In human serum there exists proteinase active in an alkaline medium, namely plasmin and proteinase which are capable to hydrolyze casein, hemoglobin and fibrin in an acidic medium, the optimal pH ranging from 5.0 to 6.0. Proteinase having slightly acidic pH may be considered as a cathepsin-like enzyme, which is to be designated as catheptase.
First of all, addition of potassium cyanide increased catheptase activity but suppressed plasmin activity. Secondly, addition of 1-cysteine markedly activated catheptase activity but decreased plasmin activity. Thirdly, catheptase activity was not suppressed by epsilon aminocaproic acid. The activity was not increased by streptokinase. From the above-mentioned facts, it is certain that there exists in human serum an acidic proteinase different from plasmin.
Catheptase activity in normal subjects was determined to be 34.3±14.1 μg per ml. tyrosine. Determination in 280 patients with various disorders revealed that higher values were seen in hypertension, urticaria, arteriosclerosis, diabetes, nephritis, neoplastic diseases, hematologic disorders such as leukemia, purpura and the like.
The diseases with the lower values comprised liver cirrhosis and old cerebrovascular injury. There was a positive correlation between fibrin degradation products (FDP) and catheptase activity (X²=8.05, p<0.01).
As to the relation between plasminogen activity and catheptase activity, in patients with urticaria, 25% of 16 cases found a higher titer of FDP. On the other hand, in patients with liver cirrhosis and/or chronic hepatitis, 60% of 17 cases, and in bleeding tendency patients, 60% of 22 cases a higher titer of FDP was observed, respectively.
It was found that the increase in catheptase activity and a high titer of FDP was twice in urticaria patients than that in the patients with other diseases.
From these results, it was concluded that catheptase is involved in fibrinolysis in the initiation of both blood and/or tissue proteinase.

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In our previous paper, we reported that 13-18% of dipropyl acetate (DPA), a new anticonvulsant drug, was excreted in expiratory air during 24 hour afters oral administration. We have since studied the degradative pathway of DPA by means of gas chromatography and gas chromatography-mass spectrometry.
Twenty four hour urine specimens of Wister male rats after oral administration of DPA were extracted with ethyl acetate and ethyl ether, combined and concentrated in vacuo, and trimethylsilylated with bis-(trimethylsilyl)-acetamide and trimethylchlorosilane for gas chromatography and gas chromatography-mass spectrometry. Six peaks characteristic of the DPA-treated rats were detected by gas chromatography, and the compounds were identified by gas chromatography-mass spectrometry. Peaks 1 and 2 were thus shown to represent DPA and succinic acid, and peaks 4 and 6 were the oxidation products of DPA and identified as ω-hydroxy dipropyl acetate and ω-carboxy dipropyl acetate, respectively.
Thus it is established that DPA is metabolized by ω-oxidation to ω-carboxy dipropylacetate. In addition, it is also probable that DPA is degradated by β-oxida tion.

