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Supplement: 11th International Symposium on Trace Elements in Man and Animals

Exchangeable Zinc Pool Size in Infants Is Related to Key Variables of Zinc Homeostasis ^{1 ,2 ,3}

Nancy F. Krebs^*,4, K. Michael Hambidge^*, Jamie E. Westcott^*, Leland V. Miller^*, Lei Sian^*, Melanie Bell and Gary Grunwald

Section of Nutrition, Departments of ^* Pediatrics and Preventive Medicine and Biometrics, University of Colorado Health Sciences Center, Denver, CO 80262

⁴ To whom correspondence should be addressed. E-mail: nancy.krebs{at}uchsc.edu.

	ABSTRACT

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The exchangeable zinc pool (EZP) is the sum of the combined pools that exchange with zinc in the plasma within 48–72 h and is thought to be critical for zinc-dependent biological processes. The size of the EZP in adults has been found to be positively related to dietary zinc intake, daily absorbed zinc and fecal excretion of endogenous zinc. In this study, we examine data on EZP size in relation to variables of zinc homeostasis in normal infants on different feeding regimens. Data from 45 male infants who participated in one of four different studies contribute to the analysis. The feeding regimens include exclusive breastfeeding (n = 9; age, 2–5 mo); breastfeeding plus modest supplementation with cow's milk–based formula (n = 16; age, 3–4 mo); exclusive formula feeding (n = 4; age, 3–4 mo) and exclusive breastfeeding plus complementary foods (n =16; age, 7 mo). Fractional absorption was determined by fecal monitoring after oral administration of zinc-stable isotopes. Urine enrichment 4–8 d posttracer was used to determine endogenous fecal zinc (7-mo-old infants excepted) and EZP size. Univariate correlations and multivariate regression analyses were performed between EZP and age, weight, dietary zinc intake, fractional absorption, total absorbed zinc and endogenous fecal zinc. Results include no significant relationship between EZP size and age, body weight or fractional absorption but a positive relationship with daily absorbed zinc and endogenous fecal zinc excretion. We conclude that the amount of absorbed zinc is not tightly regulated, and that endogenous fecal zinc is dependent on and responsive to the zinc status of the organism.

KEY WORDS: • exchangeable zinc pool • zinc homeostasis • fractional absorption • absorbed zinc

In 1994 we described a theoretical and experimental rationale for a simple method of estimating the size of the combined pools of zinc that intermix and exchange with the zinc in plasma within 48–72 h in the adult (1). This so-called exchangeable zinc pool (EZP) ⁵, which accounts for 10% of total body zinc in adults, is considered to be particularly important for zinc-dependent biological processes. Estimates of the size of the EZP might reasonably be expected, therefore, to provide useful perspectives about zinc nutritional status and zinc homeostasis (1, 2). Although this remains to be fully evaluated, measurements of the size of the EZP in adults have added to understanding of zinc homeostasis and its limitations (1– 3). Specifically, studies of adults indicate that EZP size is positively correlated not only with dietary zinc intake but also with the quantities of zinc absorbed each day and with daily excretion of endogenous fecal zinc (3).

Estimates of EZP size can be obtained from plasma- or urine-enrichment data after intravenous administration of a zinc-stable isotope tracer (1). Estimates of EZP size can also be derived from urine enrichment after oral administration of a zinc tracer provided there is a simultaneous measurement of fractional zinc absorption (4). The latter is especially useful for healthy infants for whom intravenous administration is often not feasible.

During the course of a series of studies of zinc homeostasis in normal infants on different feeding regimens, we accumulated data on EZP size. The objective of this article is to examine these data collectively to evaluate relationships between EZP size and other key variables of zinc homeostasis. With the exception of the data from one recent study of breastfed infants who receive complementary foods (5), the EZP data in the present article have not been previously published.

	METHODS

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    Study design. In each of these studies, fractional absorption of zinc was determined using fecal monitoring techniques that are based on the fecal excretion of isotope for 8 d after oral administration of zinc-stable isotope tracer (4). Urine samples were collected between days 4–8 after tracer administration for determination of endogenous fecal zinc (studies 1–3) and estimation of the size of the EZP. Studies were undertaken in the subjects' homes using methods described previously (5– 7).

