We examined the activities of intestinal acyl-CoA: cholesterol acyltransferase (ACAT) and cholesterol esterase, enzymes regulating cholesterol absorption, in rats with streptozotocin-induced diabetes to clarify the effect of diabetes on cholesterol absorption. Three weeks after the induction of diabetes, plasma cholesterol levels were slightly increased in diabetic rats compared with controls, whereas a remarkable increase in plasma cholesterol was observed in diabetic rats fed a 1% cholesterol diet. Microsomal ACAT activity in intestinal mucosa was 3 times higher in diabetic than in control rats. However, no significant difference in the enzyme activity could be detected between diabetic animals fed control chow and those fed the cholesterol diet. Furthermore, insulin supplementation given to diabetic rats caused a reduction of enzyme activity to the levels found in controls. In contrast, cholesterol esterase activity in rat intestinal mucosa was unaffected by either the induction of diabetes or cholesterol feeding. These data indicate that enhanced cholesterol absorption via a CoA-dependent esterification process in the intestine might be one of the major factors responsible for hypercholesterolemia in diabetes.

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The effect of heparin-induced lipolysis on the high density lipoprotein (HDL) subfractions was studied in 12 normotriglyceridemic patients (NTG) and 12 mildly hypertriglyceridemic patients (HTG). HDL₂ and HDL₃ were separated by ultracentrifugation before and 10min after heparin (10 units/kg of body weight) injection intravenously. In all subjects, there were negative correlations among the changes of VLDL- and HDL₂-lipid levels after heparin injection. The changes of HDL₂-lipid levels, except for triglyceride (TG), were inversely correlated with those of HDL₃-lipid levels. These results supported that the conversion of HDL₃ to HDL₂ was induced during lipolysis of triglyceriderich lipoproteins (TGRLP) in vivo. In HTG, TG, free cholesterol (FC) and phospholipid (PL) in HDL₂ increased significantly and esterified cholesterol (EC) and PL in HDL₃ decreased significantly after heparin injection. In NTG, however, EC and PL in HDL₂ decreased significantly and PL in HDL₃ increased significantly after heparin injection. NTG differed from HTG in the heparininduced changes of HDL subfractions. In this experiment, it is not confirmed whether this difference is due to only the difference of TGRLP level between in NTG and HTG.

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Particle size of lipoproteins can be analyzed easily by High Performance Liquid Chromatography (HPLC). In this study we examined particle size of low density lipoprotein (LDL) and high density lipoprotein (HDL) of various hyperlipidemic subjects including Type I, IIa, IIb, III, IV, V hyperlipidemia, familial hypercholesterolemia (FH) and hyper-alpha lipoproteinemia. Effects of drugs (Probucol and CS-514) on particle size of LDL and HDL were also studied in heterozygous FH.
1) In homozygous FH the particle size of HDL is smaller than in control.
2) The particle size of HDL is substantially reduced by Probucol but not by CS-514 in heterozygous FH.
3) In classical Type III (Apo E 2/2) HPLC profile showed broad peak of intermediate size of lipoprotein without any peak which correspond normal LDL, while in hyperlipidemia with Type III phenotype (non Apo E 2/2) LDL peak was present in addition to intermediate lipoprotein.
4) In hypertriglyceridemia (Type I, IIb, V), the particle size of HDL is smaller and that of LDL tends to be larger than those in normal control.
5) The particle size of HDL is remarkably larger, LDL particles are substantially smaller in size in hyper-alpha lipoproteinemia.

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Probucol markedly reduces the LDL-C level in hyperlipidemic patients, but it also reduces HDL-C, which is believed to result in an anti-athero-sclerotic effect. In this study, factors useful for predicting the reduction in HDL-C by Probucol were examined in order to predict the problem before actual administration.
Probucol (250-750mg/dl) was administered to 30 patients with type II hypercholesterolemia. Among them, 23 patients showed 20% or greater decrease in the HDL-C level after administration. To evaluate the factors which permit identification of this group, linear discriminant function analysis was performed using 12 preadministration lipid levels. The analysis indicated that the Apo A-I/HDL-C ratio has the greatest predictive value (F=17.8, p<0.01) of all the parameters, and that the HDL-C level is reduced by 20% or more by Probucol administration when this ratio is 5.3 or less.

