Patient with familial high density lipoprotein deficiency, Tangier disease, found in Miyazaki had an abnormal electrophoretic mobility of serum LDL which moved faster than that of normal LDL on polyacrylamide gel electrophoresis. In consistent with this finding, gel filtration using high performance liquid chromatography showed the decrease in size of LDL particles in the patient's serum; relative elution volume of LDL to serum albumin were (mean±SD) 0.886±0.008 for homozygotes and 0.851±0.005 for normal subjects. Abnormality present in LDL was also observed in fatty acid composition of cholestryl ester. In these patients, saturated fatty acid compositions (mean±SD, %) were increased to 5.56±1.09 (2.85±0.63) in C-14, 28.17±2.10 (19.38±1.79) in C-16 and 15.19±1.04 (7.89±2.23) in C-18, compared with those obtained from normal subjects as shown in parentheses, while unsaturated fatty acid compositions were decreased to 11.83±2.07 (17.04±1.66) in C-18: 1 and 32.90±2.55 (47.69±3.93) in C-18: 2 except for the value observed in C-16: 1. On the other hand, no abnormality was observed in fatty acid composition of total serum cholesteryl ester. These results together with the normal ester ratio in cholesterol suggested that the cholesterol esterifying mechanism proceeded normally but metabolism of lipoprotein particles containing cholesteryl ester of normal fatty acid composition was disturbed in Tangier disease.
Another abnormality was found in serum HDL of heterozygotes in Tangier disease. Gel filtration showed that HDL₂, normally a main component of HDL, was decreased as well as the results on the gel electrophoresis. This fact suggested the disturbance of interchange of HDL₂ and HDL₃.

View full abstract

The clinical and pathophysiological features of a case of abetalipoproteinemia in a 34-year-old patient are described. This patient is the first case reported in Japan. The patient was diagnosed as abetalipoproteinemia by confirming the Apo-B deficiency in the patient's serum and the slightly high cholesterol level in his mother's and borderline normal level in his father's. The patient had remarkably low lipid levels, acanthocytosis, and lipid malabsorption. An unusual feature of this case was that the patient had no neuromuscular or ocular manifestations. This was possibly related to his normal plasma vitamin A and E levels.

View full abstract

The lipoprotein analysis of 4 subjects with Type III hyper lipoproteinemia associated with Apo E₃ deficiency was performed by high performance liquid chromatography (HPLC) in addition to ultracentrifugation and PAG-disc electrophoresis and the results were compared with those of 3 subjects with familial hypercholesterolemia showing the phenotype of Type III (FH-Type III) classified by β-migrating VLDL and increase of IDL but without Apo E₃ deficiency. VLDL subfraction contained increased concentration of cholesterol and triglyceride and were relatively rich in cholesterol in both classical Type III and FH-Type III. LDL subfraction in classical Type III was very low and the ratio of cholesterol to triglyceride was low even before treatment when total cholesterol was high, while FH-Type III had high level of LDL-cholesterol. HPLC for lipoprotein revealed no lipoprotein eluted at the regular LDL position and elution position of major component shifted to the position of larger particle size than LDL. In FH-Type III major fraction was eluted at the exact position of normal LDL, although PAG-disc electrophoresis showed broad-β pattern similar to classical Type III.
Clofibrate treatment decreased cholesterol and triglyceride level to normal ranges although no change was occured in electrophoretic pattern and elution pattern of HPLC in classical Type III. However, in FH-Type III broad-β pattern disappeared and was onverted to typical Type IIa by clofibrate treatment. From the data above, we conclude that the mechanism of the appearance of Type III phenotype was clearly different between classical Type III with Apo E₃ and the others and it is possible even without Apo E analysis to diagnose classical Type III (E2/2) by lipoprotein analysis using HPLC and by the mode of responses to clofibrate treatment.

View full abstract

Apolipoprotein E (apo E) is one of the major protein constituents in VLDL and has been thought to play an important role in lipoprotein metabolism. We analyzed heterogeneity of apo E in VLDL from patients with hyperlipidemia and/or atherosclerosis by isoelectric focusing (IEF).
Six different apo E phenotypes were shown by IEF in agreement with the current genetic model that the major apo E isoproteins, apo E-4, apo E-3 and apo E-2, resulted from three apo E alleles, ε4, ε3 and ε2, at a single genetic locus. All the patients with broad-β disease (classical type III hyperlipoproteinemia) were confirmed to have the E2/2 phenotype representing homozygosity for ε2 allele.
We recognized an additional apolipoprotein band basic to apo E-IV in three subjects. The new apolipoprotein component was identical with ordinary apo E with regard to an apparent molecular weight by SDS-polyacrylamide gel electrophoresis and to the interaction with Heparin-Sepharose gel as well as anti-apo E antibody. This mutant apo E isoprotein, named apo E-5, was shifted more basic isoelectric point by one unit of charge than apo E-4 isoprotein. Genetic analysis of the apo E phenotypes in family members of the patients with apo E-5 indicated the presence of a new apo E allele (ε5) at the same genetic locus as former alleles. Since many of the subjects with apo E-5 had ischemic heart disease or cerebral infarction, it was suggested that the mutant apo E possibly related to the genesis of atherosclerosis.

View full abstract

Plasma lipoprotein abnormalities in patients with two different types of familial lecithin: cholesterol acyltransferase (LCAT) deficiency, “classical” type and “partial” type were studied. The apoprotein composition in lipoprotein fractions were similar in both types, but the levels of apoprotein were different. Plasma apo A I and A II levels were low in both types, but the decrease was prominent in “classical” type. Plasma apo B 100 level remained within the normal range in “partial” type but it was low in “classical” type. The levels of apo B 48 in VLDL from “partial” type increased slightly and apo B 48 level in VLDL from “classical” type was very high.
The content of discoidal HDL was low and the content of small spherical HDL was high in “partial” type as compaired with those in “classical” type. The content of large sized LDL was high in “classical” type and that was low in “partial” type. The large-sized-LDL contained significant amount of apo B 48, and the increase in the content of large-sized LDL may be related to the pathogenesis of renal injury in patients with “classical” type.

