AN ABSTRACT OF THE THESIS OF. in Foods and Nutrition presented on February 25, This investigation consisted of two parts: A. stability, B.

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1 AN ABSTRACT OF THE THESIS OF ANNE DOLORAS PERERA fr the degree f Dctr f Philsphy in Fds and Nutritin presented n February 25, 1977 Title: VITAMIN B ENRICHMENT OF WHEAT FLOUR: STABILITY AND BIOAVAILABILITY Abstract apprved:,. T- _, «^u.j, (j (James E. Leklem) This investigatin cnsisted f tw parts: A. stability, B. biavailability f vitamin B/ in wheat. Three variables; whle wheat flur (WHW), white flur (W) and W enriched with vitamin B z (WB,) D D were tested. Stability f vitamin B, during bread making and strage f bread and flur was determined. Bread was prepared frm the three types f flur under cmmercial and hme cnditins. Tw methds, straight dugh and spnge dugh fr bread making were cmpared under hme cnditins. Vitamin B, cntent f dugh befre fermentatin, after prfing and f bread was determined by a micrbilgical methd. The WB, dugh prepared using the spnge dugh methd shwed a significant increase in the vitamin B, cntent during fermentatin (P < 0.05). A significant baking lss f 10-15% was bserved in the WHW and W breads prepared under

2 cmmercial cnditins (P < 0. 05), and f 5-12% in WHW, WB, (P < 0. 01) and W (P < 0. 05) breads made using the spnge dugh methd. Strage stability f vitamin B, was determined in the cmb mercially prepared WB, bread and all purpse flur enriched with b vitamin B,. There was n significant change in vitamin B, levels D in the bread stred under frzen and refrigerated cnditins fr seven and fur weeks, respectively. Hwever, a significant drp f 10% was bserved in the vitamin B, cntent f the bread after three days f strage at rm temperature (P < 0. 01). Vitamin B, cntent f the WB, flur did nt change when stred ver a perid f 2 weeks at rm temperature. Biavailability f vitamin B, was studied in nine men, age years. Each week ne f the three types f bread, WHW (570 g), WB. (00 g) and W (00 g) was fed daily t each subject using a 3X3 Latin square design. The WHW, WB, and W bread supplied 1.20, 1.18 and mg f vitamin B,, respectively, while mg was supplied by the cnstant diet. The daily vitamin B, intake was set at 1. 5 mg, f which apprximately 3/4 was supplied frm WHW r WB, bread. During the perid when W bread was cnsumed, the subjects als received an ral dse f mg f vitamin B, in rder t maintain a cnstant daily intake thrughut the study. fund t be pyridxine. The predminant frm f vitamin B, in the diets was

3 Twenty-fur hur urines, daily fecal cllectins and fasting bld samples three days per week were analyzed fr vitamin B, and D its metablites, using micrbilgical', chrmatgraphic and flur- metric techniques. The fecal vitamin B- level was significantly higher when WHW bread was fed as cmpared t when WB- r W bread was fed (P < 0.01). There was n significant difference in the urinary excretin f ttal and free vitamin B, in relatin t the type f bread. The predminant frm f vitamin B. in urine was fund t be pyridxal The urinary 4-pyridxic acid cntent was significantly lwer when the diet was based n WHW bread as cmpared t WB. r W bread (P < 0. 01). The percentage f the daily intake f vitamin B, accunted fr by the excretry prducts analyzed in this study was 91. when WHW bread was fed. The crrespnding percentages when WB. and W breads were fed were 81.5 and 79.8, respectively. The plasma vitamin B, and pyridxal phsphate levels were slightly lwer when WHW bread was cnsumed as cmpared t WB, r W bread. These data suggest that vitamin B, was nt as available frm WHW bread as frm WB, and W bread. The availability f vitamin B, frm WB, and W bread as determined in the present study was similar. Fr the ppulatins wh are dependent n refined wheat prducts, enrichment f flur with vitamin B, will be f advantage.

4 Hwever, enrichment f refined wheat prducts cannt replace cmpletely the benefits f cnsuming whle wheat prducts.

5 ANNE DOLORAS PERERA ALL RIGHTS RESERVED

6 Vitamin B/ Enrichment f Wheat Flur: Stability and Biavailability by Anne Dlras Perera A THESIS submitted t Oregn State University in partial fulfillment f the requirements fr the degree f Dctr f Philsphy June 1977

7 APPROVED: Assistant (Prfessr f Fds and Nutritin Head f Depayrtiment f Fds and Nutritin Dean f Graduate Schl tr Date thesis is presented February 25, 1977 Typed by A & S Bkkeeping/Typing fr Anne Dlras Perera

8 TO MY MOTHER

9 ACKNOWLEDGEMENTS I wish t express my sincere gratitude t Dr. James E. Leklem, my majr prfessr, fr his excellent guidance, cnstant encurage- ment and supprt thrughut the research. His valuable time spent and suggestins made during the preparatin f this thesis are gratefully appreciated. My thanks are als extended t Dr. Lrraine T. Miller fr her help and suggestins during the research. I wuld like t thank Mrs. Margaret Edwards and Mrs. Eva Bensn fr their kind assistance and cperatin in ding the labratry wrk. The help f Terry Shultz is als appreciated. I wish t extend my appreciatin t the nine yung men wh served as subjects in the present study. Diane Peffers and Esther Shen are especially thanked fr sharing the wrk lad f the human study. Mrs. Wilda Retter is especially thanked fr her kind assistance in the typing f the initial draft f the manuscript. A very special wrd f appreciatin ges t Dr. Margy J. Wdburn, Head f the Department f Fds and Nutritin, fr her unceasing supprt and encuragement frm the admissin t the cmpletin f my studies at O. S. U.

10 My sincere thanks are als extended t Miss Helen Charley (Prfessr Emeritus, Fds and Nutritin), my ex-majr prfessr, fr her cntributin t my prgress. I wish t express my gratitude t the American Hme Ecnmics Assciatin fr awarding me their Special Internatinal Award. Withut such supprt my educatin in the United States wuld nt be pssible. I als thank the ther rganizatins and the individuals wh thrugh financial supprt enabled me t cmplete my studies at O. S.U. My appreciatins are als extended t thse faculty members in the Schl f Hme Ecnmics, my fellw graduate students in the Department f Fds and Nutritin and my friends in the cmmunity wh cntributed in varius ways tward my happiness. I wish t ffer my sincere gratitude t my belved husband, Cnrad. His lve, encuragement, supprt and patience made my wrk mre meaningful and wrthwhile. Finally I thank every persn wh supprted me with prayer, especially during my trials. Abve all, I thank Gd fr being my surce f strength and guidance at all times.

11 TABLE OF CONTENTS Page INTRODUCTION 1 REVIEW OF LITERATURE 4 Imprtance f Vitamin B/ 4 Metablism f Vitamin B/ Human Requirements fr Vitamin B/ 11 Manifestatins f Vitamin B^ Deficiency 15 Excretin f Vitamin B, 21 Studies n Biavailability f Vitamin B/ and Other Nutrients 29 Vitamin B/ in Fds 33 Cereal Enrichment Prgram 37 Stability f Vitamin B/ and Other Nutrients during Fd Prcessing 39 MATERIALS AND METHODS 47 Stability 47 Preparatin f Breads 47 Strage f Vitamin B/ Enriched Bread 51 Strage f Vitamin B, Enriched Flur 51 Biavailability 52 Subjects 52 Experimental Design 53 Diet 54 Metablic Study 58 Analytical Methds 0 Analysis f the Diet fr Vitamin B, 0 FecaL Vitamin B. 1 Urinary Vitamin B, 2 Urinary 4-Pyridxic Acid 3 Statistical Analysis 4 RESULTS AND DISCUSSION 5 Stability 5 Stability f Vitamin B^ During Bread Making 5 Stability f Added Vitamin B/ During Bread Strage 70 Stability f Added Vitamin B, During Flur Strage 73

12 Page Biavailability Subject Respnse t the Diets Dietary Intake f Vitamin B/ Fecal Excretin f Vitamin B^ Urinary Excretin f Vitamin B^ Urinary Excretin f 4-Pyridxic Acid Vitamin B, Balance RECOMMENDATIONS SUMMARY BIBLIOGRAPHY APPENDIX 1. Cmmercial Bread Making Hme Baking Methds Infrmed Cnsent Frm Diet and the Cmpsitin List f Abbreviatins 158. Data frm ANOVA Tables 159

13 LIST OF TABLES Table Page 1. Estimated recmmended dietary allwances f vitamin B. fr varius age grups Enrichment levels fr cereal prducts Vital statistics f the subjects Cnstant diet with partial cmpsitin Vitamin B, cntent f mix, prf and bread frm WHW, WB^ and W flur, prepared under cmmercial and hme cnditins,.. Vitamin B. cntent f cmmercial WB, bread stred under three different cnditins Vitamin B, cntent f WB, flur stred under three different cnditins Vitamin B/ f flur stred under different cnditins expressed as a percentage f the C cntrl flur analyzed at the same time Bdy weights and extra calries cnsumer per day Ttal vitamin B, level in the different cmpnents f a day^s diet Levels f the three frms f vitamin B, in cmpnents f the diet Assayed and calculated percentages f PAL, PIN and PAM f vitamin B, f the diet cmpnents Vitamin B, cntent and weight f feces in subjects fed diets based n WHW, WB, and W breads. 8

14 Table Page 14. Vitamin B, cntent and weight f feces during the experimental perids Urinary excretin f ttal vitamin B, Urinary excretin f free vitamin B Urinary vitamin B, expressed n different basis Pyridxal (PAL), pyridxine (PIN) and pyridxamine (PAM) in the urines f five subjects Urinary pyridxal (PAL), pyridxine (PIN) and pyridxamine (PAM) as a percentage f the ttal vitamin B, Urinary 4-pyridxic acid Urinary 4-pyridxic acid expressed as a percentage f the basal level Urinary 4-Pyridxic acid excreted during the'three experimental perids, expressed as percentage f vitamin B, intake Intake, excretin and balance f vitamin B, during the three experimental perids. 117

15 LIST OF FIGURES Figure Page 1. Metablic intercnversins f vitamin B, and the frmatin f 4-pyridxic acid Prpsed pathway fr the cnversin f pyridxine t pyridxal in bld Latin square design fr three breads: whle wheat, white enriched with vitamin B, and white ver three experimental perids fr nine subjects Graphic presentatin f mean vitamin B, levels in mix, prf and bread Three frms f vitamin B, in the diet cmpnents. 84. Scatter diagram f fecal weight against fecal vitamin B,. 88a 7. Urinary excretin f PAL, PIN, and PAM as a percentage f ttal intake f vitamin B b 8. Mean urinary excretin f 4-PA versus bdy weight during the experimental perids. 115a 9. Daily intake, excretin and balance f vitamin B,. 120

16 VITAMIN B, ENRICHMENT OF WHEAT FLOUR: STABILITY AND BIOAVAILABILITY INTRODUCTION Wheat prducts cmprise abut 30 percent f the cereals cn- sumed by the ppulatins arund the wrld (1). Almst 19 percent f the calries and 20 percent f the prteins in the average wrld daily per capita intake cme frm wheat (2). Whle grain wheat is a rich surce f B cmplex vitamins including B, (3, 4, 5). Cncen- tratin f the vitamins is highest in the aleurne layer and germ fractin. Abut 85 percent f vitamin B, is lst when wheat is cnverted t refined flur (). White bread, which is lwer in vitamin B. (40 (j-g/loog) than whle wheat bread (180 (ig/loog), is cnsumed quite extensively (3). With the imprtance f bread in the diet f all peple, it becmes imprtant t cnsider the lss f vitamin B, as well as ther nutrients during milling. Fllwing the discvery f certain vitamin deficiency diseased in the United States, effrts were initiated t meet the nutritinal needs f the peple (7). Early attempts t use vitamin supplements were unsuccessful as were thse t pursuade peple t use whle grain prducts. Frm this experience, the cncept f enrichment prgrams was develped in the United States during the 1930s and

17 early 1940s. The first step tward this was the mandatry enrich- ment f white flur with thiamin, ribflavin, niacin and irn which came int effect frm January 1, 1942 (8). Accrding t the Fd and Nutritin Bard f the Natinal Academy f Sciences, there seems t be evidence f ptential risk f deficiency f vitamin A, vitamin B., flacin, irn, calcium, magnesium, zinc, thiamin, ribflavin and niacin amng sme grups f the ppulatin (9). Because f this, the Natinal Academy f Sciences in 1974 prpsed the additin f vitamin A, vitamin B, flic acid, magnesium and zinc alng with an increase in the amunt f the ther nutrients added t the cereal prducts. The level f enrichment recmmended fr vitamin B. was 2 mg (as pyridxine) per pund f refined flur. Befre any enrichment prgram is adpted, it becmes impr- tant t test the stability f the nutrient under custmary cnditins f strage and use. Hwever stable a nutrient may be, the nutri- tinal value f a fd is gverned in part by its biavailability in a given fd prduct. The availability f vitamin B, frm fds may b be influenced by a number f factrs, ne f which is digestibility f prteins (10). Prteins f plant rigin are knwn t be digested less cmpletely than thse f animal rigin. Hwever, in a study n wheat flur as a surce f prtein fr adult human subjects, Blurchi, Friedmann and Mickelsen (11) reprted that the digesti- bility f the prtein in a diet cntaining animal prducts and ne

18 cntaining wheat were almst the same, as evidenced by fecal nitrgen excretin. Vitamin B, in nature exists mstly as cenzymes bund t prteins and the difference in digestibility may lead t a lwer bi- availability f the vitamin frm certain fds f plant rigin cm- pared t thse f animal rigin. Justificatin fr assessing the biavailability f vitamin B, frm a cereal surce is based n the dependency f a significant prprtin f the wrld ppulatin n these fds fr their nutrients, the current trend t replace animal prducts with plant surces because f fd shrtages (12), the high csts f fd and the increasing number f vegetarians (13). The infrmatin btained frm this study can be used t make recmmendatins n the enrichment f wheat flur and bread with vitamin B,. Such data may als be utilized t arrive at a mre accurate recmmended dietary allwance (RDA) fr this nutrient, since the present level f 2 mg fr an adult is an estimatin based n results btained frm studies in which the crystalline frm f vitamin B. was given t crrect bichemical disrders in subjects depleted f the vitamin (14).

19 REVIEW OF LITERATURE Imprtance f Vitamin B^, Vitamin B,, as a distinct member f the B cmplex grup, was first recgnized by Gyttrgy as early as 1934 (15). Since that time, numerus metablic functins fr vitamin B, have been identified (1). Hwever, the mechanisms f these are nt all well established. Vitamin B, has been referred t by sme authrs as the "sleeping giant f nutritin" (17). The majr frm in which vitamin B, functins is as a cenzyme, pyridxal-5-phsphate (PLP). It acts in a wide spectrum f bi- chemical reactins related primarily t the metablism f amin acids (18). Sme f the reactins catalyzed by these enzymes invlving amin acids include transaminatin, racemizatin, decarbxylatin, dehydratin and desulfhydratin (19). The transaminases represent a majr grup f PLP catalyzed enzymes which are respnsible fr the transfer f the a-amin grup f the amin acids t ket acids (20). After transaminatin the metablites may be cnverted t fats, carbhydrates r ther amin acids. Pyridxal phsphate dependent decarbxylatin f tyrsine, histidine, dihydrxy-phenylalanine (DOPA) and 5-hydrxy-tryptphan results in the frmatin f amines f bilgical imprtance in human

20 bichemistry. Fr example, histamine and 5-hydrxy-tryptamine r sertnin are required fr the functinal activities f nervus tissue. Pyridxal phsphate is als imprtant as the cenzyme fr -amin levulinic acid synthetase which decarbxylates -amin-p- ketadipic acid t -amin-levulinic acid. The latter cmpund is an intermediate in the frmatin f prphyrin which is required fr hemglbin synthesis (21). Pyridxal-5-phsphate is an essential cnstituent in glycgen phsphrylase which catalyzes the breakdwn f glycgen t glucse- 1-phsphate (22). It has been reprted that PLP plays a rle in lipid metablism, especially in the cnversin f linleic acid t the mre unsaturated arachidnic acid (23, 24). A recent study has shwn that arachidnic acid levels in liver decreased in rats made pyridxine deficient (25). The reasn fr this decrease was reprted t be the degradatin f arachidnic acid rather than lwering f the cnversin f linleate t arachidnate. Vitamin B. als appears t play an imprtant rle in the maintenance and functining f the immune system, prbably thrugh its influence n nucleic acid systhesis (2, 27). The effect f vitamin B, deficiency n immune respnses was discussed in a recent review (28). Hwever, the mechanisms invlved in the sup- pressive effect f the vitamin B, deficiency n the immune system are nt yet established.

21 A relatinship between the vitamin B, dependent metablism D f tryptphan and the ccurrence f tumrs was shwn by Yshida, Brwn and Bryan (29). Their data suggested that tryptphan metab- lites may play a rle in recurrence f bladder tumrs in patients with abnrmal tryptphan metablism. The reasn fr this has been speculated t be due t either the prducts f tryptphan having sme direct carcingenic actin r pr vitamin B- status, which leads t abnrmal tryptphan metablism, resulting in the develpment f tumrs, prbably thrugh the influence f lw vitamin B / n the b immune respnse (30). Althugh the exact mechanisms f vitamin B, metablism are b nt clearly established, attempts have been made t study the prbable pathways in animal tissues (31, 32). Metablism f Vitamin BA Vitamin B. is widely distributed in fds, bth as the free and phsphrylated frms (5). The phsphrylated frms f the vitamin frm the ingested fd are prbably hydrlyzed in the in- testine by phsphatases (33, 34). In the free state, vitamin B, is absrbed in the upper intestinal tract. Bth and Brain (31) bserved that there was virtually n absrptin f vitamin B, thrugh the stmach wall. A greater part f the pyridxine that was adminis- tered rally t rats was absrbed in the jejunum, and a small

22 amunt in the ileum. Their data supprted the cncept that absr- ptin f pyridxine was by passive diffusin rather than active transprt. Vitamin B, exists as a grup f clsely related cmpunds which include pyridxine (PIN), pyridxal (PAL) and pyridxamine (PAM) (35). These three frms, which are intercnvertible, can be transfrmed in viv t their phsphrylated frms (3), as shwn in Figure 1. The xidatin f PAL by aldehyde xidase results in the direct frmatin f 4-pyridxic acid (4-PA), the majr urinary metablite f vitamin B, (37). A review n the intercnversins f vitamin B, in mammalian tissues has been presented by McCy and Clmbini (32). Ingested PIN was reprted t be either phsphrylated t pyridxine phsphate (PNP) by the actin f pyridxal phsphkinase and adensine triphsphate (ATP) r xidized t PAL by pyridxine xidase. Pyridxal is phsphrylated directly t PLP by pyridxal phsph- kinase. Pyridxine phsphate can als be xidized t PLP by pyridxine phsphate xidase (39)«Brain, liver and kidney were reprted t have cntained ne f the highest cncentratins f pyridxal phsphkinase while muscle had very lw cncentratins (40). This suggests that nn-phsphrylated frms f vitamin B, are cnverted int phsphates mainly in the brain, liver and kidney (41).

