Several types of modifications of serum lactic dehydrogenase (LDH) isoenzymes were previously reported, such as modifications due to complex formation with immunoglobulins, binding of NAD, and existence of an inhibitory factor to LDH-H subunit. This report is presenting two types of new modifications of LDH isoenzymes.
The first case concerned a 69 year-old male with rectal carcinoma and impending myocardial infarction. Serum LDH activity was within the normal limits and the enzymogram on Cellogel showed the normal electrophoretic mobilities except that LDH-III was broadened. and skewed to the cathode. On Sephadex G-200 gel filtration (0.02 M phosphate buffer pH 7.4, or Veronal buffer pH 8.6μ=0.06), the LDH activities were detected only between G and M fractions (M. W. 21×10⁴), but not between A and G where LDH of the normal molecular size was expected to elute. On the other hand, under the condition of a higher ionic strength (0.02M phosphate buffer +0.5M NaCl pH 7.4), the enzyme activities disappeared in an abnormal position and newly appeared between A and G (11×10⁴). These findings suggest that all of LDH in this case form high molecules and dissociate under a high ionic strength. The patient's serum was observed to convert LDH of normal size of the other serum into high molecules. Some component other than the enzyme was suspected to be responsible for formation of the macro-molecular LDH. However, by the method of immunoelectrophoresis followed by activity staining, LDH activities were not detected on the all of the precipitin lines by using anti-human whole serum or anti-immunoglobulins serum. This fact and the mild condition for dissociation may suggest that some proteins other than immunoglobulins would be involved in formation of the macro-molecular LDH. Both of the separated fractions of an abnormal and normal molecular size gave the same electrophoretic pattern and the same mobility. This case was thought to be a new type of macro-molecular LDH with such properties as normal electrophoretic mobility and the mild condition for dissociation.
The second type of LDH modifications was revealed in the survey of patients with a low activity of serum LDH-V which was considered to be not corresponding to elevation of GPT. One of these cases concerned a 61 year-old male with cardiac failure. Serum LDH activity was 100 W. U. and only LDH-I was electrophoretically observed with the traces of other isoenzymes. But hemolysate of the same patient was normal in isoenzyme pattern. The patient's serum was observed to inhibit an isoenzymes containing M-subunit of the other serum. Another case was a 69 year-old male with cardiac failure, whose serum was also inhibitable to M-subunit of the other serum. After dialysis of serum with a high LDH-V activity against this patient's serum, the isoenzymes containing M-subunit of the former were strongly inhibited. When the patient's serum was dialysed against 0.02 M Tris buffer, the ability of inhibition was completely lost. This ability was preserved under the condition of 80°C for 15 min.
The first type of LDH anomaly was estimated as a modification, either due to homo-dimer formation of LDH isoenzyme molecules with some binding factor of a small molecular size, or to complex formation with some protein which is of the molecular weight of about 10×10₄ and has no contribution to the electrophoretic mobility of LDH.
The second type was proved to be a modification due to existence of M-subunit inhibitor which is dialyzable and heat stable.
These modifications may offer new problems to interpretation and clinical application of serum LDH isoenzymes.

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Zymograms of urinary isoamylase were obtained by means of agar electrophoresis on patients with total pancreatectomy, acute pancreatitis, acute parotitis, gallstone and pancreatic cancer, and were compared with those of pure pancreatic juice, pancreatic extract and saliva. Amylase activity in each piece of agar, which was cut into 25 pieces after electrophoresis, was determined by the iodometric method.
It was suggested that the main fraction of pancreatic and salivary isoamylases of urine could be separated. In addition, the increase in activity of pancreatic amylase was found in patients with acute pancreatitis and the decrease in activity in patients with pancreatic cancer with jaundice after pancreozymin-secretin sequence of injection as compared with that of control.
The increase in the urinary pancre atic amylase activity was found in patients with both cholesterol gallstone and pigment gallstone, and there was long-standing high pancreatic amylase activity in patients with cholesterol gallstone after causal operation. On the other hand, the reduction of the activity to normal was found in patients with pigment gallstone.
There was an increa se in urinary pancreatic amylase activity in a hamster with pure cholesterol gallstone experimentally produced by a 20% butter diet or a fat-free diet for 4 weeks. There was a decrease in pancreatic amylase activity in the hamster, which showed histologically some pancreatic fibrosis produced by a 20% butter diet for 8 weeks, as compared with control.
These data show one of the evidences that nutritional factors are of etiologic significance in pathogenesis of pancreatitis combined with gallstones.

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Branched chain amino acid transaminase (EC 2.6. 1.6) can be separated in to 3 types (enzyme I-III) by DEAE cellulose column chromatography or polyacrylamide disc gel eletrophoresis. Enzyme I is ubiquitously distributed in various tissues of rats, while enzymes II and III are found only in liver and brain, respectively. Enzymes I and III are very similar in their properties and are active for all three amino acids (valine, leucine and isoleucine), while enzyme II is active only for leucine and its Km is very high (25 mM). These isozymes are, however, distinguishable immunochemically. They are, enzyme II in particular, inducible in rats by various physiological conditions, and change of their activities coincides well with the amount of ketone body formed from leucine. These findings shows that transamination is a rate limiting step of leucine metabolism.
In fetal rat liver only enzyme I is present and enzyme II appears rapidly after birth. Regenerating liver has essentially the same isozyme pattern as normal adult liver, and in neither has enzyme III yet been detected. Various Yoshida ascites hepatomas, which are poorly differentiated, contain enzymes I and III, but not enzyme II. The enzyme III in hepatoma (AH 130) is indistinguishable from that found in normal brain, both enzymologically and immunochemically. Various Morris hepatomas show a variety of isozyme patterns; enzyme I only (fetal pattern), enzymes I and II (normal pattern) or a mixture of all three isozymes, but not the pattern found in Yoshida ascites hepatomas. There is no definite correlation between the isozyme pattern and rate of tumor growth. Primary hepatomas induced by 3'-methyl DAB also contain enzymes I and X and benign adenomas show the normal isozyme pattern. These findings suggest that the appearance of enzyme III may be due to aberration of gene expression caused by carcinogenesis. An alternative explanation is that it is simply due to selection of a cell population. To test this possibility we examined cultured rat hepatocytes before and after their transformation by chemical carcinogens. A cell line isolated and cloned from liver of a 5 day old rat, contains only enzyme I, but after transformation by either 4-NQO or DAB, the cells acquired both enzyme X and tumorigenicity. Among 12 other cell lines examined only those showing great deviation from euploid chromosomal numbers contained enzyme III. These results indicate that the appearance of enzyme III is indeed due to aberration of gene expression and that these isozymes may be very suitable markers of cellular differentiation and dedifferentiation as follows:
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In our previous report, a new method was presented for estimation of leucine aminopeptidase (LAP) by means of a direct colorimetric determination of ammonia reported by Fujii and Okuda.
Leucin e amide was used for determination of LAP in our method. On the other hand, leucyl-β-naphthylamide was widely used for assay of this enzyme.
LAP from various rat tissues and plasma was determined by using these substances as substrates.
Tissue homogenates were sonicated and treated with Bromelain, followed by centrifugation for 60 min (105,000×g, at 4°C). The resultant supernatant was used for enzyme solution.
Bromelain treatment caused solubilization of LAP from rat liver from its precipitate fraction, which was proved to exist by gel filtration on Sephadex G-200 column.
TEAE-cellulose column chromatography and isoelectric focusing of LAP from various tissues provide an evidence that there exists this enzyme various forms characteristic to each tissue.
It was demonstrated that various rat tissues contained several fractions which showed different substrate specificities to leucine amide and leucyl-β-naphthylamide.
It was also clarified that the LAP elevated in plasma of CCl₄ treated rats contained several fractions which showed different substrate specificities to leucine amide and leucyl-β-naphthylamide.

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Many reports on LAP isozymes in human serum and human organs have been published, since Lawrence and his coworkers confirmed existence of multiple forms of leucine aminopeptidase. But the number of LAP isozymes and their electrolysed position were found different according to the investigators and the methods of electrophoresis, and their clinical significance remanis not clarified. Moreover, it is not clear whether the LAP in multiple forms is a true isozyme controled by gene, or consists of different enzymes which hydrolyze L-leucyl peptide.
In this paper, the authors studied on the LAP isozymes in human serum and human organs with the electro-focussing method of Vesterberg and Svensson, and on the kinetics of the isozymes. LAP of the normal human serum was separated into 3 fractions at pI 3.9, 4.5 and 5.1. The supernatant of the liver was separated into 5 fractions at pI 3.9, 4.5, 5.1, 5.7 and 6.3. The authors call these fractions as LAP₁, LAP₂, LAP₃, LAP₄, and LAP₅, starting from the anode side. LAP₁ and LAP₂ were very strongly active in comparison with LAP₃, LAP₄ and LAP₅ in the liver and gall bladder. In the kidney, LAP₂ and LAP₃ were very strongly active, but LAP₁, LAP₄ and LAP₅ were slightly active. In the pancreas, LAP₃ was only strongly active fraction, whereas LAP₁, LAP₄ and LAP₅ were slightly active, and LAP₂ was not observed in the pancreas.
Investigation on kinetics of LAP has been hampered on account of lack of the adequate methods, however, in this paper, the method of Goldberg was used which is widely used in the clinical field.
The effects of substrate concentration on the activity of LAP fractions (LAP₁, LAP₂, LAP₃ and LAP₄) were shown as similar sigmoid curves and same Km values (=8×10^-5M) in 0.1M phosphate buffer pH 7.0. And the activity of these fractions was inhibited by the high substrate concentration (4×10^-3M). The effects of different pH were represented in the form of similar curves which showed that the optimal pH of the 5 fractions was 6.5. The effects of metal ions on these 5 fractions were of a similar tendency, that is, Ca⁺² and Mn⁺² inhibited the LAP activity.
With respect of these results, it is very difficult for the authors to think that these 5 fractions are a complex of different enzymes which hydrolyze L-leucyl peptides.
The multiple forms of LAP in serum of the patients of some diseases were very different from those of normal subjects' serum. The authors separated the LAP isozymes with the electro-focussing method. In the serum from a cholelithiasis patient, LAP₁, LAP₂, LAP₃, LAP₄ and LAP₅ were remarkably detectable.
LAP₂ was more strongly active in comparison with normal serum. 5 fractions were also found in the cases of acute hepatitis and subacute pancreatitis. In liver carcinoma, LAP₁ was very strongly active. LAP₂ and LAP₅ were not observed in serum from patients of liver cirrhosis.
On the ground of these results, it is suggested that more researches on LAP multiple forms may make diagnosis of hepate-biliary disease possible.

