The Journal of Nutrition

Methodology and Mathematical Modeling

The “Super-Child” Approach Is Applied To

Estimate Retinol Kinetics and Vitamin A Total
Body Stores in Mexican Preschoolers
Veronica Lopez-Teros,1 Jennifer L Ford,2 Michael H Green,2 Brianda Monreal-Barraza,3
Lilian García-Miranda,1 Sherry A Tanumihardjo,4 Mauro E Valencia,1 and Humberto Astiazaran-Garcia3

1
Posgrado en Ciencias de la Salud (PCS), Universidad de Sonora, Hermosillo, Sonora, Mexico; 2 Department of Nutritional Sciences, The
Pennsylvania State University, University Park, PA, USA; 3 Department of Nutrition, Research Center for Food and Development,

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Hermosillo, Sonora, Mexico; and 4 Department of Nutritional Sciences, University of Wisconsin-Madison, Madison, WI, USA

ABSTRACT
Background: Retinol isotope dilution (RID) and model-based compartmental analysis are recognized techniques for
assessing vitamin A (VA) status. Recent studies have shown that RID predictions of VA total body stores (TBS) can be
improved by using modeling and that VA kinetics and TBS in children can be effectively studied by applying population
modeling (“super-child” approach) to a composite data set.
Objectives: The objectives were to model whole-body retinol kinetics and predict VA TBS in a group of Mexican
preschoolers using the super-child approach and to use model predictions of RID coefficients to estimate TBS by RID in
individuals.
Methods: Twenty-four healthy Mexican children (aged 3–6 y) received an oral dose (2.96 μmol) of [13 C10 ]retinyl acetate
in corn oil. Blood samples were collected from 8 h to 21 d after dosing, with each child sampled at 4 d and at 1 other
time. Composite data for plasma labeled retinol compared with time were analyzed using a 6-component model to obtain
group retinol kinetic parameters and pool sizes. Model-predicted TBS was compared with mean RID predictions at 4 d;
RID estimates at 4 d were compared with those calculated at 7–21 d.
Results: Model-predicted TBS was 1097 μmol, equivalent to ∼2.4 y-worth of VA; using model-derived coefficients,
group mean RID-predicted TBS was 1096 μmol (IQR: 836–1492 μmol). TBS at 4 d compared with a later time was
similar (P = 0.33). The model predicted that retinol spent 1.5 h in plasma during each transit and recycled to plasma 13
times before utilization.
Conclusions: The super-child modeling approach provides information on whole-body VA kinetics and can be used
with RID to estimate TBS at any time between 4 and 21 d postdose. The high TBS predicted for these children suggests
positive VA balance, likely due to large-dose VA supplements, and warrants further investigation. J Nutr 2020;150:1644–
1651.

Keywords: children, mathematical modeling, model-based compartmental analysis, retinol isotope dilution, vitamin
A kinetics, vitamin A metabolism, WinSAAM

Introduction (3), which includes assumptions (coefficients) that account

Both vitamin A (VA) deficiency (1) and higher-than-optimal
stores (2) are public health challenges in vulnerable groups such
as children aged <5 y; thus, it is critical that researchers have
practical and reliable methods for assessing VA status, that is,
total body stores (TBS). Retinol isotope dilution (RID) and
model-based compartmental analysis have both been effectively
used to estimate VA status (3). Application of RID involves
collection of a single blood sample at a specified time after
subjects have ingested a known amount of stable isotope-
labeled VA; the ratio of labeled to unlabeled retinol in plasma
is determined and equated to TBS using a prediction equation
for dose absorption, mixing, and catabolism. In the case of
model-based compartmental analysis (4), serial blood samples
are obtained after isotope ingestion and the modeling process
provides information on whole-body retinol kinetics as well
as VA TBS (5–7). Importantly, compartmental analysis has
been shown to improve RID predictions of TBS by providing
group-specific values for the RID equation coefficients (4, 8).
For example, Green et al. (6) applied compartmental analysis
to retinol tracer data obtained from healthy young adults
(9) and found excellent agreement between model-predicted
TBS for the group and the value calculated by RID at 4–5 d
postdosing.

