Variation in Tissue Carnitine Concentrations with Age and Sex in the Rat

Transcription

1 Bichem. J. (1978) 176, Printed in Great Britain 677 Variatin in Tissue Carnitine Cncentratins with Age and Sex in the Rat By PEGGY R. BORUM Divisin fnutritin, Department fbichemistry, Vanderbilt University Schl fmedicine, Nashville, TN 37232, U.S.A. (Received 24 April 1978) Diabetes, starvatin and varius hrmnal treatments are nwn t alter drastically camitine cncentratins in the bdy. Befre the mechanisms cntrlling carnitine metablism culd be determined, it was necessary t establish nrmal carnitine cncentratins in bth sexes at different ages. Carnitine was assayed in plasma, liver, heart and seletal muscle f rats frm birth t weaning. The plasma carnitine increased rapidly during the first 2 days after birth. Carnitine in bth heart and seletal muscle increased, whereas liver cncentratins declined during the first wee f life. A carnitine-free diet cntaining sufficient precursrs fr carnitine bisynthesis was fed t weanling rats. Grups f ten male and ten female rats were illed each wee fr 1 cnsecutive wees. Carnitine was determined in plasma, liver, heart, seletal muscle, urine and epididymis in the male. There was n difference in carnitine cncentratins between the sexes at weaning. Plasma, heart and muscle cncentratins were higher in adult male rats than in adult females. Hwever, liver carnitine and urinary carnitine cncentratins were higher in adult female than in adult male rats. The epididymal carnitine cncentratin increased very rapidly during 5 t 7 days f age and the differences in carnitine cncentratins between the sexes als became apparent during this time. Thus bth the age and the sex f the human subject r experimental animal must be cnsidered when investigating carnitine metablism. [(3-carbxy-2-hydrxyprpyl)trimethyl- Carnitine ammnium hydrxide] is required fr the transprt f lng-chain fatty acids frm the cytsl t the site f f-xidatin in the mitchndrial matrix (Wlf, 1965). Thus, in a tissue that depends n fat as an imprtant surce f fuel, the cncentratin f carnitine wuld be expected t play an imprtant rle in the metablism f that tissue. The results f Cederblad et al. (1976) indicate a relatinship between the cncentratin f carnitine and the capacity fr lngchain fatty acid xidatin in human seletal muscle (Cederblad et al., 1976). The heart and epididymis f newbrn rats are nwn t cntain much less carnitine than the same rgans f the adult (Wittels & Bressler, 1965; Marquis & Fritz, 1965). Infusin f anti-insulin serum r glucagn fr 3h, starvatin fr 24h, and the inductin f severe diabetic etacidsis with allxan, all result in prnunced elevatins in hepatic carnitine cncentratins in the rat (McGarry et al., 1975). Elevated cncentratins f hepatic carnitine may indeed be required fr the initiatin f etgenesis (McGarry & Fster, 1977). Preliminary experiments suggested there were significant differences in tissue carnitine cncentratins due t age r sex. Hwever, the factrs cntrlling these cncentratins are nt well understd, and in many experiments n carnitine metablism, transprt r Vl. 176 functin, the age, the sex and the diet f the experimental animals have nt been stringently cntrlled. The purpse f this study was t investigate the rle f age and sex in determining the cncentratins f carnitine in plasma, liver, cardiac muscle, seletal muscle and epididymis in nrmal rats. A brief accunt f these investigatins has appeared (Brum & Brquist, 1977a). Experimental Animal care and diet A grup f pregnant Sprague-Dawley rats frm Harlan Industries (Bx 29176, Indianaplis, IN 46229, U.S.A.) were fed n labratry chw during pregnancy and lactatin. The pups were illed at varius times frm befre birth t 24 days f age. A number f samples were btained during the first 48 h after birth, and these time pints were carefully determined. Fr the experiments 1 male and 1 female 22- day-ld Sprague-Dawley rats were purchased frm Harlan Industries and maintained in suspended cages with screen bttms. These rats were fed ad libitum with a defined diet that cntained n detectable carnitine, but sufficient precursrs fr carnitine bisynthesis (Brum & Brquist, 1977b). Bdy weights determined twice each wee indicated that

