The Journal of Japan Atherosclerosis Society
Online ISSN : 2185-8284
Print ISSN : 0386-2682
ISSN-L : 0386-2682
Volume 6, Issue 1
Displaying 1-19 of 19 articles from this issue
  • Shigeru TAKAMATSU, Shigeru SAKUTA, Kei SATOH, Kazuho HENMI, Yuichi TAM ...
    1978 Volume 6 Issue 1 Pages 1-8
    Published: April 01, 1978
    Released on J-STAGE: September 21, 2011
    JOURNAL FREE ACCESS
    Recent advances in immunological method have facilitated the determination of serum lipprotein in easy. The clinical usefullness of the determination of β-lipoprotein by this method is widely recognized and now the immunological assessment of α1 lipoprotein which means α-lipoprotein with α1 mobility is also inclined to be adopted practically. The purpose of this study is to establish the most suitable procedure to assess serum α1lipoprotein by single radial immunodiffusion method and to clarify the significance of α-lipoprotein in atherosclerotic disorders with special reference to cerebrovascular diseases.
    The best procedure established was as follows:
    Three μl of sera diluted to 1:10 with veronal-HCl buffer (pH 8.6) were dropped into the hohle on one plate which consist of 5ml of 3% agarose and 5ml of 20% anti α1lipoprotein rabbit serum in the buffer. The plate was incubated for 48 hours at 37°C. Area of precipitation ring formed on the plate was designated as the concentration of serum α1lipoprotein. The coefficient of variation in this method was 1.62% in ten replicate on one and the same day.
    Concentration of serum α1lipoprotein decreased markedly following storage at 4°C and even-18°C indicating the necessity of instant assessment (Fig. 1).
    Subjects examined consist of 145 cerebrovascular patients and 245 non-cerecrovascular controls. In controls with neither hypertension, abnormal ECG findings nor hyperlipidemia, serum α1lipoprotein levels were significantly higher in the aged over 60 than that in the aged under 39 (Fig. 2), and the values in hyperlipidemia were higher compared to normolipidemics (Fig. 3). In normolipidemic controls with hypertension, the value was lower than that in normotensives aged over 60.
    Patients' levels were not affected by their age, blood pressure, and other serum lipid levels, and significantly lower than that of controls (Fig. 2). The value of patients complicated with abnormal ECGs and renal impairment was significantly lower than that of patients without these findings (Fig. 4). The values of patients with cerebral infarction was lower than that with cerebral hemorrhage, however, the difference was not significant.
    α1Lipoprotein was directly proportional to total cholesterol, malondialdehyde and antithrombin III in patients (Fig. 5).
    These results indicating the low level of serum α1lipoprotein in cerebrovascular disorders were in agree with those in previous observations of HDL by the ultracentrifugal method. Therefore, the results obtained comfirmed further the close relation of α-lipoprotein to atherosclerotic disorders including cerebrovascular diseases and the clinical usefullness of single radial immunodiffusion method.
    It has been emphasized that the inhibitory effect of α-lipoprotein to atherogenesis is based on the removal of cholesterol from arterial wall. The directly proportional relationship between α1lipoprotein and total cholesterol in controls in our results supports this concept. In addition, the significant relationship between α1lipoprotein and malondialdehyde suggests inhibitory effects of α1 lipoprotein on thrombogenesis which is affected by lipoperoxides. Possible important role of α-lipoprotein in the regression of atherosclerosis is also assumed by many evidence. Therefore, further intensive observation of α1lipoprotein in atherosclerotic disorders should be performed.
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  • Takashi OGAWA, Kiyoshi INADA
    1978 Volume 6 Issue 1 Pages 9-12
    Published: April 01, 1978
    Released on J-STAGE: September 21, 2011
    JOURNAL FREE ACCESS
    Lipids of plasma, erythrocyte and arterial wall in patients with occlusive arterial diseases of the extremity were measured and analyzed in detail. The following results were obtained.
    1. There were found no differences in plasma lipids lvels as cholesterol and triglyceride between patients with thromboangitis obliterans (TAO) and arteriosclerosis obliterans (ASO), althought level of triglyceride is usually higher than cholesterol.
    There were found no correlation between plasma lipids level and both diseases in contrast to myocardial infarction.
    2. Level of lipoperoxides was significantly raised in ASO compared to normal, although there was found no difference between TAO and normal.
    3. Incidence of hyperlipoproteinemia was higher in ASO with occlusive lesion of central artery than with distal occlusion. Type IIa and b were frequently found in ASO with aorto-iliac occlusion.
    4. Hyperlipoproteinemia was found more frequently in TAO than in ASO.
    5. The pattern of fatty acid composition of plasma lipids was same in both TAO and ASO, although changes were more significant in ASO. 6.
    There was no marked changes in lipids of erythrocyte membrane in both diseases.
    7. Cholesterol, especially cholesteryl ester, was markedly increased in arterial wall in ASO. Phospholipids was also increased, although low in percentage compared to other lipids.
    8. Increase of phospholipids in arterial wall was mainly resulted from increase of sphingomyelin, while phosphatidylethanolamine, phosphatidylserin and phosphatidylinositol were markedly decreased.
