a clinical history of virus hepatitis B and C or serum alanine aminotransferase and/or aspartate aminotransferase twice the upper limit of normal values, morbid obesity, rheumatologic disorders (as systemic lupus erythematosus), hyperbilirubinemia (such as Gilberts syndrome), active cancer and pregnancy. Gender, mean age, HD vintage and cause of end-stage renal disease are shown in Table 1.
Clinical parameters. Hemoglobin (Hb) was determined with an automated hematology analyzer XE-2100 (Sysmex, Kobe, Japan). Albumin, BUN and hs-CRP measurements were performed by an automated method using Dimension VISTA 1500 (Siemens Healthcare Diagnostics, Milano, Italy).
EPO dose and ERI. In order to normalize the amount of EPO required depending on the severity of anemia, we calculated an ERI, dened as the weekly EPO dose divided by Hb level (g/dl). Both the EPO dose and ERI were divided by end-dialysis body weight to indicate the required EPO dose per kilogram of body weight. A ratio of 1 : 200 was used to convert darbepoetin alpha to the EPO equivalent dose (1 mg of darbepoetin alpha 200 IU of epoetin alpha or beta).36
Dialysis therapy. MHD patients were divided in two subgroups, based on dialysis technique: 44 out of 103 patients underwent to HD and 59 patients were on HDF therapy. HD patients were treated with 1.52.0 m2 surface area polysulphone or polyamide hollow-ber hemodialysers, whereas 1.42.1 m2 polysulphone or polyamide hollow-ber dialyser membranes were used in the HDF group. All patients underwent 4 h three times/week dialysis protocol, with a well-functioning native arterovenous stula or a cuffed internal jugular indwelling venous catheter, as vascular access. All uremic patients were characterized according to sex, age and dialytic vintage; hemodialysis adequacy was assessed with spKt/Vurea and total weekly Kt/Vurea using the Daugirdas second-generation formula.37
e-GST activity. e-GST activity was determined with a spectrophotometric assay at 340 nm (37 1C), using a Modular P800 (Roche, Basel, Switzerland)
automated apparatus recently described.24 Briey, one volume (40 ml) of whole blood was diluted in 25 volumes (1 ml) of bi-distilled water and after 5 min introduced into the Modular P800, nal volume containing 1 mM glutathione (GSH), 1 mM 1-chloro-2,4-dinitrobenzene (CDNB) in 0.1 M potassium phosphate buffer, pH 6.5. Results were expressed as enzyme units (U) per gram of Hb (U/ gHb): one unit represents the amount of enzyme that catalyzes the conjugation of 1 mM of GSH to CDNB in 1 min at 37 1C. The Hb level was determined with an automated hematology analyzer XE-2100 (Dasit, Milano, Italy). To calculate Kt/
Vurea, urea was assayed by nephelometric methods (BN IITM BNHTM nephelometer, Siemens Healthcare Diagnostics, Milano, Italy).
Purication of e-GST and its reaction with DNDGIC. e-GST purication from hemolyzed erythrocytes of healthy and dialyzed patients was performed with a single-step afnity chromatography method using S-hexylglutathione Sepharose 6B.38 Protein concentration was determined using the procedure described by Lowry et al.39 Dinitrosyldiglutathionyliron complex
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(DNDGIC) was prepared as described previously by reacting S-nitrosoglutathione with ferrous sulphate in the presence of 10 mM GSH.29,30 Its reaction with puried
e-GST, from healthy or dialyzed subjects, was performed in 0.01 M potassium phosphate buffer, pH 7.0 by adding variable amounts of DNDGIC. After 1 min of incubation, the degree of inhibition was evaluated on aliquots with the standard assay procedure for GST activity.
Statistical analysis. All data were expressed as meanS.E.M. P values reported in the text and in tables have been estimated on the basis of the meanS.D. Unpaired t test was employed to compare the data between various groups; non parameter variables were analyzed by MannWhitney test. A value of Po0.05 was considered statistically signicant. To study the linear relationship between e-GST activity and other variables, non parametric correlation (Spearman p) was used. Data were processed using statistical software MedCalc (Mariakerke, Belgium).
Conict of InterestThe authors declare no conict of interest.
Acknowledgements. We thank Professor Jens Z Pedersen for critical reading of the manuscript and Dr. Erica Del Grosso, Dr. Laura Morici and Dr. Renato Massoud for technical assistance.
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Cell Death and Disease is an open-access journal published by Nature Publishing Group. This work is licensed under the Creative Commons Attribution-NonCommercial-No Derivative Works 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/3.0/
Cell Death and Disease
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Copyright Nature Publishing Group Aug 2012
Abstract
Kt/Vurea ratio is commonly used to assess the delivered dose of dialysis in maintenance hemodialysis (MHD) patients. This parameter only reflects the efficacy of dialytic treatments in removing small toxins, but not middle and protein-bound toxins. Erythrocyte glutathione transferase (e-GST), an enzyme devoted to cell depuration against a lot of large and small toxins, is overexpressed in uremic patients. Aim of the present study is to verify whether e-GST may represent a novel biomarker to assess the adequacy of different dialytic techniques complementary to Kt/V urea parameter. Furthermore, it will be investigated whether e-GST could reflect the 'average' adequacy of multiple dialytic sessions and not of a single one treatment as it occurs for Kt/Vurea . One hundred and three MHD patients and 82 healthy subjects were tested. Fourty four patients were treated with standard bicarbonate hemodialysis (HD) and 59 patients were on online hemodiafiltration (HDF). In all MHD patients e-GST activity was 60% higher than in healthy controls. In HDF, e-GST activity was lower than in HD subgroup (8.2±0.4 versus 10.0±0.4 U/g Hb , respectively). Single-pool Kt/Vurea and total weekly Kt/Vurea were higher in HDF than in HD, but no correlation was found between e-GST activity and Kt/Vurea data. e-GST, whose level is stable during the erythrocyte life-span, provides information on the long-term depurative efficacy of dialysis treatments.
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Neither ProQuest nor its licensors make any representations or warranties with respect to the translations. The translations are automatically generated "AS IS" and "AS AVAILABLE" and are not retained in our systems. PROQUEST AND ITS LICENSORS SPECIFICALLY DISCLAIM ANY AND ALL EXPRESS OR IMPLIED WARRANTIES, INCLUDING WITHOUT LIMITATION, ANY WARRANTIES FOR AVAILABILITY, ACCURACY, TIMELINESS, COMPLETENESS, NON-INFRINGMENT, MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. Your use of the translations is subject to all use restrictions contained in your Electronic Products License Agreement and by using the translation functionality you agree to forgo any and all claims against ProQuest or its licensors for your use of the translation functionality and any output derived there from. Hide full disclaimer