ORIG INAL RESEARCH CURRENT DEVELOPMENTS IN NUTRITION
Nutritional Requirements and Status
Inflammation Adjustments to Serum Retinol and Retinol-Binding Protein
Improve Specificity but Reduce Sensitivity when Estimating Vitamin A
Deficiency Compared with the Modified Relative Dose-Response Test in
Ghanaian Children
Devika J Suri,1 James P Wirth,2 Seth Adu-Afarwuah,3 Nicolai Petry,2 Fabian Rohner,2 Jesse Sheftel,1 and Sherry A Tanumihardjo1
1Department of Nutritional Sciences, University of Wisconsin-Madison, Madison, WI, USA; 2GroundWork, Fläsch, Switzerland; and 3Department of Nutrition and Food
Science, University of Ghana-Legon, Legon, Accra, Ghana
ABSTRACT
Background: Serum retinol and retinol-binding protein (RBP) concentrations are commonly used biomarkers of vitamin A deficiency (VAD);
however, evidence indicates that they are not always accurate, especially in populations with high exposure to inflammation.
Objective: The aim was to assess sensitivity and specificity of serum retinol and RBP concentrations to predict VAD, with and without adjustment
for inflammation (using categorical and regression-adjusted approaches), using the modified relative dose-response (MRDR) as the reference
standard for liver reserves.
Methods: This secondary analysis of diagnostic accuracy used inflammation and RBP data and analyzed serum retinol and MRDR from a
subsample of women of reproductive age (n = 178) and preschool children (n = 166) in the cross-sectional 2017 Ghana Micronutrient Survey.
Results: Inflammation (elevated C-reactive protein and/or α1-acid glycoprotein) was present in 41% of children and 16% of women. Among
children, estimates of VAD prevalence were as follows: 7% (MRDR), 40% (serum retinol), 29% (categorical-adjusted serum retinol), 24% (RBP), 13%
(categorical-adjusted RBP), and 7% (regression-adjusted RBP). Sensitivity (95% CI) ranged from 22.2% (2.81%, 60.0%; both adjusted RBPs) to 80.0%
(44.4%, 97.5%; serum retinol), whereas specificity ranged from 63.3% (54.7%, 71.3%; serum retinol) to 93.5% (88.0%, 97.0%; regression-adjusted
RBP). Among women, VAD prevalence ranged from 1% (RBP) to 4% (all others); sensitivity was 0% and specificity was >96% for all indicators.
Conclusions: Serum retinol and RBP had varying accuracy in estimating VAD, especially in children; adjustment for inflammation increased
accuracy by increasing specificity at the expense of sensitivity. Effects of inflammation adjustment in the context of high inflammation and VAD
prevalence need to be further explored. Especially in populations with high inflammation, the MRDR test should accompany serum retinol or RBP
measurements in a subsample of subjects in population-based surveys. This trial was registered with the Open Science Framework registry (doi:
10.17605/OSF.IO/J7BP9 ). Curr Dev Nutr 2021;5:nzab098.
Keywords: biomarkers, modified relative dose-response, RBP4, sensitivity and specificity, serum retinol, vitamin A deficiency, children, women of
reproductive age
©C The Author(s) 2021. Published by Oxford University Press on behalf of the American Society for Nutrition. This is an Open Access article distributed under the terms of the Creative Commons
Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original
work is properly cited. For commercial re-use, please contact journals.permissions@oup.com
Manuscript received April 6, 2021. Initial review completed July 9, 2021. Revision accepted July 12, 2021. Published online July 15, 2021.
Supported by UNICEF and Canada’s Ministry of Foreign Affairs, Trade, and Development through a grant between UNICEF Ghana and the University of Ghana (grant number 43210308).
Author disclosures: The authors report no conflicts of interest.
Address correspondence to DS (e-mail: dsuri@wisc.edu), or SAT (e-mail: sherry@nutrisci.wisc.edu).
Abbreviations used: AGP, α1-acid glycoprotein; BRINDA, Biomarkers Reflecting Inflammation and Nutrition Determinants of Anemia; CRP, C-reactive protein; GMS, Ghana Micronutrient Survey;
MRDR, modified-relative dose response; RBP, retinol-binding protein; TLR, total liver vitamin A reserve; VAD, vitamin A deficiency.
Introduction et al. (1)]. The gold-standard biomarker is total liver vitamin A re-
serves (TLRs; reported in μmol vitamin A/g liver), where “total” refers
Vitamin A is an essential nutrient for growth, reproduction, vision, to the sum of interconvertible retinol and retinyl esters; TLRs can be
and immune function, with particular importance throughout preg- directly assayed by liver biopsy or estimated by retinol isotope dilution
nancy and early childhood. Assessing vitamin A status of vulnerable (2, 3). Neither of these methods are currently feasible in population-
populations is important to guide public health interventions, such as based health surveys and, thus, more practical biomarkers are applied.
high-dose vitamin A supplementation, food fortification, and bioforti- The most commonly used biological indicators of vitamin A deficiency
fication. While there are several biomarkers of vitamin A status, each (VAD) in large surveys are circulating concentrations of retinol and
has advantages and drawbacks [reviewed in depth by Tanumihardjo retinol-binding protein (RBP) in the blood serum/plasma of subjects.
1
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2 Suri et al.