Copyright  C The Author(s) on behalf of the American Society for Nutrition 2020.
Manuscript received November 23, 2019. Initial review completed January 8, 2020. Revision accepted February 12, 2020.
1644 First published online March 5, 2020; doi: https://doi.org/10.1093/jn/nxaa048.
 Because it is generally not feasible to collect serial blood
samples in children, a “super-child” approach has been
developed (10, 11) that facilitates retinol kinetic studies in this
age group. For this technique, 2 blood samples are collected
from each child and data from all subjects are pooled into
a composite data set for modeling, providing information on
retinol kinetics, TBS, and the RID coefficients for the group
(11). This approach has been applied in infants from Mexico
(10); results showed that mean RID predictions calculated at
3–6 d agreed well with model-predicted TBS. Recently, an
improved super-child design, in which all children were sampled
at a common time (4 d), was applied in infants and children
from Bangladesh, Guatemala, and the Philippines (12); group
predictions of TBS by RID are expected to agree with the model.
In addition, model-predicted group-specific RID coefficients can
be used to estimate TBS in individual subjects (8), whereas
traditional RID has generally been applied to predict a mean
value for the group.
The optimal time for applying RID has been a topic of
discussion among users of the method. In studies done over
the past 30 y, RID has been done at 21 d after isotope
administration in adults and at 14 d in children (13, 14).
However, researchers have long been interested in developing
equations that could be applied at an earlier time (e.g., 3 d),
reducing the possibility that subjects might develop an infection
during the test or move from the study site; an earlier sampling
time might also allow for smaller sample volumes, because
isotopic enrichment in plasma would be higher, as well as
result in less burden on study participants and reduced costs
for field operations. In previous studies in adults (8, 15, 16)
and children (17, 18), RID predictions at 3 d were positively
correlated with estimates at 21 d; in school-age children (19),
7-d predictions were correlated with estimates at 14 d (19). In
the super-child analyses previously mentioned (12), blood was
collected from all participants at 4 d plus 1 additional time,
and RID coefficients were calculated by the model at all times
from 4 to 28 d; individual subject values for TBS are calculated
by RID at both times, thus strengthening the power of the
approach.
Here we extend our interest in VA status in Mexican
children by applying compartmental analysis and a super-child
approach to estimate whole-body retinol kinetics and TBS in
preschoolers from a geographic area similar to that of the
infants studied earlier (10); children in the current experiment
were followed for 21 d after isotope administration. Our
results allow us to look at age-related changes in VA status
in this population. In addition, by using model-predicted time-
variant RID coefficients, we were able to make within-subject
This research was supported by the International Atomic Energy Agency
(research contract 16880 to HA-G); BM-B and LG-M received fellowships from
the National Research and Technology Council (CONACyT) of Mexico.
Author disclosures: The authors report no conflicts of interest.
Supplemental Methods, Supplemental Results, Supplemental Tables 1–3,
Supplemental Figure 1, and Supplemental WinSAAM Deck are available from
the “Supplementary data” link in the online posting of the article and from the
same link in the online table of contents at https://academic.oup.com/jn/.
Address correspondence to VL-T (e-mail: veronica.lopez@unison.mx) or HA-G
(e-mail: hastiazaran@ciad.mx).
Abbreviations used: DT, delay time; FD, fraction of the dose; FDp , fraction
of dose in plasma; FSD; fractional standard deviation; RAc, retinyl acetate;
RAE, retinol activity equivalent; RID, retinol isotope dilution; SAp , plasma retinol
specific activity; TBS, total body stores; VA, vitamin A; WinSAAM, Windows
version of Simulation, Analysis and Modeling software.
comparisons of TBS at 4 d when all children were sampled to
values predicted at other times.
Methods
13
C-labeled VA and oral dose
All-trans-8,9,10,11,12,13,14,15,19,20-[13 C10 ]retinyl acetate (RAc)
was synthesized by Cambridge Isotope Laboratories to a chemical