View full abstract

Lipid and lipoprotein metabolism was studied in patients with recessive X-linked ichthyosis, a disease in which cholesterol sulfatase activity was absent.
1. Polyacrylamide disc-gel electrophoretic mobility of plasma LDL in these patients was greatly increased, as compared with control subjects. This finding could be caused by strong electronegativity of an accumulated cholesterol sulfate containing in plasma LDL.
2. Lipid and protein compositions of VLDL, LDL, HDL₂ and HDL₃ separated ultracentrifugally in these patients were similar to those of control subjects. In these patients, no abnormal change of serum Apo B level was found. Although serum Apo A level was slightly low, ratio of Apo A-I level to Apo A-II level was almost normal.

View full abstract

In man, studies on VLDL apo-B metabolism indicate that the small particles are derived entirely from larger particles. On the other hand, there is in vivo evidence that triglyceride (TG) in small particles is not derived entirely from larger particles. The above information raises the possibility that newly made TG can be transferred from hepatocyte directly into pre-existing TG-rich particles. In order to persue this possibility we incubated TG-prelabelled rat hepatocytes with human lipoprotein fractions. ³H-glycerol was injected via portal vein 20min before liver perfusion with collagenase. Collected cells were washed 4times and incubated at density ca 4×10⁶ cells/ml with or without a lipoprotein fraction in Tyrode's solution, pH 7.4 with 1% BSA. After 45min incubation at 37°C, the medium was separated from hepatocytes by centrifugation. TG-glycerol specific activities in the medium containing VLDL (sf 60-400) was significantly higher than in medium without lipoprotein addition. The labelled TG appeared to be part of the fraction as judged by ultracentrifugation, hep-Mn and anti-apo-B separation. This transfer of TG from hepatocyte to VLDL did not depend on the synthesis and release of new VLDL particles as it was not accompanied by a change in the production of ¹⁴C-leucine labelled VLDL protein and it was not blocked by chloroquine. Less transfer was found from hepatocyte to LDL (sf 0-12) than to VLDL (sf 60-400). These data suggest that newly synthetized TG can directly enter circulating TG-rich particles independent of endogeneous VLDL secretion.

View full abstract

In vitro perfused liver from rhesus monkey generated very low density lipoprotein (VLDL) similar to serum VLDL, except for the existance of three kinds of high molecular weight apoproteins. These three kinds of high molecular weight apoprotein were considered to be isoproteins of apoprotein B, because of their molecular weight, amino acid composition, and their immunological character. They were designated as B100, B74, and B42 in a centile system on SDS polyacrylamide gel electrophoresis.
Although the B42 ran a little bit faster than the B48 did, which was found in hyperlipemic monkey VLDL, it was considered that the B42 could be a modified B48.
Oleic acid supplement in perfusate resulted in the increase of triacylglycerol secretion in perfusate VLDL, indicating that fatty acid supplement results in the secretion of larger VLDL from perfusedliver.
The larger VLDL produced by the liver perfused with fatty acid supplement contained the more B42. Considering that the specific activity of B42 decreased with time over the course of perfusion, while that of B100 increased, the B42 is not considered to be degradative product of B100.
Besides, with increase of B42 in VLDL secreted by the liver perfused with fatty acid supplement, the ratio of apoprotein E to apoprotein B in VLDL increased, indicating that enlargement of VLDL secreted by the liver results in enrichment of apoprotein E and B42.
It is very important to explore the metabolic fate and the role in atherogeneity of such B42 and apoprotein E rich VLDL produced by fatty acid supplement.

View full abstract

A variety of invasive stimuli such as bacterial, parasitic infections, tumor and endotoxemia have been shown to induce a suppression of lipoprotein lipase (LPL) resulting in hypertriglyceridemia. In endotoxin-responsive C3H/HeN mice, the LPL activity in adipose tissue was markedly suppressed 16h after endotoxin administration. In endotoxin-resistant C3H/HeJ mice, the LPL activity was suppressed to a much lesser extent than in C3H/HeN mice. In a remarkable contrast, the blood from C3H/HeN mice obtained 2h after the injection with endotoxin suppressed LPL activity in adipose tissues for both of C3H/HeJ mice and C3H/HeN mice. Accordingly, a transferable factor capable of suppressing LPL would be secreted into the blood of C3H/HeN mice treated with endotoxin. Conditioned medium from peritoneal macrophages of endotoxin-responsive mice cultured in the presence of endotoxin suppressed adipose tissue LPL in C3H/HeJ mice also. The transferable factor which appeared in blood and suppressed LPL activity, therefore, appeared to be secreted by macrophages in response to endotoxin. In addition, the factor did not directly inhibit LPL activity nor accelerate the rate of decay of LPL. These results have indicated that the factor suppresses LPL activity by inhibiting its synthesis or processing in the cells.
Employing 3T3-L1 cell culture, the mechanism by which the factor suppresses LPL activity has been studied. The factor suppressed LPL activity in 3T3-L1 adipocyte in culture. In view of the known requierment of insulin and glucose for the maximal experssion of LPL activity, the effect of the factor on insulin insulin receptor interaction, the glucose uptake and protein synthesis were studied. Neither insulin binding to insulin receptor nor glucose transport was affected by the factor. However, the incorporation of ³⁵S-methionine into protein in particulate fraction of 3T3-L1 cells decreased by 50% after the cells were exposed to the factor; little change was observed in the incorporation into the soluble fraction. Comparison of the proteins obtained from cells with or without endotoxin treatment on SDS polyacrylamide gel electrophoresis has demonstrated that the factor modulates the synthesis of proteins in both of soluble and particulate fractions in a selective manner.
On the basis of these results, one of the primary effect of the factor seems most likely to be exerted on protein synthesis.