However, in otherwise healthy individuals, these biomarkers are insen- tions, nonpregnant by self-report, considered a householdmember, and
sitive to mild-to-moderate deficiency because they are homeostatically gave written consent. The full survey successfully recruited 1165 chil-
maintained in the blood until TLRs are nearly exhausted (4). Further- dren and 973 women. In each census enumeration area, the first 2 eligi-
more, concentrations of both indicators are temporarily decreased by ble children≥18–59 mo of age and the first 2 nonpregnant women who
inflammation and infection (5, 6), which may overestimate VAD in were enrolled in theGMSwere recruited forMRDR testing. In total, 166
some populations. children and 178 nonpregnant women were recruited for the MRDR
The modified-relative dose response (MRDR) test is not affected by analysis. Eligible children were older than the standard survey partici-
the acute phase response to inflammation as assessed by C-reactive pro- pants due to the increased blood volume required for MRDR analysis.
tein (CRP) and α1-acid glycoprotein (AGP) (7, 8), and the complexity of
the laboratory analysis techniques is similar tomeasuring serum retinol Dosing, blood sampling, and analysis
concentrations by HPLC. The biological basis of the MRDR test is that Participants undergoing the MRDR test received 5.3 (children) or 8.8
hepatic apo-RBP is continually synthesized, but secretion is diminished (women) μmol 3,4-didehydroretinyl acetate dissolved in soybean oil
during vitamin A depletion [defined as low TLR (1)], leading to accu- dispensed orally by positive displacement pipette ∼5 h before blood
mulation in the absence of ligand (4). When a dose of retinol (i.e., vita- sampling. Following administration of the dose, participants were asked
min A2 in the MRDR test) is administered, accumulated RBP binds the to consume 15 g of a fatty snack (Nutella®; Ferrero) on a biscuit to facil-
ligand and the complex is secreted, causing a large amount of vitaminA2 itate the absorption of didehydroretinyl acetate. Blood was collected by
to appear in the serumwithin 5 h if the person has VAD (9). Conversely, venipuncture into 6-mL polyethylene terephthalate (PET) serum tubes
a vitamin A–sufficient subject will not have accumulated apo-RBP and containing clot activator (Becton Dickinson). No adverse events were
thus will have a low amount of vitamin A2 secreted into serum by 5 h reported fromperforming the blood draw. Immediately after collection,
post-dose due to RBP turnover (10). The MRDR value is measured as blood collection tubeswere placed into a dark cold box at∼4◦C.Whole-
the serum ratio of vitamin A2 to endogenous vitamin A in the blood 5 h blood samples were centrifuged at 2800 x g for 7 min at room temper-
after dosing with vitamin A2, determined by HPLC (1). TheMRDR test ature on the same day; serum was separated, separated into aliquots,
is a reliable indicator of low TLRs and was validated with liver biopsy and immediately stored frozen at −20◦C. Samples were later stored at
in animals (11–16) and in comparison to its predecessor, the relative the University of Ghana at−20◦C until shipment on dry ice to interna-
dose-response test, which is based on the same biological principle and tional laboratories. SerumRBP, CRP, and AGPwere analyzed in a single
validated using human liver biopsy (17, 18). In addition, intervention run at the VitMin-Lab (Wilstaett, Germany) using a sandwich ELISA
studies in humans have demonstrated that the MRDR value changes in method (27). Because blood samples were drawn after the dose of 3,4-
response to VA supplementation (19–23). In Zambian children, adjust- didehydroretinyl acetate was given, the measured RBP concentrations
ing for inflammation improved the specificity of VAD estimates when captured both the RBP that was bound to retinol and the RBP that was
compared with TLR determined by retinol isotope dilution (24). bound to 3,4-didehydroretinyl.
To our knowledge, no studies have previously compared estimates Serum aliquots from these subgroups of children and women were
of VAD prevalence using serum retinol and RBP, with and without ad- further analyzed by JS, who was blinded from other study information
justment for inflammation, against the MRDR test. A random MRDR at the time of analysis, for theMRDR test at theUniversity ofWisconsin-
test subsample supports population surveys by better defining the un- Madison (Madison, WI, USA), with the intent of comparing the results
derlying vitamin A status (1), and this study adds to the evidence con- with serum retinol and RBP data by DJS to support the findings of the
cerning the accuracy of vitamin A biomarkers. This study used data survey. For theMRDR test, serum3,4-didehydroretinol and retinolwere
on the micronutrient status of Ghanaian women and preschool chil- measured simultaneously byHPLC as previously described (11). Briefly,
dren to compare the sensitivity and specificity of inflammation-adjusted 250 μL (or all available) serum was separated into aliquots into dis-
serum retinol and RBP in estimating VAD in this population by using posable glass tubes. Forty microliters C23-β-apo-carotenol was added
theMRDR test as the reference standard for low liver vitaminA reserves. as an internal standard to determine extraction efficiency, and 250 μL
ethanol was added to denature proteins. Organic compounds were ex-
tracted 3 times by addition of 300 μL hexane, mixed with a vortex, and
Methods centrifuged. Pooled hexane layers were dried under N2; the residue was
resuspended into 40 μL 75:25 methanol:dichloroethane and 35 μL was
Participants and enrollment manually injected onto an HPLC system with a reverse-phase Waters
This secondary analysis of diagnostic accuracy used demographic in- Resolve C18 column (5-μm, 3.9× 150 mm) with 89:11 methanol:water
formation and inflammation and vitamin A status biomarker data (0.73 g/L trimethylamine) solvent run isocratically at 1 mL/min. Both
from a subsample of nonpregnant women of reproductive age and retinol and 3,4-didehydroretinol were measured at 350 nm and quan-
preschool children in the cross-sectional 2017 Ghana Micronutrient tified using calibration curves constructed with HPLC-purified stan-
Survey (GMS; registered with Open Science Framework, doi: 10.17605 dards.
/OSF.IO/J7BP9). Methodological details related to stratification, cluster
selection, and recruitment of households and individuals have been de- Vitamin A and inflammation indicators
scribed elsewhere (25, 26). Children were eligible if they were between 6 Vitamin A indicators in this analysis included serum retinol, RBP,
and 59 mo at the time of data collection, considered a household mem- and the MRDR test as a proxy for low TLRs. To determine
ber, and had written consent from their mother or caretaker. Women VAD, the following cutoffs were used: serum retinol, <0.7 μmol/L
were eligible if they were between 15 and 49 y at the time of data collec- (28); RBP, <0.7 μmol/L (29); MRDR value, serum molar ratio of
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Accuracy of biomarkers for vitamin A deficiency 3
TABLE 1 Descriptive characteristics of Ghanaian children (n = 166) and women (n = 178) recruited for MRDR testing as a
subsample of a national micronutrient survey1
Children Women
n Values n Values
Age2 162 40.0 ± 12.23 175 29.7 ± 8.8
Female 165 94 (57)4 — —
Height-for-age z-score 154 −0.87 ± 1.56 — —
Weight-for-height z-score 152 −0.51 ± 0.91 — —
Weight-for-age z-score 155 −0.83 ± 1.11 — —
BMI, kg/m2 — — 169 23.9 ± 4.7
Given a vitamin A capsule in past 6 mo5 166 20 (12) — —
Household has adequately fortified oil (≥10 ppm RE) 112 57 (51) 119 60 (50)
Inflammation category6 157 167
None 93 (59) 140 (84)
Incubation 4 (3) 7 (4)
Early convalescence 19 (12) 8 (5)
Late convalescence 41 (26) 12 (7)
1AGP, α1-acid-glycoprotein; CRP, C-reactive protein; MRDR, modified-relative dose response; RE, retinol equivalents.
