Information has been obtained concerning the extracellular control of energy metabolism of liver by changing portal blood supply.
For this purpose, male adult rabbits were used for the following three different surgical treatments: (1) ligation of left portal branch (60% segment),(2) portacaval shunt (side to side anastomosis) and (3) combination of portal branch ligation with portacaval shunt.
(1) In the ligated lobe with portal blood supply only from the hepatic artery, it was seen that both activities of energy generation and utilization markedly decreased coordinately, in spite of a higher level of energy charge being shown, which is considered to arise from the result that ATP utilization was more depressed than generation.
(2) Reduction of portal blood flow via portal vein following anastomosis resulted in light atrophy of the whole liver, in a similar way to the energy balance shown in the ligated lobe.
(3) However, an increased portal blood supply via hepatic artery into the ligated lobe inhibited the atrophic alteration of the ligated lobe brought about in the first type of operation.

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In conventional in vitro systems, in which the main constituents of reaction media are composed of polyols (mannitol and sucrose) or chloride salt of monovalent cations (KCl and NaCl), the energy conservation system of isolated rat-liver mitochondria responded to added ADP with an extraordinary high sensitivity. Values as low as 10-20μM were regularly obtained as the apparent Km value for ADP, irrespective of whether the mitochondrial response was measured by the activity of oxygen consumption or by the oxidative shift of the oxidation-reduction state of endogenous pyridine nucleotides. The ADP concentration in the liver cell, as estimated by various methods, ranges between 150 and 1,500μ moles per kg wet weight, thus, far exceeding the Km value of mitochondria in the in vitro systems. Two alternatives seem to be possible as the explanation for these facts: the first is that the changes in the ADP concentration is not a physiological determinant in the oxidative phosphorylation activity of mitochondria in situ, and the second is that the Km value for ADP is much higher in mitochondria in situ, thus allowing the ADP concentration still to function as the most important factor in regulation of cell respiration. Direct measurements of the oxidation-reduction state of pyridine nucleotides in the liver in situ by means of in situ organ microfluorometry disclosed a distinctive oxidative response to 10μmoles of dicoumarol administered via the portal vein. Because this uncoupler did not induce the oxidative response in other cell components than mitochondria, it is inferred that the in situ mitochondria, are not in the functional state of “state 3 (as defined by Chance and Williams)”, but in a state between states 3 and 4. This is in keeping with the contention that in situ mitochondria can be reversibly activated by raising the effective concentration of ADP in the cell.
We therefore examined the possibility that the Km value for ADP is much higher in mitochondria in situ than that in mitochondria incubated in conventional systems. This concept was supported by several lines of evidence. In the first place, mitochondria, when combined with the cytosol fraction, exhibited considerably elevated Km values for ADP. Many of the (poly) anionic constituents of the cytosol fraction, such as albumin, ATP and glutathione, mimicked the cytosol effect in that they raised the apparent Km value for ADP. The mode of action of these polyanionic large molecules on the mitochondrial function was analyzed by employing dextran sulfate as an experimental model reagent. The results of such experiments indicate that polyanions which are not permeant to the inner mitochondrial membrane react with the protonated cationic charges on the membrane by the electrostatic force and lead to a competitive inhibition of the activity of adenine nucleotides translocase.
From the foregoing discussions and experimental evidence, it is concluded that (1) ADP can still function as a major regulatory stimulant of the energy conservation reaction in mitochondria in situ,(2)(poly) anionic compounds present in the cytosol component, after an electrostatic interaction with positive charges on the membrane, may competitively inhibit the activity of adenine nucleotides translocase, and this can be one of the mechanisms by which the apparent Km value for ADP is elevated in mitochondria in situ, and (3) thus, the activity of electron transport system can be stimulated either by raising the ADP concentration or by modulating reversible inhibition of the activity of adenine nucleotides translocase.

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According to Verkade1), the role of ω-oxidation was to make bilateral β-oxidation possible, which was more efficient than unilateral β-oxidation discovered by Knoop. We found that ω-oxidation was enhanced in liver preparations from starved and diabetic rats2). It is known that in starved or diabetic rats, utilization of glucose decreases along with increased utilization of fatty acids and, subsequently, formation of ketone bodies is increased in the liver. On β-oxidation, the last four carbon atoms of fatty acids become acetoacetyl-CoA, while those of dicarboxylic acids become succinyl-CoA. The latter is a member of TCA cycle and can be used for oxidation of acetyl-CoA and for gluconeogenesis in the liver of starved or diabetic rats.
So we examined the effect of dicarboxylic acid administration on ketone body concentration in the blood of these rats. As shown in TABLE I, administration of stearic acid, palmitic acid or capric acid to starved rats did not decrease the concentration of ketone bodies in blood, while administration of 1, 18-octadecadioic acid, 1, 16-hexadecadioic acid or sebacic acid did. In addition, we found that 14C-incorporation from dicarboxylic acid to glucose in blood was much more remarkable than that from monocarboxylic acid to glucose(TABLE II). These results suggest that the physiological significance of ω-oxidation may be to produce succinyl-CoA from fatty acids in mitochondria. From the experimental results shown in TABLE II, it was calculated that about 16 % of palmitic acids injected to starved or diabetic rats were subjected to ω-oxidation and further metabolized by β-oxidation.

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It is widely accepted that in mammals, liver and kidney play main roles in gluconeogenesis. We have already revealed that one of key gluconeogenic enzymes, phosphoenolpyruvate carboxykinase (PEPCK) activities in liver and kidney showed a diurnal variation, and the variation in liver inversely related to that in kidney, in other words the enzyme in liver showed the highest activity in the afternoon and the lowest one in the morning, and that in kidney showed the lowest activity in the afternoon and the highest one in the morning. These suggested that the inverse relationship of the diurnal variations of PEPCK activity in liver to those in kidney might indicate a cooperation of these two organs so as to maintain blood glucose level in a narrow range. The animals used in the above experiments were fed ad libitum. Under these conditions, glucagon increased this enzyme activity in liver and decreased in kidney, and either insulin or carbachol decreased this enzyme activity in liver and increased in kidney1).
In this experiment, we want to clarify the correlation between liver and kidney in the activity of PEPCK especially from the viewpoint of homeostasis.
Male Wistar strain rats, weighing 110-130g initially, were fed on 25% casein diet ad libitum or only in the daytime for more than one week, then sacrificed. The activity of PEPCK was assayed by the method of Seubert and Huth2).
The diurnal variation of the enzyme in the liver of the rats that were used to be fed only in the daytime for more than one week, was reversed to that of the ad libitum fed rats. And that in kidney of these former rats tended to be reversed to that of the ad libitum fed rats. These findings suggest that these diurnal variations may associated with their feeding behavior. Moreover, according to this feeding schedule, either epinephrine or glucagon increased this enzyme activity in liver and decreased that in kidney. We have reported the induction of this enzyme in liver of rat by l-thyroxine3). Thus, we injected l-thyroxine (50μg/100g body weight/day) for three days to thyroidectomized rats which were fed ad libitum. This treatment increased the enzyme activity in liver and decreased in kidney.
Thus far the rats, in these experiments maintained their rhythms in the body. But we want to state another case in which the animals' rhythms were disturbed.
Recently, we also revealed that in the early stage of cold-exposure rats which fed ad libitum, their diurnal variation of this enzyme activity in liver was blocked4). So we examined the effect of cold-exposure on the enzyme activities of the rats fed ad libitum. This time, these enzyme activities in both the organs increased correspondingly on cold-exposure. This suggests that these two organs may respond to the requirement for gluconeogenesis in a cold environment.
While administration of such a great deal of 3, 3', 5-triiodothyronine as 100μg per 100g body weight daily to intact rats for 6 days, resulted in an increase in the enzyme activities in both the organs unlike the case of the above thyroxine treatment. These findings lead to the following suggestion. Liver and kidney in rats may respond inversely to stresses not disturbing their rhythms in the body. Thus, the enzyme activity may show diurnal variations in these organs keeping their inversibility in order to maintain their blood glucose levels. On the other hand, to large or urgent stresses bloking their rhythms in the body, liver and kidney may respond correspondingly by other mechanisms.
Accordingly, we must take these facts into consideration, that there are diurnal variations and inverse relationship in PEPCK activity in liver to that in kidney, and those diurnal variations are blocked by large or urgent stresses.

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To elucidate the possible role of acetylglutamate, known as the natural activator of ammonia-dependent carbamyl phosphate synthetase, in regulation of urea biosynthesis, studies were made on physiological factors affecting the hepatic acetylglutamate level as well as on the relationship between the level and the ureogenic capacity of mouse liver.
Examination of diurnal variation of the acetylglutamate level showed a sharp peak immediately after dietary feeding. During the other period of the day, the level remained low and relatively constant. It was found that the variation in the acetylglutamate level after meal is greatly affected by protein content of a given diet: the value changed from the minimum of 10 n moles/g liver to the maximum of over 200 n moles/g liver by varying the dietary casein content from 0 to 60 %, as far as examined.
The ureogenic capacity of the liver, as studied in vitro by using the tissue slices with NH4Cl as the substrate, also exhibited a marked variation after dietary feeding and was shown to have a direct and positive correlation with the acetylglutamate level in the original tissue.
These results provide the first unequivocal evidence that acetylglutamate functions as a limiting factor for the activity of carbamyl phosphate synthetase in vivo and plays a vital role in the metabolic control of urea synthesis.
Based on the previous information as to the regulatory property of acetylglutamate synthesizing enzyme of the hepatic mitochondria, it is assumable that the observed variation in the acetylglutamate level is due to a possible enhancement of the enzyme activity by increased supplies for the tissue of glutamate as the substrate and arginine as the activator upon dietary uptake of proteins.

