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In Vitro Estimation of Food Iron Bioavailability

DENNIS D. MILLER and BRIAN R. SCHRICKER

Cornell University, Department of Food Science, Ithaca, NY 14853
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Publication Date: November 1, 1982 | doi: 10.1021/bk-1982-0203.ch002

A simple, rapid, and inexpensive in vitro

method for estimating food iron availability is de-
scribed. The method involves a simulated gastro-
intestinal digestion using commercially available
enzymes. Soluble, low molecular weight iron is
used as an indicator of iron availability. Similar
results are obtained when the soluble iron is in-
trinsic food iron or added extrinsic radioiron.
The method is designed to be used with single foods
or complex meals. Results obtained with this
method compare favorably with published results
from human studies using extrinsic radioiron tag
methods. The method resembles several published
in vitro methods but is unique in two ways:
1. pH adjustment is achieved by dialysis. 2. Low
molecular weight soluble iron rather than total
soluble iron is used to estimate available iron.

I t i s w e l l e s t a b l i s h e d t h a t e v a l u a t i o n of d i e t s f o r i r o n
adequacy r e q u i r e s knowledge of both the amount and the a v a i l -
a b i l i t y of the i r o n present (1). While i n f o r m a t i o n on the i r o n
content of foods i s reasonably adequate, knowledge of food i r o n
a v a i l a b i l i t y i s incomplete. T h i s gap i n our understanding o f the
p o t e n t i a l of d i e t a r y i r o n to meet n u t r i t i o n a l needs e x i s t s be-
cause a number o f complex and i n t e r a c t i n g f a c t o r s i n f l u e n c e food
i r o n a v a i l a b i l i t y and because i r o n a v a i l a b i l i t y i s d i f f i c u l t to
measure. I r o n a b s o r p t i o n from a food i s a f f e c t e d not only by the
chemical form of the i r o n i n the food but a l s o by the i r o n s t a t u s
of the person consuming the food, the presence of other foods i n
the same meal, the amount of a c i d s e c r e t e d by the stomach, the
r a t e of passage of the food through the d i g e s t i v e t r a c t , and,
most l i k e l y , other f a c t o r s . The experience o f numerous i n v e s t i -
gators has shown that accurate measurement of i r o n a v a i l a b i l i t y
i s a d i f f i c u l t , expensive and time consuming process. The occur-
rence i n the l i t e r a t u r e of f r e q u e n t l y c o n f l i c t i n g data a t t e s t s to

0097-6156/82/0203-0011$06.00/0
© 1982 American Chemical Society

In Nutritional Bioavailability of Iron; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1982.
 12
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the f a c t that food i r o n a v a i l a b i l i t y measurements are fraught
with d i f f i c u l t i e s .
The o b j e c t i v e of t h i s paper i s t o d i s c u s s an i n v i t r o method
we have developed f o r e s t i m a t i n g food i r o n a v a i l a b i l i t y . The
paper w i l l be presented i n three s e c t i o n s : 1. R a t i o n a l e f o r the
design and u t i l i t y of an i n v i t r o method. 2. A d e s c r i p t i o n o f
the i n v i t r o method. 3. R e s u l t s and e v a l u a t i o n of the method.

Rationale

Current methodology f o r e s t i m a t i o n of food i r o n a v a i l a b i l i t y

u s u a l l y i n v o l v e s one of three approaches: animal bioassays,
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human bioassays, or i n v i t r o measurements.

The most f r e q u e n t l y used animal bioassay i s the r a t hemoglo-
b i n r e p l e t i o n t e s t . F r i t z , et a l . (2), M i l l e r (3), and Rotruck
Publication Date: November 1, 1982 | doi: 10.1021/bk-1982-0203.ch002

and Luhrsen (4) have d e s c r i b e d the method i n d e t a i l . The method

depends on the a b i l i t y of an i r o n source to i n c r e a s e the hemoglo-
b i n c o n c e n t r a t i o n i n anemic r a t s . P r i n c i p a l advantages of t h i s
method i n c l u d e : 1. the use o f an i n t a c t b i o l o g i c a l system, 2. the
r e l a t i v e s i m p l i c i t y o f the method, and 3. the a v a i l a b i l i t y of the
method to a l a r g e number o f researchers. The drawbacks of t h i s
method are: 1. the problems a s s o c i a t e d with the e x t r a p o l a t i o n of
r e s u l t s from r a t s to humans, 2. the requirement that anemic a n i -
mals must be used ( r e l a t i v e i r o n a v a i l a b i l i t i e s may d i f f e r be-
tween anemic and nonanemic animals), 3. requirement f o r graded
l e v e l s of i r o n i n the d i e t s (when whole foods are used t h i s means
that the composition of the d i e t s i s u s u a l l y not constant between
groups), and 4. the expense a s s o c i a t e d w i t h the method.
A second animal bioassay method i s the whole body counting
method which has been d e s c r i b e d by Welch and Van Campen (5). In
t h i s method, food c o n t a i n i n g a r a d i o a c t i v e t r a c e r i s given t o the
animals i n a s i n g l e dose and r e t e n t i o n of the t r a c e r i n the whole
animal over time i s measured. Advantages i n c l u d e : 1. the use of
an i n t a c t b i o l o g i c a l system, 2. the s i m p l i c i t y o f the method ( f r e -
quent blood sampling and food consumption measurements are not
r e q u i r e d ) , 3. the f l e x i b i l i t y of the method (animals i n d i f f e r e n t
treatment groups may or may not r e c e i v e i d e n t i c a l d i e t s through-
out the experiment, depending on the study design requirements),
and 4. the o p t i o n to use e i t h e r anemic or nonanemic animals.
Disadvantages i n c l u d e : 1. questions regarding e x t r a p o l a t i o n of
r e s u l t s t o humans, 2, requirements f o r s p e c i a l i z e d equipment
(whole body c o u n t e r ) , and 3. questions about extent of exchange
of t r a c e r and endogeneous i r o n when an e x t r i n s i c l a b e l i s used.
The most f r e q u e n t l y used human bioassay method i s the two
pool e x t r i n s i c r a d i o i r o n tag method (6). The method i s based on
two assumptions. One, that food i r o n exchanges w i t h two common
pools i n the gut (heme and nonheme i r o n pools) and two, that ad-
ded r a d i o l a b e l e d heme i r o n exchanges completely with food heme iron
and added r a d i o l a b e l e d nonheme i r o n exchanges completely with food

