Diets high in cholesterol (Ch) have been shown to cause significant elevations in plasma Ch in several animals including man. Elevated plasma Ch has been shown to be an independent risk factor for coronary heart disease (CHD). Dietary Ch has been considered to be one of the factors which contribute to the recent decrement of mortality from CHD in USA and European countries.
Consistent features of Ch-induced hypercholesterolemia in several animals are the occurrence of β-VLDL in the d<1.006g/ml fraction, an increase in the IDL and LDL, a decrease in the typical HDL (HDL without E) and the appearance of HDL_c (HDL with E). β-VLDL from Ch-fed animals stimulate accumulation of cholesterylester in macrophage which might be a precursor of foam cell in atherosclerotic lesion.
Plasma lipoprotein abnormalities are common in patients with diabetes mellitus (DM). They are of considerable clinical interest because of their relationship to atherosclerosis. The higher incidence of CHD and other cardiovascular diseases associated with diabetes has been attributed to the disordered lipoprotein metabolism.
The present study were designed to compare the effect of dietary Ch in non-diabetic control rats and diabetic rats treated with or without insulin. Control rats and streptozotocin-induced diabetic rats treated with insulin 4U/rat/day or without insulin were fed the diet containing 1% Ch, 1% lard and 0.3% sodium taurocholate for 4 weeks. Hypothyroidism was induced by additional feeding of 0.1% propylthiouracil (PTU). Without insulin treatment, Ch-fed diabetic rats were severely hypercholesterolemic and had higher concentrations of lipoproteins of VLDL (d<1.006g/ml), IDL (1.006<d<1.019g/ml) and LDL (1.019<d<1.063g/ml) as compared with Ch and PTU-fed control rats (plasma Ch: 2466±1503.6 vs 76±11.9mg/100ml, VLDL-Ch: 1316±1246.7 vs 24±15.6mg/100ml, IDL-Ch: 691±266.7 vs 218±76.6mg/100ml, LDL-Ch: 437±145.3 vs 102±26.4mg/100ml). Concentration of HDL (1.063<d<1.210g/ml) was decreased as compared with chow-fed control rats (HDL-Ch: 22±7.1 vs 65±10.9mg/100ml). However, HDL concentration of Ch-fed diabetic rats was comparable with Ch-fed control rats (HDL-Ch: 21±9.4mg/100ml). β-VLDL increased predominantly in Ch-fed diabetic rats, whereas IDL increased mainly in the Ch and PTU-fed control rats. According to sodium dodecyl sulfate (SDS)-10% polyacrylamide gel electrophoresis (PAGE), VLDL and IDL from Ch-fed diabetic rats were unusual in that they contained more apolipoprotein (Apo) E, A-I, and A-IV. Insulin treatment greatly decreased the concentrations of VLDL, IDL and LDL and distribution of apolipoproteins in lipoprotein subfractions changed toward normal. β-VLDL from Ch-fed diabetic rats consisted of relatively uniform spherical particles and these size were smaller than VLDL from chow-fed control rats (37.8±18.6 vs 47.6±20.0nm).
In order to investigate the site of synthesis of β-VLDL, we subdivided Apo B species with SDS-3.5% PAGE. β-VLDL contained both faster migrating Apo B with an apparent molecular weight of 220, 000 and slower migrating Apo B with an apparent molecular weight of 360, 000. The pattern was comparable with that of control VLDL. It has been indicated that the liver synthesizes both Apo B subspecies in rat. Therefore, our observations indicate that at least the liver contributes to β-VLDL synthesis.
Catabolism of β-VLDL in the liver was investigated. Scatchard analysis of binding characteristics of β-VLDL prepared from Ch-fed diabetic rats to the isolated rat liver cells showed the existence of saturable and specific binding sites. The Bmax was decreased in chow-fed diabetic rat as compared with chow-fed control rat (1830 vs 776ng protein/10⁶ cells), whereas the Kd

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Changes of plasma levels of apoproteins A-I, A-II and C-III were determined after oral or intravenous fat administration and oral glucose intake. The A-I and A-II levels increased in most of the subjects after fat ingestion but no changes or even a slight decrease in the levels of A-I and A-II were observed after intravenous fat infusion. The levels of C-III increased concomitantly with the increase of triglycerides levels after fat ingestion as well as fat infusion. Oral glucose administration did not affect on the levels of A-I and A-II whereas a slight increase in the levels of C-III was observed. These observations suggest that the increase in the levels of A-I and A-II after fat ingestion is a consequence of an increase in apoprotein synthesis in the intestine during fat absorption. The increase in the levels of C-III after fat ingestion as well as fat infusion seemed to be related to the capture of C-III in the triglyceride-rich particles, i. e. C-III accumulated in the circulation with triglyceride-rich particles. However, it appeared also to be possible that the rate of C-III synthesis increases during hyperlipidemia induced by fat ingestion or fat infusion.

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To understand the catalytic sequence of LPL reaction and the mechanism by which apo C-II increases the LPL activity, the role of phospholipid vesicles and the effect of apo C-II on esterase activity of LPL which hydrolized short chain fatty acid ester (p-nitrophenyl butyrate) were investigated. Esterase activity was enhanced by the addition of dipalmitoyl phosphatidyl choline vesicles (DPPC) and the enhanced rate was dependent on the physicochemical properties of the phospholipids; below transitional temperature [DPPC 42°C, dimyristoyl phosphatidyl choline (DMPC), 24°C], both phospholipid vesicle enhanced esterase activity of LPL at the same extent (800%). The addition of DPPC vesicles increased the Vmax value of LPL for PNPB, but did not change Km values.
The DPPC-triolein vesicles which contained 0.5% tri[1-¹⁴C]oleoyl glycerol were prepared and the effect of apo C-II on the rates of hydrolysis of trioleoyl glycerol and of PNPB were simultaneously determined. The rate of PNPB hydrolysis decreased, and the rate of trioleoyl glycerol hydrolysis increased reciprocally as the added amount of apo C-II increased.
These results suggest that LPL undergoes conformational change on the surface of phospholipid so that esterase activity increased, and then apo C-II increases the affinity of catalytic site for trioleoyl glycerol, such that reciprocal effects of apo C-II on lipase and esterase activity of LPL were observed.

