We have developed a simple, rapid assay method for chylomicron and very low density lipoprotein (VLDL) remnants, using an immunoaffinity gel mixture of anti apo B-100 and apo A-I monoclonal antibodies coupled with Sepharose 4B. The standard assay contains 5μl of serum and 50μl of mixed gel, which adsorbs lipoproteins containing apo A-I quantitatively as well as most of the lipoproteins that contain apo B-100 (virtually all low density lipoprotein (LDL) and most VLDL). After 60 mins of shaking at room temperature, unbound cholesterol and triglyceride present in the supernatant are measured enzymatically. Unbound lipoproteins (RLP: remnant-like particles) are enriched in apo E, apo C and contain essentially all of the apo B-48 as well as some of the apo B-100 of the triglyceride-rich ipoproteins.
The concentration of serum RLP-C was determined in the fasting state as follows: in 419 healthy normolipidemic subjects (male: 265, female: 154, age: 15-76) with a mean±SD of 2.1±1.6mg/dl. In 5% of the 419 healthy normolipidemic subjects and in 20.3% of the 454 normolipidemic patients (CAD, diabetes, etc.) the RLP-C concentration exceeded 5mg/dl.
The levels of serum RLP-C among patients with hyperlipidemia. coronary artery disease, diabetes, obesity, fatty liver and chronic renal failure were shown to be high and over 40% of the patients exceeded 5mg/dl. Probucol was administered to the diabetic patients with relatively high RLP-C levels. After 2 months, RLP-C levels were decreased significantly by the probucol and remained at low levels.
These results indicate that the quantification of serum RLP-C levels in a fasting state is a new, useful diagnostic marker for remnant hyperlipoproteinemia.

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Diffuse intimal thickening (DIT) has developed in the human coronary artery even in infants. Eccentric intimal thickening (EIT) evolves from DIT and may develop into atheromatous plaque. The purpose of this study is to clarify histological changes in the intima during the development of DIT to EIT or plaque in the human coronary artery.
Materials for histometrical examination were obtamed from 253 autopsy cases. Histometrical studies were carried out on the proximal portions of the right coronary artery (seg. 1) and the left anterior descending artery (seg. 6). Thicknesses of the intima and the media and the I/M ratio were measured in all cases. In cases over 20 years of age, volume fractions (Vfs) of SMCs, collagen fibers, and elastic fibers in the intima were analyzed by the point-counting method. The number and density of SMCs were counted within the same area. DIT in both segs. 1 and 6 tended to increase with age. The I/M ratio in DIT increased with age but did not exceed 2. EIT increased more markedly with age than did DIT. Collagen fibers were the major component of the intima. Vfs of collagen fibers increased with successive changes from DIT to EIT and finally to plaque in contrast to the decrease in the density of SMCs when compared within the same age groups.
In immunohistochemical studies, anti-muscle antigen (HHF35) positive SMCs were present in DIT as well as in EIT. A great number of anti-macrophage antigen positive foam cells were present in the plaque, particularly in plaque shoulder. Proliferating cell nuclear antigen (PCNA) positive cells were predominantly distributed in plaque shoulder, and prolyl hydroxylase (PH) positive cells were present within the same area. Anti-collagen Type I antibody reacted intensely to fibers in plaque, but fibers which reacted with the anti-collagen Type III antibody were randomly distributed in every layer of the arterial wall.
In conclusion, DIT, EIT and plaque thickening in the coronary artery were chiefly due to SMC proliferation in the younger subjects; however, after the 4th decade, thickening was mainly due to an accumulation of collagen fibers.

