In an attempt to establish optimal conditions for the hydrolysis of estrogen conjugates in urine with Brown's method, the study was made of the hot acid hydrolysis, enzyme (β-glucuronidase) hydrolysis and solvolysis. The modification of the micro-method according to Salokangas & Bulbrook was reinvestigated. urinary estrogens in fifteen normal male subjects were meseaured with some modified micro-method involving two step hydrolysis (β-glucuronidase hydrolysis and solvolysis). The following results were obtained.
1. Optimal conditions for the hot acid hydrolysis were in 15v/v%HCL concentration at 100°C for 60minutes incubation. For β-glucuronidase hydrolysis, those were in 2, 000units per ml enzyme concentration at 37°C for 72 hours incubation and at 50°C, incubation time was 48 hours. Optimal incubation time for the solvolysis was 72 hours at 37°C in 2% H₂SO₄ concentration with continuous ether extraction.
2. Compared with the hot acid hydrolysis and two step hydrolysis involving β-glucuronidase hydrolysis and solvolysis, quantities in total estrogens were mostly equal but estradiol fraction in the hot acid hydrolysis diminished little.
3. With the modified micro-method according to Salokangas & Bulbrook, the smallest amounts of pure estrogen could be estimated to 0.015μg and the mean ratios of recovery in this procedure were as follows; estrone 76.7%, estradiol 65.1%, estriol 82.8%. By this method, it was possible to dtermine urinary estrogens untill 0.5μg per day.
4. Urinary estrogens in eleven normal male subjects with 24 to 35 years old were as follows; estrone 1.7±0.5μg per day (mean±standard deviation), estradiol 1.7±0.7, estriol 3.5±0.6, total estrogen 7.0±1.0, and in fourth with 60 to 70 yers old, estrone 1.3±0.4μg per day, estradiol 2.5±0.4, estriol 2.6±1.1, total estrogen 6.4±1.2.

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Urines of seventy one male patients with liver diseases were analyzed for estrogens by modified Brown's method and Salokangas' micromethod, including fractionations of estrogens to estrone, estradiol and estriol. The following results were obtained.
1) No evident correlation between urinary excretion of estrogens and the results of routine liver function tests occured, but ratios of estriol/estrone+estradiol appeared to decrease on aggravation of liver functions.
2) Urinary excretions of estrogens and ratios of estriol/estrone+estradiol were not in distinct correlation with histological classification of liver diseases: acute hepatitis, chronic hepatitis and liver cirrhosis.
3) Urinary excretions of total estrogens and estradiol were distinctly increased to reduce ratios of estriol/estrone+estradiol in patients with deposition of bile pigment in liver cells and bile plugs in bile ducts as notable histological findings. On the other hand, urinary excretions of both estrogens and ratios of estriol/estrone+estradiol appeared to increase in patients with regeneration of liver cells, distortion of lobular architecture, and increase of connective tissue accompanied with its collagenation.
4) At acute icteric stages of most acute hepatitis urinary excretions of total estrogens were increased. On improvement of jaundice, estradiol decreased; estriol increased; and ratios of estriol/estrone+estradiol increased to their normal ranges in all four cases of acute hepatitis. On deterioration of four cases of liver cirrhosis, in two cases of which estrone and estriol in urines increased; and in another two cases decreased, and estradiol increased in three out of four cases. In all four cases of aggrevated liver cirrhosis, increases of total estrogens in urines paralleled with pronounced decreases of ratio of estriol/estrone+estradiol.
5) In patients with vascular spider, significant increases of estrone; estradiol; and total estrogens in urines, accompanied with significant decreases of ratios of estriol/estrone+estradiol, were observed. In patients with palmar erythema and gynecomastia, ratios of estriol/estrone+estradiol were decreased without alterations of etrogens excretion in urines.

