Sergio Piomelli

In vivo lability of glucose-6-phosphate dehydrogenase in GdA- and Gdmediterranean deficiency

A decreased level of glucose-6-phosphate dehydrogenase might result from decreased rate of synthesis, synthesis of an enzyme of lower catalytic efficiency, increased lability, or a combined mechanism. To test the hypothesis of increased lability, the rate of decline of the enzyme in vivo was measured in three groups of individuals, controls, Gd(-),A-males, and Gd(-), Mediterranean males, by the slope of decline of activity in fractions containing erythrocytes of progressively increasing mean age. These fractions were obtained by ultracentrifugation on a discontinuous density gradient of erythrocyte suspensions free of contaminating platelets and leukocytes. The rate of in vivo decline of pyruvate kinase (another age-dependent enzyme) was also measured and found very similar in the three groups. The in vivo decline of glucose-6-phosphate dehydrogenase was found to follow an exponential rate, with a half-life of 62 days for controls and 13 days for Gd(-),A- erythrocytes. The activity in normal reticulocytes was estimated at 9.7 U and in Gd(-),A- reticulocytes at 8.8 U. These estimates were confirmed by direct measurements in reticulocytes isolated from patients with extreme reticulocytosis. In Gd(-),Mediterranean erythrocytes activity could be demonstrated only in reticulocytes, which were estimated to average 1.4 U. The rate of decline is so extreme that no activity could be detected in mature erythrocytes. These data suggest that the glucose-6-phosphate dehydrogenase deficiency of both the Gd(A-) and the GdMediterranean variant results from different degrees of in vivo instability of the abnormal enzyme.

Erythropoietic protoporphyria and lead intoxication: the molecular basis for difference in cutaneous photosensitivity. I. Different rates of disappearance of protoporphyrin from the erythrocytes, both in vivo and in vitro.

In lead intoxication photosensitivity is usually absent, despite concentrations of protoporphyrin in the erythrocytes equal to or greater than in erythropoietic protoporphyria. Profound differences in the distribution of protoporphyrin in aging erythrocytes were demonstrated by age-dependent fractionation of cells on discontinuous density gradients. In erythropoietic protoporphyria the concentration of protoporphyrin declined extremely rapidly with erythrocyte age; the bulk of the protoporphyrin was lost in less than 3 days and the concentration of fluorescent erythrocytes in the gradient paralleled the decline of protoporphyrin. In lead intoxication the protoporphyrin concentration declined only slightly with cell aging and erythrocytes of all ages fluoresced. In the bone marrow from a patient with erythropoietic protoporphyria all reticulocytes, but only occasional late normoblasts, fluoresced, suggesting a single population. Sterile incubation in plasma (pH 7.5) demonstrated rapid diffusion of protoporphyrin from the erythrocytes in erythropoietic protoporphyria, but not in lead intoxication. Plasma protoporphyrin was elevated in erythropoietic protoporphyria, but not in lead intoxication. Estimates of the daily loss of protoporphyrin from erythropoietic tissue in erythropoietic proporphyria suggested an order of magnitude similar to the total blood protoporphyrin. Therefore, it is not necessary to postulate a preponderant extraerythropoietic source to explain the amount of fecal excretion. A significant amount of the diffused protoporphyrin probably reaches the skin with resulting photosensitivity. In contrast, in lead intoxication protoporphyrin remains within the erythrocyte throughout its life span ; there is no diffusion into the plasma and hence no photosensitivity.

Erythropoietic protoporphyria and lead intoxication: the molecular basis for difference in cutaneous photosensitivity. II. Different binding of erythrocyte protoporphyrin to hemoglobin.

