Department of Human Life Sciences, Kochi Gakuen College, Asahitenjinmachi , Kochi , Japan (Received September 5, 2012)

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1 J utr Sci Vitaminol, 59, 9 15, 2013 Contents of All Forms of Vitamin B 6, Pyridoxine-b-Glucoside and 4-Pyridoxic Acid in Mature Milk of Japanese Women According to 4-Pyridoxolactone-Conversion High Performance Liquid Chromatography Toshiharu Yagi 1, Saya Iwamoto 1, Rie Mizuseki 1, Michi Furuya 2 and Kazuko akayama 2 1 Faculty of Agriculture and Agricultural Science Program, Graduate School of Integrated Arts and Sciences, Kochi University, Monobe-Otsu 200, ankoku, Kochi , Japan 2 Department of Human Life Sciences, Kochi Gakuen College, Asahitenjinmachi , Kochi , Japan (Received September 5, 2012) Summary The contents of six vitamin B 6 forms, pyridoxine-b-glucoside, and 4-pyridoxic acid in mature milk of 20 Japanese lactating women consuming ordinary Japanese foods were determined by a 4-pyridoxolactone-conversion HPLC method. These compounds were determined with the average recovery rate of 83.9% or more. The average total content of vitamin B 6 forms was (mmol/l). Pyridoxal and pyridoxal 5 -phosphate were found in all of the samples, and their average contents were (mmol/l) and (mmol/l), respectively. Pyridoxamine, pyridoxine, pyridoxamine 5 -phosphate, pyridoxine 5 -phosphate, and pyridoxine-b-glucoside were found in 15, 14, 13, 9, and 7 samples, respectively. The presence of pyridoxine 5 -phosphate was for the first time found in human milk. A method for the determination of 4-pyridoxic acid, which is the excretion form of vitamin B 6, was modified to quantitate it by isocratic HPLC. 4-Pyridoxic acid was found in all samples, and its average content was (mmol/l), which was only 12% of its content in cow (Holstein) milk. The total content of vitamin B 6 forms, and predominant presence of pyridoxal among other vitamin B 6 forms in the Japanese women s milk samples shared similar characteristics with American women s milk samples. Key Words vitamin B 6, Japanese women s milk, pyridoxal, pyridoxine-b-glucoside, 4-pyridoxic acid Foods contain the family of vitamin B 6 compounds which consists of six forms, i.e., pyridoxine (P), pyridoxal (PL), pyridoxamine (PM), pyridoxine 5 -phosphate (PP), pyridoxal 5 -phosphate (PLP), and pyridoxamine 5 -phosphate (PMP) (Fig. 1). PLP is a coenzyme for many enzymes involved in amino acid and carbohydrate metabolism, and plays a key role in the nutritional function of vitamin B 6. The other forms show the same nutritional efficiency because they are converted into PLP in cells. Plants contain a storage form of vitamin B 6, pyridoxine-b-glucoside (PG) (1), which is not counted as a vitamin B 6 form although its utilization is 58% relative to P (2). 4-Pyridoxic acid (4-PA), which exhibits no vitamin B 6 activity (3), was found in some foods, such as milk. The composition of vitamin B 6 and the related compounds in human milk has been extensively studied, yagito@kochi-u.ac.jp Abbreviations: BDHQ, brief-type self-administered diet history questionnaires; HPLC, high performance liquid chromatography; 4-PA, 4-pyridoxic acid; PL, pyridoxal; PLA, 4-pyridoxolactone; PLP, pyridoxal 5 -phosphate; PM, pyridoxamine; PMP, pyridoxamine 5 -phosphate; P, pyridoxine; PG, pyridoxineb-glucoside; PP, pyridoxine 5 -phosphate. because breast milk is the sole source of nutrients for almost all infants up to 4 6 mo. The total content of vitamin B 6 and the distribution of the vitamin B 6 forms in the milk of American women have been investigated by high performance liquid chromatography (HPLC) in several laboratories (1, 4 7). The studies showed that the mature milk samples contained approximately 1 mmol/l of total vitamin B 6, in which PL was the major ( % of the total content) form in the milk. PLP was the second ( %) in the mature milk although it was not found in the transitional milk (7). The contents of the other forms were very low: PM (0 5.2% and 11.1% in the mature and transitional milk, respectively), PMP (0 1.9% and 0%), and P (0 4.4% and 11.8%). So far, PP has not been found. 4-PA content was approximately 0.2 (mmol/l) (8). In contrast to the detailed studies on the milk of American women, only one study has reported on the total content of vitamin B 6 and the distribution of vitamin B 6 forms in milk of Japanese women. Average total content of vitamin B 6 in 25 mature milk samples determined by HPLC was approximately 1.45 (mmol/l) (9). The contents of PLP and PL were 1.15 and 0.30 (mmol/l), respectively; the relative content of PLP and PL was the reverse of that found in the American wom- 9

