recombinant pl 4-dehydrogenase ( pldh ) , pm - pyruvate aminotransferase ( ppat ) , and pn 4-oxidase ( pnox ) were prepared as described previously ( 1012 ) . recombinant e. coli alkaline phosphatase ( alp ) was prepared according to a standard protocol ( 13 ) . almond -glucosidase ( gls ) , pm , and pl ( 99.5% ) were purchased from sigma chemical co. ( st . louis , mo ) , pn and plp from nacalai tesque , inc . the pmp was a gift from daiichi fine chemicals co. ( takaoka , japan ) and png was prepared enzymatically from d - glucose and pn based on a transglucosidase activity of a bacterial -glucosidase ( 15 ) . food samples were bought at local markets and chosen at random in order to test and validate the method . the basic principle is to convert all vitamin b6 compounds specifically into a highly fluorescent vitamin b6-derivative 4-pla ( see fig . 1 ) . thus , seven reaction mixtures were used to determine seven vitamin b6 compounds . the filtration was done with a dismic 13 syringe filter ( pore size of 0.2 m , advantech , tokyo , japan ) . to make a control ( i.e. no - reaction test sample ) , hcl was added to the reaction mixture before addition of the food sample . for the determination of plp , the food sample was at first hydrolyzed with alp in the reaction 4 ( fig . 2 ) , and then the pl produced was determined as shown under the reaction for plp ( fig . 3 ) . both pmp and pnp were determined in the same way just like plp but using the additional enzymes and the substrate or the cofactor as shown under the reactions for pmp and pnp , respectively ( fig . first hydrolyzed with -glucosidase in the reaction mixture containing nah2po4 , which inhibited phosphatase activity in the almond -glucosidase preparation , and then pn was determined as shown under the reaction for png ( fig . 3 ) . the reaction mixtures used for determination of seven vitamin b6 compounds . illustrations of analytical procedures of seven reactions . see table 1 for quantification of the seven compounds from the hplc elution profiles of the seven reactions described above . the 4-pla was measured by a reversed - phase isocratic hplc method using a cosmosil 5c18ms - ii column ( 2504.6 mm ; nacalai tesque , kyoto , japan ) , and a mobile phase consisting of 20 mm potassium phosphate buffer ( ph 7.0 ) and 10% ( v / v ) methanol . the separation was made with a jasco hplc system ( jasco , tokyo , japan ) equipped with an as-2055 autosampler , a pu-2080 pump , and an fp-920 fluorescence detector . the fluorescence intensity of the eluted 4-pyridoxolactone was monitored at 430 nm after excitation at 360 nm . the pldh ( 10 ) , pnox ( 12 ) , ppat ( 11 ) , alp ( 16 ) , and -glucosidase ( 17 ) were assayed as described previously . one unit of enzyme was defined as the amount that catalyzed the formation of 1mol of product per min . the pldh , pnox , alp , and -glucosidase catalyze irreversible reactions ; ppat catalyzes a reversible reaction . however , when coupled with pldh the reversible reaction is forced to produce 100% conversion . for example , the enzymatic reactions shown in fig . 1 are 100% conversion of the substrates . the activities of the enzymes were assayed every time just before usage for enzymatic conversion of vitamin b6 compounds into 4-pla . chicken white meat and liver , egg yolk and white , dried anchovy , carrot , and garlic were analyzed . the edible wet part of the foods was used , with the exception of the dried anchovy , which was ground with a mini blender ( osaka chemical co. , osaka , japan ) . foods ( 0.11.0 g , dry or wet ) were homogenized in 5ml of 0.1 m hcl with a polytron homogenizer , and then the homogenate was incubated at 100c for 30min to liberate protein - bound plp . the suspension was cooled on ice , then 0.1 ml of 50% ( w / v ) trichloroacetic acid ( tca ) was added , and followed by incubation at 100c for 5min . after cooling on ice , ph of the mixture was adjusted to 7.5 by the addition of 0.5ml of 0.5 m tris - hcl ( ph 7.5 ) and 0.57ml of 1.0 m naoh . the mixture was centrifuged at 8,000g for 5min at 4c , and then the supernatant ( 550l ) was used for the enzymatic reactions . samples that were added the seven vitamin b6 compounds were similarly treated to assess recovery . spiking level was 2 pmol ( the amount in 100l applied to the hplc column ) . recombinant pl 4-dehydrogenase ( pldh ) , pm - pyruvate aminotransferase ( ppat ) , and pn 4-oxidase ( pnox ) were prepared as described previously ( 1012 ) . recombinant e. coli alkaline phosphatase ( alp ) was prepared according