Purine nucleoside phosphorylase(PNP), the enzyme in the purine salvage pathway, is necessary for the normal human T cell function of the immunological system, since PNP deficiency is associated with severely defective T cell immunity but with normal B cell immunity. Therefore, PNP is speculated to be useful as an enzyme marker for the differentiation and maturation of T cells. In order to test this possibility, the PNP activity in T, B and null cells from healthy subjects and patients with lymphoproliferative disorders has been investigated by using a new technique developed for the enzyme cytochemical staining. In this method, a suspension of unfixed-lymphocytes in phosphatebuffered saline is mixed on a glass slide directly with agarose sol containing the reagents for detection of the PNP activity. The mixture solidifies, is incubated and then dried in the form of a thin layer on the slide for a light-microscopic observation. The normal morphological shape was clearly retained in more than 90% of observed cells. The cytochemical reaction is based on the method reported by Kishi (Bull. Tokyo Med. Dent. Univ.16: 289,1969). Results of negative controls without the substrate, inorganic phosphate or xanthine oxidase provide evidence for staining dependent on the PNP activity. In healthy adults, the mean percentages of PNP-positive cells were 95% (n=7),92% (n=6) and 91% (n=3) in unseparated lymphocytes, EACrosette forming cells and E-rosette forming cells, respectively. These considerably high percentages of PNP-positive cells indicate that PNP activity is present not only in T cells but also in B cells. Contrary to the data, more than ninety six percent of the lymphocytes in 2 of 5 patients with B cell chronic lymphocytic leukemia (CLL) was PNP-negative. A slight decrease of the activity was found in the lymphocytes of the other CLL patients. The lymphocytes of a patient with null cell acute lymphocytic leukemia showed greatly decreased PNP activity. These results suggest that PNP activity is expected to be a possible enzyme marker to differentiate tumor cells or immature lymphocytes from matured cells.
6-mercaptopurine(6-MP), the hypoxanthine analog, has been found to be a potent inhibitor of human leukemia cells. For this reason, the biochemical mechanism of action of this compound has been the subject of many investigations. It became apparent from early studies that 6-MP was catalyzed by hypoxanthine guanine phosphoribosyltransferase (HGPRTase) to 6-thioinosine 5'-phosphate(6-TIMP) which inhibited the enzymes, PRPP amidotransferase, IMP dehydrogenase, adenylosuccinate synthetase and adenylosuccinate lyase. Such metabolic blockade would be expected to decrease the concentration of purine nucleotides in target cells, thereby resulting in inhibition of cell-growth. An increasing production of 6-TIMP in leukemia cells leads to enhance the inhibitory effect of 6-MP. HGPRTase activity and PRPP content in leukemia cells were already reported, but the mode of transport of this compound to cell-membrane was not reported because of difficulty to investigate on account of the possibility of efflux of metabolites during wash-out of the incubation medium. We made the quantitative investigation of incorporation of this compound possible by use of new method of Wohlhueter et al using silicone oil. In this method, cell component could be separated from the incubation medium without washing. The result showed that the radioactivity of incorporated 6-MP reached at its maximum approximately at 10 min. of incubation, thereafter reduced rapidly. This result suggests the possibility of efflux of 6-MP or metabolites of 6-MP such as 6-TX and 6-TUA. Especially, efflux of 6-TUA may affect remarkably the concentration of 6-MP in the cells. The kinetic examination of intracellular metabolites of 6-MP supported this efflux. After this rapid increasing and decreasing phase, the radioactivity in the cells showed gradual increment. This increment is supposed to reflect the phosphorylation of incorporated compound. The mode of transport of Ara-C to cell-membrane was also investigated, Under the condition that most incorporated Ara-C was converted to Ara-C nucleotide, the radioactivity in the cells showed linear increment until 30 min. of incubation. It is apparent, as compared 6-MP with Ara-C, that the mode of transport to cell-membrane is affected markedly by intracellular phosphorylation of these compounds. The present investigation suggests that 6-MP should be contacted to cells for a longer period for its effective administration even if a low dose.
