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NDT Advance Access originally published online on February 7, 2006
Nephrology Dialysis Transplantation 2006 21(6):1648-1651; doi:10.1093/ndt/gfk093
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© The Author [2006]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Original Articles: Dialysis and Transplantation

On-line haemodiafiltration with and without acetate

Francesco Pizzarelli, Tiziano Cerrai, Pietro Dattolo and Giuseppe Ferro

Nephrology and Dialysis Unit, SM Annunziata Hospital, Florence, Italy

Correspondence and offprint requests to: Dr Francesco Pizzarelli, Head of Nephrology Unit, SM Annunziata Hospital, Via dell’ Antella 58, 50011 Antella-Firenze, Italy. Email: fpizzarelli{at}yahoo.com



   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background. In patients on on-line convective treatments, given the considerable quantity of dialysis fluid re-infused, the small amount of acetate present in bicarbonate dialysis fluid as a pH stabilizing factor may allow a significant transfer of that anion to the patient, possibly inducing cytokine activation.

Methods. To verify this hypothesis, we performed on-line haemodiafiltration (OL-HDF) with (3 mmol/l) and without acetate in dialysis fluid in a cross-over randomized order on 12 prevalent patients.

Results. In comparison with the pre-treatment values, plasma acetate levels were unchanged during and after acetate-free OL-HDF, while they were 5–6 times higher in the course of OL-HDF containing acetate in dialysis fluid; plasma acetate levels returned to basal values 2 h after the end of the procedure. The total increase of bases in the patient attributable to acetate was 36%. Plasma bicarbonate values at the end of treatment were significantly lower in treatments without acetate, as compared to those with acetate. Interleukin-6 plasma levels were super-imposable at the beginning and in the course of the two methods compared, but there was a tendency towards a greater increase at an interval of 2 h following OL-HDF with acetate.

Conclusions. Our preliminary results confirm the assumption that body gain of acetate is particularly high in convective treatments, while acetate-free OL-HDF slows down acetate burden. Clinical advantages due to these effects should be evaluated in properly designed prospective studies.

Keywords: acetate; acetate-free dialysate; cytokines; inflammation; on-line haemodiafiltration



   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
In the 1990s, evidence accumulated that in bicarbonate dialysis, the small quantity of acetate present in the dialysis fluid as a pH stabilizing factor can lead to a significant transfer of that anion to the patient [1], inducing cytokine activation with production of nitric oxide, with potentially negative effects on uraemic cardiovascular disease [2–4].

In patients treated with on-line haemodiafiltration (OL-HDF) the transfer of acetate to the patient should be even greater given the considerable quantity of dialysis fluid that is infused into the patient after being properly filtered. In line with this thesis, a French experience found significantly higher post-treatment plasma acetate levels in patients treated with OL-HDF in comparison with standard bicarbonate haemodialysis [5].

Given these assumptions, we designed a phase 3 study to assess feasibility of acetate-free OL-HDF, to quantify the acetate burden assumed by the patient in the course of standard HDF and to assess whether or not the absence of that anion is associated with lower cytokine activation.



   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Twelve of our dialysis patients gave their informed consent to participate in the study. These were six men and six women, aged 61±14 years and in dialysis treatment for 83±46 months, all of them with native fistulas.

The patients were asked to abstain from food and alcohol prior to treatments because in human beings the increase in acetatemia may also be linked to the consumption of alcohol [6]. Patients were separated by simple random sampling into two groups: six of them underwent one session of standard OL-HDF and one session of acetate-free OL-HDF, the others received the two treatments in the opposite order. One patient in the group acetate-free OL-HDF as first treatment, presented with particularly elevated basal acetatemia values (1.5 mmol/l). Given this anomalous behaviour, resulting from large alcohol consumption, the patient was excluded from the study. Thus, 11 patients were included in the final analysis.

