Clinical Science (1992) 83, 213-219 (Printed in Great Britain)
213
Absorption of nophaem iron in normal women measured
by the incorporation of two stable isotopes into
erythrocytes
J. F. R. BARRETTI, P. G. WHITTAKER', J. G. WILLIAMS2 and T. LINDI
'University Department of Obstetrics, Princess Mary Maternity Hospital,
Newcastlwpon-Tyne, U.K., and WERC ICP-MS Facility, Royal Holloway and
Bedford New College, Egham, Surrey, U.K.
(Received 3 January/23 March 1992; accepted 2 April 1992)
1. Iron absorption has been quantitatively measured
as the incorporation of physiological doses of stable
iron isotopes into erythrocytes. Five milligrams of
"Fe (orally) and 250218 of =Fe (intravenously) were
given to five healthy women on 2 consecutive days.
Fourteen days later the changes in the 57Fe/56Feand
%Fe/%Feratios in the erythrocytes of each subject
were measured using an inductively coupled plasma
mass spectrometer. Isotope ratios were also measured
in two subjects who were not given any enriched
isotope. Concomitant measurements of plasma
volume using a dyedilution technique enabled the
estimation of body iron mass and the calculation of
iron absorption.
2. The mean coefficients of variation for the
"Fe/%Fe ratio and the %Fe/%Fe ratio were 0.22%
and 0.47%, respectively. This precision allowed
enrichments of basal ratios to be reliably detected in
all cases. The mean change in the "Fe/"Fe ratio was
0.00116 (SD 0.00052, P<O.OOl) and the mean change
in the %Fe/%Fe ratio was 0.00035 (SD 0.00004,
P< 0.001). Control subjects showed no enrichment.
3. The calculated iron absorption ranged from 10%
to 34%, and the amount of absorption was related to
the iron stores of the subjects. Percentage iron
absorption was identical when estimates of the
plasma volume (derived from a body mass equation)
were used instead of the plasma volume determined
by dye-dilution measurements. Incorporation of intravenous iron into erythrocytes was on average 81%
(range 68-93%).
4. The method is especially applicable to the study of
iron absorption during pregnancy when incorporation
into erythrocytes cannot be predicted.
INTRODUCTION
The incorporation of iron isotopes into erythrocyte haemoglobin (Hb) is a well-established method
of measuring iron absorption [l]. Owing to the
perceived dangers of radioisotopes, especially in
studies of infants and pregnant women, the use of
stable isotopes is becoming favoured. Oral administration of a single stable isotope and subsequent
enrichment of erythrocytes allows semi-quantitative
estimates of iron absorption; however, accurate
measurements require the intravenous administration of a second isotope to account for the
redistribution of the orally absorbed iron between
erythrocytes and the rest of the body C2-41. The
simultaneous enrichment and detection of two
stable iron isotopes in the Hb of erythrocytes
requires good measurement precision in order to
detect the small amount of isotope enrichment that
can be achieved after the administration of physiological doses of iron. Our recent work has shown
that stable iron isotope ratios in aqueous preparations of whole blood may be measured with a
precision of <0.5% by inductively coupled plasma
mass spectrometry (ICP-MS) [5]. A clinical study
demonstrating the measurement of iron absorption
by this method is presented here.
METH0DS
Preparation of isotopes
The stable isotopes were obtained in the form of
'iron wire' (Techsnabexport, London, U.K.). The
abundances of the different iron isotopes in the
wires, as measured by the manufacturer and by
ourselves, were: enriched 57Fe, 54Fe, 0%; 56Fe,
0.57%; 57Fe, 95.93%; 58Fe, 3.5%; and enriched "Fe,
54Fe, 0%; 56Fe, 0.21%; 57Fe, 6.56%; "Fe, 93.23%.
The natural abundances in elemental iron are: 54Fe,
5.8%; 56Fe, 91.72%; S7Fe, 2.2%; 58Fe, 0.28% [6].