In Nutritional Bioavailability of Iron; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1982.
 In Vitro Estimation 13
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nonheme i r o n . I n c o r p o r a t i o n o f t h e r a d i o i r o n tag i n t o hemoglobin
i s used as the response parameter. T h i s method has proven t o be
very s u c c e s s f u l (7). Advantages i n c l u d e : 1. the use of human
subjects and, t h e r e f o r e , e l i m i n a t i o n of questions regarding ex-
t r a p o l a t i o n , 2. the s i m p l i c i t y o f the method (a s i n g l e dose con-
t a i n i n g the r a d i o l a b e l e d food i s administered and a blood sample
i s drawn two weeks l a t e r f o r counting). Disadvantages i n c l u d e :
1. the a d m i n i s t r a t i o n of r a d i o i s o t o p e s t o human s u b j e c t s , 2. r e -
s t r i c t i o n s i n the use o f the method ( r e l a t i v e l y few i n v e s t i g a t o r s
are l i c e n s e d t o administer r a d i o i s o t o p e s to human subjects f o r
p u r e l y research purposes.), and 3. the wide i n t e r - s u b j e c t v a r i -
a b i l i t y i n i r o n absorption. (This v a r i a b i l i t y can be l a r g e l y
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overcome by using each subject as t h e i r own c o n t r o l and by ad-

m i n i s t e r i n g a reference dose).
In v i t r o methods have been used t o estimate i r o n a v a i l a b i l i t y
Publication Date: November 1, 1982 | doi: 10.1021/bk-1982-0203.ch002

f o r a t l e a t 50 years (8). Two approaches w i t h v a r i o u s m o d i f i c a -

t i o n s have been used. One approach i s t o measure " i o n i z a b l e " o r
" i o n i c " i r o n i n foods. T h i s i s done by determining the f r a c t i o n
of the t o t a l i r o n i n a food that w i l l r e a c t with a complexing
agent such as a , a ' - d i p y r i d y l (8) o r bathophenanthroline (9) t o
form a chromagen which can be q u a n t i t a t e d s p e c t r o p h o t o m e t r i c a l l y .
A second approach i s t o subject the food t o a simulated g a s t r i c
or g a s t r o i n t e s t i n a l d i g e s t i o n using p u r i f i e d p e p t i c and/or pan-
c r e a t i c enzymes with subsequent measurement o f the s o l u b l e i r o n
released by the d i g e s t i o n (10-13). Advantages o f i n v i t r o
methods i n c l u d e : 1. t h e i r low cost and speed, 2. t h e i r reduced
v a r i a b i l i t y compared to i n v i v o methods ( v a r i a b i l i t y caused by
d i f f e r e n c e s i n i r o n s t a t u s o f animals and humans i s avoided),
and 3. the a b i l i t y t o p r e c i s e l y c o n t r o l c o n d i t i o n s during the
determinations. Disadvantages i n c l u d e : 1. u n c e r t a i n t i e s over
the use o f an a r t i f i c i a l system, 2. the l e s s than exact d u p l i c a -
t i o n o f i n v i v o c o n d i t i o n s , 3. the i n a b i l i t y t o account f o r e f -
f e c t s o f a c t i v e t r a n s p o r t , brush border binding p r o t e i n s , e t c .
I t i s c l e a r that each approach has i t s advantages and d i s -
advantages and that a l l three approaches have provided and w i l l
continue t o provide information on food i r o n a v a i l a b i l i t y . It i s
a l s o c l e a r that f u r t h e r development and e v a l u a t i o n o f the three
approaches w i l l enhance the usefulness o f the data generated by
them.
Development o f c o n d i t i o n s s u i t a b l e f o r a s u c c e s s f u l i n v i t r o
method r e q u i r e s c a r e f u l a t t e n t i o n to c o n d i t i o n s present i n the
gut during d i g e s t i o n and t o the behavior of i r o n i n s o l u t i o n .
The environment i n the GI t r a c t and the chemical form o f the i r o n
both i n the food and i n the d i g e s t a i n t e r a c t t o determine the
f r a c t i o n of the food i r o n t h a t i s a v a i l a b l e f o r absorption.
The primary determinants o f food i r o n a v a i l a b i l i t y are:
1. the extent o f i r o n r e l e a s e from food and 2. the s o l u b i l i t y ,
molecular weight, and s t a b i l i t y o f complexes formed from the r e -
leased i r o n . A l a r g e number o f f a c t o r s i n t e r a c t to i n f l u e n c e
these determinants.

