Age and Sex Dependency of Carnitine Concentration in Human Serum and Skeletal Muscle

Carnitine plays an essential role in fatty acid metabolism. It can be synthesized in the liver, but additional intestinal resorption is necessary (1). Carnitine mediates the transport of activated acyl residues via the carnitine palmityl transferase system into mitochondria for β-oxidation (2). Whereas primary carnitine deficiency is attributable to mutations in OCTN2 [a carnitine transporter of plasma membranes (3)], several other conditions can cause secondary deficiency (4). The leading symptom of either primary or secondary carnitine deficiency is weakness of skeletal muscles. In addition to these pathologic conditions in some animal models, a physiologic decline of carnitine concentration with aging has been reported (5)(6). In addition to increasing muscular carnitine concentrations (6), oral treatment with l-carnitine or its acetyl ester has also been shown to restore many of physiologic impairments that accompany aging (7)(8)(9)(10). These data indicate the important role of carnitine in the aging process, at least in mice and rats. For human skeletal muscle, confusing results exist with respect to the age dependency of carnitine content: Whereas Costell et al. (5) found a “drastic” age-dependent decrease of carnitine in human skeletal muscle and Gonzalez-Crespo et al. (11) detected reduced free carnitine concentrations in elderly patients undergoing hip surgery, an age-dependent decrease in carnitine concentration could not be confirmed by Starling et al. (12). The answer to the question of whether carnitine in human muscle is also age dependent is hampered by the different populations investigated and the different methods used. No reliable data exist concerning the sex dependency of a possible age-dependent variation. The present study seeks to clarify whether carnitine concentrations in human skeletal muscle and serum depend on age and sex. The physiologic relevance is discussed. Analysis of carnitine in serum was performed in samples from healthy blood donors (n = 80; 18–57 years) from the local blood-donor service of the University Hospital Halle (Saale). For determination of carnitine in skeletal muscle, routine diagnostic muscle biopsies from patients (n = 52; 18–74 years) were analyzed. Healthy controls were selected among patients who had no muscle disease, as determined by combined clinical, electrophysiologic, histologic, electron microscopic, biochemical, and genetic criteria. Only specimens of proximal skeletal muscles (vastus lateralis or biceps brachii) obtained by open biopsy at standardized locations were included in this study. Biopsy was performed without anesthesia of muscle tissue. The sample was immediately frozen in liquid nitrogen and stored until further use. Written informed consent was obtained from all patients before biopsy. Frozen tissue was homogenized 1:30 (by weight) in a solution containing 50 mmol/L Tris (pH 7.5), 100 mmol/L KCl, 5 mmol/L MgCl2, and 1 mmol/L EDTA with a 2-mL glass/glass homogenizer (0.025-mm clearance; Kontes Glass Co.) as described previously (13). Total and free carnitine were radiochemically assayed directly in either serum or skeletal muscle homogenates with a modified method of Barth et al. (14). The samples were incubated 30 min at 30 °C with 35 μmol/L 14C-labeled acetyl-CoA in the presence of carnitine-acetyl-transferase (2 kU/L) and 3.5 mmol/L N-ethylmaleimide. Thereafter, the 14C-labeled acetyl-carnitine was separated from the 14C-labeled acetyl-CoA by ion-exchange chromatography with AG® 1-X8 resin (100–200 mesh, chloride form; Bio-Rad Laboratories). The eluate was measured in a 10-mL scintillation mixture with a LS-6500 counter (Beckman Instruments Inc.). For analysis of total carnitine, the sample was boiled with an equal volume of 0.2 mmol/L KOH at 56 °C for 1 h before analysis. Protein was determined as noncollagen protein by the bicinchoninic acid protein assay (Pierce) (15) in the supernatant after a 24-h digestion in 50 mmol/L NaOH and sedimentation at 13 000g. Bovine serum albumin was used as the calibrator. All chemicals were of analytical grade. l-Carnitine-l-tartrate was from Lonza, 14C-labeled acetyl-CoA from NEN Life Science, nonlabeled acetyl-CoA as trilithium salt from Merck (Merck Eurolab GmbH); all other chemicals were obtained from Sigma. Each variable was tested for normality with the Kolmogorow–Smirnow test. If this criterion was fulfilled, statistical significance was tested with the t-test for unpaired variables. Otherwise the Wilcoxon test (Mann–Whitney U-Test) was applied. P <0.05 was considered significant. Serum concentrations of total and free carnitine, as well as the ratio of both analytes, showed sex-dependent differences (Table 1 ). Serum carnitine (free and total) was ∼25% higher in men than in women (P <0.001). All analytes measured were gaussian within the population. An age-dependent increase of free (r = 0.48; P <0.01) and total (r = 0.37; P = 0.02) serum carnitine could be observed in women, whereas in men, there was a slightly positive, but nonsignificant correlation (free, r = 0.22, P = 0.17; total, r = 0.06, P = 0.73). These data are shown in Fig. 1A . Both regression lines converge at the age of ∼80 years. The ratio between free and total carnitine in serum increased significantly (P <0.05) with increasing age in both sexes, but was more pronounced in men (data not shown). Concentration of carnitine in sera and skeletal muscle homogenates. s.w., sample weight; NCP, non-collagen protein. Statistically significant difference between males and females and sex-independent value (t-test; P<0.05). Statistically significant difference between males and females (t-test; P <0.05). Concentration of carnitine in sera and skeletal muscle homogenates. s.w., sample weight; NCP, non-collagen protein. Statistically significant difference between males and females and sex-independent value (t-test; P<0.05). Statistically significant difference between males and females (t-test; P <0.05). Age- and sex-related variation of total carnitine in serum and skeletal muscle of males (•) and