291
Atherosclerosis, 29 (1978) 291-299 @ Elsevier/North-Holland Scientific Publishers,
Ltd.
REGULATION OF CHOLESTEROL SYNTHESIS IN THE HYPERLIPOPROTEINAEMIAS Polymorphonuclear Leucocyte Hypercholesterolaemia
Abnormality
Specific to Familial Type II
W.F. BREMNER, JANE L.H.C. THIRD, B. CLARK, C. CORSTORPHINE and T.D.V. LAWRIE University Department (Great Britain)
of Medical Cardiology, Royal Infirmary,
(Received 12 August, 1977) (Revised received 17 November, (Accepted 12 January, 1978)
Glasgow G4 OSF
1977 and 12 January, 1978)
Summary A simple procedure has been devised to give virtually pure preparations of polymorphonuclear leucocytes. This has permitted study of the regulation of cholesterol biosynthesis at cell level, Freshly isolated cells from donors with various forms of hyperlipoproteinaemia have been shown to have very low levels of cholesterol synthesis, presumably due to high circulating levels of apoprotein-B in donor plasma [l]. The activity of the rate-limiting enzyme for cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A reductase, rapidly increases as the cells are incubated in lipoprotein-deficient medium, until, by 12 h, cells from patients heterozygous for familial type IIa hypercholesterolaemia are clearly distinguished from other hyperlipoproteinaemias. The possible significance of this finding is discussed in relation to the causation and treatment of atherosclerotic disease. Key words:
Cholesterol biosynthesis -Familial hypercholesterolaemia olaemia - Polymorphonuclear leucocyte
- Hypercholester-
Introduction Tissue culture that the activity m
communications
studies of propagated human fibroblasts [2--51 have shown of the rate-limiting enzyme for the cholesterol biosynthetic should be addressed to: Dr. W.F. Bremner. Royal Infirmary, Glasgow. Great Britain.
292
pathway, 3-hydroxy-3-methylglutaryl coenzyme A reductase, is regulated by the concentration of low density lipoprotein (and very low density lipoprotein) in the cultured medium. This regulation has been shown to be exerted by way of receptors, apparently specific for the B-apoprotein moiety. The attachment of the lipoprotein to the receptor leads to internalisation of the lipoprotein, its degradation, release of cholesterol and consequent regulation of several aspects of cellular function such as de novo receptor synthesis, cholesterol biosynthesis and cholesterol esterification [3,6]. Specific receptors are either lacking or severly deficient in individuals who are homozygous for the autosomal dominantly inherited form of familial type IIa hypercholesterolaemia, and are some 50% deficient in the heterozygote. This approach has since been extended to leucocyte [7,8] and lymphocyte preparations [ 91. This paper describes the extension of such studies to polymorphonuclear leucocytes. Regulation of cholesterol biosynthesis has been examined in such cells isolated from subjects with various hyperlipoproteinaemias. Materials and Methods Subjects
The subjects had been referred to our Lipoprotein Clinic for characterisation and treatment of lipoprotein abnormalities [lo] with or without overt atheromatous disease. The groups consisted of 15 normo-lipidaemic controls, 14 subjects with familial hypercholesterolaemia (type IIa), 8 subjects with Type IIb abnormality [ 111, 4 subjects with Type III abnormality (2 were on dietary therapy during the time of study) [ 12],12 patients with a type IV lipoprotein abnormality, 5 subjects with Type V abnormality, and 4 subjects with dietary hypercholesterolaemia. The 14 subjects with familial hypercholesterolaemia (FH) were heterozygous members of 4 families with an inheritance pattern consistent with an autosomal dominant characteristic. Ten of the 14 had tendon xanthomata and a history of premature myocardial ischaemic or peripheral vascular disease or both. Triglyceride levels were normal in this group. Normolipidaemic controls were similar in age to this group. The 8 patients with the IIb abnormality were some 8 years older than the FH group and had cholesterol levels over 8.5 mmol/l and triglyceride levels over 3.8 mmol/l. The 4 patients with the type III abnormality had palmar planar xanthomata, peripheral vascular disease or a family history of the same. The diagnosis was made on electrophoresis and ultracentrifugal quantification of the lipoprotein constituents [ 121. Two of the subjects were on dietary therapy when studied, but the Type III abnormality persisted in their electrophoretic strips. The 12 type IV patients had fasting triglyceride levels consistently over 3.9 mmol/l. None was grossly obese, 3 were considered to have possible familial hypertriglyceridaemia, though family studies were not sufficiently complete on this point to permit firm conclusions. In the majority of these patients alcohol was considered to play a significant role in the aetiology of the abnormality. Five patients with the type V pattern had related diabetes (1 patient), excess
293
alcohol ingestion (3 patients), and in one to be a primary abnormality. Four patients were considered to have This conclusion was based on a dietetic history (with an elevated LDL cholesterol
case the abnormality
was considered
a pure dietary hypercholesterolaemia. history plus the absence of a family above 9 mmol/l).
