ClinicalScience (1919), 56,313-315
SHORT COMMUNICATION
Acyl-coenzyme A-cholesterol acyltransferase activity in human liver
S. B A L A S U B R A M A N I A M , K . A. M I T R O P O U L O S , N . B. M Y A N T , M. M A N C I N I A N D A. P O S T I G L I O N E Medical Research Council Lipid Metabolism Unit, Hammersmith Hospital, London, and Faculty of Medicine and Surgery, University of Naples, Naples, Italy
(Received 7 August 1978; accepted 5 December 1978)
Summary
fatty acids, plays an important role in the regulation of cholesterol metabolism in rat liver and is probably responsible for the synthesis of the cholesteryl esters secreted by the liver in very-lowdensity lipoproteins (Goodman & Shiratori, 1964; Balasubramaniam, Venkatesan, Mitropoulos & Peters, 1978b). However, attempts to demonstrate cholesterol acyltransferase activity in human liver have been unsuccessful (Stokke, 1972, 1974). In this communication we demonstrate the presence of the enzyme in human liver and we compare the activity and properties of the human enzyme with those of the rat liver enzyme.
1. In the presence of CoA and ATP, human liver microsomes catalyse the incorporation of [ 14C]oleate or [ 14Clcholesterol into cholesteryl oleate, thus demonstrating the presence of acylcoenzyme A-cholesterol acyltransferase (cholesterol acyltransferase) in human liver. 2. The enzyme has properties similar to those of rat liver enzyme and with both the concentration of endogenous cholesterol in the microsomal fraction is adequate to support a constant initial rate of esterification. However, unlike the rat liver enzyme, the human cholesterol acyltransferase does not efficiently utilize added cholesterol as substrate. 3. The activity of cholesterol acyltransferase in human liver was 25% of that measured in rat liver under similar conditions of assay. Key words: acyl-coenzyme A-cholesterol transferase; cholesteryl esters; liver.
Methods
Samples of liver (500 and 1500 mg) were obtained during abdominal operations from two male subjects aged 40 and 59 years, and by needle biopsy (20 and 40 mg) from a 43 year old male and a 40 year old female. The operations were carried out in Naples for diagnostic exploration of the abdomen (gall-stones in one case and carcinoma of the colon in the other) and pieces of normal liver were removed with the consent of the patients. The needle biopsies were taken for routine diagnosis of suspected liver disease with the consent of the patients and were found to be histologically normal. The liver samples were either used immediately or kept frozen at -6OOC and thawed just before they were used for the enzyme assay. The tissue was homogenized in sucrose (0-25
acyl-
Abbreviation: CoA, coenzyme A. Introduction
Acyl-CoA-cholesterol acyltransferase (cholesterol acyltransferase; EC 2.3.1.26), an enzyme catalysing the esterification of cholesterol with long-chain Correspondence: Dr S. Balasubramaniam, Medical Research Council Lipid Metabolism Unit, H’mmersmith Hospital, London W12 OHS. 27
373
S. Balasubramaniam et al.
374
mol/l)/EDTA (1.5 mmol/l) (pH 7.4) and the microsomal fraction was prepared as described by Mitropoulos & Balasubramaniam (1972). The activity of cholesterol acyltransferase was assayed from the rate of incorporation of [l-14C]oleic acid (97 000 d.p.m./nmol) into cholesteryl oleate by the microsomal fraction incubated in the presence of coenzyme A (CoA) and ATP, as described by Balasubramaniam, Mitropoulos & Venkatesan (1978a). T o measure the extent of incorporation of added cholesterol into cholesteryl oleate, [414Clcholesterol, as a solution in acetone or as a suspension in Tween-80, was added to the standard incubation mixture containing [9,10(n)-3H]oleic acid instead of [ l-14C]oleate(Balasubramaniam et al., 1978a). The mass of cholesteryl oleate formed during the incubations was calculated from the specific radioactivity of the added oleic acid and the amount of radioactivity incorporated from oleate into cholesteryl ester.
