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Anal. Chem. 1980, 52, 216-218
(6) C. C. Schnetzler, H. H. Thomas, and J . A. Philpotts, Anal. Chem., 39, 1888 (1967). (7) R. E. Honig and H . 0. Hook, RCA Rev., 21 (3), 363 (1960).
RECEIVED for review, August 9, 1979. Accepted October 15,
1979. This research was performed on equipment on loan from NASA/Goddard Space Flight Center under agreement No. GSFC 77-5(E). The support of research grants NSG 9071 from NASA and EAR 79-06950 from the National Science Foundation is gratefully acknowledged.
Stable Tracer Iron-58 Technique for Iron Utilization Studies J. J. Carni and W. D. James" Research Reactor Facility, University of Missouri, Columbia, Missouri 652 1 1
S. R. Koirtyohann Environmental Trace Substances Research Center and Department of Chemistry, University of Missouri, Columbia, Missouri 652 1 1
E. R. Morris Nutrition Institute, United States Department of Agriculture, Beltsville, Maryland 20705
Iron is the element most commonly deficient in humans. I t has been estimated that more than 5 million adult women in the United States have iron deficiency anemia, even though typical diets contain several times the quantities of iron necessary to maintain balance (1). The deficiency is prevalent among women of child-bearing age. In early studies (2, 3) of iron absorption in infants, absorption efficiencies, especially in babies less than 3 months old, differed markedly from those in older children. More recently it was shown that fortification of infant foods with iron in a form that was not absorbed served no useful purpose and that further study of the absorption of iron by infants was essential for prevention of iron deficiency ( 4 ) . Studies of the bioavailability and absorption of dietary iron are clearly warranted. Currently the common practice in the study of iron absorption is the application of a radiotracer. Oral or intravenous doses of radioiron (55Feor 59Fe)followed by a delay of about two weeks and a count of nuclear radiation emissions from a blood aliquot can provide useful information regarding gastrointestinal absorption of iron and its red blood cell incorporation efficiency. Unfortunately the use of a radiotracer is difficult to justify for infants, pregnant women, and other radiation-sensitive subjects. This is especially true for experimental studies when the individual's medical status does not warrant the absorption evaluation. In 1963 Lowman and Krivit (5)used the stable iron isotope, 58Feas an in-vivo tracer to determine plasma clearance. The tracer 58Fein the plasma samples was quantified by neutron activation analysis and NaI(T1) y spectroscopy. An attempted extension of this study to red blood cell incorporation proved futile because the small quantities of 58Fetracer could not be determined in the presence of the much larger quantities of the isotope in natural iron. Later Dyer and Brill (6) and King et al. (7,8) overcame that problem by using higher resolution, solid state y detectors and successfully determined iron utilization parameters in women. However, both of these studies were primarily concerned with the application of the technique and did not document precision and accuracy characteristics of the method. T h e limited use (6, 9) of the 58Fe stable isotope tracer technique for iron utilization studies reflects the need for thorough evaluation and documentation of the capabilities of the method. Calculations show that 10% utilization of a 5-mg tracer dose of 58Feby an adult human would result in a maximum relative change in %Fe of about 7.5% in the blood. Since this measurement requires the determination of total iron for the purpose of subtracting natural 58Febackground, 0003-2700/80/0352-0222$01 OO/O
in addition to the 58Fe determination, the precision requirements are obviously high. In this investigation, analytical techniques have been developed to measure both with a high degree of confidence (*1-3% relative). These techniques have been applied to multiple analyses of a single whole blood sample to evaluate reproducibility. Experiments have been performed to determine the accuracy with which spike material can be recovered.
EXPERIMENTAL Apparatus. In this work, the neutron activation analysis facilities at the University of Missouri's Research Reactor were
used. Samples were encapsulated in cleaned quartz irradiation vials fabricated from TO8 quartz obtained from Heraeus Amersil Co. Samples were irradiated for 50-60 h in a 1-inch reflector position having a thermal neutron flux of n cm-* s-'. Gamma emissions were detected and quantified with an Ortec VIP series Ge(Li) detector (2.2 keV resolution @ 1332 keV) and a Nuclear Data 4410 multichannel pulse height analyzer. A Perkin-Elmer 576 spectrophotometer was used for the colorimetric determination of total iron. Reagents. Spiking solutions enriched in 58Fewere prepared by dissolving Fe203(73.26% 5sFe)in HCl. The enriched iron was obtained from the Isotope Sales Division of Oak Ridge National Laboratory. All comparisonstandards were prepared by a similar dissolution of high purity (99.999%)iron oxide obtained from Spex Industries, Inc., Methuen, N.J. Collection of Blood Samples. Samples were drawn from healthy males directly into heparinized containers and were immediately frozen and stored at -20 "C. One sample was thawed, subdivided into some 100 aliquots and refrozen. The sample was kept well mixed during the aliquoting procedure by frequent shaking; aliquots were considered t o be identical homogeneous portions of the sample. These aliquots were designated and used as a reference standard. A second sample was drawn, thawed, and subdivided in Beltsville, Md., by one of us (E.R.M.) who was not involved in the actual analysis of the samples. Carefully measured quantities of enriched 5sFewere added to these aliquots. These samples along with a portion of the spiking solution were refrozen and transferred to the analytical facilities in Columbia, Mo., for analysis as blind spiked samples. Selection of Total Iron Measurement Technique and Precision Evaluation. Atomic absorption analysis and colorimetry were considered as possible analytical techniques for total iron. Either technique provided adequate sensitivity so we compared their reproducibilities with instruments available. Precision was superior with a modified 1,lO-phenanthroline-iron complex colorimetric technique (10) and a Perkin-Elmer 576 Spectrometer. Two-hundred microliters of each sample was transferred t o a 250-mL Erlenmeyer flask containing a "OB1979