A possible effect of biogeneamines on the concentration of plasma uric acid in oxonate-treated rats was studied as a model for evaluating drug-induced hyperuricemia. We previously reported that catecholamine stimulated a rapid breakdown of tissue adenine nucleotides, resulting in a marked increase of plasma uric acid and allantoin in rats.The increase of plasma uric acid by catecholamine could be observed more sensitively in oxonate-treated rats, in which further metabolism of uric acid to allantoin was effectively inhibited. The oxonate-treated rats showed an increase of plasma uric acid not only upon administration of catecholamine, but also with various biogeneamines. Acetylcholine given through the tail vein of animals caused only a transient increase of plasma uric acid, while its effect was markedly potentiated by pretreatment with cholinesterase inhibitor such as physostigmine. Physostigmine or neostigmine alone also increased plasma uric acid. Carbachol produced a more stable increase of plasma uric acid than acetylcholine, and the increase was obviously abolished by pretreatment with methylatropine, while hexamethonium had no effect on the action of carbachol. Serotonin and histamine also increased plasma uric acid, and their effects were obviously blocked by the administration of cyproheptadine or dimetotiazine, which are well-known blockers of the pharmaclolgical actions of serotonin and histamine. The increases of plasma uric acid caused by carbachol, serotonin and histamine depended upon their doses and had time courses similar to that caused by catecholamine. Thus, a number of biogeneamines can play a role in the increase of plasma uric acid. Considering the importance of biogeneamines in the pathogenesis of disease and in treatment with diverse drugs, we should further try to elucidate the induction mechanism of hyperuricemia by each amine.
Uric acid is the end-product of purine metabolism, and the purines are nitrogenous bases derived from the breakdown of nucleic acids. It was reported by the authors that CSF uric acid concentrations were increased in patients with histologically malignant brain tumor or microcephaly of atrophic type, but decreased in patients with microcephaly of hypopastic type. The purpose of this paper is to find out the diagnostic value of uric acid concentrations in cyst fluids of brain tumor or head injury with cyst formation. Cyst fluid, CSF and serum were investigated for uric acid concentration, lactate dehydrogenase activity and total protein concentration in 13 cases of brain tumor and in 7 cases of head injury. In cases of brain tumor, comparison of uric acid concentrations and lactate dehydtogenase activities between in cyst fluid and in serum can be a comment to the grade of histological malignancy. The finding of cyst fluid levels higher than the serum levels rase a suspicion of histologically malignant brain tumors such as glioblastoma, etc The finding of cyst fluid levels lower than the serum levels raise a suspicion of histologically benign brain tumors such as astrocytoma, craniopharyngioma, epidermoid cyst, etc. But lactate dehydrogenase activities in cyst fluids were higher than the serum levels in 2 cases of craniopharyngioma which were histoloically benign. Total protein concentrations in cyst fluids were thought to be of no diagnostic value to decide the grade of histological malignancy. In cases of head injury, materials are chronic subdural hematoma and encapsulated subdural hygroma. Lactate dehydrogenase activities in cyst fluids were higher than the serum levels in cases of chronic subdural hematoma, and lower than the serum levels in cases of encapsulated subdural hygroma. In cases of chronic subdural hematoma, lactate dehydrogenase in the erythrocytes is thought to come out due to hemolysis, and to show increased activity. On the contrary, uric acid concentrations in cyst fluids were lower than the serum levels in both cases of chronic subdural hematoma and encapsulated subdural hygroma. Because uric acid does not exist in the erythrocyte and is not influenced by hemolysis. This phenomenon prove that uric acid concentrations in cyst fluids of brain tumor is not easily influenced by intracystic hemorrhage compared with lactate dehydrogenase activities in cyst fluids. Total protein concentrations in cyst fluids were higher than the serum levels in 2 cases of chronic subdural hematoma, though total protein concentrations in cyst fluids of brain tumor were lower than the serum levels in all cases.
In an effort to study whether drug therapy is essential in the treatment of hyperuricemia, the authors examined the serum levels of uric acid and related substances in a total of 112 males who had undergone annual checkups for five consecutive years. Statistical analyses showed that the uric acid levels at the 5th year's checkup were not necessarily higher than those at the 1st checkup and except in a limited number of subjects with exceedingly high initial levels of 9.0 mg/dl or more, fell within normal ranges. No convincing cause-effect relationship was obtained between hyperuncemia and various clinical manifestations except gout. Principle component analyses of test results including serum uric acid showed that the serum uric acid falls in the elderly people-oriented category which includes Rohler's index, LDII, hemoglobin, and blood sugar.
