Short-term effects of nocturnal haemodialysis on carnitine metabolism

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1 Nephrol Dial Transplant (2006) 21: doi: /ndt/gfl312 Advance Access publication 5 July 2006 Brief Report Short-term effects of nocturnal haemodialysis on carnitine metabolism Daljit K. Hothi 1, Denis F. Geary 1, Lawrence Fisher 2 and Christopher T. Chan 3 1 Division of Pediatric Nephrology, 2 Department of Biochemistry, Hospital for Sick Children and 3 Department of Nephrology, Toronto General Hospital, Toronto, Canada Abstract Background. Functional carnitine deficiency [as indicated by an abnormal acyl-carnitine/free-carnitine (AC:FC) ratio] is commonly seen in patients with endstage renal disease (ESRD), resulting in significant clinical detriments including anaemia, cardiomyopathy and muscle weakness. Nocturnal haemodialysis (NHD) (5 6 sessions per week, 8 h per treatment) has been reported to reverse several surrogate markers of uraemia. Conversely, as a consequence of increased dialysis dose, NHD may have the potential to aggravate plasma nutrient deficiencies. Our objective was to determine the effects of NHD on plasma freecarnitine levels and carnitine metabolism. Methods. We conducted an observational cohort study with a before and after design. Nine ESRD patients (age: 47 3; mean SEM) were studied. Routine biochemical, haemodynamic and carnitine metabolic products were analysed at baseline while on conventional haemodialysis and 2 months post-conversion to NHD. Free-carnitine and total-carnitine levels were generated by colorimetric assays. The difference between total- and free-carnitine concentrations was estimated to be the acyl-carnitine level. Paired t-test was used to ascertain statistical significance. Results. After conversion to NHD, there was a significant increase in urea clearance in all patients. Plasma free-carnitine levels fell from to mmol/l (P < 000.1). A similar reduction in plasma acyl-carnitine levels was observed (from to mmol/l (P < 0.001)). The AC:FC ratio improved from to (P < 0.005) (Normal < 0.25). Conclusion. NHD is associated with an improvement in AC:FC ratio. Further research is needed to examine the longitudinal clinical impact of this metabolic correction and to examine whether this effect is sustained. Correspondence and offprint requests to: Dr Christopher T. Chan, 200 Elizabeth Street, 8N room 842, Toronto, Ontario M5G 2C4, Canada. Christopher.Chan@uhn.on.ca Keywords: carnitine; metabolism; nocturnal haemodialysis; uraemia Introduction Carnitine is a low-molecular-weight compound principally known for its obligate role in the mitochondrial oxidation of long-chain fatty acids, but its function extends beyond this [1]. Carnitine is essential for sustaining human life, and as a consequence of tightly regulated metabolic pathways, plasma and tissue levels are maintained within relatively narrow limits. Disturbances in carnitine homoeostasis can be detected biochemically through analysis of the ratio between plasma levels of carnitine acyl esters (acyl-carnitines) and free-carnitine concentrations (AC:FC ratio). In a normal individual, the normal AC:FC ratio is <0.25. Altered carnitine metabolism, free-carnitine deficiency and prolonged fasting that causes systemic ketoacidosis can result in an abnormally elevated AC:FC ratio. End-stage renal disease (ESRD) is associated with an increased esterified fraction of L-carnitine and incomplete fatty acid oxidation. In addition, it is speculated that gradual depletion of free-carnitine is achieved through chronic dialytic removal. Nocturnal haemodialysis (NHD) (5 6 sessions per week, 8 h per treatment) is a novel mode of renal replacement therapy, which has been reported to have several metabolic advantages over conventional haemodialysis [2 4]. Conversely, due to increased dialysis duration and frequency, NHD may have the potential to