Metode Biospesifik Penentuan Asam Pantotenat,Biotin, Folat, Vit B6 Dan B12

8/11/2019 Metode Biospesifik Penentuan Asam Pantotenat,Biotin, Folat, Vit B6 Dan B12

1/11

Food Chemistry

49 (1994) 191-201

Analyt ical Method s Section

i,

A p p l i c a t i o n o f b i o s p e c i f i c m e t h o d s t o

t h e d e t e r m i n a t i o n o f B - g r o u p v i t a m i n s

in food a review

P . M. F i n g l a s & M. R . A . Mo rg a n

A FR C Insti tute of Foo d Research, Norwich Laboratory, N orwich Research Park, Colney,

Norwich, Norfolk, UK, NR4 7UA

(Received 20 Feb rua ry 1993; revised version received and accepted 14 April 1993)

Biospecific methods using either antibodies or naturally occurring vitamin

binding prote ins for the de terminat ion of pantothenic ac id , b iot in , fola te and

vitamins B6 and BI2 in food are reviewed. Both types of assay are based on the

96-well microtitration plate format, which offers the potential for high sample

throughput. Another feature of these techniques is their flexibility to produce

either highly specific assays (suitable fo r the analysis o f individual forms), or

much broader specificity assays where the analysis of groups of vitamins is

required. The availability of both polyclonal and monoclonal vitamin antisera is

a lso out l ined. The ELISA format us ing ant ibodies has a grea ter potent ia l for

the detec tion of specific vitamers whereas EP BAs are useful for assays of broa d

specificity where the analysis of a g roup of vitamers is required, as in the case of

fola te or b iot in .

I N T R O D U C T I O N

T h e r e i s a g r o w i n g n e e d f o r m o r e r a p i d a n d s p e c i f i c

m e t h o d s f o r v i t a m i n a n a l y s i s , i n p a r t i c u l a r t o m e e t t h e

d e m a n d s o f n u t r i t i o n a l l ab e l li n g o f f o o d s a n d o t h e r n u -

t r i t i o n a l s t u d i e s . A s a n a l t e r n a t i v e t o t h e m o r e c o n v e n -

t i o n a l m e t h o d s o f a n a l y s i s b i o s p e c i f i c p r o c e d u r e s u s i n g

e i t h e r a n t i b o d i e s o r v i t a m i n b i n d i n g p r o t e i n s a r e w e l l -

s u i t e d t o m e e t i n g t h e s e d e m a n d s .

B i o s p e c i f i c m e t h o d s o f a n a l y s i s a r e u s e d t o d e s c r i b e

t w o d i s t i n c t t y p e s o f p r o c e d u r e : f i r s t l y , t h o s e b a s e d o n

t h e s p e c i f i c i n t e r a c t i o n o f a n a n t i b o d y w i t h i t s a n t i g e n ,

f o r e x am p l e , t h e r a d i o i m m u n o a s s a y ( R I A ) o r t h e e n -

z y m e - l i n k e d i m m u n o s o r b e n t a s s a y ( E L I S A ) ; s e c o n d l y ,

t h o s e b a s ed o n t h e u s e o f n a t u r a l l y o c c u r r in g v i t a m i n

b i n d i n g p r o t e i n s w i t h e i t h e r r a d i o l a b e l s ( a s i n t h e r a d i o -

l a b e l l e d p r o t e i n b i n d i n g a s s a y , R P B A ) o r e n z y m e l a b e l s

( a s i n t h e e n z y m e p r o t e i n b i n d i n g a s s a y , E P B A ) . T h e

R P B A i s a v a r i a n t o f t h e i s o t o p e - d i l u t i o n m e t h o d a n d

i s o f t en re fe r red to a s a ' r ad ioa s s ay ' . T he re a re s eve ra l

c o m m e r c i a l l y a v a i l a b l e ' r a d i o a s s a y ' k i t s f o r t h e d e t e r -

m i n a t i o n o f v i t a m i n s i n b l o o d s a m p l e s , b u t t h e i r a p p li -

c a t i o n t o f o o d a n a l y s i s is l im i t e d . E x a m p l e s o f b i o -

s p ec i fi c m e t h o d s u s e d f o r t h e d e t e r m i n a t i o n o f v i t a m i n s

a re g iven in T ab le 1 .

A l t h o u g h R I A h a s b e e n u s e f u l t e c h n i q u e i n c l i n i c a l

a n a l y s i s , i t h a s t h e m a j o r d i s a d v a n t a g e t h a t i t u s e s

Food Chemistry 0308-8146/93/$06.00 1993 Elsevier Science

Publishers Ltd, England. Printed in Great Britain

191

r a d i o a c t i v i t y , w h i c h p r e s e n t s p r o b l e m s a s s o c i a t e d w i t h

i t s s a f e - h a n d l i n g a n d d i s p o s a l . T h i s m a k e s t h e u s e o f

R I A i n r o u t i n e f o o d a n a l y s i s u n a t t r a c t i v e . I n a d d i t i o n ,

s c i n t i l l a t i o n c o u n t i n g o f t h e r a d i o a c t i v i t y c a n b e s t o w

a n d e x p e n s i ve .

T h e E L I S A p e r f o r m e d i n t h e p l a s t i c w e l l s o f m i c r o -

t i t r a t i o n p l a t e s h a s t h e a d v a n t a g e o f u ti l i si n g s ta b l e a n d

n o n - r e a c t i v e r e a g e n t s w h i c h c a n b e s t o r e d f o r l o n g

p e r i o d s . T h e s e a s s a y s c a n b e r e a d i l y a m e n a b l e t o

a u t o m a t i o n b u t c a n a l s o b e c a r r i e d o u t w i t h i n e x p e n -

s iv e e q u ip m e n t . T h i s t y p e o f i m m u n o a s s a y h a s b e e n

w i d e l y e x p l o i t e d i n f o o d a n a l y s i s ( M o r g a n , 1 98 5) . T h e

t y p e o f E L I S A s y s t e m c o m m o n l y u s e d f o r v i t a m i n s i s

t h e i n d i r e c t t y p e , w h e r e v i t a m i n - p r o t e i n c o n j u g a t e

( k n o w n a s t h e h a p t e n ) i s i m m o b i l i s e d t o t h e w e l l

s u r f a c e. T h e p r o t e i n u s e d i s d i f f e r e n t f r o m t h a t u s e d f o r

t h e i m m u n o g e n . T h e f o r m a t f o r t h i s t y p e o f a s s a y is

s hown in F ig . 1 .

P r i m a r y a n t i - v i ta m i n a n t i b o d y a n d v i t a m i n a re a d d e d

t o e a c h w e l l a n d t h e a n t i b o d y b e c o m e s d i s t r i b u t e d

b e t w e e n i m m o b i l i s e d a n d f r e e v i t a m i n a c c o r d i n g t o

h o w m u c h f r e e v i t a m i n i s p r e s e n t i n i t i a l l y . A f t e r p h a s e

s e p a r a t i o n , a c h i e v e d b y w e l l e m p t y i n g a n d w a s h i n g ,

b o u n d p r i m a r y a n t i b o d y i s d e t e c t e d b y t h e a d d i t i o n o f

a s pec ie s - s pec i f i c , enzyme- labe l l ed s econd an t ibody .

