We reported the seventh case of the internalization defective type of homozygous familial hypercholesterolemia (FH). LDL receptor activity was examined by cultured skin fibroblasts. LDL binding activity of the patient fibroblast was as much as normal fibroblasts at 37°C but at 4°C, the binding activity was about a half or one third of that of normal fibroblasts. Scatchard analysis of this binding activity revealed the dissociation constant of the patient fibroblasts was almost the same as that of normal fibroblast. But the numbers of receptor of the patient fibroblasts per cell was a half as much as normal fibroblasts. Bound LDL at 37°C was left from the cell surface into the medium rapidly when the temperature of the medium changed from 37°C to 4°C. The molecular weight of LDL receptor of patient's fibroblasts was about 130, 000 dalton determined by ligand blotting, which was the same as that of normal fibroblasts. It is suggested that some kinds of molecular defects exist in membrane spanning lesion or intracellular lesion of LDL receptor but this defect is not so big one as influencing molecular weight detected by SDS-PAGE.

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Familial hypercholesterolemia (FH) is an autosomal dominant disease due to abnormality of the low density lipoprotein receptor (LDL-R), and is characterized by hypercholesterolemia and premature coronary sclerosis.
In order to clarify the gene abnormalities of FH, we tried to identify some restriction fragment length polymorphism of the human genomic LDL-R gene with a cDNA (pLDLR-2HH 1) probe (a kind gift from Dr. Russell).
The results were as follows:
1) After XbaI digestion, the pattern of fragments of LDL-R gene was same (10 kb and 7 kb length) in two homozygous patients with FH and one normolipidemic subject.
2) After PvuII digestion, we found two fragments (19 kb and 3.5 kb length) in one of the homozygous patients. While, in the normolipidemic subject, four fragments (19 kb, 7.5 kb, 6.3 kb and 3.5 kb length) were detected. This polymorphism of the LDL-R gene is different from those reported by Humphries et al.

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The effects of the apo E alleles on plasma lipid, lipoprotein and apo E levels were determined in middle-aged subjects by the rapid flat gel isoelectric focusing method that we developed previously. The frequency of hyperlipoproteinemia was highest in the apo E3/2 group (54.5%), followed by the apo E4/3 (39.1%) and E3/3 (25.8%) groups in this order. The subjects with the apo E3/2 phenotype had significantly higher levels of plasma TG, VLDL-TG and apo E, and significantly lower levels of plasma HDL-C than those with the apo E3/3 phenotype.Furthermore, the apo ε2 allele elevated the plasma TG, VLDL-TG, VLDL-C and apo E levels and VLDL-C/VLDL-TG ratio, and lowered the plasma TC, LDL-C and HDL-C levels, whereas the apo ε4 allele slightly elevated the plasma TC, LDL-C and HDL-C levels. These results indicate that the apo ε2 and ε4 alleles are associated with the lipid abnormalities in middle-aged subjects.

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To clarify the significance of apo E phenotypes on intermediate density lipoprotein (IDL) metabolism, the metabolism of IDL from the subjects with apo E phenotype E 2/2, E 3/2, E 4/7 or E 3/3 were studied by using rat isolated hepatocytes and rat peritoneal macrophages.
1) Binding of IDL with apo E 3/2 to isolated hepatocytes was almost the same as IDL with apo E 3/3. Uptake of IDL with apo E 3/2 into macrophages was the same as IDL with apo E 3/3.
2) Deficiency of binding to rat isolated hepatocytes was observed in IDL with apo E 2/2. Uptake of IDL with apo E 2/2 into macrophages was the same as IDL with apo E 3/3.
3) Binding of IDL with apo E 4/7 to isolated hepatocytes was about 80% of that of IDL with apo E 3/3. The cholesterol ester content in macrophages which were incubated with IDL with apo E 4/7 was increased by 1.7-fold compared with control macrophages which were incubated with IDL with apo E 3/3.
4) The binding activity of IDL, depending on apo E level, to isolated hepatocytes was diminished to 50% degree in apo E 3/2 compared to apo E 3/3.
5) Serum apo E level was inversely correlated to the binding activity of IDL, depending on apo E level, to isolated hepatocytes.

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Liver has two distinct lipoprotein receptors; one is apolipoprotein (apo) B, E receptor and the other is apo-E receptor. We have already found that the 60k and 36k proteins in solubilized rat liver membranes bound apo-E HDLc and the latter bound apo-E HDLc in a specific and saturable manner. In this study, we characterize the lipoprotein-binding manner of the 60k proteins.
Rat liver membrane was prepared by ultracentrifugation as the 8, 000 to 100, 000×g pellets after the homogenation. The liver membranes were solubilized in the presence of 40mM octylglucoside, and the membranes were fractionated using DE52 chromatography. The 60k proteins were separated from both of the apo-B, E receptor proteins and the 36k proteins using preparative gel electrophoresis. The binding of ₁₂₅I-apo-E HDLc to the isolated 60k proteins showed a saturable manner, and was inhibited only by apo-E HDLc competitively but not by human LDL and canine HDL. These findings suggest that the 60k proteins are the apo-E receptor.
It is, however, remained to be determined whether the 36k proteins are the degradative products of the 60k proteins or the 60k proteins are the dimer of the 36k proteins.

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The extent of atherosclerosis in the population is drawing attention recently, as it would predict trends of atherosclerotic cardiovascular diseases. In order to obtain comparable and poolable data, rigid standardization in the measurements of atherosclerosis is mandatory, but such an experience has so far been seldom documented. The purpose of the present paper was to assess the magnitude of observer variations in the measurements of atherosclerosis, to examine their reasons and to find out possible corrective measures.
Eight centers in a multi-center cooperative study participated. Twelve aortas and 10 coronary arteries actually used for the main study were circulated to each center either blindly or providing some reference values.
So-called point-counting method was used for quantitative assessment. A transparent screen with printed dots at an equal interval was superimposed on the arterial surface. Pathology beneath each dot was classified into normal, fatty streak, fibrous plaque and complicated lesions. The number of dots for various pathology, their sum and weighted sum were computed.
Inter-and intra-observer variations were assessed using differences between means and standard deviations of indices of atherosclerosis measured by individual observers with a series of artery specimens and using correlation coefficients of these indices between observers.
Automatic counting of fatty streak using a color image processor and a computor was also tested.
The following results were obtained:
1. Significant inter- and intra-observer variations and mostly high but occasionally very low correlation coefficients were observed, when tested blindly.
2. Agreement in the mean values and correlation coefficients increased when some specimen with other person's readings were provided as reference.
3. Variations in the measurement were greater when the involved surface area was smaller. (The coronary arteries showed greater observer variations than the aorta. Observer variations were greater in weighted sum of atherosclerotic lesions (A. I.), sum of atherosclerotic areas (S. I.) followed by total surface area in this order and complicated lesions, fibrous plaque, fatty streak and no atherosclerotic lesions in this order.)
4. A color image processor assessed surface area with fatty streak with same level of betweenobserver correlation coefficients as the pointcounting methods.

