Clinical Science and Molecular Medicine (1976) 50, 307-310.
The distribution of [14C]cholesterolin muscle and skin of Rhesus monkeys after intravenous injection C. D . M O U T A F I S ' ~ ) A N D N. B. MYANT Medical Research Council Lipid Metabolism Unit, Hammersmith Hospital, London
(Received 6 January 1976) Samuel et al., 1972; Simons 8c Myant, 1974; Reichl, Simons, Myant, Pflug 8c Mills, 1973). Tissuelplasma specific radioactivity ratios greater than unity have also been observed in muscle and adipose tissue of baboons 10-12 weeks after intravenous [14C]cholesterol (Wilson, 1970). These iindings indicate the presence of tissue pools of cholesterol with slower turnover than that of the plasma cholesterol.However, a more detailed analysis of the exchangebetween plasma and tissue cholesterol requires a knowledge of the time-course of the change in specific radioactivity of the cholesterol in tissues after pulse labelling of the plasma. We have therefore measured the specific radioactivity of the cholesterol in biopsies of skeletal muscle and skin of Rhesus monkeys taken at frequent intervals after intravenous injection of [14C]cholesterol. The findings in two of these monkeys have been described briefly elsewhere (Myant, 1971).
1. The specific radioactivity of [ 14C]cholesterolin plasma and in serial biopsies of muscle and skin was measured in Rhesus monkeys for 156 days after a single intravenous injection of [14C]cholesterol. 2. Analysis of the specific radioactivity-time curves in terms of a two-compartment system indicated that all the cholesterol of muscle is exchangeable with the plasma cholesterol and that local synthesis does not contribute significantly to the cholesterol in muscle. 3.:Analysis of the curve for spec& radioactivity of skin cholesterol suggested the presence of a small pool of cholesterol with slow turnover. A contribution to skin cholesterol from local synthesis could not be excluded. Key words :cholesterol, monkey, muscle, skin.
Introduction When radioactive cholesterol is injected intravenously into human subjects, the specific radioactivity of the cholesterol in certain tissues exceeds that in the plasma at long intervals after the injection. This phenomenon has been noted in xanthomatous skin lesions (Moutafis 8c Myant, 1969; Samuel, Perl, Holtzman, Rochman 8c Lieberman, 1972) and in normal skin, skeletal muscle, adipose tissue and peripheral lymph (Moutafis BE Myant, 1971; (I'Present address: Department of Medicine, Hippokration Hospital, Athens. Correspondence: Dr N. B. Myant, MRC Lipid Metabolism Unit, Hammersmith Hospital, Du Cane Road, London W12 OHS. 307
Methods Three male monkeys (5.3-7.3 kg) were used. They were kept in separate cages and were given MRC diet no. 41B (Bruce 8c Parkes, 1946) ad libitum. Each animal was given an intravenous injection of its own plasma labelled with 50 pCi of [4-14c]cholesterol by the method of Whereat & Staple (1960). Blood was obtained on days 2 , 4 , 8 and 14, and then at the intervals shown in Fig. 1. Serial biopsies of muscle and skin (100-250 mg) were taken from the thigh under pentothal sodium anaesthesia. Afder removal of visible fat by dissection, the tissues were saponified in KOH (2 mol/l) in 80% ethanol for 6 h at 90100°C and the sterols were extracted from the hydro-
C. D. Moutafs and N.B. Myant
308
with a half-life of 22 days until day 116. The mean value obtained on day 156 suggested that the slope of the plasma specific radioactivity curve decreased towards the end of the period of observation. The specific radioactivity of skin and muscle cholesterol rose to 81 % and 64% respectively of that of the plasma cholesterol at 8 days. Thereafter the curves for the two tissues diverged. The curve for skin cholesterol followed closely that for plasma cholesterol until day 116. The curve for muscle cholesterol, on the other hand, crossed the curve for plasma cholesterol between day 8 and day 21 z1 and then fell more slowly than the plasma cholesterol curve, so that by day 156 the musclelplasma cholesterol specific radioactivity ratio was 6.8. The curve for muscle cholesterol reached its maximum approximately at its intersection with the curve for plasma cholesterol. The specific radioactivity of 500 muscle cholesterol decreased between days 36 and 116 at a rate corresponding to a half-life of 39 days. As with plasma cholesterol, the values obtained on day 156 suggested a decrease in slope of the loglinear specific radioactivity curve for muscle and 50 I00 I50 skin cholesterol