Joseph D. Schulman

A derangement in B12 metabolism associated with homocystinemia, cystathioninemia, hypomethioninemia and methylmalonic aciduria☆

Abstract An infant is described who died at seven and one half weeks of age and whose clinical manifestations included poor feeding, hematemesis, melena, normocytic anemia and failure to gain weight. He was found to accumulate abnormally large amounts of cystathionine, homocystine and homocysteine-cysteine mixed disulfide and to have low concentrations of methionine in blood and urine. In addition he manifested a marked methylmalonic aciduria that did not respond to the intramuscular administration of vitamin B, (cyanocobalamin). Enzymatic analyses of liver, kidney and brain obtained at autopsy revealed specifically deficient activity in the B,-dependent enzyme, N,-methyltetrahydrofolate-homocysteine methyltransferase. Studies of fibroblasts grown from skin obtained ante mortem showed that the activity of the B--dependent enzyme, methylmalenyl-CoA (coenzyme A) isomerase, also was deficient, but this activity could be restored by growing the cells in medium containing excess hydroxocobalamin. Analysis of liver and kidney revealed that the concentration of deoxyadenosyl cobalamin (coenzyme-B,) was abnormally low whereas the concentration of total B, was high in serum and normal in liver. Apparently this infant could not adequately metabolize B, and as a result failed to accumulate coenzymatically-active derivatives of vitamin B, in normal amounts. It is suggested that therapeutic benefit may derive from the administration of forms of 6, that could circumvent the metabolic block, by giving substances that could stimulate the non-B, -dependent methionine-synthesizing system or by the administration of methionine in large doses.

Cystine: Compartmentalization within Lysosomes in Cystinotic Leukocytes

The large amount of cystine compartmentalized in cystinotic leukocytes cosediments in isopycnic sucrose density gradients with dense lysosomal particles, within which it is presumably contained. Such cystine appears to be primarily noncrystalline in these organelles.

A new variant of maple syrup urine disease (branched chain ketoaciduria). Clinical and biochemical evaluation.

Mental retardation in a twenty month old girl was associated with an unusually mild form of maple sugar urine disease (MSUD), thus constituting a novel clinical variant of this disease. Her intact leukocytes and cultured skin fibroblasts incubated with carboxyl-labeled C 14 amino acids showed an incomplete deficiency of decarboxylase activities for the keto acids of leucine, isoleucine and valine, with values approximately 15 to 25 per cent of normal. Hyperuricemia was also a manifestation of the disease, apparently resulting from an inhibition of renal excretion of urate by the accumulated branched-chain keto acids. Severe protein restriction alone partially corrected the aminoacidemia in this patient, but a semisynthetic diet was utilized, as in classic MSUD, for optimal biochemical control.

Adrenoleukodystrophy Very long‐chain fatty acid metabolism in fibroblasts

We studied very long-chain fatty acid (VLFA) metabolism in cultured fibroblasts from patients with adrenoleukodystrophy (ALD). Total hexacosanoate (C26:0) content of ALD fibroblasts was sixfold higher than normal and did not return to normal when cells were grown in lipid-free medium. When normal or ALD fibroblasts were grown in medium containing 10% ALD serum (which is enriched in C26:0), there was no further increase in C26:0 content compared with cells grown in 10% normal human serum. Uptake and loss of 1-14C-palmitate (C16:0) and 1-14C-lignocerate (C24:0) by ALD fibroblasts were similar to normal fibroblasts. Catabolism of exogenous H-C26:0 to H2O was about 307 of normal. Oxidation of exogenous 1-14C-hexacosanoate, 1-14C-lignocerate, and 1-14C-palmitate in intact ALD fibroblasts was 42%, 27 ± 13% (SD), and 73 ± 47%, respectively, of normal. These results are consistent with, but do not conclusively prove, a VLFA oxidation defect in ALD fibroblasts.

Cystinosis and the Fanconi Syndrome

Cystinosis is a recessively inherited metabolic disorder characterized biochemically by a high intracellular content of free (nonprotein) cystine which appears to be compartmentalized within lysosomes. This results in crystal deposition in the cornea, conjunctiva, bone marrow, lymph nodes, leukocytes, and internal organs. The primary metabolic defect which leads to cystine accumulation is unknown.

Adverse reactions to oral cysteamine use in nephropathic cystinosis.

