Use of Compartmental Modeling and Retinol Isotope Dilution to Determine Vitamin A Stores in Young People with Sickle Cell Disease Before and After Vitamin A Supplementation.

BACKGROUND: Suboptimal plasma retinol concentrations have been documented in US children with sickle cell disease (SCD) hemoglobin SS type (SCD-HbSS), but little is known about vitamin A kinetics and stores in SCD. OBJECTIVES: The objectives were to quantify vitamin A total body stores (TBS) and whole-body retinol kinetics in young people with SCD-HbSS and use retinol isotope dilution (RID) to predict TBS in SCD-HbSS and healthy peers as well as after vitamin A supplementation in SCD-HbSS subjects. METHODS: Composite plasma [13C10]retinol response data collected from 22 subjects with SCD-HbSS for 28 d after isotope ingestion were analyzed using population-based compartmental modeling ("super-subject" approach); TBS and retinol kinetics were quantified for the group. TBS was also calculated for the same individuals using RID, as well as for healthy peers (n = 20) and for the subjects with SCD-HbSS after 8 wk of daily vitamin A supplements (3.15 or 6.29 µmol retinol/d [900 or 1800 µg retinol activity equivalents/d]). RESULTS: Model-predicted group mean TBS for subjects with SCD-HbSS was 428 µmol, equivalent to ∼11 mo of stored vitamin A; vitamin A disposal rate was 1.3 µmol/d. Model-predicted TBS was similar to that predicted by RID at 3 d postdosing (mean, 389 µmol; ∼0.3 µmol/g liver); TBS predictions at 3 compared with 28 d were not significantly different. Mean TBS in healthy peers was similar (406 µmol). RID-predicted TBS for subjects with SCD-HbSS was not significantly affected by vitamin A supplementation at either dose. CONCLUSIONS: Despite differences in plasma retinol concentrations, TBS was the same in subjects with SCD-HbSS compared with healthy peers. Because 56 d of vitamin A supplementation at levels 1.2 to 2.6 times the Recommended Dietary Allowance did not increase TBS in these subjects with SCD-HbSS, further work will be needed to understand the effects of SCD on retinol metabolism. This trial was registered as NCT03632876 at clinicaltrials.gov.

Filipino Children with High Usual Vitamin A Intakes and Exposure to Multiple Sources of Vitamin A Have Elevated Total Body Stores of Vitamin A But Do Not Show Clear Evidence of Vitamin A Toxicity.

Background: Young children exposed to high-dose vitamin A supplements (VAS) and vitamin A (VA)-fortified foods may be at risk of high VA intake and high VA total body stores (TBS). Objectives: TBS and estimated liver VA concentration were compared among children with adequate or high VA intake and different timing of exposure to VAS, and associations between estimated liver VA concentrations and biomarkers of VA toxicity were examined. Methods: Children 12-18 mo of age (n = 123) were selected for 3 groups: 1) retinol intake >600 µg/d and VAS within the past mo, 2) retinol intake >600 µg/d and VAS in the past 3-6 mo, and 3) VA intake 200-500 µg retinol activity equivalents (RAE)/d and VAS in the past 3-6 mo. Dietary intake data were collected to measure VA intakes from complementary foods, breast milk, and low-dose, over-the-counter supplements. TBS were assessed by retinol isotope dilution, and VA toxicity biomarkers were measured. Main outcomes were compared by group. Results: Mean (95% CI) VA intakes excluding VAS were 1184 (942, 1426), 980 (772, 1187), and 627 (530, 724) µg RAE/d, in groups 1-3, respectively; mean VA intake was higher in groups 1 and 2 compared with group 3 (P < 0.05). Geometric mean (GM) (95% CI) TBS were 589 (525, 661), 493 (435, 559), and 466 (411, 528) µmol, respectively. GM TBS and GM liver VA concentrations were higher in group 1 compared with group 3 (liver VA concentration: 1.62 vs. 1.33 µmol/g; P < 0.05). Plasma retinyl ester and 4-oxo-retinoic acid concentrations and serum markers of bone turnover and liver damage did not indicate VA toxicity. Conclusions: In this sample, most children had retinol intakes above the Tolerable Upper Intake Level (UL) and liver VA concentrations above the proposed cutoff for "hypervitaminosis A" (>1 µmol/g liver). There was no evidence of chronic VA toxicity, suggesting that the liver VA cutoff value should be re-evaluated. This trial was registered at www.clinicaltrials.gov as NCT03030339.

