Catecholamines in normal human plasma and rat serum are determined by gas chromatographic method 1, 2). In one ml of human plasma, only dopamine conjugates 3) are detected at the level of a few ng, while 3 catecholamines are detected in rat plasma in the free form at a higher level4) .

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The effect of catecholamine on formation of cyclic 3', 5' -AMP in incubated slices of brain was examined either by using the slices pre-labeled with ¹⁴C-adenine and by measuring the conversion rate of the incorporated ¹⁴C to ¹⁴C-cyclic AMP or by assaying the cyclic AMP content in the incubated slices after enzymic conversion to ATP. A comparative study with 6 different mammals indicated that adenyl cyclase of human cerebral cortex is most sensitive to norepinephrine, having a characteristic of the β -adrenergic receptor. Norepinephrine, histamine and serotonin showed synergistic effects each other as to stimulation of cerebral adenyl cyclase of a guinea pig. Mem-brane depolarization elicited by high potassium ions or depolarizing agents, such as veratridine and ouabain, potentiated the effect of norepinephrine in both guinea pig and human cerebral cortex.
Synergism between membrane depolarization and catecholamine suggests that catecholamine would be a secondary modifier of brain adenyl cyclase while the electrical activity would be a primary one. Significance of occurrence of norepinephrinesensitive adenyl cyclase in human brain was discussed in relevance to pathogenesis of affective psychosis.

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(1) Relative contribution of phenylalanine and tyrosine as precursors of catecholamine in man
Tracer amounts of L-phenylalanine-U-¹⁴C (7.7 mμ Ci) and L-tyrosine 3, 5-³H (91 mμ Ci) were given (i. v.) simultaneously to a patient of neuroblastoma (5 yrs) and his urine was collected for 48 hours after the injection. DOPA, VMA, and HVA were purified chromatographically from the urine and the ³H/¹⁴C ratios in the metabolites were analyzed. In the DOPA fraction, the ¹⁴C content was in a range of marginal detection and the ³H/¹⁴C ratios in VMA and HVA were 61 and 64, respectively, while the expected ratio was 5.07 on the assumption of an instantaneous conversion of phenylalanine to tyrosine after the injection and of possible loss of ³H from tyrosine during its hydroxylation. Essentially identical results were obtained from anotherneuroblastoma patient (4 yrs) The results suggest that phenylalanine in vivo is not utilized, to a significant extent, as a substrate for tyrosine hydroxylase in sympathetic tissues.
(2) Identification and quantification of 3-methoxy-4-hydroxyphenylglycol (MHPG) in human urine
Contrary to the generally accepted reports that sulfate is only a urinary conjugate of MHPG, a glucuronide of MHPG was isolated from urine of a ganglioneuroma patient (58 yrs) in a relatively large quantity. Purification and analysis of the glucuronide from the urine after simultaneous administration of ¹⁴C-norepinephrine and ³H-MHPG to the patient revealed that the glucuronide is a new metabolite of norepinephrine. Relative amounts of free and conjugated MHPG in various human urine specimens (e. g. pheochromocytoma and neuroblastoma) were also estimated.
(3) Selective O-methylation of physiological catechols by catechol-O-methyl-transferase (COMT) In vitro, O-methylation of physiological substrates of COMT, such as (nor) epinephrine, dopamine, 3, 4-dihydroxyphenylacetic acid, 3, 4-dihydroxymandelic acid., 4-dihydroxyphenethanol, 3, 4-dihydroxyphenylglycol, 3, 4-dihydroxybenzoic acid, N-acetyldopamine, N-acetylnorepinephrine and DOPA, gave mixtures of meta- and para-O-methyl derivatives. The extent of para-methylation relative to meta-methylation was low with substrates containing an ionized moiety in the side chain, i. e., amino acid, acids, and amines, and increased as the polar (hydrophilic) character of the side chain is decreased. With a non-polar substituent, such as in 4-ethylcatechol, the metal/para ratio was around unity. The results were interpreted as suggesting the presence of a hydrophobic region in the vicinity of the catechol-binding site of COMT which migrates against binding of polar substrates in the orientation necessary for Para-methylation, while non-polar substrates would appear to bind in a random fashion resulting in formation of nearly equal amounts of meta- and para-O-methylated products. No difference in the metal/para ratios was observed with COMT of liver and brain and no change in the ratios during more than 400 fold purification of liver COMT, which suggest that only one enzyme is involved in both meta-and para-O-methylation.

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The effects of catecholamines on gluconeogenesis were studied with key gluconeogenic enzymes such as serine dehydratase and phosphoenolpyruvate carboxykinase as markers.
Phosphoenolpyruvate carboxykinase activity in liver showed a diurnal variation with the lowest value at 700h and the highest at 1800h.
When animals received an intraperitoneal injection of epinephrine or norepinephrine at 700h and were killed 2 hours later, phcsphoenolpyruvate carboxykinase in liver was significantly induced by epinephrine, whereas it was not by norepinephrine. When the animals were treated with catecholamines at 1700h and killed at 1900h, however, the enzyme responded neither to epinephrine nor to norepinephrine.
Serine dehydratase activity in liver exhibited no statistically significant 24 hour rhythm under the same condition as described above, but almost the same tendency was observed in regard to the response to the enzyme to catecholamines.
These findings suggest that epinephrine is a regulatory factor for glucose formation from amino acids, but its regulation is closely related to diurnal rhythm in gluconeogenesis.
On the other hand, diurnal variation of liver phosphoenolpyruvate carboxykinase was affected neither by the adrenergic blockers such as chloropromazine, dibenzylin (α -blocker), propranolol (β -blocker) nor by surgical operations such as adrenalectomy and thyroidectomy.
Phosphoenolpyruvate carboxykinase activity in kidney also showed diurnal variation with the highest value at 700h and the lowest at between 1500h and 2100h. This pattern is just as the mirror image with the pattern of the liver enzyme.
Daily injections of carbachol for 6 days produced a marked increase in the activity of the kidney enzyme, whereas this treatment repressed the liver enzyme. From these findings, it can be concluded that homeostasis in blood glucose was maintained through interrelationship of liver and kidney in such ways as that the liver enzyme activity is enhanced by epinephrine, a neurotransmitter of the sympathetic nervous system, while the kidney enzyme activity is enhanced by acetylcholine, a neurotransmitter of the parasympathetic nervous system.

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Subcellular distribution of catecholamines in 12 cases of pheochromocytoma, 3 of neuroblastoma and their adrenal gland was investigated by the method of ultracentri-fugation. Catecholamine pool was divided into 4 fractions: coarse fraction (sediment by 800xg 10 minutes), heavy granules (sediment by 5, 000xg for 15 minutes), light granules (sediment by 100, 000xg for 60 minutes) and cytoplasmic sap (supernatant).
Most of catecholamines were found in the heavy granules and the cytoplasmic sap in these tumors and the adrenal. An increase of catecholamines in cytoplasmic sap of tumors occurred due to breakdown of the cells before or after operation. Catecholamines found in the coarse fraction of tumors showed somewhat higher values in comparison with that of the adrenal. By the method of sucrose continuous density gradient, catecholamine granules were studied more minutely and separated from mitochondria. Catecholamine granules of the tumors and the adrenal were widely distributed from the bottom (2. 0 M sucrose) to almost the same density of mitochondria, and catecholamines content was found to be relatively high in the upper part. In some tumors, catecholamine distribution shifted a little to the bottom. The same shift was observed in the adrenal of the rat injected L-dopa or in the adrenal kept in such condition as freezing. L-dopa can increase synthesis of catecholamines and probably raise catecholamines store in granules. Freezing destroyed the light part of catecholamine granules, then the heavier part of catecholamine granules probably remained after partial destruction of the cell.
The pool of catecholamines in those tumors resembled that of the adrenal. However, some tumors had a little heavier granules than those of the adrenal.