2Age is presented in months for children and years for women.
3Mean ± SD (all such values).
4Frequency (%) (all such values).
5Other responses: No, 128 (77%); Don’t know, 18 (11%).
6Inflammation categories based on cutoffs of CRP >5 mg/L and AGP > 1 g/L: none, neither CRP nor AGP elevated; incubation, elevated CRP only; early convalescence,
elevated CRP and AGP; late convalescence, elevated AGP only.
3,4-didehydroretinol to retinol ≥0.060 (2). The WHO criteria (28) for Statistical analysis
severe, moderate, and mild public health problems using the serum Descriptive statistics for the children andwomen included in this analy-
retinol cutoff are ≥20%, 10–19%, and 2–9%, respectively. sis were tabulated, including age, sex, nutritional status, selected vitamin
The presence of inflammation was also taken into account for serum A exposure variables, inflammation status, and vitamin A biomarkers.
retinol and RBP concentrations, using a categorical adjustment method The unweighted prevalence of VADwas determined for each biomarker
(both indicators) and a regression adjustment method (RBP only). For by calculating the percentage of children or women above or below the
the categorical adjustment, elevated values of CRP (>5 mg/L) and AGP cutoff for deficiency; theVADprevalence for serum retinol andRBPwas
(>1 g/L) were used to determine 4 inflammation categories among chil- calculated with and without adjustment for inflammation. Because our
dren and women included in the MRDR subsample: no inflammation, objective was to compare the results of multiple biomarkers of vitamin
incubation (elevated CRP only), early convalescence (elevated CRP and A status, no statistical weights were used during data analysis.
AGP), and late convalescence (elevated AGP only). Using these inflam- Sensitivity and specificity were calculated for deficiency cutoffs of
mation categories, serum retinol and RBP concentrations were adjusted unadjusted and inflammation-adjusted serum retinol and RBP using
using correction factors calculated according to the method of Thurn- MRDR as the reference standard. Sensitivity refers to the probability
ham and colleagues (5) for both the children’s and women’s values. of a test correctly identifying those who have the condition (i.e., VAD)
RBP concentrations were also adjusted using the regression ap- among all who truly have the condition (as determined by a reference
proach developed by the Biomarkers Reflecting Inflammation and Nu- or gold standard), whereas specificity is the probability of a test cor-
tritional Determinants of Anemia (BRINDA) project (30). Because RBP rectly identifying people who do not have the condition among those
is an acute phase protein that temporarily decreases during an inflam- who truly do not have it (32). Thus, a highly sensitive test will identify
matory response, the BRINDAapproach adjusts (i.e., increases) individ- true positives while minimizing false negatives and a highly specific test
uals’ RBP concentrations based on the severity of their inflammatory will identify true negatives whileminimizing false positives. Confidence
response. Specifically, the BRINDA approach yields a continuous ad- intervals for sensitivity and sensitivity were calculated using exact con-
justment that is based on an individual’s CRP and AGP concentrations fidence intervals for proportions. Data analysis was performed in Stata
and the association, estimated using linear regression, betweenCRP and (version 15.1; StataCorp LLC).
RBP and AGP and RBP. The BRINDA adjustment for RBP was made
only on the children with RBP, CRP, and AGP values (n = 147). The
BRINDA method only recommends that RBP be adjusted in preschool Results
children (31), and as such, no inflammation adjustment was used for
women in our analysis. Subjects’ descriptive characteristics are shown in Table 1. Nutritional
status was in the normal range for children and women. Only 12%
of children had received vitamin A supplementation in the past 6
Ethics mo, and about half of households (both for children and women)
Ethical approval was obtained from the Ghana Health Service Ethics had adequately vitamin A–fortified oil. Inflammation (CRP >5 mg/L
Review Committee (GHS-ERC number: GHS-ERC 15/01/2017). and/or AGP >1 g/L) was present in 41% of children and 16% of
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4 Suri et al.
TABLE 2 Serum indicators of vitamin A and prevalence of VAD as estimated by each indicator, among Ghanaian children and
women1
Children Women
Minimum, Minimum,
Biomarkers of VA n2 Values maximum n Values maximum
MRDR ratio3 149 0.03 ± 0.024 0.006, 0.17 158 0.02 ± 0.02 0.003, 0.09
Serum retinol, unadjusted,5 μmol/L 149 0.80 ± 0.25 0.33, 1.69 159 1.38 ± 0.52 0.46, 3.37
Serum retinol, Thurnham adjustment,6 μmol/L 147 0.87 ± 0.28 0.34, 1.82 153 1.35 ± 0.51 0.46, 3.25
RBP, unadjusted, μmol/L 157 0.92 ± 0.28 0.38, 1.83 167 1.59 ± 0.65 0.52, 4.00
RBP, Thurnham adjustment,6 μmol/L 157 1.02 ± 0.31 0.45, 1.94 167 1.55 ± 0.64 0.52, 4.00
RBP, BRINDA adjustment,7 μmol/L 157 1.11 ± 0.32 0.52, 1.99 — — —
Prevalence of VAD, by indicator, frequency (%)
MRDR3 149 10 (7) — 158 6 (4) —
Serum retinol, unadjusted5 149 59 (40) — 159 6 (4) —
Serum retinol, Thurnham adjustment6 147 43 (29) — 153 6 (4) —
RBP, unadjusted5 157 37 (24) — 167 1 (1) —
RBP, Thurnham adjustment6 157 21 (13) — 167 1 (1) —
RBP, BRINDA adjustment7 157 11 (7) — — — —
1BRINDA, Biomarkers Reflecting Inflammation and Nutritional Determinants of Anemia; MRDR, modified-relative dose response; RBP, retinol-binding protein; VAD,
vitamin A deficiency.