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As previously reported 5), human serum alkaline phosphatase (Alp) isozymes are separated into 5 isozyme bands on thin layer polyacrylamide gel electrophoresis. According to their anodal migration, they are numbered as Alp I, II, III, IV and 0, respectively. The results are:
Neuraminidase released neuraminic acids from the polysaccharide chains of Alp isozymes except one (Alp IV).
Anti Lewis b serum formed complexes with Alp IV, and it is supposed that fucose instead of neuraminic acid is the terminal sugar of Alp IV and that a part of endpolysaccharide chain of Alp IV has the same sugar sequence as that of blood type Lewis b substance.
Phytohemagglutinin formed complexes with Alp III and it suggests that a part of endpolysaccharide chain of Alp III has the same sugar sequence of polysaccharide which is required by the receptor sites of phytohemagglutinin.
These data suggest that the endpolysaccharide chains of Alp isozymes are different from each other and are controled by different modifying genes together with the structure genes.

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It is evident that no agreement is found in the literature about the nature of alkaline phosphatase. For instance, Binkley, in purifying an alkaline phosphatase from swine kidney, claimed that the enzyme contains no protein, or if any, it is not essential for the activity of the enzyme, whereas Mathies prepared the enzyme containing 12% nitrogen from the same source.
The activity of the enzyme prepared by Mathies was 10,500 units according to the assay of King et al., and that prepared by Binkley was 300,000 Roche units per mg of total nitrogen. According to Schramm and Armbrustar, a factor of about 7 is required to convert King units into Roche units, and thus, the preparation of Mathies would have a specific activity of 10,500×7×1.4 or 102, 800 Roche units, the factor of 1.4 being a coefficient for conversion of the result obtained at 25° into that at 37°.
Ahmed and King isolated an alkaline phosphatase fraction from placenta by the butanol extraction method. They obtained a sample with 1,150 King-Armstrong units/mg N (K-A units/mg N), and further purified it by paper electrophoresis up to 1,750 K-A units/mg N. However, they did not mention the composition of the enzyme. Ghosh and Fishman purified human placental alkaline phosphatase and reported that variant A and crystalline B were obtanined, their molecular weight being 70,000 and 200,000, respectively, and that the activity of the former was 1,71 and the latter 212 units/mg. Harkness reported a crystalline alkaline phosphatase of specific activity of 4,200 units/mg. Since the definition of the specific activity of Ghosh et al. and that of Harkness are different each other, no direct comparison of purity of their preparations are possible.
We purified the enzyme from human placenta under mild condition without using any organic solvents, and obtained two kinds of alkaline phosphatase, A and B. The specific activity of A was 132,000 K-A units/mg N and that B was 38,000 K-A units/mg N. Both A and B samples were glycoprotein. A contains 32% of glucose and 2.8% of sialic acid, and B 1.5% of sialic acid. The optimum tempera-ture, optimum pH and Km were 60°, 10.0 and 3.85×10_-3M in A and 37°, 8.8 and 2×10_-3M in B, respectively. A shows wave shaped change in activity, accompanying change in optical rotatory power during incubation at 37°, and the activity gradually rises as a whole as time passes. On the contrary, the activity of B decreases linearly with time.

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Human serum alkaline phosphatase (ALP) is separated into six bands (ALP₁-ALP₆) by agar-gel electrophoresis'.
ALP₂ originates from the liver, ALP₃ from the bone, ALP₄ from the placenta and ALP₅ from the intestine, respectively. ALP₁ and ALP₆ are probably ALP₆ which has been modified.
They are separated into two groups: one comprises ALP₂ and ALP₃, and another ALP₄ and ALP₅, according to their enzymological characteristics (TABLE I ). Then, the physicochemical properties of purified placental and intestinal alkaline phosphatases were comparatively studied.

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It has been shown that pyruvate kinase is distributed as isoenzymes in the mammarian tissues. M₁ is present in skeletal muscle, heart and in brain; L in liver parenchymal cells; M₂ in liver parenchymal as well as in extraparenchymal cells, leucocytes, spleen, adipose tissues, lung, kidney, testis, ovary, tumor tissues and in fetal muscle. The activity of pyruvate kinase (PK) was measured in sera from patients with muscular dystrophy, other muscle diseases, acute myocardial infarction, and liver diseases. This activity was also measured in liver biopsy samples from insuloma, islet cell adenoma and from lipoatrophic diabetes.
In Duchenne and Limb girdle type muscular dystrophy, serum PK activity was elevated 28-fold: elevations were found even when serum aldolase and CPK activities were not elevated. In the late stage of progressive muscular dystrophy, the activity tended to diminish, but remained elevated. In acute myocardial infarction, the activity rose within the first three days. Magnitude of this rise (average 7-fold) and duration was similar to the rise of GOT. In acute hepatitis, where serum GOT or GPT activity was mildly elevated, the activity of serum PK was increased by 50%. In one case with chronic active hepatitis, the activity was found to be elevated when measured in fresh serum. In liver cirrhosis, serum PK activity was significantly lowered.
In progressive muscular dystrophy and in acute myocardial infarction, serum PK activity was kinetically M₁ since a high ratio of PK activity at (0.2mM/2mM) phosphoenolpyruvate and little or no effect with ATP, FDP and alanine were observed. In acute hepatitis, serum PK activity was activated by FDP, and inhibited by ATP and alanine, but ATP inhibition was completely reversed in 15mM MgC12. The mildly elevated serum PK activity in active hepatitis was most likely M2, although further study with electrophoresis would be necessary to confirm this.
Pyruvate kinase activity in liver biopsy samples from patient with insuloma exhibited a 2-to 3-fold elevation of the total activity with a high ratio of type L/M, indicating induction of liver PK by a high level of endogenous insulin.
These results indicate that determination of serum PK activity is useful for the diagnosis and follow-up of progressive muscular dystrophy, other active muscular diseases, myocardial infarction and liver diseases. As for liver diseases, serum PK activity has to be measured from fresh sera while in muscle and heart diseases, the activity can be measured from stored frozen sera. The determination of pyruvate kinase isozymes in biopsy sample from liver enables us to estimate the metabolic state of the liver concerning glucose metabolism.

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Our previous studies1, 2) have covered key glycolytic and gluconeogenic enzyme activities in biopsied liver tissues obtained from the patients with liver diseases and in experimentally injured rat livers. It has been reported then that increased activities of low-Km hexokinase and glucose 6-phosphate dehydrogenase, which are nonspecific to liver, and decreased activities of glucokinase, glucose 6-phosphatase and fructose 1,6-diphosphatase, which are specific to liver, were observed in those injured livers. Thus, the altered enzyme pattern in liver injuries resembled that in undifferentiated cells, such as hepatomas, regenerating liver and fetal liver.
Similar studies were made of human and rat (male Sprague-Dawley) liver pyruvate kinase (PK), which exists in two isozyme forms, PK-L and PK-M₂. Relative activities of these two isozymes in the rat were determined either by differential inhibition by 2.5mM ATP in the presence of 40mM MgCl₂ (kinetic analysis), PK-L being inhibited and PK-M₂ uninhibited, or by cellogel electrophoresis and densitometry. The latter method was used exclusively for determination of human liver PK isozyme activities. PK-L and PK-M₂ activities were calculated on the basis of the relative activity ratios and the total PK activities.
PK-L, which is the liver-specific major component in fed rat and is considered to be a differentiated type in liver, decreased in activity by fasting and increased by refeeding glucose-casein, whereas PK-M₂, which is the minor component and is considered as the prototype in liver, did not change in activity by dietary alteration but increased in fetal and partially hepatectomized rat livers, and AH 130 hepatoma. This confirms the results reported by other workers5, 6).
In several experimental liver injuries of rats, such as acute intoxication with CCl₄ or thioacetamide, chronic intoxication with CCl₄ and feeding on a choline deficient diet, the activity of PK-L was reduced, while that of PK-M₂ significantly increased to result in an isozyme pattern similar to that observed with AH 1301). Following intraperitoneal administration of a single dose of 0.2ml of CCl₄ per 100g body weight to the rat, the total PK and PK-M₂ activities increased 4 and 10 fold, respectively, over the controls in 3 days and returned to the initial values in 1 week. In contrast, PK-L activity decreased by one third of the control value in 2 days and returned to the original level in 1 week. These alterations were more marked when the activities determined by the electrophoretic method were compared, because contamination of PK-L or PK-M₂ with each other inherent to kinetic analysis could be avoided by this method.
Administrations of cycloheximide (100μg/100g body weight in 24, 27 and 30 hours after CCl₄ treatment) suppressed the increase of PK-M₂ activity in CCl₄ liver injury, suggesting the requirement of de novo synthesis of PK-M₂ protein for the increased activity. Involvement of humoral factor in the CCl₂-mediated induction of PK-M₂ was neglected from the results of parabiosis experiments.
PK-L and PK-M₂ activities in biopsied human liver were studied in patients with various liver diseases. Although the total PK activity failed to show any significant differences among the patients with liver diseases studied, the activity of PK-L decreased and that of PK-M₂ increased inversely in chronic hepatitis (active form), especially in that accompanied by sublobular hepatic necrosis and cirrhosis of the liver. The PK-L activities in fatty livers were significantly high. The altered pattern of PK isozymes in chronic hepatitis and cirrhosis of the liver resembled that in primary hepatoma.

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1. Three types of PKs are distributed in human tissues and some of those PKs were characterized. Tissue distribution of those PKs was almost identical to that of rat. Furtheremore, electrophoretic, immunological and kinetic studies on erythrocyte PK gave the suggestive evidence that PK might be a hybridized form of L and M2 subunits.
2. On the basis of that fundamental evidence, two typical types of PK deficiency were analyzed not only on erythrocyte but also other tissues (muscle, spleen and adipose tissue). In the case of M. I.(Type I), quantitative abnormality of PK occurred not only in erythrocyte but also in liver L-PK, while M₁ (muscle) and M₂ (spleen and adipose tissue) were normal. In the cases of Y. K. and F. K.(Type II), qualitative abnormality of PK occured not only in erythrocyte but also in liver L-PK, while MI (muscle) and M₂ (spleen and adipose tissue) were normal like the case M. I.

