Zinc and Health: Current Status and Future ... - Journal of Nutrition

Zinc and Health: Current Status and Future Directions Kinetics of Zinc Metabolism: Variation with Diet, Genetics and Disease1,2 Meryl E. Wastney,*3 William A. House,† Ramon M. Barnes** and Kolinjavadi N. Siva Subramanian* *Division of Neonatology, Department of Pediatrics, Georgetown University Medical Center Washington, D.C. 20007, †Plant, Soil and Nutrition Laboratory, U.S. Department of Agriculture ARS, Ithaca, NY 14853-2901 and **Department of Chemistry, University of Massachusetts, Amherst, MA 01003-4510 ABSTRACT Kinetic studies are used to investigate metabolic processes. By adding an isotope to a system and measuring its movement in the system over time, pool sizes and transport rates can be determined by mathematically modeling the data. This approach enables rate differences to be determined in conditions that have been modified by diet, environment, genetics or disease. Kinetic studies in humans have shown that there are multiple pools of zinc that turnover from minutes to years and that processes, including zinc absorption and excretion, are regulated to maintain tissue levels when zinc intake varies. Animal studies allow for greater understanding of kinetics because more tissues can be sampled and environmental and genetic factors can be controlled. Kinetic studies in animals will provide information on the overexpression or the deletion of genes coding for specific proteins involved in zinc transport and metabolism. The advances that have been made in our understanding of the role of zinc in metabolism have been aided by the development of techniques for measuring isotopes in biological materials. In the future, the kinetics of zinc bound to different compounds will be measured. Modeling will enable this information, at the molecular level, to be integrated with knowledge of zinc metabolism at the cellular, organ and whole body level. To understand more fully the role of zinc in human health, kinetic studies are needed in healthy and disease states to identify differences in metabolic processes. This knowledge can be used as a basis for dietary and therapeutic recommendations. J. Nutr. 130: 1355S—1359S, 2000. KEY WORDS:

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humans

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rats

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zinc mathematical modeling

Kinetic studies are used to investigate the processes of absorption, distribution and metabolism of compounds in vivo as well as in vitro. By adding a tracer, such as a stable or radioactive isotope, to a system and measuring its movement in the system over time, pool sizes and transport rates can be determined by mathematical modeling of the data (Wastney et al. 1998). The power of this approach is that it enables differences, in terms of sites and rates of metabolism, to be determined in conditions that have been modified by diet, environment, genetics or disease. We first outline a kinetic study and then review in vivo

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compartmental model

studies in humans and animals. Next, new analytical techniques for increasing the information obtained from kinetic studies are discussed. Finally, some areas that can now be researched using this approach are presented. Kinetic studies There are three components to a kinetic study: (1) tracer administration, (2) measurement of the tracer, and (3) data analysis. First, to measure a compound of interest, it must be tagged, usually by administering a tracer. Tracers are either stable or radioactive isotopes (e.g., 70Zn or 65Zn) that act in the same way as the material of interest (zinc) but can be distinguished by physical and chemical properties. Stable isotopes have a number of advantages over radioisotopes, such as safety and chemical stability (Barnes 1993), but they have some disadvantages. For example, the amount of a stable isotope administered to obtain sufficient enrichment may be such that the system being studied may be perturbed by the mass of tracer administered. Also, some stable isotopes are very expensive. A second component of a kinetic study is measurement of the tracer in the system (e.g., various tissues) over time. This is necessary to determine the shape of the curves associated with each tissue. The appearance of tracer in a tissue indicates how rapidly it is being taken up, whereas the disappearance indicates how rapidly it is being lost and whether there are

1 Presented at the international workshop “Zinc and Health: Current Status and Future Directions,” held at the National Institutes of Health in Bethesda, MD, on November 4 –5, 1998. This workshop was organized by the Office of Dietary Supplements, NIH and cosponsored with the American Dietetic Association, the American Society for Clinical Nutrition, the Centers for Disease Control and Prevention, Department of Defense, Food and Drug Administration/Center for Food Safety and Applied Nutrition and seven Institutes, Centers and Offices of the NIH (Fogarty International Center, National Institute on Aging, National Institute of Dental and Craniofacial Research, National Institute of Diabetes and Digestive and Kidney Diseases, National Institute on Drug Abuse, National Institute of General Medical Sciences and the Office of Research on Women’s Health). Published as a supplement to The Journal of Nutrition. Guest editors for this publication were Michael Hambidge, University of Colorado Health Sciences Center, Denver; Robert Cousins, University of Florida, Gainesville; Rebecca Costello, Office of Dietary Supplements, NIH, Bethesda, MD; and session chair, Nancy Krebs, University of Colorado School of Medicine, Denver. 2 Supported by National Institutes of Health R01-DK53787. 3 To whom correspondence should be addressed.

