Arteriosclerotic disorders are frequently observed in diabetics and obviously play an important role in the prognosis in these patients. It is unclear whether the diabetes affects the arteries in an independent, characteristic risk factor fashion or whether the susceptibility to other risk factors is involved. The frequency of cardiovascular death in diabetics is over 70% in the U.S.A. while the percentage is 40-50 in Japan. In statistics found in the autopsy reports published by the Japan Society of Pathology in 1975, deaths due to myocardial and cerebral infarction in diabetics were, respectively, two to three times higher than in non-diabetics. This ratio was compatible with that of most other countries. The Framingham study indicated that a high incidence of cardiovascular disease in diabetics could not be explained simply by the frequency of each risk factor. Thus, diabetes is considered to play the role of an independent risk factor in arteriosclerotic-related diseases.
Specific mechanisms of regulation of blood pressure and hypertension in diabetics have been well discussed by Christlieb and other workers. Hyaline degeneration of renal arterioles which often parallels the low renin states often occurs in the initial stage of diabetic nephropathy. It has been suggested that hypertension in diabetics is caused by an aggravation of the changes in renal arterioles and the glomeruli, there by inducing a resistance to renal blood flow and disorders in the compensatory regulation of the renin-angiotensin-aldosterone system. From our observations, both basal and stimulated renin activities were lower in diabetics, especially those who had an accompanying retinopathy and proteinuria, as compared to non-diabetics. The ratio of aldosterone to renin, however, was rather high in diabetics, as determined after upright position and intravenous injection of furosemide. In the regulation of blood pressure in diabetics, disorders in aldosterone and renin secretion were thus considered to play an important role and the clinical application for anti-aldosterone agents should probably be reevaluated.
Attention has been focused on platelet function as related to mechanisms of thrombus formation in arteriosclerotic lesions. Recently, we determined thromboxane B₂ levels in plasma from diabetics by a radioimmunoassay method of our own design. These levels in diabetics were found to be significantly higher than in the non-diabetics controls, and such findings imply that the turnover of platelets and the release of thromboxane A₂ from platelets are accelerated in diabetics. On the other hand, Silberbauer reported that the content of prostacyclin in the vessel wall was decreased in diabetics. Thus, prostaglandin metabolism in platelets and vessels plays an important role in diabetic angiopathy.
Enhancement of adhesion and aggregation of platelets have long been evidenced in diabetics. In our patients also there was an enhancement of platelet aggregation, particularly in those with retinopathy and this enhacement decreased in the insulin-treated patients to a much greater extent than in those treated with only diet or oral agents. Enhancement of platelet aggregation was also found in the low insulin response group during 50g OGTT and was improved after intravenous administration of insulin, 0.05 units per kilogram of body weight, while there were no significant alterations in blood sugar and plasma insulin levels. The addition of 100 and 1, 000μU of insulin to platelet rich plasma suppressed significantly adrenaline-induced platelet aggregation. Further experiments revealed the existence of specific binding sites of insulin, insulin receptors, in the platelets themselves. Platelet aggregability was followed up for two years in diabetics treated with diet alone and in those given insulin.

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The effect on making myocardium metabolism better has been observed for 1-12 months with treatment of Cholexamin. The heart rate and electrocardiogram's findings were taken note as it's pictures before and after the Master's two step test.
Elsewhere, the changings of free fatty acid (FFA) and human growth hormone (HGH) on exertion were also observed.
The results were as followed.
1) When Cholexamin was given, 11 subjects (73%) had a decreased heart rate before and/or after the exertion, but 4 subjects (27%) had an increased heart rate.
2) 4 cases of arrhythmia which was auticular fibrillation, auricular premature systoles, ventricular premature systoles and paroxysmal auricular tachicardia were maked better.
3) One case of effort angina had been unhealty than before treated, and in another case of myocardial infarction was onset in 21th day after the administration. The cause at first hand of myocardial infarction after the treatment was unclear.
4) The relations between heart rate and changing of FFA and HGH was unclear.

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Effects of Y-9738 [ethyl 2-(4-cholorophenyl)-5-ethoxy-4-oxazole acetate] on low HDL cholesterolemia were studied in 18 patients with serum HDL cholesterol(ch) of less than 54mg/dl. Y-9738 was administered at 900mg/day for 4 months, and serum HDL-ch, total-ch and lipoprotein fraction were measured.
The HDL-ch level of 43.3mg/dl (average) before treatment significantly increased (p<0.05) one month after administration, and reached the peak of 56.0mg/dl after 3 months. LDL-ch showed no significant change and HDL-ch/LDL-ch ratio increased from 0.347 to 0.423. the α/β ratio of lipoprotein fraction also increased. Total-ch did not change significantly.

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Hypocholesterolemic effects of ML-236B, a potent inhibitor of cholesterol synthesis, have been studied in several animal species. In rodents like mice, rats and hamsters, this drug produced no detectable changes in serum cholesterol levels at doese as high as 500-2, 000mg/kg. On the other hand, hypocholesterolemic effects were obtained in hens, rabbits, dogs, monkeys and man at levels of 1-50mg/kg. The lack of effectiveness in the rat was shown to be, at least. partly, explained by the unexpected results that levels of hepatic 3-hydroxy-3-methylglutaryl-CoA reductase activity were extremely elevated in this species by the treatment with ML-236B while the excretion of fecal bile acids was markedly reduced.

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ML-236B, a competitive inhibitor of 3-hydroxy-3-methylglutaryl CoA reductase, was administered to eleven patients with primary hypercholesterolemia. After 4-8 weeks of the drug treatment at doses of 50-150mg/day, serum cholesterol levels were reduced by 22-35% (28% in average) in cases of heterozygous familial hypercholesterolemia and combined hyperlipidemia.
A marked reduction of tuberous xanthomas was noticed in a homozygous case of familial hypercholesterolemia, while the drug was less effective in reducing serum cholesterol level and a higher dose was required for the treatment. Softening of Achiles tendon xanthomas was observed in a case of combined hyperlipidemia and the anginal attack was diminished in another case. There was essentially no change in triglyceride concentration. Up to date, no remarkable side effects had been observed.

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Bile acid metabolism was examined in Sprague-Dawley male rats on 2 per cent colestipol diet, 5 per cent cholesterol diet or both. In the colestipol group, there was an about three folds increase in fecal bile acids and a slight increase in fecal neutral sterols. The composition of bile acid was altered to precominantly cholic acid group bile acid. In the cholesterol group, there was a increase in fecal bile acids and composition of bile acid was altered predominantly chenodeoxycholic acid group bile acid.
The composition of bile acid in the large intestine was similar to that of feces, which suggested that bile acids were scarcely absorbed in the large intestine. Bile acids of small intestine slightly decreased in the colestipol group and significantly decreased in the cholesterol -colestipol group. Secretion of bile acids descreased in the colestipol group. There was an eminent increase in the primary bile acid of the bile and small intestine in the colestipol and cholesterol-colestipol group.
Bile acid pool size decreased in the colestipol group and the cholesterol-colestipol group and did not change in the cholesterol group. The activities of 3-hydroxy-3-methylglutaryl coenzyme A reductase and cholesterol-7-hydroxylase increased in the colestipol group.
These findings suggested that colestipol administration altered the bile acid metabolism including bile acid pool size, composition and distribution.

