Production of dialysable and reduced iron byin vitro digestion of chicken muscle proteinfractionsMariana Diaz, Dhiraj Vattem and Raymond R Mahoney*Department of Food Science, University of Massachusetts, Amherst, MA 01003, USA

Abstract: Muscle foods enhance the absorption of non-haem iron. We studied the effect of chicken

muscle proteins on the production of reduced and dialysable iron following in vitro digestion. At equal

protein levels, both soluble and insoluble muscle proteins increased dialysable iron 4–5-fold. Removal

of low-molecular-weight components from the soluble fraction caused a 17% drop in dialysable iron. In

contrast, egg white had little effect and whey protein was inhibitory. Both soluble and insoluble

proteins increased ferrous iron by 8–9-fold and dialysable ferrous iron by 10–13-fold. There was an

excellent correlation between dialysable iron and sulphhydryl content for all the proteins tested. The

results indicate that soluble and insoluble proteins in chickenmuscle are equally effective at producing

dialysable and reduced iron because of their similar sulphhydryl content.

# 2002 Society of Chemical Industry

Keywords: iron; chicken muscle; sulphhydryls

INTRODUCTIONIt is widely accepted that muscle foods enhance the

absorption of non-haem iron,1,2 and this effect has

come to be known as the ‘meat factor’. In contrast,

animal proteins not derived from muscle tissue, such

as those from milk and egg, have a neutral or some-

what inhibitory effect.3,4

The mechanism of the meat effect is not certain, but

most studies indicate that it is due to the action of

peptides resulting from digestion of the muscle

proteins. These peptides could chelate iron which

would otherwise be insoluble at intestinal pH and may

then release it to mucosal acceptors. In addition, they

may reduce ferric iron to the more soluble and more

bioavailable ferrous form,5 probably through the

action of cysteine residues.6–9

While there have been several studies using various

muscle/meat sources, the identity of the proteins

involved and the extent of their contributions to iron

bioavailability are unclear. Some in vitro studies have

indicated a primary role for the insoluble myofibrillar

proteins in the production of soluble and dialysable

iron.10,11 However, the effect of the soluble protein

fraction which includes both proteins and peptides is

unknown.

The objective of this research was to study and

compare the effects of both soluble and insoluble

muscle components on the production of dialysable

and reduced iron following in vitro digestion. These

forms of iron have been shown to be useful indicators

of relative iron bioavailability.5,12 Egg white and whey

protein were included in the study, both for compari-

son and to validate the methodology used.

MATERIALS AND METHODSMaterialsSkinless, boneless chicken breast was obtained from a

local supermarket and trimmed to remove fatty tissue.

Fresh eggs (USDA grade A) were also obtained from a

local supermarket. Dried whey protein isolate (BiPro)

containing 97.6% protein (dry weight basis) was

obtained from Davisco (Le Sueur, MN, USA).

Spectra/Por 1 dialysis membranes (Spectrum Labs,

Gardena, CA, USA) with a diameter of 20.4mm and a

molecular weight cut-off of 6000–8000Da were cut

into 20cm lengths, soaked in water and rinsed several

times before use.

ChemicalsPepsin, pancreatin, bile extract, PIPES buffer, ferro-

zine and bovine serum albumin were from Sigma

Chemical (St Louis, MO, USA). All other chemicals

were reagent grade. Distilled, deionised water was

used throughout.

Pepsin, porcine crystallised and lyophilised (P 7012),

was dissolved in 0.01N HCl at a concentration of

20g l�1.

Pancreatin solution was prepared by suspending

100mg of porcine pancreatin (P 1750) and 600mg

(Received 22 January 2002; accepted 19 June 2002)

* 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, Extension, Education Service, US Department of Agriculture, MassachusettsAgricultural Experiment Station; contract/grant number: 852

# 2002 Society of Chemical Industry. J Sci Food Agric 0022–5142/2002/$30.00 1551

Journal of the Science of Food and Agriculture J Sci Food Agric 82:1551–1555 (online: 2002)DOI: 10.1002/jsfa.1219

of bile extract (B 8631) in 50ml of PIPES buffer at pH

6.5.

Iron reference solution (Fisher Chemical, Fairlawn,

NJ, USA) contained 1000ppm ferric iron.

Hydroxylamine hydrochloride was dissolved in 0.2N

HCl at a concentration of 100g l�1.

Ferrozine (3-(2-pyridyl)- 5,6-bis(4-phenylsulphonic

acid)-1,2,4-triazine monosodium salt) was dissolved

in water at a concentration of 9mM.

Reducing protein precipitant solution contained 100g

of trichloroacetic acid (TCA), 50g of hydroxylamine

hydrochloride and 100ml of 12N HCl in 1 l of water.