after day 116. Time (days) After a single injection of radioactive cholesterol FIG. 1. Specific radioactivity of cholesterol in plasma (O), into the circulation, radioactivity will appear in the skin ( 0 ) and skeletal muscle (A) in three monkeys after exchangeable cholesterol of the tissues, the specific intravenous injection of [14CJcholesterol (mean+ SD for radioactivity of the total cholesterol in each tissue mean values of more than two results). rising to a maximum and then declining. If the lysate with light petroleum. The extracts were behaviour of the exchangeable cholesterol in the evaporated to dryness and submitted to thin-layer whole body conforms to the two-compartment chromatography with methanol/acetone/chloroform model discussed by Wilson (1970), the maximum (1:5:14, by vol.) as solvent. The cholesterol band was specilic radioactivity of the total cholesterol in any located by stainingWith iodine vapour, scraped from tissue will occur at the intersection of the specific radioactivity curves for plasma and tissue, unless the plate and eluted with chloroform/methanol (2:1, v/v). Cholesterolwas extracted from the plasma (1) a significant fraction of the tissue cholesterol is with chloroform/methanol (2:1, v/v). Known not exchangeable, or (2) a significant fraction of the portions of the extracts were taken for assay of cholesterol in the tissue is synthesized in situ, or cholesterol (Clark, Rubin & Arthur, 1968) and for (3) both conditions (1) and (2) apply (see the radioassay. [4-14C]Cholesterol was obtained from Appendix). Hence, the fact that the specific radioThe Radiochemical Centre, Amersham, Bucks, and activity curves for muscle and plasma cholesterol was purilied by thin-layer chromatography. (Fig. 1) intersect when the curve for muscle reaches its maximum shows that all the cholesterol in the monkey’s skeletal muscle is exchangeable and that Results and Discussion synthesis of cholesterol in situ is negligible. Our serial observations on muscle therefore support Measurements were made on each monkey over a the general conclusion of Wilson (1970), based on period of 156 days. Mean values from the three analysis of the plasma specific radioactivity curve monkeys, plotted on a log-linear scale, are shown in after pulse labelling and a single post-mortem Fig. 1. The specific radioactivity of the plasma cholesterol fell rapidly during the first 3-4 weeks determination of the total mass of exchangeable after the injection and then decreased exponentially cholesterol in baboons, that synthesis of cholestero L
v)
-
Tissue cholesterol in monkeys
in the slowly exchangingcompartment (compartment B) does not contribute significantlyto the exchangeable cholesterol of the whole body. Our conclusion that cholesterol synthesis in muscle is negligible and that all muscle cholesterol is exchangeable is compatible with the conclusion that the bulk of the cholesterol required for the maintenance of cell membranes in skeletal muscle is derived from the plasma, with little or no contribution from local synthesis. The specific radioactivitycurve for skin cholesterol gives little indication of a compartment in which turnover is slow, though the high skinlplasma specific radioactivity ratio at 156 days is consistent with the presence of a small compartment with very slow turnover. A contribution to skin cholesterol from local synthesis cannot be excluded, since there are not enough early values to show precisely when the maximum specific radioactivity of skin cholesterol occurred in relation to the plasma specific radioactivity curve. Interpretation of the curve for skin is also complicated by the likelihood that exchangeable cholesterol is lost from the skin of monkeys by direct transfer to the surface of the body rather than by re-entry into the plasma (A. Magide & N. B. Myant, unpublished observations). Mout&s & Myant (1971)noted a higher specific radioactivity in non-xanthomatous skin cholesterol than in plasma cholesterol 414 days after an intravenous injection of [14C]cholesterol into a human subject with familial hypercholesterolaemia. This does not necessarilyreflecta species difference in the behaviour of skin, since the interval between injection and biopsy was much longer in the human subject than in the monkeys used for the present work. Acknowledgmemts
We are very grateful to Sir Edward Pochin for invaluable discussion and for the derivation of equations (1) and (2) of the Appendix, and to Mrs Rosemary Fordham for technical assistance.
