K. E. Creek

In vivo formation of tritium-labeled lactic acid from [2-3H]mannose or [15-3H]retinol by hamster intestinal epithelial cells

In studies designed to reexamine the in vivo occurrence of retinyl phosphate mannose we injected hamsters intraperitoneally with either [2-3H]mannose or [15-3H]retinol and sacrificed the animals 15 min later. The small intestine was removed, the epithelial cells were scraped, and a methanolic extract of the labeled cells was prepared and chromatographed on a Mono Q anion-exchange column. Intraperitoneal administration of either [2-3H]mannose or [15-3H]retinol lead to the formation of a tritium-labeled anionic compound with a retention time on the Mono Q column similar to that of standard retinyl phosphate mannose. However, the biochemical properties of this labeled anionic compound were those expected of an organic acid and not retinyl phosphate mannose. The compound was resistant to both strong acid hydrolysis and mild base hydrolysis, as well as digestion with alpha- or beta-mannosidase, phosphodiesterase I, nucleotide pyrophosphatase, or beta-glucuronidase. When chromatographed on an Aminex HPX-87H organic acid analysis column or a silicic acid column the labeled anionic compound derived from either [2-3H]mannose or [15-3H]retinol comigrated with standard lactic acid. Treatment of the anionic compound derived from [2-3H]mannose with lactate oxidase or L-lactate 2-monooxygenase resulted in the formation of a tritium-labeled product that cochromatographed, respectively, with pyruvate or acetate on the Aminex HPX-87H column. However, treatment of the anionic compound derived from [15-3H]retinol with these same two enzymes resulted in a labeled product that migrated on the Aminex column at the same position as tritiated water. This result demonstrated that the labeled hydrogen was removed during enzymatic digestion and suggested that it was present on the second carbon of lactic acid. During the course of these studies no evidence for the in vivo labeling of a compound with the properties of retinyl phosphate mannose was found. Since [2-3H]mannose leads to labeled lactic acid in vivo the tritium label must not always be lost, as expected, during the entry step into glycolysis in which mannose 6-phosphate is converted to fructose 6-phosphate. The results suggest that an intramolecular hydrogen transfer from the C-2 position of mannose 6-phosphate to the C-1 position of fructose 6-phosphate can occur during the phosphomannose isomerase reaction. The finding that the position of the tritium label on lactic acid derived from [15-3H]retinol is on the second carbon is consistent with it coming from NADH labeled with tritium in the transferable hydrogen which was formed intracellularly during the NAD+-linked oxidation of retinol to retinaldehyde.

Reduced mannose incorporation into GDP-mannose and dolichol-linked intermediates of N-glycosylation in hamster liver during vitamin A deficiency

SummaryThe molecular mechanism of reduced incorporation of radioactively labeled mannose into hamster liver glycoconjugates during the progression of vitamin A deficiency was investigated. In particular the in vivo incorporation of [2-3H]mannose into GDP-mannose, dolichyl phosphate mannose (Dol-P-Man), lipid-linked oligosaccharides, and glycopeptides of hamster liver was examined. Hamsters maintained on a vitamin A-free diet showed a reduction in the incorporation of mannose into GDP-mannose about 10 days before clinical signs of vitamin A deficiency could be observed. The decrease in [2-3H]mannose incorporated into GDP-mannose was accompanied by a reduction in label incorporated into Dol-P-Man, lipid linked oligosaccharides and glycopeptides, which became more severe with the progression of vitamin A deficiency. By the time they reached a plateau stage of growth, hamsters fed the vitamin A-free diet showed a 50% reduction in the amount of [2-3H]mannose converted to GDP-mannose, and the radioactivity associated with Dol-P-Man and glycopeptides was reduced by approximately 60% as compared to retinoic acid-supplemented controls. These results strongly indicate that the reduced incorporation of mannose into lipidic intermediates and glycoproteins observed during vitamin A deficiency is due to impaired GDP-mannose synthesis.