2Some sample sizes reduced due to insufficient serum for analysis.
3Cutoff for VAD: ratio of 3,4-didehydroretinol to serum retinol ≥0.060.
4Mean ± SD (all such values).
5Cutoff for VAD: serum retinol or RBP <0.7 μmol/L.
6Using the method of Thurnham and colleagues (5).
7Adjusted using the BRINDA regression approach.
women. Table 2 describes serum indicators of vitamin A and preva- In children, the unweighted prevalence of VAD among indicators
lence of VAD among the subjects. Due to insufficient serum for anal- ranged from a low of 7%using theMRDR test to a high of 40%using un-
ysis, 17 children and 25 women were missing serum retinol and adjusted serum retinol concentrations. Adjustment of RBP and serum
MRDR values and 11 women and 9 children were missing RBP values retinol concentrations for inflammation reduced VAD prevalence esti-
(Figure 1). mates for these 2 indicators, but still categorized them differently with
FIGURE 1 Flow diagram of participants through the study. The most common reason for missing an index or reference test was
insufficient serum available. BRINDA, Biomarkers Reflecting Inflammation and Nutrition Determinants of Anemia; MRDR, modified-relative
dose response; RBP, retinol-binding protein; SR, serum retinol.
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Accuracy of biomarkers for vitamin A deficiency 5
regard to public health relevance according to WHO criteria (28): “se- ysis of MRDR data from Ugandan children suggested that survey tim-
vere” for unadjusted and adjusted serum retinol and unadjusted RBP, ing should consider time since high-dose supplementation; however, a
“moderate” to “mild” for adjusted RBP, and “mild” when using MRDR relation between vitamin A status and days since high-dose supplemen-
results. In these groups, the BRINDAadjustment for RBP resulted in the tation was not noted (33).
closest VAD estimates as compared with MRDR. Among the women, Comparing the prevalence of VAD among children with other stud-
the prevalence of VAD was very low, ranging from 1% using RBP to 4% ies, one in Ghana found baseline MRDR values for infants in inter-
(all other biomarkers), with no changes after adjustment for inflamma- vention and control groups at 0.032 ± 0.018 (6.9% VAD) and 0.031 ±
tion using serum retinol or RBP concentrations. 0.018 (11.8% VAD), respectively (34), which is quite similar to our
The relationships between the referenceMRDRvalues and the unad- mean values, and which are also consistent with values found among
justed serum retinol or RBP concentrations among children andwomen well-nourished American children residing in the United States (10)
are shown in Figure 2. The majority of subjects were true negatives— and in Ugandan children (35). Previous work in Indonesian children
that is, cases where both MRDR and serum retinol or RBP found no has shown generally higher MRDR values (indicating lower TLR and
VAD. The children (Figure 2A) also had many false positives, where higher prevalence of VAD). An intervention study among infants and
serum retinol or RBP found deficiency butMRDR did not. After adjust- preschool children (0.6–6.6 y) found baseline MRDR values of 0.054±
ing RBP using the BRINDAmethod, the values shifted higher, resulting 0.038 to 0.065 ± 0.059 (31–40% with VAD) and serum retinol VAD
in a decrease in false positives as well as true positives (Figure 2B). The prevalence of 38–59% among the study groups (20). Two groups of
women (Figure 2C) had more false negatives, where serum retinol and preschool-aged children (0.7–6.5 y) had MRDR values indicating 12%
RBP found no VAD but MRDR did (1 woman had VAD according to and 48% VAD (19). A mean MRDR value of 0.10 ± 0.06 (72.3% VAD)
RBP; however, she did not have MRDR data). and serum retinol VAD prevalence of 58.2% were found in a cohort of
Results of the sensitivity and specificity analysis among the children infants (8). This last study in Indonesia is an interesting example of a
are shown inTable 3. UsingMRDRas the reference test, we foundhighly populationwith a high prevalence of bothVADand inflammation (CRP
variable sensitivity and specificity results for VAD prevalence estimated and AGP were elevated in 15% and 20% of infants, respectively). In this
using unadjusted and inflammation-adjusted serum retinol and RBP. case, if serum retinol were to be adjusted for inflammation, it might re-
Sensitivity (95% CI), the percentage of children with VAD who were sult in an even lower estimate of VAD, further reducing its accuracy in-
correctly identified as having VAD, ranged from 22.2% (2.81%, 60.0%; stead of improving it as it did in our population. Comparing our results
Thurnham- and BRINDA-adjusted RBP) to 80.0% (44.4%, 97.5%; un- in women with others, 2 studies in postpartumGhanaian women found
adjusted serum retinol). Confidence intervals for sensitivity estimates mean MRDR values of 0.048 ± 0.037 (21.5% VAD) (21) and 0.09 ±
were quite large due to the low number of true VAD cases. Specificity, 0.05 (51% VAD) (36), respectively, which are higher than our current
the percentage of children without VAD who were correctly identi- findings. It is interesting to note that serum retinol did not differ be-
fied as not having VAD, ranged from 63.3% (54.7%, 71.3%; unadjusted tween the women in these 2 studies [i.e., 1.4 ± 0.5 (21) and 1.5 ± 0.6
serum retinol) to 93.5% (88.0%, 97.0%; BRINDA-adjusted RBP). Over- μmol/L (36)], even though there was a much higher prevalence of VAD
all, as inflammation adjustment improved specificity, sensitivity de- in the latter group. This example best illustrates the homeostatic control
creased. Results of the sensitivity and specificity analysis among women of serum retinol concentrations over a wide range of liver vitamin A re-
showed low sensitivity and high specificity (Table 3). Sensitivities of un- serves because serum retinol was maintained while liver reserves were
adjusted and inflammation-adjusted serum retinol and RBP were 0% more depleted in the second group. A study of nonpregnant Indonesian
(0%, 45.9%) due to the nonexistence of true positive cases of VAD in this women found a mean MRDR value of 0.034 ± 0.015 (7% VAD), just
group [6 cases of VAD identified by MRDR were classified as sufficient slightly higher than the women in our study (37). Nonpregnant Ugan-
(false negative) by all testmeasures]. Specificity of all 4 testmeasureswas dan women had a lower adequate median MRDR value of 0.014 (95%
high (>96%), with slightly higher values among RBP measures com- CI: 0.009, 0.019) (35).
