C.-H. De Verdier

A METHODOLOGICAL STUDY OF THE ENZYMATIC DETERMINATION OF GLUCOSE IN BLOOD.

A methodological investigation of the glucose oxidase method for determination of glucose is described. The importance of eliminating substances reducing H2O2 generated from glucose and other substances causing unspecific oxidation of the chromogen is emphasized. On the basis of this study a method has been constructed which gives accurate glucose values in whole blood, plasma, and red blood cells.

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.

Restoration of Defective Oxygen-transport Function of Stored Red Blood Cells by Addition of Inosine

During storage of acid-citrate-dextrose-blood the oxygen affinity of the haemoglobin is increased; i.e. the oxygen dissociation curve is shifted to the left. In the meantime the red cell ATP and 2,3-diphospho-glycerate levels are decreased. In the present investigation it is shown that these changes are reversed by addition of inosine. Thus the oxygen-transport function of stored blood seems to be related to the intracorpuscular ATP and 2,3-diphosphoglycerate levels.

Low Binding of 2,3-Diphosphoglycerate to Haemoglobin F: A Contribution to the Knowledge of the Binding Site and an Explanation for the High Oxygen Affinity of Foetal Blood

The binding of 2,3-diphosphoglycerate to human haemoglobin A and F was determined using an ultracentrifugation technique. The binding of the phosphocompound was much lower to haemoglobin F than to haemoglobin A, both in the oxygenated and deoxygenated states. It is concluded that this difference may explain, at least partly, the higher oxygen affinity of foetal blood. The result supports previously advanced arguments that binding of 2,3-diphosphoglycerate to haemoglobin A takes place at βH21 His.

Affinity of Human Hemoglobin a to 2,3—Diphosphoglycerate. Effect of Hemoglobin Concentration and of pH

The affinity of human oxy- and deoxyhemoglobin A for 2,3-diphosphoglycerate was measured in purified hemoglobin solutions at 37 °C and with near-physiological ionic composition. The affinity of both oxy- and deoxyhemoglobin for 2,3-diphosphoglycerate decreased markedly as the hemoglobin concentration increased from 0.4 to 5.5 mmol/1. The data show that, under physiological conditions, about 15 % of erythrocyte 2,3-diphosphoglycerate is bound in oxygenated cells and about 35 % in deoxygenated cells. Previous observations of the pH-dependence of the affinity of hemoglobin to 2,3-diphosphoglycerate were extended to show that the affinity of the oxygen-linked site is very similar to that found for the 2,3-diphosphoglycerate-mediated decrease in the deoxygenation rate of hemoglobin.

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.

Purine metabolism in normal and ITP-pyrophosphohydrolase-deficient human erythrocytes

Fresh and stored erythrocytes from normal and ITP-pyrophosphohydrolase (ITP-ase, EC 3.6.1.19) deficient individuals were incubated with hypoxanthine, guanine, allopurinol, and inosine. Differences in the purine metabolism between the normal and the ITP-ase deficient erythrocytes were observed only in the IMP-ITP cycle. Hypoxanthine, guanine and allopurinol were converted to nucleotides at the same rate. Hypoxanthine (2.5 mumol/l) inhibited the salvage of allopurinol (40 mumol/l). A slow decrease (0.7%/day) in salvage rate was observed in both types of cells upon storage at +4 degrees C. Erythrocyte ITP-ase activity was measured in a reference sample group of 48 healthy volunteers. Two distinct groups were found with mean activities equal to 48.3 +/- 13.1 nkat/g Hb (means +/- SD, n = 38) and 11.4 +/- 4.3 nkat/g Hb (n = 10). In two previously selected subjects, the ITP-ase activity was 0.2 and 2.4 nkat/g Hb. A hypothetical genetic mechanism is discussed. The maximal energy turnover in the IMP-ITP cycle during hypoxanthine incubation was found to be less than 10% of the basal erythrocyte energy turnover.

Transferability of clinical laboratory data within a health care region

Analytical data for S-Creatinine and S-Urate are presented from seventeen laboratories in the Swedish Uppsala-Orebro regional quality assessment program. The bias and imprecision as well as the instability of the measurement procedures in the participating laboratories were estimated over three 14-week periods. Bias was estimated by a linear least squares fit of the difference between measured and assigned values vs. assigned values, and expressed in absolute and relative terms. Instability of the measurement procedures was estimated by comparing slope and intercept of regression lines of measured vs. assigned values from three fourteen week periods. According to our experiences we recommend regression analysis to describe the performance of the analytical methods of a laboratory over time. The results show that most laboratories fell within the limits of +/- 15% bias for S-Creatinine above 100 mumol l-1 and +/- 17% for S-Urate at concentrations above 250 mumol l-1. Various steps to reduce the inter-laboratory variability are suggested, including numerical correction of individual laboratory results using correction functions. In a few laboratories, instability was too high to allow for numerical corrections of analytical results.

Inter-laboratory quality control with investigation of different methodological characteristics.

A classification system was designed which gives a brief description of the analytical methods used by laboratories participating in an external quality control program. Together with the analytical results several characteristics of the methods are to be reported by the laboratories, e.g. principle, special variant, commercial reagent, type of calibration and of instrumental equipment. The system is logically structured to be easy to learn and to facilitate the transfer of the information to a computer. To test this system, a survey was made with forty-two participating large or middle sized Nordic laboratories. Control material at two concentration levels was analysed on ten different occasions. A computer program was used which allowed statistical treatment of the data and grouping of the results according to different outlined characteristics of the method. For some methods, but not all, the use of calibration standards, prepared with weighed amounts of the components were superior to calibrations usi...

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.