Determination of serum IgG was studied and a new method was devised. After application of 50μl of serum to a mini-column with Sephadex A-50, 0nly lgG was rapidly eluted in the non-adsorbed fraction. This lgG fraction was determined as the amount of protein by biuret method. By the use of this mini-column chromatography, IgG may be rapidly fractionated with a constant volume of the eluate despite the use of various serum samples. By the use of standard lgG, 98.5% of the applied sample was recovered, with Iittle influence by other proteins in serum. The results obtained by this method were found to exhibit an excellent correlation with those obtained by the well known SRID method. This method thus appears to be useful in daily clinical test.

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A new mini-column method, which we devised previously for determination of IgG, was used for quantitative determination of the enzyme activities of GOTm in human serum. When 50μl of serum was applied to this mini-column with DEAE-Sephadex A-50, GOTm was not adosorbed and eluted rapidly. The caliblation curve of purified human GOTm was determined with Karmen's, Babson's and Reitman-Frankel method, and Karmen's method was the best one of these method. When several sera with addition of purified GOTm was applied to this column, GOTm was recovered about 100% in eluate. The correlation of this method and electrophoresis method was well at low activity of GOTm, but not well at high activity of GOTm by the reason why electrophoresis method can not determined GOTm accuratly. The correlation of this method and immunoadsorbance method was very well. This method thus appeares to be useful in daily clinical test.

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Serum GOT activity is elevated in hepatitis and myocardial infarction patient. Boyd1) showed that by means of starch gel electrophoresis there were two kinds of GOT isozyme in the sera from hepatitis patient and each of them was established as GOT-m and GOT-s from human liver. However the starch gel is not suitable for the routine method because of time wasting and incomplete quantative estimation. This paper reports our studies to separate and determine the GOT isozyme in sera.
1. The determination of each GOT isozyme activity was done after the separation using the method of plate electrophoresis.
2. It is possible to obtain the separation and determination of each isozyme activity between 5 and 200Karmen unit/ml within one hour and to estimate 32 samples (sera) at the same time.
3. The distributions of the isozymes invarious organs were reinvestigated using this method.
4. GOT isozyme activities were separated and determined in sera of hepatitis and myocardial infarction by using this method.

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A sensitive and rapid spectrophotometric method for the determination of elastase (EC3.4. 4.7) by its elastoproteolytic activity with insoluble elastin as a substrate, is described.
Elastase activity was not found in human serum and elastase inhitor capacity in serum could be determined by this method. It was possible to determine elastase activity in pancreatic Juice of dogs by this method.
These results suggest that this method will be a useful test for detecting pancreatic dysfunction.

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The rate of hydrolysis of steroid glucuronides (poregnanediol-, and 17-hydroxycorticosteroid- glucuronides) is increased by an addition of sodium sulfate to the incubation medium, when usinng β-glucuronidase preparations from bovine liver.
But, sodium sulfate inhibits the enzyme hydrolysis of pregnanediol-, and estriol-glucuronides with preparations from Helix pomatia and from E. coli.
The rate of hdrolysis of nonsteroid glucuronides (P-nitrophenyl-and phenolphthaleinglucuronides) is increased by sodium sulfate when using Preparations from bovine liver and Helix pomatia, it is inhibited with preparations from E. coli.
The preparation from Helix pomatia consists of four glucuronidase fractions, none of which hydrolyzes steroid glucuronides specifically.

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We have previously reported that there was a great difference between urinary creatine values obtained by our enzymatic method and by the Folin method. In order to clarify causes of the discrepancy, specificity of the both methods was investigated.
A satisfactory recovery of creatine addewsd to urine specimens suggested that substances other than creatine did not interfere with the enzymatic method.
On the other hand, guanidinoacetic acidand guanidinosuccinic acidwhi chmight occur in urine specimens were demonstrated to give positive error in the Jaffé reaction when treated with hydrochloric acid under the conditions of creatine determination by the Folin method. The reason why guanidinoac etic acid and guanidinosuccinic acid gave the positive error in the Folin method was also discussed.