to a standard protocol ( 13 ) . almond -glucosidase ( gls ) , pm , and pl ( 99.5% ) were purchased from sigma chemical co. ( st . louis , mo ) , pn and plp from nacalai tesque , inc . the pmp was a gift from daiichi fine chemicals co. ( takaoka , japan ) and png was prepared enzymatically from d - glucose and pn based on a transglucosidase activity of a bacterial -glucosidase ( 15 ) . food samples were bought at local markets and chosen at random in order to test and validate the method . the basic principle is to convert all vitamin b6 compounds specifically into a highly fluorescent vitamin b6-derivative 4-pla ( see fig . the filtration was done with a dismic 13 syringe filter ( pore size of 0.2 m , advantech , tokyo , japan ) . to make a control ( i.e. no - reaction test sample ) , hcl was added to the reaction mixture before addition of the food sample . for the determination of plp , the food sample was at first hydrolyzed with alp in the reaction 4 ( fig . 2 ) , and then the pl produced was determined as shown under the reaction for plp ( fig . 3 ) . both pmp and pnp were determined in the same way just like plp but using the additional enzymes and the substrate or the cofactor as shown under the reactions for pmp and pnp , respectively ( fig . first hydrolyzed with -glucosidase in the reaction mixture containing nah2po4 , which inhibited phosphatase activity in the almond -glucosidase preparation , and then pn was determined as shown under the reaction for png ( fig . horizontal arrows mean the addition of the solutions to the reaction mixtures . see table 1 for quantification of the seven compounds from the hplc elution profiles of the seven reactions described above . the 4-pla was measured by a reversed - phase isocratic hplc method using a cosmosil 5c18ms - ii column ( 2504.6 mm ; nacalai tesque , kyoto , japan ) , and a mobile phase consisting of 20 mm potassium phosphate buffer ( ph 7.0 ) and 10% ( v / v ) methanol . the separation was made with a jasco hplc system ( jasco , tokyo , japan ) equipped with an as-2055 autosampler , a pu-2080 pump , and an fp-920 fluorescence detector . the fluorescence intensity of the eluted 4-pyridoxolactone was monitored at 430 nm after excitation at 360 nm . the pldh ( 10 ) , pnox ( 12 ) , ppat ( 11 ) , alp ( 16 ) , and -glucosidase ( 17 ) were assayed as described previously . one unit of enzyme was defined as the amount that catalyzed the formation of 1mol of product per min . the pldh , pnox , alp , and -glucosidase catalyze irreversible reactions ; ppat catalyzes a reversible reaction . however , when coupled with pldh the reversible reaction is forced to produce 100% conversion . for example , the enzymatic reactions shown in fig . 1 are 100% conversion of the substrates . the activities of the enzymes were assayed every time just before usage for enzymatic conversion of vitamin b6 compounds into 4-pla . chicken white meat and liver , egg yolk and white , dried anchovy , carrot , and garlic were analyzed . the edible wet part of the foods was used , with the exception of the dried anchovy , which was ground with a mini blender ( osaka chemical co. , osaka , japan ) . foods ( 0.11.0 g , dry or wet ) were homogenized in 5ml of 0.1 m hcl with a polytron homogenizer , and then the homogenate was incubated at 100c for 30min to liberate protein - bound plp . the suspension was cooled on ice , then 0.1 ml of 50% ( w / v ) trichloroacetic acid ( tca ) was added , and followed by incubation at 100c for 5min . after cooling on ice , ph of the mixture was adjusted to 7.5 by the addition of 0.5ml of 0.5 m tris - hcl ( ph 7.5 ) and 0.57ml of 1.0 m naoh . the mixture was centrifuged at 8,000g for 5min at 4c , and then the supernatant ( 550l ) was used for the enzymatic reactions . samples that were added the seven vitamin b6 compounds were similarly treated to assess recovery . spiking level was 2 pmol ( the amount in 100l applied to the hplc column ) . the chromatograms for the analysis of pl , pm , and pn in the chicken liver sample are shown in fig . 4 : they showed similar but definitely different patterns of the fluorescent peaks of 4-pla . the 4-pla was eluted at about 11 min as shown with an arrow , together with unknown fluorescent compounds in the sample . the control reaction showed a tiny peak of 4-pla ( pl , no - r in fig . 4a ) , its area increased when the liver sample was subjected to the enzymatic conversion with the reaction 1 in fig . 