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Serum amyloid A protein (SAA) is an acute-phase protein, mainly associated with high-density lipoproteins (HDL). We developed a direct binding enzyme-linked immunosorbent assay for SAA, and investigated the involvement of SAA in HDL metabolism among Patients with inflammatory diseases. The assay is simple, accurate and reproducible without denaturation of samples. By this method, SAA concentration in the plasma of 35 normal subjects was 0.14±0.07mg/dl (mean±SD). SAA concentra. tions were increased (39.6±64.0mg/dl)in the plasma of 86 patients with inflammatory diseases, and positively correlated with CRP levels (r=0.805). During the course of the acute inflammatory diseases, the increase in SAA concentration accompanied by the decrease in HDL-cholesterol, apo A-I, and apo A-II levels, recovered after the decrease of SAA concentration. The SAA levels in plasma and lipoprotein fractions were measured in 20 patients with acute inflammatory diseases and in five normal subjects. In the normal subjects whose plasma SAA levels were very low, SAA was mostly distributed in the lipoprotein free fraction (d>1.210). However, in cases with acute inflammatory diseases, the greater part of SAA was distributed toward HDL (especially the HDL₃ subfraction) and to some extent in the LDL and VLDL fractions with increased plasma SAA levels. These results suggest that increased SAA replaces apo A-I and A-II on the HDL particles, so that restructured HDL and the apo A-I and A-II freed from HDL might be catabolized faster. Plasma SAA levels increased to Various levels (19.3±23.9, 0.17-118.3mg/dl) in the plasma from 42 patients with rheumatic diseases, and chronic inflammatory conditions. On the other hand, HDL-C (40±11mg/dl), apo A-I (124±20mg/dl) and apo A-II (22±8mg/dl) levels decreased compared with those of 30 healthy adults (51±10, 152±17, 29±4mg/dl, respectively). Furthermore, in the rheumatic diseases, SAA showed a significant inverse correlation to HDL (r=-0.409), apo A-I (r=-0.642) and apo A-II (r=-0.545). Therefore, this study demonstrated that the increase of plasma SAA is one cause of the low levels of HDL in patients with chronic inflammatory diseases.

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To investigate the relationship between abnormal glucose metabolism and LDL receptor activity (LDL-RA), we, studied LDL-RA in 20 Japanese-Americans who had no symptoms or history of familial hypercholesterolemia. LDL-RA was measured using lymphocyte PHA response according to the method reported by Lipsky and Cuthbert.
75g oral glucose tolerance test (OGTT) was also performed on all subjects. The OGTT results proved to be normal in 11 subjects, and showed impaired glucose tolerance (IGT) in five subjects and diabetes in two subjects. In two subjects, the OGTT had to be stopped because of vomiting.
We compared LDL-RA with several factors, and found that LDL-RA was significantly correlated with the LDL level and with the LDL cholesterol level (r=0.46, 0.44, respectively, p<0.05).
We also determined that LDL-RA was frequently low in subjects with abnormal OGTT results, and that the ∑SG level apparently influenced LDL-RA more than the ∑IRI level did.
These results suggest that LDL-RA is partly affected by disorders in glucose metabolism.

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The relationships among the apolipoproteins (apo), physical variations, nutrition and energy expenditure were studied by using 169 healthy men who had been randomly selected from among 1, 084 male participants in 1989 in Tanushimaru, Kyushu, Japan.
The results of the univariate analysis showed that the concentration of apo A-I was positively related to the percentage of calories from alcohol (alcohol (%)), and that the concentration of apo A-II was positively related to obesity and alcohol (%), and negatively related to age and energy expenditure, while, the concentration of apo B was positively related to obesity. Concerning apo C-II, C-III and E, significant relations to energy expenditure and obesity were observed.
Multiple linear regression analysis was performed to exclude those factors which had been overestimated by the univariate analysis. Significant correlations between apo A-I and obesity, alcohol (%), smoking and daily cholesterol intake were observed. The concentration of apo A-II was well indicated by age, obesity, energy expenditure, alcohol (%) and smoking. The concentration of apo B was correlated only with obesity, while the apo B/A-I ratio showed a correlation with both obesity and alcohol consumption. Apo C-II, C-III and E were well indicated by daily energy expenditure.
Therefore, it is safe to conclude that the metabolism of serum apolipoproteins is influenced by various environmental factors. Further detailed studies on these factors are required to help prevent atherosclerotic diseases through the modification of lifestyles.

