Charles Bishop

Uric Acid in Two Patients with Wilson's Disease (Hepatolenticular Degeneration).∗

Summary A brother and sister with Wilsons disease (hepatolenticular degeneration) were found to have low serum urate levels and were injected with isotopic uric acid for pool size and turnover rate determination. The pool size of both was well below normal and the turnover rate was approximately twice the normal value. Benemid accentuated these findings in the female but was not tried in the male because of its adverse clinical effect. The rapid rate of removal of uric acid is compared to the phenomenon of aminoaciduria that is common in this disease. When ribose nucleic acid was fed to the male subject there was a statistically significant increase in urinary uric acid and this “extra” uric acid was equivalent to about 55% of the calculated purine ingested. There was no significant rise in the excretion of nonuric acid purines after nucleic acid feeding and this suggests that exogenous purine is mainly converted to uric acid before excretion.

Changes in the Nucleotides of Stored or Incubated Human Blood

Human blood was stored in ACD solution (add citrate dextrose) for up to eight weeks at 4 C. or up to 72 hours at 37 C. The changes in the blood nucleotides were followed serially by fractionation on Dowex‐1‐formate resin. The breakdown of ATP in aging blood followed the pattern, ATP→ ADP→ AMP→ IMP→ hypoxanthine, causing a steady decrease in ATP concentration and a continuing rise in hypoxanthine concentration with a transient rise and fall in the intermediate nucleotides. The reactions were roughly similar at both temperatures but required eight weeks at 4C. as against only three days at 37 C. The changes in the nucleotide pattern of stored blood are so characteristic that they may be used to date stored blood or to compare different methods of storage. The exact correlation between the nucleotide pattern of stored blood and red cell survival is not known, but it generally is accepted that both are related to the length of time and storage conditions of blood.

Maintenance of ATP Level of Incubated Human Red Cells by Controlling the pH

Fresh human blood was incubated at 37 C. in ACD solution, heparin‐glucose solution, and heparin‐glucose solution with pH controlled by automatic titration with sterile NaHCO3 solution. In ACD solution glycolysis of the red cells slowed down with time and concomitantly the ATP level decreased sharply. In heparin‐glucose solution, glycolysis continued more vigorously and the ATP levels were maintained longer. In the flasks in which the lactic acid was neutralized continuously, glycolysis proceeded almost unabated, and the ATP levels were correspondingly well maintained. Red cell viability and survival are presently assumed to be related to glycolytic maintenance of adequate levels of ATP. These studies demonstrate that storage of blood in heparin‐glucose may prolong the red cell viability over that in ACD, and that neutralization of the lactic acid produced in glycolysis gives such dramatic improvement that a whole new system of blood storage may be predicated on this principle.

Some in Vitro Effects of Adenine Added to Stored Blood

When adenine was added to freshly collected ACD blood and the blood incubated at 37 C. for one or two days, the concentration of ATP and adenine nucleotides (AMP + ADP + ATP) was higher than in the same blood without adenine. The addition of inosine with adenine was even more effective. Adenine was used by the red cells and the effective amounts of adenine to be added varied with the experimental conditions, for example, length of incubation. When adenine or adenine and inosine were incubated with outdated ACD blood, the ATP and adenine nucleotide concentrations rose, indicating that synthetic abilities were still present in red cells which had deteriorated energetically as a result of storage in citrate in the cold. When outdated red cells were returned to a more normal pH, the ATP levels increased. When adenine addition was included along with neutralization, the effects were remarkable, the ATP and adenine nucleotide levels going even above normal fresh blood levels. These experiments suggest that added adenine enhances ATP synthesis and allows formulation of a scheme for the participation of adenine in the metabolic reactions of stored cells.

THE MAINTENANCE OF ATP IN STORED BLOOD BY ADENOSINE AND INOSINE.

In order to resolve a controversy concerning the relative effectiveness of adenosine and inosine for maintaining the high energy phosphates of incubating red blood cells, experiments were performed in which either of the nucleosides was added, sometimes at the beginning of incubation and sometimes later. The results indicated that the relative effectiveness varied according to the concentration and time of addition of the nucleoside. The variability of the results with time is attributed to the fact that adenosine and inosine affect different ATP synthetic reactions whose rates change during incubation. Experiments with adenine indicated that adenine moiety is important for ATP maintenance.