2 for converting pl into 4-pla ( pl , r ) in the sample . thus , the subtraction of fluorescence intensity of 4-pla ( pl , no - r ) from that of 4-pla ( pl , r ) gave the amount of 4-pla corresponding to the amount of pl in the sample . the chicken liver sample spiked with pl showed a higher peak ( pl+sd , r ) because the corresponding amount of 4-pla was additionally produced by the enzymatic conversion . in contrast , the chicken liver sample spiked with pl without the enzymatic conversion did not show this increase ; its peak height was the same as that of the control reaction mixture ( pl+sd , no - r ) . thus , pl in the chicken liver was satisfactory determined as shown in table 1 . elution patterns of the two control reaction mixtures and reaction mixture spiked or nonspiked with pm are shown in fig . two controls ( pm , no - r , and pm+sd , no - r ) showed the same peak height as those of ( pl , no - r and pl+sd , no - r ) , respectively . in contrast , the peak height of the reaction mixture ( pm , r ) was higher than that of the reaction mixture ( pl , r ) because the former peak was a total of pl and pm . standard pm ( 2 pmol ) was quantitatively converted into 4-pla and determined ( pm+sd , r ) . 4c . like pl and pm , pn was also quantitatively converted into 4-pla and determined ( pn+sd , r ) . the peak area of the reaction mixture ( pn , r ) was almost the same as that of the reaction mixture ( pl , r ) , showing that the chicken liver sample contained a very low amount of pn . hplc chromatograms for the analyses of free forms of vitamin b6 in the chicken liver sample . the food sample ( 5l ) was subjected to the enzymatic conversion ( the procedure for pl in fig . 3 ) and applied to hplc ( pl , r ) . the food sample , to which standard pl had been added , was subjected to the enzymatic conversion ( pl+sd , r ) . their controls were also done ( pl , no - r , and pl+sd , no - r ) . ( b ) pm was similarly determined as shown in the procedure for pm in fig . ( c ) pn was similarly determined as shown in the procedure for pn in fig . 5a , b , and c shows the chromatograms for the analyses of plp , pmp , and pnp . the standard plp , pmp , and pnp spiked could be accurately determined ( plp+sd , r ; pmp+sd , r ; and pnp+sd , r ) . the peak area of pmp , r was high because it contained 4-pla produced from pl , pm , plp , and pmp . thus , pmp content was calculated as shown in table 1 . although the chromatograms obtained in the png analysis is not shown , hplc chromatograms for the analyses of phosphoester forms of vitamin b6 in the chicken liver sample . arrows show 4-pla peaks . ( a ) plp was determined . the food sample ( 5l ) 3 ) and applied to hplc ( pl , r ) . the food sample , to which standard plp had been added , was subjected to the enzymatic conversion ( plp+sd , r ) . their controls were also done ( plp , no - r , and plp+sd , no - r ) . ( b ) pmp was similarly determined as shown in the procedure for pmp in fig . ( c ) pnp was similarly determined as shown in the procedure for pnp in fig . the results showed that the method could be applied for the determination of contents of individual vitamin b6 compound in the chicken liver sample . the contents of seven vitamin b6 compounds in the foods were successfully determined ( table 2 ) . the values are in nmol / g , except for the total content that also is calculated as pn , hcl . although the recovery varied a lot among foods , the recovery tests could be determined for all the foods , showing that the present method for preparation of the food samples was almost adequate for the presented method : the standard deviation of the recovery for chicken liver , egg yolk and white , and dried small anchovy are shown in ( table 2 ) . the recovery of plp in the egg yolk and the dried anchovy was only 14 and 29% , respectively . these results indicate that plp in these foods bind so strongly to the other food component(s ) that alp can not 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 . another explanation could be that the egg yolk and dried anchovy contain some compounds that inhibit alp . although it is not clear what happens , the bioavailability of plp in the egg yolk and anchovy may be low , because it is well known that alp activity correlates significantly with in vivo luminal disappearance of plp ( 19 ) . this method may not be applicable for the foods that give a too low recovery of the vitamin b6 compounds . in such cases , it is necessary to develop an improved preparation method for the food samples . contents of vitamin b6 compounds and png in chicken liver and white meat , egg yolk and white , dried small anchovy , carrot , and garlic ; recoveries of the standards are also shown nd means = not detectable . total contents ( as pn - hcl ) of vitamin b6 compounds and png , which have been determined by a microbiological method , are described in the japanese food table . they are 35.5nmol / g for chicken white meat ; 38.4 , chicken liver ; 15.4 , egg yolk ; 0 , egg white ; 6.5 , carrot ; 6.5 , garlic ; and 16.6 , dried small anchovy . the values in nmol / g were converted from values given as mg/100 g of an edible part in the japanese food table . the microbiological and the present methods gave similar values for the contents in the chicken white meat and liver . in contrast , higher values were obtained for the other samples with the present method ; in particular , the small dried anchovy had about 9-fold higher value . because its content is in nmol / g , dry weight , in contrast to the other samples , the value obtained by the present method seems to be rational , suggesting that vitamin b6 compounds in the dried small anchovy could not be measured accurately by the microbiological method . the preferred comparison would be to measure the same food samples by the microbiological method and the present method . the relative contents of png were 19 and 42% of the total content in garlic and carrot , respectively . the png contents in plant - derived food have been reported to vary from 0% ( cauliflower ) ( 3 ) to 92% ( orange juice ) ( 20 ) . the contents of pn , which is generally used as a supplement of vitamin b6 for fortified foods , were very low in the foods examined ; actually pn was not found in either chicken white meat or egg yolk . although analytical data for many other foods are required , the results suggest a need for studies to determine which of the vitamin b6 compounds should be used for fortification of the food . each of the foods analyzed showed a characteristic pattern of the seven vitamin b6 compounds . the dried anchovy contained high amounts of plp , pmp , and pnp ; the free forms were very low . traditionally , the dried small anchovy has been used for making stock soup in japan . however , recently , a new preparation for taking the whole body as a food has been developed , which is the sample used in this study . because pmp should be absorbed as pm in the intestine after the hydrolysis by the alp , which shows reactivity toward pmp as well as plp , pmp should have the same efficacy as pm in the anti - diabetic complications . thus , the chicken liver and the dried small anchovy may be candidates for foods functioning to prevent diabetic complications . the time needed for determination of seven vitamin b6 compounds in one food by a trained student was approximately 10 days . although the first analysis was finished within 2 days , the additional days were required to determine recoveries of the recovery test . thus , the development of a determination kit consisting of the seven reaction mixtures and the seven standard compounds would be necessary to facilitate easier routine analysis of the food samples . the chromatograms for the analysis of pl , pm , and pn in the chicken liver sample are shown in fig . 4 : they showed similar but definitely different patterns of the fluorescent peaks of 4-pla . the 4-pla was eluted at about 11 min as shown with an arrow , together with unknown fluorescent compounds in the sample . the control reaction showed a tiny peak of 4-pla ( pl , no - r in fig . 4a ) , its area increased when the liver sample was subjected to the enzymatic conversion with the reaction 1 in fig . 2 for converting pl into 4-pla ( pl , r ) in the sample . thus , the subtraction of fluorescence intensity of 4-pla ( pl , no - r ) from that of 4-pla ( pl , r ) gave the amount of 4-pla corresponding to the amount of pl in the sample . the chicken liver sample spiked with pl showed a higher peak ( pl+sd , r ) because the corresponding amount of 4-pla was additionally produced by the enzymatic conversion . in contrast , the chicken liver sample spiked with pl without the enzymatic conversion did not show this increase ; its peak height was the same as that of the control reaction mixture ( pl+sd , no - r ) . thus , pl in the chicken liver was satisfactory determined as shown in table 1 . elution patterns of the two control reaction mixtures and reaction mixture spiked or nonspiked with pm are shown in fig . two controls ( pm , no - r , and pm+sd , no - r ) showed the same peak height as those of ( pl , no - r and pl+sd , no - r ) , respectively . in contrast , the peak height of the reaction mixture ( pm , r ) was higher than that of the reaction mixture ( pl , r ) because the former peak was a total of pl and pm . standard pm ( 2 pmol ) was quantitatively converted into 4-pla and determined ( pm+sd , r ) . 