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Many investigators have studied various aspects of the relationship between the progression of atherosclerosis and lipid metabolism. However, mechanism of progression of atherosclerosis has not yet been fully elucidated. In the present study, the contents of cholesterol, triglyceride, phospholipid and fatty acids composition in human normal aorta and atherosclerotic lesion examined in order to clarify the mechanism of progression of atherosclerosis. Human aorta obtained at autopsy was frozen by liquid CO₂ and sectioned into intima, intimal media and media by cryostat. The each section was homogenized and the lipids were extracted with chloroform-methanol mixture and analyzed using thin-layer chromatography and gas-liquid chromatography.
In normal human aorta, the contents of cholesterol, triglyceride, and phospholipid decreased in proportion to the distance from luminal surface, and cholesterol/phospholipid (C/P) ratio was nearly 1. In atherosclerotic lesion, the contents of cholesterol and phospholipid increased in intimal media and media, but triglyceride contents increased in intima and intimal media. C/P ratio was about three times larger than in normal aorta, respectively. In fatty acids composition of cholesterol ester, oleic acid increased and the ratio of poly unsaturated fatty acid to saturated fatty acid (P/S ratio) increased. In phospholipid, on the contrary, oleic acid decreased and lysolecithin increased in phospholipid fractions. In triglyceride, the ratio of saturated fatty acids increased with the progress of atherosclerosis.
From above findings, it was suggested that the changes of fatty acids compositions of cholesterol, triglyceride and phospholipid in the arterial wall played important role in the progression of atherosclerosis.

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The aorta is a blood vessel in which atherosclerosis occurs frequently. The blood pumped out from the heart flows inside the aorta in a varying direction as it travels along the ascending aorta, aortic arch, and descending aorta. In a vessel of such curvature, secondary flow inevitably occurs, causing a change in the distribution of shear stress in the vessel. It seems that, due to these factors, the endothelial cells are injured so that their permeability increases, leading to the deposition of lipid and protein on the vascular wall and, eventually, to atherosclerosis.
In the present study, we prepared rabbits with hypercholesterolemia induced by a 1% cholesterol diet, and investigated the relationship between the site of initial atherosclerotic lesions and the secondary flow or the distribution of shear stress in the animals under constant-pressure perfusion fixation. As a result, in the area from the aortic arch to the descending aorta in hypercholesterolemic rabbits, a high incidence of cholesterol deposits and atherosclerosis was found to coincide with the inner side of the vessel, where the shear stress was minimal.

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Atherosclerosis progresses silently taking a long period of time, and its clinical manifestations do not appear until middle or late period of life. The purpose of this study is to elcidate on what alterations occur in the initial stage of atherosclerosis in infants and young adults in particular respects to differences in organ arteries, sex and living areas.
Atherosclerotic index (A. I.) tends obviously to increase with age in both aortae and coronary arteries, but fat deposition is significantly stronger and to start much earlier in the aortae (418 cases) than in the coronary arteries (326 caces). In contrast, intimal thickening of the arteries tends to be stronger and to start earlier in the coronary arteries (185 cases) than in the aortae (246 cases). Coronary arteries are generally characterized by intimal thickening with fibrocellular proliferation but less lipid deposition, whilst the aortae tends to have lipid deposition in the intima, even though the intimas are not so much thickened. On the other hand, in the coronary arteries, fenestration of internal elastic laminae is more significant than in the aortae. In these areas, it is often observed that medial smooth muscle cells of coronary arteries are arranged longitudinally towards the widened windows in the intima.
Immunohistochemical examinations reveal that apolipoprotein B is demonstrated in the intima of the coronary arteries and the aortae even in the infants. In the aortae, apolipoprotein B is often demonstrated in the media intracellularly.
Regarding a sexual difference in the atherosclerotic index, atherosclerotic indices of both aortae and coronary arteries tend to be higher in males than in females over 20 years of age.
Compareing A.I. of the aortae and the coronary arteries in Kanto and Tohoku areas, it seems reasonable to say that A. I. is apparently higher in Tohoku area than in Kanto area in both aortae and coronary arteries aged between 30 and 40 years.

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The characteristic localization of the atherosclerosis at the cervical bifurcation of the carotid artery has been discussed with its relation to the hemodynamic stresses. However, precise histopathological study on its structure is not enough to make precise correlation between the structure and function. We study the lateral wall of the orifice of the internal carotid artery by longitudinal section 275 branches from 0 years to 90 years of age histopathologically. There are two layers of Intimal thickening: focal elastosis (near the internal elastic lamina) and loose fibrous layer (beneath the endothelial layer). Focal elastosis is already found at birth and reaches full development as early as 10 years of age. It keeps its thickness (about 0.1mm) in 80 years of age. It is thought to have a kind of physiological nature. Loose fibrous layer, on the contrary, progresses with age. It becomes 0.3mm in 30 years of age and becomes 0.7mm in 60 years of age. Furthermore, it contains foamy cells, fatty droplets, cholesterine crystals, invasion of blood vessels and calcification. This complex character of loose fibrous layer is a typical atherosclerotic lesion. It is considered that, in the lateral wall of the orifice of the internal carotid artery, the reaction of the arterial wall has two ways : 1. physiological as focal elastosis and 2. pathological as loose fibrous layer.

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The preventive effect of chromium against fat deposition in aorta was certified using cholesterolfed Japanese and New Zealand White male rabbits. The deposition area of arterial intima in the cholesterol-fed rabbits untreated with chromium was as large as 96.1%, while the fat deposition area in the cholesterol-fed rabbits which were injected 50 μg/ml of chromium intraperitaneally every other day was decreased to 56.8%.
Chromium injection brought about a remarkable decrease of serum lipids in the experiment with Japanese White rabbits (TC 444.7mg/dl in cholesterol+chromium group and TC 1, 044.1mg/dl in cholesterol alone group). However, in the experiment with New Zealand White rabbits the level of serum cholesterol was higher in the cholesterol plus chromium group than in the cholesterol alone group.
Although the serum cholesterol level was significantly different in New Zealand White and Japanese White rabbits, chromium exhibited a clear preventive effect against fat deposition in aorta in both rabbit groups.
HDL fractions, estimated by a polyacrylamidegel electrophoresis, decreased along with the duration of cholesterol feeding and they disappeared almost completely when the cholesterol level reached about 300mg/dl.
It was suggested that chromium could prevent the fat deposition by a manner independent of the serum lipid level.

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Effects of elastase on vascular characteristics of the aorta were observed. Elastase was administered orally with dose of 10, 800 E. S. units daily for 12 months to fifteen patients with atherosclerosis.
Radioisotope angiography and bio-electrical local impedance changes from disc electrodes were recorded before and after administration of elastase.
Aortic volume curves of aortic arch were derived from equilibrium images. The height of aortic volume curve was termed as H_RI and minimum aortic volume were termed as A₀min. Volume elasticity (V_E) is calculated by following formula: V_E=ΔP/(ΔV/V₀), ΔP was measured from cuff pressure on upper arm and ΔV/V₀ was calculated as H_RI/A₀min. Therefore, V_E was calculated as follows:
V_E=(Systolic Blood pressure-Diastolic Blood pressure)/(H_RI/A₀min)
Disporsable disc electrodes (NI. 601 V, Nihon Koden Co., Ltd., Tokyo, Japan), have a diameter of 3.6cm, were attached on the chest wall at V₈ of ECG chest lead positions and posterior median line of second-thoracic vertebral level (x point). This lead was termed as A lead. Alternating current of 50kHz, 350mA was applied to the outer circle electrodes, and the impedance change was recorded from the inner circle electrodes. Impedance waves and aortic flow velocity curves obtained by Millar micro-tip velocity probe at the thoracic artery were recorded simultaneously during cardiac catheterization test. The heights of dz/dt curves (the value of h_IMP)were correlated well to the peak aortic flow velocity (r=0.88).
The amplitudes of Zo curves were termed as H_IMP. H_IMP were correlated well to the aortic volume changes. Therefore, aortic compliance were calculated as follows aortic compliance=ΔV/ΔP=H_IMP/(Systolic Blood pressure-Diastolic Blood pressure)
These h_IMP and H_IMP/(SBp-DBp) as aortic compliance were compared before and after the administration of elastase.
V_E decreased from 1, 894±1, 273.4 to 1, 253±516.6 (p<0.05), while h_IMP increased from 6.5±2.7Ω/sec to 8.3±3.0Ω/sec(p<0.02)and H_IMP/(SBp-DBp) increased from 0.10±0.06 to 0.13±0.08 (p<0.02).
After the administration of elastase, aortic volume elasticity decreased, and aortic flow velocity and aortic compliance increased. It may be concluded that elastase improves elasticity of the aorta.