View full abstract

Vitamin E was transported through lipoproteins metabolism and HDL vitamin E (HDL-E) had been suggested to be highly related with tissue or cell levels of vitamin E (for example red blood cell). The following our results coincide with the above suggestion, that the correlationship between HDL-E (0.49±0.15mg/dl) and RBC-E (2.01±0.27μg/ml packed cell) was significant in control group, then reduced RBC-E (1.63±0.22) in aged group was linked with reduced HDL-E (0.36±0.07). On the other study, when red blood cells were fractionated by ultra-centrifugation (15, 000rpm, 90min.), the relative content of vitamin E in top fraction (young RBC) was 1.213/cell or 1.128/Ht against that in bottom fraction (old RBC).
These results let us have speculation that the tissue or cell vitamin E may be exchangeable most easily with HDL-E, however, cell has its own regulating (control) mechanism, furthermore.
In this work, electrochemical detector was proved to be able to detect 100ng or more low concentration of vitamin E in HPLC system with addition of Tocol as internal standard.

View full abstract

In this study, 600mg/day or 1200mg/day of Clinofibrate (Lipoclin^®) was orally administered to 42 hyperlipidemic patients during three months. Clinofibrate decreased serum triglyceride about 32% by 600mg/day and 48% by 1200mg/day. It decreased serum cholesterol 5% by 600mg/day and 7% by 1200mg/day. Those patients were classified to 28 Type IIb, 9 Type ha and 5 Type IV hyperlipidemics according to the WHO pheno-types' classification of hyperlipidemia. The lipid lowering effects in each lipoprotein fraction were studied. The lipoproteins were fractionated by ultracentrifugation using Beckman Lp-42 Ti rotor. In Type IIb hyperlipidemics, 600mg/day of Clinofibrate lowered VLDL and LDL cholesterol and VLDL triglyceride and increased HDL cholesterol. However, 1200mg/day of Clinofibrate lowered VLDL cholesterol and triglyceride but increased LDL cholesterol, slightly. Clinofibrate was known to increase lipoprotein lipase activity in peripheral adipose tissue. So, this difference between 600mg/day and 1200mg/day might be due to that of the amount of VLDL catabolized by lipoprotein lipase. The clinical usefulness of 1200mg/day administration of Clinofibrate was partly confirmed but must be further studied in the cases of longer trial by more precise observation of lipoprotein metabolism. No adverse reaction was observed in this trial.

View full abstract

Fifty-four patients were treated with clinofibrate (600mg/day) for 16 weeks to observe changes in serum lipid levels and apo A_I, A_II. The serum concentration of total cholesterol, triglyceride and phospholipid were reduced significantly, while HDL-cholesterol was increased significantly, resulting in reduction of atherogenic index significantly.
In patients with serum cholesterol above 250mg/dl, triglyceride above 150mg/dl and phospholipid above 250mg/dl, their average serum concentration were significantly reduced after administration of clinofibrate. The average serum concentration of HDL-cholesterol, however, was increased significantly in patients with HDL-cholesterol under 40mg/dl.
Serum concentration of apo A_I was increased significantly following medication, on the other hand, no change in serum concentration of apo A_II was recognized.
The reduction of atherogenic index in IIb predominated that of IIa and IV types after administration of clinofibrate.
Side-effects caused by this drug were not found in any of the patients.

View full abstract

The cooperative and prospective study on the prognostic value had been conducted by using clofibrate, aiming to observe whether clofibrate could low the incidence and mortality of atherosclerotic diseases for 7 years.
Since the trial was not in double blind fashion, blood pressure was lower and cholesterol level was higher in clofibrate group (2560 cases) than those of the control group (1427 cases).
Severity index in cerebrovascular disease tended to increase in both groups during the period, but that in cardiovascular disease appeared to regress in clofibrate group.
Cummulative incidence of cerebrovascular disease seemed to be depressed and recurrence of cardiac event, especially fatal accident, was significantly decreased.
Incidence of malignant diseases during the period was not significantly different.
Proportion of surviving according to the life table analysis was significantly greater in the clofibrate group.
Therefore, it can be concluded that clofibrate would be useful to the secondary prevention of cerebrovascular and cardiovascular diseases.

View full abstract

Hyperlipidemia can be often seen in diabetic subjects. In this study, the effect of clinofibrate on serum lipids level was investigated in 35 hyperlipidemic diabetics.
Clinofibrate (600mg per day) induced a significant decrease in serum total cholesterol levels (p<0.001) from the first month to the 6th month after its administration.
Triglycerides also decreased by 25-30% compared with the initial levels. Clinofibrate increased HDL-cholesterol, although the average of increasing rate was not statistically significant, whereas the atherogenic index decreased significantly (p<0.01) during the observation period.
Clinofibrate was well tolerated by all patients. Nearly all laboratory parameters analyzed were unchanged.
Clinofibrate has been proven to be an effective lipid-lowering drug in hyperlipidemic diabetics from this study.

View full abstract

Peroxisomal catalase and hepatic oxygen consumption were monitored non-invasively in perfused rat liver.
1) The effects of CPIB on ethanol oxidative metabolism were studied in perfused rat livers. In some of the experiments, S-8527 was compared with CPIB.
2) When the rats were fed for two weeks either with CPIB (0.5%) or S-8527 (0.1%), CPIB induced hepatomegaly, but otherwise revealed the similar hypolipidemic effects to S-8527.
3) The rate of oxygen consumption in CPIB-treated liver was higher than that of the control group.
4) The role of alcohol dehydrogenase system in ethanol metabolism was considered to be of less significance in CPIB-treated liver.
5) The catalase heme content was increased approximately up to nine-fold in CPIB-treated liver. This result indicated that H₂O₂ generation rate in CPIB-treated liver was significantly increased. Catalase H₂O₂ (compound I) was also monitored by organ surface spectrophotometry.
6) Chronic treatment with CPIB caused hepatic peroxisomal and microsomal induction. S-8527 (0.1%), however, affected neither O₂ consumption nor H₂O₂ generation rate.
These findings suggest that the hypolipidemic mechanisms of CPIB are not exactly the same as those of S-8527.

