Time Since Dose and Dietary Vitamin A Intake Affect Tracer Mixing in the 13C-Retinol Isotope Dilution Test in Male Rats.

BACKGROUND: Retinol isotope dilution (RID) estimates total liver vitamin A reserves (TLRs), the gold-standard vitamin A (VA) biomarker. RID equation assumptions are based on limited data. OBJECTIVES: We measured the impact of tracer choice, mixing period, and VA intake on tracer mixing [ratio of tracer enrichment in serum to that in liver stores (S)] in VA-deficient, -adequate, and hypervitaminotic rats. METHODS: Study 1 was a 3 × 2 × 3 design (18 groups, n = 5/group). Male Sprague-Dawley rats (21 d old) received 50, 100, or 3500 nmol VA/d for 21 d, were administered 52 nmol 13C2- or 13C10-retinyl acetate orally, and killed 5, 10, or 15 d later. Unlabeled VA (50 nmol/d) was given on days 11-14. Study 2 used 100 nmol VA/d for 21 d with 3 groups (n = 6-7): 52 nmol 13C2- or 13C10-retinyl acetate and 100 nmol VA/d throughout 14-d mixing, or 13C2-retinyl acetate without VA. Repeated-measures, 1-factor, and 3-factor ANOVAs were used for analysis. RESULTS: Mean ± SD TLRs (µmol/g liver) reflected intake: 0.11 ± 0.04 (50 nmol VA/d), 0.16 ± 0.04 (100 nmol VA/d), and 5.07 ± 1.58 (3500 nmol VA/d) in Study 1 and 0.24 ± 0.08 (100 nmol VA/d) in Study 2. In Study 1, mean ± SD S was 1.65 ± 0.26 (5 d), 1.16 ± 0.09 (10 d), and 0.92 ± 0.08 (15 d). The interactions tracer*VA intake and time*VA intake were significant between days 10 and 15 (P < 0.05). In Study 2, mean ± SD S was 1.07 ± 0.02 without VA during mixing, and 0.81 ± 0.04 (13C2) and 0.79 ± 0.03 (13C10) with VA intake throughout. Estimated:measured TLRs varied by VA intake and time in Study 1 but not between groups in Study 2. CONCLUSIONS: The 13C-content effect on RID through S is inconsistent. S is highly variable at 5 d, contraindicating early-time point RID. VA intake effects on S vary with timing and quantity. Assuming S = 0.8 at 14 d with consistent VA intake in human studies is likely appropriate.

Chronic and acute hypervitaminosis A are associated with suboptimal anthropometric measurements in a cohort of South African preschool children.

BACKGROUND: Excessive vitamin A (VA) can cause bone resorption and impair growth. Government-mandated VA supplementation (VAS) and adequate intake through dietary fortification and liver consumption led to excessive VA in South African children. OBJECTIVES: We evaluated the relation between VAS and underlying hypervitaminosis A assessed by retinol isotope dilution (RID) with measures of growth and bone turnover in this cohort. METHODS: Primary outcomes in these children (n = 94, 36-60 mo) were anthropometric measurements [height-for-age (HAZ), weight-for-age (WAZ), and weight-for-height (WHZ) z scores], serum bone turnover markers [C-terminal telopeptide of type I collagen (CTX) and N-terminal propeptide of type I procollagen (P1NP)], and inflammation defined as C-reactive protein (CRP; ≥5 mg/L) and/or α1-acid glycoprotein (AGP; ≥1 g/L). VA status was previously measured by RID-estimated total body VA stores (TBSs) and total liver VA reserves (TLRs), and serum retinol and carotenoid concentrations, before and 4 wk after children were administered 200,000 IU VAS. Serum 25-hydroxyvitamin D3 was measured by ultra-performance LC. RESULTS: In this largely hypervitaminotic A cohort, HAZ, WAZ, and WHZ were negatively associated with increasing TLRs, where TLRs predicted 6-10% of the variation before VAS (P < 0.05), increasing to 14-19% 4 wk after VAS (P < 0.01). Bone resorption decreased after VAS (P < 0.0001), whereas formation was unaffected. Neither CTX nor P1NP were correlated with TLRs at either time. Serum carotenoids were low. One child at each time point was vitamin D deficient (<50 nmol/L). CRP and AGP were not associated with growth measurements. CONCLUSIONS: Excessive TLRs due to dietary VA intake and VAS are associated with lower anthropometric measures and bone resorption decreased after supplementation. VA supplementation programs should monitor VA status with biomarkers sensitive to TLRs to avoid causing negative consequences in children with hypervitaminosis A. This trial is registered at clinicaltrials.gov as NCT02915731.