Preparation of cells About 60 ml whole blood was withdrawn on several occasions from each subject in the fasting untreated state (exceptions noted above). The blood was transferred to Sterilin sterile plastic containers with added 2.5 ml EDTA as anticoagulant. The blood and anticoagulant were mixed and 2 ml of isotonic Dextran T500 in 0.9% saline was added to accelerate clumping of erythrocytes [ 131. The tubes were mixed and allowed to stand for some 45 min, during which time the erythrocytes largely sedimented. Using a sterile siliconised wide bore needle and syringe, the supematant was transferred to 2 X 50 ml sterile, siliconised, centrifuge tubes. With a long steel needle 2 X 5 ml of sterile Ficollhypaque solution (density 1.077 g/ml) were placed under the supernatant with minimal disturbance of the interface. The tubes were spun at 400 X g for 20 min and the supernatant containing a discrete layer of lymphocytes was sucked off the cell button of granulocytes and residual erythrocytes [ 131. The cell button was twice washed with isotonic Krebs solution and then suspended in 5 ml of 0.88% NH&l solution (taken from the refrigerator at 0°C) for 10 min to lyse the erythrocytes [14]. The tubes were spun at 400 X g for 5 min, the supernatants sucked off and the granulocytes washed 4 times in Krebs solution. The cells were then suspended in a suitable volume of culture medium and gassed with 95% 02-5% COz to maintain pH. The culture medium consisted of RPM1 1640 medium (supplied by GibcoBiocult) containing penicillin (100 units/ml), Streptomycin (100 pg/ml), 1% non-essential amino-acid supplement and 10% lipoproteindeficient human serum (density > 1.215 g/ml). The cells were maintained at room temperature. Cell viability and function were assessed by histological appearances using haematoxyhn and eosin, trypan blue exclusion, phagocytosis of Indian ink particles and by the ability to convert labelled glucose to labelled carbon dioxide. H and E staining showed that the cells were nearly all polymorphonuclear leucocytes. At least 90% of cells excluded trypan blue at the end of the 12-h period of study; they also showed phagocytosis of Indian ink particles over the same period and their capacity to convert glucose to carbon dioxide was unchanged. Preparation 0 f lipoproteins Human low density lipoprotein (LDL; density 1.019= 1.063 g/ml) was isolated by sequential flotation in a Beckman ultracentrifuge, using solid potassium bromide for density alterations. The isolated LDL was dialysed with frequent changes against a buffer containing isotonic saline, Tris-HCI (pH 7.4) and 0.3 mmol EDTA. Protein concentration was assayed by the Lowry method [ 151 with bovine serum albumin as standard. LDL was labelled with carrier-free [1251]sodium iodide (Radio Amersham) by the method of MacFarlane (1958) [ 161. Unattached iodide was removed by column chromatography and the iso-
294
lated labelled lipoprotein fraction was filtered using a 15 pm Millipore filter, then stored, diluted with culture medium or unlabelled lipoprotein fraction. It was found necessary to filter the labelled solutions 3 times before use. The lipoproteins tended to clump, which subsequently caused precipitation with centrifuged cells and, thus, distorted non-specific binding values. Assay procedures Granulocytes were incubated in a lipoprotein-deficient medium for the stated times and then centrifuged, the medium was suckked off, the cells were washed 3 times and covered with an appropriate concentration of labelled and unlabelled ‘lipoprotein fraction for 20 min at 4°C. Enzymatic degradation of the low density lipoprotein is minimalised at 4°C and permits measurement of membrane uptake of lipoprotein [ 31. Lipoprotein fractions were filtered 3 times through Millipore filters [15 pm] before use to prevent distortion of results by protein aggregates. The cells were then centrifuged and washed 3 times. The cell pellet was counted in a well-type gamma counter and protein concentration was assayed by the Lowry method after solubilisation in 0.1 N NaOH. Total binding of [ 12’1]LDL is the amount of label bound to the cells in the absence of any unlabelled LDL. High affinity binding is that fraction of the total that is competitively inhibited by the presence of a 20-fold excess of unlabelled LDL and is measured as the difference between the amount of LDL bound in the presence of 15 c(g [12sI]LDL/ml and that bound in the presence of 15 pg [ 12sI]LDL/ml + 300 c(g unlabelled LDL/ml [ 171. Cholesterol biosynthetic activity assay Activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase was measured by a modification of the method used by Goldstein et al. [2]. In our hands, it was necessary to extract the enzyme by sonication of the cells. KyroEOB (a generous gift from Procter and Gamble) was used to solubilise plasma membranes (but not endoplasmic reticulum). Activity was then assayed by incubation for 2 h with labelled substrate, DL-[3-14C]HMG CoA. Labelled product, [3-14C]mevalonate, was extracted together with [ 5-3 Hlmevalonalactone as a measure of extraction losses. Results were expressed as pmole/h per mg cell protein. Sonication was necessary in our hands to give adequate enzyme activity. The cholesterol content of the medium and granulocytes was assayed on cells lysed by freezing and thawing, chloroform-methanol extraction and gas-liquid chromatographic assay. Results The activity of HMG CoA reductase in the normo-lipidaemic and FH groups was compared (Fig. 1). Activity was low in both groups on initial separation but increased with culture time in lipoprotein-free medium. While the results showed a fairly wide scatter the mean values for the two groups diverged significantly with time until, by the end of 12 h, the two groups showed a highly significant difference (P < 0.01). Activities, measured at zero time (initial extraction) and after 12 h in lipo-
295
0
4
8 HOURS
24
12 in CULTURE
Fig. 1. HMG CoA reductase activity in the normolipidaemic and FH cells in lipoprotein-deficient medium. On initial extraction, activity was low in both groups after incubation of 2 b but increased with culture time in lipoprotein-deficient medium. The two groups were significantly different after 12 h culture.
protein-deficient medium in the various hyperlipoproteinaemias, are illustrated in Fig. 2. Patients with type IIb abnormality and patients with type III abnormality did not significantly differ from the normolipidaemic groups. However, they were significantly different from the FH group, thus permitting clear distinction of FH subjects from these particular hyperlipoproteinaemias. The values on initial extraction and after 12 h incubation in the 4 patients with dietary hypercholesterolaemia did not significantly differ from those in the normal group. .f B h
lf 1 P
z >
IQ n-15
200 Activities
at
12 hours
150
F Y z :!
n-5
3 0 z 2
U
t LDL
I loo
Chol
( Dietary) nc4
50
0 z =
0
INITIAL
Activitior
Fig. 2. This illustrates HMG CoA reductase activity in various hyperlipoproteinaemias and after 12 h culture in lipoprotein-deficient medium.
on initial extraction
296
HOURS
in CULTURE
Vertical bars represent 2 standard detitim
Fig. 3. The mean jects. The values nificant after 12 normolipidaemic
high affinity-specific binding values are shown for 15 normolipidaemic and 14 FH subdiffer significantly on initial extraction (P < 0.05). while separation is even more sigh culture in lipoprotein-deficient serum (only 1 FH subject remains within 2 SD of the mean value).