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Activities of key glycolytic and gluconeogenic enzymes in rat liver were determined following intraperitoneal or intragastric administration of a single dose of 0.1-0.2ml CCl₄/100g body weight to male Sprague-Dawley rats under various experimental conditions. Hexokinase (HK), pyruvate kinase (PK), glucose 6-phosphate dehydrogenase (G6PDH), and phosphofructokinase (PFK) activities increased significantly in 24 hours, reaching 10, 5, 5 and 2 folds in 72 hours, over respective controls, and returned to the initial values in one week. In contrast with this, glucokinase (GK), glucose 6-phosphatase (G6Pase), and fructose 1, 6-diphosphatase (FDPase) activities decreased significantly, GK activity being undetectable in 48-72 hours. On the other hand, a bifunctional enzyme, aldolase (ALD), showed little change in total activity. The hepatic enzyme pattern in CCl₄ liver injury was apparently different from that of fasting and refeeding a glucose-casein mixture. The pattern was similar to that found in undifferentiated cells of regenerating and fetus livers, and of an ascites hepatoma, AH 130. This was supported by the studies of isozyme patterns of some glycolytic enzymes in CCl₄-injured rat livers, i. e. loss or decrease of liver specific GK, PK-L, and ALD-B and increase of nonspecific HK-I, II, III, PK-M₂, and ALD-A. A single molecular species of G6PDH was involved in increase of its activity under the above experimental conditions of animals.
Elevations of HK, PK and G6PDH activities after CCl₄ administration were found before the increase in ¹⁴C-orotate incorporation into liver DNA became evident. The extent of enhanced DNA synthesis following CC14 liver injury was less than that in liver regeneration after partial hepatectomy. Thioacetamide administration produced a liver enzyme pattern similar to that obtained by CCl₄ treatment. However, the mitotic index found in thioacetamide-injured liver was much smaller than in CCl₄-injured or hepatectomized liver. Thioacetamide caused also less liver cell necrosis and fatty change. These results apparently indicate that the increase in liver glycolytic enzyme activities in CCl₄ intoxication is not a reflection of liver regeneration following hepatic cell necrosis, in terms of DNA synthesis and cell division, and has no relevance to liver cell necrosis and fatty change per se. Parabiosis experiments partially excluded involvement of a humoral factor in increasing the glycolytic enzyme activities following CCl₄ treatment. Thus, it appears most likely that the enhanced enzyme activities in CCl₄ intoxication represent a positive response of parenchymal liver cells to the injury, this being directed to survival and repair of the damaged hepatic cells.
Cycloheximide administration suppressed the increases in HK, PK and G6PDH activities in CC14-injured liver, suggesting that the increase in enzyme activity is due to enzyme induction. On the other hand, actinomycin D treatment twice a day in a single dose of 50μg/100g body weight failed to suppress the increase in enzyme activity. The dietary induction of G6PDH was impaired, even though G6PDH could be induced by thioacetamide intoxication, indicating the difference existing between these two induction mechanisms. Pathophysiological significances of the enzyme induction by liver injury were discussed.

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Polynucleotide ligase, which catalyzes covalent joining of two segments of an interrupted strand in a DNA duplex, was studied with normal rat liver, regenerating rat liver and hepatoma induced by N-2-fluorenylacetamide. The activity is located in both nuclei and soluble fractions. Its optimal pH was 8.0. The enzyme requires ATP as a cofactor. Its activity was completely dependent on the presence of Mg²⁺. The activities of these enzymes in regenerating rat liver were 4 times higher, in both fractions, than those in normal rat livers, whereas in hepatoma they were about 5 times higher in soluble fraction, and about 3 times higher in nuclear fraction than those in normal rat liver.

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The capacity of adaptation of the liver in the metabolism of biological active hormones, such as adrenal cortical hormones, insulin, was studied in men as well as in animals, in association with fluctuation in their levels in blood. Possibility of existence of an auto-regulatory system for adrenal cortical hormones and insulin degradation in the liver, secondary to fluctuation in their blood levels, is presented in this paper. In addition, the mechanism of this auto-regulatory system of adrenal cortical hormones and insulin degradation in the liver was also studied.
Intravenous infusion of cortisol resulted in an increase in renal creatinine clearance associated with an increased urinary excretion of corticosteroids. Despite the increase in excretion of metabolites of cortisol in urine, the blood levels of cortisol metabolites were also markedly increased, following infusion of cortisol, what sug Zgests an increase in the capacity of the liver to metabolize corticosteroids, following an increase in their blood levels. Therefore, it was suggested that the liver possesses an auto-regulatory mechanism for the control of concentration of corticosteroids in blood, which can change the rate of metabolism of hormones secondarily to fluctuation in the blood levels.
Nonprotein bound corticosteroids in serum were more rapidly metabolized in the liver than protein bound corticosteroids. At lower corticosteroid levels, a proportionally larger amount of corticosteroids in blood was bound to serum proteins, and less available to the liver for metabolic degradation than would be at higher corticosteroid levels. At higher corticosteroid levels, the reverse was seen. Since binding of corticosteroids with serum proteins very rapidly takes place, the binding capacity of serum proteins with steroids plays an important role in rapid regulation of steroid inactivation in the liver, in addition to a slower regulation by induction of Δ⁴-3-hydrogenase activity by adrenal cortical hormones.
Degradation of insulin was decreased in liver homogenates obtained from rats kept on fast or alloxan diabetic ones in comparison with that in normally fed controls. On the other hand, degradation of insulin in liver homogenates or slices was stimulated when rats had received an insulin injection. These findings indicate that there is an auto-regulatory system for the control of degradation of insulin in the liver, secondary to fluctuation in its blood level.
The degradation rate of insulin was well correlated with the reduced glutathione level. Furthermore, decreased induction of insulinase activity was observed in the livers of these rats kept on fast and alloxan diabetic ones. Since insulin is very poorly and slowly bound with serum proteins, the auto-regulatory system of insulin degradation in the liver may be operated primarily by induction of the activity of insulinase in the liver.
The major control mechanism for secretion of hormones can be considered, namely, open loop and closed loop (negative feedback) systems. In both these systems of control, an auto-regulatory process of hormone degradation by the liver, in addition to renal regulation, would seem to operate in supplement to regulation of hormonal homeostasis.

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Recently several cases of specific phospholipid fatty liver were reported, also the relationship of these cases with 4, 4'-diethylaminoethoxy hexestrol dihydrochloride (Dh drug) was discussed, and this relationship was confirmed by experimental animal study.
A case at our hospital was 66-year-old woman who had been medicated with Dh drug for the treatment of hypertension and heart disease. She was hospitalized with marked hepatomegaly, and laboratory examination revealed an accelerated erythrocyte sedimentation, CRP positive, marked hyperlipidemia and increase of acid phosphatase value. Lucent hepatic foam cells and blood foam cells were found by histological examination, and electronmicroscopic examination revealed numerous myelin-like “lamellar materials” in hepatic cells and in bone marrow cells.
Liver homogenate and acetone powder of rat liver were prepared, and were used as enzymes for metabolic study of Dh drug. Phosphate buffer and glycylglycine buffer with ATP were used as a medium of reaction, of which the latter system showed a higher hexestrol production, a reaction product, than the former, using TLC analysis.
Diethylaminoethyl alcohol as a substrate of choline phosphokinase, and the roleof this compound in the phospholipid synthesis were discussed. Also MER-25 which has diethylaminoethoxy phenyl in its chemical structure was mentioned in relation to anti-estrogen and phospholipid biosynthesis.

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Hepatic tryptophan pyrrolase decreased rapidly its activity within 6 hours after administration of CCl₄ (0.05 ml-0.25 ml/kg, p.o.) to rats and fell to 30-40 96 of the basal level of the control in 24 hours. On the contrary, tyrosine transaminase increased its hepatic level by several times over the basal activity in 6 to 10 hours and returned to the basal level in 24 hours after poisoning. The mechanisms concerned with such an inverse change in activity were studied.
I. Inhibition of Tryptophan Pyrrolase Synthesis by CCl₄
1) Induction of tryptophan pyrrolase by glucocorticoid together with L-tryptophan was markedly inhibited by CCl₄.
2) Decrease in activity and reduction of inducibility of pyrrolase were caused neither by an inhibitor- or an activator-effect, nor by change in Km value for the substrate of the enzyme in the poisoned liver.
3) When degradation rates of pyrrolase in the liver were determined by measuring the rate of decline of the activity after administration of puromycin, no significant enhancement of degradation was observed in intoxicated rats.
4) Actinomycin D-mediated superinduction of pyrrolase was inhibited by concomi- tant administration of CCl₄.
5) These results suggest that the possible reason for the diminished functional activity of pyrrolase should be loss of synthetic activity of the enzyme due to defect of translational level in the poisoned liver.
11. Tyrosine Transaminase Induction by Differential Inhibitory Effects of CCl₄.
1) Elevation of tyrosine transaminase observed after administration of glucororticoid together with L-tryptophan was lower in the poisoned liver than that in the intact liver, what indicates that the induction of transaminase by hormone was inhibited by CCl₄.
2) The results of an immunological analysis carried out in addition to an admixture experiment indicated that increase in basal activity and reduction in capacity of hormone-mediated induction of the enzyme was derived from a net change in immunologically crossreactive transaminase protein.
3) An immunochemical method was employed to study pulse labeling of transaminase and to “chase” the degradation of prelabeled enzyme protein. The results suggested that the rise of transaminase in the intoxicated liver resulted from a marked decrease in the rate of enzyme degradation. Synthesis of the enzyme was also inhibited by CCl₄ but relatively slowly.
4) Further rise in transaminase levels was observed following administration of CCl₄ either alone or together with actinomycin D to adrenalectomized rats which had been induced by cortisol. By an isotopic-immunochemical analysis, it was elucidated that such a CCl₄-mediated superinduction of transaminase was also caused by the differential inhibitory effects of CCl₄.
5) These results suggested also that the inhibition of synthesis and of degradation of transaminase by CC14 appeared to occur at the translational level.

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Lecithin: cholesterol acyltransferase (LCAT) is produced in liver and secreted into circulation. It catalyzes esterification of plasma cholesterol and formation of lysolecithin from lecithin, and is the major source of human plasma esterified cholesterol.
In the present study, LCAT was purified 1478 folds from human plasma by means of affinity chromatography, and was subjected to the study of enzyme-substrate complex formation. Purified LCAT was shown to bind delipidized protein of high density lipoproteins (apo HDL) which was coupled to Sepharose beads. However, LCAT had a higher affinity to apo HDL-Sepharose complex which had been relipidized by plasma lecithin.
When human plasma proteins were fractionated by Sephadex G 200 column, three main peaks were eluted. The LCAT present in D> 1.21 fraction appeared between the second and the third peaks. However, when D> 1.21 fraction was first incubated with VLDL, LDL and inactivated HDL and the incubation mixture was put to Sephadex G 200 gel filtration, the main peak of LCAT activity shifted to the area between the first and the second peaks, and it corresponded to HDL zone. On the other hand, almost no enzyme activity was found at the fractions where VLDL and LDL were eluted. The results are compatible with our earlier findings which showed substrate specificity among the three major lipoproteins.
LDL was coupled to cyanogen-activated Sepharose, and these fixed LDL were incubated with LCAT and HDL for 18 hr. at 37°C When LCAT reaction proceeded, the total content of cholesterol in LDL-Sepharose decreased significantly. LDL-Sepha rose is an artificial model of fixed lipoproteins, however the experimental design will be extended to the study of LDL bound to arterial wall.

