Roles of histamine, complement and xanthine oxidase in thermal injury of skin.

The pathogenesis of burn edema in the skin of rats appears to be related to a role for histamine, xanthine oxidase and oxygen radicals. Histamine and its metabolic derivatives increase the catalytic activity of xanthine oxidase (but not xanthine dehydrogenase) in rat plasma and in rat pulmonary artery endothelial cells. In thermally injured rats levels of plasma histamine and xanthine oxidase rise in parallel, in association with increases in uric acid. Burn edema is greatly attenuated by treatment of rats with the mast cell stabilizer, cromolyn, by complement depletion and by treatment with the H2 receptor antagonist, cimetidine, but is unaffected by neutrophil depletion. These studies suggest the following pathogenesis of burn edema: thermal trauma causes complement activation with anaphylatoxin release and mast cell secretion of histamine, leading to enhancement of xanthine oxidase activity and increased production of oxygen radicals which damage endothelial cells.

[Effect of tranquilizers of the 1,4-benzodiazepine series on the xanthine oxidase activity of the rat brain]. / Izuchenie vliianiia trankvilizatorov 1,4-benzodiazepinovogo riada na aktivnost' ksantinoksidazy golovnogo mozga krys.

Experiments in vivo and in vitro on 90 rats were made to study the influence of 1,4-benzodiazepine tranquilizers (phenazepam, nitrazepam and diazepam) on cerebral xanthine oxidase activity. Phenazepam, nitrazepam and diazepam in the dose of 5 mg per 200 g bw were shown to reduce xanthine oxidase activity by 80.4%, 64.3% and 55.8%, respectively 2 h after intraperitoneal injection. 6 h after the injection of benzodiazepines the enzyme activity grows, but control values are achieved only after nitrazepam injection. In vitro experiments revealed direct influence of the tranquilizers on xanthine oxidase. Phenazepam inhibits xanthine oxidase activity in concentration as long as 10(-10) M (to 36.6%), and practically completely in 10(-6) M concentration. Nitrazepam and diazepam inhibit xanthine oxidase activity within concentration range between 10(-8) M (to 51.5% and 33.2%, respectively), and 10(-4) M (practically completely). The inhibition of xanthine oxidase activity is shown to be caused by the competition between hypoxanthine, the reaction substrate, and tranquilizer, to bind with the active site of the enzyme.

Inherited superactivity of phosphoribosylpyrophosphate synthetase: association of uric acid overproduction and sensorineural deafness.

PURPOSE: Superactivity of 5-phosphoribosyl 1-pyrophosphate (PP-Rib-P) synthetase, inherited as an X chromosome-linked trait, has been reported in nearly 20 families in which overproduction of uric acid is invariably present in hemizygous affected males. Clinical manifestations of PP-Rib-P synthetase superactivity are mainly limited to gout in early adulthood. Neurologic deficits, including sensorineural deafness, have rarely been described. We herein document the association of PP-Rib-P synthetase superactivity, gout with excessive uric acid synthesis, and sensorineural deafness in an additional family. PATIENTS AND METHODS: Two members of a Spanish family were studied: an eight-year-old boy (Patient 1) with tophaceous gout, purine nucleotide and uric acid overproduction, and sensorineural deafness, and his 27-year-old mother (Patient 2), who had gout. Fibroblast cultures were initiated from skin biopsy specimens, and measurements of PP-Rib-P and purine nucleotide metabolism in the fibroblasts were performed. RESULTS: A labile but superactive PP-Rib-P synthetase was demonstrated in the fibroblasts cultured from both Patients 1 and 2. The kinetic basis of PP-Rib-P synthetase superactivity in this family was resistance to purine nucleotide inhibition of enzyme activity. More severe derangements in the enzyme and in PP-Rib-P and purine synthesis in Patient 1's cells than in Patient 2's cells suggest that Patient 1 is hemizygous and Patient 2 is heterozygous for an X chromosome-linked genetic defect. Limited pedigree data support this view. Compared with affected members of seven other families with PP-Rib-P synthetase superactivity, these patients are intermediate in the range of clinical expression and in the severity of the enzyme defect as measured by the degree of aberration of PP-Rib-P and purine nucleotide synthesis in fibroblasts. Metabolic abnormalities were more severe in Patient 1's cells than in the cells of most male patients (in whom clinical expression is limited to early adult-onset gout) but were less severe than in the cells of two patients in whom more complex enzyme defects were associated with uric acid overproduction and neurodevelopmental abnormalities (including deafness) in male children and adult women. CONCLUSION: Certain defects resulting in PP-Rib-P synthetase superactivity may be causally related to neurologic impairment, most commonly sensorineural deafness.

[Pathways of inosine monophosphate transformation in the chicken liver]. / Izuchenie putei prevrashcheniia inozinmonofosfata v pecheni tsypliat.