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Excretion of succinic acid in large amounts has been demonstrated by the use of gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) in urine of a 9 year-old girl of Japanese extraction with low normal intellegence, exophthalmos, congenital contraction of elbows and dislocation of hip. A twenty four hour urine specimen was diluted to 1,000 ml with distilled water and prepared for gas chromatography and GC-MS in the following fashion. Firstly, 8 ml of urine was half-saturated with an equal volume of saturated NaCl solution and acidified with 6 N-HCl, then extracted with 8 ml of ethyl acetate and 8 ml of ethyl ether, twice respectively. The extracts were combined and dehydrated with magnesium sulfate prior to evaporation. Since the extraction procedure is, however, not necessarily of sufficient efficacy as far as fatty acids other than aromatic fatty acids are concerned, two ion-exchange chromatographic methods were applied as well. A DEAE-Sephadex column, 1.3 cm i. d. and 9 cm in height, was equilibrated with 0.5 M pyridine-acetate solution and 10 ml of urine was loaded. The adhering fatty acids were eluted with 150 ml of 1.4 M pyridine-acetate solution. The other chromatographic column contained 3.0 g of Dowex 2-×8 resin, 200-400 mesh, in formate form, to which 5 ml of urine, deproteinized with 30% perchloric acid, was transferred. Elution was performed with 20 ml of 12 N-formic acid. The eluates were then lyophilized and the residues were redissolved with methanol, dryed out in nitrogen stream, then trimethylsilylated for GC and GC-MS. The gas chromatograph used was equipped with dual columns, 2 m in length, packed with Gas chrom Q coated with 5% OV-1, and with hydrogen flame ionization detectors. The column temperature was programmed by 3°/ min from 80° up to 250°.
A characteristically high peak detected by GC in urine preparation from the patient in comparison with those from control subjects. Mass spectrum of the compound showed parent ion of m/e 262 corresponding to that of succinic acid, of which two carboxyl groups were trimethylsilylated. Elemental analysis based on either the high resolution mass spectrometry or the medium resolutional electrostatic field mass spectrometry were also compatible with succinic acid.
Physiological significance of massive excretion of succinic acid in urine of this patient remains obscure, though lesions in TCA cycle, especially in succinyl CoA synthetase and/or succinate dehydrogenase, resulting in reduction in energy production and presumably affecting GABA shunt secondarily, appear most persuasive.

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In order to investigate distribution of maltose intravenously administered, a mixture of 10 μCi per kg of ¹⁴C-U-maltose and 1 g per kg of 10% maltose solution was injected to normal rabbits. The levels of blood maltose and ¹⁴C-maltose rapidly decreased and reached the initial levels within 4 hours. To the contrast, circulating ¹⁴C-gluose rose gradually and exceeded ¹⁴C-maltose in 90 minutes. In urine, 24% of the total radioactivities excreted was ¹⁴C-maltose, 6% being ¹⁴C-glucose and 12% other ¹⁴C-compounds, respectively. Approximately 10% of total radioactivities was observed in the skeletal muscle 60 minutes after maltose injection, and the radioactivities were measured at different organs in the order of the kidneys, large intestine, liver, small intestine, lungs and heart.
Several experiments were carried out to study on the role played by the kidney in the metabolism of maltose. When maltose was administered to nephrectomized rabbits, ¹⁴C-maltose disappeared from blood stream very slowly and increase in ¹⁴C-glucose was the smallest. When maltose was injected to the rabbits pretreated with phloridzin, ¹⁴C-maltose decreased rapidly in blood and ¹⁴C-glucose did not increase. The difference in blood maltose levels in the artery from those in vein across the kidney was widened after maltose had been injected to normal rabbits, whereas blood glucose concentration in the renal vein exceeded that in the renal artery. In phloridzinized rabbits, the A-V difference in maltose was narrower as compared with normal rabbits and blood glucose levels in the renal vein did not exceed those in the artery. When the isolated kidney was perfused for 30 minutes with 0.5% maltose solution, the levels of maltose and glucose in the renal vein rose and not only maltose but also glucose were excreted in urine. Maltose activities in the cortex of the kidney were determined to be 10 times as high as in the medulla.
These results suggest that the kidney plays an important role in the metabolism of maltose intravenously administered. The mechanism as to urinary excretion of glucose after maltose injection was discussed.