0022-3166/00 $3.00 © 2000 American Society for Nutritional Sciences.

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multiple pools in the tissue. Obtaining data at a single time point after tracer administration provides no information on the kinetics of a system. The third component relates to analysis of the kinetic data. To interpret kinetic curves in a quantitative manner requires computer modeling, in which the model consists of one or more mathematical equations. There are a number of approaches to modeling, and the approach chosen depends on the purpose for the study and the volume of data (i.e., length of study, number of sites sampled, and so on). Mechanistic models, such as compartmental models, are useful for describing the behavior of a system in terms of pools and transfer pathways. Compartmental models may be large if the purpose is to explore the underlying physiology or pathophysiology. If the purpose of the study is to determine a specific characteristic, e.g., the turnover based on whole body data, a simpler model with fewer parameters may be sufficient. Both types of models are useful, but it is important to understand the limitations of each. Complex models have heuristic value in indicating areas in which data are lacking and in making predictions for further experimental testing even though not all parameters may be identifiable from a particular experiment. Simple models tend to lump together various processes. One example is the use of fecal monitoring, where absorption is determined as the difference between orally administered dose and the dose recovered in feces. As described by Janghorbani et al. (1980) and simulated by Wastney and Henkin (1989), accurate calculation of absorption using this model is affected by transit times through the intestine. Transit times affect how quickly unabsorbed tracer is excreted and how much absorbed tracer is resecreted. Although simpler models may be well determined (i.e., all parameters of the model can be determined with statistical confidence), the values are reliable only if the model is a correct representation of the physiological system. Previously computing and software limitations favored the use of smaller models, but these limitations no longer need apply. Kinetic studies in humans. Some kinetic studies (Babcock et al. 1982, Fairweather-Tait et al. 1993, Foster et al. 1979, Henkin et al. 1984, Lowe et al. 1993, 1997, Miller et al. 1998, Scott and Turnlund 1994, Wastney et al. 1986, 1991, 1996) involving zinc that include the three criteria described above (use of tracers, collection of data over time and analysis of the data with the use of a model) are listed in Table 1. Earlier studies used radioactive isotopes, and this allowed data collection external to the body and for the studies to be performed for a longer duration. After the development of techniques for their analysis in biological tissues, stable isotopes have been used in many studies conducted during the past decade. Fewer sampling sites are available with stable isotopes, and some investigators have used models with lumped compartments to analyze the data. It also is appropriate to apply the more extensive models developed from radioisotope data to the studies with stable isotopes. Studies have been performed using oral tracer administration to define the absorption pathways and using intravenous administration to define the distribution and endogenous excretion pathways. Short-term studies (hours to days) have been used to examine the pools of zinc that exchange rapidly with serum zinc (Fairweather-Tait et al. 1993, Lowe et al. 1997), whereas longerterm studies (weeks to months) have been used in addition to study the tissue pools that turnover more slowly (Babcock et al. 1982, Foster et al. 1979, Miller et al. 1998, Wastney et al. 1986). These studies have shown that there are multiple pools of zinc that turnover within minutes to years. The faster pools are lo-

