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.

The acute phase response affected traditional measures of micronutrient status in rural Zambian children during a randomized, controlled feeding trial.

The acute phase response (APR) to infection can alter blood-based indicators of micronutrient status. Data from a 3-mo randomized, controlled feeding trial in rural Zambian children (n = 181, aged 3-5 y) were used to determine the impact of the APR on indicators of vitamin A and iron status using baseline and final blood samples. Concentrations of acute phase proteins were categorized as raised C-reactive protein (CRP; >5 and >10 mg/L) only, both raised CRP and α1-acid glycoprotein (AGP; >1.2 g/L), raised AGP only, and neither CRP nor AGP raised to identify the respective stages of infection: incubation, early convalescence, convalescence, and healthy state. Data were insufficient to examine the incubation stage of infection. A CRP concentration of >5 mg/L was an effective elevation cutoff point in this population to show impact on micronutrient markers. Time did not affect hemoglobin, serum ferritin, or serum retinol concentrations (P > 0.05). During early convalescence, hemoglobin decreased (14-16%; P ≤ 0.05), serum ferritin increased (279-356%; P ≤ 0.05), and serum retinol decreased (20-30%; P ≤ 0.05). Serum retinol concentrations did not change during convalescence; however, hemoglobin remained depressed (4-9%) and serum ferritin was elevated (67-132%) (both P ≤ 0.05). Modified relative dose response values were unaffected by the APR (P > 0.05) but increased between time points (16%; P ≤ 0.05), indicating a decrease in liver vitamin A reserves on the background of a semiannual vitamin A supplementation program. The observed prevalence of anemia and vitamin A deficiency assessed by serum retinol concentration was higher during the APR (P ≤ 0.05). It is important to consider the impact of infection on dietary interventions and to adjust for acute phase proteins when assessing iron status or vitamin A status by serum retinol concentration alone in children.

3, 4-Didehydroretinol kinetics differ during lactation in sows on a retinol depletion regimen and the serum:milk 3, 4-didehydroretinol:retinol ratios are correlated.

3, 4-Didehydroretinol (DR) metabolism was previously followed in vitamin A (VA)-replete lactating sows. This study followed DR appearance and clearance after dosage in serum and milk during 2 lactation cycles in sows (n = 8) fed VA-free feed for 3 gestation-lactation cycles. During lactations 2 and 3, 35 µmol 3, 4-didehydroretinyl acetate was given orally after overnight food deprivation. Blood and milk were collected at 0, 1.5, 3, 5, 7, 9, 16, 24, 36, 48, 60, and 72 h; livers were obtained at kill. Samples were analyzed for DR, retinol (R), and 3, 4-didehydroretinyl esters. During lactations 2 and 3, the 5-h serum DR:R ratios were 0.028 ± 0.017 and 0.069 ± 0.042, respectively, and serum R concentrations were 0.75 ± 0.23 and 0.86 ± 0.37 µmol/L, respectively. The DR:R ratio and serum R were 0.018 ± 0.013 and 0.94 ± 0.12 µmol/L, respectively, in VA-replete sows from the same herd. After lactation 3, liver VA was 0.23 ± 0.05 µmol/g, indicating low-normal VA status. Serum DR area-under-the curve from 0 to 48 h increased as liver stores decreased. Thirteen to 23% of DR dose was secreted into milk, consistent with VA-replete sows. Milk DR concentrations were greater during lactation 3 than 2. Peak concentration occurred earlier and the half-life was shorter for milk DR in the more VA-depleted sows. The milk and serum DR:R were correlated from 3 to 9 h (r = 0.70; P < 0.0001) and increased as VA stores decreased regardless of serum R concentration. Milk DR:R may replace serum measurements during lactation.

α-Retinol is distributed through serum retinol-binding protein-independent mechanisms in the lactating sow-nursing piglet dyad.

α-Retinol (αR) is a structural isomer of retinol [vitamin A (VA)] that does not bind to serum retinol-binding protein (RBP). In this study, α-retinyl acetate (αRA) was synthesized and given orally (35 µmol) to VA-deficient lactating sows (n = 11) to assess its potential to trace RBP-independent retinol transport and tissue uptake. The αRA dose primarily appeared in sow serum as 4 α-retinyl esters (αRE) with peak serum total αR concentrations (the sum of the alcohol and ester forms) detected at 2 h (70 ± 23 nmol/L, mean ± SEM) postdose. From 0 to 40 h postdose, the percentage of serum total αR in the alcohol form did not increase. Rapid αR uptake into sow milk was observed with peak concentrations (371 ± 83 nmol/L) at 7.5 h postdose, consistent with the uptake of αRE from chylomicra. A high percentage of the αRA dose (62 ± 15%, mean ± SD) was present in the livers of sows (n = 6) killed 22-28 d postdose. Approximately 15-26% of the sow αRA dose was transferred to the livers of the nursing piglets (n = 17) after 3 d. In piglets and sows, a similar percentage of hepatic total αR was detected in the ester form as that of hepatic total retinol. Taken together, these data suggest that an oral dose of αRA effectively traces the uptake, esterification, chylomicron transport, and hepatic storage of retinol and may be useful for deciphering the role of RBP-independent delivery of retinol to other tissues.