In Nutritional Bioavailability of Iron; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1982.
 14
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The nonheme i r o n i n food i s most l i k e l y bound to food com-
ponents, probably p r o t e i n . The r e l e a s e of i r o n from these com-
ponents i s a complex process. I t i s reasonable to assume that
the b i n d i n g a f f i n i t y of the food components f o r i r o n , the d i g e s t -
i b i l i t y of the food components, the presence of reductants and
accepting l i g a n d s , the pH i n the GI t r a c t are a l l f a c t o r s which
i n f l u e n c e r e l e a s e of the i r o n from the food. Binding a f f i n i t y
and d i g e s t i b i l i t y are c h a r a c t e r i s t i c s of the food and may vary
s i g n i f i c a n t l y between foods. Reductants and accepting l i g a n d s
are a l s o l a r g e l y c o n t r i b u t e d by the food. D i g e s t i o n products
may a c t as reductants and/or accepting l i g a n d s . Food components
such as ascorbate, c i t r a t e , and simple sugars may c o n t r i b u t e sub-
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s t a n t i a l l y to the r e l e a s e of the i r o n from food.

Since f a c t o r s which are c h a r a c t e r i s t i c s of the food would
not d i f f e r between i n v i v o and i n v i t r o s i t u a t i o n s , the major
Publication Date: November 1, 1982 | doi: 10.1021/bk-1982-0203.ch002

v a r i a b l e s t h a t must be c o n t r o l l e d i n an i n v i t r o s i m u l a t i o n are
pH, enzyme concentrations and a c t i v i t i e s , and d i g e s t i o n times.
The observation that i r o n absorption i s reduced when g a s t r i c
a c i d s e c r e t i o n i s compromised (14) suggests that g a s t r o i n t e s t i n a l
pH does i n f l u e n c e food i r o n a v a i l a b i l i t y . The explanation f o r
t h i s appears to be r e l a t e d to the importance of a c i d f o r the r e -
lease of i r o n from food. Bezwoda et a l . (15) measured the capa-
c i t y to s o l u b i l i z e i r o n i n bread of g a s t r i c j u i c e from normal and
i r o n d e f i c i e n t s u b j e c t s . G a s t r i c j u i c e s with pH values above 2
had l i m i t e d c a p a c i t y to s o l u b i l i z e bread i r o n w h i l e below pH 2,
s o l u b i l i z a t i o n of i r o n increased l i n e a r l y with decreasing pH.
Even though the s t a b i l i t i e s of metal complexes i n foods are un-
known, i t i s to be expected that low pHs w i l l r e s u l t i n g r e a t e r
r e l e a s e of food i r o n s i n c e metal c h e l a t e s t a b i l i t i e s decrease
with decreasing pH (14). I t i s apparent, t h e r e f o r e , that pH
must be c a r e f u l l y c o n t r o l l e d i n any i n v i t r o method designed to
estimate food iron availability. S e l e c t i o n of an appropriate
pH f o r use i n i n v i t r o p e p t i c d i g e s t i o n s i s d i f f i c u l t s i n c e the
pH i n the i n v i v o s i t u a t i o n i s q u i t e v a r i a b l e . However, i t i s
g e n e r a l l y accepted that i n g e s t i o n o f food stimulates g a s t r i c
a c i d s e c r e t i o n and that g a s t r i c a c i d i t y i s one f a c t o r i n v o l v e d
i n the r e g u l a t i o n of g a s t r i c a c i d s e c r e t i o n . Malagelada et a l .
(16) showed t h a t i n g e s t i o n of a meal by human s u b j e c t s increased
g a s t r i c pH from about 2 to about 5. The peak pH of a 5 was
reached r a p i d l y . I t then g r a d u a l l y f e l l with time and s t a b i l i z e d
at pH 1.5 to 2 by the end of the second hour. Walsh et a l . (17)
found t h a t the r a t e of g a s t r i c a c i d s e c r e t i o n i n normal human
subjects i n response to a meal i s suppressed at a g a s t r i c pH of
2.5 compared to a g a s t r i c pH of 5.5. T h i s suggests that the
stomach " t i t r a t e s " i t s contents to an a c i d pH and that stomach
pHs f o l l o w i n g d i f f e r e n t meals should be s i m i l a r . I t seems
reasonable, t h e r e f o r e , to assume that adjustment of the pH to 2
p r i o r to i n v i t r o pepsin d i g e s t i o n would approximate the i n v i v o
situation.
Ferrous and f e r r i c ions are present i n the hydrated form i n

In Nutritional Bioavailability of Iron; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1982.
 In Vitro Estimation 15
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a c i d i c aqueous s o l u t i o n s (18). As the pH of a s o l u t i o n of these
aquated ions i s i n c r e a s e d , deprotonation of the complexed water
molecules occurs and hydroxo- and/or oxo-aquo s p e c i e s are formed
(19). T h i s process i s c a l l e d h y d r o l y s i s and may be represented
as (19)

( 3 x y ) +
x[Fe(H 0) ]2 6
3 +
+ y H0
2 ^ [Fe (0H) (H 0)J
x y 2 ~ + y H 0
3
+

where n i s the degree of h y d r a t i o n of the h y d r o l y s i s product. As

the above equation suggests, p o l y n u c l e a t e d s p e c i e s may be p r o -
duced i n the r e a c t i o n . Polymers with a molecular weight of
150,000 have been observed (19). C h e l a t i n g agents such as c i -
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t r a t e may prevent p o l y m e r i z a t i o n i f added i n s u f f i c i e n t excess

