(1) Characterization and purification of canine gastric glucagon.
Among the gastrointestinal tissues of the dog, upper stomach contains the highest concentration of gastrointestinal glucagon (IRG). In order to purify and characterize it, IRG was routinely extracted from mucosal scrapings of the fundus of the stomach from 5 dogs, using an acid-alcohol solution containg 0.01 M benzamidine. Ether-ethanol precipitates of these acid alcohol extracts were subsequently purified by gel-filtration and ion-exchange chromatography. The protein content in the coulumn effluent was monitered at either 280nm or 254nm. IRG was measured using two antisera; 30-K, a serum highly specific for pancreatic glucagon (PG), and K-4023 which strongly crossreacts with gut glucagon. IRG was eluted as one major peak corresponding to PG by gel-filtration on Bio-gel P 30 in 3M acetic acid and then on the same gel in 0.05 M NH₄HCO₃. Immunoassay dilution curve of the major peak was parallel to the dilution curve of PG standard. The IRG peak obtained after the second gel-filtration was resolved by polyacrylamide gel disk electrophoresis (PGDE) in urea at pH 8.7 into three immunoreactive components. The main component was identical to PG in its mobility. The faster minor component was very likely desamido-glucagon. A slower component, barely entering the gel, reacted more strongly with K-4023 and therefore presumably contains hydrolized fragments of glucagon. Thus the IRG peak obtained after gel-filtration was not immunologically homogeneous. Therefore the IRG peak from the alcaline Bio-gel coulumn was rechromatographed on an ion-exchange column DEAE-Sephadex A-25. Two or three immunoreactive peaks were obtained. The peak, which had the highest specific immunoreactivity was eluted similarly to the PG.
This major IRG peak was then subjected to PGDE. Only one immunoreactive component was identfied and it reacted equally well with the specific antiserum 30-k and the nonspecific K-4023.
This indicates that this IRG component from the ion-exchange column was immunologically pure.
When the gel was stained with coomassie-blue one stainable band was seen which has an electrophoretic mobility similar to standard PG. This peak was also tested for receptor binding activity using liver plasma membrane and it was found that the displacement of I¹²⁵-glucagon by serial dilution of this material was similar to that obtained with standard PG.
The IRG peak from the ion-exchange column DEAE-Sephadex A-25 was rechromatographed on an ion-exchange coulmn CM-Bio-gel A. Only one immunoreactive peak was obtained. This IRG peak is tested its biological activity.
(2) Biosynthesis of glucagon in canine gastric mucosa.
2g of gastric mucosa were incubated in 3 ml Krebs-Ringer bicarbonate buffer which contained trasylol and antibiotics, gassed with 95% O₂-5% CO₂ at 37°C for 5 hrs in the presence of ³H-tryptophan. Gastric mucosa was rinsed with Krebs-Ringer bicarbonate buffer, then homogenized in 10% trichlor acetic acid added 10mg of porcine pancreatic glucagon (Lilly). IRG was extracted and purified by the same method discribed above. One of the peaks of ³H-tryptophan was observed in the PG region. PGDE of this peak showed that one major peak of ³H-tryptophan was comigrated with PG.

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Effects of VIP on cyclic nucleotides formation in various central nervous tissue of rat were examined. The tissue including anterior pituitary prepared from rat brain after decapitation were incubated in Krebs-Ringer bicarbonate buffer containing 0.1% glucose and 0.1% bovine serum albumin and synthetic hormone to be tested. Cyclic AMP was determined with competitive protein binding assay and cyclic GMP was assayed by specific radioimmunoassay system. Hormones released into incubation medium were determined with radioimmunoassay system specific for each hormone.
Synthetic VIP increased cyclic AMP concentration, not cyclic GMP concentration, in brain medulla, hypothalamus, pons and anterior pituitary, but not in brain cortex, cerebellum and posterior pituitary. Minimum effective dose of synthetic VIP was 1μg/ml. Other synthetic gastrointestinal hormones (Gastric Inhibitory Peptide, Motilin, Bombesin, Substance P and Gastrin) did not affect cyclic AMP concentrationin every tissue. Synthetic VIP strongly stimulates cyclic AMP production, but it (in the concentration between 100μg and 10μg) did not stimulate hormone release into incubation medium from anterior pituitary (GH, TSH, LH, FSH and Prolactin) as well as from hypothalamus (TRH, LHRH and Somatostatin).
Although somatostatin decreased basal cyclic AMP level in anterior pituitary and inhibited TRH and PGE stimulation of cyclic AMP formation, it did not inhibit stimulatory effect of synthetic VIP on cyclic AMP formation in anterior pituitary and hypothalamus, and also propranolol (10^-4M) did not.
These results suggest strongly that VIP may have some physiologic actions on specific portions of rat central nervous system and, in anterior pituitary and hypothalamus, its action may be different from stimulation of hormone release from these two tissues, and those may indicated the possibility of presence of intracellular compartment of functioning cyclic AMP.

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Synthetic gastrointestinal hormones (VIP, GIP, Substance P and Bombesin) were infused at a dose of 20 pmol/kg/min for 10 min respectively.
Plasma IRI concentrations in the pancreatic vein and net release of insulin from the pancreas were significantly augmented during infusion of all of these peptides. The effect of substance P on IRI release was the most remarkable and its effect was transient compared with those of other peptides.
Plasma IRG concentration and net release of glucagon from the pancreas were also stimulated by VIP, substance P and GIP. However, bombesin did not increased glucagon secretion from the pancreas, but stimulated gut glucagon-like immunoreactivity.
These results indicate that many gastrointestinal hormones may play a role at the regulation of insulin and glucagon secretion through the specific action respectively.

