white paper Citrate vs. Acetate In Bicarbonate-Based Dialysis Fluid – What Does it Mean Clinically? Prepared by Lars-Göran Nilsson Medical Strategy Director at Gambro Medical & Safety Office

Introduction One objective of dialysis is to provide buffer base to neutralize the acids that accumulate in body fluids between dialysis sessions. Today the dominating buffer in dialysis fluid is bicarbonate, which is also the most abundant physiological buffer in plasma [1]. The use of bicarbonate in hemodialysis (HD) fluid is technically challenging as carbonate, the base form of bicarbonate, easily forms precipitates with calcium and magnesium ions that also are present in dialysis fluid. To minimize the risk of precipitate formation sodium bicarbonate is kept separate (B-concentrate) and not mixed with the divalent cations until immediately before use. In addition, the cation-containing concentrate solution (A-concentrate) is acidified to ensure that the prepared dialysis fluid gets a neutral pH that limits the presence of carbonate. Today, acetic acid is commonly used to acidify the A-concentrate. When such an A-concentrate is mixed with water and B-concentrate in the fluid path of the dialysis machine acetic acid reacts with bicarbonate to form carbon dioxide and acetate. Thus, bicarbonatebased dialysis fluid generally contains acetate. In Europe, it is common to use A-concentrates that give 3–5 mmol/l of acetate in the prepared dialysis fluid, while dialysis fluid in Japan may contain 8–11 mmol/l of acetate. Alternatives to acidify the A-concentrate have recently become available, responding to reports on acetate intolerance. Citric acid is attractive as replacement for acetic acid as it leads to dialysis fluid containing citrate. Several clinical studies have been conducted with citrate-containing dialysis fluid, showing results that indicate significant patient benefits. In addition, therapy alternatives like acetate-free biofiltration (AFB) have been developed to eliminate the need for acidifier by keeping bicarbonate separate from the dialysis fluid [2]. This paper deals primarily with potential issues of acetate in dialysis fluid and the benefits of substituting citrate for acetate.

Bicarbonate-based dialysis fluid commonly contains acetate. Citrate is an attractive alternative; studies indicate that the use of citrate-containing, acetate-free dialysis fluid is associated with significant patient benefits

Acetate in dialysis fluid Acetate concentration in blood Acetate is normally present in plasma at a level well below 100 μmol/l but increases after alcohol intake and may then reach 500 μmol/l or more. The presence of 3–5 mmol/l acetate or more in dialysis fluid leads during dialysis to diffusive transfer of acetate into blood, generally resulting in a 5- to 10-fold increase in plasma acetate level during dialysis [3,4]. The postdialysis acetate level varies greatly among patients, ranging from below 100 to above 500 μmol/l, primarily related to varying ability to metabolize acetate. Acetate as buffer source Acetate is a normal metabolite in the human body. The liver is the most important metabolic site at low acetate concentrations, while muscle cell metabolism becomes increasingly important as acetate levels increase. The metabolism of acetate generates bicarbonate and acetate therefore adds to the buffer gain during dialysis. Potential issues of acetate in dialysis fluid For some 20 years, from the late 60s to the mid 80s, acetate was typically used as the sole buffer in dialysis fluid, at concentrations in the 35–45 mmol/l range (acetate dialysis). The diffusive acetate gain from such a high acetate level in dialysis fluid typically exceeded the body’s metabolic capacity. The resulting plasma acetate level varied by body size and muscle mass but was towards the end of dialysis mostly in the 2–10 mmol/l range [5,6]. Nausea and related symptoms were common and impaired task performance during and after dialysis was demonstrated [5]. Vascular and

1

HCEN15429_1 © 2012.03. Gambro Lundia AB

respiratory complications were frequent and increased inflammatory responses were reported [7]. Some symptoms apparently related to a higher plasma acetate level, others more to a high rate of acetate metabolism. The switch to bicarbonate dialysis in the late 80s made dialysis more physiological and significantly reduced intradialytic symptoms and improved task performance [5,8]. However, since then the patient population on dialysis has changed to become older, more comorbid, and more vulnerable to the unphysiology of dialysis. The hemodialysis treatment has also changed to be more ‘intense’ with more effective dialyzers and higher blood flows leading to higher clearances and, in some cases, increased treatment time and/or frequency. This raises concerns that also the lower acetate content in contemporary bicarbonate-based dialysis fluids may cause or worsen dialysis-related morbidity. More and more ask for acetate-free dialysis fluid, especially for patients who are malnourished and/or have hepatocellular dysfunction that limits their capacity to metabolize acetate [9,10].