pared with serum retinol measures due to decreased false positives. Few studies have examined the sensitivity and specificity of serum
retinol and/or RBP to determine VAD. Our results in children are con-
sistent with a previous analysis, which found that adjusted serum retinol
Discussion had better specificity than unadjusted serum retinol (83% vs. 97%)
among Zambian children, using TLRs determined by retinol isotope di-
This study examined the sensitivity and specificity of serum retinol lution as the reference test for VAD (24). Our unadjusted findings are
and RBP, with and without adjusting for inflammation, to identify similar to another study that examined the accuracy of (unadjusted)
cases of VAD among Ghanaian children and women of reproductive serum retinol and RBP compared with the relative dose-response test
age using MRDR as the reference test. We found a low prevalence of among children with liver disease and found sensitivity and specificity
VAD identified by any indicator among women; however, among chil- for serum retinol of 90% and 78% and 40% and 91% for RBP (38). This
dren, estimates of VAD prevalence varied widely by indicator and the evidence indicates that serum retinol andRBP can have varying levels of
inflammation-adjustment procedure used. The VAD prevalence esti- sensitivity and specificity to determine VAD according to factors unre-
mated using MRDR would be classified as a mild public health signifi- lated to vitamin A status. Among the children in this study population,
cance, which might be considered a limitation in the analysis. However, the inflammation adjustments made to serum retinol and RBP values,
this information suggests that the population is mostly meeting their which increased specificity and decreased sensitivity, resulted in an im-
vitamin A needs, which may include intake from intervention sources provement in the percentage of cases correctly classified as vitamin A
such as high-dose supplementation and oil fortification. A recent anal- deficient and sufficient. Mathematically, this is due to the decrease in
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6 Suri et al.
FIGURE 2 (A) Serum retinol and RBP concentrations compared with the MRDR ratio in Ghanaian children (n = 149). (B) Serum retinol and
RBP (regression-adjusted for inflammation) concentrations compared with the MRDR ratio in Ghanaian children (n = 147). (C) Serum retinol
and RBP concentration compared with the MRDR ratio in Ghanaian women (n = 153). Horizontal dashed lines represent the cutoff for VAD
defined by serum retinol and RBP concentration at <0.7 μmol/L; vertical dashed lines represent the cutoff for VAD defined by an MRDR
ratio of ≥0.060. Using MRDR as the reference test for VAD, data points in the upper left quadrant are true negatives, points in the lower
left quadrant are false positives, points in the lower right quadrant are true positives, and points in the upper right quadrant are false
negatives. MRDR, modified-relative dose response; RBP, retinol-binding protein; VAD, vitamin A deficiency.
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Accuracy of biomarkers for vitamin A deficiency 7
TABLE 3 Sensitivity and specificity of serum retinol and RBP, unadjusted and adjusted for inflammation, to determine VAD using
MRDR as the reference test among Ghanaian children (n = 147) and women (n = 152)1
Vitamin A Vitamin A status per MRDR Correctly
status per test3 classified,
indicator2 Deficient Sufficient Sensitivity,4 % Specificity, % %
Children
Serum retinol Deficient 8 51 80.0 (44.4, 97.5) 63.3 (54.7, 71.3) 64.4
Sufficient 2 88
Thurnham-adjusted serum retinol5 Deficient 7 36 77.8 (40.0, 97.2) 73.9 (65.8, 81.0) 74.2
Sufficient 2 102
RBP Deficient 4 29 44.4 (13.7, 78.8) 79.0 (71.2, 85.5) 76.9
Sufficient 5 109
Thurnham-adjusted RBP5 Deficient 2 18 22.2 (2.81, 60.0) 87.0 (80.2, 92.1) 83.0
Sufficient 7 120
BRINDA-adjusted RBP6 Deficient 2 9 22.2 (2.81, 60.0) 93.5 (88.0, 97.0) 89.1
Sufficient 7 129
Women
Serum retinol Deficient 0 5 0 (0, 45.9) 96.7 (92.5, 98.9) 93.0
Sufficient 6 147
Thurnham-adjusted serum retinol5 Deficient 0 5 0 (0, 45.9) 96.6 (92.2, 98.9) 92.8
Sufficient 6 141
RBP Deficient 0 0 0 (0, 45.9) 100 (97.5, 100) 96.1
Sufficient 6 146
Thurnham-adjusted RBP5 Deficient 0 0 0 (0, 45.9) 100 (97.5, 100) 96.1
Sufficient 6 146
1BRINDA, Biomarkers Reflecting Inflammation and Nutritional Determinants of Anemia; MRDR, modified relative dose-response; RBP, retinol-binding protein; VAD,
vitamin A deficiency.
2Cutoffs for VAD: serum retinol <0.7 μmol/L; RBP <0.7 μmol/L.
3Cutoff for VAD: MRDR test ratio of 3,4-didehydroretinol to serum retinol ≥0.060.
4Values in parentheses are 95% CIs; intervals starting with 0 or ending in 1 are 1-sided, 97.5% CIs.
5Using the method of Thurnham and colleagues (5).
