Apo-ALA synthetase obtained from patients with pyridoxine responsive anemia showed marked increment of the susceptibility to the new serine protease in mitochondria of erythroblasts, Almost all features characteristic to this anemia could be ascribed to this result.
Patients with primary sideroblastic anemia including those with pyridoxine responsive anemia usually show hypochromic, microcytic anemia, increased serum iron with high saturation of total iron binding capacity, and the presence of a large number of ring sideroblasts with erythroid hyperplasia in the bone marrow. Ferrokinetic studies show somewhat increased plasma iron disappearance and reduced red cell utilization. Almost all symptoms characteristic to primary sideroblastic anemia could be ascribed to the decrease of δ-arninolevulinic acid synthetase (ALAs) activity in erythroblasts. ALM activity in erythroblasts of two patients with pyridoxine responsive anemia before therapy was also decreased. Administration of a large amount of pyridoxal phosphate to the patients caused the increment of the enzyme activity to nearly normal level, and parallel improvement of anemia was observed. The enzyme activity in erythroblasts of both patients before treatment was measured both with and without addition of pyridoxal phosphate to the incubation mixture. The result showed that the amount of both apo-and holo-ALAs was extremely decreased in erythroblasts of this disease before therapy. Apparent affinity to pyridoxal phosphate of apo-ALAs in erythroblasts of both patients was almost the same as that of normal controls. These results indicate that in contrast to other pyridoxine dependency syndromes which are considered to be caused by the decreased affinity of certain apo-pyridoxal enzymes to their cofactor, pyridoxal phosphate, pyridoxine responsive anemia is not caused by the reduced affinity of apo-ALAs to pyridoxal phosphate. On the other hand, ALAs obtained from erythroblasts of this disease showed marked inactivation during the procedure for the conversion to apoform by dialysis against 0.2 M Tris-HCl (pH7.0) containing 0.1 mM hydroxylamine. These results seems to indicate easy inactivation of apo-ALAs in erythroblasts of this disease.
Recently we found a new protease in mitochondria of bone marrow cells including erythroblasts and granulocytes. Its molecular weight is 31, 800, and optimum pH 8.5. The protease inactivated specifically apoforms of certain pyridoxal enzymes, and is considered to be engaged in the regulation of ALAs levels in erythroblasts through degradation of apo-ALAs. The protease activity in erythroblasts of the patients was within normal levels, however, apo-ALAs from erythroblasts of the patients showed marked increment of the susceptibility to the protease as compared with that from normal controls. Therefore, the following mechanisms are considered as the pathogenesis of this disease. Patients with pyridoxine responsive anemia show severe hypochromic anemia because of impared heme synthesis due to decreased ALAs activity caused by the increased degradation of apo-ALAs by the protease in mitochondria of erythroblasts. Administration of a large amount of pyridoxal phosphate causes increased holo-to apo-ALAs ratio in erythroblasts, which diminishes degradation of the enzyme, by the protease, causing the elevation of ALAs levels, and improving the disturbance of heme synthesis in erythroblasts. Therefore, anemia could be improved by the administration of a large amount of pyridoxal phosphate.
This is the first instance of the disease which is considered to be caused by the hypercatabolism of an enzyme due to its increased susceptibility to an intracellular protease.

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Plasma trypsin concentrations was measured using a specific radioimmunoassay in 50 normal subjects and in patients with 99 diabetes mellitus, 17 pancreatitis and 10 chronic liver disease. Decreased plasma trypsin levels were observed in diabetes mellitus and chronic pancreatitis, whereas significantly elevated plasma trypsin levels were observed in diabetic patients with nephropathy, and in patients with acute pancreatitis and chronic liver disease. In diabetic patients treated with diet and sulfonylurea plasma trypsin was 242±18ng/ml and 228±17ng/ml respectively which was not significantly different from the value of 270±18ng/ml in normal subjects. However, plasma trypsin in insulin-treated diabetic patients was 170±12ng/ml which was significantly lower than that in normal subjects. A negative correlation between plasma trypsin levels and either blood glucose levels or insulinogenic index, that is increment of plasma insulin from base line to 30 min value after 50g oral glucose tolerance test was observed in diabetic patients. Elevated plasma trypsin in patients with diabetic nephropathy and chronic liver disease may reflect the disorder of trypsin metabolism. High plasma trypsin level in acute pancreatitis is caused by uncontrolled leakage, and decreased plasma trypsin in chronic pancreatitis is due to the destruction of pancreatic tissue. These results suggest that the measurement of plasma trypsin is good parameter of not only exocrine pancreatic function but also endocrine pancreatic function.

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The distribution of human pancreatic elastase 2 in normal and pancreatitis serum was studied and a radioimmunoassay for determination of human pancreatic elastase 2 was developed.
The distribution of human pancreatic elastase 2 was investigated by gel filtration on Sephadex G-200 column. The mixture of ¹²⁵I-labeled elastase 2 (2μg) with normal serum (0.2ml) was subjected to gel filtration. Approximately 60% of elastase 2 formed a2 macroglobulin (α₂M)- elastase 2 complex and remaining 40% formed al antitrypsin (α₁AT)-elastase 2 complex. When the amount of elastase 2 was increased to 10μg, the amount of elastase 2 bound to a2M was approximately 30% and the amount of elastase 2 bound to al AT was 70%. In the case of acute pancreatitis serum (0.2ml), the distribution of elastase 2 was similar to that of 10μg elastase 2 mixed with normal serum. It was thought that when the elastase 2 increased in serum, excess elastase 2 formed complex more likely to α₁ AT than to α₂M.
In the development of radioimmunoassay, radioiodinated tracer of elastase 2 was inactivated with phenylmethylsulfonyl fluoride (PMS), inhibitor of serine proteinase in order to prevent binding of elastase 2 to α₂M and al AT in serum. α₂M-elastase 2 complex showed no immunoreactivities, whereas al AT-elastase 2 complex was immunoreactive. Radioimmunoassayable elastase 2 in human serum was a part of α₁ AT-elastase 2 complex.
The minimum radioimmunoassayable concentration of elastase 2 was 8ng/ml. The average elastase 2 level in normal serum was 294ng/ml with a standard deviation of 82ng/ml. The concentration of serum elastase 2 in acute pancreatitis was elevated and the maximum amount was 4,200ng/ml. The concentration of serum elastase 2 in chronic pancreatitis was almost the same amount as normal serum.