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A sensitive radioimmunoassay for ACTH was established on the basis of discovery of an increase in binding of ¹²⁵I-ACTH in proportion to an increase in unlabelled ACTH when a small amount of ¹²⁵I-ACTH was added to antisera of a relatively high concentration (Matsukura et al.). Talc was used to separate free from bound ¹²⁵I-ACTH. The minimal detectable value was 1 to 2 pg. By using this assay procedure, plasma ACTH levels were measured in normal subjects and patients with pituitaryadrenal dysfunction.
In normal subjects, plasma ACTH levels were high in the morning and low in the evening, showing a marked diurnal rhythm. Insulin-induced hypoglycemia or administration of pyrogen significantly raised plasma ACTH levels in all the normal subjects tested. Intravenous infusion of 5 g of metyrapone caused a significant rise in plasma ACTH levels. These results support the hypothesis that ACTH secretion in normal subjects is regulated by 3 factors, namely circadian rhythm, stress and negative feedback mechanism. Plasma ACTH levels also rose significantly following intravenous administration of 1 mg of glucagon with the mean peak levels of 284.5 ± 81.1 pg/ml in normal subjects. They increased slightly upon administration of 1 mg of synthetic 1-24 ACTH (tetracosactide), but not upon that of 10 U of oxytocin. The mechanism by which certain peptides stimulate secretion ofACTH remains unsolved.
In patients with Addison's disease, plasma ACTH levels were extremely elevated in the morning, although the circadian rhythm was maintained. Intravenous infusion of 100 mg of corticosterone or cortisol significantly lowered plasma ACTH levels in all the Addisonian patients tested. Cortisol was more effective than corticosterone in suppressing ACTH secretion.
In patients with untreated Cushing's syndrome due to adrenal hyperplasia, basal morning plasma ACTH levels were only moderately elevated, although normal diurnal rhythm was lacking. On the contrary, plasma ACTH were undetectable in patients with Cushing's syndrome due to adrenal adenoma. Plasma ACTH responses to insulin-induced hypoglycemia and to administration of glucagon or synthetic ACTH were either significantly blunted or absent in patients with Cushing's syndrome due to adrenal hyperplasia. However, plasma ACTH responded significantly to administration of lysine-8-vasopressin.
Patients with hypopituitarism had low basal plasma ACTH levels and showed no response of plasma ACTH to a single oral administration of metyrapone (30 mg/kg) at midnight, whereas normal subjects showed a remarkable increase in plasma ACTH.
Assay of plasma ACTH may prove to be of value in differentiating primary adrenocortical insufficiency from the secondary one and especially in helping to evaluate etiology of Cushing's syndrome.

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It is well known that one of the actions of ACTH is fat mobilization. We previously proposed “ Calcium theory” on the action of ACTH ; ACTH stimulates calcium uptake to adipose tissue, and incorporated calcium ions stimulates lipolysis in the tissue. It has been clarified in this report that the stimulation of lipolysis via calcium ions results from increase of formation of lipase-fat (enzyme-substrate) complex.
This communication also examins the characters of adipose tissue lipase such as hormone-sensitive lipase and lipoprotein lipase. It was found that liver esterase antibody effectively precipitated both hormone-sensitive lipase and the lipoprotein lipase. In the presence of serum, a hormone-sensitive lipase prepared by the method of Dr. Vaughan shows the optimum at pH 8.0. On the other hand, the optimum decrease at pH 6.8 in the presence of NaCl or in the absence of serum. The lipoprotein lipase activity was clearly inactivated by acetone treatment.
From these results, it is suggested that hormone-sensitive lipase and lipoprotein lipase are the same entities, and not different ones.

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To investigate the secretory mechanism of growth hormone (GH) in rat, plasma GH levels under diverse experimental conditions were determined by radioimmunoassay.
All studies were carried out with male Wistar rats (200-250 g). The rats were housed, 6 per cage, in the room under constant temperature and light-dark cycles.
Plasma GH levels in gentled rats were significantly higher than those in nongentled rats. Basal plasma GH levels were considerably fluctuated over a wide range of values. No statistically significant diurnal variation in plasma GH were demonstrated, although the lowest GH levels were generally observed at 5 p.m. and corresponded to peak values for plasma corticosterone. Pentobarbital (PB) anesthesia caused a significant rise in plasma GH in both gentled and non-gentled rats. On the other hand, many stimuli provided with much stress, such as ether anesthesia, insulin induced hypoglycemia, hypertonic glucose, 2-deoxyglucose and catheterization, resulted in a marked suppression of plasma GH. Chronic administration of reserpine accentuated the effect of insulin. All of these suppressive effects of various stresses on plasma GH were blocked partially or totally by PB anesthesia except the case of catheterization. Intensity of the stress produced by catheterization seemed to be higher than that by other stimuli provided with much stress.
Dexamethasone also caused a marked decrease in plasma GH even under PB anesthesia. This fact indicated that GH and ACTH release were regulated independently, although in many cases, GH suppression was accompanied by an increase in plasma corticosterone. Furthermore, it suggested that suppression of GH release could be caused by some other factors than stress.
Neither arginine nor vasopressin infused into the carotid artery in PB anesthetized rat affected plasma GH concentration at all. Although in the first experiment, dibutyryl cyclic AMP infused through the same route together with aminophylline caused an increase in plasma GH level, no effect of the same combination was demonstrated in the second experiment. The results obtained from infusion of SME extract were also not reproducible. In the first experiment with crude extract, a significant rise of plasma GH concentration was observed. However, in both of the 2 nd and the 3 rd experiments, no increment in plasma GH level was observed. It is too early at the present time to decide whether these test materials were inactive or the method was insensitive.
The results of the present study confirmed that the secretion pattern of GH in rat is quite unique and that a variety of stimuli provided with much stress depress plasma GH in the rat in contrast to their stimulatory effect in other species.

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Radioimmunoassay for human TSH by means of double antibody technique was investigated.
Human pituitary thyrotrophic hormone standard was a gift from the National Institute for Medical Research, London, and both human TSH for a radioiodination and anti-human TSH rabbit serum were gifts from the National fnstitute of Health, Bethesda, Md., U. S. A.
Labeling of human TSH was achieved with ¹²⁵I in accordance with Hunter and Greenwood's method. Isolation and purification of the labeled HTSH were performed by using Sephadex G-50 and G-75 chromatocolumn.
In a preliminary study, the optimal ratio between labeled hormone and antiHTSH serum in various concentrations was determined by DEAE paper chromato-electrophoresis, and the ratio for 1: 200,000 dilution of antiserum vs. 20,000 cpm of labeled hormone or 1: 300,000 dilution of antiserum vs. 15,000 cpm of labeled hormone were adopted for the radioimmunoassay.
As a rule, the immunoassay was performed in accordance with the method reported by Odell et al., however the following two points were modified. Firstly, purified bovine serum albumin (Armour) was added to a buffer solution (0.01 M phosphate, 0.15 M NaCl, 0.1 % Na-azide) instead of normal rabbit serum, and 0.1 ml of normal rabbit serum was added when anti-rabbit γ goat serum was added to complete the assay system, in much the same manner as radioimmunoassay methods for other hormones such as insulin and growth hormone. Secondly, 0.1 ml of various concentrations of standard TSH was added to each assay system instead of adding varied volumes from a constant HTSH solution.
1. Fundamental investigations. Sufficient counts percent in B/T was not obtained in normal rabbit serum added buffer system, and the method reported by Hales and Randle failed to give any good results. A high sensitive standard curve was obtained by addition of EDTA solution, and a higher sensitive standard curve was also obtained when labeled hormone was added 24 hours after addition of non-labeled HTSH as reported by Odell et al., as compared with simultaneous addition of both labeled and non-labeled HTSH.
No influence was seen on the sensitivity of the standard curve by an addition of TSH negative serum to the assay system.
A dilution test of hyper-TSH serum showed an exact relation.
2. Clinical investigation.
Hyperthyroidic sera showed the lowest TSH level, and relatively high levels were seen in Hashimoto's and chronic thyroiditis and the highest levels were seen in hypothyroidic sera. A negative correlation between TSH levels and T7 values was seen in various thyroid diseases.
Little or no change in TSH levels was seen in comparison between pre and post treated patients with hyperthyroidism, while a remarkable decrease of TSH levels was observed in hypothyroidic patients after administration of desiccated thyroid powder.
Acromegaly, diabetes insipidus and dwarfism were analysed, and the TSH level in cord serum showed higher values than in pregnant serum.
Influences of administrations of vasopressin, glucagon, adrenocortical steroid, and synthetic ACTH upon TSH and corticosteroid levels in serum were investigated. Elevations in corticosteroid levels were observed in synthetic ACTH and glucagon administration, and on the other hand, no influence has so far been seen in TSH levels in the serum.

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There was a significant rise in serum TSH levels in normal subjects given TRH either intravenously or subcutaneously. Serum TSH was also increased by oral administration of TRH but the response was considerably lower. The serum TSH increased rapidly to reach the maximum level about 30 min after intravenous administration of 50-800μg of TRH and the response appeared to be related to the quantity of TRH. The maximum response ranged from 10 to 40μU/ml by 500μg of TRH. Day to day variation of the response in a single individual was smaller than variations in different subjects. Serum GH, LH and cortisol levels did not show any significant changes following administration of TRH. Intravenous administration of 500 to 800 pg of TRH failed to alter serum thyroxine levels, but 10mg of TRH given orally caused a slight but consistent rise in the serum thyroxine level.
A clinical test, designated as TRH test, was standardized on this basis and performed in patients. The response to the test varied considerably from patients to patients with hypothalamico-pituitary diseases, i. e. hypopituitarism resulted from postpartum necrosis, meningitis, pituitary dwarfism, chromophobe adenoma, acidophile adenoma, craniopharyngioma and chordoma. In general, no response at all or a low one was noted in patients with secondary hypothyroidism. No response was noted in two female siblings with non goitrous cretinism due to isolated thyrotropin deficiency. In patients with untreated Graves' disease, the base line TSH was undetectable in most cases and did not respond to TRH. The patients rendered euthyroid by antithyroid therapy had also no response at all or a low one to TRH. Serum TSH levels which were very high in patients with primary hypothyroidism were markedly more increased following TRH administration. During treatment with desiccated thyroid, both the base line and the response to TRH were lowered. Dilution curves in radioimmunoassay with sera obtained from a patient before and 30 min after TRH administration were parallel with a standard curve with the standard TSH.