purity ≥98%. For the oral dose, aliquots of the isotope (2.96 μmol)
were dissolved in 200 μL corn oil and stored at −80◦ C until
administered to participating children. The daily VA intake
recommendation for this age group is 300–400 μg retinol activity
equivalents (RAE) per day (see below), and although the VA dose
provided here is larger than that (∼2 times), it is considered a small,
physiological oral dose of stable isotope-labeled VA that does not
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perturb the system (3).
Subjects and study design
Twenty-four preschool-aged children (3–6 y) were recruited in North-
west Mexico from families with middle socioeconomic status. The
selection of subjects was intentional and nonprobabilistic. Exclusion
criteria included the presence of malnutrition (20), diarrhea or fever
during the previous week, the presence of subclinical inflammation
(C-reactive protein ≥6 mg/L; CP3885 Latex Immunoturbidimetric,
Randox), or the presence of anemia (based on hemoglobin <110 g/L
for children aged 6–59 mo, and <115 g/L for children aged 5–11 y;
HemoCue Hb 201+ System) (21, 22); no screened children needed to
be excluded based on these criteria. Because intestinal parasites are
common among children in this area and because such parasites can
interfere with the absorption of VA (23, 24), 400 mg albendazole was
administered as a suspension to all subjects 11 d prior to the study. This
project was approved by the bioethics committee of the Research Center
for Food and Development, Hermosillo, as well as by the Sonora State
Education Secretary. Protocols were explained to parents and informed
consent was obtained.
After an overnight fast, the labeled RAc dose was administered to
each child using a positive displacement pipette (day 0). Children were
then offered a small serving (80 g) of french fries to improve absorption
of the dose. Our original intention was to carry out a 28 d study that
included 10 sampling times, with 2 blood samples collected from each
child. Because a limited amount of isotope was available, we reduced
the duration to 21 d with 9 sampling times (8 and 12 h, and 1, 2, 4, 7,
11, 14, and 21 d); blood was collected from all children (except 1) at 4 d
and from each child at 1 additional time; thus 3 children were sampled
at each of the non-4 d times. For each sample, 5 mL blood was collected
by venipuncture into a BD Vacutainer SST tube. Serum was separated
and frozen at −80◦ C for subsequent analysis. To provide the data on
13 C natural enrichment that was applied as a baseline correction, blood
was collected from 5 additional local children of the same age (19).
Children’s dietary information was collected through a series of
interviews with the parents or caregivers, who completed 3 non-
consecutive 24 h dietary recalls (2 weekdays and 1 weekend day).
Nutrient intake was calculated using the USDA’s food composition
tables (25); the RDA and the Estimated Average Requirements for
macro- and micronutrients were used to estimate dietary adequacy
based on recommendations from the Institute of Medicine (26, 27). For
VA, the estimated average requirement is 210 μg RAE/d for children
aged 1 to 3 y and 275 μg RAE/d for children aged 4 to 8 y (27).
Although Mexican children aged <5 y are provided with a large-dose
VA supplement (210 μmol retinol as retinyl palmitate) semiannually,
included children had not received a VA supplement during the 3 mo
prior to the study.
Analyses
Serum samples were extracted using a modification of the method
described by MacCrehan et al. (28) and retinol concentrations were
determined by HPLC using a 3 μm C18 column (Microsorb 4.6 mm
Vitamin A stores and kinetics in Mexican children 1645
× 10 cm), methanol:water (90:10, v/v) as mobile phase, and RAc
as internal standard. For quality control, we used National Institute
of Standards and Technology standard reference material 968e (Fat-
Soluble Vitamins, Carotenoids, and Cholesterol in Human Serum);
results were within the acceptable range. Additionally, we analyzed
3 aliquots of pooled serum per run to assess intra- and interanalysis
variation and found a CV of 2.7%. The ratio of serum labeled:unlabeled
retinol (13 C/12 C) was determined by GC-combustion-isotope ratio MS
(29) at the University of Wisconsin-Madison; the ratio was corrected
using the natural abundance of 13 C (1.08% of total C) measured in
serum of nonparticipating children (19).