2 10 Yagi T et al. H 2 C CH 2 H 2 C CHO H 2 C CH 2 H 2 Pyridoxine (P) Pyridoxal (PL) Pyridoxamine (PM) H 2 O 3 P 2 C CH 2 H 2 O 3 P 2 C CHO H 2 O 3 P 2 C CH 2 H 2 Pyridoxine 5'- phosphate (PP) Pyridoxal 5'- phosphate (PLP) Pyridoxamine 5'-phosphate (PMP) HO HO CH 2 CH 2 O H 3 C Pyridoxine- -glucoside (PG) O CH 2 CO H 2 C O O 4-Pyridoxic acid (4-PA) 4-Pyridoxolactone (PLA) Fig. 1. Vitamin B 6 forms, pyridoxine-b-glucoside, 4-pyridoxic acid and 4-pyridoxolactone. en s milk. o other forms or vitamin B 6 -related compounds were found. Recently, the total but not the individual content in Japanese women s milk was reported to be 1.36 (mmol/l) (10). Thus, the distribution of vitamin B 6 forms, PG and PA in Japanese women s milk has not been analyzed. Recently, we developed a new method (a 4-pyridoxolactone-conversion HPLC method) for determination of individual vitamin B 6 forms and PG. In the method, all vitamin B 6 forms and PG are specifically converted into 4-pyridoxolactone (PLA) (Fig. 1), a highly fluorescent derivative of vitamin B 6, by combinations of five enzymes, and then the PLA produced is quantitated by isocratic reversed-phase HPLC (11). Because of high substrate specificities of the enzymes used, and of the high fluorescence of PLA, the method needs only smallsized analytical samples, and all of the vitamin B 6 forms and PG in several food samples were determined with high recoveries (12, 13). 4-PA in biological samples has been determined in several laboratories by HPLC. A highly sensitive method involves a conversion of 4-PA into PLA by treatment with HCl (14). Then, PLA produced is quantitated by cation-exchange HPLC. Here, we have modified the method to quantitate by isocratic reversed-phase HPLC. In this report, we have determined all forms of vitamin B 6, PG, and 4-PA in 20 mature milk samples from healthy Japanese women, who answered a brief diet history questionnaire (BDHQ) (15). PP, which so far had not been detected in milk samples, were found in several Japanese women s milk samples. 4-PA was found in all samples. Its contents were very low compared to that in cow milk. MATERIALS AD METHODS Materials. Six forms of standard vitamin B 6, PG, five kinds of enzymes, and their substrates used in this study were obtained as described previously (11). PLA was prepared biotechnologically (16). 4-PA was purchased from Sigma-Aldrich Japan Inc. (Tokyo, Japan). All other chemicals used in this study were of the highest purity available. Cow (Holstein) milk was purchased from SunShine Co. (Kochi, Japan). Mature milk samples. The milk samples were collected from 20 healthy Japanese women living in or near Kochi City: age, (average6sd, ); BMI, ( ); and lactation stage, d (134643) postpartum. Their heights and weights are shown in Table 1. Mothers participating in the study had no medical history and were not receiving any vitamin B 6 supplements during their pregnancy or postpartum. Informed consent was obtained in written form from the subjects prior to enrollment in this study. Ethical approval was obtained from the Kochi Gakuen College Ethics Committee and was conducted in compliance with the Helsinki Declaration. Approximately 10 ml of the women s milk was obtained at an intermediate time during suckling at 10:00 14:00 hours from May 26th to June 21th, 2011, and placed in a 500 ml sterilized plastic beaker (Sanplatec, Tokyo) without preservatives. The milk (1 ml, each) was immediately transferred into an ice-cold Eppendolf tube, and then stored at 230 C until use. Dietary intakes of the women were assessed by a BDHQ system (15). This system gives the history of deduced diet intake of the previous 1 mo. Determination of vitamin B 6 forms and PG. The thawed milk sample was mixed thoroughly on a shaker to prevent separation of fat and aqueous phases. To the milk sample (100 ml) was added 1 ml 0.1 m HCl, and then the mixture was incubated at 100 C for 30 min in a SCIICS ALB-121 aluminum dry block bath (Tokyo, Japan). After cooling, 40 ml of 50% (w/v) trichloroacetic acid was added to the mixture. The mixture was then mixed thoroughly, and additionally incubated at 100 C for 5 min in the bath. After cooling, the ph of the