309
cholesterol. Although a two-compartment model must be an oversimplication, its use is justified in practice by the demonstration (Wilson, 1970) that this model predicts the total amount of exchangeable cholesterol determined post nwrtem in b a b n s in which the exchangeable cholesterolis labelled before death. For the two-compartment model it may be shown that, to a first approximation: (1) If a fraction F of the cholesterol in a tissue is exchangeable with labelled cholesterol in the plasma and if there is no synthesis within the tissue,
where Sm... is the maximum specific radioactivity of the tissue cholesterol after a single intravenous injection of radioactive cholesterol and P is the specific radioactivity of the plasma cholesterol at sm..
.
(2) If all the cholesterol in a tissue is exchangeable and if synthesis of cholesterol occurs within the
tissue,
where Ki is the rate constant for synthesis of cholesterol within the tissue (the fraction of the tissue cholesterol replaced in unit time by synthesis in situ) and Kzis the rate constant for transfer of exchangeable cholesterol from the tissue into the plasma. Eqn. (1) shows that if a significant fraction of muscle cholesterol is not exchangeable, S , d P will be less than unity, i.e. the maximum will occur before intersection of the two curves. In the extreme case,the curve for muscle cholesterolwould not cross the plasma curve at any time. Eqn. (2) shows that if significant synthesis of cholesterol occurs in muscle, Sm.x /Pwill, again, be less than unity. If S,,./P = 1, both possibilities are therefore excluded. Ref--
APPENDIX Wilson (1970) has shown that the plasma specific radioactivity-time curve obtained after a single intravenous injection of [14c]ch~lester~linto baboons conforms approximately to a two-compartment model, with exit of cholesterol from the system only via the compartment which includes the plasma
BRUCE,H.M. & PARKIIS, A.S. (1946) Feeding and breeding of laboratory animals. 11. Growth and maintenance of rabbits without fresh green food. Journal of Hygiene. 44, 501-507. CLARK,B.R., RUBIN,R.T.& ARTHUR, R.J. (1968) A new micro method for determination of cholasterol in serum. Analytical Biochemistry, 24,27-33. Movr~prs,C.D.& MYANT,N.B. (1969) The metabolism of cholesterol in two hypercholssterolaemicpatients treated with cholestyramine.Clinical Science, 37,443454.
310
C. D. Moutafs and N. B. Myant
MOUTAFIS, C.D. & MYANT,N.B. (1971) Effects of nicotinic acid, alone or in combination with cholestyramine, on cholesterol metabolism in patients suffering from familial hyperbetalipoproteinaemia in the homozygous form. Metabolic Eflects of Nicotinic Acid and its Derivatives, pp. 659-676. Ed. Gey, K.F. &Carlson,L.A. HansHuber, Bern. MYANT,N.B. (1971) The transport and turnover of the plasma cholesterol. Plasma Lipoproteins, Biochemical Society Symposium no. 33, pp. 99-121. Ed. Smellie, R.M.S. Academic Press, London. REICHL,D., SIMONS,L.A., MYANT, N.B., PFLUG, J.J. & MILLS, G.L.(1973) The lipids and lipoproteins of human peripheral lymph, with observations on the transport of cholesterol from plasma and tissues into lymph. Clinical Science and Molecular Medicine, 45, 3 13-329.
SAMUEL, P., PERL,W., HOLTZMAN, C.M., ROCHMAN, N.D. & LIEBERMAN, S. (1972) Long-term kinetics of serum and xanthorna cholesterol radioactivity in patients with hypercholesterolemia. Journal of Clinical Investigation, 51, 266-278.
SIMONS, L.A. & MYANT,N.B.(1974) The effect of mthyroxine on the metabolism of cholesterol in familial hyperbetalipoproteinaemia. Arherosclerosis, 19, 103-1 17. WHEREAT, A.F.& STAPLE,E. (I 960) The preparation of serum lipoproteins labeled with radioactive cholesterol. Archives of Biochemistry and Biophysics, 90, 224-228. WILSON,J.D. (1970) The measurement of exchangeable pools of cholesterol in the baboon. Journal of Clinical Investigation, 49, 655-665.