View full abstract

Coenzyme Q₁₀ was administered to 8 diabetic patients in a dose of 90mg/day for 3 consecutive weeks, and before and after treatment fasting blood was drawn to determine the levels of Coenzyme Q₁₀ in the lipoprotein fractions of serum. Following blood collection the lipoprotein fractions, VLDL, LDL and HDL, were separated by ultra-centrifugation.
The content of Coenzyme Q₁₀ in the lipoprotein fractions was, in decreasing order, LDL, VLDL, and HDL.
At the end of 3-week treatment period the Co-enzyme Q₁₀ content was increased by 54.3% for VLDL, 45.9% for LDL, and 6.7% for HDL, as compared to the pre-drug levels. A similar tendency was noted with respect to the Coenzyme Q₁₀/cholesterol ratio. After the administration of Coenzyme Q₁₀ its levels in total and VLDL fractions showed a significant increase, as compared to the pre-drug values.
It is an interesting subject of inquiry how serum Coenzyme Q₁₀ is transported to organs and tissues.

View full abstract

It has not been well established the effect of clofibrate on glucose tolerance and its glucoselowering mechanism in non-insulin dependent diabetes mellitus (NIDDM).
We studied, therefore, the effect of 4 weeks clofibrate treatment on glucose tolerance together with its glucose-lowering mechanism in 15 NIDDM patients. Long-term effect of clofibrate on glucose tolerance was also followed for 7 months in 37 NIDDM patients. All patients were treated with ordered diet and/or exercise before and during the administration of clofibrate, 1500mg daily after every meals, and the studies were started after the establishment of the constant body weight and plasma glucose levels. 75g oral glucose tolerance test (75g OGTT) and insulin tolerance test (ITT) were carried out before and at 4 weeks of the treatment.
1) Fasting plasma glucose values were 168.1±9.5mg/dl and 136.5±6.0mg/dl (p<0.01) before and at 4 weeks of clofibrate, respectively. Plasma glucose values during 75g OGTT showed significant decrease in each point (p<0.01), especially at the latter points, and ∑ glucose was decreased by 20.6±2.3% after the clofibrate treatment. Fasting IRI levels and IRI responses were also decreased statistically.
2) However, insulinogenic index and ∑ IRI/∑ glucose ratio during 75g OGTT were not changed by the treatment. Enhanced glucose fall was observed in ITT. On the other hand, correlation was not observed between the decrease of FFA, triglyceride and total cholesterol levels, and the improved glucose tolerance.
3) Glucose-lowering effect was continued during the administration periods of 7 months, and its effect was statistically significant with the same glucose-lowering rate observed in the 4 weeks administration study.
These results suggest that clofibrate would improve the glucose tolerance in NIDDM, and the glucose-lowering mechanism might be derived from the enhanced tissue sensitivity to insulin.

View full abstract

In order to elucidate the significance of HDL-cholesterol on the severity of coronary atherosclerosis, serum lipoprotein analysis was made in 70 patients who underwent coronary cineangio-graphy and 23 healthy volunteers.
Serum lipoproteins were separated into 3 fractions of HDL, LDL and VLDL using the method of Density Gradient Ultra-centrifugation (SOR-VALL OTD-65 Ultracentrifuge and SORVALL TV-865B vertical rotor). Centrifugation was performed at 65, 000rev/min for 1hr. and 45min. (10°C).
To evaluate the reliability of this method, data were compared with those methods such as the precipitating and Agarose gel electrophoresis.
Following results were obtained;
1) The correlation coefficient between HDL-cholesterol level measured by Ultracentrifugation and that of precipitating method was r=0.9189 (p<0.001), and also the correlation coefficient between the percentages of HDL-cholesterol in this method and in Agarose gel electrophoresis was r=0.8996 (p<0.001).
2) Both Atherogenic Index (HDL-cholesterol/LDL-cholester) ol) and HDL-cholesterol level of the younger volunteers was significantly higher than those of the older ones (p<0.05).
3) Both Atherogenic Index and HDL-cholesterol level of the volunteers was significantly higher than those of patients with 2 or more diseased coronary vessels (p<0.05).
4) The correlation coefficient between serum HDL-cholesterol level and the number of diseased vessels was r=-0.5207 (p<0.001), and the more the number of diseased vessls increases, the fewer serum HDL-cholesterol level becomes.
From these results, it was shown that the Density Gradient Ultracentrifugation was easy and useful method for serum lipoprotein analysis. And it was suggested that the severity of coronary atherosclerosis was estimated more precisely by Atherogenic Index from this method.

View full abstract

Serum cholesterol (CH), triglyceride (TG), high density lipoprotein cholesterol (HDL-C) and apolipoprotein (Apo) A-I, A-II and B were measured in 41 male patients with angiographically defined coronary artery disease (CAD). The CAD patients had significantly lower HDL-C, Apo A-I and A-II and higher TG and Apo B levels than controls. However, there was no significant difference in serum CH between the two groups. First-degree relatives of the CAD patients were also investigated. The male relatives of the CAD patients also had significantly lower HDL-C, Apo A-I and Apo A-II levels than controls. The female relatives of the CAD patients had significantly lower Apo A-I level than controls, but showed no significant differences in CH, TG and HDL-C. In type IV hyperlipoproteinemia, there were no significant differences in serum CH, TG and HDL-C levels between CAD patients and controls. However, Apo A-I, A-II and B showed significant differences between two groups. These results suggest that low Apo A-I and A-II levels are a good indicator for coronary atherosclerosis, and that these abnormalities are related to genetic factors.