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Enzyme activity hydrolyzing an elastase substrate, succinyl-L-alanyl-L-alanyl-L-alanine-p-nitroanilide, was found in serum and bile of patients with obstructive jaundice. Though the enzyme was not elastolytic and was of high molecular weight, it was definitely differentiated from elastase-α₂macroglobulin complex because its activity was unaffected by phenylmethylsulfonylfluoride or diisopropylfluorophosphate which inhibited the complex completely.
The high molecular weight enzyme in bile was, as well as glutamyltranspeptidase (GGTP) and leucine aminopeptidase (LAP), changed into smaller forms by means solubilizing membrane such as detergent treatment and trypsin digestion. However, it was, in contrast to GGTP and LAP, almost unchanged by limited papain digestion. The molecular weight of the enzyme molecules obtained by trypsin digestion were 102,000 daltons for the enzyme, 91,000 daltons for GGTP and 174,000 daltons for LAP, respectively. Inhibition study using some chemicals revealed that the enzyme was metal dependent and had disulfide bridges in its structure. The serum enzyme was also changed into the smaller form by the detergent treatment, and shared the same characteristics with the bile enzyme.
These results suggest that the enzyme is associated with liver plasma membrane probably more profoundly buried in the lipid bilayer than GGTP and LAP and that it is one of so-called “biliary tract enzymes” not only because of elevation of serum level in bile flow obstruction but because of the association of liver plasma membrane.

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A peptidase hydrolyzing DNP-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg-OH (Peptide III), which was originally designed as synthetic substrate for vertebrate collagenase, was detected in human serum and its enzymatic properties were partially characterized.
1) Assay of the enzyme activity in sera from healthy adults and patients with various diseases revealed that the level of the enzyme in serum was enhanced with hepatic diseases, especially alcoholic and drug-induced hepatitis.
2) In hepatic diseases, Peptide III-hydrolyzing activity was highly correlated with Suc-(Ala)₃-pNA-hydrolyzing activity and both enzyme activities were only correlated with γ-GTP among laboratory findings of the patients examined.
3) In healthy adults, both Peptide III-and Suc-(Ala)₃-pNA-hydrolyzing activities were higher in male than in female and gradually increased with age. These enzyme activities were correlated with each other (r=0.696, n=78) and with γ-GTP (r=0.688, n=56).
4) Analysis of the molecular size of Peptide III-hydrolyzing peptidase by Sepharose 4B chromatography has shown that, in healthy adults, the break through fraction (MW: about 2,000K) and a fraction corresponding to molecular weight of about 200K contained enzyme activity. In the latter fraction, Suc-(Ala)₃-pNA, hydrolyzing peptidase and γ-GTP were coeluted.
5) In serum from a patient with carcinoma recti, however, the third peak showing a high Peptide III-hydrolyzing activity appeared around 800K fraction (coeluted with α₂-macroglobulin) where the presence of two enzymes complexed with α₂-macroglobulin [against Peptide III and Suc-(Ala)₃-pNA] was confirmed. They showed proteolytic activity against gelatin and casein after dissociation from α₂-macroglobulin.
6) All the peptidases found in these fractions were metallo-enzymes except a Suc-(Ala)₃-pNA-hydrolyzing enzyme complexed with α₂-macroglobulin which is inhibited by phenylmetnylsulfonvl fluoride.
7) The origin of serum peptidases against Peptide III and Suc-(Ala)₃-pNA was suggested to be a liver microsomal fraction, based on the analysis of subcenular distribution and properties of peptidases in the guinea pig liver.

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Recently we developed the new synthetic substrates for various proteases, α-naphthylester derivatives such as acetyl-Tyr-α-naphthylester (ATNE), tosyl-L-Lys-α-naphthylester (TLNE) and L-Pro-L-Phe-L-Arg-a-naphthylester (PPANE). We found that these substrates were highly sensitive to the various trypsin-like enzymes and chymotrypsin, and also that they could be used in preparing zymograms of various proteases.
In this work, in order to clear the possibility for clinical application, we studied on the development of micro-assay of human urinary kallikrein and Clesterase, a first component of human complement, using PPANE and ATNE as substrates, respectively. With PPANE as substrate, the minimum detectable concentration of human urinary kallikrein, was about 0.001KU. Thus use of PPANE provided a highly sensitive method for detection of human urinary kallikrein. Kallikrein could be determined with a 25μl samples of human urine. Also, with ATNE as substrate, Cl esterase could be determined with a 100μl samples of human serum.
Further, we examined the proteases in normal and metaplastic stomach mucosas which has been considered to be a possible precancerous stage by preparing zymograms of these tissue extracts with TLNE and PPANE as substrates. With both substrates, the zymogram of metaplastic mucosa showed an additional band besides those of normal mucosa.
These results suggest that the α-naphthylester derivatives are very available for the investigation of proteases in various pathological conditions.

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Diagnostic significance of measurement of serum pancreatic secretory trypsin inhibitor (PSTI) has been studied. Serum PSTI content was determined by radioimmunoassay recently developed in our laboratory. The assay range in the RIA was 0.4-100ng/ml. The dilution curves of serum and urine run parallel with the standard curve. The assay was shown to be highly specific and reproducible.
Serum PSTI level in healthy control was measured to be 10.1±2.6ng/ml (mean±S. D.). In acute pancreatitis and active stage of chronic relapsing pancreatitis, it was significantly elevated (the highest value; 3,200ng/ml). However, no correlation of serum PSTI and amylase was observed.
In a case of acute pancreatitis, elevation of serum PSTI with clinical signs was observed for about 2 weeks after onset, though, serum amylase level returned to its normal range within a few days.
Thus serum PSTI content reflects more exactly the severity and pathological course of acute and chronic relapsing pancreatitis than serum amylase activity.