Acidic solvents extract the same porphyrin-protoporphyrin-from the erythrocytes of patients with either erythropoietic protoporphyria or lead intoxication. However, extractable protoporphyrin disappears rapidly, both in vivo and in vitro, from erythrocytes in erythropoietic protoporphyria but slowly, if at all, in lead intoxication. Consistent with these observations, fluorescence spectroscopy revealed that the intracellular state of the erythrocyte protoporphyrin is different in the two diseases. Spectrofluorometric measurements coupled with fractionations and biochemical syntheses showed that in erythropoietic protoporphyria the protoporphyrin is bound as the free base to hemoglobin molecules at sites other than the heme binding sites. In lead intoxication the fluorescent porphyrin is also bound to hemoglobin but is present as zinc protoporphyrin. The data suggest that the zinc protoporphyrin is bound at heme binding sites. Acidic extraction solvents remove the chelated zinc, but zinc protoporphyrin may be extracted intact from erythrocytes with acetone, ethanol, or the detergent Ammonyx-LO.

Erythrocyte Adenosine Deaminase Deficiency without Immunodeficiency: EVIDENCE FOR AN UNSTABLE MUTANT ENZYME

Inherited deficiency of the purine salvage enzyme adenosine deaminase (ADA) gives rise to a syndrome of severe combined immunodeficiency (SCID). We have studied a 2.5-yr-old immunologically normal child who had been found to lack ADA in his erythrocytes during New York State screening of normal newborns. His erythrocytes were not detectably less deficient in ADA than erythrocytes of ADA(-)-SCID patients. In contrast, his lymphocytes and cultured long-term lymphoid cells contained appreciably greater ADA activity than those from patients with ADA(-)-SCID. This residual ADA activity had a normal molecular weight and K(m) but was markedly unstable at 56 degrees C. His residual erythrocytes-ADA activity also appeared to have diminished stability in vivo. ADA activity in lymphoid line cells of a previously reported erythrocyte-ADA-deficient!Kung tribesman was found to contain 50% of normal activity and to exhibit diminished stability at 56 degrees C. ATP content of erythrocytes from both partially ADA-deficient individuals was detectably greater than normal (12.3 and 6.1 vs. normal of 2.6 nmol/ml packed erythrocytes). However, the dATP content was insignificant compared to that found in erythrocytes of ADA(-)-SCID patients (400-1,000 nmol/ml packed erythrocytes). The New York patient, in contrast to normals, excreted detectable amounts of deoxyadenosine, but this was <2% of deoxyadenosine excreted by ADA(-)-SCID patients. Thus, the residual enzyme in cells other than erythrocytes appears to be sufficient to almost totally prevent accumulation of toxic metabolites.

Complete Suppression of the Symptoms of Congenital Erythropoietic Porphyria by Long-Term Treatment with High-Level Transfusions

CONGENITAL erythropoietic porphyria is a rare autosomal recessive disorder of heme biosynthesis that is secondary to reduced (10 to 30 percent of normal) uroporphyrinogen cosynthase activity.1 In p...

Clinical and Biochemical Interactions of Glucose-6-Phosphate Dehydrogenase Deficiency and Sickle-Cell Anemia

Abstract The extremely young red-cell population in sickle-cell anemia invariably results in increased G-6-PD activity. This effect is more marked in persons who genotypically have the sex-linked Gd A- type of G-6-PD deficiency, and may even mask the phenotypic expression of the enzyme deficiency. Electrophoresis and family studies were used to determine the genotype in patients with sickle-cell anemia. Among 15 such patients five were Gd A- as compared with 11 of 102 randomly selected Afro-American controls (0.05 0.01). These findings showing a relative preponderance of the Gd A- type in patients with sickle-cell anemia support the Lewis hypothesis that the simultaneous inheritance of both defects may be clinically beneficial.

Disparate Enzyme Activity in Erythrocytes and Leukocytes. A VARIANT OF HYPOXANTHINE PHOSPHORIBOSYL-TRANSFERASE DEFICIENCY WITH AN UNSTABLE ENZYME