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Although the epidemiological observations and in vivo studies about effects of alcohol intake on lipid and lipoprotein metabolism have been reported, little data are available which show role of ethanol in lipoprotein metabolism by cultured cells. In the present study, effects of ethanol on low density lipoprotein (LDL) metabolism in cultured human skin fibroblasts and rat aortic smooth muscle cells (SMC) were investigated. In SMC, binding, internalization and degradation of ¹²⁵I-LDL were not affected by ethanol up to 1.7×10^-3M. However, binding and internalization of ¹²⁵I-LDL were increased and its degradation was decreased at 1.7×10^-1M of ethanol concentration. Similar results were obtained in the experiments using cultured fibroblasts. Preincubation with ethanol (1.7×10^-1M) for 12 hours did not affect either binding or internalization of LDL, but decreased LDL degradation in fibroblasts. These results suggest that ethanol affects significantly intracellular lipoprotein metabolism in cultured fibroblasts and smooth muscle cells.

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The effectiveness of plasmapheresis at 2-4 weekly intervals combined with hypolipidemic drugs for 2 years on coronary atherosclerosis has been evaluated in a familial hypercholesterolemic patient (hetero IIa type). Serum cholesterol levels were 390mg/dl before therapy, 217.9±19.4mg/dl (MSD) at pre-plasmapheresis, 89.1±15.6mg/dl at post-plasmapheresis during therapy. Coronary angiography (CAG) was done 3 times; before therapy, after 1 year, after 2 years. Initial CAG showed 88% stenosis in CX seg. 13 and 47% stenosis in LAD seg. 8. Second CAG (after 1 year) showed improvements in seg. 13 to 65% stenosis and in seg. 8 to 33% stenosis. Third CAG (after 2 years) showed a much more improvement in seg. 13 to 45% stenosis, and no progression in other coronary arteries. These findings suggest that plasmapheresis combined with hypolipidemic drugs might induce the regression of coronary atherosclerosis in familial hypercholesterolemic patients.

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In this study, probucol (PB) was administered to patients with hypercholesterolemia and its effects on serum lipids and apoproteins were evaluated. The subjects consisted of 23 patients with hypercholesterolemia showing a serum total cholesterol (TC) of 230mg/dl or more. Their ages ranged between 28 and 77 years (mean, 56.8 years). PB was administered at a dose of 500mg every day. The observation period was 5 months, and serum was analyzed at 4-week intervals. TC decreased gradually from 269±30mg/dl before to 213±37mg/dl after the PB administration (about 21 reduction). HDL-C also decreased 25% after 12 weeks but tended to increase thereafter. LDL-C decreased by about 20% after 12 weeks, and TG tended to decrease. Apoproteins C-II, C-III, and E decreased significantly, Apoproteins A-I, A-II, and B tended to decrease. The ratio of apo A-I/HDL-C increased significantly from 2.75±0.46 before the administration to 3.49±0.78 after 12 weeks. The ratios of apo A-II/HDL-C and apo B/LDL-C also increased significantly. These findings show that the extent of decrease was smaller in apo A-I and A-II than in HDL-C as well as in apo B than in LDL-C. The atherogenic index did not change following the PB administration. It is concluded that, following PB administration, cholesterol decreased to a greater extent than the apoproteins in HDL and LDL.