57Fe(174mg) was mixed with 17.5ml of 0.5mol/l
H2S04 and heated to 50°C until dissolved. Ascorbic
acid (555 mg, final concentration 3 mg/ml in water)
and deaerated, deionized water were added giving a
measured iron concentration of 4.7mg in 5ml. The
solutions were sterilized by filtration into ampoules
Key words: erythrocyte, inductively coupled plasma, iron absorption, mass spectrometry, plasma volume, stable isotope.
Abbreviations: Hb, haemoglobin; ICP-MS, inductively coupled plasma mass spectrometry; NAA, neutron activation analysis; ZPP, zinc protoprphyrin.
Correspondence: Dr P. G. Whittaker, University Department of Obstetrics, Princess Mary Maternity Hospital, Great North Road, Newcastle-upon-Tyne NE2 3BD, U.K.
214
J. F.
R. Barrett et al.
and were sealed under nitrogen. 58Fe for intravenous use was made up similarly, except that
10mg of iron was mixed with 3ml of 0.5mol/l
H2S04; after dissolution, 234 mg of ascorbic acid
was added and the final iron concentration was
256pg in 2ml. The final pH of the intravenous
solution was 1.7.
(4.52mg/ml) of the dye was compared with that of a
serum sample obtained from the opposite arm
10min after injection. The plasma volume (ml) was
calculated from two formulae, one using measured
A and one derived from body mass [9]:
Plasma volume (ml) =amount of dye given (mg)
x
Subjects
Five healthy women between the ages of 24 and
31 years volunteered for the study. They had no
history of any medical illnesses or menstrual disturbance. None was taking an oral contraceptive agent
or iron supplementation, and all were non-smokers.
All recruits gave their informed consent and the
project had ethical approval from the Combined
Ethics Committee of the Newcastle Regional Health
Authority and Newcastle University.
Study protocol
For 3 days before and 2 days during the study,
each subject followed a diet plan allowing the
ingestion of not more than 5mg of iron/day. The
diet plan was formulated by the Dietetic Department of the Royal Victoria Infirmary, Newcastleupon-Tyne, with food iron content determined from
McCance and Widdowson’s Food Composition
Tables [7]. After an overnight fast, the subjects
attended the research unit and were seated comfortably in a warm room for 15min before an intravenous cannula was inserted into each arm. A blood
sample of 20ml was taken and 10ml was put into a
lithium heparin tube for the measurement of basal
isotope ratios. The remainder was divided into
sample tubes for measurement of the subject’s Hb
level, serum ferritin concentration and erythrocyte
zinc protoporphyrin (ZPP), as well as to provide
blank serum for the determination of the subject’s
plasma volume. Haematological indices were determined on the same day using a Coulter counter
(model STKS) calibrated daily using ‘4C‘ (Coulter
Electronics Ltd, Luton, Beds, U.K.). Serum ferritin
concentration was measured by radioimmunoassay
using a ferritin monoclonal antibody solid phase
component system (normal range 3-99 ng/ml;
Becton Dickinson, Cowley, Oxon, U.K.) and ZPP
was measured using a haematofluorimeter (normal
(3.0pg of ZPP/g of Hb; AVIV Biomedical,
Lakewood, NJ, U.S.A.).
The plasma volume was then measured using the
Evans Blue dilution technique [S]. Approximately
15mg of Evans Blue in lOml of saline (150mmol/l
NaCl) was given intravenously into the left arm; the
syringe was then flushed ‘clean’ by an infusing rider
of saline via a three-way tap. Each ampoule contained 22.6mg of Evans Blue in 5ml of saline and
the exact amount given was determined by weighing
the ampoule before and after injection. The absorbance ( A ) at 620nm of a standard preparation
1”
L
A (serum)
.
x 0.0036
- - I
Plasma volume (ml)
=(height x 9.03) +(weight x 24.13)-766
An ampoule of 58Fe (256pg) was taken up into a
syringe with 10ml of saline and was given by
intravenous injection in a similar fashion to the dye;
5min later an ampoule of 57Fe (4.7mg) was emptied into a glass containing 50ml of fresh orange
juice (25mg of vitamin C), which was drunk by the
subject. No food, tea or coffee was allowed for 2 h.
