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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. 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