Results The microsomal fraction obtained from human liver incorporated [ 14Cloleic acid into cholesteryl
PH
oleate at a rate that was linear with time for the first 8 min and with concentration of microsomal protein up to 250 pg (Fig. la and Ib), indicating that the concentration of endogenous nonesterified cholesterol is adequate to support a constant initial rate of esterification. The apparent K , value of the human enzyme for oleate, measured in two microsomal preparations, was 35 (Fig. Id) and 60 pmol/l; these values are similar to those obtained for the rat liver enzyme (Balasubramaniam et al., 1978a). Human liver cholesterol acyltransferase exhibited maximal activity at pH 7.4 (Fig. Ic), similar to the pH optimum of the rat liver enzyme. Under optimum conditions of assay and at the concentration of endogenous cholesterol in the microsomes, the activity of cholesterol acyltransferase was 26.5 SD 9.8 pmol min-' mg-* of microsomal protein in the livers of the four human subjects, whereas the activity of the rat liver enzyme measured under the same conditions was 101 f 15 pmol min-' mg-l of microsomal protein (four observations). No activity could be detected in the human or rat liver preparations in the absence of ATP and CoA.
IOleatel (pmol/l)
FIG. 1. Assay of cholesterol acyltransferase activity in human liver. Samples of microsomal fractions (containing 250 pg of microsomal protein) and I4Cloleate (0.1 mmol/l) were incubated at 37°C in the standard incubation mixture in a final volume of 0.2 ml. In all except (a) the incubation time was 8 min. In (b) the amount of microsomal protein per flask was varied; in (c) the pH was varied; in ( d ) the amount of [l4C1oleateper flask was varied. All results are expressed as the amount of cholesteryl [ ''Cloleate
formed per flask.
Human hepatic cholesterol-ester fying enzyme When non-radioactive cholesterol (20 pg in acetone) was added to incubation mixtures containing rat liver microsomes, the incorporation of [ 14C]oleate into cholesteryl esters was significantly increased, but no significant increase occurred with incubation mixtures containing human liver microsomes. However, the addition of tracer amounts of [4-14Clcholesterol resulted in the incorporation of radioactivity into the cholesteryl oleate by the human preparations, but the extent of incorporation was much lower than that by the rat liver microsomal preparations. Thus the specific radioactivity of cholesteryl [14C]oleate formed during the incubation of human liver microsomes was 4600 d.p.m./nmol, whereas that obtained during the incubation of rat liver microsomes was 35 000 d.p.m./nmol. Discussion The present results demonstrate activity of cholesterol acyltransferase in human liver. The enzyme has properties similar to those of the rat liver enzyme in that both are isolated with the microsomal fraction of a liver homogenate and both have adequate non-esterilied cholesterol in their environment to support a constant initial rate of esterification. Moreover, the kinetic properties and the pH optimum of the enzyme of human liver are similar to those of the enzyme of rat liver. However, unlike the rat liver enzyme, human liver cholesterol acyltransferase does not utilize added cholesterol efficiently. This may be due to a higher concentration of non-esterified cholesterol in the environment of the human enzyme, or to a difference in the composition in the lipid environment of the human enzyme that results in reduced accessibility to exogenous cholesterol. In the present work, cholesterol-esterifying activity could only be detected in the presence of ATP and CoA. This shows that the enzyme activity demonstrated in human liver was due to cholesterol acyltransferase, rather than to an esterase acting in reverse or phosphatidylcholinecholesterol acyltransferase, since cholesterol acyltransferase is the only cholesterol-esterifying enzyme that uses activated fatty acids. Since human liver is known to contain fatty acyl-CoA synthetase with high capacity (Stokke, 1972) it is likely that, as in rat liver (Balasubramaniam et a[., 1978a), the activity of this enzyme in human liver is not rate-limiting for the esterification of cholesterol. If so, it would follow that the activity of the cholesterol acyltransferase in human liver mic-
3 75
