Emmanuel Shapira

Long-term outcome in treated combined methylmalonic acidemia and homocystinemia

Purpose: To describe the clinical and biochemical features and long-term outcome of a cohort of eight patients with methylmalonic acidemia and homocystinuria (cblC).Methods: Documentation of clinical features at birth and longitudinal follow-up of the biochemical and clinical response to treatment with daily oral carnitine and intramuscular hydroxocobalamin observed during continuous follow-up for an average of 5.7 years.Results: Our patients had an increased incidence of congenital malformations including microcephaly (<5%) at birth (2 of 8), congenital heart disease (2 of 8), dysmorphic facial features (1 of 8), and thyroglossal duct cyst (1 of 8). Postnatal hydrocephalus (2 of 8) and hip dislocation caused by ligament laxity (1 of 8) were also noted. One patient had profound visual impairment before 6 months of age secondary to cblC retinopathy, and two patients had abnormal retinal pigmentation with normal visual function. All patients presented with poor growth, feeding problems, and/or seizures. No patients had acute acidotic crises before or after treatment. All patients had dramatic reduction of plasma free homocystine and urine methylmalonic acid excretion after initiation of therapy with carnitine, intramuscular (IM) hydroxocobalamin (OHcbl) and, in two cases, oral betaine. Growth was significantly improved in most cases after the initiation of therapy, and microcephaly was resolved in one patient. All patients were developmentally delayed regardless of age of treatment onset, although two patients had relatively mild developmental delay.Conclusion: cblC patients may have an increased incidence of congenital malformations suggesting prenatal effects of abnormal cbl metabolism. Treatment with IM OHcbl and carnitine successfully corrects the biochemical abnormalities and improves growth. Developmental delay of variable severity is always present regardless of age at diagnosis or treatment onset.

Biochemical and clinical response to hydroxocobalamin versus cyanocobalamin treatment in patients with methylmalonic acidemia and homocystinuria (cblC)

OBJECTIVEnTo compare the therapeutic effectiveness of hydroxocobalamin and cyanocobalamin in patients with combined methylmalonic acidemia and homocystinuria.nnnSTUDY DESIGNnAnalysis of urine methylmalonic acid, plasma homocystine, and growth of two unrelated patients with cobalamin C disease who were initially receiving cyanocobalamin and were subsequently switched to hydroxocobalamin.nnnRESULTSnEach patient had a significant decrease in urine methylmalonic acid excretion while receiving cyanocobalamin, but levels remained at least 10 times normal. Cyanocobalamin treatment resulted in a decrease of plasma homocystine to near normal in one patient but had no effect on plasma homocystine in the second patient. Each patient was switched to hydroxocobalamin and urine methylmalonic acid levels decreased to the limit of detection. Plasma homocystine values while taking hydroxocobalamin remained < 5 nmol/ml in both patients. In patient 1, who continued to receive cyanocobalamin therapy for more than 1 year, growth rates (height, weight, and head circumference) were very poor. After initiation of hydroxocobalamin, growth parameters normalized with growth rates above normal.nnnCONCLUSIONnIntramuscular cyanocobalamin treatment is inadequate in the treatment of patients with cobalamin C disease. Appropriate management of cobalamin C disease should include only the hydroxocobalamin form of cobalamin.

Enzymatically Inactive Red Cell Carbonic Anhydrase B in a Family with Renal Tubular Acidosis

An inactive mutant form of red cell carbonic anhydrase B is described in three members of a large kindred who manifest infantile renal tubular acidosis and nerve deafness. A combination of enzymatic and immunologic investigations permitted its detection, despite the fact that both antigenic and electrophoretic properties of the mutant were identical to those of the normal form.

Enzymatic and Immunologic Quantitation of Erythrocyte Superoxide Dismutase in Adults and in Neonates of Different Gestational Ages

Summary: Human erythrocyte superoxide dismutase (SOD) was purified and specific antiserum was raised in rabbits. Enzyme preparations from adults and from newborns were shown to be indistinguishable in their immunologic and electrophoretic properties. Erythrocyte SOD was quantitated in blood specimens from adults and in cord blood specimens from neonates of different gestational ages, using both an immunologic and an activity assay. The mean values of SOD concentration and SOD activity for adults and for newborns of average size for gestational age (AGA) showed no significant difference. Adult red cells contained 28.0 ± 8.3 SOD units/mg hemoglobin (Hgb) whereas AGA neonatal red cells had 28.5 ± 8.3 SOD units/mg Hgb. Immunologic quantitation by single radial immunodiffusion revealed 0.69 ± 0.07 μg SOD/mg Hgb in adults and 0.70 ± 0.14 μg SOD/mg Hgb in the AGA neonates; however, the SOD concentrations from both small for gestational age (SGA) and large for gestational age (LGA) neonates were significantly lower than those of the AGA neonates and the adults (SGA: 0.57 ± 0.24 μg SOD/mg Hgb, P < 0.05; LGA: 0.59 ± 0.16 μg SOD/mg Hgb, P < 0.05).Speculation: The relatively small quantitative differences in cytosolic superoxide dismutase are unlikely to account for an increased susceptibility to oxygen therapy. The possibility of a decrease in the mitochondrial superoxide dismutase isoenzyme has yet to be studied.