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In order to investigate the effect of vasoactive intestinal peptide (VIP) upon the secretion of insulin and glucagon, experimental studies were carried out using a local circulation of the pancreas of anesthetized dogs. VIP was infused into the pancreatic artery in doses of 20, 100, 200 and 400ng for 10 minutes. The blood glucose level in the femoral artery did not change during and after VIP infusion. Blood flow rate in the pancreatic vein did not change throughout the experiment with graded doses of VIP. The secretion rate of insulin in the pancreatic vein increased transiently during the infusion of 20ng of VIP but a constant and significant increase in insulin secretion was observed in the experiment with a dose of 400ng of VIP. In contrast, the glucagon secretion in the pancreatic vein increased significantly following the infusion of VIP in doses of 20 to 400ng for 10 minutes.
To determine the regulation of VIP secretion, the plasma levels of VIP were measured in the animal experiment as well as in human study. Radioimmunoassay for VIP used in the present study was established by Yanaihara and in the assay system 10pg of VIP could be detected. The fasting levels of plasma VIP did not exceed 200pg/ml in patients with diarrhea or diabetes mellitus, except for the cases with renal failure. Plasma VIP did not change during glucose tolerance test. A small rise in VIP was observed in diabetic patients during arginine infusion test. When glucose (2g/kg) or amino acids mixture (1g/kg) was infused into the duodenum of the conscious dogs, plasma VIP in the mesenteric vein as well as in the vena cava increaed by 30 minutes. However, delayed rise in plasma VIP was observed in the mesenteric vein after butter Administration in dogs. From the present studies, it was suggested that VIP secreted from the gut during the absorption of nutrients might modulate the secretion of insulin and glucagon from the pancreas.

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Using radioimmunoassay systems respectively specific for substance P (SP), somatostatin, motilin and VIP, distributions of these hormones in porcine, human, canine, monkey and tupaia tissues were investigated in terms of the immunoreactivities of the tissue extracts. The radioimmunoassay systems had been developed exclusively with highly purified synthetic polypeptides. SP, somatostatin and VIP immunoreactivities were found throughout the porcine intestinal extracts and remarkably high concentration of VIP was detected in the colon, 340pg/mg (wet weight of tissue). The largest amount of immunoreactive motilin was found in the porcine duodenum, 109pg/mg, and in the jejunum, 131pg/mg. The human gastrointestinal tract extracts were also found to contain high level of VIP immnoreactivity, especially in the duodenum, 286 and 270pg/mg, and jejunum, 225pg/mg. In the canine and monkey tissue extracts, immunoreactive SP, somatostatin and motilin were detected throughout the intestines, and the largest amounts of SP and somatostatin were found in the canine antrum, 2.7±2.0 and 38.9±17.9pg/mg, respectively, but significant quantities of the two hormones were also present in the duodenum, jejunum and colon of both the animals. Relatively high concentration of somatostatin-like immunoreactivity was detected in the lower part of jejunum and ileum. This concentration of immunoreactive motilin in the canine and monkey duodenum was significantly lower than that of porcine and human tissues. This may be partially due to the species variation in the primary structure of this hormone. In the case of the tupaia, the four kinds of hormone immunoreactivities were found throughout the intestinal tissue extracts, and the distribution patterns of them were similar to those in the canine and monkey tissues, although some variations were found in their concentrations. The level of immunoreactive SP in the tupaia tissue extracts was higher than that in the other animals examined. SP was also detected in the lung. As in the porcine case, the highest level of immunoreactive VIP, 150pg/mg, was found in the tupaia colon extracts. In the pancreas, all of the hormones examined were found and the amount of somatostatin immunoreactivity, which was relatively large, was of the same order as those of SP and VIP, but the concentration of immunoreactive motilin was considerably low, 3.1±1.0pg/mg. In the canine brain tissue extracts, relatively high concentration of immunoreactive SP, somatostatin and VIP was found, while the amount of immunoreactive motilin was extremely low. In the pituitary extracts, the concentration of immunoreactive motilin in the anterior lobe, 139.9±37.4pg/mg, was higher than that in the posterior, 70.8±18.6pg/mg, while SP-, somatostatin- and VIP-like immuroreactivities were rich in the posterior lobe, 184.4±44.6, 581.0±140.4 and 195.0pg/mg, respectively. These results revealed that SP-, somatostatin-, VIP- and motilin-like immunoreactivities are distributed in the manner characteristic to each of the hormones over the regions examined.

