Isotopic Methods in the Study of Mineral Metabolism of Infants with Special Reference to Stable Isotopes

Home , Isotopes of selenium

Trace Elements in Nutrition of Children, edited by R. K. Chandra. Nestle Nutrition, Vevey/Raven Press, New York €> 1985.

Morteza Janghorbani, Vernon R. Young, and *Richard A. Ehrenkranz

Massachusetts Institute of Technology, Cambridge, Massachusetts 02139; and *Yale University School of Medicine, New Haven, Connecticut 06510

Of the 90 naturally occurring chemical elements, 26 have been suggested as essential for animal life (1). In addition to these, a number are of toxicological importance (e.g., Pb), and some others are of interest because of their special properties (e.g., as components of therapeutic agents). A summary of the biological significance of these elements is given in Fig. 1. The listed elements are mineral elements (2) (in contrast to C, H, O, and N). Fomon considered all except Na, Cl, K, Ca, P, Mg, and S as trace elements (2). Because of the potential importance of the isotopic techniques in the study of mineral metabolism in general, and especially in relation to the emerging importance of mineral nutrition of the premature infant, we discuss the isotopic techniques in relation to the broad category of minerals. Examination of the literature on the subject of trace elements in relation to human development indicates many gaps in the fundamental aspects of their metabolism (2-5). Although there are some limited data on various aspects of Cu, Fe, and Zn nutriture in infants and children, major questions remain for many features of their metabolism, especially with respect to premature infants (5,6). Even less is known about other trace elements (e.g., Se) in the human infant. Furthermore, available information on such important fundamental issues as body composition during development is either very fragmentary (7) or completely lacking (3). Requirements for low-birth-weight (LBW) infants have been, by and large, based empirically on composition of human milk in combination with balance studies (8). Investigation of many aspects of mineral metabolism requires the use of suitable isotopic tracers (9). Radiotracers have of course made a major impact in our understanding of mineral metabolism in animals (10) and have been and continue to be employed extensively for medical diagnostic purposes in man (11) and fairly extensively in the study of mineral metabolism in adults (especially the sick) (12). In infants, radiotracers have been used for such studies as measurement of RBC 63 64 STABLE ISOTOPE METHODS

r Li Be B F O y Na Mg Al Si P Cl

X y ox K Co Ti V Cr Mn Fe Co Ni Cu Zn As Se Br 0 0 X V Rb Sr Mo Ag Cd In Sn Sb I O 0 X o Cs Ba La W Hg Pb

V I ESSENTIAL EH] INTERACTIVE FUNCTIONS F=| TOXIC f~\ THERAPEUTIC AGENT

FIG. 1. Biological significance of minerals. The elements marked with (x) have potential as nonabsorbable markers. The chart was constructed from various sources (1,2). survival using 51Cr-labeled RBCs (13) or for issues related to blood loss employing either 51Cr- or 59Fe-labeled RBCs (14,15). However, their application to the study of mineral metabolism has again been limited (16), undoubtedly partly due to unwillingness to expose the infant to internal radiation. More recently there has been limited application of stable isotope methodology to selected aspects of mineral metabolism in the pediatric group (17-19). Chromium-50-labeled RBCs have been used to study RBC survival time, although this does not fall strictly within the realm of mineral metabolism (14). Limited use has been made in relation to calcium absorption studies (18) and the bioavailability of zinc in neonates (19). Considering the obvious need for a much better understanding of all aspects of mineral metabolism especially during the perinatal period and early infancy, the general requirement for safe and noninvasive tracer methods which avoids issues of internal radiation exposure, and the present state of knowledge and development of stable isotope tracers (Table 1), it is timely to ask the following question: What can stable isotopes do in the realistic setting of a pediatric study, and how should they be used? In this chapter we address this question.

ISOTOPIC CONSTITUTION OF MINERALS A chemical element is characterized by the invariant number of protons in its nucleus. However, the number of neutrons present may vary, giving rise to different isotopes. The isotopic pattern is characteristic of a chemical element (Fig. 2) (19a). Depending on the innate nuclear stability of these isotopes, some are unstable (radioisotopes) and others are stable (stable isotopes). In general, the naturally occurring element consists of one or more stable isotopes present in a characteristic, and in most cases constant, ratio. The isotopic constitution of minerals has been discussed in detail previously (9). Figure 2 summarizes the isotopic characteristics of the minerals of interest to the present discussion. For each element, all stable isotopes as well as radioisotopes of potential usefulness in metabolic tracer studies have been included. STABLE ISOTOPE METHODS 65

TABLE 1. Some important characteristics of stable isotope tracers

1. They are nonradioactive a. Radiation exposure is not an issue b. They do not decay so that samples can be processed when convenient c. They do not permit external organ monitoring 2. They are safe a. They are natural constituents of foods b. They do not pose problems of radiation exposure 3. They permit multiple-labeling studies a. Several labels (of different minerals) can be given simultaneously, allowing studies of mineral-mineral interaction b. Multiple isotopes of the same mineral can be given simultaneously (e.g., intravenously and orally or two separate dietary pools) c. Longitudinal studies may be done in the same individual, allowing such studies as gastrointestinal maturation versus age 4. They permit studies related to group comparisons (e.g., dynamics of metabolism of selenium in infants versus adults and the elderly) 5. They permit field studies (e.g., investigations under home feeding conditions) 6. They permit investigations related to food processing under realistic field conditions

From Fig. 2 it becomes clear that some elements consist of only a single stable isotope (Na, P, Mn, Co, I), and for these elements stable isotope tracer methods do not apply. The remaining elements occur either as two (Cl, Cu) or multiple stable isotopes. In principle, then, the stable isotope method is applicable to Mg, S, Cl, K, Ca, Cr, Fe, Ni, Cu, Zn, Se, Br, and Mo. It should be noted at this time, however, that the natural isotopic abundance varies over a wide range (e.g., ^Ca, 96.97%; ^Ca, 0.0033%) and that this introduces an important experimental parameter with respect to the optimum design of human metabolic studies, from the point of view of optimization as well as isotope cost (20-25). The radioisotopes included in Fig. 2 have been limited to those possessing a radioactive half-life of greater than 10 hr, as a shorter half-life generally would limit the value of an isotope for human metabolic studies. Furthermore, radioisotopes are, of course, of limited use in relation to studies with children, and their application can be justified only under very restricted circumstances. Stable isotopes possess several important characteristics whose understanding in relation to isotopic studies in children is a prerequisite and must be taken into account carefully. A wide variety of studies can be carried out with stable isotopes, but the experimental requirements are specific to the individual mineral and the issues to be investigated, as is discussed in the following sections.