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HMG Co-A reductase activity was measured in freshly isolated peripheral blood mononuclear cells from 8 normolipidemic healthy volunteers before and 4 hours after 50g margarine ingestion. These studies have demonstrated that LDL-cholesterol levels and HMG Co-A reductase activity before margarine loading were positively correlated (p<0.05). And a significant decrease (p<0.02) has been demonstrated in mononuclear cell HNG Co-A reductase activity in responders in which LDL-cholesterol levels decreased after margarine loading. In non-responders LDL-cholesterol levels and HMG Co-A reductase activity dose not change significantly. In responders LDL-triglyceride, LDL-phospholipid and LDL-Apo-B levels decreased as much as LDL-cholesterol.

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Human umbilical vein endothelial cells in culture synthesize platelet-activating factor (PAF) in response to the stimulation with thrombin. The most part of newly-synthesized PAF remains cell-associated even in the presence of physiological concentration of extracellular albumin which strongly binds PAF. Therefore, PAF may be incorporated into the surface membrane of endothelial cells as a part of the compositional phospholipids and thereby may lower the antithrombogenic property of these cells; and endothelial PAF generation may result in the activation of effector cells such as leukocytes and thrombocytes contacting with the surface. PAF is known to be inactivated by a specific enzyme called PAF acetylhydrolase, and previous studies have revealed the existence of this enzyme in HDL and LDL. This study was intended to clarify the effects of plasma lipoproteins on thrombin-induced production and extracellular release of PAF by endothelial cells.
Confluent monolayers of endothelial cells were incubated with [³H]acetate and thrombin in the presence or absence of either LDL or HDL (500μg protein/ml) of which PAF acetylhydrolase had been inactivated completely by the pretreatment with diisopropylfluorophosphate. [³H]PAF was separated by thin-layer chromatography and quantified by scintillation spectrometry.
Thrombin stimulated endothelial cells to produce 1, 200±616.4 dpm/10⁶ cells PAF (n=3). Approximately 2-fold enhancement of PAF production was observed in the presence of LDL (2, 507±125.9 dpm/10⁶ cells, n=3). About 7% of the total [³H]PAF was detected in extracellular medium in the presence of LDL, whereas the release in its absence was estimated to be 3%. HDL displayed no effect on endothelial PAF production and the extracellular release.
In conclusion, the atherogenicity of LDL may be mediated, in part, by its enhancing effect on endothelial PAF production. PAF may exert its biological activities being retained by the cells.

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This study was designed to provide aerobic power level to keep a lower levels of coronary atherosclerotic heart disease (CAHD) risk factors of female. Predicted maximal oxygen uptake per kilogram of body weight (VO₂ max/wt) and percent body fat (%Fat), blood pressure (SBP/DBP), total cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL-C) and TC/HDL-C were examined in 207 middle-aged female. The subjects were divided into two groups by VO₂ max/wt corresponding to border line values of CAHD risk factors; the upper was good group and the lower was poor group, and CAHD risk factor status were compared between two groups.
The findings were as follows;
1. VO₂ max/wt were correlated with %Fat, SBP, DBP, TG, HDL-C and TC/HDL-C (p<0.001), but not with TC. After adjusted for the effects of age and %Fat, 102 max/wt were correlated with SBP and HDL-C (p<0.05).
2. %Fat, SBP, DBP, TG and TC/HDL-C in good group were lower than in poor group (p<0.001), while HDL-C was higher (p<0.01).
3. The subjects with plural CAHD risk factors and with isochemic change on ECG were mainly observed in poor group.
From these results, we concluded that VO₂ max/wt is an effective parameter to quantify the CAHD risk factors status, and that VO₂ max/wt corresponding to border line values of CAHD risk factors is necessary to keep CAHD risk lower in female as well as in male.