We presented two patients associated with hypouricemia. Their serum uric acid levels were 1.6 and 1.3 mg/100ml, respectively. One patient,28 aged female, had a history of urolithiasis and showed microhematuria. Another,47 aged female, had mild hypertension and microhematuria. No other family members have been proven to have hypouricemia. These two patients gave markedly increased urate clearance and normal G F R. Other findings which indicate renal tubular dysfunction, such as glycosuria, amino aciduria, decreased phosphate reabsorption and renal tubular acidosis, were not demonstrated in them. Hypercalciuria was also not be detected. In pyrazinalnide (PZA) suppression test, these two showed different response of urate excretion. In one patient urate excretion was slightly suppressed following PZA administration. Following benzbromarone, which inhits urate reabsorption in the tubule, urate excretion almost unchanged. This patient is considered to have a defect of urate reabsorption in the proximal tubule, resulting renal urate wasting. Another patient, on the other hand, showed marked decrease of urate excretion following PZA. This result suggests that increased urate clearance in this patient is due to either enhanced secretion or diminished reabsorption of secreted urate in the tubule. Uricosuric response to benzbromarone was far lesser than in seven normal controls. Suppression rate of urate excretion following PZA under benzbromarone-induced uricosuria was similar to the value of normals. These results suggest that this patient has a defect of postsecretory reabsorption of urate in the tubule, but not enhanced secretion of urate. Renal uricosuria with hypouricemia is a rare disease and it is considered that there are some subtypes of urate transport defect which cuases renal urate wasting.
The antihypertensive and hypouricemic effects of eight-weeks administration of a uricosuric diuretic, tienilic acid (FR 3068), were examined in 18 hypertensive patients with hyperuricemia aged 38-78years. The antihypertensive effect of FR 3068 was compared with the effect of trichlormethiazide (TCMZ) in 10 paired studies, and its hypouricemic effect was also compared with the effect of xanthine oxidase inhibitor, allopurinol, co-administered with TCMZ in 10 patients. All values of blood pressure and the results of clinical examinations including serum uric acid levels were evaluated statistically using the paired t-test. The blood pressures, both systolic and diastolic, in hypertensives were significantly decreased during the daily administration of 250mg FR 3068. The mean blood pressure 157.3 ± 9.7/90.0 ± 5.1 mmHg (mean ± S. E. ) under the control with 4 mg TCMZ was decreased to 147.0 ± 5.9/84.8 ± 3.4 mmHg during the administration of 250 mg FR 3068, but the difference was not significant. Consequently, the antihypertensive effect of FR 3068 was estimated to be almost equivalent or slightly superior to the effect of TCMZ. The mean serum uric acid level in untreated hypertensives was significantly decreased from 8.43 ± 0.38 mg/dl to 5.59 ± 0.43 mg/dl during the treatment by 250 mg FR 3068. The mean uric acid level in those treated by 100 mg allopurinol with TCMZ was 6.91± 0.59 mg/dl,but it was decreased to 5.42±0.24 mg/dl when the regimen was changed to a single administration of 250 mg FR 3068. The mean levels of serum sodium, potassium, chloride, total cholesterol, triglyceride, urea N, creatinine, sGOT, sGPT and alkalinephosphatase were not significantly changed during the 8-weeks administration of FR 3068, but fasting blood sugar was slightly but significantly elevated (101.4± 10.3 mg/dl before FR 3068 vs.115.0 ±8.3 mg/dl during FR 3068) in 5 paired studies. In a patient who received 500 mg FR 3068 for 3 weeks complained of general malaise when his serum potassium level was 2.7 mEq/l, and in another patient who received 250 mg FR 3068 for 8 weeks serum potassium was decreased to 3.2 mEq/l. No other side-effects were detected throughout this study. FR 3068 appears to be a useful agent because of its unique combined action of both antihypertensive and hypouricemic effects in the treatment of essential hypertension with hyperuricemia.
A series of 46 folate and the related compounds including those already evaluated as enzyme inhibitors was tested for their inhibition of bovine milk and mouse liver xanthine oxidase in vitro. We have reconfirmed the inhibitory effect of folic acid on this enzyme N10-Formyl folic acid was even stronger inhibitor than folic acid.Reduced forms of folate and its derivatives, namely N5, N10-methenyltetrahydrofolic acid, tetrahydrofolic acid and dihydrofolic acid were also effective inhibitors of this enzyme, indicating that the biological active forms (e.g. one-carbon transfer) are not necessary for the enzyme inhibitory action. Some pteridine compounds including Neopterin, Lumazine were showed to have comparative effects to folic acid, while p-aminobenzoyl glutamic acid and p-aminobenzoic acid and glutamic acid, and their derivatives studied were without significant activity.