    9. Fatty acid composition of cholesteryl ester in arterial wall was similar to that of plasma in ASO, suggesting of their origin from plasma. However, fatty acid composition of phospholipids was definitely different with that of plasma, suggesting of their synthesis in arterial wall.
    10. Composition of phospholipids and total lipids in arterial wall in TAO was similar to those in normal artery, suggesting of its different nature of occlusive lesion in ASO.
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  • Soichiro TAKAHASHI, Fumiko NAGANO, Takashi TSUCHIDA, Hideaki SAITO
    1978 Volume 6 Issue 1 Pages 13-19
    Published: April 01, 1978
    Released on J-STAGE: September 21, 2011
    JOURNAL FREE ACCESS
    Progress in our knowledge of the lipoprotein metabolism has generated increasing evidence that atherosclerosis may be related not only to the beta- and pre-beta-lipoproteins but also, in particular, to chylomicron or remnants of chylomicron.
    There are acute changes of serum lipids and/or lipoproteins during the day, and obviously the degree and duration of alimentary lipemia are more severe and persistent when compared with that in the fasting state. The clinical study was undertaken to detect the dietary lipemia.
    For this purpose, a butter loading diet composed of two eggs, the yolk of three eggs and fifty grams of butter was ingested by the subjects, and the concentration of serum lipids and lipoproteins was determined at two-hour intervals. The feeding of the diet containing a large amount of long chain saturated and monounsaturated fatty acids produced marked hyperlipidemias and hyperlipoproteinemias in all subjects.
    However, the increment of both lipids and lipoproteins accepted as major risk factors for atherogenesis was more considerable in the atherosclerotic and/or hypertensive subjects than in the young normal subjects. In the atherosclerotics, the relative increments of long chain saturated and monounsaturated (oleic) fatty acids were observed in the fourth hour when the concentrations of serum triglyceride, cholesterol as well as beta- and pre-beta-lipoproteins reached maximum levels after the butter loading.
    Similar changes of serum lipids (eccepting the free fatty acid as well as total fatty acid composition) and lipoproteins were induced by a fructose loading diet containing two eggs, the yolk of three eggs, ten grams of butter and ninty grams of fructose.
    According to the recent concept of serum lipid and its relevance to atherosclerosis, it was concluded that the acute dietary changes in serum lipids and/or lipoproteins might have an essential role in the pathogenesis of atherosclerosis and its clinical consequence.
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  • Yoshiaki TAKIKAWA, Konin YAMADA, Yasunori SATO, Kiichi URASAWA, Akira ...
    1978 Volume 6 Issue 1 Pages 21-25
    Published: April 01, 1978
    Released on J-STAGE: September 21, 2011
    JOURNAL FREE ACCESS
    Studies were carried out on the pathogenesis of hyperlipidemia from angles of the roles of insulin and glucagon in plasma, and the results are obtained as follows:
    1) Basal plasma insulin (IRI) levels were higher in subjects with the hyperlipidemia of type IIb, IV and V than in normolipidemic subjects. However, there were hardly any difference of plasma glucagon (IRG) levels between normolipidemic subjects and those with hyperlipidemia.
    2) The increase of the plasma IRI levels after 50g GTT or after arginine loading test was higher in hypertriglyceridemic subjects with normal glucose tolerance than in normal controls, but it was lower in hypertriglyceridemic subjects with abnormal glucose tolerance than in normal controls.
    3) There were no significant difference of patterns of IRG levels after 50g GTT or after arginine loading test among normal controls, hypertriglyceridemic subjects with normal glucose tolerance, and hypertriglyceridemic subjects with abnormal glucose tolerance.
    4) These observations suggest that the cause of increase of serum pre-β lipoprotein may differ between subjects with normal glucose tolerance and those with abnormal glucose tolerance.
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  • Masafumi KOGA, Kazuto MATSUMIYA, Yuichi YAMAMURA, Yoshihisa YAMAGUCHI, ...
    1978 Volume 6 Issue 1 Pages 27-32
    Published: April 01, 1978
    Released on J-STAGE: September 21, 2011
    JOURNAL FREE ACCESS
    The uptake of cholesterol by the isolated hepatocytes of rats at 4°C from the incubation medium containing rat serum was low and did not increased by the prolongation of the incubation time up to 2hrs. However, the hepatocytes took up cholesterol very actively at 37.5° from serum as well as cholesterol/phospholipid dispersion. The amount of the uptake increased linearly as a function of the incubation time. The cholesterol uptake from the liposome dispersion by the cells increased linearly to the increase of the cholesterol concentration of liposome, but that from serum reached to the maximum at 70mg/100ml of cholesterol concentration and remained a plateau level at the higher concentration. Cholesterol was tansfered from every serum lipoproteins, though the uptake ratio was the highest in low density lipoprotein fraction. The uptake ratio decreased in like manner observed in the incubation with the whole serum, as lipoprotein concentration was elevated.
    The labeled ester cholesterol in the serum decreased more rapidly than the free cholesterol during the incubation, but the cholesterol in the hepatocytes was mainly found as the free form, suggesting that ester cholesterol taken up by the cells was hydrolyzed rapidly.
    The outflux rate of synthesized cholesterol increased as the elevation of the concentration of serum and liposomes in the medium and reached to a plateau level as high as 50-70mg/100ml of cholesterol concentartion. But there was no significance difference between the outflux rate from the incubated hepatocytes into serum and liposome suspension.
