(gan-lit) am j clin nutr-1976-newmark-645-9 (stability of b12)

8/12/2019 (GAN-Lit) Am J Clin Nutr-1976-Newmark-645-9 (Stability of B12)

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R ecen tly , H erbe rt and Jacob (1 ) rep ortedtha t in c rea sing leve ls o f a sco rb ic ac id , add edto h om o gen ized m ea ls p rio r to incu ba tion at37 C fo r 3 0 m m as a lab orato ry m im ic o f th egastric env iron m en t in m an , p rodu ced in -c rea sin g destruc tion of v itam in B 1 2 . T h is w asde term in ed b y rad io as say aga inst a se rie s o fin tr in sic fac to r con cen tra te s tan da rd s. F romth ese re su lts they in fe rred th at h igh do ses o fa sco rb ic acid , pop u la rly used as a h om erem ed y aga ins t th e com m on co ld , destroysu bstan tia l am o un ts o f v itam in B 1 2 w hening ested w ith fo od . If th is is true , the tim ingo f su pp lem en tary asco rb ic ac id in gestionw ou ld have to b e ad justed to a m in im u mex posu re to v itam in B 12 in the d igestive trac t.

R ev iew of the H erbe rt an d Jacob pape rrev ea led tha t the reported v itam in B 1 2 assayv alu es o f th e m eals tha t w ere used w ereco nsid erab ly low er fo r the food s inv o lved

The A m er ica n Jou rna l o f C l in ic a l N u trit ion 2 9: JU N E 19 76 , p p . 64 5-6 49 . P rin ted in U .S .A . 64 5

S tab ility o f v itam in B in th e p re sence o fasco rb ic ac idH aro ld L . N ew m a rk , M .S ., Jacob S chem er, M .S ., M artin M a rcus, M .S .,a nd M ukun d P rab hud esa i, M .D .

A B S T R A C T E x per im ents w e re pe rfo rm e d in tw o in de pen den t labo rato rie s, ea ch us ing th eiro w n m ea l pre pa rations w h ich w e re ex act ly sim ilar in co m p osi tio n to th e m e als describ ed by H erber tan d Jac ob (J. A m . M ed . A sso c. 23 0 : 2 41 , 1 974 ), in order to ch ec k the ir re po rt that incu bat ing m eals(por tio ns o f d aily fo od in take b y m a n) o f m ode st or h ig h v ita m in B 3 co nte n t w ith inc reas ingle vels o f a dde d L -a sco rb ic ac id (v ita m in C ) p rod uce d increas ing de stru ction of v itam in B 12 . T heprese n t stu d ie s w ere p erfo rm ed w ith s tan dard iz ed and off ic ia l m e tho ds. V itam in B 1 2 w a s determ ine dm icrob io lo g ic ally an d by rad ioa ssa y m eth od . T he resu lts show ed that 1) th e v itam in B 1 2 co nte n ts ofth ese m eals w e re in gen era l ag ree m e nt w ith va lue s calcu la ted fro m the litera tu re fo r th e foo dsinvo lved , 2) the va lues o b tained w ere m an yfo ld h ig he r tha n those repo rte d b y H e rbe rt and Ja cob ,an d 3) there w as n o de lete rio us e ffec t o f ad de d ascorb ic a cid on the v i tam in B 12 c onten t of m e als ,co n trary to the ir pu b lished resu lts . A m . J . C /in . N u tr . 2 9: 64 5-649 , 197 6 .

than w ere calcu la ted va lues tak en fro m p rev i-o usly pub lished foo d com pos ition tab le s(2 -5 ) , w h ich in tu rn led to conce rn abou t theana ly tica l m eth odo logy em plo yed in thein vestiga tion . In v iew o f these ob se rv atio nsw h ich co u ld have in f luenced the resu lts o fH erbe rt and Jacob , it w as dec ided to reexam -in e the e ffec t o f su pp lem en ta ry asco rb ic acidon v itam in B 12 in m ea ls w ith stand ard izedan d offic ia l ex trac tion p ro cedu re s, u sing bo thm ic ro b io lo g ical an d rad io assay m e thod s.