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The Subunits of the C and E apoprotein of VLDL was studied in 21 subjects with IHD and 15 normal subject whose age, plasma lipid and coronary risk factors were matched.
VLDL of fasting plasma from subjects were separated into subfractions by ultra-centrifugation. The tetramathylurea-soluble apolipoprotein were separated by electrofocus-electrophoresis and the relative proportions of apo E_II and E_III determined.
We found the characteristic of the E apoprotein in IHD with hyper-VLD lipoproteinemia (VLDL-cholesterol≥15mg/dl) as follow;
1) Apo-E/apo-C ratio of VLDL in IHD was lower than that in control. In these difference between IHD and control, a significantly low value of this ratio was found in IHD when VLDL-Ch/TG ratio was under 0.24.
2) In distribution of E apoprotein of VLDL, apo E_III in IHD was higher than that in control. But high value of apo E_III/E_II ratio was found only in low value of apo E/C ratio.
3) The subjects with both low value of apo E/C ratio and high value of apo E_III/E_II ratio had significantly decrease in HDL-cholesterol (p<0.025).
It was suggested that the characteristics of proportions of E apoprotein might cause an inhibition of VLDL-catabolism and might be part of the pathogenesis of intermediate density lipoprotein found in ischemic heart disease.

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To elucidate the role of apolipoproteins in the evolution of hyperlipidemia, VLDL apolipoproteins E & C from human plasma, especially of the patients with alcoholic hypertriglyceridemia, was analyzed by isoelectric focusing.
VLDL prepared from fasting plasma by ultracentrifugation was delipidated with acetone-ethanol. Apo VLDL was applied on the 7.5% polyacrylamide gel containing 8M urea and 2% ampholite, and isoelectric focusing was done with 150V for 16 hours at 4°C, as described by Warnick, except that holizontal plate gel and the wider pH range (pH 3.5-pH 10.0) were adopted. After fixation, staining with Coomassie Brilliant Blue and destaining of the gels, relative proportions of apolipoproteins and their isoproteins were determined by densitometry at 580Å. In alcoholics, Apo E₃/Apo E₂ ratio in VLDL were lower than those of others and tended to decrease as their plasma triglyceride levels increased. Alcohol tolerance test resulted in inconsistant changes in the apolipoprotein or isoprotein destribution, partly because of the insufficiency of the amount of loaded alcohol. Apo E isoprotein patterns were examined in 42 cases. Among them, the incidence of “variant pattern” with apo E₄ isoprotein was significantly higher in patients with alcoholic hypertriglyceridemia (type IV+type IIb) than in other hyperlipoproteinemics and normal controls.
Although the special function of apo E₄ isoprotein has not been reported, it is suggested that the exsistence of apo E₄ per se or the decrease of total apo E or the relative decrease of apo E₃ may modify the lipoprotein metabolism and constitute one of the factors which facilitate hyperlipoproteinemia, triggered such as alcohol ingestion.

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Average serum apo A-I and A-II levels, but not apo B level, of cerebrovascular patients, either with cerebral infarction or with cerebral hemorrhage, were lower than those in healthy controls. Although each of these apoprotein levels was not different between the two groups of patients, A-I/A-II ratio in patients with cerebral infarction was larger than those in hemorrhagic patients and in controls. Apo A-I and A-II levels in hypertensive patients were not significantly different from those in controls. However, among hypertensives, cases under anti-hypertensive treatment exhibited larger A-I/A-II ratio and higher apo B level than those in untreated cases.
From these result, lipoprotein metabolism in patients with cerebral infarction can be characterized not only by hypo-high-density-lipoproteinemia but by the qualitative change of HDL that manifested in large A-I/A-II ratio. Since this ratio was also large in hypertensives under the treatment, lipoprotein metabolism in such cases may be also distinguished from normal by the same qualitative change. This suggests that consideration of lipoprotein metabolism may be necessary during antihypertensive measures for the final object of such measures: prevention of vascular diseases.
Platelet aggregatory activity correlated negatively with serum apo A-I level, and platelet adhesiveness correlated positively with A-I/A-II ratio.
Hypertension ranks first among risk factors for the development of cerebrovascular diseases, besides lipoproteins may be involved in the pathogenesis of cerebral infarction in conjunction with other factors such as hypertension and thrombotic tendency.

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Eighty-eight autopsied cases were analyzed in order to clarify the significance of papillary muscle fibrosis in ischemic heart diseases without typical myocardial infarction. The photographs of the sections from the coronary arteries were taken to measure stenotic grades of the lumen by the manual optical pictures analyzing system. The specimens from each papillary muscle devided into five vertical sections. Quantities of fibrosis within these stained sections were measured by the point counting method.
Results were as follows;
1) The fibroses within papillary muscles were more frequent in the distal portion of the left ventricular free walls than in the proximal portions.
2) There was no significant difference of fibroses between the anterior and posterior papillary muscles.
3) The papillary muscle fibroses well correlated with the coronary sclerosis and the aging.

