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Evaluation of a nonisotopic technique for studies of in vivo cholesterol metabolism in mini-pigs using inhibition of 7-dehydrocholesterol reductase by AY 9944

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Research in Experimental Medicine

Summary

The feasability of a nonisotopic method for assessing the approximate rates of sterol fluxes and its regulations was studied within the plasma pool of intact animals. Appropriate daily intravenous administration of AY 9944 leads to a constant, incomplete inhibition in the enzymatic conversion from 7-dehydrocholesterol. Thereby a unique intermediate in cholesterol synthesis such as 7-dehydrocholesterol can be accumulated in the plasma. The established intermediate pool of 7-dehydrocholesterol in the plasma represents, under certain conditions, an indicator pool for the cholesterol pool in the animal. Our data show that the metabolic behaviour of the accumulated 7-dehydrocholesterol during the administration of AY 9944 is not different from cholesterol and that 7-dehydrocholesterol does not induce a feed-back inhibition in overall sterol synthesis. The rate of appearance of the endogenous sterol tracer 7-dehydrocholesterol and its removal from the plasma pool create a constant plasma 7-dehydrocholesterol level during chronic AY 9944 administration and under steady state conditions during the experimental period. The values determined for the artificially built-up intermediate pool of 7-dehydrocholesterol in the plasma are a relative measure for the complex process of the cholesterol metabolism in the animal. Manipulations in cholesterol metabolism with its resulting alterations in the “input” or “output” of sterols in the rapid exchangeable plasma pool lead to changes in the intermediated (tracer) 7-dehydrocholesterol plasma pool.

A great advantage of 7-dehydrocholesterol as an endogenous tracer is its absence from the diet. The absorption of this sterol from the intestine can be neglected. The continous measurement of 7-dehydrocholesterol concentrations in plasma during chronic AY 9944 administration, and therefore constant inhibition of 7-dehydrocholesterol reductase, allows the detection of influences on the cholesterol metabolism and the resulting adjustments in the level of the tracer pool. The method allows one to observe changes in the rate of in vivo cholesterol synthesis and in the elimination of cholesterol from the plasma pool under physiological and experimental conditions in the same animal during an extented period of observation. This method applied to the mini-pig as experimental animal might provide useful informations on influences and mechanisms in the metabolism of cholesterol in the intact animal.

Zusammenfassung

Die Untersuchungen befassen sich mit einer Methode, die den Sterolumsatz und die Regulation des Cholesterolpools aus dem Plasma-Cholesterolpool lebender, intakter Versuchstiere abschätzen läßt. Tägliche intravenöse Gabe von AY 9944 in geeigneter Dosierung führt zu einer konstanten, inkompletten Hemmung der enzymatischen Umwandlung von 7-Dehydrocholesterol zu Cholesterol. Im Plasma des Tieres erscheint 7-Dehydrocholesterol als einzige, quantitativ erfaßbare Zwischenstufe des Cholesterol-Syntheseweges. Der dabei entstehende „Zwischenpool“ aus 7-Dehydrocholesterol ist unter bestimmten Bedingungen ein Indikator für die Höhe und die Regulation des Cholesterolpools im Versuchstier. Unsere Untersuchungen zeigen, daß sich 7-Dehydrocholesterol im Stoffwechsel wie Cholesterol verhält und keine Feedback-Hemmung der Gesamt-Sterolsynthese hervorruft. Bei täglicher Verabreichung von AY 9944 entsteht unter steady state-Bedingungen durch das Ein- und Ausschleusen des endogenen Tracers 7-Dehydrocholesterol ein konstanter 7-Dehydrocholesterolspiegel im Plasmapool während der Versuchsdauer. Eingriffe in den Cholesterolstoffwechsel und die damit verbundenen Änderungen im „input“ oder „output“ der Sterole im rasch austauschbaren Plasmapool führen zu Veränderungen im „Zwischenpool“ von Plasma-7-Dehydrocholesterol. Der 7-Dehydrocholesterolpool im Plasma gibt Hinweise auf Höhe und Änderungen im Cholesterolpool des Tieres.

