Download Comparison of Stable Isotopes and Radioisotopes

Clinical Science (1994) 87. 91-95 (Printed in Great Britain)
91
Comparison of stable isotopes and radioisotopes in the
measurement of iron absorption in healthy women
Jon F. R. BARREll, Paul G. WHITTAKER*, John D. FENWICK?, John G. WILLIAMS$
and Tom LIND*
*University Department of Obstetrics and Gynaecology, Royal Victoria Infirmary,
Newcastleupon-Tyne, U.K.. ?Department of Medical Physics,
Newcastle General Hospital. Newcastleupon-Tyne, U.K., and $NERC ICP-MS Facility,
Imperial College at Silwood Park, Ascot, Berkshire, U.K.
(Received 15 October 1993/17 January 1994; accepted 25 January 1994)
1. Stable isotope methods are being used to investigate the absorption of dietary iron. In order to be
certain that this new methodology is accurate, we
have compared results obtained using stable isotopes
and inductively coupled plasma mass spectrometry
with those determined using a radioisotope and whole
body counting.
2. The stable isotope 54Fe (2.8mg) was given to 10
healthy non-pregnant women. Six women received the
isotope in aqueous form, and four took it with a meat
meal. The 54Fe served as a carrier for long of the
radioisotope 59Fe. An ampoule (200pg) of the isotope
"Fe or '*Fe was then given intravenously, and in
serum samples taken over the next 10 h the ratios of
the stable iron isotopes were measured by inductively
coupled plasma mass spectrometry and the oral iron
absorption was calculated. This was then compared
with the results obtained by using a whole body
counter to measure (on day 0 and day 14) the
y-activity emitted by the radioisotope.
3. The mean iron absorption measured by both
methods ranged from 8% to 45%. Measurement of
the post-absorptive serum enrichment of the stable
isotopes provided estimates of absorption from both
aqueous and food iron which were similar to that
yielded by whole body counting, the mean difference
being -1.5% (95% confidence interval -5.2 to
2.1%). Absorption estimated by stable isotopes exhibited the same inverse relationship with the serum
ferritin level (body iron stores) to that known to exist
with whole body counting. Similar estimates of food
iron absorption were obtained irrespective of the type
of isotope used as an extrinsic label, implying that
stable isotopes are as valid as radioisotopes in reflecting intrinsic food iron absorption.
4. This study validates the use of stable isotopes and
post-absorption curves as a new and accurate technique in the measurement of iron absorption.
INTRODUCTION
Owing to the perceived dangers of radioisotopes,
especially in studies of infants and pregnant women,
stable isotope methods have been used increasingly
to evaluate many aspects of trace element metabolism [l]. We have used a stable isotope method in
which one isotope was given orally and the other
intravenously to measure the absorption of iron in
pregnant women; the use of two isotopes allows for
correction of variations in iron clearance. Sample
analysis was by inductively coupled plasma mass
spectrometry (ICP-MS) [2]. In order to be certain
that the stable isotope method yielded accurate
absorption measurements we have conducted a
study to validate this new method against a wellaccepted radioisotope and whole body counting
method [3]. Concern has been expressed that the
stable isotopes may not reflect the metabolism of
the non-haem iron intrinsic to food [4]; unlike
radioisotopes, stable isotopes have not been validated as extrinsic labels of food iron. Therefore we
have measured the absorption of food iron which
had been labelled with both radioisotopes and
stable isotopes and given to non-pregnant women.
MATERIALS A N D METHODS
Preparation of isotopes
The stable isotopes were obtained in the form of
'iron wire' (Techsnabexport, London). The abundances of the different iron isotopes, as measured by
the manufacturer and by ourselves, were: enriched
54Fe, 54Fe 99.85%, 56Fe 0.13%, 57Fe 0.02%, "Fe
0%; enriched 57Fe, 54Fe OX, 56Fe 0.57%, 57Fe
95.93%, "Fe 3.5%; enriched "Fe, 54Fe O%, 56Fe
0.21%, 57Fe 6.56%, "Fe 93.23%. The natural abundances of isotopes in elemental iron are: 54Fe, 5.8%;
56Fe, 91.72%; 57Fe, 2.2%; "Fe, 0.28% [ S ] . The
Key words: absorption. female, iron isotopes, mass spectrum analysis, whole body counting.
