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Technical Briefs

Although the present overall results confirm the previous good reproducibility data obtained with the same immunometric method (11 ), the data reveal marked imprecision for lower renin concentrations. Blood concentrations of renin are particularly valuable and useful at low concentrations (18 ) and when the renin-angiotensin system is activated (15 ). Because various immunometric assays for active renin are used for clinical research (15, 16, 19 ), it would be advisable to validate each technique with a thorough quality-control program.

Age and Sex Dependency of Carnitine Concentration in Human Serum and Skeletal Muscle, Jens Ru ¨ diger Opalka,* Frank-Norbert Gellerich, and Stephan Zierz (Muskellabor, Universita ¨ tsklinik und Poliklinik fu ¨ r Neurologie, MartinLuther-Universita ¨ t Halle-Wittenberg, Ernst-Grube-Strasse 40, D-06097 Halle (Saale), Germany; * author for correspondence: fax 49-345-557-3505, e-mail jens.opalka@ medizin.uni-halle.de) 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 ␤-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 (7–10 ). 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 ϭ 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 ϭ 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

We are indebted to our colleagues at 48 Italian centers who participated in the present study.
References
1. Sealey JE, Laragh JH. RIA of plasma renin activity. Semin Nucl Med 1975;5:189 –202. 2. Sealey JE. Plasma renin activity and plasma prorenin assays. Clin Chem 1991;37:1811–9. 3. Derkx FHM, De Bruin RJA, Van Gool JMG, Van den Hoek MJ, Beerendonk CCM, Rosmalen F, et al. Clinical validation of renin monoclonal antibodybased sandwich assays of renin and prorenin, and use of renin inhibitor to enhance prorenin immunoreactivity. Clin Chem 1996;42:1051– 63. 4. Simon D, Hartmann DJ, Badouaille G, Caillot G, Guyenne TT, Corvol T, et al. Two-site direct immunoassay specific for active renin. Clin Chem 1992;38: 1959 – 62. 5. Sealey JE, Trenkwalder P, Gahnem F, Catanzaro D, Laragh JH. Plasma renin methodology: inadequate sensitivity and accuracy of direct renin assay for clinical applications compared with the traditional enzymatic plasma renin activity assay. J Hypertens 1995;13:27–30. 6. Menard J, Thanh-Tam G. Commentary: renin assays: a debate for clinicians, not only for specialists. J Hypertens 1995;13:367–9. 7. Nystrom F, Karlberg BE, Ohman KP. Serum angiotensin-converting enzyme activity correlates positively with plasma angiotensin II. A population-based study of ambulatory blood pressure and the renin-angiotensin system. J Hum Hypertens 1997;11:301– 6. 8. Sealey JE, Laragh JH. Commentary: plasma renin methodology. J Hypertens 1995;13:371. 9. Sealey JE, Laragh JH. Renin and prorenin: advanced and declines in methodology. Clin Chem 1996;42:993– 4. 10. Deinum J, Derkx FHM, Schalekamp MADH. Improved immunoradiometric assay for plasma renin. Clin Chem 1999;45:847–54. 11. Morganti A, Pelizzola D, Mantero F, Gazzano G, Opocher G, Piffanelli A. Immunoradiometric versus enzymatic renin assay: results of the Italian Multicenter Comparative Study. J Hypertens 1995;13:19 –26. 12. Morganti A, Pelizzola D, Mantero F, Gazzano G, Opocher G, Piffanelli A. On the Italian Multicenter Study for Standardization of Renin Measurement. [Authors’ Reply]. J Hypertens 1995;13:31. 13. Ekins RP. Immunoassay standardization. Scand J Clin Lab Invest 1991; 51(Suppl 205):33–9. 14. Sadler WA. Estimating imprecision of radioassays and related procedures: some considerations. Eur J Nucl Med 1991;18:863– 65. 15. Hurwitz S, Potter B, Weiss RJ, Jeunemaitre X, Hopkins PN, Hunt SC, et al. Influence of sodium intake on the reliability of active renin as a measure of the renin-angiotensin system in essential hypertension. Am J Clin Pathol 2001;115:304 –12. 16. Giacche ` M, Vuagnat A, Hunt SC, Hopkins PN, Fisher NDL, Azizi M, et al. Aldosterone stimulation by angiotensin II. Influence of gender, plasma renin, and familial resemblance. Hypertension 2000;35:710 – 6. 17. WHO. Expert Committee on Biological Standardization (1974). 26th Report. WHO Technical Report Series No. 565. Geneva: WHO, 1975:11pp. 18. Kruger C, Rauh M, Door HG. Immunoreactive renin concentrations in healthy children from birth to adolescence. Clin Chim Acta 1998;274:15–27. 19. Danser AHJ, Vankestern CAM, Bax WA, Tavenier M, Derkx FHM, Saxena PR, et al. Prorenin, renin, angiotensinogen and angiotensin-converting in normal and failing human hearts— evidence for renin binding. Circulation 1997;96:220 – 6.

