J. Anim. Sci. 2006. 84:E73-E78
© 2006 American Society of Animal Science
Mineral bioavailability and metabolism determined by using stable isotope tracers1
J. R. Turnlund2
USDA/ARS/Western Human Nutrition Research Center, University of California, Davis 95616
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Abstract
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Definitive data on mineral bioavailability in humans and animals can be obtained by using isotopic tracers. The use of stable isotope tracers to study important issues in mineral nutrition has expanded rapidly in the past two decades, particularly in human nutrition studies. Stable isotopes have a number of advantages over radioisotopes. There is no exposure to radiation with stable isotopes, and some minerals have no radioisotope that can be used satisfactorily as a tracer. Multiple stable isotopes of one mineral and isotopes of multiple minerals can be administered simultaneously or sequentially. The analytical methods of choice for stable isotopes are thermal ionization mass spectrometry and inductively coupled plasma mass spectrometry (ICPMS). Thermal ionization mass spectrometry offers the greatest precision and accuracy, but it is slower, more labor intensive, and more costly than ICPMS. Bioavailability data are critical to establishing reliable dietary mineral requirements and recommendations. Combined with a computer program for compartmental modeling, mineral kinetics can be studied, including mineral turnover, pool sizes, and transfer rates between compartments. Our laboratory conducts studies using stable isotopes of Zn, Cu, Fe, Ca, Mg, and Mo. We have studied the effect of the amount of dietary intake of minerals on bioavailability and use, pregnancy and aging, and interactions among minerals. The research resulted in establishing new dietary recommendations for Cu and Mo and developing compartmental models for these minerals. Although stable isotopes have been used more extensively to date in humans than in animals, the techniques applied to humans can be used to study a number of issues important to optimizing feeding strategies for animal production.
Key Words: mineral bioavailability • mineral metabolism • stable isotope
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INTRODUCTION
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Stable isotopes are valuable tools for studies of mineral metabolism in humans. When minerals in the diet are consumed, they enter the gastrointestinal tract. Varying fractions of the quantities consumed are absorbed and enter into systemic circulation, where they mix with minerals consumed earlier, and are deposited in various organs and tissues in the body. Fractions of the absorbed minerals are excreted along with endogenous minerals via bile and pancreatic secretions into the gastrointestinal tract, where they mix with unabsorbed dietary minerals. Fractions are also excreted via the urine and other miscellaneous routes. Stable isotopes of a mineral occur in nature in fixed abundances; the isotopes can be separated and collected. Isotopes of low abundance can be used as tracers to follow the metabolic fate of minerals in a specific diet or dose, and to separate them from endogenous minerals and those consumed at other times. The objectives of this review are to describe how stable isotopes of minerals are used in human experiments and to explore potential applications of these tracers for research in nonruminant animals.
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HISTORY
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The use of tracers to study the metabolic fate of a mineral was introduced when Hahn et al. (1939)
used a radioisotope of Fe in dogs to study anemia. This was followed by human studies with radioisotopes of Fe (Balfour et al., 1942; Hahn et al., 1945; Darby et al., 1947) and other minerals, such as Cu (Schubert and Riezler, 1947), Ca (Bellin and Laszlo, 1953), Zn (Daniels et al., 1956), Mg (Aikawa et al., 1958), Mo (Rosoff and Spencer, 1964), and Se and Cr (Rechowicz, 1970). When the risk associated with exposure to radiation was recognized, scientists turned to stable isotopes to study mineral metabolism. They had been used for human studies of compounds containing hydrogen, carbon, oxygen, and nitrogen beginning in 1935 (Schoenheimer and Rittenberg, 1935). The potential of stable isotope tracers for studies of mineral metabolism was first explored using neutron activation analysis (NAA) in 1963. The approach was tested for Fe, Ca, Au, Cr, and Se (Lowman and Krivit, 1963). Neutron activation analysis was the only method reported for stable isotope studies of minerals in humans for the next 10 yr (McPherson, 1965; Bethard et al., 1967; Donaldson et al., 1968; Dyer and Brill, 1972); however, NAA has a number of serious limitations. Not all isotopes can be measured with NAA, it usually lacks necessary analytical precision, and it requires access to a nuclear reactor.
Mass spectrometry had been used to measure isotopic ratios of minerals since the 1950s by geochemists and in nuclear chemistry, but it was not applied to studies of mineral metabolism until the mid 1970s. The first stable isotope study of mineral metabolism to apply mass spectrometric methods was reported by Rabinowitz et al. (1973). The group described a comprehensive investigation of Pb metabolism in men using 204Pb as a tracer. The field has expanded greatly since 1980, when nutritionists began to use mass spectrometry for analysis.