females (□). (A), serum carnitine in relation to age. The regression lines are plotted for males (solid line; r = 0.06, P = 0.73) and females (dashed line; r = 0.37, P = 0.02). (B), muscle carnitine in relation to age. The regression lines are plotted for males (solid line; r = −0.51, P <0.01) and females (dashed line; r = −0.02, P = 0.92). Linear regression was performed by the Pearson test. In the skeletal muscle of men, a significant decrease of either free or total carnitine (r = −0.53 and r = −0.51, respectively; P <0.01) was found. Free and total carnitine in men >60 years (n = 7) was 1.7 ± 0.3 μmol/g of sample weight and 2.5 ± 0.3 μmol/g of sample weight, respectively, whereas in men <60 years (n = 45), the values were 2.7 ± 0.7 μmol/g of sample weight and 3.5 ± 0.8 μmol/g of sample weight, respectively. These values remained constant, regardless of age, in women (Fig. 1B ). Additionally, there was no significant variation of carnitine esters in muscle among the different ages in either men or women. Generally, it is difficult to obtain reference values from a cohort of an ideally healthy population, especially in tissues that have to be taken in an invasive manner. In this study, we handled this problem with muscle biopsies taken for diagnostic purposes in patients who were found to be healthy by the criteria described above. This is supported by the fact that carnitine concentrations were gaussian within our population, and the observed values of carnitine were on the same order of magnitude as observed by others (16)(17)(18). In contrast to other investigations that use preferably young, healthy, and active adults, our population showed a more homogeneous distribution of concentrations across a broader age range (18–74 years). Here we report mean serum carnitine concentrations ∼25% higher in men than in women. This argues for the determination of different reference values for both sexes in serum, but the sex-specific differences decrease with increasing age (Fig. 1A ), suggesting no difference at ∼80 years. This is attributable to an increase of serum carnitine in females with increasing age, which does not seem present in men. Similar results were found by Chiu et al. (19) in sera from a majority of 216 North Americans. In those healthy adults, Chiu et al. found an increase in either free or total carnitine with age, which was much more pronounced in women. The same study reported a decrease in dehydroepiandrosterone sulfate, which was higher in men than in women. Therefore, it can be speculated that sex hormones and their precursors may be involved in changes of carnitine metabolism. Indeed the differences between males and females seem to be minimal after menopause. This is further supported by findings in rats (20)(21), in which serum carnitine concentrations increased after ovariectomy, and a report in humans (22) that describes a significant negative correlation between serum free carnitine and estradiol in females, but no significant relation to testosterone in men. Because serum carnitine is influenced by other factors, including synthesis in the liver, intestinal resorption, renal excretion, and tissue distribution, changes in serum concentrations are very difficult to interpret and are of minor diagnostic relevance. Muscular carnitine content is more reliable than serum concentration because ∼98% of total carnitine in humans is contained in muscles (23). Therefore, the intramuscular concentration, rather than the serum concentration, reflects the body content of carnitine. In contrast to serum, no mean variation in carnitine with respect to sex could be observed in skeletal muscle (Table 1 ). This is consistent with results obtained for abdominal muscles (18). With respect to age, we found inverse results between serum and muscle carnitine. In contrast to serum, there was an age-dependent decrease in muscle, but only in men. This findings may seem confusing, but can be explained by hormonal differences between men and women. Sex hormones are thought to be a major modulator of manifold cellular pathways. It is well known that aging, especially in men, is accompanied with a loss of muscle strength and lower testosterone in blood (24) and that testosterone in men controls skeletal muscle protein synthesis (25). Additionally, activation of precursor hormones, such as dehydroepiandrosterone, is quite different in men and women: Whereas ∼75–100% of estrogens are formed in peripheral tissue in women, particularly after menopause, this holds true for only 30–50% of androgens in men (26). Hence, muscle metabolism in females is mostly independent of circulating sex hormones, in contrast to men. This might contribute to the observed age-dependent decrease in muscle carnitine in males. The down-regulation of carnitine transporters attributable to reduced serum testosterone may be crucial in males (6) because human skeletal muscle cannot synthesize carnitine. A decrease in muscular carnitine could be associated with a loss of muscle strength, although the definition of a carnitine deficiency is not fulfilled. This would be in line with reports (27)(28) on sarcopenia in men and women >50 years in age. These reports describe a more pronounced loss of skeletal muscle and muscle strength in men compared with women. Therefore, oral supplementation with l-carnitine might help to slow the physiologic process of sarcopenia, especially in men. In summary, this study provides evidence that there is a sex-dependent decrease in carnitine concentrations in skeletal muscle, affecting only men. Men >60 years showed significantly lower free and total carnitine in skeletal muscle than did younger controls. In sera from females, there was an age-dependent increase of carnitine concentrations, but mean values were higher in men. Therefore, age- and sex-dependent reference values have to be considered. It is not clear whether the observed effects are only an indicator of age-dependent changes, or whether, in addition, they are a cause of functional impairments accompanying aging. This study was supported by the Medical Faculty of the University Halle (Saale), Germany.