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Eleven male patients with liver diseases were examined for conjugated estrogens in urines, which were fractionated to the estrogen glucuronides by hydrolysis with β-glucuronidase and estrogen sulfates by solvolysis. Both of them were fractionated further to estrone, estradiol and estriol by modified Brown's method and Salokangas' micromethod. The results were as follows.
1) The ratios of estrogen glucuronides to estrogen sulfates were decreased in all three fractions of estrogen, particularly in estriol in liver diseases, and the extents of decreases of ratios were paralleled with severity of the liver diseases. These decreases came from the relative increases of estrogen sulfates to estrogen glucuronides.
2) Neither levels of estrogen glucuronides and estrogen sulfates in urines, nor ratios of estrogen glucuronides to estrogen sulfates were in distinct correlation with the results of routine liver function tests.
3) The ratios of estrogen glucuronides to estrogen sulfates fluctuated in accordance with the course of the diseases. In convalescence of acute hepatitis, when the results of routine liver function tests had returned to normal ranges, the ratios of estrogen glucuronides to estrogen sulfates were still sometimes decreased, and returned to their normal levels after some delay to the results of other function tests.
4) No distinct correlation between the ratios of estrogen glucuronides to estrogen sulfates and histological findings of liver tissues could be discerned.
5) In a case of Dubin-Johnson's syndrome, the same disordered conjugation and urinary excretion of estrogens were observed as in liver damages.

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Electrophysiological studies on evoked cortical potentials following ventrolateral (VL) thalamic stimulation of man were performed with averaging computer technique for 41 patients of parkinsonism and other involuntary movement disorders during stereotaxic surgery for last 2 years.
1. Single stimulation of the VL nucleus demonstrated bilateral cortical activity with I-PN (first positive negative), II-PN (second positive negative), III-PN (third positive negative) and IV-PN (fourth positive negative) waves. III-P wave was frequently superimposed on negative response between II-N and III-N wave, and IV-PN waves were frequently feeble in single stimulation of the VL nucleus.
2. Peak latencies of these waves were estimated 3.4±1.2 msec in I-P, 10.6±1.8msec in I-N, 29±5msec in II-P, 55±6msec in II-N, 71±11msec in III-P, 90±11msec in III-N, 115±19msec in IV-P and 160±24msec in IV-N wave in ipsilateral central lead, and 4.0±1.5msec in I-P, 11.1±2.1msec in I-N, 31±4msec in II-P, 57±6msec in II-N, 74±13msec in III-P, 92±12msec in III-N, 118±24msec in IV-P and 156±29msec in IV-N wave in contralateral central lead.
3. The first deflection time of I-P wave, which meant beginning of the evoked response, was 1.5-1.8msec in stimulated side of central cortex and 2.2-2.9msec in contralateral central lead, which were obtained in 5 cases precisely measured.
4. The impulse, which provoked I-P N waves, was thought to be conducted from stimulated VL nucleus to the contralateral cortex directly via corpus callosum with 36-43m/sec of velocity.
5. Cortical evoked responses following suprathreshold low frequency stimulation (5-12Hz) of the VL nucleus showed invariably augmenting response and recruiting-like augmenting response.
Augmenting response, which consisted of a train of growth in both. positive and negative components, was evoked when each stimulus was given on the descending phase from the peak of III-N or augmented negativiy to the bottom of following positive wave of the preceding response. Analysis of augmentation suggested that synchronization of preceding evoked IV-P and present II-P wave would be occured in augmented positivity and synchronization of preceding evoked IV-N and present II-N or III-N wave would be occured in augmented negativity.
Recruiting-like augmenting response was obtained when each stimulus was given on the ascending phase from the bottom of deep IV-P to the following negative wave of the preceding response. Recruiting-like augmentation was shown to be a similar response as augmenting response in the fundamental pattern of averaged evoked activity, although development of negativity and attenuation of positivity caused recruiting-like pattern. Component analysis of recruit-ing-like augmenting response revealed that predominant development of negativity was thought to be the result of the synchronization of preceding evoked IV-N and present II-N or III-N wave. And attenuation of II-P wave was thought to be the result of the desynchronization of preceding IV-N and present II-P wave.
Responses following lower frequency stimulation with 4Hz or less were similar to responses following single stimulation.
Subthreshold stimulation were thought to be difficult to induce any growth of negative cortical response.
6. It was clarified that cortical evoked response following stimulation of the VL nucleus was influenced by components and phases of the preceding cortical response, suggesting that human specific thalamic system would play a possible important role in modulation upon cortical electrical activity.