aggravate metabolic deficiencies. Whereas the evidence for secondary carnitine deficiency as a result of conventional dialysis is mounting [5], the only report of carnitine status in NHD patients is one of four children showing falling plasma free-carnitine levels after commencing NHD [6]. In this study, we aimed to evaluate the short-term effects of carnitine metabolism before and after conversion from conventional haemodialysis (CHD) (three sessions per week, 4 h per session) to NHD. ß The Author [2006]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please journals.permissions@oxfordjournals.org

2 2638 D. K. Hothi et al. Subjects and methods Consecutive ESRD patients undergoing NHD at Toronto General Hospital, University Health Network, were assessed. The Research Ethics Board at Toronto General Hospital approved this prospective observational cohort study. Written, informed consents were obtained. None of the patients had any acute illness. We employed a spectrophotometric enzymatic assay to quantify free- and total-carnitine as described previously [10]. Our biochemical analysis was calibrated using aqueous standards that were prepared from a carnitine standard solution (320 mm), with concentrations ranging from 0 mmol/l (water only) to 160 mmol/l. The normal ranges assessed in our laboratory for free-carnitine is mmol/l, acyl-carnitines is 0 19 mmol/l and total-carnitine is mmol/l. Dialysis prescriptions Conventional haemodialysis was performed using F80 polysulfone dialysers (Fresenius Medical Care, Lexington, MA, USA). The dialysate composition was as follows: Na þ 140 mm, K þ 1 3 mm, Ca 2þ mm and HCO3 40 mm. A blood flow rate of 400 ml/min and a dialysate flow rate of ml/min was used. NHD was performed using either F80 dialysers or Polyflux dialysers (Gambro Inc., Lund, Sweden). The dialysate composition was similar to that used in CHD, but often required phosphate supplementation to maintain normal plasma phosphate levels. A blood flow rate of ml/min and a dialysate flow rate of 350 ml/min were used. For both CHD and NHD, vascular access was achieved through a long-term internal jugular catheter, an arterio-venous fistula or an arterio-venous graft. Dialysate bicarbonate was adjusted to maintain a pre-dialysis plasma bicarbonate >22 mmol/l, as recommended by Dialysis Outcomes Quality Initiatives (DOQI) [7]. Dialysis dose per treatment was estimated by equilibrated Kt/V (ekt/v) as described by Daugirdas and colleagues [8] where ekt/v ¼ spkt/v 0.6(spKt/V)/t þ 0.03 (spkt/v ¼ single-pool Kt/V, K ¼ delivered clearance, t ¼ dialysis time and V ¼ urea distribution volume). Single-pool Kt/V was determined using the blood urea reduction ratio [9]. Study protocol The laboratory staffs were blinded to the study and patient details. Baseline plasma samples were obtained after a midweek conventional haemodialysis session. To minimize circadian variation, and replicate steady state NHD conditions, subsequent NHD plasma samples were obtained at the same time of day (a minimum of 4 h after the regular NHD session). All subjects were advised to abstain from tobacco and limit their caffeine consumption. All samples were kept at 708C until biochemical analysis. Biochemical analysis of carnitine metabolism Serum of 500 ml was pipetted into the top portion of a Centrifree Õ Micropartition Device. Samples were capped and centrifuged at 3500 min 1 for 30 min. After centrifugation, 80 ml of filtrate was pipetted into a Mira sample cup. This sample was then used to measure free carnitine. The remaining filtrate of 80 ml was pipetted into a glass tube. To this, 8 ml of 1N NaOH was added to each tube, capped and then placed in heating block at 608C for 1 h. The samples were then removed from the heating block, allowed to cool in room temperature and then neutralized with 4 ml 2N HCl. We then pipetted the hydrolysed samples into Mira sample cups, and this