23 COOH CH 2 OH 4-Pyridxic Acid HO CH. CH OH x v V «CH^OH 2 CH 2 OH I Pyridxine-5- phsphate Pyridxine Oxidase (NADP) "^ CH 2 OP03H 2 Pyridxine Phsphate xidase (FMN) CHO 0) J3 M J3 I s CHO CH 2 OH Pyridxine Trans aminase JVridxal phsphate Oxidase (FMN) CH CH 2 OP03H 2 JPyridxine Phsphate A. N^ "^ xidase (FMN) Pyridxal-5- phsphate CH 2 NH 2 HO Y'NTCH.OH '3 N CH^NH 2 HO V^V CH 2 OP0 3 H 2 CH, x 43 a (A LX ^N Pyridxamine-5- phsphate Figure 1. Metablic intercnversins f vitamin B fi and the frmatin f 4-pyridxic acid. see Appendix 5. (Adapted frm 3 and 38). ATP, FMN, NADP 00

24 9 The pssible rle f circulating erythrcytes in these inter- cnversins was suggested by Hamfelt (42), and Yamada and Tsuji (43). Andersn et al. (44) prpsed a pathway fr the cnversin f pyridxine t the active frm f vitamin B, in the red cells (Figure 2). These bservatins suggest that PIN is cnverted t PLP in red cells by a pathway previusly demnstrated (41) t ccur in ther tissues, and that PLP is cnverted t PAL, which then enters the plasma frm the red bld cell. Pyridxal was reprted t be the majr transprt frm f the vitamin (44, 45). Hwever, it still remains t be determined t what extent PAL in bld plasma enters ther tissue cells fr cnversin t the active cenzyme, r the extent t which it may be cnverted t 4-PA. Schwartz and Kjeldgaard (37) reprted that PAL culd be xidized in the presence f liver aldehyde xidase t 4-PA. Several investigatrs have shwn that 4-PA was the predminant metablite f vitamin B, in urine when the vitamin was made available either thrugh fd r as supplements f PAL, PIN and PAM (4, 47, 48). Urinary excretin f 4-PA generally accunts fr abut 30-50% f the vitamin B. ingested (49). The exact site and mde f cnversin f PAL t 4-PA have nt been fully established. Cntractr and Shane identified 4- pyridxic acid-5- phsphate (4-PAP) in urine and tissues f rats (50). This cmpund was fund in rat tissues fllwing an

25 14 intra-peritneal injectin f C- tagged PIN. The same authrs bserved a buildup f 4-PAP and sme 4-PA after the appearance 10 f PLP in liver, kidney and brain f these rats (51). that 4-PA was mre prevalent in bld than 4-PAP. They bserved These findings led them t prpse the fllwing pathway fr the catablism f PLP in animal tissue: PLP 4-PAP 4-PA. PYRIDOXINE (enters red cells) Pyridxal Kinase i PYRIDOXINE PHOSPHATE xidase PYRIDOXAL PHOSPHATE Phspjaatase 1 PYRIDOXAL (frm red cells t plasma) Figure 2. Prpsed pathway fr the cnversin f pyridxine t pyridxal in bld (44).

26 Urinary 4-PA has been determined In a few investigatins as 11 a measure f the metablism f vitamin B,. Wachstein suggested that the cncentratin f this metablite in urine culd be used as a measure f the quantity f vitamin B. metablized in the bdy (52). Bxer, Pruss and Gdhart reprted that the human bdy can apparently cnvert 4-7 mg f vitamin B, per day int PLP (53). b Higher intakes f the vitamin did nt cause any further increase in the PLP cntent in the bld. Accrding t Baker ejt al., the vitamin B, cntent stred in the liver was 5-20 (J-g/g, in muscle 2- \ig/g and D in brain \ig/g (54). The amunt f vitamin B, in the whle bdy has been estimated at mg (55). Abut half f the PLP in the bdy appeared t be bund t a-glycgen phsphrylase in the muscles (22). The daily turn ver f vitamin B, was reprted t be % with a 2-3% depletin f the bdy's reserves (55). Human Requirements fr Vitamin B^ Althugh several attempts have been made t determine the vitamin B. requirements f man, data n this are still meager (5). b Estimates f the state f vitamin B, nutriture in man have been based n the prductin r cure f clinical signs f the vitamin deficiency. The recmmended dietary allwances f vitamin B. f nrmal peple f varius ages are shwn in Table 1.

27 12 Table 1. Estimated recmmended dietary allwances f vitamin B^ fr varius age grups (mg/day). Individuals Age Grup (yr) Amunt Infants Children Adlescents } 2.0 Adults 19 Upward is Surce (14) There has been much cncern ver the pssible increase f vitamin B/ requirement f wmen during pregnancy, particularly during the last trimester (57). It has been bserved that mst pregnant wmen have decreased levels f vitamin B. in bld and urine (58), lwer bld transaminase activities (59) and increased levels f tryptphan metablites in urine, particularly fllwing a trypfcphan lad test (0). The placenta seems t actively transprt vitamin B, t the fetus t maintain a five-fld cncentratin gradient favuring the fetus (1). Because f this, there is a great tendency t develp deficiency f vitamin B, during pregnancy. Studies n the require- ment f vitamin B. during pregnancy have been cvered in a recent

28 review (2). The RDA set by the Natinal Academy f Sciences (14) 13 fr pregnant and lactating wmen is 2. 5 mg/day. In a majrity f wmen studied, daily ral dses f 5-10 mg f vitamin B, crrected the bichemical signs f deficiency f this vitamin (57). The benefits derived frm the higher levels f intake are still nt fully understd and require further study. Increased levels f tryptphan metablites in urine have als been bserved in ral cntraceptive users (3). Hwever, the ther indices f vitamin B. status, namely, bld transaminase activities, plasma PLP, bld and urinary vitamin B. levels and urinary 4-PA f the ral cntraceptive users were nt significantly different frm the cntrl grup, the nn-users f ral cntraceptives. This suggests that there may nt be an extra demand fr vitamin B, in wmen using ral cntraceptives. The establishment f the RDA fr vitamin B, is cmplicated D by the fact that the requirement appears t be increased when high prtein diets are cnsumed (4, 5). A daily intake f 1.25 mg f vitamin B, has been fund t be adequate fr healthy male subjects n a lw prtein diet prviding 30 g prtein per day. On a high prtein intake f 100 g daily, 1.5 mg f vitamin B, appeared t be the daily minimum while 1.75 t 2 mg was cnsidered ptimum (). The relatinship between vitamin B. and prtein seems t be critical even at the stage f infancy. Accrding t Filer and

29 14 Martinez (7^ metablic requirements fr vitamin B/ culd be satisfied at mnths f age if the vitamin is present in amunts f 20 ug/g f dietary prtein. The vitamin B, requirement f lder infants and yung children has been estimated t be 0. 5 t 1. 0 mg/ day (14). Infrmatin n the vitamin B, requirement f infants, children D and adlescents is limited. Hwever, there has been sme evidence t shw that requirement increases with age (8). Hamfelt fund that plasma pyridxal phsphate (PLP) levels decreased with increasing age. It is pssible that lder persns have a reduced absrptive capacity fr vitamin B. at physilgical levels. The nrmal newbrn infant has sufficient tissue stres f vitamin B. t meet his needs during the nenatal perid (1). The cncentratin f vitamin B. in human milk is apprximately fig/liter during the first mnth f lactatin and gradually increases t 100 j.g (5). The daily requirement f vitamin B, f infants can be met by adequate b cnsumptin f human milk. Clinical abnrmalities have appeared in infants when the vitamin B, cntent failed t rise t a level f at b least 0-80 jig/liter f human milk (1). In develping cuntries where marginal r deficient vitamin B, intakes are cmmn, children may nt develp clinical signs f deficiency (9). This is because the amunt f prtein in the diet is relatively lw and vitamin B, requiring enzymes are usually

30 15 adapted t a lwer level f activity. Hwever, lng term deficiency f vitamin B, may lead t detrimental effects n physical and mental develpment. Data available t make an accurate estimatin f vitamin B, requirements f lder children and adlescents are insufficient. Hwever, the RDA fr this grup has been set in the range f 1.5 t 2 mg/day (70). Althugh the vitamin B, requirement culd usually be met by an adequate diet (5), there may be sme individuals wh might manifest clinical and bichemical signs f deficiency. This is due t an increased vitamin B, requirement r t a disturbance f the vitamin B, metablism in these individuals. D Manifestatins f Vitamin B/ Deficiency Symptms f vitamin B, deficiency vary greatly with the species and age f the individual (35, 71). A variety f symptms have been bserved in bth animals and in humans. Dermatitis r acrdynia is the characteristic sign f a vitamin B- deficiency in the rat. In additin t this, pr grwth, muscular weakness, fatty livers, cnvulsive seizures, anemia and nerve degeneratin are amng the cmmn deficiency symptms. The first evidence f vitamin B. deficiency in humans was reprted by Spier, Bean and Ashe (72). Patients n pr diets wh were suffering frm weakness, irritability, nervusness, depressin

31 and difficulty in walking were relieved f these symptms within 24 1 hurs f administratin f 50 mg f vitamin B,. Dietary deprivatin f this vitamin in infants results in cnvulsive seizures, nervus irritability and mental retardatin (7 3). This was evident in apprxi- mately 300 infants wh received an autclaved cmmercial liquid milk frmula lw in vitamin B, (7 4, 75). Additin f vitamin B, t the frmula reversed sme f the abve symptms. Anther study has shwn that infants placed n a vitamin B, deficient diet fr several mnths ceased t gain weight (7). B, and 4-PA were reduced t very lw levels. Their urinary vitamin Symptns f vitamin B, deficiency in man have been btained with human adults placed n diets lw in this vitamin (71). Abnr- malities f the tryptphan t niacin pathway as indicated by the excretin f xanthurenic acid have been bserved in these subjects. Abnrmally high excretin f xanthurenic acid and related metablites f tryptphan were als fund in pregnant wmen (52), as well as in wmen using ral cntraceptives (3). Bichemically, vitamin B, deficiency is recgnizable by the increased excretin f xanthurenic acid and ther tryptphan metablites in urine, especially fllwing an ral dse f tryptphan (77, 78). This is referred t as the tryptphan lad test, which has been a very useful prcedure fr detecting r in evaluating vitamin B, deficiency, particularly in clinical cases and in cntrlled research studies (7 9).

32 17 Miller and Linkswiler studied the effect f prtein intake n the develpment f abnrmal tryptphan metablism by men during vitamin B, depletin (4). After 40 days f depletin, men given 54 5 g f prtein and 0. 1 mg f vitamin B, excreted 0-29% f the 2 g lad f L-tryptphan as the metablites. In cntrast, men given 150 g f prtein and 0. 1 mg f vitamin B, excreted 29% f the D tryptphan lad as the metablites after 14 days f depletin. ever, with a daily vitamin B, intake f 1. 5 mg the excretin f Hw- tryptphan metablites was nrmal whether prtein intake was 54 r 150 g. Canham et al. studied the rapidity f nset and the severity f the bichemical manifestatins f vitamin B, deficiency with differing prtein intake. They fund a direct relatinship between these tw cnditins (5). The assciatin f vitamin B, deficiency with a frm f micr- cytic hypchrmic anemia has been reprted (8 0). A case study n pyridxine-respnsive anemia was reprted by Hrrigan (81). In a man, 54 years ld, anemia which was fllwed fr 18 years had been unaffected by flic acid, vitamin B, multivitamin preparatins cntaining niacin, ribflavih and thiamin; but respnded t 12.5 mg f pyridxine hydrchlride (PIN-HCl) given by muth daily fr 5 days. Keyhani t al. studied erythrpeisis in pyridxine-deficient mice and bserved that the mice deprived f vitamin B, develped prgressive anemia characterized by hypchrmia, micrcytsis,

33 18 reticulcytsis and erythrid hyperplasia f bne marrw and spleen (82). The effect f pyridxine deficiency n sme aspects f carbhydrate metablism in rats was investigated by Angel and Mellr (83). Vitamin B, deprivatin decreased liver and muscle glycgen cntent and the activity f glycgen phsphrylase in bth tissues. They cncluded that alteratin f amin acid metablism due t vitamin B, deficiency may have limited the supply f carbn fr glucnegenesis, thereby restricting the availability f glucse fr glycgenesis. Alng the same line, Angel and Sng studied the effect f pyridxine deficiency n lipgenesis, and shwed that there was decreased availability f glucse fr lipgenesis in vitamin B, deprived rats (84). in lipid metablism. As mentined earlier, vitamin B, plays a rle The effect f vitamin B. n the fatty acid cm- psitin f liver (25) and phsphlipids (85) has been studied. DeLrme and Lupien bserved a decrease in the prprtin f arachidnic acid and an increase in the linleic acid in the majr phsphlipids in the rats deprived f vitamin B, (85). A similar trend was bserved by Dussault and Legape in the liver fatty acids (25). The exact rle vitamin B, plays in these reactins is nt clear. Vitamin B, deficiency seems t be invlved in a number f physilgic cnditins. The frmatin f renal calculi (8) and

34 19 increased tendency fr dental caries (8 7) are tw examples,, Calhun, Jennings and Bradley reprted that rats receiving a vitamin B, deficient diet and a sulfa drug (phthalylsulfathiazle) exhibited hematuria assciated with abnrmally high xalate excretin via kidney (88). Pyridxine administratin prevented bth cnditins and it was suggested that xalate excretin in urine may be the result f the imprper utilizatin f the amin acid, glycine. The first human study n this aspect f vitamin B, was dne by Gershff, Mayer and Kulzycki in mnglid and nn-mnglid children (8 9). They bserved a marked reductin in urinary excretin f xalic acid when pyridxine was given. Supplementary vitamin B, has been fund t reduce dental caries in experimental animals and in humans (90). Strean suggested that a deficiency f vitamin B, in the diet might render teeth mre susceptible t dental caries and additin f this vitamin t the diet culd increase resistance prbably thrugh a change in ral flra (91). He reprted that it may be linked t the essentiality f vitainin B. as a nutritinal requirement fr heterfermentative bacteria and the nn-essentiality f this vitamin fr the hmfermentative type. Inbrn errrs f metablism resulting in vitamin B. dependency r change in the metablism f this vitamin have been studied (92). The symptms f vitamin B, dependency are limited t the central nervus system and include hyperirritability, cnvulsins and

35 20 electrencephalgraphic changes (93'). It has been prpsed that an inbrn r acquired abnrmality changes the binding capacity f the specific vitamin B, dependent enzymes t the cfactr. Symptms f vitamin B. dependency have been cntrlled by administratin f large dses f pyridxine ver and abve the requirements f the individual in rder t vercme the adverse binding kinetics. There are a number f cmpunds which are structural analgs f vitamin B, grup members which functin bichemically as antagnists interfering with nrmal functins f the vitamin and resulting in manifestatins f vitamin B, deficiency (94). Sme cmmnly used drugs are knwn t prmte vitamin B. deficiency thrugh inhibitin f cenzyme synthesis r by chemical inactivatin f PLP, r by cmpetitive displacement f PLP by the structural analgs (95). Amng these drugs are isniazid, D-penicillamine and dexypyridxine. Isnictinic acid hydrazide (INH) r isniazid is a cmmn therapeutic agent used in tuberculsis therapy (9). Dexypyridxine, nce used in the treatment f cancer, cmpetes with PLP by displacing the cenzyme in a number f different enzymes (94). D-penicillamine is used in pathlgical cnditins such as Wilsn's disease, cystinuria, certain types f heavy metal intxicatin and rheumatid arthritis (97). Levy bserved increased excretin f micrbilgically active frms f vitamin B, during administratin f high levels f vitamin B, antagnists (98).

36 21 Excretin f Vitamin B^ Evaluatin f vitamin excretin in relatin t intake has been used as an apprach t the assessment f requirements fr B vitamins (99). Such balance studies are als valuable in assessing availability f nutrients (100). Earlier studies n vitamin B. metablism in human subjects were reprted by Denk ej: al. (101); Jhnsn, Hamiltn and Mitchell (102); Rabinwitz and Snell (47). Hwever, a cmplete balance study f vitamin B. and its knwn metablites n human subjects was first undertaken by Linkswiler and Reynlds (48). Three levels f vitamin B, intake were evaluated D in terms f the sum f fecal and urinary B, excretin as well as excretin f urinary 4-PA, the majr urinary metablite f vitamin B,. They used nine human subjects and bserved that vitamin B. eliminatin exceeded the amunt given in the diet. The prbable reasn fr this accrding t these authrs was a synthesis f vitamin B, by intestinal flra, With a basal diet cntaining mg f vitamin B. per day, subjects studied by Linkswiler and Reynlds eliminated a ttal f 3.54 mg equivalent f the vitamin, f which abut 75% was accunted fr by 4-PA (48). The sum f fecal and urinary vitamin B, excreted was almst equal t r slightly in excess f the amunt f vitamin B, ingested in the basal diet. The results f Denk et al. shwed a

37 22 smewhat different trend (101). They bserved an average daily excretin f mg as urinary and fecal vitamin B, n a diet cn- taining 1. 7 mg f vitamin B, per day. D Vitamin B, in urine and feces accunted fr nly 57% f the intake. Yan and Fujita studied the synthesis f vitamin B, by intestinal bacteria as affected by cellulse (103). They used three types f diet, vegetable, meat and mixed. Filter paper served as the surce f cellulse. They bserved that abut 90% f the sum f urinary vitamin B, and 4-PA was made up f 4-PA. Because f this, they measured nly 4-PA as the urinary excretry prduce f vitamin B,. They bserved an increase in the excretin f fecal and urinary vitamin B, when the subjects were fed a vegetable diet. On the basis f amunt excreted in relatin t the intake, they cncluded that vitamin B, synthesized in the intestine increased markedly with the vegetable diet as cmpared t the meat diet in which a decrease in the abve parameters was bserved. With the additin f cellulse t either diet, there was an increase in the ttal vitamin B, excreted. Their findings supprt the idea that additin f cellulse t a diet increases the synthesis f vitamin B, by intestinal micrflra. They als reprted that the synthesized vitamin B. was absrbed int the system, as reflected by the increased excretin f 4-PA in the urine.