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The measurement of HDL cholesterol concentration (HDL-C) by phosphotungstic acid precipitation method was observed with normal subjects who were clinically healthy, without arteriosclerotic diseases, hyper-lipiclemia, obesty, overdrinking, hepatic disorders (males 62, females 73). The observation showed the tendency that those values were negatively correlated with aging(r=-0.464--0.530).
The values of HDL-C of the patients of arteriosclerotic vascular diseases were significantly lower than those of normal subjects.
The values of HDL-C of the arteriosclerotic patients with treatment of antilipidemic substance (Clofibrate) were significantly higher than those without treatment.

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We describe a modifed phosphotungstate-MgCl₂ procedure for high-density-lipoprotein (HDL)-cholesterol quantitation, especially by use of an enzymatic reagent.
In the present study, we found that an enzymatic cholesterol reagent can be used for HDL-cholesterol measurement without interaction of magnesium ion, influence of bilirubin, and ascorbic acid. When we compared the values of cholesterol content in HDL obtained by ultracentrifugal separation and phosphotungstate-MgCl₂ precipitation, correlation coefficient was 0.968 (slope, 1.11, y-intercept-5.40).
Our precipitation technique is more appropriate for routine clinical laboratory use.

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Clinical investigations on the effects of niceritrol, a nicotinic acid derivative, upon HDL-cholesterol (HDL-ch) and LDL-cholesterol (LDL-ch) were carried out.
Serum total cholesterol (s-TC) and serum triglyceride (s-TG) were significantly reduced during the entire period of the treatment with nicertirol. The lipoprotein fractionation with electrophoresis showed the increase of α/β-ratio induced by niceritrol.
HDL-ch level raised by 12.5% in the 16th dosing week, which was statistically significant. This increase was more evident in the patient with lower HDL-ch level. Despite that the female patient showed higher HDL-ch value (38.5mg/dl) than the value in the male (30.6mg/dl) at the pretreatment of niceritrol, the increase of HDL-ch in both groups were similarly significant. The increase of HDL-ch in the patient with hyperlipidemia of type IV was particularly significant. The increase of HDL-ch was significant when the initial level of s-TG was greater than 200mg/dl
LDL-ch level was depressed by niceritrol, the percentage decrease in the 16th week being 9.2%.
Both the ratios, HDL-ch/LDL-ch and HDL-ch/s-TC, were significantly increased throughout niceritrol treatment.
In conclusion, niceritrol is considered to be effective in preventing the development and progress of atherosclerosis on the basis of the findings that niceritrol raised the level of anti-atherogenic HDL-ch and depressed the level of atherogenic LDL-ch.
It was also suggested that the racing effect of niceritrol on HDL-ch might be associated with the effect of this drug on the metabolism of triglyceride or VLDL.

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The correlation between the severity of coronary artery disease and serum high density lipoprotein cholesterol (HDL cholesterol) level was examined. Precipitation method with heparin-managnese was used for the determination of serum HDL cholesterol and enzyme method for serum cholesterol. Thirty five mm. film of coronary arteriogram taken with Sones' technique were reviewed to score the severity of stenosis. The severity of coronary artery obstruction was expressed as a severity score, utlizing the grading method of AHA AdHoc Committee and the severity score by Gensini. (Fig. 1, 2 and 3)
Forty five cases were reviewed to be twenty of normal coronary anatomy and twenty five of coronary obstruction. Their ages were 41 to 67 (average 55) and 43 to 63 (average 58), respectively. The diagnosis of normal coronary coronary artery were five valvular diseases, three cardiomyopathies, two arrhythmias of unknown causes, two GXT positive Patients and eight cardiac neurosis. That of abnormal coronary arteries were nine cases of one-vessel disease, seven cases of two-vessel disease and three cases of three-vessel disease (Table 1 and 2).
The results of serum cholesterol level, HDL cholesterol and ratio of HDL cholesterol/Total cholesterol minus HDL cholesterol were shown as Table 3. The ratio of cholesterols showed the value above 0.36 in normal coronary patients and below 0.36 in abnormal coronary patients. The correlation between the severity score and the ratio of cholesterols of abnormal coronary atery patients is as shown at Table 5. Their severity scores were from four to 131 and the ratios of cholesterols were ranged from 0.35 to 0.07. The severity of coronary obstruction was revealed to correlate inversely with the ratio of cholesterols.
The results of our findings might be used for non-invasive method to manage the patients of ischemic heart disease. That is, 1) The presence or not of coronary artery disease after the age of 40, 2) The severity of their diseases, and then 3) Their prognosis.

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Many investigators have reported that plasma lipids and lipoproteins are closely related to the development of coronary atherosclerosis. Plasma lipids are carried in the lipoproteins, each of which has a different effect on atherosclerosis. Therefore, the levels of individual lipoproteins are better predictors of coronary artery disease (CAD) than those of plasma lipids. Recent studies emphasize that the concentrations of LDL positively and HDL negatively correlate with CAD, while the relationship between VLDL, IDL and CAD has been less well defined.
In the present study, the influences of serum lipids, lipoprotein lipids and nonlipid risk factors on CAD were studied in 182 consecutive patients evaluated by selective coronary cine-angiography. To examine the relationship between plasma lipoprotein concentrations and the severity of CAD, we assigned a score to each of the 15 segments of coronary artery depending on the finding of selective coronary cine-angiogram and defined coronary atherosclerosis index (CAI) as the sum of these scores.
On the univariate analysis, CAI correlated significantly and positively with serum cholesterol, VLDL-cholesterol, IDL-cholesterol and LDL-cholesterol, and negatively with HDL-cholesterol, respectively. However, CAI was not related to serum triglyceride and VLDL-triglyceride. Analysis of quartile distribution of IDL-cholesterol and LDL-cholesterol showed that the higher two quartiles for IDL-cholesterol (≥17mg/dl) were highly associated with the frequency of CAD, and higher concentrations of cholesterol-rich VLDL (VLDL-cholesterol ≥25mg/dl and VLDL-cholesterol to VLDL-triglyceride ratio≥0.25) were closely related to the degree and incidence of CAD. Moreover, step-down multiple linear regression analysis was used to test for significant relationships between CAI and individual lipoproteins and nonlipid risk factors according to the computerized program. Multivariate analysis demonstrated that IDL-cholesterol, LDL-cholesterol and HDL-cholesterol were significant variables of use in the final weighting procedure. This study showed that cholesterol-rich VLDL and IDL were closely related to the severity and frequency of CAD independent of LDL and HDL.
Recently, Utermann and associates reported that primary dysbetalipoproteinemia with or without hyperlipoproteinemia is a very common genetic disoder, in which IDL-cholesterol and cholesterol-rich VLDL are increased. Therefore, more attention should be paid to the importance of IDL and cholesterol-rich VLDL in the prevalence and management of CAD.

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Coronary risk factors including serum cholesterol have been analyzed by many investigators and recently abnormality of lipoprotein pattern-especially low High Density Lipoprotein (HDL)-cholesterol level is payed attention.
In 50 patients (male 35, female 15) with acute myocardial infarction (MI), we studied several coronary risk factors that include serum lipoprotein pattern, glucose tolerance, obesity, and blood pressure. The severity of coronary artery stenosis was also investigated by coronary angiogram and compared with these biochemical findings.
Results
1. MI patients had low HDL-cholesterol level, high VLDL+LDL/HDL ratio, and high triglyceride (TG) level, although total serum cholesterol level was not significantly different from normal control.
2. Midbands between LDL and VLDL fraction frequently (56%) appeared in polyacrylamide gel disc lipoprotein electrophoresis.
3. There was no significant correlationship between severity of coronary artery stenosis and serum concentration of lipid or lipoprotein in MI patients.
4. There was high incidence of glucose tolerance abnormalities.
5. Frequency of obesity was not so high (7%).
Patients with usual coronary risk factors were 80% of all patients. However, when low HDL-cholesterol level was added as one of coronary risk factors, the incidence of patients with any risk factors increased to 98% in MI patients.