3 Contents of Vitamin B 6 Forms in Human Milk 11 Table 1. Contents of PL, PM, P, PLP, PMP, PP, and PG in the Japanese women s mature milk samples. Sample o. Height (cm) Weight (kg) PL PM P (mmol/l) Recovery (%) (mmol/l) Recovery (%) (mmol/l) Recovery (%) D D D D D D D D D D D Sample o. PLP PMP PP PG (mmol/l) Recovery (%) (mmol/l) Recovery (%) (mmol/l) Recovery (%) (mmol/l) Recovery (%) D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D Average6SD of three experiments. D: less than mmol/l. mixture was adjusted to 7.5 by adding 40 ml of 0.5 m Tris-HCl (ph 7.5) and 28 ml of 5.0 m a. Water was then added to make the volume of the sample solution 2.0 ml. The mixture was centrifuged at 10,000 3g for 5 min at 4 C, and the supernatant (100 ml) was used for the enzymatic reaction. For recovery studies, definite amounts of standard vitamin B 6 forms and PG were separately added to the thawed milk, and then the standard vitamin B 6 -spiked milk samples were also subjected to the steps of the analytical sample preparation. The enzyme reactions for conversion of vitamin B 6 forms and PG into PLA were done with seven reaction mixtures as described previously (13) with the exception that 100 ml of the milk sample was used instead of 5 50 ml of the extract of foods: a decreased amount of water was added to make the volume of reaction

4 12 Yagi T et al. Cow milk o reaction Reaction Reaction spiked with 2 pmol 4-PA Human milk o reaction Reaction Reaction spiked with 2 pmol 4-PA Fig. 2. HPLC chromatograms for the analysis of 4-PA in the cow (Holstein) and Japanese woman s milk samples. Arrows show PLA peaks. The attenuation of the charts was 32 ma/full scale. The cow milk (5 ml) and Japanese woman s milk (o. 9, 50 ml), with or without 2 pmol (amount applied to the HPLC column) of standard 4-PA, were subjected to the HCl treatment, and then applied to HPLC. mixture the same. Additionally, the reaction protocol of reaction 7 for determination of PG was modified as follows. After the b-glucosidase reaction, 40 ml instead of 80 ml of 0.5 m a, 40 ml instead of 16 ml of 0.25 m Tris-HCl, and 95 ml of water were added, and then incubated at 30 C for 1 h. The reaction mixture (100 ml, each) of seven enzymatic reactions was used for the reversed-phase HPLC. Determination of 4-PA. To 100 ml of the thawed Japanese women s milk samples was added 200 ml of petroleum ether, and then the mixture was mixed thoroughly. After centrifugation at 9,000 3g for 1 min at 4 C, 50 ml of the lower layer solution was mixed with 200 ml of 5 m HCl. The mixture was then heated at 100 C for 20 min in the SCIICS ALB-121 aluminum dry block bath. After cooling on ice, 50 ml of 1 m potassium phosphate buffer, ph 7.0, 170 ml of 5 m a, and 530 ml of water were added to make the total volume 1,000 ml. The reaction mixture (100 ml) was used for the reversed-phase HPLC. For recovery studies, a definite amount (usually 2 pmol) of standard 4-PA was added to the thawed milk, and then the 4-PA-spiked milk samples were also subjected to the HCl-treatment to prepare the analytical sample. For determination of 4-PA in cow milk, only 5 ml of the petroleum ether-extracted sample was used, because the amount of 4-PA in the cow milk was very high compared to that in the human milk. HPLC conditions. The HPLC conditions for the determination of PLA produced from the vitamin B 6 forms and PG by the enzyme reactions were described previously (13). Briefly, the column was a Cosmosil 5C18MS- II ( mm, acalai Tesque, Inc., Kyoto, Japan); the mobile phase, 20 mm potassium phosphate buffer, ph 7.0, containing 10% methanol; flow rate and sample volume, 0.5 ml/min and 100 ml, respectively; and fluorescence detection, excitation at 360 nm and emission at 430 nm. For the quantitation of PLA produced from 4-PA by the HCl treatment, the same HPLC conditions were used with exception that the flow rate was changed to 1.0 ml/min. Because the amount of 4-PA in the human milk was very low, a better shape of PLA elution peak was obtained when the flow rate was increased. Enzyme and protein assay. The activities of five enzymes used were assayed every time just before usage for enzymatic conversion of vitamin B 6 forms and PG into PLA as described previously (13). Concentrations of protein in the petroleum-etherextracted milk were determined by the Lowry method (17). Statistical analysis. Values are expressed as average 6SD of three experiments. Correlation between two variables was examined with the Excel program. RESULTS Contents of vitamin B 6 forms and PG in the Japanese women s milk samples The contents of vitamin B 6 forms and PG in 20 mature milk samples of Japanese women were determined (Table 1). The recoveries of each of the samples are also shown. The average recoveries6sd (%) of PL, PM, P, PLP, PMP, PP, and PG were , , , , , , and , respectively. Thus, the compounds in milk samples could be determined without extraction of lipids. PL was found in all samples, and its average content in the 20 milk samples was (mmol/l). PM was found in 15 samples, and its average content was (mmol/l). P was found in 14 samples,