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Platelet-activating factor (PAF) has been identified as 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine, and is inactivated through the hydrolytic removal of sn-2 acetate by the action of a specific enzyme, PAF acetylhydrolase. This enzyme has been found in plasma in the form of complexes with low density and high density lipoproteins. Although PAF has been implicated in various pathological conditions such as inflammatory reactions, atherogenesis, thrombogenesis, etc., its precise role in these disease processes is not known due mainly to difficulty in quantifying the content of PAF or its metabolite in biological materials; and estimation of plasma PAF acetylhydrolase activity may provide an effective measure to investigate the role of PAF in various diseases. Therefore, in this study, the relationship between PAF acetylhydrolase and plasma lipoproteins in healthy adults was observed.
Thirty-nine male and fifty-five female healthy adults ranging in age from 33 to 89 years old were studied. Fasting blood samples were obtained using 1/10 volume of 3.8% trisodium citrate as an anticoagulant. Plasma PAF acetylhydrolase activity was estimated according to the method of Stafforini et al., in which [³H]acetate liberated from [2-acetyl-³H]PAF, by incubation with plasma, was quantified by scintillation spectrometry. In 29 cases, plasma lipoproteins were separated by ultracentrifugation and PAF acetylhydrolase activity in each fraction was assayed in a similar manner.
The average values of PAF acetylhydrolase activity in male healthy adults were 83±35.9nmol/ml/min, and it was significantly higher than that in female: 67±33.1nmol/ml/min (p<0.05). There was a tendency for the activity to increase with advancing age until the fifth decade in men and the sixth decade in women; and the activity plateaued thereafter. In 29 subjects, LDL and HDL were found to carry the activity of 62±34.2 and 18±8.9nmol/ml/min, respectively; these were 77±9.5% and 23±9.3% of the total plasma activity. Plasma PAF acetylhydrolase activity correlated positively with the levels of total cholesterol, LDL-cholesterol and apo B.
These results may serve as a basis for the further studies of PAF acetylhydrolase and lipoprotein metabolism.

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Serum amyloid A protein (apo SAA) is an acute phase protein associated with high density lipoprotein (HDL) in serum. SAA is the precursor of tissue amyloid A, which deposits as amyloid fibrils in secondary amyloidosis.
We obtained the specific polyclonal antibody against human apo SAA. Using immunoblotting and histochemical methods with this antibody, the following results were observed.
1) Apo SAA was detected in HDL fraction of the plasma from patients with acute myocardial infarction by immunoblotting on two-dimensional electrophoresis. Studies of four patients with acute myocardial infarction revealed that the quantity of apo SAA in the HDL₃ fraction was about 3.5 times larger than that in HDL₂. Apo SAA accounted for 18.5±3.1% (mean±SD) of the concentration of HDL₃ constituents in the patients. Moreover, each relative proportion of apo A-I, A-II and phospholipid in apo SAA-containing HDL₃ was decreased as compared with normal HDL₃ (27.1±1.7% vs. 36.0±2.5%, 5.3±0.6 vs. 8.7+1.0, 25.5±3.2 vs. 32.9±0.9, respectively). These results suggest that apo SAA displaces some of the apo A-I, apo A-II and phospholipid which exist in the surface shell of the HDL particle. Furthermore, the particle size of apo SAA containing-HDL₃ (mean radius 5.46±0.12nm) was larger than that of normal HDL₃ (5.22±0.03nm) as measured by gradient gel electrophoresis. We thought that apo SAA containing-HDL was remodeled based on its particle composition, and in consequence the function of HDL including reverse cholesterol transfer system might be affected.
2) Apo SAA was detected in the intima of the atherosclerotic lesion of the human coronary artery and abdominal aorta by immunohistochemical method, and the localization was quite similar to that of apo B. Apo SAA was also detected in the extract from the atherosclerotic lesion of the abdominal aorta by homogenization with 10% formic acid. These results suggest that apo SAA may be involved directly in the process of atherogenesis.
We conclude that apo SAA containing-HDL might play a role in the pathogenesis of atherosclerosis.