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LH and FSH releasing activity of synthetic Luteinizing Hormone Releasing Hormone (LH-RH) and its analogous peptides were studied in rats and men.
Female rats were ovariectomized in accordance with the modified method of Schally et al., and were used after estrogen-progesterone injection into their femoral vein. The minimum effective amount of LH-RH was 1-5ng (Fig. 1). Both LH and FSH reached the maximum level in 10 min. after the administration and then decreased rapidly (Fig 2). FSH releasing activity was not clear.
Analogous peptides were synthesized by substituting one amino acid radical of LH-RH (Pyr¹-His²-Trp³-Set⁴-Tyr⁵-Gly⁶-Leu⁷-Arg⁸-Pro⁹-Gly¹⁰-NH₂) or by making analogous ones of a shorter chain, i. e., The3D-LH-RH, [Phe⁵]-LH-RH, [Lys⁸]-LH-RH and Des-Gly¹⁰-LH-RH, Des-(7..10)-LH-RH. No LH releasing activity was observed with Des-(7..10)-LH-RH, but other compounds showed 40-80% of the activity of LH-RH (Fig 3). No FSH-releasing activity was noticed (Fig 4).
When 100μg LH-RH was injected intravenously to a normal human male, the blood level increased rapidly in 30 min. to about 10 times of the normal level for LH and 2-3 times for FSH, and thereafter it decreased and returned to the normal in 24 hrs. Such an increase of LH and FSH was not noticed in case of patients suffering from Sheehan's disease (Fig 5, 6).

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The effects of synthetic LH-RH and its analogues on cyclic AMP formation, LH and FSH release in anterior pituitary of Wister rat were investigated.
LH and FSH in the incubation medium and tissue were determined with radioimmunoassay by using rat LH and FSH assay system of NIH. Cyclic AMP of the pituitary was assayed by using cyclic AMP specific binding protein purified from rat liver.
Synthetic LH-RH increased significantly cyclic AMP formation of rat anterior pituitary within 1 min. incubation and, LH and FSH release into the incubation medium within 15 min. incubation.
The minimum effective dose of synthetic LH-RH stimulation cyclic AMP formation and LH release was 100ng/ml. and 10ng/ml., in vitro respectively. A larger dose of synthetic LH-RH (100μg/ml. or more) showed a smaller effect on cyclic AMP formation and LH release than that obtained by a smaller dose of synthetic LH-RH.
Several analogues of LH-RH were synthetized by a step-by-step method and their effects on cyclic AMP and LH release were tested.
A nonapeptide (H-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH₂) in which N-terminal pyro-glutamic acid of LH-RH was removed had no effect on cyclic AMP formation and LH release, and a decapeptide in which position 8 (arginine) was changed for position 9 (proline) of LH-RH had no effect.
Only one of the LH-RH analogues examined (pyro-Glu-His-Pro-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH₂) had some effect on cyclic AMP formation and LH release both in vitro and in vivo, but its effect was 1/100 to 1/200 lower in comparison with that of synthetic LH-RH.
In summary, these results show that synthetic LH-RH acts through adenyl cyclase-cyclic AMP system in anterior pituitary, and N-terminal amino acid of synthetic LH-RH may be biologically the most important structure of LH-RH.

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Mode of existence of synthetic human gastrin-I (SHG-I, I. C. I.) is studied on some animal plasma both in vivo and in vitro. Radioactivity of ¹³¹I-labelled SHG-I loaded rat plasma is found in the TCA-precipitate of the plasma. By separation with Sephadex G-100 and G-50 gel column, two peaks are observed: the first is near the protein fractions of lower molecular weights, the second is at those of free SHG-I. The same phenomena are observed on ¹³¹I-SHG loaded human plasma in vivo. With Sephadex G-50 gel column fractions of SHG-I loaded human plasma in vivo, immunological and biological activities are demonstrated both on protein fractions and on those of free gastrin.
Sephadex G-100 and G-50 gel chromatography of the incubates of ¹³¹I-SHG and human plasma in vitro revealed two peaks of radioactivity: on the protein fractions and on those of free gastrin, both of which keep their antigenicity as gastrin. Disc electrophoresis and sucrose density gradient centrifugation are used to study on those protein fractions with radioactivity, and both revealed one peak on radioactivity near albumin.
Moreover, radioactivity in the protein fractions can easily be migrated to the precipitate, after incubation with human albumin specific antiserum. Then, 4% of some animal albumins are used to detect their binding abilities which are found to be as high as with their whole plasma proteins; 1×10⁴c. p.m. of ¹³¹I-SHG is incubated with 0.5 ml of plasma or with the same volume of protein solutions at 37° for 60 minutes, at pH 7.4, protein fractions are collected with Sephadex G-50 gel column chromatography, and their radioactivity is counted. Binding ratio by B/T% are: human plasma, 12.9 to 10.6; rat plasma, 18.7; dog plasma, 20.1; rabbit plasma, 17.7; guinea pig plasma, 12.8 ; and 4% bovine serum albumin (Cohn's fraction V), 23.6; 4% rat albumin, 40.0; 4% human albumin, 13.8. On the contrary, they are very little capable of binding with the following proteins: egg albumin, 0.1; human serum gamma globulin, 2.3 and bovine serum gamma globulin, 1.4.
Such bindings are easily cleaved by incubation with 0.05 N NaOH or with 0.1 N HCl or through boiling at 100°, but incubation at 56° for 40 minutes, or overnight storage at 4° or a lower temperature show no effect on this binding. The optimal condition for in vitro binding is: pH 7.0 to 8.5, at 37° for about 120 minutes, with 4% of protein under shaking. More than 60% of the bound form is cleaved by Disc electrophoresis. A competitive inhibition in binding is observed between labelled and non labelled SHG-I of graded dose.
Conclusion: Thus, synthetic human gastrin, both ¹³¹I-labelled and non-labelled, is bound to plasma albumin of some mammals in vivo and in vitro. Those bound and free forms of gastrin maintain their immunological and biological abilities as native gastrin. The binding rate differs by species of animals.
As we have no data on endogenous gastrins or extracted ones, it is impossible to discuss about the physiological meaning of our observations, although it is interesting that the logarithmic curve on the disappearance rate of the bound form in plasma in vivo seems to be enough correlated with that of their biological activity.

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We have examined angiotensin-like substances produced by incubating renal and extrarenal tissue extracts with homologous plasmas in representative species of vertebrate classes comprising from teleosts to mammals. SE-Sephadex column chromatograms, ratios of oxytocic to pressor activity (O: P ratio), and susceptibilities to proteases were used as the criteria. Angiotensin-like substances from renal and extrarenal tissues in mammals did not differ from Asp¹-Ile⁵-angiotensin I. The active substance derived from avian kidney showed a peak at a more acid site in the chromatogram, a low O: P ratio, and greater susceptibility to proteases. The substance from reptilian kidney was eluted at a position similar to Asp¹-Ile⁵-angiotensin I, but it had an increased O: P ratio. It was also more resistant to enzyme treatments. The substance from amphibian kidneys was also eluted at a similar position, but a minor activity appeared later. The main peak for bullfrog material had a lower oxytocic activity, and was more susceptible to proteases. In teleosts, two peaks were always seen in the chromatograms. They were more susceptible to proteases than known angiotensins I or II. Some peaks had increased the oxytocic activity. The substances produced by aglomerular kidney of Lophius litulon and by the corpuscles of Stannius of carp had a common peak at an extremely basic site. Angiotensin-like substances of avian, reptilian, amphibian and teleostean origins are probably different from the known angiotensins I or II.
Among these unknown angiotensins, rat angiotensin was employed as a test material for further idenfication by dansyl procedure. The dansyl derivative or rat angiotensin and its tryptic or chymotryptic fragments showed the same chromatographical behaviors as that of Asp¹-Ile⁵-angiotensin I on a thin layer of Silica gel H. This result shows that angiotensin of human, hog, horse and rat, except bovine, has the same sequence. The dansyl method was capable enough with 5 pg of the peptide.

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A procedure for assay of amino acids, peptides, and other primary amine containing compounds has been developed on the basis of their interaction with ninhydrin and phenylacetaldehyde to yield highly fluorescent ternary products. The fluorescent ninhydrin procedure is 10 to 100 times more sensitive than the colorimetric ninhydrin procedure. The fluorometric reaction has been successfully automated for column chromatography of amino acids and peptides and has been used for detecting primary amine containing compounds on paper and thin-layer chromatograms.
An application has been made for measuring spermidine and spermine contents of rat brain.

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Circular Dichroism (CD) of hemoglobin and its derivatives (including abnormal hemoglobin) was investigated. It is difficult to distinguish HbA, its α, β chains and myoglobin of horse heart from each other in absorption spectrum, but it can easily be accomplished in CD except between α chains and myoglobin. Peaks of CD of deoxygenated HbA are greater than the arithmetic mean of those of isolated chains, especially in the Soret region. The phenomenon could be considered as a representation of heme-heme interaction. As an example of application of CD on abnormal hemoglobin, we investigated CD of Hb Rainier α₂β₂^145Tyr→Gys2145 TyrCys, an abnormal hemoglobin with diminished cooperativity. In the deoxygenated form of Hb Rainier, ellipticity at 433 nm is smaller than that of HbA. For the study of abnormal hemoglobins, especially for those with altered functions, usefulness of CD is suggested