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.

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).

Differential diagnosis of vitamin D-related hypercalcemia using serum vitamin D metabolite profiling.

Genetic causes of vitamin D-related hypercalcemia are known to involve mutation of 25-hydroxyvitamin D-24-hydroxylase CYP24A1 or the sodium phosphate co-transporter SLC34A1, which result in excessive 1,25-(OH)2 D hormonal action. However, at least 20% of idiopathic hypercalcemia (IH) cases remain unresolved. In this case-control study, we used precision vitamin D metabolite profiling based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) of an expanded range of vitamin D metabolites to screen German and French cohorts of hypercalcemia patients, to identify patients with altered vitamin D metabolism where involvement of CYP24A1 or SLC34A1 mutation had been ruled out and who possessed normal 25-OH-D3 :24,25-(OH)2 D3 ratios. Profiles were compared to those of hypercalcemia patients with hypervitaminosis D, Williams-Beuren syndrome (WBS), CYP24A1 mutation, and normal subjects with a range of 25-OH-D levels. We observed that certain IH and WBS patients exhibited a unique profile comprising eightfold to 10-fold higher serum 23,25,26-(OH)3 D3 and 25-OH-D3 -26,23-lactone than normals, as well as very low serum 1,25-(OH)2 D3 (2-5 pg/ml) and elevated 1,24,25-(OH)3 D3 , which we interpret implies hypersensitive expression of vitamin D-dependent genes, including CYP24A1, as a general underlying mechanism of hypercalcemia in these patients. Because serum 25-OH-D3 and 24,25-(OH)2 D3 remained normal, we excluded the possibility that the aberrant profile was caused by hypervitaminosis D, but instead points to an underlying genetic cause that parallels the effect of Williams syndrome transcription factor deficiency in WBS. Furthermore, we observed normalization of serum calcium and vitamin D metabolite profiles at follow-up of an IH patient where 25-OH-D was reduced to 9 ng/ml, suggesting that symptomatic IH may depend on vitamin D nutritional status. Other hypercalcemic patients with complex conditions exhibited distinct vitamin D metabolite profiles. Our work points to the importance of serum vitamin D metabolite profiling in the differential diagnosis of vitamin D-related hypercalcemia that can rationalize expensive genetic testing, and assist healthcare providers in selecting appropriate treatment. © 2021 American Society for Bone and Mineral Research (ASBMR).

Vitamin A deficiency has declined in Malawi, but with evidence of elevated vitamin A in children.

BACKGROUND: Reduction of vitamin A deficiency (VAD) in Malawi coincided with introduction of vitamin A-fortified staple foods, alongside continued biannual high-dose vitamin A supplementation (VAS). OBJECTIVE: We describe coverage of vitamin A interventions and vitamin A status in the 2015-2016 Malawi Micronutrient Survey. METHODS: Food samples and biospecimens were collected within a representative household survey across 105 clusters. Retinol was measured using ultraviolet excitation fluorescence (sugar) and photometric determination (oil). Preschool children (PSC, aged 6-59 mo, n = 1102), school-age children (SAC, aged 5-14 y, n = 758), nonpregnant women (n = 752), and men (n = 219) were initially assessed for vitamin A status using retinol binding protein (RBP) and modified relative dose response (MRDR). Randomly selected fasted MRDR participants (n = 247) and nonfasted women and children (n = 293) were later assessed for serum retinol, retinyl esters, and carotenoids. Analyses accounted for complex survey design. RESULTS: We tested sugar and oil samples from 71.8% and 70.5% of the households (n = 2,112), respectively. All of the oil samples and all but one of the sugar samples had detectable vitamin A. National mean retinol sugar and oil contents were 6.1 ± 0.7 mg/kg and 6.6 ± 1.4 mg/kg, respectively. Receipt of VAS in the previous 6 mo was reported by 68.0% of PSC. VAD prevalence (RBP equivalent to <0.7µmol retinol/L) was 3.6% in PSC, and <1% in other groups. One woman and no children had MRDR ≥0.060 indicating VAD. Among fasted PSC and SAC, 18.0% (95% CI: 6.4, 29.6) and 18.8% (7.2, 30.5) had >5% of total serum vitamin A as retinyl esters, and 1.7% (0.0, 4.1) and 4.9% (0.0, 10.2) had >10% of total serum vitamin A as retinyl esters. Serum carotenoids indicated recent intake of vitamin A-rich fruits and vegetables. CONCLUSIONS: Near elimination of VAD in Malawi is a public health success story, but elevated levels of vitamin A among children suggests that vitamin A interventions may need modification.