The distribution of [14C]cholesterolin muscle and skin of Rhesus monkeys after intravenous injection C. D . M O U T A F I S ' ~ ) A N D N. B. MYANT Medical Research Council Lipid Metabolism Unit, Hammersmith Hospital, London
(Received 6 January 1976) Samuel et al., 1972; Simons 8c Myant, 1974; Reichl, Simons, Myant, Pflug 8c Mills, 1973). Tissuelplasma specific radioactivity ratios greater than unity have also been observed in muscle and adipose tissue of baboons 10-12 weeks after intravenous [14C]cholesterol (Wilson, 1970). These iindings indicate the presence of tissue pools of cholesterol with slower turnover than that of the plasma cholesterol.However, a more detailed analysis of the exchangebetween plasma and tissue cholesterol requires a knowledge of the time-course of the change in specific radioactivity of the cholesterol in tissues after pulse labelling of the plasma. We have therefore measured the specific radioactivity of the cholesterol in biopsies of skeletal muscle and skin of Rhesus monkeys taken at frequent intervals after intravenous injection of [14C]cholesterol. The findings in two of these monkeys have been described briefly elsewhere (Myant, 1971).
1. The specific radioactivity of [ 14C]cholesterolin plasma and in serial biopsies of muscle and skin was measured in Rhesus monkeys for 156 days after a single intravenous injection of [14C]cholesterol. 2. Analysis of the specific radioactivity-time curves in terms of a two-compartment system indicated that all the cholesterol of muscle is exchangeable with the plasma cholesterol and that local synthesis does not contribute significantly to the cholesterol in muscle. 3.:Analysis of the curve for spec& radioactivity of skin cholesterol suggested the presence of a small pool of cholesterol with slow turnover. A contribution to skin cholesterol from local synthesis could not be excluded. Key words :cholesterol, monkey, muscle, skin.
Introduction When radioactive cholesterol is injected intravenously into human subjects, the specific radioactivity of the cholesterol in certain tissues exceeds that in the plasma at long intervals after the injection. This phenomenon has been noted in xanthomatous skin lesions (Moutafis 8c Myant, 1969; Samuel, Perl, Holtzman, Rochman 8c Lieberman, 1972) and in normal skin, skeletal muscle, adipose tissue and peripheral lymph (Moutafis BE Myant, 1971; (I'Present address: Department of Medicine, Hippokration Hospital, Athens. Correspondence: Dr N. B. Myant, MRC Lipid Metabolism Unit, Hammersmith Hospital, Du Cane Road, London W12 OHS. 307
Methods Three male monkeys (5.3-7.3 kg) were used. They were kept in separate cages and were given MRC diet no. 41B (Bruce 8c Parkes, 1946) ad libitum. Each animal was given an intravenous injection of its own plasma labelled with 50 pCi of [4-14c]cholesterol by the method of Whereat & Staple (1960). Blood was obtained on days 2 , 4 , 8 and 14, and then at the intervals shown in Fig. 1. Serial biopsies of muscle and skin (100-250 mg) were taken from the thigh under pentothal sodium anaesthesia. Afder removal of visible fat by dissection, the tissues were saponified in KOH (2 mol/l) in 80% ethanol for 6 h at 90100°C and the sterols were extracted from the hydro-
C. D. Moutafs and N.B. Myant
308
with a half-life of 22 days until day 116. The mean value obtained on day 156 suggested that the slope of the plasma specific radioactivity curve decreased towards the end of the period of observation. The specific radioactivity of skin and muscle cholesterol rose to 81 % and 64% respectively of that of the plasma cholesterol at 8 days. Thereafter the curves for the two tissues diverged. The curve for skin cholesterol followed closely that for plasma cholesterol until day 116. The curve for muscle cholesterol, on the other hand, crossed the curve for plasma cholesterol between day 8 and day 21 z1 and then fell more slowly than the plasma cholesterol curve, so that by day 156 the musclelplasma cholesterol specific radioactivity ratio was 6.8. The curve for muscle cholesterol reached its maximum approximately at its intersection with the curve for plasma cholesterol. The specific radioactivity of 500 muscle cholesterol decreased between days 36 and 116 at a rate corresponding to a half-life of 39 days. As with plasma cholesterol, the values obtained on day 156 suggested a decrease in slope of the loglinear specific radioactivity curve for muscle and 50 I00 I50 skin cholesterol after day 116. Time (days) After