Sheldon Orloff

Hyperphenylalaninemia Due to a Deficiency of Biopterin

We studied the components of the hepatic phenylalanine hydroxylating system in a child with phenylketonuria who showed substantial neurologic impairment despite early dietary control of elevated blood phenylalanine levels. Phenylalanine hydroxylase, dihydropteridine reductase and dihydrofolate reductase activities were normal. In contrast the level of hydroxylation cofactor, tetrahydrobiopterin, in liver was only 10 per cent of normal. In addition to this hepatic deficiency, serum and urinary levels of biopterin-like compounds were low, and the serum biopterin did not increase in response to a phenylalanine load as it does in normal and phenylketonuric subjects. The phenylalanine hydroxylase activity in this child, as determined by an in vivo tritium-release assay, was 2.3 per cent of the normal value. These results indicate that the child suffers from a variant form of phenylketonuria--a deficiency of a functional phenylalanine hydroxylating system secondary to a defect in biosynthesis of biopterin.

Placental Transfer of Analogs of Glucose and Amino Acids in Experimental Intrauterine Growth Retardation

Summary: We have employed the model in which one uterine artery is ligated to study maternofetal transport and tissue uptake of glucose and amino acids in the intrauterine growth-retarded rat. On the 18th day of gestation, the artery supplying one uterine horn was ligated. Two days later the rats received [3H]2-deoxyglucose and |14C;[alpha;-aminoisobutyric acid iv. One hour later the growth-retarded and control fetuses were delivered by Cesarean section and appropriate blood samples were obtained. The growth-retarded fetuses had an average weight reduction of 27%, significantly increased placental to fetal weight ratio and brain to body ratio, and a significantly reduced liver to body ratio. Total radioactivity derived from tritiated deoxyglucose in whole fetal tissues, placenta, liver, and brain were significantly decreased in the intrauterine growth-retarded (IUGR) fetuses; this was also true per gram of tissue except for liver. Liver to plasma, brain to plasma, and whole fetal tissue to plasma 3H ratios were significantly increased in the IUGR group. The radioactivity derived from [14C;[alpha;-aminoisobutyric acid was significantly reduced in whole fetal tissues, placenta, liver, and brain in the IUGR fetuses whether expressed per whole organ or per gram of tissue. Significant differences in liver to plasma, brain to plasma, and whole tissue to plasma 14C ratios were not observed.Speculation: The large reduction in maternofetal transfer of deoxyglucose and α-aminoisobutryric acid when uterine blood flow is reduced raises the possibility that one of the major causes of retarded growth in utero is a diminished supply of glucose and amino acids to the fetus. The fetal liver appears to be especially effective in increasing its extraction of 2-deoxyglucose from fetal plasma during this period of nutrient restriction. Since a major cause of fetal growth retardation in man is an impairment in maternal circulatory supply to the placenta, a similar mechanism of growth retardation may contribute to human IUGR. The possibility that nutritional supplementation may be of value in improving the growth conditions for the fetus when uterine blood flow is restricted would seem worthy of further investigation. More information is also required about the mechanisms responsible for the apparent improved extraction of glucose from plasma in IUGR.

Pantetheinase Activity and Cysteamine Content in Cystinotic and Normal Fibroblasts and Leukocytes

Summary: Cysteamine is the most effective agent known for the reduction of the elevated cystine content of cells from patients with cystinosis. A defect in endogenous cysteamine generation could account for many of the metabolic features of this disorder. To test this hypothesis, we have developed improved methods for measuring pantetheinase (cysteamine-generating) activity and intracellular cysteamine levels and used these methods to measure such parameters in cystinotic and normal leukocytes and cultured skin fibroblasts. Pantetheinase activity as defined in the text was similar in extracts of cystinotic and normal cells [leucocytes, normal, 78 ± 15 (S.E.), cystinotic, 56 ± 6.4; fibroblasts, normal, 9.4 ± 1.5; cystinotic, 7.7 ± 1.7]. Cysteamine levels were normal in leukocytes from cystinotics receiving no cysteamine or doses of oral cysteamine too low to reduce leukocyte cystine content. The results indicate that the cause of cystinosis is unlikely to be related to a failure to generate or sustain normal intracellular cysteamine levels.Speculation: cystearnine is an extremely effective cystine depleting agent for cystinotic fibroblasts in vitro and can greatly reduce cystinotic leukocyte cystine content in vivo. Its pharmacologic properties suggest that it might prove to be of value in the therapy of cystinosis. However, we do not believe that a defect in endogenous cysteamine generation is a characteristic of cystinotic cells. The eventual elucidation of the cystinotic defect may require analysis of the permeability characteristics of cystinotic lysosomes or the discovery of presently unidentified pathways for lysosomal metabolism of cystine.