5 Contents of Vitamin B 6 Forms in Human Milk 13 Table 2. Contents of 4-PA and protein in the Japanese women s nature milk samples. Sample o. 4-PA Protein (nmol/l) Recovery (%) (g/l) Average6SD of thre experiments. and its average content was (mmol/l). PLP was also found in all samples, and its average content was (mmol/l). PMP was found in 13 samples, and its average content was (mmol/l). PP was found in 9 samples, and its average content was (mmol/l). PG was found in 7 samples, and its average content was (mmol/l). Thus, the average relative amount of vitamin B 6 forms and PG was: PL, 70.3%; PLP, 15.7; P, 5.0; PG, 3.0; PM, 2.0; PMP, 2.0; and PP, 2.0, although P, PG, PM, PMP, and PG were found in limited milk samples. Total contents of vitamin B 6 forms plus PG were in a range of mmol/l: average total content was mmol/l. The correlation between contents of each of the vitamin B 6 forms and PG was analyzed because analyses of sushi toppings showed interesting correlations between contents of vitamin B 6 forms (12). In contrast to sushi toppings, the correlation between contents of PL, PM, P, PLP, PMP, PP, and PG in milk was not found: the contents of PP and PG only showed a positive correlation with a determination coefficient of 0.48 (a correlation coefficient, r50.69, p,0.01): linear correlation curve, y x Contents of 4-PA and protein The HCl-treatment quantitatively converted 4-PA into PLA, which was eluted at around 7 min under the HPLC conditions described above. Figure 2 shows HPLC chromatograms obtained when 5 ml and 50 ml of the cow and Japanese woman s (sample o. 9) milk, respectively, were analyzed. The milk samples did not PM content (nmol/l) originally contain PLA because the peak of PLA was only found in HCl-treated samples. 4-PA-spiked samples showed higher peaks, corresponding to the increase of PLA produced from the spiked 4-PA. Thus, the modified isocratic HPLC was applicable for quantitation of 4-PA in the cow and Japanese women s milk samples. The contents and recoveries of the Japanese women s milk samples are shown in Table 2. The contents of 4-PA in the Japanese women s milk samples were (nmol/l), and the average was PA contents in the American women s milk samples were (nmol/l) (8). The 4-PA content of cow milk was (nmol/l). It was much higher than that of the human milk. The high content of 4-PA ( nmol/l) in cow (Holstein) milk has been reported (8). The correlation between the content of 4-PA and those of vitamin B 6 forms and PG in the Japanese women s milk was examined. PM contents only showed a negative correlation with 4-PA contents with the determination coefficient of (r520.67, p,0.01) as shown in Fig. 3. The protein concentration of the Japanese women s milk samples are shown in Table 2. The average protein concentration of the samples was g/l ( g/l). The protein concentration showed no correlation with the content of the vitamin B 6 forms (PL: r50.005, p.0.1; PM: r50.35, p.0.1; P: r50.39, p.0.05; PLP: r50.09, p.0.1; PMP: r50.06, p.0.1; PP: r50.16, p.0.1), PG (r50.19, p.0.1), or the total content of vitamin B 6 (r50.004, p.0.1). BDHQ analysis To examine the normality of milk samples used, dietary habits of the Japanese mothers were assessed using BDHQ. BDHQ analysis showed that the mothers consumed ordinary Japanese foods and no alcohol or supplement. The deduced average intake of energy, protein, fat, and vitamin B 6 (pyridoxine equivalent determined by a microbiological method) were 1, kcal/d, g/d, g/d, and mg/d, respectively. The deduced average intake of vitamin B 6 was higher than the Recommended Daily Allowance (1.1 mg/d) of Japan (18). DISCUSSIO y = x r² = r = -0.67, P< PA content (nmol/l) Fig. 3. Correlation between contents of PM and 4-PA in the Japanese women s milk samples. The main forms (PL and PLP) and the total content

