ABSTRACT
The aim of the present study was to examine the effects of cobalamin (Cbl) on the activity and expression of L-methylmalonyl-CoA mutase (MCM) in rat liver and cultured COS-7 cells. The MCM holoenzyme activity was less than 5% of the total (holoenzyme+apoenzyme) activity in the liver although rats were fed a diet containing sufficient Cbl. When weanling rats were maintained on a Cbl-deficient diet, the holo-MCM activity became almost undetectable at the age of 10 weeks. In contrast, a marked increase in the total-MCM activity occurred under the Cbl-deficient conditions, and at the age of 20 weeks it was about 3-fold higher in the deficient rats than in the controls (108 (SD 14.5) v. 35 (SD 8.5) nmol/mg protein per min (n 5); P<0.05). Western blot analysis confirmed that the MCM protein level increased significantly in the Cbl-deficient rats. However, the MCM mRNA level, determined by real-time PCR, was rather decreased. When COS-7 cells were cultured in a medium in which 10% fetal bovine serum was the sole source of Cbl, holo-MCM activity was barely detected. The supplementation of Cbl resulted in a large increase in the holo-MCM activity in the cells, but the activity did not exceed 30% of the total-MCM activity even in the presence of Cbl at 10 micromol/l. In contrast, the total-MCM activity was significantly decreased by the Cbl supplementation, indicating that Cbl deficiency results in an increase in the MCM protein level in COS-7 cells as well as in rat liver.
Subject(s)
Kidney/enzymology , Liver/enzymology , Methylmalonyl-CoA Mutase/analysis , Vitamin B 12 Deficiency/enzymology , Animals , Blotting, Western/methods , COS Cells , Carnitine/administration & dosage , Chlorocebus aethiops , Dietary Supplements , Gene Expression , Methionine/administration & dosage , Methylmalonyl-CoA Mutase/genetics , Methylmalonyl-CoA Mutase/urine , RNA, Messenger/analysis , Random Allocation , Rats , Reverse Transcriptase Polymerase Chain Reaction/methods , Vitamin B 12/administration & dosage , Vitamin B Complex/administration & dosage , WeaningABSTRACT
BACKGROUND: Propionic aciduria (PA) and Methymalonic aciduria (MMA) result from an inherited abnormality of the enzymes propionyl CoA carboxylase and methylmalonyl CoA mutase respectively. This produces marked increases in the amino acids methionine, threonine, valine and isoleucine (MTVI). Their clinical presentation can be neonatal or late onset forms. AIM: To report 23 children with organic acidurias. MATERIAL AND METHODS: Twenty three cases of organic acidurias diagnosed since 1980 (17 PA and 6 MMA) and followed at the Institute of Nutrition and Food Technology, are reported. RESULTS: The average age of diagnosis was 3.9 days for the neonatal form and 8.3 months for the late onset form. The most frequent symptoms were hypotonia, lethargy and vomiting. Neonatal PA had mean ammonemias of 1089 +/- 678.3 micrograms/dl. The figure for MMA was 933 +/- 801.9 micrograms/dl. Seven children were dialyzed and 30% died. 16 children are followed and 81.2% have normal weight for age. Seven children required gastrostomy because of anorexia and failure to thrive. The nutritional treatment is based on natural and artificial proteins without MTVI, with periodical controls, amino acid and ammonia quantification. Some patients were submitted to enzyme assays and molecular studies. CONCLUSIONS: An early diagnosis and a very strict follow up allows a normal development of children with organic acidurias. There is a relationship between prognosis and the presentation form, the nutritional status and the emergency treatment during acute episodes. The importance of the enzymatic and molecular studies is emphasized because they facilitate treatment, accurate diagnosis and allow an adequate genetic counseling.
