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

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Isotopic Methods in the Study of Mineral Metabolism of Infants with Special Reference to Stable Isotopes Trace Elements in Nutrition of Children, edited by R. K. Chandra. Nestle Nutrition, Vevey/Raven Press, New York €> 1985. Isotopic Methods in the Study of Mineral Metabolism of Infants with Special Reference to Stable Isotopes 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 me- tabolism (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 pa- rameter 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).
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