38 23 The methds f 4-PA determinatin used by Linkswiler and Reynlds (48) and Yan and Fujita (103) were thse f Huff and Perlzweig (4) and Fujita and Fujin (104), respectively. Reddy, Reynlds and Price (105) and Wdring, Fisher and Strvick (10), in their cntributin t the 4-PA methdlgy, pinted ut the unreliability f the lder methds, mainly due t the incmplete eliminatin f flurescent substances ther than 4-PA. Thus, the lder methds wuld lead t an verestimatin f 4-PA. In a study n drug-induced vitamin B, deficiency, Levy bserved that his subjects were in psitive balance fr vitamin B, (intake exceeding excretin) during cntrl as well as the experi- mental perid (98). He determined urinary and fecal 4-PA by the methd f Wdring, Fisher and Strvick (10). Althugh the primary rute f excretin f 4-PA was thrugh the kidney (2 [im/24 hr), there was a small amunt f 4-PA in feces (0. 3 j.m/24 hr). Average values f urinary 4-PA fr healthy wmen cnsuming self-selected diets under nrmal cnditins have been reprted t range frm 3.8 t.9 ^M/24 hr (10). Urinary excretin f 4-PA by men receiving a cnstant daily intake f 1. mg f vitamin B, was mg ( JLM) per 24 hr (107). In subjects wh received mg vitamin B, frm a cnstant diet f natural b fds, the amunt f 4-PA excreted ranged frm 3. 9 t 4. 2 j.m/24 hr (105). Cntractr and Shane reprted that men and wmen

39 excreted an average f _ 4. 2 and. 1 +_ 4. 4 (j,m f 4-PA/24 24 hr, respectively (58). The male and female subjects studied by Mikac-Devic and Tmanic excreted 3. t 7.8 and 3. 1 t 5. 3 (im 4-PA per day, respectively (108). Based n such studies, Sauberlich, Skala and Dwdy suggested 3-7 j,m/day as nrmal range fr urinary excretin f 4-PA which wuld generally reflect abut 30-50% f the daily vitamin B, intake (49). There seems t be a number f dietary factrs that influence the excretin f vitamin B, and its metablites. was discussed by Yan and Fujita (103). The rle f cellulse In a review f the effect f different carbhydrates n vitamin and amin acid requirements, Harper and Elvehjem have discussed that when a less sluble carb- hydrate is substituted fr a mre sluble ne in the diet, the require- ments fr mst members f vitamin B cmplex and fr essential amin acids decrease (109). They reprted that the effect f cmplex carbhydrates in lwering the requirement fr B vitamins was related t changes in the intestinal micrflra. Sarma, Snell and Elvehjem bserved a higher grwth rate in rats when dextrin was used in place f sucrse in a pyridxine-deficient diet and cncluded that it was due t synthesis f vitamin B, by the intestinal micrflra in the presence f dextrin (110)..With the diet cntaining dextrin they als fund an increase in the amunt f urinary 4-PA. These findings were similar t thse f Yan and Fujita wh studied the effect f

40 cellulse n the synthesis f vitamin B, by human intestinal bacteria D (103). The effect f ascrbic acid Intake n the urinary excretin f 4-PA was studied by Selivanva (111); Selivanva, Agasin and 25 Pljakva (112). On basal diets cntaining a mean value f 8.8 _+ 0. mg f vitamin C per day, 38 men excreted an average f 40. jig f 4-PA/hr (range ) in their urine cllected befre breakfast. After receiving a supplement f 100 mg f vitamin C daily fr almst 50 days, they shwed a significant increase in the urinary 4-PA, with an average f 234. p.g/hr (range ). The authrs cncluded that, as the urinary excretin f vitamin C increased as a result f increased vitamin C intake, the urinary 4-PA als increased. Under nrmal cnditins the level f urinary 4-PA has been recgnized t reflect the intake f vitamin B, (47). In sme cases f abnrmal vitamin B, metablism, urinary 4-PA drps cnsiderably and cannt be used as an index f vitamin B- intake (93). Extensive studies have nt been cnducted t investigate the influence f graded intakes f vitamin B, n the excretin f 4-PA (49). Sauberlich et al. therefre feel that measurement f 4-PA, at present, cannt be recmmended as a reliable index f evaluating vitamin B, nutritin especially in clinical cases (49). Levels f 4-PA, as with levels f urinary vitamin B,, prbably prvide infrmatin n the

41 immediate dietary intake f the vitamin and may nt reflect the bdy reserves (79). Althugh the vitamin B, requirement f the human is dependent D n the level f prtein cnsumed, dietary prtein seems t have little effect n the urinary excretin f vitamin B, (5). Vitamin B, in b urine can be measured by several methds (113, 114). Of these, micrbilgical assays, using Saccharmyces carlsbergensis as the test rganism, have been cmmnly emplyed. The free frms f vitamin B, in urine can be measured by its 2 ability t supprt grwth f S. carlsbergensis (115). The bund r phsphrylated frms can be cnverted t the free frm by acid hydrlysis r urine and the ttal vitamin B. measured by the same micrbilgical methd. Kelsay et ah studied the effect f vitamin B, intake n urinary excretin f vitamin B, in adult males (107). A lw level f urinary vitamin B- was nticed in subjects when they received nly 0. 1 mg f the vitamin daily. Cntinuatin f this level f intake fr several days results in excretin levels f the free vitamin f j.g/24 hr, cmpared t ig prir t depletin when the subjects received a daily supplement f 1. 5 mg PIN. Dnald et al. btained smewhat similar results in yung wmen (11). These subjects excreted a daily average f 1 ^j.g free vitamin B, n a nrmal diet. "When they were given a diet cntaining mg f vitamin B, per

42 day fr 43 days, their levels f urinary vitamin B, drpped t b [j.g. On increasing the daily intake t 0.94 mg, the urinary vitamin B. rse t ag per day, after 10 days. When the intake level was further increased t 1. 5 mg, the excretin f vitamin B, in D urine increased t 55.8 j.g which was clse t the riginal level f 1 (j.g/24 hr bserved by Dnald et_ al. With a supplement f 30 mg PIN-HC1, urinary vitamin B. in the same subjects reached a level 27 as high as 75.5 fig/24 hr. In healthy female subjects n nrmal diets, Kkkeler bserved an excretin f 8 3 +_ 0 and 1 3 +_ 7 3 j.g/24 hr, as free and ttal vitamin B., respectively (117). b Sauberlich et al. reprted a urinary excretin f p.g f free vitamin B, per day, r 20 [ig/g creatinine, as being nrmal fr individuals with a vitamin B intake f apprximately 1. 5 mg daily (49). Urinary B, levels less than 20 ug/g creatinine are cnsidered t reflect inadequate dietary intakes f the vitamin (79). In cases where cllectin f a 24 hr urine is nt practical, fasting urine samples have been used. In such instances the results are generally expressed per gram f creatinine. Little infrmatin is available n the distributin f PAL, PIN and PAM in urine. One reasn fr this may be the lack f methds sensitive enugh t determine these frms (107). The dif- ferential micrbilgical assay f Rabinwitz and Snell resulted in lw recveries f pyridxine added t human urines (47). Fujita and

43 28 Fujin prpsed a flurmetric methd fllwing chrmatgraphic separatin f the frms and xidatin f these t 4-pyridxic acid (104), Hwever, pr reprducibility f the methd as well as difficulty in cnverting the different frms int 4-pyridxic acid and the lactne have set limitatins n this methd. Tepfer and Lehmann reprted n a methd fr assay f the three frms f vitamin B, in fds, in which chrmatgraphic separatin (using Dwex 50 in exchange resin) int different frms was fllwed by micrbilgical assay using S^. carlsbergensis (118). Their methd was later imprved (119) and is established as the AOAC methd (120). Kelsay, Baysal and Linkswiler bserved that PAL was excreted in the largest amunt when their subjects were n self-selected diets r when experimental diets were supplemented with 1.5 mg PIN-HCl (107). When assayed by the methd f Tepfer and Lehmann (118), PAL accunted fr-apprximately 5% and PAM abut 30% f the urinary vitamin B,, with nly a negligible amunt excreted as b PIN. The percentage f PAL, PIN and PAM in urinary vitamin B- reprted by Cntractr and Shane was 13.9, and 2.73, respectively (58). They used a phsphcellulse clumn t separate the free and phsphrylated frms f vitamin B, as well as 4-PA, fllwed by flurmetric determinatin f the cmpnents. The

44 29 difference in the percentages f PAL, PIN and PAM reprted by Cntractr and Shane (58) and Kelsay e^ al. (107) may be due t the difference in the methds f separatin and determinatin. Further studies using standardized techniques will help in btaining a clear picture f the distributin f the three frms f vitamin B, in urine. The percentage f dietary vitamin B, reflected in the urinary excretin f the vitamin is very small (98, 103). Hwever, when evaluated tgether with ther excretry prducts, this may help in assessing the availability f the vitamin ingested. Studies n Biavailability f Vitamin B, and Other Nutrients There is a grwing interest in assessing the bilgical availability f nutrients frm fds. Several reprts have appeared n irn with reference t wheat and enriched bread (121, 122, 123, 124), and ther fd prducts (125, 12, 127, 128, 129). Many f these studies emplyed respnse f bld hemglbin, hemglbin repletin and tissue irn levels t assess the biavailability f irn.

45 30 The biavailability f irn frm surces cmmnly used in bread enrichment was studied in anemic rats fed enriched bread cntaining 20 ppm irn (121). Based n the amunt f hemglbin repleted in 30 days, the availability f irn frm different surces as well as at different stages in breadmaking was determined. The authrs reprted that fermentatin and baking steps caused sme increase in the bilgical availability f irn, which culd prbably be due t a breakdwn f phytic acid r t a denaturatin f irn binding sites r bth. Miller used ttal hemglbin, hematcrit, red bld cell cunt, cerulplasmin, serum irn, irn and cpper levels in tissues and carcasses t assess the utilizatin f irn frm enriched white bread by nrmal and anemic rats (124). The anemic grup was prvided with irn by additin f either ferrus sulfate r irn- enriched bread t their diets. Bread appeared t stimulate the prductin f red bld cells t a greater extent even thugh irn was less available frm bread than frm ferrus sulfate fr the synthesis f hemglbin r fr strage in slid tissues. One ps- sible interpretatin was that bread cntained sme cmpnent which specifically stimulated the prductin f erythrcytes frm erythrblasts in anemic rats. Biavailability f magnesium frm wheat flur and varius rganic and inrganic salts v/as investigated by Ranhtra, Lewe and

46 31 Puyat (100). They based their assessment n the apparent absrptin, using fecal excretin and tissue cncentratin f magnesium. Their results demnstrated that magnesium was equally available frm all surces tested, de Muelenaere, Chen and Harper assessed the availability f lysine in cereal prducts using grwth and fecal analysis methds (130). They fund that lysine f crn and rice prtein was highly available. Biavailability f sulfur amin acids in crn prtein was demnstrated by Sasse and Baker using slpe- rati technique and a standard curve methd (131). Dcumented reprts n the availability f vitamins are rather limited. Pelletier and Keith studied the biavailability f synthetic and natural ascrbic acid, utilizing cumulative urinary excretin and serum levels as indices fr cmparisn (132). A similar tech- nique was used by Tamura and Stkstad t study the availability f fd flate in man (133). The similarity in absrptin f vitamin C and flic acid frm range juice and a synthetic surce was reprted by Nelsn, Streiff and Cerda (134). They used the methd f triple lumen perfusin f the human small intestine t determine the rate and amunt f absrptin f the vitamins. In a recent study, using the same intestinal perfusin methd, Nelsn, Lane and Cerda shwed that the mean vitamin B, absrptin was significantly greater frm a synthetic than frm a natural surce, which was range juice (135). They used a triple lumen tube with a

47 32 30 cm study segment t determine the uptake f vitamin B, in 15 nrmal subjects. The intestinal absrptin was expressed as jxg/ cm/hr. They als cmpared, in six subjects, the absrptin f vitamin B, frm range juice with that f the synthetic surce with and withut added glucse. Althugh they bserved sme variatin in the absrptin f water frm these three surces, the difference between range juice and slutin cntaining glucse was nt significant. Water absrptin frm the vitamin slutin withut glucse was significantly lwer than that f either f the ther tw. Despite the fact that absrptin f water was quite high frm range juice, the mean percent absrptin f ttal vitamin B, frm this surce was significantly lwer than that frm the synthetic surces. This indicates that factrs which prmte water transprt in the intestine may nt necessarily increase the bilgical availability f water sluble vitamins frm fd surces. Biavailability f vitamin B, frm fd surces has been studied in animal mdels. Lantz fund that cking f pint beans increased the availability f vitamin B-, suggesting that it may naturally exist in a bund frm which is released n cking (13). Yen, Jensen and Baker assessed the biavailability f vitamin B, in crn and sybean using day ld chicks (137). A standard curve fr vitamin B, respnse was prepared using diets cntaining increasing levels f crystalline PIN-HC1, fr which availability was assumed t be 100%.

48 33 They examined the grwth patterns f the chicks as well as the plasma and serum glutamic xalate transaminase (SGOT) activities. The frmer was fund t be mre sensitive than the latter, as an indicatr f the availability f vitamin B, frm these fd surces. D Their results suggested that crn was significantly lwer in avail- able vitamin B, cntent than sybean. Dry rasting f crn at 10 C significantly reduced the availability f the vitamin cmpared t rasting at 80 and 120 C. Similarly, autclaved full fat sybean had a lwer cncentratin f available vitamin B, than did the sy- meal. Tissue transaminase activities and vitamin B, levels were D studied by Thiele and Brin as indices f vitamin B, availability (138). They fed male rats fr 14 days n a diet devid f vitamin B,, with and withut daily supplements f 5 r 15 jig f PAL, PIN and PAM and bserved that large amunts f each frm gave higher cncen- tratins f ttal vitamin B. and transaminase activities in liver, kidney, brain, muscle and heart f the rats that received vitamin B, cmpared t thse that did nt receive vitamin B-. Their data als indicated that all the vitamers appeared t be equally available t the rats. Vitamin B/ in Fds Vitamin B, in fd ccurs as a grup f related cmpunds in

49 the free frms, PIN, PAL and PAM r chemically bund frms, PLP 34 and PMP (139). Distributin f PIN, PAL and PAM in sme natural prducts was first reprted by Rabinwitz and Snell (140). Since then several methds have been develped fr the determinatin f the three frms f vitamin B,. Chemical prcedures prpsed fr the determinatin f vitamin B. in fds have mainly been based n flurmetrie methds (141, 14Z, 143). Micrbilgical methds based n the grwth f rganisms are als widely used (144, 145). Tepfer and Lehmann described a technique t separate PIN, PAL and PAM Ln fds using in exchange chrmatgraphy (118). The cmpnents were then assayed by micrbilgical methd. Biassay techniques have als been used t evaluate the activity f vitamin B, frm different fds (14, 137). A micr- bilgical methd has been widely used in analysis f fd fr vitamin B,. D A semiautmated system fr micrbilgical vitamin assays has been attempted (147), and applicatin f this in deter- mining vitamin B, wuld cnsiderably imprve the efficiency f the b methd. Mst f the data reprted in literature n the vitamin B, cn- D tent f fds have been btained using micrbilgical methds. Saccharmyces carlsbergensis has been generally used fr the determinatin f vitamin B, activity in fds (120). The bund frms f the vitamin are inactive fr this micrrganism (10, 148). The

50 respnse f the micrrganisms t vitamin B. requires prir hydrlysis f mst prducts assayed. 35 Different hydrlysis prcedures have been discussed in the literature (10, 149). These prcedures vary depending n whether a fd prduct is f animal r plant rigin (120). Plant prducts generally need t be autclaved with 0.44 N_ HC1 fr 2 hr at 121, whereas thse f animal rigin require autclaving with N HC1 fr 5 hr at the same temperature. The frms PAL and PAM are predminant in fds f animal rigin while the majr frm in plant prducts is PIN (150, 151). Althugh vitamin B, is widely distributed in fds, sme fds are richer surces f this vitamin than thers. Muscle meat, liver, legumes, whle grain cereals and especially the bran frm cereal grains are amng the best surces f vitamin B, (152). The vitamin B, cntent f fds and the distributin f the three frms are presented by Orr (5). Mst f the whle grain prducts cntain 2-4 ag f vitamin B, per gm and are cnsidered better surces f vitamin B, than refined prducts which cntain less than 1 fig/g (4). Plansky and Tepfer reprted that durum wheat cntained mre vitamin B, (4. 3 jig/g) than hard r sft wheats (3. 4 \ig/g) (4). Whle wheat flur was analyzed in the cllabrative study f vitamin B. methdlgy (145). Amng the 13 labratries that participated, the levels f vitamin

51 B, determined varied frm 1. 4 t j.g PIN-HC1 per g, with an average f Pyridxine accunts fr abut 2/3-3/4 f the ttal vitamin B z in wheat while PAL and PAM tgether frm the remaining 1/3-1 /4 (153). Vitamin cntents f air-classified high and lw prtein flur fractins were determined by Jnes, Fraser and Mran (154). Their data indicated that vitamin B, levels in the fine fractins were higher in hard than in sft flurs. The same fractins were als reprted t be prprtinately higher r lwer in prtein, suggesting that vitamin B. may be bund t the prteins. Vitamin B, cntent f wheat bran has been reprted t be quite high, arund 13.7 fig/g 3 whereas that f whle grain was 3.7. In the patent flur the level was as lw as 0.5 j.g/g (155). tent f whle wheat is under genetic cntrl (15). Harris reprted that vitamin B. cn- Because f this and f the different effects f varius milling practices, the vitamin B, cntent f different flurs at any ne extractin rate may nt be the same. The mechanical prcess f milling and separatin f varius cmpnents f wheat grain have a definite effect n vitamin B, level f the flur as indicated by the data f Plansky and Tepfer (155). The lss f vitamin B, during milling f wheat int refined b flur was reprted t be arund 85% (). T cmbat this, as well as fr the reasns discussed later in this review f literature, the

52 37 Natinal Academy f Sciences has prpsed additin f vitamin B,, alng with sme ther nutrients, t white flur (9). Cereal Enrichment Prgram Cereals make an imprtant cntributin t the nutrients f the wrld's fd supply. Cereal prducts are als cnsidered suitable carriers fr the additin f nutrients t the diet f many peple (9). There is evidence f ptential risk f deficiency f certain nutrients amng sme segments f the ppulatin such as lw incme grups and pregnant wmen. Because f this, the Fd and Nutritin Bard f the Natinal Academy f Sciences has recmmended bradening the cereal enrichment prgram that was started in early 1940s (8). The nutrients recmmended fr enrichment f cereal prducts were selected mainly n the basis f their rle in meeting the needs f significant grups f peple, susceptible t nutritinal deficiencies (157). The currently used nutrients and their levels as revised recently t be effective frm 1977 (158), as well as thse prpsed by the Natinal Academy f Sciences (9). are reprted in Table 2. Amng the cereal prducts, abut 30% f thse cnsumed in the wrld and 82% f thse in the U.S. are reprted t be f wheat rigin (1, 159). Breads made frm wheat flur are cnsumed in almst every cuntry f the wrld and wuld serve as a suitable carrier fr added nutrients.

53 38 Table 2. Enrichment levels fr cereal prducts. ia b Presently used Prpsed Nutrient mg/lb mg/lb mg/100 g Thiamin 1.8 Ribflavin 1. 1 Niacin 15 Irn 25 Calcium 00 Vitamin A - Vitamin B. - Flic acid - Magnesium c Zinc _ a Federal Register, 197(158) u Natinal Academy f Sciences, 1974 (9) A Federal Standard f Identity fr enrichment f white flur was first established and made effective n January 1, 1942 (8). This was amended in 1943 t prvide the standards, t enrich flur with thiamin, niacin, ribflavin and irn with the ptin f including calcium and vitamin D (10). Recently the Standard f Identity f baked fd was revised t establish new levels f enrichment (158).

54 39 The prpsed level f enrichment fr vitamin B. is 2 mg (pyridxine) per pund r 0.44 mg per 100 g f flur (9). Pyridxine hydrchlride (mlecular weight 205.) is the cmmnly available synthetic frm f the vitamin. It ccurs as white platelets and is readily sluble in water (35). Althugh cereal enrichment prgram appears t be technically feasible, it is recmmended that studies be cnducted t determine the unifrmity f distributin f the nutrients, their stability during preparatin and strage, the availability f the nutrients and the cnsumer acceptance f the fds prepared frm these cereals. Stability f Vitamin B^ and Other Nutrients during Fd Prcessing Much attentin has been paid t vitamin B, studies in humans b in the recent years. This can be attributed t a number f factrs, ne f which is the lss f vitamin B, during fd prcessing (49). Expsure t direct sunlight, diffused daylight r artificial light causes inactivatin f the three frms f vitamin B., with the b direct sunlight prducing the greatest destructin (11). Fr this reasn, during its analysis in the labratry, precautins are taken t avid expsure t light f the samples cntaining vitamin B, (149).