Use of stable isotopes to study bioconversion and bioefficacy of provitamin A carotenoids.

Provitamin A carotenoids (pVACs) are important contributors to vitamin A status and in reducing vitamin A deficiency in vulnerable populations. However, there are uncertainties about the effectiveness of dietary pVACs to provide sufficient amounts of vitamin A due to large variations in provitamin A bioefficacy, and if provitamin A carotenoids other than ß-carotene could be utilized in biofortification programs. Although animal studies have compared the bioefficacy of ß-cryptoxanthin and ß-carotene in biofortified maize by measuring liver retinol concentrations after dietary exposure, it is unclear whether the higher bioavailability of ß-cryptoxanthin can be offset by the lower conversion of ß-cryptoxanthin to retinol, and how post-intestinal conversion of ß-cryptoxanthin may contribute to the total bioefficacy of ß-cryptoxanthin. Here, we present a method that, for the first time, uses stable isotope labeled ß-cryptoxanthin and ß-carotene to quantify bioconversion and bioefficacy of both pVACs simultaneously in human volunteers. The paper describes how positioning of the [13C] labels around the centric 15,15' double bond on either the ß-carotene or ß-cryptoxanthin molecule allows quantification of retinoids from both pVACs, and details the procedure for sample preparation and analysis using LC-MS/MS. Finally, we apply and discuss recent approaches to quantify bioconversion and bioefficacy of isotopically labeled ß-carotene and ß-cryptoxanthin in humans.

Determination of Vitamin A Total Body Stores in Children from Dried Serum Spots: Application in a Low- and Middle-Income Country Community Setting.

BACKGROUND: The retinol isotope dilution (RID) method has been used to evaluate vitamin A (VA) status in healthy adults and children in low- and middle-income countries (LMIC) and to assess the efficacy of various VA interventions. OBJECTIVE: The study was designed to examine whether dried serum spots (DSS) can be applied to RID when conducting VA total body store (TBS) assessments in community settings. METHODS: Four days after an oral dose of 0.4 mg [13C10]retinyl acetate was administered to Filipino children (12-18 mo), a single blood draw was divided to isolate both serum and plasma. Serum (40 µL) was spotted and dried on Whatman 903 cards and shipped at ambient temperature whereas liquid plasma (LP) was frozen at -80°C and shipped on dry ice. The VA tracer to tracee ratio from DSS and LP was quantified by LC-MS/MS. Comparisons between DSS and LP paired samples (n = 72) were made for [13C10]retinol specific activity (SAp) by Pearson's correlation and for VA TBS by Bland-Altman analysis. RESULTS: The sum of 3 coextracted DSS were required to consistently detect [13C10]retinol above the LC-MS/MS limit of quantitation (LOQ). [13C10]retinol SAp from DSS was highly correlated with SAp from LP (r = 0.945; P < 0.01). A comparison of methods for TBS determination using Bland-Altman analysis indicated agreement with an intraindividual difference of 24.7 µmol (4.6%). Mean total liver reserve (TLR) values from DSS and LP were 1.7 µmol/g (± 0.6 SD) and 1.6 µmol/g (± 0.6 SD), respectively. CONCLUSIONS: VA TBS can be determined from DSS thereby reducing the logistics and cost of maintaining a cold chain by shipping samples at ambient temperature and, thus, making the RID technique more feasible in LMIC community settings. This trial was registered at https://clinicaltrials.gov as NCT03030339.

