Å. Ericson

A Modified Method for the Determination of 2,3-Diphosphoglycerate in Erythrocytes

A modified assay for 2,3-diphosphoglycerate in erythrocytes is described. 3-phosphoglycerate, specifically formed by the action of glycerate phosphomutase modified by 2-phosphoglycolate, is determined by using a sequence of enzymes starting with phosphoglycerate kinase and ending with glycerol 1-phosphate dehydrogenase. This procedure gives a high sensitivity (2 moles NADH per mole DPG) and a favourable overall equilibrium for the assay. The effect of other metabolites and of changing the volume of the protein-precipitating solution (0.6 mol/1 PCA) was investigated. The accuracy of the method was tested by comparison with a chromatographic method and by determination of the mean value from 29 healthy non-anaemic men.

Studies on the Mechanism of Human Red Cell Loss of Viability during Storage at +4 °C in vitro

Abstract. Red cells stored in SAGM medium for 42 days at +4°C were rejuvenated by bicarbonate, pyruvate and adenosine. Autologous 24‐hour posttransfusion survival was determined in untreated as well as rejuvenated cells and showed an improvement from 77.4±4.7 to 89.2±7.2%. The erythrocyte adenylate energy charge decreased relatively more than the total adenylate concentration during storage, but the latter correlated better with posttransfusion red cell survival. Considerable deteriorations in red cell morphology (expressed as morphology index) and in deformability (measured as red cell fluidity) were observed during storage but were partly reversed by rejuvenation. The morphology index and the posttransfusion survival showed a significant correlation (r = 0.95, p<0.005) after, but not before, rejuvenation, indicating that the remaining changes are more permanent and decisive of survival. It is suggested that, in the proportion of stored erythrocytes which respond to rejuvenation, the capacity and time dependence of recovery of normal shape and flexibility are important.

A systematic study of nucleotide analysis of human erythrocytes using an anionic exchanger and HPLC

A two-step procedure for rapid HPLC analysis of nucleotides from human erythrocytes is described. A strong anion exchanger Partisil-10 is used as column material. Elution of monophosphates requires about 12 min and the elution of di- and triphosphates 21 min. The elutions are performed separately and with different injections as such a procedure will save time otherwise used to re-condition the columns. In addition to the three adenylates other nucleotides such as GTP, GDP, IMP and NAD can be recorded as isolated, well-defined peaks, which can be subject to quantitative analysis. The mean value of ATP concentration for 12 healthy individuals was 1.55 +/- 0.05 mmol/l, about 25% higher than generally reported in the literature. Accurate estimates of ADP and AMP concentrations allowed calculation of mean values for the physiologically interesting ratios: adenylate energy charge (0.945 +/- 0.002) and equilibrium constant for adenylate kinase (1.205 +/- 0.053). GDP and GTP are present in concentrations that are about 4% of those for corresponding adenylates. The analysis of nucleotides in human erythrocytes is a useful way of studying erythrocyte preservation and investigating patients with hemolytic disorders.

Plasma concentration and renal excretion of adenine and 2,8-dihydroxyadenine after administration of adenine in man

A new method of high performance liquid chromatography (HPLC) which makes it possible to analyse 2,8-dihydroxyadenine (DOA) in plasma in concentrations exceeding 0.25 mumol/l is described. The method was used to study the renal elimination of DOA. For comparison, the renal handling of adenine was also investigated. The results from an analysis of the experimental data support the assumption that more than one concentration-dependent mechanism exists in the renal tubuli for each of the two purines, adenine and DOA. In general the clearance values are higher for DOA than for adenine and indicate net secretion for both substances.

Effects of Oxygen on Red Cells during Liquid Storage at +4°C

Red cells collected in CPD and suspended in SAGM medium were stored in plastic (PVC) containers for 42 days at +4°C. Comparison was made between aerobic storage (normal air exposure) and anaerobic storage (exposure to nitrogen gas). The air‐exposed units showed a strong increase in pO2 and oxygen saturation as a result of oxygen penetration into the bags from outside. This resulted in a decrease in ATP and adenylate energy charge, a slower metabolization of adenine and hypoxanthine to AMP and IMP, respectively, and a faster decrease in red cell fluidity. To explain the findings it is concluded that aerobic storage causes an increased need of high‐energy phosphate groups, possibly used for replacement of the phospholipid membrane bilayer or in repair of phosphate bonds in the cytoskeleton. It is further proposed that a slight formation of hydrogen peroxide from free oxygen radicals moderately increases the oxidation of reduced (GSH) to oxidized (GSSG) glutathione and slightly enhances the need for reduced nicotinamide‐adenine dinucleotides mainly provided by increased flux through the pentose phosphate shunt.