55 40 Of the three frms, PIN is quite stable in acid slutins, but rapid destructin by light and heat ccurs at neutral r alkaline ph (12, 13). Pyridxal and pyridxamine are reprted t be relatively less stable than pyridxine (14). Abut 90% f the vitamin B. in fresh milk was reprted t be PAL, bth free and as the phsphate (140). Heating and strage lsses f this vitamin in milk and in infant frmula were high (15). Added PIN-HCl was much mre stable in infant frmula than vitamin B, in milk under the same prcessing cnditins. Spray-drying caused sme lss f vitamin B,, but less than that bserved during sterilizing f liquid milk. Davies, Gregry and Henry reprted a substantial lss f vitamin B, (45-70%) during prcessing f evap- rated milk. Additinal lsses were bserved 'when this milk was stred mnths at rm temperature (1). The effect f the prcessing methd n vitamin B. retentin was reprted by Eversn et al (17). Percent retentin f this vitamin in bean, beef, and tmat juice when prcessed by cn- ventinal methd was 8.7, 94.2 and 100, respectively. When prcessed by high temperature shrt time (HTST) methd and canned aseptically, the crrespnding retentin figures were 93.0, 98.3 and 100%. In the cnventinal methd, heat transfer is very slw particularly when the prducts are nn-liquid. Since heat penetra% tin int the prduct is thrugh the periphery, the material clse t

56 41 the edge gets mre drastic heat than required t achieve sterility. The HTST methd and aseptic canning are receiving increasing attentin due t better retentin f nutrients in the prducts prcessed this way (18). Raab, Luh and Schweigert studied the effects f heat prcessing n the retentin f vitamin B, in lima beans (19). Assuming the vitamin B, retentin f dry beans at 100%, they bserved 7-81% and 83-87% retentins f vitamin during water and steam blanching, respectively. Althugh steam blanching may have had imprved retentin, the results were nt significantly different frm thse f water blanching. Several reprts have appeared n the vitamin retentin f different meats during prcessing and strage. Vitamin B, is cn- sidered fairly stable during frzen strage (170). Abut 82% retentin in beef liver and 77% in bned chicken were bserved. Lushbugh, Weichman and Schweigert reprted 42-7% retentin f vitamin B. in meat after cking, with 1-13% in the drip (171). High lss f vitamin B, has been reprted during heat prcessing and irradiatin f bned chicken (170). Hwever, the retained vitamin appeared t be stable n strage as lng as 15 mnths at C. The amunt f vitamin retained seems t vary with the type f meat and the methd f cking. Meyer, Mysinger and Wdarski did vitamin B, retentin studies n ven-rasted beef lin and ven

57 42 braised beef rund (172). The retentin in the lin averaged 72% with 1% in the drip; rund averaged 49% with 34% in the drip. Micrwave heated chicken breasts retained mre vitamin B, (n a drip weight- basis) than cnventinally rasted meat (173). Bwers, Fryer and and Engler bserved a similar pattern with turkey breasts and sug- gested that higher percentage f retentin fr micrwave cking may be due t greater lss f misture by this methd than by cnventinal heating (174). The same authrs reprted that n a cked weight basis, cnventinal and micrwave heated prk differed little in vitamin B, cntent (175). D On a dried weight basis, hwever, they bserved mre retentin f this vitamin in cnventinally heated muscle. Studies n sme f the B vitamins in cereal prducts have been reprted since the 1940s. Early wrk was dne mainly n thiamin, niacin and ribflavin. Meckel and Andersn studied thiamin retentin and cmpsitin f fur types f U. S. army breads made frm enriched white flur (17). Thiamin lsses varied frm 14 t 24% depending n the nature f the laf and the treatment t which it was subjected. There was n lss f thiamin, ribflavin and niacin in the field type f laf after string at rm temperature fr as lng as tw weeks. Stability f these three vitamins during fermentatin, baking and strage f canned bread was investigated by Brenner, Dunlp and Wdicka (177). Ribflavin and niacin were retained almst cmpletely during fermentatin, baking and strage. Thiamin hwever, was decreased by apprximately 15% during baking and 20-50% during mnths strage at 72 and 100 F, respectively.

58 43 They further nted that prtein quality f whle wheat bread was significantly superir t that f enriched white bread. Lss f thiamin in tasting f breads made frm whle wheat, enriched and unenriched white flur was studied by Dwns and Meckel (178). The percent lss f thiamin in tast made frm unenriched white flur was higher than either f the ther tw types. They thught that this apparent lss may have been due t a discrepancy intrduced by the assay methd and the difficulty in accurately measuring small cncentratins. Maleki and Daghir reprted the effect f baking n retentin f thiamin, niacin and ribflavin in bth white and brwn arable bread (179). Their data suggested that lss f thiamin was greater in brwn bread than in white bread; lss f ribflavin was similar in bth types and there was nly a negligible lss f niacin in the tw breads. They als bserved a higher retentin f ribflavin in vitamin enriched samples cmpared t unenriched samples. Niacin was fund t be stable in bth enriched and unenriched samples. Mrgereidge studied the retentin f vitamins in enriched bread under practical retail cnditins t determine the effect f transparent versus semi-paque wrapping material (180). He shwed that regardless f the type f wrapper, there was n lss f thiamin, ribflavin and niacin when bread displays were subjected t nrmal intermittant illuminatin fr perids up t five days.

59 44 A recent investigatin n stability f native and added flic acid in flur during bread prcessing and strage was dne by Keagy, Stkstad and Fellers (181). Native flic acid in flur -was stable when stred at 84 F with 8% retentin after 12 mnths, but shwed prgressively increasing lsses as the temperature increased. Retentins f 78% in 4-5 mnths at 100 F and 2% in 1 mnth at 120 F were reprted. Synthetic pterylglutamic acid added at levels f 1 r 5 jj.g/g f flur shwed very small lsses. Average baking lsses f 11% fr added and 31% fr native flacin were bserved. They als reprted that flacin synthesis by yeast during fermentatin made up fr the lss f native flacin during bread prcessing. Accrding t these investigatrs, fermentatin time was a majr factr in determining the final native flacin cntent f bread. Gd recveries f PIN-HC1 added t flur and baked int bread were reprted by Hennessy et al. (182). They have presented vitamin B. values btained by bth flurmetric and micrbilgical methds. In the frmer methd, nly PIN respnded, whereas all three frms f vitamin B, were accunted fr in the micrbilgical methd. Hwever, the results btained by these tw methds agreed relatively well. This culd result because abut 3/4 f the vitamin in wheat is in the PIN frm (153). Gd stability f added vitamin B. t flur during baking and strage was reprted in a recent study

60 45 by Crt et al. (183). They baked regular yeast-risen bread using flur frtified with vitamins including vitamin B.. Vitamin B, was stable during baking and after 5 days f strage f bread at rm temperature. The breads baked frm vitamin-frtified flur stred fur mnths at rm temperature als had a vitamin B. level similar t that f the regular laf baked frm freshly enriched flur. Stability f vitamin B. added t crn meal and macarni was studied by Bunting (184). He bserved excellent stability f the vitamin during strage and cking. In strage 90-95% f the added plus natural vitamin B. 'was recvered frm enriched crn meal, and 100% frm enriched macarni after ne year at 100 F and 50% relative humidity. During the baking f crn bread made with enriched and unenriched crn meal, almst 100% f the vitamin B. was retained. After macarni was cked, abut 50% f the natural and added vitamin B, remained in the slids while 50% leached int the cking water. It was suggested that these percentages shuld be presented n the packages f the uncked prducts in rder t fulfill the Federal Standards f Identity. Dry rasting f crn t 10 C significantly reduced the cn- centratin f available vitamin B, cmpared t the samples in which the rasting temperature did nt exceed 80 and 120 C (137). Sme lss f heat susceptible cmpnents f vitamin B. may have ccurred when heated t the highest temperature f 10 C. Crn has 82-91%

61 4 PAL and PAM which are mre susceptible t heat than PIN (153). This may be the reasn fr greater lss f vitamin B. bserved at 10 C by Yen, Jensen and Baker (137). There seems t be a hst f factrs which determine the stability f vitamin B, during fd prcessing. The predminant frm f the vitamin and the temperature f prcessing appear t be f particular imprtance.

62 47 MATERIALS AND METHODS Stability Preparatin f Breads Breads were prepared using whle wheat (WHW), white enriched with vitamin B. (WB-) and white (W) flur, under bth b cmmercial and hme cnditins. Cmmercial breads were baked n a large scale at a lcal bakery (Albertsn's, Crvallis). These breads were used in assessing the biavailability f vitamin B. in human subjects (discussed later). Brminated bakers patent flur was used in cmmercially prepared white breads, WB, and W, and hundred percent whle wheat flur in the WHW bread. These breads were prepared by a straight dugh type methd with frmulae generally used in the bakery. Details are given in Appendix 1. Cmmercially baked breads were machine slided and packaged in plyethylene bags t prtect against desiccatin, packed in cardbard bxes and stred in a freezer (-40 Q until used. Brminated bakers patent flur prvided curtesy f Centennial Mills, Prtland, Oregn.

63 Straight-dugh and spnge-dugh methds were cmpared 2 under hme cnditins. All purpse flur and 100% whle wheat 3 flur were used in hme style baking. The flurs were purchased 48 frm a retail market. Straight dugh breads were prepared using the Finney and Barmre frmula (185), with the slight mdificatin f replacing 2% cmpressed yeast with 1% active dry yeast (n flur basis). Spnge dugh breads were made by the apprved methd f American Assciatin f Cereal Chemists (18) with the missin f yeast fd. The quantities f the ingredients were kept at the same levels as in the case f straight dugh frmula. mula and prcedures are given in Appendix 2. Details f the fr- The majr difference between these tw methds was in the time f fermentatin and prf. These times were 235 min and 330 min fr the straight dugh and spnge dugh methds, respectively. 4 Crystalline pyridxine-mnhydrchlride was used as the surce f pyridxine in the enrichment f WB, breads. The level f enrichment recmmended by Natinal Academy f Sciences (9), 2 mg 2 All purpse flur was unbleached and enriched with thiamin, ribflavin, niacin, and irn levels indicated in Appendix 1, prduct f Centennial Mills, Prtland, Oregn % whle wheat flur, prduct f Fisher Mills, Inc., Seattle, Washingtn. 4 Pyridxine mnhydrchlride, Lt number 50155, Calbichem, San Dieg, Califrnia.

64 pyridxine per pund f flur -was used in the WB, breads prepared 49 under hme cnditins. In the case f cmmercially prepared breads, WHW and W were baked first and their vitamin B levels were determined befre the level f enrichment fr WB, bread was D arrived at. A level f nly mg pyridxine per pund f flur was used in the cmmercially prepared WB- bread. This amunt was used t attain a level f vitamin B, in the cmmercial WB, bread b b clse t that f WHW, such that the quantities f the breads given t the human subjects in the biavailability study wuld be similar. During the curse f bread making, representative samples were taken in triplicate at three stages; after mixing all the ingredients (mix), at the end f prf just befre baking (prf) and after baking (bread). These were analyzed fr vitamin B. and misture cntent. Samples f mix and prf taken fr vitamin B, analysis were weighed and immediately frzen between tw slabs f dry ice t stp fermentatin and held frzen until hydrlyzed. In the case f bread, five slices were taken frm different psitins within the laf. Waring blender. Crumbs were made frm these slices using a Three representative samples f apprximately ne gram f the crumbs were weighed and hydrlyzed. The samples were hydrlyzed by autclaving in 0.44 N HC1 fr 2 hr at 121 C. These samples were assayed fr vitamin B. using a micrbilgical methd (120). The standard methd was slightly

65 50 mdified in the fllwing manner. In place f 100 ml f acid 5 hydrlyzed casein slutin, 10 g f vitamin free casamin acid were used in the preparatin f the basal medium stck slutin. The inculum f Saccharmyces carlsbergensis (als knwn as S. uvariurm) ATCC N was prepared by incubating the cells in liquid culture medium fr hr instead f 20 hr. The methd invlves the grwth f micrrganisms. The percent transmittance f the cell suspensin was read n an Evelyn Phtelectric Clrimeter set at 0 nm. The unknwn cncen- tratins f vitamin B, were read against a standard curve prepared with graded levels f standard pyridxine hydrchlride. A range f 0 t 5 ng f pyridxine was used. Althugh a drawn standard curve served as a check n the distributin f pints, the unknwn 7 cncentratins were determined using a calculatr interpret unequally spaced data. prgrammed t Misture cntents f the mix, prf and bread samples were determined in triplicate using a vacuum ven (120). Vitamin B- values f the samples were expressed n a dry weight basis. The 5 Vitamin free casamin acid, Difc Labratries, Detrit, Michigan. Evelyn Phtelectric Clrimeter, Rubicn Cmpany, Philadelphia, Pennsylvania. 7 Prgrammable Calculatr, 9810 A Mdel 10, Hewlett Packard, Lveland, Clrad.

66 means f mix and prf, prf and bread were tested fr significance using Student's t test at 5 and 1% levels f prbability. 51 Strage f Vitamin B, Enriched Bread Stability f vitamin B, during strage f cmmercially prepared WB, bread was investigated. Several randmly selected laves frm the cmmercial batch were placed under three different strage cnditins; three laves at rm temperature (25-27 C) with mderate lighting, six laves under refrigeratin (4-5 C) and nine laves in a chest type freezer (-5 C). The weight f each laf was nted initially and at the time f sampling. The bread strage study started n the same day the cm- mercial WB, bread was baked. Zer day samples were btained after the laves had remained under respective strage cnditins fr three hurs. Sample preparatin and vitamin B. analysis were similar t that f bread described in the previus sectin. Breads stred at rm temperature were analyzed at three day intervals ver a perid f ne week. Thse stred in the refrigeratr and the freezer -were sampled and analyzed at weekly intervals fr fur and eight weeks, respectively. Strage f Vitamin B, Enriched Flur Crystalline pyridxine mnhydrchlride was added t all

67 g purpse flur at the level f 2 mg pyridxine per pund f flur. 52 An even distributin f the vitamin was attained by mixing the flur in small quantities. Initial mixing was dne by rtating the vitamin and flur in a glass jar cntaining glass beads t facilitate thrugh mixing. This flur-vitamin mixture was transferred t a bwl f a Kitchen Aid mixer equipped with a wire whip. As the mixing cntinued, the rest f the flur was blended int the first mixture. Mixing was cntinued fr ne hur after the additin f all the flur. Cld twels were placed arund the bwl t minimize the heat prduced due t mechanical actin. Flur was stred in amber-clred, tightly sealed glass jars. These were placed under three strage cnditins; rm temperature (25-27 C) under mderate lighting, cld strage (5-7 C) and frzen (-40 C). Triplicate samples f flur frm each strage lt were analyzed fr vitamin B, after 0, 1, 4, 8, 12, 19 and 2 weeks. Biavailability Subjects Nine healthy cllege men, age 21 t 35 years, were recruited by advertisement r persnal cntact. Each participant was g All purpse unbleached, enriched flur, Centennial Mills, Prtland, Oregn.

68 53 interviewed initially fr medical and diet histries. The prtcl f the study was utlined and their rle in it was explained clearly. Persnal infrmatin, activity and the cnsent f the selected subjects were btained in writing n the frms given in Appendix 3. The vital statistics f the subjects are presented in Table 3. This study had been apprved by the Human Subjects Cmmittee at Oregn State University. Experimental Design The study invlved three variables--the breads; whle wheat (WHW), white enriched with vitamin B. (WB.) and white (W). D D The experiment was based n a three 3x3 Latin square design which is illustrated in Figure 3. The subjects were randmly assigned t ne f the nine clumns. Diet The three week experimental perid was preceded by six days f adjustment during which time the diet was gradually changed frm ne cntaining prteins f animal rigin t ne based primarily n wheat prtein. The majr fd item in the experimental diet was bread. The three types f bread, WHW, WB. and W required fr the entire study were prepared at a lcal bakery (Albertsris, Crvallis) and

69 54 Table 3. Vital statistics f the subjects. Subject (N.) Age (Years) Starting Bdy- Weight (kg) Height (cm) Range Mean + SD Mean + Standard deviatin 8 r I 11 III WHW WB W WB, 0 W WHW w WHW WB 1 1 WHW WB w 1 W WHW WB WB, 1 W WHW 1 WHW WB^ W 0 w WHW WB WB, 0 W WHW > 1 SUBJECT Figure 3. Latin square design fr three breads; whle wheat (WHW), white enriched with vitamin B, (WB ( fi ) and white (W) ver three experimental perids, I, II, and HI, fr nine subjects 1-9.

70 55 kept under frzen strage as described earlier, until they were thawed and served t the subjects. The level f vitamin B, fr the enrichment f white bread was selected after analyzing the whle wheat and white breads fr their vitamin B, cntents. Even thugh the prpsed level f enrichment is 2 mg f pyridxine per pund f flur (9), nly a 1, 12 mg level was used in rder that the amunt f vitamin B. in the WB, bread wuld be clser t that f whle wheat bread. The vitamin B. cn- D tents f the three types f bread as determined by micrbilgical assay using Saccarmyces carlsbergensis were 0. 21, 0.20, and 0.0 mg f pyridxine per 100 grams f WHW, WB, and W breads, respectively. One reasn fr the selectin f the level f vitamin B, intake b was that 2/3 t 3/4 f vitamin B, wuld be cntributed by the bread. Six hundred grams (22 slices) was selected as the daily quta f bread. This amunt f WB. bread cntained 1. 2 mg f vitamin B.. b On this basis, 1. 5 mg was set as the daily intake level f vitamin B, in this study, althugh the recmmended dietary allwance (RDA) is 2 mg (14). Accrding t Sauberlich (190), 1. 5 mg f vitamin B. is a level that marginally meets the requirements f human adults. This level f intake usually prevents excretin f abnrmal levels f tryptphan metablites in the urine after a lad test (49).

71 5 In rder t maintain 1. 2 mg f vitamin B, frm bread, 570 g f WHW bread was used. nly 0. 3 mg f vitamin B,. Six hundred grams f W bread cntained In rder t maintain the level f vitamin B, equivalent t that frm bread, mg f pyridxine hydrchlride was supplied t the subjects in the frm f an ral dse distributed equally amng the three meals when they received W bread. The daily quta f bread was cnsumed by all subjects with the exceptin f ne (Subject N. 5), wh was unable t cnsume the bulk f the entire quantity. His intake f bread was decreased t 2/3 f that f thers n day six f the adjustment perid. The ral dse f PIN-HC1 was als reduced prprtinately when he received W bread. His ttal daily intake f vitamin B, was 1. 2 mg cmpared t the 1. 5 mg given t the thers. The type f bread was the nly variable in the study during the three experimental perids. The remaining diet, which was cn- stant thrughut the study, cnsisted f the fd items given in Table 4. The cmpsitin is fund in Appendix 4. This diet was adequate in all nutrients knwn t be required by man accrding t the RDA (14). In additin t these fds, margarine, jelly, beverages, (cffee

72 tea, Tang, Lemnade, 7 -UP ), candy and sugar were als made 57 available t the subjects. A recrd f the daily cnsumptin f these items was maintained. They were encuraged t maintain their bdy weights by regulating the amunts f these fds cnsumed. A three meal regimen was fllwed. All meals were prepared and served at the metablic unit in the Department f Fds and Nutritin at Oregn State University. Table 4. Cnstant diet with partial cmpsitin. Amunt K Prtein Vitamin B^ Item g Cals g mg (PIN) Orange juice Cream f Wheat (cked) Milk Peaches Rice Casserle: Rice Carrt Celery Olive Onin (dehy.) Tmat juice Ttal Tang, General Fds Crpratin, White Plains, New Yrk. 10 Lemnade, Wyler Fds, Brden, Inc., Nrthbrk, Illinis. 7-UP, Bttled under the authrity f 7-UP Services Inc., St. Luis, Missuri.

73 Nn perishable fds were purchased in bulk s that prducts 58 frm the same lt were used thrughut the study. Perishable items such as milk were purchased n a weekly basis. Cmpsites f the cnstant diet were prepared every week and analyzed fr vitamin B, cntent by the micrbilgical methd (120). analyzed fr vitamin B. at weekly intervals. Breads were als The ral dse f vitamin B. was prepared each week by disslving 28 mg f crystalline PIN-HCl in 500 ml f 2% acetic acid. In additin t the determinatin f ttal vitamin B, cntent f the cnstant diet and the breads, these samples were als analyzed fr the amunt f the three frms f the vitamin- -pyridxal, pyridxine and pyridxamine by the methd f Tepfer and Plansky (119). Metablic Study The adjustment perid was helpful in acquainting the subjects with the rutine f the study, s that they were well prepared fr the experimental perids that fllwed. They were asked t main- tain their nrmal activities during the study. Bdy weights were recrded each day. At the beginning f each experimental perid, the subjects were given a fecal marker f FDA Blue N. 1 dye in a gelatin capsule t mark the beginning f each dietary perid. All the fecal specimens were cllected in dispsable cartns labelled with the subject's

74 59 initials, date and time f sample cllectin. Fecal cntent was weighed daily and stred frzen (-20 C) until they were analyzed fr ttal vitamin B.. Cmplete 24-hr urine cllectins were made thrughut the study. All daily urine specimens were stred refrigerated at 4 C under a layer f tluene. They were mixed and measured fr ttal vlume the fllwing mrning. Abut 15% f the ttal urine vlume f each subject was stred frzen (-20 C) in plastic cntainers until it was analyzed fr creatinine, ttal and free vitamin B, and 4- pyridxic acid,, On the 1st, 3rd and 5th days f each perid, fasting bld samples were drawn frm the antecubital veins f the subjects by a medical technician. Heparinized vacutainer tubes were used fr this purpse. determined. Hemglbin cncentratins and the hematcrits were Plasma was separated and stred frzen (-50 C). Erythrcyte transaminase activities and plasma vitamin B. as well as pyridxal phsphate were determined by anther investigatr as a separate part f this study. Amng the varius analyses dne n the bilgical material, urinary and fecal vitamin B., and urinary 4-pyridxic acid will be discussed in this thesis.