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Among several protease inhibitors (PI) in the body, α₁-antitrypsin (α₁AT) and α₂-macroglobulin (α₂MG) are supposed to be the major with regard to their plasma concentration and activities. Though these PI are able to inhibit several proteases in the body such as plasmin or trypsin in vitro, their physiological function in serum are unknown. Furthermore, it is not clear why there is an increase of these proteins in certain physiological and pathological conditions. Recently proteolytic enzymes such as thrombin or neutral proteases derived from polymorphonuclear leukocytes has been demonstrated on the surface of lymphocytes and also shown to be capable of inducing mitogenesis in lymphocytes. Further, the presence of α₁AT or α₂MG have been demonstrated on lymphocytes.
The present study was performed firstly to know the fate of these PI in the body which were raised by inflammation or tumor bearing. Secondary, the possible mechanism that the control of proteolytic activity may be involved in the regulation of lymphocyte blastogenesis was studied. As a results, imrnunoregulatory effect of α₂MG has been proposed.
Upon turpentine injection serum α₁AT rapidly increased to the maximum, which being 1.7 times that of normal control, within 24hr in rats. α₂MG increased to maximum value of about 400mg/dl between 24 and 72 hr after turpentine injection. This maximum value is calculated to be about 100 times that of normal control.
Radiolabeled α₁AT and α₂MG were administered intravenously into the rats bearing inflammatory granuloma or sarcoma and their distributi0ns into various tissues were examined as a function of time. Considerable amount of α₁AT remained in blood for 24hr, while very few amount of α₂MG and its degradation products were found in the circulation at 6hr after administration. It may be presumed from this result that intravenously administered α₂MG was rapidly degraded and disappeared from circulation. α₁AT accumulated to a higher extent and persisted longer in the tumor or inflammatory tissues than in any other normal tissues during 72 hr period. Similar results were obtained in the case of α₂MG though the level of the protein in the tissues were much lower than those of α₁AT.
Among normal tissues examined, the highest distribution of α₁AT was found in lung, followed by kidney, muscle, spleen and liver in that order. Considering the anatomical characteristics of the lung that this organ is directly open to outside environment being exposed to the toxic agents and that severe tissue destructions are seen in patients with α₁AT deficiency, the accumulation of al AT in lung may be expected to prevent the tissue damage. The distribution of α₂MG into normal tissues were far low compared to α₁AT and little differences were observed among each tissue. These results suggest that α₁AT and α₂MG may play some roles in the site of inflammation or tumor tissues to regulate inflammatory processes or tumor proliferation. α₁AT would preferably act in the tissue while α₂MG in the circulation.
The influence of PI on the immune reaction system was examined in vitro using lymphocytes culture. Con A or PHA induced lymphocyte blastogenesis was inhibited by addition of α₂MG in the culture medium. This inhibition was not observed when the concentration of α₂MG in culture was physiological level, while remarkable inhibition was occurred when the protein concentration increased up to the serum level in rats with inflammation.

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A 42-year-old woman with severe deficient variant of α₁-antitrypsin (α₁-AT) wasfound in Nichinan City, Miyazaki Prefecture, in May 1978.
She was asymptomatic and gave no previous history of recurrent bronchitis or liver disease. Physical examination on admission and function tests of the lung, liver and pancreas showed nothing remarkable.
The proband had an α₁-AT concentration of 17.9mg per 100ml, approximately 10% of the normal level, and had low trypsin inhibitory capacity, 0.104 trypsin mg per ml, in contrast with normal average level, 0.653, determined in our laboratory. Genetic phenotyping by acid starch gel, crossed immunoelectrophoresis and isoelectric focusing showed a Pi M-like phenotype.
Liver tissue obtained by peritoneoscopic biopsy was subjected to periodic acid-Schiff (PAS) staining after diastase digestion, and revealed the unusual PAS positive intracytoplasmic globules in the periportal hepatocytes, typical of those seen with severe α₁-AT deficiency, Pi ZZ. The globules were antigenically identical with or related to α₁-AT by immunofluorescent technique.
Family studies revealed that her parents were second cousins, and her younger sister had the same severe α₁-AT deficiency, and ten other relatives had intermediate deficiency.
None of the family members examined had clinical signs or symptoms of pulmonary or liver disease.
This new variant of α₁-AT was named Pi M_Nichinan according to Fagerhol's nomenclature.

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It has been well known that inactive form of renin in human plasma can be converted in vitro into active renin by acid treatment of plasma (acid activation), by cold treatment (cryoactivation) or by enzyme treatment, however the mechanisms of conversion of inactive to active renin and the clinical significances of plasma inactive renin have not been elucidated. In the present study, plasma active and acid activatable inactive renin was measured in healthy subjects and in patients with various diseases, and the relationship among those activation procedures above described was also studied.
Acid activation was performed in 24 hr dialysis of plasma to pH 3.3 at 4° followed by additional dialysis to pH 7.2. Plasma renin activity (PRA) was measured by radioimmunoassay. The angiotensin I generated from the incubation of nonacidified plasma with pig renin substrate was expressed as plasma renin concentration (PRC) and that from activated plasma was expressed as total renin concentration (TRC). The inactive renin concentration (IRC) was calculated as TRC minus PRC. With regard to PRA and PRC no significant difference was found between normal and other groups. The values of TRC and IRC were significantly higher in patients with nephrotic syndrome and were lower in those with hyperthyroidism than those in normal subjects. A significant correlation was found between IRC and BUN, and IRC and P. S. P. excretion in 15 minutes. Although PRA and PRC were increased by standing in normal subjects and patients with nephrotic syndrome, no significant changes of TRC and IRC were found in both groups. In order to investigate on the relationship between acid activation and cryoactivation, following studies were performed. Plasma was incubated at -4° for 4 days in cryoactivation and TRC was measured by the same method as the acid activation in this study. We have found that although TRC in acid activation (TRC_acid) is higher than TRC in cryoactivation (TRC_cryo), there is a significant correlation between the two measurements (r=0.72, p<0.005). The values of TRC_cryo for 30 days, however, was approximately equal to those of TRC_acid. Trasylol, lima bean trypsin inhibitor (LBTI), diisopropylfluorophosphate (DFP) and phenylmethylsulphonylfluoride (PMSF) were added to the plasma which had been dialyzed to pH 3.3 previously, then those were dialyzed to pH 7.2. DFP and PMSF were added to the pH 7.2 dialysate at the same concentration. Those inhibitors were also added to plasma prior to cryoactivation. Both acid activation and cryoactivation were inhibited by Trasylol, LBTI, DFP and PMSF. Normal human plasma was applied on Blue Sepharose CL-6B and was separated to the passthrough fraction and the 1M-NaCl fraction. The effects of acid activation, cryoactivation, activation by trypsin and kallikrein were compared in the two fractions, respectively. No activation was found in the passthrough fraction, while activation was found in the 1M-NaCl fraction by all four activation procedures. These results show that active and inactive renin in human plasma can be separated by affinity chromatography with Blue Sepharose CL-6B.
These data suggest that these activation procedures have an enzymatic basis in common and serine protease plays an important role on activation of plasma inactive renin.