4c . like pl and pm , pn was also quantitatively converted into 4-pla and determined ( pn+sd , r ) . the peak area of the reaction mixture ( pn , r ) was almost the same as that of the reaction mixture ( pl , r ) , showing that the chicken liver sample contained a very low amount of pn . hplc chromatograms for the analyses of free forms of vitamin b6 in the chicken liver sample . the food sample ( 5l ) was subjected to the enzymatic conversion ( the procedure for pl in fig . 3 ) and applied to hplc ( pl , r ) . the food sample , to which standard pl had been added , was subjected to the enzymatic conversion ( pl+sd , r ) . their controls were also done ( pl , no - r , and pl+sd , no - r ) . ( b ) pm was similarly determined as shown in the procedure for pm in fig . ( c ) pn was similarly determined as shown in the procedure for pn in fig . 5a , b , and c shows the chromatograms for the analyses of plp , pmp , and pnp . the standard plp , pmp , and pnp spiked could be accurately determined ( plp+sd , r ; pmp+sd , r ; and pnp+sd , r ) . the peak area of pmp , r was high because it contained 4-pla produced from pl , pm , plp , and pmp . thus , pmp content was calculated as shown in table 1 . although the chromatograms obtained in the png analysis is not shown , hplc chromatograms for the analyses of phosphoester forms of vitamin b6 in the chicken liver sample . arrows show 4-pla peaks . ( a ) plp was determined . the food sample ( 5l ) 3 ) and applied to hplc ( pl , r ) . the food sample , to which standard plp had been added , was subjected to the enzymatic conversion ( plp+sd , r ) . their controls were also done ( plp , no - r , and plp+sd , no - r ) . ( b ) pmp was similarly determined as shown in the procedure for pmp in fig . ( c ) pnp was similarly determined as shown in the procedure for pnp in fig . the results showed that the method could be applied for the determination of contents of individual vitamin b6 compound in the chicken liver sample . the contents of seven vitamin b6 compounds in the foods were successfully determined ( table 2 ) . the values are in nmol / g , except for the total content that also is calculated as pn , hcl . although the recovery varied a lot among foods , the recovery tests could be determined for all the foods , showing that the present method for preparation of the food samples was almost adequate for the presented method : the standard deviation of the recovery for chicken liver , egg yolk and white , and dried small anchovy are shown in ( table 2 ) . the recovery of plp in the egg yolk and the dried anchovy was only 14 and 29% , respectively . these results indicate that plp in these foods bind so strongly to the other food component(s ) that alp can not 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 . another explanation could be that the egg yolk and dried anchovy contain some compounds that inhibit alp . although it is not clear what happens , the bioavailability of plp in the egg yolk and anchovy may be low , because it is well known that alp activity correlates significantly with in vivo luminal disappearance of plp ( 19 ) . this method may not be applicable for the foods that give a too low recovery of the vitamin b6 compounds . in such cases , it is necessary to develop an improved preparation method for the food samples . contents of vitamin b6 compounds and png in chicken liver and white meat , egg yolk and white , dried small anchovy , carrot , and garlic ; recoveries of the standards are also shown nd means = not detectable . total contents ( as pn - hcl ) of vitamin b6 compounds and png , which have been determined by a microbiological method , are described in the japanese food table . they are 35.5nmol / g for chicken white meat ; 38.4 , chicken liver ; 15.4 , egg yolk ; 0 , egg white ; 6.5 , carrot ; 6.5 , garlic ; and 16.6 , dried small anchovy . the values in nmol / g were converted from values given as mg/100 g of an edible part in the japanese food table . the microbiological and the present methods gave similar values for the contents in the chicken white meat and liver . in contrast , higher values were obtained for the other samples with the present method ; in particular , the small dried anchovy had about 9-fold higher value . because its content is in nmol / g , dry weight , in contrast