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Phosphofructokinase from chicken liver has been purified to yield a homogeneous protein whose homogenity was confirmed by disc electrophoretic and ultracentrifugal techniques. The purification procedure involves activation of the crude extract of chicken liver by heat treatment, ammonium sulfate fractionation, DEAE-cellulose chromatography and molecular sieve filtration on Biogel 1.5M. The purified phosphofructokinase has been crystallized in the presence of 1mM fructose-1, 6-diP and its specific activity is 134 units per mg.
In contrast to other phosphofructokinase, this enzyme undergoes a reversible inactivation at a low temperature. The rate of the inactivation increases with decrease in temperature. The cold inactivation depends on a variety of conditions such as protein concentration, pH, kind and concentration of salt. At 2mg/ml or above the enzyme at 0° is completely stable for at least one day, but below 0.3mg/ml the activity is lost very rapidly at this temperature. At 0° the enzyme is most stable at pH 8.3, but the stability decreased considerably above or below this pH. The cold inactivation is enhanced by monovalent anions such as Cl- or Br-, but polyvalent anions such as phosphate or sulfate tend to protect against inactivation. The enzyme is protected from cold inactivation by fructose-6-P, fructose-1, 6-diP, cyclic AMP or AMP, but ATP, ADP or citrate enhances the inactivation.
The cold inactivated enzyme can be activated by rewarming. The conditions which favor or disfavor the cold inactivation inhibit or promote the heat activation, respectively. The phenomena of cold inactivation and heat activation were demonstrated with phosphofructokinase in the crude extract of chicken liver as well as the purified enzyme.
The cold inactivation is accompanied by dissociation of the enzyme (13.9S) to a protomer with a S_20,w of 5.4 as shown by sucrose density gradient centrifugation and gel filtration. Reactivation by warming of the cold inactivated enzyme results in reassociation of the protomers to an aggregate with molecular weight of about 400,000, similar to the calculated value for the native enzyme (13.9 S).
The process of dissociationand association of the enzyme induced by temperature is influenced by the kinds and concentrations of salt, pH and allosteric effectors, which suggests that this process is a complex phenomenon and that a variety of bonds seem to be involved in holding together the associated form of the enzyme.

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Rat liver glucose-6-phosphate dehydrogenase (G6PD) can be induced by entirely different stimuli, i. e. refeeding high carbohydrate diets and liver injuries caused by CCl₄ and other hepatotoxic agents. This led us to examine a possible involvement of different molecular species of the enzyme in the induction under these experimental conditions.
G6PD in the soluble fraction of rat liver was resolved into three major components, I, II and III, in the order of decreasing mobility on polyacrylamide gel disc electrophoresis. Fresh liver supernatants from normal rats had only II and III while those from CCl₄-injured and glucose-casein-refed rats revealed all of the three components. Artifactual resolution could be excluded for the present experiment with G6PD by demonstrating an identical electrophoretic pattern of these forms on both Cellogel and pre-washed or riboflavin-polymerized polyacrylamide gel. Molecular weights of these forms were found similar, and the resolution on disc electrophoresis appeared to be mainly due to charge differences. Each of three forms was identically precipitated in the Ouchterlony test by rabbit antiserum prepared against purified G6PD, which was composed of I exclusively.
Conversion of slower migrating forms into faster ones, finally to sole Component I, without any significant change in activity was demonstrated by treatment with p-chloromercuribenzoate(pCMB) or HgCl₂. Upon treatment with high concentrations of these sulfhydryl reagents or N-ethylmaleimide (NEM), however, decrease in intensity of Component I from refed rat livers was accompanied by loss of the enzyme activity. Therefore, at least two readily reactive but catalytically not essential sulfhydryl groups are present per a dimer (mol. wt.=110,000) and are involved in sequential interconversion of I, II and III forms of G6PD. A similar sequential transformation of slower moving components into faster ones occurred upon depletion of GSH in liver supernatants by treatment with diamide, a specific GSH oxidant, at 0° and its reversal was demonstrated by regenerati on of GSH following incubation of the diamide-treated supernatants at 37°. Liver supernatants prepared from CCl₄-treated rats showed decrease in liver GSH concentration with concomitant increase of the faster moving G6PD bands. Addition of GSH to the supernatants restored the normal disc electrophoretic pattern having the components II and III. The increased intensity of the faster moving components observed in liver and other tissues under varying in vivo conditions was found to be closely associated with the increased ratio of G6PD activity to GSH level.
Among three interconvertible forms of X-linked human G6PD (type B⁺),B⁺-I, B⁺-II and B⁺-III in the order of decreasing mobility on disc electrophoresis, only two components,B⁺-II and B⁺-III, were demonstrated in fresh supernatants of intact livers. Faster moving components of G6PD increased in intensity in livers from patients with chronic hepatitis, liver cirrhosis and primary hepatoma, resulting in appearance of component B⁺-I in some of these cases. The altered G6PD pattern was closely associated with concomitant increase in G6PD activity and decrease in GSH level of liver, hence with the increased ratio of G6PD/GSH. The most marked changes inthese parameters were found in a case of primary hepatoma with elevated concentration of serum α-fetoprotein. Hepatitis and cirrhosis patients with increased faster migrating components of G6PD were characterized by extensive necrosis and degeneration of parenchymal liver cells and by relatively high levels of serum α-fetoprotein. Concentration of GSH, activity of G6PD and its electrophoretic pattern in erythrocytes were not significantly altered inthese subjects with liver diseases.

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At least 13 cases from 10 different families with glucosephosphate isomerase (GPI) deficiency have been observed. Recently two additional cases of nonspherocytic hemolytic anemia with GPI deficiency in different families were investigated and one GPI variant without GPI deficiency was found in our laboratory. In the first patient, pedigree studies and starch-gel electrophoresis revealed that he was homozygous for a particular identical variant gene. There were two incidences of consanguineous marriages in his family. The propositus K. S. had 3 isozyme bands, one of which migrated faster toward the cathode than the normal main band in red cell, white cell, liver, spleen and muscle. His parents and other heterozygous family members had almost a half GPI activity of the normal and had four isozyme bands, one of which migrated between the normal main band and the propositus' main band. These findings were quite different from those of the previously reported cases. In the second patient, the proposita T. H. and heterozygous parents and a sister had the same electrophoretic migration with lower GPI activity. A thermostability test revealed that the proposita T. H. had thermolabile GPI. In pH-GPI activity relation, the proposita's curve was shifted to the acidic side. The family had consanguineous marriage and the parents' isozyme had the same migration as the normal. There seems to be no reports so far on the same GPI deficiency as this case. These two cases were completely pure homozygous whereas the previously reported cases were double heterozygous for two different GPI deficient variant genes. GPI of the propositus K. S. was designated as GPI “Narita” and that of the proposita T. H. as GPI “Matsumoto”.
In the third case, total GPI activity was within the normal limits. However, GPI isozyme was different from the normal, in that there were four isozyme bands, and the second and third bands migrated faster than the normal ones, and the first and fourth bands were stained less densely than the second and third bands.
This abnormal isozyme was very similar to Detter's PHI (GPI) 2-1. The third case seems to be heterozygous for a non-deficient GPI variant gene. Detter et al. postulated that GPI molecule may be composed as a dimer with two subunits. Our findings strongly support his hypothesis. Pedigree studies and kinetic study in the third case are currently in progress in our laboratory.

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For analysis of creatine kinase (CK) isoenzymes, the original method according to Van der Ween et al. was modified, and an improved agar gel electrophoresis with a good sensitivity was brought in use.
CK isoenzymes were investigated on sera from patients with muscular dystrophies (47), polymyositis (4), myocardial infarction (6) and cerebral hemorrhage (3).
In 47 cases of progressive muscular dystrophies (32 Duchenne, 5 benign Duchenne, 6 congenital and 4 limb-girdle type), the relation between the serum total CK activity and age of the patients in each type showed the same tendency, i. e. a higher activity in the earlier stage, as previously reported by other authors. In the congenital type, the total CK activity elevated not so much as in the Duchenne type, however, the ratio of MB isoenzyme fraction was higher than in the latter. In 4 cases of polymyositis with a high serum total CK, the activity decreased rapidly after steroid therapy, while the MB isoenzyme fraction maintained a relatively high level.
At the peak stage in acute myocardial infarction, serum MB isoenzyme was detected independently of the total CK activity, and disappeared within about 80 hours.
In 3 cases of cerebral hemorrhage with a high serum total CK, isoenzyme analysis revealed no BB but MM fraction which is considered to be of the muscular origin.
The BB isoenzyme fraction was not detected in this series except in one sample from fluid contents of a cystic brain tumor.

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By using polyacrylamide gel disc electrophoresis, we developed a simple and sensitive stain method for glycogen phosphorylase (EC 2. 4. 1. 1). The method is highly sensitive for demonstration of phosphorylase activity which could not be detected by any conventional method.
In view of electrophoretic mobility, rabbit tissue phosphorylases could be classified into two groups: the faster moving ones of brain and kidney, and the slower moving ones of skeletal muscle and liver. With electrophoresis gel containing glycogen to a considerable concentration, the mobilities were retarded. The retardation was more marked for the muscle and the brain phosphorylases than for those of liver and kidney. Therefore, all four tissue phosphorylases might be organ-specific.
Retardation of mobility of phosphorylase fraction was increased in proportion to glycogen concentration in the gels. On the other hand, in regard to protein fractions other than phosphorylase, no retardation was observed. On the basis of variation in mobility as a function of glycogen concentration, the dissociaiton constant of phosphorylase with glycogen could be calculated. The values obtained were 6.6×10^-4 M, 24×10^-4M, and 13×10^-4M for phosphorylases of rabbit skeletal muscle, liver and brain, respectively.
In some pathological conditions, phosphorylase activity was demonstrated in human urines. In the extract of human kidney, two phosphorylase fractions were demonstrated. In the urines of hydronephrosis associated with renal calculi, of unilateral renal tuberculosis, and of ureteral carcinoma, one phosphorylase activity was demonstrated in the position, corresponding to the slower moving fraction of kidney phosphorylase. In the urine of plumonary carcinoma, another phosphorylase fraction was demonstrated at the position a little faster than the former. It corres-ponded to the faster moving fraction of kidney phosphorylase. In regard to ureteral carcinoma, the catheterized urine, which was collected 2 weeks after surgical removal of the affected kidney and tissues, showed phosphorylase activity. In contrast, the urine of renal tuberculosis showed no phosphorylase activity after removal of the affected kidney.

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In an attempt to determine whether dehydroepiandrosterone (DHEA) and its sulfate (DS) are secreted by human adrenal and testis, steroids in human adrenal and spermatic venous effluent collected in surgery are analyzed by utilizing gas-liquid chromatography. It was demonstrated that both DHEA and DS levels in adrenal venous blood are definitely higher than in peripheral plasma. In sharp contrast, testis secreted, if any, little DS, although various steroids including pregnenolone, progesterone, 17α-hydroxypregnenolone, 17α-hydroxyprogesterone, DHEA, androstenediol, androstenedione and testosterone were identified in human spermatic venous blood. Taken together with our previous finding that the circulating levels of DS show several folds increase in response to the administration of ACTH and are depressed to the undetectable level after the long-term treatment of patients with glucocorticoid, or in patients with Addison's disease, whereas gonadal stimulation with HCG does not affect the plasma levels of DS in healthy subjects, estimation of the circulating levels of DS could be a valuable indicator of adrenal secretion of Cs steroids.
Plasma DS was determined in 32 patients with Cushing's syndrome. In all of the 16 cases of benign adrenocortical adenoma, the plasma DS levels were significantly suppressed, however, the levels were elevated in 16 patients with bilateral adrenal hyperplasia with three exceptions who showed normal plasma DS values. These results indicate that the circulating DS could reflect the plasma levels of ACTH and determination of plasma DS would be of help in differential diagnosis of the pathological lesion responsible for Cushing's syndrome.