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The relationship between the clinical course of sarcoidosis symptoms and serum angiotensin converting enzyme (ACE)(EC 3. 4. 15. 1) was analyzed through the observation of 51 sarcoidosis patients over the period of more than one year. Before this study, the normal value of serum ACE activity was determined by using normal subjects whose chest roentgenogram, ECG, serum creatinine, serum urea N, serum uric acid, serum cholesterol, serum Na and K, blood pressure, vital capacity and % FVC_1.0 were normal. Serum ACE activity in 311 normal subjects were examined, and the results was 18.7±5.0 nmol/ml/min. The normal range was settled as between 10 and 29 nmol/ml/min. The sarcoidosis patients were grouped into three categories according to their clinical course as follows; (A) sarcoidosis lesions in the chest roentgenogram disappeared within one year,(B) sarcoidosis lesions in the chest roentgenogram remained more than one year with or without little improvement,(C) sarcoidosis lesions in the chest roentgenogram disappeared within a year but then relapsed. In group A (33 cases) serum ACE activity level was 35.9±2.0 nmol/ml/min at the time before treatment. Twenty five of 33 cases showed higher value than 29 nmol/ml/min. The serum ACE level dropped to 22.0±1.3 nmol/ml/min after clearing of chest roentgenogram (p<0.005). In group B (12 cases) the levels of serum ACE activity before and after one year were 41.9±5.3 nmol/ml/min and 34.8±3.2 nmol/ml/min respectively. Eleven of 12 cases showed higher ACE activity than normal range after one year of treatment. In group C (6 cases) a good correlation was observed between the ACE activity and the clinical course.
Through the observation, serum ACE activity was proved to be a good indicator of the sarcoidosis clinical course.

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Chromogenic substrates were used to determine anti-thrombin III (AT-III) and prothrombin (Pro-T)in plasma. As an activator for prothrombin, ecarin was proved to be better than tissue thromboplastin, for it was not affected when assaying samples from patients with V-K deficiency.
Two substrates (S-2238 & RM601-2) for thrombin gave similar results with correlation coefficient of 0.97. In comparison with single radial inununodiffusion method and with fibrinogenagar plate method, these chromogenic substrate methods were seemed to be simpler in manual, shorter in time for completion and more accurate in results.
When there is no particular disorder in clotting states, AT-III and Pro-T levels in plasma may be measures for synthesizing activities of the liver. To evaluate AT-III and Pro-T values clinically, they were compared with other routine liver function tests using six groups of patients with liver diseases classified by needle biopsy.
In acute hepatitis, AT-III and Pro-T showed different behavior with other tests suggesting their potent usefulness in prognosis. Of the six groups, patients with cirrhoses (non-compensated phase) showed lowest values reflecting severe damages in the liver.
It was also demonstrated that repeated assays of AT-III and Pro-T with short time intervals could give characteristic measures to follow up observations in the course of liver diseases.

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In order to identify the eluate, or to identify the series of metabolites of different chemical characteristics by a single liquid chromatography, it is awaited to establish the method to analyze the eluate by reasonable and different analytical methods at once. In this paper a simple device for the multi-parallel detection method in the liquid chromatography is presented.
The device was made of multi-way joint (flow separator) and pressure coils. Pressure coils were contributed for the constant separation of the flow. Suppose that after the separation of the flow, one of the flow line was subjected to the analytical reaction using suction pump and another flow line was subjected analytical reaction under the back pressure. Then, the pressure coils were needed to minimize the change of flow rate in each line due to the different pressure in the different flow line.
One of the best merit of this method was that each detection method worked at it's best sensitivity evenso the eluate was separated into several lines. Of cause, the flow separator separates the absolute amount of the eluate, but at before and after the separation, the concentration of the eluate should be the same. This is the reason that the detection sensitivity should be able to be the same with both the multi-parallel detection method and the single detection method.
Another merit of this method is that wide range of analytical methods are able to be used with almost no restriction, for each flow line was devided independently by the pressure coil, in contrast to the multi-series detection method.
Application of the multi-parallel detection method for the analysis of catecholamines, using detection methods of the trihydroxyindole (THI) method, o-phthalaldehyde (OPA) method and the electro-chemical method, is presented.