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Brown et al. isolated a new gastrointestinal active substance, motilin. However, there are still some discrepancies in motilin research. Recently Yanaihara et al. have synthesized motilin and succeeded in making antisera raised in guinea pig and rabbit against synthetic motilin. So we measured plasma motilin with dextran-charcoal radioimmunoassay method using antiserum GP1103. Since it is important to elucidate the significance of pathophysiological role of motilin, we studied in the present experiment the relationship between plasma motilin level and gastric motor activity, and have measured plasma motilin levels in normal subjects and patients with diabetes mellitus, hyperthyroidism and chronic liver diseases. In seven healthy conscious dogs plasma motilin level was measured while the gastric contractile activity was recorded by means of a chronically implanted force transducer. When the gastric contractile activity was in the interdigestive state, plasma motilin level was always elevated in all dogs. The high plasma concentration of motilin was lowered by ingestion of food and this low level of motilin lasted as long as the gastric motor activity was in the digestive pattern. However, plasma motilin level returned to the high level again by the time when the gastric motor activity was in the interdigestive state. Furthermore, we observed that plasma motilin concentration fluctuates in complete association with gastric contractions during the interdigestive state. In human study the similar findings wereobtained. Plasma motilin concentration during the prolonged fasting also fluctuated at approximately 100 min intervals and the episodic secretions appeared to parallel the occurence of the interdigestive gastric contraction. Besides, in 8 normal subjects we studied the change of plasma motilin levels before and after meals. In fasting plasma motilin fluctuated as described above, and plasma motilin rised transiently within 30 min after meal and then decreased gradually from 30 min after meal. After administration of either 50g of glucose orally or 25g of glucose intravenously plasma motilin decreased gradually in normal subjects. In normal subjects, following the intravenous administration of 0.1U/Kg regular insulin, plasma motilin decreased in accordance with diminished blood glucose. Fasting plasma motilin levels in normal subjects were widely distributed ranging from less than 50pg/ml to 540pg/ml, with the mean of 224±36 (SE) pg/ml. In hyperthyroid patients fasting plasma motilin was 314±64pg/ml, which was not different from that of normal subjects. Patients with diabetes mellitus and with chronic liver disease had fasting plasma motilin levels of 505±89pg/ml and 391±71pg/ml, respectively, both of which were significantly higher than those in normal subjects (p<0.05). Following 50g oral glucose loading, plasma motilin levels decreased in all three groups as well as normal subjects. These results suggest that motilin is released in the fasting state and plays an important role in the gastric activity: especially interdigestive motor activity of the stomach may be controlled by plasma motilin concentration in the dog and man. We have proposed therefore the “interdigestive hormone” for this newly identified gut hormone.

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We developed a method of radioimmunoassay and immunohistochemical technique to study the physiological action of somatostatin (SRIF) in the hypothalamus, the pancreas and the gastrointestinal tract. We obtained the antiserum following immunization with synthetic SRIF which had been conjugated with human a-globulin. Synthetic N^α-tyrosylated SRIF was labelled with 125I using the lactoperoxidase method and purified on a Sephadex G-10 column. Each organ was homogenized in ice-cold 2N-acetic acid, then suspended in boiling water, centrifuged, and then lyophylized. The sensitivity of the assay varied from 3.9pg to 7.8pg, with the tracer having a specific activity of 100μCi/μg. The displacement curve of extracts from the pancreas and the hypothalamus was shown to be parallel to the standard curve. This antiserum was highly specific for SRIF with negligeble or no cross reactivity. We performed the immunohistochemical study by the enzyme labelled antibody method of Nakane using this anti-SRIF, rabbit anti-glucagon and guinea pig anti-insulin serum.
It was shown using specific RIA that SRIF was contained in the gastrointestinal tract, and especially in the stomach and pancreas. In normal rats, SRIF containing cells were found to be located mainly in the periphery of the pancreatic islets.
The SRIF content of both the pancreas and the isolated islet of streptozotocin-diabetic rats was significantly greater than the content of control rats. SRIF containing cells in islet of the pancreas of diabetic rats were increased in number and scattered into the central portion as well as in the periphery. In the streptozotocin-diabetic rats with daily injections of 1 unit of lente insulin for 4 weeks, the SRIF content of the isolated islet was significantly decreased in comparison with the content of streptozotocin-diabetic rats without insulin.
In C57BL/Ksj dbdb diabetic mice aged from 18 to 28 weeks, the content of SRIF of the pancreas had greatly increased when compared to the content of litter mates. SRIF containing cells in the islet of diabetic mice were increased in number and scattered in the central portion. In C57BL/6J obob diabetic mice, again aged from 18 to 28 weeks, the content of SRIF of the pancreas had only shown a slight increase. In chinese hamsters diabetic due to insulin deficiency, SRIF containing cells were scattered throughout the pancreatic islet.
Because in the diabetic animals described above the pancreatic SRIF had increased, it is conceivable that pancreatic SRIF may be an important factor in the pathogenesis of diabetes mellitus.

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The present investigation was undertaken to make clear the differences of the responsiviness of the rat exocrine and endocrine pancreas to caerulein in vivo and in vitro. Caerulein, an active decapeptide found in the skin of Australian amphibia Hyla caerulea, posesses remarkable similarities and biological activities to cholecystokinin-pancreozymin (CCK·PZ).
(1) Caerulein strongly stimulated the rate of flow and amylase output both in vivo and in vitro.
(2) The minimum doses which elicited the maximum secretion of the pancreatic juice and enzyme from the isolated and perfused rat pancreas and in vivo experiments were 0.05ng/ml and 10mg/ml/min, respectively.
(3) Supramaximal stimulation with caerulein resulted in significantly decreased pancreatic juice and enzyme secretion.
(4) Increase of insulin and glucagon were not observed by the doses which stimulated the rate of amylase output and flow of pancreatic juice, but they were elicited by only supramaximal dose of caerulein (more than 10ng/ml, in vitro and more than 100ng/kg/min, in vivo).
(5) Discrepancy of the effective dose of caerulein between on pancreatic exocrine and endocrine function was observed.
These results suggested that the exocrine responses of the pancreas may be due to the physiological effect of CCK·PZ, while the endocrine responses may be resulted from the unphysiological doses. It is important to differentiate the physiological effects from the pharmacological ones before coming to the condusion of the effects of G. I. hermones on the exocrine and endocrine pancreas.