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This study aims to clarify the electron accepting nature of thiazide diuretics, which nature was previously pointed out by the authors' quantum chemical (Hückel's Molecular Orbital Method) calculation.
The ability of thiazide diuretics to quench pyrene fluorescence was measured to demonstrate their electron accepting nature. Sulfanilamide, chlorothiazide, trichlormethiazide, polythiazide, cyclopenthiazide and diazoxide were dissolved in a pyrene (4×10^-4 M/1) ethanol solution, respectively (6×10^-4M and 2.5×10³ M/1). In these solutions, intensity of fluorescence of pyrene (380 nm) was measured by 360 nm excitation under a nitrogen gas phase by using an AMINCO-BOWMAN or HITACHI MFP-2 fluorospectrophotometer.
All thiazide diuretics could quench fluorescence of pyrene, but sulfanilamide and diazoxide could not.
Then, 2×10^-4, 4×10^-4, 8×10^-4, 1×10^-3, 2×10^-3 and 1×10^-2 M/1 of hydrochlorothiazide were dissolved in 1×10^-4 M/1 of pyrene ethanol solution, respectively.
Intensity and life time of pyrene fluorescence were measured by a fluorospectrophotometer and ruby laser excitation in a vacuum phase.
The quenching constant of hydrochlorothiazide was estimated to be 1.3×10⁸-6.6×10⁹/mole, sec by this experiment. Since there was no remarkable 360 nm, spectral absorption in all molecules experimented and no spectral changes by mixing thiazide molecules and pyrene, it was fully suggested that an electron of the excited pyrene would be transferred to the thiazide molecule but not to sulfanilamide and diazoxide.
Though the quenching constant of hydrochlorothiazide seemed to be rather smaller, the electron accepting nature of thiazide diuretics was experimentally demonstrated and the authors' proposal of the quantum chemical nature of thiazide diuretics obtained by Hückel's Molecular Orbital Method appeared to be accepted.
It seems to be very important to correlate the electron accepting nature of thiazide diuretics to their pharmacological action.
It might be suggested that thiazide could interfere with the biological electron transfer system, and then could block the energy formation or conversion for the renal tubular electrolyte reabsorption. Otherwise, a thiazide molecule could interfere with the electron donating group of the renal tubular membrane and cause the change in permeability of the membrane.
Further studies would be needed.

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In our previous paper1), we reported that phosphofructokinase (PFK) in a Trissoluble form is converted into a Tris-insoluble form upon relaxation or contraction of the muscle, and that this conversion might be presumably mediated by ATP. The present studies were undertaken in an attempt to elucidate what a Tris-soluble form and an insoluble one are, and also what role ATP plays in the conversion mechanism.
The partially purified enzyme1), one ml of which contained the enzyme prepared from 4 w. w. g. of the rabbit backmuscle (white muscle), was dialysed at 4° for 90 minutes against Tris-HCI (20 mM, pH 8.0) buffer with mercaptoethanol (MSH-10 mM). The activities of the enzyme dialysed decreased at 30°. However, addition (5 mM) of MgSO₄ or (NH₄)₂SO₄ instead of NH₄Cl protected the decrease in activities. This indicates that SO₄^-- is a stabilizing factor for this enzyme.
By using Tris-maleate buffer (pH 5.5-8.5) instead of Tris-HCl, the dialysed enzymes were centrifuged at 10⁴ rpm for 10 minutes. The activities after activation (preincubation with ATP at 30°C for 30 minutes, pH 8.0) in the supernatants (sup) and precipitants (ppt) were measured. It was demonstrated that the activities in the sup decreased, while those in the ppt increased according to lowering of pH, and that the enzymes dialysed at pH 5.5 completely precipitated. The enzymes were extracted from ppt by a buffer containing ATP (2 mM) or MgSO₄(10 mM) at pH 5.5, and also by a buffer containing some stabilizing factors at pH 8.0.
The activities dependent on pH were examined in Tris-maleate buffer including MgSO₄ (10 mM) or ATP (2 mM), in which the enzymes were soluble even at a low pH. In the buffer including ATP, the activities at pH 5.5 were maintained at the same value as at pH 8.0. In the buffer including MgSO₄, the activities were lost at pH 5.5, although the value at pH 8.0 was equivalent to one when ATP was added. But the activities after activation showed an equal highest value in both buffers at pH 5.5-8.5.
In Sephadex G-200 column chromatography using the buffer at pH 8.0 including MgSO₄ as an eluant, the enzyme appeared in a high molecular fraction with the activities as PFK. By using the buffer at pH 5.5, the enzyme appeared in a low molecular fraction, of which the activities as PFK were lost but appeared after activation. By using the buffer at pH 5.5 including ATP instead of MgSO₄, the enzyme appeared in a high molecular fraction with the activities as PFK. Under the same conditions as described above, S_20W of the purified enzymes (from Boehringer Mannheim GmbH-Mannheim, Germany) were 14.3S, 5.4S and 22.5S, respectively.
The ppt at pH 5.5 was suspended in K₂HPO₄ (50 mM) and MSH(10 mM), or in Tris-maleate (20 mM, pH 8.0) and MSH (10 mM) including MgSO₄ (10 mM) or ATP (2 mM), and then these activities immediately followed. The result was that every suspension showed a gradual increase in activities. On the other side, when the ppt was suspended only by Tris-maleate buffer, the activities appeared only by additions of ATP or ADP, but not by AMP, K₂HPO₄ or MgSO₄.
From these studies, it was concluded that phosphofructokinase was insoluble by decreasing amounts of ATP and lowering of pH(muscle contraction) and soluble by increasing amounts of ATP and ascending of pH (muscle relaxation), and that the activities of this enzyme were regulated by conversion between the inactive(5S) form and the active (14S-22S) one, and also that this enzyme was solubilized and aggregated to the active form by ATP even at a low pH and, in addition, might be mediated specifically by ATP-probably phosphorylation of protein.