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This study was undertaken to evaluate the relationship between serum lipoprotein concentration and angiographic severity of coronary artery sclerosis in adult male.
HDL-, LDL-, and VLDL-cholesterol were measured in normal subjects (N=47, average age 44 y/o), non-coronary heart disease (Non-CHD, N=23, average age 38 y/o) and coronary heart disease (CHD, N=72, average age 52y/o) by means of heparin-CaCl and O-phthaladehyde method.
All patients of Non CHD and CHD were studied by selective coronary cine-angiography. And as an index of severity of coronary artery sclerosis, coronary score of modified Friesinger method was applied to those who underwent selective coronary cine-angiography. The results were as follows:
1) HDL-cholesterol concentrations of CHD group was 28.5±7.3mg/dl and apparently lower than that of normal subject (47.5±10.4mg/dl) or of Non-CHD group (45.4±6.5mg/dl). LDL-cholesterol concentrations of CHD group, normal subjects and Non-CHD group were 140.3±30.7mg/dl, 121.1±27.1mg/dl, 117.9±27.4mg/dl, respectively.
2) No significant correlation was found between HDL-cholesterol and coronary score among CHD group. However HDL-C/LDL-C ratio was inversely related to severity of coronary artery disease.
In CHD group, all patients with less than 2.50 of the ratio (HDL-C/LDL-C×10) showed severe coronary artery sclerosis more than coronary score 5, whereas no patients with the ratio of over 4.0 showed severe coronary artery sclerosis.

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In recent years, many attentions have been paied to high serum level of HDL cholesterol as an anti-atherogenic factor since the report of Framingham Study in 1977. As the mechanism of the antiatherogenetic action of HDL, Miller and Miller postulated that HDL facilitated the uptake of cholesterol from peripheral tissues and its transport to the liver for catabolism and excretion.
In the present study, the role of HDL when passing through the organ (in this study, the lung) was investigated to prove Miller's concept that HDL increased the uptake of cholesterol from the peripheral arterial wall.
Materials and Methods
Of the patients those who underwent cine coronary angiography, consecutive 57 adult men were selected for this study (coronary heart disease CHD group: 40 cases, non CHD group: 17 cases). The blood sampled simultaneously from pulmonary artery (venous blood) and brachial artery (arterial blood). The concentration of HDL cholesterol and LDL cholesterol were mesured with use of heparin, Ca²⁺, and an anion-exchange resin reported by Noma and his associates. Modified Friesinger's coronary score was used to to assess the angiographic severity of coronary atherosclerotic changes.
Results and Conclusions
1) Non CHD group: there was no difference in the concentration of HDL cholesterol levels and HDL cholesterol/LDL cholesterol ratio between venous and arterial blood samples.
2) CHD group: in the subgroup of under 49 years old, the concentration of HDL cholesterol levels and HDL cholesterol/LDL cholesterol ratio were significantly higher in the arterial blood than in the venous blood. On the contrary, in the subgroup of over 50 years old this tendency was not pertinent. This finding suggests that in the subgroup of under 49 years old, HDL can easily uptake cholesterol from the peripheral arterial wall since the deposition of cholesterol is relatively fresh and loose.
3) Each age group in CHD were divided into three subgroup according to coronary score (0-5, 6-10, 11-16). In the group of coronary score 6-10, and in the group of under 49 years old HDL cholesterol levels and HDL cholesterol/LDL cholesterol ratio were significantly higher in arterial blood than in venous blood.
In this subgroup, the quantity of peripheral cholesterol is suposed to be accumulated moderatly in the arterial wall. As mentioned above, effective uptaking cholesterol from peripheral arterial wall by HDL were thought to be associated with the quantity and the freshness of deposition of peripheral cholesterol.

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Quantitative analysis of serum apo HDL and HDL-cholesterol was carried out in 75 human subjects. The subjects consisted of the following four groups: myocardial infarction, angina pectories, arrhythmia probably due to myocardial ischemia and normal controls. To avoid possibly indefinite factors, age matched healthy men and women who have the same occupation and have been in similar circumstances were subjected as normal control.
The concentration of apo HDL was measured by rocket immuno-electrophoresis using anti-HDL-serum (anti-α₁-lipoprotein-serum, BEHR-INGWERKE AG). HDL-cholesterol was determined by the precipitation method using heparinmanganese chloride.
Mean levels of both apo HDL and HDL-cholesterol were significantly lower in two groups of myocardial infarction and angina pectoris than those in the control group. In the group of arrhythmia apo HDL level was low, but no significant decrease of HDL-cholesterol was seen.
Fourteen of 41 normal controls has low levels of HDL-cholesterol, but low levels of apo HDL were seen in only 3 control subjects.
Although there was a low statistical correlation (r=0.570, p<0.001) between apo HDL and HDL-cholesterol levels, the statistics in terms of 90% confidence interval for the regression line indicated difficulties in estimating HDL-cholesterol level from apo HDL level, i. e. the levels of HDL-cholesterol and apo HDL are variable to each other under the contribution of unknown factors.
In this study, apo HDL level was a valuable discriminator to characterize HDL abnormalities in myocardial infarction. These results bring forward an interesting problem in apo HDL metabolism, especially in atherosclerotic disease.

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Plasma α₁-lipoprotein and HDL-cholesterol were studied in coronary heart disease (CHD). HDL-cholesterol was determined by enzymatic method of cholesterol when LDL and VLDL were precipitated with use of MgCl₂ and dextran sulfate.
α₁-lipoprotein was assayed rocket immuno-electrophoresis with use of the special antiserum.
The following results were obtained;
1) In a healthy control group, HDL-free cholesterol level was found to be higher in females than in males, particularly in the aged population over fifty-year-old.
2) In a CHD group, total cholesterol, free cholesterol and cholesterol ester ratio in the HDL fraction were found to be lower than those in the control group.
Significant reduction in HDL-cholesterol/α₁-lipoprotein ratio was also observed in the CHD group. These results strongly suggest some qualititative changes of the plasma HDL obtained from patients with CHD.
3) In 4 out of 7 cases who showed the high HDL-cholesterol levels over 60mg/dl, the low ratio of HDL-cholesterol to total cholesterol was observed.
4) HDL-cholesterol level was elevated one week after the first onset of acute myocardial infarction, despite reduction in α₁-lipoprotein level. The discrepancy may be due to intravenous injection of heparin which was usually done for 3 days after the onset.