Biofortified and fortified maize consumption reduces prevalence of low milk retinol, but does not increase vitamin A stores of breastfeeding Zambian infants with adequate reserves: a randomized controlled trial.

BACKGROUND: Replacement of conventional staples with biofortified or industrially fortified staples in household diets may increase maternal breast milk retinol content and vitamin A intakes from complementary foods, improving infant total body stores (TBS) of vitamin A. OBJECTIVES: To determine whether biofortified or industrially fortified maize consumption by Zambian women and their breastfeeding infants could improve milk retinol concentration and infant TBS. METHODS: We randomly assigned 255 lactating women and their 9-mo-old infants to a 90-d intervention providing 0 µg retinol equivalents (RE)/d as conventional maize or ∼315 µg RE/d to mothers and ∼55 µg RE/d to infants as provitamin A carotenoid-biofortified maize or retinyl palmitate-fortified maize. Outcomes were TBS, measured by retinol isotope dilution in infants (primary), and breast milk retinol, measured by HPLC in women (secondary). RESULTS: The intervention groups were comparable at baseline. Loss to follow-up was 10% (n = 230 mother-infant pairs). Women consumed 92% of the intended 287 g/d and infants consumed 82% of the intended 50 g/d maize. The baseline geometric mean (GM) milk retinol concentration was 1.57 µmol/L (95% CI: 1.45, 1.69 µmol/L), and 24% of women had milk retinol <1.05 µmol/L. While mean milk retinol did not change in the biofortified arm (ß: 0.11; 95% CI: -0.02, 0.24), the intervention reduced low milk retinol (RR: 0.42; 95% CI: 0.21, 0.85). Fortified maize increased mean milk retinol (ß: 0.17; 95% CI: 0.04, 0.30) and reduced the prevalence of low milk retinol (RR: 0.46; 95% CI: 0.25, 0.82). The baseline GM TBS was 178 µmol (95% CI: 166, 191 µmol). This increased by 24 µmol (± 136) over the 90-d intervention period, irrespective of treatment group. CONCLUSIONS: Both biofortified and fortified maize consumption improved milk retinol concentration. This did not translate into greater infant TBS, most likely due to adequate TBS at baseline. This trial was registered at clinicaltrials.gov as NCT02804490.

Reduced plasma carotenoids in individuals suffering from metabolic diseases with disturbances in lipid metabolism: a systematic review and meta-analysis of observational studies.

This review summarises the association between serum carotenoids, serum retinoids and dietary intake outcomes with obesity/overweight and individuals with metabolic diseases with disturbances in lipid metabolism. Observational studies reporting dietary intakes and serum concentrations of carotenoids and retinol were collected from Medline and Web of Science. Mean differences were calculated between "cases" (classified as obese, overweight or having a metabolic disease with disturbances in lipid metabolism; i.e. non-alcoholic fatty liver disease, type 2 diabetes, dyslipidaemia or metabolic syndrome) and "comparator group" (classified as normal weight healthy individuals) and summarised in meta-analyses. Significant summary measures were observed for most serum provitamin A and non-provitamin A carotenoids. Studies reporting total serum carotenoids had shown the greatest decrease (-0.28 µmol/l [-0.33, -0.23], p<.001, I2=62.5%, n = 7). There were no significant summary measures for dietary outcomes, suggesting a physiological role of low serum carotenoids in the development of obesity and associated diseases.

Use of Model-Based Compartmental Analysis and a Super-Child Design to Study Whole-Body Retinol Kinetics and Vitamin A Total Body Stores in Children from 3 Lower-Income Countries.