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A method for the determination of catecholamine-O-sulfate isomers (CA-S)was made by using high-performance liquid chromatography (HPLC) and newly developed photo-induced fluorogenic reaction. CA-S and 3, 4-dihydroxybenzylamine 3-O-sulfate (DHBA-3-S: an internal standard) were synthesized chemically and separated from each other by HPLC using silica gel column. They were found to become fluorescent compounds with ethylenediamine under UV-light. This reaction was applied to the post-column labelling of CA-S separated by HPLC for sensitive and selective detection of CA-S, and a special equipment was made with UV lamp and reaction coil of teflon tube inside of a temperature-controlled reaction box. With this system CA-S were separated and could be detected in the level of ten pmol. Urine samples were pre-treated with small ionexchange resin columns for the removal of interfering substances. The peaks corresponding to CA-S on the chromatogram obtained from a urine sample were identified by two ways; the elution patterns of CA-S in urine from Dowex 1 (AcO) column were coincident with those of the sample added with standard CA-S and with no irradiation in the post-column labelling reaction no peaks were detected in the elution time on HPLC corresponding to CA-S. CA-S in urine of healthy men were measured and the existences of all CA-S were certificated in the physiological level.

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High performance liquid chromatography with native fluorescence detection is shown to be readily applicable to the selection determination of biogenic amine in urine. Measured via their native fluorescence emitted with an excitation wave length of 280nm, the emission filter 315nm. The maximum sensitivity of this method is in the pico-gram range.
This method is applied to alumina adsorption method. In this study we present our finding regarding the change of urine catecholamine.
The concentration values of urine catecholamine (NA, DA) in the depressive patients (16 cases) were lower than those in control group, and there was negative correlation between urinary noradrenaline excretion level and clinical symptom (Hamilton Rating Scale).

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Ion-exchange chromatographic systems for separation of catecholamines were developed using a carboxylic acid type ion-exchange resin (Amberlite IRC-50) as a stationary phase and buffers of pH 6.3 or 4.0-4.3 containing boric acid as eluents. At the higher pH (pH 6-7), catecholamines were eluted faster when an eluent containing higher concentration of boric acid was used, although at the lower pH (e. g. pH 4), increase of boric acid concentration had little effect on the elution volume of catecholamines. This result seems to be due to formation of borate complex of catecholamines having less net charge at the higher pH. We used eluents containing 0.35M or 0.66M boric acid. At pH 6.3, epinephrine, norepinephrine and dopamine were eluted in the order described, whereas at pH 4, norepinephrine was eluted first, followed by epinephrine, dopamine and deoxyepinephrine. Isoproterenol overlapped with deoxyepinephrine. Since isoproterenol and deoxyepinephrine do not occur in human plasma and in urine, these amines could be used as the internal standard. Size of the column was 0.8cm in diameter and 12-28cm in length, and 1.0ml of sample solution could be injected without loss of resolution. Flow rate of buffer through the column was 0.7-1.0ml per min and separation of catecholamines was completed within an hour.
Amines were extracted from a deproteinized and neutralized extract or urine by adsorption on an ion-exchange column (Amberlite CG-50, 0.4×12 or 0.5×12cm, buffered at pH 6.5). The column was washed with water and catecholamines were eluted with 0.66M boric acid solution (pH 6.1). pH of the eluate was adjusted to the pH of the eluent to be used for chromatography and diluted to 3 or 4 ml, and when it is not used promptly, it could be stored in a refrigerator for several days. Catecholamines in the eluate were determined fluorimetrically by a modified ethylenediamine condensation method, trihydroxyindole method or 4-aminobenzoic acid-hexacyanoferrate (III) oxidation method. The former method was used for the determination of catecholamines in human urine, the second method was used for the determination of epinephrine, norepinephrine and the third method for dopamine with higher sensitivity. 20-40 picograms of each catecholamine in a sample solution (1.0ml) could be detected.

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Using the high performance liquid chromatography, a quantitative, specific, and direct method for urinary catecholamines [adrenaline (Adr.), noradrenaline (NA) and dopamine (DA)] was established. Principle of our present method is that with high performance liquid chromatograph, catecholamines were selectively separated from other urinary components by gradient elution method. For continuous detection of catecholamine fractions trihydroxyindole procedure was employed. Each catecholamine was separated definitely from other urinary components. With this method we tried to standardize some of problems concerning pretreatment of urine for analysis of catecholamines. The present results showed that pH≤53.0,?≤4°C were suitable for storage and that pH≤1.0, 100?°C, 30 min. for hydrolysis of the urine. In comparison of the determined values of urinary catecholamines with the clinical courses of the patients, a correction to devide determined values by glomerular filtration rate was found to be useful for clinical application. Using this method of the correction, prognosis of patients with brain aneurysm under surgical procedure has been studied and following results were obtained: catecholamine values (corrected) were found much higher (over 1,000pg/ml) in patients with aneurysm of unfavourable prognosis than those of good or in patients under surgery of unilateral carotid artery occlusion as controls. On the basis of our present observation, it might be suggested that urinary catecholamines were available as a parameter to evaluate prognosis of patients with brain aneurysm under surgical procedure.

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A sensitive method for the quantitative determination of all guanidino compounds in human plasma, blood cell and tissue, including basic compounds such as methylguanidine, was developed by the adaptation of the automatic amino acid analyzer JLC-6AH (JEOL). Automatic quantitative determination of the guanidino compounds was performed with 9, 10-phenanthrenequinone, a specific reagent for the guanidino group. With this method, the detection of the 2ng/ml of guanidino succinic acid and 5ng/ml of methylguanidine can be possible.
The measurement of the tissue concentration of guanidino compounds revealed that guanidinosuccinic acid could be synthesized in the normal human liver, data of which abnormally increased in acute and chronic renal failure. Guanidinoacetic acid present in the normal kidney, we disclosed that it decreased in accordance with deterioration of renal failure. Methylguanidine developed especially in renal failure, and its tissue accumulation was proved in our study.

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Guanidino compounds which are one of the uremic toxins are determinated by High Performance Liquid Chromatography (HPLC).
We investigate some conventions to perform with small volume of sample and within short time using HPLC. A length of column is shortened to 1/3 of conventional one, that is 5cm of column length and the analysis of peaks from taurociamine to methylguanidine is shortened to 1/5 (26 min) of conventional one.
A volume of sample used only 1000μl for an analysis. The lineality of standard compounds and precision are also good as well as results with conventional method, so that this method could be used for clinical test.

