C. H. M. M. de Bruyn

Purine metabolism in relation to leukemia and lymphoid cell differentiation

Abstract A number of inborn errors of purine metabolism have been associated with immunodeficiency diseases. From studies to the possible mechanism(s) leading to the defects in the immune system, it appeared that the accumulation of deoxyATP and deoxyGTP and the subsequent inhibition of ribonucleotide reductase played an important role. The inhibition of methylation pathways through the accumulation of s -adenosylmethionine seems to be a second valid concept. The amount to which certain subtypes of lymphoid cells were affected by the enzyme deficiencies was strongly related to the enzymatic make-up of the cells. Lymphoid cells from different maturation stages could be affected in a specific way, depending on the different enzyme activities of these cells. Studies on human lymphoblastic leukemias showed that, related to the immunological subtype, the different leukemias could be characterized by a different enzymatic make-up. In this paper we discuss the possibilities for a specific enzyme directed chemotherapy, directed against specific subtypes of human lymphoblastic leukemias. Experimental evidence indicates that for example the adenosine deaminase inhibitor 2′-deoxycoformycin can be used as a specific drug against acute lymphoblastic leukemia with the T cell phenotype.

A patient with purine nucleoside phosphorylase deficiency: Enzymological and metabolic aspects

1. Enzymological and metabolic data in a patient with nucleoside phosphorylase (NP) deficiency are described. 2. Incubation of intact NP-deficient red cells with [14C]adenosine showed a rapid uptake and conversion to inosine. Almost no radioactivity was incorporated in the adenosine nucleotides and no hypoxanthine labeling could be detected. 3. Incubation with [14C]inosine resulted in a rapid conversion to IMP in the normal intact red cells but in an accumulation of inosine in the medium with the erythrocytes of the patient, proving again that a NP deficiency is present. 4. The high PRPP level found may result from impaired consumption due to lack of substrates for the salvage enzyme HGPRT. 5. Incubation with [14C]hypoxanthine and [14C]adenine showed that normal HGPRT and APRT activities were present in the NP-deficient red cells. 6. In serum and urine of the patient the levels of inosine and guanosine were considerably increased, while the serum and urinary levels of uric acid were very low. In the two deceased sisters NP deficiency was also strongly suggested by analyses of the serum purines, of stored deep frozen samples.

Quantitative radiochemical enzyme assays in single cells: Purine phosphoribosyl transferase activities in cultured fibroblasts

Abstract An ultra-microchemical method using radioactive substrates has been developed for enzyme activity measurements at the single cell level. In order to demonstrate the possibilities of this radiochemical microassay, activity measurements of hypoxanthine-guanine phosphoribosyl transferase (HG-PRT) and of adenine phosphoribosyl transferase (A-PRT) in isolated human fibroblasts are described. There was a linear relationship between the number of cells incubated and the enzyme activities found. It was observed that the HG-PRT activity in single, skin derived, fibroblasts did not differ from that in amniotic fluid derived fibroblasts, thus providing a new, quantitative assay for rapid prenatal diagnosis in the Lesch-Nyhan syndrome.

In Vivo Induced Malignant Hyperthermia in Pigs. I. Physiological and Biochemical Changes and the Influence of Dantrolene Sodium

The effects of an induced malignant hyperthermia (MH) crisis have been studied in the intact pig. Both physiological and biochemical changes in skeletal muscle were studied. MH was induced with 3% halothane plus a bolus injection of succinylcholine. In the prechallenge period a significant difference was observed in the concentration of certain muscle metabolites, comparing the MH‐susceptible (MH+) with the non‐susceptible (MH‐) pigs. A lower level was measured for phosphocreatine (PCr), inosine monophosphate (IMP) and an increased level of lactate and creatine (Cr) in the susceptible pigs (MH+). The challenge caused a significant reduction of the level of PCr and adenosine in MH+ pigs, compared to the prechallenge period. After administration of dantrolene sodium, a significant decrease was measured in the level of lactate, compared to the prechallenge period as well as during the challenge. In contrast, in the control pigs no significant changes were observed in muscle metabolites, either after induction of MH or after the administration of dantrolene sodium. Enzyme activity determinations of muscle adenylate kinase and adenosine monophosphate (AMP)‐ deaminase did not show any difference in activity either before or during the MH crisis or after treatment with dantrolene sodium. The earliest physiological change during an induced MH crisis in our study was thrapid increase of the end‐tidal CO2. Within 5 min after MH induction, end‐tidal CO2 was doubled. It is concluded that the monitoring of the end‐tidal CO2 is essential to diagnose MH at a very early stage.

A second case of inosine phosphorylase deficiency with severe T-cell abnormalities.

Recently, Giblett et al. described a patient with severe T-cell immunodeficiency, associated with inosine phosphorylase deficiency (1). We found a similar case which detailed clinical stories will be published later.

Purine Metabolism in Intact Erythrocytes from Controls and HG-PRT Deficient Individuals

The present paper reports on studies on the purine metabolism in normal and HG-PRT deficient human erythrocytes.

Determination of uric acid in serum using isotachophoresis

An operational system is described for the isotachophoretic determination of uric acid in serum, making use of column coupling. The method has been compared with a standard enzymatic procedure. With the present technique small amounts of serum (ca. 3 microliter) can be applied without any pretreatment. Urate recovery was 99.0-100.5%. Under the non-physiological measuring conditions used, 12-28% of control serum uric acid was bound to macromolecules of molecular weight exceeding 25,000. The day-to-day variations of the isotachophoretic procedure were smaller than those of the enzymatic method, whereas standard deviations were comparable. The isotachophoretic procedure is less influenced by certain metabolites.

Characterisation of purine nucleoside phosphorylase from fibroblasts using ultra-microchemical methods.

A new technique to quantitate nucleoside phosphorylase (NP) activity in single or small numbers of counted visually selected cells is presented. Fibroblasts were cultivated on the plastic film bottom of culture dishes. After lyophilisation in situ, plastic film leaflets carrying a counted number of cells were cut out and tested for NP activity. Some properties of NP, including temperature stability, pH optimum and substrate affinity, have been studied. The data obtained suggest that Np might play a regulatory role in the purine interconversion pathway.

A new ultramicrochemical assay for purine nucleoside phosphorylase

Abstract An ultramicrochemical method using radioactive substrates has been developed for the quantitation of purine nucleoside phosphorylase (NP) activity in single cells. NP activity can reproducibly be measured in both directions of the enzyme reaction. A quantitative relationship between the number of cells incubated and the NP activity was found. Fibroblasts from a patient with impaired T-lymphocyte function showed a substantial reduction of NP activity (both directions). In contrast to previously available methods, the present ultramicro approach makes it possible to analyze normal and immunodeficient single B and T lymphocytes.

Molecular and tissue-specific heterogeneity in HPRT deficiency

In several patients with different degrees of HPRT deficiencies, residual activities have been determined in both lysed and intact erythrocytes. No close correlation could be found between the degree of HPRT deficiency and the severity of the clinical expression. Unless HPRT activity in both intact and lysed erythrocytes was below detection level, the residual activity in intact red blood cells was higher than in lysates. Tissue-specific heterogeneity was illustrated with a patient suffering from X-linked gout. Lysates from erythrocytes, leukocytes, and cultured fibroblasts showed 1%, 8%, and 100% of normal HPRT activity, respectively. Characterization of the erythrocyte and fibroblast HPRT from this patient showed no kinetic abnormalities. However, there was a decreased heat stability. It is concluded that for a better understanding of the pathophysiology in HPRT deficiency studies on nucleated cells from the different tissues are needed.