Evidence obtained from incubation of corn (Zea mays cv. Golden Bantam) seedings in DL-lbenzene ring-U-'4Citryptophan, L-15_3Hitryptophan, L-IU-14Claspartate and IU-'4CIglycerol indicates that niacin is synthesized in these plants via oxidative degradation of tryptophan. Aspartate and glycerol do not appear to be precursors of niacin in corn seedings. Extraction. Following incubation, the tissues were washed with distilled H20, homogenized, and extracted four times with methanol:chloroform:water (12:5:3, v/v/v) to extract free niacin into the alcohol-aqueous phase (2). To release bound niacin, an additional 100 to 200 mg tissue were homogenized and autoclaved 30 min in 1.8 ml of 1 N H2SO4 (4). The autoclaved mixture was neutralized with 0.41 N Ba(OH)2, centrifuged, and the pellet was extracted four times with methanol:chloroform:water, using the procedure described above. Niacin content of the acid extract is reported as total niacin. Aqueous extracts were evaporated in a flash evaporator, and the residue was dissolved in the appropriate chromatographic eluant.Analysis. The extracts were analyzed by reverse-phase HPLC, using a Waters Associates system consisting of a ,uBondapak C18 chromatography column, 0.39 x 30 cm, a Model 6000A pumping system, a Model 440 UV absorbance detector set at 254 nm, a Model R401 differential refractometer, and Model U6K injector. Monitor outputs were recorded on a Houston Omni-Scribe dual pen chart recorder (1 1).In the analysis of tryptophan metabolites, the initial eluant was 10 mm Na-acetate (pH 4.84), followed by 3% (w/v) methanol in 10 mm Na-acetate (pH 4.84). The change in eluants was initiated 35 min after sample injection and the second eluant reached the detectors 9 min later. Flow rate was 1.0 ml/min, maintained by a pressure of 6,200 kPa (900 p.s.i.). This system resolved all major intermediates of the tryptophan-niacin pathway except quinolinic acid.Niacin was determined in tissues incubated with [14C]aspartate or [14C]glycerol by reverse-phase ion-pair HPLC, using 50 mM sodium tetrabutylammonium phosphate (pH 7.0; Eastman), in 10%o (v/v) methanol as eluant (6). Flow rate was 1.5 ml/min, maintained by a pressure of 9,700 kPa (1,400 p.s.i.). Quinolinic acid was resolved in standard solutions by this system but was not measurable in extracts due to interference from other radioactive products.The chromatographic system was maintained at 20 (±2)°C during operation. To complete each analysis, the column was washed briefly with 75% methanol. Compounds were quantified by measurement of areas of their UV absorption peaks.Eluates were collected in 0.5-min fractions, and radioactivity in these and all other samples was determined in a Beckman LS7000 liquid scintillation counter. Scintillation fluids consisted of 0.4% PPO and 0.05% POPOP in toluene:Triton X-100 (2:1, v/v) for extracts and chromatographic fractions, and 0.5% PPO and 0.01%