rosomes is about 25% of that in rat liver microsomes. Previous failure to detect the enzyme in human liver (Stokke, 1972, 1974) may have been due to a combination of three unfavourable factors, namely: the use of labelled cholesterol as substrate for the assay, incubation in a medium containing the total supernatant from a 6000 g centrifugation of the liver homogenate and a long incubation period. As shown in the present work, the enzyme in human liver microsomes is not readily accessible to exogenous cholesterol, and any cholesterol [ I4C]oleate formed initially would have been exposed to the action of esterases in the relatively crude system used by Stokke (1972). Although it is generally agreed that the bulk of the cholesteryl esters in the plasma of fasting human subjects is formed by phosphatidylcholine-cholesterol acyltransferase, the plasma of fasting patients with familial deficiency of this enzyme does contain small amounts of esterilied cholesterol with oleate as the predominant fatty acid (Norum & Gjone, 1967). The formation of cholesteryl esters in the liver by cholesterol acyltransferase, and their possible secretion into the circulation in very-lowdensity lipoproteins, may provide a partial explanation for this. Acknowledgment We thank Dr V. S. Chadwick (Department of Medicine, Royal Postgraduate Medical School) for the two needle biopsies. References BALASUBRAMANIAM, s., MITROPOULOS, K.A. & VENKATESAN. S. ( I 978a) Rat liver acyl-CoA :cholesterol acyltransferase. European Journal of Biochemistry, 90,377-383. BALASUBRAMANIAM, S., VENKATESAN, S.,MITROPOULOS. K.A. & PETERS,T.J. (1978b) The submicrosomal localisation of acyl-coenzyme A: cholesterol acyltransferase and its substrate, and of cholesteryl esters in rat liver. Biochemical Journal, 114,863-872. GOODMAN, DE W.S. & SHIRATORI, T. (1964) In UiVO turnover of different cholesterol esters in rat liver and plasma. Journal of Lipid Research, 5,578-586. MITROPOULOS, K.A. & BALASUBRAMANIAM, s. (1972) Cholesterol 7d-hydroxylase in rat liver microsomal preparations. Biochemical Journal, 128, 1-9. NORUM, K.R. & GJONE, E. (1967) Familial plasma lecithin :cholesterol acyltransferase deficiency. Biochemical study of a new inborn error of metabolism. Scandinavian Journal of Clinical and Laborarory Investigarion, 20, 23 I243. STOKKE,K.T. (1972) The existence of an acid cholesterol esterase in human liver. Biochimica Biophysica A d a , 270, 156-1 66. STOKKE,K.T. (1974) Cholesteryl ester metabolism in liver and blood plasma of various animal species. Atherosclerosis. 19, 393-406.
SHORT COMMUNICATION
Acyl-coenzyme A-cholesterol acyltransferase activity in human liver
S. B A L A S U B R A M A N I A M , K . A. M I T R O P O U L O S , N . B. M Y A N T , M. M A N C I N I A N D A. P O S T I G L I O N E Medical Research Council Lipid Metabolism Unit, Hammersmith Hospital, London, and Faculty of Medicine and Surgery, University of Naples, Naples, Italy
(Received 7 August 1978; accepted 5 December 1978)
Summary
fatty acids, plays an important role in the regulation of cholesterol metabolism in rat liver and is probably responsible for the synthesis of the cholesteryl esters secreted by the liver in very-lowdensity lipoproteins (Goodman & Shiratori, 1964; Balasubramaniam, Venkatesan, Mitropoulos & Peters, 1978b). However, attempts to demonstrate cholesterol acyltransferase activity in human liver have been unsuccessful (Stokke, 1972, 1974). In this communication we demonstrate the presence of the enzyme in human liver and we compare the activity and properties of the human enzyme with those of the rat liver enzyme.
1. In the presence of CoA and ATP, human liver microsomes catalyse the incorporation of [ 14C]oleate or [ 14Clcholesterol into cholesteryl oleate, thus demonstrating the presence of acylcoenzyme A-cholesterol acyltransferase (cholesterol acyltransferase) in human liver. 2. The enzyme has properties similar to those of rat liver enzyme and with both the concentration of endogenous cholesterol in the microsomal fraction is adequate to support a constant initial rate of esterification. However, unlike the rat liver enzyme, the human cholesterol acyltransferase does not efficiently utilize added cholesterol as substrate. 3. The activity of cholesterol acyltransferase in human liver was 25% of that measured in rat liver under similar conditions of assay. Key words: acyl-coenzyme A-cholesterol transferase; cholesteryl esters; liver.