constituted the samples used to measure the total carnitine. Statistical analysis Primary variables of interest were free-, acyl- and totalcarnitine levels at baseline and at 2 months after conversion to NHD. Functional carnitine deficiency is analysed via the AC:FC ratio. Descriptive analyses are presented as mean SEM. All statistical tests were performed using the SPSS statistical package (SPSS-10, Chicago, IL, USA). Student s t-test and repeated measure analysis of variance were used to evaluate changes in biochemical parameters. All statistical tests were two-tailed with a P-value <0.05 taken to indicate significance. Results The study population consisted of nine consecutive adult haemodialysis patients (age: 47 3; five males). Their baseline demographic characteristics are summarized in Table 1. Principal variables are summarized in Table 1. As expected, 2 months after conversion to NHD, equilibrated Kt/V (ekt/v) per session increased significantly (from to ; P < 0.05). After conversion to NHD, plasma free-carnitine levels fell from to mmol/l (P < 000.1). A similar reduction in plasma acyl-carnitine levels was observed [from to mmol/l (P < 0.001)]. The acyl/free-carnitine ratio improved from to (P < 0.005) (Normal < 0.25) (Figure 1). Discussion To our knowledge, this is the first study to directly evaluate the short-term impact of converting from CHD to NHD on carnitine metabolism. Our results indicated that NHD is associated with reductions in plasma free-, total- and acyl-carnitine levels. Despite the lower free-carnitine concentration, the AC:FC ratio fell, towards the normal range, perhaps suggesting an improved carnitine metabolic state. L-carnitine is a low-molecular weight solute that is water soluble and exists in the plasma largely unbound to proteins [11,12]. In normal subjects, carnitine levels are maintained by gastrointestinal absorption of dietary carnitine, endogenous synthesis and renal tubular reabsorption of L-carnitine combined with selective excretion of short-chain acyl-carnitines [13]. In ESRD, all three processes are disrupted and carnitine metabolism is further aggravated by CHD.

3 Nocturnal haemodialysis and carnitine metabolism 2639 Table 1. Patient characteristics and biochemical indices before and after conversion to NHD Patient Gender Age (years) Underlying renal disease Comorbid diseases Pre-dialysis Bicarbonate (mmol/l) Dry weight (kg) Albumin (g/l) Glucose (mmol/l) CHD NHD CHD NHD CHD NHD CHD NHD 1 M 57 GN Hyperparathyroidism M 40 IgA Hypertension F 34 Diabetes Diabetic neuropathy CAD M 52 APCKD Hypertension F 41 Diabetes CAD PVD Diabetic neuropathy 6 M 57 GN Hypertension F 44 GN Hypertension F 44 GN Hypertension M 50 APCKD Hypertension Hyperparathyroidism APCKD, adult polycystic kidney disease; CAD, coronary artery disease; CHD, conventional haemodialysis; GN, glomerulonephritis; IgA, IgA nephropathy; NHD, nocturnal haemodialysis; PVD, peripheral vascular disease. In a typical CHD session, up to 75% of plasma free carnitine can be removed [5] but non-acetylated, protein-bound, acyl-carnitines with greater than 12 carbon chain lengths tend to accumulate due to lower dialysance. This can result in a form of functional carnitine deficiency arising from a total body deficiency in L-carnitine, raising the AC:FC ratio. The latter impacts on the mitochondrial capacity for energy production through an inhibitory effect on enzymes such as pyruvate dehydrogenase [14]. After switching to NHD, we noted a reduction of both the plasma free carnitine and AC:FC ratio. We hypothesize that our observation is best explained by a combination of increased dialysis duration and frequency. It is reasonable to assume that NHD may augment clearances of free-carnitine and proteinunbound acyl-carnitines. There was, however, a large inter-patient variability such that, compared with the baseline CHD values, the average percentage fall