    Cholesterol accumulation was not observed in the hepatocytes incubated in the medium containing serum, though cholesterol content was increased in the cells after the incubation in the medium with liposome of the high cholesterol concentration. The cholesterol/phospholipid ratios of the liposome between 0.2-0.8 did not influence the increase of cholesterol contents of the hepatocytes by the increase of cholesterol concentration in the medium.
    The uptake of cholesterol diminished in old rats or hypothyroid rats who had high serum cholesterol levels. The isolated hepatocytes from rats hyperresponding to a high cholesterol diet took up more cholesterol than those from the hyporesponders. The difference of the amount of cholesterol uptake between two groups became larger as cholesterol concentration in the incubated medium increased.
    In conclusion, the uptake of cholesterol by the hepatocytes from serum is probably carried out by different process from that from liposome dispersion, which is considered to be a physicochemical phenomenon. The uptake of cholesterol by the hepatocytes may be one of the important regulatory mechanism of serum cholesterol concentration.
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  • Nozomu TAKEUCHI, Masafumi KOGA, Kazuto MATSUMIYA, Yuichi YAMAMURA, Fum ...
    1978 Volume 6 Issue 1 Pages 33-38
    Published: April 01, 1978
    Released on J-STAGE: September 21, 2011
    JOURNAL FREE ACCESS
    Thiobarbituric acid reactive substances (TBARS) increased in hepatic microsome fractions from rats aged 3 months as compared with such fractions from 1-month-old rats, though serum TBARS concentration did not differ between both groups. Serum TBARS concentrations became significantly higher in adult rats aged 6-7 and 12-13 months than in those 1 month old. TBARS in microsome fractions increased further by the aging up to 12-13 months and those in mitochondria did not show such clear age-related change, though rats aged 12-13 months had the high TBARS values of mitochondria.
    Administration of a diet containing 0.5% α-tocopherol to rats for 1-3 months, lowered the TBARS concentrations in microsomes concomitantly with a decrease of serum TBARS concentration. In these rats, synthetic activities of cholesterol and fatty acid in the hepatic microsomes from acetate or mevalonate were similar in both treated and non-treated control groups. However, the activity was higher in the treated group than in the non-treated control after long-term (up to 6-12 months) feeding of tocopherol. Serum cholesterol, phospholipid and triglyceride concentrations in rats treated with tocopherol for 6-12 months were significantly lower than those in control rats. Hepatic total lipid and triglyceride in rats aged 7-13 months which were higher than those of 1-month-old were tended to decrease by the long-term feeding of tocopherol. By contrast, the hepatic phospholipid content decreased in the non-treated aged rats was less than that in tocopherol treated rats.
    After the feeding of a tocopherol deficient diet for 2-16 weeks, TBARS in serum and hepatic subcellular organelles of rats were increased markedly. Although serum lipid concentrations from the rats fed the deficient diet for 2 weeks were significantly higher than those from the controls, there was no significant difference between the two groups after feeding more than 4 weeks. However, a supplement of 2% cholesterol in these diet increased the serum cholesterol and phospholipid concentrations in such tocopherol deficient rats.
    Refeeding of a tocopherol-rich diet to the deficient animals reduced the serum lipid concentrations concomitantly with the decrease of TBARS in serum and hepatic subcellular fractions.
    The tocopherol deficient animals showed lower synthetic activities of hepatic cholesterol and fatty acid as well as glucose-6-phosphatase in microsomes. These enzyme activities of cholesterol and fatty acid synthesis in the deficient animals tended to be restored by the tocopherol administration.
    Nordihydroguaiaretic acid, which was one of the antioxidant drug, did not influence the TBARS in serum and hepatic subcellular organelles, serum lipid concentrations, glucose-6-phosphatase activity and cholesterol and fatty acid synthesis of the microsomes of tocopherol deficient rats.
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  • Akio KUDO, Naoto NAKAMURA, Nobuyuki TANAKA, Tatsuru NIIMURA
    1978 Volume 6 Issue 1 Pages 39-42
    Published: April 01, 1978
    Released on J-STAGE: September 21, 2011
    JOURNAL FREE ACCESS
    Ultracentrifugal separation of plasma lipoproteins by discontinuous sucrose gradient was tried. Lipid and apolipoprotein composition of each fraction was compared with those obtained by the method of Havel. 0.5gm of sucrose, 0.4gm of KBr and 0.1gm of NaCl were added to 2.0ml serum to make final d=1.21 and 1.0ml of d=1.21 solution with same composition was piled gently. After the centrfugation for 20 hours at 50, 000rpm, 2.0ml of d=1.063 solution (6w/v% in sucrose) and 2.5ml of d=1.006 solution (2.5w/v% in sucrose) were overlayered and recentribuged for 4 hours. Each lipoprotein floated in the solvent corresponding to each density class. Samples were collected by capillary pipette and dialyzed to physiological saline containing 1mg/ml EDTA (pH 7.2) for more than 12 hours.
