1. Insulin, rabbit IgG and its Fab' fragments were coupled to β-D-galactosidase (EC 3. 2. 1. 23) from Escherichia coli by a two-step procedure with aid of the reaction between sulfhydryl groups and maleimide residues. Maleimide residues were introduced into insulin molecules with N, N'-o-phenylenedimaleimide after mercaptosuccinylation or directly with the active ester of 4-(maleinimidomethyl) cyclohexane-1-carboxylic acid or 4-(maleinimidomethyl) benzoic acid. Maleimide residues were also introduced into molecules of reduced IgG or Fab' fragments using N, N'-o-phenylenedimaleimide. Maleimide residues attached to molecules of insulin, IgG or Fab' fragments were allowed to react with sulfhydryl groups of β-D-galactosidase.
2. Sepharose 4B, glass rods and silicone pieces were used as solid phase. Rabbit IgG was coupled to CNBr-activated Sepharose 4B.
Small rods of aminoalkylsilyl glass were loaded covalently (using glutaraldehyde) or noncovalently with insulin, rabbit IgG or its (Fab')₂ fragments. Small rods of plainglass or small silicone pieces were also loaded with rabbit IgG or its (Fab')₂ fragments by simpleadsorption.
3. The enzymoimmunoassay was performed by using the insulin- or antibody-β-D-galactosidase complex and the insulin- or antibody-loaded solid phases. Antigens and antibody subjected to the assay were insulin, anti-insulin antibody, ornithine δ-aminotransferase (EC 2. 6. 1. 13) from rat liver, human IgG, 2, 4-dinitrophenyl human IgG and α-fetoprotein.
4. Studies performed thus far indicated that attomole (10^-18 mole) quantities of antigens could be measured with presently available techniques if antibodies of adequate quality were provided.

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Antibody immobilized polystyrene vials were used for the determination of corresponding antigen with enzymoimmunoassay.
Polystyrene vials were silanized by γ-aminopropyltriethoxysilane, followed by glutaraldehyde treatment. Anti-IgG (rabbit) was immobilized to the treated polystylene vials andhuman IgG was assayed by the competitive binding method using anti-IgG immobilized vials and IgG-peroxidase conjugates.

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A novel crosslinking reagent N-(m-maleimidobenzoyloxy) succinimide (MBS) was introduced to enzyme immunoassay of peptide hormons such as angiotensin I, insulin and humanchorionic gonadotropin. Ten of the crosslinking reagents analogous to MBS were synthesized and examined by their stabilities and reactivities. The most stable pH of these reagents were approximately 6.0 and the best condition of their N-acylation was chosen as 30° for fourty minutes in phosphate buffer pH 7.5. For enzyme immunoassay of viomycin, specific antiserum to viomycinhad to be obtained. For this, the strategy of preparing viomycin protein conjugate was first the selective protection of?β-amino group of β-lysine residue in viomycin with acetyl group by three steps. The second process consist of hemisuccinylation of the N^θ-amino group followed byamide bond formations of the introduced carboxylic acid of hemisuccinyl function with amine groups of bovine serum albumin (BSA) by mixed anhydride method. The obtained viomycin-BSA conjugate gave a single band on its sodium dodecylsulfate disk electrophoresis and from itselectophoretic distance, nine of viomycin residues were calculated to be introduced per moleculeof BSA. The serum obtained from femail rabbits injected with the conjugate was kept at -20° until used. Viomycin was coupled with β-D-galactosidase easily by using the crosslinking reagent MBS and the obtained conjugate retained the enzyme activity and immuno reactivity of their components. Competitive binding test between the conjugate and viomycin to the antiserum to viomycin resulted that viomycin could be detected from 100pg to 4ng.

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Angiotensin I was coupled very easily with a-β-galactosidase (EC 3. 2. 1. 23) using a novel coupling reagent, N-(meta-maleimidobenzoyloxy) succinimide. No decrease in enzyme activity was observed during coupling procedure. Angiotensin I-β-D-galactosidase conjugate was proved to possess enough enzyme activity and immune reactivity for enzyme immunoassay of angiotensin I. By the competitive binding test, 1.5pg to 50pg of angiotensin I could be detected with a fairly good precision. The cross reactivity of angiotensin II, angiotensin III, 1-Sar-8-Ileu-angiotensin II and deamino-dicarba-Arg-vasopressin were compared to angiotensin I by the present enzymeimmunoassay and also usual radioimmunoassay using the same antiserum to angiotensin I. The results indicated that these peptides were distinguished clearly from angiotensin I but the specificities to the tested analogues by enzyme immunoassay were different from those by radioimmunoassay. This difference was diminished by an addition of a small amount of angiotensin I in the system for enzyme immnoassay. The present method was applied to measure plasma renin activity of dogs and humans. The results were compared to those obtained by standard radioimmunoassay. The both methods showed a fairly good coincidence (r=0.94, dog, r=0.90, human).

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Enzyme-linked immunoassay was applied to the determination of human chorionic gonadotropin (hCG), a glycoprotein hormone usually assayed with radioimmunoassay. For this purpose, enzyme hormone conjugate was prepared by reacting hCG with β-D-galactosidase (β-Gal.) of E. Coli in the presence of meta maleimidebenzoyl N-hydroxysuccinimide ester (MBS) as coupling reagent. The reaction mixture was then passed through a Con A-Sepharose 4B column to remove unreacted β-Gal. followed by the removal of intact hormone by means of gel filtration through Ultrogel AcA 44. The conjugate thus purified was shown to exhibit sufficient enzyme activity and immunoactivity. The immunoassay of hCG was performed by the double antibody method and using assay 0.4-250mIU/ml of hCG were detectable. This was about ten times as sensitive as radioimmunoassay.
However, difficulty was experienced when this method was utilized for the determination of hCG of plasma samples from patients. Since it was considered that the presence of the plasma affected this assay method, the following improvements were made; 1) the same volume of hormone free plasma was added to standard solutions of hCG, 2) volume of plasma sample was desirable as much as 10μl which is twenty times less than that for radioimmunoassay. The performance and validity of this assay were comparable to radioimmunoassay using 125I-hCG astracer. The dose/responce curves of both assays have the same slope and there was no significant differences between the values (correlation coefficient=Y=0.96X+1.53).

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A competitive binding enzyme immunoassay (EIA) for human serum phenytoin (DPH) has been developed. β-D-Galactosidase- s-_??_N-CH2CONHCH2CH2CH (C6H5) 2 prepared by reaction of N-(3, 3-diphenyl-l-propyl) maleylacetamide with β-D-galactosidase fromE. coliwas used as an enzyme labeled antigen. The insolubilized antibody used was prepared by combining yeast cell wall with immunoglobulin from sera of rabbits immunized with α, α-diphenylacetylated bovine serum albumin.
After 30min competitive immunoreaction between the enzyme labeled antigen and free DPH for the insolubilized antibody, the activity of enzyme bound to the antibody was measured by using o-nitrophenyl β-D-galactoside as a substrate. One to 500ng of DPH in 10μl of sera from patients could be successfully measured by this EIA. The values of DPH concentrations measured by the EIA and the conventional gas-liquid chromatography method were found to be correlated well (r=0.96, n=100).
Phenobarbital, primidone, trimethadione and carbamazepine in the sera did not interfere with the EIA, whereas 5 (4-hydroxyphenyl)-5-phenylhydantoin, when added more than 25ng per tube, interfered with the method. The method can be practically used in the ordinal clinical laboratories.