    Subjects and diets. All subjects recruited for the four studies were healthy male infants that ranged in age from 2–7 mo (Table 1). The principal caregivers of all infants gave their informed consent, and the studies and consent forms were approved by the Colorado Multiple Institutional Review Board of the University of Colorado Health Sciences Center.

    Isotope preparation and administration. Zinc oxide powder enriched with ⁷⁰Zn or ⁶⁷Zn was obtained from Oak Ridge National Laboratories (Oak Ridge, TN) and prepared for oral administration as previously described (6, 7). For the infants in study 2, the human milk and formula were labeled separately with one of the two isotopes. An accurately weighed quantity of the isotope solution was added to a weighed volume of human milk or formula that approximated the infants' 24-h intake. After a minimum 4-h period for equilibration, the labeled milk or formula was quantitatively fed to the infants in approximately six feeds over a 24-h period. For the infants in study 4, a single test meal that consisted of a complementary food (pureed beef or iron-fortified rice cereal) was labeled (5). Research personnel were present to supervise the administration of all labeled feeds.

    Sample collection. Complete fecal collections were obtained for 8 d after isotope administration. Spot urine samples with a typical volume of 100 mL were collected twice daily (morning and evening) for days 3–8 posttracer administration. These samples were collected as previously described using an adhesive zinc-free plastic bag (6, 7).

    Sample preparation and analyses. Individual fecal samples were ashed in a muffle furnace for 24 h at 450°C. Preparation of individual urine samples included wet digestion with concentrated nitric acid and H₂O₂. Dried samples were then ashed in a muffle furnace as were the fecal samples. After determination of zinc concentrations by atomic absorption spectrophotometry, reconstituted ash was placed on a chelating resin column to remove major minerals. The resulting eluents were then placed on ion-exchange resin columns, and isotopic enrichment was determined by fast-atom bombardment mass spectrometry as described previously (5– 7). Zinc concentrations in the human milk and formula samples were determined using the same procedures as the fecal samples.

    Data processing and statistical analyses. The variables examined in relation to EZP size included total dietary zinc (volume of intake multiplied by zinc concentration), fractional absorption (determined by fecal monitoring), total absorbed zinc (zinc intake multiplied by fractional absorption) and endogenous fecal zinc (determined by isotope dilution method) (4, 6). For the infants in study 4, only dietary zinc intake values from complementary foods, fractional absorption and EZP were available, because the isotope was given with only a single test meal (5). For all studies, the orally administered quantity of isotope was adjusted for fractional absorption to estimate the size of EZP. After calculation of the quantity of tracer absorbed, EZP size was estimated from division of this quantity by the enrichment value at the y-intercept of the linear regression of a log-transformed plot of the urine-enrichment data for days 3–8 posttracer administration (1).

Statistical analyses were carried out using SAS software (SAS/STAT User's Guide, Version 8, 1999, SAS Institute Inc., Cary, NC). Multiple linear regression was used to study relationships among the zinc variables total dietary zinc, total absorbed zinc, endogenous fecal zinc and EZP. All four zinc variables were log-transformed due to indications of nonnormality and unequal variance in residual analyses. Adjustments for body weight, age and study were made by including these variables as predictors in regressions involving the zinc variables. Results of regression analysis were considered significant at P < 0.05.

	RESULTS

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The means (± SD) for dietary zinc intake, total absorbed zinc, endogenous fecal zinc and EZP size for each study are shown in Table 2. The results of univariate correlations with EZP are shown in Table 3. The negative association between age and EZP was primarily due to the effect of subjects in study 4, who were older and had quite low zinc intakes. When age versus EZP was adjusted for weight, dietary zinc and study, there was no longer a significant effect (P = 0.68). Fractional absorption was not significantly related to EZP by either univariate or multivariate analyses. Multivariate analysis of total absorbed zinc versus EZP, adjusting for weight, age and study, indicated a marginally significant positive relationship with the coefficient of 0.63, r² = 0.56 and P = 0.05 (Fig. 1). Multivariate regression, adjusting for weight, age, diet zinc and study, indicated a significant effect between EZP and endogenous fecal zinc with r² = 0.71 and P = 0.02 (Fig. 2).

View larger version (17K): [in this window] [in a new window]   FIGURE 1  Total absorbed zinc versus exchangeable zinc pool (n = 29). Key to legends: BF, exclusively breastfed infants (6); BF + FF, breastfed infants receiving supplemental formula (8); FF, exclusively formula-fed infants (7). Multivariate regression analysis adjusting for weight, age and study indicates significant positive relationship (P = 0.05).