ANALYTICAL METHODOLOGY OF STABLE ISOTOPES In contrast to radiotracers, accurate measurement of stable isotopes in the fluids relevant to these studies (feces, urine, blood) is a relatively complex task (9) involving issues of trace analysis and stable isotope measurements. Two methods of analysis are applicable: neutron activation analysis (9) and mass spectrometry (26). Here we discuss only briefly the main characteristics of these two techniques 3 xlO3y ' 740

!20rJ

50 54 Ca 20 9697 0,64 0 145 2 06 163d 0.0033 0.185 B- T K 43 rT308 48 19 93 15 0,012 6 84 I2.4h

Cl 39 y 1460 42 17 75 57 3.0xl05y 24 42

5 V 37 16 95 0 0 760 4 22 872d 0 014 B- ve stable isotope olope 34 36 P f h 15 100 14 3d 25.3 d chemical j B- symbol 31 33 V 1 uf o 1 ob Mg f 11 2 6Oy 100- 12 76 70 II 20 (a om %) 0,15 21 1 h / M35O tomic n 22 23 — ss no 24 28 V 100. 22 23 Mass No — i mode of decoylfl* posilror , 0" beia minus, EC Electron capture , I T Isometric Tronsitiont FIG. 2. Isotopic constitution of selected minerals (19a). STABLE ISOTOPE METHODS 67 in specific relation to accurate stable isotope analyses of materials derived from pediatric studies.

Neutron Activation Analysis Neutron activation analysis has been described in detail for stable isotopes of Ca (21), Zn (20), Fe (27), Cu (28), and Se (22). The method can measure absolute contents of selected stable isotopes with precision and accuracy in the range of 1 to 10% (29). It is capable of relatively large sample throughput. It has been successfully applied to the measurement of absorption of Ca (18) and Zn (19) in neonates. The major limitations of the method relate to its inability to measure all stable isotopes (9). For example, of the five stable isotopes of Zn, neutron activation analysis can measure three ("Zn, 68Zn, 70Zn) (30). For copper both can be measured (28), and for iron 54Fe and 58Fe have been measured simultaneously (31). Of the calcium isotopes, ^Ca and 48Ca have been measured, whereas for Se three stable isotopes (74Se, 76Se, 80Se) are routinely measured (22). In some instances this technique can measure only a single stable isotope (e.g., 26Mg and 41K), and its application to stable isotope methodology must then be coupled with an elemental analysis method (e.g., atomic absorption spectroscopy). The second potential limitation of neutron activation analysis is its practical inability to achieve measurement precision lower than about 1% (9). The need for such precise measurements may arise in relation to long-term kinetic studies for which residual enrichment of the isotope in a particular matrix (e.g., plasma or urine) is low. For instance, in the study of plasma zinc turnover, it has been shown (32) that neutron activation analysis can be effectively employed to follow plasma appearance curves after single oral administration of physiological doses to human adults for as long as about a week. However, beyond that point the residual enrichment cannot be accurately measured with the precision inherent in this technique. In the studies related to the gastrointestinal tract, this is usually not a limiting factor so that this technique is well suited for this purpose.

Mass Spectrometry There has generally been an unfortunate misconception with respect to the use and capabilities of mass spectrometry in regard to accurate analyses of stable isotopes of minerals in human-derived materials. Of the various mass spectrometers available, electron bombardment coupled with chelate mass spectrometry has proved ineffective for this application (33). This has been due to problems in relation to preparation of suitable chelates, basic limitations due to overlapping of minor isotopes of C, H, and O (34), and poor precision of the method (33). On the other hand, thermal ionization/mass spectrometry (TI/MS) is capable of a high degree of precision in the measurement of isotope ratios (35), but unfortunately it currently suffers from major problems associated with sample preparation and throughput (35). It has been employed to a limited extent, however, for the measurement of Zn and Cu (33,36) in absorption studies in the human adult. Although of potentially 68 STABLE ISOTOPE METHODS major application, its routine use remains to be fully exploited and must await resolution of practical problems related to accurate analysis of absolute isotope contents for a relatively large number of samples. A recent development, coupling of a quadruple mass spectrometer with inductively coupled plasma (ICP/MS), may prove of major potential value for this area of research, as it now appears that this technique could overcome the sampling problems inherent in the present-day TI/ MS. However, this may, at least, occur initially at the expense of reduced precision so that unless new developments take place achievement of a high degree of precision in isotope ratios will still be left to the TI/MS methodology. It should be emphasized at this point that application of either neutron activation analysis or any form of mass spectrometry requires considerable chemical manip- ulation of the sample, as both methods inherently suffer from various types of interferences. In the case of mass spectrometry, however, it should additionally be emphasized that in its traditional application the method can measure only isotope ratios (in contrast to absolute quantities). Because metabolic studies, in general, require measurement of absolute quantities of stable isotopes, mass spectrometric data are by necessity coupled to an elemental analysis technique (33,36). This required coupling largely negates the major inherent feature of TI/MS, i.e., the high degree of precision, because the overall precision of the coupled measurements determines the usefulness of the technique. In some cases, where the requirement for absolute quantities does not exist, mass spectrometry alone can be an effective tool. Isotope dilution principles can of course be applied to eliminate the additional need for an elemental analysis capability, but this is applicable only to situations where two isotopes of unaltered natural abundance can be measured in the in vivo enriched human-derived material. This limits application of isotope dilution for such cases as Cu, which has only two stable isotopes and in many other studies involving multiple in vivo labeling (25). It can be argued, however, that redundant measurements (before and after in vitro spiking) can be applied even in the case of in vivo enriched samples of Cu, but this of course requires twice the number of analyses, which relates to the major difficulty with TI/MS, i.e., sample throughput. The above brief critique of the two analytical methods is meant to put the problem in a realistic perspective. Obviously, both neutron activation analysis and mass spectrometry have been and will continue to be successfully employed for this application. New developments might well be forthcoming to permit major advances in the practical usefulness of these techniques.

AREAS OF APPLICATION Stable isotope methods can potentially be employed in a wide range of tracer studies. It is our intent to set forth some of these applications in this section. These methods can provide pediatric research with major experimental capabilities not available otherwise. It should also be pointed out, in all fairness, that stable isotopes cannot permit such in vivo dynamic studies as body counting (whole or regional), and these capabilities are left to the domain of radiotracers. STABLE ISOTOPE METHODS 69

In this section we deal with what can be done in principle. Many of the suggested applications, especially in relation to the dynamics of body composition, have not been carried out previously and despite the inherent safety of stable isotopes must still be proved experimentally. We discuss the materials of this section under two general headings: fecal (isotope) balance procedures and body compositional studies.