BACKGROUND: Model-based compartmental analysis has been used to describe and quantify whole-body vitamin A metabolism and estimate total body stores (TBS) in animals and humans. OBJECTIVES: We applied compartmental modeling and a super-child design to estimate retinol kinetic parameters and TBS for young children in Bangladesh, Guatemala, and the Philippines. METHODS: Children ingested [13C10]retinyl acetate and 1 or 2 blood samples were collected from each child from 6 h to 28 d after dosing. Temporal data for fraction of dose in plasma [13C10]retinol were modeled using WinSAAM software and a 6-component model with vitamin A intake included as weighted data. RESULTS: Model-predicted TBS was 198, 533, and 1062 µmol for the Bangladeshi (age, 9-17 mo), Filipino (12-18 mo), and Guatemalan children (35-65 mo). Retinol kinetics were similar for Filipino and Guatemalan groups and generally faster for Bangladeshi children, although fractional transfer of plasma retinol to a larger exchangeable storage pool was the same for the 3 groups. Recycling to plasma from that pool was ∼2.5 times faster in the Bangladeshi children compared with the other groups and the recycling number was 2-3 times greater. Differences in kinetics between groups are likely related to differences in vitamin A stores and intakes (geometric means: 352, 727, and 764 µg retinol activity equivalents/d for the Bangladeshi, Filipino, and Guatemalan children, respectively). CONCLUSIONS: By collecting 1 or 2 blood samples from each child to generate a composite plasma tracer data set with a minimum of 5 children/time, group TBS and retinol kinetics can be estimated in children by compartmental analysis; inclusion of vitamin A intake data increases confidence in model predictions. The super-child modeling approach is an effective technique for comparing vitamin A status among children from different populations. These trials were registered at www.clinicaltrials.gov as NCT03000543 (Bangladesh), NCT03345147 (Guatemala), and NCT03030339 (Philippines).

Does selection for growth rate in broilers affect their resistance and tolerance to Eimeria maxima?

Chickens exhibit varied responses to infection with Eimeria parasites. We hypothesise that broilers selected for increased growth rate will show lower resistance and tolerance to a coccidian challenge. 288 chickens of fast (F) or slow (S) growing lines were inoculated with 0 (control), 2500 (low-dose), or 7000 (high-dose) sporulated E. maxima oocysts at 13 days of age in two consecutive rounds. Gain and Intake were measured daily and their values relative to BW at the point of infection were calculated over the pre-patent (days 1-4 post-infection), acute (d5-8 pi), and recovery (d9-12 pi) phases of infection to assess the impact of infection. Levels of plasma carotenoids, vitamins E and A, long bone mineralisation, caecal microbiota diversity indices, and histological measurements were assessed at the acute (d6 pi) and recovery stage (d13 pi). In addition, we measured the levels of nitric oxide metabolites and the number of parasite genome copies in the jejunumat d6pi. In absolute terms F birds grew 1.42 times faster than S birds when not infected. Infection significantly reduced relative daily gain and intake (P < 0.001), with the effects being most pronounced during the acute phase (P < 0.001). Levels of all metabolites were significantly decreased, apart from NO which increased (P < 0.001) in response to infection on d6pi, and were accompanied by changes in histomorphometric features and the presence of E. maxima genome copies in infected birds, which persisted to d13pi. Furthermore, infection reduced tibia and femur mineralisation, which also persisted to d13pi. Reductions in measured variables were mostly independent of dose size, as was the level of parasite replication. The impact of infection was similar for S and F-line birds for all measured parameters, and there were no significant interactions between line x dose size on any of these parameters. In conclusion, our results suggest that line differences in productive performance do not influence host responses to coccidiosis when offered nutrient adequate diets.

Intestinal ß-carotene bioconversion in humans is determined by a new single-sample, plasma isotope ratio method and compared with traditional and modified area-under-the-curve methods.