T h e s e a r e r e a d i l y a v a i la b l e c o m m e r c i a l l y , a c ti v e a g a i n s t

d i f fe ren t s pec ie s and l abe l l ed wi th a va r i e ty o f enzymes .

A f t e r a n a p p r o p r i a t e i n c u b a t i o n p e r i o d , e x c e s s u n -

b o u n d m a t e r i a l i s r e m o v e d a n d s u b s t r a t e a d d e d . W e l l

o p t i c a l d e n s it i e s ar e d e t e r m i n e d a f t e r a c e r t a i n t i m e a n d


2/11

192

P . M . F i n g l a s , M . R . A . M o r g a n

T able 1 . Bios pec i f i c met hods o f v i t amin ana ly s i s in f ood and b io log ica l mat er ia l s

Type Vitamin Reference

Radioimm unoassay Pantothenic acid

(RIA) Pteroylmonog lutamic acid

P G A )

2 5 - O H - D 3 ~

Wyse et al. (1979)

DaCosta & Rothenberg

(1971); Hend el (1981)

Incstar Ltd (Incstar Corporation,

Sti llwater, M I 55080, USA)

Enzym e-linked Pantothenic acid Mo rris et al . (1988)

immu nosorbent assay Biotin Alcock

(ELISA) Pyridoxamine (PAM ) Alcock et al . (1990)

Folate Finglas, P. M. & Morgan, M. R. A.,

unpublished

Vitamin B~2

5-methyl-THF

Biotin

25- & 24,25-OH-D 3

1,25-(OH)2-D

Radiolabelled protein

binding assay (RPB A)

Enzym e protein Vitamin B12

binding assay (EPBA ) Biotin

Fotates

Lau et al . (1965)

Gutcho & Mansbach (1977)

Ho od (1975); Dakshinamurti &

Allan (1979)

Reinhardt & Ho rst (1988)

Reinhardt

et al .

(1984)

Alcock et al . (1992)

Finglas et al. (1986)

Finglas

et al.

(1888b)

a Comm ercially available k it.

u n k n o w n s a m p l e s q u a n t i f i e d b y r e f e r e n c e t o s t a n d a r d

curves.

T h e a n t i b o d y i s t h e m o s t i m p o r t a n t c o m p o n e n t o f

a n y i m m u n o a s s a y s y s t e m ( s u c h a s R I A a n d E L I S A ) ,

and ant ib ody speci f ic i ty i s usual ly depen dent o n the

immunogen used . Vi t amins a lone a s sma l l molecu l e s

( low molecular weight ) a re not immunogenic , i .e . they do

no t s t imula t e an t i body p roduc t ion i n an ima l s . In o rde r

to ob t a in an t i bod i e s , t he v i t amin mus t f i r s t be coup led

to a la rger , immunogenic carr ie r molecule , normal ly a

p ro t e in . The w ay in which t he v i t amin-p ro t e in con juga t e

E <


3/11

Biospeci f ic methods fo r B -group vi tamins

Ta ble 2 . Co m m erc ia l ly a v a i la ble v i ta m in a nt i sera

193

Antibody to Type Host animal Specificity

D-Biotin Polyclonal, enzyme labelled Goat D-Biotin, avidin (?)

(alkaline phosphatase, peroxidase)

Vitamin BI2 Monoclonal (IgG1) Mouse

Folic acid Monoclonal (IgG2b) Mouse

Folic acid Polyclonal Sheep

5-Methyl-THF Monoclonal (IgG2b) Mouse

5-Methyl-THF Po ly cl on al Sheep

5-Formyl-THF Po ly cl on al Sheep

Vitamin B12 Polyclonal Sheep

Free and protein bound biotin,

adenosylcobalamin

Free and protein bound PGA + 5-

methyl-THF

Free or protein bound PGA

Free or protein bound 5-methyl-THF

Free or protein bound 5-methyl-THF

Free or protein bound 5-formyl-THF

'Vitamin B12

response in the animals. Amounts of conjugate injected

can vary from as little as 100 /zg per animal to much

larger amounts. After a booster injection, normally 4-6

weeks after the primary injection, the animal is bled

and the serum checked for the required antibody

content, avidity and specificity. The animals can be

further boosted and bled until the required antiserum

is obtained. In the case of antibody production to

pantothenic acid, five to six booster injections at

6-weekly intervals were needed before a suitable

antiserum was obtained (Morris et al. , 1988).

The antibodies produced by the above procedures

are polyclonal, i.e. the antiserum consists of a large

number of different antibodies against the hapten,

each with slightly different properties relative to it.

O

i n c u b a t i o n

r e m o v i n g u n b o u nd

f r a c t io n b y w a s h i n g

a d d i t i o n o f

s u b s t r a t e

Fig. 2. EPBA format performed on a microtitration plate.

P-E, enzyme-vitamin binding protein conjugate; O, free

vitamin; O, immoblised vitamin; S, substrate; P, coloured

end-product.

Monoclonal antibodies have also been developed

(Kohler & Milstein, 1975) which are produced

in vitro

from one cell line or clone and are therefore identical.

The advantages of using monoclonal antibodies are

that they are available in unlimited amounts and that

the method of production can sometimes result in an

antibody of improved characteristics.

Most of the antibodies to vitamins are polyclonal,

although there are commercially available monoclonal

antibodies to specific vitamin forms (s ee below).

Commercially available antisera (both polyclonal and

monoclonal) to a range o f vitamin forms have also been

developed primarily for clinical use (Table 2). Although

antisera specificities have been given in most cases, their

application in the ELISA format to the analysis of

vitamins in food has not been examined fully.

The use of naturally occurring vitamin binding

proteins in place of the antibody can also result in

suitable assays. These assays exhibit slightly different

characteristics to the antibody based procedures. How-

ever, most of the advantages of ELISA are applicable

to PBA. Both assay systems are based on the 96-well

microtitration plate format, which is extremely easy

to use and ideal for the batchwise analysis of food

(Finglas & Morgan 1988a).

In the case of EPBA, the binding protein is labelled

directly by chemically coupling to an enzyme such as

horseradish peroxidase. The protein-enzyme replaces

the antibody, and can be used either directly in the

assay (after appropriate dilution), or after further

purification. The format for the EPBA is similar to the

ELISA but contains one incubation step less (Fig. 2).

The development and application of biospecific

methods has largely focused on the water-soluble, B-

group vitamins, where existing methodology is in most

need of improvement. For these vitamins a range of

binding proteins are readily available and a number of

RPBAs have been used for clinical work. The assays

for the individual vitamins will be discussed in more

detail as follows.


4/11

194 P . M . F i n g l a s , M . R . A . M o r g a n

APPLICATION OF BIOSPECIFIC METHODS

P a n t o t h e n i c a c i d

Pantothenic acid occurs in food in the free form and in

larger amounts bound predominantly as coenzyme A

and other protein derivatives (Fox, 1984). Much of the

data on the pantothenate content of food has been

obtained previously by microbiological assay using

L a c t o b a c i l l u s p l a n t a r u m after a double enzyme extrac-

tion procedure to release the bound vitamin forms.