Vitamin A concentrations in piglet extrahepatic tissues respond differently ten days after vitamin A treatment.

Periodic supplementation to infants and young children is encouraged in developing countries by the WHO. We investigated vitamin A (VA) in extrahepatic tissues of piglets after supplementation with retinyl acetate to determine long-term storage. 3, 4-Didehydroretinyl acetate (DRA) as a tracer was used to evaluate uptake from chylomicra in 4 h. Sows were fed a VA-depleted diet throughout pregnancy and lactation. Male castrated piglets (n = 28, 11.6 +/- 0.5 d) from these sows were weaned onto a VA-free diet for 1 wk, assigned to 4 groups, and dosed orally with 0, 26.2, 52.4, or 105 micromol VA. After 10 d, 5.3 micromol DRA was administered to determine short-term uptake of 3, 4-didehydroretinol (DR). Four hours later, piglets were killed; adrenal glands, kidney, lung, and spleen were collected and analyzed for retinol and DR. Retinol concentrations of kidney and adrenal gland were higher than control, but treated groups did not differ. Retinol concentration was highest in kidney (1.70-2.52 nmol/g), followed by adrenal gland (0.30-0.48 nmol/g), lung (0.15-0.21 nmol/g), and spleen (0.11-0.15 nmol/g). Total retinol in kidney and spleen was different among the groups (P < 0.05). Unesterified retinol was the major VA form; the percent retinol of total VA was lowest in adrenal glands. DR did not differ among the groups. In 4 h, the minimum estimated chylomicron contribution to tissue DR was 63-280% higher than the maximum DR exposure from retinol-binding protein. Constant dietary intake may be important in maintaining VA concentrations in extrahepatic tissues.

Vitamin A deficiency causes metaplasia in vocal fold epithelium: a rat study.

OBJECTIVES: The roles of vitamin A in the vocal fold epithelium are not well documented, although vitamin A has been used as a conservative treatment for laryngeal leukoplakia. The purpose of this study was to analyze the roles of vitamin A in vocal fold epithelial differentiation. METHODS: Vitamin A-deficient (VAD) rats were generated, and the abnormality of their vocal fold epithelium was examined by hematoxylin and eosin staining and immunohistochemical analysis for keratin 10 and transglutaminase (TGase) 1. RESULTS: The VAD experimental rats exhibited orthokeratosis of the vocal fold epithelium. Keratin 10 and TGase 1 were up-regulated in the epithelium of the VAD rats. CONCLUSIONS: It is suggested that vitamin A suppresses TGase 1 expression in normal vocal folds to inhibit keratinization, and that the TGase 1 up-regulation caused by vitamin A deficiency may be related to the formation of metaplasia in the laryngeal epithelium.

Roles of vitamin A and macula flava in maintaining vocal folds.

OBJECTIVES: Vitamin A plays important roles in development, growth, and regeneration. Vitamin A-storing stellate cells have been identified in several organs. The functional roles of vitamin A in the vocal folds are still unknown, although vitamin A-storing vocal fold stellate cells have been observed in the macula flava of human and rat vocal folds. The purpose of this study was to investigate the roles of vitamin A in vocal folds. METHODS: Vitamin A-deficient rats were generated, and the vocal folds were examined histologically. Messenger RNA was extracted from the vocal folds and analyzed by real-time polymerase chain reaction. RESULTS: Immunohistochemical analysis of normal vocal folds revealed expression of retinoic acid receptor a in vocal fold stellate cells. The cells in the macula flava of vitamin A-deficient rats showed a larger nucleus/cytoplasm ratio than did those of vitamin A-sufficient rats, but messenger RNA expression of major extracellular matrix components in the macula flava of vitamin A-deficient rats did not present a remarkable change except for procollagen type I. Expression of hyaluronic acid, collagen types I and III, and elastin did not show a significant change in vitamin A-deficient rat vocal folds. CONCLUSIONS: These results indicate that vitamin A is not essential to maintaining the extracellular matrix of normal adult vocal folds, although vocal fold stellate cells participate in vitamin A storage.

Prenatal vitamin A deficiency causes laryngeal malformation in rats.