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Serum alkaline phosphatase (Al-P) is separated into 6 active bands by agar-gel electrophoresis1, 2). Among these bands, Al-P₄ (the 4th band from the cathode) has been found in the trimester of pregnancy, as well as in some cases of malignant tumor and other advanced diseases. All of the Al-P₄ of such different origins are similar to placental Al-P in their enzymological properties and are shown to be stable to heat, being not inactivated even at 65°. This investigation is to find out any difference among these Al-P₄ of different origins, by means of the radioimmunoassay method for placental Al-P.
Method:
Purification of placental Al-P and preparation of its antiserum were carried out in accordance with the previous reports3, 4), and iodination of the purified placental Al-P by Hunter-Greenwood method 5)(TABLE I). By using these materials, the radioimmunoassay (RI) method for placental Al-P was devised in accordance with double antibody method with anti-rabbit gama;-globulin serum (TABLE II).
Serum Al-P was separated by agar-gel electrophoresis 1) and determined by modified Kind-King method and heat-stable Al-P was measured after heating the sera at 65° for 10 minutes.
Results and Discussion:
1) The standard curve obtained by the RI method can be applied in detecting more than 1 ng of purified placental Al-P, which can be satisfactorily available within the range of 5 to 25 ng (Fig. 1).
2) Although the sera shown in TABLE III were tested, no reaction were seen in Al-P₁, Al-P₂, Al-P₃. and Al-P₅ by this RI method. However, dose dependence curve obtained on the heat-stable serum Al-P by this method was quite similar to that of placental Al-P. Besides, no difference was found among the heat-stable ones in pregnancy and patients with malignant tumor and non-malignant tumor (Fig 1). On the other hand, Fishman et al. reported that Regan isoenzyme originated from cancer tissue was similar to placental Al-P from the viewpoints of enzymology and of immunology6, 7). They also noticed that the heat-stable Al-P appearing in non-cancer patients showed no cross reactions to placental Al-P immunologically. Therefore, our results mentioned above are different therefrom: the heat-stable Al-P in non-malignant patients also shows the same pattern as placental Al-P immunologically.
Although another report using the electrophretic method shows a cross reaction between intestinal Al-P and placental Al-P antiserum, no reaction was revealed in both types of Al-P by this RI method.
3) Correlation between the amount of Al-P by using RI method (y) and activity of heat-stable Al-P (x) is as follows: in serum in pregnancy, y=1.99x-1.28 (r: 0.92) and non-pregnancy, y =1.96x-0.43 (r: 0.89)(Fig. 2). This means that the heat-stable Al-P in pregnancy and that in non-pregnancy are almost the same each other immunologically. For the purified placental Al-P, 33 ng of it was equivalent to 10 King-Armstrong (K-A) units, but 20ng was for the serum Al-P. The enzymatic activity of heat-stable serum Al-P was higher than that of purified placental Al-P in comparison with the same amount of Al-P by RI method. Ratio of the amount of Al-P by RI method to the activity of heat-stable Al-P remained unchanged in the following materials, i. e. water extract of placenta, crude placental Al-P by Morton's method and purified placental Al-P adding serum. Therefore, the decreased enzymatic activity of placental Al-P compared with serum heat-stable Al-P was not due to the damage caused in the course of its purification or the lack of activators. Further investigation will be needed in this mechanism.
Heat-stable Al-P can be determined for more than 0.5 K-A units which is equivalent to 1 ng/ml by RI method. The RI method is sensitive to the same degree as the enzymatic method, but is much more excellent as to specificity.

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The conditions of spectrophotometric initial rate analysis of serum alkaline phosphatase and leucine-aminopeptidase were investigated with three synthesized substrates; α-naphthylphosphate for alkaline phosphatase, L-leucyl-β-naphthylamide and L-leucyl-p-nitroanilide for leucine-aminopeptidase, respectively.
This study attempted to establish a new multi-channel automated method attached with a single light source and a detector system, for the assay of many sorts of serum enzymes including alkaline phosphatase and leucine-aminopeptidase, such as LDH, GOT, GPT, etc. which can be measured at 340 nm with coupling of NAD-NADH system.
(A) Alkaline phosphatase
(1) The results of the kinetic analysis suggest that a-naphthylphosphate would be superior as a substrate to βA-naphthylphosphate which has been used up to now.
(2) The conditions of the assay method using this substrate were as follows; a) buffered substrate, 5 mM α-naphthylphosphate in 2-amino, 2-methyl, 1, 3-propandiol. 1 M, pH 10.6. b) temperature at 37°. c) reaction mixture, buffered 0 substrate 2.9ml., serum 0.1ml. d) initial velocity was measured at 333 nm.
(3) The phenomenon observed for βA-naphthylphosphate that the optimum pH is dependent on the substrate concentration was similarly detected with this substrate.
(4) The activity ratio of α-naphthylphosphate to phenylphosphate was calculated and any different sort of sera was observed to have a different value for this ratio. This fact may offer a new concept on clinical evaluation of alkaline phosphatases which are composed of many isoenzymes.
(B) Leucine-aminopeptidase
As a substrate, L-leucyl-p-nitroanilide would be superior to L-leucyl-β-naphthylamide, in that the former was observed to have a higher molar extinction coefficient and a lower Km value than the latter.

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Fluorometric initial rate analyses of serum alkaline phosphatase and leucine aminopeptidase were studied. In these studies, attempt has been made to establish the assay condition for multi-enzyme analysis system with a common single detection system.
Commercially available naphthol derivative phosphates, α-naphthylphosphate, β-naphthylphosphate, naphthl-ASBI-phosphate, and naphthyl-ASMX-phoaphate, were tested as substrates for alkaline phosphatase. These substrates were cleaved by the enzyme to highly fluorescent products, β-naphthol, α-naphthol, naphthol-ASBI and naphthol-ASMX.
The effect of pH on fluorescence spectra and intensity was examined. The fluorescent intensity of α-naphthol and βA-naphthol were highly dependent on pH, but that of naphthol-ASBI and ASMX was not. Kinetic analysis of the enzyme gave the suitable condition for analysis of α-naphthyl phosphate: 5 mM in 2-amino, 2-methyl, 1, 3-propandiol 0.1M, pH 11.0, excitation 335 nm and emission at 460nm, temperature at 37°.
Change of activity ratio of test substrates for conventional standard substrate (phenylphosphate) was observed in several types of abnormal sera.
L-Leucyl-β-naphthylamide was also cleaved to a highly fluorescent product, β-naphthylamide by leucine aminopeptidase.
Fluorometric assay condition was 0.647mM L-leucyl-β-naphthylamide in 0.05M Tris pH 7.2, excitation 335nm and emission 410nm, temperature at 37°.
Fluorometric initial rate analysis was studied, but the data were strikingly different from the usual diazo-coupling method. This contradiction was based on the low substrate concentration in the assay condition restricted by the solubility of the substrate.
This result suggests reorientation of leucine aminopeptidase normal values obtained in the past.

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In order to evaluate the plasma LPL activity after intravenous administration of heparin, the authors established a new method determining FFA which was liberated through LPL by modified Ui-Itaya's colorimetric method by using Intralipid activated by human serum as the substrate.
The optimal conditions for this enzyme reaction with regard to Intralipid concentration, albumin concentration, human serum concentration, pH, temperature and time course were evaluated. The LPL activity showed the correlation to the volume of post-heparin plasma. 1 M sodium chloride showed inhibition of 40. 5% on lipolysis in post-heparin plasma.
Precision of this method showed a coefficient of variation of±5.0%.

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A new method was presented for estimation of various deaminases such as leucine aminopeptidase (LAP), adenosine deaminase, trypsin, benzylamine oxidase and so on.
The principle of the method consists in measurement of ammonia liberated by the actions of deaminases with a direct colorimetric determination of ammonia reported by Fujii and Okuda.
Leucine amide was used for estimation of LAP in our new method. On the other hand, leucine naphthylamide was widely used for the assay of this enzyme in the present clinical examination.
It was demonstrated that rat liver contained several isozymes which showed different substrate specificities toward leucine amide and leucine naphthylamide.
In human hepatitis and CCl₄-treated rats, elevated plasma LAP hydrolyzes preferentially leucine amide as compared with leucine naphthylamide.
The result suggests that leucine amide is a preferable substrate for estimation of plasma LAP in clinical examination.

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Variation of galactose, glucose, lactate, pyruvate, NEFA and IRI in blood during galactose loading test was investigated in this study.
Oral galactose loading test was examined for 40 patients with diabetes mellitus, 40 with liver diseases and 20 normal subjects. Galactose in blood was measured by the method using galactose dehydrogenase. Furthermore, the galactose loading test by intravenous administration (TengstrOm) was also investigated for 5 patients with liver diseases and 5 normal subjects. The data were explained in accordance with the criteria of judgement reported previously.
About 70 % of the patients with liver diseases and about 40 % of those with diabetes mellitus were shown to be abnormal in the oral galactose loading test. Degree of abnormality was more remarkable in liver cirrhosis. Blood glucose level in the normal subjects slightly increased by galactose loading and returned to the initial level after 2 hours, but in the patients with diabetes mellitus and liver diseases, the increase in blood glucose level continued for a long time. And proportion of increase was the most marked in the diabetic patients. NEFA level in blood decreased not in relation to blood galactose level in all the cases of normal subjects, diabetes mellitus and liver diseases.
The changes of IRI in blood that are usually observed during glucose loading test were not proved to take place in loading of galactose by both oral and venous administration. It was thought that release of insulin by galactose was not marked.
It seems that redox state of NAD and NADH in liver takes part in galactose metabolism. In this study, lactate, pyruvate and lactate/pyruvate in blood were examined with a view to studying in some way the redox state of liver cytoplasma. Normal groups for galactose loading test showed a transient increase of lactate and also elevation of lactate/pyruvate. On the other hand, in abnormal groups of the loading test, increase of lactate was not marked and lactate/pyruvate was also indefinite. It may be said that galactose loading test was advisable to clarify the function of co-enzyme level in liver cytoplasma.
By galactose loading test, an abnormal result was obtained not only in liver diseases but also in diabetes mellitus, and changes of some other substances for carbohydrate metabolism in blood were observed. From this viewpoint, it will be certain that NAD and NADH in liver cell take part in galactose metabolism.

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Since insulin-induced hypoglycemia or a rapid fall of blood glucose (cataglycemia) stimulates the secretion of pancreatic glucagon in dogs, changes in plasma glucagon were investigated in normal subjects following intravenous administration of insulin (0.1 U/kg of body weight) or discontinuance of glucose infusion. A significant rise in plasma glucagon was observed in hypoglycemia induced by insulin injection, whereas cataglycemia did not induce any rise in plasma glucagon in man.
To observe the effect of sugar on the secretion of pancreatic glucagon, 5 % solution of each sugar was infused into the femoral vein of mongrel dogs, and insulin and glucagon in the pancreaticoduodenal vein were measured. The level of pancreatic glucagon was decreased after glucose or xylitol infusion. No change in glucagon level was observed upon infusion of fructose, mannose or ribose. Insulin level in the pancreaticoduodenal vein increased following infusion of glucose, ribose or xylitol. Fructose or mannose did not produce any changes in plasma insulin or glucagon in the pancreatic vein. These results do not support the intra-insular action of insulin and glucagon.