75 0 Analytical Methds Prir t the assay f vitamin B, by micrbilgical methd, the material t be assayed first requires hydrlysis. This step varies smewhat frm material t material. Sample preparatin and the hydrlysis prcedures used with the diet, feces and urine samples will be discussed in the fllwing sectins. Analysis f the Diet fr Vitamin B-. The diet cmpsite was prepared by blending tgether a day's cnstant diet f knwn weight in a Waring blender. After additin f each item, the weight was nted and the cmpsite was pureed fr tw minutes. Finally the ttal weight f the cmpsite was nted and 1-2 gm f a well- hmgenized sample was accurately weighed int a beaker. This was fllwed by hydrlysis n the same day t release the bund frms f vitamin B,. Samples f each bread were analyzed separately fr vitamin B, cntent. Five representative slices f bread were taken frm a randmly selected laf. One half f each f these 5 slices was placed in a Waring blender and blended until it frmed finely divided, well mixed bread crumbs (generally set at "puree 11 and blended fr 3-4 minutes). A representative sample (1-2 g) f this was weighed and hydrlyzed.

76 1 The hydrlysis step fr bth the fd cmpsite and breads were carried ut by autclaving the sample with apprximately 150 ml f 0.44 NHC1 at 15 lbs pressure fr 2 hurs. After cling t rm temperature, the ph was adjusted t 4. 5 with ptassium hydrxide. The slutin was then made up t a knwn vlume and filtered thrugh Whatman N. 40 filter paper. This filtrate was used in the assay f vitamin B. by micrbilgical methd as dis- cussed previusly. The extracts f the fd cmpsite as well as f the three types f bread frm ne week were used in the separa- tin f the three frms f vitamin B, by the prcedure f Tepfer and Plansky (119). Each frm was assayed by the micrbilgical methd, using Saccarmyces carlsbergensis. A standard curve f each frm f vitamin B, was used t determine the unknwn cncen- tratins f the crrespnding frm. Fecal vitamin B,. The fecal cllectins f each subject during ne experimental perid were pled tegher in a large cntainer. They 'were thawed and then mixed n a rtary shaker fr 2-3 hurs. The pling f cllectins was dne in rder t minimize the number f samples that had t be analyzed fr vitamin B-. A representative sample prepared in the abve manner'was subjected t hydrlysis, by autclaving at 15 lb pressure with 0.44 N HC1 fr 2 hurs. This was fllwed by adjustment f ph t 4. 5,

77 2 making up t a knwn vlume with redistilled water and filtering. The filtrate was used in the micrbilgical assay. Urinary Vitamin B,. Ttal and free vitamin B. was deter- mined in the urine samples cllected n the 2nd, 4th, th and 7th days during each f the three experimental perids. The days, 7, 2 and 4 respectively represented the days immediately befre the days n which fasting bld samples were drawn. Urine samples frm days 1 and f the adjustment perid were als analyzed fr ttal and free vitamin B.. b Prir t the determinatin f ttal vitamin B, in urine, the b samples were subjected t acid hydrlysis. Ten milliliters r 1% f the ttal vlume f urine (whichever was smaller) was autclaved with 50 ml f 0. 1 N HCl at 15 lb pressure fr 30 minutes. In the determinatin f free urinary vitamin B,, 50 ml f redistilled water were added t the urine sample. N autclaving was invlved. Frm this stage n, bth samples were treated identically. The ph was adjusted t 4. 5 using either KOH r HCl. The vlume was made up t 100 ml with redistilled water and the mixture was filtered thrugh Whatman N. 1 filter paper. This filtrate, after subsequent dilutins, was used in the micrbilgical assay fr vitamin B,. A sample f urine was analyzed with every ther assay t determine the variability f the methd as well as t determine if there

78 3 was a change in the vitamin B, cntent f the urine during strage f the sample. The recvery f a knwn amunt f the vitamin B, added t a urine sample was determined with each assay. The hydrlyzed extracts f the urines f five selected subjects (1, 2, 4, and 7) n the sixth day f adjustment and f each f the experimental perids were used fr determinatin f pyridxal, pyridxine and pyridxamine. Recveries f the three frms when chrmatgraphed alne and when mixed with a sample f urine were determined. The prcedure described by Tepfer and Plansky (119) was fllwed. Urinary 4-Pyridxic Acid. Samples f urine cllected n days 2, 4,, and 7 f each f the experimental perids and days 1 and f the adjustment perid were analyzed fr 4-PA. The methd f Reddy, Reynlds and Price was used (105). This invlved in exchange chrmatrgaphy t separate 4-PA frm ther flurescent cmpunds in the urine, fllwed by flurmetric determinatin. 12 Aminc-Bwman spectrphtflurmeter was used t read the An flurescence. 12 Aminc Bwman Spectrphtflurmeter, American Instrument C., Inc., Silver Spring, Maryland.

79 4 Statistical Analysis The data were statistically analyzed by the methd f analysis f variance (ANOVA) using the system f statistical package fr the scial sciences (SPSS). The analysis was dne by a statistician using the cmputer at Oregn State University. The pertinent data frm ANOVA tables are given in Appendix fr all the variables analyzed.

80 5 RESULTS AND DISCUSSION Stability Stability f Vitamin B, During Bread Making The ttal vitamin B, cntent f mix, prf and bread prepared frm WHW, WB. and W flur, under cmmercial and hme baking (straight dugh and spnge dugh methds) cnditins is given in Table 5. The means presented in Table 5 are graphically shwn in Figure 4. The difference in the vitamin B, cntent between mix b and prf, prf and bread were tested fr significance at 5% and 1% using Student's t test. Amng the three types f flur used, WHW and W shwed a decrease in the vitamin B, cntent during fermentatin. This decrease was bserved in breads prepared under bth cmmercial and hme cnditins. In the case f WB. flur, hwever, there -was b an increase f 2-8% in the vitamin B, cntent at the prf stage as cmpared t that f mix in bth hme and cmmercially prepared breads, f which the ne prepared using the spnge dugh methd was significant (P <0.05). The increase is prbably due t vitamin B, synthesis accmpanying yeast grwth during fermentatin. Keagy et_ al. bserved a similar increase in flacin cntent during fermenta- tin, and attributed this increase t synthesis f flacin by yeast (181).

81 Table 5. Vitamin Bg cntent f mix, prf and bread frm WHW a, WBg and W flur, prepared under cmmercial and hme cnditins {ng pyridxine/100 g sample, dry basis). WHW ^ ^2 R 3 Mean + SD C R l R 2 WBg R 3 Mean + SD R l R 2 W R 3 Me; an + SD COMMERCIAL Mix Prf Bread HOME MADE a) Straight-dugh methd Mix Prf Bread b) Spnge-dugh methd Mix Prf Bread a. WHW, WBg and W - see Appendix 5. b. Rj, R2 and R3 represent the three replicatins taken frm the same mix, prf r bread preparatin. c. Mean + Standard Deviain. O O

82 400 i- COMMERCIAL &* ^.70'' kv r '^ i22 ^t 7F> 00 ^2 400 a) Straight dugh methd HOME MADE F* S: Q. '.-'. /a -* S , -.- CP O CO CD iia,-io -sn» b) Spnge dugh ^ 00 methd ^r MIXED ^s PROOF I' - '-< - s S^>.i'.-i..';'/ 'r'^ ^ ^ VI'-. BREADS "*te n '.vlv'iv ^s^ y--\' #:',-> ^SJ WHW W-B, W Figure 4. Graphic presentatin f mean vitamin B, levels in mix, prf and bread.

83 8 The reasn that such an increase was bserved nly in WB. prf in the present study may be because the synthetic frm f the vitamin B, (PIN-HC1) used in the enrichment f white flur was readily available fr yeast grwth. Althugh the vitamin B. cntent f WHW flur was fairly high, there was n increase in the vitamin B. after fermentatin as -was bserved with WB, bread. A decrease in the vitamin B, levels after b b fermentatin was bserved fr bth WHW and W breads. These changes in vitamin B, cntent during fermentatin were nt signifi- cant. As seen in Figure 4, the vitamin B, level f cmmercially prepared WB, bread was lwer than that f hme baked breads. This difference was due t the lwer level f vitamin B, enrichment (1. 12 mg PIN/lb f flur) f cmmercially prepared WB- bread cmpared t that f hme baked bread which cntained the level f enrichment (2 mg PIN/lb f flur) recmmended by the NAS (9). The reasn fr the lwer level f enrichment f cmmercial WB, D bread was discussed in detail earlier. The mean vitamin B, level bserved at different stages in the cmmercial bread making prcess using WHW flur was slightly lwer than the crrespnding level bserved under hme baking cnditins. These differences, hwever, were nt significant. ^There was a higher level f vitamin B, at all stages in the three

84 9 types f bread prepared by the spnge-dugh methd cmpared t the straight dugh methd under hme cnditins. This culd be attributed t the grwth f yeast and prbably synthesis f vitamin B, during the fur hurs f fermentatin f the spnge cntaining 0% f the flur, water and all the yeast, befre being mixed with the 'dugh' ingredients (Appendix 2). The ttal fermentatin time fr spnge-dugh methd was 330 min cmpared t 235 min fr the straight dugh methd. Baking lsses in the cmmercial breads ranged frm 10-15% (mean 13%). Of these breads, the nes made frm WHW and W flurs had a significant baking lss (P< 0.05), cmpared t the ne made frm WB. flur. There was a 0. 5 t % (mean 3. 5%) lss due t baking f the bread made using straight dugh methd under hme cnditins, but this was nt statistically significant. Hwever, when spnge dugh methd was used a significant baking lss f 5-12% was bserved in WHW, WB, (P< 0 01) and W (P< 0.05) D breads. In the flacin stability study (181), Keagy et al. fund an 8% baking lss f added flic acid when the straight dugh methd was used as cmpared t 14% using the spnge dugh methd. These lsses were cnsidered t be fairly lw. The average baking lss bserved by Keagy et al. fr the native flacin was 31%. The baking lsses f the added vitamin B, in the present study were 3. 5 and 8%

85 70 fr the straight dugh and spnge dugh breads, respectively, cmpared t a 13% lss when baked under cmmercial cnditins. The cmparatively greater lss f vitamin B. in the latter case, may be due t expsure t light during baking, cling and subsequent handling f the bread. Under hme baking cnditins, precautins were taken t minimize undue expsure f the dugh and bread t light. It is seen that bth flacin (181) and vitamin B, (present study) were lst t a greater extent when the spnge dugh methd was used, cmpared t the straight dugh methd. A higher mean level f vitamin B, was bserved in the mix, prf and bread prepared by spnge dugh methd as cmpared t straight methd. Perhaps, the vitamin B. synthesized by the yeast during the lnger fermentatin perid using the frmer methd was mre easily destryed by the baking prcess as cmpared t the added vitamin B,. Gd stability f added pyridxine in bread baked frm enriched white flur has been reprted (182, 183). The greater stability f pyridxine, which is predminant in wheat and which is als the frm used fr enrichment may have cntributed t the lw baking lsses bserved during bread making. Stability f added Vitamin B. during Bread Strage Vitamin B. cntent f the bread stred frzen (-5 C), under refrigeratin (5 C) and at rm temperature (27 C), ver varying

86 71 lengths f time is presented in Table. The mean vitamin B, level f bread stred frzen ver a perid f seven weeks ranged frm 18 t 210 jjlg/100 g. There was n trend bserved in the distributin f these means. Similarly n definite pattern was bserved in the vitamin B. levels f the bread stred under refrigeratin. These results indicate that there was n lss r gain f vitamin B, when the WB, bread was stred under frzen cnditins r in a refrigeratr. On the ther hand, in the case f bread stred at rm temperature, vitamin B. analyses were dne n the days 0 and 3. Greenish and black types f mld, prbably Penicillium and Aspergillus species, respectively, were bserved n the bread n the fifth day f strage at rm temperature. These breads did nt cntain any preservative, therefre, it was nt unexpected t find mld grwth after three t fur days f strage at rm temperature. The temperature f the rm where the bread was stred was C. The mean f vitamin B, level decreased frm 192 n day 0 t 172 \ig/ 100 g n day 3 f strage--a 10% lss at rm temperature. This change in the vitamin B. level was statistically significant cmpared t the changes bserved in the vitamin B, levels f the bread stred under frzen and refrigerated cnditins. In cntrast t this bservatin, the results f Crt et al. indicated that there was n lss f vitamin B, even after five days f strage

87 Table. Vitamin B cntent f cmmercial WB- bread stred under three different cnditins ( j.g pyridxine/100 g). Time Strage Cnditins f Fre, Mfi!=52Cl Refrigeratr r5 0 C^ Analysis V R 2 R 3 Mean + SD b R l R 2 R 3 Mean + SD Ri..Rppm tsm R 2 R 3 t27 0 C) Mean + SD day day week week week week week week week a. R,, R. and R, refer t the three replicatins taken frm the same laf f bread. b. Mean + Standard Deviatin

88 73 at rm temperature (183). The 10% strage lss f vitamin B. bserved in the present study may be due t the temperature and/ r expsure t light. The results f this sectin f the study suggest that strage f bread under frzen cnditins r in a refrigeratr will retain vitamin B, better than strage at rm temperature. Bread stred in the refrigeratr tends t stale faster than the bread stred either at rm temperature r under frzen.cnditins (188). Hwever, as indicated by the results f this study, retentin f vitamin B, is greater when stred in a refrigeratr than when kept at rm temperature. Stability f Added Vitamin B, during Flur Strage Vitamin B, levels f all purpse flur enriched with pyridxine hydrchlride and stred under three different cnditins ver a perid f 2 weeks (six mnths) are presented in Table 7. The means f the triplicate analysis indicate that there was practically n lss f vitamin B, f the WB, flur when stred at rm temperature, in a cld rm r under frzen cnditins. Althugh flur is generally nt stred frzen, this cnditin was selected in rder t establish a cntrl fr each analysis. Vitamin B, f the flur samples were determined at the times indicated in Table 7. The value determined fr the flur stred in

89 Table 7. Vitamin B cntent f WBg flur stred under three different cnditins (u_ g pyridxine/loo g). Time Strage Cnditii n f Frzen (-45 0 C) Cld strage (5 C >C) Analysis R 2 R V R 2 R 3 Mean + SD b R Mean + SD l R 3 l Rm tern perature (27 0 C» Mean + SD R 2 R 3 week week week week week week week a. R,, R and R- are the three replicatins taken frm the same lt f flur. b. Mean + Standard Deviatin. O ^

90 75 the cld rm and at rm temperature were expressed as a percentage f the -45 C cntrl flur analyzed at the same time. These percentages are given in Table 8. Table 8. Vitamin B, f flur stred under different cnditins expressed as a percentage f the C cntrl flur analyzed at the same time. Time f Analysis (Wk) Cld Strage (5 C) Rm Temperature (27 C) There was n trend bserved in the changes f the vitamin B, levels f the flur. This bservatin was similar t that f Keagy et al. (181), wh tested the stability f the added flacin during flur strage. Hwever, the native flacin f the flur in their study shwed an expnential decay pattern. In the present study, the stability f native vitamin B, in flur was nt tested. Frm the results btained n the stability f added vitamin B, t flur, under the cnditins f the present study, ne culd cnclude that there was gd stability f vitamin B. in flur during strage. The stability bserved f the added vitamin B. in bth bread and flur may be due t the inherent stability f PIN, the frm generally used in the enrichment with vitamin B..

91 7 Biavailability Subject Respnse t the Diets The calrie cntent f the cnstant diet tgether with the bread was 2029 and 2272 K cal fr whle wheat and white bread diets, respectively. In additin t this, the subjects were allwed t cnsume extra calries in the frm f margarine, jelly, sugar, candy and beverages. When necessary, these extra calries were cn- sumed t maintain bdy weight. Hwever, sme fluctuatin in bdy weight was bserved fr sme subjects during the study. The initial weight and the weight at the end f each experimental perid are given in Table 9. The average additinal calries cn- sumed per day by each subject during the three week experimental perid are als listed. Data listed in Table 9 are presented withut taking int accunt the rder in which the bread was fed. Subjects 2, 4, 7 and 8 shwed a reductin in weight during the experimental perid as cmpared t their initial bdy weight. Hwever, these reductins were nt significant. Of the abve subjects, 2 and 8 cnsumed nly 90 and 210 extra calries/day, respectively althugh they were encuraged t increase their calries in rder t maintain weight. The RDA fr men f the age categry 21 t 33 years is calries (9).

92 Table 9. Bdy weights and extra calries cnsumed per day. Subject Mean + SD a Bdy weight (kg) Initial End f perid WHW b WBg W Average extra calries/day (K-cal) a. Mean + Standard Deviatin b. WHW, WBg and W - see Appendix 5.

93 78 In spite f cnsuming apprximately 2350 and 175 extra calries per day, subjects 4 and 7, respectively, shwed sme lss in bdy weight. This was prbably due t their physical activities. Subject 3, wh was als physically active, shwed a reductin f 1 Kg with WB. bread during the first experimental perid, fllwed by a gain in weight f abut 2. 5 Kg with WHW diet and finally a weight lss. This same subject als cmplained f fatigue and was examined 13 by a physician. He was administered an ral supplement f irn, three times a day frm day 4 f the first experimental perid. Subject 5 gained Kg f weight in spite f cnsuming nly 3/4 f the daily quta f bread. This weight gain prbably ccurred because the calrie value f fd eaten during the study was far mre than what he had been previusly cnsuming. In spite f cnsuming an average 3829 extra calries, N. did nt gain weight because f his heavy physical activity. In general, all the subjects were maintained in gd physical cnditin. Since each subject was his wn cntrl, the weights f all nine were averaged. The mean and standard deviatin fr the starting weight was Kg cmpared t , and fr WHW, WB, and W bread diets, respectively. The changes in the bdy weights during the three experimental 13 5 g FeSO, b 4

94 79 perids were nt significant (Appendix ). The average weight at the start f the experiment was higher than the average weight at the end f each experimental perid. This indicates that either the bread diets r lack f calries caused sme reductin in weight f mst f the subjects. Hwever, there was n set pattern in weight reductin in relatin t the type f bread. Claims have been made that diets cntaining a substantial amung f bread can cause a significant lss in bdy weight (189). This was fund t be s in a study invlving tw grups f eight-men wh were n diets based n tw types f bread, a cmmercially prepared supermarket type and a specially prepared lw fat, high fiber type fr 8 weeks. The grup receiving the latter type f bread lst mre weight n an average (19.4 lb) cmpared t the ther grup (13.7 lb). These subjects received 12 slices f bread daily, and ther fds f their chice. Bread prvided bulk and reduced the appetite fr many f the ther fds. In the present investigatin, the subjects cnsumed 22 slices f bread daily alng with the ther fd items listed in Appendix 4. Of the ttal calries fr the cnstant diet tgether with the breads 7. 5% and 71. 0% were supplied by WHW and the white breads, respectively. Abut 80% f the prtein in the diet was derived frm the bread. In a study n wheat flur as a surce f prtein fr adult human, Blurchi et al. reprted that subjects were held in nitrgen balance

95 80 when fed a diet in which white flur prvided 90-95% f the daily- prtein intake (11). They als demnstrated that a 2500 K-cal diet high in bread wuld prvide mre than an adequate amunt f all the essential amin acids. Dietary Intake f Vitamin B, Althugh the RDA fr vitamin B, fr human adults is 2 mg, a level f 1. 5 mg was selected as the daily intake, fr reasns dis- cussed earlier in this thesis. A level f 1. 5 mg f vitamin B, was reprted as being marginally adequate fr human adults (89, 190). The requirement fr vitamin B, depends n the level f prtein intake (4, 5); a daily intake f 1. 5 mg f vitamin B. is cnsidered a minimum when the diet cntains 100 g f prtein (). In the present study, the daily intake f prtein was 5- g, and the 1. 5 mg intake f vitamin B, was therefre adequate t meet the requirements f the subjects. The amunt f vitamin B. cntributed by the different cmpnents f the diet as determined by the micrbilgical methd (120) is presented in Table 10. The vitamin B, cntent (as pyridxine) f the cnstant diet (bread nt included), when calculated using a fd cmpsitin table was mg (5). The assayed values ranged frm t ( mg). Such a difference between the calculated and

96 Table 10. Ttal vitamin B. level in the different cmpnents f a day's diet. Variable WHW WB, W D Bread Cnstant diet 0.38+^ ^0.01 Oral dse TOTAL Vitamin B,, expressed as mg f pyridxine (means f at least three assays).