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Plasma testosterone concentration (PTC) was estimated in normal adults and in cases of various endocrine diseases, by using the competitive protein binding method described by Mayes and Nugent (1968) with slight modifications.
PTC of normal female and male adults under the basal conditions were 0.30±0.15 (SD) and 6.23±1.69 (SD)ng/ml, respectively, which had already been reported elsewhere (Japan. J. Urol., 61: 1088, 1970 and 62: 708, 1971).
PTC from the spermatic vein revealed a variety, namely roughly 40 to 200 times as high as the levels from peripheral male plasma. There was no significant differences in the mean PTC between normal and paraplegic men, though the testicular sizes were considerably smaller in the latters. A lowered PTC following surgical stress recovered to the preoperative levels in several weeks in major surgery, and in several days in moderate one where plasma LH increased as early as half an hour after operative incision, and then decreased below the preoperative levels on the second day of surgery. PTC was well reduced by injections of progestins in cases of advanced renal tumors and in male precocious puberty. PTC was lower than normal in cases of male Cushing's syndrome, delayed puberty in male teenagers, eunuchoidism and various hypogonadism. PTC higher than the normal was found in the cases of congenital adrenal hyperplasia with 21-hydroxylase deficiency, virilizing adrenal adenoma, and polycystic ovary syndrome. As a result of the stimulation test with HCG 3, 000iu+PMS 3,000 iu (daily) of three or four successive days injections, PTC incresed to a slight extent in Klinefelter's syndrome and did not sufficiently in hypogonadotropic hypogonadism. Upon infusion of LH-RH, a decapeptide, both plasma LH/FSH and PTC increased in a case of cryptoorchidism; only LH/FSH did in Steinert's syndrome and neither did in hypogonadotropic hypogonadism.

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Plasma testosterone in normal subjects and patients with various endocrine disorders has been studied.
A sensitive assay for plasma testosterone was based on the method of competitive protein binding analysis (Maeda et al).
In 113 normal subjects, plasma testosterone levels of five to ten year-old males and females were found to range from 20 to 75ng/dl, and 10 to 50ng/dl, respectively. Normal range of the adult male (age 20 to 70) was between 350 and 1,150ng/dl, and that of the normal female between 27 and 108ng/dl.
Plasma testosterone of a few males over 70 and 80 years of age tended to decrease below 350ng/dl.
Plasma testosterone and 11-OHCS at 8a.m. and 8p.m. were measured in 13 normal males and females, respectively.
In the males, plasma testosterone was high in the morning and low in the evening, which shows a significant diurnal rhythm.
In the females, there was a similar tendency, significance of which is, however, questionable. But adrenal suppression by dexamethasone revealed a difinite presence of the diurnal rhythm of plasma testosterone in the females as well as in the males.
In patients with hypothalamo-pituitary disorders, panhypopituitarism and hypophysectomy, when complete, showed extreme low levels which were even below the normal female range.
High testosterone level of 435ng/dl was demonstrated in a case of male precocious puberty (3 years of age), not suppressible by dexamethasone and responded to HCG stimulation up to 943ng/dl.
In this case, plasma testosterone was diagnostic and proved to be a good index of androgenicity.
Among patients with adrenal disorders, a female with 21-hydroxylase deficiency showed a male-range testosterone level of 546ng/dl, which showed the importance of not only adrenal androgens but also testosterone as a cause of marked virilism. A patient with 17α-hydroxylase deficiency showed a low baseline of 79ng/dl, 170 after ACTH and 144ng/dl after HCG stimulation.
On the basis of these results and other steroid analysis, it was suggested that 17α-hydroxylase was deficient both in testes and adrenals in this case.
Among patients with gonadal disorders, in a complete type of testicular feminization, plasma testosterone was at the normal male level of 537ng/dl, suggesting unresponsiveness of target tissue to testosterone, and decreased to 57ng/dl after resection of intraabdominal testes.
Almost no response to HCG stimulation was observed in 3 cases of Klinefelter's syndrome and one case each of myotonic dystrophy and Werner's syndrome.
In conclusion, plasma testosterone reflects well the function of Leydig cell and seems to be a good index of androgenicity.
It is, therefore, useful in the study of pathophysiology and diagnostic problems of various endocrine disorders.

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A simplified radioimmunoassay for measurement of plasma testosterone was established. Antiserum for testosterone was prepared in rabbit by immunization with testosterone-3-oxime linked to bovine serum albumin. The antiserum was diluted to 1: 10,000 with a borate buffer containing 0.1 % BSA. Plasma samples (0.1-0.5ml) were washed with 5 volumes of n-hexane and then extracted with 10 volumes of ether-hexane (2: 8) solvent. Extraction residue was dried directly in an assay tube when a higher value of plasma testosterone was expected. Further purification procedure with alumina column chromatography was desirable in samles with lower levels of plasma testosterone. Testosterone containing fractions were dried and incubated for 12 hours at 4° with 0.5ml 1/10,000 antitestosterone antiserum and testosterone-³H (20,000 dpm) in 0.5ml buffered saline. To each tube, 0.5 ml of dextran coated charcoal solution (250mg of Norit A and 250mg of Dextran T-80/100 ml buffer) were added ; mixture was shaken, incubated for 10 minutes at 4° and centrifuged for 10 minutes at 2,500 rpm, and the supernatant was taken for liquid scintillation counting. Duplicate testosterone standards (0, 50, 100, 200, 500, 1,000pg) were obtained in a similar fashion through the entire procedure and a standard curve was traced. Sensitivity of this method was 10ng/100ml when 0.5ml of plasma sample was assayed. Coefficient of variation was about 10 percent.
Plasma testosterone levels were measured in normal men and patients with pituitary Leydig cell dysfunction by using this assay procedure. Plasma LH levels were also measured simultanously by radioimmunoassay. The normal range of plasma testosterone in adult men were 450 to 1,150ng/100ml, which showed little diurnal variation, reduced to the half values upon 2 day oral administration of 15mg of fluoxymesterone and returned to the normal level upon an additional injection of 1,000 IU of human chorionic gonadotropin (HCG). The plasma testosterone levels in aged men (60-80 years old) did not show any significant difference from those in younger men (20-40 years old).
Responsiveness of Leydig cells to HCG was estimated by measuring the plasma testosterone levels after injection of HCG. Three thousands IU of HCG were injected intramusculary for 4 days to 4 young men, 3 aged men and 6 patients with hypogonadism. An average of the plasma testosterone values before HCG injection in young men, aged men and patients with hypogonadism were 680±120, 580±80 and 170±80ng/100ml (Mean±SD), respectively. The plasma testosterone response to HCG showed a 100 (1) increase after 4 day successive injection in young men, a 50 96 increase in aged men and no increase in hypogonadism.
One hundred microgram of LH-releasing hormone (LH-RH) was injected intravenously to 12 young men, 11 aged men and 8 patients with hypogonadism. In young men, the plasma testosterone levels reached to the peak of 50% increment in 120 minutes after injection of LH-RH, while the plasma LH levels were increased by more than 5 times values in 30 minutes after injection. In aged men, no increase in the plasma testosterone levels by administration of LH-RH were shown despite plasma LH were increased to the same levels as those in young men by LH-RH injection. In hypogonadic patients, neither plasma testosterone nor plasma LH showed any response to LH-RH.
The plasma testosterone and LH levels showed no significant difference between young men and aged men. However, responsiveness to HCG and to endogenous LH were cleary decreased. These findings suggest that the control mechanism of hormonal secretion in hypothalamo-pituitary-gonadal axis may be changed by aging.
Assay of plasma testosterone using a simplified radioimmunoassay may prove to be valuable as an indicator of Leydig cell function, especially upon stimulation with HCG and LH-RH.

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Hypothalamic luteinizing hormone releasing hormone (LH-RH) has been synthesized recently, by means of which the hypothalamic-pituitary-gonadal dynamics have been elucidated with much useful informations.
We have studied the effect of LH-RH on plasma testosterone levels both in men and rats.
Health adult men, aged 20-30, apparently being of normal gonadal function, were given 100μg and 400μg of LH-RH by a single intravenous injection.
Plasma testosterone was measured serially before and after the injection by a specific radioimmunoassay using an antiserum produced against testosterone-3-bovine serum albumin. Cross-reactivity of this antiserum with other steroids is shown in TABLE I.
The injection of 100μg of LH-RH was not effective to elevate plasma testosterone level in all the normal subjects, in spite of a significant increase in circulating LH concentrations. On the contrary, 400μg of LH-RH raised plasma testosterone significantly in all the normal subjects tested, although the amount of the increase in testosterone was not so large even when 400μg of LH-RH was administered (Fig. 1). It seems necessary, therefore, to use 400μg of LH-RH when plasma testosterone is to be measured as an index of the gonadal function.
Further studies were carried out to clarify the the reason why plasma testoste- rone response to LH-RH is not so marked.
For this purpose, male Wister rats, from 70 to 90 days of age, weighing between 150 and 200 gm, were used.
I. Testes removed immediately from normal rats were decapsulated and transferred to incubation vials containing 4.0ml of Krebs-Ringer bicarbonate buffer with glucose (0.2%) and bovine serum albumin (0.5%). Incubation was performed at 37°in a metabolic shaker under 95% O₂-5% CO₂. After 60min, the media were replaced by fresh buffer added hormone samples and the final incubation was performed.
Testosterone was determined by radioimmunoassay.
The following results were obtained.
(1) Ten pituitary halves removed from intact male rats were incubated by the method of Saffran and Schally, in 10ml of Krebs-Ringer bicarbonate buffer containing 10ng of LH-RH/ml. After 4 hours, the LH released into the incubation medium was measured by bioassay using rat testis in vitro steroidogenic system. As shown in Fig. 2, the incubation medium of pituitary halves possessed the ability of testosterone production. This suggests that the LH released from the pituitary by exgenous LH-RH has a steroidogenic potency in the testis.
(2) Dibutiryl cyclic AMP caused an increase in testosterone production by rat testis in vitro (Fig. 3). This finding coincides with the previous studies which suggested that cyclic AMP mediates at least some parts of the action of LH.
(3) The time course of testosterone production by rat testis in response to HCG was studied in vitro. No response in testosterone production was observed within 60min, but a marked response after 60min (Fig. 4). The reason for this delayed response is still obscure. It may be due to the experimental condition or to the age of the rats.
II. Rats were anesthetized with pentobarbital and the jugular veins on both sides were exposed. LH-RH was given through a jugular vein and blood sample was drawn from the opposite one. Plasma testosterone began to increase 30 min after LH-RH administration, and a higher response was observed (Fig. 5)
In summary, LH-RH, 400μg in dose, caused a rise in plasma testosterone in normal men, although the rise was slow and not so marked. Testosterone production of rat testis in vitro in response to HCG was also delayed. The reason for this delayed response is still obscure. Evidence was obtained which suggests a possible role of cyclic AMP in mediating LH action in testis.