The next morning, again after an overnight fast, and
following the same diet plan, the two isotopes were
given in a similar fashion The exact quantity of
isotope given by each route was calculated by
weighing the ampoules before and after administration. After the second test dose of iron, the
subjects reverted to their normal diet.
After 14 days a lOml sample of blood was taken
from each subject for measurement of enriched iron
isotope ratios. Blood samples, also 2 weeks apart,
were taken from two male control subjects who did
not receive any enriched isotope.
Preparation of samples for ICP-MS
Aqueous 1:25 solutions of whole blood were
prepared according to the following method. To a
50ml volumetric flask was added lOml of deionized double-distilled water, 2 ml of ‘matrix modifier’ [0.14mol/l ammonia solution, 0.003 mol/l
EDTA (sodium salt) and 0.029 mol/l ammonium
dihydrogen phosphate in water], 2ml of whole
blood, 10ml of Triton X-100 (50g/l of H,O) solution and deionized double-distilled water to volume.
After preparation the samples were stored at -20°C
in plastic bottles.
ICP-MS
Samples were analysed using the ICP-MS instrument PlasmaQuad PQ2 + (VG Elemental,
Winsford, Cheshire, U.K.) Details of the operating
conditions have been published elsewhere [5] and
include a mass range of 51-56 Da and an analysis
time of 2min per sample replicate. In order to
obtain Fe isotope ratios with a precision of <0.5%,
the ICP-MS instrument had to be optimized such
that 40Ar’60 was reduced to a minimum. In addition, the Fe concentration in the samples had to be
sufficiently high to produce the signal necessary to
obtain the required precision, without saturating the
215
Incorporation of stable iron isotopes into erythrocytes
Table 1. Change in the "Fe/"Fe ratio measured in erythrocytes 2 weeks after oral administration of the stable isotope
We. Subjects A-E were five women given a mean dose of 8.48mg of s7Fe orally; subjects 1-2 were two men not given any isotope.
The analysis used 15 replicates. The detection limit was set at mean +3SD.
Subject
Basal 57Fe/S6Feratio
SD
Coefficient of variation (%)
Detection limit
2-week 57Fe/56Fe ratio
SD
Coefficient of variation (%)
...
A
B
C
D
E
I
2
0.02410
O.ooOo7
0.31
0.02433
0.02390
O.ooOo5
0.22
0.02406
0.02397
0.02390
O.MX104
0.16
0.02402
0.02389
0.00006
0.24
0.02406
0.02427
0.02382
O.ooOo5
0.21
0.02397
0.02588
O.OW04
0.17
0.02435
O.ooOo6
0.23
0.02540
0.00004
0.15
0.02507
0.00003
0.13
detection system. A preparation of natural iron
(atomic absorption standard; Sigma, Poole, Dorset,
U.K.) at a concentration of 10pg/ml was analysed
(10 replicates) and the mean isotope peak areas
(from here on called counts) were used in the
calculation of iron concentration in the samples.
The mean isotope ratios of this standard preparation were used for any bias correction of isotope
ratios in the samples. The standard has no certified
isotopic composition, but deviation from the internationally accepted representative isotopic composition [ 6 ] , in practice an average of 4% for
57Fe/56Fe and 15% for 58Fe/56Fe, was used to
adjust the isotope ratios of the unknown samples.
The bias correction significantly affects the final
absorption result, but enables across-occasion consistency. Blank samples consisting only of matrix
modifier, Triton X-100 and water were measured in
quintuplicate and the mean counts were used as
blanks which were subtracted from the mean sample
counts. Each blood sample was subject to 15 replicate analyses. The mean count for each isotope (56,
57 and 58Da) was then used to calculate the
isotope ratios, percentage abundances and total iron
concentrations.