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An energy requirement for support of macromolecule synthesis is well realized. However, although a number of works have been made to clarify the close correlation between polynucleotide synthesis and energy supply, no convincing evidence has been provided for an important role of mitochondrial metabolism in the regenerating process. Most studies on liver regeneration have been conducted on partial hepatectomized rats, but in view of alteration in energy metabolism which may occur soon after hepatectomy, the hepatectomized rats appear to be unsuitable for studying energy metabolism in the regenerating process. Consequently, we have selected the ligation method of a portal vein branch in rabbits. This method is not only a milder treatment than hepatectomy, but offers advantages that both regeneration and atrophy processes can be followed to test for a possible humoral mechanism and that enhancement in the rate of DNA synthesis is very similar to the one which has been increased by subtotal hepatectomy.
In this study, it has been suggested that (1) a highly integrated stimulation of mitochondrial energy-generating capacity precedes DNA synthesis and (2) alteration of the quantitative balance between an available factor in portal blood and respiratory assemblies induces enhancement or depression of mitochondrial metabolism, resulting in regeneration and atrophy, respectively.

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Mitochondrial inner-membrane is impermeable to acyl CoA. Thus, for a fatty acid to be oxidized, this step is bypassed via palmitylcarnitine which readily reaches β-oxidation site1). In order to fulfil its role, carnitine palmitoyltransferase (CPT) has to be present at both outside and inside the permeable barrier of the innermembrane. Indeed, it has been lately shown that CPT is present as isozymes2). One is loosely bound to outside of the inner-membrane and is inhibited by bromoacy 1CoA in the presence of carnitine (CPT 1). The other form is firmly bound to the mitochondrial inner-membrane and is not inhibited by the above mentioned inhibitor (CPT 2)3, 4).
Therefore, in this study, in order to identify the possible regulatory role of CPT (1) and (2) enzymes in fatty acid oxidation, CPT (1) and (2) activities were measured in liver mitochondria from diabetic, fetal and neonatal rats. Under these conditions, hepatic fatty acid oxidation has been reported to be either enhanced or diminished5, 6).
A simplified new method has been developed to measure both CPT (1) and (2) activities by using DTNB.
An effect of glucagon in the developing rat on liver CPT activities was also studied.
In rat liver mitochondria, palmitate was oxidized by either carnitine-independent or dependent pathway. However, the former capacity is about one fourth of the latter indicating the predominat role of carnitine-depenent pathway. In liver mitochondria from diabetic rats, both of the pathways were stimulated. Regarding carnitine-depdndent pathway, both CPT (1) and (2) activities were elevated by 50-70% in liver mitochondria from diabetic rats. Insulin treatment reversed these increases. In fetal rat liver, aftty acid oxidation as well as CPT (1) activity remained low, but within 2-3 days after birth, 3 to 5- fold elevation was found. CPT (2) activity preceded these increases in the late fetal life (20-21 days gestation). Glucagon injection to the fetases of 21 days gestation induced CPT (1) activity after five hours.
These results indicate that CPT (1) activity is a rate-controlling step for longchain fatty acid oxidation in the liver and is under hormonal control especially by insulin. The appearance of hepatic fatty acid oxidation in the neonatal rat paralleled to that of CPT (1), but not of CPT (2) activity. The development of CPT (1) activity may be an induction mediated by cyclic AMP.