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The effects of synthetic LH-RH were studied on serum radioimmunoassayable LH and FSH levels in 38 normal subjects (20 males, 11 females with menstruation, 7 postmenopausal women) and patients with isolated gonadotropin deficiency as well as various hypothalamo-pituitary disorders). The maximum response of LH was observed in 30 minutes after administration of 100μg synthetic LH-RH in both of the males and females, but in 60 minutes in the postmenopausal women.
The maximum response of FSH was observed in 60 minutes after LH-RH administration in all the subjects.
Synthetic LH-RH had no effects on serum GH, TSH and cortisol levels. The effects of LH-RH were definitly suppressed by preadministration of estrogen and testosterone. The ratio of FSH and LH (F/L ratio) were greatly decreased after administration of LH-RH in both males and females, but the F/L ratio was slightly decreased in the postmenopausal women.
No difference was seen in change of F/L ratio between the follicular phase and the luteal phase. All of three patients with chromophobe adenoma had a normal or slightly below normal response to LH-RH, while all patients with solid type of craniopharyngioma showed no or a very low response.
A patient with Sheehan's syndrome showed a small but significant response, while 2 patients with FrOhlich's syndrome showed no response.
A patient with Laurence-Moon-Biedle's syndrome and a case of isolated gonadotropin deficiency had a normal response and below normal response, respectively.
Thus it was demonstrated that there is a case of isolated gonadotropin deficiency which respond to LH-RH.

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With the development of the E-3 Kit, routine screening assay of estriol in high risk pregnancy has recently become easier.
A new equation has been developed for a single voided urine specimen which can be substituted for technically difficult 24 hour urine collection.
This new equation is more useful than another well-known equation using creatinine value, for the following two reasons:
(1) The resulting value obtained by this equation has a closer relation to the 24 hour urine value than does that of the creatinine equation.
(2) Creatinine assay is not required.
Good relationship between this value and that of 24 hour specimens were obtained in various cases of chronic fetal distress.
Practical cases are presented and discussed.

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Free [kestrone (E₁) +estradiol (E₂) in pregnant serum was measured by specific and sensitive E₂ radioimmunoassay (RIA) system.
Serum was washed with petroleum ether and estrogen fraction was extracted by. ether. The extract was further washed with 3% sodium carbonate solution in order to remove estriol (E₃) and then free (E₁+E₂) fraction was extracted by a mixture of petroleum ether: ether (2: 1).
The method of RIA was as follows: (1) 0.4ml of sample or standard E₂,(2) 0.1ml (6m μCi) of ³H-E₂ and (3) 0.1ml of anti E₂-BSA-γ-gl, diluted to 1: 1600, were mixed and incubated. ³H-E₂ bound to the antibody was precipitated at “one-half saturation” of a saturated ammonium sulfate solution and radioactivity of the precipitates was measured.
The range of measurement was 0.3-5.0ng/ml. Cross reaction of various steroids to E2 was 100% for E₁, 4% for E₃ and less than 0.03% for testosterone, hydrocortisone and progesterone. Coefficients of variations in the course of the repeated assays were 13.1-22.5% in early pregnancy, and the recovery rate was revealed to be 41.3±2.0 (S. D.).
Concentrations of free [E₁+E₂] of 69 cases of normal pregnant serum (9-42 weeks of pregnancy) showed a gradual increase according to the gestation periods. The mean values in the 4th, 7th and 10th months of pregnancy were 9.5, 16.6 and 29.5ng/ml, respectively. Blood samples were taken from 11 cases of normal puerperal women at various intervals after placental delivery and the serum levels of free [E₁+E₂] and HCG were measured. Their half-lives were 14.2 min and 3 hrs 42 min, respectively.
Estimation was made also on 29 cases of complicated pregnancy (hydatidiform mole, intrauterine fetal death, threatened abortion, anencephalus, twin, toxemia, overterm).
In conclusion, it may be safely said that the levels of serum free [E₁+E₂] are a more suitable index for placental function in the diagnosis or prognosis of threatened abortion due to hypofunction of placenta or determination of the timing of induction of delivery, since its half-life is extremely shorter than HCG.

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In order to understand clinical usefulness for monitoring the function of fetoplacental-maternal system, serum hPL, hCG and urinary estriol were measured with systematic serial estimation in comparison with the secretory behavior of these three hormones during pregnancy.
Furthermore, DHA-S dynamic test was performed to evaluate the feto-placental function. Following are the summarized data.
In normal pregnancy, both estriol and hPL were increased gradually from the beginning of pregnancy to the term in a very close correlation.
In threatened abortion which terminated in complete abortion, both estriol and hPL were low, or lowered even though they had been in the normal range at the beginning.
In the cases of intrauterine fetal death, both hormones were markedly low.
In pregnancy with anencephalus, only estriol was low. On the contrary, hPL was low in the cases of placental dysfunction. In a severe case of Rh sensitization, estriol was abruptly lowered in the course of the 40 th week of gestation.
By drip infusion of DHA-S, a remarkable increase of estriol was observed in the second and third trimesters of normal pregnancy, pregnancy with anencephalus and placental dysfunction, but no increase was found in the cases of intrauterine fetal death, hydatidiform mole and normal puerperium.
From these results, the authors may conclude that the measurement of estriol contributes to evaluation of the feto-placental unit, the hPL level represents directly the placental function and the DHA-S dynamic test is of much help in evaluating the fetal function.

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Establishment of new reaction systems for rapid and precise analysis are urgently desired in clinical chemistry. In this report, the conditions of reaction rate analysis of uric acid and creatinine were studied so as to establish the rapid analysis systems.
In reaction rate analysis with enzyme systems, the first order velocity must be proportional to the substance to be measured (substrate). Thus, the dynamic range of calibration depends upon the Michaelis constant of the enzyme for substrate.
Uricase and uricase-catalase systems were examined for analysis of uric acid in biological fluids. The Michaelis constant of uricase for uric acid was small (9.4×10^-6M) so that the dynamic range of the uricase system is narrow in calibration. Thus, it is required that the sample size of a specimen must be decreased to as small volume as possible to be suitable for sensitivity of the detector and precision of pipette sampling. Assay condition was as follows: 10 microliters of sample was incubated at 37°C with 2.9ml of borate buffer pH 8.5. After 2minutes incubation, 0.1ml of uricase solution was added and decrease of optical density at 293nm was measured by a Hitachi 124 double beam spectrophotometer equipped with a scale expanded recorder.
On the other hand, in case of reaction rate analysis without enzyme system, when concentrations of the reagents were high enough for the substances to be measured, the reaction velocity at zero time could be proportional to the concentration of measured substances.
Reaction rate analysis of picrate formation of creatinine was investigated. Picrate and sodium hydroxide concentration was determined to give a linear time course within setted reaction time (one or two minutes). Creatinine in biological fluids was measured with the following conditions: 0.2ml of serum sample was incubated at 37°C with 2.0ml of picric acid solution (0.13g/dl). After 2 minutes incubation, 0.1ml of 1N-NaOH was added and increase in absorbance at 510nm was measured by a Hitachi 124 spectrophotometer. In this case, the final concentration of picric acid and sodium hydroxide were 4.93mM and 0.044M, respectively.
Under these conditions, the linear calibration curve of creatinine was obtained up to concentration of 20mg/dl. Each serum examined gave the same affinity for picric acid and recovery of creatinine was about 100% in this system. This condition was easily applied to an LKB 8,600 Reaction Rate Analyzer without modification.

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A new method for quantitative determination of polyamines in tissues is developed in comparison with the alkaline butanol method which has been widely employed for extraction of polyamines from tissues.
The procedures consist of isolation of polyamines with a small column of Dowex-50 and their separation on paper electrophoresis followed by colorimetric determination after ninhydrin staining.
Recovery by the present method is constantly higher than 90%, with an average value of 95%, whereas the butanol extraction results in variable recoveries ranging from 70 to 90%. Our method is also superior to the butanol method from the standpoint of purity of the isolated polyamines and simplicity of the procedures.
The column method was applied to quantitative estimation of polyamines in various tissues of rat.

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3-Hydroxykynurenine (3-OHKY) and 3-hydroxyanthranilic acid (3-OHAA) are metabolites of tryptophan which have been described as “endogenous carcinogens”, and the relation between these substances in urine and bladder cancer has been discussed. In this work, the method of determination of 3-OHKY, 3-OHAA, 3-OHAA glucuronide (3-OGAA) and 3-OHAA sulfuric ester (3-OSAA) were devised and urinary excretion of these substances in humans and animals was estimated.
3-OHKY was determined as follows. To 12ml of diluted urine, 16ml of p-tolue-nesulfonylchloride solution in acetone (0.5g in 100ml of acetone) and 4ml of NaHCO₃ aq.(5g in 100ml of water) were added. The mixture was allowed to stand for 30 min and 4ml of (NH₄)₂ CO₃ aq.(10g in 100ml of water) was added. After 30 min, the mixture was acidified with 4m1 of 4N H₂SO₄. The fluorescent product was extracted with 40ml of ether, separated on alumina G thin layer with the lower layer of a mixture of CHCl₃: acetone: EtOH: 1.5% NH₄OH (2:2:1:1) and determined fluorometrically (ex. 360mμ, fl. 465mμ) in a mixture of acetorle: water (1:1).
3-OHAA was extracted with ether at pH 3, Separated on cellulose powder thin layer with a mixture of benzene: EtOH: water (50:10:1) and determined fluorometrically (ex. 348mμ, fl. 410mμ) in a mixture of MeOH: dioxane (1:9).
3-OGAA and 3-OSAA were determined as follows. The urine was desalted by acetone and MeOH and adsorbed on a column of DEAE Sephadex CO₃ (1×20cm). After the column was washed with 125ml of dil.(NH₄)₂CO₃ (1g in 100ml of water), 3-OGAA was eluted with 80ml of conc.(NH₄)₂CO₃ (15g in 100 ml of water), separated on cellulose powder thin layer with a mixture of n-BuOH: n-PrOH: EtOH: water (3:6:4:2) and determined fluorometrically (ex. 340mμ, fl. 420mμ) in MeOH. 3-OSAA was eluted with further 80ml of conc.(NH₄)₂CO₃ from the column, separated on silica gel H thin layer with a mixture of n-BuOH: n-PrOH: EtOH: water (6:3:21) and determined fluorometrically (ex. 340mμ, fl. 420mμ) in MeOH.
The results showed that 3-OHAA in human urine increased in patients with bladder cancer. The principal urinary metabolite of tryptophan determined by these methods was 3-OHAA in humans, 3-OSAA in rats and 3-OGAA in guinea pigs.