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Serum complement levels in 200 patients with hepatic disorders or jaundice were determined in succesion by the 50% hemolysis method of Mayer.
The following results were obtained.
1) In cases of acute hepatitis, serum complement levels (CH₅₀) were high during 12 weeks after the onset and gradually returned to normal range in 4 weeks with the improvement of clinical symptoms. While, CH₅₀ levels in acute persistent hepatitis showed a slightly elevated value over 8 weeks after the onset of illness and fluctuated with exacerbations.
2) In cases of chronic hepatitis, CH₅₀ levels at the stage of remission remained in normal range and showed an indefinit fluctuation by relapsing.
3) CH₅₀ levels in liver cirrhosis were low and in some cases unable to be estimated and with exacerbations became lower gradually.
4) In cases of drug induced (toxic) hepatitis, CH₅₀ levels showed an markedly elevated value and remained high during a few weeks after jaundice had faded.
5) In cases of intrahepatic cholestasis with persistent jaundice, CH₅₀ levels were high and correlated well with serum bilirubin variation.
6) It wes observed that CH₅₀ levels showed a tendency to decrease in cases of chronic hepatitis and liver cirrhosis in which serum γ-globulin increased and RA factor or/and antihepatic antibody appeared.

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Various factors to influence serum complement activity were studied in hepatic disorders.
The results were as follows:
1) The definit correlation was observed between serum complement hemolytic activity (CH₅₀) and immune adherence activity (CIA₅₀). As to the complement components in sera with low level of CH₅₀, C 1, C 2 and C 4 activity were reduced and C 3 activity remained relatively normal.
2) CH₅₀ levels in patients with hypersplenism were subnormal and returned to normal range after splenectomy.
3) In 5 cases out of 72 patients with active chronic hepatitis, CH₅₀ levels in the normal range became suddenly unable to be measured by routine method and recovered normal after several weeks or months showing no change in the clinical features. The sera, at the stadium impossible to measure CH₅₀ levels, inactivated the complement activity of fresh human serum and guinea pig serum, and showed anticomplementary activity suggesting of Ag- Ab- C complex formation.
4) Most of anticomplementary activities in liver cirrhosis were suspected to be non-specific reaction due to abnormal protein metabolism.
5) There was a tendency of the low CH₅₀ level in cases of 6MP administration, while no influences on CH₅₀ level fluctuation in clinical course were found with glucocorticosteroids or azathioprine administration.

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Recently there have been developed various activators of the brain metabolism as therapeutic agents for the treatment of functional deficiency of the brain in residual lesions due to traumatic and vascular disturbances of the head and in geriatric mental disorders. I have investigated the effects of these agents on brain glucose metabolism, cerebral blood flow, systemic blood pressure, EEG, cerebral oxygen consumption, cerebral carbon dioxide production, cerebral lactic acid release, and cerebral glucose uptake by means of the brain perfusion and in vivo chronic administration of these agents.
The agents studied were four in all; namely, 1) 5'Cytidine monophosphate-2 Na, 2) CDP-choline, 3) Pyrithioxin, and 4) Meclophenoxate.
For the brain perfusion I used the method which has advantage of studying the direct effect of the agents on the brain tissue only without involvement in the metabolism of organs other than brain, in which the brain circulation is shunted from the systemic circulation and artificial blood of known composition is perfused. Furthermore, since the constant radioactivity through the experiments of [U-¹⁴C] glucose in the blood always maintained, it is easy to know its involvement in the brain metabolism. The results may briefly be summarized as follows.
1) When the brain perfusion is done with the blood containing Cytidine monophosphate, the incorporation of the blood [U-¹⁴C] glucose into the cerebral metabolites is enhanced as compared to the perfusion of blood without cytidine monophosphate.
2) In the experiments where Pyrithioxin or Meclophenoxate is acutely administered, there can be observed no effect on the metabolic rate of the brain nor on the incorporation of blood [U-¹⁴C] glucose into cerebral glucose related substances.
3) There can be seen no effect of chronic administration of Pyrithioxin or Meclophenoxate on the [U-¹⁴C] glucose metabolism of mouse brain.
4) When the brain is perfused with the blood containing CDP-choline, the rate of incorporation of [U-¹⁴C] glucose (RSA) into the cerebral glucose metabolites is enhanced and it acts as to activate the brain glucose metabolism, when compared with the standard perfusion corresponding to the EEG level.
5) To the rate of cerebral blood flow, CDP-choline acts as to increase it temporarily, while Pyrithioxin or Meclophenoxate has no effect at all.
6) The systemic blood pressure is raised temporarily by Pyrithioxin or Meclophenoxate, while it is decreased temporarily by CDP-choline.