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Pregnancy is characterized by an activation of the renin-angiotensin system. In the present study we compared the level of renin activity, angiotensin and angiotensin-degrading activity in pregnancy sera with each other, and discussed the role of placental aminopeptidase as an angiotensinase in the regulation of blood pressure.
Plasma renin activity (PRA) and angiotensin I (A-I) were determined using a radioimmunoassay technique in sera of normal pregnant women at different stages of gestation. Plasma A-I level in normal pregnant women was higher than that in non-pregnant women, but the difference was much less striking than PRA, which may be related to notably increased renin substrate level. A-I and PRA levels gradually rose with the extension of the period of pregnancy in the early stage of pregnancy, either leveled off or lowered in the middle stage and significantly reduced at the 7th month, and tended to rise again in the late stage.
It is well known that serum leucine aminopeptidase (LAP) or oxytocinase (cystine aminopeptidase, CAP) increases progressively with advancing gestation. Our previous studies revealed that the estimation of LAP in the presence of 0.02M L-methionine has permitted exclusive determination of placental LAP (P-LAP) activity existing in pregnancy serum regardless of the presence of apparently normal levels of serum LAP. Thus, it was shown that the assays of P-LAP and CAP manifest an identical enzyme activity.
The serum levels of LAP activity using colorimetry and angiotensinase activity estimated based on the blood pressure of rat using bioassay were compared in pregnancy sera. It was found that angiotensinase appearing in pregnancy sera had the same properties as P-LAP, with respect to heat inactivation at 60° for 30 minutes and 0.02M L-methionine inhibition. Subsequently, studies were made to see the relationship between P-LAP activity as angiotensinase and blood pressure level in 10 pregnant women. Blood pressure was lower in the middle or late stage of pregnancy than in the early stage, but rose again in the late stage of pregnancy or immediately before delivery. Both PRA and A-I levels were in accordance with the number of months of pregnancy. The activity of P-LAP levels determined simultaneously with PRA and A-I levels displayed sharp increases in the middle and late stages of pregnancy, but slightly decreased before delivery. These results suggest that P-LAP may be of protective value for elevation in blood pressure. Furthermore, these fmdings are highly suggestive of Yman's hypothesis that P-LAP (CAP) as an angiotensinase would be involved in the regulation of blood pressure.

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Two angiotensin H analogues are now available in clinical study. Comparative studies of the antagonistic potency and the agonisic effect between these two analogues, i. e., [1-Sarcosine, 8-Isoleusine] angiotensin II and [1-Sarcosine, 8-Alanine] angiotensin II, were done in normal subjects on various sodium balances and in hypertensives on sodium depletion. Both analogues had an agonistic pressor effect in normals. There was found agonistic effects in both compounds not only on blood pressure, but also renin and aldosterone secretion and creatinine clearance in normal subjects on regular diet. The agonistic pressor effects changed with different sodium balances. In low sodium phase, these pressor effects were minimized. Agonistic effect of [1-Sar, 8-Ile] A II was greater than that of [1-Sar, 8-Ala] A II in all sodium balances. The antagonistic effects of both compounds were also varied by changing sodium balance, being greatest in low sodium phase. In hypertensives on sodium depletion, blood pressure responses (δBP) to these A II analogues were significantly correlated (r=0.8, n=20). These results indicate that pretreatment of sodium depletion is necessary to prevent the side effects caused by agonistic pressor effects of these analogues, and also to predict renin dependency in hypertensive patiens efficiently.
Results of clinical application of [1-Sar, 8-Ile] A II are as follows:
(1) This compound showed a pressor response in low reninemic state, and a depressor response in some cases with high plasma renin activity (PRA). There was a negative correlation between pre-infusion PRA and change in blood pressure following the infusion of this compound. In most high reninemic patients, a depressor response was observed after sodium depletion.
(2) In subjects with Cushing's syndrome and pheochromocytoma, a marked pressor response was observed.
(3)[1-Sar, 8-Ile] A II reduced blood pressure in a part of normotensive secondary aldosteronism, such as liver cirrhosis with ascites, Bartter's syndrome, congestive heart failure and renal tubular acidosis, and a case of Addison's disease.
(4) Subjects taking oral contraceptives with or without hypertension showed no reduction of blood pressure in our limited study. These results indicate that R-A-A system play an important role in the maintenance of normal blood pressure in normotensive secondary aldosteronism and of high blood pressure in a part of high reninemic hypertension.
Application of these A II inhibiting analogues should be useful for the understanding the role of R-A-A system in physiological and pathophysiological conditions in human.

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Angiotensin I-converting enzyme (peptidyl dipeptide hydrolase, EC 3.4.15.1) was solubilized from the membrane fraction of human lung using trypsin treatment and purified using columns of DE 52-cellulose, hydroxyapatite and Sephadex G-200. The purified enzyme was shown to convert angiotensin I to angiotensin II and also to inactivate bradykinin. The specific activity of the enzyme was 9.5 units/mg protein for Hippuryl-His-Leu-OH and 0.665 pmol/min per mg protein for angiotensin I. The enzymic activity obtained after trypsin treatment (1mg/200mg protein) for 2h could be divided into three components:(i) an enzyme of molecular weight 290000 (peak I),(ii) an enzyme of molecular weight 180000 (peak II) and (iii) an enzyme of molecular weight 98000 (peak III), by columns of DE 52-cellulose and Sephadex G-200. Km values of peak I, II and III fraction for Hippuryl-His-Leu-OH were identical at 1.1mM. pH optimum of the enzyme was 8.3 for Hippuryl-His-Leu-OH.