View full abstract

Familial hypercholesterolemia (FH) is an autosomal dominant disease due to the disorder of LDL-receptors characterized by marked hypercholesterolemia and atherosclerosis. It is important to establish the therapeutic procedures, since the disease has high fregneucy of ischemic heart disease. Up to the present, various drugs and procedures have been developed for the reduction of serum cholesterol, but the efficiency of long term therapy, especially whether regression of atherosclerotic lesions by reduction of serum cholesterol occurs or not, has not been reported in details. Previously we reporteid that Achilles tendon thickness (ATT) is related to the incidence of ischemic heart disease and may be regarded as an indicatorof lipid accumulation in tissues.
In this study, we measured ATT exactly with xeroradiography which is usually used to examine soft tissues, and investigated the relation between the changes of ATT and the reductions of serum cholesterol after long term therapy with drugs, probucol and cholestyramine.
Consequently, we confirmed the efficiency of drugs on the reduction of serum cholesterol in FH, particularly in FH homozygote, in whom drug therapy has not been thought to he effective, because remarkable reduction of serum cholesterol was observed by drugs under a low calorie and low fat diet. By means of xeroradiography we could measure ATT exactly and observed regression of ATT in substantial numbers of FH patients after long term therapy. The regression of ATT required reduction of serum cholesterol, but the degree of reduction of serum cholesterol wasn't always in proportion to the degree of regression of ATT. Therefore we concluded that not only the level of serum cholesterol but other factors might be important to lipid accumulation and regression in tissues.

View full abstract

We have reported that the porous glass was able to bind a maximum of 26.0mg of LDL per g of porous glass and the plasma cholesterol (CHOL) and LDL-CHOL levels were reduced in the WHHL rabbit and the cholesterol-fed rabbit by the extracorporeal treatment of plasma over porous glass column.
We reported here a 33 year-old homozygous patient with familial hypercholesterolemia who was treated for 5 months by adsorption chromatography with porous glass. The plasma CHOL levels decreased from 481±7 (Mean±SE) mg/dl to 287±5mg/dl (-40% decrease) and the LDL-CHOL levels from 344±16mg/dl to 221±10mg/dl (-35% decrease). Also, the IDL-CHOL and HDL-CHOL levels slightly decreased. Similar changes were noticed in triglyceride and phospholipid levels. The concentrations of various serum enzymes and electrolytes showed no changes, and there were no changes in the levels of the cellular elements of blood. But, the serum albumin and immunoglobulin levels slightly decreased. The decrease of serum albumin and immunoglobulins in adsorption chromatography with porous glass were smaller than plasma exchange and double filtration methods. There were no side effects in adsorption chromatography with porous glass.
These data suggest that the extracorporeal technique involving adsorption chromatography with porous glass is a safe and effective method for reducing the plasma LDL in a homozygous patient with familial hypercholesterolemia.

View full abstract

Cholesterol synthesis and LDL receptor activity were examined in circulating human lymphocytes freshly isolated from venous blood ofthe patients with or without familial hypercholesterolemia (FH).
Mononuclear cells were freshly isolated by the method using the Ficoll-Paque^® at 4°C and suspended in RPMI-1640 medium containing 4% bovine fatty acid free albumin. Incorporation from 2-¹⁴C-acetate into digitonin precipitable sterols and proteolytic degradation of ¹²⁵I-LDL at 37°C were measured.
The rate of ¹⁴C-incorporation into sterol was linear with time for at least 3h and the high affinity ¹²⁵I-LDL degradation showed the initial lag phase and the saturable curve in the cells isolated from normal subjects. There was no difference in the rate of cholesterol synthesis among the control, the heterozygous FH and homozygous FH. High affinity ¹²⁵I-LDL degradation from homozygous FH was significantly low (p<0.05), but not in the cells from heterozygous FH in comparison with that from control subjects.¹⁴C-lncorporation into sterol negatively correlated with VLDL-cholesterol (p<0.05). High affinity ¹²⁵I-LDL degradation negatively correlated with LDL-cholesterol (p<0.05).
These results suggest that in vivo LDL receptor activity in the mononuclear cells isolated from hetrozygous FH showed no difference from that in the cells isolated from control subjects, but LDL receptor activity from homozygous FH was significantly low in comparison with that from control subjects. The rate of cholesterol synthesis in the cells from heterozygous FH and from homozygous FH were within the normal range. As there was a significantly negative correlation between cholesterol synthesis and VLDL-cholesterol, the regulation of cholesterol synthesis and LDL receptor activity in circulating human lymphocytes may depend not only on LDL-cholesterol, but on VLDL-cholesterol.

View full abstract

1) The effect of 4 weeks administration with immunomodulator PSK (Krestin) on the lipoprotein lipids contents in serum, was investigated in 10 patients with hyperlipidemia including three FH heterozygous patients. There was a significant decrease of T-cholesterol, β-lipoprotein in serum. HDL-cho, and triglyceride showed no significant alterations.
2) In a series of in vitro experiments, the effects of PSK on the LDL receptor activity of normal and FH heterozygous fibroblasts were investigated. In normal subject, PSK increased the uptake of ¹²⁵I-LDL by+31% at 100μg/ml, while in FH heterozygous cells, the uptake was increased by +117% at 10μg/ml.
3) One of the reasons why β-lipoprotein in serum is decreased in patients of hyperlipidemia by PSK administration is considered to be that PSK enhanced the LDL receptor activity of fibroblasts. It was also suggested that PSK has stimulated more effectively LDL receptor activity of fibroblasts from hyperlipidemia patients than from normal subject.

View full abstract

Glycosylated-LDL was prepared by incubation of human LDL with glucose and galactose in vitro. The glycosylated-LDL containing more than 20 to 25 percent of free amino group modified, was metabolized at a slow rate by both human skin fibroblasts and mouse peritoneal macrophages. From the fractional catabolic rates of glucose-LDL and galactose-LDL, catabolism by a receptorindependent process occured 41 and 30 percent respectively. These results suggested that glycosylated-LDL was a useful tool in estimating receptor-independent LDL catabolism.