there was a slightly positive. NCP.48.1 Ϯ 5.c 74.9–92.05 was considered significant.2 mmol/L KOH at 56 °C for 1 h before analysis.53 and r ϭ Ϫ0.0b 49.2 48. P Ͻ0. statistical significance was tested with the t-test for unpaired variables. No.17. Bovine serum albumin was used as the calibrator.). Free carnitine. The samples were incubated 30 min at 30 °C with 35 ␮mol/L 14Clabeled acetyl-CoA in the presence of carnitine-acetyltransferase (2 kU/L) and 3.2 Ϯ 5.c 30.) as described previously (13 ).06. The eluate was measured in a 10-mL scintillation mixture with a LS-6500 counter (Beckman Instruments Inc.9–40.3 7.8–33.8c 72.3 1.5 55.05). Thereafter. total.3–87. and 1 mmol/L EDTA with a 2-mL glass/glass homogenizer (0.2 25. P Ͻ0.1 Ϯ 10.w.4 26.9 3.8 2.5 55. r ϭ 0.Clinical Chemistry 47. P Ͻ0.9 Ϯ 8.2 0.c 34. Bio-Rad Laboratories).8 1. as well as the ratio of both analytes. ␮mol/L Free carnitine.4 76.6 Ϯ 9.4 19. Kontes Glass Co.8 2.3 78. P Ͻ0. 5 mmol/L MgCl2.3b. whereas in men.a Total carnitine. ␮mol/g NCP Ratio of free/total carnitine.4 24. 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.9–40.w. .02) serum carnitine could be observed in women. l-Carnitine-l- tartrate was from Lonza. sample weight.4b 38. Each variable was tested for normality with the Kolmogorow–Smirnow test. non-collagen protein..1 Ϯ 0.3b. In the skeletal muscle of men. chloride form. P ϭ 0.5 Ϯ 0.7 Ϯ 7. Concentration of carnitine in sera and skeletal muscle homogenates. % Both M F Both M F Both M F Both M F Both M F Both M F Both M F Both M F 52 25 27 52 25 27 52 25 27 52 25 27 52 25 27 80 41 39 80 41 39 80 41 39 3.0c 1. ␮mol/L Ratio of free/total carnitine.37.8–46.8–59.9 24.7 Ϯ 5.9 1. All chemicals were of analytical grade.2–53.3 19.01) and total (r ϭ 0. For analysis of total carnitine.9 Ϯ 8. r ϭ 0.6 Ϯ 10. 1A. Both regression lines converge at the age of ϳ80 years. showed sex-dependent differences (Table 1).7 45. 12.1 Ϯ 7. These data are shown in Fig.2 Ϯ 6.8–67.9 19.1–4.025-mm clearance.7–4. c Statistically significant difference between males and females (t-test. An age-dependent increase of free (r ϭ 0. If this criterion was fulfilled.5 2.4–53.1–4.9 12. the sample was boiled with an equal volume of 0.001). 100 mmol/L KCl. 2001 2151 nitrogen and stored until further use.2 76. The ratio between free and total carnitine in serum increased significantly (P Ͻ0. P ϭ 0. 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.8–67.9 3.2 34. ␮mol/g s.2 Ϯ 6.2 Ϯ 0. All analytes measured were gaussian within the population.22.4 Ϯ 0.5–46.73). all other chemicals were obtained from Sigma. Frozen tissue was homogenized 1:30 (by weight) in a solution containing 50 mmol/L Tris (pH 7.7–4. Sample type Analyte Sex n Mean ؎ SD Range Skeletal muscle Total carnitine.5). Total and free carnitine were radiochemically assayed directly in either serum or skeletal muscle homogenates with a modified method of Barth et al. Serum carnitine (free and total) was ϳ25% higher in men than in women (P Ͻ0.9–29. but nonsignificant correlation (free.5 Ϯ 5.7 19.1 Ϯ 7. nonlabeled acetyl-CoA as trilithium salt from Merck (Merck Eurolab GmbH).9–88.6 48. ␮mol/g s.05) with increasing age in both sexes.4 Ϯ 0. Serum concentrations of total and free carnitine.6 25. Otherwise the Wilcoxon test (Mann– Whitney U-Test) was applied.05).51.9–92.6 9.7 17.5 mmol/L N-ethylmaleimide.c 41. Table 1.1 Ϯ 5.4b.3b. % a b s.2 19.7 12.6 7.w. Written informed consent was obtained from all patients before biopsy. Statistically significant difference between males and females and sex-independent value (t-test.2–44.5 63.0–92.0 Ϯ 6.5 75.2 0.7–4. (14 ).9–92.3 52.1 Ϯ 0.5 Serum Total carnitine.3 Ϯ 10. a significant decrease of either free or total carnitine (r ϭ Ϫ0.0 Ϯ 6. P ϭ 0. ␮mol/g NCP Free carnitine.9 25.0–4. but was more pronounced in men (data not shown). 14C-labeled acetyl-CoA from NEN Life Science.