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There appears to be a complex relationship between the renin-angiotensin system and pro- staglandin generating systems. Recently, it has been reported that the use of inhibitors of pro- staglandin (PG) synthesis in Butter's syndrome resulted in correction of hypersecretion of rien, angiotensin and aldosterone as well as hypokalemia seen in this syndrome. This strongly suggested that PGs play a primary role in sodium regulation of the body, secondarily influencing renin-angiotensin-aldosterone system.
The present study was undertaken critically to examine these points.
Indomethacin suppressed PGs and the body weight increased due to the retention of sodium. Renin-angiotensin-aldosterone system was also suppressed at the same time. However, when the body fluid increased to a certain level, sodium excretion from the kidney began to rise in spite of the sustained suppression of PGs. This sequence of events is analogous to the escape phenomenon seen in mineralocorticoid excess syndrome. However, an interesting thing was that indomethacin seemed to inhibit the increase of potassium excretion accompanying the rise of sodium excretion at that time. In contrast, another inhibitor of PGs, ibuprofen, though simulating mostly the action of indomethacin, did not reveal the inhibition of potassium excretion. Thus, it did not seem that the effect of indomethacin was directly related to its effects as a PG inhibitor. Rather, indometha- cin may primarily cause an intrarenal circulatory change, which in turn causes various changes in all the parameters including PGs, renin-angiotensin-aldosterone system. It is not clear whether the effect of indomethacin to suppress sodium potassium exchange in the renal tubules is related to the intrarenal circulatory changes.
Furthermore, during the prolonged treatment with indomethacin, PGs as well as renin-angiotensin-aldosterone systems tended to return to and even to surpass the initial levels. Similar perplexing results were also seen during the treatment of essential hypertension with diuretics. Once elevated PGs and renin-angiotensin-aldosterone systems all tended to return to the initial state under the sustained influence of diuretics. The behavior of these parameters in congestive heart failure was also to be noted because of their peculiar movements. PG generation system and renin-angiotensin-systems dissociated from each other in this pathological condition. This may be due to the abnormal distribution of intra-renal blood flow. These observations when taken altogether, request us to return to the primitive question; What are the role of renin-angiotensin- aldosterone systems and that of PGs in the sodium regulation of the body?

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Radioimmunoassay for two hypothalamic peptides, somatostatin (somatotropin-release inhibiting factor: SRIF) and neurotensin (NT), was described. Tyr₁-SRIF was radioiodinated by a modification of Chloramine T method and purified on Sephadex G-25 column using acetic acid as an eluant. Labelled tyr₁-SRIF was absorbed to Sephadex G-25 and eluted broadly after free 1251. The elution pattern of labelled tyr₁-SRIF was dependent to the molar concentration of the eluant. K_D value of labelled tyr₁-SRIF in each concentration of 1.0M, 0.5M, 0.1M and 0.05M acetic acid was 0.95, 1.61, 2.56 and 3.51, respectively. NT was labelled with 1251 by Chloramine T method. Labelled NT was purified on Sephadex G-25 using phosphate buffer saline with 0.1% gelatin (pH 7.6). In both assays, the separation of bound and free was performed by double antibody method.
Anti-SRIF serum was produced by the method of Arimura et al. and assayed in a final dilution of 1: 4000. The sensitivity of the assay was 10pg/tube. The dilution curves of various rat tissue extracts run parallel with standard curve.
Anti-NT serum was generated by immunizing rabbits with neurotensin-bovine thyroglobulin conjugates according to the method of Carraway and Leeman. In the radioimmunoassay, anti-NT serum was finally diluted at 1: 56000. The minimum detectable level was 10pg/tube. The dilu- tion curves of different regional extracts of the rat brain gave excellent parallerism with standard curve.
Brain and gastrointestinal distribution of immunoreactive SRIF (IR-SRIF) and NT (IR-NT) was examined in adult male rats. Tissues were extracted with 1ml of acetone per 100mg tissue weight. Total contents of brain IR-SRIF and IR-NT were 89.8±3.2ng and 71.6±1.8 ng, respectively. Hypothalamic contents were 24.4% of total in IR-SRIF and 11.4% in IR-NT, respectively. The remainders were found widely in other brain regions. The distribution of IR-SRIF and IR-NT in gastrointestinal tracts revealed the remarkable differences. IR-SRIF was found abundantly in antrum of stomach and pancreas. On the other hand, IR-NT was detected predominantly in jejunum and illeum.
The different distribution of IR-SRIF and IR-NT, especially in gastrointestinal tracts, may be closely related to local physiological roles of these peptides.

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Intravenous injection of synthetic substance P caused a significant increase in plasma prolactin (PRL) and growth hormone (GH) in urethane-anesthetized male rats. Simultaneous administration of either ι-dopa or nicotine significantly suppressed plasma GH increase induced by substance P, whereas plasma PRL responses to substance P were blunted by ι-dopa but not by nicotine. Plasma PRL and GH were also elevated by intravenous injection of neurotensin and xenopsin. Both β-endorphin and α-endorphin injected into the lateral ventricle significantly elevated plasma PRL and GH. β-endorphin is more potent than α-endorphin. Plasma PRL and GH responses to these opioid peptides were significantly blunted by naloxone, an opiate antagonist. Intraventricular injection of Vasoactive Intestinal Polypeptide (VIP) caused a significant and dose-related increase in plasma PRL, whereas plasma GH were not affected at the dose examined. Increases in plasma PRL induced by VIP were significantly blunted not only by ι-dopa but also by naloxone injected intravenously. These results suggest that substance P, neurotensin and endorphins stimulate the secretion of both PRL and GH, whereas VIP may stimulate PPL but not GH secretion in the rat.

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A gas-liquid chromatographic method for the simultaneous determination of phenobarbital (PB), carbamazepin (Carb), primidone (Prim) and diphenylhydantoin (DPH) in human biological fluids following therapeutic doses has been described.
After extraction with chloroform, the anticonvulsant drugs were methylated with phenyl-trimethylammonium hydroxide in dimethylformamide at 85° for 10 min. Liner temperature programming of a 1% OV-17 column was used to achieve separation and quatitation. The procedure described in the present paper is relatively simple, highly specific and sufficiently sensitive for use in routine clinical assays.
The anticonvulsant drug levels were measured not only in plasma but in saliva. Furthermore, the unbinding drugs in plasma were measured by means of ultrafiltration techniques. Ultrafiltration with using plasma aliquots (1 ml) was performed by an Amincon Centriflo membran cone (CF50) at 20°, 1,000 g for 25 min. It is applicable to screening large samples of patients.
Although it was found a patient having plasma level of 25 μg/ml, which has been considered to be the upper limit of the therapeutic range for DPH, the unbinding fraction in plasma was only 0.7 μg/ml. Thus, it should be noted that a knowledge of unbinding drugs would be more desirable for a therapeutic level than the total drugs level in plasma.