The vitamin A value (bioefficacy) of provitamin A carotenoids is determined by absorption of the carotenoid (bioavailability) and its subsequent conversion to retinol (bioconversion). Here we show that intestinal bioconversion of ß-carotene can be estimated based on analysis of a single plasma sample collected 6 h after subjects ingest a test dose of stable isotope-labeled ß-carotene from the ratio of retinyl esters to retinyl esters plus ß-carotene. Plasma isotope ratio predictions of bioconversion ranged from 50 to- 93% (mean 76%) for 45 healthy young adults with low vitamin A stores. Results were the same as predictions made by a traditional area-under-the-curve method calculated from 0 to- 8 h or a modified area-under-the-curve method calculated from 0 to- 12 h. The modified method may provide better estimates of bioconversion between 8 and 24 h after ingestion of a carotenoid dose when stable isotopes cannot be used due to cost or logistics. Furthermore, because the plasma isotope ratio method requires only one blood sample and no isolation of triglyceride-rich lipoproteins, its use will facilitate estimation of provitamin A carotenoid bioconversion in human subjects and especially children, in whom repeated blood sampling is not feasible.

A Simple Plasma Retinol Isotope Ratio Method for Estimating ß-Carotene Relative Bioefficacy in Humans: Validation with the Use of Model-Based Compartmental Analysis.

Background: Provitamin A carotenoids are an important source of dietary vitamin A for many populations. Thus, accurate and simple methods for estimating carotenoid bioefficacy are needed to evaluate the vitamin A value of test solutions and plant sources. ß-Carotene bioefficacy is often estimated from the ratio of the areas under plasma isotope response curves after subjects ingest labeled ß-carotene and a labeled retinyl acetate reference dose [isotope reference method (IRM)], but to our knowledge, the method has not yet been evaluated for accuracy.Objectives: Our objectives were to develop and test a physiologically based compartmental model that includes both absorptive and postabsorptive ß-carotene bioconversion and to use the model to evaluate the accuracy of the IRM and a simple plasma retinol isotope ratio [(RIR), labeled ß-carotene-derived retinol/labeled reference-dose-derived retinol in one plasma sample] for estimating relative bioefficacy.Methods: We used model-based compartmental analysis (Simulation, Analysis and Modeling software) to develop and apply a model that provided known values for ß-carotene bioefficacy. Theoretical data for 10 subjects were generated by the model and used to determine bioefficacy by RIR and IRM; predictions were compared with known values. We also applied RIR and IRM to previously published data.Results: Plasma RIR accurately predicted ß-carotene relative bioefficacy at 14 d or later. IRM also accurately predicted bioefficacy by 14 d, except that, when there was substantial postabsorptive bioconversion, IRM underestimated bioefficacy. Based on our model, 1-d predictions of relative bioefficacy include absorptive plus a portion of early postabsorptive conversion.Conclusion: The plasma RIR is a simple tracer method that accurately predicts ß-carotene relative bioefficacy based on analysis of one blood sample obtained at ≥14 d after co-ingestion of labeled ß-carotene and retinyl acetate. The method also provides information about the contributions of absorptive and postabsorptive conversion to total bioefficacy if an additional sample is taken at 1 d.

A Retinol Isotope Dilution Equation Predicts Both Group and Individual Total Body Vitamin A Stores in Adults Based on Data from an Early Postdosing Blood Sample.

BACKGROUND: Retinol isotope dilution (RID) is used to determine vitamin A total body stores (TBS) after an oral dose of a vitamin A stable isotope. The generally accepted prediction equation proposed by Olson's group in 1989 (Furr et al. Am J Clin Nutr 1989;49:713-6) includes factors related to dose absorption and retention, isotope equilibration in plasma compared with stores, catabolism during the mixing period, and the optimal time for measuring plasma isotope enrichment. OBJECTIVES: The objectives were 1) to develop a modified RID equation and identify an earlier sampling time for predicting TBS and 2) to improve prediction in individuals as well as groups. METHODS: To develop a modified RID equation, we used results of model-based compartmental analysis [the Simulation, Analysis and Modeling software (WinSAAM version 3.0.8; http://www.WinSAAM.org)] of plasma [13C10]retinol kinetic data from 32 previously studied, healthy young adults of European ancestry who had moderate vitamin A intakes and who ingested 2.95 µmol [13C10]retinyl acetate. RESULTS: We examined the time dependence of factors in the prediction equation related to absorption/retention (Fa) and isotope equilibration (S) and determined that 4 or 5 d postdosing was the optimal sampling time. TBS calculated by the equation TBS = Fa x S x (1/SAp), where SAp is plasma retinol specific activity (fraction of dose/µmol), were highly correlated with model-predicted TBS (r = 0.95 and 0.96 for 4 and 5 d, respectively; P < 0.001); predictions for individuals were also highly correlated (Rs = 0.94 and 0.94; P < 0.001). CONCLUSION: The equation TBS ≈ 0.5 × (1/SAp) accurately predicted vitamin A TBS in this group of 32 healthy young adults and its individual members with the use of data from 1 blood sample taken 4 d after isotope administration.

