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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
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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 <
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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.
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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
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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
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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
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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
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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.]
8/11/2019 Metode Biospesifik Penentuan Asam Pantotenat,Biotin, Folat, Vit B6 Dan B12
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 .
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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,
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Anderson , B. B. , Newmark , P . A. , Rawl ins , M. & Green , R.
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