ABSTRAa. Metabolism of propionate in human subjects was studied using bolus administration of l-'3C-propionate i.v. or orally. The study population consisted of five patients with propionic acidemia (PA), eight with methylmalonic acidemia (MMA, four responsive to vitamin BIZ), one each with multiple carboxylase deficiency and transcobalamin-I1 deficiency, and five healthy volunteers. Concentrations of I-I3C-propionate were measured in blood in three patients with PA, two with MMA, and two controls. Breath samples were obtained at intervals during 3 h after the dose, isotopic enrichment of I3CO2 was measured, and the cumulative percentage of recovery of I3C was calculated from the individual's predicted resting energy expenditure. Recovery of I3CO2 and half-time of I-I3C-propionate in PA were significantly less than normal. The same parameters in MMA were below normal, but significantly greater than in PA. Recovery of I3CO2 was well correlated with clinical severity in PA, but did not correlate in MMA. Differences between MMA and PA may indicate different distribution of propionate pools, differences in inducibility of residual enzyme activities, or an alternate pathway for decarboxylation of propionate available in MMA but not PA. Only one patient with PA demonstrated increased 13C02 production during biotin treatment. In a B12-responsive MMA patient, no differences were noted within 2 d of initiating treatment with BIZ, but there was an increase in I3CO2 production after 4 mo. Recovery of I3CO2 was normal in the patient with transcobalamin-I1 deficiency before and after treatment with vitamin B17. In the ~a t i e n t with muletiple carboxylase deficiency, 13C02 generation was nearly normal while he was receiving his maintenance dose of biotin, and was not significantly changed after 3 and 7 d without biotin treatment, despite a decrease of 30% in lymphocyte propionyl-CoA carboxylase activity. (Pediatr Res 30: [15][16][17][18][19][20][21][22]1991)

A patient with methylmalonic acidemia was found to have a persistent hyperchloremic acidosis. Investigation documented the presence of a proximal renal tubular acidosis. Between 14 and 18 months of age the urinary pH was as high as 8.0 when the serum bicarbonate was 17 mEq/liter and the threshold for bicarbonate was at 16-17 mEq/liter. When restudied at 33 months of age, the threshold had risen to 20 mEq/liter, but this was still abnormal and supplemental treatment was required to keep the serum concentration of bicarbonate above 20 mEq/liter. It is postulated that organic acid metabolites which accumulate in this and related disorders may interfere with renal tubular function as has been shown for maleic acid in experimental animals.

Multiple esterases from individual leukocyte types of normal adult human beings have been demonstrated after separation by cationic disc gel electrophoresis. Two substrates, l-naphthyl N-acetyl-dl-alanine (NAcAla) and l-naphthyl butyrate, were used routinely in a simultaneous coupling azo dye method to demonstrate the esterases. Cytochemically, the neutrophil granules stain very poorly with α-unsubstituted carboxylic acid esters such as 1-naphthyl butyrate or 1-naphthyl acetate but stain selectively and intensely at pH 7.0 with two classes of naphthol esters; α-halo-substituted esters such as 1-naphthyl 2-bromobutyrate or naphthol AS-D chloroacetate or α-amino-substituted carboxylic acid esters such as l-naphthyl N-acetyl-dl-alanine or l-naphthyl N-acetyl-l-alanyl-l-alanyl-l-alanine. Cationic zymograms showed three major neutrophil esterases which hydrolyze NAcAla and l-naphthyl N-acetyl-l-alanyl-l-alanyl-l-alanine vigorously and l-naphthyl butyrate very poorly, which provided us with the first evidence that these enzymes might be proteolytic. Cationic zymograms of a mixture of purified enzymes (reference enzymes), pancreatic elastase, chymotrypsin and trypsin, were compared to zymograms of neutrophil extracts after assaying with NAcAla. The neutrophil esterases and elastase hydrolyzed NAcA1a vigorously; chymotrypsin and trypsin hydrolyzed it moderately. More precise characterization of the neutrophil esterases was obtained with chloromethyl ketone inhibitors which have the advantage of being irreversible and also can be synthesized with a substrate-like moiety which confers specificity. Neutrophil extracts and the mixture of reference enzymes were preincubated before electrophoresis with either N-acetyl-l-alanyl-l-alanyl-l-alanine chloromethyl ketone (NAcAla3CK), N-acetyl-l-alanyl-l-phenylalanine chloromethyl ketone (NAcAlaPheCK) or N-tosyl-l-lysine chloromethyl ketone (TLysCK). Elastase was selectively inhibited by NAcAla3CK, chymotrypsin by NAcAlaPheCK and trypsin by TLysCK. Neutrophil "lysosomal" and neutrophil-enriched cell extracts which were treated before electrophoresis with the chloromethyl ketone inhibitors at the same concentrations as used for the reference enzymes were almost completely inhibited by the NAcAla3CK except for one enzyme fraction. This fraction was inhibited by NAcAla3CK when neutrophil preparations were electrophoresed and then exposed to the inhibitor before assaying for activity. The neutrophil enzymes were much less sensitive to the TLysCK and NAcAlaPheCK. The evidence from these studies strongly supports the conclusion that the major esterases in human neutrophil granules are elastase-like esterases.

We have documented the presence of five mitochondrial enzymes in samples of chorionic villus tissue and measured the levels of activity. Three of the enzymes catalyse biotin-dependent reactions. These are propionyl-CoA carboxylase, 3-methylcrotonyl-CoA carboxylase and pyruvate carboxylase. The other enzymes, 4-aminobutyric acid aminotransferase and succinic semialdehyde dehydrogenase, are involved in the degradation of the central inhibitory neurotransmitter GABA. Distinct diseases in which there is deficiency of each of these enzymes have been documented in man. Significant levels of activity were observed for all five enzymes in chorionic villus tissue. This methodology should permit early prenatal diagnosis of deficiencies of these enzymes by chorionic villus biopsy in the first trimester.