Patients with type IV and type V lipoprotein abnormalities showed enzyme activity levels on initial extraction that were lower than in the other groups. (For the type IV patients P < 0.025 and for the type V patients P < 0.05). After 12 h incubation in lipoprotein-deficient medium the mean values for the type IV and type V groups approached those of the IIb, III and normal groups. The binding data in the 15 normal lipidaemic subjects and the 14 heterozygotes, is illustrated in Fig. 3. The mean high affinity specific binding values are shown. On initial extraction of cells, the binding values for the two groups differed (P < 0.05) but results showed too wide a scatter to permit accurate separation of several heterozygotes from the normal group. After 12 h of culture, however, the differences between the two groups increased and all but one of the 14 FH heterozygotes lie outside two standard deviations of the normal lipidaemic mean value. Discussion This paper describes an extension of previous work which has examined the regulation of cholesterol metabolism in other cells in the FH abnormality [2-g]. Polymorphonuclear leucocytes would seem to offer potential advantages compared with fibroblasts and lymphocytes: they have a defined function and morphology, they may be obtained repetitively from the same patient, thus permitting sequential metabolic studies. In addition they appear to be homogeneous unlike lymphocytes, which comprise at least two cell types (B and T lymphocytes) of different lifespans and functions. Use of leucocytes obviates the prolonged period of culture required in the isolation of fibroblasts. Another aspect of potential usefulness is that polymorphonuclear leucocytes exhibit marked nuclear change with ageing and it may be feasible to isolate leucocytes of varying ages, perhaps by differential centrifugation, and so examine the effects of ageing on cholesterol biosynthesis at cell level. This particular methodological approach is relatively simple, obviaties the
297
need for prolonged subculturing techniques and permits fairly rapid differentiation of the FH abnormality. The results described here are interesting in that they indicate that the FH heterozygotes (type IIa) are clearly distinguishable from other forms of hyperlipoproteinaemias. It seems plausible that the values for cholesterol biosynthetic activity on initial extraction do not differ significantly between the FH group and the normals, because the FH have higher circulating levels of LDL which presumably have inhibited cellular cholesterol synthesis. After 12 h of incubation, however, the FH group significantly differs from all the other groups, and this permits phenotypic differentiation of this group of patients. This should be particularly useful where the family history is inadequate for delineation of the pattern of inheritance or where a lipoprotein pattern consistent with the FH abnormality is found but without the usual clinical stigmata. Of particular interest is the finding that eight type IIb patients did not show the abnormality that appears to be characteristic of FH type IIa. The abnormality that is present in these individuals is presumably a separate entity from the FH disorder. This conclusion has been reached on the basis of genetic and epidemiological studies [11,18] which suggest that there is a discrete entity, “combined hyperlipidaemia”, that usually manifests itself as combined elevations of cholesterol and triglyceride levels but sometimes as elevations of cholesterol or triglyceride alone. In this study we were particularly careful to select patients with unequivocal type IIb patterns, as there is a very high prevalence of hypertriglyceridaemia in this area, if one uses the cut-off criteria [lo]. Indeed, we have several subjects whom we suggested by Fredrickson believe to have the FH abnormality, but who have elevated triglyceride levels by the present cut-off criteria. Moreover, examination of 4 of these subjects, using this approach, indicates that they do have the type IIa abnormality and any triglyceride elevation appears to be unrelated to the abnormal cellular regulation of cholesterol biosynthesis. Our studies suggest that a proportion of subjects with a IIb phenotype form an entity discrete from the FH abnormality. The patients of the dietary group are similar to those of the normolipidaemic group in terms of cellular cholesterol control, indicating that they do not have an endogenous abnormality of cholesterol regulation. This appears to be confirmed by the rapid normalisation of their cholesterol levels after dietary restriction of saturated fat and cholesterol. Four type III patients did not significantly differ from the normal group in terms of cellular cholesterol biosynthetic control. This is consistent with studies that suggest that the primary abnormality here is deranged breakdown of lipoproteins before the production of low density lipoprotein [ 191. The type IV and type V groups are of interest in that their activities on initial extraction appear to be significantly lower than the other groups, including normolipidaemic subjects. Examination of the individual patients in fact suggested that the lowest values were found where there was a definite or strong suspicion of excess alcohol intake as a contributing factor to the hyperlipoproteinaemia. It may be that alcohol in some way reduces cellular cholesterol synthesis, perhaps by delivering some dissolved cholesterol to the cells in
298