Retinoic acid treatment of fibroblasts causes a rapid decrease in [3H]inositol uptake

NIH 3T3 fibroblasts treated with all-trans-retinoic acid (RA) showed a dramatic decrease in the uptake of [3H]inositol compared to solvent-treated controls. The onset of RA-induced inhibition of [3H]inositol uptake was rapid with a 10-15% decrease occurring after 2-3 h of RA exposure and 60-70% reduction after 16 h of RA treatment. A progressive dose-dependent decrease in inositol uptake was found as the concentration of RA increased from 10(-8) to 10(-5) M and the effect was fully reversible within 48 h after RA removal. The Vmax and Kt for the controls were 10 nmol/2.5 x 10(6) cells/2 h and 51 microM; and for RA-treated cells the values were 4 nmol/2.5 x 10(6) cells/2 h and 52 microM. The decreased [3H]inositol uptake was not due to a change in the affinity (Kt) of the transporter for the inositol but to a decrease in the Vmax. The maximal effect on inositol uptake was dependent on RA treatment of the cells after they reached saturation density or if made quiescent by serum starvation. RA was the most active of the different retinoids examined in the order RA greater than 13-cis-RA = retinyl acetate greater than all-trans-retinol greater than 5,6-dihydroxyretinoic acid methyl ester greater than N-4-hydroxyphenyl retinamide. In contrast to this effect on inositol, the uptake of fucose, mannose, galactose, and glucose was either not affected or enhanced (for mannose and fucose) by RA treatment. RA inhibition of inositol uptake was also observed in 3T3-Swiss and Balb/3T3 cells but not in two virally transformed 3T3 cell lines. Phlorizin, amiloride, and monensin inhibited inositol uptake by 66, 74, and 58%, respectively, and this inhibition was additive when the cells were treated with RA as well as these inhibitors. A decreased incorporation of [3H]inositol into polyphosphoinositides was also observed in RA-treated cells but not to the same extent as for [3H]inositol uptake. In conclusion, RA treatment of 3T3 fibroblasts decreases the uptake of [3H]inositol by up to 70% within 8 to 10 h at near physiological concentrations in a reversible and specific manner.

Enzymatic synthesis and separation of retinyl phosphate mannose and dolichyl phosphate mannose by anion-exchange high-performance liquid chromatography.

Publisher Summary This chapter describes the enzymatic synthesis and separation of retinyl phosphate (Ret-P) mannose and dolichyl phosphate (DoI-P) mannose by anion-exchange high-performance liquid chromatography. Retinyl phosphate is chemically synthesized by the phosphorylation of all- trans -retinol by bisditriethylamine phosphate. GDP-mannose (Sigma) or GDP-[U- 14 C] mannose is used as the mannose donor for the preparation of unlabeled or [ 14 C]mannose-labeled Ret-P-Mannose. Unlabeled Ret-P-Mannose is prepared. Ret-P-Mannose is purified from the extract by high performance liquid chromatography (HPLC) on a Mono Q HR 5/5 column as described in detail in the chapter. The concentration of Ret-P-Man is determined spectrophotometrically at 325 nm in 99% methanol. [ 14 C]Mannose-labeled Ret-P-Man is prepared similarly to that for the synthesis of unlabeled Ret-P-Man. [ 14 C]Man is purified from the extract by HPLC on a Mono Q HR 5/5 column is also described in the chapter. Typically 30–40% of the added [ 14 C]mannose is transferred from GDP -[ 14 C]mannose to exogenous Ret-P under these incubation conditions. [ 14 C]Mannose-labeled Dol-P-Man may be prepared using incubation conditions similar to those described for the preparation of [14C]mannose- labeled Ret-P-Man. The major difference among the incubations is the inclusion of Triton X-100 which is required for the solubilization of the polyisoprenoid Dol-P. HPLC system for the separation of Mannose, Dol-P-Man, Ret-P-Man, and Ret-P is based on observation that Dol-P-Man, Ret- P-Man, and Ret-P could be separated by anion exchange chromatography on DEAE-Sephacel columns, utilizing ammonium acetate in 99% methanol as the eluent. Mannose, Ret-P-Man, mannose phosphate, and GDP-mannose are separated based on their difference in anionic charge on the anion exchange Mono Q HR 5/5 column. Dol-P-Man is eluted in this system with approximately the same retention time as Ret-P-Man but in very poor yields due to poor solubility of Dol-P-Man in 70% methanol.