Non-reducing protein precipitant solution contained

100g of TCA and 100ml of 12N HCl in 1 l of water.

MethodsAll glassware was rinsed in 2N HCl and then rinsed

with several changes of distilled, deionised water

before use.

Sample preparationDilute salt-soluble protein (DSSP). A 50g portion of

lean chicken breast muscle containing 11.67g of

protein13 was cut up and homogenised in 150ml of

0.15M NaCl for 3min in a Sorvall Omni-Mixer. The

suspension was centrifuged at 10400�g for 20min at

4°C. The supernatant represented the DSSP fraction

and was assayed for protein content. A volume

containing 3.0g of protein (�128ml) was used for

digestion.

Dialysed DSSP (D-DSSP). DSSP solution containing

3.0g of protein was dialysed for 10h against two

changes each of 1 l of 0.15M NaCl to remove non-

protein materials. After dialysis the volume increased

from �128 to �135ml and the total protein content

decreased from 3.0 to 2.75g.

Dilute salt-insoluble protein (DSIP). A 22.5g portion of

chicken breast muscle containing 5.25g of protein13

was cut up and homogenised in 150ml of 0.15M NaCl

for 3min. The suspension was centrifuged at

10400�g for 20min at 4°C. The supernatant was

removed and the precipitate was again homogenised

with 150ml of 0.15M NaCl and centrifuged as above

to remove remaining soluble proteins. The second

precipitate was homogenised with 100ml of 0.15M

NaCl for 3min and the protein in the suspension was

calculated by subtracting the protein content in the

two supernatants from that in the original sample. The

amount of suspension containing 3.0g of protein was

brought to �140g with added NaCl solution.

Whey and egg white. Whey protein isolate was dissolved

in 0.15M NaCl, and a volume containing 3.0g of

protein was brought to �140g with added NaCl

solution. Egg white was carefully separated from the

yolk. A weighed aliquot was dissolved in 0.1N NaOH

and assayed for protein content. An amount contain-

ing 3.0g of protein was then suspended in 0.15M

NaCl, pH 9.0 and brought to �140g with added

NaCl solution.

Pepsin digestionProtein samples containing 3.0g of protein (except for

D-DSSP which contained 2.75g of protein) were

adjusted to pH 2.5 with 6N HCl. Iron (37.5mmol) was

added, followed by sufficient water to bring the sample

weight to 145g. The pH was then adjusted to 2.0,

checked after 10min, and 5ml of pepsin solution was

added. The samples were then incubated in a shaking

water bath at 37°C for 2h. The sample flasks were

shaken at �100strokesmin�1 with an arm movement

of 1.5cm.

At the end of the digestion period the samples,

whose pH ranged from 2.7 to 3.2, were stored in an ice

bath for 90min while the titratable acidity was

measured.

Titratable acidityA 20g aliquot of pepsin digest was brought to room

temperature and 5ml of pancreatin solution was

added. The sample was then titrated with 0.5N NaOH

until the pH reached 6.5 After 30min the pH was

checked, readjusted to pH 6.5 if necessary, and the

final volume of 0.5N NaOH used was recorded. The

number of NaOH equivalents used was calculated and

a solution of NaHCO3 was prepared which contained

the same number of equivalents in 20ml of NaHCO3

solution.

Pancreatin digestionAliquots (20ml) of pepsin digest were placed in wide-

necked 250ml Erlenmeyer flasks; each flask now

contained �400mg of protein and 279mg of iron. A

dialysis bag containing 20ml of NaHCO3 solution

(whose concentration was determined from the

titratable acidity) was added to each flask, and the

samples were shaken for 30min at 37°C. At the end of

that period the pH rose to �4.6.

Pancreatin solution (5ml) was added to each flask,

and the pH rose to �5.5. The flasks were then shaken

for a further 2h at 37°C. At the end of the digestion

period the bags were removed, rinsed and weighed.

The digest outside the bag (retentate) was also

weighed. The pH of both retentate and dialysate was

6.5�0.1.

ControlsAn iron-only control was run using the procedures

described above but with water in place of the protein

sample.

In order to compensate for endogenous iron in the

samples, proteins were digested as above without

adding ferric iron. The resulting iron values were

subtracted from the values with added iron to correct

for the contribution of endogenous iron.

AnalysesAfter digestion the dialysate and retentate were

1552 J Sci Food Agric 82:1551–1555 (online: 2002)

M Diaz, D Vattem, RR Mahoney

centrifuged at 1750�g for 10min to remove insoluble

iron. Aliquots of the supernatants containing soluble

iron were mixed with equal volumes of reducing

protein precipitant and of non-reducing protein

precipitant and left overnight. The next day the

samples were centrifuged again at 1750�g for

10min to remove TCA-insoluble protein. The final

supernatants were analysed for protein and iron.