OBJECTIVES: Our previous research demonstrated that vitamin A might be related to vocal fold development. The purpose of this study was to determine whether vitamin A deficiency affects prenatal laryngeal development in rats. METHODS: Two considerations were necessary in designing a study using a rat model: for embryonic survival, vitamin A is necessary through day 10 of gestation, and laryngeal formation occurs primarily after day 11. Thus, we created a rat model that developed vitamin A deficiency after embryonic day 11. Ten pregnant rats (5 vitamin A-deficient rats and 5 control rats) were studied. Embryos were collected at embryonic day 18.5 and analyzed histologically. RESULTS: Eighteen percent of the vitamin A-deficient embryos were alive and demonstrated laryngotracheal cartilage malformation, incomplete separation of the glottis, and/or laryngoesophageal clefts. CONCLUSIONS: These results document the important role played by vitamin A in laryngeal development.

One-time graded doses of vitamin A to weanling piglets enhance hepatic retinol but do not always prevent vitamin A deficiency.

BACKGROUND: Vitamin A supplements are administered to infants in developing countries at immunization contacts; doses of 50000 IU vitamin A are recommended. Doses of 100000 IU are given to children aged 0.5-1 y. The efficacy of these doses has not been adequately determined. OBJECTIVE: We aimed to quantify liver vitamin A after the administration of vitamin A doses to piglets. Piglets are a good model for infants because of their similar size, gastrointestinal anatomy, and vitamin A requirements. DESIGN: Castrated male piglets born to sows fed a vitamin A-depleted diet throughout 1 (parity A) or 3 (parity B) pregnancy and lactation cycles were randomly assigned to receive 1 of 4 oral vitamin A doses (ie, 0, 25000, 50000, or 100000 IU) at weaning (days 9-14). A vitamin A-depleted diet was fed until the piglets were killed on day 10. Serum retinol was measured on days 1, 2, 4, 7, and 10. The modified relative dose response was measured before supplementation and at the time of killing, and liver vitamin A concentration was measured. RESULTS: In both parities, 25000 IU did not result in a mean liver retinol reserve > 0.07 micromol/g liver (the deficiency cutoff). The 50000-IU dose increased mean reserves above 0.07 micromol/g only in parity A. Liver vitamin A reserves with the 100000-IU treatment were only 5% above those with the 50000-IU treatment. The modified relative dose-response test reflected differences in liver vitamin A stores in parity B, and the 0-IU group differed significantly from the 100000-IU group (P = 0.011). CONCLUSION: This piglet model suggests that, for supplementation to infants <6 mo old, a 50000-IU dose is likely to be more efficacious in mitigating deficiency than is a 25000-IU dose.

The modified-relative-dose-response values in serum and milk are positively correlated over time in lactating sows with adequate vitamin A status.

The modified-relative-dose-response (MRDR) test, which requires a blood sample after dosing with 3,4-didehydroretinyl acetate (DRA), has been used to determine vitamin A (VA) status of individuals and groups worldwide. Less invasive methods using milk are in development in a swine model. Swine are a good choice for studying VA metabolism because their gastrointestinal anatomy, morphology, physiology, and VA requirements are similar to those of humans. In this study, DRA was used as a VA tracer in lactating sows to follow the metabolism of newly ingested VA. Lactating sows (n = 6) were administered 35 micromol DRA after overnight food deprivation. Blood and milk were collected at 0, 1.5, 3, 5, 7, 9, 24, and 48 h; livers were obtained at the time of killing. Samples were analyzed for didehydroretinol (DR), retinol (R), and didehydroretinyl esters (DRE). Serum DR:R was compared with that in milk and other VA indicators. DRE rapidly increased in serum, corresponding to chylomicra, whereas DR increased at a slower rate corresponding to the holo-DR:retinol-binding protein complex released from the liver. An estimated 10-20% of the dose was irreversibly lost in milk over 48 h. The mean MRDR value was 0.018 +/- 0.013 at 5 h and the mean liver VA was 0.73 +/- 0.21 micromol/g, both signifying sufficient stores. Milk and serum DR:R values were directly correlated (r = 0.64, P < 0.0001). Thus, DR:R values in milk may be a potential alternative to serum in determining VA status in lactating women. Future work is required in VA-deficient sows and women of varying VA status to determine DR trafficking and to compare DR:R values in milk with those in serum.

Carotenoid profiles and consumer sensory evaluation of specialty carrots (Daucus carota, L.) of various colors.

Five different colored carrots were analyzed for their carotenoid profile and underwent sensory evaluation to determine consumer acceptance (n = 96). Four major carotenoids were identified and quantified by use of HPLC methods. High beta-carotene orange carrots were found to contain the greatest concentration of total carotenoids. Except for the white, all the carrots are a significant source of bioavailable carotenoids. Sensory evaluation showed the high beta-carotene orange and white carrots to be favored over the yellow, red, and purple carrots in both blind and nonblind treatments (P < 0.01). However, all the carrots were well accepted by the consumer panel. With this information, carrot growers should be encouraged to cultivate specialty carrots to provide sources of both vitamin A precursors and phytochemicals.