Abbreviations: AUC, area under curve; ICP-MS, inductively coupled plasma mass spectrometry; ZPP, zinc protoporphyrin.
Correspondence: Dr Paul G. Whittaker. University Department of Obstetrics. 4th Floor. Leazes Wing, Royal Victoria Infirmary. Newcastle-upon-Tyne N E I 4LP. U.K.
92
1. F. R. Barrett et al.
radioisotope 59Fe as ferric citrate was obtained
from Amersham International (Amersham, Bucks,
U.K.)
54Fe for oral use was mixed with 0.5mol/l H,SO,
(ratio lOmg/lml) and heated to 50°C until dissolved. Ascorbic acid (at a final concentration of
3 mg/ml) and deaerated, deionized water were
added, giving a final iron concentration of 2.83mg
in 5 ml. The solutions were sterilized by filtration
into ampoules and were sealed under nitrogen. 57Fe
and 58Fe for intravenous use were made up similarly, except that 10 mg of iron was mixed with 3 ml
of 0.5mol/l H 2 S 0 4 and the final Fe concentrations
were 200 pg in 2 ml. The final pH of the intravenous
solution was 1.7 to ensure stability during storage,
but was diluted in saline immediately before
injection.
Subjects
Ten healthy non-pregnant women between the
ages of 39 and 43 years volunteered for the study.
They had no history of any medical illness, and
none was taking iron supplementation; all were
non-smokers. All women had completed their families, and used a permanent method of contraception,
either tuba1 ligation or vasectomy, hence the reason
for asking them to volunteer for the radioisotope
studies. All subjects gave their informed consent,
and the study had the approval of the ethics
committee of the Newcastle Regional Health
Authority.
Administration of isotopes and labelling of food
For 3days before and on the day of the study,
each subject followed a diet plan to provide a daily
intake of 13mg of iron (on average 4mg from
meat). The diet plan was formulated by the Dietetic
Department of the Royal Victoria Infirmary, Newcastle, with the food iron content determined from
McCance and Widdowson’s food composition tables
[6]. After an overnight fast, the subjects attended
the Medical Physics Department of the Newcastle
General Hospital. The subject’s height and weight
was measured, and a background count was
obtained by scanning the subject from head to foot
at 300mm/min on a Shadow Shield whole body
counter. An intravenous cannula was inserted into
each arm and was flushed with heparinized saline to
prevent clotting. A 20ml blood sample was taken
for measurement of basal isotope ratios and full
blood count, serum ferritin and erthrocyte zinc
protoporphyrin (ZPP). Haematological indices were
determined on the same day using a Coulter
counter (Coulter Electronics Ltd, Luton, Beds,
U.K.). Serum ferritin level was measured by
radioimmunoassay (normal range 3-99 ng/ml; Becton Dickinson, Cowley, Oxon, U.K.) and the Z P P
was measured using a haematofluorimeter (normal
range <3.Opg of ZPP/g of Hb; AVIV Biomedical,
Lakewood, NJ, USA.).
Aqueous iron absorption (six subjects)
The cannula in the left arm was connected to a
500ml bag of warmed normal saline, which was
allowed to infuse slowly. An ampoule (200pg) of the
stable isotope for injection was taken up into a
syringe with lOml of saline and was then given
intravenously into the left arm of each subject. The
syringe was then flushed clean from the infusing
rider of normal saline using the three-way tap. 57Fe
was used in three subjects and 58Fe in three subjects
due to a temporary delay in preparation of 57Fe.