View full abstract

The medium, in which macrophages were incubated with acetylated LDL (a-LDL) for 24 hours was collected. The medium was added to the medium in which rabbit aortic smooth muscle cells were incubated. The medium derived from the macrophages incubated with a-LDL remarkably enhanced the incorporation of ³H-cholesterol into smooth muscle cells and increased the content of total cholesterol in smooth muscle cells.
Above these results, it was suggested that the factor which was released from macrophages incubated with a-LDL might regulate the lipid metaholism in smooth muscle cells and might cause the formation of foam cell originated from smooth muscle cells.

View full abstract

Purpose of the present study is to develop of a simplified intravenous fat tolerance test. A single dose of fat emulsion (0.25ml of 10% Intralipid/kg of body weight) was injected intravenously through the catheter inserted into the antebrachial vein taking for 90 seconds and the blood samples were drawn at 5, 8, 11, 14, 17, 20, 23 and 29 minutes after the midpoint of the injection. The test was done on 28 normal, diabetic or hyperlipidaemic subjects after an overnight fasting and at supine position.
Lipoprotein fraction of d<0.95g/ml was separated with ultracentrifugation according to Hatch's method. Light scattering index of serum and the lipoprotein fraction of d<0.95g/ml were estimated with nephelometer (Nepherotic DN-2110, Kyoto Dai-ichi Kagaku Co., Ltd.) after dilution with physiological saline by 100 times or 48 time respectively.
Disappearance curves were exponential as shown in Fig. 2. Fractional removal rate of Intralipid (K₂) of the serum and the lipoprotein fraction of d<0.95 g/ml were calculated by the method of least squares using logarithmic value of the light scattering index (Fig. 3).
The K₂ values of the serum and the lipoprotein of d<0.95 g/ml showed good agreement in magnitude and range, and well correlated (r=0.855, p<0.001) as shown in Fig. 4. K₂ obtained in the present study was apparently higher than that obtained from other fat tolerance tests using large amount of Intralipid. A hyperbolic relationship was found between K₂ of the serum or the lipoprotein of d<0.95g/ml and fasting serum triglyceride concentration (Fig. 5). The inverse relatioship between K₂ values and fasting serum triglyceride became linear when the datas were transfered to their logarithm (Fig. 6).
These results revealed that the simplified fat tolerance test with small amount of fat emulsion and short sampling time is valuable to obtain K₂ value and a convenient tool to study pathogenesis of hypertriglyceridaemia.

View full abstract

A case of familial lipoprotein lipase (LPL) deficiency is reported. The patient was a 34 year-old man of Japanese, whose parents were first cousins. In 1977 at his age of 29, he was diagnosed as primary idiopathic hypogonadism and hyperlipemia. Plasma concentration of triglyceride (TG) was 2000mg/dl, in spite of the medication of clofibrate and clinofibrate. In 1982 he was admitted because of severe epigastralgia after excessive drinking and eating. On physical examination there was neither skin xanthomas, hepatosplenomegaly, nor lipemia retinalis. The serum taken after an overnight fast was turbid. On standing overnight at 4 C, a creamy layer of chylomicrons appeared on the top. The serum TG concentration varied from 577 to 3704mg/dl, according to dietary fat intake. The serum cholesterol level was moderately elevated, the range being from 132 to 436mg/dl. Lipoprotein electrophoresis and ultracentrifugation showed the presence of excessive ammounts of chylomicrons in the fasting serum. The concentration of the VLDL was also elevated, but LDL and HDL levels were depressed. Postheparin plasma LPL activity was 0.8μmoles FFA/ml/hour (Normal control; 6.4±2.1). The level was less than 20% of normal controls, so it was diagnostic for LPL deficiency. The LPL activity of his elder sister at the age of 38 was alsolow, being 1.2μmoles FFA/ml/hour (Serum TG; 675mg/dl). The LPL activity of his abdominal addipose tissue was 0.20μmoles FFA/hr/gm wet tissue. It was lower than the level of normolipemic controls of similar age and sex. Immunochemical measurement of apoproteins showed that the levels of apoproteins C-II, B and E were elevated. But the levels of apo A-I and A-II decreased. Restriction of dietary fat to 10 gm or less has been useful to reduce serum TG levels and keep him free of symptoms. Available lipidl-owering drugs were not effective.