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Lipoprotein (a) [Lp (a)] is known to appear in high levels in patients with ischemic heart disease (IHD). The purpose of this study was to determine Lp (a) levels and to investigate the association of Lp (a) and atherosclerotic risk factors in diabetic patients. Thirty-three women and 27 men, aged 27-72 years, were studied. The mean (±SD) levels of plasma Lp (a) were elevated in the diabetics compared to the controls (34.0±36.7vs. 18.3±10.4mg/dl, P<0.01). Lp (a) concentrations increased significantly as the urinary albumin concentrations (mg/g·creatinine) increased; the groups with concentrations below 30mg/g·cr, 30-300, and above 300 showed increases in Lp (a) concentrations (mean±SD) of 18.8±3.8, 29.8±10.2, and 35.1±31mg/dl, respectively. Therefore, plasma Lp (a) concentrations were elevated in diabetic patients from the microalbuminuria stage. The levels of Lp (a) remarkably increased in the diabetics with chronic renal failure (80.3±48mg/dl). There were, nonetheless, significant positive correlations between Lp (a) and urinary albumin concentrations. However, no correlations were noted between Lp (a) and other factors such as age, duration of diabetes, ideal body weight, fasting plasma glucose, glycosylated hemoglobin, BUN, creatinine, lipoprotein lipids or apoproteins. In patients with chronic renal failure, Lp (a) levels tended to decrease during hemodialysis treatment. An increased rate of Lp (a) synthesis and/or decreased catabolism may have contributed to the elevated levels of Lp (a) in patients with diabetic nephropathy. Along with other known cardiovascular risk factors such as elevated blood pressure, atherogenic abnormalities of plasma lipids and lipoproteins, increased levels of Lp (a) may play an important role in accelerating the development of atherosclerosis in this condition.

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The acute effects of submaximal ergometry exercise on HDL metabolism were studied in 10 healthy males. The bicycle ergometry exercise was performed with 50W of incremental loading every 3 minutes until an end point of 90% of the target heart rate was reached. Changes in the components and particle size of the HDL subpopulations were studied during the exercise protocol. Blood samples were obtained before breakfast after 14 hours of fasting (8:00a.m.), before exercise (10:30a.m.), immediately after submaximal exercise (11:00a.m.), and both one and two hours after exercise. After one week, blood samples were collected from the same subjects over the same time intervals without exercise. HDL₂ was separated from HDL₃ by ultracentrifugation. The total cholesterol (TC), free cholesterol (FC), triglyceride (TG), and phospholipid (PL) in the HDL subfractions were measured by applying enzymatic methods. The protein content was estimated with bichinchoninic acid. The total lipoprotein mass was determined by adding the TC, TG, PL and protein contents. The particle size of the HDL subfractions was measured by gradient gel electrophoresis.
Two hours after the exercise, TC, esterified cholesterol, PL and the total mass in HDL₂ and HDL₃ were significantly decreased, while the particle size of both HDL₂ and HDL₃ were apparently increased relative to thier values before the exercise (both p<0.05). A new HDL subpopulation corresponding to HDL₁ immediately appeared after exercise, and disappeared 2 hours after the exercise.
The results obtained in this study indicate that submaximal exercise promoted HDL metabolism, and consequently enhanced cholesterol reverse transport.

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The aim of this study is to investigate the effects of dietary fiber intake on serum lipid concentration in the aged. The amount of daily food intake for three consecutive days was measured and the serum levels of total-cholesterol (TC), triglyceride (TG), HDL-cholesterol (HDLC) were determined for 270 subjects (135 males and 135 females), 65 years and over, living in a local community. The LDL-cholesterol (LDLC) was calculated by (TC—HDLC)—TG×0.2.
Dietary fiber intake showed little difference by sex.
Dietary fiber intake had a significantly positive correlation with HDLG, the intake of carbohydrates and protein and total fat, but had a significantly negative correlation with LDLC, LDLC/HDLG.
Multiple regression analysis showed that dietary fiber intake had a statistically significant relationship with HDLG, LDLC/HDLG, and LDLC.
Since HDLC, known as an antiatherogenic, was related to the increase in the dietary fiber intake, and LDLC, known as an atherogenic, was related to its decrease, it was suggested that dietary fiber intake might have an important effect on the genesis and prevention of atherogenic diseases.