the body. In this respect it is of interest that several studies have suggested that high alcohol intake may protect against the complications of atherosclerosis [20,21] though other studies suggest that alcohol per se is a risk factor for arterial disease [ 221. The major drawback to the data presented here is that the levels of LDL in culture medium that exert a significant inhibition of cholesterol biosynthetic activity are much lower than levels normally found in human plasma. Other studies which we have undertaken show that as little as 15 c(g LDL/ml of culture medium will reduce cholesterol biosynthetic activity by as much as half after 12 h of culture in lipoproteindeficient medium. This compares with levels of circulating apoprotein B in human plasma which average 83 f 16 mg/dl in normals and 162 f 52 mg/dl in type II subjects, Type IV subjects having values intermediate between these two figures [ 11. It may be that cells in the FH abnormality synthesise excess cholesterol until circulating LDL levels reach a sufficient value to shut off cellular cholesterol biosynthesis. An alternative explanation, as yet unproven, may be that cholesterol biosynthetic activity in hepatocytes is regulated at much higher LDL levels. The data presented here suggest that it is important to delineate patients with the FH abnormality and to regard their response to therapy as possibly unique to them. As the FH heterozygote is relatively common and occurs in increased numbers in infarction survivors, particularly among younger sufferers [ 181, failure to delineate this particular group may bias results of therapy which one would expect to be beneficial in other forms of hyperlipidaemia, particularly dietary hyperlipidaemia. References 1 Schonfeld. G.. Lees, R.S.. George, P.K. and Pfleger. B., Assay of total plasma apolipoprotein B concentration in human subjects, J. Clin. Invest., 53 (1974) 1468-1467. Coenzyme A 2 Brown, M.S.. Dana, S.E. and Goldstein, J.L.. Regulation of 3-hydroxy-3-methylglutaryl reductase activity in cultured human fibroblasts. J. Biol. Chem.. 249 (1974) 789-796. 3 Brown, M.S. and Goldstein, J.L., Familial hypercholesterolaemia - Defective binding of lipoproteins to cultured fibroblasts associated with impahed regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductasc activity, Proc. Nat. Acad. Sci. (U.S.A.), 71 (1974) 788-792. 4 Goldstein. J.L. and Brown, M.S., Binding and degradation of low density lipoproteins by cultured human fibroblasts, J. Biol. Chem.. 249 (1974) 6153-5162. 6 Goldstein. J.L. and Brown, M.S.. Familial hypercholesterolaemia - A genetic regulatory defect in cholesterol metabolism, Amer. J. Med., 58 (1975) 147-160. 6 Brown, M.S. and Goldstein, J.L., Receptor-mediated control of cholesterol metabolism - Study of human mutants has disclosed how cells regulate a substance that is both vital and lethal, Science, 191 (1976) 160-154. 7 Fogehnan. A.M., Edmond. J., Polito, A. and Popjak. G., Control of lipid metabolism in human leucocytes, J. Biol. Chem., 248 (1973) 6928-6929. a Higgins, MJ.P., Lecamwasam. D.S. and Galton, DJ.. A new type of familial hypercholesterolaemia, Lancet. 2 (1976) 737-740. 9 Kayden. H.J.. Hatam. L. and Beratis. N.G. Regulation of 3-hydroxy-3methylglutaryl coenzyme A reductasc activity and the esterification of cholesterol in human long term lymphoid cell lines, Biochemistry, 16 (1976) 521. 10 Fredrickson, D.S. and Levy, R.I., Familial hyperlipoproteinemia. In: J.B. Stanbury. J.B. Wyngaarden and D.S. Fredrickson (Eds.). The MetaboUc Basis of Inherited Disease. 3rd edition, McGraw-Hill. New York. 1972, PP. 545-614. 11 Goldstein, J.L., Schrott. H.G. and Hazzard. W.R., Hyperlipidaemia in coronary heart disease Genetic analysis of lipid levels in 176 families and delineation of a new inherited disorder, combined hyperlipidaemia, J. CBn. Invest., 62 (1973) 1644-l 568.
299 12
Fredrickson, D.S.. Morganroth. J. and Levy, R.I., Type III hyperhpoproteinemia -An analysis of two contemporary definitions, Ann. Intern. Med., 82 (1975) 150. 13 Boyum, A., Separation of blood leucocytes. granulocytes and lymphocytes, Tissue Antigens, 4 (1974) 269-274. 14 Roes. D. and Loos, J.A. Changes in the carbohydrate metabolism of mitogenically-stimulated human peripheral lymphocytes. Biochem. Biophys. Acta, 222 (1970) 565-582. 15 Lowry, O.H.. Rosebrough. N.J.. Farr. A.L. and Randall. R.J.. Protein measurement with the Fohn phenol reagent, J. Biol. Chem.. 193 (1942) 265-275. 16 McFarlane. A.S., Efficient trace-IabeBing of proteins with iodine, Nature, 182 (1958) 53-54. 17 Scatchard, G., The attraction of proteins for smaB molecules and ions, Ann. N.Y. Acad. Sci.. 51 (1949) 660-672. 18 Slack, J. and Nevin. N.C., Hyperhpidaemic xanthomatosis. Part 1 (Increased risk of death from ischaemic heart disease in first degree relatives of 53 patients with essential hyperlipidaemia and xanthomatosis), J. Med. Genet., 5 (1968) 4-8. 19 Chait. A.. Brunzell. J.D., Albers. J.J. and Hazzard. W.R., Type III hyperhpoproteinaemia (“remnant removal disease”). Lancet. 1 (1977) 1178-1180. 20 Caste& W.P.. Doyle, J.T.. Gordon, T., Hames. C.G., Hjortland, M., HuIIet. S.B., Kagan. A. and Zukel, W.J., Alcohol and blood lipids, Lancet. 11 (1977) 153. 21 Barboriak, J.J., Rimm, A.A.. Anderson, A.J., Schmidhoffer. M. and Tristani. F.E., Coronary artery occlusion and alcohol intake. Brit. H. J., 39 (1977) 289-293. 22 KIatsky, A.L., Friedman, G.D.. SiegeIaub, A.B. and Gerard, M.J., Alcohol consumption and blood pressure, New EngI. J. Med., 296 (1977) 1194-1199.