View larger version (17K): [in this window] [in a new window]   FIGURE 2  Endogenous fecal zinc versus exchangeable zinc pool (n = 29). Multivariate regression analysis adjusting for weight, age, diet zinc and study indicates significant positive relationship (P = 0.02).

	DISCUSSION

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It is reasonable to hypothesize that the size of the EZP is dependent on zinc nutritional "status," and this premise is supported by consistent observations of positive correlations between dietary and/or absorbed zinc (milligrams per day) and EZP size (1, 3, 11, 12). The apparent discrepancy between our findings in multiple studies and those reported by other research groups (9, 13, 14) is not readily explicable but emphasizes the need for more extensive research. Blood sampling was not undertaken in the infants included in the studies that provide the basis for this report. Hence, no comparison is possible with other potential biomarkers of zinc status.

In our own experience, the relationships observed between EZP size and both absorbed zinc and endogenous zinc excreted via the intestine are of most interest, because they provide evidence of interrelationships that are of cardinal importance to the maintenance of whole-body zinc homeostasis (11). In this context, these data both confirm and extend previous observations and are consistent with the following conclusions. First, the quantity of zinc absorbed each day does not seem to be regulated in response to changes in zinc "status" as reflected by EZP size. Otherwise, an inverse relationship between EZP size and absorbed zinc would be expected, whereas in fact the reverse is observed. This is compatible with the conclusion that regulation of total absorbed zinc is limited and results in an EZP size that varies directly with the quantity of zinc ingested and absorbed. Whether and at what upper and lower levels of absorption and EZP size this relationship might cease is presently unknown. The lack of any apparent relationship between fractional absorption and EZP size also suggests that fractional absorption is more reflective of the amount of zinc in the lumen as well as presence of any affectors of absorption (e.g., phytic acid) than of zinc status of the host per se.

Second, the quantity of endogenous zinc excreted via the intestine has a positive linear relationship to the size of the EZP over the very wide range of zinc intake (0.6–10 mg/d) of the normal infants included in these studies. A similar positive correlation was observed in adult women with a smaller range of dietary zinc intakes (3). We thus propose that the quantity of endogenous zinc excreted via the intestine is dependent on and responsive to the host's zinc status.

The risks of post hoc analyses of relationships between variables obtained from somewhat heterogeneous groups of infants are acknowledged. The consistency, however, of the findings reported herein with observations in adults provides stimulus for further investigations. If the conclusions herein are supported by data obtained under controlled experimental conditions, they offer important insight into the processes responsible for maintaining zinc homeostasis and may also provide clues for conditions associated with its disruption.

	FOOTNOTES

 
¹ Published in a supplement to The Journal of Nutrition. Presented as part of the 11th meeting of the international organization, "Trace Elements in Man and Animals (TEMA)," in Berkeley, California, June 2–6, 2002. This meeting was supported by grants from the National Institutes of Health and the U.S. Department of Agriculture and by donations from Akzo Nobel Chemicals, Singapore; California Dried Plum Board, California; Cattlemen's Beef Board and National Cattlemen's Beef Association, Colorado; GlaxoSmithKline, New Jersey; International Atomic Energy Agency, Austria; International Copper Association, New York; International Life Sciences Institute Research Foundation, Washington, D.C.; International Zinc Association, Belgium; Mead Johnson Nutritionals, Indiana; Minute Maid Company, Texas; Perrier Vittel Water Institute, France; U.S. Borax, Inc., California; USDA/ARS Western Human Nutrition Research Center, California and Wyeth-Ayerst Global Pharmaceuticals, Pennsylvania. Guest editors for the supplement publication were Janet C. King, USDA/ARS WHNRC and the University of California at Davis; Lindsay H. Allen, University of California at Davis; James R. Coughlin, Coughlin & Associates, Newport Coast, California; K. Michael Hambidge, University of Colorado, Denver; Carl L. Keen, University of California at Davis; Bo L. Lönnerdal, University of California at Davis and Robert B. Rucker, University of California at Davis.

² Presented in part as oral abstract presentation at Experimental Biology meeting, San Diego, 2000 [Krebs NF, Westcott JE, Miller LV, Herrmann TS & Hambidge KM (2000) Exchangeable zinc pool size in normal infants: correlates with parameters of zinc homeostasis. FASEB J 14: A205 (abs.)].