The development and validation of a radioimmuno-

assay for free pantothenic acid was first reported in the

late 1970s (Wyse

e t a L ,

1979). This procedure was

based on the competitive binding of an antibody

specific for pantothenic acid between the unlabelled

vitamin in the sample or standard and radiolabelled

antigen. Although the assay was found to be specific

and sensitive for pantothenate levels in food, and

results correlated highly with results obtained by micro-

biological assay (Walsh et a l . , 1980, 1981), the use of

radiolabelled tracers is not always desirable in the food

context.

A direct, enzyme-linked immunosorbent assay

(ELISA) for pantothenic acid has also been developed

using specific antibodies active to the vitamin which

have been covalently linked to alkaline phosphatase en-

zyme (Smith et a l . , 1981). The immobilised phase o f the

ELISA consisted of a human serum albumin-pan-

tothenate conjugate passively adsorbed to the surface

of polystyrene assay tubes. The binding of the enzyme-

linked antibody to the pantothenate on the surface

of the tubes depends on the amount of free vitamin

present initially. The application of ELISA for the

determination of pantothenic acid in foods was not

reported.

More recently, an indirect ELISA has been devel-

oped, particularly directed to food analysis (Morris e t

a l . , 1988). Pantothenic acid-bovine serum albumin

(BSA) immunogen was synthesised by two different

conjugation procedures utilising either ends of the vita-

min molecule for conjugation in order to generate anti-

sera of different characteristics. In the first of these, the

primary alcohol was derivatised using bromoacetyl bro-

mide to form the bromoacetyl vitamin derivative which

was subsequently reacted with reduced and denatured

protein (Fig. 3). Radiotracer studies using sodium

[1-~4C]-pantothenic acid indicated a covalent attachment

C H ? H 0

~ - ~ ~ H O - CH 2 - C - C -- C - N H - C H 2 - C H 2 - C / /

\

CH H OH (~

Fig. 3. Coupling of pantothenic acid to protein to use as im-

munogens for preparation of anti-pantothenic acid antisera:

(1) bromoacetyl derivatisation and conjugation; (2) mixed an-

hydride procedure. [From: Morris, H. C.

et al.

(1988). The

development of an enzyme-linked immunosorbent assay

(ELISA) for the analysis of pantothenic acid and analogues,

Part I--Production of antibodies and establishment of

ELISA systems.J. Micronutr. Anal. , 4, 33~t5.]

T a b l e 3 . D i f f e r e n t E L I S A f o r m a t s f o r t h e d e t e r m i n a t i o n o f

pa nto thenic a c id ( PA) a nd a na lo g ues

Compound Antiserum Dilution Solid- Limit

typea (v : v) ph as e detectionb

(PA-KLH) (/zg per well)

c o n e .

(/xg ml l)

Pantothenic BA 1 : 4 000 0.1 500

acid

Pantothenic MA 1 10 000 1-0 5 000

acid

Pantetheine MA 1 10 000 1.0 5

Panthenol MA 1 : 10 000 1-0 50

a BA, Bromoacetyl bromide antiserum; MA, mixed anhydride

antiserum.

b Calculated by subtracting three standard deviations from

the mean zero value.

of the vitamin to the protein and a hapten-protein

ratio of 20 : l. The second immunogen was prepared by

linking pantothenic acid to BSA via the carboxylic acid

group by means of the mixed anhydride procedure

(Erlanger et a l . , 1959). A hapten-protein ratio of 17:1

was found by spectrometry for this conjugate. Two

corresponding pantothenic acid-keyhole limpet haemo-

cyanin conjugates were synthesised using the above

coupling procedures and used to form the ELISA solid

phases by passive adsorption to the wells of the mi-

crotitration plates.

Both immunogens generated antisera of differing

qualities (Table 3). The bromoacetyl antiserum was

extremely specific for pantothenic acid with low cross-

reactivities for the compounds tested. It is well-known

that antibodies exhibit the greatest specificity for

portions of the hapten distal to the point of coupling

to the protein carrier. This is clearly illustrated in the

lack of antibody recognition for pantothenol and

pantetheine when the primary alcohol group was used

for conjugation, as the differences in structure occur at

points furthest away. In contrast, the antibody raised

by coupling at the other end of the molecule, i.e. via

the carboxylic acid group, showed greater recognition

for pantetheine and pantothenol compared with pan-

tothenic acid. Both antisera fail to recognise co-enzyme

A, presumably because this contains pantothenic acid

derivatised at each end of the molecule.

The characteristics of the two ELISA systems for the

determination of pantothenic acid and analogues are

given in Table 3. The bromoacetyl antiserum and

ELISA system was chosen for the determination of the

pantothenate content of a range of foods because of its

greater specificity for the free vitamin, lower sensitivi-

ties and backgrounds. The ELISA results were com-

pared to those obtained by microbiological assay

(Table 4). The foods chosen were representative of the

major sources of pantothenic acid in the UK diet and

offer a range of food matrices with different interfer-

ences. The agreement between the two procedures was

generally good, as was the agreement with the food

table data. Earlier work also demonstrated that the


5/11

Biospeci fi c me thods for B -group v i tamins 195

Ta ble 4 . Resul t s fo r pa nto thenic a c id co ntent ( m g per 100 g ) in

f o o d s

Food ELISA Microbiological Food

assay tablesa

Milk (whole cow's) 0.36 0.28 0.35

Potato (maincrop, raw) 0.23 0.23 0.30

Bread (white) 0.23 0-23 0.30

Eggs 1.74 1.46 1.80

Liver (lamb's) 8-25 8-65 8.20

Lettuce 0.18 0.07 0.20

a McCance and Widdowson's

The Composition of Foods,

Paul

and Southgate (1978).

agreement between RIA and microbiological assay for

pantothenic acid in a much larger number of foods was

good (Walsh

et al. ,

1980, 1981).

During the validation of the ELISA procedure, it

was further demonstrated that there was no interfer-

ence (or matrix effect) with the foods tested. The analy-

sis of different volumes of sample extract gave the

correct proportionate responses and the addition of

standard amounts of the vitamin to extracts gave

satisfactory recovery data.

A comparison of the ELISA with both the micro-

biological assay and RIA is given in Table 5. Assuming

the sample extraction is common to each procedure,

then the ELISA can be performed in a working day,

whereas the microbiological assay takes 2-3 days. RIA

requires a 7 h scintillation counting step which will in-

crease if larger numbers of samples are analysed. RIA

also uses more expensive reagents and chemicals but

labour costs are comparable to ELISA. However, the

major drawback of RIA is the use of radiochemicals

which can be a serious limitation to its use in the food

laboratory.

B i o t i n

Biotin is one of the lesser known and most stable of

the B-group vitamins. Conventional determination of

biotin in food and biological materials is usually by

T a b l e 5 . C o m p a r i s o n o f E L I S A , m i c r o b i ol o g ic a l

microbiological assay with

L . p lan tarum.

Biotin can

exist in eight different stereoisomers but only one

contains vitamin activity, D-biotin. The stereoisomer,

L-biotin, is biologically inactive. The organism L.

p lantarum

exhibits a growth response to both biotin

D-sulphoxide (100 ) and O,L-oxybiotin (50 ), in

addition to that for o-biotin (Bonjour, 1991).