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The effects of elastase on coronary arteries were examined in experimental hypertensive and hypercholesterolemic rats. The body weight, the heart weight and the blood pressure did not show any differences between the elastase administrated rats and the control rats. The level of serum lipid was not lowered by the administration of elastase in the hypertensive and hypercholesterolemic rats.
The incidence of intimal thickening, fragmentation or disappearance of internal elastic lamina and medial fibrosis of the coronary arteries of the elastase administrated group was small in number in comparison with the control group.

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To clarify the mechanism by which elastase administration prevent atherosclerosis, rabbits were kept for 20 weeks as follows : Group I; Normal diet group, Group II; Normal diet-elastase (10mg/kg B.W./day) administered group, Group III; High-cholesterol diet group, Group IV; High-cholesterol diet-elastase administered group. Serum total cholesterol level was not different between Group I and Group II. But, that in Group IV was lower than that in Group III. As for cholesterol ester content in aorta, it was not different between Group I and Group II. But it was lower in Group IV than in Group III. This decrease in cholesterol ester content in aorta was not sufficiently explained by the decrease in serum total cholesterol. So, in order to investigate the effect of elastase, cholesterol ester in aorta was examined by dividing into three fractions, elastin-bound, collagen-bound and free form fractions. All these fractions were decreased in Group IV comparing with those in Group III. The ratio of cholesterol ester in free form was higher in Group IV than in Group III. These results suggested that elastase might convert the fiber-bound cholesterol ester into free form, such that it could be easily removed from the tissue.

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The fragmentation, loss and calcification of an elastic fiber, an arterial wall constituent, are important changes relative to the development and progress of atherosclerosis. The loss and fragmentation of elastic fibers is attributed to a decrease in elastin level in artery tissue, while the mechanism of calcification is yet to be elucidated. This paper describes the relationship between a morphological classification of artery calcification and pathologies of a sclerotic artery. Fibrous plaques, atheromas and surrounding tissue specimens from autopsies of 70 or older patients preserved at the Department of Pathology, Tokyo Medical College were used in the human study. In the animal study thoracic aortas from rats with atherogenic dietinduced atherosclerosis were investigated in comparison with counterparts of atherosclerotic rats fed an atherogenic diet and those of rats coadministered with a Ca²⁺-antagonist and elastase.
Results Human artery calcification was morphologically classified into five types: (1) With the powder size type calcification was noted at sites distal to lesions of atheroma or at sites removed from the intima of the artery; (2) with the sandgrain size type collagen fibers were found proliferating at sites of atheroma or at sites removed from the intima of the artery and fissured elastic fibers were present at sites of decreased elastin level; (3) the mass size type was found the same as the sand-grain size type except that fissured elastic fibers were localized in lesions of atheroma and its surrounding areas; (4) with the belt-linear type calcification was noted in the subintimal region, fissured elastic fibers or in the area of transition from deep intima to media in lesions of atheroma; and (5) with the mixed type the area of transition from lesions of atheronecrosis to media was found calcified.
Whereas with rats with experimental atherosclerosis calcification of belt-like or linear form was localized mainly in the media, calcification was insignificant in rats coadministered with elastase and a Ca²⁺-antagonist.
Conclusion Human aorta calcification morphologically classified into five types differed that in atherosclerotic rats, whose calcification was suppressed with the coadministration of elastase and a Cat-antagonist.

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The effects of pancreatic elastase on lipid metabolism in experimental atherosclerotic rats were examined by biochemical approaches. The experimental atherosclerosis (Ch group) was induced in rats by administering Vitamin D₂, 400, 000 units/kg B.W./day, for 5 days and then with atherogenic diet for 6 weeks.
Elastase, 450 EL units/kg B.W./day, (Ela group) was administrated for 6 weeks.
The following results were observed:
1) Serum cholesterol level of Ela group was slightly lower than that of Ch group.
2) Cholesterol level especially cholesterol ester in aortic elastin fr. was significantly lower in the Ela group than in the Ch group.
3) Excretion of cholesterol in feces was promoted in the Ela group.
It was suggested from the result of this study that the effect of lowering cholesterol deposition on aortic elastin was caused by externally administerd elastase.

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The arterial pulse wave velocity (PWV) has been considered as a useful indicator of arteriosclerosis (AS). We developed the new method to evaluate AS by accumulation of the vascular sound.
In this new method, two microphones were placed on the abdomen along the aorta at 7-8cm distance. The vascular second sound (VSS) of more than 50 beats were accumulated using of R wave of ECG as a trigger. The time interval was measured between proximal and peripheral peaks of VSS and PWV was calculated.
PWV by the new method was compared with PWV by the conventional method and the invasive method that the microtip catheter (Miller) was withdrawn from the ascending aorta to the abdominal aorta during diagnostic catheterization. PWV measurements were carried out in 14 male and 4 female subjects, ranging in age from 23-68 yo.
Significant correlation was found between PWV by the new method and that by the invasive method in the abdominal aorta (y=0.850x-68.35, r=0.792, p<0.001) and in the whole aorta (y=0.843x-41.12, r=0.660, p<0.005). PWV by the new method was correlated with that by the conventional method (y=0.763x+8.61, r=0.685, p<0.005).
Since this new method was simple and accurate, we thought that PWV calculated by VSS was useful as an indicator of regional arteriosclerosis in particular.

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The significance of pulse wave velocity (PWV) was investigated in relation to the clinical findings showing arteriosclerosis (aortic calcification diagnosed by chest X-ray, ischemic change of ECG and cerebral infarction diagnosed by computor tomography), and clinical data in patients with diabetes mellitus.
Prevalence of aortic calcification was increased in proportion to the increase in PWV value. A marked increase in prevalence (78.8-100%) of aortic calcification was noted when PWV values were over 9m/sec. However, prevalence of ECG abnormality was about 30% when PWV values were over 9m/sec. PWV value in patients with aortic calcification was significantly higher than that in patients with cerebral infarction. Therefore, PWV value was most significantly correlated with aortic calcification among these arteriosclerotic findings. The above findings indicated that PWV measurement (noninvasive method) is useful as one of the indices for evaluation of arteriosclerosis.
Mean values of PWV gradually increased in normals as age advanced. Moreover, PWV values in patients with diabetes mellitus were greater than those in age matched controls. The longer the duration, the higher the PWV values. PWV values were higher in poor controled patients than those in good controled patients. PWV values were higher in patients with hypercholesterolemia than those with normocholesterolemia.
These data suggest that blood glucose control, duration of diabetes and hypercholesterolemia could be risk factors for arteriosclerosis.

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Chronic diabetic state was produced in monkeys (Macaca fuscata) with i.v. streptozotocin and they were kept untreated for up to 41 months. Hypercholesterolemia (313mg/dl v.s. Normal; 151mg/dl) and hypertriglyceridemia (477mg/dl v.s. Normal; 70mg/dl) together with hyperapoproteinemia (B, C-II) were observed. The major subfraction for cholesterol increase was attributed to LDL; and for TG to mostly (VLDL+IDL) fraction. HDL-chol and apo A-I were normal. The changes seemed to be greater in the monkeys with longer duration of diabetes.
Microscopically, endotherial thickening, appearance of form cells and proliferation of smooth muscle cells in the median layer of abdominal arteries were observed. Degeneration of cardiac muscle fibers was also observed. Impairment of contractility and ventricular dilatation were noted by ultrasonic cardiography.
Long-standing insulinopenia leads to hypercholesterolemia (LDL) and hypertriglyceridemia (VLDL+IDL), which in turn may have contributed to the development of early lesions of macroangiopathy in chronic diabetic monkeys.