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A novel method for preparations of hapten-protein conjugate was investigated using a novel coupling reagent N-(m-maleimidobenzoyloxy) succinimide (MBS) or its analogue. Haptens having amino group such as viomycin or 6-aminopenicillanic acid were introduced by a maleimide residue by the acylation with MBS under the mild aqueous condition. A hapten having hydroxyl groups but no amino group such as estradiol was introduced a maleimide residue by the reaction of the hapten and m-maleimidobenzoic acid with the presence of dicyclohexylcarbodiimide. Thus, a maleimide residue was easily introduced to haptens. The disulfide bonds of cystine residues in a protein such as bovine serum albumin (BSA) were reduced to thiol groups by the treatment of dithiothreitol. The thiol groups of the reduced BSA was coupled with the maleimide residue of the modified haptens to give hapten-protein conjugates. The yields of each conjugate was fairly good and every canjugate gave almost a single band on their sodium dodecylsulfate disk electrophoreses. Also, the introduced numbers of the hapten per molecule of BSA were easily calculated by the electrophoretic distances.

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The activation of enzyme was carried out by the periodate treatment. Enzyme immunoassay using antigens conjugated with activated different enzymes including Peroxidase (Horse radish), Alkaline phosphatase (Calf intestine) and Glucose oxidase (Aspergillus niger) was established for routine method. In this system, the antigen determination was carried out by the titration of enzmye activity of supernatant. The liquor or high diluted serum that the neglisible for the endogeneous enzyme activities were easily estimated by this system.
However in this system to titrate the small amount of antigen, for example Alpha-fetoprotein, endogeneous enzyme influenced the enzyme actitity of supernatant, especially Alkaline phosphatase and Peroxidase. To remove Peroxidase activity from serum was accomplished by addition of small amount of perioxid to serum followed by addition of glutaraldehyde fixed sheep red cell. To remove Alkaline phosphatase except heatstable Alkaline phosphatase, for example Reagan phosphatase, was accomplished by heating.

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This paper describes highly sensitive enzyme immunoassayes for insulin and cortisol using peroxidase as the label enzyme. The fluorescence reaction of homovanillic acid-H₂O₂ and chemiluminescence generating reaction of luminol-H₂O₂ with peroxidase are used as a means of determining peroxidase activity. The faint chemiluminescence is measured by a photon-counter (HTVC767). The peroxidase conjugate was synthesized according to NAKANE's method with slight modifications. The peroxidase-cortisol conjugate was also prepared by the mixed anhydride method. These conjugates were chromatographed on a Bio-Gel P-60 column. Various techniques for separating the bound and free labeled molecules were examined. Good results were obtained by the solid-phase method using anti-insulin or anti-cortisol-γ-globulin linked Sepharose 4B.
The sensitivity of the assay, i.e., the amount of antigen displacing 10% of the initially bound tracer was fbund to be 0.2 to 0.5μU per tube and 25 to 30 pg of cortisol per tube. Upon testing a panel of 28 human plasma by the radioimmunoassay and the enzyme immunoassay (immuno-sorbent method) a coefficient of correlation between these two methods of r=0.916 was found. The recovery of 0.25 and 0.50ng of cortisol/ml was 85 and 83%, respectively.

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A method for enzyme immunoassay of TSH is described. TSH is conjugated with horse-radish peroxidase according to Nakane's method 3), and separation of bound and free is obtained by solid-phase technique using Sepharose 4B-anti TSH complex. Enzyme activity is determined by fluorometry. The working curve for TSH standards is satisfactory to recognize TSH concentrations as 0. 25μ U/tube. The method requires only 18 hours incubation and minimal equipment, but its sensitivity is lower than that of ordinary radioimmunoassay. Further development is. expected in this method.

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Enzyme immunoassay (EIA) methods of TSH, cortisol and testosterone were developed and evaluated for clinical use. TSH-alkaline phosphatase (ALP), cortisol-21-hemisuccinate-ALP and testosterone-3-oxime-glucoamylase conjugates were used and B-F was separated by double antibody technique. Values for serum TSH, cortisol and testosterone determined by EIA correlated with those determined by radioimmunoassay. The good recovery of these hormones added to serum and the low coefficient of variation indicate that these methods are applicable for routine tests.

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A method for preparation of antigens of catecholamines and L-DOPA as haptens was developed. These haptens were conjugated with carrier proteins by the Mannich reaction after Nmaleylation of the haptens for protecting the amino groups of the side chain, then the maleyl groups were removed by acid hydrolysis under a mild condition. By immunization of rabbits with these conjugates antibodies were produced. In a cross-reactivity test by Ouchterlony's method the antibodies to each one of the conjugates selectively reacted with the conjugate of that hapten even if the carrier protein was bovine or rabbit serum albumin. The binding activity of the antibody to L-epinephrine was tested by Millipore filtration. The binding of 3 H-epinephrine was specifically inhibited by epinephrine but only slightly by the other catecholamines and their metabolites. The preliminary experiment of enzyme immunoassay for epinephrine was attempted using the antibody.

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Turnover of various size of endogeneously labeled very low density lipoprotein VLDL, d (1.006) was studied in the fasting rabbits. Triglyceride fatty acid of very low density lipoprotein (VLDL-TGFA) was labeled by either ¹⁴C- or ³H-palrnitate and labeled VLDL was subfractionated on 2% agarose column according to their size. Dose VLDL was prepared by mixing either large ¹⁴C-VLDL and small ³H-VLDL or small ¹⁴C-VLDL and large ³H-VLDL and was injected to recipient rabbits intravenously. Specific activity curves of VLDL-TGFA and LDL-TGFA (d 1.006 to 1.063) were obtained from the blood samples obtained at certain time intervals after the administration of Dose VLDL. In specific activity curves cif VLDL-TGFA two exponential components were ovserved; the rapid removal phase was followed by the slower removal phase. The values for half-life of VLDL-TGFA were taken directly from the observed slopes of initial rapid disappearance. The following results were obtained;
(1) Half-life of Dose VLDL-TGFA was ranging from 4.0 to 23.0 min.
(2) In every rabbit large VLDL disappeared faster than small VLDL from the circulation and there was statistically significant negative correlation between half-life of Dose VLDL and average diameter of Dose VLDL (n=20, r=-0.512, p<0.05). This result may suggest that the large VLDL are not all metabolized via small VLDL, for if that were so the half-life of each fraction would be the same.
(3) Statistically significant positive correlation was observed between half-life of Dose VLDL and VLDL-TG level of recipient rabbit (n=20, r= 0.671, p<0.01)
(4) Specific activity curves of VLDL-TGFA and LDL-TGFA was suggesting precursor-product relationship between these two lipoprotein fractions.
Some rabbits were exanquinated 15 and 30 min after the administration of Dose VLDL and distribution of radioactivity in VLDL subfractions were compared with those of Dose VLDL. The result was suggesting that VLDL-TG was not completely hydrolyzed by the enzyme when VLDL particles first bind to the capillary wall but they were partially hydrolyzed and decreased in their size gradually in the process of metabolism.