E xp erim en ta l m e thod sM ic rob io log ica l assays

F ou r se ts ea ch o f the m o des t (T able I an d h igh(T a ble 2) v itam in B 1 2 m eals of th e com po sition an dprepa ration pre vious ly de scr ibe d (1) w ere p rep are d in th eP rod uc t D e velopm en t L ab ora to r ies, H o ffm an n- L aR oc he In c., a nd in cub ate d at 37 C fo r 30 m m w ith 0 , 0 .1 ,0 .2 5 , o r 0 .5 g L -a sco rb ic a cid (v ita m in C ). Im m ediate lyafter th is in cub ation the m ea ls w ere fro zen and k ep tf roz en un til assaye d . T h e die ts w e re assaye d e ithe r I)

direct ly , 2) af ter e x tractions by the A sso cia tion o fO ffic ia l A n aly tic al C hem is ts (A O A C ) p roc edu re (6 ), o r3) b y the proce du re pu blishe d by the B ritish A n aly tica lM e tho ds C om m itte e (7 ). T h e fo llo w in g q ua ntit ies ofA O A C e xtractan t w ere a dd ed to 20 g of th e h om og enizedm e als : m ode st B 12 , 25 m l; h igh B 12 m e al , 3 00 m l.T he m e als w ere th en ex trac ted for 1 0 m m at 1 5 ps i s tea mpre ssu re (1 21 C ) , b ro ug ht to app rop ria te vo lum e, ce n-trifuge d , the p H of al iqu o t w as a d ju ste d to 6 .0 an d d i-lu ted to assay rang e. F o r th e B ritish m ethod , 4 m l of a Im g /m I K C N solu tion and 16 m l w ater w ere ad de d to 20 gof ho m o gen ize d m eal; th e p H w a s a d ju ste d to 4 .6 to 5 .0 .A f ter sta nd ing fo r 30 m m w ith oc cas ion al sti rrin g , thepH w as re ad juste d to 4 .6 to 5 .0 as nec essary . T heco nta ine rs w ere p lac ed in a boiling w a ter bath an d w erelef t in th e b ath for 3 0 m m a fte r th e in te rna l te m p era tu rereac he d 90 C . T he m eal sam ple s w e re co ole d and broug htto a vo lum e of 10 0 m l. A p ort ion of th e sam ples w asce n tr ifu ged and an aliqu o t w a s a d ju ste d to pH 6 .0 a nddilu ted to a ssa y ran ge. A ll a ssay s for v itam in B 1 3 w ererun by the A O A C m icro b io log ical p roce dure (6) , u sin gLa cto bac illu s le ich m ann ii.

F ro m th e P rod uc t D ev elo pm ent D ep artm en t, H off-m an n-L a R oc he In c., N utley , N e w Je rse y 07 110 , an d th eD ep artm en t o f P ath o lo gy , F ord ham H o spita l, M iser i-co rd ia-F ord ha m A ff ilia tion , B ron x , N ew Y o rk 10 458 .


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64 6 N E W M A R K E T A L .

A v era ge o fd ata in pa per .

dup lic ate as say s. C a lcu late d fro m

RadioassaysF or the rad ioas say , se pa rate prep ara tio ns sim ilar to

tho se use d for the m ic rob io lo g ic al a ssa ys of b o th them ode st B 12 c onten t m eal an d a h ig h B 1 2 co nte n tm e al w e re p rep are d in a F o rdh am H ospita l labo rato ry .P rop or tion al a m o unts o f fresh ly prepa red ascorb ic ac idso lu tion w e re a dd ed to 20 g p ortions o f the se h om oge -n ize d m eals equ iva len t to 0 . 1 , 0 .25 , an d 0 .5 g per m ea l.T he se pre parations , as w ell as con tro l m e al aliq uo tsw ith ou t asc orb ic acid , w e re inc uba ted for 3 0 m m at 3 7 C .A fte r incu bat ion , K C N w as ad ded to o ne set an d th e pHw a s ad justed to 4 .8 . T h is set w a s p la ced in a b oil ing w ate rbath an d tim ed for 30 m m af ter the te m p era tu re re ach ed90 C . T h e pH ofthe o ther set w as ad ju ste d to 4 .8 and th esam ple s w ere k ep t at ro om tem pe ratu re .