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Determination of serum lipids (TC, TG and HDL-TC) and apolipoprotein (apo A-I, A-III, B, C-I, C-II, C-III and E) levels and disc gel electrophoresis of serum lipoproteins were performed in 46 patients of coronary hearts disease and their 43 roughly-age-matched controls. Coronary heart disease (CHD) group with mean age of 61 years old had 75% or greater stenosis in one or more major coronary arteries. Among them 28 patients were suvivors of acute myocardial infarction which occured not less than 12 months before the study. Controls with mean age of 62 years old showed no evidence of significant stenosis in major vessels on selective arteriography. All female patients experienced menopause at least 5 years before the study.
Serum levels of apo A-I, A-II, B, C-II, C-III and E were determined by electroimmunoassay and apo C-I by radial immunoassay. HDL was isolated by precipitating VLDL and LDL with heparin-manganese chloride and cholesterol (HDL-TC) was measured. Following results were obtained: (1) CHD group showed significantly higher levels of TC, HDL-TC apo A-I, apo C-I, apo C-II and ratio of (TC-HDL-TC)/HDL-TC and apo B/apo A-I than those of control group. (2) midband lipoprotein was detected in 54% of CHD group and 30% of control group; the difference of frequency was statistically significant. Serum lipid and apolipoprotein levels, however, did not correlate with the presence of midband lipoprotein.(3) Levels of apo C-I, C-II, C-III and E showd highly significant correlation with those of TC and TG.(4) In CHD group, survivors of acute myocadial infaction had significantly lower levels of HDL-TC, ratio of (TC-HDL-TC)/HDL-TC and apo B/apo A-I than those who had no history of myocardial infarction which may imply that LDL/HDL ratio was one of the important factor for the development of myocardial infarction.

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A newly established immuno-precipitation using the specific goat antiserum (purified γ-globoline fraction of it) against human β-lipoprotein was applied to the fractionation of lipoprotein in human serum. The lipids in α-lipoprotein fraction, cholesterol (α-LPC), glyceride (α-LPG) and phospholipid (α-LPPL) were determined by the enzymic methods for each sort of lipids, respectively. Contents of the lipids in serum were also determined by the same methods (TC, TG and PL), and the lipid contents in β-lipoprotein fraction were calculated (β-LPC, β-LPG and β-LPPL).
Results on the contents of the lipids, fractionated, in serum of myocardial infarction are as follows.
I) Comparison between the lipids of acute myocardial infarction and those of lipid matched controls:
α-LPC and α-LPPL at the acute stage of myocardial infarction were lower than those of lipid matched controls. α-LPG, on the contrary, was elevated at the acute stage.
II) Comparison of the acute and convalescent stage:
α-LPC and α-LPPL at the convalescent stage were increased as compared with those at the acute stage. As far as α-LPG concerned, there exist no remarkable differences between acute and convalescent stage; but the elevation of share of glyceride in α-fraction was evidently demonstrated (α-LPG/TG).
III) Difference of distribution of each sort of the lipids between acute and convalescent stage:
The elevation of share of lipids in α-fraction (α-LPC/TC, α-LPG/TG and α-LPPL/PL) was clearly observed at the convalescent stage compared with those at the acute stage; almost the same degree of elevation in share of each sort of lipids was noticeable. On the contrary, the distinct but not the same degree of decline in share of each sort of the lipids was observed in β-fraction at the reconvalescence; β-LPG was markedly declined (34.6%), followed by β-LPPL (33.0%) and β-LPC (8.9%).

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α-cholesterol was measured on fasting blood samples from 611 men and women aged 35 years and older in WARA, GIFU.
The average concentration of serum α-cholesterol levels of 217 women were significantly higher (59.9±14.6mg/dl) than those of 228 men (58.1± 15.0mg/dl) in the inhabitants between 35 and 69 years of age.
In male habitants the α-cholesterol/total-cholesterol ratio remained invariable except the group older than 70, in whom it was significantly higher than the other age groups. On the other hand, α-cholesterol/total-cholesterol ratio in the female group in the age from 35 to 39 was considerably higher than that in male group of corresponding age, and it decreased gradually reading the lowest in the age group from 60 to 69, where it was significantly lower than that in the male group of corresponding age.
Incidence of coronary heart disease diagnosed on ECG findings with Minnesota cord 1, 4 and 5, was significantly associated with total cholesterol and low α-cholesterol/total-cholesterol ratio but not with α-cholesterol value. Serum α-cholesterol levels were inversely related to cigarette smorking and serum triglycerides levels, and directly related to total cholesterol and alcohol consumption. However, incidences of the peoples with ischemic ECG findings were not markedly differences in both group consuming cigarette more than 20 daily and ingesting alcohol everyday habituelly.

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It is clear that accelerated atherosclerosis and its clinical consequence cardiovascular disease is a major risk to long-term survivors on chronic hemodialysis. Several risk factors for this complication have been proposed.
In the present study we focused the interest on the possible relationship between the plasma lipids, especially high density lipoprotein cholesterol (HDL-C) and ischemic heart disease (IHD). Studies were carried out on 36 patients undergoing maintenance hemodialysis (20 males and 16 females), ranged from 39 to 72 years of age.
Nineteen of them (53%) had IHD, showing much higher incidence of the complication than for normal and hypertensive subjects of comparable age. When compared with the same age and sex, the HDL-C levels were significantly lower in the dialysis patients (DP) than in healthy subjects (p<0.001). Among the DP, subjects with existing clinical IHD had significantly lower levels of HDL-C than those without IHD (p<0.01). On the contrary, there was no significant relation between total cholesterol (TC) levels and incidences of IHD in the DP. TC-HDL-C/HDL-C(β/αC) ratios were higher in the DP than in the normal subjects, but the significant difference of these ratios was recognized only in females (p<0.05). Among the DP, the β/αC ratios were significantly higher in the patients with IHD than those without that (p< 0.001).
The results suggested that the reduced serum HDL-C concentrations and the increased β/αC ratios might be the most important factors for the development of atherosclerosis, and hence of IHD, although DP were subject to a number of other cardiovascular risk factors such as hypertension, hyperuricemia, glucose intolerance, secondary hyperparathyroidism and others.
The HDL fraction is considered to include the preferential substrate lipoproteins for lecithin cholesterol acyl transferase (LCAT) in plasma. However, there were no consistent correlations between the lipid concentrations in the HDL fraction and the LCAT activities in plasma.
The plasma LCAT activities were positively correlated to the serum triglyceride, phospholipids and free cholesterol levels in the DP.

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Dietary fatty acids are incorporated into chylomicrons and the chylomicron triglycerides are degraded by the action of lipoprotein lipase. The resultant particles, remnants, are selectively metabolized by the liver. This study was undertaken to examine the effects of chylomicrons and their remnants on fatty acid synthesis in isolated rat hepatocytes.
Chylomicrons in the medium containing postheparin rat serum significantly inhibited fatty acid synthesis in hepatocytes (p<0.001). On the other hand, chylomicrons in the medium containing preheparin rat serum caused very small inhibition (n. s.). Chylomicron remnants prepared in vitro caused significant inhibition (p<0.01) and remnants prepared from the chylomicrons injected into functionally hepatectomized rats, in vivo, inhibited fatty acid synthesis to a greater extent than unmetabolized chylomicrons at an equivalent protein concentration (p<0.05) and at an equivalent triglyceride concentration (p<0.01). Thus, the hepatocytes recognize the remnant but not the unmetabolized chylomicron. The chylomicron remnants produced by the action of lipoprotein lipase may play an important role in the regulation of hepatic fatty acid synthesis.