to the other samples , the value obtained by the present method seems to be rational , suggesting that vitamin b6 compounds in the dried small anchovy could not be measured accurately by the microbiological method . the preferred comparison would be to measure the same food samples by the microbiological method and the present method . the relative contents of png were 19 and 42% of the total content in garlic and carrot , respectively . the png contents in plant - derived food have been reported to vary from 0% ( cauliflower ) ( 3 ) to 92% ( orange juice ) ( 20 ) . the contents of pn , which is generally used as a supplement of vitamin b6 for fortified foods , were very low in the foods examined ; actually pn was not found in either chicken white meat or egg yolk . although analytical data for many other foods are required , the results suggest a need for studies to determine which of the vitamin b6 compounds should be used for fortification of the food . each of the foods analyzed showed a characteristic pattern of the seven vitamin b6 compounds . the dried anchovy contained high amounts of plp , pmp , and pnp ; the free forms were very low . both the plp and pl were high in the garlic . traditionally , the dried small anchovy has been used for making stock soup in japan . however , recently , a new preparation for taking the whole body as a food has been developed , which is the sample used in this study . because pmp should be absorbed as pm in the intestine after the hydrolysis by the alp , which shows reactivity toward pmp as well as plp , pmp should have the same efficacy as pm in the anti - diabetic complications . thus , the chicken liver and the dried small anchovy may be candidates for foods functioning to prevent diabetic complications . the time needed for determination of seven vitamin b6 compounds in one food by a trained student was approximately 10 days . although the first analysis was finished within 2 days , the additional days were required to determine recoveries of the recovery test . thus , the development of a determination kit consisting of the seven reaction mixtures and the seven standard compounds would be necessary to facilitate easier routine analysis of the food samples . the content of pn that is used for fortification of foods was very low in the foods examined . this work was partially supported by the ministry of education , culture , sports , science and technonogy- japan , grant - in - ais for exploratory research , 18650215 , 2007 , and by a kochi university president 's discretionary grant .
backgroundthere is a need for a reliable and accurate method for quantification of each of the seven individual vitamin b6 compounds including pyridoxine--glucoside in foods.objectiveto determine pyridoxal ( pl ) , pyridoxamine ( pm ) , pyridoxine ( pn ) , pyridoxal 5-phosphate ( plp ) , pyridoxamine 5-phosphate ( pmp ) , pyridoxine 5-phosphate ( pnp ) , and pyridoxine--glucoside ( png ) in foods.designby specific enzymatic treatment , each of the seven vitamin b6 compounds was all converted into 4-pyridoxolactone , which is a highly fluorescent compound . in total , seven separate , enzymatic steps were performed for each sample . 
 separation and quantification were performed with reversed - phase high performance liquid chromatography ( hplc ) coupled with fluorescence detection . for each sample type 
 the result was corrected for the recovery based on spiked samples . 
 the method was applied for analyses of chicken liver , chicken white meat , egg yolk , egg white , dried anchovy , carrots , and garlic.resultsthe recovery varied from 14 to 114% in chicken liver , chicken white meat , egg yolk , egg white , dried anchovy , carrot , and garlic . 
 each food showed a characteristic distribution of the seven vitamin b6 compounds . 
 the png was only found in low amounts ; that is , 1729nmol vitamin b6/g in the plant - derived foods , carrot and garlic . 
 only egg white showed a lower content , 3nmol / g . 
 overall the content in chicken liver , chicken white meat , and egg yolk had a total content of vitamin b6 between 42 and 51nmol / g . 
 both pm and pmp were high in the chicken liver . 
 in contrast , pl and plp were high in the chicken white meat . 
 the main vitamin b6 in the egg yolk was plp . 
 the dried anchovy contained high amounts of plp and pmp and a total content of 144nmol / g.conclusionsthe enzymatic - based hplc method was applied for the determination of seven vitamin b6 compounds in foods . 
 their distribution in the foods varied significantly .