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A case of lactate dehyrogenase (LDH) deficiency and a case of phosphoglycerate kinase (PGK) deficiency have been comparatively studied.
The propositus with LDH deficiency is a 64-year-old male with mild diabetes, not anemic and suffering from neither reticulocytosis nor hyperbilirubinemia. It is shown that the propositus has no obvious evidence of hemolysis.
On the other hand, the propositus of PGK deficiency is a 6-year-old male with mild mental retardation, which suffers from anemia and reticulocytosis or hyperbilirubinemia.
Although hematologic findings of these cases are different each other, their biochemical data are similar in some respects.
It is of special interest that the erythrocyte fructose-1, 6-diphosphate (FDP), dihydroxyacetone phosphate (DHAP) as well as glyceraldehyde 3-phosphate (GA3P) concentrations in both cases are found to be markedly increased.
However, the red cell concentration of 2, 3-diphosphoglycerate (2, 3 DPG) is elevated and ATP level is clearly decreased in PGK deficiency, whereas ATP and 2, 3-DPG level are normal in LDH deficiency.
Red cell phospholipids compositions from these cases are normal.
Although the precise mechanism of hemolysis remains to be defined, these results suggest that the decreased amount of ATP and accumulation of 2, 3-DPG might lead to a symtomatic hemolytic anemia.

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Retinol - binding protein (RBP), a specific transport protein for vitamin A in human blood, has been shown to be excreted in large amounts in urine from patients with chronic cadmium poisoning and “Itai-Itai” disease.
In this report, biochemical abnormalities of serum constituents, renal function tests and urinary clearance of albumin, prealbumin and RBP were studied to elucidate the pathological mechanism for the increased RBP excretion.
Elevated level of serum creatinine and reduction of endogenous creatinine clearance were demonstrated in all of the cases with the disease. In about a half of them, retention of urea and elevation of alkaline phosphatase level in serum were observed. Serum levels of total protein, albumin and prealbumin were within the normal ranges with few exceptions. RBP level tended to be high in the cases of moderate nitrogen retention, and significant correlation of RBP and prealbumin levels was demonstrated in this experiment including the patients and their family members.
Clearance values of the 3 serum proteins of the patients were calculated in terms of μl/min. and compared with the average values in normal persons shown in parenthesis: albumin clearance 1.2 (0.14); prealbumin clearance 26 (0.1); RBP clearance 1260 (2.0). The major increase of the RBP clearance compared with that of the albumin clearance favors the grossly impaired reabsorption in renal tubules in the patients with the disease. However, a more complicated mechanism could be involved for the excretion of heterogenous forms of RBP and the increased prealbumin clearance.

