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

Abstract

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.