Theoretical considerations and detection limits
The ability to detect a change in isotope ratio is
dependent upon the precision with which the isotope ratios can be determined. If the SD, and hence
the coefficient of variation, of the determinations are
small, and the limits of detection are set at three
times the SD above basal [lo], then it can be
ascertained that changes in isotope ratios are a
result of individual isotope enrichment and are not
due to the imprecision of measurement. Our recent
work [5] has shown that the determination of iron
isotope ratios in whole blood can be made with
sufficient precision by ICP-MS. We calculated then,
that provided the coefficients of variation of the
measured ratios are less than 0.9%, then a 10mg
dose of 57Fe given orally (assuming a 10% absorption) and a 500pg dose of 58Fe given intravenously
(assuming 93% erythrocyte incorporation) should
enrich the basal isotope ratios in whole blood
0.00004
0.15
0.02408
0.02486
O.ooOo8
0.33
O.OO010
0.43
0.02458
0.02410
0.02393
O.oooO5
0.00004
0.21
0.17
sufficiently to detect a change in these ratios, and
hence allow calculation of the iron absorption.
Calculation of iron absorption
The absorption of the oral dose of iron is calculated by measuring the change in the 57Fe/56Fe
ratio over the 2-week period as well as the basal
iron mass. This is then adjusted for the redistribution of absorbed isotope by the amount of 58Fe
recovered 2 weeks after the intravenous injection
and for the small amount of 57Fe present in the
intravenous preparation and conversely the small
amount of 58Fe present in the oral preparation. The
derivation of this formula is explained in the
Appendix.
Total absorption of 57Fe
=
[(
A57Fe/56Fe x 58Fe given (i.v.+p.o.)
A5 Fe/56Fe)
1
- 57Fe(i.v.)
Statistical comparison was performed by Student's
paired t-test and the results are presented as means
(SD). The SD and coefficient of variation of the final
absorption estimate were calculated using the SD of
both basal (ratio,) and 2-week-enriched isotope
ratios (ratio,) [a].
Coefficient of variation =
(SD, ratio, + SD, ratio,)
100 x J
ratio, -ratio,
RESULTS
The amount of 57Fe given to the subjects varied
between 8.04 and 9.00mg, and of 58Fe between
0.51mg and 0.53mg. The mean basal and 14-day
57Fe/56Feand 58Fe/56Feratios in the five subjects
(A-E) and two control subjects (1,2) are shown in
Tables 1 and 2. The average coefficient of variation
of the 57Fe/56Fe ratios (0.22%) was better than
that of the 58Fe/56Fe ratios (0.47%), as expected
from counting statistics.
In all the five subjects given the stable isotopes
J. F. R.
216
Barrett et al.
Table 2. Change in the aFe/% ratio measured in erythrocytes 2 weeks after intravenous administration of the stable
isotope We. Subjects A-€ were five women given a mean dose d 0.52mg of BFe intravenously; subjects 1-2 were t w o men not
given any isotope. The analysis used 15 replicates. The detection limit was set at mean +3SD.
Subject...
Basal uFe/s6Fe ratio
SD
Coefficient of variation (%)
Detection limit
2-week "Fe/sbFe ratio
SD
Coefficient of variation (%)
A
B
C
D
E
I
2
0.00296
o.ooo02
0.61
0.00301
0.00301
o.ooo02
0.54
0.00306
0.00289
o.ooo01
0.35
0.00292
0.00298
0.00291
0.00302
o.oooo1
o.oooo1
0.48
0.35
0.00294
0.00306
o.ooo02
0.5 I
0.003 II
0.00332
o.ooo02
0.53
0.00330
o.ooo02
0.13
o.oooo1
0.00289
o.ooo01
0.43
0.00322
0.38
Table 3. Percentage oral iron absorption derived from both dyedilution measurement and body mass calculation of the subjects'
iron mass. The total amount of iron of mass 56 in five women was
measured by using Evans Blue e6Fe mass,)
or was calculated from a
standard formula ( V e mass,). Oral I'Fe absorption was then calculated
using the formulae given in the Appendix. The percentage recovery of the
intravenous "Fe indicates incorporation into erythrocytes using measured
iron mass.