B oth se ts w ere su bdilu te d w ith d eio n iz ed w ate r so th atex pec ted va lue s w ou ld l ie w ith in th e line ar por tion of thes tan dard curve. F ou r m l of a so lu tion co nta in in g 0 .6 go f g lu tam ic ac id an d 2 m g of K C N pe r 10 0 m l ad justedto pH 3.3 w e re a dd ed to 1 .0-m I sam ple a liq uo ts. P rel im i-na ry a ssay s re vea led the p rese nc e o f h ig h non spe cif icb ind ing w h ich w a s e lim ina ted by he atin g the sam ple to9 0 C fo r 2 1 h r. C yan oco ba lam in s tan dards that w ere s im i-la rly tre ate d w ere una ffe cted b y th is p ro ced ure . A liq uo tsof the final d ilu tio n w e re u sed for the satu ra tion an aly sisw hic h fo llow ed . W ith the ex cep tio n o f e x te nde d h eat ingtim e th e m ethod w a s e sse n tia lly that of W ide an d K il-lan der (8) .

Resu l tsT he m a jo r so u rces o f v itam in B 12 in th e

m od est m eal (T ab le 1 ) a re co ttage cheeseand m ilk , each of w h ich sup p ly ab ou t 1 g ofv itam in B 12 to the m ea l. In th e h igh B 12 m eal (T ab le 2 ), the m a jo r sou rce o f th ev itam in is liv er , w ith m ilk ad d ing a sm a llfrac tion of th e to ta l con ten t. A com pariso n ofcalcu la ted va lues and the va rio us assay va lueso f the tw o m ea ls is g iv en in T ab le 3 . C lear lyth e resu lts o f the m ic rob io log ica l a ssay s an drad ioassays b y stand a rd ized ex trac tion p ro ce -du re s em ploy ed in th is study a re in roug hag reem en t w ith th e v alu es ca lcu lated fro m th elite ratu re . O n the o the r hand , the resu lts o fH erbe rt an d Jacob (1 ) are o n ly a sm all frac-tion o f the exp ec ted v alu es.

T A B L E IC o m p osi tion of m ode st v itam in B 12 m eal

C o m p o n e n t A m o u n tC rea m of po tato sou p 1 77 m lC ottage c he ese 1 00 gLettuce lO gC an ned p eac hes 360gCrackers 6gBread 25 gButter 5gM ilk 23 7 m l

F or th e m od est B 12 con ten t m eal, w ithand w itho u t ad ded asco rb ic ac id , m icrob io -log ica l a ssays in d ica te th at the A O A C extrac -tion procedure g ives som ew ha t h ighe r re su ltsthan the B ritish p rocedu re , w h ile th e re su ltsw ith ou t ex trac tion a re abou t /2 to of theto ta l B 12 P < 0 .01 ; T ab le 4 ) . I t is n o tew orth ytha t the va rio us leve ls o f add ed asco rb ic ac idhave sign ifican t e ffec t ne ithe r on the freev itam in B 12 (no ex tractio n afte r hom ogen iza -tio n ) con ten t o f the m ea l no r on to ta l v itam inB 12 a fter ex trac tion .

T A B L E 2C om po sition of h ig h vitam in B 1 2 m e al

C o m p on ent A m oun tC h ick en no odle so up 1 77 m lC rack ers 6 gG rille d be ef live r 90 gC a tsu p 15 m lW ho le ke rne l c orn 9 0 gE gg sa lad 45 gA pp le l7 5gB read 2 5gB u tte r 5 gM ilk 23 7 m l

T A B L E 3C alc u la ted a nd assay v alu es for m od est v i tam inB 12 a nd high v itam in B 12 m ea ls

M e a l Cal-cu lated

Microb io log icala ss ay e xt ra ct io n

procedure O Brit ish

{ 49}Radio-assay

Her b e r tan d

Jacob(1)

M od est B 12H igh B 1 2

ca 2 .0ca73

g /me a l2 .7 2 .0

9 6 7 81. 6

460 .378.9

B ased o n va lue s in H o m e E con om ics R esearchR ep ort 36 , U nited S ta tes D epa rtm en t of A gr icu ltu re ,1 96 9 , W ash in g to n , D .C . A ssum in g 90 g gr ille d liv er

had B 12 c onten t s im ilar to 90 g fresh liver .

