Changes in lactate dehydrogenase activity in cultured rat aortic smooth muscle cells with the growth and effect of iron on these changes were investigated.
When smooth muscle cells were grown in the medium which was changed on days 3, 5 and 7 after plating, LDH activity increased with the growth. But LDH activity decreased when the medium was not changed for more than two days.
Maximum proliferation occurred when iron concentration was physiological, whereas increase in LDH activity occurred at about 10% of physiological concentrations and the activity decreased with increasing concentrations of iron.

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We studied the influences of short-term (2-week) cholesterol feeding on the proliferative activity of enzyme-dispersed rabbit aortic smooth muscle cells in primary culture. The cells were cultured in Eagle's minimum essential medium containing 10% fetal bovine serum. Marked subendothelial edema was observed in the aortas of cholesterol-fed rabbits by pathological examination. The enzyme-dispersed smooth muscle cells from control rabbits had a lag phase of 4-5 days before they started to proliferate. On the other hand, cells from cholesterol-fed rabbits started to divide 2-3 days after seeding and tended to grow to higher densities than controls.
Hyperlipidemic serum from cholesterol-fed rabbits slightly enhanced the proliferation of subcultured smooth muscle cells compared to normolipidemic serum when the serum was added at the concentration of 5% in the medium, but rather suppressed it when added at 10 or 20 concentration.
Our results indicate that the enzyme-dispersed smooth muscle cells in primary culture from cholesterol-fed rabbits have greater proliferative activity independent on the presence of hyperlipidemic serum and that the cells themselves have acquired increased proliferative abilities.

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Many investigators have studied the effect of low density lipoprotein (LDL) on the proliferation of arterial smooth muscle cells (SMC). The methodology is not well studied. The purpose of this study was to investigate the method for analysis of the effect of LDL on DNA synthesis in cultured human arterial SMC.
Human arterial SMC were cultured from splenic artery with MEM containing 5% of LPDS (lipoprotein deficient serum from normal subjects). LDLs were ultracentrifugally prepared from normal subjects (n=8) and diabetics (n=12). 0.2×10⁵ cells/dish were cultured with 5% LPDS-MEM for 48 hours. This medium was replaced with fresh 5% LPDS-MEM containing LDL (25 microgr./ml) and 0.5 micro Ci of ³H-Thymidine. After 6 hours intracellular radioactivity was measured and DNA synthesis calculated as cpm/microgr, cell protein. This experiment was repeated for 6 times during 36 hours. In another experiment DNA synthesis in these SMC was studied during 36 hours after ³H-Thymidine addition.
DNA synthesis in SMC cultured without LDL addition was almostly constant during 36 hours, however, the value of cpm/microgr, protein was variated in each 7-experiment. Therefore, we calculated the value of Index as shown below: Index (%)
This Index was useful in comparison of data among 7-experiments. The time point of the maximum value of Index in the culture with each LDL was variated from 18 to 36 hours after LDL addition in the experiment repeatedly performed for 6 hours during 36 hours. The maximum Index was higher in the culture with diabetic LDL than in that with normal LDL. When DNA synthesis was studied during 36 hours after ³H-Thymidine addition, Index in the culture with diabetic LDL was significantly (p<0.05) higher than that in the culture with normal LDL.
We concluded that (1) Index of DNA synthesis was useful for analysing the effect of LDL on SMC in vitro, (2) DNA synthesis should be studied for 36 hours after addition of LDL and ³H-Thymidine.

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High-cholesterol diet induced fatty liver in rats (SHR). Cholesterol ester and triacylglycerol accumulated in large amounts in liver, but the increases of these lipids in plasma were relatively small, suggesting that the step before the secretion of lipids from liver is limited under these conditions. Plasma cholesterol ester consisted of mainly arachidonate species in rats fed normal diet but oleate and linoleate esters increased significantly by feeding high-cholesterol diet. At the 2-position of phosphatidylcholine in plasma and liver, oleate and linoleate increased slightly by feeding high-cholesterol diet. The enzymatic bases for inducing such changes were examined. Microsomal glycerophosphate acyltransferase in liver increased 2 to 3-fold to meet the increased supply of oleate, the synthesis of which was stimulated by 10-fold increase in microsomal Δ⁹-desaturase activity. Endogenous cholesterol in microsomes increased 2-fold and the acyl-CoA: cholesterol acyltransferase activity also increased 3-fold whereas plasma lecithin: cholesterol acyltransferase activity was unchanged. Thus, the increase in cholesterol oleate was due mainly to the increases in acyl-CoA: cholesterol acyltransferase and Δ⁹-desaturase activities in liver.

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An in vitro incubation procedure was established which enabled to estimate the cholesterol translocation between α- and β-lipoprotein regardless of TC levels in serum. This method is based on two principles. The first is the estimation of decrease of free cholesterol in β-lipoprotein fraction of sample serum; the decrease was calculated as the difference between the FC quantities in β-lipoprotein before incubation (βLpFC b. i.) and after incubation (βLpFC a. i.). The second is the esterification during incubation; the esterification arise only in α-lipoprotein particles. The EC quantity, transferred from α- to β-lipoprotein factors, was calculated as follow:
(αLpEC b. i.+EC e. d. i.)-αLpEC a. i. =EC (α→β) (LpEC stands for EC in α-lipoprotein, αLpEC a. i., for EC in α-lipoprotein after incubation, and EC e. d. i., for EC esterfied during incubation.)
The transfer of cholesterol was found to be influenced by factors such as follows: 1) LCAT activity, 2) cholesterol level, 3) triglyceride level, 4) imbalance of β-lipoprotein and α-lipoprotein, 5) imbalance of cholesterol and phospholipid, 6) ratio of βLpPL/αLpPL as well as Atherogenic-Index, and 8) age. In addition, some factors of unknown nature also appeared to influence the cholesterol transfer.