The rates of distribution of the radioactive precursor label de novo and of the reutilization pathways of inosine monophosphate biosynthesis between adenine and guanine nucleotides in the acid-soluble fraction and RNA from chicken liver were determined. The activity of enzymes involved in inosine monophosphate conversion into adenine or guanine nucleotides or in uric acid biosynthesis was determined. It was shown that in chicken liver the activity of IMP-dehydrogenase and the rate constant for the label ([14C]glycine) incorporation into the guanine nucleotide pool is 3.5 times as high as that of adenyl succinate synthetase and the rate constant for the label liberation into the adenine nucleotide pool. The rate of xanthine synthesis is by one order of magnitude more than that of hypoxanthine and uric acid. The rate of IMP synthesis from [14C]hypoxanthine via the guanine nucleotide pathway is also higher in this case. Hence, in chicken liver IMP oxidation to XMP seems to proceed with a high efficiency and can thus be used, in addition to dephosphorylation, for ammonia extrusion.

Copper(II)ethylenediaminetetraacetate does disproportionate superoxide.

The superoxide dismutase (SOD) mimetic reactivity of Cu(II)EDTA was studied in the pH range of 6.0 to 8.0. Cu(II)EDTA disproportionated superoxide without inhibiting superoxide production by xanthine oxidase, as a result of bonding sites becoming available on the copper complex with increasing acidity. This disproportionation by Cu(II)EDTA is offered as evidence that the addition of EDTA to biological preparations for the purpose of complexing copper and thereby inhibiting copper-dependent superoxide disproportionation and promoting superoxide-dependent reactions is not a valid practice.

Increase of urate formation by stimulation of sympathetic hepatic nerves, circulating noradrenaline and glucagon in the perfused rat liver.

In the isolated rat liver perfused in situ stimulation of the nerve bundles around the portal vein and the hepatic artery caused an increase of urate formation that was inhibited by the alpha 1-blocker prazosine and the xanthine oxidase inhibitor allopurinol. Moreover, nerve stimulation increased glucose and lactate output and decreased perfusion flow. Infusion of noradrenaline had similar effects. Compared to nerve stimulation infusion of glucagon led to a less pronounced increase of urate formation and a twice as large increase in glucose output but a decrease in lactate release without affecting the flow rate. Insulin had no effect on any of the parameters studied.

Purine synthesis de novo in cultured lymphoblast cells derived from patients with gout.

Rates of de novo purine synthesis in lymphoblast cell cultures derived from ten patients with gout were compared with those from control individuals. Since the growth rate of the culture, an assay procedure was developed to account for the variation in lymphoblast growth rates and to permit valid quantitative comparison between purine synthesis in each cell line. Clear differences were demonstrated between the rates of purine synthesis in cells from normal control subjects and those from patients with a deficiency of hypoxanthine-guanine phosphoribosyltransferase activity (HPRT-deficient). Lymphoblasts from the gouty patients showed purine synthesis either within the normal range or intermediate between this and the HPRT-deficient cells. In patients having normal renal function, de novo purine synthesis of lymphoblast cells correlated with the degree of urate production as reflected by the urinary excretion of urate over a 24 h period. Three patients, with demonstrable excessive production of urate in vivo, exhibited increased purine synthesis in lymphoblasts. This increased synthesis did not appear to result from any of the enzyme mutations currently recognized as responsible for abnormal purine metabolism.

Oral zinc therapy normalizes serum uric acid level in Wilson's disease patients.

The authors investigated changes in the serum uric acid (s-UrA) level seen in a Wilson's disease patient who had to undergo oral zinc therapy because of the occurrence of D-penicillamine-induced acute sensitivity reactions, including neutrophilic agranulocytosis, thrombocytopenia, and skin eruptions. Although s-UrA levels were low before oral zinc therapy (mean +/- SD, 1.60 +/- 0.20), they increased (mean +/- SD, 2.63 +/- 0.32) to within normal range (2.8-8.0 mg/dl) after therapy. There were no significant changes in the renal tubular reabsorption of UrA during oral zinc therapy. This therapy also produced an improvement of the decreased cholinesterase (ChE) values usually seen in Wilson's disease. These results suggest that oral zinc therapy can normalize UrA metabolism in Wilson's disease by improving liver dysfunction and increasing UrA synthesis.

Allopurinol in renal failure and the tumour lysis syndrome.