T A B L E 4E ffe ct o f e x tra ction pro ced ure on app are n tv ita m in B 1 2 co nte n t of m o de st B 12 m eal

A sc o rb ic a cida dde d to m eal

p rio r toincubat ionat 3 7 C

M ic ro b io lo g ic a l assa ye xt ra ctio n p ro ce du re H e rb e r tan d

Ja c o b (I)N o ne A O A C B rit ish

g00. 10 .250. 5

g m e a I0 .98 I 2.7 I 2.00 .9 0 3 .1 2 .10 .9 2 3 .0 2 .20 .9 9 3 .5 2 .0

0 .370 .21O . 0 7 b0 .02


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T A B L E 5E ffe ct o f ex tract ion p roc edu re on a ppa ren tv i tam in B 1 2 co nte n t of h ig h B 1, m e al

{ 7 6 }A ve rage of d uplicate assays . C alc u la ted fro md ata in pa per .

S T A B IL IT Y O F V IT A M IN B 12 IN T H E P R E S E N C E O F A S C O R B IC A C ID 64 7

{ 176} alc u la ted from d ata in p ape r.

F o r the h igh B 1 2 co n ten t m eal w ith an dw ithou t add ed asco rb ic acid , th e resu lts o b -tam ed by m ic ro b io lo g ical as says fo llow asim ila r pa tte rn (T ab le 5 ). T he da ta ind ica teonce aga in tha t n e ith e r th e free v itam in B 1 2leve ls no r the to tal B 12 lev els b y A O A Cextrac tion sh ow sign ifican t ch anges a fter in -cuba tion a t 37 C fo r 30 m m w ith g raded leve lso f ad ded asco rb ic ac id . T h e resu lts o b ta inedby th e B ritish ex tractio n an d m ic ro b io lo g icalas say ind ica te tha t th e re su lts fo r a ll th reelev els o f a sco rb ic ac id ad d ition do no t d iffe rsign if ican tly , co nsid ering th e no rm al v ariab il-ity o f th is m e tho d of a ssay .

A s a fu rthe r check o n th e va lid ity o f th eassay re su lts , ex trac ts o f b o th the h igh B 12an d m odest B 12 d iets p rep ared b y th eA O A C p ro cedu re w ere h yd ro ly zed at p H1 2 fo r 3 0 m m at 12 1 C . T h is p ro cedu re iskno w n to d es troy v itam in B 1 2 (9 , 1 0 ) and an ym ic ro b io lo g ical ac tiv ity rem a in ing a fte r suchtrea tm en t cou ld be co nside red as m ic ro b io -log ica l g ro w th fac to rs fo r the as say organ ismtha t a re no t cob alam in s. A ssays o f thesetrea ted ex tracts in d icated tha t at best o n ly asm a ll frac tio n (e .g ., le ss than 20% ) of th ev itam in B 12 ac tiv ity a s m easu red by L. leich-mannii in the d ie ts m igh t be du e to non spe -c ific stim u la tion of the tes t o rgan ism . T he re-fo re , the m ic rob io log ica l ac tiv ity reported ise ssen tia lly v itam in act iv i ty .

T he rad io as says in d icate tha t low er resu ltsa re o b ta ined in the absence of in itia l hea t an dcyan ide trea tm en t than w hen th ey a re used P< 0 .2 ; T ab le 6 ) . H en ce by ano th e r ana ly tical

app roach , ne ithe r se t o f re su lts fo r the m od-est B 12 m eal o r the h ig h B 12 m ea l in d i-cate s any los s o f v itam in B 12 ac tiv ity in thep resence o f the ad ded leve ls o f a sco rb ic acid .

s co r b i c a c i dadd ed to m e alp r i o r t o

incubat ionat 37 C

M c r ob i o l o g i c a l a ss aye xtra ctio n p ro ce du re H e r b ean d

Jac ob (I)N o ne A O A C B ritish

g00.10 .250. 5

pg/meal37 9 6 78

35 8 9 6834 I 83 I 6137 9 6 6 0

8.9ca8 .5b

6 .7ca5 .l

DiscussionA rev iew of the lite ra tu re in d icated tha t the

re su lts ob tain ed w ith L . l ei chmanni i fo r theh ig h B 12 and m odest B 12 m eals w ere inag reem en t w ith asse ssm en ts o f the v itam inB 1 2 con ten t a s assay ed by a va rie ty o fm e tho ds. T he v itam in B 12 of th e h ig h B 1 2m eal cam e a lm os t en tirely from liv er . In th iscon tex t it had b een show n by K am ik ubo an dO gu n i (3 ) and by L ich ten ste in e t al. (4 ) tha tfo r beef liv e r the re su lts o f L . l ei chma nni iassays w ere es sen tia lly sim ilar to the resu ltsof Ochr omonas malhamensis assays , th e lat-te r o rgan ism also be ing m ore spec ific fo rcyano co ba lam in an d hyd ro xycob alam in thanfo r pseud o co ba lam ins (1 1 ) . T he v itam inB 1 2 o f the m od es t B 12 d ie t cam e from m ilkand co ttage ch eese . In th is ca se G rego ry (5 )and L ich ten ste in e t a l. (4 ) had sho w n tha tsim ila r re su lts w ere ob tain ed fo r m ilk an dcheese , re sp ec tive ly , by b o th o f the abo veassay org an ism s.