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The clinical features and the pattern of plasma lipoprotein abnormalities were investigated in six cases of hyperlipoproteinemia which were suspected to be broad β disease.
Plasma samples containing ethylenediamine tetraacetic acid were obtained after an overnight fast. Samples were fractionated by two steps of ultracentrifugation at d=1.006 and 1.019 into four fractions; 1) VLDL or the top fraction obtained by the first centrifugation at d=1.006, 2) the second fraction (density class near 1.006) which was obtained from 1ml portion just beneath the VLDL fraction, 3) LDL₁ fraction or the top fraction at the second centrifugation (1.006 <1.019), 4) the last bottom fraction which contained LDL₂ and HDL(d >1.019).
Lipoproteins were analyzed on polyacrylamide gel (PAG, 3%, 4mm×5cm) electrophoresis. The lipid composition was also determined for each fraction.
PAG electrophoresis revealed one or more peaks of abnormal lipoproteins with different migration rates for each fraction. Lipoproteins in VLDL fraction had a peak at the position identical to the ordinary VLDL, but the peak was accompanied by long trailing or a group of several small components, which showed a higher migration rate than the ordinary VLDL. Corresponding to the fast migrating part of the VLDL fraction, one or more irregular peaks appeared also in the second fraction. In LDL₁ fraction, an abnormal lipoprotein appeared as a single symmetrical peak with a relatively narrow base which showed an intermediate migration rate between VLDL and LDL. In normal subjects, no lipoprotein peaks were found in the second and third (LDL₁) fractions. Even LDL₂fraction appeared as a group of several peaks in these cases. The result shows that the abnbrmal limroteins in the plasma were distributed through a wide range of density classes.
The ratio of cholesterol to trig1ycerid in VLDL fraction in these cases ranged from 0.20 to 0.62. These values were significantly higher than the control value (=0.20). The ratio became increasingly higher through the second (density near 1.006) and the third (LDL₁) fractions toward LDL₂. The continuous changes in the density as well as in the lipid composition showed that the abnormal lipoproteins are intermediates between VLDL and LDL.
With an exception of case 6, these cases are diagnosed as the primary form of hyperlipoproteinemia. Although broad β disease is strongly suggested, it must be pointed out that all of the cases showed normal ECG and no one had an evidence of ischemic heart disease nor any kind of xanthomas. Although the ratio of cholesterol to triglyceride in VLDL fraction was increased, a half of our cases were excluded from the definite type III, if the criterion suggested by Hazzard (>0.42) was applied. Applying the criterion introduced by Fredrickson and his associates (VLDL-cholesterol to total triglyceride>0.25), only one of our cases fell under the category of broad disease. Estimation of the lipoprotein lipase activity and the analysis of apoproteins are now going on in our laboratory.
One of these cases (case 3) was defmitely familial. One of his brother and three of his five children had also hyperlipoproteinemia. The patient had a history of acute illness at his age of 50 showing abdominal pain, diarrhea and anemia. Soon after the recovery from the crisis, hyperlipemia was noticed which was enhanced by the intake of alcoholic biverage. When the triglyceride level was markedly increased (2,500 mg/100ml), hyperlipoproteinemia of Type V was suggested by the presence of chylomicron and the increased amount of VLDL on plasma lipoprotein electrophoresis. The intake of low fat diet decreased the plasma concentration of triglyceride to approximately 500mg/100ml, when lipoprotein electrophoresis revealed a pattern of broad βdisease.

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Type III hyperlipoproteinemia (HLP) is clinically manifested by palmar xanthomatosis and a high incidence of premature atherosclerosis. However, regarding the defmition and diagnosis of type III HLP there are many conflicting reports. Proving “β-VLDL” by ultracentrifugation and lipoprotein electrophoresis has been a specific diagnostic test for type III HLP. However, electrophoretic determination of the presence of β-VLDL requires subjective interpretation and yields only qualitative information. In fact, β-VLDL has recently been proved not to be specific for type III HLP. Thus, various chemical criteria have been proposed to be more specific and sensitive for type III HLP by many authors, but none of these criteria are satisfactory at present.
In an effort to find a more specific and sensitive chemical criteria, we made serial determinations of serum lipoproteins in three patients with type III HLP.
The VLDL-chol/VLDL-TG ratios were between 0.27 and 1.93, and were found to be useful in differentiating type III from normal, type IIa and IIb HLPs. However, it was impossible to differentiate type III from type IV by the ratio.
The VLDL-chol/TG ratios in type III HLP were greater than 0.20, but a few patients with normal lipids, type IIa and IIb HLPs showed a ratio greater than 020. Ratio above 0.30, which is the criterion for type III HLP introduced by Fredrickson et al.,was proved to be definite for type III HLP. However, in 13 out of 28 samples of type III the ratios were less than 0.30.
From the present study, it seemed to be logical to conclude that the VLDL-chol/VLDL-TG ratio above 0.33 should be considered as diagnostic of type III HLP, when the VLDL-chol is above 55mg/dl.
Several clinical studies were made to elucidate some aspects of pathophysiology of type III HLP.
In a case of type III HLP associated with Sheehan's syndrome, postheparin lipase activity (PHLA), particularly hepatic PHLA was low. During replacement therapy with hydrocortisone and desiccated thyroid, the serum cholesterol and triglyceride levels decreased to the normal levels, however, broad-β band on agarose gel electrophoresis and β-VLDL remained.
In 5 cases of hypothyroid with type III HLP, hepatic PHLA were low. and after thyroid hormone replacement PHLA restored to the normal level.
Administration of heparin to type III HLP resulted in rapid clearance of rapid-moving VLDL and accumulation of β-VLDL.
These results suggested that hepatic PHLA may some roles in the pathogenesis of type III HLP.
High carbohydrate diet converted type IIa HLP associated with hypothyroidism to type III HLP, and after high fat diet type III HLP returned to type IIa HLP. Thus, carbohydrate inducibility in type III HLP was proved to be pronouned in hypothyroidism.