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A method for high-performance liquid chromatography of carbohydrates was developed by using Hitachi 2633 and 2634 strong anion exchange resins of borate form, and Fluoresugar (acyanoacetamide) as the fluorogenic detecting reagent. Ten carbohydrates were completely separated in 100min by stepwise elution with two borate buffers, and the lower limit of detection was 10 picomole for each carbohydrate.
For routine analysis of the monosaccharide compositions of carbohydrate materials in sera and urines, a single-buffer system was used, which allowed rapid determination of xylose, fucose, galactose, glucose, and mannose. The fucose/galactose molar ratios for the non-dialyzable urinary carbohydrate materials of several cancerous patients were found to be lower than those of normal subjects.

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In using high pressure liquid chromatographic technique nucleosides, cyclic 3', 5'-AMP, cyclic 3', 5'-GMP and 5' mono-, 5' di-, 51trinucleotides could be analyzed. All nucleotides could be quantitated in a single step separation within 45min employing 1m mole/l potassium phosphate (pH 3.0) between 0.4mole/l potassium phosphate (pH 3.1) gradient. Half ml of plateletrich plasma with 1/10 volume of ACD or 3.8 % sodium citrate furnished an adequate sample for analysis.
Nucleotide levels in platelets from 12 healthy donors expressed in 1011 platelets were as follows: AMP 0.56 (0.22) mean (SD), ADP 2.6 (0.6), ATP 4.0 (0.5), GMP 0.32 (0.22), GDP 0.48 (0.2), GTP 0.32 (0.08), UDP 0.28 (0.04) and CTP 0.11 (0.08) micromoles.
Upon platelet aggregation without release of ADP, ATP and platelet factor 4 (primary aggregation) induced by lower concentration of ADP and epinephrine, no remarkable change of nucleotide profiles was observed. Amount of ATP, ADP, GTP and GDP in platelets was decreased down to 50% of initial values during aggregation with release reaction (secondary aggregation) by collagen, arachidonate and higher concentration of ADP and epinephrine. Ratio of ATP/ADP in platelets showed no significant change before and during release reaction. As amount of released ADP was usually higher than ATP released, these nucleotides would decrease not only by the extrusion from the platelets in the release but also by consumption during release reaction.
It is concluded that release reaction of platelets always accompanies the decrease of nucleotide levels and platelet aggregation itself does not necessarily accompany the change of nucleotide contents. Loss of nucleotides during release reaction is derived from the extrusion from the platelets and consumption in the platelets.

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A sensitive and reproducible fluorescence high-performance liquid chromatography is described for the determination of corticoids and 17-oxosteroids in biological fluids. Δ4-3-oxosteroids and 17-oxosteroids are labelled with dansyl hydrazine in HCl-EtOH solution and in trichloroacetic acid-benzene solution, respectively. In the case of corticoids, steroids are extracted with methylene chloride from 0.1 ml of plasma or 1 ml of urine, labelled with dansyl hydrazine, and then chromatographed on a microparticulate silicagel column (Hitachi gel, No.3042, 4 ×250 mm) with fluorophotometric detector at 350 nm (excitation) and 505 nm (emission) using CH₂Cl₂- EtOH-H₂O (985: 15: 5 or 948: 35: 17) as eluent. 17-oxosteroids are extracted after enzymatic hydrolysis (Helicase) or solvolysis, and assayed by the same manner except the eluent. In this case, CH₂Cl₂-EtOH-H₂O (400: 1: 2) is used as eluent. Linearity of the fluorescence intensity with the amount of all steroids are obtained-greater than 0.5ng. The recoveries were between 90 and 106%, and the coefficient of variations were between 3 and 6%. Comparisons with radioimmunoassay gave a correlation coefficient of 0.978 (n=36) and 0.964 (n=81), for serum cortisol and dehydroepiandrosterone sulfate, respectively. A simultaneous determination of various 17-oxosteroids in urine was successfully carried out by the method. Urinary free cortisol could be analyzed without interference of carbamazepine. After administration of metyrapone, 11-deoxycortisol levels in serum could also be measured rapidly by this method. The method is a clinically useful one for the routine analysis of oxosteroids in blood and urine.

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High performance liquid chromatography (HPLC) was proven to be useful for the analysis of corticoid (CS) extracted from plasma, urine or adrenal tissue.
In the suitable condition of HPLC (LC-2 type) with Zorbax SIL or CN column, organic solvent (cyclohexane: dichloromethane: ethanol=9: 4: 1 or 50% methanol), and UV 254 nm detector, CS fractions both of polar (6β-OHF, E_R, epiE_R etc.) and less polar group (progesterone, 11-desoxycorticosterone etc.) were separated quantitatively in extracts from plasma and urine. The ratio of each CS-fractions on chromatogram was varied with age and/or diseases of patients.
The serial changes of HPL-chromatogram of CS and its metabolites. in plasma were analyzed after oral administration of prednisolone, betamethasone or triamcinolone and their plasma concentrations contrasted with those of ACTH and endogenous F (cortisol).