W hen the v itam in B 12 con ten t o f the m ea lsa s de te rm in ed by H erb e rt and Jacob (1 ) w ascom pared to the am ou n t ca lcu la ted fromlite ra tu re va lues o f th e m eal com p onen ts (2 ) ,it w as ev iden t tha t the va lues repo rted byth ese investiga to rs w ere cons ide rab ly low er.T hus , the m odest m ea l w as on e-fif th an dthe h igh m ea l on e-e igh th o f the calcu latedva lues. T h ese d isc repanc ie s in v itam in B 1 2con ten t su gg ested th e po ssib ility o f in ad e -qu ate m eth odo logy .

I t is now accep ted tha t a co m m o n erro r inthe ea rly y ea rs o f v itam in B 12 resea rch in toT A B L E 6R adioas say s on m od es t B 1 2 and h igh B 12m e als a fter in cub ation at 37 C

A sco rb ic ac ida d d e d

N o add ed A dd edc ya n id e cy an ide

Herber tan d

Jac ob (I)g pg/meal

0Modest B12

0 .84 1 .6 0 .3 70 .1 1 .1 2 .0 0 .2 1 { 17 6}0 .25 1 .4 1 .7 0 .07 {176}0 .5 0 .9 1 .9 0 .0 2 { 17 6}

0H igh B12

32 4 6 8 .90 .1 3 6 4 9 c a8 .5 { 17 6}0 .25 3 1 6 2 6 .7 {176}0. 5 3 1 47 c a5 .I { 17 6}


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6 48 N E W M A R K E T A L .

the coba lam in con ten t o f fo od w as inadequ ateex trac tion from the tig h tly -bo und coba lam info rm s . S eve ra l ex ten sive ex trac tion proce -du re s hav e becom e u n ive rsa lly accep ted fo rfoo d assays fo r co ba lam in co n ten t ov er thelas t tw o d ecades. H o w eve r , H erbe rt andJacob (1 ) used a m eth od w hich w as deve lop edfo r a ssay s o f v itam in B 1 2 in se ru m , w herem ild ex trac tion appea red to be adeq ua te bythe re fe rences cited . O n the o the r hand ,consu lting th e n ow ex tensive lite ra tu re rev ea lstha t a m o re ex tended ex trac tion procedu re isrequ ired to relea se th e m ore tig h tly -bo undcoba lam ins p re sen t in m any foo ds (7 , 9 , 12 ) .In add ition , acco rd in g to S k eg gs (9 ) , m anyd isc rep anc ies in assay va lues de te rm ined byrad ioac tive m e tho ds are th e re su lt o f failu reto ex trac t the to ta l cob alam in p re sen t in th esam ple an d con ve rt it to cyano co ba lam inag a inst w h ich it is to b e com p ared . T hesho rt ex trac tion tim e ap pa ren tly u sed byH erbe rt and Jaco b (1 ) w as co nside rab ly le ssth an th at foun d necessary b y R osenb lu m (1 2)fo r to ta l co ba lam in in live r, the recom m en dedex tractio n tim e be ing bo iling fo r 1 h r a t p H4 .0 in th e p re sence of sod iu m nitr ite andpo ta ssium cy an ide .

In the p resen t stu dy th e v itam in B 1 2 va lueso b ta ined by stan da rd ized ex trac tion andassay m e tho ds o f bo th m ea ls w ere in ro ughagreem en t w ith tho se ca lcu lated from lite ra -tu re va lues and m any fo ld h ig he r than tho serep orted b y H erbe rt and Jacob . N o s ign if i-can t de le ter iou s e ffec t o f supp lem en ta ry as-co rb ic ac id on th e v itam in B 1 2 con ten t o f th em ea ls w as foun d , con tra ry to tha t in d ica tedby H erbe rt and Jacob .