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Two kinds of the midband lipoproteins (Lps) were studied. The one was a midband which occurred very closely to β Lp and appeared as a double-β Lp profile on polyacrylamide gel (PAG) disc electrophoresis. The other was those appeared as the braod-midband Lp profile, which occurred frequently in uremic patients on hemodialysis.
The midband Lp of the double-β Lp-emia was detected not in LDL which migrates at β Lp position usually but in HDL (d 1.063-1.21) which migrates at α Lp position usually, exclusively (HD-midband Lp). As isolated by PAG block electrophoresis, HD-midband Lp showed a lipid composition intermediate between β Lp and pre-β Lp. This HD-midband Lp occurred in 1.8% of a population of approximately 1100 and showed familial corroboration.
LDLs isolated from the patients with the broad-midband Lp profile produced 2-3 Lp bands on PAG block electrophoresis and 2-3 peaks in ultracentrifugal Schlieren analysis. One of such LDLs which showed 3 Lp bands and 3 peaks was subjected to a series of ultracentrifugal analyses to determine buoyant densities of each peak-component. On the basis of these analyses, the 3 peak-components were isolated ultracentrifugally and it was revealed that they are consisted of Sf 13, 10, 7 and 6 LDL-subfractions. These LDL-subfractions demonstrated electrophoretic behaviors and chemical compositions intermediate between pre-β-VLDL and β-LDL, suggesting abnormal accumulation of catabolic remnant(s) of VLDL.
It was concluded, at least tentatively, that for the most adequate identification of abnormal Lps which appear as the midb and Lps and may have atherogenic potential or any other pathological significance, which is obviously the primary purpose of such study as this, carefull characterization of the midband Lps seemed to be essential and productive.

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Serum concentration of apolipoprotein A-I (apo A-I) and B (apo B) was determined by rocket immunoelectrophoresis. The monospecific antiserum for the assay was prepared in the rabbits by immunizing with isolated and purified apo A-I or LDL. The supporting medium for the electrophoresis was prepared with 1% agarose-gel which contained 1.5% anti-apo A-I or 2% anti-apo B. Serum samples were carbamylated before electrophoresis. A normal serum usually required 1: 12 dilution for apo A-I and 1: 6 dilution for apo B. The electrophoresis was carried out at constant voltage of 2V/cm for 16 hours at 15°C. The buffer was 0.02M veronal at pH 8.6. The immunoprecipitates were stained with amido black 10B and measured the hights. The apoprotein concentrations were expressed as arbitrary units/100ml where the serum used for references assumed to contain 100 units/100ml of each apoprotein.
Apo A-I and apo B levels in 9 young healthy males revealed 102 ± 18 units/100ml and 99 ± 29 units/100ml, respectively. In 11 young females, apo A-I and apo B were 111 ± 7 and 102 ± 21 units/100ml, respectively (TABLE I). Increased levels of apo B was observed in hyperlipidemic subjects. In 7 patients with type II hyperlipidemia mean apo B lavel was 203± 26 units/100ml and in 16 type IV patients the level was 208 ± 16 units/100ml. These values were significantly higher than 18 controls with apo B concentration of 124 ± 24 units/100ml. Apo A-I in hyperlipidemics showed slight decrease in type II patients but no significant changes in type IV cases (TABLE II).
Serum concentration of apo B significantly correlated with the cholesterol levels. On the other hand, apo A-I levels correlated with cholesterol concentration in normolipidemic subjects but the correlation was inversed in hyperlipidemics (TABLE III). These observation suggested that in hyperlipidemics the serum apo A-I was relatively deficient for their cholesterol content.

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Apo-C has been isolated from VLDL of severe hypertriglyceridemic serum in western contries where this type of disease is rather common. Because of a very low incidence of hypertriglyceridemia among Japanese population, we developed a method to isolate Apo-C from normolipemic or mild hyperlipemic serum. Pooled serum was spun at d, 1.125 for 2.67 × 10⁸ g-min twice and d <1.125 fraction was isolated and delipidated. Apoprteins were disolved in Tris-HC1 buffer of pH 8.2 containing 6M urea and subjected to chromatography on Sephadex G-150 column to separate Apo-C fraction. Subsequently, Apo-C fraction was rechromatographed to remove the contaminating Apo-A and fractionated on DEAE Cellulose column with a density gradient of TrisHC1 of pH 8.2, rainging from 0.01 to 0.12M. Reasonably purified C-I, C-II, and C-III-2 fractions were obtained. They were further purified by polyacrylamide gel electrophoresis and visualized under ultraviolet light after staining with fluorescent dye. Antisera were raised in rabbits by the administration of the fluorescent bands which were cut from the gels and emulsified with Freund's complete adjuvant.

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Significant decrease of α-lipoprotein concentration was noted in subjects with hyperlipidemia of type IV of WHO classification compared with those in controls and in hyperlipidemia of type Ha and type llb. Ratio between concentrations of phospholipid and total cholesterol in α-lipoprotein fraction separated by centrifugation of the mixture of serum and heparin-CaCl₂ solution, was higher in subjects with hyperlipidemia of type IV than those in controls and in the other type hyperlipidemic subjects. Decrease of lecithin and increase of lyso lecithin were also noted in the phospholipid fraction of sera from subjects with hyperlipidemia of type IV compared with those in controls and in the other type hyperlipidemias.
These differences in the lipid constituents, however, were unable to see in HDL_{2, 3} fraction of sera from controls and hyperlipidemic subjects. Therefore, it is suspected that the discrepancies of these results might be due to the absence of very high density lipoprotein (d>1, 201) in HDL_{2, 3}. There found no differences on the electrophoretical patterns of apo-protein in HDL_{2, 3} between controls and the hyperlipidemic subjects.

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Investigations on lipide metabolism have been performed on a seventy years old woman bearing Ig-A, lambda type multiple myeloma with hyperlipidemia of type II-b or type III classified according to Fredrickson. She showed increased serum, levels of cholesterol, cholesterol-ester, triglyceride and phospholipide, and she was accompanied with retention of vitamin-A by vitamin- A tolerance test, delayed FFA release by PHLA test and increased LCAT. The serum of the patient was divided into several fractions by differential ultracentrifugation. The bottom-fraction (d>1.21) made precipitate with the LDL-fraction by ouchterlony's double diffusion technique and also precipitated with other LDL-fractions obtained from a healthy control or a multiple myeloma without hyperlipidemia. In the mixture of patient's bottom- and LDL-fraction some new larger complexes, which were not observed in each fraction, were found by polyacrylamide gradient-gel electorphoresis. The bottom-fraction made precipitin arcs against LDL-fraction and anti Ig-kantiserum by immunoelectrophoresis, and the location of the bottom-LDL precipitate coincided with that of bottom-anti-Ig-A. These results suggest that the Ig-A of the patient serum has some affinity for LDL-fraction making complexes to interfere with lipide metabolism.