With respect to age. respectively. (B). It is well known that aging. Hence.and sex-related variation of total carnitine in serum and skeletal muscle of males (F) and females (Ⅺ). In this study.01) was found. Indeed the differences between males and females seem to be minimal after menopause. Chiu et al. which was higher in men than in women. intestinal resorption.2152 Technical Briefs respectively. This findings may seem confusing. 1B). found an increase in either free or total carnitine with age. the intramuscular concentration. but no significant relation to testosterone in men. such as dehydroepiandrosterone.7 Ϯ 0. This is consistent with results obtained for abdominal muscles (18 ). in women (Fig. Age. and tissue distribution.3 ␮mol/g of sample weight. 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 ). P Ͻ0.06. P Ͻ0. in which serum carnitine concentrations increased after ovariectomy. activation of precursor hormones.02. our population showed a more homogeneous distribution of concentrations across a broader age range (18 –74 years). In contrast to serum. reflects the body content of carnitine.5 Ϯ 0. suggesting no difference at ϳ80 years. muscle carnitine in relation to age. 1A). gaussian within our population.5 Ϯ 0. we found inverse results between serum and muscle carnitine. (A). r ϭ 0. Free and total carnitine in men Ͼ60 years (n ϭ 7) was 1. muscle metab- . serum carnitine in relation to age. no mean variation in carnitine with respect to sex could be observed in skeletal muscle (Table 1). Generally. r ϭ Ϫ0.02). Therefore. In contrast to serum. The regression lines are plotted for males (solid line.51. 21 ). This is further supported by findings in rats (20. Here we report mean serum carnitine concentrations ϳ25% higher in men than in women. Additionally. particularly after menopause. Therefore. This is supported by the fact that carnitine concentrations were Fig.7 Ϯ 0. Linear regression was performed by the Pearson test. Additionally. These values remained constant.92). In contrast to other investigations that use preferably young.01) and females (dashed line. respectively. r ϭ Ϫ0. which does not seem present in men. P ϭ 0. (19 ) in sera from a majority of 216 North Americans. Muscular carnitine content is more reliable than serum concentration because ϳ98% of total carnitine in humans is contained in muscles (23 ). Similar results were found by Chiu et al. but only in men. especially in men. rather than the serum concentration. and a report in humans (22 ) that describes a significant negative correlation between serum free carnitine and estradiol in females. In those healthy adults.73) and females (dashed line. regardless of age. changes in serum concentrations are very difficult to interpret and are of minor diagnostic relevance. healthy. 1. we handled this problem with muscle biopsies taken for diagnostic purposes in patients who were found to be healthy by the criteria described above.3 ␮mol/g of sample weight and 2. and the observed values of carnitine were on the same order of magnitude as observed by others (16 –18 ). it can be speculated that sex hormones and their precursors may be involved in changes of carnitine metabolism. there was no significant variation of carnitine esters in muscle among the different ages in either men or women. This argues for the determination of different reference values for both sexes in serum. but can be explained by hormonal differences between men and women. it is difficult to obtain reference values from a cohort of an ideally healthy population. but the sex-specific differences decrease with increasing age (Fig. this holds true for only 30 –50% of androgens in men (26 ).37. the values were 2. Sex hormones are thought to be a major modulator of manifold cellular pathways. Because serum carnitine is influenced by other factors. The same study reported a decrease in dehydroepiandrosterone sulfate. including synthesis in the liver. r ϭ 0. P ϭ 0. renal excretion. there was an age-dependent decrease in muscle. The regression lines are plotted for males (solid line. whereas in men Ͻ60 years (n ϭ 45). P ϭ 0.8 ␮mol/g of sample weight. This is attributable to an increase of serum carnitine in females with increasing age. which was much more pronounced in women. is quite different in men and women: Whereas ϳ75–100% of estrogens are formed in peripheral tissue in women. and active adults. especially in tissues that have to be taken in an invasive manner.7 ␮mol/g of sample weight and 3.