A family is reported in which each of two sisters has a son with no detectable hypoxanthine phosphoribosyltransferase (HPRT) (EC 2. 4. 2. 8) in his erythrocytes, a finding considered pathognomonic of Lesch-Nyhan disease. However, neither has the stigmata of the disease. One boy is neurologically normal, and the other is moderately retarded. There was only a slight increase in urinary uric acid, but the amounts of hypoxanthine and xanthine, and their ratios, were similar to those found in Lesch-Nyhan disease, strongly indicating that excesses of these last two oxypurines are not responsible for the symptomatology in that disease. In contrast to the nondetectable HPRT activity in the red blood cells, leukocyte lysates from the two boys have 10-15% of normal activity, possibly reflecting continuing synthesis of an unstable enzyme. This hypothesis is supported by the demonstration that at 4 degrees C HPRT activity was rapidly lost in the propositus while the activity increased in control subjects. The mothers cells were intermediate between the two. The intact and disrupted leukocytes of the hemizygote, in the absence of added phosphoribosyl converted as much hypoxanthine to inosinate as the normal cell, and appropriate tests indicated that under these circumstances enzyme concentration is not rate limiting whereas the concentration of the cosubstrate, phosphoribosyl pyrophosphate, is. The capacity for normal function in the intact mutant cell is more representative of in vivo conditions than the lysate, which may explain the important modification of clinical symptomatology, the relatively mild hyperuricosuria, and the presence of mosaicism in the circulating blood cells of the heterozygotes. A similar explanation may apply to other genetic diseases in which incomplete but severe enzyme deficiencies are found in clinically normal individuals. An associated deficiency in glucose-6-phosphate dehydrogenase in this family permitted confirmation of previous observations on linkage with hypoxanthine phosphoribosyltransferase.

Chelation therapy in β-thalassemia major. III. The role of splenectomy in achieving iron balance

Transfusion requirements for 1978 were compiled for 79 patients with thalassemia major (ages 1 to 29 years) who were maintained at hemoglobin concentrations of greater than 10 gm/dl. In 46 patients with intact spleens, the mean transfusion requirement was 258 ml/kg/year, and there was a clear increase with age. The transfusion history prior to 1978 had no influence on the increase of transfusion requirement with age. In contrast, in 33 splenectomized patients, the mean transfusion requirement was 203 ml/kg/year and it did not increase with age. Urinary iron excretion in response to deferoxamine increased with age, with no obvious difference between splenectomized and nonsplenectomized patients. The ability to achieve iron balance with a daily dose of 20 mg/kg of deferoxamine was a function of the transfusion requirement splenectomized patients with lower blood requirements generally achieved negative iron balance, whereas nonsplenectomized patients did not. We conclude that the spleen should be removed when the transfusion requirement exceeds 250 ml/kg/year, which usually occurs between 6 and 8 years of age. In young patients with intact spleens, a higher dose of deferoxamine may be use in order to prevent hemosiderosis.

Planning an exchange transfusion in patients with sickle cell syndromes

Partial exchange transfusions are performed in sickle cell patients for a variety of reasons. An algorithm to plan a nonautomated exchange in patients with sickle cell syndromes was developed and validated by a study of 40 such procedures. Formulas that can be used to explore alternatives, by assessing at any point during the exchange the current concentration of sickleable cells and the hematocrit, were devised: a computer program in BASIC is available for maximum versatility. The two most important determinants of the exchange are the patients initial hematocrit and the desired final concentration of sickleable cells; the rate and type of exchange (continuous or discontinuous) are not important. The final hematocrit depends on the type of blood product used. An exchange can be performed with packed red blood cells (PRBC), whole blood (or its equivalent, PRBC reconstituted with albumin), or it can be started with PRBC and continued with whole blood. Whole blood decreases the concentration of sickeleable cells rapidly and increases the hematocrit slowly; it does not markedly increase the viscosity. PRBCs decrease the concentration of sickleable cells more slowly and increase the hematocrit faster; thus, they may increase the blood viscosity to dangerous levels.

Cystic fibrosis in a black child with hemoglobin S-D disease

A 9-year-old black patient with the simultaneous occurrence of cystic fibrosis and hemoglobin S-D disease is reported. She was placed on chronic transfusion therapy from 1 year of age because of her rapidly worsening pulmonary condition. Her pulmonary function is now stable. The role of this therapy in the overall management of this unusual syndrome is discussed.