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To elucidate the initial compositional changes in cell lipids and the progress of atherogenesis, we analyzed lipid fractions in intima-media tissues separated from the arch, renal and abdominal portions from 8 non-diabetic and 4 type-2 diabetic autopsy cases. Intima-media was homogenized in 0.13M Tris-HCl buffer and centrifuged. Total lipids, lipid fraction (TLC) and protein (Lowry's method) in supernatant were measured.
Total lipid content was 8.1±0.8mg/g wet weight (M±SE) in non-diabetics and it raised significantly (p<0.005) to 15.0±3.3mg/g wet weight in diabetics. Cholesterol esters were dominant but triglyceride raised most remarkably to 30.9±10% in diabetics (non-diabetics: 13.3±4.4 %). Phospholipids reduced significantly (p<0.005) to 12.5±2.7% in diabetics (non-diabetics: 27.2±2.6%). Free cholesterol also reduced in diabetics. These changes were especially predominant in the abdominal portion.
We supposed that the decrease of phospholipids might cause the disorder of permeability in the aortic cell membranes, resulting in the accumulation of the TG-rich lipoproteins that are known to increase in diabetes.

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There are several kinds of tests to investigate the abnormal plasma lipoprotein profiles. The aim of this study is to investigate whether OGTT is a useful test for abnormal lipoprotein catabolism.
Sixteen normal subjects and eleven patients with diabetes mellitus (DM) were examined. Blood sugar, immunoreactive insulin (IRI) and serum lipid levels—total cholesterol (TC), triglyceride (TG), HDL-cholesterol (HDL-C)—were measured at fasting and 30, 60, 120 minutes after 75g of oral glucose administration. VLDL-cholesterol (VLDL-C), LDL-cholesterol (LDL-C) were calculated based on Friedwald's formula.
Results obtained are as follows:
1. Serum TC was not changed after 75g of oral glucose administration.
2. Serum TG was significantly reduced 120 minutes after the oral glucose administration.
3. VLDL-C was also reduced significantly 120 minutes after the oral glucose administration. LDL-C and HDL-C were increased but not significantly.
4. There was no significant change in serum lipid levels between normal subjects and patients with DM during OGTT.
These results suggest that the acute effect of oral glucose administration on serum lipids is mainly caused by catabolism of VLDL.

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Diabetic mice C₅₇ BL/ksj-db/db mice are well known with their genetically obese condition. They show hyperinsulinemia and resistance to insulin from their early stage, and they soon become to disclose the same symptons as those observed in patients with maturity onset diabetes mellitus.
Using these adult models we studied changes in serum glucose and triglyceride (TG) levels by various levels of insulin, which were achieved by the daily injection of insulin or streptozotocin (STZ). Lean mice were used as a control. Three unit/kg of insulin was injected intraperitoneally to 8db/db mice and the same number of lean mice for 7 days.
Daily insulin treatment to lean mice made 53 decrease of their serum glucose levels, as compared with those in non treated lean mice. On the contrary, db/db mice whose glucose level exhibited 518±24mg/dl, did not show any change, though they were administered the same dose of insulin as lean mice.
Concomitant treatment of AS-103 (40mg/kg p.o), a derivative of ascocholorin, and insulin in 8db/db mice resulted in a statistically significant decrease of glucose level as compared with those of insulin treated control db/db mice.
On the other hand, intraperitoneal administration of STZ (180mg/kg) to db/db mice caused 29% decrease of IRI, 25% decrease of TG and in turn, 18% increase of glucose level in serum and concomitant administration of AS-103 exhibited the reduction of serum glucose, as compared with that level in STZ treated control db/db mice, indicating that endogenous insulin may work in AS-103 treated mice more effectively in the controls.
Effect of insulin on adrenaline-stimulated glycerol release was studied using epididymal fat pads slices taken from AS-103 treated and untreated db/db mice and their lean litter mates. Fat pads slices were incubated in Krebs-Ringer bicarbonte buffer containing adrenaline (1μg/ml) and insulin (10, 100, 1, 000μU/ml). Glycerol release in epididymal fat pads from db/db control mice was not affected in spite of the presence of insulin. On the other hand, the fat pads from AS-103 treated db/db mice released less glycerol than those from the non treated db/db . control mice at 100, 1, 000μU/ml of insulin.
These data may suggest that AS-103 have an effect to improve the sensitivity and responsiveness to insulin in the insulin resistant, diabetic animal model.

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The present study was undertaken to elucidate the correlation with platelet vitamin E and lipoprotein vitamin E. The subjects comprised 30 type II diabetic patients (17 male, 13 female) and 14 healthy subjects (7 male, 7 female). In these patients, each lipoprotein fraction (VLDL, LDL, HDL) was isolated from fasting plasma by ultracentrifugation. Vitamin E concentrations of serum and each lipoprotein fraction were measured by the method of Abe and Katsui. The level of platelet vitamin E was measured fluorometrically using a modification of the Hatan-Kayden's method.
Platelet vitamin E level was 0.78±0.23μg/10⁹ platelets (Mean±SD) in diabetic patients. This value was significantly higher than the vitamin E (0.51±0.17 μg/10⁹ platelets) in healthy subjects (p<0.001). In diabetic patients, platelet vitamin E level was correlated with the level of serum vitamin E or VLDL vitamin E, but not significantly. In diabetic patients, platelet vitamin E level was significantly correlated with the ratio of LDL vitamin E to LDL cholesterol (r=0.365, p<0.05). These results suggested that platelet vitamin E level might be influenced by the level of VLDL vitamin E or LDL vitamin E.

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In order to assess the mechanism of accumulation of remnant lipoproteins in diabetes mellitus, we examined the lipoprotein metabolism in streptozotocin-induced diabetic rats. Cholesterol-fed diabetic rats showed a marked hyperlipoproteinemia associated with an elevation of chylomicrons and remnant lipoproteins. In VLDL fraction of the cholesterol-fed diabetic rats apolipoprotein E (apo E) was increased and apo A-I, which is absent from normal VLDL, was present. This might suggest the increase of chylomicron remnants in choles-terol-fed diabetics, indicating the impairment of exogenous cholesterol pathway in diabetes. When these animals were treated with a pharmacological dose of estrogen or triiodothyronine (T₃), elevated remnants were cleared from plasma. Apo E was decreased in VLDL and HDL fractions by these treatments. We, furthermore, measured postheparin lipolytic activity. Lipoprotein lipase (LPL) activity was increased in cholesterol-fed diabetics, although hepatic lipase (HL) activity was not changed. When estrogen or T₃ was injected to these rats, LPL and HL activities were suppressed. Therefore, the clearance of remnant lipoproteins by estrogen or T₃ was not due to activition of lipolysis. The binding activity of hepatic lipoprotein receptor was also measured. The activity was enhanced by estrogen treatment in diabetics as well as in normal rats. However, there was no difference between the binding activity of hepatic receptor in diabetics and that in non-diabetics.
These data suggest that the chylomicron remnant accumulation was not due to inactivation of lipolysis. But we speculate at least in part an impaired interaction of remnant lipoproteins and hepatic receptor might contribute to the remnant lipoprotein accumulation.

