Betty Revsin

A simple, rapid method for prenatal detection of defects in propionate metabolism.

Incorporation of radiolabel from propionate‐l‐i4C into protein (TCA insoluble material) in fibroblasts or amniotic fluid cells, provides a rapid, simple means of detecting fetuses with inborn errors of propionate metabolism using small numbers of cells. Controls were easily differentiated from mutant lines over differing media pH conditions. This method was successfully used to diagnose correctly a normal fetus at risk for methylmalonic acidemia. This method can be used as an adjunct in diagnosis, but cannot replace direct enzyme analysis.

Glycine transport in normal and non-ketotic hyperglycinemic human diploid fibroblasts☆

Uptake, inhibition and kinetic studies in control and non-ketotic hyperglycinemic human fibroblasts show that glycine transport is mediated by the three neutral amino acid carriers A, ASC, and L as described for ascites tumor cells by Oxender & Christensen [5]. NKH fibroblasts lines transported glycine at a reduced rate, and the final intracellular concentration of glycine was less than that of the controls. In both control and NKH lines, transport activity required metabolic energy, SH groups, Na+ and K+ ions. Kinetic data indicated that both control and non-ketotic hyperglycinemic lines had a similar KM but that the Vmax in the NKH cells was always lower suggesting that movement of the carrier and/or the dissociation of the carrier and glycine was depressed in the mutant lines. These data suggest that patients with non-ketotic hyperglycinemia have a defect in glycine transport instead of or in addition to a defective glycine cleavage reaction.

Imino acid transport in human diploid fibroblasts

Abstract These studies describe the transport of proline and hydroxyproline in human diploid fibroblasts. Inhibition and kinetic analysis demonstrate that proline is actively transported by the “A” neutral amino acid carrier. Proline transport is Na + dependent and is particularly sensitive to sulfhydryl inhibitors and ouabain. Hydroxyproline is also actively transported but its transport is mediated by a system different from those described previously for other neutral amino acids. Hydroxyproline transport requires the presence of Na + and is sensitive to sulfhydryl inhibitors and ouabain. There is little inhibition of hydroxyproline transport in the presence of other amino acids with the exception of methionine. The methionine inhibition of hydroxyproline transport is of the non-competitive type. Little cross-reactivity was exhibited by the systems which transport proline and hydroxyproline. These studies indicate that human skin fibroblasts do not possess an iminoglycine transport system as has been described for many other tissues. The iminoglycine transport system has been identified as the genetic transport defect in iminoglycinuria. Consequently, skin fibroblasts are not an appropriate system for use in diagnosis of this disorder.

Effect of Valine on Propionate Metabolism in Control and Hyperglycinemic Fibroblasts and in Rat Liver

Summary: Measurement of methylmalonyl-CoA mutase and propionyl-CoA carboxylase activities in lysates from fibroblasts derived from control, nonketotic hyperglycinemia, propionic acidemia, and both vitamin B12-responsive and -nonresponsive variants of methylmalonic acidemia showed only one abnormality: a 59% decrease in carboxylase activity in the nonketotic hyperglycinemic lysates (P < 0.01). When fibroblasts from all cell types were grown on valine-supplemented (24 mM) media, mutase activity was generally inhibited. As for carboxylase activity, control lines were inhibited 35% as compared to controls without valine and propionic acidemia activity was undetectable. On the other hand, carboxylase activity in both methylmalonic acidemia variants was increased 40% and nonketotic hyperglycinemia carboxylase activity was increased 80% (P < 0.01) when grown on valine-supplemented media. Isoleucine could not substitute for valine in producing increased carboxylase activity in these mutants.Glycine cleavage activity in fresh rat liver homogenates (11.1 μmol/mg protein/90 min) did not vary significantly when 24 mM valine was added to the reaction (9.9 μmol/mg protein/90 min). Therefore, the hyperglycinemia observed in both ketotic and nonketotic forms is probably not caused by a direct effect of valine on the glycine cleavage reaction.These data suggest that the presence of increased amounts of propionic acid in scrum or urine does not necessarily rule out the possibility of nonketotic hyperglycinemia due to the decreased activity of the carboxylase enzyme.Speculation: Decreased activity of the propionyl-CoA carboxylase enzyme in nonketotic hyperglycinemia fibroblasts suggests altered propionate metabolism in this mutation. Additionally, growth of nonketotic hyperglycinemia fibroblasts on high concentrations of valine results in greatly increased carboxylase activity. This effect of valine is also evident in methylmalonic acidemia lysates although no decrease in carboxylase activity in these mutants grown under normal conditions is observed. Although the valine effect is not presently understood, it may be possible that nonketotic hyperglycinemia can be diagnosed from fibroblasts grown in tissue culture based on the combination of these two findings: (1) decreased propionyl-CoA carboxylase activity in nonketotic hyperglycinemic lysates grown under normal culture conditions and (2) increased propionyl-CoA carboxylase activity in nonketotic hyperglycinemic lysates grown on valine-supplemented media.