Biotin occurs in food both as the free vitamin and in

bound forms where it can be attached to a simple

amino acid, for example, as in biocytin, or more

complex proteins and peptides. The free vitamin is

present in high levels in foods of plant origin. The

complete liberation of the bound vitamin requires acid

hydrolysis at elevated temperatures (Bonjour, 1991).

In addition to the microbiological assay, isotope

dilution assays based on the competition between radio-

labelled and unlabelled vitamin for the binding sites on

avidin, a naturally occurring biotin binding protein,

have been developed. Radiotracers reported include

[~4]C-biotin (Hood, 1979), [3H]-biotin (Dakshinamurti

& Allan, 1979) and [125I]-biotin (Livaniou

et al. ,

1987)

derivatives. These assays are sensitive, analysing 1-

10 ng biotin, but the application of these techniques

for the determination of biotin in food is limited and

not conclusive. Although RIA results were reported to

agree reasonably well with the microbiological assay

for the biotin content in animal feed samples, RIA

values were 20-50 higher than the microbiological

assay in the case of wheat samples (Hood, 1975).

An ELISA and an EPBA assay have also been devel-

oped for the analysis of biotin in food. The EPBA

utilises the high affinity of the ureido group of the

molecule by avidin. An antibody has been produced for

biotin using a biotin-BSA conjugate synthesised

through the carboxylic acid group of the side chain

(Alcock & Morgan, M.R.A. , 1988, pers. comm.) The

ant ibody is extremely specific for D-biotin, and shows

no cross-reactivity with dethiobiotin.

The two assays produce slightly different calibration

curves for biotin reflecting the differences in the affinity

of the vitamin for binding protein on one hand, and

the antibody on the other. The curves are shown in

a ssa y ( MA) a nd RIA fo r pa nto thenic a c id

Method detail ELISA Microbiological assay RIA

Equipment Microtitration plate Autoclave, spectrometer, incubator Scintillationounter

washer and reader, incubator

Reagents Antisera (primary and Organism, cultures, Antisera, radiolabelled

secondary), coated p l at es mic roi noc ulu mroth, media pantothenic

Assay preparation 2 7 3

(h)

Assay length (h) 4 24~36 7

End-method of Ab sor ban cet 450 nm Turbi dity /absor bancet Scintillation

determination 620 nm counting

Sample throughput 125 sps per week 40 sps per week

Cost Fairly low once primary Reagent costs low but

antiserum is available labour high

100 sps per week

Reagents/equipment

high, labour low


6/11


7/11

Bios pe c i fi c m e thods fo r B-g r oup v i tam ins 197

T a b l e 6 . C r u s s - r e a c t i o n a o f v a r i o u s fo r m s o f v it a m i n B 6 f o r

d i f fe r e n t a n t i s e r a p r e p a r a t i o n s

Antiserum PM PL PL PMP PLP 4-PA

type

1 1 0 0 < 0 . 1 < 0 - 0 1 0 . 8 < 0 . 0 1


8/11

198 P . M . F i n g l a s , M . R . A . M o r g a n

c o u p l i n g c y a n o c o b a l a m i n t o K L H u s i n g t h e b r o m o -

a c e t y l b r o m i d e p r o c e d u r e ( W y s e

et a l . ,

1979), i.e. via

the p r imary a l coho l g roup . Th i s was i n con t ra s t t o

p rev ious r epor t s whe re con juga t e s we re syn thes i sed

us ing t he ca rboxy lamide g roups on t he co r r i n r i ng

(Olesen

et a l . ,

1971 ; Van de W e i l

et a l . ,

1974) . Improved

assay sens i t i v i t i e s we re found us ing t he b romoace ty l

c o n j u g a t e c o m p a r e d w i t h t h e e a r l i e r w o r k w h i c h

a l l owed the use o f t he enzyme l abe ll ed R-pro t e in a t

much h ighe r d i l u t i ons . The coa t ed mic ro t i t r a t i on p l a t e s

and enzyme l abe l l ed R-pro t e in so lu t i ons we re found t o

be s t ab l e fo r seve ra l months wi thou t apprec i ab l e l oss

of ac t ivi ty .

The l im i t o f de t ec t i on o f t h i s a ssay i s 9 pg pe r we l l

and t he re i s a l ow l eve l o f non-spec i f ic back grou nd

b i n d i n g . T h e P B A w a s u s e d t o d e t e r m i n e t h e a m o u n t

o f a d d e d c y a n o c o b a l a m i n i n b r e a k f a s t c e re a ls . G o o d

a g r e e m e n t w a s f o u n d b e t w e e n t h e a n a l y s e d a m o u n t

de t e rmined by EPBA and t he t heore t i c a l l eve l . Ex t rac t s

o f non- fo r t i f i ed sample s w e re t e s t ed fo r i n t e r fe rence i n

t h e a s s a y a n d g a v e s u p e r i m p o s a b l e c u r v e s c o m p a r e d

wi th s t anda rd so lu t i ons . I t was a l so obse rved t ha t

shor t e r i ncuba t i on t imes o f abou t 1 h a t 37C gave

s imi l a r r e su l t s t o t hose ob t a ined a t 4C wi th an

ove rn igh t i ncuba t i on .

I t w a s c o n c l u d e d t h a t t h e E P B A f o r c y a n o c o b a l a m i n

ana lys i s i n fo r t i f i ed foods had a number o f advan t ages

ove r bo th t he mic rob io log i ca l a ssay and RIA. I t was

eas i e r t o pe r fo rm , had po t en t i a l fo r improved rep ro -

duc ib i l i t y and much- reduced a ssay t imes compared

wi th mic rob io log i ca l p rocedure s . Al though the l im i t o f

de t ec t i on fo r EPBA i s su f f i c i en t fo r use wi th fo r t i f i ed

foods , i n o rde r t o measure na tu ra l l eve l s i n foods ,

fu r the r improvement s i n a ssay sens i t i v i t y us ing a l t e rna -

t i ve end-po in t de t ec t i on sys t ems and ex t rac t i on / c l ean-

u p p r o c e d u r e s m a y b e r e q u i re d .

F o l a t e

The fo l a t e s rep re sen t an impo r t an t g roup o f t he B-

group v i t amins . A l a rge numbe r o f fo l a t e v i tamers a re

know n, d i ff e r ing i n t he ex t en t o f the r educ t i on o f t he

p t e roy l g roup , t he p re sence o f subs t i t uen t s and t he

num ber o f g lu t amyl r e s idues . In o rde r t o measure t he

po lyg lu t ama te fo rms p re sen t i n b io log i ca l ma te r i a l s ,

p re l im ina ry enzyma t i c decon juga t i on i s r equ i red .

At p re sen t t he mos t wide ly used and accep t ed p ro -

cedure fo r fo l a t e s i s t he mic rob io log i ca l a ssay us ing L .

r h a m n o s u s

( A T C C 7 4 6 9 ) a n d

E n t e r o c o c c u s h i r a e

(A TC C 8043), whe re a r e sponse o f t he o rgan i sm to t he

mix tu re o f fo l a t e s p re sen t i n t he sample i s measured .