Atherosclerosis, 29 (1978) 291-299 @ Elsevier/North-Holland Scientific Publishers,
Ltd.
REGULATION OF CHOLESTEROL SYNTHESIS IN THE HYPERLIPOPROTEINAEMIAS Polymorphonuclear Leucocyte Hypercholesterolaemia
Abnormality
Specific to Familial Type II
W.F. BREMNER, JANE L.H.C. THIRD, B. CLARK, C. CORSTORPHINE and T.D.V. LAWRIE University Department (Great Britain)
of Medical Cardiology, Royal Infirmary,
(Received 12 August, 1977) (Revised received 17 November, (Accepted 12 January, 1978)
Glasgow G4 OSF
1977 and 12 January, 1978)
Summary A simple procedure has been devised to give virtually pure preparations of polymorphonuclear leucocytes. This has permitted study of the regulation of cholesterol biosynthesis at cell level, Freshly isolated cells from donors with various forms of hyperlipoproteinaemia have been shown to have very low levels of cholesterol synthesis, presumably due to high circulating levels of apoprotein-B in donor plasma [l]. The activity of the rate-limiting enzyme for cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A reductase, rapidly increases as the cells are incubated in lipoprotein-deficient medium, until, by 12 h, cells from patients heterozygous for familial type IIa hypercholesterolaemia are clearly distinguished from other hyperlipoproteinaemias. The possible significance of this finding is discussed in relation to the causation and treatment of atherosclerotic disease. Key words:
Cholesterol biosynthesis -Familial hypercholesterolaemia olaemia - Polymorphonuclear leucocyte
- Hypercholester-
Introduction Tissue culture that the activity m
communications
studies of propagated human fibroblasts [2--51 have shown of the rate-limiting enzyme for the cholesterol biosynthetic should be addressed to: Dr. W.F. Bremner. Royal Infirmary, Glasgow. Great Britain.
292
pathway, 3-hydroxy-3-methylglutaryl coenzyme A reductase, is regulated by the concentration of low density lipoprotein (and very low density lipoprotein) in the cultured medium. This regulation has been shown to be exerted by way of receptors, apparently specific for the B-apoprotein moiety. The attachment of the lipoprotein to the receptor leads to internalisation of the lipoprotein, its degradation, release of cholesterol and consequent regulation of several aspects of cellular function such as de novo receptor synthesis, cholesterol biosynthesis and cholesterol esterification [3,6]. Specific receptors are either lacking or severly deficient in individuals who are homozygous for the autosomal dominantly inherited form of familial type IIa hypercholesterolaemia, and are some 50% deficient in the heterozygote. This approach has since been extended to leucocyte [7,8] and lymphocyte preparations [ 91. This paper describes the extension of such studies to polymorphonuclear leucocytes. Regulation of cholesterol biosynthesis has been examined in such cells isolated from subjects with various hyperlipoproteinaemias. Materials and Methods Subjects
The subjects had been referred to our Lipoprotein Clinic for characterisation and treatment of lipoprotein abnormalities [lo] with or without overt atheromatous disease. The groups consisted of 15 normo-lipidaemic controls, 14 subjects with familial hypercholesterolaemia (type IIa), 8 subjects with Type IIb abnormality [ 111, 4 subjects with Type III abnormality (2 were on dietary therapy during the time of study) [ 12],12 patients with a type IV lipoprotein abnormality, 5 subjects with Type V abnormality, and 4 subjects with dietary hypercholesterolaemia. The 14 subjects with familial hypercholesterolaemia (FH) were heterozygous members of 4 families with an inheritance pattern consistent with an autosomal dominant characteristic. Ten of the 14 had tendon xanthomata and a history of premature myocardial ischaemic or peripheral vascular disease or both. Triglyceride levels were normal in this group. Normolipidaemic controls were similar in age to this group. The 8 patients with the IIb abnormality were some 8 years older than the FH group and had cholesterol levels over 8.5 mmol/l and triglyceride levels over 3.8 mmol/l. The 4 patients with the type III abnormality had palmar planar xanthomata, peripheral vascular disease or a family history of the same. The diagnosis was made on electrophoresis and ultracentrifugal quantification of the lipoprotein constituents [ 121. Two of the subjects were on dietary therapy when studied, but the Type III abnormality persisted in their electrophoretic strips. The 12 type IV patients had fasting triglyceride levels consistently over 3.9 mmol/l. None was grossly obese, 3 were considered to have possible familial hypertriglyceridaemia, though family studies were not sufficiently complete on this point to permit firm conclusions. In the majority of these patients alcohol was considered to play a significant role in the aetiology of the abnormality. Five patients with the type V pattern had related diabetes (1 patient), excess
293
alcohol ingestion (3 patients), and in one to be a primary abnormality. Four patients were considered to have This conclusion was based on a dietetic history (with an elevated LDL cholesterol
case the abnormality
was considered
a pure dietary hypercholesterolaemia. history plus the absence of a family above 9 mmol/l).