Fecal (Isotope) Balance Procedures

Studies related to the issues of gastrointestinal absorption and endogenous secretions of minerals are of fundamental importance not only in relation to dietary management but also for a more complete understanding of gastrointestinal function in relation to development. Little is known about the quantitative aspects of the two fundamental processes contributing to the net fecal excretion of minerals in the pediatric group (3): (a) unabsorbed dietary sources; and (b) sources of endogenous origin. Because of the significance of gastrointestinal tract in the overall homeostatic regulation of many minerals (e.g., Fe, Zn, Ca) and the issues related to the state of maturity of gastrointestinal function, especially in LBW infants (37), direct measurement of absorption and endogenous secretion of minerals is an important experimental capability. Thus the stable isotope approach can provide a significant methodology for this purpose. Measurement of the extent of endogenous secretions into the gastrointestinal tract has traditionally been carried out by appearance of radiotracer in stools following injection of an appropriate preparation (38). This method is not suitable for use with infants and children because of the issues of radiation exposure, the need for venipuncture (especially for LBW infants), and of course the underlying assumption that injected tracer is excreted in a manner identical to that of the native mineral. An alternative approach, avoiding the intravenous route completely, is possible using the stable isotope balance method. To understand this, we have employed the simplified scheme given in Fig. 3. In this scheme, a known quantity of an appropriate dose of the enriched stable isotope (shown as the small box in the intake pool, Fig. 3) is administered during a well-defined balance period during which the intake of the mineral of interest is accurately monitored (by use of appropriate markers). The mineral of interest and the enriched preparation enter the preabsorptive pool of the mineral, which also contains the endogenously secreted component (here shown as three separate entry points to indicate continuous secretion along the length of the gastrointestinal tract). A fraction of the mineral from the preabsorptive pool is then absorbed, and the remainder appears in the fecal pool defined by the two diet markers. If we assume that fractional absorption of the mineral from the preabsorptive pool is independent of its origin, i.e., the absorptive process cannot distinguish among the endogenous and dietary sources of the mineral, the following relationship applies: 70 STABLE ISOTOPE METHODS

INTAKE

Gl TRACT

FECAL POOL

FIG. 3. Simplified model illustrating the experimental methodology of fecal isotope balance (see text for explanations).

Af.i = Ao>, i(l - F) ^r- A* -F) -f Ae i(l - F) = (1) A,,2 Ao,2 .(1 - F) -<- Ao,2,2(1 -F) -h Ae,2(1 - F)

In this set of equations (Eq. 1), Af, and Af>2 are quantities of each isotope of mineral recovered in the fecal pool; AOil-1 and AO)2il are the corresponding isotopes in the diet (the ratio of AoAJIAo2t\ is known as the natural abundance ratio); A*o , 2 and Ao 2 2 are the two isotopes in the enriched preparation (the asterisk indicates that the preparation is enriched with respect to isotope 1); and Aea and Ae 2 are the two isotopes originating from endogenous secretions (Ae 1/Ae2=Ao,M/Ao21). Be- cause the ratio Ae^/Ae_2 is known, the only unknowns are F (fractional absorption) and Ae (endogenous secretions before reabsorption takes place). Thus solution of the set of equations in Eq. 1 yields data directly on fractional absorption of the mineral from the preabsorptive pool and the absolute magnitude of endogenous secretions (prior to reabsorption). Application of this method to the measurement of mineral absorption and endogenous secretions involves several assumptions whose validity is briefly discussed below.

1. Absorption from a common pool: The assumption is made that absorption takes place from a common pool. The validity of this assumption has been investigated in relation to adult nutrition for Fe (39), Zn (25), and to some extent Se (40), at least as far as the dietary sources of the mineral are concerned. It is now STABLE ISOTOPE METHODS 71 accepted that all nonheme sources of iron form a common pool, whereas heme iron is absorbed via a different mechanism (39). The limited work that has been carried out with Zn also seems to indicate that in the rat model (41) and perhaps also in the human (25) food zinc (intrinsic tag) forms a single pool with inorganic zinc (extrinsic tag) prior to absorption. The available data for selenium are less conclusive partly because of the complexity of selenium biochemistry resulting from the multiple oxidation states and the labile nature of many selenium com- pounds (40). Work with biologically labeled foods employing the stable isotope method indicates that when absorption of selenium from chicken meat is compared with an extrinsic selenite tag given simultaneously the former is more completely absorbed (40). However, when the biologically labeled food is either egg white or egg yolk, absorption is identical between the food Se and the Se from selenite (42). Thus because of the critical nature of this assumption, significant further work is needed before the issue for selenium is satisfactorily resolved. In relation to infant foods, we are unaware of any work focusing directly on this. The validity of the commonality of absorption between the endogenously secreted mineral and that originating from the diet has not been investigated as far as we are aware. However, even if the assumption is not valid, the parameter At (1 - F) corresponds to the net amount of unabsorbed mineral of endogenous origin which appears in the fecal pool. Thus its magnitude should be directly proportional to the quantity of the endogenous secretions into the preabsorptive pool. When estimating the accuracy with which endogenous secretions can be determined employing the present proposed concept (Eq. 1), it is important that the amount of endogenous contribution to the fecal pool not be greatly different from that derived from unabsorbed dietary sources. Accurate information on the relative contributions to the fecal pool of the diet and endogenous sources of minerals is not available for adult man or for infants and children. Extrapolations based on limited available data in the human adult (43—46) indicate that for such minerals as Ca, Zn, Cu, Fe, and Se the endogenous component of gastrointestinal content is similar in magnitude to the dietary contribution. If this is correct, the proposed approach provides an accurate means for directly measuring both sources of mineral. This in turn permits evaluation of many possible factors contributing to homeostatic control of mineral nutrition at the gastrointestinal level. 2. Transit times and reentry. Two important issues regarding the accurate measurement of mineral absorption employing the stable isotope approach are related to the gastrointestinal transit time of the ingested label and the reentry of the absorbed dose. The former determines the length of time required for complete collection of stools inclusive of all the unabsorbed isotope and is a critical experimental parameter for accurate estimation of absorption, whereas the latter determines the magnitude of underestimation in the value of true absorption if reentry is neglected. Typical data for two infants given single oral doses of 70Zn and ^Ca simultaneously (19) are presented in Figs. 4 and 5. These data indicate a number of important similarities as well as differences between these two minerals. First, as might be expected, transit time appears to be very similar, with about 50 hr as 72 STABLE ISOTOPE METHODS

°/o EXCRETED =26.3 F=73.7%

Y = 26.288 +.0009551 (r2 =.117)

°/o EXCRETED =10.96 Co = 89.0% = 10.9594 +.022795t

I I I I I 0 10 20 30 40 50 60 70 80 90 100 TIME (H) FROM ADMINISTRATION OF DOSE

FIG. 4. Fecal excretion plots for 7°Zn and ^Ca administered simultaneously to a premature infant, C8/17 (19).