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Small molecular weight apo B_S (B-48) originates from intestine and has different genetic regulation from large molecular weight apo B_L (B-100). Apo B_S will be a marker of exogenous lipoprotein. We have investigated apo B_S concentration in VLDL of primary (especially type IIb and IV hyperlipoproteinemia which are considered to be caused by metabolic derangement of endogenous lipoprotein) and secondary hyperlipoproteinemia associated with diabetes mellitus. Concentrations of lipids in fasting plasma and lipoprotein fractions (VLDL, IDL, LDL and HDL) were determined enzymatically. Apo B subspecies in VLDL were analyzed by SDS-3-15% grandient polyacrylamide gel electrophoresis. Apo B_S ratio (percentage of apo B_S in total apo B) in normal control subjects was 2.34±0.36 and did not show age and sex differences. Apo B_S ratio in primary type IV (4.77±1.48) and type IIb (3.67±0.57) hyperlipoproteinemia were significantly higher than control. Significant positive correlations were observed between apo B_S ratio and plasma concentrations of triglyceride (TG), apo C-II and apo E. There was a significant negative correlation between apo B_S ratio and plasma concentration of apo B. Type II diabetic patients showed a significantly higher apo B_S ratio (4.82±0.49) than control. In the diabetics, significant correlations were not observed between apo B_S ratio and plasma concentrations of TG, apo C-II and apo E. Most of apo B_S ratios in the diabetics were plotted in the positions higher than the regression line in control and primary hyperlipoproteinemia. The data presented suggested the usefulness of apo B_S ratio for understanding of metabolic derangement of exogenous lipoproteins.

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Small molecular weight apo B_S (B-48) originates mainly from the intestine and will be a metabolic marker of exogenous lipoprotein. We reported hypertriglyceridemia and high level of VLDL in MSG rats. In this study we have investigated abnormalities of lipoprotein metabolism by estimating percentage of apo B_S, which will be a metabolic marker of exogenous lipoprotein, in total apo B (apo B ratio) and absolute amounts of apo B_S, apo B_L and apo E in MSG rats. At the 24th week after subcutaneous injection of MSG (4mg/g body weight) neonatally for 5 days, concentrations of lipids and lipoprotein fractions (VLDL, IDL, LDL and HDL) in fasting plasma were determined enzymatically. Apo B subspecies in VLDL were analyzed by SDS 3-15% gradient polyacrylamide gel electrophoresis. MSG rats showed a marked increase of plasma triglyceride and a slight but significant increase of phospholipids. Lipids and protein concentrations of VLDL were significantly higher in MSG rats than in control rats. But there were no changes in the composition of these increased VLDL. Apo B_S ratio was significantly higher in male MSG rats (57.5±1.5%) than in male control rats (30.2±2.5%). However, this ratio was not different in female rats.
Absolute amounts of apo B_S were extremely higher in MSG rats than in control rats (male: 5.0±0.8 vs. 1.1±0.2mg/dl, female: 9.3±1.4 vs. 1.3±0.1mg/dl.) Absolute amounts of apo B_L were higher in female MSG rats (5.9±1.1mg/dl) than in female control rats (0.7±0.1mg/dl), but not different in male rats. Absolute amounts of apo E were higher in MSG rats than in control rats (male: 7.8±0.4 vs. 3.9±0.4mg/dl, female: 13.2±1.8 vs. 4.1±0.5mg/dl). The data presented indicate that MSG-induced obese rats have the metabolic derangement of exogenous lipoprotein, such as apo B_S.

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We studied in vitro and in vivo effect of Triton WR-1339 (T) on canine plasma lipoproteins. We established that T has a unimer molecularr weight of 4, 500, a micellar molecular weight of 180, 000 and a critical micellar concentration (CMC) of 0.018mM. T (2-10mg/ml) induced concentration-dependent structural changes in HDL which were characterized by a progressive displacement of apo A-I from HDL surface without loss of lipids. The addition of T to HDL particle caused an increase in size (95±5Å to 114±7Å). Intravenous injection of T(150mg/kg) caused the partial displacement of apo A-I from HDL surface, followed by the decrease of apo A-I level in the plasma. These structural changes of HDL may be responsible, at least in part, for the hyperlipidemia attending the intravenous administration of T into experimental animals.

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Isolated livers from Japanese monkeys were perfused in order to assess the nature of apolipoprotein A-I secreted directly by the liver and to determine the effect of cholesterol feeding on the production of apolipoprotein A-I and on the apo A-I isoprotein pattern. Perfusate containing ³H-leucine was recirculated for 60 min, followed by additional 2 hours perfusion with fresh perfusate. Cholesterol feeding resulted in an increase in serum cholesterol level and in an decrease in HDL-cholesterol. Regression analysis between the serum cholesterol and HDL-cholesterol levels revealed the negative correlation (r=-0.72). Hepatic production of apo-HDL, however, was increased by the cholesterol feeding. In order to assess the nature of apo A-I secreted by the liver, two dimensional electrophoresis of apo A-I was employed. The control perfusate HDL contained isoprotein 2 (basic and immature apo A-I) and 4 (mature one) present in roughly equivalent amounts. On the other hand cholesterol feeding resulted in an increase in basic apo A-I of perfusate HDL. These observations indicate that cholesterol feeding may increase in the catabolism of HDL, which may be dependent on the conversion of proapo A-I to mature apo A-I.