Subject ... A
Y e mass, (mg)
Y e mass, (mg)
Oral I'Fe absorbed, (%)
Coefficient of variation of
absorption (%)
?Fe recovery (%)
B
C
D
E
1390
1355
34.3
1296
1203
10.3
1396
1373
17.8
1187
1092
24.9
1492
1315
23.1
5
79
17
10
4
68
80
84
6
93
there was an increase in both isotope ratios at 14
days of at least nine times the SD, whereas in the
two control subjects the change in the isotope ratios
lay at or within the limit of detection. The mean
change in the 57Fe/56Fe ratio was 0.00116 (SD
0.00052; P<O.OOl) and the mean change in the
ssFe/56Fe ratio was 0.00035 (SD 0.00004, P <0.001).
The iron mass of each subject derived from both
the measured (dye dilution) and calculated (body
mass) plasma volumes and the percentage of the
oral iron dose absorbed is shown in Table 3.
Although estimates of the subject's erythrocyte mass
vary by up to 12%, the percentage iron absorption
calculated using either of these values is identical
and ranged from 10 to 34% (geometric mean 20.3%).
The coefficient of variation of the calculated iron
absorption was shown to vary inversely with the
percentage absorption. The percentage of the intravenous dose that was incorporated into the erythrocytes ranged from 68 to 93% (mean 81%).
Table 4 shows the haematological parameters and
the percentage of the oral iron dose that was
absorbed by each subject. All subjects had Hb levels
within normal limits; however, the subject with the
highest percentage absorption had a low serum
ferritin concentration, suggestive of storage iron
depletion.
0.00302
0.00339
o.ooo01
0.34
0.00328
0.00313
o.oooo1
o.oooo1
0.40
0.42
o.oooo1
0.47
0.00306
Table 4. Haematological parameters of each subject relating the
percentage of oral absorption to the subject's iron stores
Subject. .. A
Hb (g/lM)ml)
Serum ferritin concn. (pg/l)
Mean cell volume (f/l)
Erythrocyte ZPP (pgglg of Hb)
Oral 57Feabsorbed (%)
12.5
8
86
2.0
34.3
B
C
D
E
14.2
89
89
1.9
10.3
13.9
53
93
1.6
17.8
13.5
32
87
1.9
24.9
12.9
42
90
1.9
23.1
DISCUSSION
The fact that most absorbed iron is incorporated
into the Hb of the erythrocytes within 2 weeks of
administration [l 11 provides a useful technique for
the measurement of iron absorption. The accuracy
of the method has been validated by comparison
with the 'gold standard' measurement of whole-body
counting using radioisotopes [12, 131. Furthermore,
the clinical protocol is simpler and more accurate
than the faecal balance method [l, 121. Assumptions of how much of the absorbed iron will be
present in the erythrocytes 14 days after administration have varied from 74% to 94% [14, 151.
Comparison of food absorption with a reference
dose absorption may remove the need for this
assumption, but still leaves errors owing to the
considerable across-occasion variation in iron
absorption within individuals if not determined on
the same occasion with two isotopes. This source of
error has been overcome by the use of an intravenous isotope administered concurrently with the
oral isotope, and the ratio of these two isotopes in
the circulating erythrocytes 2 weeks after administration allows a quantitative measure of iron
absorption [12, 151. Our value of 81% for incorporation of the intravenous dose into erythrocytes is
close to the generally accepted value of 80%, but
individual variation is considerable.
The application of this principle to the field of
stable-isotope research has been hampered by
concern over the extreme precision that would be
required to detect the small enrichments that would
be obtained in a clinical experiment, and as such
have been confined to studies mainly on the com-
Incorporation of stable iron isotopes into erythrocytes
parison of iron absorption from different foods.
Most of these studies have used the single isotope
"Fe administered orally, and have measured the
change in the 58Fe/57Feratio in the erythrocytes by
ICP-MS [2, 16-18], neutron activation analysis
(NAA) [19, 201 or fast atom bombardment mass
spectrometry [21].
This present study has shown that, in clinical use,
ICP-MS allows precision of measurement on a
simple preparation of whole blood that approaches
that of counting statistics. Enrichment of both isotopes were easily detected, suggesting that the isotope doses were adequate. The oral dose of 10mg of
57Fewas spread over 2 days, 5mg being equivalent
to a typical meal, and other iron intake was reduced
to 5mg daily so that the total intake remained
undisturbed. In the two control subjects the changes
in the basal isotope ratios were at or well within the
pre-set detection limit. The average coefficient of
variation of the 57Fe/56Feratios (0.22%) is equivalent to a minimum detectable absorption of 3%.