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Radioimmunoassay method for determination of retinol-binding protein (RBP), originally reported by Smith et al. and modified by Muto et al., was studied to measure the low level of the protein in urine without prior concentration process. For purification of labelled RBP, prepared by iodination using chloramine T method, affinity column chromatography of prealbumin coupled to Sepharose 4-B was successfully applied to. Double antibody technique was used and the method yielded reproducible results in the ranges of 5-50ng per assay.
In order to extend the results of the previous study, differentiation of the patterns of proteinuria was further studied by measuring urinary excretion of total protein, albumin and RBP. Four types of proteinuria (normal, glomerular, tubular and mixed glomerular-tubular proteinuria) were differentiated by the double logarithmic plot of albumin and RBP excretion in urine. Serial determinations of the pattern of proteinuria in the same patient were found to be useful for following the course of the disease and involvement of the site of lesion in the kidney.
In this experiment, the glomerular pattern was found in the cases of chronic nephritis, nephrotic syndrome and diabetic nephropathy; the tubular pattern mainly in the cases of “Itai-Itai” disease; the mixed pattern in uremic patients with diabetic nephropathy, multiple myeloma and other etiologies.
It was concluded that the measurement of low molecular weight protein as RBP together with albumin excretion in urine would be very important for the earl y detection of tubular dysfunction and the differentiation of proteinuric patterns.

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The Department of Medicine, Medical School, Osaka University Recently, attention has been drawn to hypoprotein diet therapy on patients with chronic renal failure, because hemodialysis management could attain a good clinical result with an adequate hypoprotein diet therapy.
But, it still remains not clarified whether any difference in amino acid metabolism and renal tubular transport exists between patients with chronic renal failure and healthy subjects.
This study aims to clarify any difference in serum amino acids concentration in the patients with chronic renal failure and an effective nitrogen balance therapy using essential amino acid transfusion under the hypoprotein fed patients.
Material and Method;
1) Blood was obtained from 11 patients with chronic renal failure (BUN 33-149 mg/dl, protein 25-40g fed) and 10 healthy subjects (20-35 years old) after 10 hours fasting.
Serum amino acids concentration was dertermined by a Yanagimoto Amino Acids Autoanalyser.
2) A 2 weeks' essential amino acid transfusion was given (5%, 200ml) daily to the subject H. N.(BUN 95mg/dl, protein 13g fed).
During the 2 weeks' amino acid transfusion and five days pre and post therapeutic stages, daily nitrogen output has been estimated by measurement of daily urinary nitrogen excretion and by using the amount of fecal nitrogen excretion reported by Prof. Koishi.(under the hypoprotein diet stage, 0.77g/day.) Then, a nitrogen balance was obtained.
Serum creatinine, BUN and other clinical and chemical examinations were carried out.
Results
1) Valine, leucine, methionine, serine and tyrosine in the patients with chronic renal failure were in serum concentration significantly lower than those in the healthy subjects, respectively. Glutamic acid, taurine and asparatic acid in the patients with chronic renal failure were in serum concentration significantly higher than those in the healthy subjects, respectively. There were no significant differences in other serum amino acids concentration. Phenylalanine/tyrosine ratio was increased in all the patients, as reported by Josephson, Shear and Loeper.
3-methyl histidine was found in all the patients, but not in the healthy subjects.
There was no correlation between an individual amino acid concentration, total amino acids concentration and BUN.
2) Transfusion of essential amino acids solution brought out a positive nitrogen balance and lowering of BUN, leading to comfortable physical condition.
Discussion
Some differences in serum amino acids concentration were observed between the patients with chronic renal failure and the healthy subjects. However, a complete agreement with the previous reports could not be obtained. This would be resulted from the differences in the measurement technique, the diet or the renal amino acid transport mechanism. Toxicity of 3- methyl - histidine should be examined. As already reported by Giovanetti and others, essential amino acid transfusion on the patient with chronic renal failure under hypoprotein diet brought out a good clinical result.
A more practical device should be arranged on nitrogen nutrition in patients with chronic renal failure.
Summary
Serum amino acids concentration in patients with chronic renal failure was examined and some. differences were found out in comparison with that in healthy subjects.
A positive nitrogen balance was obtained by transfusion of essential amino acid in the patients with chronic renal failure.

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