Ein großer Vorteil des endogenen Tracers 7-Dehydrocholesterol liegt darin, daß bei den Untersuchungen die Darmresorption der Sterole und damit ihr Einfluß auf die Höhe des Sterolpools vernachlässigt werden kann, da 7-Dehydrocholesterol nicht in der Nahrung vorhanden ist und dementsprechend nicht unmittelbar in den „Tracer-pool“ eingehen kann. Fortlaufende Messungen des Plasmaspiegels von 7-Dehydrocholesterol während täglicher intravenöser Gabe von AY 9944, und der daraus resultierenden konstanten Hemmung der 7-Dehydrocholesterol-Reduktase, zeigen eventuelle Einflüsse auf den Cholesterolstoffwechsel des Tieres indirekt durch entsprechende Änderungen im Plasmaspiegel des „Tracerpools“ an. Mit dieser Methode ist es möglich, Änderungen in der Geschwindigkeit der in vivo-Cholesterolsynthese und in der Elimination von Cholesterol aus dem Plasmapool unter physiologischen und experimentellen Bedingungen am gleichen Tier während einer langen Versuchsperiode zu beobachten und Informationen über den Mechanismus und über Einflüsse auf den Cholesterolstoffwechsel der Versuchstiere zu erhalten.

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References

  1. Alexande, C., Day, C. E.: Distribution of serum lipoproteins of selected vertebrates. Comp. Biochem. Physiol.46B, 295–312 (1973)

    Google Scholar 

  2. Back, P., Hamprecht, B., Lynen, F.: Regulation of cholesterol biosynthesis in rat liver: diurnal changes of activity and influence of bile acids. Arch. Biochem.133, 11 (1969)

    PubMed  Google Scholar 

  3. Bar-on, A., Kook, I., Stein, O., Stein, Y.: Assembly and secretion of very low density lipoproteins by rat liver following inhibition of protein synthesis with cycloheximide. Biochim. Biophys. Acta306, 106–114 (1973)

    PubMed  Google Scholar 

  4. Brechenmacher, D. A.: Untersuchungen zur in vivo Cholesterinsynthese beim Miniaturschwein. Inauguraldissertation (1975) München

  5. Bricker, L. A., Siperstein, M. D.: Demonstration of cholesterol feedback deletion in intact hepatoma-bearing rats. J. Clin. Invest.48, IIa (1969)

  6. Bricker, L. A., Weis, H. J., Siperstein, M. D.: In vivo demonstration of the cholesterol feedback system by means of a demosterol supression technique. J. Clin. Invest.51, 197–205 (1972)

    PubMed  Google Scholar 

  7. Brown, M. S., Goldstein, J. L.: Supression of 3-hydroxy-3-methylglutaryl Coenzym A reductase activity and inhibition of growth of human fibroblasts by 7-ketocholesterol. J. Biol. Chem.249, 7306–7314 (1974)

    PubMed  Google Scholar 

  8. Brown, M. S., Dana, S. E., Goldstein, J. L.: Regulation of 3-hydroxy-3-methylglutaryl CoA reductase in cultured human fibroblasts. Comparison of cells from a normal subject and from a patient with homozygous familial hypercholesterolaemia. J. Biol. Chem.249, 789–796 (1974)

    PubMed  Google Scholar 

  9. Dickson, L. G., Patterson, G. W.: Inhibition of sterol biosynthesis in Chlorella-ellipsoidea by AY 9944. Lipids7, 635–643 (1972)

    Google Scholar 

  10. Dickson, L. G., Patterson, G. W.: AY 9944 inhibition of sterol biosynthesis in Chlorellaemersonii. Lipids8, 443–445 (1973)

    PubMed  Google Scholar 

  11. Dickson, L. G., Patterson, G. W., Cohen, C. F., Dutky, S. R.: Novel sterols from inhibited Chlorella-ellipsoidea. Phytochemistry11, 3473–3477 (1972)

    Google Scholar 

  12. Dowling, R. H., Mack, E., Picott, J., Berger, J., Small, D. M.: Experimental model for the study of the enterohepatic circulation of bile in Rhesus monkeys. J. Lab. Clin. Med.72, 169–176 (1968)

    PubMed  Google Scholar 

  13. Dvornik, D., Kraml, M., Bagli, J. F.: A cholesterol biosynthesis inhibitor (AY 9944) as tool in studies of sterol metabolism. Progr. Biochem. Pharmacol.2, 46 (1967)