Raimundo et al.and sex-dependent reference values have to be considered. age and oxidative status. Van ’t Veer-Korthof ET. The down-regulation of carnitine transporters attributable to reduced serum testosterone may be crucial in males (6 ) because human skeletal muscle cannot synthesize carnitine. Schmidt MJ. 19. Effects of dehydroepiandrosterone sulfate on carnitine acetyl transferase activity and L-carnitine levels in oophorectomized rats. J Clin Chem Clin Biochem 1990. especially in men. FEBS Lett 1993. 25. An X-linked mitochondrial disease affecting cardiac muscle. Arenas J. Schatzberg. Transport and function of carnitine in muscles. Lundholm K. J Neurol Sci 1977. J Clin Chem Clin Biochem 1990. J Gerontol A Biol Sci Med Sci. there was an age-dependent increase of carnitine concentrations. Measures of bioavailable serum testosterone and estradiol and their relationships with muscle strength.71:143– 6. To accurately predict debrisoquine phenotype from .edu) The CYP2D6 gene. Belanger A. Wang ZM. Duran M. Scholte HR. Campos Y. Mutations of OCTN2. et al. Labrie F. Carter AL.17:211– 6. Jr. 9. 18. In sera from females. Different sensitivity of rabbit heart and skeletal muscle to endotoxin-induced impairments of mitochondrial function. lead to deficient cellular carnitine uptake in primary carnitine deficiency. Sheffield-Moore M. Zierz S. Fitzpatrick J. Clin Chim Acta 1974. 12.32:181– 6. Gallagher D. and blood in normal subjects. e-mail gmurphy@stanford. Opalka JR. CA 94305. 10. or whether. although the definition of a carnitine deficiency is not fulfilled. Rebouche CJ. Quagliariello E. Stanford University School of Medicine. Roelops RJ. Lindstedt S. Effect of carnitine feeding on the levels of heart and skeletal muscle carnitine of elderly mice. Mollica MP. Petrosillo G.2 Alan F. Siliprandi N. skeletal muscle and neutrophil leucocytes. Costell M.1 Nina Pascoe. fax 650-7255714.454:207–9. 15.28:303– 6. 28. Cusan L. 22. 13. Schmidt MJ. Palo Alto. Wu X. 2 Department of Veterans Affairs Sierra-Pacific Mental Illness Research. Biochem Biophys Res Commun 1989. 2001 2153 olism in females is mostly independent of circulating sex hormones. Gadaleta MN. Sartorelli L. Lamberts SW. et al.56: B140 –1. Eur J Biochem 2001. CA 94304. Broquist HP. J Am Coll Nutr 1998. Ann Rev Nutr 1986.26:2229 –32. Anal Biochem 1988. in addition. Iossa S. et al. Wiechelmann K. Ketting D. oral supplementation with l-carnitine might help to slow the physiologic process of sarcopenia. age. Gravenstein S. Keller ET. Am J Physiol Endocrinol Metab 2000. Shug AL. Braun R. Gellerich FN. Paradies G. Simard J.62:327–55. Rani PJ. Chiu KM. References 1. * address correspondence to this author at: Neuroscience Research Laboratories. Borstein B. Soboll S. Liverini G. Claassen. Van der Klei-Van Moorsel JM. DHEA and the intracrine formation of androgens and estrogens in peripheral target tissues: its role during aging. Baumgartner RN. Ciman M. is involved in the metabolism of a large number of medications (1 ). 4. muscle tissue. et al. Steroids 1998. Grisolia S.28:297–301. Luu-The V. No. Heymsfield SB. Therefore. Weight stability masks sarcopenia in elderly men and women. Matsumoto K. van den Beld AW. Binkley N. Wang J. Trumbeckaite S.161:1135– 43. 14. Biochim Biophys Acta 1997.28:211– 6. Age-and sex-related differences of serum carnitine in a Japanese population.58:477– 84. FEBS Lett 1999.279:E366 –75. Murphy. Carnitine measurements in liver. Correlation of serum L-carnitine and dehydro-epiandrosterone sulphate levels with age and sex in healthy adults. Visser M. Cederblad G. and body composition in elderly men. J Neurol Sci 1983. Therefore. 2. O’Connor JE. Ruts E. Relationships between muscle carnitine. 1344:201–9. an organic cation/carnitine transporter. 20. in contrast to men. Shug AL. Skeletal muscle mass and distribution in 468 men and women aged 18 – 88 years. 