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It is well known that lipase from either bacteria or mammalian pancreas has no ability to hydrolyze serum triglyceride. However, it was observed that when serum sample was treated by phospholipase C prior the application of lipase, the hydrolysis of triglyceride with lipase was appeared markedly.
And also the similar results was observed on the reaction process of cholesterol esterase, in which incubated with cholesterol oxidase in advance.
These findings suggest that the enzymatic ability of hydrolysis is profoundly related to a structure of lipoprotein in serum.
A enzymatic determination for the lipids in serum should be considered such reaction process.

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A woman and her brothers with LCAT deficiency are reported. They had typical signs and symptomes of the disease, such as corneal opacity, moderate anemia with a slight hemolytic component and proteinuria.
The concentration of plasma esterified cholesterol was very low in all patients, the concentration of total cholesterol was very low in the proband and a brother, and that of triglyceride was high in the brothers. Plasma LCAT activity was about 10% of normal in all patients.
The erythrocyte lipid pattern was abnormal: the cholesterol content was 1.5 times of normal erythrocyte. In the agarose electrophore sis, αband was absent, and the pre-β band could not be separated forrn β band which moved slowly. In the polyacryl amide gel disc electrophoresis the abnormal bands were observed between α and β bands.
The lipid pattern of the major plasma lipoprotein fractions was abnormal. In chylomicron, VLDL and LDL1 (1.006-1.019), the concentration of lipid was too low to be measured. In LDL₂ (1.019-1.063) and HDL, the concentration of free cholesterol and triglyceride was very high and the cholesterol e ster ratio was very low.
Electron rnicroscopic examination reve aled morphological abnormalities in all major plasma lipoproteins. VLDL contained particles which were varied in size and shape. LDL₁ showed similar appearance to VLDL. LDL₂ contained discoidal particles. HDL fraction contained characteristic particles which formed rouleaux.
The post-heparin lipolytic activity was very low in the proband and a brother.
Unlike the patients with LCAT deficiency so far reported, these two patients had obvious jaundice and peripheral neuropathy. The UDP-glucuronyl transferase activity in a biopsied liver was very low in all patients, compared to normal, suggesting low UDP-glucuronyl transferase activity in the liver as one of the causes of jaundice.
Only these three patients in the family had haptoglobin subtype 1-1.
The proband was given ³H-labelled cholesterol perorally. The specific radioactivity of free and ester cholesterol was determined at appropriate intervals. The specific radioactivity of ester cholesterol increased at 5-11 hours after the ingestion and then decreased rapidly. On the other hand that of free cholesterol increased at 5-24 hours and decreased very slowly.
The fatty acid pattern of ester cholesterol was determined by gas liquid chromatography. The ratio of oleic acid was very high and that of linoleic acid was very low, compared to normal. The results suggested that most of the esterified cholesterol in the plasma observed in LCAT deficiency was not formed from free cholesterol in the circulation but in the intestine as a constituent of chylomicron.

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In order to investigate the origin of LP-X, a characteristic lipoprotein appeared in the blood of patients with cholestasis, the nature of bile-lipid was compared with that of LP-X.
Structural analysis of bile-lipid isolated with agarose chromatography showed that, like LP-X, bile-lipid was a kind of lipoprotein containing albumin at the core of its structure.
LP-X-like lipoprotein, characterized by the migration to the cathodal side on agar electrophoresis, was not detected in bile alone, but it appeared in the appropriate mixture of bile and plasma or bile and albumin. It was also observed that on agar electrophoresis the proportion of albumin and bile acids in the mixture was an important factor for the cathodal migration of the bile-lipid.
As for the surface structure of LP-X-like lipoprotein, although LP-X-like lipoprotein formed by mixing bile and albumin did not react with anti apo C, LP-X-like lipoprotein formed in the mixture of bile and plasma did react with anti apo C as LP-X did.
Particles with the diameter of 30 to 40nm were observed on the electron micrograph of the bile-lipid and the size of the particles was compatible with that of LP-X or bile-lipid particles reported by other investigators.
LP-X-like lipoprotein was observed on agar electrophoresis in the blood of rat which was obtained 12 hours or more after the anastomosis between bile duct and vena iliaca.
These results show the close relationship between bile-lipid and LP-X, and suggest the possibility that LP-X may originate from bile-lipid regurgitated into blood in cholestasis.

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In order to find out the effect of diabetic metabolism on plasma cholesterol esterification reaction, the plasma LCAT activities were studied on streptozotocin-treated diabetic rats. The plasma LCAT levels increased as the increase in plasma cholesterol, triglycerides and fasting blood sugar; however, a significant negative correlation was found between LCAT and plasma IRI, suggesting this enzyme reaction is associated with diabetic metabolism.
To investigate further the problems, the plasma cholesterol esterification activities (CEA) were measured in the treated-diabetics and in the non-diabetic subjects. The enzyme levels were not significantly elevated in hyperglycemic diabetics whose plasma TG levels stayed within the normal range. However, they were significantly elevated in hypertriglyceridemics in both groups. These results indicate that the diabetic metabolism per se is not connected to the plasma cholesterol esterification reaction, but it does so through the derangement in plasma TG (VLDL) metabolism.
Our in vitro study revealed net transfer of cholesterol from LDL-Sepharose into rabbit plasma under the presence of active LCAT, however these transfer reaction was not demonstrated when fresh human plasma was incubated with LDL-Sepharose. It seems reasonable to conclude that plasma containing higher CEA relative to cholesterol content can absorb more cholesterol from the insoluble lipoproteins.

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The concentration of plasma triglyceride (TG) will tend to rise or fall as its flux in or out of plasma is varied. Therefore, to determine if a rise in plasma TG concentration is caused primarily by increased secretion or by decreased removal, it is necessary to measure the removal rate and TG secretion rate.
Clinical Study
First, we demonstrated the relationship between plasma TG concentration and the fractional removal rate of exogenous TG by using the intravenous fat tolerance tests. Secondly, we reported here the new method for measuring TG secretion rates in humans. Intravenous fat tolerance tests according to the Carlson's method were performed in 3 control subjects and 10 hypertriglyceridemic patients. The fractional removal rates (K₂ value) of Intralipid negatively correlated with fasting plasma TG concentraion. Furthermore, after the injection of heparin, the clearance of Intralipid was enhanced and K₂ value increased. It is likely that we can use these fractional removal rates as a index of the removal rates of endogenous TG. These results suggest that intravenous fat tolerance test is suitable for assessing the removal rate and this method is possible to serve as a clinical routine test.
We demonstrated the new method for measuring TG secretion rates in control subjects and hyertriglyceridemic patients by using protamine sulfate which is known to inhibit TG removal from plasma. Since the administration of protamine sulfate may not completely inhibit the clearance of VLDL-TG, the fractional removal rates of Intralipid before and after the treatment with protamine sulfate were calculated by intravenous fat tolerance tests.
The elimination of exogenous TG from blood was delayed till 30 minutes after the administration of protamine sulfate. The inhibition of TG clearance was not complete and the inhibition rates showed the variability on individual subjects. Their rates varied from 34% to 83%. Then, estimating the TG secretion rates by using protamine sulfate, we should consider the inhibition rates of clearance by protamine sulfate and correct those values. Then, on another day, heparin (20u/kg) was injected followed 5 minutes later by protamine sulfate (1mg/kg). Thereafter blood samples were taken every 10 minutes over a period by one hour. VLDL was separated by ultracentrifugation and both LDL and HDL were separated by the precipitation's method. TG concentration were estimated with Fletcher's method.
Both whole plasma TG and VLDL-TG concentration increased almost linearily till 30 minutes after the administration of protamine sulfate. The increments in whole plasma TG and in VLDL-TG for the hypertriglyceridemic patients were more pronounced than those of control subjects.
The increments in whole plasma TG and VLDL-TG actually obained from experiments correlated with fasting TG concentration. However, considering the inhibition rates of clearance by protamine sulfate, only the corrected increments in VLDL-TG showed the correlation with fasting plasma TG concentration. TG secretion rates were calculated from those increments in whole plasma TG and VLDL-TG with and without considering the inhibition rates by protamine sulfate. There was positive correlation between fasting plasma TG and these TG secretion rates except one obtained from the corrected increments in whole plasma TG.
These studies suggest that the administration of protamine sulfate may provide a valuable tool for estimating TG secretion rates. However, TG secretion rates obtained from our experiments are higher than those which have been previously calculated by the isotopical technique. The explanation for these results might be that TG secretion rates were enhanced under heparin injection. It is concluded that the cause of hypertriglyceridemia is due to metabolic alterations both in splanchnic production and clearance from the blood.
Animal Experiment

