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Characterization of Pyridoxal 5'-Phosphate Disappearance from In vivo Perfused Segments of Rat Jejunum^1,2,

Gastroenterology Research Laboratories, Veterans Administration Medical Center—Downtown Division, Augusta, GA 30910 and the Medical College of Georgia, Augusta, GA 30912

The disappearance of pyridoxal 5'-phosphate (PLP) from the lumen of in vivo perfused segments of rat jejunum was evaluated utilizing a single-pass technique. Net water flux was monitored by [¹⁴C] dextran, a nonabsorbable volume marker. Unlabeled PLP was measured by the tyrosine apodecarboxylase assay. PLP disappearance was linear with respect to PLP concentration at concentrations below 300 µM but was saturable at high concentrations (3 mM). PLP disappearance was significantly inhibited by 1 mM pyridoxamine 5'-phosphate and 5 mM and 10 mM L-phenylalanine but not by 1 mM pyridoxamine. Both in vivo disappearance (during perfusion) and in vitro PLP decay (exiting perfusate used as medium) correlated with the measured alkaline phosphatase activity of exiting perfusate under low-phosphate (1.1 mM) conditions. In contrast, in vivo PLP disappearance was not correlated with perfusate alkaline phosphatase activity under high-phosphate (80 mM) conditions. When exiting perfusate was ultracentrifuged at 105,000 x g for 1 hour, only 35% of the initial alkaline phosphatase activity remained in the supernatant. Conclusions were: 1) PLP disappearance from the lumen of an in vivo perfused segment of rat jejunum is saturable and inhibited by L-phenylalanine; 2) PLP disappearance appears in part to be a function of intraluminal alkaline phosphatase; and 3) A major portion of the alkaline phosphatase activity measured in the exiting perfusate represents membrane-bound enzyme.

KEY WORDS: • pyridoxal 5'-phosphate • alkaline phosphatase • jejunum

¹ Supported by the Medical Research Service of the Veterans Administration.

² Presented at the FASEB meetings in Atlanta, GA, April 12–17, 1981. Also published in abstract form in Fed. Proc. 40, 863, 1981.

Manuscript received 23 June 1981.