Physiological Requirements for Zinc

Abstract

The estimates of zinc physiological requirements by the International Zinc Nutrition Consultative Group (IZiNCG) in 2004 were conspicuously low in comparison with those estimated by the Institute of Medicine (IOM) in 2001. The objective of this review is to explore the reasons for this gap and to reflect on lessons learned. All estimates of inevitable losses of endogenous zinc, especially intestinal excretion of endogenous zinc, were reviewed. An error in zinc menstrual losses, as well as a minor error in the linear regression of endogenous fecal zinc (EFZ) vs. total daily zinc absorption (TAZ) by IOM, were corrected. The review revealed an error by IZiNCG in selecting two data points for the linear regression of EFZ on TAZ. A second major reason for the "gap" is attributable to weighting of the data in the regression analysis by number of subjects per study by IZiNCG. Adjusting for these factors, together with use of the same reference data for body weights, resulted in satisfactory agreement between the two estimates of physiological requirements. The lessons to be learned from this review are discussed together with suggestions for future action by IOM as well as a constructive role for IZiNCG.

Full text

Int. J. Vitam. Nutr. Res., 81 (1), 2011, 1 – 14 1
DOI XXXXXXXXXX Int. J. Vitam. Nutr. Res., 81 (1), 2011, © Hogrefe & Huber Publishers
Original Communication
Physiological Requirements
for Zinc
K. Michael Hambidge, Leland V. Miller and Nancy F. Krebs
Received: January 18, 2011; Accepted: March 21, 2011
Abstract: The estimates of zinc physiological requirements by the International Zinc Nutrition Con-
sultative Group (IZiNCG) in 2004 were conspicuously low in comparison with those estimated by the
Institute of Medicine (IOM) in 2001. The objective of this review is to explore the reasons for this gap
and to reflect on lessons learned. All estimates of inevitable losses of endogenous zinc, especially intes-
tinal excretion of endogenous zinc, were reviewed. An error in zinc menstrual losses, as well as a minor
error in the linear regression of endogenous fecal zinc (EFZ) vs. total daily zinc absorption (TAZ) by
IOM, were corrected. The review revealed an error by IZiNCG in selecting two data points for the
linear regression of EFZ on TAZ. A second major reason for the “gap” is attributable to weighting of
the data in the regression analysis by number of subjects per study by IZiNCG. Adjusting for these fac-
tors, together with use of the same reference data for body weights, resulted in satisfactory agreement
between the two estimates of physiological requirements. The lessons to be learned from this review are
discussed together with suggestions for future action by IOM as well as a constructive role for IZiNCG.
Keywords: Zinc, physiological requirements, factorial estimation
Introduction
This paper offers a critical review and resolution of
major discrepancies in two recent estimates of physi-
ological requirements for zinc [1, 2]. An accurate
estimate, or reasonable agreement among multiple
estimates, of physiological requirements is of criti-
cal importance for our understanding of human zinc
nutrition and homeostasis. Beyond the core impor-
tance of physiological requirements for estimating
and understanding human dietary zinc requirements,
a strong argument can be made for their value in con-
tributing to our understanding of the zinc status of
populations when combined with reliable dietary data.
Furthermore, they provide critical reference points for
bioavailability studies of zinc in biofortified crops [3].
In 2001, the Institute of Medicine (IOM) published
new estimates of physiological zinc requirements [1].
Three years later, the International Zinc Nutrition
Consultative Group (IZiNCG) elected to publish al-
ternative estimates [2]. The conspicuous differences
between the IZiNCG and IOM estimates have cast
a pall over our understanding of zinc homeostasis,
and especially zinc requirements, through the latter
half of the past decade. The estimated physiological
requirement for young children, a critical age group
for preventing zinc deficiency, is approximately 30 %
lower as estimated by IZiNCG, compared to the figure
estimated by IOM [2]. A recent example of the confu-
sion resulting from the disparities between these two
estimates has been provided by a recent study in Ban-
gladesh, in which it was calculated that 0 % of young
children had zinc-deficient diets based on IZiNCG
reference data, while 50 % of these diets were zinc-
deficient based on IOM reference data [4]. These are
examples of the confusion prevailing at this time. Un-