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The immunochemical determination of serum apolipoprotein C-III was carried out by the rocket immunoelectrophoresis using monovalent antiserum against apo C-III. The antisera were prepared in rabbits injecting apo C-III-2 obtained by preparative Disk electrophoresis (8M urea) of delipidated human very low density lipoprotein (VLDL).
Apo C-III concentration in normal human subjects at age 20-60 years old revealed 104±27unit/dl (100 unit for reference serum). After intravenous administration of intralipid (0.1g/kg B. W.), the immediate transfer of apo C-III from high density lipoprotein (HDL) to VLDL was observed. In the hypertriglyceridemic patients of fatty liver, nephrotic syndrome, and diabetic patients, the marked increase of apo C-III was demonstrated, whereas in the liver disease, apo C-III was decreased in proportion to the severity of the parenchymal injury. The concentration of apo C-III correlated to serum triglyceride in diabetic patients, but not in the liver diseases.

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In the present study, using heparin as the model associated with enhanced lipoprotein lipase activity, we studied the correlation of lipoproteins and the change of apoprotein subunits when catabolism of VLDL was accelerated. 33 male patients with ischemic heart disease, hyperlipidemia, essential hypertension and simple obesity were investigated.
Blood samples were drawn following an overnight fast and in 10 min. after heparin Na (10U/kg body weight) injection.
The results have been obtained as follows:
when VLDL catabolism was accelerated by heparin Na infusion,
1) Following decrease in VLDL-TG, FC and apo CII, CIII groups in VLDL were changed simultaneously and FC in HDL was increased reciprocally.
2) The subjects were divided into subgroup according to TC/TG ratio in VLDL.
Change of lipid composition in VLDL is similar. However as concerns apoprotein, cholesterol-rich VLDL subgroup has an increase in contribution of HDL apo A-I.

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The concentration of vitamin E (VE) in very low density lipoprotein (VLDL), low density lipoprotein (LDL) and high density lipoprotein (HDL) was determined with fluorometric method in 34 diabetic patients with and without diabetic macroangiopathy (14 and 20 patients, respectively) to elucidate the role of VE in diabetic macroangiopathy. Each value of the diabetics was statistically compared with that of 19 age-matched controls. The concentration of cholesterol (Ch) in each plasma lipoprotein fraction was determined with Abell's method in 31 male diabetic patients with and without diabetic macroangiopathy (8 and 23 patients, respectively) to compared with the distribution of VE in each plasma lipoprotein fraction.
The total concentration of VE in plasma (Tot-VE) was 1.58±0.10mg/dl (mean±SE) in the diabetics, that was significantly higher than 1.30±0.07 mg/dl in the controls (p<0.05). In the diabetics, the concentration of VE in VLDL (VLDL-VE) and LDL (LDL-VE) was high, and that in HDL (HDL-VE) was low, as compared with each concentration in the controls, but each value has no statistical significance between both the groups. In the diabetics with ischemic heart disease, HDL-VE was significantly lower than that in the diabetics without it; 0.31±0.05mg/dl, 0.48±0.04mg/dl, respectively (p<0.01).
The distribution of Ch was similar to that of VE in each plasma lipoprotein fraction.
There was a positive correlation between HDL-VE and HDL-Ch. On the other hand, a negative correlation between HDL-VE and plasma lipid peroxide was found.
The above results indicate that VE in the plasma lipoprotein fractions, especially, HDL-VE has a close relationship to diabetic macroangiopathy, as well as HDL-Ch and lipid peroxide.

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The study was performed on 12 male rabbits, being devided into two groups; one group of 7 rabbits fed on the commercial rabbit pellet to which 2mg of Glibenclamide per kilo of the food was mixed (G-group), and the other of 5 rabbits fed on the rabbit pellet only as a control group (C-group). The food intake was limited to 200g daily.
Three months later, blood was collected from these rabbits in the fasting state for lipoprotein analysis, and then the aorta was taken out for determination of cholesterol and cholesterolester. Cholesterol content in the aorta of G-group (2.68±0.12mg/g wet weight) was significantly higher (p<0.05) than that of C-group (2.28±0.12mg/g wet weight).
The high density lipoprotein cholesterol level was similar in both groups, while the low density lipoprotein cholesterol level of G-group (10.8±2.3mg/dl) was higher than that of C-group (7.3±1.5mg/dl).
As a result of this study, it is suggested that Glibenclami de may have atherogenic action in the normal rabbit, probably because of an increment of low density lipoprotein cholesterol.

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To study about the cause of increased serum glycoprotein in arteriosclerotic diseases, column chromatography of Sephadex G-100 and electrophoresis were performed on sera of 10 cases of diabetics (DM), 11 cases of hypertensives (HT), 5 cases with aortic arch calcification (AAC), and 10 healthy persons (HP), totaling 36 cases.
1) Serum glycoproteins were fractionated into 2 fractions of FrI and FrII by column chromatography of Sephadex G-100. The molecular size is in order of FrI>FrII.
Serum FrI concentration is higher than that of HP in DM, HT and AAC, but there is no difference in FrII. Namely, the cause of higher level of total serum glycoproteins in arteriosclerotic diseases is due to the higher level of FrI concentration in total serum glycoproteins.
2) The electrophoretic patterns of FrI by the PAS staining were separated into three fractions of α₂-, β-, and γ-glycoprotein.
The concentrations of α₂- and γ-glycoprotein were higher in DM, HT and AAC than that in HP, but β-glycoprotein was higher only in DM.
3) The electrophoretic patterns of serum of FrI by the ozone schiff staining were separated into two fractions of α-and, β-lipoprotein, but no staining of lipoprotein was found on FrII.
The β/α ratio is higher in HT and AAC than that in HP, but no difference is found on the β/α ratio between DM and HP.
4) There are clear differences among DM, HT and HP in the further precise study of the serum glycoproteins by Sephadex G-100 column chromatography and electrophoresis.
Usually, hypertension and diabetes mellitus have been understood by many investigators as similar factors related to arteriosclerosis. However, from the view point of the constituents of serum glycoprotein and lipoprotein, these two diseases should be understood as arteriosclerotic factors different from each other.

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Polyenphosphatidylcholine was administered 1, 500mg a day to each 33 diabetics, whose blood sugar control were stable by diet and oral antidiabetic drugs for a year, and the serum lipids, lipoprotein fractions and fasting blood sugar were examined.
Two to three months after its administration, serum triglycerides and, β-lipoprotein decreased. Six and twelve months after its administration, serum triglycerides, β-lipoprotein and pre-β-lipoprotein fractions decreased and α-lipoprotein fraction increased significantly compared with the level before its administration.
It seemed that polyenphosphatidylcholine induced the increment of α-lipoproteins, the easier transportation of lipids and then lowering serum triglycerides, β-lipoprotein and pre-β-lipoprotein fractions.