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It has been well known that both pheochromocytoma and neuroblastoma developed from primitive elements of the neural crest. In 1957, Mason et al. first described an increased catecholamine excretion in an infant with neuroblastoma. Although many biochemical studies have been reported on patients with pheochromocytoma, only a few papers on neuroblastoma have appeared. Since the most significant clinical features in neuroblastoma were anemia, skin nodules, palpable large tumors in the abdomen and cachexy, the biochemical studies on catecholamine metabolism in this neural tumor might have been overlooked. The purpose of this paper is to report on abnormal excretion patterns of urinary catecholamines and their metabolites in children with neuroblastoma and on the usefulness of one spot test which was developed previously in this laboratory for screening of neuroblastoma.
There were 7 cases of neuroblastoma studied. Diagnosis was established by histologic identification of biopsy, autopsy and surgical specimens of the tumor.
Twenty-four hour urine was collected under acidic condition. Adrenaline (A) and noradrenaline (NA) were measured by a modification of the method of Euler et al. Dopamine was determined by the method of Drujan et al. Metadrenaline and normet-adrenaline (3-methoxyamine, MA) were determined by the method of Yoshinaga et al. Vanillylmandelic acid (VMA) was estimated by the modification of the method of Studnitz et al. Abnormally high catecholamine excretion was observed in 4 of the 7 patients. Dopamine was increased in 2 of the 3 patients estimated. So far as tested, MA was significantly elevated in all 4 patients. High excretion of VMA was found in 6 of the 7 patients, as shown in TABLE I. From these data, neuroblastoma could be sub-classified into the following various types: (1) elevated excretion of both catecholamine and metabolite ; (2) elevated excretion of catecholamine with nearly normal metabolite ; (3) nearly normal catecholamine excretion with increased excretionof metabolite and (4) normal excretion of both catecholamine and metabolite. A demonstrable abnormality of catecholamine metabolism was also found in these patients when excretion ratio of catecholamine: MA: VMA was compared with that in urine from normal subjects, patients with pheochromocytoma and subjects who received noradrenaline infusion. The ratio in normal subjects was 1: 10: 140, while in patients with neuroblastoma was 1: 190: 1, 040. Almost the same ratio was obtained in both urine specimens from patients with pheochromocytoma and from subjects during noradrenaline infusion (Fig. 1, 2).
The date were not yet sufficient to explain why different patterns of catecholamine and metabolite appeared in the urine from these children. The urinary excretion of MA and VMA exceeded that of catecholamine by 100 to 2, 000 times. Therefore, it appeared that metabolism of catecholamine in these children might be performed in a different manner from the usual one, which would lead to an excessive formation of methylated products, MA and VMA. Possible explanation might be that the metabolic activity in tumor tissue or non-tumor peripheral tissue was abnormally increased.
One spot test for qualitative detection of increased methylated products of catecholamine was performed in these 7 neuroblastomas. Since 1961, the test has been routinely used in our clinic for screening of pheochromocytoma, neuroblastoma and argentaffinoma. Up to the present, 18 more cases of neuroblastoma were subjected to this test in other medical institutions. Positive results were reported in 22 of these 25 patients (83%). It is our hope that this simple test be used more widely for screening of these amine-producing tumor.

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Twenty-seven cases of pheochromocytoma, consisting of 17 patients with sustained hypertension and 10 with paroxysmal hypertension, were studied. The patients, 17 males and 10 females, ranged in age from 10 to 48 years with the mean of 30. 1 years. Abnormal laboratory findings including proteinuria, glycosuria, increased blood sugar, high serum cholesterol, elevated basal metabolic rate and severe retinal changes were found in these cases, particularly in the sustained type. Regitine and histamine test constituted a valuable screening procedure, but tyramine test gave false-negative results more frequently in the paroxysmal type. One spot test1) was positive in 22 of the 27 cases (TABLE I). The tumors consisted of 18 unilateral, of which 11 in the right and 7 in the left, and 6 bilateral and 3 extra-adrenal ones. Four of the 6 bilateral cases were siblings from two different families and 5 had medullary carcinoma of the thyroid. One patient with the biggest bilateral adrenal tumors died preoperatively from cardiac arrest and 3 died from peripheral shock shortly after removal of the tumor. Remaining 23 cases were cured completely.
Abnormally high excretion of catecholamines and their metabolites was found in all of the 27 cases. There was no direct relationship between 24 hour catecholamine excretion and the tumor size (Fig. 1). Most patients with paroxysmal hypertension excreted less than 500 μ g per day of catecholamine, while the patients with sustained hypertension had catecholamine excretion in excess of 500μ g. Like Crout et al. 2, 3), excretion pattern of catecholamines and their metabolites in these patients could be classified into two categories: catecholamine-dominant and metabolite-dominant types (Fig. 2). However, most patients with the catecholamine-dominant pattern had sustained hypertension. The metabolite-dominant pattern was found in almost all the patients with paroxysmal hypertension. Noradrenaline infusion study showedthat the catecholamine-dominant pattern was observed in urine collected during infusion and the metabolite-dominant pattern was found in urine voided 2 to 4 hours after the infusion4). Linear relationship between catecholamine and metabolite excretion was demonstrated in the patients with paroxysmal hypertension. However, the sustained type of pheochromocytoma showed a lower metabolite/catecholamine ratio than the paroxysmal type (Fig. 3). These results suggested that there was the maximal up-take and metabolism of catecholamine in the sustained type. Overflow of catecholamine could be the cause for producing the catecholamine-dominant pattern. On the other hand, tissue up-take in the patients with paroxysmal hypertension might not be fully saturated with catecholamine, and metabolism in the peripheral tissues might be active enough. Thus the latter patients showed the metabolite-dominant excretion pattern.
Based on determination of catecholamine in tumor and urine, Crout et al.³) postulated that a pheochromocytoma of a large size had a slower rate of catecholamine release than a small tumor and escaped detection until the tumor has become large enough. In our study, however, urinary catecholamine excretion and severity in clinical manifestations were not related to the tumor size. We also found that catecholamine content in large tumors differed considerably from portion to portion since they had multiple areas of hemorrhage, necrosis and cysts5). Degenerative changes may reduce the number of tumor cells and catecholamine synthesis, but remaining tumor cell continued to grow. If a small tumor repeated these processes in escaping detection, it would become large and necrotic. We found that patients with large tumors had a more prolonged clinical course (Fig. 4), and all tumors, either small or large, were detected when catecholamine excretion reached certain levels.

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Clinical and hemodynamic characteristics of 7 patients with hyperdynamic β -adrenergic circulatory state were studied and the following results were obtained.
The subjects studied consisted of 7 patients with tachycardia and labile hyper-tension, 3 males and 4 females, ranging from 18 to 65 years old. The chief complaint was palpitation associated with tachycardia due to mental stress or following postural change. The results of laboratory examinations, including thyroid, liver and renal function and values for catecholamines in plasma and urine,. were normal, except high BMR in some cases.
In the hemodynamic studies, an increase of heart rate, cardiac index (measured by external counting method with RISA) and forearm muscle blood flow (measured by capacitance plethysmography) was observed in these patients. Following an intravenous infusion of isoproterenol (0.02 g/kg/min), the heart rate, cardiac index and forearm muscle blood flow were more remarkably increased than those observed in the control subjects. Following an intravenous injection of propranolol (10 mg), a decrease of the heart rate, cardiac index and forearm muscle blood flow was significantly larger in these patients. When the patients were tilted at an angle of 60°, the heart rate increased remarkably in most of the patients, the stroke volume index and cardiac index increased in 2 of the 5 cases, while these were reduced moderately in the control subjects. These abnormal responses to postural change were normalized by pretreatment with propranolol. Furthermore, the complaints and hemodynamic changes disappeared after the propranolol treatment (30-60 mg/day, orally).
In addition to the 7 patients described above, we observed 10 atypical or borderline patients. They were divided into two groups. The first group consists of7 patients in whom hemodynamic responses to isoproterenol were exaggerated while those to propranolol were normal. The second one consists of 3 patients in whom responses to propranolol were augumented while those to isoproterenol were in a normal range.
The cause of this state remains still unknown, and we discussed it by analyzing the patients' past history.