sufficient length for complete collection of unabsorbed isotope. We have explored this issue in additional subjects for 70Zn, and our data (19) permit us to conclude similarly that within 50 hr the unabsorbed dose appears in the stools. Based on these data and the knowledge that limited overpooling introduces no undesirable problems in the preparation of fecal pools, we recommend a 72-hr collection from the time of administration of the dose (19). Additionally, our observations of relative transit behavior in adults for stable isotopes of Fe (27), and Se (22) lead to similar conclusions for these two minerals also. An important difference between the data given in Figs. 4 and 5 for Zn and Ca is in regard to the reentry of the absorbed dose into the gastrointestinal tract. The data clearly show a positive slope in the cumulative fecal excretion curve for 46Ca after the unabsorbed isotope has been eliminated, amounting to about 0.5% of the administered dose per 24-hr period, whereas for 70Zn the slope is insignificantly small. This is also consistent with the known behavior of these minerals (38,47). The significant reentry of ^Ca introduces the issue of the relative error (underestimation) introduced in the measurement of true gastrointestinal absorption should this effect for the initial 72 hr be neglected. (Correction requires isotopic analyses of individual stools as in Figs. 4 and 5 which is not practical for routine studies.) Our estimates of absorption of 46Ca in three infants by both methods [method 1: single pooling for 72 hr; method 2: linear extrapolation to zero transit time (47)] are summarized in Table 2. The data clearly show that despite the consistent systematic underestimation using method 1 the error is only 2% of the true value STABLE ISOTOPE METHODS 73

.OO2835t = .691)

19.703 + .02039 t (r2 = .984)

I I I 10 20 30 40 50 60 70 80 90 100 TIME (H) FROM ADMINISTRATION OF DOSE

FIG. 5. Fecal excretion plots for 7°Zn and 46Ca administered simultaneously to a premature infant, S7/20 (19). of absorption. Thus it appears to us that the simple pooling method should provide a sufficiently accurate estimate of true absorption for Ca (and certainly for Zn and Fe) and that the more elaborate method is not justified. We have made similar observations for absorption of Se in adults whose reentry can also be observed (48). Whether these conclusions apply to the case of Cu remains to be established, the uncertainty being related to the known significant reentry of absorbed Cu via the bile. It should be mentioned here that reentry of the absorbed dose is not quantitatively the same as the amount of endogenous secretions, as this equivalency requires isotopic equilibration in the body pools responsible for endogenous secretions. This is in fact a major reason for the observed negligible reentry of the absorbed 70Zn label (Figs. 4 and 5) in contrast to the known significant endogenous contribution of this mineral via the pancreatic and intestinal secretions. 3. Issues related to the required dietary enrichment. Stable isotopes are natural constituents of foods and body fluids so that proper use of stable isotope methodology requires an understanding of the required degree of dietary isotopic enrichment. We have discussed this in detail for the adult population for Zn (30), Se (22), and Ca (21). The amount of the enriched isotope required to permit satisfac- 74 STABLE ISOTOPE METHODS

TABLE 2. Comparison of two methods of determining gastrointestinal absorption of ^Ca in premature infants

Percent absorption Infant code Method 1a Method 2"

C8/3 86.0 87.1 C8/17 86.9 89.0 S7/20 78.5 80.3

•Simple 72-hr pooling. bExtrapolation method of Lutwak (47).

TABLE 3. Relationship between recommended dietary allowances (RDA) and the required isotope enrichment for a 2-month-old infant

Cost of RDA Stable isotope isotope/feeding Mineral (mg/day) and level (dollars) Ratio label/RDA

Ca 330 «Ca, 54 M-g 230 0.00016 Mg 50 ^Mg, 28 mg 50 0.56 Fe 1.2 ^Fe, 19 ^g 1 0.016 Zn 3.0 ™Zn, 100 n-g 3 0.033 MZn, 3 mg 6 1.0 Cu 0.4 ^Cu, 0.62 mg 0.2 1.6 Se 0.05 74Se, 2.2 jxg 0.5 0.044 76Se, 22 (jig 0.1 0.44 tory estimation of gastrointestinal absorption and endogenous secretions depends on a number of parameters, including its natural abundance, the dietary intake, the precision of isotopic analyses, and whether the enriched isotope can be given as a single dose or spread over several consecutive feedings. In the simple case of a single feeding with an isotope whose natural abundance is relatively low (e.g., 70Zn, 58Fe, 48Ca, or 46Ca) an adequate isotope enrichment is about five times its daily intake (20-22). This has been summarized for a number of minerals for a typical 2-month-old infant (Table 3). This is only a small fraction of the daily intake of some of the minerals (e.g., 46Ca, 58Fe). Therefore if other issues related to the use of "extrinsic tag" do not preclude the method (25,40), administration of the label will not influence the total level of the infant's mineral intake. For some others, however (e.g., 65Cu, 26Mg, ^Zn), administration of the required amount adds significantly to the day's intake of the mineral, and this might be inadvisable. Should this be the case, the required dose can be spread over several days' feeding. Alternatively, if it is possible to improve the precision of isotopic measurements, a smaller dose of the isotope could be effectively utilized. STABLE ISOTOPE METHODS 75

Some Applications of Fecal Isotope Balance Procedures As indicated earlier, application of fecal balance with stable isotopes is in relation to the measurement of absorption and endogenous secretions. The majority of investigations examining absorption of minerals in infants and children to date have been based on the mineral balance procedure. The recommendation of mineral intakes are also generally based on these data (3,5,8). Thus little is known about the components of a net fecal balance (true absorption and endogenous secretion) and factors influencing their behavior. This is a complex problem because many dietary-host factors (49) influence their magnitudes. In particular reference to the premature infant, the degree of maturity of the gastrointestinal system is known to influence fat absorption (37), but its effect on minerals has not been studied (2,19). Additionally, functional maturity of such organs as liver and pancreas, whose secretions are known to influence absorption of other nutrients and which undoubtedly contribute to varying degrees to the endogenous component of the preabsorptive pool, might exert significant modulating influence on both absorption and endogenous secretions of the relevant mineral (e.g., liver for Cu and pancreas for Zn). Availability of the stable isotope methodology provides a powerful experimental tool for studies that directly investigate absorption-secretion in relation to maturity of the gastrointestinal system. Furthermore, this might be worthy of special note because such studies can be repeated longitudinally in the same individual (unlike with radiotracers) and/or simultaneous multielement investigations carried out. We have investigated absorption of Zn and Ca employing 70Zn and ^Ca as the "extrinsic tag" in a number of premature infants. The data for zinc have been described previously (19), but some examples from this work might be usefully illustrative here and have been summarized in Table 4. In this study an aqueous 70 46 solution of ZnCl2 and CaCl2 was administered intragastrically with a single bolus gavage feeding of the indicated diet. The data illustrate the relatively high absorption of the extrinsic tag in comparison with that in human adult males and employing the same methodology (50) (Table 5). In the human young adult, absorption of the extrinsic tag varied from 30 ± 3% when soy isolate provided the sole source of dietary protein to 41 ± 4% for milk. In the infant study, the range of mean values for the three diets was 58.0±6.1% to 68.6 ±4.0%. Although detailed comparative interpretations are not possible at this time because of the many confounding issues related to gastrointestinal absorption between infants and adults, they do point out that a unified methodology is at hand to address issues of comparative gastrointestinal handling of minerals in various age groups. In a preliminary way these data may also be indicative of a more effective absorptive mechanism in the premature neonate for zinc as compared to the young adult. This is consistent with the theoretical requirements (2). Our data for absorption of 70Zn in premature infants, although not definitive because of some needed refinements, do seem to indicate a general lack of corre- lation with postnatal and postconceptual age as well as body weight and the nature TABLE 4. Absorption of 70Zn "extrinsic tag" in premature infants*