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Serum lipoprotein and apoprotein were determined in 154 non-insulin dependent diabetes aged from 17 to 78 years. The 154 patients were divided into 3 groups, by their age: those below 39, those 40 to 59 and those above 60. Each of the 3 groups was divided again into 2 subgroups by hyperlipemia (TC≥250mg/dl and/or TG≥150mg/dl) and normolipemia (TC<250mg/dl and TG<150mg/dl). Control Subjects for apoprotein were 7 healthy volunteers aged below 39, and also normal controls from the references No. 6. The results obtained were as follows:
1) In the normolipemia with and without arteriosclerosis the serum levels of VLDL, LDL, HDL-cholesterol (HDL-C) and apoprotein (A, C and E) were in the normal range. But only apo B was higher than the normal levels in all diabetic groups.
2) The levels of VLDL and LDL were increased and HDL-C was decreased significantly in the hyperlipemia when compared with in the normolipemia. Apo B, C and E in the hyperlipemia showed significantly higher than in the normolipemia, while apo A-I and apo A-II didn't differ statistically between in the normolipemia and hyperlipemia.
3) Atherogenic Index (AI) was increased more significantly in the patient with hyperlipemia than with normolipemia.
On the other hand the apo B/apo A-I ratio were higher in both diabetic groups with hyperlipemia and with normolipemia than in controls. Apo B/apo A-II ratio also showed the same results.
We conclude that apo B and apo B/apo A-I ratio or apo B/apo A-II ratio were increased in all diabetic patients, especially even in the normolipemic diabetics aged under 39. High levels of apo B might have atherogenic effects even on the young normolipemic diabetic patients.

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In order to clarify the role of apoproteins on the pathogenesis of diabetic micro- and/or macroangiopathy, serum total cholesterol (t-chol), triglycerides (TG), HDL-cholesterol, apoprotein A-I (apo A-I), A-II (apo A-II) and B (apo B) were measured in 10 normal subjects and 22 patients with type II diabetes mellitus (DM). DM were divided into two groups as follows: 1) DM without proliferative retinopathy (DM-ret (-)), 2) DM with proliferative retinopathy (DM-ret (+)).
Both serum t-chol and TG of DM were significantly higher than those of normal subjects, but there was no significant differences in both t-chol and TG between DM-ret (-) and DM-ret (+).Serum HDL-cholesterol levels of both DM-ret (-) and DM-ret (+) were within normal range. Serum apo A-I of normal subjects, DM-ret (-) and DM-ret (+) was 128±7mg/dl, 179±48mg/dl and 202±60mg/dl, respectively, and serum apo A-I of DM was remarkably higher than that of normal subjects (p<0.05). Serum apo A-II of DM was higher than that of normal subjects (40±1mg/dl), but there was no significant differences between DM-ret (-) (55±21mg/dl) and DM-ret (+) (50±16mg/dl). Serum apo B of normal subjects, DM-ret (-) and DM-ret (+) was 85±15mg/dl, 166±59mg/dl and 155±99mg/dl, respectively. Serum apo B of DM was significantly higher than that of normal subjects (p<0.001), but there was no significant differences between DM-ret (-) and DM-ret (+).
These data suggest that elevated apo A-I, A-II and B due to the disturbance of apoprotein metabolism in type II diabetes mellitus may play an important role on the progress of diabetic atherosclerosis, but may be little importance for microangiopathy such as proliferative retinopathy.

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We previously reported that nifedipine did not suppress atherosclerosis in WHHL rabbit which lacks LDL receptors and is a unique model of human familial hypercholesterolemia. This time we studied the effect of nifedipine, a calcium antagonist, on atherosclerosis of cholesterol-fed rabbits. Japanese white rabbits were fed 2% cholesterol rabbit chow, and divided into two groups with similar mean values of serum cholesterol levels and body weights. Nifedipine was given 20mg orally twice a day (nifedipine group), and in control group placebo was given in the same way (placebo group).
Rabbits were sacrificed at the end of 12th week. Body weights and serum cholesterol levels were not different between nifedipine and placebo group. The percentage of aortic intimal surface area covered by atherosclerotic lesions (%AS) was 25.9±7.6% (mean±S.D.) in nifedipine group (n=7), and 55.6±22.8% in placebo group (n=8), showing significant differences (p<0.01). The aortic cholesterol and calcium contents showed similar tendency to the results of %AS.
That nifedipine suppressed atherosclerosis in cholesterol-fed rabbits but not in WHHL rabbits seems to give important suggestions to the mechanisms of antiatherosclerotic effect of nifedipine.

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Recently it has been reported that Ca²⁺-antagonists suppressed the experimental atherosclerosis caused by high cholesterol diet. And it is suggested that the calcium metabolism in the arterial wall may be involved in the initiation and the development of arteriosclerosis. In order to investigate whether Ca²⁺-antagonists can also suppress another experimental model of arteriosclerosis, we employed the method of placing a polystyrene cuff around the common carotid artery of rabbits to cause an arteriosclerotic lesion, and applied this method to study the anti-arterioscle-rotic effect of Ca²⁺-antagonist, diltiazem and flunarizine.
We designed two experiments. In experiment I 20 rabbits were invested with cuffs in their right common carotid arteries and then divided into two groups, control group (n=10) and diltiazem group (n=10). In experiment II, 18 rabbits were treated similarly and divided into two groups, control group (n=9) and flunarizine group (n=9). Then a daily oral dose of 100mg/kg of diltiazem in the diltiazem group, and 5.7mg/kg of flunarizine in the flunarizine group were administered. After 21 days all the rabbits were sacrificed and their right common carotid arteries were excised and examined.
In both experiments intimal thickening was found over the segments invested with the cuff, and disruption of internal elastic lamina (I. E. L.) was also observed. Then the measurements of intimal thickness were carried out. The results showed that, in experiment I, although the mean value of the diltiazem group was somewhat lower than that of the control group, the difference was not significant, and in experiment II, there was no difference between the flunarizine group and the control group. The incidence of the disruption of the I. E. L. in each group was also examined. The incidence in diltiazem group was significantly lower than that in the control group, whereas there was no significant difference between flunarizine group and its control group. The reason why diltiazem suppressed the disruption of the I. E. L. seemed that it might improve the hemodynamic changes of the invested segments, or might prevent the increasing affinity of elastic fibers to calcium and inhibit elastolysis.
It was concluded that in this experimental model diltiazem was effective in inhibiting the disruption of the I. E. L., but not so effective in suppressing the intimal thickening, while flunarizine had no effect on these two findings.

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Effects of captopril on the peripheral hemodynamics, platelet function, plasma prostaglandins and other blood parameters were studied in 9 patients (mean age 68.4 years) with essential hypertension. Blood flow and maximum venous out flow of the bilateral forearm and calf were increased 8 weeks after treatment with oral administration of 37.5mg of captopril per day. There was a tendency that collagen- and arachidonic acid-induced platelet aggregation were decreased, plasma thromboxane B₂ was decreased, plasma triglyceride was decreased and plasma renin activity was increased 8 weeks after the captopril treatment. The level of serum insulin was significantly decreased and plasma fibrinolytic activity was significantly increased 8 weeks after the treatment.
These results show that captopril may have beneficial effects on peripheral hemodynamics, platelet function and plasma fibrinolysis to prevent thrombotic complication in the treatment of patients with essential hypertension.

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Prostaglandin E₁ (PGE₁) is a potent vasodilator as well as an inhibitor for platelet aggregation. Because of these properties, PGE₁ has clinically been used for the treatment of several vascular disorders such as arteriosclerosis obliterans (ASO). Since PGE₁ is rapidly inactivated in the lung and the distribution of PGE₁ to a whole body induced its systemic effects as diarrhea and hypotension. Moreover, PGE₁ causes an irritation in the blood vessels near the site of injection. To avoid these therapeutic problems of PGE₁, we incorporated PGE₁ in lipid microsphere with an average particle size of 0.2μm, whose suspension is in widespread use for parenteral nutrition in man. We tried to compare effects of lipo PGE₁ (PGE₁ incorporated in LM) with those of commercially available PGE₁.
The results was that lipo PGE₁ showed a much more beneficial effect than free PGE₁ for several vascular diseases with atherosclerosis. Improvement of intermittent claudication, rest pain and skin ulceration in patients with ASO. Decrease of frequency of chest pain in patients with angina pectoris. Slight improvement of hemiparesis and aphasia in patients with cerebral infarction. Improvement of numbness, spontaneous pain, hypesthesia and sensation of cold in patients with diabetic neuropathy. No side effects were recognized in lipo PGE treatment.