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STABLE AND RADIOACTIVE ISOTOPE TRACERS
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The stable isotopes of minerals nutritionally important to man and animals are shown in Table 1. The elements Na, F, P, Mn, and I have only one naturally occurring isotope. Thus, because stable isotopes cannot be enriched, stable isotope tracer studies cannot be conducted. Others, such as Cu and Mg, have only relatively abundant isotopes, so larger doses of isotopes and high-precision analytical methods are required to achieve adequate enrichment. However, the radioisotopes of these elements have very short half-lives (12 to 62 h; Lide, 2005), which can be an even greater disadvantage than the limitations associated with relatively abundant stable isotopes. Others (Ca, Se, and Mo) have 6 or 7 naturally occurring isotopes, resulting in a wide variety of opportunities for metabolic studies.
Radioisotope Tracers
Radioisotopes have several advantages over stable isotopes in tracer studies. Analysis is rapid and little sample preparation is usually necessary. Analysis is simple, instrumentation is considerably less expensive, and the isotopes themselves are usually less expensive. As a result, studies with radioisotopes are less costly and can be completed more quickly. In addition, radioisotopes can be localized in vivo, whereas stable isotopes cannot. The primary drawback of radioisotopes is exposure to radiation. Some studies can be conducted with minimal exposure, but this exposure limits their use considerably. In addition, some minerals have no suitable radioisotope for tracer studies.
Stable Isotope Tracers
Stable isotope tracers have several limitations. As noted previously, some minerals have only one stable isotope or only abundant isotopes. As the abundances of isotopes increase, the amounts that must be administered increase and doses greater than true tracer doses are often required. This introduces a risk of perturbing metabolism with the doses required for adequate enrichment. Stable isotopes cannot be localized in vivo. Sample preparation can be laborious and costly and instruments are more expensive than those used for radioisotopes. There is a broad range in the cost of stable isotopes, from a few dollars to over a thousand dollars per milligram, and the cost of a sufficient quantity of an isotope for a study can be prohibitive. Availability of enriched isotopes can be limiting and instrumentation may not be readily available.
The overwhelming advantage of stable isotopes over radioisotopes is that they can be used as tracers with no exposure to radioactivity. Therefore, they can be used safely in infants and pregnant women. They do not decay, so long-term studies can be conducted, and sample analysis can be delayed. Another major advantage is that multiple isotopes of multiple minerals can be used simultaneously without interfering with one another. The cost, complexity, and time-consuming procedures of stable isotope research are being decreased rapidly with advances in instrumentation including the wider availability of lower-cost inductively coupled plasma mass spectrometry (ICPMS) instruments.
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ANALYTICAL METHODS
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The first analytical method used for stable isotope studies of mineral metabolism, NAA, is not used extensively now due the limitations mentioned above. A variety of mass spectrometric methods are available for measuring isotopic ratios. Several methods were tried in early attempts at using stable isotopes to study mineral metabolism, usually because instruments were available in laboratories, but many of these have since been abandoned. They include spark-source mass spectrometry and electron-impact mass spectrometry. Gas chromatography-mass spectrometry has limited applications, but can be used for Se and Cr (Reamer and Veillon, 1981). Fast atom bombardment mass spectrometry has been used for minerals such as Ca (Smith, 1983) and Zn (Peirce et al., 1987), but this technique is usually used only when the instrument is available to investigators and more appropriate instruments are not. Thermal ionization mass spectrometry (TIMS) is considered the "gold standard" for determination of isotopic ratios due to its extremely high precision. It has been used extensively for studies of Zn, Cu, Fe, Ca, and Mo metabolism (Turnlund and Keyes, 1990; Crews et al., 1994); however, the instruments are very expensive, and sample preparation and analysis are slow. The most recently developed approach for analysis of stable isotope ratios is ICPMS, which was first introduced to quantify minerals (Houk et al., 1980). The instrumentation has advanced greatly since its introduction, leading to its use for measuring isotopic enrichment. It is usually the method of choice today because of its advantages over TIMS (Crews et al., 1994; Turnlund and Keyes, 2002). Less sample preparation is required than for TIMS, it is much faster, and smaller sample sizes are needed. Nonetheless, when the greatest precision is required, TIMS is the method of choice. Moreover, for some isotopes of elements such as Ca and Fe, interferences limit the use of ICPMS, and TIMS is preferred.