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pLDLR-1 is a cDNA clone for the LDL receptor. This clone was obtained from a bovine adrenal cDNA library that was constructed by Okayama-Berg method and corresponds to approximately half of the mRNA for the bovine adrenal cortex LDL receptor.
In order to analyze the gene abnormality of the familial hypercholesterolemia, we have gotten this clone from Dr. Russell and studied about it.
We determined the nucleotide sequence of pLDLR-1 with the dideoxy chain terminating method using a general and a reverse M13 primer respectively. The new two nucleotides (Adenine, Cytosine) were revealed at its 5'end.
Then we analyzed human genomic DNA by molecular hybridization with pLDLR-1 as a probe, pLDLR-1 cross-hybridized to the human genomic DNA.
From these studies, we conclude that it will be possible to check the DNA polymorphism in familial hypercholesterolemic patients and normal controls with the pLDLR-1.

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It is well known that atherosclerosis is accelerated in patient with diabetes mellitus (DM). If some plasma proteins undergo non enzymatic glycosylation in DM and certain chemical modifications of LDL give the alterations in their interactions with certain cells, it is easy to understand that atherosclerosis may be relevant in DM. We found new simple method for analysis of plasma Glycosylated beta-lipoprotein (G-b-lipo) by affinity column chromatography and measured the plasma G-b-lipo in DM or non diabetic subjects. The Value of plasma G-b-lipo was higher in DM than non diabetic subjects. The higher levels of plasma G-b-lipo in DM appear to reflect hyperglycemia for the past several days and correlate with other established parameters of hyperglycemia such as glycosylated hemoglobin.
Also, we observed that the clearance rate of G-b-lipo prepared from rabbit plasma was lower as compared to native b-lipo in identical rabbit.
These results gave us special interests with regard to the role which G-b-lipo may play on the hyperlipidemia and angiopathies in DM.

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Hypertriglyceridemia seems to be a risk factor for coronary artery disease. The accumulation of triglycerides in the bloodstream could result either from oversecretion of very low density lipoprotein (VLDL) triglycerides from the splanchnic region or be due to impaired clearance by peripheral tissues. Although there is already evidence to suggest that splanchnic secretion of VLDL triglycerides is increased in type IV hyperlipidemia, this has come from studies with isotope tracers measuring the kinetics of incorporation into plasma triglycerides. Because of difficulties in interpretation of data from tracer studies, not all studies have demonstrated increased rates of VLDL-triglycerides turnover in patients with hypertriglyceridemia. For this reason Kayes and Galton have developed a method to measure rates of VLDL-triglycerides secretion which principle is to inhibit a lipoprotein lipase by protamine sulphate and to leave inflow of VLDL-triglycerides unimpaired.
Then we have used a high performance liquid chromatography (HPLC) to analysis lipid compositions of lipoproteins.
Six patients with hypertriglyceridemia and 3 normal subjects were examined. A significant rise in serum triglycerides and VLDL-triglycerides (p<0.001) was observed at 1 hour in patients with hypertriglyceridemia.
HPLC has been shown to be an useful method for analysis the lipoprotein from the serum during the course of heparin and protamine sulphate administrations.

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Serum concentrations of α₁-antitrypsin (α₁-AT), α₂-macroglobulin (α₂-MG) and elastase I (IRE) were assayed in 133 patients with type 2 diabetes mellitus in relation to diabetic vascular complications. Serum α₁-AT and α₂-MG were assayed by single radial immunodiffusion. IRE was assayed by radioimmunoassay. Hypertension (HT), ischemic heart disease (IHD) and calcification of the aortic arch (CAL) were used as parameters for macroangiopathy. Diabetic retinopathy (RP) was used as a parameter for microangiopathy.
The findings may be summarized as follows; α₁-AT was significantly higher level in the group with HT, IRE was significantly lower level in the group with IHD demonstrated by ECG, and α₂-MG had a tendency to be higher level in the group of higher plasma glucose level or in the group treated with insulin. All of them had no significant relation with RP.
These observations may suggest that α₁-AT and IRE levels correlate with the presence of diabetic macroangiopathy.

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The purpose of this study is to determine effects of insulin or oral antihyperglycemic agents of plasma apoprotein levels and plasma lipoprotein composition in the diabetics.
Thirty diabetics were divided into 3 groups; Group-I=10 patients treated with insulin, Group-II=10 patients treated with Glibenclamide, Group-III=10 patients treated with Gliclazide. Control group was constituted by 10 healthy volunteers. All of the subjects had no evidences of cardiovascular disease and hyperlipidemia. After an over-night fasting, blood samples were collected into tube with EDTA. Plasma lipoproteins were separated into each lipoprotein fraction by the ultracentrifuge. Lipid composition of each fractions were measured by the enzymatic methods. Plasma apoprotein A-I, A-II, and B concentrations were measured by the method of single radial immunodifusion.
HDL-cholesterol levels of the diabetics were significantly higher than those of the control men. However, cholesterol concentrations of the HDL subfraction were different among the groups. Plasma apo A-II of the patients showed the levels as same as that of the healthy men, whereas plasma apo A-I and B concentrations were significantly lower and higher in the Glibenclamide-group, respectively. Lipid composition in each fraction was also different among the groups.
According to this study, it is possible that a treatment with oral antihyperglycaemic agent for diabetics accelate the development of atherosclerosis, especially in normolipidemia. In this paper, we want to discuss effects of a agent to control blood glucose level on plasma lipoprotein metabolism.