Relation between Timing of High-Dose Vitamin A Supplementation and Modified-Relative-Dose-Response Values in Children 12-23 Months in Uganda.

BACKGROUND: High-dose vitamin A (VA) supplements (VAS) can temporarily affect VA status. Hence, micronutrient surveys might need to be timed around VAS campaigns to accurately estimate VA deficiency (VAD) prevalence. Little is known about optimal timing of micronutrient surveys when the modified-relative-dose-response (MRDR) is used as a VA indicator. OBJECTIVES: We evaluated the association between days since the end of a VAS campaign and MRDR values in children aged 12-23 mo in Uganda. METHODS: We pooled data from 2 cross-sectional, population-based surveys in eastern Uganda conducted in 2015-2016 (n = 118 children). We estimated the prevalence of VAD (MRDR ≥0.060). Days since the end of a VAS campaign ("days since VAS") was calculated as the interview date minus the end date of the VAS campaign. The MRDR value was assessed using HPLC. We excluded children whose MRDR values were below the limit of detection (<0.007). We used linear regression to evaluate the association between days since VAS and log-transformed MRDR. In adjusted analyses, we controlled for potential confounders. Statistical analyses accounted for the surveys' complex design. RESULTS: The prevalence of VAD was 5.2% (95% CI: 1.1%, 9.3%). Mean days since VAS was 54.1 d (range 39-68 d). Days since VAS was not associated with log-transformed MRDR in unadjusted analyses ($\hat{\beta } = \ $0.0055; 95% CI: -0.009, 0.020; P = 0.45) or adjusted analyses ($\hat{\beta } = $ -0.0073; 95% CI: -0.024, 0.010; P = 0.39). CONCLUSIONS: MRDR measurement through a nutrition survey began as early as 1.3 mo after the end of a VAS campaign in eastern Uganda. Days since the end of a VAS campaign was not associated with MRDR in Ugandan children aged 12-23 mo. Future studies should consider longitudinal designs and evaluate time since VAS and MRDR in children of different ages and in regions with higher VAD prevalence.

Retinol-binding protein, retinol, and modified-relative-dose response in Ugandan children aged 12-23 months and their non-pregnant caregivers.

Retinol-binding protein (RBP), retinol, and modified-relative-dose response (MRDR) are used to assess vitamin A status. We describe vitamin A status in Ugandan children and women using dried blood spot (DBS) RBP, serum RBP, plasma retinol, and MRDR and compare DBS-RBP, serum RBP, and plasma retinol. Blood was collected from 39 children aged 12-23 months and 28 non-pregnant mothers aged 15-49 years as a subsample from a survey in Amuria district, Uganda, in 2016. DBS RBP was assessed using a commercial enzyme immunoassay kit, serum RBP using an in-house sandwich enzyme-linked immunosorbent assay, and plasma retinol/MRDR test using high-performance liquid chromatography. We examined (a) median concentration or value (Q1, Q3); (b) R2 between DBS-RBP, serum RBP, and plasma retinol; and (c) Bland-Altman plots. Median (Q1, Q3) for children and mothers, respectively, were as follows: DBS-RBP 1.15 µmol/L (0.97, 1.42) and 1.73 (1.52, 1.96), serum RBP 0.95 µmol/L (0.78, 1.18) and 1.47 µmol/L (1.30, 1.79), plasma retinol 0.82 µmol/L (0.67, 0.99) and 1.33 µmol/L (1.22, 1.58), and MRDR 0.025 (0.014, 0.042) and 0.014 (0.009, 0.019). DBS RBP-serum RBP R2 was 0.09 for both children and mothers. The mean biases were -0.19 µmol/L (95% limits of agreement [LOA] 0.62, -0.99) for children and -0.01 µmol/L (95% LOA -1.11, -1.31) for mothers. DBS RBP-plasma retinol R2 was 0.11 for children and 0.13 for mothers. Mean biases were 0.33 µmol/L (95% LOA -0.37, 1.03) for children, and 0.29 µmol/L (95% LOA -0.69, 1.27) for mothers. Serum RBP-plasma retinol R2 was 0.75 for children and 0.55 for mothers, with mean biases of 0.13 µmol/L (95% LOA -0.23, 0.49) for children and 0.18 µmol/L (95% LOA -0.61, 0.96) for mothers. Results varied by indicator and matrix. The serum RBP-retinol R2 for children was moderate (0.75), but poor for other comparisons. Understanding the relationships among vitamin A indicators across contexts and population groups is needed.