Gestational age Postnatal age Postconceptual ™Zn absorption Diet" No. (weeks) Birth wt (g) (days)0 age (days)c Body wt (g)c (%)

PTHM 29.0 ± 0.7 1,102 ± 115 34.8 ± 5.9 239 ± 2 1,431 ± 54 68.4 ± 4.6 Fortified PTHM 30.5 ± 0.6 1,192 ± 26 22.0 ± 1.2 235 ± 4 1,570 ± 42 58.0 ± 6.1 Formula 29.2 ± 0.4 1,116 ± 55 27.3 ± 3.6 230 ± 3.6 1,318 ± 75 68.6 ± 4.0

•From Ehrenkranz et al. (19). bPTHM, preterm human milk; Fortified PTHM, 1 ;1 v/v mixture of PTHM and a proprietary preterm formula; Formula, a proprietary preterm formula. cAt the time of isotope administration. STABLE ISOTOPE METHODS 77

TABLE 5. Absorption of nZn in adult men consuming different diets*

Diet No. ™Zn absorption (%)

Study 1 Soy isolate 5 34 it 4 Milk 5 41 it 4 Soy/milk (50/50) 5 41 it 7 Study 2 Soy isolate 8 30 dt 3 Beef 7 41 dt 4

•From Solomons et al. (50). of the diet (19). One potentially significant point that must be made clear is that these measurements relate to the absorption of the "extrinsic tag," and inasmuch as the issues of complete exchange between the administered "extrinsic tag" and the Zn native to the diet remain unresolved the results of all "extrinsic tag" studies must be interpreted with caution. The resolution of this issue, central to all bioavailability studies of this type, has been technically overcome in adults (25,40), and the same approach should be applicable to the infant. This will require a biologically labeled formula diet similar to the meat diet used in the adult studies (25,40).

Studies of Body Content and Turnover Studies related to dynamics of whole body content of minerals and their turnover, especially in relation to the changes occurring during rapid growth in early infancy (2), have not made much progress partly because of the inavailability of a safe isotope methodology. The published data are primarily based on analyses of a few cadavers and date back to the period 1940 to 1950 (2,7). For the newer trace minerals (e.g., Se) there are no data on children as far as we know. Dynamics of body content during early life can be investigated with a combination of the metabolic balance and the isotope dilution principle. These methods have been applied extensively to the measurement of K-space, body water, and Br- and Cl-space (51) in adults using primarily radiotracer methodology. The stable isotope approach appears to be especially well suited for this purpose during the early period of life, although its use has not yet been explored. Based on available data on body composition at various ages, we have summarized the body content of several stable isotopes at three ages in Table 6: the premature infant at 29 weeks' gestation, the term infant (40 weeks' gestation), and a 2-month-old infant delivered at term. At 29 weeks, a fetus (or premature infant) would contain about 0.31 mg ••^a compared with 1.12 mg for a term infant and 1.52 mg for a 2-month old, corresponding to a net gain of 0.81 and 0.40 mg 46Ca for each interval, respectively. Thus it is clear that during this period total body calcium can be readily enriched significantly even with a single oral dose of the isotope, especially as retention of 78 STABLE ISOTOPE METHODS

TABLE 6. Isotope content of whole body at various ages

Isotope content (mg) 29 Weeks 40 Weeks 2 Months Isotope (gestation) (gestation) (postnatal)

"Ca 0.31 1.12 1.52 41K 152 445 796 27 94 100 37CI 803 1,590 1,895 »Fe 0.26 1.22 1.29 ^Cu 1.26 5.59 6.31 *>Zn 0.127 0.400 0.530 74Se 82 ng 2.46 pig 4.10 \LQ

ingested Ca is known to be high during this period (18). Following such a protocol and establishment of isotope dilution equilibrium, urine can be effectively employed for dilution studies, removing the additional difficult constraint related to multiple blood withdrawals with infants. Unfortunately, the cost of enriched 46Ca is high (about $5,000/mg 46Ca) so that conduct of such studies, although feasible technically and needed urgently, is not likely to be pursued aggressively unless some major adjustments are made with regard to the isotope cost factor. Measurement of K-space is an important parameter of body composition (46) and has been carried out previously, especially in adults, with isotope dilution of the radiotracer 42K (half-life 12 hr). In addition to the issues related to radiation exposure, the relatively short half-life of the radiotracer has limited the time allowed for dilution equilibrium to less than that ideally required (52). We have summarized the data on body content of 4IK (stable isotope) for three age groups in Table 6. At 29 weeks total body content of the isotope is about 152 mg, increasing to 445 and 796 mg, respectively, for the full-term and the 2-month-old infant. Assuming advisable daily potassium intake of about 400 mg, it becomes clear that significant enrichment of body potassium can readily be achieved with oral administration of 41K employing a number of realistic dosing protocols. Again, the major limitation for this application at present appears related to the cost of the isotope (about $6.50/mg). In contrast to Ca and K, cost does not appear at this time to be a major limiting factor in the application of stable isotopes to the dynamics of body composition in the infants for other minerals. For Cl or Na space, bromine (either radiobromine or naturally occurring bromine) has been utilized previously and appears to be the accepted method (52). Two stable isotopes of bromine are available for these studies (79Br, natural abundance = 50.54%; 81Br, natural abundance = 49.46%). Because of the low bromine content of human body, application of either bromine isotope followed by measurement of its dilution in urine appears to present little difficulty at this time. With respect to Fe, measurement of total body Fe can be readily carried out by the simple procedure of oral administration of 58Fe in a readily absorbed form (iron ascorbate or ferrous sulfate) followed by dilution of the STABLE ISOTOPE METHODS 79