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It is well known that induction of metabolic acidosis leads to an increase in renal production and excretion of ammonia from glutamine, which plays an important role in the mechanism to maintain the acid-base equilibrium. In acidosis, the increase in renal ammonia production is related to an increased renal glucose production.
Glucose production in renal cortical slice and nitrogen excretion in urine were studied in alloxan-diabetic rats. Gluconeogenesis was increased in the renal cortex from diabetic rats, but not in the cortex from diabetic rats simultaneonly given sodium bicarbonate. The ratio of ammonia to urea in urine was higher in the diabetic rats than in those given sodium bicarbonate. The increase of gluconeogenesis found in the cortex from diabetic rats may be caused by acidosis and followed by an increase in renal ammonia production and excretion. Activities of several enzymes in kidney and liver from NH4 Cl-acidotic or alloxandiabetic rats were assayed. In the acidotic kidney, activities of glutamic dehydrogenase and phosphate dependent glutaminase were increased, but not in the acidotic liver. In the diabetic liver, activities of supernatant aspartate transaminase and serine dehydrogenase were increased, but not in the diabetic kidney. Ammonia and aspdrtate formation from glutamate or glutamine in mitochondria from liver and kidney were studied. Mitochondria from kidney produced more ammonia than aspartate, while mitochondria from liver produced more aspartate. Oxidation of glutamate in kidney mitochondria appears to be involved in a deamination reaction, whereas the oxidation in the liver to be involved in a transamination reaction. These differences in enzyme inducibilities and oxidation of glutamate between liver and kidney maybe related to difference in the physiologic functions of gluconeogenesis in liver and kidney. Although hepatic glucose pioduction is of an important relation to glucose homeostasis in blood, the physiologic role played by renal glucose production consists in maintenance of acid-base equilibrium.
Glutamic dehydrogenase of kidney and liver were compared in its crystalline and homogeneous preparation. No difference was found; crystalline forms, sedimentation patterns, electrophoretic behaviors, kinetic and immunodiffusion were identical. It has been obscure what are the different mechanisms which control the differences of enzyme inducibilities and oxidation of glutamate, resulting in organ specificity of physiological functions.

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In our previous investigations, it was elucidated that oral administration of polychlorinated biphenyls (PCB) brought about triglyceridemia in human beings and atrophy of adipose tissue in rats. The purpose of the present study was to investigate, by using rats as an experimental animal, the disturbance of fat metabolism, caused by a long time PCB administration of a small dose.
The rats were fed on 1 mg of PCB daily for 30 days and 1-¹⁴C acetate of 20 μCi was intraperitoneally to the animals thus treated. The rats were killed by decapitation. The back skin and perirenal adipose tissue were cut off. After having been extracted with chloroform-methanol solution from these tissues, the lipid was separated by TLC into phospholipid, free sterol, free fatty acid, triglyceride, and sterol ester. Analysis of lipid by GLC followed. The results obtained were as follows:
(1) Oral administraion of PCB to rats resulted in reduction of free fatty acid and triglyceride in the skin lipid, but it exerted no influence upon the content of phospholipid and total sterol in the rat skin.
(2) Oral administration of PCB to rats revealed a reduction of incorporation of [1-¹⁴C] of acetatate into triglyceride and free fatty acid of the skin lipid, but it promoted the incorporation of ¹⁴C) into total sterol.
(3) Nonsaponifiable matters of skin, analyzed and detected by TLC and GLC, were squalene, cholesterol, Δ⁷-cholestenol, desmosterol, dihydrolanosterol, and lanosterol. The amount of cholesterol was the highest, and dihydrolanosterol and lanosterol were so little as unable to determine the amounts.
(4) Incorporation of ¹⁴C-labeled acetate into squalene was the highest among the nonsaponifiable matters of the rat skin, followed by Δ⁷-cholestenol and desmosterol. The incorporation into cholesterol was the lowest.
(5) Upon oral administration of PCB, incorporation of [¹⁴C] into squalene was reduced to one third of the control group. When incorporation of [¹⁴C] into Δ⁷-cholestenol and desmosterol was compared with that of the control group, more [¹⁴C] was incorporated into Δ⁷-cholestenol than into desmosterol, but this relation was reversed upon administration of PCB. The incorporation of [¹⁴C] into cholesterol was the same both in PCB and control groups.
(6) Incorporation of [¹⁴C] into cholesterol ester fatty acid of rat skin was enhanced by administration of PCB, but other fatty acids in the skin lipid was not influenced
(7) Triglyceride content of the perirenal adipose tissue was reduced markedly upon administration of PCB and when [1-¹⁴C] acetate was injected intraperitoneally, radioactivity was found only in palmitic acid, whereas it was distributed further in longer and desaturated fatty acids in the control group.

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Successful separation of amylase-isoenzyme into fast-γ, pre-γ and three other unidentified fractions by agar gel electrophoresis prompted us to investigate isoenzyme pattern in patients with various clinical disorders or in post-surgical or post-mortem stage where an elevation of the enzyme was noticed.
The elevated enzyme activity was mainly located in the pre-γ fraction when the sera or urine of the patients with mumps was analyzed. A similar pre-γ-dominant isoenzyme pattern was noticed in the sera, pleural fluid or in the neoplastic tissue of the patients with bronchial carcinoma showing a considerable elevation of the enzyme activity, and also in some sera of post-surgical patients showing an elevation of the enzyme. The enzyme level in post-mortem sera separated from 1-atrium was generally higher than that from r-atrium and was mainly of pre-γ type.
These results are at least compatible with the notion that pre-γ isoenzyme of amylase is secreted from the bronchial tissue, accounting for the elevation of the enzyme in some patients with bronchial carcinoma or in post-mortem sera from 1-atrium. The elevation of the pre-γ amylase in post-surgical stage clearly indicates that the origin is not pancreas, but the salivary gland or bronchial tissue.

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A case of complete deficiency of lactate dehydrogenase subunit H has been presented. Deficiency of LDH H-subunit of this case is one of inborn errors of metabolism. The propositus (J. A.) is a 64 year-old male with mild diabetes, which has been under good control by restricted diet and torazamide. Low serum LDH activity was noticed upon recent laboratory check-up, and finally, complete absence of LDH H-subunit was discovered by serum isoenzyme study. In addition, the erythrocytes, leukocytes, platelets as well as saliva of the propositus were found to be deficient in LDH H-subunit. LDH of the propositus was composed only of M-subunit. Hence, the LDH isoenzyme patterns of the serum, blood cell and saliva in the propositus showed only one band, LDH-5 (MMMM tetramer). It is of special interest that erythrocytes fructose 1, 6-diphosphate, dihydroxyacetone phosphate, as well as glyceraldehyde 3-phosphate concentration in the propositus were found to be definitely increased. These data strongly suggest that disturbance in erythrocyte glycolysis exists at the glyceraldehyde 3-phosphate dehydrogenase step. However, the erythrocyte glyceraldehyde 3-phosphate dehydrogenase activity in this case was within the normal range, which suggests indirectly that the cause of this disturbance in glycolysis might be due to high NADH and relatively low NAD caused by block of LDH step. In the whole blood of the propositus, lactate concentration came out to be low, whereas that of pyruvate was within the normal range.
The studies on reduction of methemoglobin with glucose and lactate suggest that LDH activity plays only a minor role in the reduction of methemoglobin in human erythrocytes.

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It has been shown that three unique types of PK, namely L, M₁ and M₂, are present in both rat and human tissues. In this report, the relationship of enzyme abnormality due to hereditary PK deficiency to the gene expression was investigated by using PK isozymes as a marker. Acrylamide gel electrophoresis, immunological study using anti-rat M₁ and M₂ sera (since both sera perform cross reaction with M₁ and M₂ PKs of human tissues), and kinetic studies were made on six cases with PK deficiency. In two cases of them (Y. K. and F. K.), the same experiments were carried out about not only PKs from erythrocyte but also those from biopsy samples (liver, muscle and adipose tissue) taken during splenectomy. In a normal erythrocyte, the activity level of PK is 4-5 U/10¹⁰ cells, and Km for PEP in the presence and the absence of FDP are 0.11 and 0.53 mM, respectively. The six cases were able to be classified into three groups on the basis of activity level and kinetic properties of erythrocyte PK (R. B. C.-PK). In the first group (T. O. and T. S.), the activity level is very low, however, Km for PEP in the presence or the absence of FDP is within the normal range. In the second (Sapporo and Nagasaki-types), the activity level is normal or rather higher than then normal, however, Km for PEP in the presence or the absence is much higher than that of the normal. In the third (A. N., and Y. K., F. K. sisters), both the activity level and the Km for PEP are abnormal (very low and very high, respectively). The present investigation was focussed on the third group, especially Y. K. and F. K. sisters.(1) Zymogram of PK from the normal tissues showed that R. B. C.-PK was separated from L-type PK, migrating to the anode more slowly than that. It has been thought that R. B. C.-PK is L-type, however, this suggests a possibility that normal R. B. C.-PK is a heterogeneous hybrid of M₂- and L-type. In two deficient cases(Y. K. and F. K.), R. B. C.-PK from them migrated to the anode more slowly than the normal one, and was separated. PK isozymes from other tissues, muscle (M₁-type), adipose tissue and spleen (M₂-type) of the patients were not distinguishable electrophoretically from those of the normal. In contrast, a comparative zymogram of PK from the patients' and the normal liver showed that L-type was not detected in the patients liver and the activity band corresponding to L-type was separated from normal L-type migrating to the anode at a slower rate, while M₂-type as a minor component in liver was not different from each other. Then, L-type in the patient's liver was designated as L'-type.(2) Kinetic properties of PKs from R. B. C. and liver of the patients were different from those of the normal, especially Km for PEP in the presence and the absence of FDP were higher than those of the normal. On the other hand, M₁ (muscle) and M₂-type (spleen and adipose tissue) were not different kinetically from each other as well as the electrophoretic results.(3) Immunoreactivity of R. B. C.-PK from both the patient and a normal subject with anti-M₂ serum greatly differed; the amount of neutralization of the patient R. B. C.-PK by anti-M₂ serum was very low (0-15%), on the other hand, that of normal R. B. C.-PK was very high (50-60%). Both the PKs were not neutralized by anti-M₁ serum. Both the M₁ and M₂-types of PK from the patients and the normal did not differ qualitatively. Therefore, if one assumes that R. B. C.-PK is a hybridized form of L-and M₂-type subunits, then one would expect that the qualitative abnormality of the enzyme (L'-type) due to mutation of structure gene concerned in L-type subunit formation occurs in R. B. C. and liver of PK deficient subjects, and that, as a result, R. B. C.-PK of the patients is a L'-M₂ type hybrid

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A serial and simultaneous determination of urinary levels of VMA and HVA by means of thin-layer chromatography was carried out in ten cases of neuroblastoma, and significance of these assays was assessed in the follow-up of these patients. Other CA metabolites such as VLA and MHPG were qualitatively assayed at times by using paper chromatography of Armstrong et al.
The findings obtained are:
1) At the time of diagnosis, both levels of VMA and HVA were abnormally high in eight cases, only HVA was elevated in one case with the upper limit of VMA, and these two metabolites were within the normal ranges in one case.(TABLE I)
2) A rapid fall of markedly elevated VMA and HVA was observed following the complete removal and, even inoperable cases, good responses to chemotherapeutic agents.(Fig. 1)
3) No fall of HVA and VMA after operation indicated incomplete extirpation of the tumor and/or presence of massive metastases. It was also suggested that the surgical intervention might be a possible trigger for spread of metastases, because of the postoperative rapid rise of these compounds in urine.(Fig. 2)
4) A serial and simultaneous follow-up of VMA and HVA was of help for an early biochemical detection of any development of recurrent masses in a patient whose illness was silent.(Fig. 4)
5) The cases investigated were tentatively classified as follows (Scheme 1):
Group A (4 cases): Following the treatment, markedly elevated VMA and HVA returned to the normal level with the clinical improvement.(Fig. 1)
Group B (4 cases): Unresponsive to any treatment, VMA and HVA remained high or even became more elevated in association with clinical exacerbations.(Fig. 2)
Group C (1 case): Through his entire course, VMA was within the normal range. Significantly elevated preoperative HVA fell gradually to normal, during the period when he developed wide spread bone metastases.(Fig. 3)
Group D (1 case): At the onset, VMA and HVA were both normal and VLA only was elevated. Postoperatively, the rise of HVA was followed about a month later by a palpable recurrent mass. Eventually, HVA and VMA were markedly elevated at the terminal stage of his illness.(Fig. 4)
Group A revealed a favorable prognosis, even in the inoperable cases. All three cases under the age of one year were in this group. Patients in Group B were all above two year old and had a poor prognosis. Two cases in Group C and Group D were also expired.