Findings in 3 clinical trials challenge the accuracy of the Institute of Medicine's estimated average requirements for vitamin A in children and women.

BACKGROUND: Vitamin A (VA) estimated average requirements (EARs) for women and children are extrapolated from rats and adult males. The retinol isotope dilution (RID) test can sensitively characterize VA status and intake requirements. OBJECTIVES: These studies evaluated current EARs for children 4-8 y and women 19-30 y old. METHODS: Zambian children (n = 133, ages 5-7 y), US women (n = 51, ages 19-27 y), and Indonesian women (n = 29, ages 19-30 y) were provided diets or supplements containing 30%-155% of VA EARs for 42-90 d. RID was performed before and after the intervention to quantify changes in total body VA stores (TBSs) and total liver VA reserves (TLRs). Linear regression was performed between VA intake and change in TBSs or TLRs. RESULTS: Baseline mean ± SD TLRs were hypervitaminotic in Zambian children (1.13 ± 0.41 µmol VA/g liver), optimal in US women (0.46 ± 0.32 µmol/g VA/g liver), and deficient to marginal in Indonesian women (0.10 ± 0.08 µmol VA/g liver). VA intakes, resulting in no change in TBSs or TLRs, were 185 (95% CI: 18, 288) or 257 (95% CI: 124, 411) and 285 or 330 (CIs undefined) µg retinol activity equivalents (RAE)/d in the Zambian and US trials, respectively, but inconclusive in Indonesian women. The regression was not significant in either group of women. CONCLUSIONS: Point estimates of VA intakes to maintain stores were below the current EARs of 275 (children) and 500 (women) µg RAE/d despite the TLRs being higher than the EARs were formulated to maintain (i.e., 0.07 µmol VA/g liver). Interventions based on these EARs may need to be scaled back. Lack of change in VA stores in women taking lower doses may result from physiological adaptation resulting in lower VA utilization. Longer, larger, and controlled studies are needed to accurately define EARs for VA.These trials were registered at Clinicaltrials.gov as NCT04123210 and NCT01814891.

Breast Milk-Derived Retinol Is a Potential Surrogate for Serum in the 13C-Retinol Isotope Dilution Test in Zambian Lactating Women with Vitamin A Deficient and Adequate Status.

BACKGROUND: Vitamin A (VA) deficiency (VAD) affects ∼19 million pregnant women worldwide. The extent of VAD in Zambian women of reproductive age is unknown owing to lack of survey inclusion or the use of static serum retinol concentrations, a low-sensitivity biomarker. OBJECTIVES: This cross-sectional study employed isotopic techniques to determine VA status with serum and milk among women aged 18-49 y (n = 197) either lactating with infants aged 0-24 mo or nonlactating with or without infants. METHODS: Assistants were trained and piloted data collection. Demographic data, anthropometry, and relevant histories were obtained including malaria and anemia. For retinol isotope dilution (RID), baseline fasting blood and casual breast milk samples were collected before administration of 2.0 µmol 13C2-retinyl acetate and 24-h dietary recalls. On day 14, blood (n = 144) and milk (n = 66) were collected. Prevalence of total liver VA reserves (TLR) ≤0.10 µmol/g was defined as VAD with comparison to the DRI assumption of 0.07 µmol/g as minimally acceptable for North Americans. RESULTS: When a 20% adjustment for dose lost to milk was made in the RID equation for lactation, mean total body VA stores (TBS) for lactating women were 25% lower than for nonlactating women (P < 0.01), which was not the case without adjustment (P = 0.3). Mean ± SD TLR for all women were 0.15 ± 0.11 µmol/g liver. Using retinol purified from breast milk instead of serum for RID analysis yielded similar TBS and TLR, which were highly correlated between methods (P < 0.0001). Serum retinol ≤0.70 µmol/L had 0% sensitivity using either VAD liver cutoff and milk retinol ≤1.0 µmol/L had 42% sensitivity for VAD at 0.10 µmol/g. CONCLUSIONS: Determining accurate VA status among women of reproductive age, especially lactating women, forms a basis for extrapolation to the general population and informing policy development and program implementation.