on NHD of free carnitine was 38% (range: 2 64%) and acyl-carnitine 49% (range: from an 82% fall to a 1% increase). Part of this difference could be explained by the fact that the plasma free-carnitine and acylcarnitine levels were measured within 1 h after dialysis on conventional haemodialysis and 4 h post-nhd. The former is likely to be more reflective of dialysis effects, whereas the latter that of a post-dialysis rebound effect from tissue plasma equilibration and L-carnitine gain from the diet. Alternatively, an improvement in AC:FC ratio may reflect a restoration of carnitine metabolism. In attempts to manage protein and phosphate balance, carnitine-rich foods such as meats, fish and dairy products are typically only available in moderation to CHD patients. NHD is associated with improved phosphate control [15,16], and although no food diaries were available, dietary restrictions were liberalized in all 9 patients. Raj et al. [17] demonstrated an increase in the plasma levels of methionine and lysine in NHD patients, which suggests the potential of enhanced endogenous carnitine synthesis [18,19]. Superficially, our results appear to have an opposing effect on overall carnitine metabolism. A reduced plasma free-carnitine would imply a deterioration in carnitine metabolism, but the restored AC:FC ratio suggests an improvement. Both variables are at risk of error from improper handling of samples [19] and imprecision of different laboratory techniques [20,21]. In normal adults, the L-carnitine pool is 120 mmol [22] and the plasma content only constitutes a small proportion of the total amount. Therefore, short-term changes of plasma free-carnitine do not translate into alterations in the total body carnitine status. The AC:FC ratio is felt to be a more robust measurement of overall carnitine metabolism but no direct comparison data exist. In recognition of these difficulties, the National Kidney Foundation Interdisciplinary Consensus Panel has in fact recommended that irrespective of biochemical findings, treatment be based on clinical evidence of dialysis-related carnitine disorder characterized by anaemia, intra-dialytic

4 2640 D. K. Hothi et al. Free carnitine (µmol/l) Acyl/Free carnitine ratio CHD NHD Fig. 1. Reduction in plasma free-carnitine and acyl-free-carnitine levels after conversion from CHD to NHD. hypotension, skeletal muscle dysfunction and easy fatiguability [23]. Interestingly, NHD has been associated with an improvement in these symptoms: (i) a rise in the plasma haemoglobin levels and a fall in the erythropoietin requirement [24], (ii) stepwise increase in peak exercise capacity and exercise duration [25] and (iii) improved blood pressure control and cardiovascular outcomes such as regression of left ventricular hypertrophy [26]. Taken together, it appears that NHD may have the potential to improve the functional carnitine deficiency typically associated with haemodialysis practice. The results of our study should be interpreted within the limitations of its observational design. We did not compare our NHD cohort with a control conventional dialysis group as this pilot project, given the lack of published data in carnitine metabolism in NHD patients, was designed as a first step to characterize the natural biochemical shift in carnitine biology in patients switched to NHD. In conclusion, 2 months after conversion from CHD to NHD, we have demonstrated a reduction of the AC:FC ratio. We believe this reflects an improvement in carnitine metabolism and improved acyl-carnitine clearance. More studies are required both to ascertain whether this effect is sustained and to determine the mechanism for the apparent improvement in carnitine metabolism. Conflict of interest statement. None declared. References 1. Arduini A, Mancinelli G, Radatti GL, Dottori S, Molajoni F, Ramsay RR. Role of carnitine and carnitine palmitoyltransferase