E v e n w i t h t h e i n t r o d u c t i o n o f s e m i - a u t o m a t e d m i c ro -

b io log i ca l p rocedure s i nc lud ing t he mic ro t i t r a t i on p l a t e

f o r m a t ( N e w m a n & T s a i, 1 9 8 6 ; H o r n e & P a t te r s on ,

1988) , t h i s approach i s bo th t ime -consuming and

demanding i n execu t ion . In add i t i on , t he r e sponse o f

the o rgan i sm to t he d i f f e ren t fo l a t e fo rms i s no t a lways

identical .

H P L C p r o c e d u r e s u s i n g b o t h U V a n d f l u o r o m e t r i c

de t ec t i on fo r t he ana lys i s o f food fo l a t e s have been

wide ly r epor t ed i n r ecen t yea r s . Al though adequa t e

re so lu t i on fo r s t anda rd mix tu re s can be ob t a ined , t he

low fo l a t e l eve l s found i n mos t foods can l e ad t o

prob l ems in t he de t ec t i on and quan t i f i c a t i on o f t he

v i t amers us ing t h i s t e chn ique . Th i s ha s been demon-

s t r a t ed i n a r e cen t EC in t e r l abora to ry compara t i ve

s tudy fo r t he de t e rmina t i on o f fo l a t e i n Brusse l s

sp rou t s (F ing l a s et a l . , 1993). A g r oup o f expe r i enced

v i t amin l abora to r i e s t ook pa r t u s ing mic rob io log i ca l ,

H PL C and b iospec i f i c p rocedure s . Al tho ugh the ag ree -

m e n t b e t w e e n t h e m i c r o b i o l o g i c a l m e t h o d s w a s q u i t e

e n c o u r a g i n g w i t h a b e t w e e n - l a b o r a t o r y C V o f a b o u t

2 0 % , t h e r e w a s p o o r a g r e e m e n t b e t w e e n t h e H P L C

procedure s , pa r t i cu l a r l y i n t he t ypes o f fo l a t e v i t amers

measured (Tab l e 8 ) .

Seve ra l r epor t s i n t he l i t e ra tu re can be found o f t he

ana lys i s o f fo l a te s i n food us ing RIA , an d t he i r com-

pa r i son t o t he mic rob io log i ca l a ssay (Graham et a l . ,

1980 ; Re ingo ld

et a l . ,

1980 ; Kle in & Ku o , 1981) . I t

wou ld appea r t ha t t he r e su l t s ob t a ined be tween t he

RIA and mic rob io log i ca l p rocedure s a re f r equen t ly

con t rad i c to ry .

A nu mb er o f f a c to r s a re know n to a f fec t t he fo l a te -

bindin g aff ini ty of the prote in used, e .g . tempe ra ture ,

i ncuba t i on t ime and pa r t i cu l a r l y pH. In add i t i on , t he

ca l i b ran t used i n t he a ssay mus t be s t ab l e and have an

aff ini ty for the binding prote in tha t i s the same as the

fo l a t e fo rm found in t he sample s . Al though PGA i s

more s t ab l e t han 5 -me thy l -THF, i t shou ld no t be used

as t he a ssay ca l i b ran t i n a ssays run a t pH 7-5 , because

of it s g rea t e r a f f in i ty fo r t he F BP c om pared t o t ha t o f

5 -me thy l -THF a t t h i s pH (Givas & Gutcho , 1975) .

How eve r , a t t he h ighe r pH of 9 .3 , PG A i s p re fe r red be -

cause o f it s g rea t e r s t ab i l it y and equ iva l en t b ind ing

c o m p a r e d w i t h 5 - m e t h y l - T H F . A l t h o u g h t h e u s e o f

comm erc i a l RI A k i t s fo r the ana lys i s o f b lood l eve ls o f

5 -m e thy l -T HF can be j us ti f i ed wi th ca re fu l s t anda rd i sa -

t i on o f t he p rocedure s , t he ap p l i ca t i on o f the se a ssays

to food sys t ems i s l im i t ed and no t conc lus ive (Gregory ,

1985).

I n o n e o f t h e f e w r e p o r t s o f a n t i b o d y p r o d u c t i o n t o

f o la t e c o m p o u n d s , b o t h P G A - B S A a n d p - a m i no -

benzo i cg lu t amic ac id+BSA were used a s immunogens

in r abb i t s (R icke r & Sto l l a r, 1967 ) . The an t i se rum

obta ined f rom the fo rmer , i . e . whe re t he con juga t i on

was t h rough the g lu tamic ac id end o f t he molecu l e

(F ig . 6 ) , exh ib i t ed c ross - reac t i v i t i e s no t on ly t o PGA

(100%) bu t a l so t o two non- fo l a t e ana logues (p t e ro i c

ac id, 71% and 6 -aminop te r in , 24%). By con t ra s t , whe re

con juga t i on was t h rough the p t e r i d ine r i ng , a s i n t he

second con juga t e , even b roade r spec i f i c i t y an t i se rum

w a s p r o d u c e d .

As an a l t e rna t i ve , an EPBA assay was deve loped fo r

fola te ut i l i s ing an enzyme label led fola te binding

pro t e in f rom cow' s m i lk (F ing l a s et a l . , 1988b). The

b ind ing p ro t e in i s commerc i a l l y ava i l ab l e . S t anda rd

c u r v e s f o r P G A , 5 - f o r m y l - T H F a n d 5 - m e t h y l - T H F

u s i n g P G A - K L H c o a t e d p l a t e s a t a c o n c e n t r a t i o n o f

1 /xg ml ~ and an enz ym e-F BP d i l u t ion o f 1 :100 (v /v )

a re shown in F ig . 7a . Al l t h ree cu rves exh ib i t good


9/11

B i o s p e c i f ic m e t h o d s f o r B - g r o u p v i t a m i n s 199

Ta ble 8 . Fo la te l ev e l s ( / ~g per 100 g ) in Brusse l s spro uts by

different procedures*

Meth o d b

Deco n ju g ase en zy m e Mean fo l a te

co n t en t

(numb er o f labs)

Microbio log ical Hu m an p lasma 824 (6)

assay Chicken p lasma 984 (6)

EP BA Hu m an p l asm a 7 39 (3)

Chicke n plasm a 1 320 (2)

RP BA Hu m an p l asm a 2 0 9 3 (2)

Chicke n plasm a 1 290 (2)

HP LC ( to tal ) Hum an p lasma 762 (1)

Chicke n plasm a 729 (1)

F ro m F in g las et al. (1993).

b

EPBA: en zym e labelled protein binding assay. RPBA :

rad io label led pro tein b ind ing assay (commercial ly avai lab le

'radioassay' kits.

