Carol Seaman

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.

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...

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.

376 THE THRESHOLD OF LEAD TOXICITY IN CHILDREN

The elevation of erythrocyte protoporphyrin (EP) is a sensitive indicator of childhood Pb poisoning, reflecting interference of Pb with mitochondrial function. This study was directed to establish the minimum blood Pb level (BPb, μg/dl) at which this metabolic evidence of toxicity becomes apparent in children. Venous blood samples were obtained for screening purposes from 1944 apparently normal N.Y.C. children (aged 2-12, median 4.7). BPb was measured by atomic absorption and EP by fluorometry; 1816 children had BPb < 30, the accepted “normal” limit. Up to BPb 15 there was no correlation with EP; above BPb 15 the EP increased exponentially, with a slope similar to that reported for children with BPb > 30. The threshold BPb for EP elevation was determined, by probit analysis and by two different segmented curve-fitting techniques, to be 15.5 (c.l. 14.7 to 16.7). To exclude that the elevation of EP could be due to greater absorption of Pb by children with Fe deficiency, the Fe status of 164 children was estimated by serum ferritin (SF) and transferrin saturation (TS). There was no significant difference in either SF or TS between children with BPb below or above 15, confirming that the elevation of EP observed in the latter group results from the Pb toxicity. This study indicates detectable metabolic Pb toxicity at a level of BPb which is well below the value presently accepted as “normal.” This evidence, and the recent demonstration that in remote human populations BPb is negligible, indicate an unacceptable degree of exposure to Pb of urban children.

ELEVATION OF δ -AMINOLEVULINIC ACID DEHYDRATASE (ALAD) AND ERYTHROCYTE PORPHYRINS (EP) IN SICKLE CELL SYNDROMES

In patients with sickle cell syndromes, EP levels may be markedly elevated. In 249 patients with sickle cell syndromes, we measured EP, ALAD, and ALAD after reactivation with dithiothreitol (ALADSH), in parallel with reticulocytes and pyruvate kinase (PK), two age-dependent parameters. EP, ALAD, ALADSH, reticulocytes and PK were much higher than normal in all patients, with the most marked elevation in patients with homozygous sickle cell disease. In many cases, the elevation of EP resulted from free protoporphyrin base, rather then Zn-protoporphyrin. The logarithm of EP was correlated both with reticulocytes and with the logarithm of PK; the regression lines intersected the normal ranges for EP and reticulocytes and for EP and PK respectively. For ALAD and ALADSH, there were similar correlations; however, the regression lines remained well above the normal ranges without intersecting them. The in vivo t1/2 in the erythrocytes of both ALAD and ALADSH were estimated on discontinuous gradients of arabino-galactane at 43 days, indicating an age-dependent loss of enzyme activity, and not just oxidation of SH-groups with time. The in vivo t1/2 of EP is 60 days (JCI 56: 1519, 1975). These data indicate that both EP and ALAD are increased in sickle cell syndromes as a result of young red cell age. However, the higher ALAD values and the unusual protoporphyrin species suggest additional factors, probably some degree of dyserythropoiesis or the presence of large numbers of normoblasts in these asplenic individuals.

THE INTERACTION OF IRON DEFICIENCY AND LEAD POISONING IN NORMAL CHILDREN. AN EPIDMIOLGICAL STUDY

In children the levels of erythrocyte porphyrins (EP) increase exponentially with the blood lead (BPb) above a threshold of 18 μg/dl (P.N.A.S. 79: 3335, 1982). The EP levels are also elevated by iron deficiency. This study was directed to assess the response of normal and Fe deficient children to low levels of Pb. In addition to BPb and EP, serum ferritin (SF) and mean corpuscular volume (MCV) were measured in 3459 children age 1 to 6 years. The normal range for SF and MCV were first established for each year of age. Children with SF < 2 σ below the mean or both SF and MCV < 1 σ below the mean were considered certainly Fe deficient; those with both SF and MCV > 1 σ below the mean were considered certainly Fe sufficient. Children who did not fulfill any of these criteria were left unclassified and excluded from the analysis. The threshold BPb for EP elevation was established by the segmented line technique. The threshold BPb for the entire group of 3459 children was 18 μg/dl; for the 331 Fe deficient children it was 12.5 μg/dl and for the 2314 Fe sufficient children it was 20.3 μg/dl; all slopes of elevation were parallel. Because of the lower threshold BPb, Fe deficient children have a greater increase in EP in response to Pb than Fe sufficient children. These findings demonstrate the greater sensitivity of Fe deficient children to the effect of Pb, probably mediated by the competitive inhibition between Pb and Fe at the ferrochelatase step (Blood 66: 47a, 1985). Fe deficiency increases the sensitivity of children to Pb; Fe supplementation may protect them from the adverse effects of Pb on heme synthesis.

A MORE ECONOMICAL AND EFFECTIVE BLOOD PRODUCT FOR CHRONIC TRANSFUSIONS

Frozen red blood cells (RBCs) are commonly used in chronic transfusion programs for patients with homozygous β -thalassemia, sickle cell anemia after stroke, etc., to avoid the severe transfusion reactions due to sensitization to WBCs. An alternative technique for removal of WBCs consists of filtration on cotton wool. We have used or transfusion RBCs first filtered on cotton wool filters (Imugard IG500, Terumo Corp., Tokyo, Japan) and then washed with a solution containing NaCl 0.8%, Dextrose 0.2%, buffered with phosphate to pH 7.4. These cells appear superior to frozen RBCs, as they are less expensive to prepare (

937 INTERACTION OF PYRUVATE/LACTATE AND NAD/NADH RATIOS IN THE REGULATION OF RED CELL (RBC) GLYCOLYSIS

55 vs.