1.2

1.0 f 11

1.2 r- .8 x [1 - 1 1 |! ! \r 1.0 - il 6 ( I 0 - II \ i I \ 1 1 .8 - 1 x .4 en ji ii i .6 - x .2 ? Q 1i | ,-Unenriched I ! / ratio o \ or A i i i i l i l l I I 10 40 80 120 160 200 240 o - x Time from Admin, of Isotope, (hours) !X « 2 .2 - ?-x— o Unenriched -x x-°--*> xo x . x /ratio

1 1 1 1 1 1 | 1 1 1 1 1 1 1 1 1 1 1 l -3 3 7 II 15 19 23 27 31 Time from Administration of Isotope,(days)

FIG. 6. Plots illustrating kinetics of plasma-urine isotope equilibration in human adults following 74 2 a single oral administration of Se03 - (48). Open circles are plasma enrichment data; the (x) indicates data for urine. The inset refers to the initial 10 days of the 31-day study. absorbed 58Fe in the RBCs after establishment of isotope equilibrium. Iron intake for this population group is advised at 1.2 mg/day, of which perhaps about 10% is absorbed (3,5). Thus addition of 58Fe in the readily absorbable form (ferrous ascorbate or sulfate) at 0.5 to 1 mg/day for a day or two should easily enrich the body iron pool very significantly so that its incorporation in the circulating mass of RBCs can be measured accurately. A small sample of blood (0.1 ml) would suffice for simultaneous measurements of both isotopes 58Fe and 54Fe using a special procedure developed at MIT (31). It should be noted here that in order for this procedure to be successful, absorption of the 58Fe dose must of course be measured accurately, and this can readily be accomplished by the fecal isotope balance procedure (27) especially at relatively high expected values of iron absorption for ferrous sulfate or ascorbate. Similarly, compositional studies of other trace elements can be carried out realistically as seen from body isotopic data given in Table 6. The major issue for these studies relates to the feasibility of achieving significant isotopic enrichment in the body. Relative to daily intake of the trace minerals, body content of their appropriate isotopes (Table 6) is small so that significant enrichment can be achieved via the simple method of oral supplementation. To illustrate the feasibility of these concepts, a typical kinetic curve for plasma/urine dilution of orally administered 74Se (in adults) is shown in Fig. 6. Of course, for the infant, 80 STABLE ISOTOPE METHODS urine would be the medium of choice in such studies because of the need for multiple sample withdrawals unless a single timed plasma sample were deemed sufficient.

SUMMARY AND CONCLUSIONS The available data related to mineral nutriture of infants and children indicate the existence of major gaps in our understanding of important aspects of their metabolism. The intensive and successful effort in regard to the development of stable isotope tracer methodology for application to human studies has paved the way for application of this safe isotopic technique to pediatric research. Applica- tions to date have been limited, however, to the studies of gastrointestinal absorption of Zn and Ca in infants. These methods can in general be employed to investigate gastrointestinal physiology and dynamics of body composition for a majority of important minerals.

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19a. Walker FW, Kirouac GJ, Rourke FM. Chart of the nuclides. 12th ed. Schenectady, New York: General Electric Company, 1977. 20. Janghorbani M, Ting BTG, Young VR. Accurate analysis of stable isotopes 68Zn, 70Zn, and !8Fe in human feces with neutron activation analysis. Clin Chim Acta 1980; 108:9-24. 21. Janghorbani M, Sundaresan A, Young VR. Accurate measurement of stable isotopes ^Ca and 48Ca in human feces, plasma, and urine in relation to human nutrition of calcium. Clin Chim Acta 1980;113:267-80. 22. Janghorbani M, Ting BTG, Young VR. Use of stable isotopes of selenium in human metabolic studies: Development of analytical methodology. Am J Clin Nutr 1981;34:2816-30. 23. Janghorbani M, Ting BTG, Steinke FH, Young VR. Intrinsic labeling of chicken meat with stable isotopes of zinc, for intended use in human feeding studies; feasibility and design considerations. BrJ Nutr 1981;46:395-402. 24. Janghorbani M, Christensen MJ, Steinke FH, Young VR. Feasibility of intrinsic labeling of poultry meat with stable isotope of selenium (74Se) for use in human metabolic studies. J Nutr 1981:111:817-22. 25. Janghorbani M, Istfan NW, Pagounes J, Steinke FH, Young VR. Absorption of dietary zinc in man: comparison of intrinsic and extrinsic labels using a triple stable isotope method. Am J Clin Nutr 1982:36:537-45. 26. Gramlich JW, Machlan LA, Brletic KA, Kelly WR. Thermal-ionization isotope-dilution mass spectrometry as a definitive method for determination of potassium in serum. Clin Chem 1982:128:1309-13. 27. Janghorbani M, Ting BTG, Young VR. Absorption of iron in young men studied by monitoring excretion of a stable iron isotope (58Fe) in feces. J Nutr 1980;110:2190-7. 28. Ting BTG, Kasper LJ, Young VR, Janghorbani M. Copper absorption in healthy young men: studies with stable isotope 6SCu and neutron activation analysis. Nutr Res 1984;4:757-69. 29. Janghorbani M, Young VR. Advances in the use of stable isotopes in minerals in human studies, AIN symposium, Atlanta, Ga. Fed Proc 1982:41:2702-8. 30. Janghorbani M, Istfan NW, Young VR. Stable isotope approaches for measurement of dietary zinc availability in man. In: Nutritional bioavailability of zinc. ACS Symposium Series 210. Washing- ton, DC: American Chemical Society, 1983:41-59. 31. Ting BTG, Pagounes J, Janghorbani M, Young VR. Radiochemical neutron activation analysis of stable isotopes in relation to human mineral nutrition, modern trends in activation analysis, Toronto, Canada. J Radioanal Chem 1982;70:133-44. 32. Janghorbani M, Young VR. Stable isotopes in studies of dietary mineral bioavailability in humans, with special reference to zinc. In: Prasad AS, ed. Clinical, biochemical, and nutritional aspects of trace elements. New York: Alan R. Liss, 1982:447-68. 33. Turnlund JR, King JC. Assessment of bioavailability of dietary zinc in humans using the stable isotopes ™Zn and 67Zn. In: Inglett GE, ed. Nutritional bioavailability of zinc. ACS Symposium Series 210. Washington, DC: American Chemical Society, 1983:31-40. 34. Johnson PE. A mass spectrometric method for use of stable isotopes as tracers in studies of iron, zinc and copper absorption in human subjects. J Nutr 1982;112:1414—24. 35. Janghorbani M, Young VR, Gramlich JW, Machlan LA. Comparative measurements of zinc-70 enrichment in human plasma samples with neutron activation and mass spectrometry. Clin Chim Acta 1981;114:163-71. 36. Turnlund JR, Michel MC, Keyes WD, Yves Schutz MA, Margen S. Copper absorption in elderly men determined by using stable 65Cu. Am J Clin Nutr 1982:36:587-91. 37. Lebenthal E, ed. Textbook of gastroenterology and nutrition in infancy. Vol. 1. New York: Raven Press, 1981. 38. Spencer H, Rosoff B, Feldstein A, et al. Metabolism of Zn-65 in man. Radiat Res 1965:24:432- 45. 39. Hallberg L. Bioavailability of dietary iron in man. Annu Rev Nutr 1981; 1:123—47. 40. Christensen MJ, Janghorbani M, Steinke FH, Istfan NW, Young VR. Simultaneous determination of absorption of selenium from poultry meat and selenite in young men: application of a triple stable isotope method. Br J Nutr 1983;50:43-50. 41. Evans GW, Johnson PE. Determination of zinc availability in foods using the extrinsic label technique. Am J Clin Nutr 1977:30:873-8. 42. Sirichakwal R Selenium nutrition and metabolism in young men as influenced by dietary forms [Thesis]. Cambridge, Massachusetts: MIT, 1983. 82 STABLE ISOTOPE METHODS