Systematic Review and Meta-Analysis of the Relative Dose-Response Tests to Assess Vitamin A Status.

Vitamin A (VA) is an essential nutrient often lacking in the diets of people in developing countries. Accurate biomarkers of VA status are vital to inform public health policy and monitor interventions. The relative dose-response (RDR) and modified-RDR (MRDR) tests are semi-quantitative screening tests for VA deficiency that have been used in Demographic and Health Surveys and VA intervention studies. A systematic review and meta-analysis of sensitivity and specificity were conducted to summarize the physiological evidence to support the RDR tests as methods to assess VA status and investigate the impact of different pathological and physiological states on the tests. A total of 190 studies were screened for inclusion, with 21 studies comparing the RDR tests with the gold-standard biomarker, liver VA concentration (68% and 80% sensitivity and 85% and 69% specificity for the RDR and MRDR, respectively). Nearly all studies with VA interventions in VA-deficient populations demonstrated a response of the tests to VA intake that would be expected to improve VA status. The impacts of chronic liver disease, protein malnutrition, age, pregnancy and lactation, infection and inflammation, and various other conditions were examined in 51 studies. The RDR and MRDR tests were reported to have been used in 39 observational studies, and the MRDR has been used in at least 6 national micronutrient surveys. The RDR and MRDR are sensitive tests for determining population VA status and assessing VA interventions. Although they are robust to most physiological and pathological states, caution may be warranted when using the tests in neonates, individuals with chronic liver disease, and those with protein or iron malnutrition. Research on further improvements to the tests to increase accessibility, such as sampling breast milk instead of blood or using intramuscular doses in subjects with malabsorption, will allow wider adoption. This review was registered with PROSPERO as CRD42019124180.

Overlapping Vitamin A Interventions with Provitamin A Carotenoids and Preformed Vitamin A Cause Excessive Liver Retinol Stores in Male Mongolian Gerbils.

BACKGROUND: Vitamin A (VA) deficiency is a public health problem in some countries. Fortification, supplementation, and increased provitamin A consumption through biofortification are efficacious, but monitoring is needed due to risk of excessive VA intake when interventions overlap. OBJECTIVES: Two studies in 28-36-d-old male Mongolian gerbils simulated exposure to multiple VA interventions to determine the effects of provitamin A carotenoid consumption from biofortified maize and carrots and preformed VA fortificant on status. METHODS: Study 1 was a 2 × 2 × 2 factorial design (n = 85) with high-ß-carotene maize, orange carrots, and VA fortification at 50% estimated gerbil needs, compared with white maize and white carrot controls. Study 2 was a 2 × 3 factorial design (n = 66) evaluating orange carrot and VA consumption through fortification at 100% and 200% estimated needs. Both studies utilized 2-wk VA depletion, baseline evaluation, 9-wk treatments, and liver VA stores by HPLC. Intestinal scavenger receptor class B member 1 (Scarb1), ß-carotene 15,15'-dioxygenase (Bco1), ß-carotene 9',10'-oxygenase (Bco2), intestine-specific homeobox (Isx), and cytochrome P450 26A1 isoform α1 (Cyp26a1) expression was analyzed by qRT-PCR in study 2. RESULTS: In study 1, liver VA concentrations were significantly higher in orange carrot (0.69 ± 0.12 µmol/g) and orange maize groups (0.52 ± 0.21 µmol/g) compared with baseline (0.23 ± 0.069 µmol/g) and controls. Liver VA concentrations from VA fortificant alone (0.11 ± 0.053 µmol/g) did not differ from negative control. In study 2, orange carrot significantly enhanced liver VA concentrations (0.85 ± 0.24 µmol/g) relative to baseline (0.43 ± 0.14 µmol/g), but VA fortificant alone (0.42 ± 0.21 µmol/g) did not. Intestinal Scarb1 and Bco1 were negatively correlated with increasing liver VA concentrations (P < 0.01, r2 = 0.25-0.27). Serum retinol concentrations did not differ. CONCLUSIONS: Biofortified carrots and maize without fortification prevented VA deficiency in gerbils. During adequate provitamin A dietary intake, preformed VA intake resulted in excessive liver stores in gerbils, despite downregulation of carotenoid absorption and cleavage gene expression.