2 678F P. R. BORUM these animals maintained a nrmal grwth rate n this diet. Ten female rats and ten male rats were illed n the day when the animals were received and each wee thereafter fr 1 wees. Thus the nutritinal state f the animals in the first time pint f the fllwing Figures is nt as well cntrlled as in the remaining time pints. Urine was cllected by using suspended metablic cages. Assay prcedures Tissue carnitine cncentratins were measured by a mdificatin f the methd f Cederblad & Lindstedt (1972) as described by Brum et al. (1977). Plasma prtein cncentratin was determined by the micr biuret methd (Itzhai & Gill, 1964). Nncllagen prtein was slubilized by the methd f Lilienthal et al. (195), and the cncentratin assayed by the micr biuret methd. Urinary creatinine was measured by a mdificatin f the Flin and Wu prcedure as described by Technicn Bulletin N-llb (1969). r.g e 7 5 (a) Statistical analysis The t-test prgram in the 22 General Library Statistics/Engineering f Wang Labratries Inc., Tewsbury, MA 1874, U.S.A., was used t analyse the data n a 22 series calculatr. Results Carnitine cncentratins in perinatal rats Since there are n differences in carnitine cncentratins between male and female rats befre weaning, s the data fr bth sexes are presented tgether. Data presented in the fllwing Figures are based n nn-cllagen prtein. The same cnclusins are reached when the carnitine cncentratins are expressed per g wet wt. f tissue. As shwn in Fig. 1(a), there are lw carnitine cncentratins in fetal liver, with a high cncentratin at birth, which decreases thrughut the sucling perid. These values agree well with thse f Rbles-Valdes et al. (1976). Carnitine cncentratins are lw in the heart f the fetus at birth, but increase during the sucling perid. The reprted large increase in heart carnitine cncentratins between 5 and 15 days f age (Rbles- Valdes et al., 1976) was nt bserved in these studies. The mst rapid increase in heart carnitine appeared t ccur during the first 4 days after birth (see Fig. lb). The increase in carnitine cncentratins after 3- t. 4 9 (b) 7- I r. cdi F O Fig. 1. Carnitine cncentratins in the liver and heart f preweaned rats The assay methds and animal treatments are described in the Experimental sectin. Pints fr animals up t 6 days f age are the means fr triplicate measurements f a pled sample frm animals. (a) Liver carnitine cncentratins. The S.E.M. are: 9 days,.3 (n=5); 1 days,.3 (n=7); 13 days,.2 (n=); 14 days,.2 (n=4); 16 days,.2 (n=4); 19 days,.1 (n=6); 21 days,.3 (n=4); 24 days,.1 (n=6). (b) Heart carnitine cncentratins. The S.E.M. are: 9 days,.3; 1 days,.3; 13 days,.3; 14 days,.3; 16 days,.5; 19 days,.7; 21 days,.2; 24 days,.7. O:.Q t. = cȯ c) s: ^ O O - 4 r _.- E a- 21 c (a) a 1 (b) F Fig. 2. Carnitine cncentratins in the muscle andplasma f preweaned rats The animals are described in the legend t Fig. 1. (a) Muscle carnitine cncentratins. The S.E.M. are: 9 days,.2; 1 days,.3; 13 days,.3; 14 days,.5; 16 days,.2; 19 days,.5; 21 days, 2.; 24 days,.5. (b) Plasma carnitine cncentratins. The S.E.M. are: 9 days, 3.; 1 days, 3.; 13 days, 1.8; 14 days, 1.1; 16 days, 1.1; 19 days, 2.; 21 days, 1.5; 24 days,

3 VARIATION IN TISSUE CARNITINE CONCENTRATIONS birth bserved in cardiac-muscle and seletal-muscle tissue are very similar (Fig. 2a). Plasma carnitine cncentratins are presented in Fig. 2(b). There is a very rapid increase in plasma carnitine cncentratins during the first 48 h after birth. Plasma carnitine cncentratins As bserved in Fig. 3, the plasma carnitine cncentratins d nt change appreciably in female rats frm 22 t 85 days f age. Hwever, they increase dramatically in male rats, such that the adult male rat has apprximately twice that fund in the plasma f the female rat. Liver carnitine cncentratins The adult male