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We conducted a retrospective study to examine the effects of beta-blockers on serum lipid metabolism. In 11 patients propranolol at a mean dose of 39.1mg daily significantly elevated triglyceride and atherogenic index and lowered HDL cholesterol, but total cholesterol was not altered significantly. No significant change of lipid levels was observed in 20 patients treated with pindolol. Acebutolol significantly lowered total cholesterol by 8.1 per cent in 26 patients. Triglyceride and HDL cholesterol levels were not altered significantly. A trend of reduced atherogenic index, although not significant statistically, was found in the patients prescribed acebutolol. These results suggest that minimal dose of propranolol causes atherogenic changes of lipid metabolism. Pindolol has no harmful effect on serum lipid levels. Acebutolol possesses a favorable hypolipidemic effect to prevent atherosclerosis. Since one of the important purposes of prescription of beta-blockers to the patients with hypertension and/or coronary heart disease is prevention of progression of atherosclerosis of the coronary vessels, beta-blockers should be selected on the base of lipid metabolism as well as hemodynamic effects.

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Combined effect of pantethine and probucol on serum lipoprotein metabolism and on the incidence of atheromatous lesions in aorta and coronary artery was studied in cholesterol-fed rabbits.
Probucol treatment (0.5% in diet) resulted in reducing HDL-cholesterol and serum apo A-I levels significantly, while pantethine treatment (0.25-0.75% in diet) tended to increase HDL-cholesterol and serum apo A-I levels. Combined treatment with two drugs showed a significant prevention in the reduction of HDL-cholesterol and serum apo A-I levels by probucol alone. Probucol or pantethine treatment reduced (V) LDL-cholesterol and serum apo B levels effectively and these effects were accelerated additively when the two drugs were given concurrently.
Atheromatous lesions in aorta and coronary artery in cholesterol-fed rabbits were prevented by probucol (0.5% in diet) or pantethine (0.75% in diet) for 24 weeks. The combined treatment with two drugs showed more marked prevention than either drug alone.
From these findings, it is concluded that the combined treatment of probucol with pantethine is effective for improvement of serum lipoprotein disorders and for prevention of the incidence of atheromatous lesions in aorta and coronary artery in cholesterol-fed rabbits.

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The hypolipidemic action of NIP-200, 3, 5-dimethyl-4, 6-diphenyl - tetrahydro-2H-1, 3, 5-thiadiazine-2-thione, was studied in three models of cholesterol-fed rats. In lipids emulsion-induced hyperlipidemic rats, oral administration of 300mg/kg of NIP-200 for 3 days lowered serum total cholesterol by 48% and elevated HDL-cholesterol by 36%. NIP-200 showed no significant effect on serum triglyceride. In the prevention study on cholesterol-fed rats, oral administration of NIP-200 for 14 days decreased serum total cholesterol, free cholesterol and phospholipid in a dose dependent manner, and moreover elevated HDL-cholesterol at all doses. NIP-200 decreased liver total cholesterol by 16% at 150mg/kg, but showed no effect on other doses. The hypolipidemic effect of NIP-200 was considered to be about two to three times as great as that of clofibrate. In the treatment study on cholesterol-fed rats, oral administration of 300mg/kg of NIP-200 for 7 days decreased serum total cholesterol and triglyceride by 44% and 48%, respectively, and increased HDL-cholesterol by 48%.

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The male heterozygous familial hypercholesterolemia were treated by repeated plasma exchange (PE) and plasmapheresis (PP) at intervals of 3 weeks for one and a half year. One patient was 57 and another was 51 years old whose initial plasma cholesterol level were 490 and 442mg/dl, respectively. Both patients had angina pectoris and their plasma cholesterol were resistant to the conventional drug therapy. PE was performed using IBM Cell Separator (model 2997). Plasma (1, 500ml) was removed and replaced by an equal volume of human serum albumin. PP was performed using IBM Cell Separator and cellulose acetate hollow fiber filter (cascadeflo AC-1760). We reported the changes of plasma apolipoproteins concentrations during long-term PE in these two patients previously. In the present study, we compared the changes of plasma apolipoproteins concentrations during repeated PP with those during repeated PE. Plasma concentrations of apo A-I, A-II, B, C-II, C-III and E were measured by single radial immunodiffusion method. Plasma apolipoproteins decreased significantly by two different methods (PE and PP). Apo B was equally removed during PE and PP. owever, removal of apo A-I, A-II, C-II and E were less during PP than PE. Concentrations of plasma apolipoproteins before PP were more stable than those before PE. These data suggested that PP was better therapy than PE for familial hypercholesterolemia from the point of specific removal of apo B-containing lipoproteins.

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The effectiveness of long-term (5-year) repeated plasma exchange treatment in a homozygous patient with familial hypercholesterolemia has been evaluated. Plasma exchange at fortnight intervals reduced mean serum cholesterol levels from 618mg/dl to 342mg/dl (45% reduction) and mean LDL cholesterol levels from 463mg/dl to 251mg/dl (46% reduction). Plasma exchange at a week interval reduced mean cholesterol level to 217mg/dl (65% reduction) and LDL cholesterol levels to 128mg/dl (72% reduction). Resolution and disappearance of xanthoma and reduction of Achilles tendon thickness were observed. However, serial coronary angiographic findings showed a progression of coronary stenosis.

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Male Wistar rats were made hyperinsulinemic by the subcutaneous inplantation of osmotically driven minipump which delivers approximately 6.4 U/day of porcine insulin for up to 2 weeks. Five percent sucrose solution was given as drinking water for hyperinsulinemic rats to prevent from hypoglycemia. Semisynthetic diet with or without 2% cholesterol, 0.5% cholic acid and 0.2% propylthiouracil were prepared.
Plasma HDL-cholesterol level was elevated by the combination of insulin infusion and sucrose supplementation and suppressed by the cholesterol-rich diet. However, the combination failed to raise HDL-cholesterol level in rats with cholesterol-rich diet.
Since cholesterol loading suppresses endogenous cholesterol synthesis, the failure of this combination to raise HDL-cholesterol level in cholesterol-fed rats suggests the important role of endogenous cholesterol synthesis for elevating HDL-cholesterol level in rat plasma.

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We have examined the changes of plasma and lipoprotein lipids, plasma apolipoproteins (apo A-I, A-II, B, C-II, C-III and E) and subpopulations of VLDL particles separated through heparin affinity column by cholesterol overloading in Japanese man.
This study was carried out in 4 healthy men (38±4 of age) within 10% of diserable body mass index, being fed diet with 6 eggs a day (1, 650mg of cholesterol daily). Blood samples were obtained before and 1, 2 and 3 weeks after cholesterol loading.
As a results, plasma cholesterol and HDL-cholesterol were increased slightly (plasma cholesterol 193±18 to 208±23, HDL-cholesterol 58±15 to 64±16mg/dl, mean±S. D.) and so as plasma phospholipids and LDL-phospholipid (plasma phospholipids 218±29 to 249±43, LDL-phospho-lipids 48±7.2 to 61±14mg/dl) on cholesterol loading but these changes in plasma and lipoprotein lipids were not statistically significant. Plasma apolipoprotein A-I, A-II, B and E, C-II concentration were changed from 164±18.1 to 166±8.98mg/dl, from 38.97plusmn;4.21 to 38.7±3.26mg/dl, from 84.3±8.50 to 88.0±11.2mg/dl, from 4.85±1.04 to 5.48±1.48mg/dl and from 11.3±2.63 to 12.7±3.19, respectively. But these changes were not significant. Apo C-II was increased significantly from 3.53±1.66 to 5.11±2.07mg/dl (p<0.05) by cholesterol loading for 3 weeks.
The subpopulation of VLDL rich in apo E, which is characteristically similar to β-VLDL and chylomicron remnant, was increased significantly (p<0.005) after 2 weeks of cholesterol rich diet. On the final day of this 3 weeks experiment, apo E rich VLDL particle (heparin bound VLDL) was increased 1.98-fold over the initial value. We concluded cholesterol rich diet increase apo E rich VLDL separated through heparin affinity column eventhrough no remarkable increase of plasma and lipoprotein lipids are detected. Therefore the further determination into the subfraction of heterogenous lipoprotein is presumably important to consider the relationship of abnormal lipid metabolism and atherosclerosis.