(19) . Many of the c h e l a t e s are, however, q u i t e unstable and ad-
d i t i o n of concentrated base can cause p r e c i p i t a t i o n even when
Publication Date: November 1, 1982 | doi: 10.1021/bk-1982-0203.ch002

c h e l a t i n g l i g a n d s are present. Upward pH adjustment with NaHCO^

r a t h e r than NaOH appears to be l e s s l i k e l y to cause p r e c i p i t a t i o n
(20) .
As the i r o n i s r e l e a s e d from food i n the a c i d i c environment
of the stomach, l i g a n d s r e l e a s e d i n the d i g e s t i o n process combine
with the i r o n to form c h e l a t e s . These c h e l a t e s should i n h i b i t
p o l y m e r i z a t i o n and p r e c i p i t a t i o n of i r o n as the stomach contents
are n e u t r a l i z e d i n the duodenum provided that l o c a l i z e d regions
of h i g h pH caused by too r a p i d a d d i t i o n o f concentrated base are
avoided. T h i s suggests that pH adjustment i n an i n v i t r o simula-
t i o n i s a c r i t i c a l step.
When the products of g a s t r i c d i g e s t i o n reach the duodenum,
bicarbonate s e c r e t e d by the pancreas begins to n e u t r a l i z e the
stomach a c i d . The c o n c e n t r a t i o n of bicarbonate i n p a n c r e a t i c
j u i c e ranges from about 70 to about 150 meq/£ (21). In a study
i n v o l v i n g human s u b j e c t s , Murthy e t a l . (22) found the pH o f duo-
denal a s p i r a t e s to range from 4.7 to 7.2 f o l l o w i n g a d m i n i s t r a t i o n
of a Lundh Test meal.
S e l e c t i o n of a p p r o p r i a t e concentrations f o r d i g e s t i v e en-
zymes i s a d i f f i c u l t undertaking. A l a r g e range of concentra-
t i o n s used i n i n v i t r o s t u d i e s have been reported. Akeson and
Stahmann (23) used 15 mg pepsin and 40 mg p a n c r e a t i n per gram of
p r o t e i n . Narasinga Rao and Prabhavathi (11) used pepsin concen-
t r a t i o n s ranging from 12 to 60 mg per g of food. They reported
no d i f f e r e n c e i n i r o n r e l e a s e when t h i s range of p e p s i n concen-
t r a t i o n s was used. Lease (24) used 20 mg of pepsin per gram of
food. H a z e l l et a l . (12) used 10 mg pepsin and 10 mg p a n c r e a t i n
per gram of p r o t e i n . While d i f f e r e n t concentrations of enzymes
w i l l produce d i f f e r e n t r a t e s o f d i g e s t i o n , the a c t u a l enzyme con-
c e n t r a t i o n s are not c r i t i c a l provided they are p r e c i s e l y d u p l i c a -
ted when comparisons between foods or meals are being made.
Appropriate d i g e s t i o n times f o r an i n v i t r o system l i k e w i s e
cannot be determined with p r e c i s i o n . Rates o f passage of d i g e s t a
are determined by s e v e r a l i n t e r a c t i n g f a c t o r s i n c l u d i n g the osmo-
l a r i t y of the meal, the r e l a t i v e amounts of l i q u i d and s o l i d i n

In Nutritional Bioavailability of Iron; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1982.
 16
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the meal, the s i z e of the meal and the carbohydrate, p r o t e i n , and
f a t content of the meal (25). However, choice of an appropriate
pepsin d i g e s t i o n time may be based on estimates of stomach empty-
ing times and r a t e s of d i g e s t i o n . Grimes and Goddard (26) showed
t h a t , f o l l o w i n g a bread and water meal, about 70% of the s o l i d
phase (bread) and from 1 to 30% of the l i q u i d phase (water) r e -
mained i n the stomach one hour a f t e r the meal was consumed by
human s u b j e c t s . Malagelada et a l . (16) showed w i t h human sub-
j e c t s that g a s t r i c volume rose q u i c k l y to the volume of the meal
and remained at that volume f o r about one hour. The g a s t r i c v o l -
ume then f e l l g r a d u a l l y u n t i l i t reached b a s a l l e v e l s about 3
hours from the time the meal was consumed. Narasingao Rao and
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Prabhavathi showed i n an i n v i t r o system that i r o n r e l e a s e d from

food was the same when pepsin i n c u b a t i o n times were v a r i e d from
50 to 180 minutes. Selecton of an i n v i t r o p a n c r e a t i n d i g e s t i o n
Publication Date: November 1, 1982 | doi: 10.1021/bk-1982-0203.ch002

time i s even more d i f f i c u l t s i n c e d i g e s t a i s c o n s t a n t l y e n t e r i n g