    Polyacrylamide gel electrophoresis demonstrated distinct separation of each lipoprotein class without contamination. Contents of apolipoproteins, total lipids, total cholesterol and triglycerides were measured. When compared with those obtained by the Havel's method, lipid and apolipoprotein contents were higher in VLDL and lower in LDL in our method. These changes suggest that VLDL contains the “remnants” of d<1.019. Recovery of lipids and apolipoproteins in HDL was higher in the present method. Shortening of ultracentrifugal time was considered to have prevented degeneration of HDL.
    Although our method would provide more rapid ultracentrifugal separation of plasma lipoproteins, more detail examinations of its physicochemical and structural properties should be required.
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  • Kiyoshi MIYAHARA, Kojiro SHOJI, Kanji MIYAKE, Masamichi SHIMONO, Takef ...
    1978 Volume 6 Issue 1 Pages 43-48
    Published: April 01, 1978
    Released on J-STAGE: September 21, 2011
    JOURNAL FREE ACCESS
    Using 20 microliter of serum, cholesterol concentration in each lipoprotein separated with agarose gel electrophoresis were determined by gas liquid chromatography. Procedure: After performing electrophoresis, alpha and beta (involved prebeta) lipoproteins were detected with Seidel's solution 2. Each portion was taken off and saponified. Heptan extracted cholesterol was trimethyl silanized together with beta-sitosterol added as internal standard. Result: Firstly, this method was compared with classical ultracentrifugation method analysis for the separation of each lipoprotein cholesterol and confirmed to be highly correlated each other. By the presented procedure in normal subjects (n=28), alpha cholesterol was 52±19mg/dl (M±SD) and beta+prebeta cholesterol was 113±28mg/dl. The ratio of cholesterol in beta+prebeta to alpha was 2.6±1.5. Alpha cholesterol decreased in cases with history of myocardial infarction. Beta+prebeta cholesterol highly increased in essential hyperlipidemias, diabetes mellitus, hypertensive disease, nephrotic syndrome and myocardial infarction. In 5 cases of type IIa hyperlipidemia treated with antihyperlipidemic agents, alpha cholesterol decreased about a month later paralleled with a decrease of total serum cholesterol. Assuming alpha cholesterol value as a factor of anti-atherogeneity, it should be noted that alpha cholesterol must be followed routinely together with total serum cholesterol, particularly in these treated patients. And the reported method must be useful owing to its accuracy, easiness and “not” time and money consuming.
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  • Toshitaka TAMAI, Tsuguhiko NAKAI, Ryoyu TAKEDA
    1978 Volume 6 Issue 1 Pages 49-55
    Published: April 01, 1978
    Released on J-STAGE: September 21, 2011
    JOURNAL FREE ACCESS
    The association between premature atherosclerosis and increased concentrations of low density lipoprotein (LDL) is well known. Miller G.J. et al proposed that a reduction of plasma high density lipoprotein (HDL) concentration may accelerate the development of atherosclerosis by impairing the clearance of cholesterol from the arterial wall. Cholesterol-HDL (C-HDL) has generally been measured using either the heparin-manganese precipitation method of Burstein and Samaille or as the lipoprotein cholesterol after ultracentrifugal fractionation of HDL (1.063<d<1.210g/ml). The purpose of the present study was to compare these two methods. The following conditions were used to assess the effects of changes in final MnCl2 and heparin concentrations on the precipitation of LDL and very low density lipoprotein (VLDL):
    1. Mn2+ concentration was varied while heparin concentration was held constant.
    2. Heparin concentration was varied while Mn2+ concentration was held constant.
    After the heparin-manganese chloride precipitation of plasma, cholesterol quantitation, agarose gel electrophoresis and immunodiffusion studies using rabbit anti-lipoprotein B serum of the supernatant were carried out. A constant amount of cholesterol in the supernatant was measured over a final MnCl2 concentration greater than 35mM and over a heparin concentration range of 46-945 units/ml. After observing no contamination of β- and pre β-lipoprotein by agarose gel electrophoresis and the absence of lipoprotein B by immundiffusion in the supernatant, optimum final concentration of heparin and MnCl2 was determined. Final heparin and MnCl2 concentration used in our study were 197 units/ml and 87mM, respectively. Lipoprotein fractions of d>1.063g/ml were isolated by ultracentrifugation. The density of plasma was adjusted to d=1.063g/ml with KBr solution and centrifuged in a Type 40.3 rotor on the Beckman L5-50 preparative ultracentrifuge for 44 hours at 105, 000 x g, 10°C. Lipoprotein fractions of d>1.063g/ml were recovered using a tube slicer to cut the cellulose nitrate tube. C-HDL quantitation by precipitation and ultracentrifugation was compared in 30 samples. Ultracentrifugally quantitated C-HDL (X±S.D.) was 48.5±11.8mg/dl, while by precipitation in parallel aliquots was 42.6±8.5mg/dl. C-HDL measured by ultracentrifugation and precipitation methods were significantly correlated, r=0.85, p>0.001. The cholesterol contents of the ultracentrifuged fraction in most samples were higher than those of the heparin/Mn2+ supernatant, presumably as a result of the contribution of the Lp(a)-lipoprotein and lipoprotein B in HDL. Ishikawa, T. T. et al reported that an additional problem with the heparin-MnCl2 precipitation method appears when the plasma triglycerides are elevated (usually>300mg/dl) and a “floating precipitate” appears in the supernatant after an addition of heparin and manganese, providing an overestimation of C-HDL by the precipitation method as compared to ultracentrifugation. We confirmed this finding in a patient whose plasma triglyceride and cholesterol were 307mg/dl and 231mg/dl, respectively. After precipitation of plasma by heparin-MnCl2, the supernatant contained β and pre-β lipoprotein other than α lipoprotein in agarose gel electrophoresis. C-HDL by precipitation method and by ultracentrifugation were 44mg/dl and 27mg/dl, respectively. When heparin-manganese precipitation method is used for the determination of plasma high-density lipoprotein cholesterol concentration, these problems should be considered.