rat des nt have a higher cncentratin f carnitine in the liver than the female, as was bserved in the plasma. Rather, carnitine cncentratins appear t be smewhat higher in female liver (see Fig. 4). Cd Ca- Cd._ C) a.) la u 7 6 [ 5 3 [ 2 [ _ en uj z X QX X II Fig. 3. Carnitine cncentratins in theplasma ffemale and male rats frm age 22 days t 85 days The assay methds and the animal treatments are described in the Experimental sectin. Each pint is the mean fr plasma carnitine determinatins n ten animals, with the S.E.M. indicated by the bars. The P values fr the difference between carnitine cncentratins fr female and male rats were determined as described in the Experimental sectin. Symbls: *, females;, males. N.S., P>.5. Vl a 2.6 = n E 1.8 a 1.6 a * 1.4 O u F F I- I. U) 6666 tn. Ce en, X. Z Z Z Z Q. Q. Q. X Fig. 4. Carnitine cncentratins in the liver ffemale and inale rats frm age 22 days t 85 days Liver carnitine cncentratins were determined in the same animals described in the legend t Fig. 3. Symbls: *, females; Cl, males. N.S., P>.5. Cardiac-muscle and seletal-muscle carnitine cncentratins Cardiac-muscle carnitine values are presented in Fig. 5(a). The carnitine cncentratins in the cardiac muscle f the adult male rat are significantly higher than the carnitine cncentratins f the adult female heart. Fig. 5(b) shws the same trend in seletal muscle. The cncentratin f seletal-muscle carnitine is higher in males than in females after abut 5 days f age. Epididymal carnitine cncentratins Epididymal carnitine cncentratins in the male rats used in this experiment are presented in Table 1. There is a dramatic increase in the epididymal carnitine cncentratins at the same time that significantly higher cncentratins f carnitine are fund in the plasma, heart and muscle f the male rat cmpared with that f the female rat.

4 68 P. R. BORUM Urinary carnitine cncentratins The urinary carnitine values in Table 2 are based n mg f creatinine in the urine. The same trend emerges when the carnitine values are expressed n the basis f a 24h cllectin perid. At the same time pints at which earlier data shwed higher cncentratins f carnitine in mst tissues f the male rat than in the female rat, the female is excreting mre carnitine in her urine than is the male rat. c a L Ln L O 6 C 5 C C) II II II 6 Zz Q. Q. z Z Z X. X. X Q. E Z.5 C:) e 4 a Fig. 5. Carnitine cncentratins in the heart and muscle f female and male rats frm 22 days t 85 days (a) Heart carnitine cncentratins were determined in the same animals described in the legend t Fig. 3. (b) Seletal-muscle carnitine cncentratins. Symbls: *, females; a, males. N.S., P>.5. Discussin Nutritinally the labratry rat must quicly switch frm the predminantly carbhydrate transplacental alimentatin t a high-fat diet f mil. During the sucling perid, the rat btains mst f its energy frm fatty acid xidatin, s the presence f an adequate amunt f carnitine in the newbrn may be f the utmst imprtance (Bailey & Lcwd, 1973). The primary surce f carnitine in nenatal tissues, at least 2-3 days pst partum, is the mther rat, whse liver and mil carnitine cncentratins are very high (Rbles-Valdes et al., 1976). It is nt nwn if the newbrn liver is capable fsynthesizing adequate amunts f carnitine when the newbrn is deprived f dietary carnitine. The cmplete series f experiments with male and female rats frm 22 t 85 days f age has been carried ut twice with identical results. An imprtant aspect f this wr is that since all rats were fed n a diet cntaining n carnitine, but sufficient lysine and methinine fr carnitine bisynthesis, the plasma carnitine cncentratins are nt simply a reflectin f recent ral carnitine intae. The high cncentratin f carnitine fund in heart, muscle, and liver n the day f shipment (Figs. 4, Sa and 5b) may indeed Table 1. Age-dependence f carnitine cncentratins in the epididymis f rats The mean epididymal carnitine cncentratins f male rats at varius ages are presented±s.e.m. fr ten animals. The increase in carnitine cncentratins ver that f the weanling is als given. Carnitine Increase ver Age (nml/mg f weanling cncentratin (days) nn-cllagen prtein) (-fld) ± ± ± ± ± ± Table 2. Age-dependence f urinarv carnitine excretin in female and male rats The mean urinary carnitine cncentratins f male and female rats are presented±s.e.m. fr 16 animals. The P value is fr female urinary carnitine values against male urinary carnitine values. N.S., P>.5. Carnitine (nml/mg f creatinine) Age (days) Females 397± ± ±36 27±23 162± ± ± 12 2± 13 Males 543±54 18±25 92±15 57±13 41± 8 59±19 55±1 61± 9 16±15 165±16 p N.S