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Patients with impaired tubular reabsorption and tubular proteinuria excrete considerable amounts of low molecular weight proteins. Recently, retinol-binding protein (RBP), a specific Vitamin A transport protein in human plasma, has been isolated and identified in the urinary protein fractions of tubular proteinuria and “Itai-Itai” disease. The four immunologically identical RBP subfractions, prepared from the urine of patients with “Itai-Itai” disease, gave a total of four zones on polyacrylamide gel electrophoresis; apo-1 in post-albumin region, holo-1 and apo-2 in alpha-1 region and holo-2 in alpha-1 to alpha-2 region just before beta-2 microglobulin and transferrin. By the use of immunoelectrophoretic technique, more than ten plasma protein subfractions have been identified, including prealbumin, albumin, alpha-1 acid glycoprotein, alpha-1 antitrypsin, alpha-2 heat-stable glycoprotein, haptoglobulin, transferrin, Ig G, Ig A and immunoglobulin light chains.
The average daily excretion in healthy female students was 0.04mg for RBP, 4.2mg for albumin and 20mg for total protein. The patients with “Itai-Itai” disease excreted large amounts of RBP, ranging from 20mg to 100mg per day, together with slightly or moderately increased amounts of albumin and total protein. The patients with glomerular disorders had a slightly or moderately increased excretion of RBP, despite a large increase in excretion of total protein and albumin The ratio of urinary albumin/urinary RBP was high in glomerular proteinuria (730-9,200), intermediate in normal persons (53-253), and low in “Itai-Itai” disease (0.5-4.6). Although the pathophysiological mechanism for urinary excretion of RBP has not been clarified as yet, the results of the present study suggest the importance of quantitative determination of urinary RBP and albumin and calculation of the ratio of the two proteins for detection of tubular disorders and differentiation of various forms of proteinurias.
It was also demonstrated that considerable numbers of family members of “Itai-Itai” disease had intermediate levels of RBP excretion and urinary albumin/RBP ratio which ranged between those in normal persons and those in patients with “Itai-Itai” disease themselves.

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Retinol circulates in rat plasma, in the form of being bound to a specific transport protein: Retinol Binding Protein (RBP). A sensitive, specific radioimmunoassay for rat RBP was developed. By means of this assay, a study was conducted to examine the effects of vitamin A (VA) depletion and deficiency, and of repletion, on the level of serum RBP, in order to explore the role of nutritional VA status in the regulation of RBP metabolism in the rat. In VA deficient rats, the serum VA levels decreased markedly (almost zero) and the serum RBP levels also declined (15 μg/ml) with a time-course similar to that observed for VA, but with a lag of about 3 days: most of the circulating RBP was present as apo-RBP. However, in vivo supplementation with retinoic acid in VA deficient rats did not recover the serum RBP to the normal level.
The level of immunoreactive RBP in livers of deficient rats was found to be 4 times higher than that in control rat livers (p<0.001). Furthermore, in vivo administration of VA to deficient rats immediataly restored the low serum RBP level toward the normal (within 5 hours).
These findings strongly suggest that VA deficiency primarily interferes with secretion, rather than with synthesis, of RBP by the liver, and that the deficient liver contains a pool of preformed apo-RBP which can be released rapidly into serum, as holo-RBP, when VA becomes available

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Back muscles of rabbits were homogenized in Tris-HCl buffer (20 mM, pH 8.0) containing mercaptoethanol (10 mM) and centrifuged at 10₄rpm for 10 minutes. The activities of phosphofructokinase in the supernatants were variable and had a tendency to be higher upon injection of a muscle relaxant (3-0-toloxy-1, 2-propanediol), to be lower only upon decapitation, in spite of an equal amount of lactic dehydrogenase and aldolase. The precipitates were subsequently homogenized in K₂HPO₄ (50 mM) solution containing mercaptoethanol (10 mM). The homogenates were centrifuged at 10⁴rpm for 10 minutes. The activities in the supernatants were lower upon injection of a muscle relaxant and higher upon decapitation.
The Tris-extracts following injection of the muscle relaxant, described above, were incubated at 30° and then centrifuged at 10⁴rpm for 10 minutes. The activities in the supernatants showed a decrease in a due course. On the contrary, the activities of phosphate-suspension in the precipitates showed an increase. But the activities in the Tris-extracts containing KF showed a little changes.
These findings indicate that phosphofructokinase may convert from a Tris soluble to a Tris-insoluble form according to either relaxation or contraction of muscle. In addition, these conversions may be presumably mediated by phosphorylation of protein. Based on this speculation, a series of studies were undertaken to determine whether muscle tissue contains the activation system associated with kinase for phosphofructokinase.
Procedure: Assay mixture for phosphofructokinase contains F-6-P, ATP, Mg, NADH₂, mercaptoethanol, auxiliary enzymes and an enzyme preparation. ATP and Mg in the above assay system is also adequate as a substrate of kinase. So, the activities may include one of the enzymes which is activated by kinase during the assay. If so, the preincubation effect with ATP and Mg on the activities will be demonstrated.
Preparation: The extract by K₂HPO₄ solution from the back muscle under the effect of the muscle relaxant were used for purification. A treatment with 0.5% streptomycin, fractionation by 33-65% and then 33-50% of ammonium sulfate resulted in a preparation with a 5 fold higher specific activity and yield of 60%.
Results: Phosphofructokinase activities of the preparation, which was kept at 4°., decreased gradually day by day. On the other side, the preincubation effects with ATP and Mg increased, and the activities after preincubation preserved the initial phosphofructokinase activity. These effects were independent of dilution of the enzyme. Radioactivities of gamma ³²P-ATP incorporated to TCA-insoluble fraction increased in parallel with the enzyme activity during preincubation.

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Tissue specificity to (cyclic 3', 5')-AMP (CAMP) binding protein and protein kinase was studied with disc electrophoresis.
Soluble fractions of rat tissue were incubated with H³-CAMP and separated by disc electrophoresis of 7.5% gel. Each gel was sliced into 30, extracted with 30% H₂O₂ and 1N NaOH, and their radioactivity was counted. Although every tissue shows a specific pattern to radioactivity, three common peaks were observed on liver, spleen, kidney, etc.. Those CAMP binding components were differentiated by Rf-ratio on disc electrophoresis: stomach type of peak at 0.76, adrenal type at 0.43, and muscle type at 0.30.
Cyclic AMP dependent protein kinases were also studied on the soluble fraction of rat gastric mucosa and liver. On the gastric mucosa, a single peak of CAMP dependent protein kinase was detected at the slice number 14 to 16. On the liver, two peaks at the slice number 1 and 7 to 10. Dissociation from CAMP dependent protein kinases are observed by incubation of each soluble fraction with additional CAMP (10^-5M) prior to electrophoretic separation. On the gastric mucosa, the peak of CAMP dependent protein kinase changed its mobility on disc electrophoresis and moved to the place of free type protein kinase at the slice number 1 and 7 to 9. On the liver, the same phenomenon was observed and they moved to the slice number 1 and 3 to 6.
With those findings on rat gastric mucosa and liver, tissue specific and electrophoretically different subunits are suspected to be the cyclic AMP binding protein and protein kinase, which compose each tissue specific cyclic AMP dependent protein kinase.

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Energy metabolism increases after protein food has been ingested. This phenomenon was called specific dynamic action (S. D. A.) by M. Rubner in 1902. The mechanism of S. D. A. is not yet clear, though different hypotheses have been proposed by various laboratories. We have devised a continuous recording analyzer for contents of oxygen and carbondioxide in respiratory gas. The following results were obtained by continuous measurement of energy metabolism and Respiratory Quotient (R. Q.) of albino rats.
(1) Increase in O₂ consumption and R. Q. began almost immediately after rats started to ingest protein diets. O₂ consumption reached to its maximum within 10-15 min, then gradually decreased and reached its constant level at about 15-30% higher than the basal metabolism in about 30 min. Thereafter this high level was maintained at least for 60 min.
(2) Passage of food ingested was inspected by X-ray in rat fed on a diet containing 25% of barium sulfate. Most of ingested food were still in stomach and only a little amount of food were in duodenum and upper jejunum over 45 min after food ingestion have been started. Accordingly changes in energy metabolism and R. Q. has occurred in rats perhaps before digestion and absorption of the nutrient.
(3) Value of S. D. A. increased in pallarel with increasing contents of protein in diet, but the maximum value of R. Q. slightly decreased inversely.
(4) Value of S. D. A. was higher in the rats which had ingested casein as a nitrogen source than in those which had ingested amino acid mixture simulated to casein composition.
(5) No significant difference in value of S. D. A. were observed among the rats having ingested various kinds of proteins, the biological value of which ranged from 24 to 65.
(6) Value of S. D. A. did not change in adrenalectomized rats. Value of S. D. A. markedly decreased in alloxan diabetic rats and, in addition, the maximum value of R. Q. also decreased in severe diabetic rats.
(7) Intraperitoneal administration of insulin causes increases in O₂ consumption and R. Q. similar to that upon food ingestion. Administration of insulin together with glucagon or pancreozymin causes a faster and greater increase in energy metabolism as well as R. Q. value.
(8) S. D. A. was not observed in rats after they had undergone a ventromedial hypothalamic lesion.