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The effects of oral fat (cream) ingestion on plasma concentrations of lipids, lipoproteins and apolipoproteins (apo) and VLDL-apo B subfractions were studied in fasted 8 healthy men aged 25-43 years (mean age: 30 years old). Cream, 200g (triglyceride 102.8g, phospholipid 7.4g, cholesterol 1.0g), was ingested within 3 minutes early in the morning. Blood was withdrawn at 0, 30, 60, 120, 180, 240 and 300 minutes after fat ingestion. Lipoproteins were fractionated by sequential ultracentrifugation. Apolipoproteins were measured by single radial immunodiffusion. Apo B subfractions in VLDL were analyzed by SDS-3-15% gradient polyacrylamide gel electrophoresis. After staining, the protein bands were evaluated at 590 nm wave length by densitometer and peak area was measured. Apo B-48 ratio (percent of apo B-48 in total apo B) was calculated.
Fasting plasma concentrations of lipids and apolipoproteins were as follows: cholesterol (Ch); 184.3 ±9.0mg/dl, triglyceride (TG); 108.7±16.3mg/dl, phospholipids (PL); 197.9±5.9mg/dl, HDL-Ch; 64.3±2.4mg/dl, apo A-I; 135.5±8.5mg/dl, apo A-II; 29.8±1.2mg/dl, apo B; 72.7±5.7mg/dl, apo C-II; 3.1±0.4mg/dl, apo C-III; 8.1±0.6mg/dl, apo E; 3.7±0.1mg/dl. After fat ingestion, plasma TG increased approximately 3 times of the value before fat loading, which was mainly due to the increments of VLDL-TG and IDL-TG. Concentrations of any apolipoproteins did not show significant changes. Apo B-48 ratio increased 2 folds 60 minutes after fat loading (4.6%-8.9%) and decreased gradually to the level before fat ingestion, indicating that apo B-48 ratio is a valuable parameter to follow-up the exogenous lipoprotein metabolism. Degree of plasma lipids (especially TG) response to fat ingestion varied from subject to subject. Increment of plasma TG concentration correlated significantly with fasting plasma levels of TG (r=0.87, p<0.005), VLDL-TG (r=0.81, p<0.01), VLDL-Ch (r=0.81, p<0.01) and apo C-III (r=0.71, p<0.05), indicating that degree of TG increase after fat load is reflected in the fasting plasma levels of lipids, lipoproteins and apolipoprotein.

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Eicosapentaenoic acid (EPA) is known to have anti-atherogenic and anti-thrombotic effects. Effects of plasma polyunsaturated fatty acids (EPA, DHA, AA) on plasma apolipoproteins (A-I, A-II, B, A-I/A-II ratio, A-I/B ratio) were examined in healthy 98 people at Hachijo Island.
The results were as follows:
1) EPA/AA ratio (0.6±0.3) of people in Hachijo Island was higher than that of city people (0.45±0.20).
2) The levels of EPA and DHA showed negative correlation with level of HDL.
3) The level of EPA showed positive correlation with levels A-I and B, DHA showed positive correlation with levels of A-II and B, AA showed positive correlation with A-I and B and EPA/AA ratio showed no significant correlation with apolipoproteins.
4) The levels of EPA showed negative correlation with A-I/B ratio, DHA showed negative correlation with A-II/B ratio, AA showed negative correlation with A-I/A-II ratio and EPA/AA ratio showed negative correlation with A-I/B ratio.
5) A-I/A-II ratio and A-I/B ratio were significantly lower in high TC group than middle and low TC group.

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We studied the relationship between plasma lipids and apo E4/2 phenotype. Apo E phenotypes were determined by the rapid flat gel isoelectric focusing method that we reported previously. The frequency of apo E4/2 phenotype was 0.92 (4/436). Three of the subjects with apo E4/2 phenotype had type IV hyperlipoproteinemia. They showed normal laboratory data except for the lipids. The other subject had diabetes mellitus of 11 years' duration and had type III hyper-lipoproteinemia. The families of three subjects had the rare phenotypes of apo E3/2, E2/2 and E4/3, and most of them had type ha, IIb or IV hyperlipoproteinemia. In conclusion, apo E4/2 phenotype may be associated with type IV or III hyperlipoproteinemia, and apo E2 and E4 may be associated with the occurrence of lipid abnormalities.

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A new variant of apolipoprotein E (apo E-Kochi) was identified in a serum of a 29-year-old man with hyperlipidemia.
This patient was found to have hyperlipidemia in September, 1984, but had no symptom of cardiovascular disease. On examination, xanthomas, corneal arcus and hepatosplenomegaly were not evident. Electrocardiogram was no abnormal finding. Liver function tests, oral glucose tolerance test and thyroid function test were normal.
His serum cholesterol and triglyceride values ranged from 214-308mg/dl and 546-1, 568mg/dl, respectively. An agarose electrophoretogram of the serum showed broad-beta band. Lipid concentrations of the VLDL and IDL were elevated, and these fractions accounted 67 % of the serum cholesterol. But the ratio of VLDL-cholesterol to VLDL-triglyceride was 0.26. The hyperlipoprotein phenotype of the patient was difficult to classify, but resembled mixed type of III and IV. Apo E level of the serum was elevated markedly, that was 22.4mg/dl.
The isoelectric focusing pattern of the VLDL from the patient revealed major double bands in the apo E3 region and minor double bands in the sialated isoform regions (E3_s1, E_s2) of the gel. In the family study, the proband, his mother, maternal uncle and grandmother were found to have the same pattern of apo E. His father, brother and sister were found to possess ordinary E3/E3 phenotype.
The cationic component of the double bands of apo E of the proband was focused exactly in the E3 region, and the more anionic component was equal in amount to cationic band and different in isoelectric point by a half charge unit (≅0.06 pH). After cysteamine modification, the double bands shifted one charge range toward cathode, indicating that each component of the double bands contain one residue of cysteine. Each component of the double bands was revealed to have equal molecular weight by SDS-polyacrylamide gel electrophoresis, and same affinity to heparin-Sepharose gel column. Purified protein from the anionic band, as well as protein from the cationic band and ordinary apo E3 showed immunochemical precipitation reaction with anti-apo E goat serum.
The results from the above have indicated that the cationic band from the patient was apo E3 and the more anionic band was a new variant isoform of apo E, that was designated as apo E-Kochi. We presume that the apo E-Kochi may has histidin/neutral amino acid interchange at residue 140 of apo E3 that are sufficient to account for the half-charge difference between apo E-Kochi and E3 isoform.