Preparation of cells About 60 ml whole blood was withdrawn on several occasions from each subject in the fasting untreated state (exceptions noted above). The blood was transferred to Sterilin sterile plastic containers with added 2.5 ml EDTA as anticoagulant. The blood and anticoagulant were mixed and 2 ml of isotonic Dextran T500 in 0.9% saline was added to accelerate clumping of erythrocytes [ 131. The tubes were mixed and allowed to stand for some 45 min, during which time the erythrocytes largely sedimented. Using a sterile siliconised wide bore needle and syringe, the supematant was transferred to 2 X 50 ml sterile, siliconised, centrifuge tubes. With a long steel needle 2 X 5 ml of sterile Ficollhypaque solution (density 1.077 g/ml) were placed under the supernatant with minimal disturbance of the interface. The tubes were spun at 400 X g for 20 min and the supernatant containing a discrete layer of lymphocytes was sucked off the cell button of granulocytes and residual erythrocytes [ 131. The cell button was twice washed with isotonic Krebs solution and then suspended in 5 ml of 0.88% NH&l solution (taken from the refrigerator at 0°C) for 10 min to lyse the erythrocytes [14]. The tubes were spun at 400 X g for 5 min, the supernatants sucked off and the granulocytes washed 4 times in Krebs solution. The cells were then suspended in a suitable volume of culture medium and gassed with 95% 02-5% COz to maintain pH. The culture medium consisted of RPM1 1640 medium (supplied by GibcoBiocult) containing penicillin (100 units/ml), Streptomycin (100 pg/ml), 1% non-essential amino-acid supplement and 10% lipoproteindeficient human serum (density > 1.215 g/ml). The cells were maintained at room temperature. Cell viability and function were assessed by histological appearances using haematoxyhn and eosin, trypan blue exclusion, phagocytosis of Indian ink particles and by the ability to convert labelled glucose to labelled carbon dioxide. H and E staining showed that the cells were nearly all polymorphonuclear leucocytes. At least 90% of cells excluded trypan blue at the end of the 12-h period of study; they also showed phagocytosis of Indian ink particles over the same period and their capacity to convert glucose to carbon dioxide was unchanged. Preparation 0 f lipoproteins Human low density lipoprotein (LDL; density 1.019= 1.063 g/ml) was isolated by sequential flotation in a Beckman ultracentrifuge, using solid potassium bromide for density alterations. The isolated LDL was dialysed with frequent changes against a buffer containing isotonic saline, Tris-HCI (pH 7.4) and 0.3 mmol EDTA. Protein concentration was assayed by the Lowry method [ 151 with bovine serum albumin as standard. LDL was labelled with carrier-free [1251]sodium iodide (Radio Amersham) by the method of MacFarlane (1958) [ 161. Unattached iodide was removed by column chromatography and the iso-
294
lated labelled lipoprotein fraction was filtered using a 15 pm Millipore filter, then stored, diluted with culture medium or unlabelled lipoprotein fraction. It was found necessary to filter the labelled solutions 3 times before use. The lipoproteins tended to clump, which subsequently caused precipitation with centrifuged cells and, thus, distorted non-specific binding values. Assay procedures Granulocytes were incubated in a lipoprotein-deficient medium for the stated times and then centrifuged, the medium was suckked off, the cells were washed 3 times and covered with an appropriate concentration of labelled and unlabelled ‘lipoprotein fraction for 20 min at 4°C. Enzymatic degradation of the low density lipoprotein is minimalised at 4°C and permits measurement of membrane uptake of lipoprotein [ 31. Lipoprotein fractions were filtered 3 times through Millipore filters [15 pm] before use to prevent distortion of results by protein aggregates. The cells were then centrifuged and washed 3 times. The cell pellet was counted in a well-type gamma counter and protein concentration was assayed by the Lowry method after solubilisation in 0.1 N NaOH. Total binding of [ 12’1]LDL is the amount of label bound to the cells in the absence of any unlabelled LDL. High affinity binding is that fraction of the total that is competitively inhibited by the presence of a 20-fold excess of unlabelled LDL and is measured as the difference between the amount of LDL bound in the presence of 15 c(g [12sI]LDL/ml and that bound in the presence of 15 pg [ 12sI]LDL/ml + 300 c(g unlabelled LDL/ml [ 171. Cholesterol biosynthetic activity assay Activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase was measured by a modification of the method used by Goldstein et al. [2]. In our hands, it was necessary to extract the enzyme by sonication of the cells. KyroEOB (a generous gift from Procter and Gamble) was used to solubilise plasma membranes (but not endoplasmic reticulum). Activity was then assayed by incubation for 2 h with labelled substrate, DL-[3-14C]HMG CoA. Labelled product, [3-14C]mevalonate, was extracted together with [ 5-3 Hlmevalonalactone as a measure of extraction losses. Results were expressed as pmole/h per mg cell protein. Sonication was necessary in our hands to give adequate enzyme activity. The cholesterol content of the medium and granulocytes was assayed on cells lysed by freezing and thawing, chloroform-methanol extraction and gas-liquid chromatographic assay. Results The activity of HMG CoA reductase in the normo-lipidaemic and FH groups was compared (Fig. 1). Activity was low in both groups on initial separation but increased with culture time in lipoprotein-free medium. While the results showed a fairly wide scatter the mean values for the two groups diverged significantly with time until, by the end of 12 h, the two groups showed a highly significant difference (P < 0.01). Activities, measured at zero time (initial extraction) and after 12 h in lipo-
295
0
4
8 HOURS
24
12 in CULTURE
Fig. 1. HMG CoA reductase activity in the normolipidaemic and FH cells in lipoprotein-deficient medium. On initial extraction, activity was low in both groups after incubation of 2 b but increased with culture time in lipoprotein-deficient medium. The two groups were significantly different after 12 h culture.
protein-deficient medium in the various hyperlipoproteinaemias, are illustrated in Fig. 2. Patients with type IIb abnormality and patients with type III abnormality did not significantly differ from the normolipidaemic groups. However, they were significantly different from the FH group, thus permitting clear distinction of FH subjects from these particular hyperlipoproteinaemias. The values on initial extraction and after 12 h incubation in the 4 patients with dietary hypercholesterolaemia did not significantly differ from those in the normal group. .f B h
lf 1 P
z >
IQ n-15
200 Activities
at
12 hours
150
F Y z :!
n-5
3 0 z 2
U
t LDL
I loo
Chol
( Dietary) nc4
50
0 z =
0
INITIAL
Activitior
Fig. 2. This illustrates HMG CoA reductase activity in various hyperlipoproteinaemias and after 12 h culture in lipoprotein-deficient medium.
on initial extraction
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HOURS
in CULTURE
Vertical bars represent 2 standard detitim
Fig. 3. The mean jects. The values nificant after 12 normolipidaemic
high affinity-specific binding values are shown for 15 normolipidaemic and 14 FH subdiffer significantly on initial extraction (P < 0.05). while separation is even more sigh culture in lipoprotein-deficient serum (only 1 FH subject remains within 2 SD of the mean value).