to effectively modulate the inflammatory response in HD patients. Replacing such fluid with acetatefree dialysis fluid has led to reduced levels of proinflammatory cytokines and CRP in some studies [19,20] but not in others [4,21]. In this context it is of interest to note that acetate, in concentrations only slightly above what patients may reach during bicarbonate HD, has been found to be an effective modulator of the inflammatory process in acute alcoholic hepatitis by enhancing acetylation of proinflammatory gene histones [22,23]. Dialysis symptoms associated with the use of acetate dialysis were - nausea - intradialytic hypotension - hypoxia - impaired task performance - pro-inflammatory cytokine release

To what extent does the acetate content of bicarbonate-based dialysis fluids contribute to

Contemporary dialysis and treatment intolerance HD patients still consider intradialytic symptoms (fatigue, hypotension, dizziness, etc.) a significant issue in dialysis and many suffer from long recovery times after dialysis [11]. Recent studies and case reports indicate that acetate may still be an important factor in dialysis intolerance [12–14]. In a single-blinded study patients perceived less malaise and showed better blood pressure stability when regular dialysis fluid was replaced with acetate-free, citrate-containing dialysis fluid [12]. In addition, the use of acetate-free biofiltration instead of regular hemodialysis generally leads to better hemodynamic stability [for review see 2]. The sensitivity to acetate may change over time, as indicated in a recent case report [13]. It has been suggested that poor tolerance to fluid removal in acetate dialysis relates to increased nitric oxide (NO) production. In vitro studies have shown that NO synthase activity in murine endothelial cells, human smooth muscle cells, and rat ventricular cardiac myocytes is enhanced in the presence of acetate, also at levels typically used in bicarbonate-based dialysis fluid, while it is unaffected by the presence of acetatefree fluids [15,16].

treatment intolerance?

Citrate in dialysis fluid Using citric acid as an acidifier in A-concentrates for hemodialysis leads to dialysis fluid containing citrate, which is attractive for several reasons. Citrate dialysis fluid may limit the thrombogenic effects of dialysis, although it leads to a citrate concentration in blood that is well below what is aimed for in regional citrate anticoagulation. The antioxidative and antiinflammatory effects of citrate [24] may further improve dialysis fluid biocompatibility. Also important, citrate is rapidly eliminated from body fluids independent of renal function. Citrate chemistry and kinetics Citrate is the anionic base of citric acid, a weak organic acid; the dominating form at physiological pH is trivalent citrate (C3H5O(COO)33−; MW 189D). Citric acid/citrate is widely used industrially in foods, beverages and pharmaceuticals, as a preservative/ conservative due to its antioxidant properties, or to add a sour taste to foods and soft drinks. Commonly applied citrate infusions for regional anticoagulation typically result in a systemic plasma citrate concentration of 1 mmol/l [25–27], after which citrate is removed from plasma with a half-life in the 30–60 minute range. The liver is the most important organ for citrate clearance. Chronic HD patients show similar removal kinetics as people with intact renal function [25], while liver failure is associated with approximately 50% reduction in whole body clearance of citrate [26,27].

Acetate and inflammation It was early realized that interleukin-1 release from human monocytes was increased in the presence of acetate in high concentrations, as those used in acetate dialysis [17]. Consequently, acetate was considered as one factor of the interleukin hypothesis aimed at explaining the high prevalence of metabolic and immunological disorders in dialysis patients [18]. It is still unclear if the lower acetate levels employed in current bicarbonate-based dialysis fluids are sufficient

2

HCEN15429_1 © 2012.03. Gambro Lundia AB

Citrate as buffer source Citrate adds considerably to the buffer gain during dialysis as each molecule of citrate metabolized is equivalent to the generation of three molecules of bicarbonate. Citrate is transported intracellularly by special plasma membrane citrate transporter proteins, where it may enter the mitochondrial Krebs cycle and be metabolized to generate bicarbonate and energy. Citrate may also be broken down in the cytoplasm in a process that fuels fatty acid and sterol synthesis.