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The contents of thiobarbituriacc id reactives ubstances (TBARs) in rat hepatic subcellularo rganellesa nd in serum increased with aging and this evidence was observed in earlierp eriod in hepatic microsomes ratherthan in serum. The TBARS leveli n hepatic microsomes from three month old rats was significantlhyi gher than that in one month old rats and its concentration in 12 month old rats reached 9 times ashigher as that in one month old rats. ln tocopherola dministrationt o rats for 12 month, the TBARS concentration in serum and in hepatic microsomes were lower than those of control rat and the tendency of increase of this concentration with aging was moderate in this group during the observation period.
Synthetic activitieosf cholesterola nd fatty acid in rat liver decreased concomitantly with agirlg. However, those activitiedsi d notdecrease significantliyn the rats fedtocopherol for long period. Elevation of serum lipid concentration with aging was moderate in tocopherol administeredr ats for 6 to 12 months and was significantlyo wer as compared with that in control rats. ln addition, the phospholipid content in liver was higher and triglyceridceo ntent was lower after long term feeding of tocopheroL
TBARS content in serum and hepatic subcellularo rganelleso f rats fed tocopherol free diet increased markedly and lower syntheticactivitieosf hepatic cholesterola nd fatty acid as well as glucose-6-phosphatase in microsomes were observed after two weeks. Refeedirlg of tocopherolrichd iet to the rats decreased the TBARS Ievels andrepaired the synthetic activitietso normal. ln tocopherol deficientr ats, supplementof 2% cholesterolt o tocopherol deficientd iet increased merkedly theserum cholesteroland phospholipid.
From these results above mentioned, it is considered that the TBARS production in hepatic microsome is prevented by tocopherol administrationvery, efficient8ywhiclhe ad to the prevention of the impairment of the function of hepatic subcellular organelles concomitantly with aging.

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Uptake of serum cholesterol by isolated hepatocytes and hepatoma cells of rats was investigated in vitro. The uptake of cholesterol by the hepatocytes at 4° was very low and did not increase by the prolongation of the incubation time. However, the amount of cholesterol taken up by the hepatocytes at 37.5° was high and increased proportionally to the incubation time and serum cholesterol concentration. Such mechanism of uptake is not considered to be a nonspecific physiochemical process but a specifi carrier-mediated process. Ester cholesterol in the medium decreased more rapidly than free cholesterol during the incubation, but cholesterol in the hepatocytes was mainly found in the free form, suggesting that ester cholesterol taken up by the cells was hydrolyzed rapidly. Cholesterol in the every serum lipoprotein fractions could be transfered to the hepatocytes in vitro. The uptake of cholesterol diminished in old rats or hypothyroid rats. The isolated hepatocytes from rats hyporesponding to a high cholesterol diet took up more cholesterol than those from the hyperresponders.
The uptakes by the isolated tumor cells (AH 130 and SLC) were very low, about one fifteenth or one twentieth of the hepatocytes, when expressed by per cell number. Purified tumor membranes, however, were as capable of binding the serum cholesterol as hepatocyte membranes. Therefore, the lack of negative feed back control of cholestero-synthesis in malignant cells is not derived from the absence or decrease of binding ability of lipoproteins to cell membranes, but probably from a lower rate of internalization of lipoproteins from surface membrane into cytoplasma.

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Effects of albumin or high molecure compound on α-amylas eactivity by use of Blue Starch Method has been widely reported. Rate of increased activity by addition of albumin was varied from 10 to 90 percent, using urine samples which are protein negative by sulfosalicyli cacid method. To investigate the cause of change in activity, analysis of chemicals, α-amylase isozyme and trace amount of protein in sample was done. On as tudy of trace amount of protein not detected by sulfosalicylic acid method, amount of protein presented in urine is in proportion to the rate of increased α-amylase activity. These results show that the certain amount of albumin for the determination of α-amylase activity in urine or pancreatic juice required to obtain a constant result in routine test.

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1. A method was developed for the separative assay of placental alkaline phosphatase in human serum from the isozymes derived from other organs using acetate membrane electrophoresis with an electrophoresis buffer containing 2×10^-3M of MgCl₂.
2. the activity of placental alkaline phosphatase was completely recovered after the electrophoresis for 40 minutes by the addition of 2×10^-3M of MgCl₂ to the electrophoresis buffer and to the applied serum.
3. It was possible to measure accurately the placental alkaline phosphatase activity between l and 30 K-AU. using this method.
4. Neale1) described that placental alkaline phosphatase activity could be determined by treating serum a 56° for 30 minutes. But it is difficalt to obtain the constant results, because it is essential to adjust the temperature accurately at 56°
5. Placental alkaline phosphatase actMty in sera could be correctly determined only when the samples were heated at 56-58° for 30 minutes after the treatment with 10^-3M of EDTA and 3×10^-3M of MgCl₂ at 37° for 20 minutes.

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Assay method of serum cortisol using high pressure liquid chromatography was established and the various assay conditions were studied. The hormone was extracted from 1.0ml of sera obtained from healthy students and hospital patients. The final extracts were applied to Zorbax Sil column in Shimazu-Du Pont 830. Cortisol fraction separated from the internal standard (prednisolone) and other fractions was estimated as the area (height×Width). Normal Value Was 10.7±3.0μg/dl (Mean±S. D.) Obtained from 15 healthy males and 3 healthy females. The recovery of internal standard (prednisolone) was an important index for the judgment of accompanying technical errors. The recovery should be over 50%, over which the assay error was within ±5 to 10%. The present method for serum cortisol assay was particularly useful in clinical medicine to test the adenohypophysis-adrenocortical hormone system, for instance in feedback inhibition test on ACTH secretion and ACTH load test on glucocorticoid secretion.

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