Iron solubilisation by chicken muscle proteindigestsAnihita Seth, Mariana Diaz and Raymond R Mahoney*Department of Food Science, University of Massachusetts, Amherst, MA 01003, USA

Abstract: The objective of this work was to compare the solubilisation of iron by in vitro digests of

soluble and insoluble protein fractions from chicken muscle. Chicken breast muscle was extracted to

provide dilute salt-soluble protein (DSSP) and dilute salt-insoluble protein (DSIP) fractions. These

fractions together with casein and ovalbumin were subjected to in vitro digestion in the presence of

ferric iron. After proteolytic digestion, soluble iron increased fourfold for DSSP, 20-fold for DSIP,

twofold for casein and 0.5-fold for ovalbumin. 64% of the soluble iron in the DSSP digest and 30% of the

soluble iron in the DSIP digest were ferrous; in the casein and ovalbumin digests, less than 6% was

ferrous. Dialysable iron was less than 5% of the soluble iron for all proteins and was mostly ferric iron.

DSIP solubilised twice as much iron as DSSP but much less than casein or ovalbumin digests. It was

concluded that muscle proteins solubilise iron by reduction and chelation to mostly large (non-

dialysable) peptides resulting from digestion.

# 1999 Society of Chemical Industry

Keywords: iron; bioavailability; chicken protein

INTRODUCTIONIron de®ciency anaemia is one of the world's most

prevalent nutritional problems. It is caused by a

combination of factors ranging from inadequate iron

intake to impaired absorption and poor bioavailability,

especially of non-haem iron. A number of studies have

shown that muscle foods enhance non-haem iron

absorption,1±4 and this effect has come to be known as

the `meat effect' or `meat factor'. In contrast, animal

proteins and foods which are not from muscle tissue,

such as egg albumin, and milk do not increase iron

absorption,3 while plant proteins such as soy inhibit

iron absorption.5 The effects of various proteins has

been reviewed by Berner and Miller.6

Although the meat effect is well documented, the

mechanism of the effect remains controversial. Several

in vitro studies suggest that peptides arising from

digestion of proteins are responsible.7±10 These could

work simply by chelating iron so as to increase its

solubility at neutral pH and/or by reduction of ferric

iron to the more soluble ferrous form.

However, the lack of non-digested controls in most

such studies means that other components not related

to digestion could be involved. Carpenter and

Mahoney11 reported that beef contained a low-

molecular-weight (dialysable) component which solu-

bilised iron without digestion.

Some studies have measured soluble iron as an

indicator to compare the effects of meat or proteins,

since solubility at neutral pH is a prerequisite for

bioavailability.8,9,11 Others have preferred to measure

dialysable iron.7,10,12,13 Most studies on the meat

effect have used whole meat samples; however,

Slatkavitz and Clydesdale9 found that an acid-inso-

luble fraction of chicken muscle solubilised more iron

than a water-soluble extract. The notion that insoluble

proteins may be important was con®rmed by Kirwan etal,14 who found that a heavy meromyosin fraction

from rabbit muscle gave the highest level of dialysable

iron from among the muscle fractions tested.

While most of the previous studies suggest that

peptides are involved, comparisons and conclusions

about the origin and nature of the peptides are dif®cult

because of differences in meat source, digestion

methodology and measurement of the various forms

of iron produced.

The aim of this study was to compare the major

protein fractions from a single muscle source involved

in solubilising iron, and the forms of iron produced.

We have also studied the kinetics of iron solubilisation

during digestion of muscle protein. Chicken breast

muscle was chosen as the source because of its low

endogenous iron content. The results will help us in

our goal of isolating peptides which appear to be

responsible for the `meat factor'. For comparison we

have included casein and ovalbumin in this study: they

are non-tissue animal proteins which do not enhance

iron absorption in vivo.3,4

Journal of the Science of Food and Agriculture J Sci Food Agric 79:1958±1963 (1999)

* Correspondence to: Raymond R Mahoney, Department of Food Science, University of Massachusetts, Amherst, MA 01003, USAE-mail: rmahoney@foodsci.umass.eduContract/grant sponsor: Cooperative State Research, Extention, Education Service, US Department of Agriculture, MassachusettsAgricultural Experiment Station; contract/grant number: 758(Received 7 December 1998; revised version received 30 March 1999; accepted 2 July 1999)

# 1999 Society of Chemical Industry. J Sci Food Agric 0022±5142/99/$17.50 1958

MATERIALS AND METHODSMaterialsProtein sources

Lean chicken breast was obtained from a local super-

market and trimmed free of fatty tissue; it contained

0.72�0.039mg iron (haem and non-haem) per

100g.15 Ovalbumin (crystallised, lyophilised, gradeV)

and puri®ed casein were from Sigma Chemical (St

Louis, MO).