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Fasting blood sugar (FBS), serum total cholesterol (TC), triglycerides (TG), HDL cholesterol (HDL-C), VLDL plus LDL cholesterol (VL-C) and LCAT activity were examined in 279 japanese diabetic patients and related with clinical features.
HDL-C was measured by phosphotungstic acid-MgCl₂-OPA method. LCAT activity was measured by Nagasaki-Akanuma method. In these patients, HDL-C was not correlated with FBS or VL-C, but inversely correlated with TG (r=-0.16, p<0.05) and with VL-C/HDL-C ratio (r=-0.57, p<0.005). LCAT activity was correlated with TC(r=0.36, p<0.005), TG(r=0.38, p<0.005) and with VL-C (r=0.36, p<0.005), but not with FBS, HDL-C or VL-C/HDL-C ratio.
In untreated diabetics, HDL-C level was lower (Male: 51.9mg/dl, Female: 55.9mg/dl) than normal subjects (M: 55.8mg/dl, F: 60.7mg/dl), while LCAT activity was higher (105.2 Units) than normal subjects (77.4 Units) (p<0.05). Mean HDL-C decreased significantly in patients with nephropathy. VL-C/HDL-C ratio was increased significantly in diabetic patients with retinopathy, neuropathy and nephropathy.
Patients with HDL-C lower than 45mg/dl or higher than 70mg/dl were selected. In high HDL-C group, LCAT activity was positively correlated with VL-C(r=0.70, p<0.005), with VL-C/HDL-C ratio (r=0.79, p<0.005) and inversely correlated with HDL-C(r=-0.45, p<0.01). On the other hand, none of these correlations exsisted in low HDL-C group. Patients with poor FBS control, with diabetic complications and with ishemic heart diseases were more prevalent in low HDL-C group; obese patients were significantly more prevalent in this group. We presume that LCAT activity increases in response to the rise of VL-C in high HDL-C group, but not in low HDL-C group. VL-C/HDL-C ratio and LCAT activity seem to be good clinical indexes. The treatment of obesity seems important in view of its correlation with VL-C/HDL-C ratio and LCAT response.

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An inversed relationship between plasma HDL-cholesterol level and the incidence of atherosclerotic disease has been demonstrated.
And patients with diabetes mellitus have a higher incident of atherosclerotic diasease than nondiabetics. The factors that increase the incidence of atherosclerotic diasease in diabetics are not well understood. Therefore, measurement of HDL-C in diabetics is important. And there are some studies about the problem whether HDL-C in diabetics is lower or higher than in healthy subjects. But the results of the studies are not yet established.
In this study, we divided diabetics into three groups treated with diet only, oral agent, and insulin group, and compared HDL-C of three diabetic groups with that of healthy group.
We determined HDL-C concentrations in plasma of 848 subjects (male 475, female 373) who received a routine medical check up in Meiji Life Insurence Shinjuku health testing center and 308 diabetics (diet only 136, oral agent 86, insulin 82) by the heparin-Mn²⁺ precipitation method. The healthy subjects, who had no abnormalities in TG, TC, obesity index, skin fold thickness, blood pressure, ECG finding, GOT, GPT, and blood sugar, were obtained from the testing center. The average HDL-C values of 127 healthy males and 160 females were 61.0±16.9mg/dl and 68.5±13.7mg/dl.
Plasma HDL-C was strongly affected by TG, TC, and obesity index in both non-diabetics and diabetics. Average plasma HDL-C value of the female diabetics treated with diet only and oral agents was lower than that of healthy females. And average plasma HDL-C value of the male diabetics treated with insulin was higher than that of healthy males.
But plasma HDL-C values adjusted with TG, TC, and obesity index were similar in non-diabetics and diabetics-in any three therapeutic groups. Therefore, we concluded that difference before adjustment was due to the influence of TG, TC, and obesity index. High incidence of atherosclerosis in diabetics was not accounted for by plasma HDL-C.

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HDL cholesterol levels were determined in the sera of normal control and of diabetics. The results obtained were as follow:
1. The mean level of HDL cholesterol in the serum of normal control was 47.9±9.0mg/dl for men and 55.9±10.5mg/dl for women. However, no difference of HDL cholesterol level in the sera between normal control and diabetics was seen.
2. A significant decrease of HDL cholesterol level was found in the sera of diabetics with hyperlipidemia type IIa or IV, and a marked decrease of HDL cholesterol level was seen in the sera of patients with diabetic nephropathy. On other hand, the level of HDL cholesterol in the sera of the diabetics treated with insulin for 10 years or more over was somewhat higher than that of normal control and of normolipidemic diabetics.
3. The decrease of the HDL cholesterol level in the sera of patients with diabetic nephropathy may be due to the release of the HDL into the urine, which was demonstrated by PAG disc electrophoresis of urine proteins.

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Plasma lipids are widely known to be elevated in obese subjects. Since Sumo-wrestlers are considered to be one example of the characteristic human experimental obesity, it could be quite interesting to examine their plasma lipids according to the obesity index.
Plasma lipids were determined on 538 currently Sumo-wriestlers with the age ranging from 15 to 37. Their mean height is 179.7cm and mean body weight is 111kg.
Plasma total cholesterol increased with advancing the obesity index and triglyceride also increased markedly with the index. HDL-cholesterol tended to decrease with increasing obesity index. There were statistically significant changes in those plasma lipid levels between the group of obesity with 9% or less and the group of extreme obesity with 60% or over.
In the same dregree of obesity, HDL-cholesterol tended to decrease in the group of elevated plasma triglyceride level. There was a strong inverse correlation between plasma triglyceride and HDL-cholesterol (r=-0.2662 for younger group, r=-0.2652 for older group. P<0.01). Therefore, plasma triglyceride was considered to be potent determinant on HDL-cholesterol.
Plasma triglyceride was significantly correlated with γ-GTP, and GOT, GPT in certain groups, while these enzyme levels were entirely indifferent to HDL-cholesterol.
Present results are important as the basal correlation for the follow-up study of the human obese subjects.

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High-density and low-density lipoprotein cholesterol were determined by a modified heparin-Ca precipitation method in 66 male and 13 female survivors of cerebral infarction (C. I.). The mean values for HDL-cholesterol concentration and HDL:LDL cholesterol ratio for both sexes of C. I. patients were significantly lower than control values (25 male, 7 female). These significant differences were also found if C. I. patients were compared with patients with various diseases excluding ischemic heart disease, peripheral arterial disease, hyperlipidemia, hepatic disease and diabetes mellitus (40 male, 37 female). When male C. I. patients were divided into two groups, C. I. in the cortical arterial system and lacune in the perforating arterial system, HDL-cholesterol concentration and HDL:LDL cholesterol ratio were significantly lower in the former group than in the latter, suggesting that these lipoprotein abnormalities play a part in the pathogenesis of C. I., particularly in the cortical arterial system.
HDL subfractions, HDL₂ and HDL₃, were ultracentrifugally isolated and their compositions were studied. HDL₃ cholesterol concentration has more clearly discriminated C. I. patient from healthy subject than HDL cholesterol concentration has.

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Serum lecithin: cholesterol acyltransferase (LCAT) rate was determined by the method of Nagasaki & Akanuma in 72 patients with cerebrovascular diseases and 147 controls. Liver function tests were within normal limits in all of these subjects.
The average values of non-obese healthy men and women, who had no abnormalities in physical findings, blood pressure, ECG, urinalysis, etc., were 107±38.0nmol/ml/hr and 82±39.9nmol/ml/hr, respectively. The value of female subjects was significantly higher than that of male. There was no significant change by age.
In male and female patients with cerebral infarction, average serum LCAT rates were 93±32.6nmol/ml/hr and 118±49.9nmol/ml/hr, respectively. The value of female patients was significantly higher than that. of age-matched healthy control. Among male patients with cerebral infarction, the cases with angiographically demonstrated obstruction of internal carotid or middle cerebral arteries showed significantly lower value as compared with those without such findings and healthy subjects. There was no such difference in the values of female patients.
The average values of male and female patients with cerebral hemorrhage were 99±31.8nmol/ml/hr and 129±35.6nmol/ml/hr, respectively. Again the value of female patients was significantly higher than that of control. No significant difference was observed between two types of disease.
In male subjects, LCAT rate correlated positively with α₁-lipoprotein level which was lower in patients as compared with normal control. These results may suggest that the mechanisms involved in the activation of LCAT in vivo is defective in patients and such mechanisms posess the more decisive role in the pathogenesis of cerebrovascular diseases than the amount of this enzyme per se. The low LCAT level, in conjunction with defective activation, may possibly bring about the more pronounced atherosclerotic lesions especially in major cerebral arteries.
In female, there was no relation between LCAT rate and α₁-lipoprotein level. In view of no significant difference in α₁-lipoprotein level between patients and controls, our results may suggest the complexity of lipid metabolism in woman.