Patients with type IV and type V lipoprotein abnormalities showed enzyme activity levels on initial extraction that were lower than in the other groups. (For the type IV patients P < 0.025 and for the type V patients P < 0.05). After 12 h incubation in lipoprotein-deficient medium the mean values for the type IV and type V groups approached those of the IIb, III and normal groups. The binding data in the 15 normal lipidaemic subjects and the 14 heterozygotes, is illustrated in Fig. 3. The mean high affinity specific binding values are shown. On initial extraction of cells, the binding values for the two groups differed (P < 0.05) but results showed too wide a scatter to permit accurate separation of several heterozygotes from the normal group. After 12 h of culture, however, the differences between the two groups increased and all but one of the 14 FH heterozygotes lie outside two standard deviations of the normal lipidaemic mean value. Discussion This paper describes an extension of previous work which has examined the regulation of cholesterol metabolism in other cells in the FH abnormality [2-g]. Polymorphonuclear leucocytes would seem to offer potential advantages compared with fibroblasts and lymphocytes: they have a defined function and morphology, they may be obtained repetitively from the same patient, thus permitting sequential metabolic studies. In addition they appear to be homogeneous unlike lymphocytes, which comprise at least two cell types (B and T lymphocytes) of different lifespans and functions. Use of leucocytes obviates the prolonged period of culture required in the isolation of fibroblasts. Another aspect of potential usefulness is that polymorphonuclear leucocytes exhibit marked nuclear change with ageing and it may be feasible to isolate leucocytes of varying ages, perhaps by differential centrifugation, and so examine the effects of ageing on cholesterol biosynthesis at cell level. This particular methodological approach is relatively simple, obviaties the
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need for prolonged subculturing techniques and permits fairly rapid differentiation of the FH abnormality. The results described here are interesting in that they indicate that the FH heterozygotes (type IIa) are clearly distinguishable from other forms of hyperlipoproteinaemias. It seems plausible that the values for cholesterol biosynthetic activity on initial extraction do not differ significantly between the FH group and the normals, because the FH have higher circulating levels of LDL which presumably have inhibited cellular cholesterol synthesis. After 12 h of incubation, however, the FH group significantly differs from all the other groups, and this permits phenotypic differentiation of this group of patients. This should be particularly useful where the family history is inadequate for delineation of the pattern of inheritance or where a lipoprotein pattern consistent with the FH abnormality is found but without the usual clinical stigmata. Of particular interest is the finding that eight type IIb patients did not show the abnormality that appears to be characteristic of FH type IIa. The abnormality that is present in these individuals is presumably a separate entity from the FH disorder. This conclusion has been reached on the basis of genetic and epidemiological studies [11,18] which suggest that there is a discrete entity, “combined hyperlipidaemia”, that usually manifests itself as combined elevations of cholesterol and triglyceride levels but sometimes as elevations of cholesterol or triglyceride alone. In this study we were particularly careful to select patients with unequivocal type IIb patterns, as there is a very high prevalence of hypertriglyceridaemia in this area, if one uses the cut-off criteria [lo]. Indeed, we have several subjects whom we suggested by Fredrickson believe to have the FH abnormality, but who have elevated triglyceride levels by the present cut-off criteria. Moreover, examination of 4 of these subjects, using this approach, indicates that they do have the type IIa abnormality and any triglyceride elevation appears to be unrelated to the abnormal cellular regulation of cholesterol biosynthesis. Our studies suggest that a proportion of subjects with a IIb phenotype form an entity discrete from the FH abnormality. The patients of the dietary group are similar to those of the normolipidaemic group in terms of cellular cholesterol control, indicating that they do not have an endogenous abnormality of cholesterol regulation. This appears to be confirmed by the rapid normalisation of their cholesterol levels after dietary restriction of saturated fat and cholesterol. Four type III patients did not significantly differ from the normal group in terms of cellular cholesterol biosynthetic control. This is consistent with studies that suggest that the primary abnormality here is deranged breakdown of lipoproteins before the production of low density lipoprotein [ 191. The type IV and type V groups are of interest in that their activities on initial extraction appear to be significantly lower than the other groups, including normolipidaemic subjects. Examination of the individual patients in fact suggested that the lowest values were found where there was a definite or strong suspicion of excess alcohol intake as a contributing factor to the hyperlipoproteinaemia. It may be that alcohol in some way reduces cellular cholesterol synthesis, perhaps by delivering some dissolved cholesterol to the cells in