and l e a v i n g the duodenum and jejunum. I t i s reasonable to s e l e c t
a d i g e s t i o n time that produces s i g n i f i c a n t d i g e s t i o n . Care must
be taken to use i d e n t i c a l d i g e s t i o n times when comparisons are
being made between foods or meals.
Once the c o n d i t i o n s f o r an i n v i t r o method f o r e s t i m a t i o n of
i r o n a v a i l a b i l i t y have been e s t a b l i s h e d , s e l e c t i o n of an appro-
p r i a t e response parameter must be made. T o t a l s o l u b l e or " i o n -
i z a b l e " i r o n present i n the f i l t r a t e o r supernate obtained from
an i n v i t r o d i g e s t i o n has been the most f r e q u e n t l y used response
parameter (10-13).
The foregoing d i s c u s s i o n provides a b a s i s f o r s e l e c t i o n of a
r e l i a b l e indicator of i r o n a v a i l a b i l i t y . F i g u r e 1 summarizes i n
schematic form the changes that occur as food moves through the
d i g e s t i v e t r a c t . The assumption i s made that absorbable i r o n i s
present i n the duodenum as a low molecular weight s o l u b l e c h e l a t e .
I t i s f u r t h e r assumed t h a t the other forms of i r o n that may be
present do not c o n t r i b u t e s i g n i f i c a n t l y to absorbable i r o n .
These assumptions seem j u s t i f i e d f o r the f o l l o w i n g reasons:
1. Iron may be absorbed as the i n t a c t c h e l a t e or the chelate
may t r a n s f e r i t s i r o n t o an acceptor on the mucosal c e l l
s u r f a c e . Absorption and exchange would be much more r a p i d
with s o l u b l e forms of i r o n s i n c e i n s o l u b l e forms would have
l i m i t e d contact with the mucosal c e l l s u r f a c e .
2. Iron bound to l a r g e molecular weight l i g a n d s may be a v a i l -
able but absorption would be l i m i t e d to an i r o n t r a n s f e r
mechanism s i n c e l a r g e molecules are g e n e r a l l y not absorbed
i n t a c t . Large molecular weight s o l u b l e c h e l a t e s of a v a i l -
a b l e i r o n would most l i k e l y i n v o l v e p r o t e i n s as the l i g a n d
and d i g e s t i o n would q u i c k l y transform them i n t o low mole-
c u l a r weight c h e l a t e s .
3. Polymerized i r o n , even when s o l u b l e , i s probably not r e a d i l y
a v a i l a b l e . Bates et a l . (20), i n s t u d i e s designed to mea-
sure i r o n exchange r a t e s between chelates and t r a n s f e r r i n ,
showed that polymerized i r o n was t r a n s f e r r e d to t r a n s f e r r i n

In Nutritional Bioavailability of Iron; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1982.
 In Vitro Estimation
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Stomach Duodenum Mucosal Cell

Publication Date: November 1, 1982 | doi: 10.1021/bk-1982-0203.ch002

[-•Fe-H 0 4 i
2 Fe-lig s

HCO3
+
Enzymes

L+Fe-LIGj ±

Figure 1. Proposed model for changes that occur in nonheme iron as food moves
through GI tract. Abbreviations: lig , low molecular weight soluble ligand; LIG ,
s a

large molecular weight soluble ligand; LIG , large molecular weight insoluble ligand;
t

Poly Fe , soluble polynuclear (polymerized) iron; Poly Fe insoluble polynuclear

s it

(polymerized) iron.

In Nutritional Bioavailability of Iron; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1982.
 18
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at a very slow r a t e while i r o n i n the low molecular weight
n i t r i l o t r i a c e t a t e c h e l a t e was almost instantaneously t r a n s -
f e r r e d t o the t r a n s f e r r i n . Polymerized i r o n may be depoly-
merized by l i g a n d s and/or reducing agents (27) and thereby
enter the low molecular weight i r o n c h e l a t e p o o l .
C o n s i d e r a t i o n of the f a c t o r s d i s c u s s e d above provides a r e a -
sonably good r a t i o n a l e f o r the design of an i n v i t r o method to
estimate food i r o n a v a i l a b i l i t y . B r i e f l y , i t seems apparent that
an i n v i t r o method should:
1. Simulate i n v i v o d i g e s t i o n c o n d i t i o n s .
2. Permit pH c o n t r o l .
3. Provide f o r gradual pH adjustment with a m i l d base.
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4. D i s t i n g u i s h between low and h i g h molecular weight s o l u b l e

iron.
5. Accommodate food mixtures (meals).
Publication Date: November 1, 1982 | doi: 10.1021/bk-1982-0203.ch002

D e s c r i p t i o n of the Method

D e t a i l s of the method have been d e s c r i b e d elsewhere (28,29).

A flow diagram of the method i s shown i n F i g u r e 2. B r i e f l y , the
method i n v o l v e s the f o l l o w i n g steps:
1. Water i s added to mixtures of foods (meals) so that the
water content of d i f f e r e n t meals i s approximately the same.
2. The meals are blended i n a food blender to a creamy c o n s i s -
tency .
3. The pH of the blended meals i s adjusted to 2 with 6 N HC1
and the samples are spiked with ^ F e .
4. Pepsin i s added and the meals are incubated i n a shaking
water bath a t 37°C f o r 2 hours.
5. A l i q u o t s of the pepsin d i g e s t are analyzed f o r a) t i t r a t -
a b l e a c i d i t y (the number of e q u i v a l e n t s of KOH r e q u i r e d t o
t i t r a t e a 20 g a l i q u o t c o n t a i n i n g 5 ml of the p a n c r e a t i n -
b i l e mixture to pH 7.5), b) nonheme i r o n c o n c e n t r a t i o n , and
5
c) ^ F e a c t i v i t y .
6. A d i a l y s i s bag c o n t a i n i n g an amount of NaHC03 i n 25 ml of
water e q u i v a l e n t to the p r e v i o u s l y determined t i t r a t a b l e
a c i d i t y i s added to a 20 g a l i q u o t of the pepsin d i g e s t .
The sample i s incubated f o r 30 minutes at 37°C i n a shaking
water bath. During t h i s time, the pH i n c r e a s e s to about 5.
7. F i v e ml of a p a n c r e a t i n - b i l e a c i d mixture i s added to each
d i g e s t i o n v e s s e l ( i t i s added to the contents o u t s i d e the
d i a l y s i s bag). Incubation i s continued f o r 2 hours.
8. The d i a l y s i s bag i s removed, r i n s e d i n d i s t i l l e d water, and
emptied of i t s contents (the d i a l y s a t e ) .
9. The d i a l y s a t e i s weighed and analyzed f o r ^ F e a c t i v i t y and
bathophenanthroline r e a c t i v e i r o n .
10. R e s u l t s are expressed as percent of t o t a l nonheme and r a d i o -
i r o n i n the o r i g i n a l a l i q u o t t h a t i s present i n the d i a l y -
s i s bag at the end of the d i g e s t i o n .
In order to evaluate the method, meals were prepared that