23. Protective efficacy of L-carnitine on acetylcholinesterase activity in aged rat brain. Daynes RA. Loof NE. Barth PG. Dorand L. this study provides evidence that there is a sex-dependent decrease in carnitine concentrations in skeletal muscle. This might contribute to the observed age-dependent decrease in muscle carnitine in males. Harper P. 11. In summary. Hui J. 16.8: 655– 60. These reports describe a more pronounced loss of skeletal muscle and muscle strength in men compared with women.53:311–21. It is not clear whether the observed effects are only an indicator of age-dependent changes.717:233– 43. diLisa F. Ross R. 24.1. 28 ) on sarcopenia in men and women Ͼ50 years in age. The effect of aging and acetyl-L-carnitine on the function and on the lipid composition of rat heart mitochondria. Gomez-Reino JJ. Paulson DJ. Acetyl-L-carnitine treatment stimulates oxygen consumption and biosynthetic function in perfused liver of young and old rats. The *2 allele is the most common allele encoding intermediate debrisoquine hydroxylase activity in Caucasians (3 ). Rapid Detection of the C؊1496G Polymorphism in the CYP2D6 *2 Allele. J Clin Chem Clin Biochem 1990.17:71– 4. affecting only men.315:43– 6. Gravenstein S. Berden JA. Androgens and the control of skeletal muscle protein synthesis. Wadstro ¨ m C. Costill DL. Labrie C. Tang NL. Hum Mol Genet 1999. Ruggiero FM. Yuen PM. Age Ageing 1999. Takiyama N. The effect of aging and acetyl-L-carnitine on the pyruvate transport and oxidation in rat heart mitochondria. Eur J Appl Physiol Occup Physiol 1995. This would be in line with reports (27. 8. Stanford. Cederblad G. A decrease in muscular carnitine could be associated with a loss of muscle strength. Heshka S. J Appl Physiol 2000.34:279 – 86.1 and Greer M. encoding debrisoquine hydroxylase. Jeremy D. Seth P. 21. J Clin Endocrinol Metab 2000. J Steroid Biochem 1982. J Rheumatol 1999. Recently. Ann Med 2000.Clinical Chemistry 47. Secondary carnitine deficiency. 26. Cell Mol Life Sci 2001. Department of Psychiatry and Behavioral Sciences. Stanford University School of Medicine.85:3276 – 82. but to date.1. 7. Havighurst TC. Clin Chem 1993. Muscle dysfunction in elderly individuals with hip fracture. Men Ͼ60 years showed significantly lower free and total carnitine in skeletal muscle than did younger controls. Borum PR. Grisolia S. Starling RD.63:322– 8. and Clinical Center. CA 94305-5485. Neuhof C. Education.28:359 – 63.2* (1 Department of Psychiatry and Behavioral Sciences. Germany. Martin MA. they are a cause of functional impairments accompanying aging. Fink WJ. MSLS P-104. Carnitine metabolism and function in humans. This study was supported by the Medical Faculty of the University Halle (Saale).89:81– 8. (2 ) described a CϪ1496G polymorphism in the promoter region of the CYP2D6 *2 allele that has a strong effect on debrisoquine metabolic phenotype when present in combination with a null allele. Costell M. Janssen I. 17. 5. et al. Sex steroid regulation of urinary excretion of carnitine in rats. Panneerselvam C. 12. de Jong FH. Chiu KM. 3.268:1–9. substantial variability among *2 carriers in metabolic activity has been reported. Gonzalez-Crespo MR. Investigation of the bicinchoninic acid protein assay: identification of the groups responsible for color formation. 6. Grobbee DE. 27.39:592–9. Age-dependent decrease of carnitine content in muscle of mice and humans. Luyt-Houwen IE. Ganapathy V. Menabo ´ R. Gadaleta MN. Ann N Y Acad Sci 1994.6:41– 66. Stanford. Pols HA. but mean values were higher in men.175:231–7. Petrosillo G. Ruggiero FM. Concentration of carnitine in human muscle tissue. Muscle carnitine levels in neuromuscular disease. bone density. The role of carnitine in intracellular metabolism. Paradies G. Bremer J. Stratman FW.

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