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100g oral glucose tolerance test was carried out in 50 patients with arteriosclerosis obliterans (ASO), 38 patients with Buerger's disease (TAO) and 44 patients as a control group. Responses of NEFA and immunoreactive insulin (IRI) were measured simultaneously.
Diabetes mellitus was complicated in 8% (4 patients) of ASO and in 5.3% (2 patients) of TAO.
As the results of 100g GTT, DM type was 40% (20 patients) of ASO and 26.3% (10 patients) of TAO, normal type was 16% (8 patients) of ASO and 7.9% (3 patients) of TAO.
The rate of impaired glucose tolerance was high in both ASO and TAO.
Response of IRI in ASO had a tendency that its peak was delayed as compared with normal type of a control group which indicated the maximum on 30min.
Response of NEFA in ASO was high and decreased slowly. But on 2 hours, there was no difference between ASO and normal type of a control group. Among each type of glucose tolerance of ASO, there were not many differences in values of NEFA.
Though the number of patients of the following case is different from the above mentioned one, serum cholesterol, triglyceride, Phospholipid and NEFA in both ASO and TAO were higher than those of a control group, but there was no difference between ASO and TAO. On HDL-cholesterol and Lipoperoxide, there was no difference among 3 groups.

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Human serum esterase was purified by affinity column chromatography on a column of covalentry linked p-trimethyl-ammoniumanilinium dichloride to Sephrose 4B. The purified preparation hydrolyzed both benzoylcholine and tributyrin.
This affinity ligand inhibited competitively the hydrolysis of benzolycholine and inhibited noncompetitively the splitting the tributyrin. This might be explained the hypothesis that there are two sites in the active center of human serum esterase, that is, catalytic and anionic sites. Then, the experiments were carried out to clarify the relationship between this esterase and lipase in post heparin plasma. Lipase and esterase activities were estimated in post heparin plasma from patients with various diseases. Lipase and esterase were well correlated with r=0.77, suggesting that some relationship might exist between the lipase and the esterase. Post heparin plasma was applied to DEAE-Sephadex column, and two separate peaks were obtained. Lipase activity was found in the former peak, which also contained slight esterase activity. Another esterase activity was found in the latter peak, which no lipase activity was detected. The esterase in the latter peak corresponded to the purified esterase obtained by the affinity column chromatography. Then, experiments were carried out to examine the substrate specificity between the former peak (lipase fraction) and the purified esterase. Lipase and esterase hydrolyzed both tri and monoacylglycerol. The chain length of fatty acids in esters susceptible to enzymes were shifted to longer side by conversion of the substrate from triacylglycerol to monoacylglycerol. The lipase hydrolyzed the ester longer chain fatty acid both tri and monoacylglycerol as compared to the esterase. From these results, the lipase possessed a tendency to act on more hydrophbic neutral esters, while the esterase was susceptible to more hydrophilic neutral esters.

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It is well known that heparin and heparinoids (HPS) play an important role in the activation of lipoprotein lipase (LPL), whereas it is not so clear whether HPS are necessary any more for the activated LPL itself on the action in the organs and/or tissue or not. On this paper, we tried to detect the relationship between heparin, heparinase and activated LPL in vitro. Citrated post-heparin plasma (PHP) from rabbit was used in this experiment. Heparin content and LPL activity (by NEFA) were detected with and/or without heparinase. Inhibitory effect of protamine sulfate on LPL activity was also detected.
(Results)
(1) Heparin content was markedly decreased by adding of heparinase in vitro.
(2) Heparin content of PHP was abruptly decreased on addition of heparinase, whereas no inhibitory changes of LPL activity were observed on this PHP.
(3) Dose-responded inhibition by protamine sulfate on LPL activity was observed in lower concentration, while in higher concentration the inhibitory effect reached in plateau.
From results mentioned above, we supposed that LPL activity in PHP was mostly induced from peripheral organs, and that heparin was no more necessary for the activated LPL.

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The catabolism of plasma triglyceride is regulated by lipoprotein lipase and lipoprotein lipase activator. Lipoprotein lipase activator was measured by a method which was based on the observation that guinea pig postheparin plasma would not hydrolyze triglyceride unless human serum (or any activator) was added. Plasma lipoprotein lipase activity, lipoprotein lipase activator, triglyceride and cholesterol were measured in 11 patients with type IV hyperlipidemia, 9 normolipidemic males and 10 normolipidemic females. All of them had neither drinking habit, obesity nor liver dysfunction. Lipoprotein lipase activity was selectively measured by an immunochemical method and was within normal limits in all subjects. Activator was significantly higher in type IV hyperlipidemic subjects (229±116%, mean±S. D.) than in normal subjects (male: 129±37, female: 92±18%)(p<0.05), and, as plasma triglyceride concentrations increase, activator (s) tends to increase. However, plasma triglyceride concentrations inversely correlated with activator/triglyceride (p<0.001), and in addition, inversely correlated with lipoprotein lipase activity/activator (p<0.001)
Further studies are necessary to understand the role of lipoprotein lipase activator for the genesis of the hypertriglyceridemia.

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To understand the role of hepatic and extrahepatic triglyceride lipase (TGL) in the metabolism of lipoproteins, we have reported the regulation of hepatic triglyceride lipase by insulin. The purpose of the present study was to determine an assay method of triglyceride lipase activity in rat heart muscle and to investigate the changes in plasma lipoproteins and triglyceride lipase activity in heart muscle of streptozotocin (STZ) induced diabetic rats. Three different methods of determining triglyceride lipase activity in rat heart muscle were studied utilizing the following preparations; 1) acetone-ether powder of heart muscle, 2) heart muscle slices, 3) heart muscle homogenates. Acetone-ether powder of rat heart muscle was prepared according to the method of Tan M. H. et al utilizing 0.05M NH₄OH-NH₄Cl buffer, pH 8.1. Release of TGL by heparin from 100mg of rat heart muscle slice was performed in 2.0ml of Krebs-Henseleit bicarbonate buffer, pH 7.4 with 2.1M glycine, 1.5% bovine serum albumin and heparin 50U/ml under 95% O₂/5% CO₂ at 37°C, based on the method of Lithell H. et al. Rat heart muscle, 200mg, was homogenized in either 4.0ml of 2.1M glycine buffer, pH 8.3, or 0.078M Tris-HCL buffer, pH 7.4 or 0.05M NH₄OH-NH₄Cl buffer, pH 8.1. Triglyceride lipase activity per mg protein of heart muscle was the highest in heart muscle homogenate utilizing 2.1 M glycine buffer, pH 8.3 among the assays investigated. TGL activity in rat heart muscle was activated by rat serum and heparin and inhibited by higher concentration of NaCl, 2.0M of which inhibited completely this activity, as is characteristic of lipoprotein lipase. Twelve-hour fasting increased heart muscle TGL activity from 2.53±0.77 to 4.09±1.28μmol FFA/hour/mg protein. However, heart muscle TGL activities in 48 hour and 72 hour-fasted rats (P<0.05) were lower than those in fed rats. TGL activities in heart muscle homogenates in diabetic rats either 3 days or 4 weeks after STZ injection, were decreased significantly compared with those of control rats. A significant negative correlation between heart muscle TGL activities and plasma triglyceride levels was observed in rats 4 weeks after STZ injection. Cholesterol, triglyceride (TG) and phospholipid in plasma, VLDL (d<1.006g/ml), LDL (1.006<d<1.063g/ml) and HDL (d>1.063g/ml) obtained from rats 4 weeks after STZ injection were studied. Cholesterol and phospholipid in plasma, VLDL and HDL of STZ diabetic rats were significantly higher than those of control rats. Plasma TG and VLDL-TG were extremely higher in diabetic rats than in control rats. Our data indicate the regulation of triglyceride lipase activities in rat heart muscle by insulin and a significant role of this enzyme in pathogenesis of hyperlipoproteinemia of diabetic rats.