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Previous investigation at our laboratory revealed that cardiovascular responses to administered isoproterenol and propranolol were markedly more intense in hyperthyroid patients than those in normal subjects. These results suggested that responsiveness of the β -adrenergic receptor in the cardiovascular system would be augumented in these patients.
In order to investigate the mechanism underlying hyper-function of the β -adrenergic receptor in hyperthyroidism, any possible influence of ferrous ion on vasoconstrictive response to catecholamines in hyperthyroid animals was studied. Rabbits and rats which had been found to be hyperthyroid animals were given thyradin for 14 to 21 days and elevation of protein-bound iodine levels were observed. Perfusion of rabbit ear vessels and rat hind limbs was performed by the method of constant perfusion pressure (Krawkow-Pissemski) and the constant perfusion flow (Perpex Pump), respectively. Response of the vessels to infusion of catecholamines were observed under various conditions. The results were as follows:
I) Experiments on rabbit ear vessels:
1) Vasoconstrictive response to noradrenaline (0.05 μ g /0.5 ml) and adrenaline (0.05 μg/0.5 ml) in the hyperthyroid animals was significantly lower than those in normal animals.
2) In normal rabbits, perfusion of 1,500 ml of 1 mM α, -dipyridyl produced a marked reduction of the vasoconstrictive responses to catecholamines.
After this procedure, ferrous ion was administrated. The vasoconstrictive response was increased by a small dose (1 mM FeSO₄, 0.1 ml) and decreased by a large dose (0.1 ml of 1/100 M) of ferrous ion. In the hyperthyroid rabbit ear, the vasoconstrictive responses to catecholamines were increased by perfusion of α, -dipyridyl. Pretreatment by both a small and a large doses of ferrous ion resulted in decrease of these responses.
II) Experiments on rat hind limbs:
1) Basal perfusion pressure in the hyperthyroid rat (16.4 ± 2.3 mmHg) was same as that in normal rat (16.3± 1.5 mmHg) under these experimental conditions (1.25 ml/ min constant perfused). Rise in pressure following administration of noradrenaline (0.5 μ g/0.1 ml) and adrenaline (0.5 μ g/0.1 ml) have tendency to be lowered in the hyperthyroid rat than in normal rats, respectively.
2) After perfusion of 37.5ml of 2 mM α, -dipyridyl, pressor responses to noradre-naline and adrenaline were decreased both in the hyperthyroid and normal rats. Thereafter, administration of a small dose of ferrous ion (0.1 ml of 1 mM FeSO_4/) resulted in augmentation of the pressor response to catecholamines in normal rats. However, this augmentation was not observed in the hyperthyroid rats. Followingadministration of a large dose of ferrous ion (0.1 ml of 1/100 M FeSO4), the response to noradrenaline and adrenaline was decreased in normal rats, while it was increased in the hyperthyroid rats. The effects of a small dose and a large one of ferrous ion were compared between the normal and hyperthyroid rats, and a statistically significant difference was observed (noradrenaline: p< 0.02, adrenaline: p< 0.05).
These findings suggest that the cardiovascular abnormalities observed in hyperthyroidism may be related, in part at least, to disorder of ferrous ion metabolism.

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It was discussed over the production of experimental inflammation and the effects of anti-inflammatory agents on it.
1. It is necessary to use many models or to use pathophysiologically similar model to human disease, because only a few information can be introduced from a simple experimental model.
2. Total anti-inflammatory response may be involved in the depressant action on the central nervous system as well as local anti-inflammatory response.
3. Since there was some discrepancy between the effectiveness of anti-inflam-matory agents in vivo and in vitro, the results obtained from the in vitro studies could not fully explain the mechanism of anti-inflammatory action.
4. A further developmental direction with anti-inflammatory agents would exsist in inhibition of the primary reaction of inflammation and in drug capable of a mild inhibition of the secondary reaction of inflammation.

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In the early stage of inflammation, various zymogens, such as Hageman factor, plasminogen, kallikreinogen and the first component of complement, Cl, are activated, and these activated enzymes produce various chemical mediators, such as histamine, serotonine, anaphylatoxin and kinin, etc. The present report deals with activation of human plasma kallikreinogen and plasminogen, concomitant kinin formation, and inhibition of plasmin, plasma kallikrein and activated Cl. by various w-amino and w-guanidino acid esters. On the activation of plasminogen, kallikreinogen and kinin formation, highly complicated mechanisms have been proposed by several authors. As described in our previous reports 7, 8), both kallikrein and plasmin release kinin directly from kininogen, and no conversion of kallikreinogen into kallikrein by purified plasmin was observed under our experimental conditions. Hageman factor, which had been purified from bovine plasma, could convert kallikreinogen into kallikrein: addition of purified Hageman factor to partially purified kallikreinogen, which had been obtained from EDTA-treatment of human euglobulin fraction, increased TAMehydrolytic activity. However, no activation of plasminogen by Hageman factor was observed.
In plasma there exist two types of kininogen I and, which are separated from each other with DEAE-Sephadex A-50 column chromatography 3). Actions of various kinin forming enzymes on these two types of kininogen were examined, and the kinin formed was determined by means of rat uterus. Both purified human plasmin and kallikrein activated by acetone released kinin mainly from kininogen II. On the other hand, the kinin formation by kallikrein activated by Hageman factor was observed mainly from kininogen I and poorly from kininogen II. These results show the different kinin forming activities of these enzymes, and particulary, between kallikrein activated by acetone and that by Hageman factor.
Saturated aliphatic esters of amino and guanidino acid extensively inhibit trypsin, plasmin and kallikrein 11). In this report, it was found that aromatic esters, such as phenyl guanidinocaproate, phenyl and carboxyethylphenyl trans-4-aminomethylcyclohexanecarboxylate, exhibited a far more extensive inhibitory effect not only on the esterolytic and kinin forming activities of plasmin and kallikrein, but also ATE-hydrolytic activity of the activated first component of human complement, Cl.

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Non steroidal anti-inflammatory drugs generally act as an uncoupler on oxidative phosphorylation. And many reports also have shown that non-steroidal anti-inflammatory drugs have the capacity of lysosomal membrane stabilization. The present authors tried to examine this stabilization by the drug by using rat liver and agarplanted granuloma. The results showed that anti-inflammatory non-steroidal drugs had effects of either stabilization or disruption of lysosomal membranes. These two effects depend on the concentration of drug used. Based upon these results, the authors present a schema which explains the inflammatory process from the biochemical viewpoint.

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In order to study the effect of some analgetics and anti-inflammatory drugs on permeability of biological membrane, such analgetics or non-steroidal anti-inflammatory drugs as acetanalide, acetophenetidine, acetaminophen, aminopyrine, phenylbutazone, oxyphenbutazone, benzydamine hydrochloride, flufenamic acid, mepirizole, salicylic acid, mefenamic acid, indomethacin and ibufenac, were added to the bicarbonate Ringer's solutions which were bathing the urinary bladder of toad, Bufo bufo japonicus; and the changes in net water flux (Bentley), SCC, P. D. and conductance (Ussing & Zerahn) were measured.
1.4×10^-3-10^-4M Benzydamine hydrochloride and flufenamic acid added to the serosal side inhibited the effect of vasopressin on water permeability, but mepirizole and salicylic acid did not show any significant effect. In contrast, all the other drugs potentiated the effect of vasopressin and, even in the absence of vasopressin, enhanced water permeability which was completely inhibited by 10^-5M epinephrine or 10^-4M 2, 4-DNP.
The effect of cyclic 3', 5'-AMP on water permeability was inhibited by addition of oxyphenbutazone and benzydamine hydrochloride to the serosal side, but slightly enhanced by aminopyrine and acetaminophen.
Furthermore 5×10^-3M oxyphenbutazone on the serosal side inhibited the effect of vasopressin, although this drug in a lower concentration enhanced the effect of vasopressin. When it was added to the mucosal side, the effects of vasopressin and theophylline were also potentiated.
Almost the same results were obtained in case of mefenamic acid. On the contrary, benzydamine hydrochloride inhibited the effect when added either to the serosal side or to the mucosal side, and also depressed the effect of theophylline.
Short-circuit current, membrane potential difference and conductance (SCC/P. D.) were depressed when 5×10^-5M mefenamic acid and 5×10^-4M benzydamine hydrochloride were added either to the serosal or to the mucosal side. In contrast, oxyphenbutazone depressed these electrical properties only when added to the serosal side.
From the above results, it could be concluded that non-steroidal anti-inflammatory drugs may be classified into at least three groups from the viewpoint of difference in their effects on osmotic flow of water which were induced by vasopressin, and also suggested that some direct effects of these drugs on cell membrane may play an important role in the mode of action of anti-inflammation.

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SMON is a new disease with characteristic clinical features. Pathologically pseudosystemic degeneration of spinal cord and peripheral nerve is prominent, which suggests its etiology to be due to toxins or nutritional deficiency. Recently green tongue and green feces have been noticed in many SMON patients, in parallel with the neurological symptoms. In last May, 1970, green urine was found in 2 SMON patients, in which needle-like crystals were detected. These crystals were confirmed by Prof. Tamura to be of unconjugated chinoform, and the green substance was its iron (III) chelate. From this detection, the idea that chinoform may be a causative agent of SMON was advocated, and the supporting results have since been obtained by us, as follows:
1) Almost all SMON patients had taken chinoform prior to the onset of the disease and appearance of SMON in the group of the patients who had not taken chinoform were exceptional.
2) Close relationship between the administered amount of chinoform and the onset of SMON was confirmed in Toda-Warabi area. This finding was supported by a survey in Tokyo area.
3) Rarity of SMON in children could be explained from the viewpoint of the dose of chinoform, because it was confirmed in 4 pediatric clinics that chinoform was rarely administered and its long term administration was exceptional in children.
4) SMON-Like features in rabbits were obtained by a small dose of chinoform i. v. and per os. The pathological findings of the peripheral nerves were similar to that of SMON.
5) Abdominal symptoms of SMON were confirmed, consisted of ordinary abdominal complaints before chinoform administration and abdominal distress after the administration.
6) Recently SMON-like patients have been reported in other countries to be suspicious of chronic chinoform intoxication. It could be suggested that more SMONlike patients may exist than supposed in foreign countries.
7) Since discontinuance of chinoform administration in September 1970, no new appearance of SMON has been reported in Tokyo area. Although some problems remain to be settled, it can be suggested that chinoform is the causative agent of SMON.