Super-child data set and kinetic analyses

Plasma enrichment of [13 C10 ]retinol in each analyzed sample was
calculated based on the 13 C/12 C ratio (see Supplemental Methods)
and converted to fraction of the oral dose in plasma (FDp ) using

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Equation 1:


FDp = [enrichment × retinol concentration (μmol/L)]

×plasma volume(L) /dose(μmol) (1)

where plasma volume was estimated for each child using the average
sex-specific hematocrit for this age group and an estimate of blood
volume (liters) calculated using the nomograms of Linderkamp et al.
(30) based on age, height, and body weight.
To generate a composite (super-child) data set, we calculated the
geometric mean FDp at each time. Data for geometric mean FDp were
plotted over time (8 h to 21 d), and model-based compartmental analysis
was applied using the Windows version of the Simulation, Analysis and
Modeling software (WinSAAM version 3.3.0) (31) and a previously
published (12) 6-component model describing whole-body VA kinetics
in children (Figure 1A; see figure legend for a description of the model
components). Model parameters include fractional transfer coefficients
[L(I,J)s, or the fraction of retinol in compartment J transferred to
compartment I each day] and delay times [DT(I), or the time (d) that
retinol spends in compartment I] for components 3 and 8. Absorption
efficiency for preformed VA was fixed at 80% (2), and an estimate of
VA intake was included as weighted data (32) to improve confidence in
model predictions such as VA TBS and disposal rate (33). Because intake
estimates included both preformed VA and provitamin A carotenoids
[as RAE based on the Institute of Medicine conversion factors (27)], we
adjusted the contribution of provitamin A to its equivalent as preformed
VA. Specifically, for each child, adjusted VA intake was calculated
as preformed VA + (provitamin A/0.8), assuming 80% absorption
efficiency for preformed VA; then the geometric mean of these values
was used as a modeling input. For modeling, geometric mean FDp data
were weighted using a fractional SD (FSD) of 0.05 at all times except
4 d when the weight was increased to 0.025 in light of the larger
number of children sampled at that time; the adjusted VA intake for
the group was weighted using an FSD of 0.05. The value for L(8,5)
(Figure 1A) was calculated, as detailed in Ford et al. (12), to reflect
50% of the disposal rate and fixed in the model; DT(8) was also
fixed, at 75 min. Data were fit to the model shown in Figure 1A by
systematically/manually adjusting the value for each parameter until
the model predictions agreed with the observed FDp mop data. Then,
weighted nonlinear regression analysis was performed in WinSAAM to
obtain final parameter values and their statistical uncertainties. Finally,
plasma retinol pool size [M(5), μmol; Figure 1A] was calculated for each
sample as plasma retinol concentration (μmol per liter) × estimated
plasma volume (liters); the geometric mean was calculated and used in
the steady state model solution in WinSAAM to predict the mass of
VA in other compartments [M(I), μmol], including TBS [M(6) + M(7)],
and VA transfer rates [R(I,J)s, or μmol in compartment J transferred to
compartment I each day], including VA disposal rate and intake [U(3)].
FIGURE 1 Conceptual compartmental model for retinol kinetics
in children (A), with results for Mexican preschoolers (B). Circles
represent compartments, the rectangles are delay components,
and interconnectivities between compartments [L(I,J)s] are fractional
transfer coefficients or the fraction of retinol transferred to com-
partment I from compartment J each day; DT(I)s are delay times
(or the time spent in delay element I). DT(3) represents the site of
input of the orally administered [13 C10 ]retinyl acetate dose (∗) and
dietary VA [U(3)]. Component 3 and compartment 4 represent the
processes of VA digestion and absorption as well as chylomicron
production and catabolism until clearance of chylomicron remnants by
liver hepatocytes (compartment 4). Retinol in compartment 4 is bound
to retinol-binding protein and secreted into plasma (compartment 5),
the site of sampling (triangle). Plasma retinol exchanges with VA in
2 extravascular pools, 1 smaller (compartment 7) and 1 larger storage
pool (compartment 6), which is also a site of irreversible loss from
the system; the sum of VA in compartments 6 and 7 corresponds to
TBS. A portion of the plasma retinol pool is also transferred irreversibly
to component 8, which represents uptake by tissues for immediate
utilization following a delay of 75 min (12). Panel B shows final model
results including L(I,J)s, R(I,J)s [i.e., transfer rates calculated as L(I,J) ×
M(J)], and DT(I)s; compartment masses [M(I)]s are indicated inside the
corresponding compartment. Absorption efficiency was fixed at 80%
and the adjusted dietary VA intake (1.59 μmol RAE/d) was included
in the model as weighted data. VA disposal rate = R(10,6) + R(8,5).
All 7 adjustable parameters were well identified (FSD < 0.5) (31); the
mean FSD was 0.187 (range: 0.0460–0.356). FSD; fractional standard
deviation; RAc, retinyl acetate; RAE, retinol activity equivalent; TBS,
total body stores; VA, vitamin A.

1646 Lopez-Teros et al.

TABLE 1 Anthropometric and biochemical characteristics of
participating children1
Characteristic Value
Age, y 4.80 ±
Weight, kg 19.98 ±
Height, cm 109 ±
Weight-for-age z-score 0.55 ±
Height-for-age z-score 0.04 ±
BMI-for-age z-score 0.75 ±
Whole blood hemoglobin,2 g/L 138 ±
Serum C-reactive protein, mg/L <6
Serum retinol, μmol/L 1.34 ±
1
Values are means ± SD for Mexican children (n = 24).
2
n = 23.
Estimation of TBS by RID
In addition to the model’s prediction of TBS for the group, we also
calculated this variable for individual children using the RID equation
presented by Green (34) and shown here as Equation 2:
   