cated in plasma, liver and red blood cells, and the slower pools are located in muscle and bone. Zinc kinetics have been determined in healthy subjects consuming either normal or relatively high amounts of zinc. By comparing the changes in the kinetics attributable to zinc intake, it was found that processes at five sites, including absorption and excretion, are regulated to maintain tissue levels during high zinc intake (Wastney et al. 1986). By comparing kinetic data in healthy adults age 20 – 84 y, it was shown that although a few values changed while subjects consumed their normal dietary intake of zinc, there were significant changes with age at four sites of zinc regulation when the subjects consumed supplemental zinc (Wastney et al. 1992). The results may indicate either a reduced need for zinc or a reduced ability to regulate the amount of zinc retained with aging. Rates of zinc absorption and retention have been ascertained in preterm infants (Wastney et al. 1996). Because the infants were growing during the study, it was necessary to model both tracer and tracee (total zinc). In addition, it was necessary to model clinical interventions, such as blood transfusions, because the administration of adult blood cells provided a significant source of zinc to the infants. The results from a population of infants who were healthy and growing at expected rates (Wastney et al. 1999) can be used to evaluate zinc metabolism and requirements in infants who are sick or growing slowly. By comparing zinc kinetics in subjects with normal, high or low copper intake, Scott and Turnlund (1994) investigated sites of interaction between copper and zinc and the degree of interaction at each site. They reported that zinc absorption was lower on high copper intake (28 versus 34%, respectively). This is the only report of which we are aware that used zinc kinetics to study element– element interactions. Kinetic studies in animals. Animal studies allow a greater understanding of kinetics because more tissues can be sampled and environmental and genetic factors can be controlled. Table 2 shows some studies of zinc kinetics in animals (Dunn and Cousins 1989, House and Wastney 1997, Lowe et al. 1991, Lowe et al. 1995, Popov and Besel 1977, Serfass et al. 1996). In one study with rats (House and Wastney 1997), zinc kinetics were measured in 15 tissues, and the data were analyzed using modeling techniques. In addition to providing baseline values for healthy animals, the study (House and Wastney 1997) revealed the existence of slow and fast pools of zinc in muscle and bone and showed that three pools observed in humans, which turned over in 4 h, 1.3 d and 50 d, were consistent with kinetics in kidney, spleen and testes, respectively, in rats. Zinc kinetics were studied in young piglets fed either a control, a zinc-adequate or a zinc-deficient diet (Serfass et al. 1996). Results showed that animals fed the deficient diet compensated by increasing fractional absorption; however, liver zinc levels remained lower in the zinc-deficient piglets than in the control animals. The latter suggested that although growth rates were equivalent over the study period, subsequent growth and overall health would probably be compromised in the zinc-deficient group. Long-term studies (⬎6 mo) in humans fed mildly zinc-deficient diets showed that fractional absorption was increased but the increase was not sustained (Lee et al. 1993). Dunn and Cousins (Dunn 1991, Dunn and Cousins 1989) studied the effect of metallothionein induction on zinc kinetics in vivo in rats. They found that induction caused increased redistribution of zinc among body tissues; there was a fourfold increase in zinc uptake by liver metallothionein and a 85%

KINETICS OF ZINC METABOLISM

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TABLE 1 Zinc kinetics in humans Study length Adults

5d

n 17

Isotopes 69mZn p.o.

and i.v. Adults

336 d, 307 d

Adults

5d

Adults

270 d, 270 d

10

65Zn p.o.

2

69mZn i.v.

32

65Zn p.o. (25)

and i.v (7)

Tissues sampled Plasma, red blood cells, urine, feces, liver, thigh Plasma, red blood cells, urine, feces, liver, thigh, whole body Plasma, red blood cells, urine, feces, liver, thigh Plasma, red blood cells, urine, feces, liver, thigh, whole body Plasma, red blood cells, urine, feces, liver, thigh, whole body Plasma

Conditions

Model

Patients with taste/ smell dysfunction

6 compartments ⫹ intestine

Foster et al. 1979

Patients with taste/ smell dysfunction, regular and high Zn (⫹100 mg/d)


Babcock et al. 1982

Patients with adrenal cortical insufficiency, off and on carbohydrateactive steroids Normal on regular and high Zn (⫹100 mg/d)

8 compartments

Henkin et al. 1984


Wastney et al. 1986

Healthy, normal diet


Wastney et al. 1991

Healthy (alcoholics)

2 compartments

Lowe et al. 1993 FairweatherTait et al. 1993 Scott & Turnlund 1994 Wastney et al. 1996

Adults

78 d

4

65Zn and 70Zn

Adults

120 min

6(7)

70Zn i.v.

Adults

10 d

2

70Zn i.v.

Plasma, urine, feces

Healthy


Adults

24 d, 42 d, 24 d

5

70Zn i.v., 67Zn

Plasma, urine, feces


Preterm infants

30 d

2

Healthy on normal, low and high Cu intake Healthy

Adults

11 d

p.o. 70Zn i.v. (1),

p.o. (1)

6

70Zn i.v., 67Zn

p.o.

Adults

9d

5

70Zn i.v., 67Zn

p.o.