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Green urine excreted by a SMON patient was treated with the separation procedure summarized in Fig. 1. The pale yellow crystals thus separated (A and B) were identified with chinoform (5-chloro-7-iodo-8-hydroxyquinoline) on the basis of the data of melting point, elemental analysis and infrared and NMR spectra. Furthermore, the green pigment obtained from urine and also from green feces of a SMON patient were identified with an iron (III) chelate of chinoform, in view of its absorption spectrum and behavior in thin-layer chromatography.
This finding indicates the possibility of circulation in human body of unconjugated chinoform, when a quantity of the drug in excess of the conjugation capacity of liver was absorbed in overdose or for other reasons; such circulation of free chinoform would spoil human health.

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In recent years, there have been many patients in Japan suffering from subacute myelo-optico-neuropathy (SMON) following abdominal disorders. At present, its etiology is not known, however, there are a few kinds of etiological hypotheses. One is that SMON may be a toxicosis caused by oral administration of chinoform, which have many contradictions in explaining the disease. Another is our viral etiological hypothesis, and the present report deals with isolation and some propertiesof the suspect virus present in stools and spinal fluid of SMON patients.
Virus was isolated, with a high frequency, in BAT-6 cell cultures accompanying a weak and incomplete cytopathic effect (CPE) from feces and spinal fluid of SMON patients living in different prefectures. Attempts to isolate the virus accompanying CPE in HeLa cells, primary monkey kidney cells, and human embryonic kidney cells were all unsuccessful. On the other hand, no virus was isolated in BAT-6 cells from control specimens except the case of aseptic meningitis. Antiserum prepared from the virus isolated from feces neutralized not only the CPE produced by other viruses from stool but also the CPE produced by all viruses from the spinal fluid of SMON patients.
Neutralizing antibody (NT) titers of 13 among 15 sera collected from SMON patients on different days after the onset of the disease were 5 to 10. In contrast, 10 sera collected from normal adults showed NT titer less than 5. Failure to detect high NT titers in patients sera may explain the subacute course and relapse of the disease. Furthermore, convalescent sera of two cases of aseptic meningitis showed NT titer of 160 to 320. The fact suggests that SMON may be a new viral infection following insufficient immunological state.
BAT-6 cells were found to be not susceptible to human enteroviruses so far tested, and the virus showed a characteristic host range in tissue culture. The virus was sensitive to ether, and 5-iodo-2'-deoxyuridine. Also, the virus was filtrable through a membrane filter with an average pore size of 220mμ, but the virus was unable to pass through a 100mμ pore filter. Studies on pathogenicity of the virus in mice are revealing that the virus seems to be a new neuropathic slow virus. Further investigations about the properties of the virus are now in progress.

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Effects of exposure to cold and hot air on hormone levels in blood, especially on growth hormone (GH), were studied in normal adult subjects.
Two male and four female subjects were exposed to cold (4°) for 1 or 2 hours in thin underclothes after overnight fasting and sitting at least for 2 hours under room temperature. Blood samples were withdrawn at an interval of 30 minutes. In the first experiment, 1 hour exposure to cold was undertaken in two male subjects. Serum GH levels showed no change nor decrease during cooling. They increased, however, from an undetectable value to 5.2mμg/ml in 30 minutes and from 2. 5 to 5.6mμg/ml in 60 minutes after cooling, respectively. Two male and four female subjects were exposed to cold for 2 hours in the second experiment. Serum GH levels did not rise during cooling but increased significantly in all the cases after rewarming under room temperature. Blood glucose and serum insulin levels did not change and NEFA increased to 113-267% of the initial values during or after cooling. Serum cortisol increased from 18.4 to 23.8μg/dl during cooling in one subject but changed little in the others.
Exposure to hot air (46-48°) for 1 hour was studied in five adult male subjects after overnight fasting. Body temperature elevated from 36.3-36.7 to 37.5-37.9° and pulse rate increased from 68-74 to 96-102/minutes in 60 minutes after exposure to hot air. Sweating was very intense and body weight decreased by 0.5 to 1.5 kg, but blood hematocrit values did not change significantly. Serum GH levels increased from 0.8-2.0 (mean 1.4) to 3.5-30 (mean 12.3) mμg/ml in 60 minutes after heating and decreased to the initial value in 60 minutes under room temperature. There was no significant change in the levels of blood glucose and serum insulin. NEFA increased to 149.5-187.6% of the initial value during or after exposure to hot air in four subjects, but the increment was little in one of the cases. Serum cortisol values did not rise significantly and serum thyroxine levels showed no change during and after exposure to hot air. Two subjects, whose serum GH increased during exposure to hot air, was administered 50 g glucose p.o. 30 minutes prior to and just before entering into the hot room and then exposed to hot air. Serum GH levels decreased from 1.0 and 1.7 to an undetectable value and 0.5 mμg/ml in 60 minutes after exposure hot air.
Serum GH levels during hypothermic surgery had been reported not to rise during the operation. However, GH secretion in this kind of operation under hypothermia might be affected by premedication with a central nervous depressant such as chlorpromazine. The present experiment of exposure to cold in normal adult subjects clarify that the exposure did not stimulate GH secretion. From the fact that serum GH levels did not rise in response to 1 or 2 hour exposure to cold and increased after rewarming, the presence of a refractory period to stimulate GH release may be denied.
The mechanism of GH release in response to exposure to hot air was not clarified. However, from the result that glucose administration completely suppresses the GH release during heating, exposure to hot air can give rise to no stress to stimulate the GH release.

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Carbohydrate intolerance has been observed in the subjects with pheochromocytoma and the decreased glucose tolerance is completely or partially reversed by removal of the tumor. Inhibition of insulin secretion by catecholamine has been reported by Porte and his co-workers. The evidence has been given by a few authors in human subjects with pheochromocytoma. Investigation has been made on insulin response to glucose and tolbutamide before and after the removal of the tumor in a patient with pheochromocytoma.
The patient, 36 years old, engineer, was admitted to hospital for hypertension of 14 years' duration and diabetes mellitus. Blood pressure ranged from 206 to 150mmHg systolic and from 110 to 90 diastolic. Fasting blood sugar was 229mg/100ml, which gradually decreased by dietary therapy. After renal biopsy, he complained of abdominal pain at the right upper quadrant and his blood pressure fell to 76 mmHg. Immediately, he underwent laparotomy. A large mass, 155 g, was removed from the right kidney. Diabetes mellitus which had been observed at the preoperative period disappeared after the removal of the tumor. Glucose tolerance curve obtained before the operation showed a severe type of diabetes mellitus, but it was completely reversed after the removal of the tumor. Insulin response to glucose, which had been depressed before the operation, was improved after the removal of the tumor. A decreased insulin response to tolbutamide at the preoperative period was corrected after the operation.
These data support the hypothesis that a decreased glucose tolerance observed in the patients with pheochromocytoma may be mediated by inhibition of insulin secretion due to chronically secreting catecholamine.

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It is not yet known whether insulin exerts its various metabolic effects only through interaction with cell surface, or by acting at a specific site within the cell.
The present study was designed to explore the reaction mechanism of insulin by using synthetic dextran-insulin complex which is soluble and poorly permeable to cell membrane.
Crystalline bovine insulin was covalently coupled at pH 9 with dextran (MW=40,000 or 70,000) which had been activated with CNBr by slight modification of procedure described by Axen and Porath. After coupling, dextran-insulin was precipitated with acetone and purified by gel filtration through a column of Sephadex G-75. Bound insulin in dextran-insulin complex was determined by a micromethod using polyacrylamide gel electrophoresis. The shape of radioimmunoassay curve of dextran-insulin indicated that its immunochemical effectiveness is lower than that of unmodified free insulin. Next, when dextran-insulin was injected intravenously into alloxan-diabetic rats, the blood glucose concentration extensively lowered at a dose as little as 0.4U/kg. Its hypoglycemic action was not only potent, but also long acting, compared with unmodified insulin. It was also found that dextran-insulin markedly induced glucokinase, pyruvate kinase, and ATP citrate lyase in the liver at a much smaller amount than that of free insulin, after injection into alloxandiabetic rats.