TBS μmol = Fa × S × 1/SAp
where Fa represents the fraction of the oral tracer dose absorbed and
found in the exchangeable extravascular pools (compartments 6 and 7)
at the time of sampling, S is the ratio of retinol specific activity in plasma
to that in the exchangeable extravascular pools, and SAp is retinol
specific activity in plasma (FDp /μmol of retinol in plasma). Group
values for Fa and S were calculated over time using the model and then
included in Equation 2 along with each child’s SAp at the corresponding
time. Using the model, Fa was calculated as [FD(6)t + FD(7)t ], where
FD(I)t is the fraction of the dose in compartment I at time t, and S
was calculated as [FD(5)t /M(5)]/{[FD(6)t + FD(7)t ]/[M(6) + M(7)]}.
TBS was calculated for all children at 4 d plus at times after 4 d
for individuals sampled later than that; predictions were compared to
evaluate time-related differences in TBS.
Statistical analyses
Data related to subject characteristics, dietary analyses, serum retinol
concentrations, and hemoglobin concentrations were analyzed using
NCSS 11 statistical software (version 11.0.6); results are presented as
arithmetic means ± SD unless otherwise specified. A paired sample t
test was used to check whether serum retinol concentrations differed in
the 2 blood samples taken from each subject. Predictions of TBS by RID
are presented as geometric means and IQRs. TBS predictions at 4 d were
compared with those at later times using the Wilcoxon signed rank test.
For all tests, P < 0.05 was considered significant.
Results
Subjects
The median age of the 24 participants (11 females) was 4.8 y.
Based on average z-scores (Table 1), all children were well
nourished; none were anemic based on serum hemoglobin con-
centration (22), and all children tested negative for subclinical
inflammation based on C-reactive protein measurements. Mean
serum retinol concentration was 1.34 ± 0.34 μmol/L; retinol
concentrations were not different in the 2 samples obtained
from each child (P = 0.48). Based on WHO guidelines for VA
deficiency [plasma retinol concentration <0.70 μmol/L (35)],
none of the children were VA deficient.
Participants’ average habitual energy intake
(1561 ± 281 kcal/d), calculated based on 3 nonconsecutive
24-h dietary recalls, was ∼300 kcal higher than recommended
(26, 27); macronutrient distribution was 51% from
0.67
6.04
6.22
1.52
0.95
1.73
12.2
0.34
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FIGURE 2 Geometric mean observed FD in plasma for
[13 C10 ]retinol vs. time in children after oral administration of
labeled vitamin A and model-simulated FD in plasma and 2 EV pools
(compartments 6 and 7; Figure 1A). Symbols are observed data and
lines are model simulations. The composite data set for 24 children
included 3 subjects per time except at 4 d when 23 children were
sampled. Observed data at 11 d include the original FD (x) and the
(2) adjusted FD (•) (see Results); the adjusted FD at 11 d was used for
modeling. EV, extravascular; FD, fraction of dose.
carbohydrates, 16% from protein, and 34% from fat. Iron
intake (11.2 ± 4.0 mg/d) was also higher than recommended
(26, 27). All but 2 children consumed the estimated average
requirement for VA [geometric mean = 407 μg RAE/d (IQR:
305–543)] (27).
Kinetic data and model predictions
Composite (geometric mean) plasma retinol isotope response
data ([13 C10 ]retinol FDp compared with time) are plotted
in Figure 2. Note that because values for FDp for the 3 children
sampled at 11 d (as well as at 4 d) all looked high in light of
data for the rest of the group, suggesting that these individuals
had lower stores, we normalized the data at 11 d based on
each subject’s relative position to the geometric mean at 4 d
to obtain a better central tendency at 11 d and to better align
with the group pattern. Specifically, for each of these 3 children,
we calculated a 4-d ratio (geometric mean FDp at 4 d/child’s
FDp at 4 d) and multiplied each subject’s FDp at 11 d by
that ratio; the geometric mean of the adjusted values was used
at 11 d for modeling. Also shown in Figure 2 is the model-
calculated fit to the data, simulated to 40 d; an estimate for
adjusted VA intake [1.59 μmol (456 μg RAE/d)] was included
as weighted data in the model, as previously described by Ford
et al. (12). As observed in other groups of children (12), the
plasma tracer response showed an early and rapid appearance of
labeled retinol in plasma, peaking at 8 h postdose, followed by
rapid disappearance as retinol was distributed to extravascular
tissues. After 7 d, the bend in the curve is related to retinol
recycling from tissues to plasma; a terminal slope, reflecting