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MAJOR CONSIDERATIONS IN STABLE ISOTOPE STUDIES OF MINERAL NUTRITION
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Applications
Stable isotope tracers are used to study a number of issues in mineral metabolism in humans. Most studies reported to date were done on mineral absorption or bioavailability. A number of approaches can be used to determine absorption. The most common method used is to administer isotopes orally followed by complete stool collections. It is assumed that the isotopes administered and not recovered in the stools were absorbed. Isotopes may be administered in a single bolus, added to a single meal or multiple meals, or incorporated into a food intrinsically. It is critical when using this approach to ensure complete stool collections. If collections are not complete, absorption will be overestimated. When using fecal monitoring to determine absorption, some of the tracer that was fed is absorbed and excreted into the gastrointestinal tract, resulting in absorption being underestimated. The degree of the underestimation varies depending on the mineral studied, and is greatest when the primary route of excretion is via the bile into the gastrointestinal tract. Correction for the excretion can be made by administering another isotope of the same mineral intravenously and determining the fraction excreted into the stools. Iron absorption can be determined in infants and children by administering an isotope orally and measuring its incorporation into erythrocytes approximately 2 wk after administration (Fomon et al., 1988). The method does not work well in adults because the pool of Fe in erythrocytes is so large that enrichment is difficult to detect. Absorption also may be determined for some minerals by giving one isotope orally and another intravenously. Analysis of plasma or urine samples at specific time points can then be used to determine absorption based on the relative amounts of each dose appearing in the samples. The approach has been evaluated for Ca (Eastell et al., 1989), Zn (Friel et al., 1992; Lowe et al., 2000), and Mg (Sabatier et al., 2003). Although this method requires an infusion in addition to the oral dose, it is simpler because complete collections are not required.
Stable isotopes are also used to estimate mineral turnover, endogenous excretion of a specific dose, and total endogenous excretion. They are used to accurately determine concentration of minerals in a sample by isotope dilution. By using isotope dilution, isotope ratios of the sample, the content of enriched isotopes, and the total mineral content can be determined in a single analysis. Stable isotopes also are used for compartmental modeling of mineral kinetics.
Isotope Selection and Dose
Some minerals have a number of stable isotopes to choose from when selecting a tracer, as shown in Table 1
. There are several important considerations when selecting the isotope to enrich. The best isotope for enrichment is usually one with a low natural abundance, because a smaller dose of isotope is needed to obtain adequate enrichment. The abundance of the reference isotope should also be considered. The best analytical precision is obtained when the ratio of the enriched isotope to the reference isotope is between 0.1 and 10. Interferences must be taken into account, and different analytical methods have different interferences. For example, the argon used in ICPMS analysis interferes with 40Ca and 56Fe. Different methods also have different levels of sensitivity and precision. The cost and availability of the isotopes also must be established when designing an experiment.
In determining the dose of an isotope to be administered, isotopic abundances must be considered, as well as the fractions of the dose expected in the samples to be collected, the total quantity of the element in those samples, the length of time detectable enrichment is required, and the precision of the ratio measurements All these factors must be considered to design and conduct a successful, cost-effective experiment.
Multiple Labeling
Multiple labeling with stable isotopes makes it possible to study a number of variables simultaneously. Multiple isotopes of a single mineral can be administered simultaneously by the same route or by different routes. Different foods, meals, or menus can be labeled with different isotopes in a single experiment. Another isotope can be used as an isotopic diluent in the analytical phase of the experiment. One or more isotopes of multiple minerals can be administered simultaneously or sequentially to study interactions between minerals. We have administered multiple isotopes of as many as 5 different minerals in a single experiment. Table 2 shows an example of stable isotopes administered in a single experiment of Mo metabolism at several levels of Mo intake. Definitive data on a number of issues of Mo metabolism and interactions with other minerals were determined (Turnlund, 1995; Turnlund et al., 1995a). By collecting blood, urine, and stool samples, absorption and turnover of Mo, Zn, and Cu were studied as a function of Mo intake in a single experiment.