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In order to assess the short-term effects of insulin on lipid metabolism, serum triglyceride and cholesterol were determined in 58 patients with NIDDM before and after 2 hour euglycemic clamp studies. The steady state of the plasma insulin level of 78.0±4.2μU/ml was produced by primed continuous infusion of human regular insulin (HI-rDNA, Lilly) at 40mU/m²/min for 120 min. 15%glucose was automatically infused to maintain the basal plasma glucose level. Serum triglyceride and cholesterol levels significantly fell to 84.7±2.2% and 91.7±1.4% of the basal level respectively during 2 hour glucose clamp studies. In the lipoprotein fraction of triglyceride (TG), VLDL (Sf60-400)-TG significantly decreased, but IDL₁ (Sf20-60)-TG and IDL₁ (Sf12-20)-TG showed no significant change. Each lipoprotein cholesterol slightly fell and in particular the decrease of LDL-cholesterol was significant in the group with good insulin sensitivity (glucose infusion rate≥6mg/kg/min).
In conclusion
(1) Insulin infusion decreased serum triglyceride and cholesterol level during 2 hour euglycemic clamp studies.
(2) Decrease in serum TG by insulin infusion was mainly due to the decrease in VLDL-TG.
(3) Significant decrease in LDL-cholesterol in the group with good insulin sensitivity during short-term insulin infusion indicates the important role of insulin on LDL catabolism.

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Diabetes mellitus is frequently complicated with atherosclerosis. PWV (pulse wave velocity) is an indicator for the sclerotic change of thoratic and abdominal aorta. We studied the relation between PWV and lipid metabolism in diabetics.
The subjects who took part in this study were 54 diabetics who were aged from 50 to 59 years old and had a duration of illness over 5 years. Fasting blood sugar level and the concentration of glycosylated hemoglobin (HbA₁) were measured. Serum total cholesterol (TC), triglyceride (TG), HDL-cholesterol (HDL-C), apo B and apo A-I were measured by enzymatic methods and SRID, respectively. PWV was measured by the methods of Hasegawa, et al.
We concluded that (1) PWV increased in poorly controlled diabetics (FBS 180mg/dl) more than in well controlled diabetics (FBS 119mg/dl), and (2) PWV had significant positive-relation to serum TC, TG, apo B, apo A-I and apo B/A-I ratio, and had significant negative-relation to HDL-C.
According to these data we should improve not only glucose metabolism but also derangement of lipid metabolism.

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It is our purpose to clarify the relationships among blood sugar level, serum lipids and obesity in the cases with glucose intolerance. In observation I, 40 male outpatients with diabetic type in 75g OGTT were 47.5±12.4 (M±SD) years old and were investigated for 5.1±2.3 months. They ingested 1, 626 kcal and 228g of carbohydrate per day on an average. When fasting blood sugar (FBS) decreased, serum cholesterol (Chol) decreased and triglyceride (TG), serum HDL-Cholesterol (HDL-C) or (ChoI-HDL-C)/HDL-C tended to decrease. Serum TG decreased when it's initial values were in the range from 150 to 250mg/dl.
In observation 2, 18 male outpatients with diabetic type in 75g OGTT were 46.7±10.5 years old, who showed no change or the increment of body weight for 3.2±0.8 months. They ingested 1, 599 kcal and 219g of carbohydrate.
When FBS decreased, serum cholesterol decreased and TG, HDL-C and (ChoI-HDL-C)/HDL-C tended to decrease. Serum TG decreased when it's initial values were in the range from 100 to 250mg/dl.
In observation 3, 45 male outpatients were investigated, who showed the more decreased ratio of ΔIRI/ΔBS (30 min), that was more than 0.1 in the decrement of this ratio, after about one year. 7 cases with borderline type in 50g OGTT showed the decrement of both body weight and serum TG, and tended to show the increment of HDL-C. 7 cases with the increased body weight tended to show the increment of TG. 9 cases with diabetic type showed the decrement of body weight, and tended to show the decrement of FBS, cholesterol or TG, while to show the increment of HDL-C.
In observation 4, one young female case with borderline type became hyperglycemia of 348mg/ dl in FBS, and showed the increments of serum TG (the percentage of increment 206.4%), VLDL (222.3%), NEFA (292.7%), Cholesterol (169.8%), LDL (142.3%) or β-Lipoprotein (266.4%).
From the above findings, there are suggested the relationships among FBS, TG or the change of body weight in the cases with glucose intolerance.

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In type V patient, premarin injection ((12mg/day)×4 days) caused an increase in VLDL-triglyceride and a decrease in LDL-cholesterol and HDL-cholesterol. Premarin inhibited lipoprotein lipase (LpL) activity in post heparin plasma and purified bovin milk LpL activity.
Premarin increased the bind of LpL to VLDL, but newly added VLDL-triglyceride was not hydrolyzed in the presence of premarin. These results suggested that premarin may directly inhibit the catabolic system of VLDL into LDL and the inhibition may be due to re-utilization failure of the LpL enzyme caused by premarin, which strengthened the binding of LpL uneffectively to the lipid surface.

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Many studies have been reported so far as to the lipid metabolism in thyroid diseases. In this report, 14 patients with hyperthyroidism (Graves' disease) have been studied divided into three stages such as (I) untreated hyperthyroid state. (II) just after the patients became euthyroid and (III) clinical euthyroid state more than 2 months after stage (II). Total cholesterol (T-chol) triglyceride (TG), phospholipid (PL), free fatty acid (FFA), VLDL, LDL and HDL-cholesterol (HDL-chol) were measured at each stage. In stage (II) T-chol, TG, PL, VLDL, LDL and HDL-chol were significantly higher than those in stage (III). In the hyperthyroid state, both anabolism and catabolism are thought to be acceralated, but catabolism is said to be more acceralated in the lipid metabolism. This may be the reason that many lipids are low in the hyperthyroid state except for FFA. The present study suggests that catabolism may be normalized much faster than anabolism in the clinical course of hyperthyroidism in the treatment with antithyroid drugs.