View full abstract

Two kinds of lipase, lipoprotein lipase (LPL) and hepatic triglyceride lipae (H-TGL) are released into plasma by intravenous administration of heparin. LPL is believed to play an important role in the degradation of triglyceride rich lipoprotein, and is also suggested to be engaged in the metabolism of HDL. On the other hand, the physiological role of H-TGL was undefined for a long time, but recently the deficiency of H-TGL was discovered and it's abnormalities in lipoprotein composition resembled Type III hyperlipoproteinemia associated with apo E3 deficiency, suggesting this enzyme may be essential for the metabolism of IDL or remnants of TG-rich lipoproteins.
Considering these roles of LPL and H-TGL in the lipoprotein metabolism, the precise measurement of postheparin lipolytic activity (PHLA) is very important.
PHLA is now commonly measured by using radioisotopically labeled substrate, but we had better restrict the use of RI if possible in order to avoid the environmental pollution. In non-RI method, PHLA was measured by using Ediol as substrate and titration method of Dole for the determination of released free fatty acid. because of its low sensitivity, we must use much more volume of postheparin plasma (PHP) for incubation than that in the RI method. Considering the property of H-TGL, which is inhibited by serum in vitro, the activity measured by this method is not accurate.
In this work, we established a new non-radioisotopic method for the measurement of LPL and H-TGL in postheparin plasma, using NEFA KitK (NIPPON SHOJI, Osaka, Japan) for the deterurination of released free fatty acid. The selective measurement of LPL and H-TGL was performed as described by M. L. Baginsky and W. V. Brown. Using this measurement system, we examined LPL and H-TGL activity of postheparin plasma (50units/kg Body Weight) in normal control group and type III hyperlipoproteinemia with apo E3 deficiency and discussed the contribution of LPL and H-TGL to lipoprotein metabolism.
Following results were obtained.
1) Extracted free fatty acid by Dole's method can be measured with linearity up to about 300nmol/tube for LPL and about 400nmol/tube for H-TGL by NEFA Kit-K. Thus, we can measure PHLA with lenearity up to about 60μmol/ml/hr for LPL and about 80μmol/ml/hr for H-TGL, since 5μl of postheparin plasma was used for the measurement of PHLA in our system.
2) H-TGL was inhibited almost completely after 10min pre-incubation of PHP with an equal volume of 100mM SDS at 26°C. The remaining activity (LPL activity) was plateau till 70min of preincubation.
3) The addition of hyperlipidemic serum up to 10μl did not interfere the purified H-TGL activity.
4) The mean activities of LPL and H-TGL of normal males are 9.4 and 20.1μmol/ml/hr respectively which are nearly equal to those reported by J. Boberg and M. Boberg.
5) The relationships between PHLA (LPL and H-TGL) and lipid composition in VLDL, IDL, LDL, HDL2 and HDL3 were not statistically significant in this study.
6) After clofibrate treatment with apo E3 deficient patient, LPL activity increased, cholesterol and triglyceride in VLDL, IDL, LDL and HDL3 decreased, and HDL2 cholesterol increased.
These results suggested that this non-radioisotopic method is accurate and useful for clarifing the etiology of various hyperlipidemia and the contribution of abnormalities of lipoprotein metabolism to atherosclerotic diseases.

View full abstract

It has been reported that peripheral monocytes in culture synthesize and secrete lipoprotein lipase (LPL). In the present study, we have cultured peripheral monocytes from two healthy subjects and obtained the findings confirming that peripheral monocytes in culture produce and secrete a lipolytic enzyme. The enzyme secreted by them had the properties very similar to or identical with LPL. Cultured monocytes appear to be unique in that they not only synthesize but spontaneously secrete LPL, even in the absence of heparin. We have also observed that monocytes from two subjects with primary LPL deficiency and a subject with acute monocytic leukemia did not secrete a significant amount of LPL.

View full abstract

Lipoprotein lipase (LPL) secretion by the monocytes obtained from a woman, aged 83, with myelomonocytic leukemia was investigated. LPL activity secreted into the incubation medium by the leukemic monocyte-derived macrophages was detected after 1 day in culture and reached a maximum level at 2 weeks. The rate of the enzyme secretion was enhanced by 2-fold in the presence of heparin. The enzymes both in heparin eluates and in cells required serum for activation and was inhibited by 1M NaCl. The enzyme was retained on the heparin-sepharose column and eluted with 1.5M NaCl.
These results indicate that certain leukemic monocytes can secrete the enzyme which fullfills the criteria for LPL. These cells could be very useful for the cellular study of LPL synthesis and metabolism.

View full abstract

The purpose of the present study was to characterize the properties of cholesterol esterase (CEase) of rabbit smooth muscle cells (SMC) and rat peritoneal macrophages because both cells seem to play an important role in foam cells formation in which lipids, especially cholesterol ester (CE) are accumulated. Acid and neutral CEase existed in lysosome and microsome, respectively in both cells. Acid CEase activity was higher than neutral CEase activity, but the ratio was different between SMC and macrophages. Neutral CEase of SMC was lower than that, of macrophages. Other properties of acid and neutral CEases of SMC and macrophages were similar with respect to Km, effect of phospholipids and divalent cations for lecithine emulsified CE. But optimal pH of CE hydrolysing activity in LDL was located at pH 4.5 and was not detected at neutral or alkaline range, whereas there was two pH optima for CEase activity when lecithin emulsified CE was used as substrate. These results suggested that LDL-CE hydrolysis could be performed only in lysosome.
Incubation with native LDL increased acid and neutral CEase in both SMC and macrophages. Acetylated LDL increased acid and neutral CEase of macrophages greater than native LDL, but did not change those of SMC. These results suggested that SMC can internalize only native LDL but macrophages can internalize more acetylated LDL than native LDL.

View full abstract

It is suggested that phospholipids play important roles in the formation of atherosclerosis. To clarify the role of phospholipid metabolisms and their regulation in relating to the formation of atheroma, the properties of phospholipase A₁ and phospholipase A₂ activities were studied compared with those in rabbit aortic smooth muscle cells (SMC) and those in rat peritoneal macrophages. 1-palmitoyl-2-[1-¹⁴C]oleoyl-phosphatidylcholine (POPC) vesicle and 1-linoleoyl-2-[1-¹⁴C] linoleoyl-phosphatidylcholine labeled LDL (LDL-DLPC) were used as the substrate for the assay of phospholipase A₁ & A₂ activities.
1. Phospholipase A₁ & A₂ activities existed in SMC and macrophages.
2. Optimal pH of phospholipase A₁ activity was pH 4.0 in both SMC and macrophages, and that of phospholipase A₂ activity was pH 4.5 in SMC and pH 6.0 in macrophages.
3. Optimal pH of phospholipase A₁ & A₂ activities greatly changed with various PC substrates; POPC vesicle, DLPC vesicle, LDL-POPC and LDL-DLPC. This seemed to imply that the intracellular localization and the mode of existence of phospholipid regulated its own hydrolysis.
4. By feeding rats with high cholesterol diet (1% cholesterol, 0.5% cholic acid) for 12 weeks, phospholipase A₂ activity of macrophages decreased, while that of aorta was slightly increased.
5. When intact SMC and macrophages were incubated with various lipoproteins, phospholipase A₂ activity in SMC was slightly increased by LDL and much more increased by acetylated LDL. On the contrary, phospholipase A₂ activity in macrophages was much decreased by both LDL and acetylated LDL. It is believed that LDL is mainly incorporated into SMC, and denatured LDL into macrophages. But in this experiment, LDL and acetylated LDL showed the similar effects on phospholipase A₂ activity in both SMC and macrophages. These results suggested that there are some pathways other than LDL pathway or scavenger pathway, in which LDL-phospholipid is metabolized.