Protein was measured by the biuret method14 using

bovine serum albumin as a standard.

Total iron was measured by mixing 1ml of sample

treated with reducing protein precipitant with 2ml of

ammonium acetate solution (100g l�1), followed by

0.5ml of ferrozine reagent. The absorbance was read

at 562nm after 1h. Ferrous iron was measured by

using samples treated with non-reducing protein

precipitant and reading the absorbance immediately

after the addition of ferrozine. Iron concentrations

were calculated from a standard curve generated using

known concentrations of FeCl3 (0–5mg ml�1) in the

presence of the reducing agent hydroxylamine hydro-

chloride.

Sulphhydryl content was determined using Ellman’s

reagent, 5,5-dithiobis(2-nitrobenzoic acid) (DTNB),

as described by Habeeb.15 Protein samples were

denatured by mixing with 22 volumes of 0.085M

Tris-glycine buffer, pH 8.0 containing 8M urea;16 for

the DSIP fraction the buffer also included 0.6M KCl.

After denaturation, 0.1ml of DTNB was added to 3ml

of diluted protein, and the absorbance at 412nm was

read after 15min. The sulphhydryl concentration was

calculated using a molar extinction coefficient of

1.36�104m�1cm�1.15

RESULTSThe effect of the proteins on dialysable iron is shown in

Fig 1. In the control about 15% of the original iron

added was soluble but only 2% was dialysable. Egg

white caused a small increase in dialysable iron, but

whey protein reduced dialysable iron by 54%. All the

muscle protein fractions increased dialysable iron 4–5-

fold compared with the control. There was no signi-

ficant difference between the effects of the insoluble

fraction (DSIP) and the soluble fraction (DSSP).

However, dialysis of the DSSP fraction to remove

soluble, low-molecular-weight muscle components led

to a small drop (�17%) in dialysable iron.

The effect of the proteins on reduction of iron is

shown in Fig 2. All the proteins caused an increase in

total ferrous iron compared with the control. Whey

protein increased ferrous iron by 50% and egg white

increased it 4-fold. However, all the muscle proteins

increased ferrous iron 8–9-fold. The soluble protein

fractions (DSSP and D-DSSP) produced slightly more

ferrous iron than the DSIP fraction. Dialysis of the

DSSP fraction had no significant effect on ferrous

iron.

Egg white and whey protein had little or no effect on

dialysable ferrous iron, but the muscle protein frac-

tions increased it 10–13-fold, with DSSP having the

greatest effect. Comparison of Figs 1 and 2 shows that

with egg white most of the dialysable iron was ferric,

but with whey protein more than half was ferrous.

With the muscle proteins about half of the dialysable

iron was ferrous.

The sulphhydryl content of the protein sources is

shown in Table 1. The muscle protein fractions all had

about 2.5–3 times as many reactive sulphhydryl groups

as the non-muscle proteins. Dialysis of the soluble

fraction (D-DSSP) reduced the total sulphhydryl

content, but some peptide material was also removed

by dialysis, so the sulphhydryl content per gram of

protein was essentially unchanged.

The relationship between various iron forms and

sulphhydryl content in the protein sources is shown in

Figs 3–5. There was an excellent correlation between

total dialysable iron and sulphhydryl content (Fig 3)

(r2=0.99) and good correlations of both ferrous iron

(Fig 4) (r2=0.96) and dialysable ferrous iron (Fig 5)

(r2=0.96) with sulphhydryl content. All correlations

Figure 1. Effect of proteins on production of dialysable iron. EW, egg white;WP, whey protein; DSIP, dilute salt-insoluble protein; DSSP, dilutesalt-soluble protein; D-DSSP, dialysed dilute salt-soluble protein. Differentletters indicate that mean values were significantly different at p <0.05(n=3).

Figure 2. Effect of proteins on production of total (shaded) and dialysable(white) ferrous iron. For abbreviations used, see caption to Fig 1. Differentletters indicate that mean values were significantly different at p <0.05(n=3).

J Sci Food Agric 82:1551–1555 (online: 2002) 1553

Chicken proteins produce dialysable iron

were significant at p<0.05. The results indicate that,

for these protein sources, sulphhydryl content is a very

good indicator of the production of both dialysable

and reduced forms of iron.

DISCUSSIONIt is widely accepted that dialysable iron is a good

indicator of relative iron bioavailability. However, for

valid comparison of samples it is important to control

pH changes during the digestion and dialysis processes

in a consistent manner.12 We found that the first pH

adjustment, after the pepsin digestion, was best con-

trolled by dialysis of bicarbonate equal to the titratable

acidity (after allowing for the effect of pancreatin).