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Int. J. Vitam. Nutr. Res., 81 (1), 2011, © Hogrefe & Huber Publishers
K. M. Hambidge et al: Physiologic requirements for zinc
til these estimates are reconciled, this confusion will
continue to handicap progress in our understanding of
how to best prevent and treat human zinc deficiency,
now regarded as a public health challenge of global
proportions. The objective of the evaluation reported
here is to explore why these differences occurred and
to seek to reconcile these disparate estimates of human
physiological zinc requirements.
Methods
The methods used to determine the total absorbed zinc
(TAZ) and intestinal loss of endogenous zinc (EFZ)
data were reviewed [5 – 16], as well as any calcula-
tions of EFZ when not reported in the publications.
Where substantial errors were detected, data were
revised accordingly. Minor errors and discrepancies
were not addressed.
The linear regression methods used to analyze the
relationship of EFZ to TAZ were evaluated in light
of data characteristics and accepted statistical practice
[17,18]. In particular, the use of sample size-weighted
regression by the IZiNCG was evaluated.
The next step was to re-evaluate estimates of excre-
tion of endogenous zinc from non-intestinal routes
and to use these estimates together with the linear
regression data for EFZ vs. TAZ in the same modified
factorial approach to estimate the physiological zinc
requirements adopted by IOM [1], and subsequently
by IZiNCG [2]. The differences in estimates of integu-
mental losses were initially ignored as these depended
on the deliberate use of different reference data for
body weight [2]. However, as a final step, these refer-
ence data were standardized to determine the effect
of this step on the gap between the two estimates of
physiological requirements.
Results
Estimates of intestinal losses of
endogenous zinc
Apparent errors in data calculations and discrepancies
were discovered in the data used to define the rela-
tionship of EFZ to TAZ by both the FNB/IOMand
IZiNCG (Figure 1). Most of these were minor in na-
ture and some involved judgments regarding use of
data from multiple metabolic study periods. The minor
errors and discrepancies, which on balance affected
physiological requirement estimates by ≤ 0.10 mg Zn/
day, have not been addressed here. The single excep-
tion to this related to the discovery that regression of
the EFZ vs. TAZ data used by the IOM produced
slightly different physiological requirement estimates
than those published (3.3 mg/day and 3.8 mg/day) and
the use in this report of the revised estimates of 3.2 mg
Zn/day for women and 3.9 mg Zn/day for men.
Data selection judgments were not reevaluated, with
the exception of one consequential data selection er-
ror. In this case two data points selected for use by the
IZiNCG were derived using a misconceived calcula-
tion that produced erroneously low EFZ values. The
calculation at issue presented EFZ as the difference
between true zinc absorption (determined by whole-
body counting) and apparent absorption of an orally
administered isotope (0.9 mg Zn/day and 0.4 mg Zn/
day for the two dietary groups [15]). This calculation
provides a measurement of absorbed isotope secreted
into the intestine, not total endogenous zinc intestinal
losses. The publication [15] also reported the correct
calculation of EFZ as the difference between true ab-
sorption and apparent elemental absorption measured
by conventional metabolic balance (1.5 mg Zn/day
and 1.8 mg Zn/day). When the erroneous data were
replaced with these values (Figure 2), the IZiNCG es-
timates of physiological requirements increased from
1.86 to 2.32 mg Zn/day for women and from 2.69 to
3.02 mg Zn/day for men (Figures 3a and 3b). These
changes were so large because there were 14 subjects
Figure 1: Data and regression lines of intestinal losses of
endogenous zinc on absorbed zinc used by the FNB/IOM
(black square symbols and solid line) and the IZiNCG (gray
circle symbols and dashed line). This graph is similar to
Figure 1.3 in the IZiNCG document [2], but shows the ac-
tual data used by the IOM which differ slightly from those
reported by the IZiNCG.

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K. M. Hambidge et al: Physiologic requirements for zinc
in this study and the IZiNCG use of sample size-
weighted regression gave these points more weight
than most of the other data.
Linear regression methods used to
characterize relation of EFZ to TAZ
The IZiNCG used regression analysis wherein the
data were weighted by sample size on the expectation
that larger samples would produce greater precision
and, therefore, ought to be accorded more weight in
the analysis [19]. Since 16 of the data were reported
with sufficient information to permit the calculation
of variance, it was possible to evaluate the appro-
priateness of the weighting scheme. The correlation
between variance and sample size was significant
(r = 0.52, P = 0.041), but the correlation was positive,
not negative as would be expected (Figure 4). When
“outliers” were removed, the remaining data were un-
correlated (r = –0.075, P = 0.81). Therefore, weighting
by sample size was not supported by the data. When
ordinary least squares (unweighted) regression was
used instead (Figure 5), the IZiNCG physiological
requirement estimates increase to 2.67 mg Zn/day
for women and 3.44 mg Zn/day for men (Figures 3a
and b). An analysis of the residuals from the ordinary
least squares (OLS) regression of the IZiNCG data
indicated that error variance was not constant, varying
directly with TAZ or EFZ magnitude (P = 0.0033). A
similar analysis of the regression residuals from the
IOM data found the same relationship, though it was
not significant (P = 0.056). With both the IOM and
IZiNCG data, new regression analyses with the data
weighted by the reciprocal of variance estimated from
the residuals produced physiological requirement es-
timates that varied by less than 0.1 mg Zn/day from
those derived with the OLS regression.
Figure 2: Revision of the intestinal endogenous zinc loss ver-
sus absorbed zinc data used by the IZiNCG to replace two
erroneous data (x symbols) with more accurate data (open
circles) from the same publication. The original IZiNCG
regression line is also shown.
Figure 3: Comparison of estimates of zinc physiological
requirements. Bars at the extreme left depict the original
IZiNCG estimates of requirements for adult men (3a) and
adult women (3b). Corrected original IOM estimates by
IOM are depicted by extreme right hand bars. This figure
shows the extent to which the gap between these estimates
is closed by correction of the IZiNCG data (Figure 2) and
the elimination of weighting by number of subjects per
data point in regression of endogeous fecal zinc versus total
absorbed zinc per day. In addition, Figure 3b shows the
extent to which the gap for females is further narrowed by
correction of an error by FNB/IOM in the estimate of zinc
losses in menses.