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Some biochemical changes in rat kidney following administration of parathyroid ormone (PTH) were examined in vivo and in vitro.
Infusion of 5μg PTH into a 150g rat increased renal parenchyma cyclic AMP by 4 times in a few minutes. Such a high level of cyclic AMP in the kidney was observed also in the kidney from a vitamin D defficient rat which was supposed to have secondary hyperparathyroidism. Infusion of calcium, phosphate, magnesium, acid or lkali could not change the cyclic AMP amount.
Among glycolytic or TCA cycle intermediates assayed, α-ketoglutarate (α-KG) concentration was lowered markedly very soon after infusion of PTH or dibutyryl cyclic AMP (DBcAMP)(3mg). Concentration of α-KG was also low after infusion of CaCl₂ and HCl, but increased by infusion of EGTA (ethylene bisoxyethylenenitrilotetraacetate), NaHCO₃ or acetazolamide.
In an attempt to know the effector and mechanism of these PTH and cyclic AMP actions on TCA cycle intermediates in the kidney, an in vitro system employing the rat renal tubules prepared by enzymatic digestion with collagenase and hyaluronidase was developed. This preparation had active respiration, and increase in cyclic AMP concentration was observed following addition of 0.2μg/ml PTH. They also formed glucose from lactate or other TCA cycle intermediates, with a higher rate than kidney slices.
When the renal tubules were incubated with lactate substrate, CaCl₂ stimulated glucose production and lowered the amount of α-KG formed. Addition of PTH in the medium containing 0.25mM CaCl₂ had the same effect, but not in the medium free from calcium. The stimulatory effect of PTH on cyclic AMP formation was not influenced by the presence or absence of calcium in the medium. Addition of DBcAMP 0.5mM stimulated glucose production and lowered α-KG only in the presence of calcium in the same way as PTH did. Thus calcium dependency of PTH or DBcAMP action on glucose production and α-KG metabolism was clear.
Possibility remained that PTH might stimulate renal glucose production through the change in H⁺ ion concentration, as PTH was known to inhibit renal tubular H⁺ excretion. The effect of lowering pH to 6.8 by decreasing HCO3 concentration in KRB buffer was compared with that of PTH, DBcAMP and CaCl2. Among the substrates examined, low pH stimulated glucose production only from citrate, α-KG and glutamate, but PTH, DBcAMP and CaCl₂ stimulated the production not only from α-KG and glutamate but also from succinate, malate, oxaloacetate (OAA) and pyruvate. Metabolism of α-KG was tested further by analyzing some of TCA cycle and glycolytic intermediates. PTH and DBcAMP stimulated α-KG consumption and glucose production and lowered the amount of OAA in the medium with calcium. This pattern was similar to that of increasing calcium concentration. H⁺ ion, on the contrary, increased OAA concentration. By using malate as a substrate, the metabolism of malate in the renal tubules was examined in the same way. PTH, DBcAMP and calcium enhanced consumption of malate and production of glucose, and induced the change in metabolites pattern with low OAA and high phosphoenolpyruvate (PEP). Increase in H+ ion concentration did not show any significant change. The typical change in metabolism of α-KG and malate induced by addition of PTH and DBcAMP could not be observed in the tubules incubated in the absence of calcium, but the effect of increasing H⁺ ion concentration on the metabolism of α-KG was clear even in the absence of calcium.

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Bromine treatment for enhancement of the reactivity of thyroxine by ceric-arsenite reaction has greatly simplified the BEI (butanol-extractable iodine) procedure or determination of serum thyroxine by anion exchange chromatography.
In the course of the study to develope a completely automated direct (non-incineration) method for serum thyroxine using the AutoAnalyzer system, it was found that about 10 % of free thyroxine is dialyzed through the cellophane membrane of the dialyzer when alkalinity of the recipient stream is higher than 0.25 N NaOH. Thus, the automated direct dialysis method has been studied. Application of the method on serum samples revealed that protein-bound thyroxine is dissociated and dialyzed just in the form of free thyroxine, and in serum there are undetermined dialyzable substances which react with the ceric-arsenite system other than thyroxine and inorganic iodide. By combination of gel filtration and the direct dialysis method, it has been developed to completely automate the serum thyroxine determination.

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3', 5'-Cyclic AMP (cyclic AMP)-dependent and independent protein kinases (protein kinase B₁ and B₂, respectively) and a cyclic AMP-binding protein (R-protein) are partially purified from rat liver. Although R-protein shows no kinase activity, it almost totally depresses protein kinase B₂ activity. Cyclic AMP relieves the kinase of the inhibition by R-protein. The treatment of protein kinase B₁ with cyclic AMP resolves the kinase into R-protein and protein kinase B₂. The evidence indicates that protein kinase B₁ is a complex of R-protein and protein kinase B₂, and that cyclic AMP may regulate the attachment of R-protein to the protein kinase.

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Galactose loading test has long been used as one of so-called liver function tests. However, the significance and mechanism (s) of the test have not been clarified from the viewpoint of liver “functions”. Galactose metabolism has been known to be inhibited by NADH at the site of epimerization from UDP-galactose to UDP-glucose. Therefore, galactose metabolism would be affected by the level of plasma FFA concentration as in the case of glucose metabolism in the liver. In order to study this possibility, the relationship between galactose and lipid metabolism was examined in human beings.

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In rabbit muscles, glycolytic enzymes become poorer and respiratory enzymes become richer in the following order: back muscle, gastrocnemius, soleus, diaphragma, and heart1-3). This aspect seems to be stationary even with changes in nutritional environment.
The present studies are undertaken in an attempt to elucidate the behavior of key enzymes in carbohydrate metabolism for differentiation of these enzyme patterns and also of contraction-relaxation of muscles.
One group of rabbits were anesthetized by intravenous injection of a relaxant-3-o-toloxy-1, 2-propanediol and then bled thoroughly. The other group of the animals were killed by decapitation. The five kinds of muscles stated above in both groups of rabbits were removed, and immediately frozen by liquid nitrogen, and then activities of phosphorylase with or without AMP and of glycogen synthetase with or without G6P were measured. The results were that the total activities of phosphorylase and the amount of active form of glycogen synthetase in both groups of animals were poorer in the above mentioned order of five muscles, and the ratio of active form to the total of both enzymes also decreased in the same order by the relaxant.
Activities of glycogen synthetase in liver and those in muscle of rat were compared under various nutritional environments. Liver enzyme decreased on starvation and increased on feeding protein free diet. But even under the same conditions, the muscle enzymes hardly showed any change. By denervation, the changes in enzyme activities in muscle could be observed and seemed to respond what were the diet. In rat whose ischiadic nerve of one side was partly excised, aldolase in gastrocnemius of the side of excision decreased and that of the opposite side was not modified. Phosphorylase of the side of excision decreased and that of the opposite side increased. Phosphofructokinase of the excision side was hardly modified and that of the opposite side decreased.
These facts may indicate that innervation would protect the changes in muscle enzyme activities, and that an especially close interrelationship would exist between nerve ending and key enzyme in view of the interesting changes in key enzymes upon injection of a relaxant and after denervation.

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(I)In vitro release of lipase from adipose.
When rat epididymal fat pads were incubated in buffer under physiological conditions, at 37°, release of lipase from the tissue was only slight in the absence of heparin, but significant on addition of heparin. Release of glucose-6-phosphate dehydrogenase under these conditions was not stimulated by addition of heparin. When epididymal fat pads were incubated in buffer under unphysiological conditions, a considerable amount of lipase was released, and addition of heparin had no significant effect on the release.
These results suggest that heparin specifically stimulates release of lipase in vitro, and that intact tissue is required for this stimulation.
Serum is required both for the optimal activity of lipase released from adipose tissue by heparin in vitro and for that of purified clearing factor lipase which appears in blood stream following intravenous heparin injection.
The former enzyme was completely precipitated by the antibody to the latter.
These results strongly suggest that the clearing factor lipase is released in blood from the adipose tissue on intravenous heparin injection. Moreover, the epididymal fat pads in vitro seem to be a suitable system to be used in the studies on the mechanism of release of clearing factor lipase by heparin.
(II)Relationship between clearing factor lipase and lipases in various tissues was examined immunochemically. Lipases extracted from the heart, lung and kidney were markedly precipitated, but pancreatic lipase was not, by the antibody to clearing factor lipase. Lipase in liver extract was slightly precipitated by the antibody to clearing factor lipase.
These results suggest that the clearing factor lipase is released in blood from such tissues as heart, lung, kidney and liver on intravenous heparin injection.