Pepsin

Pepsin (porcine, crystallised, lyophilised 1:60000

containing 2100units mgÿ1 solid, P7012) was from

Sigma. It was dissolved in 0.01M HCl at a concentra-

tion of 11.5mgmlÿ1 immediately before use.

Pancreatin

Pancreatin (porcine, puri®ed, 4�USP, gradeV) was

from Sigma. It was dissolved in tyrode buffer at a

concentration of 1.44mgmlÿ1 immediately before use.

Tyrode buffer

Tyrode buffer contained the following salts made to 1l

with water and adjusted to pH 6.0 with 6M HCl: NaCl

(8.0g), NaH2PO4 �H2O (0.0575g), NaHCO3 (1.0g),

KCl (0.2g), CaCl2 (0.2g), MgSO4 �7H2O (0.26g).

Bovine serum albumin, ferrozine, hydroxylamine

hydrochloride and iron standard solution (atomic

absorption standard containing 1020mg Fe3�mlÿ1 in

1% HCl) were all from Sigma. Trichloroacetic acid

(TCA) was from EM Science (Gibbonstown, NJ).

Dialysis tubing with a molecular weight cut-off of

6000±8000kDa and a ¯at diameter of 20.4mm was

from Spectrum Medical Industries (Los Angeles, CA).

Before use it was washed with 50% ethanol, followed

by 0.1mM EDTA and then distilled, deionised water.

MethodsAll glassware was washed in 12M HCl and rinsed

several times with distilled deionised water before use.

Extraction of proteins from muscle

Chicken breast muscle (15g) containing 3.5g pro-

tein15 was cut up and homogenised in 150ml 0.15M

NaCl. The homogenate was centrifuged at 10400�gfor 20min at 4°C and the supernatant was designated

the dilute salt-soluble protein (DSSP). The precipitate

was homogenised with another 50ml 0.15M NaCl to

exact residual DSSP, then centrifuged as above. The

second precipitate was designated the dilute salt-

insoluble protein (DSIP). Both supernatants were

assayed for soluble protein and the protein content of

the DSIP was calculated by difference. The second

supernatant from the wash with 50ml NaCl was then

discarded. Extraction and separation of the DSSP and

DSIP fractions resulted in the following protein

contents: DSSP, 1.18�0.022 (n =10)g protein per

15g muscle; DSIP, 2.32�0.024 (n =10)g protein per

15g muscle.

Preparation for digestion and addition of iron

The DSIP fraction was heated at 98°C for 20min in a

boiling water bath to aid protein denaturation and

reduce foaming, then suspended in 75ml 0.02M HCl.

The DSSP fraction was not heated, because heating

caused the soluble proteins to aggregate. 75ml of the

DSSP fraction was used for digestion. Ovalbumin and

casein were prepared by suspending 2.32g protein in

75ml 0.02M HCl.

Each protein suspension (�75ml) was adjusted to

pH 2.5 with HCl, then 4ml standard iron (1020mg

Fe3�mlÿ1) was added. The pH was adjusted to 2.0

with HCl and the volume was made to 100ml with

0.01M HCl. The fractions were refrigerated overnight

and digested the next day.

Digestion

Aliquots (25ml) of protein (DSSP, DSIP, casein or

ovalbumin) were placed in Erlenmeyer ¯asks (in

triplicate) and equilibrated at 37°C in a shaking water

bath. Pepsin solution (2ml) was added to each ¯ask

and the ¯asks were then shaken for 2h.

At the end of this stage the pH (�3) was adjusted

slowly upwards (dropwise) to �5 using 1M NaOH,

followed by 1.0 and 0.1M NaHCO3 to a pH of 6.0. A

dialysis bag containing 7ml tyrode buffer, pH 6.0, was

then placed in each ¯ask and 10ml pancreatin solution

was added. The ¯asks were then shaken for a further

2h at 37°C.

At the end of the digestion period the bags were

removed and the volumes of dialysate (inside the bag)

and retentate (outside the bag) were measured. The

dialysate was centrifuged for 12min at 1750�g at

20°C and the retentate was centrifuged for 20min at

10400�g at 4°C to precipitate insoluble iron and

protein. The supernatants were then mixed with an

equal volume of 10% TCA and centrifuged for 10min

at 1750�g to remove proteins causing turbidity. The

resulting supernatants were then analysed for protein,

total iron and ferrous iron as described below.