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Urinary kynurenine was converted into 2-aminoacetophenone by heating with barium hydroxide and was analyzed in the form of its trifluoroacetyl derivative by gas chromatography. The peak having a retention time equal to that of authentic 2-trifluoroacetylaminoacetophenone was investigated by combined gas chromatography-mass spectrometry. The procedures are shown in the following lines.
Ten ml of saturated barium hydroxide and 1g of barium hy droxide powder were added to 10ml of urine sample, and the mixture was heated in a boiling water bath under ref lux for 20min. After cooling in an ice bath, 1.0ml of chloroform solution containing p-xylene-dichloride as an internal standard was added, and the solution was extracted with twice 5ml of chloroform. The combined chloroform extract was evaporated to dryness under reduced pressure, and a drop of ethylacetate and two drops of trifluoroacetic anhydride were added to the residue, and the reaction solution was injected onto the gas chromatograph.

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Unconjugated chinoform in specimens from SMON patients was extracted with a mixture of benzene and pyridine, and adsorbed on Florisil or alumina. After extraction from the adsorbent, chinoform was acetylated and analyzed by means of gas chromatography equipped with an electron capture detector. About 10μg of chinoform was detected from 1ml of serum of a SMON patient after suspension of the drug for one month.

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The amounts of catecholamines in plasma of patients with essential hypertension and those of normal subjects were determined by gas chromatography in accordance with the large volume injection method.

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The mode of staining reactions of several AMPS to alcian blue and toluidine blue O was observed on cellulose acetate strips as well as in test tubes.
1. Alcian blue could stain 7 AMPS tested in all buffer conditions except that at pH 7. 0, which indicated its significant validity for qualitative and crude separation procedures.
2. Toluidine blue O stained heparin in reddish violet with a strong metachromasia but chondroitin sulfate A, B and C in blue-violet color with a slight metachromatic property, and heparitin monosulfate as well as keratan sulfate in blue color without any metachromasia.
Hyaluronic acid, however, resulted in differnt reactions by different commercial batches of toluidine blue O, that is, one batch did not react with this material, whereas another did positively.

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Permeability of erythrocyte membrane to PCMBS (parachloromercuriphenyl sulfonic acid), PCMB (parachloromercuribenzoic acid) and chlormerodrin (a mercurial diuretic) was investigated by using radioactive ²⁰³Hg compounds. Among these three mercurials, the rate of uptake was in the order of PCMBS<chlormerodrin<PCMB.
SITS (4-acetamido 4'-isothiocyanostilbene-2, 2'-disulfonic acid) which modifies amino groups in erythrocytes membrane inhibited PCMBS uptake, while permeability of PCMB and chlormerodrin was not affected. The effect of SITS on PCMBS permeability was decreased in a lower temperature. The activation energy of PCMBS uptake was 21.1kcal/mole, and at least two kinds of activation energy (22.7 and 10.2kcal/mole) were determined in the presence of SITS.
Superficial binding of PCMBS to red cell membrane was markedly eliminated by PCMB, while the rate of uptake of PCMBS was not affected. The PCMB uptake was accelerated at a lower pH. The optimum pH for uptake of PCMBS was shown to be a 7, which was lowered in the presence of SITS.
On the basis of these results, at least two pathways can be differenciated. One is the aqueous pathway for anionic substances such as PCMBS, which is inhibited by SITS and related to K⁺ permeability. The other is in lipid nature and provides rapid penetration for non-ionic reagents.

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The relation between the optical properties (absorption and circular dichroic (CD) spectra) and function of hemoglobin was investigated on abnormal hemoglobins found mainly in Japan. Hb Tokuyama and Agenogi, with no altered functions, show the same optical properties as those of Hb A. Hb Tochigi and Hb Hiroshima, though accompanied by clinical symptoms, have almost similar spectrophotometrical as well as functional behaviors to those of Hb A. In contrast to these abnormal hemoglobins, Hb Rainier and Ube 1, which have lost most of the cooperativity, show absorption and CD spectra distinctly different from those of Hb A.

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As a series of our studies on estrogen biosynthesis, stereochemistry of hydrogen loss from C-2 during placental and microbial aromatization of 19-norsteroid has been investigated. The substrates required for this purpose, epimeric (2-³H) estr-4-ene-3, 17-diones, were prepared along the way which had previously been developed in this laboratory. These tritiated compounds were mixed with an appropriate amount of (4-¹⁴C)-estr-4-ene-3, 17-dione and recrystallized repeatedly up to a constant isotope ratio.
The labeled substrates were incubated with human placental preparation according to the method of Ryan. The incubation mixture consisted of the steroid substrate, placental 10,000×g supernatant and NADPH-generating system suspended in 0.05M phosphate buffer, pH 7.0. After incubation at 37° for 1 hr, the mixture was extracted with ethyl acetate and the extract was submitted to thin-layer chromatography (TLC) on silica gel. The products and unchanged substrate were separated, diluted with the carrier and then recrystallized up to constant specific activity, respectively. Estrone formed from the substrate with the label in the 2 β-position exhibited a 79% loss of tritium. In contrast, the 2 α-labeled substrate lost only 20% isotope. These data unambiguously tell us that hydrogen loss from C-2 in the aromatization process is stereoselectively β and in consequence C-1, 2 hydrogen elimination is 1β, 2 β-cis. The 1 β-hydroxylated products obtained from both incubations showed almost the same tritium content with a slight decrease.
The double-isotope labeled substrates were also incubated with respiring cultures of Bacillus sphaericus for 48 hr. The incubation mixture was extracted with ethyl acetate and the extract was similarly purified by TLC. The desired estrone was eseparated, diluted with the carrier and then recrystallized until a constant isotope ratio has been attained. The aromatized product from the 2 β-tritiated substrate lost 80% of the label, while 2 α-epimer showed only an 11% loss of tritium. Elimination of hydrogen from C-2 is thus stereospecifically β; in other words loss of hydrogen at C-1, 2 is 1α, 2 β-trans. Stereochemistry of Δ¹ dehydrogenation of 19-norsteroid is then the same as that of C19 steroid.

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A case of lactate dehydrogenase (LDH) anomaly is reported. The patient is a 43-year-old female with mild subarachnoid hemorrhage. There are no abnormal findings on liver and heart functions. Her serum LDH activity has elevated persistently (1,050-1,100 Wroblewski units). Celloseacetate electrophoresis showed that LDH₂ and LDH₃ were bridging and that 86% of the total activity was found in LDH_2-3. On gel filtration through a Sephadex G-200 column, the activity was recovered in two fractions, macromolecular LDH eluted from the column somewhat after “M” fraction and normal LDH eluted after “G” fraction. LDH activity was stained overlappingly on a part of IgA precipitin lines on agarose gel immunoelectrophoresis using antisera against human whole sera and IgA globulin followed by specific stainning for LDH activity. LDH zymogram was not altered by mercaptethanol reduction and LDH-IgA complex dissociated by acidification (pH 3.4), therefore the linkage is a hydrogen bond rather than a S-S bond. The patient's serum IgA, dissociated from LDH by acidification, reacted with normal LDH_1-5 to make macromolecular LDH and reacted also similarly with dog and rabbit LDH.
It might therefore be assumed that the patient's own LDH was normal and that the abnormal LDH pattern was due to the presence of an abnormal IgA.

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Genome of Oncorna virus provides the information on carcinogenesis of an infected cell. This is evident from the fact that a different morphology of the focus is observed when a different strain (type) of a virus group would infect the same kind of host cells.
Physiological conditions of the cells, such as cell cycle, macromolecular syntheses, cell crowdiness, and media also exercise an influence upon fixation and development of the viral genome.

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Measurements of plasma renin activity (PRA) and aldosterone secretion are indispensable for preoperative diagnosis of primary aldosteronism.
Introduction of PRA assay by means of radioimmunoassay has made the PRA assay clinically available at present. However, the measurement of aldosterone secretion is still only for the investigational use account of complexities of the assay procedures.
Herein reported are our experiences on 13 cases of primary aldosteronism, in which diagnosis was based on measurements of PRA and plasma aldosterone concentration (PAC) by radioimmunoassay.
PRA is defined as angiotensin I generation by 1ml of plasma incubated at 37° for a unit hour under the condition in which plasma angiotensinases and converting enzyme are properly inhibited. The resultant angiotensin I is measured by radioimmunoassay using. a specific antibody against angiotensin I. PAC is measured by radioimmunoassay after aldosterone is extracted from the plasma with dichloromethane and partially purified by paper chromatography in Bush B₅ system.
PRA was suppressed even after 3 days of salt restriction, followed by 80mg Furosemide per os administration, while PAC was always elevated on ad lib salt intake in all the 13 cases of primary aldosteronism. In 4 cases, PAC was measured on venous plasma obtained during adrenal venography. The concentration of aldosterone was approximately 10 to 20 times higher on the ipsilateral side of adenoma.
PAC is conceivably modified by the secretory rate of aldosterone as well as metabolic clearance thereof. Nevertheless, we believe the measurement of PAC by radioimmunoassay together with PRA is useful in clinical diagnosis of primary aldosteronism, provided PAC is clearly elevated on ad lib salt intake and that PRA is suppressed on salt restriction. The measurement of PAC is also useful in deciding the laterality of the adenoma.

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Inhibitory effects of exogenous polyunsaturated fatty acids were investigated on the enzymes, which catalyze fatty acid synthesis, in vivo and in vitro.
In response to refeeding of a high-carbohydrate, fat-free diet, a striking adaptive rise in the activities of lipogenic enzymes of rat liver was associated with concomitant deficiency in essential fatty acids in both phospholipid and FFA fractions of the liver lipids. Oral administration of methylesters of polyunsaturated fatty acids recovered the supranormal activities of lipogenic enzymes toward the normal. The degree of fall in enzyme activity was a function of the dose of polyunsaturated fatty acids administered and the length of experimental periods. Moreover, dampening of the enzyme activity was closely related to exogenous input of polyunsaturated fatty acids in the liver.
Fatty acid composition of liver FFA was disclosed to be distinct from that of plasma FFA in starved rats. Total FFA concentration in the whole liver was assumed to be approximately 6×10^-4M. Most of FFA (84 %), however, was bouud to liver cell particulates.
Examination of lipogenic enzymes in vitro revealed that free fatty acids inhibited enzyme activity and that polyunsaturated fatty acids were more potent than saturated or monounsaturated fatty acids with the exception of ω 7 monoenic acids.
In vitro concentration of arachidonic acid required for 50% inhibition of acetyl CoA carboxylase activity was found to be 7×10^-5M, which was much higher than the maximum concentration of free arachidonic acid in liver supernatant. Furthermore, in vitro inhibitions of both fatty acid synthetase and malic enzyme activity by polyunsaturated fatty acids were less evident.
These results strongly suggest that exogenous polyunsaturated fatty acids may dampen the activity of the lipogenic enzyme set in its synthetic site as alsoreported by Majerus and Kilburn (9).