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  • Partial Purification and Characterization of Triglyceride Lipase from Rat Liver
    Masao YAMADA, Kunimi TANAKA, Toshio MURASE
    1978 Volume 6 Issue 1 Pages 57-61
    Published: April 01, 1978
    Released on J-STAGE: September 21, 2011
    JOURNAL FREE ACCESS
    Hepatic triglyceride lipase (TGL) was extracted from acetone-ether dried powder of rat liver with 0.05M NH4Cl-NH4OH buffer (pH 8.5) containing 0.5U/ml of heparin for 60min at 0°C. The crude enzyme extracts thus obtained were partially purified on a heparin-Sepharose affinity chromatography. This gave a 78-fold purification over crude enzyme extracts. A purified enzyme did not require serum for substrate activation and was not inhibited by 1M sodium chloride, being differed from lipoprotein lipase in rat postheparin plasma.
    A purified enzyme had a single pH optimum at 9.0, whereas crude enzyme extracts had lipolytic activities over a wide range of pH values with two maxima at pH9.0 and 4.0. The lipolytic activities in crude enzyme extracts were separated on heparin-Sepharose column into two fractions, a breakthrough fraction and a purified hepatic TGL. In an attempt to define the location of these lipolytic activities, NaCl-noninhibitable hepatic TGL in postheparin plasma and crude enzyme extracts of postheparin liver were assayed for their activities at different pH values. The lipolytic activities of crude extracts of postheparin liver had a pH optimum at 4.0 with a marked decrease at 9.0, being the same with that of the break-through fraction of the column. Heparin-released hepatic TGL in plasma, on the other hand, had a similar pH dependence to that of a purified hepatic TGL.
    These results indicate that TGL activity in the liver is heterogeneous. This enzyme activity can be separated into two forms. One, having a high affinity for heparin bound to Sepharose, is in the hepatic plasma membrane and released by heparin into the circulation. The other, having no affinity for heparin, might be present intracellularly. The functional importance of the former form of enzyme was discussed.
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  • Especially Concerned with Arterial Wall Lipase
    Kohji SHIRAI, Nobuo MATSUOKA, Yasushi SAITO, Akira KUMAGAI, Toshiharu ...
    1978 Volume 6 Issue 1 Pages 63-66
    Published: April 01, 1978
    Released on J-STAGE: September 21, 2011
    JOURNAL FREE ACCESS
    The physiological significance of serum nonspecific carboxyl esterase (tributylin hydrolase) is not clearly understood. Cucuianu et al. reported that esterase was closely related to triglyceride metabolism. Furthermore, Okuda et al. reported that nonspecific esterase might be converted to lipase (ediol hydrolysis activity) in rat liver. To clarify the relationship between serum nonspecific esterase and triglyceride metabolism, such as hypertriglyceridemia and triglyceride hydrolysis in arterial wall, the experiments were carried out in humans and rats.
    In the clinical investigation of 500 farmers at Yoshida Machi in Ehime prefecture, serum triglyceride concentrations of the farmers increased after a period of sprinkling agricultural pesticide. Serum nonspecific carboxyl esterase decreased reciprocally with the increase in triglyceide concentration. These results suggest that serum nonspecific esterase might play a role of triglyceride hydrolysis. When serum nonspecific esterase, which was purified by the method of (NH3)2 SO4 fractionation, Sephadex G-200 and DEAE Sephadex A-50 column chromatography to show a single band of protein in disc electrophoresis, was incubated with slices of rat aorta which had been treated with heparin to remove lipase, a greater extent of lipase (triolein hydrolase) activity was observed than that of the heparin treated aorta and the same extent as that of none treated aorta. Serum nonspecific esterase had no effect when it was treated in boiling water. The same results were confirmed not only chemically but also histochemically.
    These results suggested that serum nonspecific carboxyl esterase might be converted to lipase in aorta.
    Further studies are now in progress to clarify the relationship between nonspecific carboxyl esterase and lipase, such as localization of nonspecific esterase and lipase in the subcellular fraction, and immunological identification of nonspecific esterase and lipase.
    1) Cucuianu, M. et al.: Clin. Chem. Acta, 59: 19 (1975).
    2) Okuda, H. & Fuji, S.: J. Biochem., 64: 377 (1968).
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  • Kojiro SHOJI, Kiyoshi MIYAHARA, Kanji MIYAKE, Yoshito SHIMIZU, Masamic ...