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Post-heparin plasma has been shown to contain several enzymes, that is, lipoprotein lipase, hepatic triglyceride lipase, monoglyceride hydrolase, phospholipase and so on. Little is known, however, in the physiological roles of these enzymes in the catabolism of lipoprotein triglyceride. The present study was designed to help elucidate the physiological roles of monoglyceride hydrolase in the catabolic reaction of lipoprotein triglyceride.
The monoglyceride hydrolase activity was inhibited progressively with increasing amounts of serum added. However, in the case of preincubation of serum with the enzyme, the inhibitions were more slightly than those with substrate. At lower concentrations bovine serum albumin slightly activated the enzyme activity, whereas at higher concentrations it caused the significant inhibition. Subsequently, the effects of apolipoproteins on the monoglyceride hydrolase activity were investigated. In the cases of apoLp C-I, C-II and C-III, the enzyme activity was activated in the preincubation of the apolipoproteins with the enzyme, whereas without effect with the substrate. On the other hand, in the cases of apo-LDL and -HDL, the enzyme activity was inhibited in the both preincubation conditions.
Triglyceride hydrolysis by lipoprotein lipase has been shown in vitro to be activated by apoLp C-II and inhibited by apoLp In vitro evidences also indicated that the inhibition was related to the apoLp C-III-triglyceride ratio. Hydrolysis of triglyceride in VLDL would result in inhibitory levels of the apoLp C-III on a given lipoprotein unless there is a mechanism for removal of apoLp C-III from the lipoprotein or for blocking in its inhibitory activity. Brown and Baginsky suggested that monoglyceride could act in the blocking of apoLp C-III inhibition of lipoprotein lipase reaction, and that the interaction of monoglyceride with apoLp C-III could produce a complex which left the surface of the VLDL. On the other hand, the possibility of monoglyceride hydrolase forming a complex with apoLp C-III has been suggested by the present results. The present results also suggested that the monoglyceride-apoLp C-III complex was as active as monoglyceride alone for the substrate of monoglyceride hydrolase, and that the monoglyceride hydrolase-apoLp C-III complex reacted as the activated-form of the monoglyceride hydrolase. In summary, monoglyceride hydrolase also act as one of the blocking agents for the apoLp C-III inhibition of lipoprotein lipase activity.

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Experiments were carried out to purify non-specific esterase from rat plasma. The purified esterase showed a single band on polyacrylamide gel electrophoresis. When the purified esterase was incubated with slices of rat aorta which was pretreated with heparin to remove lipase, remarkable elevation of lipase activity was observed. The recovery of lipase activity by addition of the esterase was also confirmed by histochemical method. These results suggested that plasma esterase might convert to lipoprotein lipase in aorta. Experiments were now in progress to certify this possibility.

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In an attempt to elucidate the mechanism responsible for diabetic hypertriglyceridemia, the ability of patients to remove plasma triglyceride (TG) was explored. This was indirectly evaluated by measuring postheparin lipolytic activity (PHLA). In nine normal controls, PHLA reached to a peak in 10 minutes after the i. v. administration of a standard dose of heparin (10U/kg), which thereafter disappeared from the circulation following a monoexponential curve. Five diabetic subjects, who were accompanied by a common form of sustained moderate hypertriglyceridemia, were found to have a normal PHLA.
We observed four diabetic subjects who were associated with an unusual form of gross hypertriglyceridemia, which, after hospitalization, rapidly subsided in response to a proper treatment. These subjects showed characteristic features of low PHLA with rapid rate of disappearance. This suggests that such a special form of profound hypertriglyceridemia should be caused by an impaired TG removal resulting from deficient lipases.

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Normal ranges of meternal lipid concentration in 10 months pregnancy and at delivery are 210-300mg/dl of total cholesterol, 280-400mg/dl of phospholipids, 160-400mg/dl of triglycerides.
The frequency of hyperlipemia is 31.3% and hypolipemia is also 21.2% of whole pregnant women. The 72.7% of toxemic pregnant women are in hyperlipemia, and there is no case in hypolipemia. Hypertriglyceridemia is specially significant in toxemic cases.
In atempt to know the mechanism of hyperlipemia during pregnancy, the studies onthe rate of elimination from the blood stream of injected fat emulsion “Intralipid” and measurement of LPL activity were done in non-pregnant and pregnant women.
It is characteristic in pregnancy that the high concentration of endogeneous and exogeneous TG at fasting time and low K₂ value (elimination rate from blood stream of “Intralipid”). The activity of lipoprotein lipase of post heparin plasma in pregnant women is significantry decteased and is about 1/3 of non-pregnant cases. It was discussed that the clinical observation and the reason for the hyperlipemia in pregnancy.