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Recently, from our laboratory, it was suggested that serum nonspecific carboxyl esterase was converted to lipase in the arterial wall. The experiments were carried out to clarify characteristics of esterase and lipase of postheparin plasma. Lipase (triolein hydrolysis) and esterase (tributyrin hydrolysis) activities were increased in blood of rats after heparin injection. Hydrolysis of methylbutyrate by serum esterase was observed to be linearly increased at the lower concentration of methybutyrate than that of 0.153M, but no increase was observed at the higher concentration than that of 0.153M. On the other hand, post heparin plasma, to which phenylmethyl sulfonyl fluoride as an inhibitor of esterase was added, scarecely hydrolysed 0.153M of lower concentration of methylbutyrate, but linearly hydrolysed it at 0.153M to 0.459M of methylbutyrate. These results suggest that lipase acts on hydrophobic condition of methylbutyrate and esterase acts on hydrophilic condition of it as a substrate, because 0.153M methylbutyrate or lower is soluble in water and the higher concentration of methylbutyrate than 0.153M is insoluble in water.
Remarkable decrease of lipase activity was observed by trypsin treatment but esterase activity was not. Hydrolysis of 0.153M or lower methylbutyrate as a substrate was unchanted by trypsin treated postheparin lipolytic activity fraction (PHLA-F) compared with nontreated one. But decreased hydrolysis by trypsin-treated PHLA-F was observed when higher concentration than 0.153M of methylbutyrate was used as a substrate.
Lipase activity of PHLA-F, which was decreased by trypsin treatment, was recovered when serum was added. Above these results suggest that trypsin treatment or serum might modify the hydrophobicity of PHLA-F.
1) Kohji Shirai, Nobuo Matsuoka, Yasushi Saito, Akira Kumagai, Toshiharu Muraoka, Hiromichi Okuda: Hyperlipidimia and Atherosclerosis., J. Jap. Atheroscler. Soc. 6 509 (1978):

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Isolated liver parenchymal cells were prepared from adult rat liver by perfusing the livers according to the method of Berry M. N. and Friend D. S. with some modifications. The hepatocytes were cultured in the complete symthetic serum free culture media HI/WO₅/BA₂₀₀₀ under 5% CO₂/95% air at 37°C. The optimum conditions for cell attachment to Falcon plastic dishes (diameter: 60mm) were determined. When approximately (1.5-3.5)×10⁶ cells per dish suspended in HI/WO₅/BA₂₀₀₀ were incubated at 37°C for 4 hours, about 42% of cells inoculated were attached. The experiments were started after the first exchange of the culture media 4 hours after cell inoculation. During first 24 hours after cell inoculation, light microscopic study and the following metabolic function of the cells were investigated:
1. Total protein synthesis
2. Albumin synthesis
3. Hepatic triglyceride lipase (H-TGL) synthesis and its activities
4. Effect of insulin on amounts of H-TGL and its activities
The results of metabolic and morphological studies showed that integrity of cultured hepatocytes was well maintained at least for 24 hours after inoculation. A constant rate of total protein synthesis and albumin synthesis were observed up to 12 hours and then these rates were decreased. A constant rate of H-TGL synthesis which was determined by [¹⁴C] leucine incorporation to H-TGL precipitated by anti-H-TGL rabbit serum, was observed up to 12-24 hours. H-TGL activities were studied in adult rat hepatocytes cultured for 12 hours. A constant increase of H-TGL activity for 4 hours incubation and the plateau of the activity from 4-10 hours were observed and then the activity was decreased. The effect of insulin on H-TGL synthesis was studied in cultured hepatocytes prepared from adult normal and streptozotocin induced diabetic rats which were prepared by intravenous injection of streptozotocin (STZ), 65mg/Kg body weight according to the method of Junod A. et al. Addition of insulin into the culture media increased HTGL synthesis significantly in cultured hepatocytes prepared from STZ diabetic rats. A significant positive correlation was observed between [¹⁴C] leucine incorporation to H-TGL and log₁₀ insulin concentration from 0 to 10μM. However, no effect of insulin on H-TGL synthesis in the concentration up to 10μM was seen in cultured hepatocytes prepared from normal rats. The results presented indicate that H-TGL is regulated partly by insulin for optimal expression and that H-TGL might be a key enzyme for pathogenic features of hyperlipoproteinemia observed in diabetic rats.

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In studying the role of phospholipids (PL) in progression and regression of atherosclerosis, 50μCi of emulsified ¹⁴C- lecithin was injected into 21 male albino rabbits fed 0.67% cholesterol for 207 days, whose body weight averaged 3, 440g, serum cholesterol 930mg/dl and phospholipids 304mg/dl.
Serum lipoproteins were fractionated by the precipitation method using dextran sulfate and Ca into very low and low density lipoproteins (VLDL+LDL) and high density lipoproteins (HDL); VLDL+LDL cholesterol and phospholipid concentrations were 872mg/dl vs 236 mg/dl, while HDL's 48mg/dl vs 84mg/dl, respectively.
¹⁴C-Lecithin was rapidly incorporated into PL in both fractions of lipoproteins, and showed the specific activity (SA) curve consisting of two phases: a steep decline in 10 hours and a slow decrease after it. The radioactivity also appeared in the cholesteryl ester fractions (CE) of both VLDL+LDL and HDL. The SA curve of VLDL+LDL-CE revealed a two-phase increase: a rapid rise in 3 hours, then a gradual increase after it, while that of HDL a three-phase change: a sharp rise in a short time and rather a fast decrease in 3 hours followed by a slow gradual decrease after it. The SA of all the lipoprotein lipids reached an equilibrium in 24 hours, though there was a delay in reaching the equilibrium in atherosclerotic rabbits when compared with normal rabbits, probably due to the expanded serum lipid pool and the decreased LCAT activity in the rabbits fed cholesterol for a long time of period.
These indicate that there occurred an active transfer of lipid molecules between serum lipoproteins either by an enzymatic activity of LCAT or by a mechanical transfer of molecular exchange. The physiological and pathological role of the molecular transfer of lipids between serum lipoproteins was emphasized.

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