    1978 Volume 6 Issue 1 Pages 67-72
    Published: April 01, 1978
    Released on J-STAGE: September 21, 2011
    JOURNAL FREE ACCESS
    Levels of serum α-tocopherol, lipoperoxide and lipids were studied in acute stage of stroke and MI. Serum α-tocopherol were 0.73±0.43 (n=46) in stroke and 0.72±0.31mg/dl (n=10) in MI within 48 hours of the onset, which were significantly lower than the level of control group (0.99±0.22 mg/dl). Concurrently, the TBARS (TBA reactive substance) denoting the level of the lipoperoxide, were 5.64±2.72 (n=41) in stroke and 5.40±3.16 nmol/ml (n=9) in MI respectively. These were markedly higher than the level of control group (3.22±0.89nmol/ml).
    No correlation was seen in serum α-tocopherol and TBARS but serum cholesterol or the ratio of linoleic to oleic acid of FFA was correlated with α-tocopherol in stroke. Along with following studies up to one month, α-tocopherol of most cases of stroke came down to the lowest level early in the 3 to 5 days and therafter recovered gradually. The ratio of α-tocopherol to cholesterol was minimum in early phase and then took a crescendo curve later in two weeks. TBARS increased during the first week in both groups. Finally, a case of MI was presented in whom all of α-tocopherol, TBARS and FFA which had been normal the day before the accident, changed in a steep wise soon after the occurence of the attack. The results imply that α-tocopherol may be consumed, playing a role of the antioxidant against the lipoperoxide which must increase depending on the tissue damage induced by stroke or MI.
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  • Kei SATOH, Shigeru TAKAMATSU, Shigeru SAKUTA, Kazuho HENMI, Seitoku MI ...
    1978 Volume 6 Issue 1 Pages 73-81
    Published: April 01, 1978
    Released on J-STAGE: September 21, 2011
    JOURNAL FREE ACCESS
    It has been widely recognized that the determination of serum malondialdehyde (MDA), a product of lipid peroxidation, is one of the useful measure for the clinical study on the role of lipoperoxide in atherosclerotic disorders. Determination of serum MDA has been carried out mainly by the colorimetry of compound formed by coupling of MDA with thiobarbituric acid (TBA). Recently, however, it has been pointed out that sialic acids have the great influence on MDA determination in these methods. In this study, an attempt was made to establish the specific colorimetric method for the estimation of serum MDA eliminating such an influence.
    The most important point in our modification is to use the TBA reagent dissolved in 2M sodium sulfate instead of the reagent in water used in conventional methods. Using this TBA reagent, the optimum conditions for the specific serum MDA determination was searched and established.
    The procedure elaborated is as follows; MDA in serum specimen was precipitated with protein by adding 5 volumes of 20% trichloracetic acid to 0.5ml of serum in the test tube covered with aluminum foil to shut off the light. After centrifugation at 3, 500r.p.m. for 10 minutes the supernatant was decanted and the precipitate was washed once with 0.1N sulfuric acid. Then 2.5ml of 0.1N sulfuric acid and 3.0ml of TBA reagent were added to this precipitate and the coupling of MDA with TBA was carried out heating in boiling water bath for 30 minutes. After cooling in cold water the resulting chromogen was extracted with 4.0ml of n-butanol by vigorous shaking. This was followed by centrifugation at 3, 000 r.p.m. for 10 minutes and optical density of the organic phase was determined at the wavelength of 530nm.
    In the absorption spectra obtained by conventional methods, exogenously added sialic acid greatly raised the optical density at 530nm, which is specific for MDA. On the contrary, in our method, sialic acid gave little color and had no absorbance at 530nm, and addition of sialic acid did not result in any changes in serum MDA value. Heated in trichloracetic acid, sialic acids may give rise to formation of aldehyde that reacts with TBA in the conventional method. Since coupling of MDA with TBA is performed by heating in 0.1N sulfuric acid, there seems to be no aldehyde formation from sialic acids in our method. This, with the effect of sodium sulfate to facilitate the transfer of chromogen into organic phase, may enable us to eliminate the interference from sialic acids.
    The new method is specific for MDA and suitable for clinical application.
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  • Taku YAMAMURA, Hiroshi SUDO, Susumu MISUGI, Yuji MATSUZAWA, Katsunori ...
    1978 Volume 6 Issue 1 Pages 83-87
    Published: April 01, 1978
    Released on J-STAGE: September 21, 2011
    JOURNAL FREE ACCESS
    Atherosclerosis and xanthomatosis are frequently associated with familial hyperbetalipoproteinemia. In this report, we present three kindreds (SS, YS and N) with familial hypercholesterolemia and xanthomatosis. Two probands (S. S. and Y. S.) were born to consanguineous marriages. S. S., 17-yr-old female, and Y. S., 2-yr-old female, had an extremely high serum cholesterol level, 1003 and 903mg/dl respectively, being affected with severe cutaneous xanthomatosis. All of the probands' parents and many other relatives had moderate hypercholesterolemia (more than 300mg/dl). But, the inheritance of xanthomatosis and atherosclerosis was not so severe among members of the SS and YS families. Only one of them (father of proband Y. S.) had xanthomatosis and another one (an uncle of proband S. S.) had coronary insufficiency.