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In order to evaluate influences of thyroid hormone on serum lipid and apoprotein metabolism, serum lipid, lipoprotein and apoprotein levels were studied in 15 hypothyroid and 18 hyperthyroid patients before and after therapy. Lipoproteins were isolated by serial ultracentrifugation. Apoproteins were determined by SRID method.
Total cholesterol (TC), intermediate density lipoprotein (IDL), low density lipoprotein (LDL) and high density lipoprotein (HDL) cholesterol (C) levels (mean±SD) were elevated in the untreaed hypothyroid patients compared to 26 normal subjects (261±66mg/dl versus 194±37mg/dl, 13±12mg/dl versus 8±4mg/dl, 158±67mg/dl versus 105±29mg/dl and 72±26mg/dl versus 59±16mg/dl, respectively).
Otherwise, TC and LDL-C levels were significantly lower in hyperthyroid patients as compared to the control group (132±23mg/dl versus 194±37mg/dl and 59±20mg/dl versus 105±29mg/dl, respectively). When hyperthyroid patients were restudied after restoration of the euthyroid state, HDL-C increased from 56±17mg/dl to 67±18mg/dl.
The LDL-C/HDL-C ratio was 2.8 in the hypothyroid patients, compared to 1.9 in the controls and 1.1 in the hyperthyroid patients. The very low density lipoprotein (VLDL-C/VLDL-triglyceride (TG) ratio were elevated in the hypothyroid patients. Apo B, apo C-II and apo E concentrations were increased in the hypothyroid patients compared to the controls (139±39mg/dl versus 103±23mg/dl, 5.5±2.3mg/dl versus 3.6±1.1mg/dl and 5.9±1.9mg/dl versus 4.2±0.8mg/dl, respectively). Apo A-I, apo A-II and apo B levels were decreased in untreated hyperthyroid patients compared to the euthyroid state (113±29mg/dl versus 151±36mg/dl, 22±5mg/dl versus 31±9mg/dl and 70±30mg/dl versus 103±25mg/dl, respectively).
On rendering the patients euthyroid, most of these changes in the lipid and apoprotein values were reversed.

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At the last meeting, we reported that the arginase activity in erythrocyte in cases of cerebral and myocardial infarctions showed lower values than that of cerebral bleeding and in normal cases.
In this meeting, we discussed whether the erythrocyte arginase is involved in the inhibition of thrombosis in vivo or not. Erythrocyte ghosts of rabbits were prepared, and the purified arginase was enclosed in them by dialysis methods. The arginase-loaded erythrocytes were transfused back to the same rabbit, in which arachidonic acid (2mg/kg) was injected into the carotid artery after 30min. to induce experimental thrombi, and the presence of thrombosis was examined microscopically.
Next rabbits were given the arginase per as and the effect of oral administration of the arginase on platelet aggregation were observed.
It has been demonstrated that the arginase inhibits the formation of thrombus induced by arachidonic acid due to increasing its concentration in the erythrocyte. The arginase inhibited ADP induced platelet aggregation strongly with its oral administration.
Consequently, it was concluded that the arginase in the erythrocyte has an important role for the inhibition of thrombus formation.

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An emulsion of fish oil (from sardines) rich in eicosapentaenoic acid (EPA) was manufactured. One hundred ml of the emulsion contained 10g of fish oil, of which 3g was EPA. We administered 100ml of the emulsion to six rabbits intravenously on days 1 and 4. Blood samples were taken on days 0 and 7 and compared. The EPA content in serum phospholipids and phospholipids in red blood cell membranes increased about 10 and 4 times, respectively. Platelet aggregation induced by collagen (10μg/ml) and thrombin (0.3u/ml) was depressed significantly. Control experiments performed in exactly the same way except that soybean oil emulsion was used instead of fish oil emulsion produced almost no significant changes. High levels of EPA in serum and RBC membranes and depression of platelet aggregation were obtained by this method in a very short time.

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Platelet aggregation was examined in the presence of aspirin plus dipyridamole or trapidil. Platelets play a significant role in the development of thrombosis and atherosclerosis, which can be prevented by antiplatelet therapy. The most common antiplatelet agent is aspirin, which suppresses thromboxane A₂ activity in platelets, but in large doses, inhibits prostacycline (PGI₂) production in vascular endothelial cells. Thus, this study examined whether a small amount of aspirin plus dipyridamole or trapidil could inhibit platelet aggregation but not the production of PGI₂ in vascular endothelial cells. Platelet aggregation was induced with 2.2μg/ml collagen and observed using an aggregometer. The three drugs were added to the in vitro reaction mixture or administered in various concentrations.
In vitro experiments showed that each antiplatelet agent could inhibit platelet aggregation in a dose-dependent manner. A synergistic effect was noted with 10μM aspirin plus 100μM trapidil. In vivo experiments showed a similar tendency when 40mg aspirin was orally administered with 50mg dipyridamole or 50mg trapidil.
These results suggest that a small amount of aspirin combined with dipyridamole or trapidil is very effective for antiplatelet therapy. Also, in vitro experiments may be clinically useful for evaluating the efficacy of antiplatelet agents.

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We have examined following 15 risk factors for atherosclerosis on the 1, 279 persons (male 521 and female 758) who had visited health examination at Fujieda city during the period of 1981.
CRFI was newly settled as follow: (T-cholest—erol-HDL-CHO)/T-cholesterol. Age, obesity, electrocardiogram, ocular fundus, blood pressure, GOT, T-cholesterol, tryglyceride, HDL-cholesterol, white blood cells count (WBC), red blood cell counts (RBC), hematocrit (Ht), hemoglobin (HGB), blood sugar, uric acid and cholesterol risk factor index (CRFI) were determined.
Higher points of CRFI (>0.8) were counted 5.7% on male group and 5.0% on female group. A significant high co-relation between Ht and WBC was noted on the male group. In the female group, significant differences were noted among the 1) age, uric acid and blood pressure systolic and diastolic, 2) age-T-cholesterol-ocular fundus, 3) age and CRFI, and 4) TG-blood pressure systolic.
The common risk factors for both male and female groups were as follow: 1) Fundscopieage-blood pressure syst. and diast. are co-related, 2) Fundus hypertonie-ECG-blood pressure systolic, 3) Uric acid-tryglyceride-T-cholesterol-CRFI-obesity, 4) Fundus hypertonie-sclerosis-Keith Wagener finding, 5) Ht-RBC-HGB, and 6) HDL-CHO had negative co-relation to TG.