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APPLICATIONS IN HUMANS
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Stable isotope tracers have been used by my group to compare mineral metabolism in different population groups. For example, we compared absorption of Zn, Cu, and Fe in young and elderly men and found that Zn absorption (Turnlund et al., 1986), but not Cu and Fe absorption (Turnlund et al., 1988), was lower in the elderly (Turnlund et al., 1986, 1988). Endogenous losses of Zn also were lower, however, suggesting the lower absorption was compensated for by lower losses. We compared Zn and Cu absorption in pregnant and non-pregnant women and found that, although the efficiency of absorption changed little, the small increase in absorption throughout pregnancy would result in accumulation of enough of those minerals to provide the amounts needed for the fetus and products of pregnancy (Swanson et al., 1983; Turnlund et al., 1983)
A number of stable isotope studies were conducted using 65Cu to assess the effect of Cu intake on absorption, turnover, and retention. We found that the efficiency of Cu absorption decreased markedly as intake increased, but the amount absorbed still increased (Turnlund et al., 1989). Some of that Cu was retained in the intestinal tract, and then eliminated as intestinal mucosa exfoliated (Turnlund et al., 2003). The quantity of Cu, then excreted into the gastrointestinal tract, increased as the quantity absorbed increased (Turnlund et al., 2005). Excretion in the urine was very low and not affected by intake. These three points of regulation helped to decrease the risk of Cu deficiency and toxicity. The absorption and retention data, along with data on Cu status, were a major factor in establishing the new Dietary Reference Intakes for Cu (IOM, 2002a). We are currently particularly interested in the Cu intakes at which these regulatory mechanisms are overwhelmed, suggesting intakes that could result in deficiency (Turnlund et al., 1998a) or toxicity (Turnlund et al., 2005).
Stable isotope studies of Mo metabolism demonstrated that, in contrast to Cu, the efficiency of Mo absorption did not decrease as intake increased. As a result, the quantities absorbed directly reflected intake. However, urinary excretion of the absorbed Mo increased as the amount absorbed increased, demonstrating that the point of regulation of Mo retention was urinary excretion (Turnlund et al., 1995a,b). Data from this research were used to establish dietary recommendations for Mo (IOM, 2002b).
Intrinsic Labeling
Intrinsic labeling of foods allows studies that compare bioavailability of minerals from different food sources. We labeled soy with 100Mo and kale with 97Mo, and then compared absorption of molybdenum from these sources with that of 96Mo added to the diet. The 94Mo was added to samples before analysis to quantify isotopes and total Mo by isotope dilution. We found that the Mo in kale was absorbed as efficiently as the Mo added extrinsically, but the absorption from soy was lower than either. Once absorbed, the fraction of absorbed Mo excreted from the three sources was similar (Turnlund et al., 1999). This finding suggests that the source of Mo affects absorption, but once absorbed use is the same, regardless of the source.
Compartmental Modeling of Mineral Kinetics
Compartmental modeling of mineral metabolism is a means to determine the metabolic fate of minerals in tissues that cannot be measured directly. Computer modeling programs such as SAAM (1994) are available to be used for development of models. Initial models are usually based on literature from laboratory animal experiments. Stable isotope enrichment data collected from blood, urine, and fecal samples are then fitted by computer by modifying the theoretical model. Compartmental models of mineral kinetics are developed and estimates of turnover, transfer between compartments, and compartmental masses are estimated (Turnlund et al., 1998b; Cobelli et al., 2000).
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REFERENCE BOOKS AVAILABLE
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In addition to the references cited previously, several books are available that provide greater detail on applications of stable isotopes for research on mineral metabolism, their methods, instrumentation, and experimental results (Turnlund and Johnson, 1984; Mellon and Sandstrom, 1996; Lowe and Jackson, 2001; Abrams and Wong, 2003; Wolfe and Chinkes, 2004).
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IMPLICATIONS
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Enriched stable isotopes of minerals can be used as tracers in animals in the same types of experiments as those conducted in humans. In addition to studies in humans, they have been used in an increasing number of laboratory animal experiments (Frederickson et al., 1982; Dunn et al., 1991) and in ruminants (Buckley, 1991). They are especially valuable when radioisotopes cannot be used. Potential applications in nonruminant animals include determining the most bioavailable and cost-effective form of a mineral supplement; determining the optimal intake of a mineral and determining intakes that are too low or too high; determining changes in metabolism of a mineral during growth, pregnancy, and lactation; identifying and evaluating components of the diet that interfere with or enhance metabolism; evaluating interactions among minerals; and gaining a better understanding of mineral nutriture of nonruminant animals.
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Footnotes
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1 Invited review. Presented at the "Stable Isotope Tracer Techniques for Nonruminant Research and Their Practical Applications" symposium held at the American Society of Animal Science Annual Meeting, Cincinnati, OH, July 24–28, 2005. 
2 Corresponding author: jturnlun{at}whnrc.usda.gov
Received for publication August 10, 2005.
Accepted for publication August 25, 2005.
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Abstract
INTRODUCTION