In Nutritional Bioavailability of Iron; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1982.
 ML
ILER AND SCHRC
IKER In Vitro Estimation

FOODS + H 0 2

^ Blend
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^HCI+ 5 9
Fe
Publication Date: November 1, 1982 | doi: 10.1021/bk-1982-0203.ch002

pH 2 MEAL HOMOGENATE
Pepsin, 2 hr, 37°

PEPSIN DIGEST

Dialysis bag
30 min, 37°

Pancreatin-bile Titratable
2 hr, 37° a c , d , t y

Remove bag [Nonheme Fe]

Rinse, empty, weigh |-V ^ g

DIALYSATE, pH 7

i
Count
Batho assay

% Dialyzable F e
59

and nonheme Fe

Figure 2. Schematic of the in vitro method. See text for details.

In Nutritional Bioavailability of Iron; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1982.
 20
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would permit food s u b s t i t u t i o n s that might a l t e r l e v e l s of d i a l y -
zable i r o n . One meal was of our own f o r m u l a t i o n (the standard
meal) and two were r e p l i c a t e s o f meals used by Cook and Monsen
(2,30). The composition of these meals i s shown i n Table I .

Table I: Composition of Test Heals Used i n

Iron A v a i l a b i l i t y T r i a l s *

Standard STD, Cook & Monsen SS, Cook & Monsen

Beef; l e a n ground Beef; ground Albumin; egg
Bread; white, Potatoes; dry Dextrose
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enriched
Cornmeal Corn o i l
Beans; snap, f r o z e n Bread; white, CaHP0
Publication Date: November 1, 1982 | doi: 10.1021/bk-1982-0203.ch002

enriched
M i l k ; f l u i d , whole Margarine K HP0
2 4

Water Peaches Fe s o l u t i o n
Ice m i l k Water

Water

The STD and SS meals were o r i g i n a l l y formulated by Cook and

Monsen (7,30).

Results and Evaluation

The 2 hour p e p s i n i n c u b a t i o n time was based on work reported

by Narasinga Rao (11) and on l i t e r a t u r e r e p o r t s on g a s t r i c empty-
i n g time (16,26). The s l i g h t l y longer p a n c r e a t i n i n c u b a t i o n time
of 2 1/2 hours was chosen f o r two reasons. F i r s t , i t was neces-
sary to delay a d d i t i o n of the p a n c r e a t i n - b i l e mixture f o r 30 min-
utes a f t e r the d i a l y s i s bag was added and the i n c u b a t i o n begun.
The delay allowed the pH to r i s e to about 5 and thereby prevented
i n a c t i v a t i o n of the p a n c r e a t i c enzymes which does occur at lower
pHs (22). Secondly, t h i s time p e r i o d produced s u f f i c i e n t d i a l y s -
ate i r o n concentrations f o r accurate bathophenanthroline measure-
ments. F i g u r e 3 shows a time course f o r d i a l y z a b l e i r o n changes
during the second i n c u b a t i o n . D i a l y z a b l e i r o n r i s e s r a p i d l y to
e q u i l i b r i u m l e v e l s when the semisynthetic meal i s used. T h i s
suggests that p a n c r e a t i n d i g e s t i o n has l i t t l e e f f e c t on a v a i l a b l e
i r o n i n a meal composed of p u r i f i e d i n g r e d i e n t s . For the s t a n -
dard meal, on the other hand, d i a l y z a b l e i r o n i n c r e a s e s s t e a d i l y
over the e n t i r e i n c u b a t i o n p e r i o d . T h i s suggests that d i g e s t i o n
by p a n c r e a t i c enzymes does play a r o l e i n food i r o n a v a i l a b i l i t y .
Data from Malagelada et a l . (16) provided the; b a s i s f o r
choosing a pH of 2 f o r the pepsin d i g e s t i o n . A f i n a l d i a l y s a t e
pH of 7 was chosen f o r the p a n c r e a t i n d i g e s t i o n on the b a s i s of
the data of Murthy e t a l . (22). Attempts were made to adjust the

In Nutritional Bioavailability of Iron; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1982.
 In Vitro Estimation
ML
ILER AND SCHRC
IKER
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Publication Date: November 1, 1982 | doi: 10.1021/bk-1982-0203.ch002

Figure 3. Time course for changes in dialyzable iron during the second digestion
step. See text for a description of meals and digestion conditions. Key: A * standard,
colorimetric; A , standard, radioactive; | , SS, colorimetric; Q SS radioactive.
t