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Low-density lipoproteins (LDL: 1.019-1.063) were isolated by sequential ultracentrifugation over a series of weeks from 22 cholesterol-fed rabbits to study the peroxidizability of LDL due, to Cu⁺⁺.
1) Twenty-two male rabbits, each 12 weeks old and weighing, 2.5 to 3.5kg, were divided into 2 groups by diet, namely, 11 rabbits were fed standard oriental feed and 11 rabbits were fed feed with 1% cholesterol.
2) Blood was drawn at 0 week, 2 weeks, 5 weeks, 7 weeks, and 10 weeks after feeding, and thereafter LDLs were separated by Havel's method.
3) The peroxidizability was evaluated by calculation of LDL lipid peroxide per LDL-cholesterol after dialysis of LDL against 5μmol Cu⁺⁺. The lipid peroxide was estimated by the modulation of Yagi's method.
4) LDL-cholesterol, LDL-apo B and LDL-lipid peroxide increased markedly weekly in cholesterolfed rabbits, but in rabbits fed standard oriental feed the values did not change. Each LDL separated at each of the various weeks was dialyzed into Cu⁺⁺ for 1, 2, 3, 4, 5, 6, 12, 24, 48, 72 and 96 hours.
The maximum values of LDL-lipid peroxide per LDL-cholesterol in each LDL were obtained at 6 hours after dialysis in LDL of 0 week, at 24 hours in LDL of 2 weeks, at 48 hours in LDL of 5 weeks, and at 72 hours in LDL of 10 weeks.
5) The ratios of LDL-cholesterol to LDL-apo B were in the order of LDL of 10 weeks>LDL of 5 weeks>LDL of 3 weeks>LDL of 2 weeks>LDL of 0 week.
Conclusion: 1) Peroxidizability due to Cu⁺⁺ was higher in low cholesterol LDL than in high cholesterol LDL.
2) It was speculated that the formation of atheroma due to the buildup of cholesterol in rabbits was related to the higher ratio of LDL-cholesterol/LDL-apo B than to the degree of the peroxidizability of LDL.

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When endogenous synthesis, of cholesterol is blocked by an HMG-CoA reductase inhibitor, proliferation of Phytohemagglutinin (PHA)-stimulated human lymphocytes is markedly inhibited, provided an exogenous source of cholesterol is not supplied. Based on this principle, Lipsky et al. developed a simplified method to assay functional LDL receptors. However, it is still a controversial point whether HDL can be taken up by the cell via an LDL receptor. We modified Lipsky's method by using pravastatin (HMG-CoA reductase inhibitor) and lipoprotein-depleted fetal calf serum, and examined the capacity of LDL and HDL subfractions to supply cholesterol to lymphocytes. Peripheral blood lymphocytes were isolated from venous blood of 10 healthy adults and from l patient with FH homozygote. Human LDL and HDL were isolated from the serum of normal fasting adults by ultracentrifugation, and HDL_2b and HDL_2a fractions which contained Apo E but not Apo B were prepared using a heparin-Sepharose column. The ability of LDL cholesterol to reverse pravastatin-mediated inhibition of normal lymphocyte responses was found to be concentration dependent, and the suppression ability of pravastatin was reversed by the HDL_2b and HDL_2a fractions. However, the effect of these fractions was weaker than that of LDL. HDL₃ showed no capacity to recover the cells from suppression. On the other hand, lymphocytes lacking normal LDL receptors from FH homozygote could not obtain cholesterol from LDL or any subfractions of HDL. Therefore, our result suggests that both HDL_2b and HDL_2a can be taken up by lymphocytes via LDL receptors.

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It is well known that stress and hyperlipidemia are risk factors of atherosclerotic diseases. It has been reported that various types of stress accelerate the progress of atherosclerosis. However, there have been no reports on the correlation between stress due to sonic exposure alone and stress due to abnormal lipid metabolism. Therefore, many problems on the subject remain to be solved. The aim of this study is to clarify these problems. In the present study, We divided rabbits on a high-cholesterol diet into two groups—sonic stress (+) and sonic stress (-) groups. Each group was examined for fluctuations in the serum lipid levels, pathological changes, and the effects of γ-Oryzanol.
High values of serum TG were observed in the sonic stress (+) group, compared with the sonic stress (-) group, but no differences were observed in TC, HDL-C, and LDL-C. Next, pathological studies demonstrated that sonic stress promoted atherosclerotic changes in the aorta of the rabbit.
As a result, it was assumed that blood pressure was elevated and platelet aggregation was accelerated by sonic stress.
Atrophy of thymus gland observed as an influence of sonic stress exerted on the weight of the various organs. This phenomenon is considered to be interesting in thinking about immuno-reaction and progress of atherosclerosis.
In the following study, as a result of the effect of γ-Oryzanol, as anti-stress agent which improved lipid metabolism in the stress (+) group and (-) group, it was found that the agent had a stronger anti-atherosclerotic activity in the sonic stress (+) group.
The results of these studies, therefore suggested that sonic stress accelerates atherosclerosis.

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