Plasma Retinol Kinetics and ß-Carotene Bioefficacy Are Quantified by Model-Based Compartmental Analysis in Healthy Young Adults with Low Vitamin A Stores.

BACKGROUND: Model-based compartmental analysis of data on plasma retinol kinetics after administration of labeled retinol provides unique information about whole-body vitamin A metabolism. If labeled ß-carotene is coadministered, its bioefficacy relative to the retinol reference dose can also be estimated. OBJECTIVES: The objectives were to model plasma retinol kinetics after administration of labeled preformed vitamin A and provitamin A ß-carotene and to determine relative ß-carotene bioefficacy. METHODS: We used the Simulation, Analysis and Modeling software (WinSAAM version 3.0.8; http://www.WinSAAM.org) to analyze previously collected data on plasma [13C10]- and [13C5]retinol kinetics for 14 d after oral administration of 1 mg [13C10]retinyl acetate and 2 mg [13C10]ß-carotene in oil to 30 healthy young adults of European ancestry [13 men, 17 women; mean ± SD age: 24.5 ± 4.2 y; mean ± SD body weight: 65.2 ± 10 kg; mean ± SD body mass index (in kg/m2): 22.5 ± 1.9] with moderate vitamin A intakes. RESULTS: A 6-component model provided the best fit to the data, including compartments for initial metabolism of vitamin A, plasma retinol, and extravascular vitamin A storage. The disposal rate was 6.7 ± 3.1 µmol/d, fractional catabolic rate was 6.0% ± 2.3%/d, and vitamin A stores were 123 ± 71 µmol. Relative ß-carotene bioefficacy, based on the ratio of the areas under the fraction of dose curves calculated by WinSAAM, averaged 13.5% ± 6.02% (retinol activity equivalents = 7.7:1.0 µg). Interindividual variation in relative ß-carotene bioefficacy was high (CV: 44%). CONCLUSIONS: Vitamin A kinetics in these young adults were best described by essentially the same model that had been previously developed by using data for older adults with higher vitamin A stores; differences in parameter values reflected differences in vitamin A status. Estimated ß-carotene bioefficacy was relatively low but similar to previously reported estimates obtained by graphical methods. This trial was registered at the UK Clinical Research Network as UKCRN 7413.

An LC/MS/MS method for stable isotope dilution studies of ß-carotene bioavailability, bioconversion, and vitamin A status in humans.