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The present study was designed to investigate the synthesis of lipoproteins in liver, using human adult hepatocytes in primary monolayer culture.
The hepatocytes were isolated by collagenase and Dispase (Godo Shusei Co.) digestion from the liver specimens taken for a biopsy from patients. Approximately 85% or more of total isolated cells excluded trypan blue dye. They were inoculated in serum-free media, DM-170 (Kyokuto Seiyaku Co., Ltd.), supplemented with 10^-6M insulin, 10^-6M dexamethasone, human fibronectin and bovine albumin on collagen precoated culture dishes. These dishes were placed in a humidified incubator at 37°C under 5% CO₂/95% air and the medium was changed every 24 hours. Twenty four hours after cell inoculation, the initially round shape of the hepatocytes changed to a cuboidal form as they contacted neighboring cells. The fine structure of cultured hepatocytes such as Golgi apparatus, rough and smooth endoplasmic reticulum in the cytoplasm, and desmosomes and bile canaliculi in the intercellular bordary were observed on day 5 by electron microscopic pictures. A constant rate of total protein synthesis, determined by [¹⁴C]-leucine incorporation into trichloroacetic acid (TCA) insoluble material, was observed up to 96 hours of the cultured periods. Albumin and several serum proteins in the concentrated culture media were detected by double immunodiffusion and immuno-electrophoresis between anti human-serum antibody and concentrated conditioning culture media in which the hepatocytes were cultured for 24 hours.
Human plasma HDL₃ 1.110<d<1.210g/ml and LDL₂ 1, 019<d<1, 050g/ml were fractionated from pooled normal human plasma by sequential ultracentrifugation. Each lipoprotein fraction was dialyzed, and immunized into rabbits by injection with Freund's complete adjuvant. Each anti-sera was used as anti Apo A-I or Apo B antibody for immunological studies.
A single precipitin line was observed between anti Apo B sera and conc. culture media (approximately 25 times), in which the hepatocytes were cultured for 48 hours. However, Apo A-I was not detected in the same culture media (concentrated by approximately 50 times).
The hepatocytes were cultured with [¹⁴C]-leucine in DM-170 under the same condition, incorporation of [¹⁴C]-leucine into Apo A-I or Apo B in the media was investigated. After four days of culture, the culture media was pooled and analyzed by anti Apo A-I or anti Apo B sera and anti-rabbit IgG goat sera. The incorporated radioactivities to Apo A-I and Apo B was 371 (92*) and 2886 (183*) dpm/dish/4 days, respectively (control*).
In conclusion, we showed directly synthesis and secretion of Apo A-I and Apo B by the cultured adult hepatocytes of human. This culture system can be utilized for the metabolic study of not only lipoproteins but also hormones and drugs.

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There is little known on the antithrombotic effects of niceritrol, such as on platelet retension and aggregation. Methods: (1) After over night fasting, blood samples were obtained from healthy volunteers before and 4 hr after single dose of oral administration of 500mg niceritrol. Platelet retension on glass beads column and aggregation were measured. Conversion of ¹⁴C-arachidonic acid (AA) was carried out for 1 min in washed platelet. (2) Two groups of Rabbits were fed either control diet or high cholesterol diet. Each group was further divided into two subgroups. One subgroup was administered niceretrol 150mg/kg/day, and the other was not. After 4 and 8 weeks of feeding, rabbits were sacrificed. The production of prostacyclin-like substance by a thoracic aorta was measured by the inhibition of human platelet aggregation and by the RIA of 6-keto-PGF_1α. Results: (1) A single dose of oral administration of niceritrol to the volunteers significantly decreased collagen induced platelet aggregation. The conversion rate of ¹⁴C-AA to TXB₂ in platelet suspension was significantly decreased (18.9± 4.9% to 7.7±4.4%, p<0.001). Platelet retension was decreased. (2) In every subgroup, niceritrol exerted no effect on prostacycline-like substance production by aortas.

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To clarify the mechanism of cholesterol ester deposition in the arterial wall, the reconstructed LDL (rLDL) and acetylated LDL (acLDL) cholesterol ester metabolism in the rat peritoneal macrophage and the arterial wall were studied. Reconstructed LDL was prepared by the method of Krieger et al. It is reported that there are acid (pH 4.5) and neutral pH 7.5) cholesterol esterase (CEase) in the arterial wall. rLDL-cholesterol oleate and acLDL-cholesterol oleate were hydrolized by rat arterial homogenate and by rat peritoneal macrophage homogenate only at pH 4.5. Next, microsomal resterification system of cholesterol which was product hydrolized in lysosome was studied. ReLDL[³H]-cholesterol oleate were at first incubated with particulate fractions of arterial wall homogenate or macrophage homogenate at pH 4.5, then were incubated at pH7.5 with the addition of [¹⁴C]oleoyl-CoA, and formed amount of [³H]cholesterol-[¹⁴C] oleate were measure after separating with thin layer chromatography. The rate of reesterification of cholesterol was enchanced by the addition of supernatant fraction with the arterial wall, but with peritoneal mecrophage, it was decreased by the addition of supernatant fraction. These results suggested that LDL-cholesterol ester metabolism was greatly affected by the factor contained in the supernatant fractions. The hydrolysis of reLDL-cholesterol oleate by both arterial and macrophage homogenate at pH 4.5, was enhanced by the addition of tocopherol. Farther more, tocopheroltreated reLDL cholesterol oleate was much more hydrolized by arterial homogenate or by macrophage homogenate than nontreated reLDL cholesterol oleate, and also tocopherol-treated arterial wall homogenate or macrophage homogenate hydrolized reLDL-cholesterol oleate much more than untreated homogenates. On the other hand, reesterification of once hydrolized reLDL cholesterol by tocopherol-treated arterial wall and macrophage homogenates were decreased comparing to nontreated homogenates.
These results suggest that LDL-CE were mainly hydrolized at acid area in the microphage and arterial wall. And once hydrolized cholesterol oleate was resterified in the neutral area. Tocopherol may prevent the deposition of cholesterol ester by facilitating the hydrolysis of LDL-cholesterol ester in lysosome and by reducing the reesterification of once hydrolized cholesterol in microsome.

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Pantethine (PaSS) was administered per orally at the dose of 600mg per day for 3 months to hemodialysis patients who were on a strict diet control and had taken no lipid lowering drugs. Serum total cholesterol (TC), triglycerides (TG), phospholipids (PL), and high density lipoproteincholesterol (HDL-C) levels were determined at 0, 1, 2, and 3 months after the drug treatment, respectively. As a result, the following findings were observed.
1) In hypercholesteremic subjects, TC levels were decreased with months while PaSS reversed the change in hypocholesteremics.
2) PaSS decreased TG levels in most of the patients, paticularly in hypertriglyceridemics.
3) PL levels gradually increased with months after PaSS administration regardless of the corresponding TC and TG levels.
4) HDL-C levels tended to be decreased at one month and significantly elevated at 3 months after treatment with PaSS in hypertriglycemics.
From our previous and present studies, we have confirmed that PaSS partially improves serum lipid abnormalities in hemodialysis patients even though long term administration of PaSS.