³ This work was supported by research grants from the National Cattlemen's Beef Association, Ross Products Division Abbott Laboratories and Mead Johnson Nutritionals and from the following National Institutes of Health (NIH) grants: General Clinical Research Centers Program, National Centers for Research Resources, NIH grant M01 RR00069; Colorado Clinical Nutrition Research Unit, NIH grant DK48520 and NIH grant DK02240.

⁵ Abbreviation used: EZP, exchangeable zinc pool.

	LITERATURE CITED

TOP ABSTRACT METHODS RESULTS DISCUSSION LITERATURE CITED

 

1. Miller, L. V., Hambidge, K. M., Naake, V. L., Hong, Z., Westcott, J. L. & Fennessey, P. V. (1994) Size of the zinc pools that exchange rapidly with plasma zinc in humans: alternative techniques for measuring and relation to dietary zinc intake. J. Nutr. 124: 268–276.

2. Miller, L. V., Krebs, N. F. & Hambidge, K. M. (2000) Development of a compartmental model of human zinc metabolism: identifiability and multiple studies analyses. Am. J. Physiol. Regul. Integr. Comp. Physiol. 279: R1681–R1684.

3. Sian, L., Mingyan, X., Miller, L. V., Tong, L., Krebs, N. F. & Hambidge, K. M. (1996) Zinc absorption and intestinal losses of endogenous zinc in young Chinese women with marginal zinc intakes. Am. J. Clin. Nutr. 63: 348–353.[Abstract/Free Full Text]

4. Krebs, N., Miller, L. V., Naake, V. L., Lei, S., Westcott, J. E., Fennessey, P. V. & Hambidge, K. M. (1995) The use of stable isotope techniques to assess zinc metabolism. J. Nutr. Biochem. 6: 292–307.

5. Jalla, S., Westcott, J., Steirn, M., Miller, L. V., Bell, M. & Krebs, N. F. (2002) Zinc absorption and exchangeable zinc pool sizes in breast-fed infants fed meat or cereal as first complementary food. J. Pediatr. Gastroenterol. Nutr. 34: 35–41.[Medline]

6. Krebs, N. F., Reidinger, C. J., Miller, L. V. & Hambidge, K. M. (1996) Zinc homeostasis in breast-fed infants. Pediatr. Res. 39: 661–665.[Medline]

7. Krebs, N., Reidinger, C. J., Miller, L. V. & Borschel, M. (2000) Zinc homeostasis in normal infants fed a casein hydrolysate formula. J. Pediatr. Gastroenterol. Nutr. 30: 29–33.[Medline]

8. Krebs, N. F., Steirn, M. & Westcott, J. E. (2000) High iron (Fe) formula associated with lower zinc (Zn) absorption in breastfed infants. Pediatr. Res. 130: 29–33.

9. 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.[Abstract/Free Full Text]

10. 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.

11. Krebs, N. F. & Hambidge, K. M. (2001) Zinc metabolism and homeostasis: the application of tracer techniques to human zinc physiology. Biometals 14: 397–412.[Medline]

12. Hambidge, K. M., Krebs, N. F., Miller, L. V., Coleman, M., Widler, M. & Fennessey, P. V. (1993) Estimation of the total size of pools of zinc that exchange with plasma within two days in normal infants and adults. In: Proceedings of the Eighth International Symposium on Trace Elements in Man and Animals—TEMA 8, Dresden, Germany, 16–21 May 1993 (Anke, M., Meissner, D. and Mills, C. F., eds.). Verlag Media Touristik, Gersdorf.

13. King, J. C., Shames, D. M., Lowe, N. M., Woodhouse, L. R., Sutherland, B., Abrams, S. A., Turnlund, J. R. & Jackson, M. J. (2001) Effect of acute zinc depletion on zinc homeostasis and plasma zinc kinetics in men. Am. J. Clin. Nutr. 74: 116–124.[Abstract/Free Full Text]

14. Pinna, K., Woodhouse, L. R., Sutherland, B., Shames, D. M. & King, J. C. (2001) Exchangeable zinc pool masses and turnover are maintained in healthy men with low zinc intakes. J. Nutr. 131: 2288–2294.[Abstract/Free Full Text]

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