Modified relative dose response values differ between lactating women in the United States and Indonesia.

IMPACT STATEMENT: Vitamin A (VA) deficiency is a major health issue globally, and lactating women are particularly vulnerable due to increased needs for milk production. Accurate detection of VA deficiency is important; however, most population surveys measure VA status using serum retinol, which is affected by inflammation and lacks sensitivity. The modified relative dose response (MRDR) test qualitatively distinguishes between VA deficiency and sufficiency and could improve population surveys if completed in a randomly selected subsample of individuals in surveys. The original relative dose response test required two blood samples, while MRDR requires only one, a significant improvement in accessibility of the technique by decreasing burden on subjects and investigators. This work demonstrates significant deficiency in Indonesian women compared with US women. In combination with previous research using lactating sows, these human data support milk as a surrogate for blood in the MRDR, which may be less invasive, but requires further validation.

Retinol isotope dilution accurately predicts liver reserves in piglets but overestimates reserves in lactating sows.

IMPACT STATEMENT: Vitamin A (VA) deficiency and hypervitaminosis A have been reported in groups of people worldwide. Conventional biomarkers of VA deficiency (e.g. serum retinol concentration, dose response tests) are not able to distinguish between sufficiency and hypervitaminosis A. Retinol isotope dilution (RID) predictions of VA status have been validated in humans and animal models from deficiency through toxicity; however, RID during life stages with unique issues related to isotopic tracing, such as infancy and lactation, requires further evaluation. This study investigated RID in piglets and lactating sows as models for human infants and women. In piglets, RID successfully determined VA deficiency (confirmed with liver analysis), and that the tracer mixes quickly. Conversely, in lactating sows, although serum and milk enrichments were similar, traditional RID equations overestimated VA stores, likely due to losses of tracer and higher extrahepatic VA storage than predictions. These data inform researchers about the challenges of using RID during lactation.

Total Adipose Retinol Concentrations Are Correlated with Total Liver Retinol Concentrations in Male Mongolian Gerbils, but Only Partially Explained by Chylomicron Deposition Assessed with Total α-Retinol.

BACKGROUND: Liver vitamin A (VA) concentration is the gold standard for VA status, but is not routinely accessible. Adipose tissue VA concentrations, as retinol and retinyl esters, may be correlated to liver VA. α-VA (as α-retinol) is a cleavage product of α-carotene that traces postprandial VA distribution to tissues but cannot recirculate from hepatic stores, providing insight into tissue VA sources. OBJECTIVE: We performed a secondary analysis of VA and α-VA in Mongolian gerbil liver and adipose to determine the suitability of adipose tissue VA as a biomarker of VA status. METHODS: Gerbils (n = 186) consumed feeds containing 0-15.9 µg (0-55.6 nmol) retinol activity equivalents/g as preformed VA and/or provitamin A carotenoids for 36-62 d in 3 studies. Body fat percentage was determined in the final study by MRI. Serum and liver retinol, α-retinol, and retinyl esters were extracted and analyzed by HPLC or ultra-performance LC (UPLC). Epididymal and retroperitoneal adipose tissue retinol and α-retinol were determined by UPLC after homogenization, saponification, and extraction. Linear regression models with appropriate data transformations identified determinants of adipose VA concentrations. RESULTS: Liver VA did not predict serum retinol concentrations. After logarithmic transformation of adipose and liver values, liver VA positively predicted both adipose depots' VA concentrations (P < 0.001, R 2 > 0.7). Adding serum retinol or body fat percentage did not significantly increase the adjusted R 2. In liver, α-VA concentration explained much of the variation of VA (P < 0.001, R 2 > 0.7), but far less in epididymal and retroperitoneal adipose (P = 0.004 and 0.012, respectively, R 2 < 0.4). CONCLUSIONS: Adipose VA is correlated with liver VA and is a potential biomarker of VA status. It is not fully explained by chylomicron deposition and is negatively affected by serum retinol. Adipose biopsy validation is needed for human applications.