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Coronary angiography was performed in 17 male patients with myocardial infarction and under the age of 40 years. Of these, 4 (23.5%) had 2 vessel disease and 9 (52.9%) had 1 vessel disease, while no significant coronary obstruction was demonstrated in the remaining 4 cases. None had 3 vessel disease. The most common site of the lesion was at the anterior descending branch (48.9%), which was followed by the left circumflex branch (23.5%) and the right coronary (17.6%) in that order. Corresponding to the high incidence of lesions in the anterior descending branch, the myocardial infarction was located in the anterior wall in 13 cases.
A multivariate analysis dealing with the severity of the coronary sclerosis and eight risk factors, family history, obesity, smoking habbit, hypertension, diabetes mellitus, serum total cholesterol, serum triglycerides and serum uric acid, showed that the multiple correlation coefficient in this relatively young age group was 0.77 which was greater than the corresponding figure of 0.35 in the older age group. The standard regression coefficient in the young age group was 0.43 for total serum cholesterol and 0.41 for serum uric acid, respectively.

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Previously we have reported that the rate of cerebral vascular disorders as well as the cases of severe hypertension in old ages are significantly less in Hachijo island than in the whole country.
We have also examined the serum eicosapentaenoic acid (EPA), EPA/AA ratio and platelet aggregation in the groups eating fish well and seldom (the group eating fish well represents those eating fish at every meal), and found that the group eating fish well had high concentration of EPA, high EPA/AA ratio and diminished platelet aggregation.
In this paper we attempted quantitative examination of apolipoproteins of the habitants in Hachijo island. Blood samples, obtained from 45 subjects, 25 male and 21 female subjects, were analyzed. Apolipoproteins (Apo A-I, Apo A-II, Apo C-II and Apo E) were estimated by the method of Single Radical Immunodiffusion (SRID). Apo C-II in male was significantly higher than that in female. Apo A-I and apo A-II in the group of obesity were higher than those in nonobesity. There was no significant difference in the composition of apolipoproteins between the group of smoking and non-smoking, while apo A-I and apo A-II in the group of drinking were significantly higher than those in the non-drinking group. A-I/A-II ratio in the group eating fish well was higher than that in the no fish group. Significant correlation were found among the values of serum total cholesterol, triglyceride and apo E, apo C-II.

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Normal and two-kidney one-clip hypertensive (RVH) Wistar rats were fed ethanol in their drinking water in concentration of 10% for eleven weeks.
Blood pressure were monitored and levels of serum lipids (TC, EC, TG, TPL, HDL-C) were determined.
After sacrifice, the entire heart was weighed. Hydroxyproline content in aorta and left ventricle, and elastin content in aorta were measured.
There were no further augumentation in blood pressure both in normal and RVH rats fed with ethanol.
Serum TC, TPL and HDL-C decreased in RVH rats, while ethanol-fed RVH animals showed no changes.
Heart weight to body weight ratios were maximal in ethanol-fed RVH rats (p<0.01vs. RVH rats).
Hydroxyproline content in left ventricle was maximal in ethanol-fed normal rats (p<0.01vs. RVH and RVH-ethanol groups). Elastin content in aorta decreased in RVH-ethanol group (p<0.01vs. ethanol-fed group).
These data indicate that ethanol administration has quite different influences on RVH-rats from that on normotensive rats.

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Long-term cold stress, that is, exposure to 4±1°C temperature for one hour every day, was given to Sprague-Dawley rats and Wistar rats. The cold stress load was started from one month of age and carried on over several months. Changes in the systolic blood pressure and cardiovascular system of the rats were observed in this experiment.
The results were as follows:
1) Hypertension was produced in the rats.
2) Angionecrosis of the coronary artery, Fibromuscular sclerosis of myocardial artery and PN-like lesion in the heart, kidney and mesentery were observed in Sprague-Dawley rats.
3) In Wistar rats' heart, myocardial cell damage, fibrosis and inflammatory cell infiltration were detected.
4) Compared to non-stress Wistar rats, cold stress rats showed a significant thickening of the media of the myocardial artery.

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Adrenal regeneration hypertension was performed on Wistar rats, WKY and SHR by Skelton's method. For the first time, we observed glomerular foam cell in the kidneys of those experimental rats. Glomerular foam cells were seen in the rats that were markedly hypertensive and hypercholesterolemic state, and in those that showed a high secretion of protein in the urine. We suggested that those experimental rats exhibited the state of nephrotic syndrome.
Glomerular foam cells were noticed in approximately one-third of the rats. Moreover, we found glomerular foam cells in all of the Wistar rats and WKY fed a high cholesterol diet, that is, a 5 percent cholesterol and 2 percent cholic acid-including diet. An electron microscopic study of the glomerular foam cells revealed that the cell origin was probably circulating monocyte and mesangial cell. Glomerular foam cells were located in the mesangial matrix or subendothelium of the glomerular capillary.