Treatments were performed weekly apart at the same midweek interval using the paired HDF technique employing double chamber dialyzers in Polyethersulfone® [7,8]. Dialysis fluid in standard OL-HDF contained 3 mmol/l of acetate. In acetate-free OL-HDF the acid concentrate bag contained 3 mmol/l of hydrochloric acid; when acid and basic concentrates mix with treated water to form dialysis fluid, HCl neutralizes an equivalent quantity of bicarbonate, which is converted into water and carbon dioxide to stabilize the dialysis fluid pH. Other than the difference in acetate, the composition of the dialysis fluid was identical in both procedures (Na+ 138 mmol/l, K+ 2 mmol/l, Formula 32 mmol/l, Ca++ 1.5 mmol/l), with the treatment parameters being identical as well: volumes of liquid infused in pre-dilution 167 ml/min, effective blood and dialysis fluid flows 300 ml/min and 500 ml/min, respectively, duration 210–240 min.

Acid–base and electrolytes (specific ion electrodes) were determined on blood samples taken at start, mid and end treatments. Acetate (spectrophotometric method, Boehringer Mannheim GmbH, Mannheim, Germany), C reactive protein (CRP, nephelometric method, Array® 360, Beckman Coulter Inc., Fullerton, CA, USA), and interleukin-6 (IL-6, Quantikine®, R&D Systems, Minneapolis, MN, USA) were determined on blood samples taken hourly during the treatment as well as 2 h after it was terminated. For mass balance, acetate values were obtained also from the dialysis fluid continuously collected at the dialyzer outlet by means of spilling pumps. Since the dialysis fluid was not collected under paraffin, we estimated the mass balance of bicarbonate using a mathematical model [9].

Data are expressed as mean±SD. For statistics, we used the SPSS statistical package. The time dependent changes during each treatment and in the 2 h following, were analysed by ANOVA with Bonferroni post hoc test for multiple comparisons. Comparisons between the two treatments at each time point were performed by Kruskall–Wallis test for multiple independent samples. A statistically significant difference was accepted at P<0.05 (two-tailed test).



   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
There were no differences in the clinical pictures of the patients nor in mean plasma values between cross over periods. The interdialytic weight gain was 2.6±1.0 kg and 2.7±1.1 kg in acetate-free and standard OL-HDF, respectively. The progress of acetatemia in the 11 patients included in the study is shown in Figure 1. The pre-dialytic plasma acetate levels did not change during or after acetate-free OL-HDF, but were found to be 5–6 times higher in the course of OL-HDF with acetate, to return to basal values in the 2 h following.


Figure 1
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  		Fig. 1. Trend in plasma acetate during OL-HDF and in the 2 h following. {blacksquare}- - -{blacksquare} OL-HDF with infusion in pre-dilution of 40 l of filtered dialysis fluid containing 32 mmol/l of bicarbonate and 3 mmol/l of acetate. {blacktriangleup}- - -{blacktriangleup}: OL-HDF with infusion in pre-dilution of 40 l of filtered dialysis fluid containing 32 mmol/l of bicarbonate without acetate. Horizontal thick line: upper limit of physiological levels for plasma Acetate. Vertical bars: SD, *P<0.01 Ac+ HDF vs Ac– HDF.

 
In OL-HDF with acetate in the dialysis fluid, the mass balance for acetate was found to be strongly positive, 75±29 mmol, while that calculated for the bicarbonate was estimated at 132±100 mmol. Therefore, the portion of the overall base gain resulting from the acetate was 36%.

The trend in plasma electrolytes and blood gases is shown in Table 1. Neither within nor between treatments did we find differences in the parameters analysed, but the plasma bicarbonate levels were significantly lower at the end of the acetate-free OL-HDF compared to the treatment containing that buffer in the dialysis fluid.


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  		Table 1. Blood gases and electrolytes during standard OL-HDF and acetate-free OL-HDF

 
CRP and IL-6 trend are shown in Table 2. IL-6 plasma levels are super-imposable at the beginning and during the treatments, whereas there is a tendency to a greater increase within 2 h after the conclusion of OL-HDF with acetate. The difference, however, is not significant. Analysing the basal individual data, six patients had CRP values <0.6 mg/dl; three, values ranging from 0.6 to 0.9 mg/dl and two from 0.9 to 2 mg/dl. Variability in category assignment did not vary between cross-over periods.


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  		Table 2. Trend in serum CRP and IL-6 in patients during treatments and in the 2 h following

 


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
For some decades now, bicarbonate dialysis has replaced acetate dialysis, owing to the well-known collateral effects of this buffer [10–14]. In dialysis fluid containing bicarbonate, the pH of the solution is stabilized with the addition of 4–8 mmol of acetate. It should be stressed, however, that even the addition of such ‘small’ quantities produces a strong dialysate-blood diffusive gradient, the physiological plasma levels for this anion being <0.1 mmol/l.