43. Istfan NW. An approach for study of zinc bioavailability in man using stable isotopes [Thesis]. Cambridge, Massachusetts: MIT, 1982. 44. Mason KE. A conspectus of research on copper metabolism and requirements of man. J Nutr 1979;109:1981-2066. 45. Bronner F. Dynamics and function of calcium. In: Connor CL, Bronner F, eds. Mineral metabolism. Vol. IIA. New York: Academic Press, 1964:341-4. 46. Black DAK. Potassium metabolism. In: Maxwell MJ, Kleeman CR, eds. Clinical disorders of fluid and electrolyte metabolism. 2nd ed. New York: McGraw-Hill, 1972:121-49. 47. Lutwak L. Tracer studies of intestinal calcium absorption in man. Am J Clin Nutr 1969;22:771- 85. 48. Kasper LJ. A comparative analysis of selenium metabolism in the normal and selenium-depleted state using stable isotope methodology [Thesis]. Cambridge, Massachusetts: MIT, 1983. 49. Young VR, Nahapetian AT, Janghorbani M. Selenium bioavailability with reference to human nutrition. Am J Clin Nutr 1982;35:1076-88. 50. Solomons NW, Janghorbani M, Ting BTG. Bioavailability of zinc from a diet based on isolated soy-protein (Supro-620): application in young men of the stable isotope tracer, ™Zn. J Nutr 1982;112:1809-21. 51. Maxwell MH, Kleeman CR, eds. Clinical disorders of fluid and electrolyte metabolism. 2nd ed. New York: McGraw-Hill, 1972. 52. Blahd WH. Radioisotope techniques. In: Maxwell MH, Kleeman CR, eds. Clinical disorders of fluid and electrolyte metabolism. 2nd ed. New York: McGraw-Hill, 1972:613-27.

DISCUSSION

Dr. Arnaud: I generally agree with your results and your presentation not only because I have been using stable isotopes for the last 10 years but because I believe that such studies represent the future, especially in men and infants. I have three questions: The first one is related to the preabsorptive pool and the reabsorption of endogenous minerals. You made the assumption that there is a homogeneous preabsorptive mineral pool which is coming from both food and endogenous minerals. I agree that iron secreted in bile can mix very well with the iron from food; but as most of the fecal losses of iron are coming from red blood cells going into the lumen and from mucosa cells, those cells are released all along the gastrointestinal tract and I do not think that this most important part of endogenous iron can be mixed with the pool coming from food absorption. Have you data supporting this first hypothesis? I think that your fractional absorption factor is not the same for endogenous minerals as it is for those from food. I have two other questions as well. In 1982 Judith Turnlund and collaborators published a study using iron-58 where thermal ionization mass spectrometry had higher precision and accuracy than neutron activation. What is your opinion about this problem? Can we improve the accuracy of the data obtained with neutron activation when we use the mass spectrometry method? The last question is a technical one. It should be important to test stable isotopic and radioactive methods and it should be very important in adult man to administer iron- 58, iron-55, and iron-59 to be sure that there are no technological problems. I say this because I have had some problems the last 2 months in accurately determining iron-55 in the blood. It is not a very easy technique, and we can easily make a 100% error in the estimation of iron absorption. In such experiments, it should be very interesting to calculate fecal excretion of iron-59 and iron-58 and in blood to calculate iron absorption from the iron-55/iron-59 ratio. When we look at the metabolism in the rat, we can see that iron can remain in the cells, and the latter are constituents of the feces. Therefore I do not think that the iron in mucosa cells and red blood cells is available as well as food iron. STABLE ISOTOPE METHODS 83