Plasma, red blood cells, urine, feces Plasma, urine, feces

Healthy women

Plasma, red blood cells, urine, feces

Healthy

decrease in release of zinc from hepatic metallothionein. The kinetics provided an understanding of the mechanisms involved in zinc redistribution between tissues and between pools within liver after metallothionein induction. Kinetic studies in animals will provide information on the overexpression of and deletion of genes coding for specific proteins involved in zinc transport and metabolism. By comparing kinetics in wild types with those in mutants, the role of specific proteins can be determined in vivo. Studies are in progress to describe the role of metallothionein on zinc kinetics in knockout mice (House, W. A. & Wastney, M. E., unpublished data). Opportunities provided by new analytical techniques The advances that have been made in our understanding of the role of zinc in metabolism have been aided by the devel-

Reference

9 compartments ⫹ intestine (tracer), 11 compartments ⫹ intestine (tracee) 3 compartments ⫹ intestine (tracer), 4 compartments ⫹ intestine (tracee) 7 compartments ⫹ intestine

Lowe et al. 1997

Miller et al. 1998

opment of techniques for the accurate determination of isotopes in biological materials (Barnes 1993, 1996, 1998a). New developments will allow more detailed kinetics to be described in at least three ways (Barnes 1998b). First, increased levels of stable isotope detection will allow smaller doses to be administered, reducing the risk of the dose perturbing the system and reducing cost. Second, methods are being developed to measure isotope enrichment in various zinc-binding species (e.g., metalloproteins and metalloenzymes) within blood and other tissues (Lu and Barnes 1996, Lu et al. 1995). Measurement of the kinetics of zinc bound to different species may provide insight into the forms of zinc that are the most active physiologically. Third, multielement studies will be possible in which the kinetics of several elements will be determined simultaneously on the same subject. This will allow interactions to be studied more extensively and sites, as well as the degree of interaction, to be determined.

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TABLE 2 Zinc kinetics in animals1 Species

Study length

n

Isotopes

Tissues sampled

Conditions

Rat

35 d

NA

65Zn i.v.

Liver, skeleton, whole body

Zn-deficient diet

Rat

24 h

4 each

65Zn i.v.

Plasma, liver, liver-MT, feces

After cAMP induction of MT Zn-deficient, control, Zn-supplemented Control, glutensensitive enteropathy Control, marginal Zn-deficient diet Mature males

Rat

1.5 h

7–9

65Zn i.v.

Plasma, liver, small intestine, kidney

Dog

3h

12

70Zn i.v.

Plasma

Piglet

4d

15,15

70Zn i.v.

Rats

4d

6 each

65Zn i.v.

Plasma, red blood cells, liver, kidney Plasma, liver, muscle, bone, kidney, testes, spleen, skin, duodenum, jejunum, ileum, cecum, colon, whole body, urine, feces

Model

Reference

11 compartments ⫹ intestine 8 compartments 2 compartments

Popov and Besel 1977 Dunn and Cousins 1989 Lowe et al. 1991

2 compartments

Lowe et al. 1995

11 compartments ⫹ intestine 14 compartments ⫹ intestine

Serfass et al. 1996 House and Wastney 1997

1 NA, not available; each, number of animals studies at each time point.

Kinetic studies of the role of zinc in health Abnormal zinc metabolism occurs in a number of disorders, and kinetic studies can be used to identify the site or sites with altered zinc metabolism. This information can be used to determine the mechanisms by which zinc metabolism is involved in the disease as a basis for suggesting therapeutic approaches. By comparing kinetics in healthy subjects versus that in patients, the sites of abnormal zinc metabolism in diabetes, Crohn’s disease, sickle cell anemia and many other disorders could be identified (Vallee and Falchuk 1993). For example, patients with adrenal corticosteroid insufficiency have increased serum zinc and decreased urine zinc levels, and kinetic studies showed that zinc uptake by erythrocytes and liver was low in these patients (Henkin et al. 1984). Rates of zinc uptake were restored by steroid treatment, and the increase in uptake is considered to occur through metallothionein induction in these tissues. To understand the roles of zinc in metabolism, kinetic studies are needed in both healthy and disease states. For example, zinc kinetics in healthy preterm infants (Wastney et al. 1996, 1999) could be used to assess whether zinc metabolism is perturbed in infants who are growing at suboptimal rates and, if so, at which sites. By knowing whether zinc absorption, endogenous excretion or uptake by tissues is perturbed, rather than whether serum levels are altered, it may be possible to more fully understand the role of zinc in human health and to devise therapeutic strategies and dietary recommendations. Areas for research using kinetic studies We have reviewed kinetic studies only at the whole body level here. Many in vitro studies have addressed tissue and cellular uptake and the metabolism of zinc (Reyes 1996). Models will be increasingly used to integrate information from the cellular to the whole body level (Bassingthwaighte 1995). They will be refined to account for changes in metabolism in different physiological and clinical conditions. Most current models are based on kinetic studies in the steady state, but they will be increasingly used to understand the dynamics of zinc metabolism, when zinc levels change such as after a meal, during dietary perturbations or during growth. Models can be