    Google Scholar 

  14. Dvornik, D., Givner, M. L., Rochefort, J. G.: Agents affecting lipid metabolism. XXIV AY 9944, an inhibitor of cholesterol biosynthesis, as tool in the estimation of the rate of cholesterolgenesis. Circulation32, Suppl. II, 10 (1965)

    Google Scholar 

  15. Dvornik, D., Givner, M. L., Rochefort, J. G.: Tool for the estimation of cholesterol biosynthesis. Proc. Can. Fed. Biol. Soc.10, 154N. 432 (1967)

    Google Scholar 

  16. Dvornik, D., Hill, P.: Effect of long-term administration of AY 9944, an inhibitor of 7-dehydrocholesterol Δ7-reductase, on serum and tissue lipids in the rat. J. Lipid. Res.9, 587–595 (1968)

    PubMed  Google Scholar 

  17. Dvornik, D., Kraml, M., Bagli, J. F.: Agents affecting lipid metabolism. X. Endogenous formation of Δ5,7,24-cholestatrien-3β-ol. J. Am. Chem. Soc.86, 2739–2741 (1964)

    Google Scholar 

  18. Dvornik, D., Kraml, M., Bagli, J. F.: Agents affecting lipid metabolism. XVIII. A 7-dehydrocholesterol Δ7-reductase inhibitor (AY 9944) as tool in studies of Δ7-sterol metabolism. Biochemistry5, 1060–1064 (1966)

    PubMed  Google Scholar 

  19. Dvornik, D., Kraml, M., Dubuc, J., Givner, M., Gaudry, R.: Agents affecting lipid metabolism. IV. A novel mode of inhibition of cholesterol biosynthesis. J. Am. Chem. Soc.85, 3309 (1963)

    Google Scholar 

  20. Edwards, P. A., Gould, R. G.: Turnover rate of hepatic 3-hydroxy-3-methylglutaryl Coenzym A reductase as determined by use of cycloheximide. J. Biol. Chem.247, 1520–1524 (1972)

    PubMed  Google Scholar 

  21. Fidge, N.: The isolation and properties of pig plasma lipoproteins and partial characterization of their apoproteins. Biochim. Biophys. Acta295, 258–273 (1973)

    PubMed  Google Scholar 

  22. Forentin, R. A., Nam, S. C.: Dietary induced atherosclerosis in miniature swine. Exp. Mol. Pathol.8, 263–301 (1968)

    PubMed  Google Scholar 

  23. Gibbons, G. F., Mitropoulous, K. A.: Effect of trans-1,4 bis (2-chlorobenzylaminomethyl) cyclohexane dihydrochloride and carbon monoxide on hepatic cholesterol biosynthesis from 4,4-dimethylsterols in vitro. Biochim. Biophys. Acta380, 270–281 (1975)

    PubMed  Google Scholar 

  24. Givner, M. L., Rochefort, J. G., Dvornik, D. M.: Tool for the estimation of the rate of cholesterol biosynthesis. Circulation34, Suppl. III, 12 (1966)

    Google Scholar 

  25. Goodman, D. S., Noble, R. P.: Turnover of plasma cholesterol in man. J. Clin. Invest.47, 231–241 (1968)

    PubMed  Google Scholar 

  26. Goodman, D. S., Noble, R. P., Dell, R. B.: Three — pool model of the long-term turnover of plasma cholesterol in man. J. Lipid Res.14, 178–188 (1973)

    PubMed  Google Scholar 

  27. Grundy, S. M., Ahrens Jr., E. H.: Measurements of cholesterol turnover, synthesis and absorption in man, carried out by isotopic kinetic and sterol balance methods. J. Lipid Res.10, 91–107 (1969)

    PubMed  Google Scholar 

  28. Grundy, S. M., Ahrens Jr., E. H.: The effect of unsaturated dietary fats on absorption, excretion, synthesis and distribution of cholesterol in man. J. Clin. Invest.49, 1135–1152 (1970)

    PubMed  Google Scholar 

  29. Hamprecht, B., Nüssler, C., Lynen, F.: Rhythmic changes of hydroxymethylglutaryl Coenzyme A reductase activity in livers of fed and fasted rats. FEBS Letters4, 117–121 (1969)

