Joseph D. Schulman

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.

Glutathionuria: Inborn error of metabolism due to tissue deficiency of gamma-glutamyl transpeptidase

Summary A patient has been discovered with glutathionemia and marked glutathionuria. Serum concentrations of individual aminoacids were normal and percent renal resorption of these amino acids fell within or very close to the normal range under ordinary dietary conditions. Cultured skin fibroblasts from the patient manifested an extreme reduction in the activity of gamma-glutamyl transpeptidase measured with glycylglycine, cystine, or methionine acceptors and the artificial substrate gamma-glutamyl-nitroanilide, and in an assay using 35 S-glutathione as substrate. A “new” human enzyme deficiency state has thus been characterized. Further studies of this disorder should permit clarification of the possible role of this transpepti-dase in human amino acid transport and in glutathione metabolism.

Effect of homocysteine and homocystine on platelet and vascular arachidonic acid metabolism.

Summary: Normal hemostasis depends in part on the balance achieved between proaggregatory and prothrombotic platelet thromboxane A2, measured as its stable end-product thromboxane B2 (TXB2), and vascular prostacyclin (PGI2), which inhibits platelet aggregation and is antithrombotic. Cystathionine-β-synthase deficiency is characterized by a high frequency of thromboembolic disease. We therefore studied, in vitro, the effects of homocysteine and related compounds on platelet TXB2 and vascular PGI2 formation.In paired samples of platelet rich plasma, which had been preincubated with L-homocystine (1 mM), mean production of the two platelet cyclooxygenase products, TXB2 and 12-hydroxy-5,8,10-heptadecatrienoic acid increased significantly from control levels [13.6% ± 1.9 to 19.8% ± 2.1 (P < 0.02) TXB2 and 29.8% ± 4.2 to 39.4% ± 4.1 (P < 0.01) HHT]. In the presence of D.L-homocysteine (1 mM), mean platelet TXB2 and 12-hydroxy-5,8,10-heptadecatrienoic acid production was also significantly increased [12.7% ± 1.5 to 16.9% ± 1.5 (P < 0.01) TXB2 and 27% ± 4 to 31% ± 4.1 (P < 0.02) HHT]. Cystine, cysteine, or methionine (1 mM) did not have similar effects in this test system. Homocysteine and homocystine were without effect on the synthesis of vascular PGI2 by umbilical artery segments [control, 0.22 ± 0.03 to 0.21 ± 0.03 ng/mg with D.L-homocysteine and 0.20 ± 0.04 control to 0.19 ± 0.04 ng/mg with D.L-homocystine]. A homocyst(e)ine-induced increase in platelet thromboxane production in the absence of an increase in vascular prostacyclin, if present in vivo, may contribute to the vascular thromboses characteristic of human homocystinemias (homocystinurias).

Intracytoplasmic sperm injection facilitates fertilization even in the most severe forms of male infertility: pregnancy outcome correlates with maternal age and number of eggs available

OBJECTIVE To evaluate, in a prospective study, the fertilization and pregnancy rates after intracytoplasmic sperm injection (ICSI) in infertile couples with severe male infertility. DESIGN Intracytoplasmic sperm injection was performed in 229 consecutive IVF cycles on 190 couples with rigorously defined severe male infertility or proven failure of fertilization in prior IVF cycles. Neither male nor female partners were chosen from a waiting list or on any other selective basis, including age, prior or anticipated ovarian response, or oocyte number or quality. There were no upper age limits, in no instance was donor sperm used for ICSI, and cycle cancellation rate was minimal. SETTING Private genetics and fertility center in Fairfax, Virginia. MAIN OUTCOME MEASURES Fertilization, transfer, and pregnancy rates were measured in ICSI-treated couples, and comparisons were made regarding both female age and strictly defined semen categories. RESULTS Two hundred six cycles (90%) resulted in ETs, with initiation of 52 pregnancies (25% per transfer, 23% per cycle). Thirty-eight of 52 (18% per transfer) were clinical pregnancies with established gestational sacs or were ongoing or delivered. Pregnancies were achieved even in older women but were more readily established in younger women producing larger numbers of metaphase II oocytes. The severity of semen abnormalities had some small effect on fertilization rate, but only actual necrospermia was associated with markedly decreased frequency of embryo formation. Pregnancy per transfer was similar across groups. In some cases, pregnancy was initiated with fewer than 100 viable sperm in the ejaculate. CONCLUSIONS Intracytoplasmic sperm injection is a very powerful new treatment for severe male infertility. Paradoxically, egg number and probably egg quality are now the main determinants of success in treating male infertility.

Empty follicle syndrome

: Four patients who had no oocytes retrieved during an IVF cycle were studied in an attempt to identify predictors of such an occurrence and suggestions for its cause. All 30 follicles aspirated in five cycles in these four women yielded no oocytes. One patient had two cycles that produced no eggs. The empty follicle syndrome may represent a new syndrome and a cause of infertility.

Platelet Survival and Morphology in Homocystinuria Due to Cystathionine Synthase Deficiency

In homocystinuria due to cystathionine synthase deficiency thromboembolism is a major cause of mortality and morbidity. Recent studies by others identified an abnormally shortened platelet survival and increased platelet vacuolization in patients with homocystinuria. When we studied six additional patients, however, we found the platelet survival to be within normal limits for each. The mean survival (+/-1 S.D.) was 9.75+/-0.94 days (normal, 9.27+/-1.06). In addition, platelets from five patients with homocystinuria and three obligate heterozygotes could not be distinguished from those of seven normal control subjects by electron microscopy. Specifically, no increased vacuolization was observed. Genetic heterogeneity, technical differences of differences in plasma homocystine concentrations could account for these descrepant results. The mechanism of thrombosis in homocystinuria remains an open question.

Ornithine-ketoacid transaminase activity in human skin and amniotic fluid cell culture

Abstract l -Ornithine-2-oxoacid aminotransferase activity was detected in human diploid cells cultured from control skin biopsies, from amniotic fluids, and from the skin biopsy of a patient with the type of hyherornithinemia associated with homocitrullinuria and hyperammonemia. The potential usefulness of finding this enzyme activity in cultured human cells is discussed.

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.

Human in vitro fertilization.

Human In Vitro Fertilization MARK EVANS;ANIL MUKHERJEE;JOSEPH SCHULMAN; Obstetrical & Gynecological Survey

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.