Isotope dilution is currently the most accurate technique in humans to determine vitamin A status and bioavailability/bioconversion of provitamin A carotenoids such as ß-carotene. However, limits of MS detection, coupled with extensive isolation procedures, have hindered investigations of physiologically-relevant doses of stable isotopes in large intervention trials. Here, a sensitive liquid chromatography-tandem mass spectrometry (LC/MS/MS) analytical method was developed to study the plasma response from coadministered oral doses of 2 mg [(13)C10]ß-carotene and 1 mg [(13)C10]retinyl acetate in human subjects over a 2 week period. A reverse phase C18 column and binary mobile phase solvent system separated ß-carotene, retinol, retinyl acetate, retinyl linoleate, retinyl palmitate/retinyl oleate, and retinyl stearate within a 7 min run time. Selected reaction monitoring of analytes was performed under atmospheric pressure chemical ionization in positive mode at m/z 537→321 and m/z 269→93 for respective [(12)C]ß-carotene and [(12)C] retinoids; m/z 547→330 and m/z 274→98 for [(13)C10]ß-carotene and [(13)C5] cleavage products; and m/z 279→100 for metabolites of [(13)C10]retinyl acetate. A single one-phase solvent extraction, with no saponification or purification steps, left retinyl esters intact for determination of intestinally-derived retinol in chylomicrons versus retinol from the liver bound to retinol binding protein. Coadministration of [(13)C10]retinyl acetate with [(13)C10]ß-carotene not only acts as a reference dose for inter-individual variations in absorption and chylomicron clearance rates, but also allows for simultaneous determination of an individual's vitamin A status.

Single nucleotide polymorphisms upstream from the ß-carotene 15,15'-monoxygenase gene influence provitamin A conversion efficiency in female volunteers.

ß-Carotene, the most abundant provitamin A carotenoid in the diet, is converted to retinal by ß-carotene 15,15'-monoxygenase (BCMO1). However, ß-carotene absorption and conversion into retinal is extremely variable among individuals, with proportions of low responders to dietary ß-carotene as high as 45%. Recently, 2 common nonsynonymous single nucleotide polymorphisms (SNPs) within the BCMO1 coding region (R267S; rs12934922 and A379V; rs7501331) revealed reduced catalytic activity, confirming that genetic variations contribute to the low responder phenotype. Because 4 SNPs 5' upstream from the BCMO1 gene were recently shown to affect circulating carotenoid concentrations, the current study aimed to investigate the effects of these SNPs on ß-carotene conversion efficiency. Three of the 4 polymorphisms (rs6420424, rs11645428, and rs6564851) reduced the catalytic activity of BCMO1 in female volunteers by 59, 51, and 48%, respectively. The TG-rich lipoprotein fraction retinyl palmitate:ß-carotene ratio was negatively correlated with the G allele of rs11645428 (r = -0.44; P = 0.018), whereas it was positively correlated with the G allele of rs6420424 (r = 0.53; P = 0.004) and the T allele of rs6564851 (r = 0.41; P = 0.028). Furthermore, large inter-ethnic variations in frequency of affected alleles were detected, with frequencies varying from 43 to 84% (rs6420424), 52 to 100% (rs11645428), and 19 to 67% (rs6564851). In summary, a range of SNPs can influence the effectiveness of using plant-based provitamin A carotenoids to increase vitamin A status in at-risk population groups and this effect may vary depending on ethnic origin.

Importance of ß,ß-carotene 15,15'-monooxygenase 1 (BCMO1) and ß,ß-carotene 9',10'-dioxygenase 2 (BCDO2) in nutrition and health.

In humans, varying amounts of absorbed ß-carotene are oxidatively cleaved by the enzyme ß,ß-carotene 15,15'-monooxygenase 1 (BCMO1) into two molecules of all-trans-retinal. The other carotenoid cleavage enzyme ß,ß-carotene 9',10'-dioxygenase (BCDO2) cleaves ß-carotene at the 9',10' double bond forming ß-apo-10'-carotenal and ß-ionone. Although the contribution of BCDO2 to vitamin A formation has long been debated, BCMO1 is currently considered the key enzyme for retinoid metabolism. Furthermore, BCMO1 has limited enzyme activity towards carotenoids other than provitamin A carotenoids, whereas BCDO2 exhibits a broader specificity. Both enzymes are located at different sites within the cell, with BCMO1 being a cytosolic protein and BCDO2 being located in the mitochondria. Expression of BCMO1 in tissues other than the intestine has recently revealed its function for tissue-specific retinoid metabolism with importance in embryogenesis and lipid metabolism. On the other hand, biological activity of BCDO2 metabolites has been shown to be important in protecting against carotenoid-induced mitochondrial dysfunction. Single-nucleotide polymorphisms (SNPs) such as R267S and A379V in BCMO1 can partly explain inter-individual variations observed in carotenoid metabolism. Advancing knowledge about the physiological role of these two enzymes will contribute to understanding the importance of carotenoids in health and disease.

