Determination of Individual Vitamin B₆ Compounds Based on Enzymatic Conversion to 4-Pyridoxolactone

Abstract

Summary A determination method for individual natural vitamin B 6 compounds was developed. The vitamin B 6 compounds were specifically converted into 4-pyridoxolactone (PAL), a highly fluorescent compound, through a combination of enzymatic reactions and HCl-hydrolysis. PAL was then determined by HPLC. Pyridoxal was completely oxidized to PAL with pyridoxal 4-dehydrogenase (PLDH). Pyridoxine and pyridoxamine were totally converted into PAL through a coupling reaction involving pyridoxine 4-oxidase and PLDH, and one involving pyridoxamine-pyruvate aminotransferase and PLDH, respectively. The 5 ¢ -phosphate forms and pyridoxine-␤ -glucoside were hydrolyzed with HCl, and then determined as their free forms. Pyridoxine 5 ¢ -phosphate and pyridoxine-␤ -glucoside were not separately determined here. Three food samples were analyzed by this method. Key Words vitamin B 6 , 4-pyridoxolactone, pyridoxal 4-dehydrogenase, pyridoxine 4-oxidase, pyridoxamine-pyruvate aminotransferase There are six natural forms of vitamin B 6 : pyridoxine, pyridoxal, pyridoxamine, pyridoxine 5 ¢ -phosphate, pyridoxal 5 ¢ -phosphate, and pyridoxamine 5 ¢ -phosphate ( Fig. 1). In addition, plants contain pyridoxine-␤ -glucoside, a storage form of vitamin B 6 ; it is not counted as vitamin B 6 because its nutritional availability as a form of vitamin B 6 in the human body is controversial ( 1 ). The above six vitamin B 6 compounds have identical nutritional functions. On the other hand, recent studies have shown that individual vitamin B 6 compounds exhibit specific biochemical functions; pyridoxamine, in particular, can detoxify active carbonyl products and is undergoing a phase II clinical trial for the treatment of complications of diabetes ( 2 ). Vitamin B 6 compounds are strong quenchers of singlet oxygen ( 3 ), and pyridoxal 5 ¢ -phosphate and pyridoxamine 5 ¢ -phosphate, coenzyme forms of vitamin B 6 , show stronger protection of yeast cells from oxidative death than vitamin C, a representative antioxidative vitamin ( 4 ). Thus, the development of a method for the determination of individual vitamin B 6 compounds plus pyridoxine-␤ -glucoside in foods is required to estimate their functionality.Although many methods have been developed for the determination of individual vitamin B 6 compounds, no method can be satisfactorily applied to the analysis of food samples. Thus, a microbioassay involving a budding yeast, Saccharomyces cerevisiae (former name, S. uvarum ), which was developed about 40 y ago ( 5 ), has been used to determine the total content of vitamin B 6 in food samples; in the figure, the content of pyridoxine-␤ -glucoside is included because food samples are prehydrolyzed with HCl for the microbioassay, and pyridoxine-␤ -glucoside in the samples is converted into pyridoxine. The best method so far developed for individual determination may be cation-exchange HPLC with a fluorescence detection system ( 6 ), which is applicable to biological samples, such as serum, brain, liver, milk and so on, from experimental animals. The seco...

All-enzymatic HPLC method for determination of individual and total contents of vitamin B6 in foods

“…These results indicate that PLP in these foods bind so strongly to the other food component(s) that ALP cannot hydrolyze PLP bound to them. Thus, improvement of the sample preparation method is required, although it is better than HCl-hydrolysis (9), in which recoveries of PLP and PM in garlic could not be estimated. The difficulty of PLP recovery from tissue samples has been described (18).…”

“…Recently, we have developed a method for determination of individual vitamin B 6 compounds based on specific enzymatic conversion of the vitamin B 6 compounds into the high fluorescent compound 4-pyridoxolactone (4-PLA) (9). However, the method used the HCl-hydrolysis to convert the phosphoester forms into corresponding free forms.…”

“…However, the method used the HCl-hydrolysis to convert the phosphoester forms into corresponding free forms. Thus, PNG and PNP were not determined separately, and the recovery of the standards added were very low due to loss by non-specific reactions with components in the foods such as minerals (9). …”

Detection of B6 Vitamers in Grain Products: Experimental and Computational Studies

“…b Chromatogram of vitamin B in wheat bread sample (A) 2-PL (t R 4.7 min, 4.4 ng mL −1 ), 3-PN (t R 5.6 min, 32.2 ng mL −1 ) (Desai et al 2008;Eitenmiller and Landen 1999;Eitenmiller et al 2008). Nishimura et al (2008) have established a method for the determination of individual vitamin B 6 compounds on the basis that the B 6 vitamers are converted by enzymes into highly fluorescent 4-pyridoxolactone. HCl hydrolysis is also used to convert the phosphoester forms into their corresponding free forms.…”

Development of a LC-MS/MS method using stable isotope dilution for the quantification of individual B6 vitamers in fruits, vegetables, and cereals

“…The number and instability of the molecules, their high polarity, the similarity in structure, and connection to the examined matrices outline just some of the factors enriching the aggravating complexity of a specific individual determination [13][14][15][16]. Hence, a numerous variety of methods using several principles, e.g., microbiology [17][18][19], electrochemistry [20], spectrometry [21,22], and chromatography [23][24][25][26][27][28][29][30][31][32][33][34], has been developed to quantify vitamin B 6 , either as part of a multivitamin method or in a selective screening of the B 6 group. Many traditional methods pursue the detection of specifically selected vitamers or have to rely on the enzymatic transformation into PN beforehand [25,32,35].…”