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Effects of detergents, especially sodium dodecyl sulfate (SDS) and sodium taurocholate (NaTC), on some lipases were studied.
The lipase preparations used were hog pancreas lyophilized powder (PL), pseudomonas lipase (PsL) and candida cylindracea. lipase (CL).
Lipolytic activity was determined by estimation of liberated fatty acids, using tributyrin as a substrate, by pH-stat titration with 0.005N sodium hydroxide at 25°.
The pH optima were 8.0 to PL and 7.5 to PsL and CL. Inhibitions of lipolytic activities were observed at all concentrations used of SDS and NaTC, and shifting of pH optimum was not seen for all combinations except for PsL-NaTC. Inhibition by SDS or NaTC was smaller in case of CL than in others, and they were almost the same in cases of PL and PsL.
To investigate in detail inhibition of lipolytic activity, kinetic studies for the amounts of substrate were performed. With Lineweaver-Burk's plots, the types of inhibition by SDS or NaTC were miscellaneous, especially NaTC acted on PL as a noncompetitive inhibitor, on PsL as an un-competitive one, and on CL as mixed one. In case of CL, SDS inhibited lipolytic activity un-competitively in concentrations below 300μM, and competitively in concentrations higher than 400μM.
Interesting results were obtained by using coexistence of SDS with NaTC. With regard to lipolysis catalyzed by the three lipases, the inhibitory rate b coexistence with these two detergents was smaller than those by each detergent used separately. NaTC was unable to reverse the inhibition to the rate in the complete system in the presence of SDS on lipolysis. On the contrary, the reverse by SDS was complete in the presence of NaTC.

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Comparison of the substrates at the time of determination of plasma LPL activities revealed that there is a slight difference in property between Ediol and Intralipid as shown in TABLE I.
Furthermore, it was found the comparison of plasma LPL activities determined by both of the substrates that a favorable correlation can hardly be obtained and rather a tendency of dissociation is observed as shown in Fig. 3.
It is therefore necessary to realize at this moment that the plasma LPL activity varies according to the substrates used.

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1) Phospholipase A2 from aged frozen bovine pancreas was isolated and purified up to 299 folds by means of ion exchange Sephadex column chromatographr.
2) Bovine pancreatic phospholipase required special treatments to render the enzyme fully soluble in water.
3) Bovine phospholipase A2 was inhibited by EDTA and was made dependent on Ca⁺⁺ ion. The optimum pH was around 8, and the optimum temperature showed a broad range between 25°-50°. However, pretreatment with an acidic medium at pH 3 at 90° for 5 min led to a high recovery of phospholipase A₂ activity.
4) Leucine aminopeptidase containing a fraction obtained by ammonium sulfate fractionation appeared to inhibit the phospholipase activity thereafter.
5) The phospholipase A activity which appeared upon treatment with trypsin was likely to be prephospholipase.

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Unlike monogastric animals, ruminants derive most of their energy from shortchain fatty acids, mainly acetate, propionate and butyrate, which are the major end products of ruminal microbial fermentation of dietery carbohydrates in the rumen and which are absorbed from it. Therefore, the pattern of ruminant nutrition and intermediary metabolism differs in many respects from that of ordinary monogastric animals.
It was reported that infusion of several short-chain fatty acids stimulates insulin secretion in sheep and cow, but not in monogastric animals such as rats, rabbits and pigs, and that infusion of glucose was much less effective than that of short-chain fatty acids on plasma insulin secretion in sheep. It is generally accepted that propionic acid has a glycogenic function, whereas acetic acid and butyric acid have a ketogenic function in ruminants. However, it was reported on the other hand that intravenous injection of butyrate or propionate produces similar hyperglycemia, and butyrate gives rise to a greater release of insulin than propionate.
The present experiment was conducted to investigate the effect of infusion of several short-chain fatty acids on plasma insulin and glucose levels, and also to find a clue on the mechanism of insulin secretion in ruminants.
Crossbred ewes, weighing 40 to 50 kg, were used in the experiment. A saline solution of glucose, lactic, acetic, propionic, butyric, valeric, capronic and caprylic acids, respectively, neutralized up to pH 7.0 with sodium hydroxide in a final concentration of 2.5 M, were separately given intravenously within 2 minutes in a dose of 1.25 m moles per kg.
Blood glucose concentrations were not affected significantly after the injection of lactate, acetate or saline. After the injection of propionate, butyrate or caprylate, blood glucose concentration rose from approximately 40mg/dl to 70 mg/dl in all the cases within 20 minutes. Iso-valerate or capronate had a more potent effect on the hyperglycemic response than other short-chain fatty acids the highest glucose concentration was 90 to 95 mg/dl which had been attained within 15 minutes after the injections.
Plasma insulin level changed in a similar pattern to that of blood glucose concentration after administration of each substance. An injection of equimolar quantities of glucose produced a significantly smaller elevation in plasma insulin despite blood glucose load several times larger than seen after the infusion of the short-chain fatty acids. An insulin response by the injection of short-chain fatty acids was particularly striking in the case of iso-valerate or capronate.
As a result, it is indicated that the increase in plasma insulin level was associated with hyperglycemic responses by the short-chain fatty acids and that both responses increased with increasing chain length of the short-chain fatty acids.
Although the mechanism of the insulinogenic effect caused by administration of short-chain fatty acids remains unsolved, it is of significance on ruminant nutrition that short-chain fatty acids which represent the major energy source for ruminants are much more effective on insulin release rather than glucose.

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Liver, kidney, muscle and adipose tissue appear to be the major site of insulin degradation. Mirsky and Broh-Kahn (1949) investigated the insulin inactivating capacity of various tissues and postulated that insulinase might be responsible for insulin degradation. Recently, Brush, Cheng and co-workers have indicated that a soluble enzyme extracted from rat skeletal muscle rapidly degrade insulin and is of a remarkable degree of specificity for the insulin molecule.
We have carried out further experiments in order to verify the new enzyme extracted from rabbit, rat and pig muscles.
In order to facilitate the procedure of enzyme purification. The Brush's method was modified. Namely the rear leg muscles of rat, rabbit and pig were removed, minced, and then homogenized at a high speed in 0.35 M sucrose (5 ml/gm tissue). The homogenate was prepared by filteraid filtration at 4° and the supernatant was dialysed three times for at least 4 hours, each time aganist 20 volumes of distilled deionated water. After adjusting with acetic anhydride the dialysed supernatant to pH 5, a sufficient amount of 0.015 M acetate buffer, pH 5, was added to bring it up to 6.5 L. Ca₃ (PO₄)₂ gel in a sufficient amount was added to obtain a ratio of gel to protein of 0.28: 1. The gel-protein mixture was stirred at 4° for 20 minutes, followed by centrifugation at 10,000×g for 10 minutes. The supernatant was discarded and the gel resuspended and stirred for 20 minutes in 0.05M phosphate buffer, pH 6.2. Following sedimetnation of the gel by centrifugation, it was resuspended and stirred for 20 minutes in 0.05 M phosphate buffer, pH 7.5. Removal of the gel was accomplished by centrifugation, and the supernatant containing the enzyme was rapidly frozen at-70° for eventual use. Furthermore, the supernatant, from which the enzyme had been extracted, was washed by 0.05M phosphate buffer, pH 7.5, 4 times. The final protein content was obtained about 1, 000 mg from 1.5kg of muscle. Enzyme assay was carried out by the modified Mirsky's method. And also the rates of degradation were assayed for remaining immunoreactive insulin according to the method of Morgan and Lazarow.
A marked activity was found in each of pig and rat muscle, i.e. specific activity of 2.29-2.56μU/mg of the enzyme protein. The enzyme from rabbit muscle, however, showed no remarkable activity. The maximum activity was obtained at pH 8.0-8.6, declining on each side of this optimum range. The activity of the enzyme was blocked by N-ethylmaleimide, DFP and boiling treatment. The inhibitors of trypsin and chymotrypsin, however, did not impair the enzymatic activity. Furthermore, the rate of degradation of proinsulin attacked by the enzyme was from 0 to 18%, while that of insulin was 100%. It was also found to be a competitive inhibitor of insulin degradation. Km for insulin degradation, as indicated, was 1.23μM, and K₁ for proinsulin was 2.06μM. These results suggest that the enzyme is a sulfhydryl dependent protease which is of quite similar characteristics to Brush's one and has a species specificity for insulin degradation. Through this purification process, it appears that the enzyme-inhibitor most probably exists.

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With regard to pathogenesis of diabetic angiopathy, hexosamine has roused a particular interest from the fact that it is an important component of glycoproteins and mucopolysaccharides which play a role in vascular lesions. Recently we found that the level of bound hexosamine in urine in untreated diabetic patients is decreased remarkably when blood sugar is controlled by insulin, which indicates that urinary excretion of bound hexosamine is well correlated with severity of diabetes. An attempt was made in the present study to clarify the carbohydrate metabolism, insulin secretion and retinal findings in hexosamine-treated rats in comparison with streptozotocin-induced diabetic rats.
1) Male Splague-Dawley rats were individually maintained in metabolic cages on chow and water ad libitum. They were made to fast overnight and injected intravenously with 65mg/kg body weight of streptozotocin. Twenty-four hour urine specimens were collected in 0.1 % sodium azide. Urinary volume and sugar increased rapidly as severe diabetes was established. Twenty-four hour urinary bound hexosamine also increased markedly. There was a slight but significant rise in liver hexosamine content in the diabetic rats, 2 months after the injection.
2) Glucosamine (250mg/kg body weight) was injected intraperitoneally daily to normal rats for 2 months. There was a significant elevation of urinary bound hexosamine in the glucosamine treated rats, though the rate of increase was less than in diabetic rats. Oral glucose tolerance tests were performed with gastric tubing. No significant differences were observed in serum glucose levels between the normal and treated rats, except at the 3rd hour when glucose was still somewhat elevated in the latters.
There was no insulin response to administration of glucose in the glucosamine treated rats, although there was no significant change at the fasting level of insulin between the two groups. No significant decrease in glucokinase activity was observed in the treated rats in comparison with a marked decrease in diabetic rats. G 6 Pase activity increased significantly in both diabetic rats and glucosamine treated ones, though the amount of the increase was much smaller in the latter group.
3) No remarkable histological changes appeared in the liver and kidney in both diabetic rats and glucosamine treated ones compaired to normal ones. The latter showed as significant increase in urinary protein excretion as the diabetic rats. No remarkable diabetic changes such as microaneurysm were detected in the retinal capillaries of either the diabetic rats or the glucosamine treated ones. It was observed that the ratio of endothelial cells to the mural cells of the retinal capillaries increased. These data suggest that glucosamine is at least partially responsible for microangiopathy.
4) Liver hexosamine content and excretion of urinary bound hexosamine increased in the diabetic rats, but no significant changes were observed in the glucosamine synthetase activity of liver which is the main site of glucosamine biosynthesis. A prolonged administration of glucosamine increased tissue and urinary bound hexosamine as in the diabetic state. 10 μpCi of U^-14C-glucose and 15μCi of U^-14C-pyruvate were injected into normal and diabetic rats. Incorporation of ¹⁴C-glucose into urinary bound glucosamine decreased in the diabetic rats as compared with the normal in the first 24 hours urine specimen, but when dilution with high blood sugar in the diabetic rats was taken into consideration, the incorporation was not impaired but increased. Incorporation of U-¹⁴C-pyruvate into urinary glucosamine inclined to increase in the diabetic rats. Therefore, the increase in hexosamine in the diabetic specimen is most likely due to increased biosynthesis of hexosamine, from glucose and through gluconeogenesis.