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We have performed Immunocytochemical analysis on the intimal smooth muscle cells using monoclonal antibodies specific to: muscle actins [HHF35 (H)], smooth muscle actins [CGA7 (C)], vimentin [43βH8 (V)] and desmin (D). All studies were performed on methanol-Carnoy's-fixed, paraffin-embedded tissues. Intimal smooth muscle cells were divided into 3 major groups, based on qualitative aspects of immunoreactivities with antibodies. Group I (V+, H+, C+, D+): They were few in number, and mostly recognized in the fibrous thickening lesions of arterioles. Group II (V+, H+, C+, D-): Most of the intimal smooth muscle cells, including smooth muscle cell-derived foam cells, expressed this phenotype. Group III (V+, H+, C-, D-): Approximately 20 to 30% of HHF35-positive cells were non reactive with CGA7. This study demonstrates the application of monoclonal antibody technology to the study of the phenotypic state of intimal smooth muscle cells.

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To investigate the relation of daily intake of foods and nutrients on serum lipid levels or apolipoproteins, amount of daily food intake in a day were measured and serum cholesterol (TCH), HDL-cholesterol (HDL-CH), triglyceride (TG), and apolipoproteins were determined for 138 inhabitants aged above 30 under 70 living in a fishing and a farm village in a local town, Mie, Japan. In females the farm village has lower mortality rate of heart disease and higher of cardiovascular disease than the fishing village. In males the farm village has higher mortarity rates of heart disease, cardiovascular disease and hypertensive disease than the fishing village.
1. TCH and TG levels were higher tendency in the farm village than in the fishing village in male, but significantly higher in the fishing village than the farm village in female.
2. Apo A-I, apo A-II, apo C-II and apo C-III were lower in male but apo C-II, apo C-III and apo E were higher in female in the fishing village than the farm village.
3. Fish intake was more but meat and pulse intakes were less in the fishing village than in the farm village in male. Confectioneries was more but pulse and fungi intakes were less in the fishing village than in the farm village in female.
4. Apo A-I had positive correlation with vitamin A and both apo A-II and apo C-III had positive correlation with alcohol intake. Apo A-II had positive correlation with meats and negative correlation with fruits and fish intakes. Apo C-II, apo C-III and apo E had positive correlation with milk intakes.
These results suggested that the differences of the diets between the two villages effect the serum lipid levels and apolipoprotein levels.

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Pathological analysis with ultrastructural study was employed to observe and to compare in detail lesions of the coronary artery of cynomolgus and rhesus monkeys. Animals were fed individually with the same atherogenic ration under identical conditions for 4, 8, and 12 months, and controls of each species were fed with a low fat, cholesterolfree ration. Transmission electron microscopic studies of coronary arteries from these animals showed the following conclusions.
1) Synthetic smooth muscle cells (SMC) without lipid and macrophages without lipid appeared more frequently in the cynomolgus lesions than in the rhesus. Furthermore, phenotypic expression of synthetic SMCs in the cynomolgus was more active with greater diversity, while the rhesus showed less phenotypic modulation. 2) Increased percentage of both synthetic SMCs with lipid and macrophages with lipid were demonstrated in the cynomolgus lesions as compared to those of the rhesus. This indicated that foam cells including SMC and macrophage-derived foam cells, are more prevalent in cynomolgus than in rhesus. 3) Medial disruption, synthetic SMCs and macrophages containing lipid appeared more often in cynomolgus media than in rhesus media. 4) There were greater percentages on the myocardial side of both synthetic SMCs and macrophages in the intima of both species. 5) Approximately 42% of all foam cells in the cynomolgus intimal lesion was derived from SMC. There were fewer macrophages in rhesus lesions. 6) The difference in expression between the two macaque species was dependent on different responses of macrophages as reticuloendothelial system and of SMCs as medial components. The configuration of the artery wall could be one of the important atherogenic factors.