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Recently, as the secondary structure of protein structure has come to be distinctly reflected by the ultraviolet circular dichroism (CD) spectrum, an attention has been called to the protein structure of cell membrane by circular dichroism. Lenard and Singer have demonstrated by CD spectrum that the chemical fixation of the cell membrane induces changes in the structure of native red cell membrane, but it is not clear whether or not their conclusion is applicable in common to the membrane of other cells.
One of the objects of the present study was to clarify whether the conclusion of Lenard-Singer can be applied generally. For this purpose the author studied changes in the protein structure occurring after the chemical fixation of mitochondrial particles of the rabbit liver and rabbit red cell membrane and bovine serum albumin.
It seems quite significant to elucidate the general applicability of their conclusion by clarifying the conformational changes in cell membraneous proteins and other proteins induced by the chemical fixation as it would offer a great parameter to grasp the molecular structure of native cell membrane.
The second objective was to elucidate the specific phenomenon, i.e. the cause of red shift of the cell membrane, by the circular dichroism.
On the basis of the findings obtained in the observations of those changes occurring in mitochondrial particles, red cell membraines and bovine serum albumin after the chemical fixation, the author proposes here a dynamic cell membrane model as suggested by Seno. The results of the study may be briefly summarized as follows.
1) In the investigation of spectra of the BSA fixed with formaldehyde it has been clearly demonstrated that the aggregation of protein molecules induces the red-shift of the CD spectrum confirming the theory of Lenard-Singer to be valid.
2) Of the findings reported by Lenard-Singer, the facts that chemical fixation induces about 30% (corrected value, 50%) adhesion of helical structure to the cell membrane, and that the chemical fixation with KMnO₄ brings about the loss of 100% helical structure of the membraneous protein molecules were also demonstrated similarly in the rabbit liver mitochondrial particles and rabbit red cell membrane, indicating that these findings are fairly common in all kinds of cells.
3) However, the extent of conformational changes in the cell membrane induced by the fixation with KMnO₄, OsO₄ or glutaraldehyde as concluded by Lenard-Singer differs from author's own finding. Regarding this problem it seems to be desirable to study further many other kinds of cells.
4) Noting the resemblance of the CD spectrum of BSA fixed with formaldehyde to that of the cell membrane, the author has assumed that the cell membraneous protein molecules are arranged in the form of aggregation. On the basis of this assumption the author has proposed a modified form of the unit membrane model. This modified unit membrane model has the possibility of being readily transposed to a particulate unit model and it has been designed from the perspective of the circular dichroids as against the dynamic membrane model proposed by Seno.

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Characterization of the non-glucuronide conjugated bilirubin fraction showing positive phosphate-ester reaction reported by Kondo was studied. The results were as follows:
1) After ³²P-sodium phosphate was injected into the duodenum of Wister strain rat, bile was collected from the external bile fistula. Crude bilirubin was prepared from the collected bile by Sakamoto's method. ³²P-radioactivity was localized at the ester-form bilirubin fraction of the crude bilirubin. ³²P-radioactivity was detected in the bile during from 10 to 15 minutes after the duodenal injection. Total amount of the excreted ³²P-radioactivity collected up to eight hours after the loading was 1.7%.
2) Localization of the radioactivity at radioautogram and radio chromatogram obtained from the paper chromatogram of the ester-form bilirubin fraction separated by Kondo's method was found at the same spot showing positive phosphate-ester reaction reported by Kondo.
The radioactivity of the azo-pigments yielded from the ester-form bilirubin localized at the spots showing positive phosphate-ester reaction.
3) Amount of bilirubin phosphate fraction increased in the rat bile after carbon tetrachloride poisoning. Amount of bilirubin phosphate fraction in the bile collected from the patients with liver cirrhosis was much more than that from normal subject. This results suggests the bilirubin phosphate fraction increased compensatly when the bilirubin glucuronide fraction was decreased in the liver parenchymal damage.

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The bile was collected from the rat external bile fistula after the duodenal administration of ³⁵S-sodium sulfate. The exsistence of bilirubin sulfate in the bile was studied. Also clinical significance of bilirubin sulfate was studied useing jaundiced urine obtained from the patients with acute hepatitis, liver cirrhosis and obstructive jaundice. The amount of sulfate was measured by Weber & Schalm's method. The results were as follows:
1) Appearance of ³⁵S-radioactivity into the rat bile was found from 5 to 6 minutes after the duodenal administration of ³⁵S-sodium sulfate. The total amount of radioactivity up to 8 hours was 42.1% of given ³⁵S-radioactivity.
2) Crude direct bilirubin fraction obtained from the rat bile contained 88% of total bile radioactivity.
3) Crude direct bilirubin was fractionated with paper chromatography, then ³⁵S-radioactivity was dectected with radioautogram and radio chromatogram obtained from the paper chromatogram. The ³⁵S-radioactivity was found at two spots of Rf 0.32-0.41 and 0.20-0.25. The radioactivity was localized at the original spots of paper chromatogram obtained from the mixture of direct bilirubin and ³⁵S-sodium sulfate.
4) Amount of bilirubin sulfate decreased at 12 hours when the liver parenchyma was highly damaged after acute carbon tetrachloride poisoning, then increased.
5) No significant corelation was found among the ratio of bilirubin sulfate to direct bilirubin and non-specific colloid reactions, serum transaminases, alkaline phosphatase, total bilirubin and direct bilirubin.
6) Bilirubin sulfate obtained from the jaundiced urine was ranged from 2.09 to 10.0% of the direct bilirubin and averaged 5.98% when bilirubin sulfate was caliculated as bilirubin monosulfate.