After 5min an ampoule (2.83mg) of the isotope
54Fe was given orally in a glass containing 50ml of
orange juice. The 54Fe served as a carrier for
185 kBq of radioisotope 59Fe (OSmSv), which was
added to the same drink. The exact quantity of
isotope given by either route was calculated by
weighing the ampoules before and after administration, and no food, tea or coffee was allowed for
2 h thereafter. Eleven blood samples (8 ml) were then
taken over the 10h after the intravenous injection
and the serum was frozen until assay.
Food iron absorption (four subjects)
The procedure for the measurement of food iron
absorption was similar to that described above,
except that the oral isotopes 54Fe and 59Fe were
added to a meal as an extrinsic label. All subjects
received 57Fe as the intravenous stable isotope
tracer. The meal consisted of 60g of cooked lean
bacon, one bread roll and 50ml of fresh orange
juice. The total non-haem iron content of the meal
was 3.2mg [6, 71. The bacon was cooked and the
rind removed, and the roll was lightly toasted. The
ampoule of 54Fe was then emptied into the centre
of the bread roll as this constituted the bulkiest
component of the meal [8, 91. The bacon roll was
then eaten by the subject together with the 50ml of
fresh orange juice (25mg of vitamin C).
Isotope analysis
One hour after the administration of the radioisotope 59Fe, and in the interval between the 60 and
75min serum samples, the subject was scanned on
the whole body counter to determine the 100%
administration level (after subtraction of background). Fourteen days later each subject returned
to the Medical Physics Department and the oral
absorption was calculated as the ratio of the 14day
retention to the 100% level, corrected for radioactive
decay [3].
The stable isotopes of iron in each serum sample
were measured by ICP-MS with appropriate quality
control [2]. The isotope ratios were expressed by
the ratio of 57Fe or 58Fe (the intravenously administered isotopes) to 56Fe, and also of 54Fe (the oral
isotope) to 56Fe. For example, the basal 57Fe/56Fe
is 0.024 and 15min diter injection it might be 0.100,
Methods for iron absorption measurement
93
an enrichment of 0.076. The serum enrichments in
isotope ratios were plotted against time [lo], and
from the area under these curves (AUC) the amount
of the orally absorbed isotope was calculated by the
formula as previously described [2]:
The mean coefficient of variation in the measurement of the 54Fe/56Fewas 1.7%, of the 57Fe/56Fe
was 2.2% and of the 58Fe/56Fewas 4.6%.
Other ferrokinetic aspects reflected in the curves
include the time ( Tmax,)taken from isotope administration to reach maximal oral isotope enrichment in
serum, and the serum half-time (tIl2) of injected iron
isotope in the first 2 h after injection.
0
I20
Statistics
Results are given as the mean and SD or mean
and 95% confidence interval. T,,, and t1,2 were
analysed using the log and inverse values, respectively. Agreement between the two analytical methods
was tested as described by Bland and Altman [ll].
Log transformation was used for absorption results,
but the differences, reflecting measurement error
rather than subject variation, did not increase as
absorption increased and so were not transformed.
Linear correlation was calculated between the log
absorption estimates and the log serum ferritin
levels [7].
RESULTS
Stable isotope measurement and absorption curve
analysis
Fig. 1 shows the isotope ratio enrichments of the
post-absorption serum curves for two subjects given
aqueous iron. The importance of using two isotopes
is demonstrated. Subject no. 4 had a higher overall
oral enrichment than subject no. 5 (oral AUC: 52.9
versus 36. l), but with similar initial intravenous
enrichments at 15min (subject no. 4, 0.0780 subject
no. 5, 0.0743) and differing levels of iron clearance
(t1,2: subject no. 4, 119min; subject no. 5, 58min)
that gave different intravenous AUC (subject no. 4,
13.8; subject no. 5, 7.8) so that the oral absorption
of subject no. 4 was less than that of subject no. 5
(29.8% versus 34.5%). Mean t1,2 in all subjects was
92min (95% confidence interval 75-1 19 min). The
T,,,, was significantly longer in those subjects given
iron with food than in those given only aqueous
iron (mean difference 80 min, 95% confidence interval 31-149min, P<O.Ol).