I t is in te re stin g to n o te th at th e m ic rob io -lo g ical a ssay m ed ium for v itam in B ,2 used b ybo th the A O A C (6) an d the U n ited S ta te sP ha rm acope ia (13 ) con tain s a sco rb ic acidadd ed a t th e leve l o f 4 g /lite r . T h is is abo u t10 6 tim es the leve l o f v itam in B 12 a t the low est

leve l o f stand a rd used in th e m icrob io log ica la ssay . T h e v itam in B 12 and v itam in C areau to c lav ed tog eth er in th is m ed ia fo r 5 m m at1 21 C as p art o f the n orm a l test p rocedurew itho u t d estruc tion of v itam in B 1 2 .

I t h as lon g b een k now n tha t lo w leve ls o firon in so lu tion in va rious io n ic o r com p lex edfo rm s w ill m ark ed ly enhan ce v itam in B 12stab ility in the p re sence of a sco rb ic ac id .T h us, S heno y and R am asa rm a (10 ) foun d

tha t th e iro n con ten t w as the stab iliz er o fv itam in B 1 2 in live r ex trac ts . C om m erc ia lpha rm aceu tica l liqu id fo rm ula tions h av e usedlow leve ls o f iron sa lts o r com plexes tostab iliz e v itam in B 1 2 ag ain st d eg rada tio n byh igh co ncen tra tion s o f a sco rb ic ac id fo r lo ngpe rio ds o f m o n ths o r yea rs . L ev els o f iro n aslow as 1 7 pp m in the so lu tion (1 4 ) o r 1 2 p pm 15 have b een u sed success fu lly in su chsystem s . T h e U nited S ta tes F o od an d D rugA dm in istra tio n has recog n ized th is use o firon as a v itam in B 12 stab iliz er in aqueou sm ultiv itam in so lu tion s in a F o od A d d itiveR egu latio n (16 ) . M an y fo ods have iron leve lsin th is range .

T he v itam in B 1 2 in foo ds, h ow ev er , un lik eth at in p ha rm aceu tica l p repa ra tion s, is usu -a lly tigh tly bou nd . Fo r ex am ple , the m a jo rp art o f the co ba lam in pre sen t in live r is in th efo rm o f a coen zy m e bo und to a liv er p ro tein .T h is b ind in g se rv es to enhan ce fu rthe r thes tab ility o f v itam in B 12 activ ity , p re sum ab lyb y red uc ing accessib ility o f v itam in B 12 b ind -ing site s to p ro lon ged chem ical attack .

S u m m a r yH erbe rt and Jacob have reported v itam in

B 1 2 v alu es fo r tw o specif ic te st m ea ls tha t a reon ly a sm a ll po rtion (o ne -f ifth to one -eig h th )o f the lev els ca lcu la ted from lite ra tu re va lueso f th e m ea l com pon en ts . T he ir lo w resu ltsare ap pa ren tly d ue to in adequa te ex trac tio nof th e v itam in B 1 2 in the sam ple s duringprep ara tion fo r a ssay .

T he v itam in B 1 2 va lues ob ta in ed b y off i-cia lly recog n ized m e th ods o f ex tractio n andassay ed b y m ic rob io log ica l o r rad io as sayw ere m anyfo ld h ig he r than tho se o f H erbe rtan d Jacob and w ere in rou gh agreem en t w iththose ca lcu lated from lite ratu re va lues.

W hen p erfo rm ed w ith stan da rd ized an doffic ia l ex trac tion m eth ods an d assay ed e ithe rm icrob io log ica lly o r by rad ioassay , the re isno sign if ican t de lete r ious e ffec t o f addedasco rb ic ac id on v itam in B 1 2 s tab ility in foo dsw h en te sted in m ea ls and und er cond itionsreported b y H erbe rt and Jacob . a

T he authors w ish to th ank M iss A nn D ow ell and M r.S a m i W a sse f for he lp in g in pe rfo rm ing the se a ssa ys a ndD rs . M y ron B r in a nd Ja ck B a uernfe ind for the ir he lpfu lsuggest ions.


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S T A B IL IT Y O F V IT A M IN B 1 , IN T H E P R E S E N C E O F A S C O R B IC A C ID 64 9R ef e r en ces

I. H E R B E R T V ., A N D E. J A C O B D es truc tio n of v i tam inB 1 , b y ascorb ic ac id . J. A m . M ed. A sso c. 23 0 : 241 ,1 9 7 4 .