This paper illustrates several important points relating to the use of allopurinol in renal failure, or situations of purine overproduction: It is very easy to give too much allopurinol. Most of the side effects (bone marrow depression, exfoliative dermatitis, etc) are the result of overdosage due to the retention of oxipurinol, an effect exaggerated by thiazide diuretics. Monitoring of plasma oxipurinol levels (ideally less than 100 mumol/l) by high-pressure liquid chromatography is helpful for adjusting dosage in renal failure. Some estimate of the anticipated purine excess is equally vital in deciding dosage during tumour lysis if the risk of urate nephropathy is not to be substituted for the certainty of xanthine nephropathy. In this situation the use of allopurinol may even be questioned. Patients with HGPRT deficiency are exquisitely sensitive to allopurinol, and careful monitoring of the effect on urinary purine levels is essential if xanthine colic is to be avoided.

In vivo voltammetric evidence of production of uric acid by rat caudate.

Linear sweep in vivo voltammetry with carbon paste electrodes records a prominent peak at about 340 mV in the anterior caudate of rat brain. This peak is increased by microinfusion of uric acid or xanthine oxidase (which enhances conversion of hypoxanthine and xanthine to uric acid) and is decreased or eliminated by microinfusion of uricase. Allopurinol (a specific xanthine oxidase inhibitor) also decreases this peak when given either intracranially or intraperitoneally. Co-administration of uricase and allopurinol reliably eliminate the peak in question. These data suggest that uric acid, a purine metabolite that has been thought to be absent in brain, is formed locally in rat caudate and that uric acid is the sole component of the peak at 340 mV. In vivo voltammetry may be a useful new tool for studying brain purine metabolism.

Hyperuricemia in glycogen storage disease type I. Contributions by hypoglycemia and hyperglucagonemia to increased urate production.

Studies were performed to determine whether hypoglycemia or the glucagon response to hypoglycemia increases uric acid production in glycogen storage disease type I (glucose-6-phosphatase deficiency). Three adults with this disease had hyperuricemia (serum urate, 11.3-12.4 mg/dl) and reduced renal clearance of urate (renal urate clearance, 1.1-3.1 ml/min). These abnormalities were improved in one patient by intravenous glucose infusion for 1 mo, suggesting a role for hypoglycemia and its attendant effects on urate metabolism and excretion. A pharmacologic dose of glucagon caused a rise in serum urate from 11.4 to 13.0 mg/dl, a ninefold increase in urinary excretion of oxypurines, a 65% increase in urinary radioactivity derived from radioactively labeled adenine nucleotides, and a 90% increase in urinary uric acid excretion. These changes indicate that intravenous glucagon increases ATP breakdown to its degradation products and thereby stimulates uric acid production. To observe whether physiologic changes in serum glucagon modulate ATP degradation, uric acid production was compared during saline and somatostatin infusions. Serum urate, urinary oxypurine, radioactivity, and uric acid excretion increased during saline infusion as patients became hypoglycemic. Infusion of somatostatin suppressed these increases despite hypoglycemia and decreased the elevated plasma glucagon levels from a mean of 81.3 to 52.2 pg/ml. These data suggest that hypoglycemia can stimulate uric acid synthesis in glucose-6-phosphatase deficiency. Glucagon contributes to this response by activating ATP degradation to uric acid.

The use of xanthine oxidase for a specific and sensitive fluorimetric determination of 6-mercaptopurine in serum.

A specific and highly sensitive fluorimetric method was developed for the determination of 6-mercaptopurine (6MP) in serum. The method is based on the enzymatic oxidation of 6MP with xanthine oxidase to the oxypurine, followed by oxidation with acidic chromate to the corresponding 6-sulfonate. The fluorescent product has excitation and emission maxima at 330 and 400 nm, respectively. The limit of sensitivity was approximately 22 pg/ml for 6MP in water. The sensitivity limit for 6MP in serum containing azathioprine was approximately 2.2 ng/ml. The rate constants for conversion of 6MP into the final product (6-thiouric acid) and the apparent Michaelis constant were also determined by a nonlinear regression analysis based on the integrated Michaelis-Menten equation using the ultraviolet absorbance data and the simplified complementary tristimulus colorimetry.

Purine metabolism in high and low uric acid lines of chickens: de novo uric acid synthesis in isolated hepatocytes and phosphoribosylpyrophosphate amidotransferase activities.

The de novo biosynthesis of uric acid was examined in isolated hepatocytes from the high and low uric acid lines of chickens. Rates of incorporation of radiolabeled glycine into uric acid by hepatocytes from the high uric acid (HUA) line were approximately 3.6-fold greater than found in low uric acid (LUA) control hepatocytes. Uric acid synthesis rates in these cells were positively correlated with plasma uric acid levels (r = +0.77; P less than 0.01). The activity of phosphoribosylpyrophosphate (PRPP) amidotransferase was measured in acetone powder preparations from liver and kidney tissues of the HUA and LUA lines. Activities in kidney tissues were about 21% lower than those found in livers. PRPP amidotransferase activities in liver and kidney tissues did not correlate significantly with plasma uric acid levels. The increased synthesis of uric acid in the HUA line may be the result of the increased PRPP synthetase activities and PRPP pool sizes previously reported for these tissues.