    The synthesis of cholesterol and the HMG CoA reductase activity were estimated on cultured skin fibroblasts from the probands S. S. and Y. S. Both of the incorporation of 14C-acetate into cholesterol and the activity of HMG CoA reductase were elevated by approximately 10-fold. In contrast to normal fibroblasts which received a feedback control by LDL, the HMG CoA reductase activity of the fibroblasts from the probands did not show any change even after the fibroblasts were cultured in lipoprotein-free medium. According to the results of Goldstein and Brown, these probands were identified with homozygotes with familial hypercholesterolemia. The HMG CoA reductase activity of fibroblasts from Y. S.' father and the regulation of this enzyme activity by lipoproteins were just intermediate between homozygous and normal subjects, showing that he was heterozygotic with familial hypercholesterolemia.
    In both of families, hypercholesterolemia was transmitted as dominant inheritance, with many family members being affected with this disorder as heterozygote. Consanguineous marriage of heterozygotics gave rize to a homozygote with severe cutaneous xanthomatosis.
    On the other hand, the N family was characterized by dominant inheritance of tendon xanthomas and also had a tendency of atherosclerosis. Though hypercholesterolemic subjects in this family were not homozygous but heterozygous, all of them had tendon xanthomas. Furthermore, some of the other members, even if normolipidemic, had distinct tendon xanthomas. It was assumed that, in the members of the N family, the formation of xanthomas was dependent on the presence of macrophages with a strong activity of incorporating cholesterol, while in S. S. and Y. S. the deposition of cholesterol depended on the excessively high cholesterol level.
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  • Toshihiro HABA, Ryozo TATAMI, Kosei UEDA, Ryosei UEDA, Tomio KAMETANI, ...
    1978 Volume 6 Issue 1 Pages 89-93
    Published: April 01, 1978
    Released on J-STAGE: September 21, 2011
    JOURNAL FREE ACCESS
    In the previous paper, we reported that radiological examination of the Achilles tendon thickness is useful for diagnosis of familial hypercholesterolemia. To validate Achilles tendon thickness in diagnosing familial hypercholesterolemia, we examined sterol synthesis from [14C] acetate and HMG CoA Reductase activity in cultured skin fibroblasts derived from one fetus, two normal subjects and six heterozygous familial hypercholesterolemic patients.
    The Achilles tendon thickness (14.3±2.1mm, mean±SD) in the heterozygotes were significantly thicker than those of the normal subjects (6.3±1.2mm).
    Cells were grown in dishes containing 10% fetal calf serum. On Day 6, the medium was replaced with 3ml of fresh medium containing 10% human lipoprotein deficient serum (LPDS). Twenty-four hr later, human serum was added to obtain an appropriate concentration of cholesterol.
    The results were as follows.
    1. Cholesterol synthesis from [14C] acetate in normal cells decreased by 90.8±5.2% (mean±SEM) when the medium concentration of cholesterol changed from zero to 12μg/ml. While cholesterol synthesis in heterozygous cells decreased by 59.6±4.0%, homozygous cell (GM-486) showed no response at 12μg/ml of cholesterol.
    2. HMG CoA Reductase activity in normal cells decreased by 87.4±7.9% when the medium concentration of cholesterol was changed from zero to 20μg/ml. The Reductase activity in heteroxygous cells decreased by 40.6±4.7%. The homozygous cell showed 88% activity at cholesterol concentration of 120μg/ml.
    3. From these data, it is concluded that all six hypercholesterolemic patients with abnormally thick tendon are familial and radiological examination of Achilles tendon is very useful for diagnosis.
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  • Masayuki SHIRAISHI, Akinori KAMIO, Kenshi SHITAMA, Shigeo TAKEBAYASHI
    1978 Volume 6 Issue 1 Pages 95-100
    Published: April 01, 1978
    Released on J-STAGE: September 21, 2011
    JOURNAL FREE ACCESS
    At least four genetically-distinct collagen phenotypes designated type I, II, III and IV collagen are isolated from mammalian tissues. In human arterial wall, both type I and III collagens are found out, whereas the latter predominates.
    In the present study, two distinct types of colagen were extracted by limited pepsin digestion from human aortic media and vena cava at different ages including fetuses. Human aortic media contained approximately 55% type I and 45% type III collagen on the basis of dry weights of salt-fractioned precipitates. In contrast, human vena cava consisted of approximately 75% type I and 25% type III collagen. No age-related change of type I/type III collagen ratio was found between the aortic media and vena cava. The results described here indicate that the arterial smooth muscle cell which may play an important role on atherogenesis, has a sufficiently different expression in relation to collagen synthesis comparing to that of vena cava in man.
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  • Tomio KAMETANI
    1978 Volume 6 Issue 1 Pages 101-113
    Published: April 01, 1978
    Released on J-STAGE: September 21, 2011
    JOURNAL FREE ACCESS
    Effects of elastase on plasma and aortic lipoproteins in 1% cholesterol-fed rabbits were investigated.
    Elastase was injected (4mg/rabbit/day) i.m. in 8 cholesterol-fed rabbits for 12 weeks.
    Marked hypercholesterolemia and atherosclerosis were produced by cholesterol-feeding. In the cholesterol-fed rabbits, cholesterol-rich VLDL and apoprotein R-2 and 3 of VLDL, which are called as arginine-rich apoproteins, increased.