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We have developed a rapid, simple, specific and quantitative assay for plasma theophylline by high performance liquid chromatography. For reagents of deproteinization, various water-miscible organic solvents such as methanol, ethanol, I-propanol, acetone, acetonitrile and tetrahydrofuran were studied. Methanol gives best result. 50μl of plasma is mixed with 250μl of methanol solution containing 7-(2-hydroxyethyl) theophylline (4μg/ml in methanol) as an internal standard. Under these conditions, plasma proteins are precipitated and total theophylline is dissolved into the methanol solution. After the centrifugation, 20μl of the supernatant is injected into the high performance liquid chromatograph equipped with a Waters U6K injector, a 4mm×30cm μBondapak C18 column, a Model 440UV detector set at 0.01 absorbance units (280nm) and a 6000A solvent delivery system. The flow rate of the eluent of 0.01M sodium acetate (pH 4.0): acetonitrile (10:1, v/v) is 1ml/min. Elution of theophylline occurs at 9 minutes and that of internal standard at 12 minutes. The amount of theophylline is calculated from the peak height ratio of theophylline to internal standard. Recoveries are 98-102% and the standard curve obtained from theophylline spiked plasma is linear in range of 0μg/ml to 30μg/ml. Within-run and day-to-day reproducibility date for 5μg/ml are±1.0% and±1.5%, respectively. The assay can be accomplished in less than 15 minutes.

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A method for the separatory determination of d-and l-pentazocine in blood plasma by radio-immunoassay has been developed. In order to obtain the optically specific antiserum to l-pentazocine a hapten-carrier conjugate was prepared from l-pentazocine 2'-carboxymethyl ether by coupling with bovine serum albumin (BSA) by the mixed anhydride technique. The antiserum raised against this antigen proved to be highly specific to l-pentazocine by cross-reaction studies with the closely related compounds. The anti-dl-pentazocine antiserum was similarly produced by immunization with dl-pentazocine 2'-carboxymethyl ether-BSA conjugate. These antisera were used for radioimmunoassay where the B/F separation was carried out by the 50% ammonium sulfate method. The amount of the d-enantiomer was estimated indirectly by simultaneous quantitation of the l-enantiomer and total pentazocine. The blood levels of d- and l-pentazocine in man administered an officinal dose of the racemate were determined by the established method.

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A novel method for enzyme immunoassay of penicillin has been developped. The specific antiserum to penicillin was obtained by a new method. Disulfide bonds of cystine residues in bovine serum albumine (BSA) was reduced to thiol groups with dithiothreitol. An amino group of ampicillin was coupled with N-(m-maleimidobenzoyloxy) succinimide (MBS) and the resulting MBS-acylated ampicillin was coupled with the reduced thiol groups of BSA. Injections of ampicillin-BSA conjugate, emulsified with complete Freund's adjuvant to a rabbit yielded anti-penicillin serum. A new cross linking reagent N-(3-maleimidopropionylglycyloxy) succinimide was prepared and used for enzyme labeling of ampicillin. The labeling with β-galactosidase was performed in aqueous mild conditions by two stage reactions by the similar method reported previously for enzyme labeling of insulin and viomycin with MBS. The enzyme labeled ampicillin was purified by a chromatography on a Sepharose 6B column and the peak fraction in the enzyme activity was used for enzyme immunoassay. Enzyme immunoassay of penicillin was performed by the competitive binding procedure with the double antibody method. Sensitivity of the assay was 40μg/litter. Cross reactivity of penicillin G and cephaloglycin was tested and the assay method clearly distinguished cephalosporin from penicillin.

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A competitive binding enzyme-immunoassay (EIA) for phenobarbital (PB) in human sera has been devised. An enzyme labeled antigen, β -D-Galactosidase-m-diazo-phenobarbital (13-GAL-PB) was prepared by reacting m-diazonium chloride of PB with 13-galactosidase of E. coll. The insolubilized antibody was prepared by combining yeast cell walls with immunoglobulins from sera of rabbits immunized with bovine serum albumin-m-diazo-PB.
After 60 min competitive immunoreaction between the β P-GAL-PB and PB (antigen in samples) for the insolubilized antibody, the enzyme activity of free or bound β -GAL-PB in the supernatant or the precipitate separated by centrifugation, was measured by using o-nitrophenyl-β -D-galac-topyranoside as a chromogenic substrate. One to 400 ng of PB in 5 μl of serum samples (=0.25-80μ /g/ml of serum) could be successfully determined within 2 hours.
Concentrations of PB in 106 sera from epileptic patients measured by the EIA and the conventional gas liquid chromatography method were found to be correlated well (r=0.95, Y=0.96 x +1.08).
The method is very simple and can be practically used without any special equipments in the ordinal clinical laboratories.