Kinetics of iron solubilisation

The solubilisation of iron during enzymatic digestion

of the DSIP fraction was followed over time. For the

pepsin phase a 50ml aliquot of DSIP (pH 2.0) con-

taining 2040mg iron, prepared as previously described,

was incubated at 37°C. Pepsin solution (4ml) was

added and the mixture was shaken at 37°C for 3h.

Aliquots of 9ml were withdrawn at 15±30min inter-

vals, adjusted to pH 6.0 and centrifuged for 20min at

10400�g at 4°C. The supernatants were analysed for

total iron and protein as described below.

For the pancreatin phase, 50ml aliquots of DSIP

plus iron were digested for 3h as described above,

without taking samples. At the end of the pepsin

digestion the pH was adjusted to 6.0 and 20ml

pancreatin solution was added. The mixture was then

shaken at 37°C for 2h. Aliquots of 19ml were

withdrawn immediately after the pancreatin addition

(time 0) and at 30±60min intervals thereafter. Each

J Sci Food Agric 79:1958±1963 (1999) 1959

Iron solubilisation by chicken muscle

aliquot was rapidly cooled on ice and then centrifuged

for 20min at 10400�g at 4°C. The supernatants were

analysed for total iron and protein as described below.

Assays

Protein was analysed by the biuret method.16

Iron was measured by a modi®cation of the ferrozine

method.17 For determination of total iron, 1ml of

sample was mixed with 1ml 10% (w/v) hyroxylamine

hydrochloride in 0.2M HCl. After 20min; 1ml 10%

ammonium acetate was added followed by 0.5ml

9mM ferrozine and mixed well. The absorbance at

562nm was read after 30min and compared with a

standard curve for iron using standards in the range

1±5mgmlÿ1. For determination of ferrous iron the

hydroxylamine solution was replaced by 0.2M HCl

and the absorbance was read immediately after adding

ferrozine.

Dialysable iron and protein were calculated from the

concentration in the dialysate, assuming that equili-

brium was reached during dialysis. Non-dialysable

iron and protein were calculated by subtracting

dialysable iron and protein values from total iron and

protein values.

Controls

In order to see if digestion was responsible for iron

solubilisation, non-digested controls were run in

which pepsin and pancreatin were omitted.

The solubilisation of iron without sample proteins

was checked by running controls (no sample) in which

the sample protein was replaced with an equal volume

of 0.01M HCl.

The contribution of endogenous iron to the soluble

iron pool was determined by running controls without

added iron. The extrinsic iron was replaced by an

equal volume of 0.01M HCl.

Total iron was calculated as iron in the digests

minus any endogenous iron and was used for com-

parison with the no-protein controls. Net iron was

calculated as total iron minus iron in the no-protein

controls.

RESULTSEffect of digestion on iron solubilityThe total iron solubilised by digested and non-

digested chicken muscle proteins, casein and ovalbu-

min is shown in Table 1. Digestion of the chicken

muscle fractions led to large increases in total soluble

iron. After subtracting their respective controls, diges-

tion of DSSP led to a fourfold increase in soluble iron

and digestion of DSIP led to a 20-fold increase.

Compared with the no-protein controls, DSSP and

DSIP digests solubilised ®ve and 10 times as much

iron respectively. Even without digestion, both DSSP

and, to a lesser extent, DSIP solubilised more iron

than the control. However, the net iron solubilised was

small compared with the increases following digestion.

Casein and ovalbumin both solubilised large

amounts of iron without digestion. The amount of

iron solubilised rose by 90% when casein was digested

and by 48% when ovalbumin was digested.

These results show that solubilisation of iron

increased markedly after digestion of the muscle

fractions and that the digested DSIP fraction solubi-

lised twice as much iron as the digested DSSP fraction.

However, the amounts of iron solubilised were very

small compared with that solubilised by casein and

ovalbumin.

Ferrous iron in digestThe percentage of ferrous iron in the net iron

solubilised by the digests is shown in Fig 1. Almost

two-thirds of the net soluble iron in the DSSP fraction

was ferrous, while in the DSIP fraction about one-

third was ferrous. In contrast, the proportion of ferrous

iron in casein and ovalbumin digests was very low. The

total amounts of ferrous iron in the digests were:

ovalbumin, 177mg; DSIP, 80.2mg; DSSP, 78.2mg;

casein, 28.2mg.