However, bicarbonate did not effectively control the

final pH at the end of the pancreatin digestion, so

PIPES buffer was used for that purpose. Together,

these components provided consistent and repro-

ducible pH changes when comparing protein

samples and sample replicates.

The results for dialysable iron show that all the

muscle protein fractions were enhancers whereas egg

white was near neutral and whey protein was inhibi-

tory. These results agree with studies on bioavailability

in humans.2–4

For dialysable iron there was very little difference

between the muscle protein fractions, indicating that,

when isolated, the soluble sarcoplasmic proteins are as

effective as the insoluble myofibrillar proteins. The

effect of the latter has been attributed primarily to the

myosin component, which is rich in sulphhydryl

residues.11

The effect of the soluble fraction is presumably due

to its peptide components, which include enzymes,

myoglobin and small peptides. Removal of the small

peptides from the soluble fraction caused a small

decrease in dialysable iron, indicating that the peptides

have an effect, albeit a minor one compared with the

proteins. The principal peptides in the sarcoplasm of

muscle are carnosine, which does not bind iron,17 and

glutathione, which binds and reduces iron.6 It is

therefore likely that glutathione is responsible for a

small amount of the dialysable iron produced by the

soluble fraction. Most of it, however, is due to the

soluble proteins, whose major constituents are the

glycolytic enzymes.18

In this experiment, equal amounts of protein were

used in the digestions. Based on our extraction

procedures, however, the ratio of insoluble to soluble

proteins is about 2:1.10 Accordingly, it seems likely

that in whole muscle the myofibrillar proteins are the

principal enhancers of dialysable iron and that the

soluble proteins and glutathione play a lesser role,

Table 1. Sulphhydryl (SH) content of proteinsources

Protein SH content (mmolg�1) a

Whey protein 31.9�0.80

Egg white 35.8�0.73

DSSP 95.6�1.13

D-DSSP 95.8�1.6

DSIP 98.7�1.0

a Mean�SD (n=3).

Figure 3. Correlation of total dialysable iron with sulphhydryl content inprotein source. For abbreviations used, see caption to Fig 1.

Figure 4. Correlation of total ferrous iron with sulphhydryl content in proteinsource. For abbreviations used, see caption to Fig 1.

Figure 5. Correlation of dialysable ferrous iron with sulphhydryl content inprotein source. For abbreviations used, see caption to Fig 1.

1554 J Sci Food Agric 82:1551–1555 (online: 2002)

M Diaz, D Vattem, RR Mahoney

owing to their lower content rather than to their innate

effect on dialysable iron.

The effects of the proteins on reduction of iron are

similar to the effects on dialysable iron, with only small

differences between the muscle fractions. The muscle

protein fractions not only reduced more iron than

the non-muscle proteins but also produced more

dialysable ferrous iron. Since iron must be reduced19

and also be small enough to diffuse to the sites of

absorption before it can be absorbed, dialysable

ferrous iron may be the best in vitro indicator of iron

bioavailability.5 Accordingly, the effectiveness of

muscle proteins may be due not simply to their ability

to reduce iron but also to promote the formation of

low-molecular-weight reduced iron—iron which is not

bound to large peptides or polymerised by hydroxyl-

ation. In contrast, egg white produced 4 times as much

ferrous iron as the control but very little more

dialysable ferrous iron, which may explain why it does

not enhance iron bioavailability.

The reduction of iron by proteins is presumably due

to their sulphhydryl residues. For our data a good

correlation was observed between reduced forms of

iron and sulphhydryl content, which confirms a trend

previously reported.8 The only exception was egg

white, which produced almost 3 times as much ferrous

iron as whey protein yet had only 12% more sulph-

hydryl groups. Almost all the additional ferrous iron

was non-dialysable.

There was also an excellent correlation between

sulphhydryl content and total dialysable iron—which

includes significant quantities of low-molecular-

weight ferric iron. This ferric iron is very insoluble at

neutral pH and therefore must be complexed to small

ligands—such as peptides—in order to be dialysable.

These results suggest that sulphhydryl residues

could play two roles in promoting iron bioavailability:

(1) by reducing ferric iron to ferrous and2 as a

component of small peptides which bind ferric iron

either through the sulphhydryl groups alone9 or in

combination with other residues such as histidine.20

Overall, this study indicates that, at equal levels, the

soluble muscle proteins are as effective as the insoluble

proteins in producing dialysable and reduced iron.

This appears to be related to the very similar

sulphhydryl content of the protein fractions.

ACKNOWLEDGEMENTSThis material is based on work supported by the

Cooperative State Research, Extension, Education

Service, US Department of Agriculture, Massachu-

setts Agricultural Experiment Station, under Project

No 852.

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