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Pantethine (600mg/day) was administered perorally to hemodialysis patients with type IIb and IV hyperlipidemia for three months. Various lipoprotein parameters before and after the drug administration were determined. VLDL (d< 1.006g/ml) and intermediate density lipoproteins (IDL)(d=1.006-1.019g/ml) were isolated by preparative ultracentrifugation. High density lipoproteins (HDL) were separated by magnesium dextransulfate precipitation method. The measurement of sialic acid in VLDL was performed according to Warren's thiobarbituric acid assay. Lipid levels in VLDL, IDL and HDL were determined by routine methods, and protein concentration in VLDL and IDL by Lowry's method. The percent distribution of apo C-II and apo C-III in VLDL was estimated by Kane's polyacrylamide gel electrophoresis, and apo B levels in VLDL were determined by single radial immunodiffusion method.
Consequently, the following results were summarized.
1) Sialic acid content in VLDL of hemodialysis patients increased not only in the levels but also in the ratio of sialic acid to protein, while pantethine did not affect these changes. With respect to apoproteins containing sialic acid, apo B levels significantly decreased but the percentage of apo C-III tended to increase after pantethine administration.
2) Pantethine normalized an increase in the total amounts of VLDL and IDL of hemodialysis patients.
3) Pantethine increased HDL cholesterol and phospholipid levels which usually decreased in hemodialysis patients.
These results led us to a conclusion that pantethine had no effect on VLDL sialic acid levels but increased HDL in parallel to the normalization of the deranged VLDL and IDL metabolism, regardless of a decrease in the apo C-II/C-III ratio of VLDL, in hemodialysis patients.

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In studying the metabolic characteristics of human serum lipids and lipoproteins, we have investigated the relationship of the baseline level of serum lipids to the response to the drug treatment. We have analyzed the correlation between the initial lipid levels and the magnitudes of change in lipids in 80 subjects with hyperlipidemia in the niceritrol double blind study and 871 patients with hyperlipidemia in a pantethine open study.
In both studies, the reductions in total cholesterol and triglycerides revealed a positive correlation with their baseline concentrations; higher the pretreatment levels, greater the reduction. The elevation in HDL-cholesterol had a negative correlation with the initial levels; lower the initial level, greater the increase. These relationships were represented by the regression lines between them.
The ratio of the effective cases in both studies also showed a positive relation with the baseline levels; greater the abnormalities in initial levels, higher the ratio of effective case. These relationships were expressed by a logistic function curve.
These results indicated that human serum lipids and lipoproteins had a metabolic characteristics of an intrinsic tendency where the abnormally high or low lipids restore the stable optimal levels.

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Elastase complex is used to the treatment of atherosclerosis. In order to clarify such therapeutic effect of elastase complex as improving metabolism of the connective tissues and lipids, by solely elastase or in combination with the coexisting other factors, atherosclerosis was experimentally induced in rats (by administering 2.45×10⁵units/kg b.w./day of Vit. D₂ for 4 days, and then with atherogenic diet for 5 weeks). Elastase complex (elastase (E), 90 EL.U/mg) and partially purified elastase (elastase (Ep), 326 EL. U/mg, Eisai) were i. m. injected in 450-540 EL.U/kg b. w./day to different group of rats, and the effects of both elastase (E) and elastase (Ep) in various organs (aorta, heart, liver, kidney, lung) were compared.
It was found as the results that;
1) Both elastase (E) and elastase (Ep) exerted anti-hyperlipemic effects as determined by total-and free-cholesterol, phospholipids and triglycerides.
2) Cholesterol level in the arterial elastin fraction (elastin-cholesterol) was tend to be reduced by both elastase (E) and elastase (Ep) but that in the collagen fraction (collagen-cholesterol) was tend to be increased.
3) Glycosaminoglycan (GAG) content in the aorta was not altered by the both elastase (E) and elastase (Ep), but the incorporation of [³⁵S] sulfate into aortic sulfated GAG increased to approximately 3 times, and the fact suggests the metabolic turnover of GAG was stimulated.
4) Cholesterol accumulation in the heart was prevented by both elastase (E) and elastase (Ep).
5) The effects of the elastase is prominent in the heart and artery where the connective tissue (collagen and elastin) and lipid (cholesterol) contents are well correlated.
Elastase, either elastase (E) or elastase (Ep), shows same effects on the connective tissue metabolism and lipid metabolism, so that effect of elastase may be ascribable to the elastase activity itself and not to any other factors as far as the present study being concerned.

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Twelve cases of atherosclerotic patients, and four patients with drug induced-hepatitis who had hyperlipemia and high alkali-phospatase, were treated by elastase (5400 ELU daily) for 12 weeks. Serum total cholesterol decreased significantly after 8 week-treatment in the liver disease group. Serum triglyceride level decreased significantly from 131mg/dl to 84mg/dl in the liver disease group after 4 week-treatment. Serum HDL-cholesterol level was elevated from 44mg/dl to 81mg/dl in the liver disease group. Meanwhile GOT level had decreased from 180 units to 58 units after 10 week-treatment. Serum GPT was also improved from 242 units to 46 units after 50 daystreatment by elastase. In conclusion, elastase was effective for drug induced-remittend-hepatitis but the effect was enhanced by the concomitant use of steroids.