Stable isotope and radioisotope comparison
The measured absorption by each method after
the administration of aqueous and food iron is
480
240
360
Time (min)
600
Fig. 1. Serum enrichment of oral and intravenous iron isotopes in
Solid lines are oral (Ye/51Fe)
subject no. 4 ( 0 )and subject no. 5 (0).
isotope enrichments: broken lines are intravenous ("Fe/"Fe) isotope
enrichments.
Table 1. Absorption of iron measured by the stable isotopl and
radioisotope methods, compared with serum ferritin levels
Iron absorption (%)
Stable isotope
Radioisotope
Serum ferritin level
method
method
(PdU
Aqueous iron
Subject no. I
Subject no. 2
Subject no. 3
Subject no. 4
Subject no. 5
Subject no. 6
3.5
9.6
12.9
29.8
34.5
45.3
15.3
16.1
14.9
30.5
29.0
40.9
41
52
Food iron
Subject no. 7
Subject no. 8
Subiect no. 9
Subject no. 10
5.5
8.2
15.4
38.6
10.6
8. I
15.3
37.0
34
56
57
15
Geometric mean
14.9
19.1
27
25
12
18
10
shown in Table 1. Use of food or aqueous iron did
not reveal any significant bias in the differences, and
by combining the absorption measurements of both
the food and aqueous iron, the mean difference
between the two methods of measurement was
- 1.5% with a 95% confidence interval of -5.2 to
2.1%. By plotting the mean difference between the
absorption measurements against the average of
these measurements for each subject (Fig. 2), it was
seen that the 'limits of agreement' between absorption measurements (mean & 2 SD) lie between an
underestimate of 11.7% and an overestimate of
8.6%. These 'limits of agreement' would be experi-
J. F. R. Barrett et al
94
Mean+2SD
m
0
. O
A
0
U
-
0
Mean
n
.
-12
A
Mean - 2 SD
-I
0
A
- -
I
10
20
30
40
Average absorption by both methods (%)
50
14-
~
t
--
.t
..-t--+
t
t-+ti
100
10
Serum ferritin level (pg/l)
Fig. 2. Comparison of stable isotope and radioisotope methods of
measuring iron absorption. W , Aqueous label; 0,
label mixed with
food.
mentally acceptable considering the wide range of
iron absorption in normal individuals [ 1 11.
Haematology and correlation with absorption estimates
All patients had Hb concentrations greater than
12g/100ml, and all had a mean corpuscular volume
greater than 85 fl. However, there were two subjects
(nos. 4 and 6 ) with serum ferritin levels of 12ng/ml
or less, who also had elevated levels of ZPP
( > 3.0pg of ZPP/g of Hb). There was a significant
correlation between the log absorption estimates of
both stable isotope and radioisotope methods and
the log of the serum ferritin level (r=0.78; P<O.O5
and r=0.86; P<O.O5) (Fig. 3).
DISCUSSION
Whole body counting of orally absorbed radioactive iron is accepted as the reference method for
iron absorption [12]. Use of the incorporation of
oral and intravenous tracers into erythrocytes has
been shown to have a close correlation with whole
body counting [13, 141 and the use of only oral
tracer data gave a poorer correlation because of the
uncertainty of blood volumes and radio-iron utilization in new erythrocytes [14]. The use of a double
radioisotope technique with post-absorption serum
measurements had the best correlation with whole
body counting [14, 151, although debate has existed
as to whether the post-absorptive serum measurement of therapeutic ( >30mg) doses of iron accurately reflects oral iron absorption [14, 16-19]. In
order for the results of our studies investigating the
absorption of iron during pregnancy to be accepted
as accurate, a direct validation of the stable isotope
Fig. 3. Relationship between serum ferritin level and iron absorp
tion measured by the radioisotope method (squares) and the stable
isotope method (triangles). Label mixed with food is indicated by open
symbols
methodology had to be undertaken. We found that
the measurement of the post-absorptive serum
enrichment of two iron stable isotopes provided
estimates of iron absorption both from aqueous and
food iron that were similar to that yielded by whole
body counting. Furthermore, absorption estimated
by stable isotopes exhibited the same inverse relationship with the serum ferritin level (body iron
stores) to that known to exist with whole body
counting [20-221. By chance, a wide range of
absorption was encountered in the study, and the
relationship between the two methods held true
across the entire range.