6 14 Yagi T et al. (about 1 mmol/l) of vitamin B 6 in the Japanese women s mature milk was quite similar to those in the American women s mature milk so far reported (1, 4 7, 19). The American women s milk samples were analyzed by reversed-phase HPLC (1, 7, 19), ion-exchange HPLC (4, 5) or a microbiological method (6). Here, PP, which had not been found in American mothers mature milk samples, was found in 45% of Japanese women s milk samples, although the contents were low. PG, which had been reported to comprise a mean of 2.5% of the total vitamin B 6 contents (6), was found in 35% of Japanese women s milk samples, and accounted for a mean of 4% of the total vitamin B 6 contents. Although there was a correlation between PP and PG contents, it may not be significant, because only five samples contained both of them, and five samples contained either one of them. The limited detection of PG in the milk samples coincides with the results that there is little practical significance to the consumption of PG, which is contained mainly in plant-derived foods, on the content of PG in milk by free-living populations (6). The average concentrations of PL and PLP in 12 American lactating women s whole blood samples were and mmol/l, respectively (20). Because there are no practical differences in contents and kinds of vitamin B 6 forms in mature milk between the Japanese and American women, these values may be applicable to the Japanese women. Thus, the concentrations of PL and PLP in the milk may be 9-fold and 3.8-fold higher than those in the whole blood, respectively. The results suggested that PL and PLP were selectively excreted into the women s milk. Although the excretion of P (21, 22) and its transporter (23) have been reported, there is no information about PL or PLP excretion mechanisms. There was a correlation between contents of PM and 4-PA, which is the excretion form of vitamin B 6. Although the graph of correlation (Fig. 3) showed the correlation is significant, the mechanism that caused the correlation is not known. The analysis of more milk samples is required to confirm the results and to elucidate the mechanism. There was no significant correlation between contents of each of the vitamin B 6 forms or total contents of free and phosphate forms. The results were in contrast to those obtained in analyses of sushi toppings (sashimi, raw meat of fishes) (12). In the toppings, the total contents of the free and phosphate forms exhibited strong correlation with a determination coefficient of 0.938: the total amounts of the phosphate forms were about 3-fold higher than those of the free forms. The contents of PM and PMP, PL and PLP, and P and PLP moderately correlated with determination coefficients of 0.717, 0.689, and 0.556, respectively, while the contents of P did not correlate with those of PP. The correlations may be general characteristic in the cells. Thus, although we have no data about the distribution of vitamin B 6 forms in mammary gland cells, the distribution of vitamin B 6 forms in the milk may not reflect the intracellular distribution, PL being specifically excreted into the milk. The priority of PL for infants over other vitamin B 6 forms, especially P, which is generally used as a supplement for formula milk, has not been examined. Here, 20 milk samples were analyzed. There is a need to analyze a large number of milk samples from lactating women living in various regions in Japan to generalize the results obtained here. Acknowledgments We are grateful to Ms. oriko Sadahiro, the director of Sadahiro Maternity Hospital, and Ms. Yoshiko Tanimoto, the director of Tanimoto Maternity Hospital, for their kind help in the collection of milk samples. This work was partially supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan, Grants-in-Aid for Exploratory Research, , REFERECES 1) Gregory JF, Ink SL Identification and quantification of pyridoxine-b-glucoside as a major form of vitamin B 6 in plant-derived foods. J Agric Food Chem 35: ) Gregory III JF, Trumbo PR, Bailey LB, Toth JP, Baumgartner TG, Cerda JJ Bioavailability of pyridoxineb-d-glucoside determined in humans by stable-isotopic methods. J utr 121: ) Hodson AZ Vitamin assay, vitamin B-6 in sterilized milk and other milk products. J Agric Food Chem 4: ) Coburn SP, Mahuren JD A versatile cationexchange procedure for measuring the seven major forms of vitamin B 6 in biological samples. 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