Purine metabolism and urate biosynthesis in isolated chicken hepatocytes.

The metabolism of some purine compounds to urate and their effects on de novo urate synthesis in chicken hepatocytes were investigated. The purines, listed in descending order of rates of catabolism to urate, were hypoxanthine, xanthine, inosine, guanosine, guanine, IMP, GMP, adenosine, AMP, and adenine. During a 1-h incubation period, conversion to urate accounted for more than 80% of the total quantities of guanine, guanosine, and inosine metabolized, but only 42% of the adenosine and 23% of the adenine metabolism. Adenine, adenosine, and AMP inhibited de novo urate synthesis [( 14C]formate incorporation into urate), whereas the other purines, especially guanine, guanosine, and GMP, stimulated de novo urate synthesis. When hepatocytes were incubated with glutamine and adenosine, AMP, guanine, guanosine, or GMP, the rates of de novo urate synthesis were lower than the additive effects of glutamine and the purine in separate incubations. Increasing phosphate concentrations had no effect on urate synthesis in the absence of added purines but, in combination with adenosine, AMP, guanosine, or GMP, increased urate synthesis. These results indicate that the ratio of adenine to guanine nucleotides and the interaction between substrates and purine nucleotides are involved in the regulation of urate biosynthesis in chicken liver.

Incorporation of 15N from glycine into uric acid in gout: a follow-up study.

Over-incorporation of 15N-labeled glycine into uric acid indicates over-production of uric acid by de novo purine biosynthesis. This metabolic aberration, though considered to be inborn (3), may be modified by changing of life style, aging and long-term therapy. In the patient under study, the protracted use of allopurinol seems to have played the most important role. Aging contributed to a certain extent, and changing life style was the least significant factor.

Salvage of circulating hypoxanthine by tissues of the mouse.

Hypoxanthine is an inefficient precursor of purine nucleotides in mouse tissues. In vitro, mouse erythrocytes salvage less than 10% of hypoxanthine (10 microM) added to whole blood in 30 min of incubation at 37 degrees C. In vivo, circulating hypoxanthine is rapidly degraded (greater than 90% in 10 min) to allantoin and uric acid. All tissues examined (other than erythrocytes) converted small amounts of hypoxanthine to nucleotides, with kidney and lung being the most active tissues examined. It is estimated that less than 2% of circulating hypoxanthine is salvaged in the mouse; the remainder is catabolized.

Activation of xanthine dehydrogenase during the prenatal period through L-thyroxine, thiourea and cycloheximide.

Xanthine dehydrogenase activity in the liver of embryonic chicks has been shown to be inducible by L-thyroxine, thiourea, and cycloheximide. Results reported here indicate different mechanisms underlying the activation through L-thyroxine on the one hand, and through thiourea and cycloheximide on the other. Using hepatocyte suspension and homogenized liver, it has been shown that prenatal activation of xanthine dehydrogenase results in increased rate of uric acid formation from nucleic acids and purine derivatives, but not from amino acids.

Purine metabolism in high- and low-uric acid lines of chickens: hypoxanthine/guanine phosphoribosyltransferase activities.

The activity of hypoxanthine/guanine phosphoribosyltransferase (HGPRT) was examined in the livers and kidneys of two genetic lines of chickens selected for different plasma uric acid levels. Previous work demonstrated that the high-uric acid line (HUA) had significantly greater de novo uric acid synthesis rates in kidney tissue compared to the low-uric acid line (LUA). In addition, phosphoribosylpyrophosphate (PRPP) synthetase and xanthine dehydrogenase activities in livers and kidneys were significantly higher in the HUA compared to the LUA line. PRPP pool sizes were also significantly higher in both livers and kidneys of HUA birds. HGPRT activities in livers of HUA birds were significantly (P less than 0.05) greater than in LUA birds. The mean value of liver HGPRT was 7.36 +/- 0.25 pmole inosine-5'-monophosphate (IMP) and 6.05 +/- 0.27 pmole IMP produced/micrograms protein/hr, respectively, for the HUA and LUA lines. There were no significant differences (P greater than 0.05) in kidney HGPRT activities between the two groups. The mean value of kidney HGPRT was 52.87 +/- 1.62 pmole IMP and 50.72 +/- 1.62 pmole IMP produced/micrograms protein/hr, respectively, for the HUA and LUA line. Elevated liver HGPRT may serve to enhance the regeneration of PRPP in the HUA liver. Elevated liver PRPP synthetase and PRPP pool size suggest an increased flux through the de novo purine biosynthetic pathway in HUA birds. The resulting additional pyrophosphate from the glutamine PRPP amidotransferase reaction would stimulate recovery of PRPP and spare the system from a substantial loss of energy.