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s an aid for designing a drug administration procedure, isoketopinic acid loading test was carried out to search the conjugation and excretion ability of patients.
Isoketopinic acid, 300 /mole, was dissolved in 50 ml of water containing 50 mg of NaHCO₃, and orally administered to fasting subjects, and 2 and 4 hours later, urines were collected.
One ml of the urine was added with 03 mg of cinnamic acid as an internal standard, extracted twice with 4 ml of ether. The ether phase was dried with Na₂SO₄ and reacted with diazomethane. After evaporation of the solvent, the methylated sample was dissolved in 0.2 ml of acetone and analysed by gas chromatography to determine the content of free isoketopinic acid.
The content of total isoketopinic acid was similarly determined after hydrolysis of the glucuronide by adding 03 ml of 1M NaOH to 1 ml of urine and heating at 70° for 10 min. The content of conjugate isoketopinic acid was obtained by subtracting the value of the free from that of the total.
About 50% of administered isoketopinic acid was excreted in 2 hr in urine of normal subjects, about 70% in 4 hr, mainly as the conjugate (TABLE I). Larger deviation in excretion amounts of total and conjugate isoketopinic acid was observed with hepatitis patients (TABLE II, III), whileclear decrease was observed with 2/3 of liver cirrhosis patients (TABLE IV) and a half of SMON patients (TABLE VI) tested.

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Antipyrine has been used as a model drug to investigate the drug metabolism in man. The plasma half-life of antipyrine (in vivo study) and microsomal aniline hydroxylase (in vitro study) were determined in patients with various liver diseases to demonstrate wheather in vivo drug elimination represented in vitro drug metabolism or not.
One of the metabolites of antipyrine, 3-hydroxymethy1-2-methy1-1-phenyl-3-pyrazolin-5-one (AN-CH₂OH), gave 6 per cent absorbance that of antipyrine under the same colorimetric procedure of antipyrine determination. However, using GLC method, AN-CH₂OH was not detected in the extract of serum drawn from a subject given antipyrine orally. Amount of unchanged antipyrine excreted in the urine was only 3 to 5 per cent in 24 hours and the longer the half-life was, the more unchanged antipyrine was excreted. Therefore, it was evident that elimination of antipyrine from plasma indicated velocity of antipyrine metabolism.
After observing a close correlation between aniline hydroxylase and antipyrine hydroxylase activities in rat liver microsome, aniline hydroxylase activity was used as an indicator of in vitro drug metabolism in man.
The values of plasma half-life of antipyrine and the values of microsomal aniline hydroxylase activity in patients with acute hepatitis (convalescent stage), and chronic hepatitis (active form) were widely distributed, but did not significantly differed from those of normal controls. On the other hand, the drug metabolizing activity in patients with liver cirrhosis was markedly decreased both in in vivo and in vitro studies.
The velocity of antipyrine metabolism and the microsomal aniline hydroxylase activity was proved to be well correlated (r=0.79) in subjects in which in vitro and in vivo study were carried out simultaneously.
From the results obtained above it was concluded that the elimination of antipyrine from plasma reflects hepatic microsomal drug metabolism.

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Aniline hydroxylation by human red cells was studied in the absence or presence of methylene blue. In the presence of methylene blue, aniline hydroxylation by human red cells was considerably accelerated. The effect of monoiodo acetate, removal of glucose, cyanide, aminotriazol and atebrin on the rate of aniline hydroxylation was also studied in the presence of methylene blue. On the basis of these results, the mechanism of acceleration of aniline hydroxylation by human red cells in the presence of methylene blue was discussed in relation to NADPH electron flow system in the red cells. The aniline hydroxylating activity of whole red cells in the body was shown to be comparable to that of one liver when methylene blue was given.

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We investigated the possibility that red blood cells dealkylate 4, 4' -bis(β -diethylamino-ethoxy) α, β-diethyldiphenylethane (DH), which caused a peculiar drug-induced lipidosis. It is suggested that this reaction involves hemoglobin since both the hemolysate, with or without ghosts and the crystalized hemoglobin caused dealkylation of DH. The dealkylation of DH in red blood cells was reduced by the replacement of oxygen with nitrogen and enhanced by NADPH. We investigated the interaction between DH and hemoglobin, since the absorption spectra of the hemolysate and crystalized hemoglobin in the presence of DH was similar to that of cytochrome P450. We speculate that red blood cells may have an important role in the extrahepatic metabolism of drugs.

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During the course of biochemical studies on drug metabolisms in injured liver, a new technique for determining FT-207 in blood and liver was developed by using a high pressure liquid chromatography. The pharmacodynamics of FT-207 was analysed by measuring its disappearance rate (K) from the circulating blood. Since K values vary depending upon the dose of FT-207 administered, a constant dose was intravenously or orally given to rats (100mg/kg body weight) and human subjects (ca.16mg/kg). The alterations of K values in rats pretreated for 3 days with phenobarbital or indometacin suggested that the disappearance rates seemed to be well correlated with the activity of drug-metabolizing enzymes in liver microsomes. Inhibition of ³H-uridine incorporation into liver RNA by FT-207 was unchanged upon pretreatment of rats with phenobarbital and indometacin. Clearances of FT-207 from blood were much slower in hepatoma patients with cirrhosis of the liver than malignant cases without liver impairement. The results may be consistent with the fact that FT-207 levels in blood tended to gradually increase with the daily oral administration in cases with liver diseases as compared with those without hepatic dysfunction.