97 82 assayed values can be expected when dealing with fds, due t varietal differences and variatin in cking and handling. In additin, there is als a 10-15% variability inherent in the micrbilgical assay fr vitamin B,. The amunt f PAL, PIN and PAM in the cmpnents f the diet are given in Table 11. Als listed are the percentages f the ttal vitamin B, accunted fr as the three frms. D The amunt f PAL, PIN and PAM in the diet cmpnents are presented graphically in Figure 5. The percentages f the three frms f vitamin B. as assayed in the diet cmpnents, and the amunts calculated frm literature values (5) are presented in Table 12. In the case f the cnstant diet, the calculated amunts represent the levels f PAL, PIN and PAM in the three majr fd surces f vitamin B,: range juice, milk and tmat juice. Pyridxine was fund t be the predminant frm f vitamin B, b in the ttal diet fllwed by pyridxal and a smaller amunt f pyridxamine. The diets f plant rigin are generally reprted as being higher in PIN cmpared t PAL and PAM (151). Yan and Fujita determined that there was a large amunt f PIN and PAL, but nly a small prprtin f PAM in the vegetable diet given t their subjects (103).

98 83 Table 11. Levels f the three frms f vitamin B/ in cmpnents f the diet (mg) a. Frms Vitamin f B Bread Cnstant Oral C WHW b WB W diet dse PAL PIN PAM Percent f the ttal Vitamin B^ accunted fr as three frms a. Amunts adjusted t 100% recvery b. WHW, WB and W, see Appendix 5 c. Assumed t be 100% PIN Table 12. Assayed and calculated percentages f PAL, PIN and PAM f vitamin B, f the diet cmpnents. Diet A ssaye id Calculated Cmpne nt PAL PIN PAM PAL PIN PAM WHW WB * 78* 10* W Cnstant * Calculated values fr WB, bread were based n thse f W bread and the amunt f pyridxine hydrchlride added in the enrichment.

99 cd cd cd 0.3 CD OQ E 'M ss? *7n *& ezu WHW Ttal Intake 1.58 (mg) : ' ): '. iil'i'l W-B 1.5 cd: t?**^ ^ d W 1.54 PAL IS b bread PIN PAM^3 ^ ^ ^i cd cnstant diet d ral dse Figure 5. Three frms f vitamin B, in the diet cmpnents.

100 85 Fecal Excretin f Vitamin B. The fresh weight and the vitamin B. cntent f the feces fr the three experimental perids are given in Tables 13 and 14. In Table 13 the values are presented in the rder in which the three types f breads were served, whereas the values in Table 14 were tabulated fr each subject, withut taking int accunt this rder. The data in Table 14 shws that the fresh weight f feces f all subjects fr WHW bread was cnsiderably higher when cmpared t the crrespnding values fr the ther tw types f bread. The mean fecal weight fr the WHW bread diet was g/wk, cmpared t and ^448 fr WB, and W breads, respectively. The WHW bread diet was higher in fiber (11.08 g/day) cmpared t the diets based n the WB, and W breads (3. 1 g/day). The markedly higher stl weight assciated with WHW bread may be due t the higher fiber cntent and the ability f the fiber t hld water. Suthgate et al. have shwn that cnsuming a diet high in fiber causes an increase in the stl weight, bth misture and dry matter (191). They als reprted that diets high in fiber tend t have a shrter transit time in the gut. This culd cause excretin f sme nutrients which therefre wuld nt be available t the bdy.

101 Table 13. Vitamin B_ cntent and weight f feces in subjects fed diets based n WHW 0, WB and W breads. Bread Type WHW WBg W Subject Weekl Weight Vitamin B, Subject Week 2 Weight Vitamin B^ Subject Week 3 Weight Vitamin B, Overall mean + Weight Standard deviatin Vitamin B, a. Fecal vitamin B expressed as mg f pyridxine per week. b. Fecal weight in grams f fresh feces per week. c. WHW, WBg and W see Appendix 5. 00

102 Table 14. Vitamin B, cntent 3 and weight f feces during the experimental perids, Subject Mean + SD d WHW C weight Vitamin Bg W-B c weight Vitamin B, W weight Vitamin B^ D a. Fecal vitamin Bg expressed as milligrams f pyridxine per week. b. Fecal weight represents the fresh weight in grams per week. c. WHW, WBg and W - see Appendix 5 d. Mean + Standard Deviatin. 00

103 In the present study, higher levels f fecal vitamin B. were D assciated with higher fecal weights. This relatinship can clearly 88 be seen in Figure. The mean fecal excretin f vitamin B, while subjects were fed WHW bread diet was mg/wk. This level f excretin was significantly greater (P <0.01) than the and _ 0. mg/wk, when they were fed WB, and W bread diets, respectively (Appendix ). Hwever, n the basis f per gram f feces, vitamin B, values were _ 0. 4, _ 0. 7 and _ 0. 9 ng fr the diets cntaining WHW, WB, and W breads, respectively. This shws that althugh the ttal vitamin B, excreted in feces was b higher when WHW bread was fed, the cncentratin f the vitamin in feces was lwer during this same perid than during the perids when WB, and W breads were fed. Data f Yan and Fujita indicated a similar pattern (103). When the diet was changed frm an rdinary mixed type t a vegetable diet, the vitamin B. cntent f the feces increased. Additin f cellulse t the vegetable diet resulted in further intensificatin f this effect. The reasn fr a higher level f fecal vitamin B, when WHW bread was given, culd be related t the vitamin B, in whle wheat being present as phsphate frms. Sme f these frms may escape the actin f alkaline phsphatase n the brush brder f the intestine where the phsph- rylated frms are hydrlyzed t the free frms which are then available t the bdy. Fecal excretin f vitamin B, accunted fr

104 O 5. CO.g e 3, ^ 4> <!> «! f r 2900 Fecal weight (g/wk) Figure. Scatter diagram f fecal weight against fecal vitamin Bg f the nine (1-9) subjects, when fed experimental diets based n Q WB and ^ W breads. O WHW, 00

105 an average f percent f the dietary intake f the vitamin 89 when WHW bread was fed. The crrespnding percentages fr the WB, and W breads were _ 5. 0 and 28. +_ 4., respectively. These values were calculated taking int accunt the lwer level f vitamin B, intake (1. 2 mg) f subject 5. When expressed as a percentage f the intake f vitamin B,, the fecal excretin f the vitamin when the diets were based n WB- and W breads gave values which were clser t thse reprted in litera- ture. Denk et al. reprted that and 21. percent f the vitamin B, intake was excreted in feces when their subjects were n diets cntaining 1.05 and 1.7 mg f vitamin B-, respectively (101). Levy fund 23 and 18 percent f the daily intake f 2 and 3 mg f vitamin B. excreted in feces f his subjects (98). Urinary Excretin f Vitamin B, Urinary excretin f ttal vitamin B, in the samples cllected n the 2nd, 4th, th and 7th days f the experimental perids and day 1 and f the adjustment perid are presented in Table 15. The rder in which the three types f bread were served is als indicated. The starting value fr ttal urinary vitamin B z in seven ut f nine b subjects was higher than their value n the last day f the adjustment perid. The ttal vitamin B, level in urine during the experimental perids was lwer in almst all the subjects cmpared t the level

106 J3_ Q a ««ID in O CO CM «5 d tv * lo CM Tf lo O CO Tji K * ^ *-l t-h ^-H O CO O O O > \ i i 5 * O T-I CM Ol s +1 tv tv c ^K T}" t~s 00 CM CO CO CM * > Tf CO Tf tv l-h 8 CM ?l CO tv Ol Ol 00 CM CO CO tv iv tv IV t to t t "> +1 T-I T-I T-I T-I T-I ' in in * ^ CO c * tv i* m -^t" CM CM to t 00 in CO d CM CM t d rh T-t 1H 1H tv t m + 1 Oi CO CM CM t 00 1H i-t 1H CO 1-i d + 1 tv tv m CM m ^f CM t CO CM i in in 15 r-i 00 1H T-t t 00 T-t + in CM t tv t t t tv t * % CM tv i-i TJ" in * * t tv CO * U1 rt i-* Ol * CO CO CM t l-( T-t d + T-t d r-t K T-i m c ^ w ai IH t ^ ai Ot + i* tv c t tv t > \ (M DO 00 CM O in CM i-l ^ "* 5 *" CO CM CM TC * CM Ol 00 t (M CO CO i m * c ^ 00 Tl > > CM in CM CO CO CO r T-I - +i i i-i in ^ CM t tv CO CO CM pa n 1 tv.<j< O r-l c m Tf Tf CO T-I O 00 <7> a> + ai -"t i, <~> O t->? *H ih 00 CTi CO [V 00 tv ^ T-I t CM m t 0\ 0\ 0\ ^ in t CM CM m CO?»H i * tv S in T-t CM "^ TH I-( T-H T-I * Tf >-i tv t <M CO in N tv t in * * 1-4 T-l l-h 1H 2 +1 tv in t CM in (v CO CO CO CM t in CM TJ" t O O 0> CM CM CO ""t ^ > T-< T-I T-I O in c 2* i K CM i-l W O 00 i-i tv ih O O i-h If CO CO CO tv CM <S\ <3\ tv O <Ti 0\ <3\ 3 TH t Q «w < Q Q CM * to tv rt rt rt w Q Q Q Q. a Q in en + 1 CM + 1 CO a ja CM it to fv a j^ncm <(< t K 1 1 4) n «Q Q Q Q ^ Q Q a

107 Table 15. Cntinued Subject Number Overall Mean + S.D. Mean + SD b Means WHW WB W a. WHW, WB and W see Appendix 5 b. Mean + Standard Deviatin. ^D

108 92 bserved fr day 1 f the adjustment perid. Since the urinary excretin f vitamin B. is an indicatin f the level f vitamin B, b intake n a shrt term basis, the values bserved in the present study suggest that the daily intake f vitamin B. by the subjects a day r tw prir t the study was prbably greater than the 1. 5 mg level they received during the study. Subject excreted 30 ^g f ttal vitamin B, in 24 hr urine, which was higher than the amunt excreted by ther subjects. The reasn fr this higher level is related t the subject being n a vitamin supplement up t tw weeks befre beginning the present study. Subject 1, 5 and had lwer levels f ttal vitamin B, in urine during the perids they received WHW bread cmpared t the values bserved during the ther tw perids. Subject 8 shwed a tendency tward a gradual decrease in his urinary vitamin B. as the study prgressed, prbably indicating a gradual adjustment t the level f vitamin B. intake. A smewhat similar trend was als bserved in subjects 1 and 3. Tward the end f the study, the urinary vitamin B. levels f all the subjects appeared t be clser tgether, with a range f 8 t 175 (jlg/24 hr cmpared t 107 t 307 [xg/24 hr at the beginning f the study. This suggests that the subjects apprached a cmmn vitamin B, status as the study prgressed. Althugh ne bjective f the adjustment perid was t achieve such a cmmn status,

109 93 the six days f adjustment did nt seem t be sufficient. The verall mean and standard deviatin fr ttal urinary vitamin B, was , _ and (ig/24 hr fr the perids when WHW, WB, and W breads were served, respec- tively. The slightly higher value bserved with WB. bread did nt appear t be significant (Appendix ). The urinary excretin f free vitamin B, are given in Table 1. These figures als fllw a pattern very similar t that f the ttal vitamin B, in urine. The verall means fr the free vitamin were , and u.g/24 hr when WHW", WB and W breads were fed, respectively. Sauberlich, Skala and Dwdy have reprted that with a vitamin B, intake f 1. 5 mg, abut ug f free vitamin B//24 hr is excreted in the urine (49). This range is lwer than that in the present study. The ttal and free vitamin B, levels reprted in the present investigatin are mre in agree- ment with the values reprted by Kkkeler (117). The means and standard deviatins reprted in this latter study were 13 4^73 and 83 H-^ 0 Mg/24 hr fr urinary ttal and free vitamin B,, respectively. The rati f ttal t free urinary vitamin B, is presented in Table 17. The rati fr each subject remained cnstant, but there was cnsiderable variatin between subjects. Accrding t Scriver and Cullen the ttal urinary vitamin B, excreted by the healthy subjects n regular diets was five times the amunt f the free

110 Q CO + 1 s n 0) s in CO r lo s 94 \ t CM O K <0 00 Ol CO d in «>»H 1^ t-t > in CM * CM' a^ > i v in lo CO t^ tv (O > +1 "> O tx l > IO in c Oi t^ tv t-^ in in in O c en tv * lo rh CM - +1 O l^ TH T-< 0 vd CM i in in r ^i in th in -rf :> O rh Oi fv ^ CO CM Oi 00 CM CM CM r t^ t^ * CO OO Ol O O) r^ t^ in CM. 00 Ti 00 «5 CT> Oi 00 K K d + 1 i c i * in ^ fv tv tv in CM rt m in i in CM r 10 in m v 00 in h. ^ + Tj«tH <*< CM tv * * Ol th CM CO CO CM CO CO. t-( th th t-h rl + < * t^ in i * CO CO CM CM -- tv M * i CM * m K t^ i R Oi t^ CO r v th i < >D m in» C01 «> * Oi CM V3 Oi th O m 10 i ^ CM i-i c ai t^ Oi t^ CM m ^-t O O TH ^ + 00 th > \ t^ CM ^ Oi th \ m CO > rh + Cv 00 * CM th th f- 00 O O Ol Oi «3 m.g in * * in O Tf CM r rrpco O Ol CM 5 ^> m in < CM (O t-s t O th th ttf «> ( m in tx it t* c in c in in m in (U Tj< I>N CO CO 00 O in T-* t^s CM 00 O O CO -sf O ih - +1 r i CO 00 CM th CO CM th CM CM 00. th th th rh th +1 CM c * m c CM ^f O Oi 8 Oi O N in > CM CM Ol ^7 CM th m th th c in t^ t^ ^ tv CM. CM P ' tv in + 1 * in * s s s s a) a f- cu E- Q < - "3 a? CM Tf lo t-s ^3 Q CO CM re <O f^ CM Q a! CM rji «5 [>- CO «-5 Si J» Q ^

111 Table 1. Cntinued. Day Overall Mean + S.D. Mean + S.D. [ Means WHW WB W a. WHW, WB, and W see Appendix 5. b. Mean + Standard Deviatin. s

112 00 +1 «(U K O ^ v Oi CO CM CM CO lo t-. * i fvj d in ck t 00 W3 in d tv O TH OS T-t lo CM r r t t CM CO d d d rh r-t y-l CM ^-i rh th»-( *-* i-i *-* CT> ^ Oi l-h t-^ CO CM Ol r (O t-* * CO * in 00 ui CM r CM <?, d < i lo 00 lo in (?\ «3 (M rj CM & 0\ <Ti T-4 v-i y-i tv 00 K in in in Ol t-t lo TH CM T-1 th CO IH 'H ^-t CTl i v CM CM CM v 10 Tf *-t t^ > 00 v in in t in * O < a\ i rh O CM Ol CM O tv Ol CM > t TH i v <*»-l Tf t-c l-h T-I CM I-H in TH CO Oi rh Ol < O i t^ tf v O O Ol tv * (O K O t rh O CM s t CTv t TH in CM. in CM K t^ rh ^-H r-h tv t-. t CM O in in m O * in tv i K in rh t^ VO v VO ^3 CXl lo VO IO t^ CO CM O fh rh 00 t c in O «* 00 CO fo rh Ol CM rh O 0\ CO 00 rh t^ tv rh * Ol in in tv t^ Ol 1 Tf r^ N VO f~ O CM O CM v rh to * in rh O CM rh 1* CM CM O CM * O t^ TH CM O t Ol rh Ol VO t^ VO rh rh rh tv 00 K tf in * Oi 00 O 00 >;( t i t CO CM CO tv in O i t rh Ol O O O i CM CO CM i i in tv h» v m * to rh K v in K m * m i < in ^ d <J\ t^ < > in in m O 00 CM tv lo tv t O (» fv fx «p W frr 1H rh Oi * t Tt< v t t^ m Oi t m r c CM m rh t * O CM CM rh CO CO CM CM t t 00 t-n t in v v v rh VO <* Ol v in n, x v m Ol Ol Ol O rh O CM O in v O rh in N v in in in O rh CO in in in c t -^ r r r t^ in v K rh 00 Ol B > I f- s «*.-H CM? ^ 1 < a +J fs «* 2 CM <U Z2. <u H v 0) m a a fi H 4-) i «W f-h 8 bo VO(U CQ fl H a H.3 B a <u t-) h? Q) b b i v 4-> n V n CS ^> ^ fs CO v m s *

113 Table 17. Cntinued. Diet Subject Number Overall Mean + S.D. a Ttal vitamin Bg WHW as % intake WB W Free vitamin B, WHW as % intake WBg W a. Mean + Standard Deviatin b. WHW, WB and W See Appendix 5.