In Nutritional Bioavailability of Iron; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1982.
 22
NUTRT
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pH of the pepsin d i g e s t by dropwise a d d i t i o n of d i l u t e NaHC03 or
NaOH but t h i s approach produced v a r i a b l e and p o o r l y r e p r o d u c i b l e
values f o r d i a l y z a b l e i r o n . Adjustment of pH by d i a l y s i s produced
the l e a s t v a r i a b l e and most r e p r o d u c i b l e d i a l y z a b l e i r o n v a l u e s .
In a d d i t i o n , t h i s method of pH adjustment permits d i a l y s i s of iron
to occur during the n e u t r a l i z a t i o n process, a s i t u a t i o n which
more c l o s e l y resembles the i n v i v o process. Even with t h i s
method of pH adjustment, however, the f i n a l d i a l y s a t e pHs d i d
vary. In order to determine the e f f e c t of v a r i a t i o n i n f i n a l pH
on d i a l y z a b l e i r o n , an experiment was run using d i f f e r e n t amounts
of bicarbonate i n the d i a l y s i s bags. The amounts used were based
on t i t r a t a b l e a c i d i t y measurements made with d i f f e r e n t end p o i n t s .
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A l i q u o t s were t i t r a t e d to f i n a l pHs ranging from 6.5 to 8.5. Ad-

d i t i o n of NaHC03 based on these t i t r a t i o n s produced f i n a l d i a l y s -
ate pHs ranging from about 6 to about 7.5. F i g u r e 4 shows t h a t
Publication Date: November 1, 1982 | doi: 10.1021/bk-1982-0203.ch002

i n the range of pH v a r i a b i l i t y observed when d i f f e r e n t meals are

run i n the i n v i t r o procedure ( f i n a l pHs i n the a c i d i t y t i t r a t i o n
of 7.0 to 8.0), the e f f e c t of pH on d i a l y z a b l e i r o n i s s m a l l . I t
i s i n t e r e s t i n g to note that the d i r e c t i o n of pH e f f e c t s are d i f -
f e r e n t f o r the semisynthetic and standard meals. The decrease
observed w i t h i n c r e a s i n g pH i n the semisynthetic meal would be
expected i f d i g e s t i o n were not a f a c t o r s i n c e h i g h e r pHs would
cause i n c r e a s e d formation of polymerized i r o n . The i n c r e a s e ob-
served i n the standard meal can be explained by an i n c r e a s e i n
p a n c r e a t i c enzyme a c t i v i t y a t higher pHs.
Table I I shows e f f e c t s on d i a l y z a b l e i r o n caused by v a r i o u s
s u b s t i t u t i o n i n t o the standard meal. These data have been r e -
ported elsewhere ( M i l l e r et a l . 28, S c h r i c k e r et a l . 29).
* t
Table I I : Percent D i a l y z a b l e Iron ' , E f f e c t s of S e l e c t e d Foods

Substitution Colorimetric Radioactive

None, Standard meal 4.08 + 0.31 a

3.80 + 0.31 a

Ham f o r beef 5.05 + 0.80 a

5.14 + 0.23 a

Whole wheat f o r white bread 2.06 + 0.42 b

1.49 + 0.10 b

Spinach f o r green beans 5.73 + 0.33 a

5.04 + 0.19 a

Water f o r m i l k 4.59 + 0.32 ac

4.16 + 0.14 a

Tea f o r m i l k 2.80 + 0.31 b

1.52 + 0.21 b

Cola f o r m i l k 6.14 + 0.61° 7.82 + 0.34°

Orange j u i c e f o r m i l k 24.96 + 0.83 d
25.83 + 0.86 d

Values represent means + S.E. f o r three o b s e r v a t i o n s .

t
In each column, means followed by d i f f e r e n t s u p e r s c r i p t s are
s i g n i f i c a n t l y d i f f e r e n t (P<0.01).

In Nutritional Bioavailability of Iron; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1982.
 In Vitro Estimation
ML
ILER AND SCHRC
IKER
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Publication Date: November 1, 1982 | doi: 10.1021/bk-1982-0203.ch002

2-

m 1 1 1 1
B.S 7.0 7.5 B.0 B.S
FINRL PH
Figure 4. Effect of pH on dialyzable iron. Values on x axis are endpoints for the
titratable acidity measurement. Final pH of dialysate after incubation about 0.5
below the pH shown. Key: A , standard, colorimetric; A , standard, radioactive;
| , SS, colorimetric; • , SS, radioactive.

In Nutritional Bioavailability of Iron; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1982.
 Downloaded by UNIV OF NEW SOUTH WALES on August 17, 2015 | http://pubs.acs.org
Publication Date: November 1, 1982 | doi: 10.1021/bk-1982-0203.ch002

H3 Human In Vivo
• Dialyzable Iron (colorimetric)
S Dialyzable Iron (radioactive)
H Rat In Vivo

o
CO

c
o

o 2
c
0)
H
JO
< H

In Nutritional Bioavailability of Iron; Kies, C.;

Liver Chicken
I
Figure 5. Comparison of estimated absorption ratios for standard meals using different meth-
I

ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

odology. All meals are compared to the beef meal. Ratios for human in vivo trials are derived
from data from Cook and Monsen {!). Ratios are plotted on a logarithmic scale.
0