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the body. In this respect it is of interest that several studies have suggested that high alcohol intake may protect against the complications of atherosclerosis [20,21] though other studies suggest that alcohol per se is a risk factor for arterial disease [ 221. The major drawback to the data presented here is that the levels of LDL in culture medium that exert a significant inhibition of cholesterol biosynthetic activity are much lower than levels normally found in human plasma. Other studies which we have undertaken show that as little as 15 c(g LDL/ml of culture medium will reduce cholesterol biosynthetic activity by as much as half after 12 h of culture in lipoproteindeficient medium. This compares with levels of circulating apoprotein B in human plasma which average 83 f 16 mg/dl in normals and 162 f 52 mg/dl in type II subjects, Type IV subjects having values intermediate between these two figures [ 11. It may be that cells in the FH abnormality synthesise excess cholesterol until circulating LDL levels reach a sufficient value to shut off cellular cholesterol biosynthesis. An alternative explanation, as yet unproven, may be that cholesterol biosynthetic activity in hepatocytes is regulated at much higher LDL levels. The data presented here suggest that it is important to delineate patients with the FH abnormality and to regard their response to therapy as possibly unique to them. As the FH heterozygote is relatively common and occurs in increased numbers in infarction survivors, particularly among younger sufferers [ 181, failure to delineate this particular group may bias results of therapy which one would expect to be beneficial in other forms of hyperlipidaemia, particularly dietary hyperlipidaemia. References 1 Schonfeld. G.. Lees, R.S.. George, P.K. and Pfleger. B., Assay of total plasma apolipoprotein B concentration in human subjects, J. Clin. Invest., 53 (1974) 1468-1467. Coenzyme A 2 Brown, M.S.. Dana, S.E. and Goldstein, J.L.. Regulation of 3-hydroxy-3-methylglutaryl reductase activity in cultured human fibroblasts. J. Biol. Chem.. 249 (1974) 789-796. 3 Brown, M.S. and Goldstein, J.L., Familial hypercholesterolaemia - Defective binding of lipoproteins to cultured fibroblasts associated with impahed regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductasc activity, Proc. Nat. Acad. Sci. (U.S.A.), 71 (1974) 788-792. 4 Goldstein. J.L. and Brown, M.S., Binding and degradation of low density lipoproteins by cultured human fibroblasts, J. Biol. Chem.. 249 (1974) 6153-5162. 6 Goldstein. J.L. and Brown, M.S.. Familial hypercholesterolaemia - A genetic regulatory defect in cholesterol metabolism, Amer. J. Med., 58 (1975) 147-160. 6 Brown, M.S. and Goldstein, J.L., Receptor-mediated control of cholesterol metabolism - Study of human mutants has disclosed how cells regulate a substance that is both vital and lethal, Science, 191 (1976) 160-154. 7 Fogehnan. A.M., Edmond. J., Polito, A. and Popjak. G., Control of lipid metabolism in human leucocytes, J. Biol. Chem., 248 (1973) 6928-6929. a Higgins, MJ.P., Lecamwasam. D.S. and Galton, DJ.. A new type of familial hypercholesterolaemia, Lancet. 2 (1976) 737-740. 9 Kayden. H.J.. Hatam. L. and Beratis. N.G. Regulation of 3-hydroxy-3methylglutaryl coenzyme A reductasc activity and the esterification of cholesterol in human long term lymphoid cell lines, Biochemistry, 16 (1976) 521. 10 Fredrickson, D.S. and Levy, R.I., Familial hyperlipoproteinemia. In: J.B. Stanbury. J.B. Wyngaarden and D.S. Fredrickson (Eds.). The MetaboUc Basis of Inherited Disease. 3rd edition, McGraw-Hill. New York. 1972, PP. 545-614. 11 Goldstein, J.L., Schrott. H.G. and Hazzard. W.R., Hyperlipidaemia in coronary heart disease Genetic analysis of lipid levels in 176 families and delineation of a new inherited disorder, combined hyperlipidaemia, J. CBn. Invest., 62 (1973) 1644-l 568.
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Fredrickson, D.S.. Morganroth. J. and Levy, R.I., Type III hyperhpoproteinemia -An analysis of two contemporary definitions, Ann. Intern. Med., 82 (1975) 150. 13 Boyum, A., Separation of blood leucocytes. granulocytes and lymphocytes, Tissue Antigens, 4 (1974) 269-274. 14 Roes. D. and Loos, J.A. Changes in the carbohydrate metabolism of mitogenically-stimulated human peripheral lymphocytes. Biochem. Biophys. Acta, 222 (1970) 565-582. 15 Lowry, O.H.. Rosebrough. N.J.. Farr. A.L. and Randall. R.J.. Protein measurement with the Fohn phenol reagent, J. Biol. Chem.. 193 (1942) 265-275. 16 McFarlane. A.S., Efficient trace-IabeBing of proteins with iodine, Nature, 182 (1958) 53-54. 17 Scatchard, G., The attraction of proteins for smaB molecules and ions, Ann. N.Y. Acad. Sci.. 51 (1949) 660-672. 18 Slack, J. and Nevin. N.C., Hyperhpidaemic xanthomatosis. Part 1 (Increased risk of death from ischaemic heart disease in first degree relatives of 53 patients with essential hyperlipidaemia and xanthomatosis), J. Med. Genet., 5 (1968) 4-8. 19 Chait. A.. Brunzell. J.D., Albers. J.J. and Hazzard. W.R., Type III hyperhpoproteinaemia (“remnant removal disease”). Lancet. 1 (1977) 1178-1180. 20 Caste& W.P.. Doyle, J.T.. Gordon, T., Hames. C.G., Hjortland, M., HuIIet. S.B., Kagan. A. and Zukel, W.J., Alcohol and blood lipids, Lancet. 11 (1977) 153. 21 Barboriak, J.J., Rimm, A.A.. Anderson, A.J., Schmidhoffer. M. and Tristani. F.E., Coronary artery occlusion and alcohol intake. Brit. H. J., 39 (1977) 289-293. 22 KIatsky, A.L., Friedman, G.D.. SiegeIaub, A.B. and Gerard, M.J., Alcohol consumption and blood pressure, New EngI. J. Med., 296 (1977) 1194-1199.