Ineffectiveness of Ascorbic Acid Therapy in Nephropathic Cystinosis

Because high concentrations of ascorbic acid (0.57 mM) lower the free (nonprotein) cystine content of cultured cystinotic skin fibroblasts by over 50 per cent, we did a double-blind clinical trial to establish whether this drug would benefit cystinotic children. Sixty-four patients were randomized into the study; 32 received ascorbic acid (200 mg per kilogram of body weight per day), and 32 placebo. The study was terminated after approximately two years because there was no indication that vitamin C was beneficial and accumulating evidence that it might be harmful. Of 11 patients who left the study because of death or the requirement for dialysis or renal transplantation, eight were receiving ascorbic acid. The estimated relative risk (treatment vs. control) of an adverse event was R = 2.7, with a 90 per cent confidence interval of (0.8, 11.5). The serum creatinine concentration increased 0.53 mg per deciliter per year in patients receiving vitamin C and 0.24 mg per deciliter per year in patients receiving placebo (P = 0.08).

Intrauterine growth retardation in renal insufficiency: An experimental model in the rat

Experiments described here with partially nephrectomized pregnant rats with pair-fed controls and controls fed at will indicate that decreased maternal food intake is a major factor in the intrauterine growth retardation associated with moderate renal insufficiency during the last trimester of gestation. Although renal disease in human pregnancy is often associated with vascular insufficiency, the possibility that maternal undernutrition may also play a contributory role in the fetal growth failure associated with certain cases of human renal compromise merits further study.

HYPERPHENYLALANINEMIA DUE TO PHENYLALANINE HYDROXYLASE COFACTOR DEFICIENCY

A child with phenylketonuria diagnosed early developed severe hypotonia with minimal signs of spasticlty and retardation in all developmental patterns despite adequate dietary management. At 4 years of age, a liver biopsy was performed and the components of the phenylalanine hydroxylase system were measured in vitro. Normal levels of phenylalanine hydroxylase, dihydropteridine reductase and dihydrofolate reductase were found. However, there was only 5% of the normal level of active phenylalanine hydroxylase cofactor. This cofactor deficiency was also demonstrable in plasma and urine. Since the phenylalanine hydroxylase cofactor (tetrahydrobiopterin) is probably also necessary for the hydroxylation of tyrosine and tryptophan, the rate limiting steps in the biosynthesis of dopamine and norepinephrine and serotonin, this child may be suffering from a lack of these neurotransmitters.The childs phenylalanine hydroxylase activity has also been measured with a new in vivo method. In this procedure, hydroxylase activity is assayed by following the rate of formation of tritiated water from ring tritium labelled phenylalanine. Phenylalanine hydroxylase activity 1-5% of normal was detected in vivo confirming the incomplete block in the conversion of phenylalanine to tyrosine predicted by the in vitro assays on the liver biopsy.

Lipochromosome mediated gene transfer: Identification and probable specificity of localization of human chromosomal material and stability of the transferents

SummaryUsing lipochromosomes (phospholipid-entrapped chromosomes) we have transferred the human HGPRT gene into HGPRT deficient mouse cells (A9) with a frequency of approximately 1×10−5 (Mukherjee et al., Proc. Natl. Acad. Sci. USA 75: 1361–1365; 1978). Two other genes located on the long arm of the human X-chromosome were also expressed in two independently derived populations of transferents (A9/GT3 and A9/GT4). We report here the chromosomal and enzymatic composition of human HGPRT-positive clones from each subpopulation analyzed in detail with alkaline Giemsa-11 staining.All the clones expressed human PGK and HGPRT, but one (A9/GT4C6) lacked human G6PD. In each of four clones examined microscopically, a small piece of presumptive human chromatin was visible in the karyotypes of most cells. The chromatin fragment was free or attached in each cell of an individual clone. When integrated, the human chromosomal fragment in each clone appeared associated with the centromere of the same telocentric A9 chromosome (No. 6 by Q-banding).These data suggest that: (a) substantial human chromosomal fragments can be transferred into recipient cells using the lipochromosome technique; (b) clones from human HGPRT positive A9 transferent subpopulations may or may not possess other human X-linked markers; (c) the stability of lipochromosomally transferred genes varied from clone to clone and stability is generally poor in the absence of continuous selection pressure (e.g., HAT); (d) when multiple X-linked human genes were transferred to mouse cells a cytologically detectable human chromosomal fragment was identified free or attached to a host chromosome; and (e) integration of transferred human chromosomal material into mouse chromosomes may occur at preferential site(s) in the recipient genome.