When citrate is used as a dialysis fluid constituent the plasma citrate level will depend on the diffusive transfer of citrate from dialysis fluid to plasma as blood passes through the dialyzer. It can be estimated that for a 1.0 mmol/l citrate dialysis fluid the plasma citrate level in blood leaving the dialyzer will typically be in the range of 0.5–0.8 mmol/l, depending on the flow rates through the dialyzer and the effective clearance. Although significantly lower than what is achieved in RCA, this may potentially be sufficient to evoke a light anticoagulation effect within the extracorporeal circuit. In a study on well-heparinized patients, dialysis with a citrate-containing dialysis fluid did not reduce the pre-dialyzer plasma levels of thrombin-antithrombin (TAT) or prothrombin fragments 1+2 (F1+2) compared to conventional dialysis fluid [30]. No attempt to reduce heparin was done. However, in another study a 33% reduction in heparin dosage was successfully achieved with citrate dialysis fluid without loss of dialyzer efficiency [38]. Heparin-free dialysis with citrate-containing dialysis fluid was in 24 high bleeding risk patients found to be equally effective to prevent massive clotting as the conventional saline flushing technique [31].

Citrate-containing dialysis fluid The first publication on the use of citrate in bicarbonatebased dialysis fluid came in 2000, reporting on the early clinical experience of 2.4 mEq/l (0.8 mmol/l) citrate combined with 0.3 mmol/l acetate [28]. It was demonstrated that with citrate-containing dialysis fluid the plasma citrate level in arterial blood increased during dialysis to 0.17±0.05 mmol/l and returned almost to baseline level one hour after terminating dialysis. A slight but significant lowering of ionized calcium, total calcium, and magnesium levels in plasma was also seen during the dialysis session. A 12-week follow up study in 25 patients demonstrated that pre-dialysis plasma calcium levels were unaffected while pre-dialysis magnesium and potassium levels decreased slightly over time when the citrate-containing dialysis fluid was used [28].

Citrate in dialysis fluid and acid base parameters Kuragano et al., in a cross-over comparison between conventional and citrate-containing, acetate-free dialysis fluid, found that patients with a pre-dialysis bicarbonate level below 20 mEq/l at baseline showed a significantly increased bicarbonate level with the use of citratecontaining fluid while those above 20 mEq/l at baseline did not [20]. The comparison was complicated as the effective buffer content in dialysis fluid was apparently higher with the citrate-containing fluid. However, in a randomized cross-over study Grundström et al. saw that 6 weeks of HD with a citrate-containing (1.0 mmol/l) acetate-free dialysis fluid resulted in a lower degree of acidosis pre-dialysis, with greater base excess and higher arterial blood pCO2 level [32].

Following the initial study, several other studies have reported on the use of citrate in dialysis fluid at levels ranging from 2 to 4 mEq/l, in combination with acetate or without [19,21,29–32,38]. Citrate in dialysis fluid - low systemic citrate levels - rapid in vivo metabolism, generates bicarbonate - reduces ionized calcium levels within the circuit making dialysis less thrombogenic - potential positive impact on inflammatory status - potential positive impact on acid-base status

Citrate as anticoagulant Citrate is a potent anticoagulant due to its high affinity for ionized calcium, an indispensible cofactor in the coagulation cascade, and regional citrate anticoagulation (RCA) was early advocated as a way to manage dialysis in patients at high bleeding risk [33]. In RCA citrate is infused into the arterial blood entering the dialyzer, dialysis fluid is normally calcium-free, and calcium is infused to the venous line to restore the plasma calcium level before blood is returned to the patient [34,35]. The procedure typically requires that plasma ionized calcium within the extracorporeal circuit is reduced to 0.2-0.3 mmol/l, which is achieved by a plasma citrate level greater than 3 mmol/l [36]. Systemic plasma citrate concentration at end of RCA dialysis may be in the range of 0.5–1.5 mmol/l [34,37]. During RCA dialysis patients require close clinical observation and repeated measurements of the systemic ionic calcium level, adding to the laboriousness and cost of treatment.

Figure 1. Increasing pre-dialysis base excess level seen during 6 weeks use of a 1.0 mmol/l citrate-containing, acetate-free dialysis fluid compared to use of regular dialysis fluid, bicarbonate level in dialysis fluid being unchanged, in a cross-over study in stable HD patients (N=20; mean ± SEM). At 6 weeks, p