as integral components of the pathway for membrane phospholipid fatty acid turnover in intact human erythrocytes. J Biol Chem 1992; 267: Friedman AN, Bostom AG, Levey AS, Rosenberg IH, Selhub J, Pierratos A. Plasma total homocysteine levels in patients undergoing nocturnal versus standard haemodialysis. J Am Soc Nephrol 2002; 13: Yuen D, Richardson RM, Fenton SS, McGrath-Chong ME, Chan CT. Quotidian nocturnal haemodialysis improves cytokine profile and enhances erythropoietin responsiveness. ASAIO J 2005; 51: Bugeja AL, Chan CT. Improvement in lipid profile by nocturnal haemodialysis in patients with end-stage renal disease. ASAIO J 2004; 50: Evans A. Dialysis-related carnitine disorder and levocarnitine pharmacology. Am J Kidney Dis 2003; 41 [4 Suppl 4]: S13 S26 6. Geary DF, Piva E, Tyrrell J et al. Home nocturnal haemodialysis in children. J Pediatr 2005; 147: NFK KDOQI Guidelines Maintenance dialysis management of acid-base status. 8. Daugirdas JT. Second generation logarithmic estimates of single-pool variable volume Kt/V: an analysis of error. JAm Soc Nephrol 1993; 4: Borah MF, Schoenfeld PY, Gotch FA, Sargent JA, Wolfsen M, Humphreys MH. Nitrogen balance during intermittent dialysis therapy of uremia. Kidney Int 1978; 14: Maeda J, Dudrick SJ. Rapid spectrophotometric determination of plasma carnitine concentrations. JPEN J Parenter Enteral Nutr 1990; 14: Fornasini G, Evans AM. Analysis of free carnitine in plasma of ESRD patients undergoing chronic dialysis: what is the best assay? Dial Transplantation 2003; 32 [6 Suppl 1]: S1 S Marzo A, Arrigoni Martelli E, Mancinelli A et al. Protein binding of L-carnitine family components. Eur J Drug Metab Pharmacokinet 1991; 3: Rebouche CJ, Lombard KA, Chenard CA. Renal adaptation to dietary carnitine in humans. Am J Clin Nutr 1993; 58: Bremer J. Pyruvate dehydrogenase, substrate specificity and product inhibition. Eur J Biochem 1969; 8: Lindsay RM, Alhejaili F, Nesrallah G et al. Calcium and phosphate balance with quotidian haemodialysis. Am J Kidney Dis 2003; 42 [Suppl 1]: Al-Hejaili F, Kortas C, Leitch R et al. Nocturnal but not short hours quotidian haemodialysis requires an elevated dialysate calcium concentration. J Am Soc Nephrol 2003; 14: Raj DSC, Ouwendyk M, Francoeur R, Pierratos A. Plasma amino acid profile on nocturnal haemodialysis. Blood Purif 2000; 18: Vaz FM, Wanders RJ. Carnitine biosynthesis in mammals. Biochem J 2002; 361: Young GA, Parsons FM. Plasma amino acid imbalance in patients with chronic renal failure on intermittent dialysis. Clin Chim Acta 1970; 27: Brogan LR, Fornasini G, Chace DH, Evans AM. Evaluation of plasma L-carnitine levels in healthy individuals: comparison of three analytical methods [Abstract]. APSA 2002 (Australasian Pharmaceutical Sciences Association) Conference in Melbourne, Victoria, Australia 21. Stevens RD, Hillman SL, Worthy S, Sanders D, Millington DS. Assay for free and total carnitine in human

5 Nocturnal haemodialysis and carnitine metabolism 2641 plasma using tandem mass spectrometry. Clin Chem 2000; 46: Brass EP. Pharmacokinetic considerations for the therapeutic use of carnitine in haemodialysis patients. Clin Ther 1995; 17: Eknoyan G, Latos DL, Lindberg J. National Kidney Foundation Carnitine Consensus Conference. Practice recommendations for the use of L-carnitine in dialysisrelated carnitine disorder. Am J Kidney Dis 2003; 41: Schwartz DI, Pierratos A, Richardson RM, Fenton SS, Chan CT. Impact of nocturnal home haemodialysis on anaemia management in patients with end-stage renal disease. Clin Nephrol 2005; 63: Chan CT, Notarius CF, Merlocco A, Floras JS. Improvement in exercise capacity and duration after conversion to nocturnal haemodialysis. ASN, Philidelphia, PA. J Am Soc Nephrol 2005; 16: 733A 26. Chan CT. Cardiovascular effects of frequent intensive haemodialysis. Semin Dial 2004; 17: Received for publication: Accepted in revised form: