Dramatic Decrease of Carnitine Esters after Interruption of Exogenous Carnitine Supply in Hemodialysis Patients

Abstract

L-carnitine supplementation is extensively used in patients on maintenance hemodialysis (HD) to improve dialysis-related clinical symptoms. In a series of studies, we investigated the dynamics of carnitine pool in carnitine-supplemented HD patients; here we report dramatic decrease with special changes of the ester profile due to interruption of the exogenous intake after the last HD session. Serum samples were collected from 18 L-carnitine-repleted end-stage renal disease (ESRD) patients before the L-carnitine supplementation, after completion of a carnitine supplementation period treatment (12 weeks, 1 g/IV/HD), right before the HD session, and 44 h after the dialysis. Levels of free carnitine (FC) and the individual esters were determined using electrospray MS/MS technique. Normally, L-carnitine supplementation causes significant elevation of all carnitine compounds to supraphysiological levels, which reaches a standard steady-state-like profile. In this study we found a dramatic decrease in the level of FC, and in short- and medium-chain acylcarnitines (ACs) 44 h after the last dialysis. At the end of this interdialytic period, FC levels increased to only 65% of the predialysis level, whereas the amounts of C2 and C3 esters recovered to only 50%. The level of C6 was 65% of the predialysis level, whereas the amount of C8 chain length ACs returned to 72% of the predialysis level. No significant change was seen in AC concentrations above C10 chain length. Omission of one single dosage of supplemental carnitine in long-term administration schemes results in dramatic decrease and reprofiling of carnitine esters even after the usual 44 h of interdialytic period.

• carnitine esters
• hemodialysis
• L-carnitine
• mass spectrometry

INTRODUCTION

Carnitine has an indispensible role in energy metabolism via facilitating the transport of long-chain fatty acids through the mitochondrial membrane and controlling the rate of beta-oxidation of long-chain fatty acids with subsequent energy production.^1,2 Moreover, carnitine is involved in the regulation of cellular and mitochondrial acyl-CoA/CoA ratios, in the transport of activated short- and medium-chain acids from the peroxisome to the mitochondria, and in the detoxification and elimination of potentially toxic acyl groups accumulated as a result of normal and abnormal metabolism.^3–8

In healthy individuals, the plasma and tissue levels of carnitine are maintained within a relatively narrow range. In this regulation process, the kidney has a crucial role involving active tubular reabsorption of L-carnitine and preferential excretion of acylcarnitines (ACs).⁹ It is well known that one single hemodialysis (HD) session results in a decline of as much as 75% in the plasma carnitine concentration,¹⁰ and dialytic removal of ACs has been proved to be carbon chain length dependent¹¹; however, no report is available on the changes of the carnitine ester profile during the interdialytic period. The aim of this study was to examine the alterations of the individual ACs between two dialysis sessions in the sera of carnitine-repleted end-stage renal disease (ESRD) patients undergoing long-term HD treatment after interruption of the exogenous supply.

MATERIALS AND METHODS

During the entire study period the guidelines and regulations approved by the local ethics committee and in accordance with the Helsinki Declaration of 1975, as revised in 2000, were followed. Written informed consent was obtained from each participant before the start of the study.

Study Population

ESRD patients aged between 40 and 85 years were recruited at the FMC dialysis center of Pécs. Enrolment requirements included HD treatment three times a week for at least 6 months and the absence of acute illness, heart failure, or previous carnitine supplementation.

Eighteen nondiabetic patients (10 males, 8 females) with a mean age of 59.2 years were included. The underlying renal pathologies that progressed to ESRD were nephrocalcinosis (1 patient), chronic pyelonephritis (2 patients), benign nephrosclerosis (9 patients), and chronic glomerulonephritis (6 patients). Most of the patients received antihypertensive therapy (ACE inhibitors 17, calcium channel blockers 11, beta-blockers 6, others 2), but none of them was on lipid-lowering medication. Additional drugs for all individual patients were calcitriol, calcium carbonate, and iron, as well as erythropoietin. The average weekly dose of erythropoietin-beta was 4700 ± 3450 IU at week 0 and 4550 ± 3250 IU at the end of the observation period. Samples from 36 adult volunteers (19 males and 17 females, mean age: 49.7 years) served as healthy controls. The patients underwent three HD sessions per week as described previously.¹²

Study Design

The patients received carnitine treatment for 12 weeks. The supplementation was carried out after each HD session with 1 g L-carnitine intravenously. After the IV administration of L-carnitine, the venous line was flushed with saline for about 2 min.

Sample collection was performed following an overnight fast, early in the morning, for healthy controls, as well as for the patients, immediately before the beginning of the respective HD session. To avoid the effect of diet or fasting time on the circulating carnitine esters, strict postalimentary time scheduling was introduced. Serum samples were separated immediately after blood collection in a refrigerated centrifuge and stored at −70°C until analysis. For carnitine and AC measurements, blood samples of the patients were taken before the initiation of the carnitine supplementation (day 0), after completion of the 12-week carnitine supplementation period just before the first dialysis treatment, and right before the next dialysis session (44 h later).

Analysis of Carnitine and Acylcarnitines

Free carnitine (FC) and ACs were measured after derivatization as butyl-esters using the isotope dilution mass spectrometry method in a Micromass Quattro Ultima (Manchester, UK) ESI triple quadrupole mass spectrometer as described previously.¹³

Statistical Analysis

For statistical analyses, paired and unpaired Student’s t-test were applied. Variables are presented as mean ± SEM; p < 0.001 was considered statistically significant.

RESULTS

The serum circulating carnitine ester profiles are shown in Table 1.

Table 1. Serum carnitine ester profiles in HD patients before carnitine supplementation, and before and 44 h after a dialysis session following the end of carnitine supplementation and in controls (mean ± SEM, μmol/L).

		Patients on hemodialysis (n = 18)
		Before L-carnitine	After 12 weeks of L-carnitine treatment
Carnitines	Healthy controls (n = 36)	Day 0	Before last HD^*	44 h after last HD
Free carnitine	48.143 ± 1.581	24.920 ± 0.888^**	186.759 ± 12.728	122.462 ± 9.300^***
Acyl	7.723 ± 0.267	11.486 ± 0.493^**	62.752 ± 3.781	36.126 ± 2.244^***
Total	55.866 ± 1.735	36.406 ± 2.028^**	249.511 ± 16.250	158.588 ± 9.857^***
AC/FC ratio	0.160 ± 0.006	0.461 ± 0.015^**	0.336 ± 0.009	0.295 ± 0.018
Short-chain acylcarnitines
C2 carnitine	5.540 ± 0.211	7.157 ± 0.410^**	48.069 ± 3.338	24.966 ± 1.572^***
C3 carnitine	0.238 ± 0.013	0.369 ± 0.026^**	3.114 ± 0.310	1.717 ± 0.161^***
C4 carnitine	0.094 ± 0.005	0.311 ± 0.027^**	2.194 ± 0.213	1.535 ± 0.182^***
C5 carnitine	0.116 ± 0.005	0.238 ± 0.015^**	1.491 ± 0.096	1.073 ± 0.080^***
C5-OH carnitine	0.029 ± 0.001	0.059 ± 0.003	0.164 ± 0.009	0.133 ± 0.009^***
Total short-chain ACs	6.017 ± 0.221	8.134 ± 0.450^**	55.032 ± 3.683	29.424 ± 2.094^***
Medium-chain acylcarnitines
C6 carnitine	0.018 ± 0.001	0.045 ± 0.004	0.120 ± 0.011	0.079 ± 0.006^***
C8 carnitine	0.171 ± 0.009	0.219 ± 0.012^**	0.746 ± 0.047	0.534 ± 0.050^***
C8:1 carnitine	0.094 ± 0.010	0.331 ± 0.026^**	0.861 ± 0.070	0.665 ± 0.060^***
C10 carnitine	0.178 ± 0.021	0.217 ± 0.015^**	0.583 ± 0.048	0.567 ± 0.053
C10:1 carnitine	0.218 ± 0.011	0.303 ± 0.026^**	0.714 ± 0.063	0.654 ± 0.056
C10:2 carnitine	0.029 ± 0.001	0.060 ± 0.005	0.155 ± 0.012	0.126 ± 0.011
C12 carnitine	0.089 ± 0.005	0.130 ± 0.006^**	0.238 ± 0.013	0.238 ± 0.021
C12:1 carnitine	0.083 ± 0.005	0.103 ± 0.011	0.239 ± 0.025	0.240 ± 0.037
Total medium-chain ACs	0.880 ± 0.046	1.408 ± 0.063^**	3.656 ± 0.219	3.103 ± 0.315
Long-chain acylcarnitines
C14 carnitine	0.067 ± 0.004	0.121 ± 0.006^**	0.208 ± 0.020	0.201 ± 0.026
C14:1 carnitine	0.121 ± 0.008	0.132 ± 0.009	0.280 ± 0.032	0.227 ± 0.023
C14:2 carnitine	0.070 ± 0.004	0.090 ± 0.009	0.196 ± 0.030	0.146 ± 0.015
C14-OH carnitine	0.024 ± 0.001	0.059 ± 0.005	0.123 ± 0.009	0.112 ± 0.010
C16 carnitine	0.115 ± 0.006	0.116 ± 0.006	0.263 ± 0.016	0.239 ± 0.014
C16:1 carnitine	0.025 ± 0.002	0.030 ± 0.003	0.063 ± 0.005	0.059 ± 0.006
C18 carnitine	0.045 ± 0.002	0.040 ± 0.002	0.076 ± 0.004	0.073 ± 0.005
C18:1 carnitine	0.114 ± 0.007	0.083 ± 0.006	0.170 ± 0.015	0.174 ± 0.013
C18:2 carnitine	0.068 ± 0.004	0.045 ± 0.004	0.108 ± 0.010	0.104 ± 0.009
Total long-chain ACs	0.649 ± 0.030	0.716 ± 0.030	1.487 ± 0.099	1.335 ± 0.079
Dicarboxylic acylcarnitines
C4DC carnitine	0.032 ± 0.002	0.207 ± 0.016^**	0.430 ± 0.081	0.321 ± 0.037
C5DC carnitine	0.097 ± 0.006	0.779 ± 0.075^**	1.658 ± 0.111	1.538 ± 0.126
C6DC carnitine	0.009 ± 0.001	0.140 ± 0.013^**	0.209 ± 0.018	0.192 ± 0.016
C8DC carnitine	0.016 ± 0.001	0.064 ± 0.005	0.209 ± 0.018	0.154 ± 0.019
C10DC carnitine	0.023 ± 0.002	0.038 ± 0.003	0.071 ± 0.005	0.059 ± 0.004
Total dicarboxylic ACs	0.177 ± 0.007	1.228 ± 0.093^**	2.577 ± 0.137	2.264 ± 0.149

Note: ^*p < 0.001 compared with control and day 0, ^**p < 0.001 compared with control, ^***p < 0.001 compared with that before first HD.

Under baseline conditions (i.e., before L-carnitine supplementation), the ESRD patients on long-term HD treatment displayed significantly decreased free (p < 0.001) and total carnitine (p < 0.001) levels and a markedly elevated level of ACs (p < 0.001) leading to an elevation in AC-to-FC ratio compared with the healthy controls. The proportion of ACs within the total carnitine pool was 31.5% in the patients group, whereas it was only 13.9% in the healthy subjects.

As a response to the 12-week-long L-carnitine supplementation, the concentrations of FC and almost all the investigated ACs were elevated to a supraphysiological level; however, the AC-to-FC ratio remained increased. A redistribution of the plasma carnitine pool was observed: whereas 31.5% of the total carnitine pool was found in ACs under baseline conditions, only 25.2% of the total carnitine was present as ACs after a 12-week L-carnitine supplementation.

HD treatment induced a dramatic decrease in the serum carnitine pool in carnitine-repleted ESRD patients after interruption of the exogenous carnitine supply. At the end of the interdialytic period (44 h after the HD), FC concentration was about only 65% of the predialysis value. Moreover, a marked reduction was observed in the levels of almost all short-chain ACs, the most pronounced changes were seen in C2- and C3-ester concentrations, for which only approximately 50% of the predialysis value remained. In the medium-chain AC fraction, a notable decrease was detected in the level of C6-carnitine (65% of the predialysis level) and the amount of C8 chain length ACs remained at about 72% of the predialysis level, whereas the levels of C10 and C12 chain length esters remained nearly unchanged. Among the long-chain ACs no significant change was observed.

DISCUSSION

Carnitine homeostasis is altered in ESRD, reflecting the chronic metabolic derangements associated with this condition. A dramatic redistribution of the plasma carnitine pool toward ACs is characteristic of ESRD, reflecting incomplete oxidation of endogenous substrates and their accumulation in the coenzyme A and carnitine pools.¹⁴ HD in ESRD patients has a further influence on the carnitine pool. Because of its small molecular weight and the fact that carnitine is not protein-bound in the plasma, it is easily dialyzed, leading to diminished free-carnitine levels in these patients, finally resulting in an abnormal plasma AC-to-FC ratio. Long-term HD removes carnitine not only from the plasma but also from muscle stores.¹⁵

Under baseline conditions, our patients presented a reduced serum L-carnitine concentration compared with the controls. The AC-to-FC ratio was 0.461 ± 0.015, indicating a redistribution of the serum carnitine pool toward ACs. This concept is supported by the observation that in the patients group the proportions of FC and ACs within the total carnitine pool were 68.5% and 31.5%, respectively, whereas they were only 86.1% and 13.9%, respectively, in the healthy subjects. These results are consistent with Reuter et al., who found that the proportions of L-carnitine and ACs within the total carnitine pool change with the duration of dialysis. In patients who have undergone HD for at least 6 months, about 70% of the total carnitine pool comprises L-carnitine, and this value is approximately 50% in patients on HD for more than 12 months.¹⁶

Administration of carnitine to patients on long-term HD has a beneficial effect on dialysis-related symptoms such as skeletal myopathies, cardiomyopathy, poor exercise performance, and anemia, and dialytic complications like hypotension, cramps, weakness, and fatigue, and may act in at least two ways, namely, by increasing the low carnitine levels in serum and tissue and by eliminating the potentially toxic acyl groups, mainly from the liver and possibly from other organs.¹⁷ Repeated administration of L-carnitine for a long period results in its distribution into the cells and its incorporation into the slowly equilibrating deep tissue compartment, and finally into the total body carnitine pool.¹⁸ In our patients the 12-week intravenous carnitine administration increased both serum carnitine and AC concentrations to a supraphysiological level; however, the AC-to-FC ratio remained elevated.

In several studies the effect of HD on endogenous plasma carnitine concentrations was investigated. Reuter et al. examined the impact of a single dialysis session on the individual plasma AC levels and found that the short- and medium-chain esters are removed by HD and that the dialytic removal of ACs is inversely related to carbon chain length of the acyl groups.¹¹ Evans and colleagues¹⁸ found a marked reduction in plasma level of L-carnitine during dialysis, which returned to the predialysis level in the subsequent 44-h interdialytic period in ESRD patients undergoing long-term HD. Contrarily, in our study the concentration of L-carnitine and several ACs (C2, C3, C4, C5, C6) remained far below the predialysis levels 44 h after the dialysis in the carnitine-supplemented patients, although the amounts of these compounds were higher than the unsupplemented levels. However, the kinetics of these changes in the interdialytic period remains unknown, as the current design does not allow drawing further conclusions.

In conclusion, our study shows that since carnitine and short-chain ACs are removed fairly efficiently by HD, omission of one single dosage of supplemental carnitine in a long-term administration scheme results in a dramatic decrease and reprofiling of carnitine esters even after the usual 44 h of interdialytic period.

ACKNOWLEDGMENTS

The authors are grateful to Marta Hartung for her help in the technical management of the study, as well as to Sigma Tau for providing carnitine solutions.

Funding. This work was supported by OTKA: T73430, ETT: 497/2006, ETT 210-07/2009 and ÁOKKA 34039-6/2009.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.