In order to quantify absorption and incorporation, some estimate of the total iron mass is needed.
This was achieved by an estimate of plasma volume
and measurement of the amount of iron in a basal
sample of blood analysed against a sample of iron
of known concentration. Only the mass of iron at
mass 56 was used in the calculations; this was
assumed constant as the amount of 56Fe in the
isotope preparations is negligible and any natural
shift over the 2-week period would affect all isotopes in proportion to their natural abundances. It
became clear during analysis of the results that the
use of the intravenous isotope not only allowed for
a measure of incorporation into erythrocytes, but
using the ratio of the two isotopes minimized the
effect of errors in the calculation of the iron mass.
Similarly, we believe that errors which might arise
from the use of an aqueous preparation of iron as a
standard (a certified natural abundance iron reference standard in whole blood matrix is not available) would be minimal. We therefore suggest that
in further applications of this method in nonpregnant subjects standard formulae should be used
to estimate plasma volume.
Only two other studies have applied the principles of dual isotopic incorporation by erythrocytes
to stable isotopes. Lehmann et al. [21] measured
the iron absorption of five subjects after the oral
administration of between 5 and 25 mg of 54Fe[21].
In two adults the incorporation of the radioisotope
59Fe into erythrocytes was used in an analogous
fashion to the intravenous 58Fe in this study. In
three children the incorporation into erythrocytes
was estimated from the literature. The blood
volumes used in the calculations were derived from
formulae. Iron absorption ranged from 6.4% to
49.6%, the latter value in an anaemic subject.
Dyer & Brill [20] measured the iron absorption
of a group of pregnant women using "Fe and NAA
[20]. They also used "Cr to measure the plasma
217
Table 5. Iron absorption measured in fasting healthy adults using
dual-radioisotope techniques in comparison with that measured in
the present study. Abbreviation: NS, not stated. Absorption results are
given as the range of percentage absorption of the oral dose.
Authors
Year
Isotopes
Hallberg et 01.
Lunn et 01.
Svanberg et of.
Present study
1960
1967
1975
1991
s9Fe/ssFe
S9Fe/ssFe
s9Fe/ssFe
s7Fe/seFe
Dose
Sex
4
10
3
2x5
M+F
la30
NS
5-12
17-45
10-34
(w)
Iron
absorption
Ref.
6)
F
F
1261
[I21
PI
volumes of the subjects. Each patient received alternating intravenous or oral doses of "Fe 15 days
apart; thus the single isotope was used both as an
indicator of incorporation into erythrocytes (intravenous dose) and absorption (oral dose). The usefulness of this method is limited by the lack of
published data regarding the enrichments obtained
and the expense which will result from the use of
"Fe as the oral tracer, rather than the much
cheaper 57Fe as used in our study.
Most of the radioisotope studies that have used
the erythrocyte method have been applied to food
iron absorption or diseases states [22-241. The
double-radioisotope studies that have been performed on fasted non-anaemic adults are shown in
Table 5 to allow comparison with our own results.
Whereas the normal range of iron absorption is
wide, the most accurate method of measurement,
whole-body counting, has established that normal
non-anaemic women, given a 5mg dose of iron
while fasting, should absorb between 8% and 9% of
the administered dose [l]. The mean absorption in
our study was 20.3% (range 10-34%). Our subjects
were placed on a low-iron diet for the days preceding and during the test in order to stabilize iron
intake and optimize isotope absorption by making
the enriched isotope not an undue addition to the
normal dietary intake [25]. This, together with the
finding that the subject with the highest absorption
was iron-depleted (serum ferritin concentration less
than 12pg/l) [l], may explain why some of our
results are slightly higher than those obtained by
whole-body counting. The relationship between the
iron stores of the subject and the level of absorption
(Table 4), as well as the acceptable coefficient of
variation of these measurements, is further evidence
of the validity of our results.