There has been debate over the use of stable
isotopes as extrinsic labels of food iron. When
radioisotopes are used as extrinsic labels, very little
carrier iron (about long) is used to label the
intrinsic pool of food iron, and while we used a
similar method of labelling with a stable isotope to
that described using a radioisotope [8], approximately 3mg of stable isotope or 50% of the total iron
content of the meal was used as a label. Theoretically, at least, this might be less exchangeable with
the common pool of food iron [4, 231. Whilst the
production of food intrinsically labelled with 54Fe is
possible [23], it is likely to prove expensive and was
not possible within the time course or financial
constraints of this study. Therefore, in an effort to
test the reliability of the extrinsic labelling method,
both stable isotopes and radioisotopes were used as
extrinsic labels by addition to the same meal and
the absorption estimates were compared. Ferric
iron, the chemical form of the radioisotope, is less
absorbable than ferrous iron (the form of the stable
isotope) at high doses [24, 251. However, only small
doses of both forms were administered, and the
Methods for iron absorption measurement
reducing effect of the ascorbic acid contained in the
orange juice eliminated a potential difference in
absorption due to differing valencies of the iron
associated with each tracer.
When used as an extrinsic label of food iron, T,,,.
was significantly longer in the food iron absorption
profiles than in those of aqueous iron. This suggested that the stable isotope mixed with the meal
and was not absorbed in the manner of an aqueous
iron dose. Furthermore, similar estimates of food
iron absorption were obtained irrespective of the
type of isotope used as an extrinsic label. This
confirms that the stable isotope mixed and
exchanged with the intrinsic food iron in a similar
extent to the radioisotope; thus by inference, extrinsic stable isotopes are as valid as extrinsic radioisotope tracers [26] in reflecting intrinsic food iron
absorption. Cook et al. [27] stated that the ratio of
absorption derived from extrinsically and intrinsically labelled food was close to unity when the
extrinsic tag was mixed with carrier iron of similar
dose to our 54Fe dose.
The age-old obstetric dilemma regarding the need
for the routine prescribing of iron supplements
during pregnancy can only be solved when a safe,
accurate and economically feasible method of measuring food iron absorption during pregnancy has
been developed. The results of this study suggest
that such a method is now available, and its
application is under investigation.
ACKNOWLEDGMENTS
This study was supported by grants from the
Medical Research Council, the Natural Environmental Research Council, Action Research for the
Crippled Child and the Sir Jules Thorn Charitable
Trust.
REFERENCES
I. Janghorbani M, Ting BTG, Zeisel SH. Trace element research with stable
isotope tracers. In: Prassad A, ed. Essential and toxic elements in human
health and disease. New York: Alan R. Liss. 1388: 545-56.
2. Whittaker PG, Lind T, Williams JG. Iron absorption during normal human
pregnancy: a study using stable isotopes. Br J Nutr 1991; 65: 457-63.
3. Will G. Boddy K. Iron turnover estimated by a whole body monitor. Scot
Med J 1967; 12: 157-62.
95
4. Fairweather-Tait SJ. Fox TE. Wharf SG. Eagles J, Crews HM, Marrey R.
Apparent zinc absorption by rats from foods labelled intrinsically and
extrinsically with “Zn. Br J Nutr 1991; 6& 65-71,
5. Commission on Atomic Weights and Isotopic Abundances. Isotopic composition
of the elements 1989. Pure Appl Chem 1991; 63 991-1002.