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Although glutamic dehydrogenase (EC 1. 4. 1. 3) was considered to play an important role in the brain, the properties of this enzyme have remained unknown. Thus, glutamic dehydrogenase was purified from rat brain mitochondria by the method described in the legend to Fig. 1, and its properties were studied. Biochemical properties of rat brain glutamic dehydrogenase, such as pH optimum, Km values for the substrates, cofactor requirements and inhibition by estrogenic steroids, were identical with those of bovine liver glutamic dehydrogenase. In this paper are reported the inhibitory actions of psychotropic drugs on brain glutamic dehydrogenase, whereby 50 per cent inhibitions were obtained to correlate their central nervous effects with their inhibition of enzyme activity.
1) Intensity of their inhibition appeared to depend on concentration of enzyme protein. As shown in Fig. 2, there was a more intense inhibition of the reaction at lower protein concentrations.
2) The inhibitory actions of the psychotropic drugs were compared by obtaining 50 per cent inhibitions. As shown in TABLE I, the major tranquillizers had the most potent inhibitory actions, though the minor tranquillizers or antidepressants inhibited glutamic dehydrogenase to a lesser extent. Among the major ones, butyrophenone derivatives, in the form of haloperidol and triperidol, were found to be the most potent. Next to them, trifluoperazine and perphenazine (phenothiazine derivatives) were potent inhibitors. Oxypertine and clotiapine, which were fundamentally different in their chemical structures from the butyrophenone and phenothiazine derivatives, inhibited the reaction to a greater extent than chlorpromazine, perazine, fluphenazine and levomepromazine (phenothiazine derivatives). Thus the chemical structures of the psychotropic drugs were not.correlated with their inhibition of glutamic dehydrogenase. However, the evidences referred to in the TABLE I suggested that there might be a correlation between inhibition of the enzyme activity in vitro and central nervous system effects in vivo. Liver glutamic dehydrogenase, purified by the same method as described in brain, was also inhibited to the same degree with the brain enzyme by each psychotropic drug.
3) Anticonvulsants, convulsants, biogenic amines and their antagonists had no or little effects on glutamic dehydrogenase.
4) The inhibitory actions of psychotropic drugs could be prevented or reversed by addition of ADP. These results suggest that the glutamic dehydrogenase inhibitions by psychotropic drugs may be of an allosteric nature.

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It is well-known that chicken and rat are uricotelic and ureotelic animals, respectively.(1) Incorporation of ¹⁴C-serine into uric acid and allantoin by chicken liver and rat liver slices and hepatoma cells were studied in comparison. The radioactive carbon was incorporated remarkably into uric acid by chicken liver slices, but were little incorporated into uric acid and allantoin by rat liver slices. On the other hand, in the case of rat ascites hepatoma cells, a considerable amount of incorporation was observed as same as the case of chicken liver.(2) Urea cycle which is inherent in rat liver was not detected in these rat ascites hepatomas.(3) The observations mentioned above suggest that abnormal phylogenic gene expression occurs in some rat ascites hepatomas, upon assumption that genes for uric acid synthesizing enzymes and urea cycle enzymes in rat liver cells had been acquired in an earlier stage of evolution before differentiation of Ayes and Mammalia and that the expression of only the genes for uric acid synthesizing enzymes had been repressed in a successive stage. Abnormal gene expressions may exist in some hepatomas arising not only from ontogenic aspect but also from phylogenic aspect.

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Two fractions having thymidine kinase (ATP; thymidine 5 phosphotransferase, EC 2. 7. 1. 21) activity were separated from tumor cells such as Yoshida sarcoma, Ehrlich ascites tumor, Hepatoma AH 130, Morris hepatoma 7793 and Morris hepatoma 7794 A by means of DEAE-cellulose column chromatography, while only a single fraction of thymidine kinase was observed in normal cells such as bone marrow, spleen, embryonic and regenerating rat liver.
The properties of thymidine kinases from Yoshida sarcoma were compared with those of the regenerating liver thymidine kinase.
By the properties we meant Km for thymidine and for ATP, heat stability, urea effect and optimal pH.
DEAE-Cellulose column chromatography of purified thymidine kinase from the Yoshida sarcoma yields two peaks of enzyme activity.
Molecular weight of Peak I and Peak II have been estimated to be 70,000 and 120,000, respectively.

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NADH-Methemoglobin reductase was purified 40,000-fold from human erythrocytes in the form of a simple protein without a prosthetic group or a metal, having the molecular weight of about 30,000. The enzyme showed NADH-diaphorase activity, and reduced metmyoglobin and ferric cytochrome c as quickly as methemoglobin. The rate of reduction of cytochrome b₅ by the enzyme is higher than that of methemoglobin, and the methemoglobin reduction is augumented in the presence of a catalytic amount of cytochrome b₅. In view of Kms for NADH and methemoglobin, the enzyme seems to play a major role in the reduction of methemoglobin in erythrocytes.

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Heme octapeptides derived from various cytochromes c (CHP), as represented in TABLE I, have been studied by a number of workers as a model for metal conjugated enzymes. Recently we have reported on some oxidase activities besides those of peroxidase in CHP which original cytochromes c lack. On the other hand, yeast and mammalian cytochromes c have been used clinically as a remedy for cerebral apoplexia, poisoning especially due to carbon monoxide, etc., there is, however, no satisfactory answer as to the mechanism of such effects of cytochromes c on these conditions. The purpose of the present study was to produce some types of experimental hypoxia and to investigate the biological effects of CHP in vivo. The methods of producing some types of experimental hypoxia were as follows.
Exp. 1. Hemorrhage: In order to produce hemic hypoxia, male albino rabbits weighing 2.5-3.0kg were bled from the ear vein daily for 8 consecutive days, the amount of blood withdrawn being in the range from 5 up to 20ml in the course of the study.
Exp. 2. Exposure to low pressure: Five or 10 male Imamichi rats, weighing 210g on an average, were exposed to high altitude in a large well-ventilated glass chamber. The rate of ascent was 30mmHg/min, and when the altitude of 250-300mmHg was reached, the chamber was left standing for 1-3 hrs.
Exp. 3. Histotoxic hypoxia: Racemic isoproterenol hydrochloride, 5mg/kg in the form of saline solution, was injected intraperitoneally into male Imamichi rats weighing approximately 210g.
CHP in 1mg/ml saline solution was injected intravenously in Exp. 1, intraperi-toneally in Exp. 2 and Exp. 3. The usual dose for this agent is 0.1-1.0mg/kg of body weight. Control groups consisting of 5-10 animals were given injections of an equivalent volume of saline solution. Serum lipids were extracted with chloroform-methanol and weighed by a modified method of Folch et al. The serum and tissue enzymes, which were homogenized, were calculated by methods as described elsewhere. Histopathological examinations were performed by using preparation of ordinary paraffin sections succeeded by H-E and other stainings and histochemical ones including succinic dehydrogenase stain.
When the number of erythrocytes decreased to 1/3 or less of the initial level on the 6 th or 7 th day, the sera became milky due to elevation of total lipids contents, mostly triglycerides. CHP-Injections inhibited hemorrhage lipemia caused by anemic anoxia as shown in Fig. 1. The effects of exposure to altitude on serum and liver levels were indicated in Fig. 2. Serum GOT, GPT and acid phosphatase increased according to the duration of exposure, whereas liver acid phosphatase decreased apparently. Histologically, the tissue damages due to high altitude were most apparent in the liver where centrilobular cord cells degeneration as the from of vacuolic degeneration and congestion were observed, which were milder in case of animals given CHP than the controls. The rats receiving injections of racemic isoproterenol hydrochloride developed myocardiac necrosis. The tissue levels of GOT was decreased as shown in Fig. 4. Histologically, the cardiac lesions formed by this compound were characterized by formation of several small necrobiotic foci of muscle fibers accompanied by interstitial edema, congestion and cellular infiltration including neutrophills. These lesions were most significant in the subendocardiac myocard of frontar and apical portions of left ventricles. Intensity of these lesions were rather mild in the cases which were administered with CHP.
The data obtained as described above indicate the therapeutic activities of CHP for various ischemic tissue damages. It should be emphasized that the biological functions of CHP in various types of hypoxia may be connected with its characteristic enzymatic activities, but evidence thereof remains to be obtained in a future study.