    PubMed  Google Scholar 

  30. Havel, R. J., Eder, H. A., Bragdon, J. H.: The distribution and chemical composition of ultracentrifugally seperated lipoproteins in human serum. J. Clin. Invest.34, 1345–1353 (1955)

    PubMed  Google Scholar 

  31. Horlick, L.: Effect of a new inhibitor of cholesterol biosynthesis (AY 9944) on serum and tissue sterols in the rat. J. Lipid Res.7, 116–121 (1966)

    PubMed  Google Scholar 

  32. Horton, B. J., Horton, J. D., Sabine, J. R.: Repeated estimation of liver cholesterol synthesis in vivo, using the inhibitor AY-9944. Biochim. Biophys. Acta239, 375–481 (1971)

    Google Scholar 

  33. Jackson, R. L., Baker, H. N., Taunton, O. D., Smith, L. C., Garner, C. W., Gotto, A. M. Jr.: A comparison of the major apolipoproteins from pig and human high density lipoproteins. J. Biol. Chem.248, 2639–2644 (1973)

    PubMed  Google Scholar 

  34. Jackson, R. L., Taunton, O. D., Segura, R., Gallagher, J. G., Hoff, H. F., Gotto, A. M. Jr.: Comparative studies on plasma low density lipoproteins from pig and man. Comp. Biochem. Physiol.53B, 245–253 (1976)

    Google Scholar 

  35. Janado, M., Martin, W. G., Cook, W. H.: Separation and properties of pig-serum lipoproteins. Can. J. Biochem.44, 1291–1209 (1966)

    Google Scholar 

  36. Kaiser, W., Stocker, K., Erhardt, W.: Long-term measurements of biliary cholesterol and bile acid secretion in the unanaesthetized mini-pig by controlled interruption of the enterohepatic circulation. Res. Exp. Med.135, 55–66 (1978)

    Google Scholar 

  37. Kandutsch, A. A., Saucier, S. E.: Prevention of cyclic and Triton-induced increases in hydroxymethyl-glutaryl coenzyme A reductase and sterol synthesis by puromycin. J. Biol. Chem.244, 2299–2305 (1969)

    PubMed  Google Scholar 

  38. Kim, E., Goldberg, M.: Serum cholesterol assay using a stable Liebermann-Burchard reagent. Clin. Chem.15, 1171 (1969)

    PubMed  Google Scholar 

  39. Knipping, G. M. J., Kostner, G. M., Holasek, A.: Studies on the composition of pig serum lipoproteins. Isolation and characterization of different apoproteins. Biochim. Biophys. Acta393, 88–99 (1975)

    PubMed  Google Scholar 

  40. Kraml, M., Bagli, J. F., Dvornik, D.: Agents affecting lipid metabolism. IX. Inhibition of the conversion of 7-dehydrocholesterol to cholesterol by AY-9944. Biochem. Biophys. Res. Commun.15, 455–457 (1964)

    PubMed  Google Scholar 

  41. London, I. M., Schwarz, H.: Erythrocyte metabolism. The metabolic bahavior of the cholesterol of human erythrocytes. J. Clin. Invest.32, 1248–1252 (1953)

    PubMed  Google Scholar 

  42. Lutsky, B. N., Hsiung, H. M., Schroepfer, G. J. Jr.: Inhibition of enzymatic reduction of delta-14 double bond of 5 alpha-cholesta-8,14-dien-3-beta-ol by AY 9944. Lipids10, 9 (1975)

    PubMed  Google Scholar 

  43. Marsh, A., Kim, D. N., Lee, K. T., Reiner, J. M., Thomas, W. A.: Cholesterol turnover, synthesis and retention in hypercholesterolemic growing swine. L. Lipid Res.13, 600–615 (1973)

    Google Scholar 

  44. Mercer, E. I., Glover, J.: Sterol metabolism. 6. The interconversion of cholesterol, 7-dehydrocholesterol and lathosterol in the rat. Biochem. J.80, 552–556 (1961)

    PubMed  Google Scholar 

  45. Miettinen, T. A.: Detection of changes in human cholesterol metabolism. Ann. Clin. Res.2, 300–320 (1970)

    PubMed  Google Scholar 

  46. Miettinen, T. A.: Cholesterol production in obesity. Circulation44, 842–859 (1971)

    PubMed  Google Scholar 

  47. Mitoma, Ch., Yasuda, D., Tagg, J. S., Neubauer, S. E., Calderoni, F. J., Tanabe, M.: Effects of various chemical agents on drug metabolism and cholesterol biosynthesis. Biochem. Pharmacol.17, 1377–1383 (1968)