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Daily 5, 400 EL units of elastase were administered for 12 weeks to 114 patients with hyperlipoproteinemia or hypo-alphalipoproteinemia and the effect of the drug on their serum lipids and lipoproteins were investigated.
Although serum total cholesterol was not afected significantly by the treatment, HDL cholesterol increased accompanied with a decreased tendency of VLDL-LDL cholesterol. Consequentaly, VLDL-LDL/HDL cholesterol ratio decreased markedly. HDL cholesterol in both groups of the patients with high and low HDL cholesterol levels at pretreatment increased by elastase administration. By contrast, a significant decrease was observed only in a group of the patient with high VLDL-LDL cholesterol level but not in the low group.
Among the measured serum lipids, free fatty acid, peroxide, beta-lipoprotein, VLDL-LDL cholesterol and VLDL-LDL/HDL cholesterol ratio decreased significantly, and HDL cholesterol, phospholipids increased, but apo A protein, triglyceride and LCAT activity were not affected by the treatment.
Percentage of alpha-lipoprotein to total lipoprotein increased, and those of pre-beta and betalipoprotein tended to decrease. Apo C II and C III ratio increased after the treatment of elastase in the patients with low levels in these apoprotein subclass. Analysis of HDL fraction showed that there were remarkable increase in phospholipids and esterified cholesterol accompanied with slight increase of free cholesterol and triglyceride and without significant change in apo A protein. These findings suggest that the transport of cholesterol by serum HDL in circulating blood might be accerelated by elastase, because esterified cholesterol and phopholipids increased in HDL per unit apo A protein.

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We observed the hemodynamic and antisclerotic effects of Elastase (10800 E. S. units daily, orally) in 16 patients having arteriosclerotic disease for a period of 6 to 12 months.
As the indices of arteriosclerosis, we used the pulse wave velocity and the volume elasticity in the Windkessel system, and got those indices and other hemodynamic parameters by Wezler's and Blumberger-Holldack's methods non-invasively and mechanocardiographicaphically. The data were analysed at 4 stages, that is, before administation, within 3 months, from 3 to 6 months and over 6 months after administration of Elastase.
At each stage, the values of pulse wave velocity were 918±170, 784±186, 803±131, 781±102cm/sec, and volume elasticity, 2355±602, 1926± 557, 1984±462, 1950±343dyne/cm² respectively, and the decrease of the indices was statistically significant (p<0.025-0.05). Total serum cholesterol and triglyceride were almost within normal limits at each stage (208±82, 261±112, 212±41, 186±40mg/dl; 150±89, 142±86, 172±102, 148± 99mg/dl, respectively).
Total peripheral resistance was slightly decreased, consequently cardiac output was slightly increased. Blood pressure, heart rate, systolic time intervals, and the indices of cardiac function or cardiac contractility, that is, ET/PEP, SV/ET and A wave ratio of apexcardiogram, were not changed and remained within normal limits at all stages.
We conclude that Elastase has a good influence upon the arteriosclerotic patients whose levels of total serum cholesterol and triglyceride are within normal limits as the agent did in the patients with hypercholesterinemia and hypertriglyceridemia, and the ameliorating effect on the pre-existing arteriosclerosis. This conclusion in clinical study is in agreement with many reports in animal experiments.

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Isopycnic density gradient ultracentrifugation (single spin method) reported by Nilsson J. et al (Anal. Biochem., 110; 349, 1981) was employed to isolate serum lipoproteins in normal controls and patients with various diseases. Consequently, the following findings were summarized.
1) The chemical composition and apoprotein constituents of normal human lipoproteins, namely low density lipoproteins (LDL), high density lipoproteins₂ (HDL₂) and HDL₃, isolated by the method were same as those obtained by conventional ultracentrifugation.
2) The method was not useful for the separation of very low density lipoproteins (VLDL) and intermediate density lipoproteins (IDL).
3) In normal controls, the lipoprotein profiles were different from individual to individual. On the other hand, the specific lipoprotein profiles in various metabolic disorders were observed. In addition, the lipoprotein profiles changed in parallel to the clinical coarse. In some diseases, such as obstructive jaundice, chronic renal failure and hyperlipidemia, the lipoproteins shifted to abnormal density which was not seen in normal controls.
These results indicate that this method is useful for the studies on serum lipoprotein metabolism, particularly LDL and HDL, not only in normal controls but in patients with various diseases.

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Treatment of familial hypercholesterolemia with diet, medication or operation has met with discouraging results. Previous reports suggested that plasmapheresis or extracorporeal removal of low density lipoprotein (LDL) by means of heparin agarose beads could successfully treat severe hypercholesterolemia.
We studied a removal of plasma LDL by adsorption chromatography with porous glass. Following results were obtained.
1) Batch and column chromatography with porous glass decreased the concentrations of plasma cholesterol.
2) The porous glass bound LDL and high density lipoprotein (HDL), the binding of the LDL being greater than that of HDL.
3) The porous glass did not alter the concentrations of various serum enzymes and electrolytes, but, a slight decrease was noticed in the total serum protein.
4) Plasma cholesterol concentrations were reduced in WHHL rabbit after extracorporeal treatment of plasma over porous glass.
These data suggested that a removal of plasma LDL by extracorporeal treatment of plasma over porous glass might be an effective method for reducing the plasma cholesterol in familial hypercholesterolemia.

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It was reported that type III hyperlipoproteinemia showed characteristic lipoprotein changes, namely appearance of β-VLDL, and an increase of cholesterol and apoprotein E and deficiency of apoprotein E3 in VLDL. In the cholesterol fed guinea pig, plasma lipoprotein feature was similar of human type III hyperlipoproteinemia. In the present study, plasma lipoprotein changes and isoelectric focussing pattern of apo VLDL in control and cholesterol fed guinea pig were reported. Control guinea pigs were fed with commercial chow and cholesterol group animals were fed with the same basal diet added 1% cholesterol and 5% coconut oil for 2 weeks. In the cholesterol guinea pigs cholesterol in VLDL was increased (Fig. 1), agarose gel electrophoretogram of serum showed broad β band (Fig. 2) and apoprotein E in lipoprotein fractions was increased. (Fig. 3) The analytical ultracentrifugation of IDL and LDL (d: 1.006-1.063) showed increase of IDL and LDL in cholesterol fed guinea pigs as compared to control. (Fig. 4) The isoelectric focussing pattern of apo E was identified to six bands, and apo C was four. As compared with human, the first and second band from the cathod were correspond to apo E3 and E2 respectively, and the former was markedly diminished in both control and cholesterol group. (Fig. 5, 6) From these results it was suggested that the metabolism of VLDL remnant in guinea pigs was similar to that of human type III hyperlipoproteinemia. Guinea pig was considered to be a good animal model for studying human type III hyperlipoproteinemia.