During pregnancy assumptions as to the level of
incorporation of iron into erythrocytes and of the
formulae used to calculate plasma volumes are
unreliable. The erythrocyte method we have described is especially applicable to the study of iron
absorption in pregnant women. In paediatric or
field studies, where researchers may choose to rely
on assumptions of incorporation into erythrocytes,
we have shown that studies using oral 57Fe are
J. F. R.
218
feasible, allowing considerable savings over the cost
of 58Fe.
Barrett e t al.
24. Bothwell, T.H., van Doorn-Wittkampf, van W, H., du Preez, M.L. & Alper, T.
The absorption of iron. Radioiron studies in idiopathic haemochromatosir,
malnutritional cytosiderorir and transfusional hemosiderosir. J. Lab. Clin. Med.
1953; 41, 83648.
ACKNOWLEDGMENTS
This work was supported by Action Research and
the Royal Society. The ICP-MS facility at Royal
Holloway and Bedford New College is supported by
the Natural Environment Research Council.
25. Fairweather-Tait, S.J.. Swindle, T.E. & Wright, A.J.A. Effect of iron intake on
Fe bioavailability in rats. Br. J. Nutr. 1985; 54, 7946.
26. Hallberg, L. & Solvell, L. Absorption of a single dose of iron in man. Acta
Med. Scand. 19M); 358 (Suppl.), 19-42.
APPENDIX
Calculation of iron absorption after the administration of
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of three radioactive methods of measuring iron absorption. Br. Med. J. 1967;
3, 331-3.
13. Werner, E., Roth, P., Hansen, C. & Kaltwasser, J.P. Comparative evaluation of
intestinal iron absorption by four different methods in man. In: Unhizaki, I.,
ed. Structure and function of iron storage and transport proteins. Amsterdam:
Elsevier Science Publishers B.V., 1983: 403-8.
14. Finch, C.A. Ferrokinetia in man. Medicine 1970; 49, 17-53.
15. Lanen, L. & Milman, N. Normal iron absorption determined by means of
whole body counting and red cell incorporation of 19Fe. Acta Med. Scand.
1975; 198, 271-4.
16. Fomon, S.J., Janghorbani, M.. Ting, B.T.G. et al. Erythrocyte incorporation of
ingested 58-iron by infants. Pediatr. Rer. 1988; 24, 20-4.
17. Fomon, S.J., Ziegler, E.E., Rogers, R.R. et al. Iron absorption from infant
foods. Pediatr. Res. 1989; 26, 250-4.
18. Woodhead, J.C., Drulis, J.M., Rogers, R.R. et al. Use of the stable isotope,
’Ve, for determining availability of nonheme iron in meals. Pediatr. Res.
two stable isotopes
The total circulating iron mass of a subject may
be calculated from the formula:
Total iron mass ( T )=(a x 25 x b)
(1)
where a is the iron concentration of the blood
sample, b is the blood volume of the subject and 25
is the dilution factor in the preparation of the blood
sample.
The iron concentration of the sample can be
determined according to the following equation:
a=
[standard concentration] x sample counts
(2)
standard counts
The blood volume is derived from measurement of
the plasma volume and packed cell volume (PCV)
c11
1
plasma volume x (PCV x 0.88)
b=[
100-(PCV x 0.88)
(3)
+plasma volume
Thus before the
total amount of
in the form of
circulating iron
Fe:
administration of any isotope, the
iron that exists in the erythrocytes
57Fe is the product of the total
mass ( T ) and the abundance of
Amount of 57Fe= T x abundance of 57Fe (4)
1988; 23, 495-9.
19. Fairweather-Tait, S.J. & Minski. M.J. Studies on iron availability in man, using
stable isotope techniques. Br. J. Nutr. 1986; 55, 279-85.
20. Dyer, N.C. & Brill. A.B. Use of the stable tracers s8Fe and Y r for the study
of iron utilisation in pregnant women. In: Nuclear activation in the life
sciences. Vienna: IAEA, 1972 469-77.
21 Lehmann, W.D., Fischer, R. & Heinrich, H.C. Iron absorption in man
calculated from erythrocyte incorporation of the stable isotope iron-54
determined by fast atom bombardment mass spectrometry. Anal. Biochem.