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The relaxing protein was purified from the minor component of metin by DEAE cellulose column chromatography. Myosin B was usually desensitized by washing with 2mM NaHCO₃. The following results were obtained on the regulation by the relaxing protein and MgATP, of the Mg⁺⁺-activated ATPase of actomyosin.
1) In the presence of Ca⁺⁺, the ATPase of actomyosin was activated by the relaxing protein when the concentration of MgATP was low (10^-5-5×10^-4M). The maximal rate of ATP hydrolysis was observed at 10^-4M of MgATP. At higher concentrations of substrate, the ATPase was inhibited. The relaxing protein lowered the substrate concentration at which the ATPase of actomyosin was inhibited by the substrate itself. In the absence of Ca⁺⁺, the substrate inhibition was further potentiated and occurred at much lower concentrations of MgATP.
2) When actomyosin was reconstituted from F-actin and myosin A which was pretreated with PCMB and β-mercaptoethanol, its ATPase activity was greatly enhanced by the relaxing protein even at high concentrations of MgATP (-4mM). The enhancement was more prominent in the presence of Ca++ than in its absence.
3) Troponin was removed from the relaxing protein by precipitation at pH 4.6. The precipitate activated the ATPase activity at low concentrations of the substrate both in the presence and absence of Ca⁺⁺. In the presence of Ca⁺⁺, the enhancement of substrate inhibition by the precipitate was larger than that by the original relaxing protein. But in the absence of Ca⁺⁺, it was smaller than that by the parent protein.

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As already reported, thiazide diuretics are theoretically presumed to havea metal chelating action in the S-O part due to their similarity to the electronic state of the phosphate part of ATP.
This study attempted to demonstrate experimentally the metal chelation of thiazide diuretics and to investigate the effects of this metal chelating action on the activity of Na, K-dependent ATPase which had been prepared from 0.1% DOC treated microsome fragments of the rat kidney cortex.
Reduction potential of a mixed solution of 2mM thiazide diuretics (hydrochlorothiazide, acetothiazide, trichlormethiazide, polythiazide and benzthiazide) with 1mM CuCl₂ was moved to more negative positions than that of Cu⁺⁺ of ionic state. These half wave potentials were found to move to be more negative by pH change to alkaline side.
Visible absorption spectra of a mixed solution with acetothiazide (20mM-60mM) and CuCl2 (20mM) were observed to have the maximum absorption at the wave length of 650mμ. And it was revealed by absorption spectra of various molar ratios of acetothiazide and Cu⁺⁺ that the molar ratio of ligand (acetothiazide) to metal ion was 2: 1. Half wave potential was recognized polarographically in a diazoxide-CuCl₂ solution, but not in quinethazone, furosemide and ethacrynic acid-CuCl₂ solution. These findings suggest that thiazide diuretics and diazoxide have a metal chelating action in the SO₂NH part of heterocyclic ring.
The stability constant for thiazide-Cu chelate complex in 1N NaOH were calculated by the formula introduced from Heyrovsky and Ilkovic's using the polarographical and spectrophotometrical data. Magnitude of the stability constant showed that the chelating action of thiazide diuretics had a significant strength in vivo as compared with that of amino acids or antibiotics.
Inhibitory effect of various diuretics on the activity of Na, K-dependent ATPase were investigated also in this study. The enzyme activity was inhibited by ethacrynic acid, and 50% inhibition of the enzyme activity was obtained in the concentration of 10^-4M ethacrynic acid after one hour preincubation. 4×10^-4M quinethazone resulted in a slight decrease of the activity of Na, K-dependent ATPase, but 10-3M hydrochlorothiazide, 5×10^-4 M trichlormethiazide and 10^-3M furosemide had no inhibition in vitro. Oral administration of trichlormethiazide 25mg/kg b. w. to the rats was found to have no effect on inhibition of enzyme activity. But even if thiazide diuretics (1mM)removed completly Mg⁺⁺ (2mM)from the reaction media for assay of ATPase activity through their chelating action, only 0.5mM decrease of Mg⁺⁺ could not have an effect on the enzyme activity, because Na, K-dependent ATPase had the same activity in the range of 0.5mM to 2mM Mg concentration of the media. Therefore, 0.5mM Mg concentration at the lowest of the media was employed to clarify the chelating action of thiazi dediuretics. By this study, an 18% inhibition of the activity of Na, K-dependent ATPase was found by hydrochlorothiazide (1mM) preincubated.
The results suggest that thiazide diuretics would have a considerable degree of chelating action in vivo. But the effect of chelating action of thiazide diuretics on biometals could not be related directly to inhibition of the enzyme activity of Mg activated, Na, K-dependent ATPase. Membrane permeability of thiazide-biometal complex and the direct effect of ethacrynic acid on the enzyme protein of ATPase should be explored.

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1) An attempt was made to find out a method to purify plasminogen activator from rabbit kidney. The activator could be extracted from the lysosome-rich fraction with universal buffer, pH 10.5, and the proteolytic activity could be isolated from the activator by alkaline extraction. The alkaline extraction, followed by chromatography on DEAE-cellulose with stepwise increase in concentration of KCI solution, gave 51 fold purification of the activator. Three major protein peaks (peak I, peak II and peak III) were eluted with three stepwise increase in concentration of KCI. Almost 60% of the activator activity was eluted with 0.1 M-KCI sglution in peak II, which. showed esterase activity against L-histidine methyl ester, and peak I was esterolytic against L-lysine ethyl ester and eluted with 0.05M-KCI solution.
2) The purified activator (peak II) was intracutaneously active as aninducer of extravasation of trypan blue in a localized skin area of the rabbit, and, on the other hand, peak I was not active as an inducer.
3) Aminomethylcyclohexanecarboxylic acid strongly inhibited the activator activity against caseinolysis and fibrin clot lysis of plasminogen. L-Histidine hexyl and benzyl ester also inhibited the activator activity against caseinolysis, but did not inhibit fibrin clot lysis. These compounds showed the anti-inflammatory activity to the enhancement of permeability in the rabbit skin.

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Severe cardiac calcification developed spontaneously in hereditarily diabetic KK mice maintained on a stock diet. Feeding the mice upon purified diets with various mineral compositions caused two types of acute and reproducible calcification. In the first type of calcificatiori, which was induced by a diet low in magnesium and high in phosphorus, the heart and kidney were most severely affected and the calcium contents of the tissues were elevated to about 300- and 100-fold of the normal levels at 10 days of the feeding, respectively. The rapid increase of tissue calcium level was first recognized in the kidney with a simultaneous rise in plasma inorganic phosphorus after 24 hours on the diet. Addition of magnesium or reduction of phosphorus in the diet completely prevented the development of a11 these changes. Another type of experimental calcification, induced by diets low in magnesium, phosphorus and calcium, was characterized primarily by cardiac calcification and by the absence of renal calcification. Both calcifications induced spontaneously and by diet were not recognized in ICR, C57BL and CF1 mice. The present findings suggest that relative deficiency in magnesium is mainly responsible for development of the tissue calcifications in KK mice.

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It was previously reported that KK mice, when fed on a semi-synthetic diet low in magnesium and high in phosphorus, developed severe calcifications in soft tissues. Feeding this diet developed for only a few days such various changes as renal calcification, a decrease in glomerular filtration rate, a decrease in disappearance rate of plasma urea, hyperuremia, hyperphosphatemia, calcification of the heart and other soft tissues, and a decrease in alkaline phosphatase activity subsequent to a transient elevation. These changes were closely related each other and developed in the order as stated above. Since the changes were completely prevented by supplement of magnesium alone, relative deficiency of dietary magnesium is a major causal factor. However, all of these changes never developed in the control ICR mice. A systemic study on the KK mice maintained on the diet revealed that the reduction of glomerular filtration rate which has been caused by deposition of calcium salts within the renal tubules resulted in development of hyperuremia and hyperphosphatemia. Hyperphosphatemia brought about a rise in product of Ca×Pi, which was the main cause of the soft tissue calcification such as the heart and others.

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Mammals are fed only on milk after birth. The only carbohydrate in milk consists of lactose, which is hydrolyzed into galactose and glucose, absorbed and metabolized. In this paper, the phenomenon of weaning was studied from the viewpoint of carbohydrate metabolism.
The enzyme β-galactosidase of rat intestine mucosa which digests lactose was studied. The enzyme activity increased after birth, reached the maximum in about 10 days, decreased thereafter and in weaning (20 days after birth), already reached the level of matured rats.
Liver Gal-1-P uridyl transferase in galactose metabolism reached the maximum in 6 days after birth, then decreased gradually and in weaning reached the level of matured rats. On the contrary, liver glucokinase in glucose metabolism increased rapidly just before weaning (17 days after birth) and reached the level of matured rats. Liver hexokinase activity did not show any remarkable change along with development of the rats and always remains low.
The enzyme activities in carbohydrate metabolism shows similar changes in guinea-pigs whose weaning time is different (5 days after birth). Decrease in Gal-1-P uridyl transferase in weaning could not be prevented by a galactose diet given before weaning.
Cortisone, an adrenocortical hormone, inhibited liver Gal-1-P uridyl transferase activity but promoted liver glucokinase activity, but no changes in the liver hexokinase activity was brought about.
Therefore, the phenomenon of weaning is not only the process to shift the diet from milk to natural foods, but the biochemical body system is changed by adrenocortical hormone and other factors and, as a result, it is inevitable that weaning occurs.