These results show that digests of muscle protein

fractions have the ability to reduce ferric iron to the

Figure 1. Percentage of ferrous iron in the net iron solubilised by eachprotein source.

Table 1. Solubilisation of iron by digested and non-digestedproteins

Total iron1 (mg)

Sample (g protein) Digested Non-digested

DSSP (1.18) 121�26.0 2,a 31.6�9.862,b

DSIP (2.32) 226�48.72,a 18.1�8.002,b

Casein (2.32) 3860�20.5a 2020�77.4b

Ovalbumin (2.32) 3130�12.8a 2110�20.8b

Control (0) 23.2�5.4 8.40�2.32

1 Values are mean�SD (n =3) except where noted.2 Values are mean�SD (n =12).a,b Differs signi®cantly from control in that column (p<0.05).

1960 J Sci Food Agric 79:1958±1963 (1999)

A Seth, M Diaz, RR Mahoney

ferrous state, while the non-muscle proteins have little

or no effect on iron valence.

Dialysable ironThe amount of dialysable iron as a percentage of net

soluble iron is shown in Fig 2. Very little iron was

dialysable (<5%) for all sources. There was about

twice as much dialysable iron in the DSSP digest as in

the DSIP digest fraction, but there was essentially no

dialysable iron in either the casein or ovalbumin digest.

In the muscle protein digests, more than 90% of the

dialysable iron was ferric, since less than 10% of the

dialysable iron was ferrous. Consequently, most of the

ferrous iron in both DSSP and DSIP digests was non-

dialysable.

Changes in soluble iron and protein duringdigestionOf the two muscle fractions, the DSIP solubilised the

most iron, so we studied its digestion over time.

Changes in soluble iron and soluble protein during

pepsin digestion of the DSIP are shown in Fig 3. The

amount of soluble protein rose very rapidly after

addition of pepsin, reaching 90% of its maximum

value in the ®rst 15min. A similar pattern was seen for

total soluble iron, which rose sevenfold in the ®rst

15min but more slowly thereafter, reaching its maxi-

mum value at the end of the 3h digestion. Over the

course of the digestion the amount of soluble iron rose

13-fold from the zero-time value.

Changes in soluble iron and soluble protein during

the pancreatin digestion are shown in Fig 4. The ®rst

point represents the values after a 3h pepsin digestion

before any pancreatin was added.

Soluble iron rose upon addition of pancreatin and

reached a maximum in the 30min sample, after which

it declined to near starting levels. During this time the

amount of soluble protein remained essentially con-

stant, indicating that pancreatin action did not

increase the solubilisation of the DSIP. Pancreatin

action increased the solubilisation of iron, but ex-

tended digestion caused the iron level to fall from its

maximum level (reached in the 30min digest). Based

on these results, the optimum digestion conditions for

solubilisation of iron by the DSIP would be 3h with

pepsin and 30min with pancreatin.

DISCUSSIONThis study demonstrates that digestion of proteins in

the presence of ferric iron leads to increases in soluble

iron, conversion of some ferric iron to ferrous iron and

production of small amounts of dialysable iron.

Figure 2. Percentage of dialysable iron in the net iron solubilised by eachprotein source (* less than 0.01%).

Figure 3. Changes in total soluble iron (&) and soluble protein (*) duringpepsin digestion of DSIP.

Figure 4. Changes in total soluble iron and soluble protein duringpancreatin digestion of DSIP following a 3h pepsin digestion.

J Sci Food Agric 79:1958±1963 (1999) 1961

Iron solubilisation by chicken muscle

Digestion of chicken muscle protein fractions

caused large changes in soluble iron, indicating that

peptides are involved in the effect, either by binding

iron and/or reducing it to the more soluble ferrous

form. It is also possible that some iron is bound to large

insoluble proteins/polypeptides and that digestion

results in smaller iron±peptide complexes which are

then soluble.11 The DSIP fraction solubilised about

twice as much iron as the DSSP fraction. However, the

amount of DSIP in the digests was about twice as

much as the DSSP in the digests, so the relative

capacity for solubilising iron may be very similar. The

non-digested muscle proteins did solubilise more iron

than the control, so there is some evidence for a factor

in meat which does not involve digestion, eg

glutathione,18 which would be present in the DSSP

fraction. However, the effect was small compared with

the effect of digestion, so it is likely to be of minor

importance.