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K. M. Hambidge et al: Physiologic requirements for zinc
Estimates of non-intestinal losses
of endogenous zinc
As correctly noted by IZiNCG, the value for men-
strual losses used by IOM resulted from an error in
using the data from their quoted reference [20]. The
correction of female menstrual losses from 0.1 to
0.01 mg Zn/day reduced the FNB/IOM estimate of
zinc physiological requirement for adult women to
2.97 mg Zn/day (Figure 3b).
The final step of standardizing weight reference
data (when added to the modifications of IZiNCG
data) closed the gap to 4 % of the original gap for men
and 2.5 % of the original gap for women (Figures 6a
and 6b).
Discussion
The results of this review clarify the reasons for the
conspicuous gap between the IOM and IZiNCG esti-
mates of physiological zinc requirements. Two major
factors, attributable to the IZiNCG, are the selection
Figure 4: Relation of measured sample variance to sample
size for 16 of the data used by the IZiNCG for which suf-
ficient information to calculate the variances was reported.
The “2” next to a symbol indicates that the symbol rep-
resents two data having the same values. These are cases
where a pooled standard deviation was reported for two
measurements. Counter to expectation, there is a significant
positive correlation between variance and sample size (r =
0.52, P = 0.041). When the outlying points are removed, the
remaining data are uncorrelated (r = -0.075, P = 0.81).
Figure 5: Regressions of IOM (black square symbols and
solid line) and IZiNCG (gray circle symbols and dashed
line) data after correction of IZiNCG data and elimination
of weighted regression.
Figure 6: The further reduction in the difference between
FNB/IOM and IZiNCG estimates of physiological zinc re-
quirements for adult men (6a) and women (6b) if, in addi-
tion to the corrections depicted in Figure 3, the same body
weight standards used by FNB/IOM are applied (not as a
correction factor) to IZiNCG estimates. The percentages
of the gap unaccounted for are 4 % and 2.5 % for men and
women, respectively, of the IOM estimates.

