ABSTRACT
The sensitivity of fluorescent detection of the biologically active form of Vitamin B-6, pyridoxal 5'-phosphate (PLP), in biological samples has been improved approximately four-fold by adopting chlorite as a post-column derivatization reagent (instead of bisulfite) in high-performance liquid chromatography (HPLC) separation. Chlorite oxidizes PLP to the more fluorescent 4-pyridoxic acid 5'-phosphate, and avoids the toxicity and heating of the cyanide procedure. Detection of another major metabolite, 4-pyridoxic acid (4-PA), is not effected. Detection of pyridoxal (PL) is slightly lowered due to eluting at a lower pH.
Subject(s)
Chlorides/chemistry , Chromatography, High Pressure Liquid/methods , Pyridoxal Phosphate/blood , Chromatography, High Pressure Liquid/standards , Humans , Oxidation-Reduction , Pyridoxal Phosphate/chemistry , Pyridoxic Acid/analogs & derivatives , Pyridoxic Acid/blood , Reproducibility of ResultsABSTRACT
BACKGROUND: Renal insufficiency is associated with altered vitamin B-6 metabolism. We have observed high concentrations of 4-pyridoxic acid, the major catabolite of vitamin B-6 metabolism, in plasma during renal insufficiency. OBJECTIVE: The objective was to evaluate the renal handling of 4-pyridoxic acid and the effects of renal dysfunction on vitamin B-6 metabolism. DESIGN: We measured the renal clearance of 4-pyridoxic acid and creatinine in 17 nonpregnant, 17 pregnant, and 16 lactating women. We then examined the influence of vitamin B-6 or alkaline phosphatase activity on the ratio of 4-pyridoxic acid to pyridoxal (PA:PL) in plasma in 10 men receiving a low (0.4 mg pyridoxine.HCl/d) or high (200 mg pyridoxine.HCl/d) vitamin B-6 intake for 6 wk, in 10 healthy subjects during a 21-d fast, in 1235 plasma samples from 799 people screened for hypophosphatasia, and in 67 subjects with a range of serum creatinine concentrations. RESULTS: Renal clearance of 4-pyridoxic acid was 232 +/- 94 mL/min in nonpregnant women, 337 +/- 140 mL/min in pregnant women, and 215 +/- 103 mL/min in lactating healthy women. These values were approximately twice the creatinine clearance, indicating that 4-pyridoxic acid is at least partially eliminated by tubular secretion. Elevated plasma creatinine concentrations were associated with marked elevations in 4-pyridoxic acid and PA:PL. PA:PL was not affected by wide variations in vitamin B-6 intake or by the wide range of pyridoxal-P concentrations encountered while screening for hypophosphatasia. CONCLUSIONS: Plasma 4-pyridoxic acid concentrations are markedly elevated in renal insufficiency. Plasma PA:PL can distinguish between increases in 4-pyridoxic acid concentrations due to increased dietary intake and those due to renal insufficiency.