6Adjusted using the BRINDA regression approach (30).
false positives/increase in true negatives. Reducing false positives was Despite the WHO recommendation that serum retinol and its sur-
beneficial to the test’s accuracy because the population had low rates of rogate, RBP, cannot stand alone, the assessment of VAD in the field us-
VAD. However, if the population had high rates of VAD and the same ing the combination of serum retinol or RBP and MRDR has been lim-
effect of inflammation adjustment were seen, it could reduce the per- ited, likely due to the lack of widely available technical support, limited
centage of those correctly classified, because a potential decrease in sen- availability of 3,4-didehydroretinol, and the scarcity of laboratories that
sitivity would result in fewer true positives and more false negatives. can measure the serum MRDR value. Arguments for the use of serum
Thus, adjustments for inflammation can improve specificity but at retinol and RBP cite their relative ease to collect and analyze compared
the expense of sensitivity—that is, they are not improving the test per with more accurate but more complex and costly techniques requiring
se. For example, in a population that is mostly vitamin A sufficient but more blood samples, such as retinol isotope dilution. Albeit less widely
has high levels of inflammation, both serum retinol and RBP would be available, the MRDR test is similar in technical difficulty as measur-
suppressed and it would be beneficial to adjust for inflammation to im- ing serum retinol by HPLC (1). If a study’s researchers prefer RBP or
prove specificity, which would help reduce the false-positive VAD cases. serum retinol due to cost and logistical reasons, formative research prior
The children in our study appear to be this type of population, so the to the study’s implementation can be conducted to assess the underly-
VAD estimates become more accurate with adjustment for inflamma- ing prevalence of inflammation. Alternatively, the MRDR assessment
tion as the specificity increases. However, in situations where there is a for VAD risk can be conducted in other population-based surveys—as
high prevalence of VAD in addition to inflammation, this adjustment done in Ghana—to give context around the accuracy and interpretation
could potentially mask the deficiency—that is, lower sensitivity would of RBP in a variety of populations. While total replacement of serum
meanmissing true-positive VAD cases. The women in our study, on the retinol or RBP by MRDR may not be feasible in large-scale surveys, in
other hand, appear to have low VAD and relatively low inflammation; part due to the 4–6-h wait period, data generated through this work
therefore, the adjustments to serum retinol have little effect and theVAD clearly indicate that the MRDR test should be considered as a comple-
estimates are similar among the indicators. Thus, the decision to rely on ment to serum retinol/RBP, especially in the case of high inflammation
adjusted serum retinol and/or RBP to estimate VADmay vary based on and potentially high or unknown levels of VAD. In fact, none of the
the prevalence of VAD and inflammation status of the population being above-investigated biomarkers is suitable to detect hypervitaminosis A,
surveilled, but it would not be possible to know this without first hav- a risk in populations exposed to multiple sources of preformed vitamin
ing more reliable data with which to compare the estimates from these A (39–42) (e.g., high-dose supplementation, fortification) andmay have
indicators. negative implications in bone metabolism and growth (43–46). This is
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8 Suri et al.
a concern because potential hypervitaminosis A may be ignored if only References
screening for VAD is conducted (and especially if those indicators are
inflating the true VAD prevalence); other indicators have been recom- 1. Tanumihardjo SA, Russell RM, Stephensen CB, Gannon BM, Craft NE,
mended for evaluation of potential hypervitaminosis in at-risk popu- Haskell MJ, Lietz G, Schulze K, Raiten DJ. Biomarkers of Nutrition forDevelopment (BOND)—vitamin A review. J Nutr 2016;146:1816S–48S.
lations (39, 41). Given the growing evidence surrounding the risks of 2. Tanumihardjo SA.VitaminA: Biomarkers ofNutrition forDevelopment. Am
hypervitaminosis A, greater efforts to accurately target truly at-risk pop- J Clin Nutr 2011;94:658S–65S.
ulations and individuals to the extent possible are needed. 3. Gannon BM, Tanumihardjo SA. Comparisons among equations used for
A limitation to this study was the low rate of true VAD cases among retinol isotope dilution in the assessment of total body stores and total liver
both children (10) and women (6), whichmakes sensitivity calculations reserves. J Nutr 2015;145:847–54.4. Muto Y, Smith JE, Milch PO, Goodman DS. Regulation of retinol-
more volatile (hence the large confidence intervals). Future studies in binding protein metabolism by vitamin A status in the rat. J Biol Chem
populations with low VAD prevalence could use larger sample sizes to 1972;247:2542–50.
address this issue, as well as investigate the accuracy of serum retinol 5. Thurnham DI, McCabe GP, Northrop-Clewes CA, Nestel P. Effects of
and RBP in a variety of contexts of both high and low VAD and inflam- subclinical infection on plasma retinol concentrations and assessment of
mation. However, the finding of such low VAD rates in this population prevalence of vitamin A deficiency: meta-analysis. Lancet North Am Ed2003;362:2052–8.
argues precisely for the use of a more accurate indicator (or greater con- 6. Filteau SM, Morris SS, Abbott RA, Tomkins AM, Kirkwood BR, Arthur P,
fidence in an inflammation-adjustedmeasure) to assess VAD in the first Ross DA, Gyapong JO, Raynes JG. Influence of morbidity on serum retinol
place. Both young children and women of reproductive age are higher of children in a community-based study in northern Ghana. Am J Clin Nutr
risk groups for VAD, yet in this study population, that risk was found to 1993;58:192–7.
be low, perhaps due to the success of vitaminA programs, due to dietary 7. Bresnahan KA, Chileshe J, Arscott S, Nuss E, Surles R, Masi C, Kafwembe E,Tanumihardjo SA. The acute phase response affected traditional measures
changes, or a combination. As mentioned above, as VAD decreases in of micronutrient status in rural Zambian children during a randomized,
a population exposed to multiple overlapping VA interventions it may controlled feeding trial. J Nutr 2014;144:972–8.
also be important to screen for hypervitaminosis. 8. Wieringa FT, Dijkhuizen MA, West CE, Northrop-Clewes CA, Muhilal.