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K. M. Hambidge et al: Physiologic requirements for zinc
of erroneous data and the weighting of data by sample
size in the regression analysis. Together, these two
factors accounted for 58 % and 62 % of the gaps for
men and women, respectively. Correction of an error
in estimated menstrual losses by IOM has also helped
to close the gap for women.
After these corrections, the remaining difference
is attributable to the use of different reference data
for adult weights, which in turn impacted estimates
of integumental losses, leading to the difference in
the data sets. Discussion of the relative merits of the
reference data for weight is beyond the scope of this
paper; however, it is useful to understand the effect
these differences have on estimated physiological zinc
requirements.
The data relating EFZ to TAZ have a major role
in estimating physiological requirements and, from
there, to estimating dietary zinc requirements. The
current use of linear regression analysis of EFZ versus
TAZ as a core strategy first evolved in establishing the
(dietary reference intakes) DRIs for zinc and was sub-
sequently accepted and used by IZiNCG. IOM elected
to utilize only adult male data (lack of individual data
limiting this to means) because the slope of regression
analysis for female data alone was insignificant, with
wide confidence intervals. This remains so despite an
increase in number of studies [21], likely attributable
to the relatively limited range of mean TAZs for the
female studies. These calculations were then applied
to adult women with appropriate modifications for
different estimated losses from non-intestinal routes.
IZiNCG elected to use all available data for both gen-
ders combined to evaluate the relationship between
EFZ and TAZ. Of note, the regression results for
these combined data are not significantly different
from male alone [21].
Regarding the IZiNCG use of sample size-weighted
regression, an examination of measurement variability
of a large subset of the IZiNCG data demonstrated
that the assumption of a positive relation between
measurement precision and sample size was not well
founded. This may be attributable to the heteroge-
neous experimental designs and analytical methods
used in the studies from which the data originated.
Furthermore, this application of weighted regression
strays from the principal use of weighted regression
as a remedy for non-constant error variance wherein
data are typically weighted by the reciprocal of an
estimated error variance function [17, 18]. An appro-
priate relationship between error variance and sample
size would need to exist for the regression technique
used by the IZiNCG to be valid; and the measured
sample variances indicate that the necessary relation-
ship does not hold with these data. Whatever differ-
ences in opinion on the different statistical handling
of these data, all can agree that weighting by number
was one of two major reasons for differences between
the IZINCG and IOM estimates. Another incidental
effect of weighting by sample size was that the result-
ing regression line substantially favored data from
females as 62 % of the individual data included were
from female subjects.
Several additional items of discussion are relevant
to this review. There is a major need for well-designed,
stable isotope-based studies to acquire additional data
to assist in factorial estimates of zinc requirements,
especially experimental data for women, children, and
perhaps the elderly. However, an important reminder
from this experience is that great care is essential to
make appropriate and accurate use of the data already
available. This experience also serves as a reminder
of the importance of adequate internal and external
review not only of a preliminary draft, but of the final
document.
This is also an appropriate moment to reflect on
which organizations should assume the responsibility
of publishing their own versions of estimated physi-
ological and dietary requirements for any nutrient.
In this case it is reasonable to question whether it is
appropriate to claim to provide “international” esti-
mates when, with the exception of one study of two
groups of women [6], virtually all of the data utilized
in deriving these estimates were obtained from stud-
ies conducted in the United States. IOM DRIs are
widely used for international purposes, for example in
the recent development of recommendations for zinc
fortification of flour [22]. Apart from questions about
the validity of current DRI Upper Levels for zinc and
lack of a speedy mechanism for correcting an obvious,
though minor, error in losses of zinc in menses, the zinc
DRIs continue to serve well. It is, however, unfortu-
nate, that the DRI process was insular in its accepted
sources of data and it is hoped that this policy has or
will change. The Panel on Micronutrients (including
zinc) for the DRIs wisely avoided tackling the phytate
issue because the necessary data were unavailable at
that time, at least in a format that was usable. Sufficient
data and an apparently valid model of the impact of
dietary phytate on zinc absorption are now available
[23, 24] and were supported recently by an indepen-
dent analysis [25]. This would now be an excellent,
indeed urgent, time for the IOM to review the DRIs
for zinc and extend these to include the inhibitory
effect of dietary phytate. It would also be beneficial
to see the IOM less reluctant to recognize the im-
portance of the DRIs beyond North America and be

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K. M. Hambidge et al: Physiologic requirements for zinc
willing to assume some broader, overt international
responsibility as is likely to occur with the guidelines
currently being developed by EURRECA (European
Micronutrient Recommendations Aligned) [26]. IZ-
iNCG could make a useful contribution by supporting
these major established or new initiatives while giving
special attention to the impact of pathophysiological
and environmental factors, which are likely to be a
special burden on zinc requirements in poor, tropical
environments.
In conclusion, the outcome of this study designed
to determine the extent to which the widely diver-
gent estimates of physiological requirements for zinc
by IZiNCG and IOM can be reconciled, has been
very reassuring. While factorial methodology may not
appear exciting or novel, it remains the only estab-
lished means of estimating zinc requirements. With
the handicap of these differences in estimates behind
us, it is time to move on to new horizons including
incorporation of phytate into estimates, obtaining the
experimental data needed for more direct estimates
of zinc requirements for young children with a wide
range of phytate intakes, and re-addressing the issue
of upper limits for zinc as major goals. An important
prerequisite is priority attention to the acquisition of
more adequate experimental data, especially that for
excretion of endogenous zinc.
Acknowledgements
This work was supported by HarvestPlus (8030) and
in part by the International Atomic Energy Agen-
cy (IAEA 13254/R3) and the National Institutes of
Health (K24 DK083772).
Abbreviations
DRI Dietary Reference Intakes
EFZ Endogenous Fecal Zinc
EURRECA EURopean micronutrient RECom-
mendations Aligned
IOM Institute of Medicine
IZiNCG International Zinc Consultative Group
TAZ Total Absorption of Zinc
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K. Michael Hambidge, MD
University of Colorado Denver
Pediatric Nutrition
12700 E. 19th Ave
Box C225
Aurora, CO 80045
USA
E-mail: Michael.Hambidge@ucdenver.edu