Tot al fo late includ ing 5-me thyl -THF , 5-form yl-TH F and

T H F f or m s .

l i m i ts o f d e t e c t i o n o f 6 , 34 a n d 3 6 ~ g p e r w e l l, r e s p e c -

t iv e ly . T h e r e l a ti v e r e s p on s e s o f 5 - f o r m y l - T H F a n d 5 -

m e t h y l - T H F a r e a p p r o x i m a t e ly t h e s a m e a n d t h e re f o r e

e i t h e r f o r m c o u l d b e u s e d a s t h e c a l i b r a n t i n t h e a s s a y

f o r f o o d w o r k . A l t h o u g h P G A d o e s n o t o c c u r n a t u -

r a l l y i n f o o d s , i t i s u s e d f o r e n r i c h m e n t p u r p o s e s , s o i n

t h a t c a s e P G A s h o u l d b e u s e d a s t h e c a l i b r a n t . O t h e r

f o l a te f o r m s , w i t h t h e p o s s i b le e x c e p t i o n o f T H F , a r e

t h o u g h t t o b e u n s t a b l e d u r i n g e x t r a c t i o n . B y c h a n g i n g

t h e pl a te c o a ti n g c on j u g a te f r o m P G A - K L H t o 5-

f o r m y l - T H F t h e r es p o n se o f t he P G A f o la t e f o rm c a n

b e m o d i f i e d a n d b e c o m e s c l o s er t o t h e r e s p o n s e o f t h e

o t h e r t w o f o l a t e f o r m s t e s t e d ( F i g . 7 b ) .

T h e E P B A w a s u s e d f o r t he d e t e r m i n a t i o n o f f o l a te

i n a r a n g e o f r a w a n d c o o k e d v e g e t a b l e s ( F i n g l a s et a l . ,

1 9 8 8 b ) . S a m p l e s w e r e a l s o a n a l y s e d b y m i c r o b i o l o g i c a l

a s s a y a n d t h e r e s u l t s f r o m t h e t w o a s s a y s c o m p a r e d .

G o o d a g r e e m e n t w a s o b t a i n e d b e t w e e n t h e m e t h o d s

w i t h a l i n e a r r e l a t i o n s h i p o v e r t h e r a n g e 0 4 0 0 /~ g p e r

1 0 0 g f o u n d . T h e E P B A c o u l d b e c a r r i e d o u t i n o n e

w o r k i n g d a y c o m p a r e d t o 2 - 3 d a y s f o r t h e m i c r o b i o -

lo g i ca l a s say .

R e s u l t s f r o m t h e r e c e n t i n t e r l a b o r a t o r y c o m p a r i s o n

o f f o l a t e m e t h o d s ( F i n g l a s

et a l . ,

1 9 9 3 ) a r e g iv en i n

T a b l e 8 . T h e E P B A s u s e d w e r e e s s e n t i a l l y b a s e d o n t h e

s a m e f o r m a t a s d e s c r i b e d a b o v e b u t t h e r e s u l ts b e t w e e n

l a b o r a t o r i e s v a r i e d f r o m 3 7 8 t o 1 0 5 1 /~ g p e r 1 00 g

( n = 3 ) f o r h u m a n p l a s m a e n z y m e a n d f r o m 9 0 3 t o

1 6 0 1 p~g p er 1 0 0 g (n = 2 ) fo r ch i ck e n p a n cr eas d ec o n -

j u g a s e e n z y m e . T h e H P L C p r o c e d u r e s i n d i c a t e d t h e

m a j o r f o l a t e f o r m s p r e s e n t i n B r u s s e l s s p o u t s w e r e 5 -

m e t h y l - T H F ( 5 0% ) , T H F ( 30 % ) a n d 5 - f o r m y l - T H F

( 20 % ). T w o o f th e l a b o r a t o r i e s u s e d 5 - m e t h y l - T H F a s

t h e c a l i b r a n t , w h e r e a s t h e t h i r d u s e d P G A .

T h e s t u d y c o n c l u d e d t h a t t h e m a i n l i m i t a t i o n o f

b o t h t h e E P B A a n d R I A i s t h e r e s p o ns e o f t h e in d i-

~ a )

HN N N

2 ' ~ y ~ ~ 0 COOH

I | ~ H I I J H

N N S ~C . N = ~ . . ~ C--N --C --C 2--C 2--C O

OH

b )

c ) d )

O

O

e ) f )

Fig. 6. Fola te vitamins and structura lly related com pound s: (a) folic or pteroy lglutam ic acid; (b) 5,6,7,8-tetrahydrofolic acid

(R = H) , 5 -formyl tet rahydrofo l ic acid (R - - CHO), 5 -methyl tet rahydrofo l ic acid (R - - CH3); (c) p tero ic acid ; (d ) p ter ine (R ' - - H) ,

pterin-6-c arbox ylic acid (R' = COO H); (e) p-am inobe nzoic acid (PAB A) and (f) N-(p-aminobenzoyl)-L-glutamic acid (PABG A).

[From: Finglas, P. M. et aL (1988) . The developm ent and cha racter i sat ion o f a p ro tein-b id ing assay for the determinat ion of

fo late-- po ten t ial use in food analysis . J . Micronutrient Analysis, 4, 295308.]


10/11

100

0

0

J

i

1 0 p g 1 0 0 p g l n g 1 0 n g 1 0 0 n g

100

50

E

E

Folate vitamin/well

Fig. 7a. EPBA standard curves for var ious fo lates using

P G A -K LH p l a tes co a t ed a t 1 /zg m l l an d F BP -en z y m e

c o n ju g a te a t l : 1 0 0 ( v : v ) . l , P G A ; Q , 5 - C H O T H F ; A ,

5-CH3THF. Marker bars ind icate _+l SD. [From: as for

Fig. 6.]

50

0

0

~ i :

1 0 p g 1 0 0 p g l n g 1 0 n g 1 0 0 n g

2 0 0 P . M . F i n g l a s , M . R . A . M o r g a n

Folate vitamin/well

Fig.

7b. EPBA curves for var ious fo lates using 5-formyl-

tet rahydrofo l ic ac id-K LH plates coated at l0 /zg ml 1. I ,

P GA ; O, 5 -CH OTH F ; A , 5 -CH3 TH F . Mark er b a rs i n d ica t e

_+ 1 SD. [From: as for Fig. 6.]

v i d u a l f o la t e f o r m s t o t h e F B P u s e d. C a r e f u l c o n t r o l o f

t h e p H o f t h e a s s a y b u f f er u s e d a n d c h o i c e o f th e a s s a y

c a l i b r a n t i s n e e d e d , i f t h e s e a s s a y s a r e t o b e a p p l i e d t o

t h e m e a s u r e m e n t o f f o o d f o la t e s.

T h e E P B A a s s a y f o r f o l a te o f f er s t h e p o t e n t i a l

f o r b r o a d s p e c i fi c i ty a n d s u f fi c ie n t s e n s it i v it y f o r t h e

m e a s u r e m e n t o f f o o d f ol a te s . F u r t h e r w o r k , h o w e v e r ,

i s n e e d e d t o s t a n d a r d i s e t h e m e t h o d o l o g y , p a r t i c u l a r l y

i n t h e c h o i c e o f c a l i b r a n t a n d a s s a y c o n d i t i o n s , b e f o r e

r e s ul ts c a n b e c o n f i d e n t l y c o m p a r e d w i t h o t h e r p r o -

c e d u r e s s u c h a s t h e m i c r o b i o l o g i c a l a s s a y .