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The purpose of the present study was to investigate the following:
(1) Cellular and subcellular distribution of apolipoprotein after injection of ¹²⁵I-labeled canine Apo A-I into dogs.
(2) In vitro proteolysis of canine high density lipoprotein subfraction, HDL₃, by acid proteases in canine liver lysosomes.
(3) Binding, uptake and proteolytic degradation of rat high density lipoprotein subfraction, HDL3, by isolated rat liver parenchymal cells.
(1) Canine Apo A-I was purified by column chromatography of the delipidated canine high density lipoprotein subfraction, HDL₃, using Sephadex G-100. The purified Apo A-I showed only a single band on SDS polyacrylamide gel electrophoresis. The calculated molecular weight of canine Apo A-I was 28,000. The C- and N- terminal amino acid was alanine and aspartic acid, respectively. The purified canine Apo A-I was iodinated by the iodine monochloride method and injected intravenously into healthy male mongrel dogs. At one day after the injection of ¹²⁵I-Apo A-I, the liver took up 4.8 times more radioactive counts than the kidney and twenty times as many counts as the spleen. The highest radioactivity recovered in the subcellular fractions of the liver was found in the lysosomal fraction.
(2) Soluble lysosomal fraction had 23-fold greater proteolytic activity of HDL₃ than homogenate, while the mitochondrial and microsomal fractions showed almost no activity. Proteolysis of HDL₃ and bovine serum albumin by acid lysosomal proteases conformed to a hyperbolic courve. The straight line was obtained on Lineweaver-Burk plots. Apparent Km for HDL₃ was 46 micromolar and 374 micromolar for bovine serum albumin proteolysis. Iodoacetate inhibited HDL₃ proteolysis 100 percent at concentrations greater than 50 micromolar, while bovine serum albumin proteolysis was inhibited 68 percent by iodoacetate at a concentration of 100 micromolar. Maximum inhibition of HDL₃ and bovine serum albumin proteolysis by pepstatin was 45 percent and 70 percent, respectively. These observations suggest that the endopeptidase, cathepsin B, plays a more significant role in HDL₃ proteolysis.
(3) In the standard assay of ¹²⁵I-labeled HDL₃ binding, uptake and proteolytic degradation by rat liver parenchymal cells, approximately 10 million cells were incubated at 37°C? in 2.0ml of Krebs-Henseleit bicarbonate buffer, pH 7.4. The maximum rat HDL₃-binding capacity (B_max) and the dissociation constant (Kd) of the isolated rat liver cell was 31 nanograms/mg dry weight of cells and 60×80^-8M, based on M. W. 28,000 of Apo A-I, respectively. Radioactivity in cells reached a maximum at 30 minutes of incubation and this plateau persisted for at least 2 hours. There was a lag phase for 15 minutes and after that a constant rate of proteolytic degradation was observed up to 2 hours at 37°C. Proteolytic degradation of ¹²⁵I-labeled HDL₃ was inhibited by chloroquine. Inhibition of HDL₃ proteolytic degradation by chloroquine may be explained by an effect on cathepsin B in liver lysosomes.
These data suggegt that binding, uptake and proteolytic degradation of rat HDL₃ by isolated rat liver parenchymal cells are actively performed and linked in the sequence: binding, then uptake and finally proteolytic degradation. There may be a specific HDL₃ or lipoprotein A recognition sites on the plasma membrane. The important role of liver lysosomes in proteolytic degradation of HDL₃ was indicated.

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Diet has a powerful effect on plasma lipid concentration, however these levels often vary among individual cases on the same diet.
The present studies were undertaken to investigate the releasing mechanism of very low density lipoprotein (VLDL) in lanolin-induced hypercholesterolemia of rat and the role of high density lipoprotein (HDL) on catabolic pathway of VLDL in human plasma.
Material and Methods;
A) Three adult wister strain rats were loaded by 10g of lanolin (oral) before experiments. Four rats were remained fasting overnight as control. 15μCi/rat of ¹⁴C-1-palmitate and 50μCi of ³H-2-glycine were injected venously (at 3 hours after lanolin load on experimental group). At 1 hour after injection of these labeled compounds plasma lipoprotein fraction was separated by using the ultracentrifuge-method and lipid fraction of liver-lipid was separated by the thin layer chromatogram. Radioactivity of each lipid fraction and of lipoprotein-protein were counted by scintiration counter.
B) Fifteen hospitarized subjects have consumed the formula diet (1000 Cal./day containing 140g of carbohydrate and 80g of protein) for four weeks. These subjects were selected by following criterion; Relative body weight after the diet therapy was less than 120%. Plasma triglyceride (TG) level after the diet therapy was less than 117g/100ml. The body weight was decreased by more than 2% after four weeks of the diet therapy.
These hyperlipidemic subjects have been divided into two groups according to the response of low density lipoprotein (LDL) to the diet therapy. Before and after four weeks of the diet therapy blood sample was drown in the morning after overnight fasting. Lipoprotein fraction was separated by ultracentrifugation.
Results and Discussion;
A) Injected ¹⁴C-palmitate was mainly incorporated into phospholipids (PL) of liver in control group. During lanolin loading the incorporation of ¹⁴C-palmitate into hepatic fat was signif-icantly increased. The increased incorporation was caused by an increase in PL accompanied by an increase in incorporation of ³H-glycine into HDL-protein. Additionaly, incorporation of ¹⁴C-palmitate into VLDL-TG and also of ³H-glycine into VLDL-protein were significantly increased by lanolin load. Since PL has an important role of activation of lipoprotein lipase (LPL) which hydrolizes VLDL-TG in blood stream, the increased incorporation of ¹⁴C-palmitate into PL by lanolin load might be due inevitably to accelerate LPL.
On the other hand, the increasing in hepatic PL was inhibited by CCl₄-infusion which is well known that CCl₄ could induce the secretion of VLDL from liver. This result was suggested that PL might play an important role for development in liver, especially releasing mechanism of connecting unpolar lipids with VLDL-protein.
B) There are two type on the response of LDL-cholesterol to the diet therapy which diet factors were restricted. These types have not characteristic of serum lipids level. The group characterized by a decreased LDL-cholesterol has higher level of HDL-cholesterol than the group with an increased LDL-cholesterol. On the other hand, the group with an increased LDL-cholesterol has high ratio of LDL to HDL and of cholesterol to TG in VLDL. These results were suggested the enough HDL, as shown in the group with decreased LDL-cholesterol, made smoothly on catabolism of VLDL in plasma.
We posturated from these results in rat and human that the accelerated release of VLDL might be the first step when several diet factors have been loaded. On the process of VLDL-release from liver PL might play the most important role to connect lipids synthesized in liver with apo-VLDL and there appears to be accompanied by an increase in HDL for facilitating VLDL-catabolism in plasma.