The DSIP fraction was heated (unlike the other

protein sources) to reduce foaming. However, we do

not believe that heating affected the results. Our own

subsequent studies (not reported here) have shown no

difference in iron solubilisation between heated and

non-heated DSIP fractions. Other researchers have

found no differences in iron solubilised by raw or

cooked meat.10

Both casein and ovalbumin solubilised large

amounts of iron without digestion and still more after

digestion. However, these proteins do not enhance

iron uptake in vivo3,4 so solubility of iron per se is not

the only factor determining iron bioavailability, even

though it is a prerequisite. It is possible that the iron±

protein or iron±peptide complexes formed with casein

and ovalbumin are too strong to permit transfer of iron

to mucosal accepters or too large to diffuse to the sites

of absorption, or both.

Both muscle protein fractions caused conversion of

substantial amounts of ferric iron to ferrous iron, while

the non-muscle proteins had little or no effect. A

similar trend was observed by Kapsokefalou and

Miller.10 This effect may be important, since reduc-

tion of ferric iron to ferrous iron appears to be a

prerequisite for uptake into the mucosal cells of rats

and mice.19,20 The inability of casein and ovalbumin

to enhance iron absorption may be related, in part at

least, to their inability to reduce dietary ferric iron to

the more soluble and possibly more bioavailable

ferrous form.

Very little of the iron in the muscle protein digests

was dialysable, and it was almost all ferric iron.

Consequently, almost all the ferrous iron was non-

dialysable. The non-dialysable iron could consist of

large polymers of iron hydroxides (but not so large as

to be insoluble) or iron chelated to peptides too large

to pass through the membrane. Whether or not this

iron would be bioavailable is unclear. Miller and

Berner21 have argued that dialysable iron is more

bioavailable than non-dialysable iron, because of

diffusional limitations as the mucus slows the move-

ment of large molecules from the lumen to the sites of

absorption.

Very low levels of dialysable iron were also reported

by Carpenter and Mahoney11 using beef. Like us, they

added the dialysis bags after raising the pH of the

digest, manually, to neutral pH where it remained

constant during dialysis. In contrast, much higher

levels of dialysable iron have been reported in studies

where the dialysis bag is added at the end of the pepsin

digestion when the pH is around 3, and slowly rises

during dialysis.10,13,22 It may well be that the propor-

tion of dialysable iron is dependent on the pH when

dialysis begins and the ¯uctuations in pH during

dialysis.

The amount of dialysable iron is also likely to

depend on the molecular weight cut-off of the dialysis

tubing. We used tubing with a cut-off of 6000±

8000kDa based on previous work in this area,10,11

but the value is arbitrary. Use of a higher-cut-off

membrane might have led to higher values for

dialysable iron.

The kinetics of iron solubilisation during digestion

of the DSIP fraction shows that iron solubilisation

occurs as soon as soluble peptides are produced. The

peptides ®rst produced by pepsin action are likely to be

quite large, so it is likely that initially, at least, iron is

bound to these large peptides. Pancreatin produces a

further rise in iron solubility. In theory at least,

pancreatin action could create new iron-binding

peptides, but since this happens at pH 6, there would

be hardly any free soluble iron to bind to, so it is more

likely that pancreatin action reduces the size of some

large insoluble iron±peptide complexes to make them

smaller and soluble. The amount of peptide involved

must be small, since we could not detect any change in

soluble protein. Continued pancreatin action beyond

the optimum caused a decrease in soluble iron. This

was also observed by Politz and Clydesdale8 and could

be explained by assuming that further proteolysis

degrades the peptides so that they lose their ability to

bind iron. The released iron would then precipitate at

the neutral pH.

CONCLUSIONBoth the DSSP and DSIP fractions of chicken breast

muscle solubilise more iron after digestion, some of

which is reduced to the ferrous form. However, very

little of the iron was dialysable, indicating that it was

bound to peptides larger than 6000±8000kDa. At the

levels present in muscle the insoluble protein fraction

solubilised about twice as much iron as the soluble

protein fraction. Optimum digestion conditions for

iron solubilisation with DSIP were 3h with pepsin and

30min with pancreatin.

By comparison with casein and ovalbumin it is clear

that solubility per se is not a good indicator of bio-

availability. The ability of the muscle proteins to

reduce iron to the ferrous form may be a better

indicator.

1962 J Sci Food Agric 79:1958±1963 (1999)

A Seth, M Diaz, RR Mahoney

ACKNOWLEDGEMENTSThis material is based upon work supported by the

Cooperative State Research, Extension, Education

Service, US Department of Agriculture, Massachu-

setts Agricultural Experiment Station, under project

758.

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J Sci Food Agric 79:1958±1963 (1999) 1963

Iron solubilisation by chicken muscle