Methods
Samples of liver (500 and 1500 mg) were obtained during abdominal operations from two male subjects aged 40 and 59 years, and by needle biopsy (20 and 40 mg) from a 43 year old male and a 40 year old female. The operations were carried out in Naples for diagnostic exploration of the abdomen (gall-stones in one case and carcinoma of the colon in the other) and pieces of normal liver were removed with the consent of the patients. The needle biopsies were taken for routine diagnosis of suspected liver disease with the consent of the patients and were found to be histologically normal. The liver samples were either used immediately or kept frozen at -6OOC and thawed just before they were used for the enzyme assay. The tissue was homogenized in sucrose (0-25
acyl-
Abbreviation: CoA, coenzyme A. Introduction
Acyl-CoA-cholesterol acyltransferase (cholesterol acyltransferase; EC 2.3.1.26), an enzyme catalysing the esterification of cholesterol with long-chain Correspondence: Dr S. Balasubramaniam, Medical Research Council Lipid Metabolism Unit, H’mmersmith Hospital, London W12 OHS. 27
373
S. Balasubramaniam et al.
374
mol/l)/EDTA (1.5 mmol/l) (pH 7.4) and the microsomal fraction was prepared as described by Mitropoulos & Balasubramaniam (1972). The activity of cholesterol acyltransferase was assayed from the rate of incorporation of [l-14C]oleic acid (97 000 d.p.m./nmol) into cholesteryl oleate by the microsomal fraction incubated in the presence of coenzyme A (CoA) and ATP, as described by Balasubramaniam, Mitropoulos & Venkatesan (1978a). T o measure the extent of incorporation of added cholesterol into cholesteryl oleate, [414Clcholesterol, as a solution in acetone or as a suspension in Tween-80, was added to the standard incubation mixture containing [9,10(n)-3H]oleic acid instead of [ l-14C]oleate(Balasubramaniam et al., 1978a). The mass of cholesteryl oleate formed during the incubations was calculated from the specific radioactivity of the added oleic acid and the amount of radioactivity incorporated from oleate into cholesteryl ester.
Results The microsomal fraction obtained from human liver incorporated [ 14Cloleic acid into cholesteryl
PH
oleate at a rate that was linear with time for the first 8 min and with concentration of microsomal protein up to 250 pg (Fig. la and Ib), indicating that the concentration of endogenous nonesterified cholesterol is adequate to support a constant initial rate of esterification. The apparent K , value of the human enzyme for oleate, measured in two microsomal preparations, was 35 (Fig. Id) and 60 pmol/l; these values are similar to those obtained for the rat liver enzyme (Balasubramaniam et al., 1978a). Human liver cholesterol acyltransferase exhibited maximal activity at pH 7.4 (Fig. Ic), similar to the pH optimum of the rat liver enzyme. Under optimum conditions of assay and at the concentration of endogenous cholesterol in the microsomes, the activity of cholesterol acyltransferase was 26.5 SD 9.8 pmol min-' mg-* of microsomal protein in the livers of the four human subjects, whereas the activity of the rat liver enzyme measured under the same conditions was 101 f 15 pmol min-' mg-l of microsomal protein (four observations). No activity could be detected in the human or rat liver preparations in the absence of ATP and CoA.
IOleatel (pmol/l)
FIG. 1. Assay of cholesterol acyltransferase activity in human liver. Samples of microsomal fractions (containing 250 pg of microsomal protein) and I4Cloleate (0.1 mmol/l) were incubated at 37°C in the standard incubation mixture in a final volume of 0.2 ml. In all except (a) the incubation time was 8 min. In (b) the amount of microsomal protein per flask was varied; in (c) the pH was varied; in ( d ) the amount of [l4C1oleateper flask was varied. All results are expressed as the amount of cholesteryl [ ''Cloleate
formed per flask.