C O N C L U S I O N S

B i o s p e c i f ic m e t h o d s o f a n a l y s i s o f f e r t h e a n a l y s t c o n -

s i d e r a b l e p o t e n t i a l t o f u l f il t h e i n c r e a s i n g n e e d f o r

m o r e r a p i d , s e n s i t iv e a n d s p e c if i c v i t a m i n a s s a y s . T h e

t e c h n i q u e s d o n o t r e q u i r e h i g h l y s k i l l e d o p e r a t o r s

o r s o p h i s t i c a t e d d e t e c t i o n s y s t e m s a n d c a n b e e a s i l y

a u t o m a t e d .

E P B A s a r e u s e f u l f o r a s s a y s o f b r o a d s p e c i fi c i ty

w h e r e t h e a n a l y s i s o f a g r o u p o f v i t a m e r s i s r e q u i r e d ,

a s i n t h e c a s e o f f o l a t e o r b i o t i n . T h u s , t h e r e s u l t s

o b t a i n e d w i t h t h e s e a s sa y s m a y b e e x p e c t e d t o c o r r e l a t e

b e t t e r w i t h t h e r e s u l t s o f m i c r o b i o l o g i c a l p r o c e d u r e s .

H o w e v e r , t h e E L I S A f o r m a t u s i n g a n t i b o d i e s h a s t h e

g r e a t e r p o t e n t i a l f o r t h e d e t e c t i o n o f s p e c if i c v i t a m i n

f o r m s , w h i c h c a n b e u s e f u l f o r n u t r i t i o n a l s t u d i e s

i n c l u d i n g t h e a s s e s s m e n t o f v i ta m i n b i o a v a i l a b i l i t y in

f o o d a n d t h e m e a s u r e m e n t o f v it a m i n s t a tu s . T h e

s p e c i fi c i ty o f a n t i b o d i e s m e a n s t h a t t h e y c o u l d b e u s e d

i n c o n j u n c t i o n w i t h c h r o m a t o g r a p h i c p r o c e d u r e s t o

a s s i st i n t h e i d e n t i f i c a t i o n / q u a n t i f i c a t i o n o f i n d i v i d u a l

v i t a m e r s b e c a u s e n o r m a l d e t e c t i o n s y s t e m s a r e i n a d e -

q u a t e . I n a d d i t i o n , b y t h e c a r e f u l s e l e c t i on o f i m m u -

n o g e n c o n j u g a t e s , o r b y m i x i n g t w o o r m o r e d i f f e r e n t

a n t i s e r a t o g e t h e r , a n t i b o d i e s c o u l d b e u s e d t o d e t e c t

s e l ec t e d c o m b i n a t i o n s o f v i t a m e r s .

R E F E R E N C E S

Alcock, S. A., Finglas, P. M. & Morgan, M. R. A. (1990).

An enzyme-l inked immunosorbent assay for pyr idoxamine

and i t s compar ison wi th al ternate analy t ical p rocedures .

Food and Ag ricultural Immu nology, 2, 197-204.

Alcock, S. A., Finglas, P. M. & Morgan, M. R. A. (1992).

Produc t ion and pur i f ication of an R-pro tein enzym e conju-

gate for use in a micro t i t rat ion p late pro tein-b ind ing assay

for vitamin B12 in fortified food. Food Chemistry, 45,

199-203.

Anderson , B. B. , Newmark , P . A. , Rawl ins , M. & Green , R.

(1974) . P lasma b ind ing of v i tamin B6 compounds.

Nature,

2 5 0 ,

502.

Bon jour, J. P. (1991). Biotin. In: Handbook of Vitamins, 2nd

edn, ed . L. J . Machl in . Marcel Dekker , New York , pp .

393-427.

Butler, V. P. & C he n, J. P. (1967). Digoxin-specific antibod-

ies.

Proc. Nat l Acad. Sci . USA,

57, 71-8.

Chin , H . B. (198 5) . Vi tamin B I2 . In :

Methods of Vi tamin

Assay, 4th edn, ed. J. Augustin, B. P. Klein, D. A. Becker

& P. B. Venugopal. Wiley-Interscience, New York, pp.

497-514.

Cordo ba, F . , Gon zalez, C. & Rivera, P . (1966) . Ant ibodies

against pyr idoxal -5-phosphate and pyr idoxamine-5-phos-

phate.

Biochim. Biophys. Acta,

127, 151-8.

DaC osta, M. & R othenberg , S . P . (1971) . Iden t i ficat ion of

an immunoreact ive fo late in serum ext racts by rad io-

immunoassay . Br. J. Hematol., 21, 121-30.

Dakshinamurti , K. & Allan, L. (1979). Isotope dilution assay

for biotin: use of [3H]-biotin. In: Methods in Enzymology,

Vol . 62D, ed . D. McCormick & L. Wright . Academic

Press , New York , pp . 284-7 .

England ,

J. M. & Linnel l , J . C. (1980).

Lancet

,

1072-4.

E rlanger , B. F . , Borek, O. F., Beiser, S. M. & L iebermann , S .

J . (1959) . Preparat ion and character i sat ion of conjugates of

bovine serum albumin wi th progesterone, deoxycor t ico-

s terone and esterone.

J. Biol . Chem.,

234, 1090-4.


11/11

B i o s p e c i fi c m e t h o d s f o r B - g r o u p v i t am i n s 201

Farquharson, J. & Adams, J. F. (1976). The forms of vitamin

B-12 in foods. Br. J . Nutr. , 36, 127-36.

Finglas, P. M., Faulks, R. M. & Morgan, M. R. A. (1986).

The analysis of biotin in liver using a protein-binding

assay. J. Micronutr. AnaL, 2, 247-57.

Finglas, P. M. & Morgan, M. R. A. (1988a). The determina-

tion o f vitamins in food by biospecific analysis. Internat.

Industrial Biotechnology, 8(3), 9-12.

Finglas, P. M., Faulks, R. M. & Morgan, M. R. A. (1988b).

The development and characterisation of a protein-binding

assay for the determination of folate--potential use in food

analysis. J.

Micronutr. AnaL,

4, 295-308.

Finglas, P. M., Kwiatkowska, C. A., Faulks, R. M. &

Morgan, M. R. A. (1988c). Comparison of a non-isotopic,

microtitrat ion plate folate-binding protein assay and a

microbiological method for the determination of folate in

raw and cooked vegetables. J.

Micronutr. AnaL,

4, 309-22.

Finglas, P. M., Faure, U. & Southgate, D. A. T. (1993). First

BCR-intercomparison on the determination of folates in

food. Food C hemistry , 46 , 199-213.

Fox, H. M. (1984). Pantothenic acid. In: H a n d b o o k o f

Vitamins--Nutritional, Biochemical and Clinical Aspects,

ed. L. J. Machlin. Marcel Dekker, New York, pp. 437-57.

Givas, J. K. & Gutcho, S. (1975). pH dependence of the

binding of folates to milk binder in radioassay of folates.

Clin. Chem., 21,427-8.

Goodfriend, T. L., Levine, L. & Fasman, G. D. (1964). Anti-

bodies to bradykinin and angiotensin: a use of carbo-

dimides in immunology.

Science,

144, 13~A 6.

Graham, D. C., Roe, D. A. & Ostotag, S. G. (1980). Radio-

metric determination and chick bioassay of folacin in forti-

fied and unfortified frozen foods. J. Food ScL , 45, 47.