Serum retinyl esters are positively correlated with analyzed total liver vitamin A reserves collected from US adults at time of death.

Background: Minimal human data exist on liver vitamin A (VA) compared with serum biomarkers. Cutoffs of 5% and 10% total serum VA as retinyl esters (REs) suggest a VA intoxication diagnosis. Objectives: We compared total liver VA reserves (TLRs) with the percentage of total serum VA as REs to evaluate hypervitaminosis with the use of US adult autopsy samples. Secondary objectives evaluated serum retinol sensitivity, TLRs among lobes, and hepatic α-retinol concentrations, an α-carotene cleavage product. Design: Matched serum and liver samples were procured from cadavers (n = 27; mean ± SD age: 70.7 ± 14.9 y; range: 49-101 y). TLRs and α-REs were quantified by ultra-performance liquid chromatography. Pearson correlations showed liver and serum associations. Sensitivity and specificity were calculated for >5%, 7.5%, and 10% total serum VA as REs to predict TLRs and for serum retinol <0.7 and 1 µmol/L to predict deficiency. Results: Serum RE concentrations were correlated with TLRs (r = 0.497, P < 0.001). Nine subjects (33%) had hypervitaminosis A (≥1.0 µmol VA/g liver), 2 of whom had >7.5% total serum VA as REs; histologic indicators corroborated toxicity at 3 µmol/g liver. No subject had >10% total serum VA as REs. Serum retinol sensitivity to determine deficiency (TLRs <0.1 µmol VA/g) was 83% at 0.7 and 1 µmol/L. Hepatic α-retinol was positively correlated with age (P = 0.047), but removing an outlier nullified significance. Conclusions: This study evaluated serum REs as a biomarker of VA status against TLRs (gold standard), and abnormal histology suggested that 7.5% total serum VA as REs is diagnostic for toxicity at the individual level in adults. The long-term impact of VA supplements and fortificants on VA status is currently unknown. Considering the high prevalence of hypervitaminotic TLRs in this cohort, and given that many countries are adding preformed VA to processed products, population biomarkers diagnosing hypervitaminosis before toxicity are urgently needed. This trial was registered at clinicaltrials.govas NCT03305042.

ß-Cryptoxanthin and zeaxanthin are highly bioavailable from whole-grain and refined biofortified orange maize in humans with optimal vitamin A status: a randomized, crossover, placebo-controlled trial.

Background: Biofortification of staple crops with ß-carotene is a strategy to reduce vitamin A deficiency, and several varieties are available in some African countries. ß-Cryptoxanthin (BCX)-enhanced maize is currently in field trials. To our knowledge, maize BCX bioavailability has not been assessed in humans. Serum retinol 13C content and xanthophyll concentrations are proposed effectiveness biomarkers for biofortified maize adoption. Objective: We determined the relative difference in BCX and zeaxanthin bioavailability from whole-grain and refined BCX-biofortified maize during chronic feeding compared with white maize and evaluated short-term changes in 13C-abundance in serum retinol. Design: After a 7-d washout, 9 adults (mean ± SD age: 23.4 ± 2.3 y; 5 men) were provided with muffins made from BCX-enhanced whole-grain orange maize (WGOM), refined orange maize (ROM), or refined white maize (RWM) for 12 d in a randomized, blinded, crossover study followed by a 7-d washout. Blood was drawn on days 0, 3, 6, 9, 12, 15, and 19. Carotenoid areas under the curve (AUCs) were compared by using a fixed-effects model. 13C-Abundance in serum retinol was determined by using gas chromatography/combustion/isotope-ratio mass spectrometry on days 0, 12, and 19. Vitamin A status was determined by 13C-retinol isotope dilution postintervention. Results: The serum BCX AUC was significantly higher for WGOM (1.70 ± 0.63 µmol ⋅ L-1 ⋅ d) and ROM (1.66 ± 1.08 µmol ⋅ L-1 ⋅ d) than for RWM (-0.06 ± 0.13 µmol ⋅ L-1 ⋅ d; P < 0.003). A greater increase occurred in serum BCX from WGOM muffins (131%) than from ROM muffins (108%) (P ≤ 0.003). Zeaxanthin AUCs were higher for WGOM (0.94 ± 0.33) and ROM (0.96 ± 0.47) than for RWM (0.05 ± 0.12 µmol ⋅ L-1 ⋅ d; P < 0.003). The intervention did not affect predose serum retinol 13C-abundance. Vitamin A status was within an optimal range (defined as 0.1-0.7 µmol/g liver). Conclusions: BCX and zeaxanthin were highly bioavailable from BCX-biofortified maize. The adoption of BCX maize could positively affect consumers' BCX and zeaxanthin intakes and associated health benefits. This trial is registered at www.clinicaltrials.gov as NCT02800408.