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We have established a highly sensitive simultaneous determination of the individuals of human serum 3α-hydroxy bile acids, using high performance liquid chromatography (HPLC) combined with enzymatic fluorimetric method. The separation of thirteen components of bile acids, free and glycine- or taurine-conjugated bile acids, was satisfactorily attained by HPLC on a μ Bondapak phenyl column, using 0.3% ammonium carbonate solution mixed with acetonitrile (the volume ratio is 14:4 and 8:4) as mobile phase and 3α-hydroxysteroid dehydrogenase as reagent.
A significant linear correlation was obtained between the peak height and the amount of each bile acid (unconjugated bile acids; 0-125ng, glycine-conjugated bile acids; 0-187.5ng and taurine-conjugated bile acids; 0-250ng). The minimum detectable amount of each bile acid was ranged from 6.3 to 31.0ng.
The recovery rate of each bile acid added to human serum was approximately from 81.7 to 106.6%.
CV value of each bile acid in human serum was ranged 0.72-9.09% (intra-assay) and 1.39- 9.09% (inter-assay).
The concentration of bilirubin (0-40mg/dl) did not interfere to the determination by this method.
A present method made it available to separate thirteen components of bile acids simultaneously within one hour, and to determine free and conjugated bile acids inlml of even healthy human serum.
In the cases of intrahepatic cholestasis and extrahepatic cholestasis, the levels of glycocholic acid and taurocholic acid were markedly elevated and especially in the cases of intrahepatic cholestasis the level of cholic acid was significantly higher than that of extrahepatic cholestasis.
The patients with liver cirrhosis have increases of every bile acid components. Especially the level of conjugated chenodeoxycholic acid in the non-compensative stage was significantly higher than that in the compensative stage.
In the patients with acute hepatitis glycocholic acid and glycochenodeoxycholic acid was increased.
In the cases of chronic hepatitis, the level of total serum bile acids was elevated in parallel with the severity of liver cell damage, and glycochenodeoxycholic acid and taurochenodeoxycholic acid were higher in the cases of active type than that of inactive type.
Patients with a silent stone were shown to have normal patterns of bile acid components except for a slightly increased level of lithocholic acid. These results showed characteristic patterns of bile acid components in sera of patient with various diseases and might indicate their liver function or dynamic state of enterohepatic circulation.

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Bile acids determination in biological fluids have diagnostic importance on liver function test.
Recent progressive of high performance liquid chromatography have permitted the separation and determination of various kind of bile acids.
On the other hand, the enzymatic determination of the bile acids with 3α-hydroxysteroid: NAD oxidoreductase (E. C. 1.1. 1.50) were performed already, and applied to HPLC by Baba. et al.
In this paper, we describe some results showing more sensitive and inexpensive method with HPLC and immobilized enzyme.

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A method for simultaneous determination of unconjugated and conjugated bile acids in human bile has been described. Bile acids were extracted with a Sep-pack C₁₈ cartridge and separated into the unconjugated, glycine-, taurine-conjugated and sulfated groups by ion-exchange chromatography on piperidinohydroxypropyl Sephadex LH-20. Resolution of each unsulfated group into ursodeoxycholate, cholate, chenodeoxycholate, deoxycholate and lithocholate was attained into two stages by high-performance liquid chromatography (HPLC) on a Radial-Pak A column. First, 0.3% ammonium phosphate (pH7.7) /acetonitrile (19: 8, v/v) was used for the separation of the latter threee as a mobile phase. Ursodeoxycholate and cholate were efficiently resolved in 0.3% ammonium phosphate (pH7.7) /acetonitrile (23: 8, v/v). The UV (205nm) detector showed a linear response to unconjugated, glycine- and taurine-conjugated bile acids with a quantitation limit of 500, 50 and 150ng, respectively. The present method was found to be applicable to determination of bile acids in human bile with satisfactory accuracy and pre- cision. Separation of monosulfated bile acids by HPLC was also carried out. Effects of pH and salt concentration of a mobile phase on the k' values of sulfated bile acids were investigated with the reversed-phase columns. The 3-monosulfates were efficiently separated on ODS SC-02 using three aqueous ammonium carbonate/acetonitrile systems.

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A rapid method for determining simultaneously the total concentration of group of glycine-(G) or taurine-(T) conjugated bile acids was developed using high-pressure liquid chromatography on a column of an anion exchange packing, TSK Gel IEX 540 DEAE (80×8mm i. d.).
With 0.017 M sodium citrate buffer (pH 3.6)-EtOH (3: 2, v/v) as an eluent, the bile acids in each group were eluted at almost the same retention time. On this condition, G and T were per- fectly separated in less than 10 min.
Each bile acid showed the same response to the differential refractometer. The values of each group obtained by this method were coincided with those by the fluorometry using 3α-hydroxysteroid: NAD oxidoreductase-NAD system. Good reproducibility was obtained and the added bile acids to the bile were recovered quantitatively.
This method was applied to the human bile samples pretreated simply from normal subjects and from patients suffering from various hepatobiliary diseases.

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A high performance liquid chromatography with spectrofluorometer assay was applied for the determination of α-, β-, γ- and δ-tocopherols in serum, bile and intravenous fat emulsions.
The tocopherols were extracted from 0.5ml of serum or bile with 5ml of n-hexane after precipitation of proteins with ethanol containing tocol as an internal standard. Concerning fat emulsions, extractions were repeated three times from 0.2ml of emulsions. After centrifugation (3,000 r. p.m., 5min.) 4ml of the supernatant fluid was evaporated at 40° under nitrogen stream. The residue was dissolved in 50μl of n-hexane and 20μl of this was injected into the HPLC column (Nucleosil-5NH₂, 4mmφ×250mm). Isopropylether-hexane mixture was applied as mobile phase and the tocopherols were determined separately by using a spectrofluorometer (Ex. 298nm, Em. 325nm). The procedure was standardized by chromatographing standard mixtures containing known mass ratios of tocopherols and tocol. Linear relationships between peak-height ratios and mass ratios were recognized. Within-run precisions of 1.9 to 21% were obtained analyzing 10 standard samples. Analytical recoveries of tocopherols for fortified serum and bile were from 94 to 100% and for fortified fat emulsion were from 103 to 117%.
The levels of total tocopherols in normal human sera ranged from 0.78 to 1.22mg/dl; a-toco-pherol from 0.67 to 1.19mg/dl; β-tocopherol from 0.01 to 0.02mg/dl; γ-tocopherol from 0.02 to 0.13mg/dl, and δ-tocopherol was less than 0.01mg/dl. α-Tocopherol occupied more than 87% of the total tocopherol.
Serum levels of tocopherols in various diseases were examined. Pancreatic diseases and congenital choledochal cyst showed lower levels of a-tocopherol.
One to four days after operation, all the patients who didn't get blood or plasma transfusions showed lower serum levels of total and a-tocopherol than the preoperative levels (mean level was 80% of the preoperative value). They recovered within several days by sufficient food intake.
The levels of α- or total tocopherol had positive correlation with cholesterol and β-lipoprotein. Negative correlation with lipid peroxide was not recognized in this series.
Fat emulsions for intravenous injection contained from 20 to 30mg/500ml of total tocopherols and about 60% was γ-tocopherol and 30 % was δ-tocopherol. They may be derived from soybean oil which consists main compartment of the fat emulsion.
When fat emulsions were injected intravenously in the patient with T-tube drainage, the serum and biliary levels of γ- and δ-tocopherol markedly increased.