Cysteamine (2-aminoethanethiol) has been given orally to 19 patients with nephropathic cystinosis for periods of 8-24 months in doses ranging from 50 to 70 mg base/kg/day. Adverse reactions were noted in 3 patients early in the study when a rapidly increasing dosage schedule was followed. The reactions included hyperthermia, lethargy and rash. These reactions were not seen when patients were started on a very low dosage which was increased gradually at 3-week intervals to a level which depleted leukocytes of about 90% of their free cystine. All three reactions resolved within 24 h or cessation of therapy and in these cases successful readministration of drug was achieved. Chronic cysteamine administration to pediatric patients with cystinosis is feasible. The efficacy of this therapy is still being evaluated.

Liposome-mediated Transfer of Simian Virus 40 DNA and Minichromosome into Mammalian Cells

We have investigated the use of liposomes as carriers for the transfer of simian virus 40 (SV40) DNA into mammalian cells. The amount of DNA entrapped in liposomes was dependent on the input DNA concentration and lipid composition. DNA remained intact after liposome encapsulation and was resistant to deoxyribonuclease digestion. Combined transfer to and expression of liposome-entrapped SV40 DNA in monkey kidney cells was assayed by infectious plaque formation. Negatively-charged liposomes containing phosphatidylserine were more effective in DNA transfer and expression than neutral liposomes. The inclusion of carrier salmon sperm DNA inhibited liposome-entrapped SV40 DNA infectivity. Infectivity of liposome-entrapped DNA was directly related to both liposome DNA concentration and number of vesicles added. Liposome-entrapped SV40 minichromosome was 20-fold more infective than free minichromosome, but only 20% more infective than liposome-entrapped SV40 DNA. Thus, the presence of hyperacetylated histones on the DNA failed to enhance liposome-mediated DNA transfer appreciably. Incubation of cells with various modulators of endocytosis implicated the endocytotic pathway in the mechanism of liposome-mediated DNA transfer. These studies show that liposomes are suitable carriers for the introduction of viral DNA and chromatin into mammalian cells.

Adenine Therapy for Lesch-Nyhan Syndrome

Extract: A child with the Lcsch-Nyhan syndrome was identified at birth by demonstrating a gross deficiency of the enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) in cord blood. Therapy with adenine, begun during the 1st month of life, failed to prevent the development of the severe neurological damage characteristic of this disease.Speculation: Since this work was completed, Dr. Grant Bartlett of San Diego has found (unpublished work) a very poor uptake of intravenously administered adenine-14C in the brain of rabbits. This raises a possible reason for the failure of adenine to prevent the neurological dysfunction in this disease. A purine compound that is more readily taken up by brain tissue might, therefore, offer a more reasonable approach to treatment.

Identification ofheterozygous genotype for cystinosis in utero by a new pulse-labeling technique: Preliminary report

U s l x o a new method developed for the study of small numbers of cystinotic cells in vitro, we have made a probable identification in utero of the heterozygous state of nephropathic cystinosis. The data imply that diagnosis of the homozygous state may also be possible early in gestation. Schneider and associates ~ demonstrated a 100-fold increase in content of free cystine in fibroMasts cultured from the skin of cystinotic children when compared to normal children (Table I ) , and a lesser increase of about sixfold in the fibroblasts of most individuals heterozygous for cystinosis. 1 In other heritable disorders, the expression of the biochemical abnormality in cultured fibroblasts has been accompanied by a similar expression in cells cultured from the amniotic fluid of an affected fetus, 2-* although one apparent exception to this rule has recently been found. ~ However, the large number of cells required for the usual chemical determination of cystine content

Benign cystinosis: The clinical, biochemical and morphologic findings in a family with two affected siblings

Benign cystinosis is an autosomal recessive disorder characterized by the presence of cystine crystals in the eye and the bone marrow. There are no associated renal or peripheral retinal abnormalities which are typical of nephropathic cystinosis. Patients affected with the benign disorder are asymptomatic and presumably have a normal life expectancy. The youngest known patients, a girl aged eleven and a boy aged sixteen, are described. Biochemical and morphologic evidence suggests that in both forms of cystinosis storage of cystine occurs within the lysosomes, primarily of actively endocytic, mesodermally-derived cells. Although the two forms of cystinosis show points of similarity in the distribution of cystine crystals, they differ quantitatively in the amount of cystine accumulated, substantially less intracellular cystine being present in the benign than in the nephropathic form of the disorder. The abnormal gene products responsible for the accumulation of cystine in the two forms of the disease remain to be demonstrated.