As expected, in the present study we did not observe any increase in plasma acetate levels in acetate-free OL-HDF. Per contra, we have found that in OL-HDF with a concentration of acetate in the dialysis fluid of 3 mmol/l, the overall rate of increase of bases in patients attributable to acetate has been 36%. Although, we lack a direct comparison with haemodialysis, in bicarbonate haemodialysis Agliata et al. have found values of 25% using dialysis fluids containing 4 mmol/l of acetate [1]. Therefore, as compared to their data, we have found a greater acetate body burden at lower concentrations of this anion in the dialysis fluid. This difference is readily explainable by the considerable quantity of dialysis fluid we reinfuse, ~40 l per treatment. In the course of ‘high-volume’ convective treatments, the elevated rate at which acetate enters the bloodstream exceeds the capacity to metabolize it of the enzyme acetyl CoA synthetase, whose Michaelis constant (Km) for acetate is ~0.7 mmol [15], which explains the increase in acetatemia of 6–7 times over basal values that we found throughout the duration of the treatment; the 2 h following are sufficient to metabolize the accumulated acetate burden (Figure 1). At the end of standard OL-HDF, we found acetate plasma levels in the region of 0.3 mmol/l, e.g. a figure remarkably similar to that found, 0.32 mmol/l, by Fournier et al. in a large population of French patients treated with OL-HDF [5]. Unfortunately, in the French experience acetate mass balance was not studied, thus no comparison can be performed on that specific issue.

In both techniques studied, the concentrations of bicarbonate in the dialysis fluid were identical, 32 mmol/l. This implies that the two treatments were not balanced in terms of total bases, since acetate-free OL-HDF lacked the 3 mmol of that buffer present in the dialysis fluid used in the standard treatment. This is very likely the explanation of the significantly lower end-of-treatment bicarbonate plasma values in treatments without acetate, as opposed to those with it (Table 1). In acetate-free OL-HDF, therefore, the optimal concentration of bicarbonate in the dialysis fluid and/or of HCl in the concentrate bags must be reconsidered. We are conducting studies on this specific matter.

At the end of the 1980s, it was demonstrated that the high concentrations of acetate contained in acetate dialyses promoted the production of various cytokines by activated monocytes [13,14]. This also seems to be the case even with the much lower concentrations of acetate in bicarbonate haemodialysis, a technique in which an increase of TNF synthesis [2], plasma levels of IL-1ß [4], activity of nitric oxide synthetase [2,3] and activation of polymorphonucleates and monocytes [4] have been demonstrated. By contrast, changes of these kinds do not appear in acetate-free biofiltration [2–4], a diffusive-convective dialysis technique based on buffer-free dialysate and post-dilution bicarbonate infusion tailored to the patients’ requirements [16].

Pre-dialytic IL-6 serum levels in our patients are in line with expectations for the dialysis population [17,18]. The values remained stable and super-imposable between the two techniques in the course of the treatments, while we observed a tendency to increase after 2 h following completion, particularly in OL-HDF with acetate (Table 2). Although the difference is not significant, it might be clinically relevant due to the fact that plasma IL-6 levels are strongly associated with co-morbidity in dialysis patients and are a powerful predictor of cardiovascular and all-cause mortality [17,18].

In line with literature data [19], almost half of our population had greater than normal pre-dialytic CRP values. We did not observe any variation in CRP in the course of OL-HDF with and without acetate, nor in the control 2 h after the end of treatments. It should, however, be stressed that this observation period is probably insufficient to capture an acute inflammatory response since it generally takes many hours, at any rate not less than four, to observe an increase in CRP levels after tissue damage [20]. In addition, the different behaviour of serum levels of CRP and of IL-6 2 h after the end of treatment has been already described [21] and is in line with the hepatic protein synthesis following dialysis-induced inflammatory response.

In our study, we performed OL-HDF with a two-chamber dialyser [7,8]. It is therefore open to debate whether the results achieved can be generalized to other OL techniques.