the system fractional catabolic rate for VA, was predicted to be
reached at ∼30 d.
Final model predictions are presented in Figure 1B, including
fractional transfer coefficients and delay times, as well as
transfer rates and compartment masses calculated using a
steady-state solution in WinSAAM with the value for plasma
retinol pool size fixed at the geometric mean value (1.15 μmol).
Model results showed that the processes of VA digestion
and absorption plus chylomicron production and catabolism
until clearance by liver hepatocytes took 4.0 h; ∼1 pool of
Vitamin A stores and kinetics in Mexican children 1647
FIGURE 3 VA TBS and liver VA concentrations estimated at 4 d
(n = 23). In each case the geometric mean of predictions is designated
by the line. TBS, total body stores; VA, vitamin A.
hepatocyte chylomicron–derived retinol was mobilized into
plasma each day. Sixteen pools of plasma retinol were turned
over each day (18 μmol/d), with 72% of that plasma turnover
transferred to the larger exchangeable extravascular pool
(compartment 6) and 24% to the smaller exchangeable pool
(compartment 7); the remaining 4% left plasma irreversibly
via nonexchangeable tissues (component 8). Based on the
distribution of plasma retinol turnover, 77% of the dose
was absorbed and exchangeable. In addition, 1.2% of VA in
the larger exchangeable pool recycled to plasma each day,
compared with 25% in the smaller pool; irreversible loss from
the larger extravascular pool was 0.06%/d. The exchangeable
extravascular pools contributed 93% of total plasma retinol
input and the remaining 7% came from diet.
Model-predicted TBS (sum of VA in compartments 6 and
7) for the group was 1097 μmol; nearly all (98%) of TBS was
accounted for by the larger exchangeable pool (compartment
6; Figure 1B). VA disposal rate was 1.3 μmol/d for the group.
Compared with irreversible loss from compartment 6, the
system fractional catabolic rate was 2 times higher (0.12%/d),
because the model assumes half the loss occurs from plasma.
The exchangeable extravascular pools contained 28-mo worth
of VA. In addition, the transit time in plasma was 1.5 h,
compared with 81 d in the larger exchangeable pool and 4.0 d
in the smaller pool; the system residence time was 863 d. The
model also predicted that, on average, retinol recycled to plasma
13 times before being irreversibly utilized, and spent 64 d in the
exchangeable pools before cycling back to plasma.
Estimation of TBS in individuals
Using the super-child model, we calculated the composite
RID coefficient FaS at 4 d (3.01). Using this value and each
child’s SAp on day 4, TBS was calculated for individuals using
Equation 2 (Supplemental Table 1 and Figure 3), providing a
geometric mean value of 1096 μmol (IQR: 836–1492 μmol).
Also shown in Figure 3 and Supplemental Table 1 are estimated
VA concentrations in liver, calculated assuming that liver is 3%
of body weight in children and that liver contains 80% of VA
TBS (2, 13, 36). The geometric mean liver VA concentration was
1.51 μmol/g (IQR: 1.30–2.10 μmol/g).
For the 12 children whose second sampling time was after
4 d (i.e., at 7, 11, 14, or 21 d), TBS was also calculated using
Equation 2 with the time-variant values for Fa and S predicted
by the model and each subject’s SAp at the corresponding time.
The composite coefficient FaS was 1.59 at 7 d, 1.09 at 11 d,
0.911 at 14 d, and 0.739 at 21 d (see Supplemental Table 2
1648 Lopez-Teros et al.
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FIGURE 4 Ratio of TBS at each child’s second time after 4 d (either
7, 11, 14, or 21 d)/TBS at 4 d compared with TBS predictions at 4 d.
Predictions are for 12 children who were sampled at both 4 d and a
second time after 4 d; each time is designated by a different symbol.
The line indicates a ratio of 1. TBS, total body stores.
for FaS at all days from 4 to 28). Similar to predictions at 4 d,
the geometric mean TBS at times from 7 to 21 d was 1026
μmol (IQR: 768–1418 μmol). When predictions at the 2 times
were paired for individuals, TBS at times after 4 d were not
significantly different from those calculated at 4 d (P = 0.33).
Shown in Figure 4 are the ratios of TBS calculated at 7, 11, 14,
or 21 d to TBS at 4 d; the mean ratio was 0.94 (range: 0.55–
1.1). Except for the 1 child sampled at 21 d (ratio of 0.55),
predictions at these later times were within 23% of the value
predicted at 4 d. Based on the distribution of TBS, the child’s 21
d value (1316 μmol, compared with 2404 μmol at 4 d) appears
to better align with the status for the group; this is also true for
the child’s estimated liver VA concentration (4.1 μmol/g at 4 d,
compared with 2.2 μmol/g at 21 d).
Discussion
In this work, we used compartmental analysis and a super-child