114 98 vitamin excreted (192). In the case f vitamin B, dependant subjects receiving mg f pyridxine daily, the ttal urinary vitamin B, excreted was nly twice that f the free vitamin. The rati f ttal t free urinary vitamin B, in the present study ranged frm t 1. 90, which is less than that bserved by Scriver and Cullen in their healthy subjects (192). They reprted 0. 1 and mp, mles/ Kg/hr fr the ttal and free urinary vitamin B, excreted, respectively. When expressed n the same basis, the average ttal and free urinary excretin f vitamin B. in the present study are 0.42 and m jl mles/kg/hr, respectively. The rati f ttal t free urinary excretin f vitarnin B, bserved by Kkkeler (117) in healthy female subjects ranged frm 1. 5 t 2, 5, which is clser t the range bserved in the present study, than the rati reprted by Scriver and Cullen. In the study dne by the latter grup, hydrlysis f urine was carried ut by autclaving 5 t 10 ml f urine in 180 ml f N HC1 at 20 psi fr 5-7 hr in cntrast t the 30 min autclaving time used in the present investigatin as well as in the study by Kkkeler. The difference in the ratis f ttal t free urinary excretin f vitamin B. in the abve studies may be due t the difference in the autclaving time. Further, Scriver and Cullen used urine samples cllected between midnight and 8 am befre breakfast while 24 hr urine cllectins were made in the present investigatin. There was n marked difference in the ratis f ttal t free urinary vitamin B,

115 99 bserved with the three types f bread. Hwever, tw ut f nine subjects (N. 2 and^) had substantially higher ratis with WHW bread cmpared t thse bserved with the ther tw types f bread. Urinary excretin f vitamin B. can als be expressed n the basis f creatinine. This is generally dne in cases where a 24 hr urine cllectin is impractical (49). The mean ttal and free urinary vitamin B. values in the present study, expressed as J.g/g f creat- inine are given in Table 17. Expressing the data in this manner did nt indicate a significant difference amng the breads. Accrding t Sauberlich et_ al, a urinary free vitamin B. level f 20 M.g/g f creatinine generally reflects a marginal level f vitamin B, intake (49). In the present study, the free urinary vitamin B, level ranged frm a minimum f 33 t a maximum f 93 [ig/g f creatinine, with a mean value f 57 (Table 17). This indicates that the 1. 5 mg vitamin B, intake was sufficient. Cnsidering the value f the day f the adjustment perid as 'basal 1, the ttal and free urinary vitamin B, f the experimental perid were calculated as a percentage f the crrespnding basal value f each subject. The results f ttal and free vitamin B, b expressed in this manner are presented in Table 17. There were n marked differences in the ttal and free vitamin B. values during the three experimental perids, even when the data

116 100 were expressed in several different ways as presented in Table 17. This suggests that the type f bread had n appreciable effect n the urinary excretin f vitamin B, in the nine subjects under the cnditins f the present study. The values btained fr the urine sample that was analyzed seven times with alternate assays ranged frm 114 t 121 (117 +_ 3) and 7 t 75 (71 +_ 4) j.g/24 hr, fr the ttal and free vitamin B, cntents, respectively. These results indicate that the urine sample was fairly stable when stred frzen and the variability f the micr- bilgical methd frm ne assay t anther was well within the expected range f 10 t 15 percent. When the excretin f ttal vitamin B, was expressed as a percentage f the dietary intake f the vitamin B,, values f , and were btained when subjects were fed diets based n WHW, WB, and W bread, respectively (Table 17). The crrespnding percentage f free vitamin B, were. 0 +_ 2. 2,. 2 +_ 1. 9 and. 2 +_ The lwer level f vitamin B, intake (1. 2 mg) f subject 5 was taken int cnsideratin in calculating these percentages. The data suggests that nly a small percentage f the dietary intake f vitamin B, was excreted in urine as vitamin B.. Accrding t Levy, nly abut 5 percent f the ttal intake f vitamin B, is accunted fr in the sum f PAL, PIN and PAM excreted in the urine (98).

117 As mentined previusly, the predminant frm f vitamin B, 101 in the diets was PIN. Hwever PAL represented the largest per- centage f vitamin B, in the urine f the subjects. The amunts f PAL, PIN and PAM excreted in the urine n the day 7 f each perid fr five selected subjects are given in Table 18. When PAL, PIN and PAM were expressed as percentages f the sum f the three frms (Table 19), there was a significantly lwer value f fr PAL when the subjects were fed WHW bread as cmpared t 0. 8 H^ and _ 3. 84, fr WB. and W bread diets, respectively (P< 0.01). Kelsay, Baysal and Linkswiler have reprted that PAL was excreted in the largest amunt in the urine f subjects wh received a supplement f 1. 5 mg f PIN-HC1 in additin t a self selected diet (107). In their study PAL accunted fr 0-5% and PAM 30-35% f the urinary vitamin B., with nly a negligible amunt f PIN. These prprtins have als been reprted by Sauberlich, et al. as nrmal prprtins f the three frms f vitamin B. in human urine (49). The results f Kelsay et al. (107) and thse f the present study seem t be in clse agreement althugh the percentages f PIN re- prted in the latter cannt be cnsidered negligible. Smewhat different prprtins f the three frms f vitamin B, in urine have been reprted by Cntractr ans Shane (58). They bserved 13.9, and 2. 73% f PAL, PIN and PAM, respectively in the urines

118 102 Table 18. Pyridxal (PAL), pyridxine (PIN) and pyridxamine (PAM) in the urines f five subjects ( f Xg/24hr). Subject Frm f Vitamin Bg PAL PIN PAM ADJUSTMENT PERIOD PAL PIN PAM EXPERIMENTAL PERIODS WHW PAL PIN PAM WB fi PAL PIN PAM W WHW, WB and W see Appendix 5.

119 103 Table 19. Urinary pyridxal (PAL), pyridxine (PEST) and pyridxamine (PAM) as a percentage f the ttal vitamin B^-. Frm f Vitamin Bg PAL PIN PAM Percentages in urine Adjustment perid a Experimental perids WHW b WB W Literature values (107) 0-5 little r nne Percent recveries Present study Standard Added t urine Literature values (193) Standard Added t urine a. Mean + Standard Deviatin fr five selected subjects. b. WHW, WBg and W see Appendix 5.

120 104 analyzed by a methd emplying phsphcellulse clumns. This latter methd is different frm the ne emplyed in the present study as well as that used by Kelsay et al. (107). The percent recveries f PAL, PIN and PAM f the chrmatgraphed standards cmpared against the apprpriate unchrmat- graphed standards are presented in Table 19. Additinal checks were dne by mixing knwn amunts f the standards with the urine samples. The ranges bserved in the present study were clser t 100 percent than thse reprted by Kelsay (193). Urinary excretin f PAL, PIN and PAM expressed as a percentage f ttal intake f vitamin B/ frm the diets based n WHW WB, and W breads are presented in Figure 7. The amunt f urinary PAL, expressed as a percentage f the ttal intake f vitamin B,, while cnsuming the WHW bread diet was , cmpared t _ 1. 4 and _2. 5 fr WB, and W bread diets, respectively. The pssible reasn fr the lwer percentage f PAL in urine when the diet cntained WHW bread may be because the fecal excretin f vitamin B, was cmparatively higher with this type f bread and a smaller percentage was available t the bdy. The lwer PAL cncentratin f urines assciated with the WHW bread was als reflected by the urinary 4-PA levels, which will be discussed in the next sectin.

121 105 7r PAL PIN PAM ;: c b x LU O k- c a> - ^ y WHW W-B< 1 ^ W Figure 7. Urinary excretin f PAL, PIN and PAM as a percentage f ttal intake f vitamin B, (data fr five subjects).

122 10 Urinary Excretin f 4-Pyridxic Acid The urinary excretin f 4-PA by the nine subjects during the adjustment and the experimental perids is presented in Table 20. The rder in which the three types f bread were fed is als indicated. With the exceptin f subjects 4 and 8, all subjects excreted a lwer level f urinary 4-PA when they received WHW bread as cmpared t when WB, r W bread was fed. The amunt f urinary 4-PA excreted n the day f the perid when WHW bread was fed was significantly lwer (P< 0. 01) than the amunt when WB, r W bread was fed. When the amunt f 4-PA was expressed as a percentage f the basal level (Table 21), the values fr days (P< 0.01) and 7 (P< 0. 05) f the perid when WHW bread was cnsumed were significantly lwer than the crrespnding percentage fr the perid when WB, r W bread was cnsumed. The crrespnding per- centages fr the days 2 and 4 were nt significantly different fr WHW bread as cmpared t the ther tw types, which indicates that the effect f the type f bread n the excretin f 4 PA was mre significant tward the end f the perid than at the beginning. The reasn fr this difference may be that there was sme carry-ver effect t the first few days frm the previus week's diet. The mean f 4-PA fr the days 4, and 7 was ^M/24 hr when WHW bread was cnsumed. This value was significantly lwer than

123 n I CO >H CM «107 O 'O > O cyi > IE 9i»-i i w ^ i-t CM T-* IH CM in CO R > ^ "*> S1 CM 00 tv 1H?! CM CO CO rj?l «CO CM CM CO t VO in > CO CO CO (N in in * 10 in T \0<*> 00 CM IO 0 < c i-i in in in m * cri in i-i in? Sa\ m ts in m i-i ^ 4 4 ^ * tv lo CO CO Ol R CO CM 1-4 * CO* CO * * CO ID^ C\ O^ ih (35 IH Oi tv rt ^ Tl* CO CO * CM CM 3?, 2 ^ S ST tv m «3 tv Tf c c CO tv OO tv O «5 ^ O ^ i i in * * t i in +', CM CM in i-i m * p3 'OS c R in CO CO CO CO CO?l > CO CO CO i CO C\ CO tv ^. vt: ^ 00 * * «* O CO "* ^ ^ N- CO CM CM Cxi? tv i-l ih * tv ^ CM CM i-l CM 0\ CO Q CM T OO O "O 00 CM m t CM CM CM CO (M O s CM in CM?l > Oi tv i-i 8 in 00 tv CO * * CO * " * CO * CO CO CO? (Otv Tf O 00 CQ ih,h < in? * <* CO CO * CO CO ( 50 * tv 00 PQ VO '-l r-l 00 ^ CO CO * CO 8 * c*? O IS -rt * IO tv K Ol CO CM CM CM CO T in in 3! ^ Oi O c CM CM CO CO x CM <* i-< (^ m tv -^ jji ^ CO i-l 00 5 tn c * * W5 a\ * 00 Tf CM O > i-l CM in *' in * rt tv in? CM it CM * s s CO CO CO CM 't-i k i a _ O * Q O» * 1 1 ci tv ir> e S- t f-t 00 a Q < ".2 JJ S CO 00 CO in CO It * * 00 CO * * * CO *"?! w * < tv CM a ^ 2 Q ^ a M 4) Q) Q> (U inc CM tv PQ * CO CM tv ^ * * ^ * CM * IO tv CO CM t +1 CO c Jt n l-j a <u 0) v ih Q ^ s s,-1 m ^ * X m t c ^ CO c c CO

124 Table 20. Cntinued Subject N umber Day Overall Mean + S.D. Mean + S.D Mean WHW WB W a. WHW, WB and W see Appendix 5. b. Mean + Standard Deviatin. O 00

125 +1 (U 109 CM CO CM * m, t^ 00 tv VO tv +1 v < Ol Ul CO K at T-i CM O CM TH i-l y-i + t rv CO 00 t 00 * ^. CO CM l-h 1-1 ;> i-i i-i TH 1H l-l + in 00 t tx, in tv tv > S 0 tv ^ +1 <Jl 0\ 00 CM t lo t lo»?l a> d «^P, 00 <* Ol tv 01 CTl Ol S T, CM c in CM * ih O* i-i i-i +1 > a ^ > tv tv v R?, tv <* in <* <* * 5? in t * in in in St. 0) W> m CM m i i/i CM tv O CM 00 Ol O 'l *-H W VH + t CM tv tx (O CO 00 V, * in CTI tv t t \ -, CM O -H i-h i-i rt + ex, * in w i r>. t-x * <f\ m CM ^ t>. tv tx R «?, CM CM CM i-l K IV Sip, ir> IH *-i c C! ai CM,-4 tv CO "J ai in K CM > c t t O ^H, s O 00 tv i-l, * CTl TH + ^ i-i +1 s?. X (U 1 '3 CM tv 00 to to Tt< ^ CO t CO c\ O 00 V -* ^ * 00 O CM T-1 CM i*" in CM c CO ih, g a\ tv tv T-1 l-l i-h 1-1 ~> +1 > O O O 00 ^ i-h r^ T-* to fv 0\ + CD O tv Tt tv pq a. a, 2?. Oi t c in tv tv S 1 Q & t w, a TH 2 «CM c <U S -c i 4 d) QJ 43 n a ft,3 CM * «5 tv 3 J3 Q in + 1 CM.* (U a 0) 2 ^ CM * to tv Q en + 1 CO ^ a ^ CM * 10 tv Q in + 1 0

126 Table 21. Cntinued Experimental Subject Numb er Perids Overall Mean + S.D. Mean WHW WB W _+ 20 a. WHW, WB- and W see Appendix 5. b. Mean + Standard Deviatin.

127 and ^0.78, the mean f 4-PA fr days 4, and 7 Ill when WB. and W bread were fed, respectively. Althugh the mean f 4-PA fr days 2,4, and 7 was lwer when WHW bread was fed than when WB. r W bread was fed, this difference was nt significant. The amunt f 4-PA reflects, in part, the vitamin B, that is metablized in the bdy (49). The abve results suggest that the quantity f vitamin B. while the subjects received WHW bread was lwer than the amunt metablized frm either f WB. r W breads. In the metablic intercnversins f vitamin B. (Figure 1), it is seen that PAL is the nly frm that is xidized t 4-PA by the actin f aldehyde xidase. Frm the results f urinary excretin f the three frms vitamin B., it was clear that the cncentratin f PAL in the urines f the subjects while cnsuming WHW bread was less than that during the ther tw perids. This indicates that the factr r factrs which cntribute t the lwer level f urinary PAL may als have cntributed t the lwer levels f urinary 4-PA in subjects receiving WHW bread. Frm the results discussed s far, it is pssible t infer that availability f vitamin B. frm WHW bread, the natural surce, was lwer than that frm the ther tw types f bread in which vitamin B. was prvided mainly as the synthetic frm, PIN-HC1, either incrprated int bread r in WB., r given as an ral supplement

128 with W bread. A study f the absrptin c vitamin B, frm a natural and a synthetic surce was dne by Nelsn et al. (135). Their data suggest that absrptin f vitamin B. was significantly lwer frm the natural surce, range juice, than frm the synthetic slutin. The lwer availability f vitamin B, frm WHW bread seen in the present study may be because vitamin B, is either bund t ther cnstituents in the whle wheat flur and was nt all released during digestin r that digestibility f WHW bread was lwer due t the presence f mre fiber (9. 1 g) cmpared t WB, r W (1. 2 g) bread. The higher level f excretin f vitamin B. in feces when the sub- jects were fed WHW bread (4. 9 mg/wk) as cmpared t when WB, (3. 1 mg/wk) r W (3.0 mg/wk) bread was fed, supprt the abve idea. Subjects 4 and 8 excreted a higher amunt f 4-PA when WHW 112 bread was fed than when WB, r W bread was fed. These subjects were tw f three wh received WHW bread the first week after the adjustment perid. During the six day adjustment perid, all the subjects cnsumed gradually increasing amunts f whle wheat bread as part f the adjustment diet. This may have cntributed t the adaptatin f the digestive system in digesting WHW bread fr these subjects receiving this type f bread immediately after the adjustment perid. Hwever, subject 9, wh als received WHW bread the first week fllwing the adjustment perid, excreted lwer

129 levels f 4-PA during this same perid than during the perid when 113 WB, r W bread was fed. The reasn fr this difference between subjects 4 and 8 and subject 9 is nt clear. The urinary 4-PA values f subject 9 were, in general, lwer than thse f the ther subjects, which may suggest that he had an altered metablism f vitamin B,. The 4-PA level bserved n the day 1 f the adjustment perid was higher than that f the day f the same perid. The higher initial 4-PA value indicates that the level f vitamin B, the subjects b had been cnsuming prir t the study was greater than 1. 5 mg. This trend was als indicated by the urinary excretin f vitamin B, (Table 15). The diet cnsumed by the subjects the day befre the first day f the study was nt evaluated fr vitamin B, cntent. Had the vitamin B, levels cnsumed n that day been knwn, a mre definite statement culd be made in relatin t the higher excretin f urinary vitamin B, and 4-PA levels. It has been reprted that 20-50% f the ingested vitamin B, is cnverted t 4-PA in the adult while the remainder is excreted via ther rutes (49). In the present study (Table 22), _ percent f the dietary intake f vitamin B, was accunted fr as urinary 4-PA when the subjects received WHW bread. The crres- pnding (percentages fr WB, and W breads were _. 9 and , respectively. These values shw that a higher percentage f

130 Table 22. Urinary 4-Pyridxic acid excreted during the three experimental perids, expressed as percentage f vitamin B intake. Experimental Subji 2ct Number Overall Perid mean + S.D. WHW WB C W WHW, WB and W - see Appendix 5. *-

131 115 the dietary intake was metablized int 4-PA when the subjects were fed WB, and W bread cmpared t WHW bread assuming the same amunt f vitamin B, was absrbed frm all three surces. Urinary excretin f 4-PA in humans has been fund t be dependent n bdy weight (194). Yung fund a negative relatinship between bdy weight and urinary 4-PA and reprted that the lighter subjects may have had a smaller bdy pl f vitamin B,, thereby making mre vitamin B available fr cnversin t the metablite. The bdy weight versus JLM f 4-PA/24 hr f urine determined in the present study is shwn in Figure 8. Even if the data n subject 5 are disregarded, due t his lwer intake f vitamin B,, there seems t be a psitive relatinship in that heavier subjects excrete cm- paratively mre urinary 4-PA than the lighter nes. This indicates that mre vitamin B/ has been cnverted t urinary 4-PA in persns with a greater bdy weight. The larger bdy size and prbably larger size f rgans (e.g., liver) f such persns may lead t increased capacity t metablize mre vitamin B, than persns f smaller bdy size. Further research is needed befre a definite statement can be made with regard t the relatinship between bdy weight and excretin. Vitamin B, Balance D The data n the intake, excretin and balance f vitamin B,

132 5 - $ ^ <i> Q 0 T3 O X O c 2 - HV 50 T" 0 _j, T Bdy Weight (Kg) Figure 8. Mean urinary excretin f 4-PA versus bdy weight during the experimental perids; O WHW, O subjects. (Data fr N. 5 nt included because f his lwer intake f vitamin B fi.) WB and W, fr the nine (1-9)

133 11 during the three experimental perids is presented in Table 23. The daily intake f vitamin B. fr all the subjects except N. 5 was 9. 37, and \LM fr WHW, WB and W bread diets, respectively, while the crrespnding intake f subject N. 5 was. 99,. 88 and. 82 JJLM, respectively. Fr all subjects, the sum f the excretin f urinary and fecal vitamin B, and urinary 4-PA was , and ULM/24 hr, fr WHW, WB. and W bread diets, respectively. Of these excretry prducts, ttal urinary vitamin B^ and 4-PA levels reflect the amunt f vitamin B, that was absrbed. In the present study, this amunt was 4. 14, 4. 5, and 4. 5 JLM/day when subjects were fed WHW, WB, and W bread, respectively. These amunts represent 45, 52 and 52 percent f the daily intake f vitamin B, fr the WHW, WB, and W bread diets, respectively. Assuming that all the vitamin B. has been accunted fr, there was a psitive balance fr vitamin B, with the three types f bread, since the intake f vitamin B, exceeded the excretin as measured in this study. The same relatinship was true f the means bserved fr the individual subjects fr all the experimental perids, with the exceptin f N. 8, in whm the average sum f daily excretin exceeded the intake f vitamin B, by 0. 9 fxm, during the perid he received WHW bread. mean and standard deviatin fr the balance was , The verall

134 Q + 1 H «0\ >* (O * 00 t in tm t \ N t^ N m in O tx t 1H l-l s N! O O O O O O H O TH 1-1 l-t O * lo r-i *-4 Cl O m rt i ^ 00 OS CM <M CM VO ch * tm <M ef> CO t 00 IV tv l-t tv 1H t CM n t r VD t-s VO w <q i v tv > O O O N CM in ^ i-i t^ i-l CM tv t in r VO 00 * O VO fv CM (V O TH CO CM TH &i <?t <h t^ t CTI in in \ \ CM TH rti r (M tv (O Cv 0\ O 00 t-v VO 00 * CTi * i-h O O i-i O c (U tv i-i CO CM ih a> v t t t O in i-i tv v v v i-l CM CM CTl O i-i in tn CM a\ CTI CM!-< G\ r? CM CM * CM CM CO * Tf O CM CM c a x a) a) a) 43 tv O i-i CO CM i-l O Oi O 00 CM CM O * O O O r-l in Tt" < * tv 1-1 CM r in l-l l-h VO bo cl c 3 T3 VO m a 1 (^ 00 CM i Cv O i-i CO CM i-l (y\ <j\ G\ CM VO VO CO 1H l-h in v tv in CJ> TC in in in v m O) K Oi K tv tv a\ TC in CM CM CM in ps CM 00 tv CM CM CM ^ 0\ O * in v Tt* tv v tv K v v O i-i tv O i-i CO CM ih Tti in 0\ CM in CM tx 00 v in in v * t^ l-h CO Ok VO tv CM in t CM 73 0 tv t CM ih t v in t 0\ s tv VO i-l rt * O t tv O IN CM IN IN IN CO * 00 0\ Ol ^ CO CM CM t <* in IN IN 00 CM ih O IN O ii CO CM ii CTl 01 CM IN cji in m O t m v v v it t O O O t * * IN v * TH t IN O Tf IN CM 00 t O ii ii IH Q 0) v m bj Ui S t VH.2 -P 1? l t ID Tf W pa