Purification and properties of neutral β-galactosidases from human liver

Abstract Two neutral β-galactosidase isozymes were purified from human liver. The initial step of purification was removal of the acidic β-galactosidases by adsorption on concanavalin A-Sepharose 4B conjugate. Subsequent purification steps included ammonium sulfate precipitation, diethylaminoethyl cellulose column chromatography, Sephadex G-100 gel filtration, and preparative polyacrylamide-gel isoelectric focusing. The final step of purification was affinity chromatography of the separated isoelectric forms on ϵ-aminocaproyl-β- d -galactosylamine-Sepharose 4B conjugate. The purified β-galactosidase isozymes had activity toward both β- d -galactoside and β- d -glucoside derivatives of 4-methylumbelliferone and p-nitrophenol with a pH optimum around 6.2. These enzyme forms were also found to possess lactosylceramidase II activity with a pH optimum in the range of 5.4 to 5.6, but not lactosylceramidase I activity and no activity toward galactosylceramide or GM1-ganglioside. The molecular weight was found to be in the range of 37,500–39,500 for the two neutral isozymes and they had similar Km and V values; the more acidic form (designated β-galactosidase N1) was more heat stable than the other form (designated β-galactosidase N2). Antibodies evoked against the N1 and N2 β-galactosidases gave identical precipitin lines retaining enzymatic activity. No cross-reactivity was observed between the neutral and the acidic isozymes when examined with the respective antisera.

The nature of the residual arylsulfatase activity in metachromatic leukodystrophy

The residual arylsulfatase A activity in three liver specimens from patients with metachromatic leukodystrophy (two of the late infantile form and one of the juvenile form) is shown to be secondary to the residual arylsulfatase B activity in the ASA determination. The mutant enzyme, which is immunologically cross reacting with normal ASA, does not retain any residual enzymatic activity.

Presymptomatic late-infantile metachromatic leukodystrophy treated with bone marrow transplantation

At 8 months of age, before clinical neurologic deterioration, the younger of two sisters with metachromatic leukodystrophy received a transplant of bone marrow from her haploidentical, heterozygote mother. Compared with the course in the older, affected, untreated sibling, the onset of neurologic regression was delayed 1 year and progressed at a slower rate.

Absence of an |[alpha]|2-Macroglobulin-Protease Complex in Cystic Fibrosis

Extract: The present study using immunologic methodology confirms previous observations from this laboratory of an absence of a protease component with arginine esterase activity in plasma of patients with cystic fibrosis. In this study, the pooled plasma from control individuals was activated and partially purified after adsorption on columns of soybean trypsin inhibitor conjugated to Sepharose 4B followed by elution with benzamidine. The fraction was further purified by isoelectrofocusing on polyacrylamide gels. Proteins around the pI range of 5.5 were eluted and utilized to prepare an antiserum. Immunoelectrophoresis of activated plasma samples from control subjects and patients with cystic fibrosis was performed utilizing the antiserum. In controls, four precipitin arcs with residual esterase activity were observed, whereas only three were seen in plasma from patients with cystic fibrosis. Double gel diffusion experiments using specific antisera ruled out the presence of trypsin, chymotrypsin, plasminogen, prothrombin, C1 esterase, α1-trypsin inhibitor, and inter-α-trypsin inhibitor in the concentrated benzamidine eluate. The antisera to α2-macroglobulin gave an immunoprecipitate which was readily stained for proteolytic activity. On immunoelectrophoresis, the α2-macroglobulin precipitin band corresponded to the band absent in plasma of patients with cystic fibrosis. In contrast, the α2-macroglobulin levels were similar in plasma of control subjects and patients with cystic fibrosis. Using the antiserum to the protein fraction with a pI of 5.5 in cross immunoelectrophoresis, three “rockets” with proteolytic activity could be demonstrated in control plasma. One specific enzyme-active “rocket” was absent in plasma of patients with cystic fibrosis. In a double blind study of 15 control samples and 15 samples from patients with cystic fibrosis, a specific “rocket” was shown to be present in 13 control samples and absent in 14 cystic fibrosis samples. α2-Macroglobulin was determined by both an immunologic procedure and by its trypsin binding (trypsin protein esterase concentration). The ratio of the immunologic assay to the biologic activity assay was 90 for the normal plasma samples and only 65 for cystic fibrosis samples.Speculation: The absent α2-macroglobulin-protease complex in plasma of patients with cystic fibrosis might reflect a molecular defect in either a protease with arginine esterase activity or within the α2-macroglobulin molecule.

PMN chemotactic inhibition associated with a cryoglobulin

A child with recurrent pyogenic infections, eczema, markedly elevated serum concentrations IgA, and totally absent PMN chemotactic acticity is described. The chemotactic defect was characterized as a humoral inhibitor present in serumand plasma, directed toward autologous as well as homologous PMNs. Immunochemical studies revealed the purified inhibitor to be a cryoglobulin composed of an IgM-IgA complex. Incubation of the cryoglobulin complex with PMNs resulted in a quantitative dose-response curve.

Cell-directed inhibition of polymorphonuclear leukocyte chemotaxis in a patient with mucocutaneous candidiasis.

A patient with mucocutaneous candidiasis and impaired polymorphonuclear leukocyte (PMN) chemotaxis is described. The patients PMN chemotaxis was markedly decreased in the presence of autologous plasma but normal in control plasma. Cell-directed inhibitory activity was found in whole patient plasma as well as the 40% ammonium sulfate precipitate fraction of both the patients and the control plasma. The inhibitor was heat stable, reversible, nondialyzable, eluted from DEAE cellulose with 0.005 M sodium phosphate buffer, and migrated with IgG on immunoelectrophoresis. The supernate from 40% ammonium sulfate-fractionated patient and control plasma contained a cell-directed enhancer of PMN chemotaxis that antagonized the cell-directed inhibitor activity. It is possible that the patients chemotactic defect may be caused by imbalance between plasma factors that regulate chemotaxis.