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It has recently been demonstrated that bile acids are actually present in normal blood of humans and some experimental animals. In order for investigate the origin of those bile acids, ¹⁴C-dehydrocholic and 3H-7α, 12α-dihydroxy-3-oxocholanoic acids were injected into the portal vein of a rat provided with a bile fistula, through which different hydrostatic pressures were applied. The following data were obtained: 1. When ¹⁴C-dehydrocholic acid was injected into the rat under the maximum pressure corresponding to that of bile excretion (ca. 20cm H₂O), radioactivity in blood was found for the most part in conjugated bile acid (s). 2. When 3H-7α, 12α-dihydroxy-3-oxocholanoic acid was injected into the rat under the maximum pressure mentioned above, radioactivity in blood was found to be the highest and, for the most part, was in conjugated bile acid; under a lower pressure (0-9cm H₂O) applied to the choledocus, radioactivity in blood was found for the most part (60-70%) in free bile acids and the remainder in conjugated bile acids; the conjugated acid was identified as taurocholic acid, while the free acids were proved to be cholic acid and the injected material which remained unchanged.
Based on these data, it was concluded that some or most part of bile acid (s) in normal blood come directly from the liver cells through Disse's space (parapedesis).

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The bilirubin binding capacity (BBC) of human serum albumin has long been studied by many investigators, but few of them reported on the nature of binding site (s), if any, of the protein. This is mainly because of lack of the knowledge on its primary structure. Recently Swaney 1) et al. have determined the adjoining structure of the lone Trp in albumin and suggested that its apolar characteristi may play a significant role in steroids binding of albumin.
In this study, albumin was purified by DEAE-cellulose column chromatography, and characterized by cellulose acetate membrane electrophoresis and gel immunoelectrophoresis as a single component (Fig. 1). Disc electrophoresis, however, resolved it into 3 components, probably monomer, dimer and so on, according to other workers' published data2). The fraction of albumin was submitted to amino acid analysis. Its composition (TABLE I) roughly resembled that of Saifer's but differed therefrom in more Glu content and less Tyr content.
From the structure of bilirubin and the fact that bilirubin can not be bound with ovoalbumin, it is postulated that the binding site (s) would be of hydrophobic nature and have positively charged groups and Tyr.
Acetylation with acetic anhydride in 10% pyridine-water on an ice-bath yielded ε-N, acetyl (Lys)-albumin. After passage through Sephadex G-25 column and dialysis, their BBC were determined by means of absorbancy at 280nm and 460nm of proteinbilirubin complexes which had been obtained from the protein solutions in the presence of a large excess of bilirubin by gel-filtration on Sephadex G-25.
The modified proteins generally had less BBC and higher acetylation degrees which were determined on the basis of ninhydrin value and dinitrophenylation value. With 5% acetylated-albumin, BBC remained 116% of that of the native one (TABLEIII). As an albumin molecule has 65 Lys residues, it might be concluded that any Lys which were preferentially acetylated is not involved in binding.
The particular amino acid residue, Trp, as mentioned above, was chosen as a next target for modification. Koshland reagent5), 2-hydroxy, 5-nitrobenzyl bromide (HNB-Br), was made to react with albumin under the following conditions: albumin 0.1μmole, HNB-Br 1μmole, urea 8 M in 3 ml of 5% MeOH-water. The pH of the reaction mixture was kept at 3.0 by using pH-statt for 2 hrs. The modified protein was obtained by gel-filtration on Sephadex G-25 column equilibrated with 8M urea. It was then subjected to gel-filtration on Sephadex G-100 equilibrated with 0.01N NaH₂PO₄. The protein emerged as a single peak was named HNB-Alb.
In the third attempt to elucidate the bilirubin binding site (s) qualitatively, Vallee's reagent6), N-acetylimidazole (N-AI), was used for Trp modification. The conditions: albumin 0.1μmole: N-AI 100μmole: 0.1M phosphate, pH 7.0 at room temperature. The reaction proceeded under continuous stirring. Each 1.5ml of the reaction mixture was pipetted out at 1 hr and 2 hrs intervals and the aliquot was gel-filtrated on Sephadex G-10 to give the modified proteins-I and-II, respectively.
Judging from the absorption spectrum of HNB-Alb, Trp in the protein was modified by approximately 100% (TABLE II), and BBC of the modified protein was decreased but similar in behavior with respect to bilirubin concentration to that of native albumin (Fig. 2). O-AcAlbs whose amounts of unmodified Tyr were determined on the basis of absorbance at 278nm showed a marked decrease in their BBC. The specimen II which had more modified Tyr were much more decreased in BBC than I (Fig. 2). The exact number and location of the modified Tyr in the primary structure is under investigation.

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A semisynthetic antibiotic rifampicin 3-(4-methyl-piperazinyl-iminomethyl)-rifamycin SV has been noted clinically as a powerful antibacterial drug. But of metabolism of the drug in human body, many problems still remain unknown. A remarkable experimental fact was found that rifampicin was promptly oxidized by treating with human saliva under hydrochloric acid condition. As such oxidation could not be observed in the solution of this acid without saliva, it seemed that a certain enzyme should bring about this oxidation. This is a report about a special oxidation enzyme present in human saliva.
By using rifampicin as a substrate, the salivary enzyme activity could be determined tentatively by a spectrophotometric assay in accordance with the principle that by oxidation of rifampicin the absorption peak is changed from 475 mμ to 540mμ. This enzyme was relatively unstable, being destroyed gradually at 25° However, when stored at 5°, it was possible to keep the enzyme in constant activity for a long time. The enzyme activity was completely inhibited by H₂S, cysteine, cyanides, citrates and diethyldithiocarbamate. This is an evidence to show that this enzyme contains a certain heavy metal, perhaps copper which is a conspicuous oxidation catalyst. Methanol, ethanol and propanol were also very strong inhibitors, and it was further observed that pig's pancreatic lipase attacked strongly the activated enzyme under acid condition. These inactivations probably suggest that the enzyme also contains some lipid which is required perhaps for the complete activity.
With rifampicin as a substrate, the complete enzyme activity appeared at a very low pH below 3. In contrast, no activity was observed at a pH over 4. Narrowness of the pH range susceptible to convert the enzyme from inactivity into activity was also remarkable. Such pH dependent activity of this enzyme indicates undoubtedly that the original enzyme in saliva is in an inactive form, and is activated by gastric acid (HCl) when reached the stomach. The salivary enzyme at a neutral pH revealed a complete resistance to heat treatments within 30 minutes. But the activated enzyme in acid saliva was very unstable, so that the enzyme was inactivated rapidly and completely at 40° for 30 minutes, and partially at 37°. In addition, any mechanical treatments seemed to constitute hindrance to the activated enzyme.
When saliva was dialyzed in a cellophane bag against distilled water for one day at 5°, the enzyme activity was recognized in the liquid outside of cellophane bag. The enzyme was eluted in a localized manner in a peptide fraction when saliva had been gel-filtrated with distilled water on a 1.4×15cm Sephadex G-15 column. The enzyme fraction appeared to be of positive ninhydrine reaction and of negative biuret reaction. It may thus be concluded that this enzyme is a very low molecule compound, perhaps consisting of oligopeptide.
A heavy metal, which forms a black metal sulfide by reaction with ammonium sulfide, was detectable apparently in the enzyme fraction while negligible in other fractions. But Cl and urea exerting any questionable influence on the enzyme activity were both negative in the fraction. Paper chromatography of a relatively pure enzyme fraction obtained by gel-filtration revealed only one spot, which was coloured by spray of ninhydrine. However, there have remained further demonstrations to prove that the spot is identical to the enzyme itself.
The specific absorption peak of the enzyme fraction was found around 270mμ. This enzyme was distinguishable from well-known copper enzymes: tyrosinase, ascorbase and polyphenol oxidase, and also peroxidase which is a porphyrin iron compound, in view of comparison of their optimum pH, molecular weight and spectrum one with another.

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