135 _ 0. 4 and 1. +_ (j.m/day when the subjects were fed WHW, WB, and W bread diets, respectively. A psitive vitamin B. balance was bserved by Levy in his D subjects when a vitamin B. intake f 2-3 mg was cnsidered in relatin t the urinary and fecal excretin f PAL, PIN, PAM and 4-PA (98). These excretry prducts accunted fr apprximately 40% f the vitamin B, intake. On the average, 0% f the intake was unaccunted fr in their excretry prducts, and it is unlikely that the subjects Levy studied retained that much, unless they were deficient in vitamin B-. The percentage unaccunted fr by Levy was much higher than the 10 t 20 percent unaccunted fr in the present study, where the average intake f vitamin B, was 1. 5 mg. In cntrast t the abve investigatins, the studies dne by Linkswiler and Reynlds (48) and Yan and Fujita (103) revealed that excretin f vitamin B, and 4-PA by humans accunted fr mre vitamin B, than cnsumed. They cnsidered this as evidence f the synthesis f vitamin B. by the micrflra present in the human intestine. With a vitamin B, intake f 0. 5 t 1. 2 mg in nine sub- jects, Linkswiler and Reynlds bserved the ttal excretin f vitamin B, and 4-PA. t be 3. 2 t 3. 8 mg. Of this, 1. t 2. 8 mg was accunted fr as urinary 4-PA. The methd f 4-PA deter- minatin used by Linkswiler and Reynlds (48) was that f Huff and Perlzweig (4). This methd has since been fund t be unreliable

136 119 due t the incmplete eliminatin f flurescent substances ther than 4-PA. This may have led t an verestimatin f urinary 4-PA. The methd used by Yan and Fujita (103) fr the determinatin f 4-PA was that f Fujita and Fujin (104), which had a drawback similar t that f Huff and Perlzweig, and therefre wuld result in an verestimatin f 4-PA in urine. Therefre, thse studies in- vlving 4-PA determined by the lder methds (48, 103), need t be viewed with cautin. The mean intake, excretin and the balance f vitamin B, when the subjects were fed diets based n WHW, WB, and W bread are illustrated in Figure 9. Of the 10 t 20 percent f the vitamin B, intake that was nt accunted fr by the excretry prducts in the present study, sme may have entered the bdy pl f vitamin B,, while sme may have been excreted via rutes nt cvered in this investigatin; fr example, sweat and saliva. Vitamin B, cntent f sweat and saliva has been reprted as and j,g/100 ml, respectively (195). Urinary 4-PA is knwn t be the majr metablite f vitamin B, (49). Hwever, there culd be sme minr b metablites r intermediary prducts which are excreted. A further explanatin fr the unaccunted vitamin B, culd be attributed t the micrbilgical assay f vitamin B,. frm f vitamin B, f the diet in the present study was PIN. b The majr In the determinatin f ttal vitamin B,, the standard curve generally used, b is f PIN. The grwth f S^. carlsbergensis in the presence f PAM has been reprted as percent f that f PIN and PAL.

137 120 Type f Bread WHW W-Bs W Vitamin Bg Intake (%) PVWWff Percent Excreted as Fecal 45.1 w*r^ Vit. B e Urinary Vit.Bfi Urinary 4-PA n 7T?T / T/Vi X.-':-T Ttal excreted Balance unaccunted ^^X Figure 9. Daily intake, excretin and balance f vitamin B, f subjects fed diets based n WHW, WB, and W bread. D

138 121 Accrding t Shane and Snell, the uptake f PIN by S. carlsbergensis exhibits twin ph ptima f 3. 5 and, PAL being transprted primarily by the ph 3. 5 system and PAM by the ph system whereas PIN is transprted rather effectively by bth systems (19). These bservatins may explain the reasn fr PAM being less active than PIN and PAL in supprting yeast grwth, as mst f the assays are cnducted near ph Yan and Fujita reprted that PAM was the predminant frm f vitamin B, in feces even when the diet cntained mstly PIN and PAL (103). Assuming the bservatin f Yan and Fujita was true fr the present study as well, the use f PIN standard t determine fecal vitamin B, may have given a value lwer than the actual vitamin B, cntent in feces. The majr frm f urinary vitamin B, was determined t be PAL. This has als been reprted by Kelsay et al. (107) and Sauberlich_et al (79). Therefre, if a PAL standard had been used instead f PIN fr the standard curve t determine the vitamin B- excreted in urine, the ttal vitamin B, wuld have been lwer than the levels reprted in the present study. Using the apprpriate frm r a cmbinatin f frms in crrect prprtins wuld eliminate the errrs intrduced by the different respnse f the micrrganism t the three frms f vitamin B.. As mentined earlier, the bdy pl f vitamin B, culd retain sme f the ingested vitamin. Vitamin B. status is generally

139 122 reflected by the levels f vitamin B. in bld and the cenzyme activity (197). The vitamin B. parameters in the bld drawn frm the subjects in the present study will be available in detail in a separate thesis (198). The mean and standard deviatin f plasma vitamin B. were _ 1. 9, and ng/ml when the diet was based n WHW, WB, and W bread, respectively. The plasma pyridxal phsphate (PLP) levels shwed a mean and standard deviatin f , 8. 3 _ and ng/ml when the subjects were fed WHW, WB, and W bread, respectively. Hwever, these differences in the plasma vitamin B, and PLP levels in relatin t the type f bread wer nt significant. Based n the results f the present investigatin f the biavailability f vitamin B, frm WHW, WB, and W bread, the fllwing bservatins are summarized: 1. There was significantly mre vitamin B. excreted in feces when the subjects were fed WHW bread than when they were fed WB, r W bread. 2. The urinary vitamin B, cntent was nt affected by the type f bread cnsumed. 3. There was a significant decrease f PAL in the urinary vitamin B. when the diet was based n WHW bread cmpared t WB, r W bread.

140 The urinary 4-PA levels were significantly lwer when the subjects received WHW bread than when they received WB. r W bread. 5. The percentage f vitamin B, intake accunted fr, in the excretry prducts was mre when WHW bread was cnsumed than when WB, r W bread was cnsumed, b

141 124 RECOMMENDATIONS The results f this study supprt the feasibility f enrichment f wheat flur with vitamin B,, as far as stability and biavailability are cncerned. Enrichment f white flur and bread with vitamin B, will have definite advantages fr ppulatins that depend n an appreciable amunt f refined cereal prducts in the diet. Accrding t the results f phe present study, the added vitamin B, was stable during strage f flur at cld and at rm temperature, and during strage f bread under frzen and refriger- ated cnditins. Bth native and acjded vitamin B, was fairly stable during bread making. As far as stability is cncerned, enrichment f white flur with vitamin B, (PIN-HC1) wuld pse n prblem. Generally, cuntries -where wheat is nt prduced, especially thse in the trpical belt, imprt mstly refined flur as ppsed t whle wheat flur because f better strage prperties f the frmer. Enrichment f refined flur with vitamin B. will be f advantage t cnsumers f this prduct in such cuntries, mst f which als have lw per capita cnsumptin f animal prducts, a majr surce f vitamin B. in the develped cuntries. Althugh the results f the present study indicated that vitamin B. was less available frm whle wheat bread cmpared t white b bread enriched with vitamin B, r white bread plus the ral

142 supplement f vitamin B,, there are advantages f cnsuming whle wheat bread which cannt be replaced by vitamin B, enriched white 125 bread. One such advantage is the presence f fiber. Cncern fr fiber in the diet has been emphasized (199). A diet cntaining 'whle wheat bread as ppsed t white bread will cntribute fiber, especially t the typical U.S. diet which tends t be generally lw in this cnstituent. In additin, as cmpared t white bread, whle wheat bread is als a rich surce f several nutrients, especially the B vitamins and sme minerals (3). Althugh slices f bread were cnsumed daily by the subjects in the present study, 4- slices (apprximately 125 g) is a reasnable amunt an average persn wuld cnsume per day alng with ther fd items. Sme 0. 3 mg f vitamin B, wuld be cn- tributed by 125 g f WHW bread whereas the same quantity f WB, bread wuld cntribute 0.4 mg. In anaverage daily diet cntaining 2 mg f vitamin B/, the amunt f vitamin B, cntributed by bread will nt make a substantial difference in relatin t the type f bread cnsumed, i.e. WHW r WB.. In diets which are generally lw in fiber, WHW bread will supply sme fiber in cntrast t white bread. Cnsumptin f WHW bread shuld be encuraged amng the public. Perhaps, the enrichment f flur with vitamin B. might lessen the shift frm refined wheat prducts t whle wheat prducts.

143 The present study was dne in nine male subjects in a narrw 12 age range (21-33 years). The results f this study d nt lend t making a final decisin as t whether refined cereal prducts shuld r shuld nt be enriched with vitamin B,. A study f nutritinal status n a mre representative ppulatin with respect t vitamin B, enriched bread will be necessary befre a definite recmmendatin can be made. Further, the feasibility f distributin f synthetic vitamin B. in the flur alng with the enrichment mixture used at present needs t be evaluated. Since mst f the cmmercial breads use preser- vatives t prlng shelf life, the stability f added vitamin B, in the presence f such preservatives needs t be studied.

144 127 SUMMARY The bjectives f the present study were tw fld: A. T determine the stability f vitamin B, in wheat flur during bread making and strage. B. T investigate the biavailability f vitamin B. frm bread, in human subjects. Three variables; whle wheat flur (WHW), white flur (W) and white flur enriched with vitamin B, (WB.) were tested. The WB, dugh prepared using the spnge dugh methd shwed a significant increase in vitamin B, cntent at the end f fermentatin cmpared t that at the beginning (P < 0.05). The changes bserved during fermentatin in vitamin B, cntent f the remaining types f dugh were nt statistically significant. The WHW and W breads baked under cmmercial cnditins had a significant lss (P < 0. 05). Under hme cnditins, the WHW and WB, bread prepared using the spnge dugh prcedure resulted in a baking lss significant at the 1% level while that f W bread was significant at the 5% level cmpared t the baking lss bserved in breads made using straight dugh methd which was nt significant. The baking lsses deter- mined in the present study were 3. 5 and 8 percent fr the breads made using straight and spnge dugh methds, respectively, and 13 percent fr breads made under cmmercial cnditins.

145 128 The WB, bread stred under frzen and refrigerated cnditins shwed gd stability f vitamin B.. Hwever, the WB, bread stred under rm temperature had a significant lss f 10 percent in the vitamin B, cntent after three days (P< 0.01). Mld grwth was bserved n this bread after five days f strage at rm temperature. The level f vitamin B, f WB, flur stred in the cld rnn and at rm temperature did nt decrease ver a perid f 2 weeks, sug- gesting that added vitamin B. was stable during strage f flur. The results f the first part f the study indicate gd stability f vitamin B, in wheat flur during bread making and strage. Biavailability f vitamin B> frm the three types f cmmercial bread was studied in nine men, age years. The level f vitamin B, intake was 1. 5 mg, f which 1. 2 was supplied frm the bread. The majr frm f vitamin B, in the diets was PIN. Fecal excretin f vitamin B, was _ 0. 9 mg/wk when WHW bread was fed. This level f vitamin B, was significantly (P < 0. 01) higher than the levels f and mg/wk btained when WB, and W breads were fed, respectively. The greater fecal excretin f vitamin J3, during the perid when WHW bread was fed was assciated with a greater fecal weight. This was due t the high fiber cntent f the diet based n WHW bread. The percentage f daily intake f vitamin B, excreted in feces was when WHW

146 129 bread was given, cmpared t and 27.4 when WB, and W breads were given, respectively. There was n significant difference in the urinary excretin f ttal and free vitamin B, in relatin t the type f bread fed. Apprxi- mately 8 percent f the daily intake f vitamin B. was excreted in the urine as ttal vitamin B,. The predminant frm f urinary vitamin B, was PAL. Pyridxal accunted fr percent f urinary vitamin B. when WHW bread was fed. This percentage was significantly lwer (P < 0. 01) than the 0. 7 percent and 3. 1 percent f the urinary vitamin B, as PAL when WB, and W bread were fed, respectively. The mean f urinary 4-PA f days 4, and 7 f each week was H^ 1. 0, and j.m/24 hr when the diets were based n WHW, WB, and W bread, respectively. The excretin f 4-PA in subjects receiving WHW bread was lwer, with the exceptin f tw f nine subjects. When expressed as a percentage f vitamin B, intake, 37. 2, and percent was excreted as 4-PA when the diets were based n WHW, WB, and W bread, D respectively. The lwer values bserved fr the perid when WHW bread was fed suggests that the amunt f vitamin B. absrbed and/r metablized during the same perid was lwer than that with WB, r W bread.

147 130 When the excretry prducts f vitamin B, analyzed in the present study are summed, 91. percent f the vitamin B. intake was accunted fr when WHW bread was fed, cmpared t and percent fr WB. and W bread, respectively. Plasma levels f vitamin B, and pyridxal phsphate were fund t be slightly lwer during the perid WHW bread was cnsumed, cmpared t the perids WB. and W bread were cnsumed. These data suggest that vitamin B, was nt as available frm WHW bread as frm WB. r W bread. Under the cnditins f the present study, the availability f vitamin B, frm WB. bread and W bread plus the synthetic vitamin B. was similar. In cnclusin, the enrichment f refined flur with vitamin B, will have advantages fr ppulatins wh are dependent n refined wheat prducts. Hwever, such an enrichment cannt replace cmpletely the benefits f cnsuming whle wheat prducts. Therefre, cnsumptin f whle wheat prducts shuld be encuraged amng the public.

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162 Plansky, M. M., E. W. Murphy and E. W. Tepfer. Cmpnents f vitamin B/ in grains and cereal prducts. J. Assc. Offic. Agric. Chem. 47: , Jnes, C. R., J. R. Fraser and T. Mran. Vitamin cntents f air-classified high- and lw-prtein flur fractins. Cereal Chem. 37:9-18, I Plansky, M. M. and E. W. Tepfer. Effect f fumigatin n wheat in strage III Vitamin B/ cmpnents f wheat and wheat prducts. Cereal Chem. 48:392-39, Harris, R. S. Supplementatin f fd with vitamins. J. Agric. Fd Chem. 7:88-102, Brenstein, B. Ratinale and technlgy f fd frtificatin with vitamins, minerals and amin acids. CRC Critical Reviews in Fd Technlgy 2(2): , Federal Register. Title 21 - Fd and Drug. Chapter 1. Fd and Drug Administratin. Part 17 - Bakery Prducts, revisin f standard f identity. 41: , (February) Tepfer, E. W., M. M. Plansky, J. F. Eheart, H. T. Slver, E. R. Mrris, E. N. Hepburn, and F. W. Quackenbush. Nutrient cmparisn f selected wheats and wheat prducts. XI. Summary. Cereal Chem. 49:173-18, Wilder, R. M. and R. R. Williams. Enrichment f flur and bread: a histry f mvement. Natinal Research Cuncil. Bulletin N Natinal Academy f Sciences, Washingtn, D. C, Kser, S. A. Vitamin Requirements f Bacteria and Yeasts. Charles C. Thmas Pub., Springfield, Illinis, pp , Hchberg, M., D. Melnick and B. L. Oser. On the stability f pyridxine. J. Bil. Chem. 155:129-13, Brenstein, B. Vitamins and amin acids. In Handbk f Fd Additives, ed. T. E. Furia CRC Press. Chemical Rubber C., Cleveland, Ohi, 1972, pg. 89.

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164 174. Bwers, J. A., B. A. Fryer and P. P. Engler. Vitamin B- in turkey breast muscle cked in micrwave and cnventinal vens. Pultry Sci. 53:844-84, Bwers, J. A., B. A. Fryer and P. P. Engler. Vitamin B- in prk muscle cked in micrwave and cnventinal vens. J. Fd Sci. 39s42-427, Meckel, R. B. and G. A. Andersn. Thiamine retentin and cmpsitin f U.S. Army bread. Cereal Chem. 22: , Brenner, S., S. G. Dunlp and V. O. Wdicka. Effect f frtificatin f canned bread n stability. Cereal Chem. 25: 37-37, Dwns, D. E. and R. B. Meckel. Thiamine lsses in tasting bread. Cereal Chem. 20: , Maleki, M. and S. Daghir. Effect f baking n retentin f thiamine, ribflavin and niacin in arable bread. Cereal Chem. 44: , Mrgareidge, K. The effect f light n vitamin retentin in enriched white bread. Cereal Chem. 33: , Keagy, P. M., E. L. R. Stkstad and D. A. Fellers. Flacin stability during bread prcessing and family flur strage. Cereal Chem. 52: Hennessy, D., A. M. Steinberg, G. S. Wilsn, and W. P. Keaveney. Flurmetric determinatin f added pyridxine in enriched white flur and bread baked frm it. J. Assc. Offic. Agric. Chem. 43:75-78, I Crt, W. M., B. Brenstein, J. H. Harley, M. Osadca and J. Scheiner. Nutrient stability f frtified cereal prducts. Fd Technl. 30(4):52-5 and 2, Bunting, W. R. The stability f pyridxine added t cereals. Cereal Chem. 42:59-572, Finney, K. F. and M. A. Barmre. Varietal respnses t certain baking ingredients essential in evaluating the prtein quality f hard winter wheats. Cereal Chem. 22: , 1945.

165 18. AACC. Cereal Labratry Methds. American Assciatin f Cereal Chemists Inc. St. Paul, Minnesta Watt, B. K. and A. L. Merrill. Cmpsitin f fds. Agricultural Handbk N. 8. U.S. Dept. Agric. Washingtn, D.C., Pence, J. W., N. N. Standridge, T. M. Lubisich, D. K. Mecham and H. S. Olctt. Studies n the preservatin f bread by freezing. Fd Technl. 9: , Eckstein, P. M. The wrld f Science, Mechanix Illustrated. 72(575);10, Sauberlich, H. E. Human requirements fr vitamin B^. Vitamins and Hrmnes. 22: Suthgate, D. A. T., W. J. Branch, M. J. Hill, B. S. Drasar, R. L. Walters, P. S. Davies and I. M. Baird. Metablic respnses t dietary supplements f bran. Metablism. 25: , Scriver, C. R. and A. M. Cullen. Urinary vitamin B, and 4-pyridxic acid in health and in vitamin B. dependency. Pediatrics. 3:14-20, Kelsay, J. L. The effect f prtein intake n the vitamin B/ requirement f man as determined by the excretin f quinlinic acid and the niacin metablites and f vitamin B/ and fur-pyridxic acid. Ph.D. Thesis. Madisn, University f Wiscnsin, 197, Yung, J. N. Urinary excretin f 4-pyridxic acidby wmen using sterid cntraceptives and by mental retardates with and withut Dwn's syndrme. Master's Thesis. Crvallis, Oregn State University, Altman, P. L. and D. S. Dittmer. Bld and ther bdy fluids. Federatin f American Scieties fr Experimental Bilgy. Washingtn, D.C., 191, pp. 400, Shane, B. and E. E. Snell. Transprt and metablism f vitamin B/ in the yeast Saccharmyces carlsbergensis J. Bil. Chem. 251: ,

166 197. Jacbs, A., I. A, J. Cavill and J. N, P. Hughes. Erythrcyte transaminase activity. Effect f age, sex and vitamin B/ supplementatin. Am. J. Clin. Nutr. 21: , Peffers, D. E- The Biavailability f Vitamin B, frm Wheat bread in Humans. Master's Thesis, Crvallis, Oregn State University (in prgress) Spiller, G. A. an<j R. J. Amen. Dietary fiber in human nutritin. CRC Critical Reviews in Fd Science and Nutritin. 7(l):39-70,

167 APPENDICES

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