    PubMed  Google Scholar 

  48. Moore, P. R., Baumann, C. A.: Skin sterols. Colorimetric determination of cholesterol and other sterols in skin. J. Biol. Chem.195, 615 (1952)

    PubMed  Google Scholar 

  49. Nestel, P. J., Whyte, H. M., Goodman, D. S.: Distribution and turnover of cholesterol in humans. J. Clin. Invest.48, 982–991 (1969)

    PubMed  Google Scholar 

  50. Niemiro, R., Fumagalli, R.: Studies on the inhibitory mechanism of some hypocholesterolemic agents on 7-dehydrocholesterol Δ7-bond reductase activity. Biochim. Biophys. Acta98, 624–631 (1965)

    PubMed  Google Scholar 

  51. Nilsson, A., Zilversmit, D. B.: Fate of intravenously administrated particulate and lipoprotein cholesterol in the rat. J. Lipid Res.13, 32–38 (1972)

    PubMed  Google Scholar 

  52. Perl, W., Samuel, P.: Input-output analysis for total input rate total tracer mass of body cholesterol. Circ. Res.25, 191–199 (1969)

    PubMed  Google Scholar 

  53. Quintao, E., Grundy, S. M., Ahrens, E. H. Jr.: Effects of dietary cholesterol on the regulation of total body cholesterol in man. L. Lipid Res.12, 233–147 (1971)

    Google Scholar 

  54. Ratcliffe, H. L., Luginbühl, L.: Domestic pig-model for experimental atherosclerosis. Atherosclerosis13, 133–136 (1971)

    PubMed  Google Scholar 

  55. Samuel, P., Perl, W.: Long-term decay of serum cholesterol radioactivity: body cholesterol metabolism in normals and patients with hyperlipoproteinaemia and atherosclerosis. J. Clin. Invest.49, 346–357 (1970)

    PubMed  Google Scholar 

  56. Samuel, P., Holtzman, M., Meilman, E., Perl, W.: Effect of neomycin on exchangeable pools of cholesterol in the steady state. J. Clin. Invest.47, 1806–1818 (1968)

    PubMed  Google Scholar 

  57. Samuel, P., Perl, W., Holtzman, C. M., Rochman, N. D., Lieberman, S.: Long-term kinetics of serum and xanthoma cholesterol radioactivity in patients with hypercholesterolemia. J. Clin. Invest.51, 266–278 (1972)

    PubMed  Google Scholar 

  58. Scallen, T. J., Dean, W. J., Loughran, E. D., Vora, B. V.: Isolation and chemical characterization of delta 7,24-cholestatrien-3 beta-ol from pig tissues. J. Lipid Res.10, 121–127 (1969)

    PubMed  Google Scholar 

  59. Shapiro, D. J., Rodwell, V. M.: Diurnal variation and cholesterol regulation of hepatic HMG-CoA reductase activity. Biochem. Biophys. Res. Commun.37, 867–872 (1969)

    PubMed  Google Scholar 

  60. Shapiro, D. J., Rodwell, V. W.: Regulation of hepatic 3-hydroxy-3-methylglutaryl Coenzym A reductase and cholesterol synthesis. J. Biol. Chem.246, 3210–3216 (1971)

    PubMed  Google Scholar 

  61. Röschlau, P., Bernt, E., Gruber, W.: Enzymatische Bestimmung des Gesamtcholesterins im Serum. Z. klin. Chem. Klin. Biochem.12, 403–407 (1974)

    PubMed  Google Scholar 

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  1. Medizinische Poliklinik, Universität München, Pettenkoferstraße 8a, D-8000, München 2, Federal Republic of Germany

    W. Kaiser & K. Stocker

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  1. W. Kaiser
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This study was supported by the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg

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Kaiser, W., Stocker, K. Evaluation of a nonisotopic technique for studies of in vivo cholesterol metabolism in mini-pigs using inhibition of 7-dehydrocholesterol reductase by AY 9944. Res. Exp. Med. 174, 79–108 (1978). https://doi.org/10.1007/BF01851941

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