View full abstract

The extent and severity of coronary arteriographic findings associated with coronary risk factors were analyzed in relatively young patients. 100 patients who had the clinical diagnosis of coronary artery disease (90 male and 10 female) were selected out of 1, 000 patients studied in this laboratory. The average age was 43 years, range 20 and 49 years. 20 patients (20%) developed significant stenosis of 3 major coronary arteries and 18 patients (18%) had obstructions of 2 vessels and 36 patients (36%) had single vessel disease and 9 patients (9%) had lesions less than 50 narrowing of lumen diameter, while 11 patients (11%) exhibited coronary spasm during the procedure. The remaining 6 patients had normal arteriogram.
The left anterior descending was affected in 68 patients, of which 37 patients presented complete occlusion. The right coronary artery was involved in 31 patients and 10 patients of these presented complete occlusion. The left circumflex was involved in 26 patients, of which 8 patients presented complete occlusion. The main left coronary was involved in 2 patients.
In left ventriculography, 29 patients had major ventricular aneurysm and 22 patients had akinesis or hypokinesis.
The risk of coronary artery disease appeared to rise in proportion to the high level of total cholesterol, triglyceride, μ-lipoprotein and uric acid. The extent and severity of coronary artery seemed to be related to the existence of hypertension and cigarette habit.
These results indicate that coronary artery is severely affected by the atherosclerotic process even in relatively young generation and it is necessary to correct the coronary risk factors for the prevention and treatment of coronary artery disease in young patients.

View full abstract

Erythrocyte deformability and membrane fluidity in hypercholesterolemia were analysed and the relationship between the lipid composition and the function of erythrocyte were considered. Red blood cells were obtained from normal adult group, normal aged group, hypercholesterolemic aged group, normal rabbits and hypercholesterolemic rabbits, respectively. According to aging of erythrocyte, membrane lipid content was decreased but in hypercholesterolemic aged group, membrane cholesterol was increased and cholesterol/phospholipid ratio (C/P ratio) was significantly increased. Fatty acid composition of membrane phospholipid analysed using a gas liquid chromatograph revealed the significant decrease of C_20:4 in old cells. Erythrocyte deformability analysed by the method of nuclepore filtration technique revealed the impairment of deformability in hypercholesterolemic state. We obtained the same result from experimental hypercholesterolemic rabbits. Analysis of membrane fluidity using a ESR was performed and the order parameter values showed the decrease of fluidity of aged erythrocytes and of the erythrocytes in hypercholesterolemic state. These results suggested that the membrane of old erythrocytes in aged with hypercholesterolemia contained more free cholesterol and less C_20:4, leading to the damage of membrane mechanism such as deformability or membrane fluidity.

View full abstract

A new enzyme immunoassay of apolipoprotein B by sandwich method has been developed. Assay sensitivity is the highest for EIA (2ng) compared with SRID method (300ng) or Laser nephelometry (200ng). In addition, EIA has widest assay range (2-200ng) and the reproducibility of EIA was satisfied (C. V. 7.8% at 185.7mg/dl). A good correlation has been observed between values determined by EIA & SRID method or Laser nephelometry.
Clinically, apo B correlates well with LDL-cholesterol indicating that apo B may serve as an index expressing atherogenicity.
The fasting serum apo B concentration of normal subjects was 84.9±16.0mg/dl (mean±SE). Apo B levels were high in Type IIa (203±34.4mg/dl) & Type IV (145±20.3mg/dl) hyperlipidemia. In Type II Diabetic patients, apo B levels were high (125±4.4mg/dl) and also increased in borderline glucose intolerance (116±8.2mg/dl).

View full abstract

As macrophage-derived foam cells relate to the progression and regression of atherosclerosis, we attempted to acquire monoclonal antibodies against macrophages, using hybridoma technique. Use of these antibodies enables identification of macrophage-derived foam cells in atherosclerotic lesions.
Using BALB/C mice and SP2/0 mouse myeloma cell line, we established several clones of mouse hybridoma and which produce monoclonal antibodies against rabbit alveolar macrophages. ANM #43 antibody obtained in this series was confirmed to be specific for macrophages.
With this antibody, we, studied atherosclerotic lesions in aorta of rabbits fed a one percent cholesterol diet for 2, 5, 10 and 20 weeks, using labeled avidine-biotin technique. In early lesions, positive cells were frequently encountered on the surface and/or beneath the endothelial lines. Positive cells were also scattered in the atheromatous lesions of rabbits fed for 20 weeks, crowded positive cells were found especially around the necrotic foci. However large amounts of foam cells piled up lipids exhibited negative.
Medial smooth muscle cells did not react to the antibody.

View full abstract

The present study aimed to clarify the longterm effect of Soysterol on arteriosclerosis as measured by aortic pulse wave velosity (PWV). Control group consisted of 20 males and 15 females, 35 in total in ages from 46 to 82 years (average age 67.5 years), while Soysterol group consisted of 19 males and 15 females, 34 in total in ages from 33 to 79 years (average age 63.4 years). Soysterol was dosed at 1200mg per day in Soysterol group and all subjects were measured for PWV every 3 months. The period of observation were 31 to 118 months (average 82.4 months) in control group and 11 to 54 months (average 40.8 months) in Soysterol group.
PWV increased from 8.14m/sec to 11.7m/sec after 90 months for control group and from 9.14m/sec to 9.46m/sec after 50 months in Soysterol group, with regression coefficient of 0.0367 for former against lower regression coefficient of 0.0174 for the later. Significant differences (p<0.05 to 0.01) were observed in PWV between both group on and after 20 months of observation.

View full abstract