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The pyrimidine antimetabolite such as futorafur (FT-207) is widely prescribed in carcinomas of breast and gastrointestinal tract. Since 1972, it has been suggested that FT-207 act through metabolic conversion to 5-FU and represented a chemical depot form of 5-FU. The newly synthesized antimetabolic drug, FD-1, is lipid soluble and believed that it is converted to FT-207 and N-3 compound, respectively. These two chemicals are further converted to 5-FU as the active metabolite for cancer therapy. For the preparation of the program for the treatment of the patients with carcinomas by FD-1, it is essential to estimate the concentration of FD-1 and its metabolites in biological fluides.
A high speed liquid chromatography was developed for the separation and estimation of FD-1 and its metabolites. To solubilize FD-1 in water system, paired ion chromatographic method using PIC^th B7 was applied. On high speed liquid chromatogram, 5-FU, FT-207, N-3 and FD-1 were separated and eluted in this order. The determination of 5-FU with sensitivity of 1ng/ml and of FD-1 with sensitivity of 10ng/ml, respectively. The results have suggested that the method would be sufficient for the clinical studies of these anticancer therapeutics.
The effect of each drug on cellular immunity was estimated individually by means of normal human lymphocyte culture system. For this purpose, the lymphocytes were separated into T and B cells by free flow electrophoresis. The blastogenesis of T cell against each drug was carried out in the presence of phytohemaggultinin-P, and of B cell was performed in the presence of porkweed mitogen. The results suggested that FD-1 as well as FT-207 inhibited the blastogenesis of T cell in the range of 50-60%. But N-3 compound less inhibited than these two. On the other hand, N-3 compound effectively inhibited the blastogenesis of B cell. The descrepancy of the effect of N-3 compound on the blastogenesis between T cell and B cell is not known. The metabolic pathway of FD-1 in vivo and the further metabolic pathway of its metabolites must be elucidated.

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Hydrocortisone analogues have been used widely in therapy as a potent glucocorticoid, but very few studies of their plasma concentration, metabolism, properties of glucocorticoid receptor and biological effects have been published.
A sensitive and specific radioimmunoassay for betamethasone (BM) and prednisolone (P) has been developed 1, 2). Using this method plasma concentrations of BM and P were measured in normal subjects and patients with liver diseases following oral administration of BM and P. Plasma concentrations of BM were detected already at 20 min after administration in normal subjects. The peaks of the plasma concentrations were reached at 1 or 2 hours after administration but the wide variations of maximum values were observed in both normal subjects and patients with liver diseases. Peak levels of BM in patients with liver diseases were higher than those in normal subjects a little. After the peak levels plasma BM rapidly fell and disappeared at 24 hour post-administration in normal subjects. In patients with liver diseases the disappearances of BM from blood were markedly delayed and the relatively high plasma concentrations of BM were maintained even at 24 hour post-administration. The half-time of disappearance of BM from blood was 3 to 4 hours in normal subject. On the other hand it elongated to 5 to 6 hours in patients with liver diseases.
Plasma cortisol (Fk) levels were suppressed already at 20 min after administration of BM and rapidly fell in normal subjects, but the disapperance of Fk from blood markedly delayed in patients with liver diseases. The half-times of plasma Fk were 1 to 1.5 hours and more than 3 hours in normal subjects and patients with liver diseases respectively.
There was a good correlation between the severity of the liver disease as measured by the ICG retention at fifteen minutes and removals of BM and Fk from blood.
The peaks of the plasma concentrations of P were also reached at 1 or 2 hours after administration in normal subjects and patients with liver diseases, but peak levels of P in patients with liver diseases were lower than those in normal subjects. After the peak levels plasma P fell rapidly more than BM and disappeared at 24 hour post-administration in patients with liver diseaeses as well as in normal subjects. The half-time of disappearance of P from blood was 2.5 to 3 hours.

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(I) At present it is assumed that the digitalis glycosides mainly undergo biotransformation in the liver. However, since the metabolism of the glycosides in cases of liver damage has not yet sufficiently been studied, there remain many problems to be solved in the future.
In attempt to clarify digitalis metabolism in cholestasis, the plasma digitoxin and digoxin concentrations of dogs given with a single intravenous administration were determined by radioimmunoassay under the following four groups; (1) surgically prepared biliary ligation,(2) fistula,(3) ligation after phenobarbital pretreatment, and (4) sham-operation. The plasma digitoxin concentrations in ligated dogs were significantly higher than those with either fistula or sham-operation. But the plasma digitoxin concentrations in ligated dogs with phenobarbital pretreatment were significantly lower than those with the group (2) and (4), while plasma digoxin concentrations did not differ significantly between the four groups. After intravenous tritium digitoxin administration, both the dichloromethane-extractable radioactivity in plasma and the total radioactivities in the heart revealed significantly higer levels in ligated dogs than the other three groups. Biliary ligation resulted in marked decrease in the activities of drug metabolizing enzymes. The highly sustained plasma levels of digitoxin in ligated dogs appeared to correlate with the reduction of these enzyme activities. However, digoxin was excreted easily through the kidney despite cholestasis only if the renal function was normal.
These data strongly suggest that in clinical practice, digoxin should be the choice of digitalis in patients with cholestasis.
(II) There is disagreement among investigators concerning digitoxin metabolism and elimination in patients with impaired renal function.
In order to elucidate the influence of renal function on digitoxin metabolism, both the radioactivity of dichloromethane-extractable fraction in plasma and the total radioactivity in tissues were determined in dogs with experimental renal failure induced by either bilateral nephrectomy or ureter ligations after a single intravenous dose of tritium digitoxin.
The plasma dichloromethane-extractable radioactivity was remained at the higher level significantly in the nephrectomized and ureter-ligated dogs than the control, and the total radioactivity in the heart and the liver was significantly higher in the nephrectomized dogs than the control, but not significantly higher in the ureter-ligated dogs. In contrast to both the heart and the liver, the total radioactivity of the kidney was lower in the ureter-ligated dogs than the control.
Although there are some reports that the elimination of digitoxin is not significantly affected by renal impairment, since our data suggest that the kideny appeared to play a significant role in the elimination of digitoxin, caution should be used in clinical practice when digitoxin is administered to patients with impaired renal function.

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