High adenosine content in human uterine smooth muscle compared with striated skeletal muscle

We determined the concentrations of adenosine and some of its catabolic products in biopsy specimens from predetermined loci of human myometrium under different functional conditions to compare uterine muscle with rectus abdominis muscle from the same individuals. In order to achieve a good resolution in the separation of nucleosides and purine bases, a preseparation procedure was developed prior to analysis of these compounds on high performance liquid chromatography. Adenosine occurred in a nearly 70-fold higher concentration in smooth uterine muscle in comparison with striated skeletal muscle. Similarly, myometrial inosine and hypoxanthine were 7- and 2.4-times in excess over the rectus muscle, whereas xanthine was scarcely and rather evenly represented in the two types of muscles. The uterine content of adenosine and inosine was distinctly higher in pregnant women compared to non-pregnant ones. A regional difference existed for adenosine, with 3.3 times higher concentration in fundus uteri compared to the isthmic part. A reverse pattern was observed for hypoxanthine and inosine, being 2-3 times more frequent in the isthmic part. The orthophosphate concentration was not stoichiometrically related to the adenosine concentration in a simple way, being 2-3 times lower in uterine muscle compared to the skeletal muscle. A significant correlation existed between uterine contents of AMP and adenosine and similarly, significant inverse correlations were apparent between uterine ATP and ADP contents and energy charge on one hand and adenosine content on the other.

Purine metabolism of erythrocytes preserved in adenine, adenine- inosine, and adenine-guanosine supplemented media

Four different media for erythrocyte preservation have been compared by studying 16 variables mainly describing the purine metabolism and glycolysis of the erythrocyte. The concentrations of the additives in the erythrocyte suspensions were as follows: adenine, 0.25 mmol/l; adenine, 0.75 mmol/l; adenine, 0.25 and inosine, 0.50 mmol/l; and adenine, 0.25 and guanosine, 0.50 mmol/l. Evaluated from the concentrations of glycerate 2,3‐bisphosphate and purine nucleoside triphosphates, the medium with adenine‐guanosine was superior to the others. In this medium with adenine‐guanosine was superior to the others. In this medium the guanosine was rapidly split to guanine, which was slowly used for nucleotide synthesis or deaminated to xanthine. Differences between ATP and GTP in their reactivity with the two enzymes phosphofructose kinase seems to explain the beneficial effect of guanosine on preservation of erythrocytes.

Adenine metabolism in man 1. After intravenous and peroral administration

The basal plasma concentration of adenine and its renal excretion was studied in two men. For its analysis partly new chromatographic techniques were developed. The plasma concentration varied around 70 nmol/l; the renal excretion rate was, as reported earlier by other investigators, around 10 nmol/min. Loadings, intravenously during about 3 h and orally, both with about 30 nmol adenine per kg body mass revealed that most of the adenine was metabolized to nucleotide form. In the experiments with intravenous administration of adenine only about 2% of the given dose appeared in the urine as adenine and somewhat less as the easily precipitable metabolite 2,8-dihydroxyadenine. In the peroral loads, with higher plasma adenine concentrations, the ratios of the renally excreted two compounds were one to a few per cent higher.

Metabolism of Guanosine in Human Erythrocytes

Abstract. The metabolism of guanosine in human erythrocytes has been studied in two different experimental systems – direct incubation and dialysis incubation – the latter allowing continuous addition and removal of substances. Intra‐ and extracellular purine compounds were analyzed using high‐performance liquid chromatography (HPLC). At 37°C, a normal pH (7.4) and a favorably high concentration of inorganic phosphate, guanine nucleotides were synthesized at a substance rate of about 0.17 mmol · h‐1 (calculated per liter erythrocytes) when guanosine was kept at a concentration of 25 μmol · I‐1. At a higher guanosine concentration the rate of synthesis increased only moderately. Erythrocytes loaded with guanylates lost these nucleotides at a rate of 0.023 mmol · h‐1 at a normal phosphate concentration and somewhat slowlier at a higher phosphate concentration. The metabolism kept the guanylates in an equilibrium that was similar to the equilibrium between the adenylates.

Specimen handling for the assay of adenylates and glycerate 2, 3-bisphosphate in erythrocytes.

ATP, ADP, AMP and glycerate 2, 3-bisphosphate in blood are frequently analyzed in order to evaluate erythrocytes for blood donation and to diagnose hemolytic anemias. In order to find an uncomplicated and safe procedure for the preanalytical treatment of blood a freezing-storing procedure using dry ice was worked out and combined with a later PCA precipitation. The pre-freezing stability of the components was also investigated and found to be best at room temperature.