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Rabbit antisera monospecific to human Lp(a) lipoprotein were produced and used to immunochemically determine serum concentrations of Lp(a) lipoprotein in healthy subjects and patients with ischemic heart disease (IHD), cerebral infarction and various diseases. The incidence of IHD patients having high serum levels of Lp(a) was significantly higher than that of healthy controls. No significant correlations were found between serum levels of Lp(a) lipoprotein on one hand and serum levels of total cholesterol, highdensity lipoprotein cholesterol or triglyceride on the other. When the Lp(a) phenotype distribution was determined according to the agar double diffusion test of Ouchterlony, the frequency of the type Lp(a⁺) was estimated as about 30% for Japanese.
Lp(a) lipoprotein was delipidated with a mixture of ethanol and diethylether and extracted with tetramethylurea to remove any protein other than apoprotein B. Insoluble residue of protein was dissolved in 10% sodium dodecyl sulfate (SDS) solution and then subjected to SDS polyacrylamide gel electrophoresis. We found two apoprotein bands on polyacrylamide gels: one corresponded to apoprotein B in low-density lipoprotein (d=1.030-1.040g/ml) which was considered as “B-100” of Kane, and the other was believed to be a new and slightly larger subspecies of apoprotein B. The aparent molecular weights determined by SDS polyacrylamide gel electrophoresis were 320, 000 dalton for the former apoprotein and 350, 000 dalton for the latter one, respectively.

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Age and sex related alterations of serum lipids and relative distribution of individual lipid in HDL, VLDL and LDL were studied on 230 subjects of healthy adults (114 males, 116 females) including 26 octogenarian and 13 nonagenarian in Kochi district. The lipids in serum lipoprotein separated by electrophoresis on agarose were determined by use of scanning densitometer after visualization by enzymatic reagents.
The levels of total cholesterol (TCh), total phospholipid (TPL) and total triglyceride (TTG) showed continuous increase with aging, to a peak in the seventies, then decreased by eighties and nineties. Each concentration of the lipids in nineties was 168.8, 174.5 and 96.2mg/dl respectively. In every age group, the levels of TCh and TPL in females were slightly higher than those in males, but TTG level of females was lower than that of males.
HDL-C (%) of TCh did not alter in each age group, and showed 24.6% in eighties and 25.4% in nineties. LDL-C (%) increased after seventies, showed 67.6% at eighties and 68.5% at nineties. VLDL-C (%) decreased after eighties. In males, HDL-C (%) increased with aging and showed the highest value of 28.9% at nineties, but in females, it declined gradually with advancing age.
HDL-PL (%) of TPL gradually decreased to seventies, then increased after eighties and beyond. LDL-PL (%) increased gradually with aging and VLDL-PL (%) decreased after eighties.
HDL-TG (%) of TTG showed no marked change in every age group. VLDL-TG (%) remained almost 60% between the ages from thirties to seventies, but markedly decreased to 38.8% at eighties and 23.1% at nineties, while LDL-TG (%) was nearly 30% to seventies and increased to 50.0% in eighties and 67.3% in nineties.
The amount of lipids in HDL was almost constant in each age group. The content of lipids in VLDL and LDL increased to a peak in seventies and then decreased at eighties and beyond, but the amount of lipids in LDL after eighties was still higher than those of under sixties.
In conclusion, total concentration of each lipid, relative distribution of individual lipid in HDL, VLDL and LDL and lipid composition of each lipoprotein fractions significantly changed with aging, particularly altered at eighties and nineties largely due to the marked change in triglyceride concentration. From our observation of crosssectional changes with age in serum lipoproteins, the age of seventies may be a turning point of the biochemical aging process.

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Triolein particles stabilized with phosphatidylcholine were prepared in two different diameters. Binding of human plasma apolipoproteins A-I, C-II, and C-III to those particles were studied by using the technique of gel permeation chromatography in a Sepharose CL-6B column. All these three apolipoproteins bound to the surface of the lipid particles without altering major structure of the particles. The analysis of apolipoprotein binding isotherm revealed that a dissociation constant of apolipoprotein A-I for the large particle was greater than that for the small particles by more than ten times. Contrarily, apolipoproteins C-II and C-III bound to the large particles as strongly as to the small particles. The maximum binding level was similar to each other among the three apolipoproteins for the two lipid particles when those were compared on the basis of amino acids per phospholipid. These results, suggesting that those apolipoproteins share common binding sites of the lipid particles, account for the characteristic distribution of those apolipoproteins among various classes of lipoprotein in plasma.

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Apolipoprotein C-II (apo C II), a protein constituent of human very low density lipoproteins, is the activator for lipoprotein lipase (LPL). It had been established that apo C II has amino acid sequence of the 78 residues.
To determine the minimal sequence requirements for activation, we have prepared synthetic fragments of apo C II and tested them for their ability to activate LPL. The COOH-terminal synthetic fragment 76-78, 75-78, 74-78, 73-78, 72-78 were tested.
All fragements had a little ability to activate LPL. How ever, the fragment corresponding to residue 75-78 increased the hydrolysis more significantly to compare with other synthetic fragments.

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Apo E levels were measured by double antibody radioimmunoassay. Plasma apo E levels in normal subjects were 9.5±1.8mg/dl. Both LCAT and LPL deficient patients had abnormal plasma lipoprotein patterns as well as 3-4 folds higher plasma apo E levels than those of normal controls.
In order to characterize abnormally high levels of apo E in these two diseases, we analyzed the distribution of apo E in plasma lipoproteins using gel chromatography (Bio-Gel A5m). In plasma of normal subject, apo E was mainly distributed in triglyceride-rich lipoproteins (Fraction A) and large HDL (Fraction B). LPL deficient patient had large Fraction A and small Fraction B, and LCAT deficient patient had large Fraction B.
From these results, we suggested that the increase of plasma apo E in LPL deficiency is due to the retention of triglyceride-rich lipoproteins and that the increase of plasma apo E in LCAT deficiency is due to the defect of apo E transfer from HDL to triglyceride-rich lipoproteins.