This study contributes to evidence that serum retinol and RBP are Estimation of the effect of the acute phase response on indicators of
not consistently accurate indicators of VAD. Adjusting for the presence micronutrient status in Indonesian infants. J Nutr 2002;132:3061–6.9. Tanumihardjo SA, Muhilal YY, Permaesih D, Sulaiman Z, Karyadi D, Olson
of inflammation improved specificity but sacrificed sensitivity among JA. Vitamin A status in preschool-age Indonesian children as assessed
the children in this study, which could have different impacts on the by the modified relative-dose-response assay. Am J Clin Nutr 1990;52:
validity of VAD estimates depending on the underlying inflammation 1068–72.
and VAD prevalence of the population under surveillance. Especially in 10. Tanumihardjo SA, Koellner PG, Olson JA. The modified relative-dose-
populations with high inflammation, we recommend that MRDR anal- response assay as an indicator of vitamin A status in a population of well-nourished American children. Am J Clin Nutr 1990;52:1064–7.
ysis should be used in place of or in conjunction with serum retinol 11. Valentine AR, Tanumihardjo SA. Adjustments to the modified relative dose
and/or RBP in estimating VAD prevalence when possible. To render the response (MRDR) test for assessment of vitaminA statusminimize the blood
MRDR testmore available, capacity-building efforts are warranted. Fur- volume used in piglets. J Nutr 2004;134:1186–92.
thermore, if vitamin A programs overlap, more sensitive measures of 12. Valentine AR, Tanumihardjo SA. One-time vitamin A supplementation of
high status need to be undertaken to cover the hypervitaminotic range lactating sows enhances hepatic retinol in their offspring independent of dosesize. Am J Clin Nutr 2005;81:427–33.
of TLRs, such as retinol isotope dilution or retinyl ester concentrations. 13. Surles RL, Hutson PR, Valentine AR, Mills JP, Tanumihardjo SA. 3, 4-
Didehydroretinol kinetics differ during lactation in sows on a retinol
Acknowledgments depletion regimen and the serum:milk 3, 4-didehydroretinol:retinol ratiosare correlated. J Nutr 2011;141:554–9.
The authors thank Helena Bentil as survey coordinator and Matilda 14. Tanumihardjo SA, Olson JA. A modified relative dose-response assay
Steiner-Asiedu. The authors’ responsibilities were as follows—DJS: employing 3,4-didehydroretinol (vitamin A2) in rats. J Nutr 1988;118:598–
performed statistical analysis and interpretation and prepared the 603.
manuscript; JPW: provided support in statistical analysis; JPW, FR, NP, 15. Tanumihardjo SA, Furr HC, Erdman JW, Olson JA. Use of the modified
and SA-A: designed, implemented, and analyzed the main Ghana Mi- relative dose response (MRDR) assay in rats and its application to humansfor the measurement of vitamin A status. Eur J Clin Nutr 1990;44:219–24.
cronutrient Survey study and reviewed the manuscript; JS: conducted 16. Surles RL, Li J, Tanumihardjo SA. The modified-relative-dose-response
serum retinol and MRDR analysis and contributed to writing and re- values in serum andmilk are positively correlated over time in lactating sows
viewing the manuscript; SAT: contributed to interpretation, writing, with adequate vitamin A status. J Nutr 2006;136:939–45.
and reviewing the manuscript; and all authors: read and approved the 17. Amédée-Manesme O, Anderson D, Olson JA. Relation of the relative dose
final manuscript. response to liver concentrations of vitamin A in generally well-nourishedsurgical patients. Am J Clin Nutr 1984;39:898–902.
18. Amedee-Manesme O, Mourey MS, Hanck A, Therasse J. Vitamin A relative
dose response test: validation by intravenous injection in children with liver
Data Availability disease. Am J Clin Nutr 1987;46:286–9.
19. Tanumihardjo SA, Permaesih D, Dahro AM, Rustan E, Muhilal, Karyadi D,
Data described in the manuscript, code book, and analytic code will Olson JA. Comparison of vitamin A status assessment techniques in children
be made available upon request pending the agreement of the request- from two Indonesian villages. Am J Clin Nutr 1994;60:136–41.20. Tanumihardjo SA, Permaesih D, Muherdiyantiningsih, Rustan E, Rusmil
ing institution to the terms and conditions of formalized data trans- K, Fatah AC, Wilbur S, Muhilal, Karyadi D, Olson JA. Vitamin A status
fer agreements of University of Wisconsin and University of Ghana- of Indonesian children infected with Ascaris lumbricoides after dosing with
Legon/GroundWork. vitamin A supplements and albendazole. J Nutr 1996;126:451–7.
CURRENT DEVELOPMENTS IN NUTRITION
Downloaded from https://academic.oup.com/cdn/article/5/8/nzab098/6321831 by University of Ghana, Legon user on 15 November 2021
Accuracy of biomarkers for vitamin A deficiency 9
21. Tchum SK, Tanumihardjo SA, Newton S, de Benoist B, Owusu-Agyei S, high-dose vitamin A supplementation and modified-relative-dose-response
Arthur FK, Tetteh A. Evaluation of vitamin A supplementation regimens in values in children 12–23 months in Uganda. J Nutr 2021;151(4):1025–8.
Ghanaian postpartum mothers with the use of the modified-relative-dose- 34. Newton S,Owusu-Agyei S, AsanteKP,Amoaful E,MahamaE, TchumSK,Ali
response test. Am J Clin Nutr 2006;84:1344–9. M,Adjei K,Davis CR, Tanumihardjo SA.VitaminA status and body pool size
22. Manorama R, Sarita M, Rukmini C. Red palm oil for combating vitamin A of infants before and after consuming fortified home-based complementary
deficiency. Asia Pac J Clin Nutr 1997;6:56–9. foods. Arch Public Heal 2016;74:10.