Serum Carotenoids Reveal Poor Fruit and Vegetable Intake among Schoolchildren in Burkina Faso.

The health benefits of fruits and vegetables are well-documented. Those rich in provitamin A carotenoids are good sources of vitamin A. This cross-sectional study indirectly assessed fruit and vegetable intakes using serum carotenoids in 193 schoolchildren aged 7 to 12 years in the Western part of Burkina Faso. The mean total serum carotenoid concentration was 0.23 ± 0.29 µmol/L, which included α- and ß-carotene, lutein, and ß-cryptoxanthin, and determined with serum retinol concentrations in a single analysis with high performance liquid chromatography. Serum retinol concentration was 0.80 ± 0.35 µmol/L with 46% of children (n = 88) having low values <0.7 µmol/L. Total serum carotene (the sum of α- and ß-carotene) concentration was 0.13 ± 0.24 µmol/L, well below the reference range of 0.9⁻3.7 µmol carotene/L used to assess habitual intake of fruits and vegetables. Individual carotenoid concentrations were determined for α-carotene (0.01 ± 0.05 µmol/L), ß-carotene (0.17 ± 0.24 µmol/L), ß-cryptoxanthin (0.07 ± 0.06 µmol/L), and lutein (0.06 ± 0.05 µmol/L). These results confirm the previously measured high prevalence of low serum vitamin A concentrations and adds information about low serum carotenoids among schoolchildren suggesting that they have low intakes of provitamin A-rich fruits and vegetables.

Use of Stable Isotopes to Evaluate Bioefficacy of Provitamin A Carotenoids, Vitamin A Status, and Bioavailability of Iron and Zinc.

The ability of nutrition scientists to measure the status, bioavailability, and bioefficacy of micronutrients is affected by lack of access to the parts of the body through which a nutrient may travel before appearing in accessible body compartments (typically blood or urine). Stable isotope-labeled tracers function as safe, nonradioactive tools to follow micronutrients in a quantitative manner because the absorption, distribution, metabolism, and excretion of the tracer are assumed to be similar to the unlabeled vitamin or mineral. The International Atomic Energy Agency (IAEA) supports research on the safe use of stable isotopes in global health and nutrition. This review focuses on IAEA's contributions to vitamin A, iron, and zinc research. These micronutrients are specifically targeted by the WHO because of their importance in health and worldwide prevalence of deficiency. These 3 micronutrients are included in food fortification and biofortification efforts in low- and middle-income regions of the world. Vitamin A isotopic techniques can be used to evaluate the efficacy and effectiveness of interventions. For example, total body retinol stores were estimated by using 13C2-retinol isotope dilution before and after feeding Zambian children maize biofortified with ß-carotene to determine if vitamin A reserves were improved by the intervention. Stable isotopes of iron and zinc have been used to determine mineral bioavailability. In Thailand, ferrous sulfate was better absorbed from fish sauce than was ferrous lactate or ferric ammonium citrate, determined with the use of different iron isotopes in each compound. Comparisons of one zinc isotope injected intravenously with another isotope taken orally from a micronutrient powder proved that the powder increased total absorbed zinc from a meal in Pakistani infants. Capacity building by the IAEA with appropriate collaborations in low- and middle-income countries to use stable isotopes has resulted in many advancements in human nutrition.