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The composition of apolipoprotein C and E isoforms of human triglyceride-rich lipoproteins appears to be related to the metabolism of hypertriglyceridemia. We analysed the heterogeneity of apo E in very low density lipoprotein from 25 hypertriglyceridemic patients, 38 patients with diabetes mellitus, and 23 patients with ischemic heart disease. The apo E2/E3 ratio in diabetics and in patients with ischemic heart disease tended to be higher than in controls. The incidence of variant pattern with apo E4 was higher in those patients than in controls. In this study, a new series of apo E components, apo E-7, was found in three patients with hyperlipidemia. All of three patients had the phenotype E 3/7. The first case, a 68-year-old man, had ischemic heart disease. The concentration of serum cholesterol and triglyceride was 202 and 312mg/dl, respectively and not responded to dietary restriction. The agar gel electrophoretic pattern of lipoproteins demonstrated broad beta band. The values for the cholesterol in VLDL, VLDL-cholesterol to VLDL-TG ratio, and VLDL-cholesterol to total triglyceride ratio were 43 mg/dl, 0.23, and 0.14, respectively, and were not compatible with the laboratory diagnostic criteria of type III hyperlipoproteinemia. Two brothers had also apo E-7 with hypertriglyceridemia. The second case, a 52-year-old male, had a history of hypertension. The serum cholesterol and TG levels were 281 and 511mg/dl, respectively (Type IV). The third case, a 47-year-old male, had mild diabetes mellitus. The serum cholesterol and TG levels were 223 and 224mg/dl, respectively. Apo VLDL from his mother and father revealed phenotype E 3/2 and E 4/2, respec-tively. These results suggest that the mutant apo E-7 may be related to the development of hyperlipidemia and atherosclerosis. Detailed structural and functional analyses of these mutant apo E are remained to be elucidated.

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We investigated lipoprotein metabolism in eleven patients with XLI, a metabolic disease in which steroid sulfatase was deficient. Polyacrylamide gel electrophoretic mobility of LDL in all patients markedly increased compared with control subjects. In these patients, marked changes in lipid and apoprotein compositions of LDL were observed, that is, esterified cholesterol contents were decreased, in contrast, triglyceride contents were increased, and apoprotein B to cholesterol ratios were higher than those in normal control. These findings suggested that the rapidly migrating LDL in patients with XLI was accompanied by remarkable changes of lipid and apoprotein compositions probably due to increased cholesterol sulfate which had strong electronegativity.

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Serum lipoprotein, apoprotein and lipid levels were studied in 110 healthy children and 30 children with mycoplasma pneumoniae infection.
Results are as follows:
1) No dietary influence was observed on the concentrations of apoproteins (A-I, A-II, B, C-II and E) in healthy children, when the blood samples were taken, preprandial and 2 to 3 hours postprandial.
2) HDL-C levels were reduced in almost all patients with mycoplasma infection at the acute stage, but were returned to almost normal during recovery.
3) HDL₂-C level was markedly lowered, compared with that of HDL₃-C, in the patients at the acute stage, which may account for the decrease of HDL-C concentration.
4) A significant decrease of apoprotein A-I and A-II levels, and a significant increase of apoprotein B level at the acute stage returned to almost normal during the recovery.
5) The above data being compatible with the variations of lipoprotein and apoprotein observed in atherosclerotic state, mycoplasma infection might be a possible risk factor in the development of atherosclerosis.

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Arachidonic acid in phospholipids (PL) was an important factor for the initiation and progression of atherosclerosis. Lysophospholipid acyltransferase is an important pathway for polyunsaturated fatty acids (PUFA) to be incorporated into PL (Lands' pathway). Previously we reported that arterial acyltransferase (ATase) had different fatty acid specificity for different lysophospholipids such as lysophosphatidylcholine (lyso PC) and lysophosphatidylethanolamine (lyso PE) and that this specificity regulated the arachidonic acid (AA) metabolism into PL. In this paper, we studied the AA metabolism at various phospholipids through acyltransferase in atheromatous lesions.
First, we investigated the relationship between the AA content and lyso PE ATase on aorta of diabetic rats, spontaneous hypertensive rats, aging rats and rabbits on high cholesterol diet. The changes of AA content in PL of aorta and the changes of lyso PE ATase activities in comparison with controls were inversely related. These results indicate that ATase has a role of maintaining AA in PL.
Next, AA content was analyzed in individual phospholipids in atheromatous lesions. AA content in phosphatidylcholine (PC) is much more decreased than in phosphatidylethanolamine (PE) in atheromatous lesions of rabbit aorta and human aorta compared to controls. These results were considered to be caused by a different regulation of ATase related to PE and PC.
We have already reported that kinetics and fatty acid specificity of these ATase were different. The results in this paper also showed lyso PC ATase and lyso PE ATase were different in the following point; 1) effects of freezing and thawing (lyso PC ATase was decreased by 45% but lyso PE ATase was decreased by 19%), 2) effects of low density lipoprotein (lyso PC ATase was increased by 36 but lyso PE ATase was increased by 81 %.), 3) effects of supernatant of atheromatous lesions (lyso PC ATase was decreased by 79% but lyso PE ATase was increased by 13 %), 4) the changes of activities in atheroma (lyso PC ATase was increased by 36 % but lyso PE ATase was increased by 81%).
These results indicate the possibility that lyso PC ATase and lyso PE ATase are not the same enzyme. It is suggested that different regulation of ATase may explain the fact that the changes of arachidonic acid content are different among individual phospholipids in atheromatous lesions.

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Rabbits fed on a 1% cholesterol diet for 18 weeks were daily injected i. m. with porcine pancreatic elastase (10mg/rabbit/day). The enzyme activity in serum toward Suc-(Ala)₃-pNA, a synthetic substrate of elastase, was significantly higher in elastase-administered rabbits for periods of 18 weeks. This activity was considered as an elastase activity because elastatinal, a specific inhibitor of elastase, strongly inhibited not only the enzyme activity in serum but also the activity of porcine pancreatic elastase used for administration. Orceinelastin hydrolyzing activity was lower than Suc(Ala)₃-pNA hydrolyzing activity in sera of elastase-administered rabbits and a level of α2-macroglobulin, an inhibitor of elastase, was elevated in these sera. This result may suggest that elastase forms a complex with α2-macroglobulin, a high-molecular-weight inhibitor in serum and indicate that its complex is slightly active on the high-molecular-weight substrate, elastin, but retains to some extent the activity on the lowmolecular-weight substrate, Suc-(Ala)₃-pNA. Elevated serum lipids were lowered, especially on the 18th week, by elastase administration. On the 18th week, furthermore, there was a decrease in lipids in aortic tissue and elastin fraction from elastase-administered rabbits. In particular, a decrease in the content of lipids in elastin fraction was significant. This decrease in elastin-bound lipids may be a result of proteolytic action of elastase against lipid-bound elastin, probably denatured one. Scanning electron microscopic study on the endothelial surface of rabbit aorta showed a reduction or regression of plaques in elastase-administered rabbits.

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We studied acute and long-term effects of Clinofibrate on lipoprotein lipase (LPL) activity. In an acute administration study, Clinofibrate (400mg, single) was administered orally to 5 healthy subjects. On the other hand, in a long-term administration study, Clinofibrate (1, 200mg/day) was administered orally to 11 hyperlipidemic patients for about 4 to 8 weeks. We investigated postheparin lipolytic activity (PHLA), LPL and hepatic triglyceride lipase (HTGL) activity in the acute and long-term administration study, and serum lipid levels of all cases before and after Clinofibrate administration.
The results were as follows:
1. Acute administration study
Changes in LPL activity by Clinofibrate administration were variable.
2. Long-term administration study
1) Total cholesterol and triglyceride decreased significantly. High density lipoprotein-cholesterol was stable.
2) PHLA and LPL activity increased in 8 out of 11 patients. Decrease in total cholesterol and triglyceride was more marked in patients with the increased LPL activity than with decreased or unchanged. There is significant positive correlation between the percent increase in LPL activity and the percent decrease in triglyceride (r=0.77, p<0.05).

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