We acknowledge several limitations in our study, the major being the small sample size. Moreover, analysis was carried out by only one data point and the issue of possible cytokine activation was explored at limited time marks and relies on a trend and not on a significant result. Per contra, this pilot study provides for the first time suggestive evidence that body gain of acetate is particularly high in convective treatments and the negative effects of the acetate itself are well established.

In conclusion, our preliminary clinical experience demonstrates feasibility of acetate-free OL-HDF but leaves open the possibility of a lower cytokine activation. This issue needs to be assessed by a multicentre study in a broader population observed over an appropriate period of time. Benefits and harms of newer technologies over existing therapies should be well documented before their broader adoption.

Conflict of interest statement. F.P. has participated in meetings sponsored by Bellco.



   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Agliata S, Atti M, Fortina F et al. Acetate in the dialysate in bicarbonate dialysis. Blood Purif 1992; 10: 88
  2. Amore A, Cirina P, Mitola S et al. Acetate intolerance is mediated by enhanced synthesis of nitric oxide by endothelial cells. J Am Soc Nephrol 1997; 9: 1431–1436
  3. Noris M, Todeschini M, Casiraghi F et al. Effect of acetate, bicarbonate dialysis and acetate-free biofiltration on nitric oxide synthesis: implications for dialysis hypotension. Am J Kidney Dis 1998; 32: 115–124[Web of Science][Medline]
  4. Todeschini M, Macconi D, Garcia Fernandez N et al. Effect of acetate-free biofiltration and bicarbonate hemodialysis on neutrophil activation. Am J Kidney Dis 2002; 40: 783–793[CrossRef][Web of Science][Medline]
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  8. Pizzarelli F, Cerrai T, Tetta C. Paired hemodiafiltration: technical assessment and preliminary clinical results. In: Ronco C (ed.), Contributions to Nephrology, Vol. 138, Karger: Basel 2003; 99–105
  9. Fernandez PC, Cohen RM, Feldman GM. The concept of bicarbonate distribution space: the crucial role of body buffers. Kidney Int 1989; 36: 747–752[CrossRef][Medline]
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  12. Veech R. The untoward effects of the anions of dialysis fluids. Kidney Int 1988; 34: 587–597[Medline]
  13. Bingel M, Koch KM, Lonnemann G, Dinarello CA, Shaldon S. Enhancement of in vitro human interleukin-1 production by sodium acetate. Lancet 1987; 1: 14–16[Medline]
  14. Anderson J, Briefel G, Jones JM, Ryu JH, McGuire M, Pyo Yun Y. Effects of acetate dialysate on transforming growth factor ß1, interleukin and ß2-microglobulin plasma levels. Kidney Int 1991; 40: 1110–1117[Medline]
  15. Campagnari F, Webster LT. Purification and properties of acetyl coenzyme A synthetase from bovine heart mitochondria. J Biol Chem 1963; 238: 1628–1633[Free Full Text]
  16. Zucchelli P, Santoro A, Ferrari G, Spongano M. Acetate-free biofiltration: hemodiafiltration with base-free dialysate. Blood Purif 1990; 8: 14–22[Medline]
  17. Kaizu Y, Kimura M, Yonemana T et al. Interleukin-6 may mediate malnutrition in chronic hemodialysis patients. Am J Kidney Dis 1998; 31: 93–100[Web of Science][Medline]
  18. Rao M, Guo D, Perianayagam MC et al. Plasma Interleukin-6 predicts cardiovascular mortality in hemodialysis patients. Am J Kidney Dis 2005; 45: 324–333[CrossRef][Web of Science][Medline]
  19. Panichi V, Migliori M, De Pietro S et al. The link of biocompatibility to cytokine production. Kidney Int 2000; 58: S96–S103[CrossRef][Web of Science]
  20. Kushner I, Broder ML, Karp D. Control of the acute phase response: serum C-reactive protein kinetics after acute myocardial infarction. J Clin Invest 1978; 61: 235–242[Web of Science][Medline]
  21. Caglar K, Peng Y, Pupim LB et al. Inflammatory signals associated with hemodialysis. Kidney Int 2002; 62: 1408–1416[CrossRef][Web of Science][Medline]
Received for publication: 3.11.05
Accepted in revised form: 4. 1.06


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