2 . P an to th en ic A cid , V itam in B , a nd V ita m in B 1 , inF oo ds H o m e E c ono m ics R esearch R ep ort N o. 36 ,Agr icu l tu ral R esea r ch S e rv ic e , U .S . D e pt. o f A g ri-cu ltu re , W a sh ing ton , D .C ., 19 69 .

3. K A M I K U B O T ., A N D Y . O G U N I . Microb io log icalde term in atio n of v itam in B 1 , w ith Ochromonasmalhamensis. J. V itam in ol. 5 : 51 , 1 95 9 .

4. L I C H T E N S T E I N H ., A . B E L O IA N A N D H. R E Y N O L D SC om pa rat ive v ita m in B 1 , a ssa y of food s o f an im a lo rig in by L actoba di ll us l ei chma nni i an d Ochromonasmaihamensis. J. A g r . F ood C hem . 7 : 7 71 , 19 59 .

5. G R E G O R Y M . E . T h e m ic rob io log ical as say ofv ita m in B 12 in the m ilk o f d iffe ren t an im a l spe cie s.B rit. J . N utr. 8 : 34 0 , 195 4 .

6 . O ff ic ia l M ethod s of A na lysis (12 th e d). W a sh in g-ton , D .C .: A ssociatio n of O ffic ia l A na ly ticalC he m ists, 1 975 , p . 84 2 .

7 . A na ly tical M ethod s C om m ittee : T h e est im a tio n ofv ita m in B 1 2. A nalyst 8 1 : 13 2 , 1 95 6 .

8. W I D E L . A N D A . K I L L A N D E R A rad io sorb en t tec h-n iq ue for the assay o f se rum v itam in B 12 S can d . J.C lin . L a b . Inv est . 27 : 1 51 , 19 71 .

9. S K E G G S H . R . T he V ita m ins (2n d ed .) N ew Y ork :A c ad em ic P res s, 1 96 7 , v o l. 7 , pp . 27 7-2 93 .

10 . S H E N O Y K . G . A ND G . B . R A M A S A R M A I ron a s astab i lize r of v itam in B 1 , ac tiv ity in l ive r ex tra cts an dth e nature of so -ca lled a lka li sta b le fac to r. A rch .B ioc he m . B ioph ys. 55 : 29 3 , 19 55 .

11 . B R I G G S G . M . A ND F . S . D A F T . W ater so lu b lev i tam ins , par t I (V itam in B 13 , fo lic A cid , ch o line an dpa ra-a m inob en zo ic a cid ). A nn . R e v . B ioc hem . 24:33 9 , 195 5 .

12 . R O S E N B L U M C . A n aly t ica l ap p lications of rad ioac -tive v itam in B 1 2. T a lan ta II : 25 5 , 196 4 .

13 . T h e U nite d S tates P ha rm aco peia , 19 th R e v . U nitedS tate s P ha rm aco peial C o nve ntion , Inc. , R o ckv ille ,M d. 1 97 5 , p . 613 .

14 . N E W M A R K H . L . U .S . P ate n t 2 ,82 3 , 1 67 -F e bru aryII , 1 958 , S tab le v i tam in B 12-con tain in g so lu tion s.

15 . F R E E DM A N L ., M . B L I T Z D . B . S A B IN E A N D E.E I G E N U S. P ate n t 2 ,9 39 ,8 2 I-June 7 , 196 0 , Stab lev itam in B 1, so lu t ion s.

16 . F o od A dd itiv es. F ede ral R e gis ter 27 : 8 83 , 1 962 .


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r r t um

In th e a rtic le S ta b ility o f v ita m in B 1 2 in th e p re s e n c e o f a s c o rb ic a c id b y H a ro ld LN e wm ark J a c o b S ch e m e r M a rtin M a rc u s a nd M u ku n d P ra bh u d e sa i m J Cl in N ut r2 9 : 6 4 5 1 9 7 6 lin e 8 s e c o n d p a ra g ra p h p a g e 6 4 6 s h o u ld re a d 9 0 C fo r I h r in s te a d o f9 0 C fo r2 h r

93 2 T he m e rican Journal of linical N utrition 2 9 : AU G U S T 1 9 7 6 p 93 2 P rin te d in U S A

(gan-lit) am j clin nutr-1976-newmark-645-9 (stability of b12)

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