Differences in the Effect of Lactic Acid and Neutral Lactate on Glycolysis and Nucleotide Pattern in Incubated Whole Human Blood

Whole human blood collected in heparin and glucose was incubated for up to 48 hours. Glycolysis proceeded, giving rise to lactic acid formation, and in consequence the pH decreased continuously. Blood ATP levels also decreased as glycolysis slowed since glycolysis is the only energy source in the red cells of whole human blood. The decrease in glycolytic rate was shown to be entirely dependent upon the increasing acidity and not on the lactate per se, since added neutral lactate had no effect on the rate of glycolyis or the ATP level whereas added lactic acid had a profound effect on both glycolysis and ATP level.

COMPARISON OF BLOOD STORED IN ACD AND IN A SOLUTION CONTAINING HEPARIN, PHOSPHATE, GLUCOSE AND ADENINE.

Duplicate blood samples were obtained from six healthy young males, one aliquot was collected in ACD and one aliquot in a solution of heparin‐glucose‐phosphate‐adenine (HGPA). All were stored under blood bank conditions for a total of four weeks. The HGPA samples maintained their ATP and nucleotide adenine levels much better than the ACD samples and had generally higher pHs and faster rates of glycolysis. Their osmotic fragility and plasma K+ levels were similar to those of the ACD samples. Because there was considerably more hemolysis in the HGPA samples than in the ACD samples and in the absence of red cell survival data, the HGPA system is not recommended at this time as a substitute for the ACD system but as a comparative system to reveal metabolic shortcomings of the ACD storage system.

Factors in the In Vitro Maintenance of the Nucleotide Pattern of Whole Human Blood

The effects of various glycolytic inhibitors on the relatively stable nucleotide pattern of freshly drawn heparinized human blood, incubated for two hours at 37 C. was observed. Fluoride, iodoacetate, arsenate, or 2‐deoxyglucose caused a similar high‐energy nucleotide breakdown as observed on hemolysis, viz., rapid disappearance of ATP, GTP, ADP, with simultaneous increase in AMP and IMP. The maintenance of a normal blood nucleotide pattern in vitro thus depended on cellular integrity and a source of energy. The addition of compounds believed to enhance or inhibit the pentose shunt pathway had no effect on the blood nucleotide pattern. This is interpreted to mean that under essentially in vivo conditions, the major source of energy for the red cell is derived from the Embden‐Meyerhof cycle.

The fate of uric acid in the normal and gouty human being

Abstract A normal subject was intravenously injected on three occasions with a relatively small dose of N15 uric acid. The recovery of N15 as urinary uric acid was 72 to 78 per cent and as fecal nitrogen was 16 to 19 per cent. Less than 4 per cent was recovered as urinary urea and ammonia. When N15 uric acid was injected into a normal subject after cholecystectomy, N15 was found in the bile collected from a T tube in the common duct. The recovery of intravenously injected N15 uric acid as urinary uric acid in six gouty subjects was less than in the normal subject (34 to 54 per cent). In the three of these subjects who were thus studied, the recovery of N15 as fecal nitrogen was somewhat above normal (25 to 38 per cent). It is concluded that in the normal subject uric acid is mainly disposed of as urinary uric acid or fecal nitrogen. This cannot be said for the gouty subject. Whether this inability to recover completely the injected dose of uric acid in these two fractions in the gouty subject is due to an artifact of the experimental conditions or to a novel feature of metabolism cannot be decided with the present data.

Changes in activities of some enzymes during in vivo aging of mouse erythrocytes.

Summary Red cell G-6-PD, 6-PGD, and GOT were studied in mice during and after prolonged arrest of erythropoiesis by means of repeated subcutaneous injections of actinomycin D. No clear downward trend in the activities of any of these enzymes in the progressively aging cells was found except for GOT, which initially declined as the reticulocytes disappeared and then stabilized at a new lower level. Discontinuation of the drug evoked a marked reticulocytosis accompanied by a sharp increase of G-6-PD and GOT activity. These data suggest that the major factor determining the red blood cell enzymes level is the presence or absence of reticulocytes.