Gregory, J. (1982). Relative activity of the non-phosphory-

lated B-6 vitamers for Saccharomyces uvarum and

Kloechera brevis in vitamin B-6 microbiological assay. J.

Nutr . , 112, 1642.

Gregory, J. F. (1985). Folacin. Chromatographic and radio-

metric assays. In: Methods o f V i tamin Assay, 4th edn, ed. J.

Augustin, B. P. Klein, D. A. Becker & P. B. Venugopal.

Wiley-Interscience, New York, p. 485.

Gutcho, S. & Mansbach, L. (1977). Simultaneous radioassay

of serum vitamin B12 and folic acid. Clin. Chem., 23, 1609.

Hendel, J. (1981). Radioimmunoassay for pteroylmonoglu-

tamic acid.

Clin. Chem.,

27, 701.

Hood, R. L. (1975). A radiochemical assay for biotin in bio-

logical materials. J. Sci. Food Agric. , 26, 1847-52.

Hood, R. L. (1979). Isotope dilution assay for biotin: use of

[14C]-biotin. In: M eth o d s in E n zym o lo g y , Vol. 62D, ed. D.

McCormick & L. Wright. Academic Press, New York, pp.

279-83.

Horne, D. W. & Patterson, D. (1988). Lactobacillus casei

assay of folic acid derivatives in 96-well microtitre plates.

Clin. Chem., 34, 2357-9.

Klein, B. P. & Kuo, C. H. (1981). Comparison of microbio-

logical and radiometric assays for determining total folacin

in spinach.

J. Food Sci. ,

6, 552.

Kohler, G. & Milstein, C. (1975). Continuous cultures of

fixed cel ls producing antibodies of predefined specificity.

N a tu re L o n d o n ) ,

256, 495-7.

Lau, K.-S., Gottlieb, C., Wasserman, L. R. & Herbert, V.

(1965). Measurement of serum vitamin B12 level usingradio-

isotopic dilution and coated charcoal.

Blood,

26, 202-14.

Livaniou, E., Evangelatos, G. P. & Ithakissios, D. S. (1987).

Biotin radioligand assay with an J25I-labelled biotin deriva-

tive, avidin and avidin double antibody reagents. Clin.

Chem., 33, 1983-88.

Marcus, M., Prabhudesai, M. & Wassef, S. (1980). Stability

of vitamin B12 in the presence of ascorbic acid in food and

serum: restoration by cyanide of apparent loss.

Am. J . Clin .

Nu tr . , 33, 137.

Mock, D. M. & Dubois, D. B. (1986).

Anal, Biochem.,

153,

272-8.

Morgan, M. R. A. (1985). New techniques in food analysis--

immunoassays and their application to small molecules. J.

Assoc. Public Analysts, 23, 59-63.

Morris, H. C., Finglas, P. M., Faulks, R. M. & Morgan, M.

R. A. (1988). The development of an enzyme-linked im-

munosorbent assay (ELISA) for the analysis of pantothenic

acid and analogues. Part I- -Produ ction of antibodies and

establishment of ELISA systems. J. Micronutr. AnaL, 4,

33-45.

Newman, E. M. & Tsai, J. F. (1986). Microbiological analysis

of 5-formyltetrahydrofolic acid and other folates using an

automatic 96-well plate reader.

Anal B iochem. ,

154,

509-15.

Newmark, H. L., Scheiner, J., Marcus, M. & Prabhudesai,

M. (1976). Stability of vitamin B12 in the presence of

ascorbic acid. Am. J . C l in . Nu tr . , 29, 645-51.

Olesen, H., Hippe, E. & Hater, E. (1971). Nature of vitamin

B12 binding. I Covalent coupling of hydroxocobalamin to

soluble and insoluble carriers. Biochim. Biophys. Acta, 243,

66-74.

Polansky, M. M., Reynolds, R. D. & Vanderslice, J. T.

(1985). Vitamin B6. In: Methods o f V i tamin Assay, 4th edn,

ed. J. Augustin, B. P. Klein, D. A. Becker & P. B. Venu-

gopal. Wiley-Interscience, New York, pp. 417-43.

Reingold, R. N., Picciano, M. F. & Perkins. G. (1980).

Identification of folate forms in selected infant foods. Fed.

Proc., 39, 656-9.

Reinhardt, T. A. & Horst, R. L. (1988). Simplified assays for

the determination of 25-OH-D, 24,25-(OH)2-D and 1,25-

(OH)2-D. In: Vitamin D. Molecular, Cellular and Clinical

Endocrinology,

ed. A. Norman. Walter de Gruyter, Berlin,

pp. 720q5.

Reinhardt, T. A., Horst, R. L., Orf, J. W. & Hollis, B. W.

(1984). A microassay for 1,25-dihydroxy vitamin D not

requiring high performance liquid chromatography: appli-

cation to clinical studies. J.

Clin . Endocrino l . Metab . ,

58,

91-8.

Richardson, P. J., Favell, D. J., Gridley, G. C. & Jones, G.

H. (1978). Application o f a commercial radioassay test kit

to the determination of vitamin B12 in food. Analyst , 103,

865-8.

Ricker, R. & Stollar, B. D. (1967). Antibodies reactive with

specific folic acid determinants.

Biochemistry,

6, 2001-5.

Shaw, W. H. C. Bissell, C. J. (1960). The determination o f

vitamin B12. A critical review. Analyst , gs, 389--409.

Smith, A. H., Wyse, B. W. & Hansen, R.._q. (1981). The

development of an ELISA for pantothenat .. Abstract No.

3886, Fed. Proc. , 40, 915.

Tsalta, C. D. & Meyerhoff, M. E. (1987). Homogeneous

enzyme-linked assay for vitamin B12. Anal Chem. , 59,

837-41.

Van de Weil, D. F. M., Goedemans, W. T. & Woldring, M.

G. (1974). Production and purification of antibody against

protein-vitamin B12 conjugates for radioimmunoassay

purposes. Clin . Chem. Acta , 56, 143-9.

Viceps-Madore, D. J. Cidlowski, J. A., Kittler, J. M. &

Thanassi, J. W. (1983). Preparation, characterisation and

use of monoclonal antibodies to vitamin B6. J. Bio l .

Chem., 258, 2689-96.

Voigt, M. N. & Eitenmiller, R. R. (1960). Comparative

review of the thiochrome, microbial and p rotozoan analysis

of B-vitamins. J. Food Protection, 41,730-8.

Walsh, J. H., Wyse, B. W. & Hansen, R. G. (1980). A com-

parison of microbiological and radioimmunoassay methods

for the determination of pantothenic acid in foods. J. Food

Biochem., 3, 175-82.

Walsh, J. H., Wyse, B. W. & Hansen, R. G. (1981). Pan-

tothenic acid content of 75 processed and cooked foods. J.

Am. Dietet. Assoc. , 78, 140-3.

Wyse, B. W., Wittwer, C. & Hansen, R. G. (1979). Radio-

immunoassay for pantothenic acid in blood and other

tissues.

Clin. Chem.,

25, 108-1 i.

Metode Biospesifik Penentuan Asam Pantotenat,Biotin, Folat, Vit B6 Dan B12

Documents

Transcript of Metode Biospesifik Penentuan Asam Pantotenat,Biotin, Folat, Vit B6 Dan B12