Human hepatic cholesterol-ester fying enzyme When non-radioactive cholesterol (20 pg in acetone) was added to incubation mixtures containing rat liver microsomes, the incorporation of [ 14C]oleate into cholesteryl esters was significantly increased, but no significant increase occurred with incubation mixtures containing human liver microsomes. However, the addition of tracer amounts of [4-14Clcholesterol resulted in the incorporation of radioactivity into the cholesteryl oleate by the human preparations, but the extent of incorporation was much lower than that by the rat liver microsomal preparations. Thus the specific radioactivity of cholesteryl [14C]oleate formed during the incubation of human liver microsomes was 4600 d.p.m./nmol, whereas that obtained during the incubation of rat liver microsomes was 35 000 d.p.m./nmol. Discussion The present results demonstrate activity of cholesterol acyltransferase in human liver. The enzyme has properties similar to those of the rat liver enzyme in that both are isolated with the microsomal fraction of a liver homogenate and both have adequate non-esterilied cholesterol in their environment to support a constant initial rate of esterification. Moreover, the kinetic properties and the pH optimum of the enzyme of human liver are similar to those of the enzyme of rat liver. However, unlike the rat liver enzyme, human liver cholesterol acyltransferase does not utilize added cholesterol efficiently. This may be due to a higher concentration of non-esterified cholesterol in the environment of the human enzyme, or to a difference in the composition in the lipid environment of the human enzyme that results in reduced accessibility to exogenous cholesterol. In the present work, cholesterol-esterifying activity could only be detected in the presence of ATP and CoA. This shows that the enzyme activity demonstrated in human liver was due to cholesterol acyltransferase, rather than to an esterase acting in reverse or phosphatidylcholinecholesterol acyltransferase, since cholesterol acyltransferase is the only cholesterol-esterifying enzyme that uses activated fatty acids. Since human liver is known to contain fatty acyl-CoA synthetase with high capacity (Stokke, 1972) it is likely that, as in rat liver (Balasubramaniam et a[., 1978a), the activity of this enzyme in human liver is not rate-limiting for the esterification of cholesterol. If so, it would follow that the activity of the cholesterol acyltransferase in human liver mic-
3 75
rosomes is about 25% of that in rat liver microsomes. Previous failure to detect the enzyme in human liver (Stokke, 1972, 1974) may have been due to a combination of three unfavourable factors, namely: the use of labelled cholesterol as substrate for the assay, incubation in a medium containing the total supernatant from a 6000 g centrifugation of the liver homogenate and a long incubation period. As shown in the present work, the enzyme in human liver microsomes is not readily accessible to exogenous cholesterol, and any cholesterol [ I4C]oleate formed initially would have been exposed to the action of esterases in the relatively crude system used by Stokke (1972). Although it is generally agreed that the bulk of the cholesteryl esters in the plasma of fasting human subjects is formed by phosphatidylcholine-cholesterol acyltransferase, the plasma of fasting patients with familial deficiency of this enzyme does contain small amounts of esterilied cholesterol with oleate as the predominant fatty acid (Norum & Gjone, 1967). The formation of cholesteryl esters in the liver by cholesterol acyltransferase, and their possible secretion into the circulation in very-lowdensity lipoproteins, may provide a partial explanation for this. Acknowledgment We thank Dr V. S. Chadwick (Department of Medicine, Royal Postgraduate Medical School) for the two needle biopsies. References BALASUBRAMANIAM, s., MITROPOULOS, K.A. & VENKATESAN. S. ( I 978a) Rat liver acyl-CoA :cholesterol acyltransferase. European Journal of Biochemistry, 90,377-383. BALASUBRAMANIAM, S., VENKATESAN, S.,MITROPOULOS. K.A. & PETERS,T.J. (1978b) The submicrosomal localisation of acyl-coenzyme A: cholesterol acyltransferase and its substrate, and of cholesteryl esters in rat liver. Biochemical Journal, 114,863-872. GOODMAN, DE W.S. & SHIRATORI, T. (1964) In UiVO turnover of different cholesterol esters in rat liver and plasma. Journal of Lipid Research, 5,578-586. MITROPOULOS, K.A. & BALASUBRAMANIAM, s. (1972) Cholesterol 7d-hydroxylase in rat liver microsomal preparations. Biochemical Journal, 128, 1-9. NORUM, K.R. & GJONE, E. (1967) Familial plasma lecithin :cholesterol acyltransferase deficiency. Biochemical study of a new inborn error of metabolism. Scandinavian Journal of Clinical and Laborarory Investigarion, 20, 23 I243. STOKKE,K.T. (1972) The existence of an acid cholesterol esterase in human liver. Biochimica Biophysica A d a , 270, 156-1 66. STOKKE,K.T. (1974) Cholesteryl ester metabolism in liver and blood plasma of various animal species. Atherosclerosis. 19, 393-406.