In the case of iron, half of the endogenous losses consist of red blood cells entering the gut lumen, and these cells are produced all along the gastrointestinal tract, not only in the duodenum and upper jejunum where the iron from food is absorbed. If you look at whole body autoradiography in the rat, you cannot see a lot of radioactivity in the intestine several days after iron administration; the reabsorption of endogenous iron seems to be a minor pathway, and what appears in the intestine is excreted in the feces and not reabsorbed. Dr. Janghorbani: Concerning the issue of the assumptions that are made: This is of course a very important issue. Each one of the assumptions has a different degree of feasibility in relation to a different parameter of absorption and endogenous secretion. In the determination of iron absorption, the only assumption that needs to be validated, other than the fact that you measure the stable isotopes, is that the extrinsic tag mixes properly with that component of dietary iron which is either heme or nonheme. The additional issue that enters in relation to the stable isotope technique is that the level of a stable isotope tracer dose added to the dietary pool is not at trace levels. When it comes to our suggestion that one can combine the measurements of absorption with fecal balance of a mineral to get at the very important and much neglected issue of endogenous secretion, the additional assumptions come in— namely, that the fractional reabsorption of the endogenously secreted native iron, be it in the bile, pancreatic secretion, or whatever, is in fact equal to that of the extrinsic tag. If that is not valid, and its validity has not been directly tested of course, the equations still provide data with regard to the unabsorbed rather than the total original endogenous secretion. That of course is also a very important parameter that has not been measured. The issue of mass spectrometry versus neutron activation has been dealt with in a number of publications. It comes down to this: Mass spectrometry, as reported in the literature for the application of interest here, with very few exceptions at this time, is of questionable value. When it comes to iron, what has been done with mass spectrometry is rather rudimentary and in fact has confounded the issue of availability. Development of these necessarily difficult technologies requires careful assessment of analytical capabilities and limitations which I have not seen done. The issue of validity is a very important issue. When one develops a new technology, one must be conservative; one must err on the side of caution. One must always validate a new method against what is accepted, but this, of course, has been done by us, as well as others. In one experiment that was published (J Nutr 1981;111:2236), we in fact administered zinc- 70 stable isotope and zinc-65 radiotracer simultaneously to the rat. We measured absorption in the same excreta with the stable isotope and radioisotope techniques and had it also evaluated by an independent laboratory with the radioisotope technique. There is no problem with the feasibility. In the case of zinc, the two techniques yield, within the statistics of the overall measurements, identical results. Now, you could accept this for iron or you could question it; if you choose to question it, I suggest that somebody else repeat the experiment. If you accept it based on the zinc experiments and what is known from iron kinetics and absorption, then of course there is no problem. Now, concerning the question you asked about excretion of iron by exfoliation of the intestinal cells: I believe that that iron is nonheme iron. If that is the case, then of course reabsorption of that iron simply follows nonheme iron kinetics except for the portion that is exfoliated after the major sites of absorption. Dr. Golden: There are other differences between stable and nonstable isotopes, one of the major ones being that many of the stable isotopes have unpaired nuclear particles and are therefore available for assay by other techniques. The technique I am thinking about is nuclear magnetic resonance (NMR), which can be used to follow kinetics in vivo and to 84 STABLE ISOTOPE METHODS actually look at where they are going and what is happening to them in terms of their atomic associations. Would you comment on the future of NMR in the metabolism of metal tracers? Dr. Janghorbani: There is a major focus these days in the United States on development of NMR. However, I do not expect such a technology to be able to do studies on the dynamics of turnover of trace elements within a foreseeable time frame, if at all. To be NMR-sensitive requires that the nucleus possess special spins, and of course not all stable isotopes can fulfill this requirement. Additionally, I do not know if anyone has ever been able to detect trace elements in living systems such as mammalian organs. Dr. Golden: My understanding was that in vivo measurements with phosphorus, examining ATP, is already being done in England, but I do not know about the United States. I would have thought that it is quite feasible with a number of other elements, particularly if you can get sufficient enrichment, although it might cost $8,000 to do an experiment. Dr. Janghorbani: The use of NMR in relation to phosphorus is of course not feasible because phosphorus is a monoisotopic element. Dr. Gebre Mehdin: How about the use of positron emission spectroradiography for studying these issues? Dr. Janghorbani: What you mean is giving a subject a positron-emitting isotope and then following that positron emission. Those are radiotracer experiments. A positron emitter is a radioisotope that gives off a positron, which is really nothing but an electron with a positive charge, which then in vivo annihilates to provide two gamma rays at 180° and 511 keV That is not a stable isotope tracer and, of course, is not different from any other radiotracer except you detect a different parameter—the gamma rays coming from annihi- lation of a positron. It is not at all different from measuring gamma rays from zinc-65. Dr. Senterre: Fifteen years ago I measured fecal excretion of endogenous calcium by a tracer dose of calcium-45 in the milk. By combining apparent and true absorption of calcium, I got values ranging from 10 to 20 mg/kg body weight per day in very-low-birth-weight infants. What figure did you get with your stable isotope techniques? Dr. Mertz: I accept that isotopes with higher atomic numbers vary so little in mass that they are probably handled like the real thing, but as the atomic number decreases you must come to a point where the difference in mass is so important that the organism can distinguish between them in some ways. What then is the lower limit of atomic number that you trust? Do you trust lithium, for example? Dr. Janghorbani: That of course is a very important question, and there are certainly data for light elements. When you do tracer studies with such elements, these differences are observable. With regard to metals, I am not aware of any data in biological systems. There will be differences, but without having data to back this up we cannot measure with our present capabilities any differences in relation to the things we are discussing in a human subject or in an animal. However, the issue of comparing stable isotopes with radioisotopes of course makes that question unimportant because if there is an observable difference it also applies to radiotracer work. Dr. Picciano: If I remember your presentation correctly, you measured the absorption of zinc from preterm human milk, preterm human milk fortified with zinc, and a specially adapted premature formula. You gave infants an extrinsic dose of the stable isotope with milk feedings. What were the other experimental parameters? Dr. Janghorbani: The way this experiment was carried out was not the way that one would do it if one were to repeat it of course. The infants were given their particular feeding, half of it; then the solution was given by intubation, followed by feeding the second portion. The assumption was that in this way the tracer would have sufficient time to mix with the STABLE ISOTOPE METHODS 85 meal prior to absorption. This is a very important issue because of what I referred to a number of time before, namely, that in all these isotopic measurements what you are really measuring is something about the isotope tracer. If the tracer does not measure the actual behavior of the mineral native to the diet, you of course not only did not gain any useful information but potentially confused the issues further. Dr. Picciano: Therefore what you may in fact be measuring is the absorption of a radiotracer dose of zinc as influenced by human milk or as influenced by a formula? Dr. Janghorbani: Yes. We have carried out similar experiments in the important area of plant protein sources, i.e., soy proteins. We used our extrinsic tag approach in human adults and have studied as much as 100% nitrogen replacement of the diet with soy protein isolates and concentrates, and the results have been published. We have come to the conclusion that total substitution of nitrogen of the diet with soy isolates and soy concentrates for as many as 90 days does not adversely influence, in a nutritionally relevant way, the nutriture of zinc. If the extrinsic tag does not measure absorption of dietary zinc, then of course these conclusions are very much in error, but that is the best we can do at this time. We have validated the extrinsic tag versus the intrinsic tag using animal food systems. We have intrinsically labeled chicken meat and hen's egg, and in fact we know how to intrinsically label soy proteins also. Our studies to date seem to indicate that the extrinsic tag is a valid approach for zinc, but perhaps not for selenium. Dr. Picciano: Your calculation of iron intake during infancy is correct if you have a totally breast-fed infant, but if you have an infant who is consuming an iron-fortified formula and an iron-fortified cereal, you can be off by a factor of approximately 60. Dr. Janghorbani: Yes. I had a note in my paper to make a point that 1.2 mg is for a 2- month-old based on human milk. I think the point that I was making is valid for either situation. The RDAs are of course not as high as you suggest. Dr. Picciano: I am referring to actual consumption, not recommended dietary allowances. Actual measures of iron intake of a 2-month-old infant receiving an iron-fortified formula and iron-fortified cereal in the United States indicate values as high as 60 mg, approximately 15 mg/kg. Dr. Janghorbani: The U.S. Food and Nutrition Board's recommendation is based on 1.5 mg/kg/day for the first year of life.