1
 In Vitro Estimation 25
2. ML
ILER AND SCHRC
IKER
To f u r t h e r evaluate the method, a s e r i e s o f meals were f o r -
mulated and prepared t o d u p l i c a t e those o f Cook and Monsen (7)
(see Table I ) . These meals were chosen because they were used by
Cook and Monsen i n a study i n v o l v i n g human s u b j e c t s and, t h e r e -
f o r e , provided a means f o r comparison o f i n v i t r o and human i n
v i v o methods. A r a t i n v i v o method was a l s o used i n t h i s study.
The meals were homogenized, spiked with ^ F e , a n < j administered
to r a t s v i a stomach tube. Iron r e t e n t i o n was measured u s i n g
whole body counting. F i g u r e 5 compares r e s u l t s obtained u s i n g
these three methods.
Comparison o f r e s u l t s from the i n v i t r o and human i n v i v o
methods shows good agreement between the two methods.
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On the b a s i s of the d e s c r i p t i o n and r e s u l t s presented above,

i t i s reasonable to c h a r a c t e r i z e the i n v i t r o method described
here as:
Publication Date: November 1, 1982 | doi: 10.1021/bk-1982-0203.ch002

1. Rapid and inexpensive

2. Reproducible
3. A good p r e d i c t o r of r e l a t i v e i r o n b i o a v a i l a b i l i t y
4. P o t e n t i a l l y u s e f u l f o r :
a. Food i r o n a v a i l a b i l i t y screening
b. I d e n t i f y i n g f a c t o r s and mechanisms which may i n f l u e n c e
iron availability

Literature Cited

1. Monsen, E.R.; Hallberg, L.; Layrisse, M.; Hegsted, D.M.; Cook,

J.D.; Mertz, W.; Finch, C.A. Am. J. Clin. Nutr. 1978, 31,
134-141.
2. Fritz, J.C.; Pla, G.W.; Harrison, B.M.; Clark, G.A.; Smith,
E.A. J. Assoc. Off. Anal. Chem. 1978, 61, 709-714.
3. Miller, J. J. Agric. Food Chem. 1977, 25, 154-158.
4. Rotruck, J.T.; Luhrsen, K.R. J. Agric. Food Chem. 1979, 27,
27-33.
5. Welch, R.M.; Van Campen, R. J. Nutr. 1975, 105, 253-256.
6. Hallberg, L. Proc. Nutr. Soc. 1974, 33, 285-291.
7. Cook, J.D.; Monsen, E.R. Am. J. Clin. Nutr. 1976, 29, 859-867.
8. Shackleton,L.; McCance, R.A. Biochem. J. 1936, 30, 583-591.
9. Lee, K. Clydesdale, F.M J. Food Sci. 1979, 44, 549-554.
10. Jacobs, A.; Greenman, D.A. Brit. Med. J. 1969, 1, 673-676.
11. Narasinga Rao, B.S.; Prabhavathi, T. Am. J. Clin. Nutr. 1978,
31, 169-175.
12. Hazell, T.; Ledward, D.A.; Neal, R.J. Br. J. Nutr. 1978, 39,
631-638.
13. Lock, S.; Bender, A.E. Br. J. Nutr. 1980, 43, 413-420.
14. Forth, W.; Rummel, W. Physiol. Rev. 1973, 53, 724-792.
15. Bezwoda, W.; Charlton, R.; Bothwell, T.; Torrance, J.; Mayet,
F. J. Lab. Clin. Med. 1978, 92, 108-116.
16. Malagelada, J.; Go, Longstreth, G.F.; Deering, T.B.; Summer-
- s k i l l , W.H.J.; Go, V.L.W. Gastroenterology. 1977, 73, 989-
994.

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ACS Symposium Series; American Chemical Society: Washington, DC, 1982.
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17. Walsh, J.H.; Richardson, C.T.; Fordtran, J.S. J. Clin.
Invest. 1975, 55, 462-468.
18. Spiro, T.G.; Saltman, P. In: Jacobs, A.; Worwood, M., Eds.;
"Iron in Biochemistry and Medicine"; Academic Press: New
York, NY, 1974; pp 1-28.
19. Sylva, R.N. Rev. Pure and Appl. Chem. 1972, 22, 115-132.
20. Bates, G.W.; Billups, C.; Saltman, P. J. Biol. Chem. 1967,
242, 2810-2815.
21. Ganong, W.F. "Review of Medical Physiology", 6th ed.; Lang
Medical Publications: Los Altos, CA, 1973; p 370.
22. Murthy, S.N.S.; Kostman, J.; Dinoso, V.P. Dig. Dis. Sci.,
1980, 25, 289-294.
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23. Akeson,W.R.; Stahmann, M.A. J. Nutr. 1964, 83, 257-261.

24. Lease, J.G. J. Nutr. 1967, 93,523-532.
25. Fein, H.D. In: Goodhart, R.S.; Shils, M.E., Eds.; "Modern
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Nutrition in Health and Disease", 6th ed.; Lea and Febiger:

Philadelphia, PA, 1980; p 894.
26. Grimes, D.S.; Goddard, J. Gut. 1977, 18, 725-729.
27. Saltman, P.; Hegenaur, J.; Christopher, J. Ann. Clin. Lab.
Sci. 1976, 6, 167-176.
28. Miller, D.D.; Schricker, B.R.; Rasmussen, R.R.; Van Campen, D.
Am. J. Clin. Nutr. 1981, 34, 2248-2256.
29. Schricker, B.R.; Miller, D.D.; Rasmussen, R.R.; Van Campen, D .
Am. J. Clin. Nutr. 1981, 34, 2257-2263.
30. Cook, J.D.; Monsen, E.R. Am. J. Clin. Nutr. 1975, 28, 1289-
1295.
RECEIVED June 2, 1982.

In Nutritional Bioavailability of Iron; Kies, C.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1982.