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Regulation of cholesterol metabolism in the liver were investigated using rabbit isolated hepatocytes. CS-514, HMG-CoA reductase inhibitor, inhibited cholesterol synthesis from [¹⁴C]acetate in rabbit isolated hepatocytes, and increased uptake of LDL, but not of HDL. Dexamethasone did not have significant effect on cholesterol synthesis, bile acid synthesis and LDL uptake into isolated hepatocytes. These results were not necessarily coincident with the changes of plasma lipoprotein levels such as hypercholesterolemia in hypercortisonism. Glycyrrhizin inhibited cholesterol synthesis, and enhanced LDL uptake and promoted the synthesis of bile acid from LDL-cholesterol. These results are consistent with in vivo observation that glycyrrhizin has cholesterollowering effect and enhances excretion of bile acid.

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In order to elucidate the interrelationship between protein levels and exercise training the investigation was undertaken to determine the lipid levels in rats fed on varied contents of the dietary casein (15%, 20% or 25%) and trained by swimming for 30min a day for 3 weeks. Four-week-old male Wistar rats (the groups consisted of sedentary control and exercise training groups) were used for the experiments. Our results showed the following significant changes; In the exercise group, a significantly decreased concentration of blood glucose and contents of glycogen in the liver, soleus or gastrocnemius muscle were observed, as compared to the control group. In the plasma, the lipid levels of exercise group showed decreased TG, T-chol and PL, while FFA and HDL-C concentrations were increased, as compared to the control group. In the aorta, concentrations of TG, T-chol, FFA and PL were diminished by the exercise and protein levels (20% or 25%). In the liver and gastrocnemius muscle, the concentrations of TG, T-chol and FFA were decreased, whereas the concentration of PL was significantly increased in the exercise group. FFA and HDL-C levels in plasma tended to increased in the exercise group and these increments were remarkable in the 20% or 25% protein levels. These results suggest that exercise training and protein dietary improve the fat metabolism and help to prevent the development of atherosclerosis.

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We already reported that Elastase had a antiatherosclerotic effect caused by decrease of lipid binding ability of extracellular matrix, especially that of elastin. However, its mode of action has not yet been clarified. This study was undertaken to elucidate a site of action of Elastase.
Rabbits fed on a 1% cholesterol-diet for 16 weeks were injected iv, with ¹²⁵I-labeled Elastase. Three hours later, those aortic uptakes of ¹²⁵I-Elastase were counted, and also autoradiography of aortic tissue section was done. The results are given as under.
1. Sclerotic aortic uptake of ¹²⁵I-Elastase was higher radioactivity than sclerotic aortic uptake of ¹²⁵I-Na and non-sclerotic aortic uptake of ¹²⁵I-Elastase, 71.7±11.96, 34.5±8.58 and 25.6±8.60 (cpm/mg wet weight) respectively.
2. By autoradiography, localization of ¹²⁵I-Elastase was shown in foamy cells.

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Atherosclerosis occurs at favored sites. According to Fry's hypothesis, the early atherosclerotic lesions are to be expected in high shear stress area. On the other hand, Caro proposed that the early lessons should develop in the region compatible with low shear. We examined the intimal thickening of carotid arteries collecting from autopsy specimens grossly and microscopically.
We showed that the early lesions of atherosclerosis was more prominent on the outer walls of the carotid bifurcations, compared to the inner walls. Shear stress on the outer walls is said to be relatively lower than the inner ones. So, our result is in accordance with Caro's hypothesis.
With the same materials, we measured the bifurcation angles of the carotid arteries. The average of the bifurcation angles of left carotid arteries was 7 degrees wider than the right (p<0.05). The discrepancy between left and right angles was thought to be related to the anatomical position. Restricted to carotid arteries, degree of bifurcation angles seems no relation to atherosclerosis.
The intimal thickening on low shear regions was compared between the internal carotid and left anterior descending arteries (LAD). Shear rate of LAD is said be lower along the myocardial sites than along epicardial sites of the vessels. Intimal thickening of the inner walls of LAD was recognized even in infantile cases. Grading for the intimal thickening of LAD was 10 year-advanced, comparing to internal carotid arteries. Then, the development of atherosclerosis of internal carotid arteries would progress more slowly than coronary arteries.

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