a single injection of radioactive cholesterol FIG. 1. Specific radioactivity of cholesterol in plasma (O), into the circulation, radioactivity will appear in the skin ( 0 ) and skeletal muscle (A) in three monkeys after exchangeable cholesterol of the tissues, the specific intravenous injection of [14CJcholesterol (mean+ SD for radioactivity of the total cholesterol in each tissue mean values of more than two results). rising to a maximum and then declining. If the lysate with light petroleum. The extracts were behaviour of the exchangeable cholesterol in the evaporated to dryness and submitted to thin-layer whole body conforms to the two-compartment chromatography with methanol/acetone/chloroform model discussed by Wilson (1970), the maximum (1:5:14, by vol.) as solvent. The cholesterol band was specilic radioactivity of the total cholesterol in any located by stainingWith iodine vapour, scraped from tissue will occur at the intersection of the specific radioactivity curves for plasma and tissue, unless the plate and eluted with chloroform/methanol (2:1, v/v). Cholesterolwas extracted from the plasma (1) a significant fraction of the tissue cholesterol is with chloroform/methanol (2:1, v/v). Known not exchangeable, or (2) a significant fraction of the portions of the extracts were taken for assay of cholesterol in the tissue is synthesized in situ, or cholesterol (Clark, Rubin & Arthur, 1968) and for (3) both conditions (1) and (2) apply (see the radioassay. [4-14C]Cholesterol was obtained from Appendix). Hence, the fact that the specific radioThe Radiochemical Centre, Amersham, Bucks, and activity curves for muscle and plasma cholesterol was purilied by thin-layer chromatography. (Fig. 1) intersect when the curve for muscle reaches its maximum shows that all the cholesterol in the monkey’s skeletal muscle is exchangeable and that Results and Discussion synthesis of cholesterol in situ is negligible. Our serial observations on muscle therefore support Measurements were made on each monkey over a the general conclusion of Wilson (1970), based on period of 156 days. Mean values from the three analysis of the plasma specific radioactivity curve monkeys, plotted on a log-linear scale, are shown in after pulse labelling and a single post-mortem Fig. 1. The specific radioactivity of the plasma cholesterol fell rapidly during the first 3-4 weeks determination of the total mass of exchangeable after the injection and then decreased exponentially cholesterol in baboons, that synthesis of cholestero L
v)
-
Tissue cholesterol in monkeys
in the slowly exchangingcompartment (compartment B) does not contribute significantlyto the exchangeable cholesterol of the whole body. Our conclusion that cholesterol synthesis in muscle is negligible and that all muscle cholesterol is exchangeable is compatible with the conclusion that the bulk of the cholesterol required for the maintenance of cell membranes in skeletal muscle is derived from the plasma, with little or no contribution from local synthesis. The specific radioactivitycurve for skin cholesterol gives little indication of a compartment in which turnover is slow, though the high skinlplasma specific radioactivity ratio at 156 days is consistent with the presence of a small compartment with very slow turnover. A contribution to skin cholesterol from local synthesis cannot be excluded, since there are not enough early values to show precisely when the maximum specific radioactivity of skin cholesterol occurred in relation to the plasma specific radioactivity curve. Interpretation of the curve for skin is also complicated by the likelihood that exchangeable cholesterol is lost from the skin of monkeys by direct transfer to the surface of the body rather than by re-entry into the plasma (A. Magide & N. B. Myant, unpublished observations). Mout&s & Myant (1971)noted a higher specific radioactivity in non-xanthomatous skin cholesterol than in plasma cholesterol 414 days after an intravenous injection of [14C]cholesterol into a human subject with familial hypercholesterolaemia. This does not necessarilyreflecta species difference in the behaviour of skin, since the interval between injection and biopsy was much longer in the human subject than in the monkeys used for the present work. Acknowledgmemts
We are very grateful to Sir Edward Pochin for invaluable discussion and for the derivation of equations (1) and (2) of the Appendix, and to Mrs Rosemary Fordham for technical assistance.