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Accelerated arteriosclerosis is thought to be a major factor increasing morbidity and mortality in uremic patients. Since there is a lack of definite clinical evidence supporting this concept, the present experiments was undertaken to study the relations between uremia and arteriosclerosis using an experimental uremic model in male Wistar rats.
Chronic uremia was induced by 5/6 resection of the total kidney. Six or eight months later, blood was obtained after overnight fasting and the thoracic aorta was removed, 6 components in media of thoracic aorta, namely, smooth muscle cell (SMC), eastin (EL), collagen (CL), acid mucopolysaccharides (AMPS), glycoprotein (GP), and calcium phosphate (Cal), were stained specifically and measured by the method of microspectrophotometry (MSP). Furthermore, biochemical studies were performed on these materials. The results obtained were as follows:
1) By histochemical observation, abnormality of 6 components of media were found particularly in advanced uremic rats.
2) Increase of SMC, and decrease of EL and AMPS were observed by MSP in uremic rats. There was no significant difference in CL and GP between uremic rats and controls.
3) There was no significant difference in plasma calcium between uremic rats and controls. Plasma phosphorus was elevated in uremic rats. Aortic calcium and phosphorus contents were markedly elevated, particularly in advanced uremic rats.

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Ninety seven 2 month-old wealing swine fed either a basal ration alone or the basal ration containing different levels of vitamin D (100, 000, 200, 000 or 300, 000IU per ton diet) plus skimmed milk powder (50 or 100kg per ton diet) for 4 months. No significant changes were observed in plasma calcium, cholesterol, triglycerides or phospholipid levels between the experimental groups fed diets containing 200, 000IU of vitamin D per ton diet with or without skimmed milk powder. Marked coronary arterial lesions were found in the group which was fed the diet containing 300, 000IU of vitamin D plus 100kg skimmed milk powder per ton of diet. The intima of these coronary arterial lesions contained activated smooth muscle cells, degenerate smooth muscle cells without stainable lipid, and lipid-containing cells derived from macrophages and smooth muscle cells. These results suggest that excess dietary vitamin D plus skimmed milk powder has a synergistic effect on the development of coronary atherosclerosis.

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It was previously reported that excess iron accelerated the progress of noradrenalin-induced atherosclerosis. In the present experiment, influence of addition of beef tallow to the diet on atherosclerosis was examined.
Of 14 male Japanese white rabbits, five were fed on an iron-adequate diet with vegetable oil (I-group), four fed on an excess-iron diet with same oil (II-group) and five fed on the same diet as in the II-group to which beef tallow was added (III-group).
All of the rabbits were fed for 18 weeks. They were loaded by drip infusion of noradrenalin from the 14th week to the 16 week of the feeding period.
The results were as follow:
1) The incidence rate of grossly visible atherosclerosis was 60% in I-group, 75% in II-group and 20% in III-group.
2) The level of total-iron in liver was significantly higher (p<0.001) in II-group than in I- and III-groups and that in III-group was between those in I- and II-groups. The level in small intestine was lowest in I-group and highest (p<0.025) in III-group.
3) The level of calcium in the thoracic aorta was extremely high in II-group and that in III-group was lower than in I-group.
4) The level of total cholesterol in the abdominal aorta was significantly higher (p<0.05) in II-group than in I- and III-groups.
5) The level of triglyceride in the thoracic aorta was higher in II-group and that in III-group was lower than in I-group.
6) The severity of atherosclerosis in the thoracic aorta was parallel with both calcium and triglyceride contents.
From the above results, it was assumed that beef tallow might have inhibited development of atherosclerosis due to excess iron, because P/S ratio in the diet influenced on permiability of the membrane of cells, and as a result of that also on passing of iron through the membrane.

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In order to clarify the mechanisms underlying atherogenesis, we studied effects of drugs on the arterial intimal thickening induced in rabbits by the cuff method. Under pentobarbital anesthesia, the carotid artery was sheathed with a cuff made of a polyethylene tube (1.5cm long PE-280). Animals were given normal diet and water ad lib., and i. m. drugs 5 times a week for 3 weeks. After sacrifice, their sheathed arteries were observed histologically. Although non-treated arteries had no intimal thickening, smooth muscle cells appeared in the intima after the cuff-treatment. Intimal thickening increased gradually and reached its peak 3 weeks after the cuff-treatment, and it shrank thereafter. Inflammatory changes were observed in the adventitia throughout the experimental period.
Scanning electron microscopy revealed endothelial injury after the cuff-treatment. A number of leukocytes and platelets adhered to the exposed internal elastic laminae 1 day after the cuff-treatment. Injured endothelium began to repair within 3 days, and almost complete recovery from the damage was attained 2 weeks thereafter. Dexamethasone inhibited both intimal thickening and adventitial inflammation in a dose-dependent manner (0.01-10mg/kg), but more than 1mg/kg of dexamethasone made animals thin remarkably. A calcium antagonist FR34235 inhibited the intimal thickening in a dose-dependent manner (0.01-10mg/kg), and had no effect on animals' weight. These results suggest that passing endothelial injury and adventitial inflammation are involved in the atherogenesis by the cuff-treatment, and this method is a suitable model to estimate antiatherogenic effect of drugs. A calcium antagonist FR34235 is expected to be an antiatherogenic agent.

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The purpose of this study is to clarify the relation between the pathological changes and clinical factors. As the former, myocardial fibrosis, coronary stenosis, diameter of aortic root, pathological changes of aortic root and heart weight were taken, and as the latter, aging, blood pressure, diabetes mellitus, serum cholesterol and ECG were taken.
The subjects were 100 autopsied cases including 62 males and 38 females aged 18-93 years without congenital heart disease, valvular disease, cardiomyopathy, myocarditis and myocardial infarction.
Each pathological and clinical items was classified into two or three categories and then analyzed by multi-variate-statistical analysis.
As the results of this study, the obvious correlation were observed in following three groups:
1) severe myocardial fibrosis and hypertension.
2) increased heart weight, moderate myocardial fibrosis, diabetes mellitus and LVH in ECG.
3) severe coronary stenosis and hypercholesterolemia.