1988: 172. 151-9.
22. Svanberg,’B., Arvidsson, B., Bjorn-Rasmussen, E., Hallberg, L., Rossander, L.
& Swolin, 8. Dietary iron absorption in pregnancy-a longitudinal study with
repeated measurements of non-haeme iron absorption from whole diet. Acta
Obstet. Gynaecol. Scand. 1975; 48(Suppl.), 43-86.
23. Cook, J.D., Layrirse, M., Martinez-Torres, C., Walker, R., Monsen, E.R. &
Finch, C.A. Food iron absorption measured by an extrinsic tag. I. Clin. Invest.
1972; 51, 805-15.
The abundance of 57Fe may be calculated thus:
Percentage abundance =
mass 57 counts
total Fe counts
(5)
However, in the experiment two stable isotopes are
administered, 57Fe and 58Fe, with the latter isotope
influencing the denominator of the above equation,
thus influencing the calculation. Therefore the
change in the amount of 57Fe must be related to
that part of the iron mass which is not experimentally altered, i.e. the amount of 56Fe.
Incorporation of stable iron isotopes into erythrocytes
Thus eqn. (2) becomes:
or
1
a(56)=
[standard concentration] x sample (mass 56 counts)
standard (mass 56 counts)
(2a)
and eqn. (4):
Amount of erythrocyte 57Fe= T(56)
mass 57 counts
mass 56 counts
(44
The increase in the amount of 57Fe in the second
sample (sample,) taken at 14 days over the first
basal sample (sample,) is:
Erythrocyte 57Fe increase
= T(56)x
219
[(57Fe counts), (57Fe counts),]
[(56Fe counts), - (56Fecounts),]
T(56) A57Fe/56Fe
x A58Fe/56Fe
(A57Fe/56Fe)
(6)
58 Fe
increase
58Fegiven
[(
1
x "Fe given (i.v.+p.o.) - 57Fe(i.v.).
(7)
Thus to derive the increase in total-body 57Fefrom
that detected in the erythrocytes, eqn. (6) becomes:
(9)
From eqn. (8) it will be seen that the use of the
double-isotope technique not only accounts for the
redistribution of absorbed iron within the body but
also eliminates the error that measurement of blood
volume and erythrocyte mass will have on iron
absorption. However, the amount of iron recovered
is affected by variation in the measurement of
plasma volume.
The percentage oral absorption of iron is calculated by:
Percentage iron absorption
- total
-
amount of 57Feabsorbed(mg)
amount of Fe given(mg)
57Feincrease= T(56)x (A57Fe/56Fe)
"Fe given
58Feincrease
(8)
Total absorption of 57Fe=
The 56Fe mass can be assumed to remain constant
over the 2 weeks. A similar equation can be used to
derive the increase in 58Fe. This increase in "Fe
allows the calculation of the percentage recovery of
58Fe and reflects iron redistribution between
erythrocytes and the rest of the body in each
subject. If only 50% of the "Fe is recovered, then
the increase in the amount of 57Fe detected in the
erythrocytes represents only half of what was
absorbed into the body at the time of sampling:
Recovery of 58Fe(%) =
8 given
~
~
The total mass of 56Fe can be cancelled out.
However, although most of the detected increase in
57Fewas from oral absorption, 3.8% by mass of the
intravenously administered 58Fe dose (and thus
100% absorbed) consisted of 57Fe.The mean contribution of 57Fe resulting from intravenous administration (i.v.) was 0.04 mg. Similarly, depending upon
the oral iron absorption of each subject, a percentage of the erythrocyte "Fe will be derived from the
oral absorption, as the 57Fe dose administered
orally (p.0.) consisted of 6.6% 58Fe. The amount of
"Fe that was absorbed orally ranged from 0.02mg
to 0.09mg. Therefore eqn. (8) needs to be modified
thus:
A58Fe/56Fe)
= T(56)x
5
REFERENCE:
I. Paintin, D.B.The haematocrit
Commonw. 1963; 70, 807-10.
ratio in pregnancy. J. Obrtet. Gynaecol. Br.