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Serum apolipoprotein A-I (A-I) and A-II (A-II) levels were measured in normal subjects by electroimmunoassay. Both A-I and A-II levels increased with age, had the peak in the young and decreased. A-I levels were higher in females than in males in the young. A-II levels were not different between males and females in any age group.

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The concentrations of serum apolipoproteins A-I and A-II were determined in 64 hyperlipidemic subjects by the method of SRID and rocket immunoelectrophoresis. Further determinations were carried out on the levels of apoprotein B (rocket technique), the concentration of triglycerides, total cholesterol and HDL cholesterol.
The concentrations of apo A-I and A-II in the type IV cases were lower than in type II subjects. The A-I and A-II levels were significantly correlated with the levels of HDL cholesterol but not with the levels of triglycerides or total cholesterol.
The concentrations of apo B in the hyperlipidemic subjects were increased markedly and correlated with the levels of total cholesterol but not with the levels of triglycerides.
The concentrations of apo A-I in hyperlipidemic subjects determined by SRID method were correlated with the values obtained by the rocket technique (r=+0.76, n=63), and indicated the validity of the method.

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Serum HDL₂- and HDL₃-cholesterol (HDL₂-C and HDL₃-C) levels were measured in 96 healthy subjects (44 men, age 43±13 yrs; 52 women, age 40±11 yrs) in order to clarify the role and metabolism of HDL subfractions. The healthy subjects, who had no abnormalities in triglyceride, total cholesterol, obesity index, blood pressure, ECG findings, blood sugar, liver functions, and renal functions, were obtained from the health testing center.
1) Mean serum HDL₂-C level was significantly higher in healthy women than in healthy men (p<0.001). However there was no difference between the mean serum HDL₃-C level of healthy men and that of women (Fig. 1).
2) There was a negative correlation between smoking and HDL₂-C in healthy men (r=-0.34, p<0.05). And a possitive correlation between drinking and HDL₂-C was observed (r=0.36, p<0.02). However there was no correlation between smoking or drinking and HDL₃-C (Fig. 2).
3) HDL₂-C level has a tendency to decrease paralled to HDL-C level, on the other hand there are no apparent changes in HDL₃-C level except under the range of less concentration of 40mg/dl of HDL-C level in healthy women. However in the range of less than 40mg/dl of HDL-C, HDL₃-C also its decrease with the increasing value of HDL-C level (Fig. 3).
4) Fig. 4 shows the relationship between HDL₂-C and HDL₃-C in healthy momen. HDL₃-C level shows its increase with the increasing value of HDL₂-C under the range of less than 20mg/dl of HDL₂-C. But in the range of more than 20mg/dl of HDL₂-C, HDL₃-C level shows no apparent changes (Fig. 4).
Fig. 1 and Fig. 2 indicate that in healthy subjects HDL-C level depends mainly on HDL₂-C level. But Fig. 3 and Fig. 4 indicate that under the range of less than 40mg/dl of HDL-C or 20mg/dl of HDL₂-C, HDL₃-C level shows its decrease with the decreasing value of HDL-C or HDL₂-C. In a previous report we showed that in patients with atherosclerotic disease both of HDL₂-C and HDL₃-C were lower than in healthy subjects. Considering these results not only HDL₂-C level but also HDL₃-C level are important in atherosclerosis.

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1) There is a group of people in Japan who make it a habit of drinking a mixture of 3 egg yolkes and green tea (12g) (abbreviated as Egg Tea) which is one of folk remedies. We could get a chance of clinically examining 10 of these people (average year, 60) who had continued on this habit for quite many years (average 21 years). Their Tcho was 213.7mg/dl and T-G, 120.7mg/dl, which were both in their normal ranges. HDL-cho was 74.4mg/dl, atherogenic index (A. I) 2.0, which is low. Besides, their clinical results by ECG and chest XP examinations showed they were scarcely atherosclerotic.
2) In order to confirm the results of I-a), the egg tea was loaded for 3 months to stroke patients who had arrived at stable stage and their lipoprotein methabolism was examined, T-cho tended to rise in the first one month (In A Group who had egg tea 3 times a day, 38mg/dl), after which it reached a plateau and gradually declined. HDL-cho gradually rose (In A group, it revealed to 82mg/dl in the 2nd month) and the increase was dependent to the dose of administration and AT significantly declined in the 2nd month.
-b) Increase in ApoA concentration and LCAT activity was recognized.
-c) Loading of egg alone caused increase of T-cho and HDL-cho and a slight decline tendency of AI.
3) A long-term egg-tea loading coursed suppression of cholesterol synthesis, which worked more specifically strong on LDL-cho and VLDL-cho while HDL-cho was least suppressed. This suggests that in the cholesterol synthesis suppression mechanism the LDL system and HDL system are suppressed through different routes so that possible existence of two suppression pathways was suggested.
4) The fact that the long-time egg tea users had very few atherosclerotic symptoms clinically is considered due to suppression of LDL synthesis leaving HDL synthesis under less suppression so that its high value is maintained.

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Serum lipids and red cell deformability in 76 patients with arteriosclerosis obliterans (ASO) and in 31 patients with thromboangitis obliterans (TAO) were analyzed. The following results were obtained.
1) Among serum lipids, only triglyceride level was significantly increased in ASO and slightly in TAO.
2) HDL-cholesterol and Apoprotein-A were significantly decreased in TAO as well as ASO, without any change in the ratio of HDL-cholesterol and Apoprotein-A.
3) Decrease of red cell deformability was observed in both ASO and TAO.
4) In ASO with the sooner manifestations appeared the more remarkable changes were observed in serum lipids.
5) Less remarkable changes in serum lipids were observed in TAO than in ASO.