6. Paul AA, Southgate DAT. McCance and Widdowson’s the composition of
fwds. 4th ed. London: HMSO, 1978.
7. Geigy scientific tables. 8th ed. Bale: Ciba-Geigy, 1981.
8. Bjorn-Rasmussen E. Hallberg L, Magnusson B, Rossander L, Svanberg B,
Arvidsson B. Measurement of iron absorption from composite meals. Am J
Clin Nutr; 1976 Y. I31 1-19,
9. Svanberg B, Arvidsson B, Bjorn-Rasmussen E, Hallberg L, Rossander L, Swolin
B. Dietary iron absorption in pregnancy-a longitudinal study with repeated
measurements of non-haem iron absorption from the whole diet. Act, Obstet
Gynaecol Scand 1975; 48 (Suppl.): 43-86.
10. Gibaldi M, Perrier D. Pharmacokinetics. New York: Marcel Dekker, 1982:
145-98.
I I. Bland MI, Altman DG. Statistical methods for assessing agreement between
two methods of clinical measurement. Lancet 1986; i: 307-10.
12. Bothwell TH. Charlton RW, Cook JD. Finch CA. Iron metabolism in man.
Oxford: Blackwell. 1979 425-38.
13. Lunn ]A. Richmond J, Simpson ID, Leask ID, Tothill P. Comparison of three
radioactive methods of measuring iron absorption. Br Med J 1967; Ill: 331-3.
14. Werner E, Roth P, Hansen C, Kaltwasser JP. Comparative evaluation of
intestinal iron absorption by four different methods in man. In: Urshizaki I,
ed. Structure and function of iron storage and transport proteins. Amsterdam:
Elsevier. 1983: 403-8.
IS. Werner E, Hansen C. Wittmaack K, Roth P, Kaltwasser JP. The application of
stable isotopes of iron as tracers in investigations of iron metabolism. INSERM
Symp Ser (Paris) 1983; I13 201-24.
16. Bothwell TH, Mallett B, Oliver R, Smith MD. Inability to assess absorption of
iron from plasma radioiron curves. Br J Haematol 1955; I: 352-7.
17. Hallberg L, Solvell L. Absorption of a single dose of iron in man. Acta Med
Scand 1960; 358 (Suppl.): 1 H 2 .
18. Ekenved G. Serum iron increase as a measure of iron absorption-studies on
the correlation with total absorption. Scand J Haematol 1976 Suppl. 28: 31-63.
19. Heinrich HC, Fischer R. Correlation of post-absorptive serum iron increase
and erythrocyte T e incorporation with the whole body retention of absorbed
Y e . Klin Wochenshr 1982; (0: 14934.
20. Kuhn IN, Monsen ER, Cook ID, Finch CA. Iron absorption in man. J Lab Clin
Med 1971; 71: 715-21.
21. Charlton RW, Lynch SR, Bothwell TH. Proteins of iron metabolism. New
York: Grune and Stratton, 1977 387-92.
22. Cook ID, Lipschitz DA, Laughton EMM, Finch CA. Serum ferritin as a measure
of iron stores in normal subjects. Am J Clin Nutr 1974; 27 681-7.
23. Janghorbani M. Young VR. Advances in the use of stable isotopes of minerals
in human studies. Fed Proc Fed Am Soc Exp Biol 1982; 41: 2702-8.
24. Brise H, Hallberg L. Absorbability of different iron compounds. Acta Med
Scand 1962; 171 (Suppl. 376): 23-38.
25. Moore CV, Dubach R, Minnich V, Roberts HK. Absorption of ferrous and
ferric radioactive iron by human subjects and by dogs. J Clin Invest 1944; U:
75-7.
26. Martinez-Torres C, Layrisse M. Iron absorption from veal muscle. Am J Clin
Nutr 1971; U: 531-40.
27. Cook ID. Layrise M, Martinez-Torres C, Walker R. Monsen ER. Finch CA.
Food iron absorption measured by an extrinsic tag. J Clin Invest 1972; 51:
805-15.