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Despite suggestions on the role of insulin in triglyceride metabolism, the relationship betweeni nsulin secretory function and familial form of endogenous hypertriglyceridemia has remained unclear. To elucidate this relationship, 83 family members of familial endogenous hypertriglyceridemia including 9 index cases as well as 90 controls matched with age, sex and relative body weight were investigated. All probands revealed primary, endogenous hypertriglyceridemia, hyperinsulinemia, biopsy-proven hepatic steatosis (triglyceride accumulation) and normal activityof post heparin lipoprotein lipase (protamin-inactivated form). A significantly increased insulin secretion expressed as sum of plasma insulin values during oral glucose tolerance test was noted frequently in all age-groups of relatives, usually accompanied by glucose intolerance and hypertriglyceridemia. The first degree relatives of the index cases showed the increased insulin secretion more frequently (52%) than the second degree relatives (21%). Seven pedigrees obviously showed vertical transmission of the combined disorders of hyperinsulinemia and hypertriglyceridemia. H owever, the possibility of monogenic dominant inheritance proved unsupportable, since frequency distribution curves for adjusted blood glucose and plasma insulin and triglyceride in the relatives were similarly shifted to higher values with no apparent bimodality. Obesity was frequent but was not confined to the relatives. Impaired liver function was often noted even in the minors and liver biopsy performed in 2 of them revealed steatosis. Therefore, the insulin resistance expressed as hyperinsulinemia and hyperglycemia seems to be the essential genetic disorder. However, the insulinogenic indices 30 minutes after oral glucose load were significantly higher in the young relatives as compared to the aged; thus, the increased response of pancreatic beta cells to glucose might be more essential in this form of familial hypertriglyceridemia.

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To detect perturbations of the membrane structure and properties of mammalian erythrocyte induced by increased lipid content of the blood plasma, cellular shape changes based on conformational change of the membrane was utilized as the sensitive indicator.
Erythrocytes from genetic “fatty” rat with remarkable increased VLDL level or those from nephrotic patients with increased LDL level, did not show any appreciable shape change. In contrast, elevation of the plasma level of lysolecithin or free fatty acid induced varying degrees of echinocyte-spherocyte shape change, depending on the amount of the free lipid (s) incorporated into the membrane, which was accompanied by the changes in the membrane surface area, surface charge, osmotic fragility, etc. Partial removal of these membrane lipids normally present in the erythrocyte membrane induced a stomatocyte-type shape change.
From these results and from consideration of the known clinical findings on the abnormally -shaped erythrocytes in some hepatobiliary diseases and particularly in lipoprotein X cases, importance of the plasma level of the free lipids such as lysolecithin, free fatty acid and bile acids in inducing membrane perturbations on erythrocytes, and probably of some tissue cells, was emphasized. These lipids may act as “protein stabilizers” or “detergents” to give powerful effect on the membrane constituents. In contrast, the plasma lipoproteins may behave as a kind of “membrane” to fuse with the erythrocyte membrane in the presence of “detergent” lipid or to exchange the lipid constituents with it only under limited circumstances.

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The effects of genetic factor and aging on development of hypercholesterolemia were studied using the rats with different responsiveness for a high cholesterol diet and those with different ages. Labeled cholesterol in various serum lipoprotein fractions was actively taken up by isolated hepatocytes in vitro when incubated at 37.5°C. Although the uptake ratio from HDL was lower than those from other lipoprotein fractions, the net amount of removed serum cholesterol was larger than those from VLDL or chylomicrons, because cholesterol concentration in HDL was markedly higher than that in VLDL or chylomicrons. Therefore, serum HDL-protein seems to play a significant role for hepatic uptake of cholesterol in vivo as well as LDL-protein.
The isolated hepatocytes from the hyporesponded rats to cholesterol administration removed serum cholesterol much more than those from the hyperresponded rats in vitro. It is proposed that the reduced uptake of serum cholesterol by those cells may cause a decrease in feed back control for cholesterol synthesis and consequently an increase in synthetic activity. However, in the present experiment, the increase of cholesterol synthesis was not observed in the liver from such hyperresponded rats. The half lives of serum cholesterol were shown to delay in these hyperresponders, suggesting the decrease rate of the clearance of cholesterol from peripheral circulation. Therefore, it is suggested that the decreased uptake of serum cholesterol does not always induce the increase of hepatic cholesterol synthesis due to the impairment of feed back control, but the reduced clearance of serum cholesterol from peripheral circulation. Elevation of the serum cholesterol concentration in the hyperresponder by aging was larger than that in the hyporesponder. It means that a genetic defect concerning cholesterol metabolism was emphasized by the retardation of cholesterol metabolism by aging. The stimulation of hepatic cholesterol synthesis caused an enhancement in enzyme activities of cholesterol metabolism, for example cholesterol 7α-hydroxylase and enzymes for bile acids especially cholic acid synthesis. Cholic acid and its secondary bile acid, deoxycholic acid, are well reabsorbed from the intestine and result in an enlargement of pool size of bile acids. By contrast, the reabsorption of lithocholic acid which is formed from chenodeoxycholic acid by the intestinal micro-organism is lower than those of other bile acids and consequently easily excreted into feces. Such an increase of the pool size inhibits the enzyme activities for cholesterol synthesis as well as those for cholesterol catabolism. Thus, cholesterol synthesis and catabolism are well correlated and enhanced cholesterol synthesis is readily restored by the increased of bile acid formation to keep the homeostasis of serum cholesterol level.
Although the hepatic uptake of serum cholesterol was reduced in old rats, synthetic activity of hepatic cholesterol was not enhanced but decreased markedly. Hepatic cholesterol synthesis from acetate, mevalonic acid or squalene in microsomal fractions was decreased in old rats. Well washed microsomes showed no synthetic activity of cholesterol in vitro without the addition of hepatic cytosol fraction, which contains sterol carrier proteins. When hepatic cytosol from the old was added to the microsomes from the young animals instead of young cytosol, the cholesterol synthesis from squalene as well as mevalonic acid in the young microsomes were decreased markedly. Thus, the impairment of the function of sterol carrier protein is suggested in aging in which the activities of microsomal enzymes are decreased.

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The effects of dietary lipid and daily food-uptake on hepatic HMG-CoA reductase activity and serum cholesterol level of the normal and diabetic rats were studied. The animals were maintained on either carbohydrate diet (dextrose 75%, casein 20%, and vitamin and salts 5%) or on fat diet (dextrose 30%, casein 20%, vitamins and salts 5%, corn oil 20% and indigestible fibers 25%) for five days from 8 o'clock in the morning till 6 o'clock in the evening. Daily food consumption per animal under these conditions were nearly 60 calories per day and no significant differences were observed. The other groups of normal and diabetic animals, forcedly fed one of the two kinds of diets 76 calories daily for four days, were also studied. The measurements of the enzyme activity were done at noon and midnight. The serum samples were obtained at noon and used for cholesterol analysis.
The diurnal variations of the enzyme activity were shown on the all groups observed. The lipid feeding markedly stimulated the HMG-CoA reductase activity of both normal (6.7 folds) and diabetic (20 folds) rats. Increased consumption of the diets also stimulated the enzyme activity in most of normal and diabetic rats (1.4 folds). However, these stimulatory effects on the reductase activity by these variouse different dietary states were not accompanied by increase nor decrease of serum cholesterol level.
These discrepancies between the hepatic cholesterogenesis and serum cholesterol level suggest that the liver has rather limited contribution to the whole cholesterogenesis or that the catabolism of cholesterol is also stimulated coordinately with its production by these dietary manipulations.
The possible mechanism of the induction of hepatic HMG-CoA reductase by dietary lipid and increased food consumption were discussed.

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