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Thirteen patients with arteriosclerotic diseases and 5 normal healthy subjects were orally given 300mg aspirin once daily for 4 consecutive weeks. Examinations with a stress on platelet functions were carried out before and after the first administration and during at the end of 4 weeks of treatment to clarify the influence of aspirin on them. The following results were obtained:
1) The serum lipid (total cholesterol, triglycerides and HDL-cholesterol) levels and the atherogenic index were not significantly different before and after the consecutive daily administration of aspirin in both groups.
2) The proportion of arachidonic acid (C: _20:4) to the phospholipid (PE and PC) levels and NEFA in plasma and platelet produced no significant changes before and after the consecutive daily administration of aspirin.
3) The platelet aggregation tended to return to the pretreatment level 24 hours after the first administration in the group of normal healthy subjects, but this tendency was not clear in the group of patients with arteriosclerosis. In addition, the 24 hour values during the consecutive daily administration showed a similar tendency.
4) With regard to the influence on prostanoids, the ratio of TXB₂ to 6-Keto-PGF_1α decreased after the first administration, but it showed little alternation throughout the day and was maintained at a lower level constantly during the consecutive daily administration in both groups. When the range of fluctuation was examined by defining the pretreatment value as 100%, TXB₂ was primarily suppressed in both groups. The reduction of the ratio seemed to be mainly due to this change. Moreover, 6-Keto-PGF_1α tended to be decreased to a less degree and TXB₂ to a higher degree in the group of patients with arteriosclerosis than in that of normal healthy subjects.
5) The βTG and PF-4 levels were significantly decreased as compared with the pretreatment values, in the arteriosclerosis group but no significant changes in these parameters were observed in normal healthy subjects.
6) There was no significant correlation between the serum aspirin concentration and platelet functions.
The results indicate that aspirin exerts a satisfactory effect as an antiplatelet drug when administered at 300mg once daily and additionally that its suppressive potency is within a constant limit. Thus, continuous long-term administration of aspirin is suggested to be possible.

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The present study was carried out to determine the relationship between the fatty acid composition of platelets and platelet aggregation in ischemic heart disease and to clarify the meaning of its atherogenesis. The cases studied were 19 patients with coronary heart disease (CHD group) including 9 patients with myocardial infarction (MI group) and 10 patients of angina pectoris with positive Master's test (EA group), 9 patients with negative Master's test (HD group) and 10 healthy control. In 24 of the 38 subjects the influence from exercise was investigated using the Master's double two step test. The results are as follows.
1) The linoleic acid content (18:2) of platelets and plasma was decreased in the CHD group and, in particular, in the MI group when compared with the N group.
2) In the MI group, the arachidonic acid content (20:4) of platelets was increased when compared with the N group, and the ratio of the linoleic acid content to the arachidonic acid content (20:4/18:2) of platelets was significantly higher than that in the N group. In the HD group and EA group, the arachidonic acid content of platelets was not increased when compared with the N group, while the ratio of the linoleic acid content to the arachidonic acid content of platelets tended to be higher than that in the N group.
3) Linoleic acid or arachidonic acid contents showed a significant correlation between in platelets and plasma.
4) ADP- and ADR-induced platelet aggreagtion was significantly accelerated in the CHD group and, in particular, in the EA group when compared with the N group, while collagen-induced platelet aggregation showed no difference between the N group and the CHD group.
5) A negative correlation between the linoleic acid content of platelets and ADP-induced platelet aggregation was observed.
6) Fatty acid composition in plasma showed no change by exercise, but the stearic acid content of platelets was decreased in the N group and the CHD group.
7) ADP and ADR-induced platelet aggregation was accelerated at mean values after exercise in all groups, there was no difference between any group. Collagen-induced platelet aggregation was markedly accelerated after exercise in the MI group when compared with the N group and the HD group.
From the above results, it was suggested that the linoleic acid content of platelets was closely related to platelet aggregation, and a decrease in linoleic acid, an increase in arachidonic acid and a high ratio of the linoleic acid content to the arachidonic acid content of platelets played a part of inducing coronary atherosclerosis or thrombosis.

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A new classification is proposed for the disorders of serum lipids including not only hyperlipidemias but also hypolipidemias, of which the clinical importance has begun to be widely recognized. The lipids employed are total cholesterol (TC), triglycerides (TG) and HDL-cholesterol (HDL-c). The naming follows in principle Fredrickson-WHO system. When both TC and TG are high, it is classified as type IIb, TC high and TG normal as type IIa, TC normal and TG high as type IV, TC high and TG low as type IIc, both TC and TG normal as type N, TC low and TG high as type IVc, TC normal and TG low as type L4, TC low and TG normal as type La, and both TC and TG low as type Lb. To these added is a suffix of either -h, -n or -1, according to the levels of high, normal or low HDL-c, respectively (see Fig. 1 in the text).
The normal range of serum lipids is set, on the basis of clinical and epidemiological observations, as 160-219mg/dl for TC, 50-149mg/dl for TG, and 40-69mg/dl for HDL-c. Thus a patient whose TC is 287mg/dl, TG 169mg/dl and HDL-c 38mg/dl is diagnosed as type IIb-1, and when TC is 152mg/dl, TG 75mg/dl and HDL-c 42mg/dl as type La-n.
This classification is applied to three populations; firstly to the bank employees of 932 subjects (846 males and 86 females), secondly the university staffs and employees of 1662 persons (1102 males and 560 females), and thirdly 1556 subjects (1189 males and 366 females) who visited Keio Health Councelling Center for their health examinations in 1981. It turned out that hyperlipidemias average 48% of the total population, 16% being with high HDL-c, 27% with normal HDL-c, and 6% with low HDL-c, while hypolipidemias accounts for 17% of the population, 3% with high HDL-c, 10% with normal HDL-c and 4% with low HDL-c. The normal with all three lipids is 32% of the total, and the inbalanced type of IIc and IVc only 3%.
This system of classification may serve as a new tool for a more comprehensive study of dyslipidemias, and for a better understanding of the interrelationship of lipid metabolism. The prognosis of lipid disorders may be studied from a different viewpoint.

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