Cystinotic and normal fibroblasts: Differential susceptibility to cysteine toxicity in vitro

SummaryExtracellular cysteine concentrations between 0.5 and 2.5 mM resulted in death of normal but not cystinotic cells grown in Eagles minimal essential medium containing supplemental fetal bovine serum and antibiotics. Differential cell survival was determined by viable cell counting using Trypan Blue dye exclusion. In cocultivation experiments of [3H]thymidine-labelled cystinotic fibroblasts with nonradioactive normal fibroblasts, autoradiography confirmed the selective survival of cystinotic cells in medium containing 1 mM cysteine. At this concentration of 1 mM cysteine, intracellular cystine content increased slightly in surviving normal cells but not in cystinotic cells, which normally contain a high level of intracellular cystine. This comparative resistance of cystinotic fibroblasts to elevated extracellular cysteine concentrations forms the basis for an in vitro selective system for these mutant human cells. Further exploration of this resistance phenomenon may well expand the understanding of the molecular defect in cystinotic cells.

562 FAILURE OF ASCORBIC ACID (AA) THFRAPY IN NEPHROPATHY CYSTINOSIS

Since there is no specific therapy for cystinosis and since AA decreases the free-cystine content of cystinotic fibroblasts (Science 86:1040, 1974), a randomized double-blind trial of high dose (200 mg/Kg/day) AA was undertaken. An unblinded committee monitored the data every 4 months. At entry the AA and placebo (PL) groups were comparable in all major clinical parameters. There were 52 patient years for the AA group and 50 patient years for the PL group. Of the 11 patients who died, began chronic dialysis or were transplanted, 8 had received AA. Neither treatment affected the WBC cystine content. Patients receiving AA had a greater mean rise in serum creatinine (Cr). This was especially true in patients whose initial Cr was ≥1.0 & <3.5. Although a small benificial effect of AA could not be excluded statistically, it was more likely that AA hastened the progression of renal failure. In view of this adverse risk-benefit ratio the study was terminiated.

HYPERAMMONEMIA WITH PARTIAL CARBAMYL PHOSPHATE SYNTHASE (CPS) DEFICIENCY RESPONSIVE TO ARGININE THERAPY

The six week old son of a first cousin marriage presented with hyperammonemia, transient acidosis, hepatosplenomegaly, failure to thrive, and seizures. Quantitative plasma and urine amino acids, including lysine clearance, were normal, orotic acid excretion was not increased, and no organic acid abnormalities were evident by GLC-mass spectroscopic analyaia. Notably, during periods when the patient was placed on a diet containing 1 gm/kg/day protein intake, arginine supplementation (100 mgm/kg/day) consistently and significantly lowered plasma ammonia (on arginine, 82 μgm% ± 3, S.E.; off arginine, 113 ± 8; p<.01). The mean value for plasma ammonia during periods on a 2gm/kg/day protein diet without arginine (80 ± 17) was lower than on 1 gm/kg/day protein (p=0.1). Liver biopsy was examined by light and electron microscopy. Enzymes of the urea cycle were assayed by Dr. P. J. Snodgrass, revealing a partial deficiency of CPS (0.49 μmol/mg prot/hr; normal neonatal mean 1.13, range 0.81-1.41), assayed in the presence of excess N-acetylglutamate. Since arginine activates glutamate N-acetylase and N-acetylglutamate produced by this reaction is a cofactor for CPS, we postulate that arginine, supplied as such or through moderate amounts of dietary protein, lowered plasma ammonia in our patient by stimulating residual CPS activity.