American Chemical Society
52, NO. 1, JANUARY 1980
HCIOl mixture digested in a microwave oven (11). The sample mixture and digestate were heated to dryness, then dissolved in HCl, buffered with sodium citrate, reduced with 1% hydroquinone, and complexed by the addition of 5% 1 , l O phenanthroline. After the delay of 6-8 h, absorbancies were measured and compared to appropriate standards. Neutron Activation Analysis for Total 5sFe. When 58Fe (0.3% of natural iron) is exposed t o thermal neutron irradiation, a radioactive species is produced by the nuclear transformation, 68Fe(n,y)59Fe.This species can be used to measure the quantities of 58Fepresent in blood samples. For each determination,three or more 400-pL replicate portions of the thawed blood sample were pipeted into cleaned quartz vials. Forty yL of a standard cobalt solution (10 pg/mL) also was added to the vials as a flux monitor. All additions were quantitative by weight. These vials along with similarly prepared blanks and iron standards were lyophilized and irradiated. Samples were held for 2-3 weeks to allow for the reduction of shorter lived matrix activities. Each vial was opened and crushed in a 250-mL Erlenmeyer flask and the sample solubilized using the HN03-HC104, microwave oven digestion procedure. After heating to dryness, the sample was dissolved with 25 mL of 6 N HC1. A 20-mL portion was then transferred to a plastic scintillation vial for Ge(Li) y spectroscopicevaluation of the induced 59Fe. For determination of @Fe,spectral peak areas obtained in the 1292 keV y ray of 59Fe during a 2000-s counting period were compared to those obtained for standards. These peak areas were generally in excess of 20000 counts, indicating that statistical errors in counting were less than 1%. Analyses of Bloods Spiked with These experiments were designed to simulate the analysis of bloods taken from subjects who had received 58Feoral doses. Both total iron and 6sFe were determined. The spiked samples were prepared to simulate a wide range of iron absorption. Total 5sFeadded varied from 1.1-10 pg per sample, which represented a relative increase of 58Fe over natural levels of 15-140%. R E S U L T S A N D DISCUSSION Calculations. Routine comparator standard calculations were employed in the determination of total iron by spectrophotometric or activation techniques. Excess 58Fe per gram blood over natural levels, which would represent the quantity of spike or dose present in the sample, was calculated with the following equation:
5sFe- 0.0033(Fe total - 58Fe excess) = 58Feexcess
(1)
where 58Fe is the total concentration of the isotope in the sample as measured by neutron activation analysis, 0.0033 is the isotopic mass ratio in natural iron of 5BFeto total Fe, Fe total is the concentration of the element in the sample as measured by spectrophotometry and 5sFe excess is the concentration of the isotope in the sample in excess of natural background levels. Percent iron utilization of dose is calculated as follows: % F e utilization =
bg 58Feexcess/g blood pg 58Fedose
X
TBM (g)
100 (2) where Total Blood Mass (TBM) is determined from a total X
blood volume estimate and measured density, and 58Fedose is the quantity of the isotope in the dose as determined by neutron activation analysis. Some uncertainty exists in the value of the isotopic mass ratios for iron (12,13). The uncertainty does not enter directly into the determination of iron utilization by this method. The isotopic ratio for 58Feis used in the calculation of 5sFe present in the administered dose as well as the total iron and 58Fein the blood sample. Therefore, small errors due to the value used will cancel out. Precision Evaluation. The colorimetric technique for total iron was used for 33 determinations of iron in the reference standard over a time period of about 2 years. Total
440
217
I
Flgure 1. Multiple iron analysis of reference standard
Table I. Normalized and Unnormalized Comparisons of Standard Results to Constant Flux
standard number
unnormalflux flux ized normaliza- normalized result, tion result, counts/pg factor counts/pg
2 3
39200 38200 37200
mean std. dev., %
38200 2.62
1
0.927 0.888 0.814
42300 43000 42500 42600 0.88
iron concentration also was determined repeatedly by neutron activation. Figure 1 details the variability of the results of these analyses. Each point represents the mean of triplicate analyses in one neutron activation irradiation can or colorimetric run and the error bars represent the standard deviation of their distribution. A value of 486 f 9 pg total iron per gram of blood was derived f r o y t h e mean of those determinations. Relative standard deviations of replicate analyses in the same run averaged 0.9 and 1.5% for the colorimetric and activation measurements, respectively. The standard deviations for all runs, calculated from individual means, were 2.0 and 1.7% respectively. It can be seen from Figure 1that error bars from all measuTements by both techniques lie within the limits of f5?4 relative deviation from the mean. With careful laboratory procedures, a total error of not larger than 3% is routinely possible by each technique. The combination of errors from both techniques in the final result computed by subtracting background 58Fefrom total 58Fe would then be