design to estimate whole-body retinol kinetics and TBS in a
group of Mexican preschool-aged children, collecting 2 blood
samples from each child and generating a composite data set
for modeling. Because obtaining serial blood samples in field
settings can be challenging, especially in children, Haskell et
al. (37) used a population-based approach for the first time
to describe retinol kinetics in Peruvian children. Later, Lopez-
Teros et al. (10) advanced the approach by using compartmental
analysis to study retinol kinetics and TBS in Mexican infants,
and more recently Ford et al. (12) applied it in Bangladeshi
and Filipino infants and young Guatemalan children. Besides
generating information on retinol kinetics and TBS in the
children studied here, we also calculated group mean, time-
variant values for RID equation coefficients using the model and
applied those in a RID equation to estimate TBS in all children
at 4 d as well as at their second post-4 d time.
In our earlier study of Mexican infants (aged 1–3 y),
model-predicted TBS was higher than expected [844 μmol
(10); equivalent to 1.73 μmol/g liver calculated assuming 80%
of VA is stored in the liver (2) and liver weight is 3% of
body weight in children (13)]. For the older children from
the same region included here, model-predicted TBS was also
high (1097 μmol; 1.46 μmol/g liver) and similar to the value
obtained for Guatemalan preschool-aged children [1062 μmol
(12); equivalent to 1.89 μmol/g liver]. For both groups of
preschoolers, >50% of VA intake is provided by fortified
foods as preformed VA (38, 39), but unlike in Guatemala, VA
fortification is not mandatory in Mexico. However, Mexican
children receive a large-dose VA supplement (210 μmol)
semiannually through their fourth year; the high TBS in this
group, compared with the infants studied earlier, suggests
that children in this region could be in positive VA balance
(∼125 μmol/y). Additionally, model-predicted TBS for the
children studied here was higher than that reported for African
preschool-aged children from Zambia [686 μmol (40)] and
South Africa [574 μmol (41)], where sugar fortification and
liver consumption, respectively, are common. Viewed together,
these data support the idea that the overlap of mandatory
fortification strategies aimed at reducing VA deficiency, along
with the fortification of commonly consumed commercial
foods, could be contributing to the increases in TBS in children
that have been observed over the last decade (42, 43).
To evaluate whether the high TBS in the children studied
here is reasonable considering their exposure to large-dose VA
supplements, we did the following simple retention calculation.
Assuming that their dietary VA intake [407 μg RAE/d (1.42
μmol RAE/d)] maintains VA balance, and that children received
a supplement containing 105 μmol of retinol at 6 mo of
age that was retained at 70% efficiency (44) and then 210
μmol of retinol every 6 mo until 4.5 y (i.e., a maximum
of 8 times) that was retained at 50% efficiency (45), VA
supplements alone could account for ∼900 μmol of positive
VA balance. This estimate does not include additional positive
balance from dietary VA or breastmilk. Overall, we conclude
that TBS predicted for these children (71% of whom were aged
>4.5 y) is reasonable and that a large portion of their VA stores
results from a positive VA balance due to frequent, large-dose
supplements. Furthermore, as more data have been obtained
on VA stores in children with varying socioeconomic status in
Northwest Mexico (10, 24, 46) over the past 10 y, it is becoming
clear that VA supplementation should be limited to those from
low- and very-low-income sectors of the populations, for whom
VA deficiency is still of public health concern, especially among
infants aged <2 y (47).
To predict TBS in individuals, we used compartmental
analysis to estimate the coefficients that are included in RID
prediction equations. Model-predicted values for the composite
RID coefficient FaS at 4 d for this group of Mexican children
aged <5 y was 3.01, a value close to that observed for
Guatemalan preschool-age children [2.93 (12)]; when this value
for FaS was used to calculate individual subject values for TBS
using Equation 2, the geometric mean was 1096 μmol (IQR:
836–1492). As noted in other publications (10–12), the super-
child modeling approach can provide group-specific values for
Fa and S at any time after dosing (Supplemental Table 2) so that
TBS can be calculated at any appropriate time (e.g., 4–28 d).
When values for the coefficients were used in Equation 2, TBS
predictions at 4 d were not significantly different from those