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Adenosine 3', 5'-monophosphate-dependent protein kinases in most tissues and organs from various mammalian species appear to be similar enzymes which show rather broad and identical spectra of phosphate acceptor proteins, and to phosphor- ylate simultaneonsly several regulatory enzymes and functional proteins, such as glycogen phosphorylase b kinase, glycogen synthetase, lipase, histone, membrane proteins, and ribosomal proteins. The evidence suggests that the action of hormone seems to be amplified by succesive activations of two consecutive enzymes, adenyl cyclase and protein kinase, resulting in the pleiotropic control of various biochem- ical reactions in each target cell.

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In the first report about the salivary oxidation catalyst detectable with rifam- picin as a substrate, the author introduced from several points of view that the catalyst should be a low molecule peptide enzyme with copper at its active center, namely, a relatively unstable substance, detectable in a salivary fraction consisting of peptide which can be eluted by Sephadex gel-filtration, pH dependent activation, and complete inhibition by H₂S, cysteine, cyanides, citrate, methanol and ethanol. The oxidation catalyst was further named tentatively naphthohydroquinone de- hydrogenase.
The present paper reports on investigations on the oxidation mechanism of rifampicin by the salivary catalyst, and a significant individual difference in the oxidation activity of saliva.
On gasometric analysis of molecular oxygen uptake in the reaction system, it was found that the oxidation activity of the catalyst fraction, which had been isolated from saliva by Sephadex G-25 gel-filtration, correlated well to the molecular oxygen uptake accompanied by catalytic oxidation of rifampicin. This finding may be an evidence to show that hydrogen liberated by oxidation of rifampicin is accepted by molecular oxygen.
Upon investigation of catalytic oxidation of rifampicin with heavy metals, it was observed that the oxidation was developed only in the presence of divalent copper out of several investigated metals: iron, copper, zinc, cobalt, lead, silver and mercury. But with divalent copper, for example CuCl₂, the catalytic oxidation was not so significant at pH below 3, necessary to activate completely salivary catalyst. In the meanwhile, an apparent oxidation was observed rather at pH over 4, unable to activate a salivary catalyst. However, the catalytic oxidation by divalent copper may suggest probably that the heavy metal contained in the salivary catalyst is copper
Rifampicin could also be oxidized promptly and completely in the presence of ferricyanide under an acid condition. However, this phenomenon seemed to be that the rifampicin oxidation was not developed catalytically, but resulted from an oxidative action of ferricyanide well known as an oxidant, and it was supported by this fact that any other ferric compound not only catalysed no oxidation of rifampicin but also was an inhibitor of a salivary catalyst.
In comparing one with another catalytic oxidation of rifampicin with various, salivas, namely those which had been sampled from different subjects under the same condition, it was noted that there was a significant individual difference in the oxida- tion activity of saliva. It was further confirmed that the oxidation activity of indi- vidual saliva was considerably reproducible in duplication tests, and that the activity order was not so varied, even though analysis was carried out in the corrected activities of which the oxidation activity was divided by the 280 mp extinction of the fraction itself, respectively.

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Vitamin D₃ and its active metabolites being insoluble in water, the transport would take place in an associated form with some kind of proteins. Specific binding proteins were identified in rat plasma and intestinal mucosa by using gel filtration (Sephadex G-200), polyacrylamide gel disc electrophoresis and sucrose density gra- dient ultracentrifugation.
The binding proteins of D₃ and 25-HCC were identified in rat plasma. Five protein peaks (designated as peaks 1, 2, 3, 4 and 5 in the elution order) were observed on the gel filtration (2. 4x65 cm). Peaks 1, 2 and 4 were bound with D₃ and 25-HCC. However, peaks 2 and 4 were bound with a considerable amount of D3 and peak 4 with 25-HCC. The properties of these peak fractions were also analyzed with poly- acrylamide gel disc electrophoresis and sucrose density gradient ultracentrifugation. D₃ was bound with more than five bands of gel stained with acid blue black 10 B. Foul bands of them contained a small amount of carbohydrate and lipid, and a band of them was a-globulin band associated specifically with 25-HCC. The sedi- mentation constants of D₃ binding protein ware calculated as 14.5 S, 10.5 S, 8.1 S, 6.2 S and 4.3 S, and the 25-HCC were as 14.5 S, 10.5 S, 6.8 S and 4.3 S. The sedi- mentation constant of the protein, to which D₃ was bound specifically, was calculated to be about 8. 1 S. The protein for 25-HCC was 6.8 S.
After intra-abdominal injection of D₃-³H to D deficient rat, in the cytoplasmic fraction of intestinal mucosa, three protein peaks appeared upon analysis of Sephadex G-200 column to be bound with radioactivities. This suggested that two other peaks might bind metabolites of D₃. After D₃-1, 2-³H, 25-HCC-26, 27-³H and 1, 25-DHCC-26, 27-³H were incubated with intestinal cytoplasm in vitro (for 20 minutes, at 2°C), the radioactivity was observed corresponding with peaks 1, 2 and 3 respectively. Each protein binds specifically D₃ and its active metabolites, respectively. Sedimentation constants of peaks 1, 2 and 3 were 13S, 8.0-8.6S and 3.5-4. OS upon sucrose density gradient ultracentrifugation.
On the other hand, a single protein fraction (10 S) was identified in the nuclei and bound both D₃ and its metabolites. Active metabolites of D₃ will be translocated by these proteins in the intestinal cell.

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The purification procedure of Cl-esterase and Cls from human plasma was described, and some properties of these enzymes were characterized.
Cl-esterase was purified from euglobulin of human plasma by successive column chromatography with DEAE-cellulose, hydroxylapatite and TEAE-cellulose. The final product was found to be homogeneous upon ultracentrifugal and disc-electrophoretic analysis. The S₂₀, w was 4.3 and the molecular weight was determined as 113, 000 by gel filtration using Sephadex G-200.
Purified Cl-esterase released kinin from human plasma kininogen I and II. This enzyme did not activate purified plasminogen or plasma kallikreinogen.
Cls, proenzyme of Cl-esterase, was purified from euglobulin fraction of human plasma by DEAE-cellulose and hydroxylapatite column chromatography. The final product was found to be homogeneous upon ultracentrifugal, disc-electrophoretic and immunoelectrophoretic analysis. The S₂₀, w was 8.4 and the molecular weight was determined as 180, 000.
No conversion of Cls into Cl-esterase was observed upon incubation of purified Cls alone or addition of purified Cl-esterase. However, the addition of Clr or trypsin caused a rapid conversion of Cls into Cl-esterase. Moreover, it was found that purified plasmin and plasma kallikrein converted Cls into Cl-esterase at a very slow rate.

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Beef heart mitochondria were treated with 2, 4-dimethylmaleic anhydride (DMMA). A structural protein fraction was selectively released from the mitochondria by DMMA treatment. When the DMMA to protein ratio was about one to one, ap- proximately 55 % of total protein was released and an increased amount of DMMA showed no further release of protein. Electron micrographs indicated that residue fraction of DMMA treated mitochondria retained a trilaminar arrangement and the inner membrane particles were disappeared from the treated mitochondrial prepara- tions. Approximately 40 % of NADH-cytochrome C reductase and cytochrome oxidase activities were retained in the DMMA treated residues. All of these activities were found in the residues and essentially no activities were recovered in the supernatant. In case of μ-hydroxybutyrate dehydrogenase, DMMA treatment (DMMA: protein= 0.25: 1, mg/mg) gave approximately 50 % decrease in the activity at pH 8.2, and it could be completely recovered if the residues were brought to pH 6.5. Additional treatment with DMMA resulted in loss of the activity and it was only partially recovered by lowered pH. Depletion of lipid from mitochondria by extraction with aqueous acetone caused no substantial effect on the amount of protein released by DMMA treatment. These results may lead to the following conclusions. The structural protein may not be an essential component for the basic architecture of mitochon- drigmembrane but loosely associated with it. It is conceivable that phospholipid is intimately associated with the basic membrane and not with the structural protein in the mitochondrion. Among respiratory enzymes, NADH-cytochrome C reductase and cytochrome oxidase activities are restored in the absence of structural protein.(Supported USPHS Grant GM-12831, AM-14632.)

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Studies were carried out on the urinary excretion and biosynthetic pathways of aminoacetophenone derivatives.
1. Administration of tryptophan to hens or rats resulted in occurrence of aminoacetophenone (AAP), 2-amino-3-hydroxyacetophenone (3 HAAP) and 2-amino- 5-hydroxyacetophenone (5 HAAP).
2. Kynurenine was enzymically converted to kynurenamine by 0.01 M phosphate buffer (pH 8. 0) extract of rat liver and then non enzymically to AAP.
3. 5HAAP was formed from 5-hydroxykynurenine via 5-hydroxykynurenamine by 0.01 M phosphate buffer (pH 8. 0) extract of rat small intestine through the similar route to that of formation of AAP from kynurenine.
4. Rat liver rnicrosomes catalyzed hydroxylation of AAP to yield 3HAAP in the presence of NADPH.

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Our previous report revealed a metal-chelating ability of thiazide diuretics upon a polarographic analysis, but failed to discover a metal-chelating ability of furose- mide, a potent, thiazide related diuretic.
Then, the question was left whether a metal-chelating ability is an essential physico-chemical property of thiazide and its related diuretics.
This study aimed to clarify a metal-chelating ability of furosemide by titration in dioxane-water solution and to calculate the stability constant (log Kc) by Bjerrum's method modified by Nozawa and Hatano.
Experiments were carried out, as follows:
1) Titration curve of 20ml of 10^-2 M/l of furosemide solution (solvent composed of 4 dioxane and 1 aq. dist.) was recorded by using 0.1-NaOH after an addition of 3 ml of 0. 1 N-HCl.
2) Titration curves of the mixture of furosemide, HCl and CuCl2 (solvent and concentration of furosemide and HCl were same as above described and concentration ratio of furosemide/Cu was 4, 3, 2, 1, respectively.) were also recorded.
3) Titration curves of the mixture of furosemide, HCl and MgCl2 or ZnCl2 were also recorded under the same experimental condition described above.
The following results were obtained:
1) In furosemide solution, pK1=3.8 (COOH group) and pK2=7.5 (NH group) were clearly demonstrated.
2) The buffering action of metal-furosemide solution was also demonstrated near pH of pK2.
3) A red shift of the color of CuCl2 -furosemide solution was observed near pH of pK2.
The following conclusions were reached:
1) Furosemide could make a metal-chelating bond with Cu, Mg or Zn ion. Bonding ratio seemed to be 1, an intramolecular metal chelation would be made between NH group and COOH group.
2) Log Kc was estimated to be 3.72.
A metal-chelating ability seemed to be an essential physico-chemical property of thiazide and its related diuretics.
Though Kc of furosemide value was lower than that of thiazide diuretics already reported by the authors, a metal-chelate complex formation of furosemide under a physiological pH would have an important role on its pharmacological action.

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