    The plasma cholesterol, VLDL-cholesterol and VLDL-apoproteins were significantly lower in the elastase-treated rabbits than in the cholesterolfed rabbits (P<0.05, P<0.02, P<0.01). The plasma triglyceride and LDL-cholesterol levels were lower in the elastase-treated rabbits (not significant). The lower plasma cholesterol level in the elastase-treated rabbits was produced through the decrease of VLDL-cholesterol level.
    Some authors report that elastase increases the catabolism of cholesterol in liver and the excretion of bile acids. Therefore, the present data suggest that elastase decreases plasma cholesterol and VLDL-cholesterol through the increase of uptake and catabolism of VLDL “remnant” in liver.
    Both VLDL- and LDL-cholesterol contents, particularily LDL-cholesterol, in the aortic intima and media increased significantly in the cholesterol-fed rabbits.
    Macroscopicaly, elastase tended to inhibit the atherosclerosis of aorta. Elastase decreased the VLDL- and LDL-cholesterol contents of the aorta, although statistically insignificant. Since LDL has more atherogenecity than VLDL, elastase might not have shown a significant antiatherogenecity. While there was no difference in plasma LDL-cholesterol between the cholesterol-fed and the elastase-treated rabbits, the aortic LDL-cholesterol content in the latter were almost half that in the former. Thus, elastase was thought to have directly inhibited the deposition of LDL in the arterial wall.
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  • Akira TANIMURA, Itsuro HAYASHI, Teruyuki NAKASHIMA
    1978 Volume 6 Issue 1 Pages 115-119
    Published: April 01, 1978
    Released on J-STAGE: September 21, 2011
    JOURNAL FREE ACCESS
    Large number of thrombus were frequently found on intimal surface of the aortas obtained from autopsy cases. Thrombi were classified morphologically into three types-microthrombus, membranous thrombus and massive thrombus as shown in our previous study.
    Organizing processes of each thrombi were discussed on this paper.
    1) Microthrombi could be found not only on sclerotic intima but also on no lesion areas. Many of them could be thought to be dissolved if they exist longer on living aortas. However, some of the microthrombi showed certainly the transformation into microfibrous elevations. The main cells that participated in organization of microthrombus were endothelial cells.
    2) Membranous thrombi were apt to be deposited on sclerotic lesions. Unlike other types of thrombus, cellular reactions were minimum in organizing processes. The organization of these thrombi resulted in hyaline thickening of intima.
    3) Massive thrombi could be found frequently on atheromatous plaques or ulcerative lesions. Their organization showed dual processes; the organizations from superficial layer and from base of thrombi. The organization from superficial layer of thrombi seemed to be more important.
    Smooth muscle-like cells seen in superficial layer of organizing thrombus were thought to play important role in organizing processes.
    The authors could not be determined whether the origin of these cells were endothelial, multipotential mesenchymal cells or from blood cells.
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  • Nubuo MATSUOKA, Masaki SHINOMIYA, Kohji SHIRAI, Nobuhiro MORISAKI, Shu ...
    1978 Volume 6 Issue 1 Pages 121-128
    Published: April 01, 1978
    Released on J-STAGE: September 21, 2011
    JOURNAL FREE ACCESS
    Rats were fed in the following manner, 1) Normal diet for 16 weeks 2) High cholesterold diet for 8 weeks and then normal diet for 8 weeks 3) High cholesterol diet for 8 weeks and then normal diet plus pantethine for 8 weeks 4) High cholesterol diet for 16 weeks 5) High cholesterol diet for 8 weeks and then high cholesterol plus pantethine for 8 weeks 6) High cholesterol diet plus pantethine for 16 weeks.
    Serum cholesterol level in rat fed a high cholestrol diet (HCD) for 16 weeks was considerably higher than in rat fed a normal diet (ND) for 16 weeks. The increased serum cholesterol was significantly lowered by administration of pantethine for 16 weeks or last 8 weeks.
    Serum triglyceride, phospholipid were not changed. Acid cholesterol esterase activity, mainly located in lysosome, in arterila wall extract from rats fed a HCD for 16 weeks was observed to be decreased than in those from rats a ND for 16 weeks
    In rats fed a HCD for 8 weeks and then ND for 8 weeks, serum cholesterol level was the same extent as normal diet for 16 weeks. However, acid cholesterol-esterase activity in those arterial wall extracts was not restored to the activity in rats fed a ND for 16 weeks. The activity was increased by administration of pantethine.
    These results suggest that pantethine might protect deposition of cholesterol ester in arterial wall. The ratio of cholesterol ester synthesizing activity to alkaline cholesterol esterase activity, mainly located in microsome, was increased in arterial wall extract from rate fed a HCD for 16 weeks than in those from rats fed a ND for 16 weeks. The ratio in arterial wall extract from rats fed a HCD administered pantethine for 16 weeks was lower than in those from rats fed a HCD for 16 weeks. These results suggest that deposition of cholesterol ester might be increased in microsome of arterila wall and pantethine might protect the deposition. Triglyceride metabolism, such as lipase and triglyceride synthesizing activities, was not observed to be changed in arterila wall extract of rats in each feeding manner. From these results, lipid metabolism in arterial wall of rats fed a HCD and the value of the pantethine on lipid metablism in prevention and regression of atherosclerosis were discussed.
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