23. Tanumihardjo SA, Muherdiyantiningsih, Permaesih D, Komala, Muhilal, 35. Whitehead RD, Jr, Ford ND, Mapango C, Ruth LF, Zhang M, Schleicher
Karyadi D, Olson JA. Daily supplements of vitamin A (8.4 μmol, 8000 IU) RL, Ngalombi S, Halati S, Ahimbisibwe M, Lubowa A, et al. Retinol-
improve the vitamin A status of lactating Indonesian women. Am J Clin Nutr binding protein, retinol, and modified-relative-dose response in Ugandan
1996;63:32–5. children 12–23 months and their non-pregnant caregivers. Exp Biol Med
24. Suri DJ, Tanumihardjo JP, Gannon BM, Pinkaew S, Kaliwile C, Chileshe J, 2021;246(8):906–15.
Tanumihardjo SA. Serum retinol concentrations demonstrate high specificity 36. Tchum S, Newton S, Tanumihardjo S, Fareed K, Tetteh A, Agyei S. Evaluation
after correcting for inflammation but questionable sensitivity compared of a green leafy vegetable intervention in Ghanaian postpartum mothers.
with liver stores calculated from isotope dilution in determining vitamin African J Food Agric Nutr Dev 2009;9(6):1294–308.
A deficiency in Thai and Zambian children. Am J Clin Nutr 2015;102: 37. Tanumihardjo SA, Muherdiyantiningsih, Permaesih D, Dahro AM, Muhilal,
1259–65. Karyadi D, Olson JA. Assessment of the vitamin A status in lactating
25. Wegmüller R, Bentil H, Wirth JP, Petry N, Tanumihardjo SA, Allen and nonlactating, nonpregnant Indonesian women by use of the modified-
L, Williams TN, Selenje L, Mahama A, Amoaful E, et al. Anemia, relative-dose-response (MRDR) test. Am J Clin Nutr 1994;60:142–7.
micronutrient deficiencies, malaria, hemoglobinopathies and malnutrition 38. Feranchak AP, Gralla J, King R, Ramirez RO, Corkill M, Narkewicz MR,
in young children and non-pregnant women in Ghana: findings from a Sokol RJ. Comparison of indices of vitamin A status in children with chronic
national survey. PLoS One 2020;15(1):e0228258. liver disease. Hepatology 2005;42:782–92.
26. University of Ghana; GroundWork; University of Wisconsin-Madison; 39. van Stuijvenberg ME, Dhansay MA, Nel J, Suri D, Grahn M, Davis CR,
KEMRI-WellcomeTrust; UNICEF.GhanaMicronutrient Survey 2017. Accra Tanumihardjo SA. South African preschool children habitually consuming
(Ghana): 2017. sheep liver and exposed to vitamin A supplementation and fortification have
27. Erhardt JG, Estes JE, Pfeiffer CM, Biesalski HK, Craft NE. Combined hypervitaminotic A liver stores: a cohort study. Am J Clin Nutr 2019;110:91–
measurement of ferritin, soluble transferrin receptor, retinol binding protein, 101.
and C-reactive protein by an inexpensive, sensitive, and simple sandwich 40. Olsen K, Suri DJ, Davis C, Sheftel J, Nishimoto K, Yamaoka Y, Toya Y,
enzyme-linked immunosorbent assay technique. J Nutr 2004;134:3127–32. WelhamNV, Tanumihardjo SA. Serum retinyl esters are positively correlated
28. World Health Organization. Serum retinol concentrations for determining with analyzed total liver vitamin A reserves collected from US adults at time
the prevalence of vitamin A deficiency in populations. Geneva (Switzerland): of death. Am J Clin Nutr 2018;108:997–1005.
WHO; 2011. 41. Mondloch S, Gannon BM, Davis CR, Chileshe J, Kaliwile C, Masi C, Rios-
29. World Health Organization. Global prevalence of vitamin A deficiency in Avila L, Gregory JF, III, Tanumihardjo SA. High provitamin A carotenoid
populations at risk 1995–2005. Geneva (Switzerland): WHO; 2009. serum concentrations, elevated retinyl esters, and saturated retinol-binding
30. Namaste SM, Aaron GJ, Varadhan R, Peerson JM, Suchdev PS; BRINDA protein in Zambian preschool children are consistent with the presence of
Working Group. Methodologic approach for the Biomarkers Reflecting high liver vitamin A stores. Am J Clin Nutr 2015;102:497–504.
Inflammation and Nutritional Determinants of Anemia (BRINDA) project. 42. Tanumihardjo SA, Mokhtar N, Haskell MJ, Brown KH. Assessing the safety
Am J Clin Nutr 2017;106(Suppl 1):333S–47S. of vitaminA delivered through large-scale intervention programs. FoodNutr
31. Larson LM, Namaste SM, Williams AM, Engle-Stone R, Addo OY, Suchdev Bull 2016;37(2 Suppl):S63–74.
PS,Wirth JP, Temple V, SerdulaM, Northrop-Clewes CA. Adjusting retinol- 43. Penniston KL, Tanumihardjo SA. The acute and chronic toxic effects of
binding protein concentrations for inflammation: Biomarkers Reflecting vitamin A. Am J Clin Nutr 2006;83:191–201.
Inflammation and Nutritional Determinants of Anemia (BRINDA) project. 44. Pease CN. Focal retardation and arrestment of growth of bones due to
Am J Clin Nutr 2017;106(Suppl 1):390S–401S. vitamin A intoxication. JAMA 1962;182:980–5.
32. Trevethan R. Sensitivity, specificity, and predictive values: foundations, 45. BinkleyN, KruegerD.Hypervitaminosis A and bone. Nutr Rev 2000;58:138–
pliabilities, and pitfalls in research and practice. Front Public Heal 44.
2017;5:307. 46. Tanumihardjo SA, Gannon BM, Kaliwile C, Chileshe J, Binkley NC.
33. Pickens CM, Flores-Ayala R, Ford ND,Whitehead RD, Jr, Tanumihardjo SA, Restricting vitamin A intake increases bone formation in Zambian children
Ngalombi S, Mapango C, Sheftel J, JefferdsMED. Relation between timing of with high liver stores of vitamin. Arch Osteoporos 2019;14:4–9.
CURRENT DEVELOPMENTS IN NUTRITION
Downloaded from https://academic.oup.com/cdn/article/5/8/nzab098/6321831 by University of Ghana, Legon user on 15 November 2021