used to represent the current understanding of a system, to identify gaps in knowledge and in experimental design to ensure that necessary and sufficient data are collected to determine parameters of interest. In the future, models will play a greater role in the design of studies as well as in the interpretation of data. With respect to nutrition, kinetic studies can be used to determine the sites where zinc interacts with other nutrients, including trace elements, vitamins and macrominerals; to determine the degree of interaction; and to predict how these interactions may alter zinc requirements. With respect to physiology, kinetic studies could address how rates of zinc metabolism and pool sizes change under different physiological and clinical conditions, such as during growth or pregnancy. By comparing kinetics in healthy and various disease states, the roles of zinc in disease may be elucidated through the identification of differences in metabolic processes. From an environmental perspective, it is important to know how zinc interacts with nonessential metals, and this information can be obtained from kinetic studies. Finally, a powerful use of kinetics will be to study and define the role of gene products in vivo by comparing kinetics in the wild type versus conditions in which the genes are overexpressed or missing. In conclusion, although tissue levels provide a snapshot of the zinc status, kinetic studies allow the exploration of mechanisms, such as the pathways of metabolism, rates of movement and sites of homeostasis, that vary with conditions such as diet, genetics and disease. LITERATURE CITED Babcock, A. K., Henkin, R. I., Aamodt, R. L., Foster, D. M. & Berman, M. (1982) Effects of oral zinc loading on zinc metabolism in humans. II: In vivo kinetics. Metabolism 31: 335–347. Barnes, R. M. (1993) Advances in inductively coupled plasma mass spectrometry: human nutrition and toxicology. Anal. Chim. Acta 283: 115–130. Barnes, R. M. (1996) Analytical plasma source mass spectrometry in biomedical research. Fresenius J. Anal. Chem. 355: 433– 441. Barnes, R. M. (1998a) Capillary electrophoresis and inductively coupled plasma spectrometry: status report. Fresenius J. Anal. Chem. 361: 246 –251. Barnes, R. M. (1998b) Plasma source mass spectrometry in experimental nutrition. Adv. Exp. Med. Biol. 445: 379 –396. Bassingthwaighte, J. B. (1995) Toward modeling the human physionome. Adv. Exp. Med. Biol. 382: 331–339. Dunn, M. A. (1991) Hormonal induction of metallothionein synthesis: its effect

KINETICS OF ZINC METABOLISM on zinc kinetics in the rat. In: Metallothionein in Biology and Medicine (Klaassen, C. D. & Suzuki, K. T., eds.), pp. 283–294, CRC Press, Boca Raton, FL. Dunn, M. A. & Cousins, R. J. (1989) Kinetics of zinc metabolism in the rat: effect of dibutyryl cAMP. Am. J. Physiol. 256:(Endocrinol. Metab.19.):E420 – E430. Fairweather-Tait, S. J., Jackson, M. J., Fox, T. E., Wharf, S. G., Eagles, J. & Croghan, P. C. (1993) The measurement of exchangeable pools of zinc using the stable isotope 70Zn. Br. J. Nutr. 70: 221–234. Foster, D. M., Aamodt, R. L., Henkin, R. I. & Berman, M. (1979) Zinc metabolism in humans: a kinetic model. Am. J. Physiol. 237: R340 –R349. Henkin, R. I., Foster, D. M., Aamodt, R. L. & Berman, M. (1984) Zinc metabolism in adrenal cortical insufficiency: effects of carbohydrate-active steroids. Metabolism 33: 491–501. House, W. A. & Wastney, M. E. (1997) Compartmental analysis of zinc kinetics in mature male rats. Am. J Physiol. 273: R1117–R1125. Janghorbani, M., Ting, B. T. G. & Young, V. R. (1980) Accurate analysis of stable isotopes 68Zn, 70Zn, and 58Fe in human feces with neutron activation analysis. Clin. Chim. Acta 108: 9 –24. Lee, D.-Y., Prasad, A. S., Hydrick-Adair, C., Brewer, G. & Johnson, P. E. (1993) Homeostasis of zinc in marginal human deficiency: role of absorption and endogenous excretion of zinc. J. Lab. Clin. Med. 122: 549 –556. Lowe, N. M., Bremner, I. & Jackson, M. J. (1991) Plasma 65Zn kinetics in the rat. Br. J. Nutr. 65: 445– 455. Lowe, N. M., Green, A., Rhodes, J. M., Lombard, M., Jalan, R. & Jackson, M. J. (1993) Studies of human zinc kinetics using the stable isotope 70Zn. Clin. Sci. 84: 113–117. Lowe, N. M., Hall, E. J., Anderson, R. S., Batt, R. M. & Jackson, M. J. (1995) A stable isotope study of zinc kinetics in Irish setters with gluten-sensitive enteropathy. Br. J. Nutr. 74: 69 –76. Lowe, N. M., Shames, D. M., Woodhouse, L. R., Matel, J. S., Roehl, R., Saccomani, M. P., Toffolo, G., Cobelli, C. & King, J. C. (1997) A compartmental model of zinc metabolism in healthy women using oral and intravenous stable isotope tracers. Am. J. Clin. Nutr. 65: 1810 –1819. Lu, Q. & Barnes, R. M. (1996) Evaluation of an ultrasonic nebulizer interface for capillary electrophoresis and inductively coupled plasma mass spectrometry. Microchem. J. 54: 129 –143. Lu, Q., Bird, S. M. & Barnes, R. M. (1995) Interface for capillary electrophoresis and inductively coupled plasma mass spectrometry. Anal Chem. 67: 2949 – 2956. Miller, L. V., Krebs, N. F. & Hambidge, K. M. (1998) Human zinc metabolism:

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advances in the modeling of stable isotope data. Adv. Exp. Med. Biol. 445: 253–269. Popov, B. V. & Besel, V. S. (1977) Mathematical model of zinc metabolism in rat: imitation of zinc deficient and zinc excessive diets. In: Proceedings of the Trace Element Metabolism in Man and Animals (TEMA3) (Kirchgessner, M., ed.), Vol. 3, Arbeitskreis Tiererna¨hrungsforschung Weihenstephan, FreisingWeihenstephan. Reyes, J. G. (1996) Zinc transport in mammalian cells. Am. J. Physiol. 270 (2 pt 1):C401–C410. Scott, K. C. & Turnlund, J. R. (1994) A compartmental model of zinc metabolism in adult men used to study effects of three levels of dietary copper. Am. J. Physiol. 267: E165–E173. Serfass, R. E., Fang, Y. & Wastney, M. E. (1996) Zinc kinetics in weaned piglets fed marginal zinc intake: compartmental analysis of stable isotopic data. J. Trace Elem. Exp. Med. 9: 73– 86. Vallee, B. L. & Falchuk, K. H. (1993) The biochemical basis of zinc physiology. Physiol. Rev. 73: 79 –118. Wastney, M. E., Aamodt, R. L., Rumble, W. F. & Henkin, R. I. (1986) Kinetic analysis of zinc metabolism and its regulation in normal humans. Am. J. Physiol. 251: R398 –R408. Wastney, M. E., Ahmed, S. & Henkin, R. I. (1992) Changes in regulation of zinc metabolism with age. Am. J. Physiol. 263: R1162–R1168. Wastney, M. E., Angelus, P., Barnes, R. M. & Siva Subramanian, K. N. (1996) Zinc kinetics in preterm infants: a compartmental model based on stable isotope data. Am. J. Physiol. 271(Regul. Integ. Comp. Physiol. 40):R1452– R1459. Wastney, M. E., Angelus, P. A., Barnes, R. M. & Siva Subramanian, K. N. (1999) Zinc absorption, distribution, excretion, and retention by healthy preterm infants. Pediatr. Res. 45: 191–196. Wastney, M. E., Gokmen, I. G., Aamodt, R. L., Rumble, W. F., Gordon, G. E. & Henkin, R. I. (1991) Kinetic analysis of zinc metabolism in humans after simultaneous administration of 65Zn and 70Zn. Am. J. Physiol. 260: R134 – R141. Wastney, M. E. & Henkin, R. I. (1989) Calculation of zinc absorption in humans using tracers by fecal monitoring and a compartmental approach. J. Nutr. 119: 1438 –1443. Wastney, M. E., Patterson, B. H., Linares, O. A., Greif, P. C. & Boston, R. C. (1998) Investigating Biological Systems Using Modeling: Strategies and Software. Academic Press, New York.