309
cholesterol. Although a two-compartment model must be an oversimplication, its use is justified in practice by the demonstration (Wilson, 1970) that this model predicts the total amount of exchangeable cholesterol determined post nwrtem in b a b n s in which the exchangeable cholesterolis labelled before death. For the two-compartment model it may be shown that, to a first approximation: (1) If a fraction F of the cholesterol in a tissue is exchangeable with labelled cholesterol in the plasma and if there is no synthesis within the tissue,
where Sm... is the maximum specific radioactivity of the tissue cholesterol after a single intravenous injection of radioactive cholesterol and P is the specific radioactivity of the plasma cholesterol at sm..
.
(2) If all the cholesterol in a tissue is exchangeable and if synthesis of cholesterol occurs within the
tissue,
where Ki is the rate constant for synthesis of cholesterol within the tissue (the fraction of the tissue cholesterol replaced in unit time by synthesis in situ) and Kzis the rate constant for transfer of exchangeable cholesterol from the tissue into the plasma. Eqn. (1) shows that if a significant fraction of muscle cholesterol is not exchangeable, S , d P will be less than unity, i.e. the maximum will occur before intersection of the two curves. In the extreme case,the curve for muscle cholesterolwould not cross the plasma curve at any time. Eqn. (2) shows that if significant synthesis of cholesterol occurs in muscle, Sm.x /Pwill, again, be less than unity. If S,,./P = 1, both possibilities are therefore excluded. Ref--
APPENDIX Wilson (1970) has shown that the plasma specific radioactivity-time curve obtained after a single intravenous injection of [14c]ch~lester~linto baboons conforms approximately to a two-compartment model, with exit of cholesterol from the system only via the compartment which includes the plasma
BRUCE,H.M. & PARKIIS, A.S. (1946) Feeding and breeding of laboratory animals. 11. Growth and maintenance of rabbits without fresh green food. Journal of Hygiene. 44, 501-507. CLARK,B.R., RUBIN,R.T.& ARTHUR, R.J. (1968) A new micro method for determination of cholasterol in serum. Analytical Biochemistry, 24,27-33. Movr~prs,C.D.& MYANT,N.B. (1969) The metabolism of cholesterol in two hypercholssterolaemicpatients treated with cholestyramine.Clinical Science, 37,443454.
310
C. D. Moutafs and N. B. Myant
MOUTAFIS, C.D. & MYANT,N.B. (1971) Effects of nicotinic acid, alone or in combination with cholestyramine, on cholesterol metabolism in patients suffering from familial hyperbetalipoproteinaemia in the homozygous form. Metabolic Eflects of Nicotinic Acid and its Derivatives, pp. 659-676. Ed. Gey, K.F. &Carlson,L.A. HansHuber, Bern. MYANT,N.B. (1971) The transport and turnover of the plasma cholesterol. Plasma Lipoproteins, Biochemical Society Symposium no. 33, pp. 99-121. Ed. Smellie, R.M.S. Academic Press, London. REICHL,D., SIMONS,L.A., MYANT, N.B., PFLUG, J.J. & MILLS, G.L.(1973) The lipids and lipoproteins of human peripheral lymph, with observations on the transport of cholesterol from plasma and tissues into lymph. Clinical Science and Molecular Medicine, 45, 3 13-329.
SAMUEL, P., PERL,W., HOLTZMAN, C.M., ROCHMAN, N.D. & LIEBERMAN, S. (1972) Long-term kinetics of serum and xanthorna cholesterol radioactivity in patients with hypercholesterolemia. Journal of Clinical Investigation, 51, 266-278.
SIMONS, L.A. & MYANT,N.B.(1974) The effect of mthyroxine on the metabolism of cholesterol in familial hyperbetalipoproteinaemia. Arherosclerosis, 19, 103-1 17. WHEREAT, A.F.& STAPLE,E. (I 960) The preparation of serum lipoproteins labeled with radioactive cholesterol. Archives of Biochemistry and Biophysics, 90, 224-228. WILSON,J.D. (1970) The measurement of exchangeable pools of cholesterol in the baboon. Journal of Clinical Investigation, 49, 655-665.