Fate of prostaglandin E2 in suckling rats after intragastric administration

[3H]Prostaglandin E2 was administered intragastrically to suckling rats at 10 micrograms and 0.1 microgram doses. At the higher dose, 91% of the radioactive label was recoverable at zero time, decreasing to 29% at 5 h. At the lower dose, 40% of the dose was recoverable at zero time, decreasing to 8% at 5 h. With time, the radioactivity in the stomach showed a steady decrease whereas it increased in the tissues. At the 10 microgram dose of [3H]prostaglandin E2 the amount of radioactivity showed a steady increase in the small intestine lumen and small intestine wall. In liver and kidney the maximum amount of radioactive label was found at 1 h. After 1 h the radioactivity began to decline in the liver, while the kidney remained at the same level for the entire 5-h period. At the 0.1 microgram dose of [3H]prostaglandin E2 the radioactivity in the small intestine lumen reached a maximum 3 h after gavage and thereafter declined. The amount of label in the small intestine wall increased for the entire 5 h. In liver and kidney the radioactivity peaked at 1 h, remained at the same level until the 3rd h, then exhibited a decline. Quantitation of the unmetabolized prostaglandin E2 reaching the various organs studied was possible 30 and 60 min after administration of the 10 micrograms dose of prostaglandin E2. At 30 min 42.9% of radioactive label present in the liver could be shown to be authentic prostaglandin E2. This corresponded to 0.64% of the original dose. At 60 min only 22.8% of the radioactive label found in the liver could be shown to be authentic prostaglandin E2, which corresponded to 0.46% of the administered dose. Similar results were found in the small intestine lumen, the small intestine wall and in the kidney. At 3 and 5h, none of the radioactivity found in these organs could be identified as authentic prostaglandin E2.

Propionate metabolism in fetal livers of 15 to 19 weeks' gestation

Extracts of hepatic tissue obtained from saline-induced abortuses were analyzed for methylmalonyl CoA carbonylmutase (MM) and propionyl CoA carboxylase (PC) activity. MM activity was similar to control values, which suggests that abortion material may be used to confirm the prenatal diagnosis of methylmalonic acidemia. Confirmation of a presumptive propionic acidemia diagnosis is more tenuous due to the instability of PC and the possibility that saline may induce PC activity.

1684 ABNORMAL PROSTAGLANDIN (PG) METABOLISM AND PLATELET FUNCTION IN CYSTIC FIBROSIS (CF) PATIENTS

Our previous studies (Lemen, et al. Am.Rev.Resp.Dis., 117:639-46, 1978) reported abnormal plasma PG concentrations in CF patients; but, the physiologic significance of these observations is unknown. We correlated plasma PGs to ADP induced platelet aggregation (PA) in 8 normal and 11 CF subjects. Platelet proaggregators (e.g. thromboxane (Tx)A2 as TxB2 and PGE2) and antiaggregators (PGI2 as 6-keto-F1α) were measured by radioimmunoassay. With parallelism between standards and plasma and with the within-sample coefficient of variation <9%, 6-keto-F1α was significantly (p <0.05) reduced in CF patients (X±SE 408 ± 105 pg/ml) compared to normal subjects (X± SE 708 ± 196 pg/ml). PGF2α was significantly (p <0.05) elevated in CF patients (X±SE, 282 ± 106 pg/ml compared to normal subjects (X±SE, 173 ± 65). TxB2 and PGE2 were not different in CF patients (X±SE, 327 ± 84 and 793 ± 239 pg/ml, respectively) compared to normal subjects (X±SE, 318 ± 92 and 995 ± 495 pg/ml, respectively). ADP induced PA was inhibited in 13/18 studies in CF patients. Platelet electronmicrographs showed decreased dense bodies/platelet profile in 4/8 studies, and PA correlated with dense bodies in 4/8 studies. PA was not correlated with drug therapy including antibiotics or vitamin E. We conclude that abnormal PG metabolism may result in abnormal platelet function in CF patients. Supported in part by grant HL 23773 and the Cystic Fibrosis Foundation.

1176 DIFFERENTIAL EFFECT OF PROSTAGLANDINS (PG) ON AMINO ACID (AA) TRANSPORT IN HUMAN FIBROBLASTS

PGs have been implicated as agents that affect membrane transport of numerous substances, yet little is known of the role of PGs on AA membrane transport. We examined the effects of PGE2 and PGF2α on AA transport by the A, ASC and L systems. Fibro-blasts were preincubated with PGE2 or PGF2α for 10 min prior to addition of the labeled AA.PGE2 stimulated the uptake of all AA carrier systems while PGF2α inhibited all AA transport except for alanine which has high affinity for the A system. Our studies suggest 1) PGE2 and PGF2α have opposing, dose dependent actions on the L and ASC systems (valine/cycloleucine and glycine respectively) but have the same effect on the A system (alanine) and 2) PGE2 and PGF2α may act as an autoregulatory mechanism controlling AA membrane transport in cells. Supported in part by NIH Grant HL 23773 and the Cystic Fibrosis Foundation.