“Total” folate in blood has usually been measured to evaluate the folate status of pregnant women. However, folate is composed of many metabolites. The main substrate is 5-methyltetrahydrofolate (5-MTHF), with folic acid (FA) representing a very small component as an unmetabolized species in blood. We longitudinally evaluated 5-MTHF, FA and total homocysteine in maternal and cord blood from Japanese pregnant women. Subjects were 146 pregnant women who participated in the Chiba study of Mother and Child Health (C-MACH) prospective cohort study. Sera were obtained in early and late pregnancy, at delivery, and from cord blood. Species levels were measured by isotope-dilution mass spectrometry. Both 5-MTHF and FA levels were lower than reported levels from pregnant women in populations from countries with mandatory FA fortification. As gestational age progressed, serum 5-MTHF levels decreased, whereas serum FA levels were slightly reduced only at delivery compared to early pregnancy. A significant negative association between serum 5-MTHF and total homocysteine was shown at all examined times, but no associations with FA were evident. At delivery, cord 5-MTHF was significantly higher than maternal levels, while FA again showed no significant correlation. These results suggest that 5-MTHF is actively transported to the fetus through placental transporters and may reflect folate status during pregnancy as a physiologically important species.

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Retinoylation (acylation of proteins by retinoic acid) is considered as one mechanism of retinoic acid (RA) action occurring in cells in vitro and in vivo. Previously, our studies showed that in rat tissues the formation of retinoyl-CoA from RA, the first step of retinoylation, required ATP, CoA and MgCl(2). In the current study, we examined whether the transfer of retinoyl-CoA into proteins, the second step of retinoylation, occurs in rat tissues. [(3)H]-Labeled-retinoyl-CoA bound covalently to proteins in rat liver, kidney, testis, and brain. The levels of incorporation of retinoyl-CoA into proteins were higher in vitamin A-deficient rats than in normal ones. The formation of retinoylated proteins depended on the incubation time, and the concentrations of retinoyl-CoA and homogenate. The reaction was suppressed by fatty acyl-CoAs and palmitic acid, but not by arachidonic acid. The Vmax and Km values for retinoyl-CoA in the formation of retinoylated proteins using a crude liver extract were estimated to be 2,597.3 pmol/min/mg protein and 9.5 x 10(-5) M, respectively. Retinoylated proteins formed from retinoyl-CoA, including a 17 kDa protein exhibiting high radioactivity, disappeared in the presence of 2-mercaptoethanol, indicating that RA was linked to the proteins through a thioester bond. These results demonstrate that retinoylation in rat tissues occurs via retinoyl-CoA formed from RA. This process may play a significant physiological role in cells.

No abstract

Retinoylation (retinoic acid acylation) is a posttranslational modification of proteins occurring in a variety of cell types in vitro and in tissues in vivo. The widespread occurrence of retinoylation suggests that it may play a role in many effects of retinoic acid (RA) on cells. One metabolic pathway for retinoylation involves the intermediate formation of retinoyl-CoA and subsequent transfer and covalent binding of the retinoyl moiety to protein. However, such reactions are not well known. To gain further insight into retinoylation, we studied the synthesis of retinoyl-CoA, the first step in this multi-stage process. The formation of [(3)H]-retinoyl-CoA was determined in incubation mixtures containing rat liver extract, [(3)H]-RA, ATP, CoA, and MgCl(2). No retinoyl-CoA was formed in the presence of boiled extract, or in the absence of ATP, CoA, or MgCl(2) (a divalent cation). A greater amount of retinoyl-CoA was obtained from microsomal fractions of rat liver than from other subfractions. The presence of retinoyl-CoA was also detected in extracts prepared from rat testis, kidney, brain, spleen, and pancreas. The level of retinoylation in various tissue extracts was related directly to the amount of retinoyl-CoA formed. V(max) and K(m) values for RA in the formation of liver retinoyl-CoA were estimated to be 1.0 x 10(-4) micromol/min/mg protein and 24 nM, respectively. Synthesis of retinoyl-CoA was suppressed by fatty acids and fatty acyl-CoAs. These results indicate that ATP-dependent generation of retinoyl-CoA occurs in rat tissues and may play a significant physiological role in RA actions mediated by retinoylation.