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When sera from several mammals put on electrophoresis in polyacrylamide gel containing urea and EDTA, albumin fractions were separated into several bands. Use of purified bovine serum albumin confirmed that the fastest migrating band (Band 1) represented fatty acids-bound albumin and the secondary migrating albumin band (Band 2) coresponds to fat-free albumin. The natures of other bands have remained- obscure so far.
In case of mouse sera, Band 1 appeared only in the fasting phase and disappe- ared at the feeding phase. Bands 3-5 which had not been observed in the fasting phase appeared in 14 minutes after the onset of food uptake and disappeared in 2 hours after food intake. Bands 6-7 appeared only in the last stage of food intake, i. e., in 2-3 hours after meal. Sera from hereditary obese hyperglycemic mice (C57 BL/6J-ob and C57 BL/Ks J-db), however, showed abnormal patterns of serum albumin sub-band, e. g., Bands 3-7 were observed instead of Band 1 in the fasting phase of the ob mice, and Bands 3-5 were not observed even in the feeding phase in the db mice.
Human sera collected at different times after morning meal from a normal male showed a pattern similar to that of the normal mice with regard to albumin sub- band appearance. But the satisfaction (feeding) phase in which Bands 3-5 had appeared continued for more than 11 hours whereas it appeared only for 1 hour in normal mouse sera.
Human sera collected in 60 minutes after oral administration of 50g glucose from diabetic patients failed to show Bands 3-7 which resembled that of the db mice

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Calcitonin, a calcium hormone which is considered to lower serum calcium and phosphate due to its inhibitory action on bone resorption, has also been shown by our hands to develop an action on the kidney that leads to alterations in efficiency of uriary excretion of calcium. Therefore, this hormone is considered to affect calcium fluxes at the plasma membrane of tubular cells either by changing its permeability or by changing the function of the Ca⁺⁺ pump. In either case, alteration in the Ca++ concentration should take place in these cells. And, if Ca++ in the cell exerts a strong influence upon the regulation of metabolic processes, calcitonin is expected to develop considerable metabolic effects.
In an attempt to make these points clear, we examined, in the rat kidney in situ, the effects of salmon calcitonin on the oxidation-reduction state of pyridine nucleotide and on the energy charge of adenine nucleotide. The effects were then compared with those of reagents that alter the Ca⁺⁺ levels in these cells.
Male rats of Sprague-Dawley strain were used after an overnight fast and ad lib intake of water. The oxidation-reduction state of pyridine nucleotide was directly and continuously recorded with an organ-microfluorometer. By this means, metabolic responses of superficial cells (proximal and distal convoluted tubulus) in the kidney in situ were detected. Energy charge was calculated from the concentration of adenine nucleotides (AMP, ADP and ATP) as determined by an enzymatic method. Calcitonin, extracted from salmon ultimobranchial body by the method of Copp, was used. All reagents were administered intravenously.
Calcitonin injection resulted in an oxidative shift of pyridine nucleotide which commenced immediately and lasted for a considerable period of time. The response showed a dose-response relationship in the dose range of 0.01 to 2 MRC units. This oxidative response was accompanied by a significant increase in the energy charge. These effects were reproduced by intravenous EGTA which lowers Ca⁺⁺ both in the extracellular fluid (ECF) and in the intracellular fluid (ICF), but not by parathyroidhormone which, in its early phase of action, lowers Ca⁺⁺ in the ECF and elevates Ca⁺⁺ in the ICF, nor by intravenous CaCl₂ that elevates Ca⁺⁺ in both compartments. It is suggested therefore that the calcitonin effects reflects a metabolic consequence of the f a11 of Ca⁺⁺ in the ICF.
The rats pretreated with the maximum doses of calcitonin (more than 2 MRC units) did not exhibit the oxidative response to pentachlorophenol, an uncoupler which selectively oxidizes mitochondrial pyridine nucleotide. They developed an oxidative response, however, to pyruvate which oxidizes NADH in the cytosol compartment. In parathyroidectmized rats, though calcitonin failed to produce the oxid-ative shift, the response became demonstrable by a prior administration of succinate which is supposed to reduce mitochondrial NAD.
These results led us to conclude that the fraction of pyridine nucleotide which responds to calcitonin consists mainly of NADH of mitochondrial compartment.
In mitochondria, because of “monopoly of the succinate route”, succinoxidase activity must be suppressed when NADH is actively oxidized. Therefore, we propose such sequence of events as that the calcitonin-induced fall of Ca⁺⁺ in cytosol of tubular cells causes suppression of succinate dehydrogenase (and therefore succinoxidase), which leads to an increase in NADH oxidation with the resultant augmentation of ATP synthesis.

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In a preceding report, we described a series of experimental evidences indicating that an intravenous administration of calcitonin to rats produces in the kidney in situ an oxidation shift of the oxidation-reduction state of pyridine nucleotide and a rise in the energy charge of adenine nucleotides on the one hand, and a fall in Ca⁺⁺ in the cells (most likely in cytosol) on the other. The former events are suggested to represent mitochondrial reactions that occur as a consequence of the latter hormone effect. Thus, a mechanism is expected to exist by which the regula- tion by cytosol Ca⁺⁺ of mitochondrial energy metabolism comes into effect. We propose that the mechanism involves the succinoxidase system of the inner mitochondrial membrane, and the data in favor of these contentions are presented.
1. The activity in isolated renal tubules of succinate oxidation was very low, in sharp contrast to the very high activity in isolated mitochondria prepared from the tubules. Because a sufficient succinate penetration into the cell appears to take place, the succinoxidase system is suggested to be functionally in a controlled state in in situ mitochondria.
2. A mitochondrial preparation, which was devoid of calcium contarriination, and rendered to have a restricted activity of succinate oxidation by a previous treatment with AMP and DNP, exhibited a definite requirement for both ATP and Ca⁺⁺ in its full activation. Succinate dehydrogenase was the step that limited the overall reaction.
3. In the presence of Ca++, ATP at 0.2mM caused full activation. With regard to Ca⁺⁺ in the presence of ATP, the activation curve exhibited a wide dose-response relationship, in which 0.5-0.7μmoles Ca⁺⁺ per mg mitochondrial protein caused a half of the maximal activation. Because the Ca⁺⁺ effect was demonstrable at a suitable concentration (-10^-6M), Ca++rather than ATP is the candidate for the possible physiological regulator of this reaction.
4. The Ca⁺⁺ effect required the integrity of the inner membrane and was preven-ted not by La⁺⁺⁺ or Pr⁺⁺⁺, but by ruthenium red or high concentrations of KCl. Thus the low affinity sites for Ca⁺⁺ binding on the inner membrane are suggested as the locus which detects any changes in cytosol Ca⁺⁺, and leads to activation or deactivation of succinate dehydrogenase. This may regulate mitochondrial energy metabolism by switching off or on the mitochondrial oxidation of DPNH.
5. Similar reactions were demonstrated in mitochondria isolated either from kidney, liver, or from brain. Thus they appear to be a universal mechanism by which alterations in cytosol Ca⁺⁺ is reflected by changes in mitochondrial metabolism.

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We previously investigated the mechanism of ACTH action in fat mobilization. These investigations appeared to indicate that this hormone stimulated calcium ion uptake into adipose tissue, and the incorporated calcium ion stimulated lypolysis in the tissue through an increase in formation of lipase-fat (enzyme-substrate) complex.
On the other hand, recent investigations on adrenal cortex, another target organ of ACTH, have revealed that the action of ACTH in vitro requires calcium ion in the medium, ACTH stimulates radioactive calcium ion uptake into this organ, and calcium ion stimulates steroidogenesis in adrenal homogenate.
Based on these results, we suggested that ACTH increased calcium ion uptake into this organ, and the incorporated calcium ion stimulated steroidogenesis. In the present investigation, we tried to clarify the mechanism of the stimulatory effect of calcium ion on steroidogenesis. It was demonstrated that sonication of adrenal homogenate did not affect the stimulatory effect of calcium ion on steroidogenesis. It was also clarified that calcium ion stimulated the reaction of 21-hydroxylation as well as 11-hydroxylation.
Stimulation of steroidogenesis was also observed with other metal ions such as alkaline earth metal ions (magnesium, barium, and strontium ions), but not with such ions as transition metal ions (manganese, cobalt, nickel, zinc ions).

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The influence of glucocorticoid administration or increased serum corticoid concentration on TRF-induced TSH release was studied in patients receiving glucocorticoids and in patients with Cushing's syndrome. In addition, the effect of gluco-corticoid on TRF-induced TSH release was investigated in vivo in dexamethasone treated rats. The TRF-induced TSH release was inhibited in patients who had received glucocorticoids for long periods or in high doses. These patients had received more than 60 mEq of cortisol per day for more than six months. Definite plasma TSH increases by TRF were observed in patients receiving short term low doses or intermittent low doses administration of glucocorticoid. Little or no rise in plasma TSH occurred following TRF administration to patients with Cushing's syndrome. TSH response of TRF in rats receiving only one dose of dexamethasone (10 ug/100g B. W.) was (394. 8±15. 5 %) greater than control (162. 7±15 %). Successive administra-tion of dexamethasone for 15 days inhibited the TSH response (159. 3±14. 0 %) to the control level. It is possible to conclude from these observations that the mechanism of the glucocorticoid suppressive action on TSH secretion after shortterm, low doses of glucocorticoid administration may be an impaired secretion of endoge-nous TRF, which results in a supernormal TSH response induced by exogenous TRF. With longterm and high doses of glucocorticoid therapy, TSH secretion appears to be inhibited not only at the suprahypophyseal level but also at the pituitary level.

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