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Coenzyme Q (CoQ) homologues in rat heart mitochondria were determined by the method of the high pressure liquid chromatography (HPLC). The mitochondria fraction was prepared from rat heart homogenate by the differential centrifugation following protease digestion. Mitochondria suspension was saponified with methanolic alkaline solution, and then extracted with n-hexane. The n-hexane extract was evaporated under N₂ gas at 30°, and the residue was dissolved in dioxane. The sample was analyzed by HPLC employing 150×4,6mm column packed with Li-Chrosorb RP-18. Ethanol: water (97:3) was used as eluent. 2, 3, 6-Trimethyl-5-nonaprenyl-1, 4-benzoquinone (TQ-9) was used as an internal standard. Total content of CoQ homologues (CoQ_7-10) of the heart mitochondria of normal rat was 6.16±0.79μg per mg protein. The predominant component isCo% which constitutes 90.5% of the total.
Adriamycin (ADM) is one of the widely used antitumor agents. However, it is known that the prolong clinical use of ADM is limited by the side effect of cardiotoxicity. To find the pathogenesis of this cardiotoxic effect, the heart mitochondria isolated from rat treated with ADM was examined and the effectiveness of exogenous CoQ₁₀ which was administered for the purpose of decreasing the side effect of ADM was studied.
Administration of ADM (daily dose of 4mg/kg body weight for 6 days) induced the uncoupling with the electron transport system. And, this was associated with a decrease in CoQ₁₀ content of the heart mitochondria. However, exogenous (i. p.) administration of CoQ₁₀ to the ADM-treated rat could partially restore the respiratory disorders. The CoQ₁₀ content of the heart mitochondria was also restored to the normal level. Therefore, it was suggested that the administration of CoQ₁₀ is useful for the protection from the side effect induced by ADM.
In addition, the described technique by using HPLC would be more simple and sensitive than the other conventional method.

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The high-performance liquid chromatographic (HPLC) procedures were developed for the determination of plasma gliclazide, a new sulfonylurea with anti diabetic property. Normal-phase HPLC on Lichrosorb SI-60 column, reversed-phase HPLC on Lichrosorb RP-2, RP-8, RP-18, Nucleosil C₁₈, Jasco SC-01 columns and anion-exchange HPLC on Diaion CDR-10 column were examined for determination of plasma gliclazide.
Among them, the following two HPLC methods were selected for high theoretical plate and good separation from plasma components.
1) Reversed-phase HPLC on Jasco SC-01 column with methyl alcohol: 0.2% acetic acid =3:2 (v/v) as mobile phase [retention time; gliclazide: 16min, IS (tolbutamide): 10min].
2) Anion-exchange HPLC on Diaion CDR-10 column with acetonitrile: methyl alcohol: 1.2M ammonium perchlorate=4:3:7 (v/v/v) as mobile phase [retention time; gliclazide: 14min, IS (methyl benzoate): 19min].
The extraction with phosphate buffer(pH 7.0) gave no interference of plasma components on chromatograms of the anion-exchange HPLC. The relationship between the plasma levels of gliclazide and the peak height ratio (gliclazide/IS) by the two methods were linear (correlation coefficient,r=1.00) in the range of 0.3 to 10μg/ml plasma. Each reproducibility (CV) of the two methods was 4.0% on reversed-phase HPLC (n=8), 3.5% on anion-exchange HPLC(n=12), respectively. The detection limits were 0.2μg/ml on the two methods.
The two methods agreed well with the GLC-ECD method. The decay curves of plasma gliclazide in seven diabetic patients given 80mg of the drug orally were determined by HPLC. Reversed-phase HPLC or anion-exchange HPLC was shown to be suitable for the assay of gliclazide in plasma. The latter was available for routine clinical examination because of good stability of packing.

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Impaired drug metabolisms patients with liver injury were investigated by measuring pharmacokinetics of several drugs on high performance liquid chromatography. The drugs tested were an anti-canceragent [1-(2-tetrahydrofuryl)-5-fluorouracil (FT-207)], an immunosuppressive agent [azathioprine (AZP)] and an antibiotics [cefazolin (CEZ)], which were metabolized differently in liver and played important effects in vivo. The K values following an intravenous injection of FT-207 (K_F) decreased significantly in cirrhotic patients with primary hepatoma. In these patients with continuous oral administrations of the drug, the blood levels gradually increased day by day to the levels higher than 10μg/ml. The K_F values were significantly correlated withKim values.
Increases in the K_F values by pretreating with tocopheryl nicotinate and reduced glutathione were observed in cancer patients without liver involvements but not in cirrhotic patients with hepatoma, indicating difficult amplification of masked-compound efficacy in patients with advanced liver injury. The K values of CEZ(K_C), which were excreted mainly in urine, decreased even in cirrhotic patients, especially, with ascites. The impaired metabolisms of CEZ may be related to altered renal functions due to associated hepatorenal syndrome but not to severity of liver injury, since endogenous creatinine clearance and urine volume were correlated with K_C measured. The metabolisms of AZP in vivo was impaired in rats pretreated with probenecid or carbontetrachloride, in which glutathione S-transferase activities markedly diminished, indicating that AZP efficacy as an immunosuppressing agent could not be expected sufficiently in subjects with severe liver injury. These results suggest that individual measurement of each drug pharmacokinetics is necessary for planning an optimal dose administered to each of patients with liver injury.

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