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Phospholipids in the aorta play an important role in the progression or regression of atherosclerosis. The properties of phospholipids are in part affected by the fatty acid composition of phospholipids, especially by polyunsaturated fatty acids (PUFA) at the second position.
1-Acylglycerophosphate acyltransferase was reported to be a key enzyme to incorporate PUFA into the second position of phospholipids. In the aorta, there are few reports on this enzyme in spite of its significance. In this study, characterization of this enzyme was carried out in the rat aorta and the role of this enzyme in the phospholipid metabolism in the aorta was discussed.
Whole homogenate enzyme activity was onefifth of that in the liver. Enzyme activities increased as a function of enzyme protein, incubation time, substrates (PUFA) and acceptor concentrations. When lyso PC was used as the acceptor, the enzyme kinetics showed a sigmoid curve with increased concentration of either the acceptor (lyso PC) or substrate fatty acids. However when lyso PE was used as the acceptor, the kinetics showed a Michaelis-Menten type curve with increased concentration of either the acceptor (lyso PE) or substrate fatty acids.
When lyso PC was used as the acceptor, linoleic acid had a higher affinity to the enzyme than arachidonic acid because the S₅₀ value of linoleic acid was lower and the V_max value of linoleic acid was higher than those of arachidonic acid. When lyso PE was used as the acceptor, the K_m and V_max values of arachidonic acid were higher than those of linoleic acid.
When linoleic acid was used as the substrate, the synthesis of phosphatidylcholine (PC) was higher than phosphatidylethanolamine (PE) at any concentration of linoleic acid. However, when arachidonic acid was used as the substrate, the synthesis of PE was higher than PC at low concentration of arachidonic acid, but that of PC was higher than PE at high concentration of arachidonic acid.
Fatty acid specificity was as follows:
When lyso PC was used as the acceptor, V_max was in the order of linoleic acid>linolenic acid>eicosapentaenoic acid. When lyso PE was used as the acceptor, V_max was in the order of arachidonic acid>eicosapentaenoic acid>linoleic acid. Saturated fatty acid (palmitic acid) and docosahexaenoic acid were poor substrates.
These results indicate that acyltransferase was regulated not only by the concentration of enzyme protein, acceptors and substrates, but also by the specificity of the acceptors and substrates. PUFA such as arachidonic acid are decreased in the phospholipids of atheromatous lesions. The impaired regulation of this enzyme might be responsible for the abnormal phospholipids in the athermatous lesions.

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Lipid droplets were prepared from atherosclerotic aorta of WHHL rabbit. Two peroxidized fatty acids, which were much more polar than unoxidized fatty acids, were recovered from saponified lipid droplets by reverse phase HPLC analysis. Maximum absorption of these peroxidized fatty acids was observed at 233nm, suggesting that they are cis-trans conjugated diene fatty acids. One of the fatty acids corresponded to 13-hydroxyoctadecadienoic acid.

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The effects of porcine pancreatic elastase on experimental atherosclerosis rabbits have been studied. Rabbits were fed 1% cholesterol for 4 months to induced experimental atherosclerosis. Two groups of atherosclerosis rabbits were kept for 5 months as follows; First group (C-group) was fed normal diets and placebo, 2nd group (E-group) was fed normal diets and orally administrated with elastase 100mg/kg/day (97units/mg). The lipids levels in plasma, in lipoprotein, and apo-protein, cholesterol esterases and Suc (ala)₃ pNA hydrolytic activity in aorta were analyzed. Plasma cholesterol was rapidly decreased in E-group, and those contents in VLDL and LDL fraction were decreased in same group comparing with C-group. Also, in lipoprotein fraction, Apo E and Apo A were increased in E-group.
Neutral cholesterol esterase activity and Suc (ala)₃ pNA-hydrolytic activity were different from those in C-group.
Pathologically, fatty streak involvement in aorta was 50% decreased in E-group. The foam cells in intima and hyperplastic intima of aorta were decreased by elastase administration. These results indicated that elastase had accelerative effect on regression of the experimental atheroscerosis.

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Elaszym® (10, 800Unit/day) was administered to 38 hyperlipidemic patients for 16 weeks. Those patients were classified to two groups according to their sensitivity to this drug in serum cholesterol level. In high responders, serum cholesterol was lowered from 273±13 to 231±11mg/dl (p<0.001) but in low responders, it was not changed significantly. About the background of these two groups, age, sex and type and level of hyperlipidemia were not different between them. Apoprotein E was decreased significantly in the both groups. Increase in apoprotein C-II and C-III were statistically significant in the low responders. Atherogenic index and ratio of total cholesterol—HDL cholesterol to apoprotein B were significantly decreased in the high responders. These changes of apoproteins might be related to anti-atherogenic action of this drug.

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We have studied the effects of elastase-like enzyme (ELE) on the lipid metabolism in rats, especially about lipoprotein metabolism and post heparin lipolytic activity (PHLA) in triglyceride metabolism. Lipids in plasma and liver were slightly decreased by administration of ELE. The LCAT activity in plasma was lowered to about 80% of the control value. In lipoprotein fraction, although VLDL-TG. decreased by ELE treatment, plasma post heparin lipolytic activity was not changed between the both groups. The decrease of Apo E in VLDL fraction and increase of Apo E in HDL fraction were observed in ELE group by SDSPAGE. It is considered that the increase of Ano E in HDL fraction is brought through an increase of Apo E rich HDL (nascent HDL) released from liver which comes from decrease of the LCAT activity in plasma. In isoelectric focusing patterns of Apo HDL, increased contents of Apo E₃, E₄ and decreased contents of Apo A-I which is activator of plasma LCAT.
These results suggest that ELE induced increase in transport of HDL into the liver together with accelerated metabolism of plasma lipoprotein.

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