calculated at times from 7 to 21 d (P = 0.33).
Another RID equation, the isotope mass balance equation,
has been used to predict TBS at 14 d in groups of children
(3, 40, 41). Thus, we compared results obtained using RID
Equation 2 and the FaS at 14 d (0.911; Supplemental Table
2) with those calculated by the isotope mass balance equation
using the equivalent FaS (0.671) for the 3 children studied
here whose second blood sample was obtained at 14 d. Using
Equation 2, the geometric mean TBS for these 3 children
was 852 μmol compared with 626 μmol using the isotope
mass balance equation. That is, TBS predictions by the mass
balance equation for these 3 individuals were ∼73% of the
value obtained using Equation 2. The difference is due to the
coefficients. Specifically, the coefficients in the isotope mass
balance equation (41) are derived as follows: Fa is 0.839
assuming 90% absorption efficiency (48, 49) and using a
correction for catabolism with a fractional catabolic rate of
0.5%/d during the mixing period (14 d) (40) and assuming the
ratio of specific activity in serum/liver (i.e., S) to be 0.8 (50).
These values compare with our model-predicted value of 0.754
for Fa, assuming an absorption efficiency of 80%, and 1.21 for
S at 14 d. Although a statistical comparison is not warranted,
this discrepancy could be related to the fact that group-specific
coefficients were used in Equation 2 whereas the values used in
the other equation are derived from the literature.
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In a relevant post hoc analysis, which is presented in detail
in Supplemental Results, we compared VA kinetics in this
group of Mexican preschoolers to previously published results
for a group of Guatemalan children of similar age using a
partially parallel modeling approach analogous to that used by
Ford et al. (12). Results showed that kinetics for the Mexican
children were similar to those predicted for the Guatemalan
group; specifically, mean plasma tracer response data could be
fit equally as well for the Mexican children if DT(3), L(5,4),
L(6,5), and L(5,7) (Figure 1A) were equal and parallel to those
for the Guatemalans. Thus, processes describing the time for
ingested/absorbed retinol to reach hepatocytes, the transfer of
diet-derived retinol in hepatocytes to plasma as well as plasma
turnover to the larger exchangeable extravascular pool, and
recycling from the smaller extravascular pool to plasma were
quantitatively the same in the 2 groups. Using this approach,
model-predicted TBS for the Mexican children was within 7%
of the value estimated when the composite data set was fit in-
dependently (Figure 1B). Interestingly, L(5,6) was faster for the
Mexican children compared with the Guatemalans; this is likely
due to the lower VA intake for the former group and suggests
that a greater contribution of recycling from stores compared
with diet was required to maintain plasma retinol homeostasis.
In summary, our results on retinol kinetics in Mexican
preschoolers add to our understanding of whole-body VA
metabolism in infants and children. As in another recent super-
child study (12), the dietary VA intake data that we had collected
as part of the nutritional assessment of participating children
were also critical for constraining the model and increasing
confidence in model predictions. To our knowledge, we report
for the first time the prediction of TBS in individual children
using RID at 4 d postdose and we show how the super-child
modeling approach can be used with RID to estimate TBS at
any time between 4 and 21 d postdose. Overall, the addition of
a super-child component to RID studies improves predictions of
group TBS and allows calculation of VA stores for individuals
in the group.
Acknowledgments
We acknowledge and thank Bertha Isabel Pacheco Moreno,
Jessica Avila Prado, and José Carlos Valenzuela Gutiérrez for
assisting with the field work; Orlando Tortoledo Ortiz for
analyzing serum samples; and Joanne Balmer Green (Penn State
University) for contributions to the writing of the manuscript.
The authors’ responsibilities were as follows—VL-T, MHG,
JLF, HA-G: designed the study; JLF, MHG: did the modeling
and its interpretation; VL-T, HA-G: supervised the study; SAT:
donated essential materials and supervised retinol isotopic
Vitamin A stores and kinetics in Mexican children 1649
enrichment analysis; BM-B, LG-M: carried out field work
and dietary analysis; MEV: donated essential materials; VL-
T, JLF, MHG: wrote the manuscript; JLF, MHG: had primary
responsibility for the modeling analysis and data interpretation;
and all authors: read and approved the final manuscript.
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