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Since the softening of the utero-vaginal tract accompanying the advance in pregnancy roughly parallels the change of estrogen in pregnancy urine, it seems that estrogen plays an important role in the softening mechanism of the delivery tract. Up to date there are many reports concerning the relationship of estrogen in the pregnancy urine, especially of estriol, to the foetal and placental mechanisms, but there seems to be no report on estrogen contents in pregnancy urine with observations on changes of cervical canal factors in one and the same individual. For the purpose to elucidate whether or not there exists an estrogen fraction that has a close relationship with conditions of the cervical canal the author conducted fractionation and estimation of 8 fractions of urinary estrogen with lapse of time, starting from 10 gravid months, while pursuing changes in the cervical canal factors in the same individual, and compared results in the individual with normal softening of the canal and those with delayed softening. In addition, the significance of estrogen in the field of obstetrics was studied from the aspects of level of 8 estrogen fractions and clinical course in individuals administered estriol agent. The results may be briefly summarized as follows.
1. The estriol fraction in the urine at 38 gravid weeks is above 95%.
2. Judging from the changes of each fraction, estriol and 16-epi-estriol are the fractions that are closely associated with the cervical canal.
3. In the individual with cervical canal of mature type from the beginning the level of urinary estriol is relatively high but it does not form peak; the estriol level in the case with the cervical canal in the process of transformation from immature type to the mature shows a peak once and later it decreases; and in the case with cervical canal which remains as immature type the peak is small or flat.
4. When estriol agent is administered to individual with cervical canal of immature type, the urinary estriol shows somewhat larger peak, resulting in the maturation of cervical canal and delivery time is shortened.

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By means of the brain perfusion slightly modified of the method by Geiger artificial blood with L-[U-¹⁴C]-alanine was perfused and circulatory disturbances was induced by such artificial decrease of brain blood flow. In this instance, alanine metabolism in the brain was compared with the metabolism observable during the standard brain perfusion. The results are briefly summarized as follows.
1) In the blood flow disturbing experiments where the blood flow was lowered by 43.0-60.0%. oxygen consumption, carbon dioxide formation and glucose uptake were markedly decreased, while the output of lactic acid from the brain was increased as compared with respective values in standard brain perfusion.
2) Despite the fact that there was no significant difference in the alanine concentration between the arterial and venous blood, there could be observed a significant decrease in the radioactivity of the venous blood when compared with that of arterial blood. This indicates clearly that alanine is being exchanged between the blood and brain. In addition, it has been demonstrated that the radioactivity of blood in the blood flow disturbing experiments was 3.8±1.10% as against 5.90±1.80% in the experiments of standard brain perfusion.
3) About 0.42-0.33% alanine in the blood was taken up and it was oxidized completely to carbon dioxide by the brain, showing no significant difference between the standard perfusion and blood flow disturbing experiment.
4) In the case of blood flow disturbing experiments there were observed a marked increase of lactic acid and an increasing tendency of alanine in the brain.
5) In both the standard brain perfusion and blood flow disturbing experiment alanine taken up by the brain within 70 minutes contained 50% of ¹⁴C in the brain. In the latter experiments the rate of ¹⁴C-incorporation into glutamic acid, aspartic acid and glutamine was decreased and its incorporation into lactic acid was increased.
6) In the blood flow disturbing experiment radioactivity in glutamic acid, aspartic acid, glutamine, alanine and lactic acid in the brain tended to decrease. The radioactivity of GABA was greater than that of glutamic acid, there could be observed a glutamic acid-GABA compartmentation phenomenon. just as in the experiments with [U-¹⁴C] glucose in the perfused blood.

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