5 VARIATION IN TISSUE CARNITINE CONCENTRATIONS 681 reflect the respnse f tissue carnitine cncentratins t starvatin (McGarry et al., 1975) and the nrmal changes brught n by the stress f shipment. Althugh the mechanism resulting in the altered tissue cncentratins after shipment are nt well understd, the data emphasize the need t cntrl stringently bth the diet and the envirnment f animals that are used in tissue carnitine studies. The results f these experiments clearly shw that tissue carnitine cncentratins in the rat are influenced bth by the age and sex f the animal. Present wr indicates that the same situatin may apply t humans. It has been reprted that in humans plasma carnitine cncentratins are lwer in the female than in the male (Cederblad, 1976). We have cnfirmed these results, and have als fund that histlgically nrmal male muscle has 27.4±1.6 (±s.e.m.; n=23) nml f carnitine/mg f nn-cllagen prtein, whereas nrmal female muscle has 2.3 ± 1.5 (± S.E.M.; n= 16) nml f carnitine/mg f nn-cllagen prtein. The difference between carnitine cncentratins in nrmal male and nrmal female muscle is significantly different (P=.5). Thus bth the age and the sex f each subject must be cnsidered, whether an investigatin invlves the effect f sme hrmnal r envirnmental factr n carnitine metablism in experimental animals, r the study f a patient with a disease characterized by abnrmal tissue carnitine cncentratins. The main site f carnitine bisynthesis is the liver, with sme carnitine being synthesized in the testes (Haigler & Brquist, 1974; Cx & Hppel, 1974). Thus the carnitine cncentratin in any ther tissue is the summatin f the transprt f carnitine int and ut f that tissue. The carnitine cncentratin f a tissue wuld therefre be regulated by factrs cntrlling the rate f carnitine bisynthesis and carnitine transprt. Preliminary studies indicate that a prduct f the varies and a prduct f the testes as well as a prduct f the pituitary have a rle in the cntrl f tissue carnitine cncentratins (P. R. Brum, unpublished wr). I than Mrs. Carla M. Yr and Mrs. M. Cllier fr excellent technical assistance and Mrs. Marie Hall fr secretarial help. This wr was supprted by NIH Grant AM-1619 and a grant frm the Muscular Dystrphy Assciatin f America. References Bailey, E. & Lcwd, E. A. (1973) Enzyme 15, Brum, P. R. & Brquist, H. P. (1977a) Fed. Prc. Fed. Am. Sc. Exp. Bil. 36, 29 Brum, P. R. & Brquist, H. P. (1977b) J. Nutr. 17, Brum, P. R., Brquist, H. P. & Relfs, R. 1. (1977) J. Neurl. Sci. 34, Cederblad, G. (1976) Clin. Chim. Acta 67, Cederblad, G. & Lindstedt, S. (1972) Clin. Chim. Acta 37, Cederblad, G., Bylund, A. C., Hlm, J. & Schersten, T. (1976) Scand. J. Clin. Lab. Invest. 36, Cx, R. A. & Hppel, C. L. (1974) Bichem. J. 142, Haigler, H. T. & Brquist, H. P. (1974) Bichem. Biphys. Res. Cmmun. 56, Itzhai, R. F. & Gill, D. M. (1964) Anal. Bichem. 9, Lilienthal, J. L., Jr., Zierler, K. L., Fl, B. P., Bua, R. & Riley, M. J. (195) J. Bil. Chem. 182, Marquis, N. R. & Fritz, I. B. (1965) J. Bil. Chem. 24, McGarry, J. D. & Fster, D. W. (1977) Arch. Intern. Med. 137, McGarry, J. D., Rbles-Valdes, C. & Fster, D. W. (1975) Prc. Natl. Acad. Sci. U.S.A. 72, Rbles-Valdes, C., McGarry, J. D. & Fster, D. W. (1976) J. Bil. Chem. 251, Technicn Bulletin N- llb (1969) Technicn Crpratin, Tarrytwn, NY Wittels, B. & Bressler, R. (1965) J. Clin. Invest. 44, Wlf, G. (ed.) (1965) Recent Research in Carnitine, M.I.T. Press, Cambridge, MA Vl. 176