The combined impact of plant-derived dietary ingredients and acute stress on the intestinal arachidonic acid cascade in Atlantic salmon (Salmo salar).

A study was conducted to assess the effect of substituting high levels of dietary fish oil (FO) and fishmeal (FM) for vegetable oil (VO) and plant protein (PP) on the intestinal arachidonic acid (AA) cascade in the carnivorous fish species Atlantic salmon. Four diets were fed to salmon over a period of 12 months, including a control FMFO diet, with varying replacements of plant-derived ingredients: 80 % PP and 35 % VO; 40 % PP and 70 % VO; 80 % PP and 70 %VO. Subsequently, fish were examined pre- (0 h) and post- (1 h) acute stress for blood parameters and intestinal bioactive lipidic mediators of inflammation (prostaglandins). Plasma cortisol responses were greatest in the FMFO group, while 80 % PP and 70 % VO fish exhibited increased plasma chloride concentrations. The n-3:n-6 PUFA ratio in intestinal glycerophospholipids from 70 % VO groups significantly decreased in both proximal and distal regions due to elevated levels of 18 : 2n-6 and the elongation/desaturation products 20 : 2n-6 and 20 : 3n-6. Increases in n-6 PUFA were not concomitant with increased AA, although the AA:EPA ratio did vary significantly. The 40 % PP and 70 % VO diet produced the highest intestinal AA:EPA ratio proximally, which coincided with a trend in elevated levels of PGF2alpha, PGE2 and 6-keto-PGF1alpha in response to stress. PGE2 predominated over PGF2alpha and 6-keto-PGF1alpha (stable metabolite of PGI2) with comparable concentrations in both intestinal regions. Cyclo-oxygenase-2 (COX-2) mRNA expression was an order of magnitude higher in distal intestine, compared with proximal, and was significantly up-regulated following stress. Furthermore, the 80 % PP and 70 % VO diet significantly amplified proximal COX-2 induction post-stress. Results demonstrate that high replacements with plant-derived dietary ingredients can enhance COX-2 induction and synthesis of pro-inflammatory eicosanoids in the intestine of salmon in response to acute physiological stress.

Sn-2-monoacylglycerol, not glycerol, is preferentially utilised for triacylglycerol and phosphatidylcholine biosynthesis in Atlantic salmon (Salmo salar L.) intestine.

Pathways of lipid resynthesis in the intestine of fish are relatively unknown. Various reports have suggested the existence of both sn-1,3-specific (pancreatic) and non-specific (bile salt-activated) lipase activity operating on dietary triacylglycerol (TAG) in the intestinal lumen of fish during digestion. Thus, sn-2-monoacylglycerol (2-MAG) and glycerol, respective hydrolytic products of each lipase, are absorbed and utilised for glycerolipid synthesis in enterocytes via two alternative routes: monoacylglycerol (MAG) and glycerol-3-phosphate (G3P) pathways. Despite different precursors, both pathways converge at the production of sn-1,2-diacylglycerol (1,2-DAG) where TAG or phosphatidylcholine (PC) synthesis can occur. To elucidate the relative activities of MAG and G3P pathways in Atlantic salmon enterocytes, intestinal segments were mounted in Ussing chambers where equimolar mixtures of sn-2-oleoyl-[1,2,3-(3)H]glycerol (2-MAG) and [(14)C(U)]glycerol, plus unlabelled 16:0 and 18:2n-6 as exogenous fatty acid sources, were delivered in bile salt-containing Ringer solution to the mucosa. The MAG pathway predominated, over the G3P pathway, synthesizing ca. 95% of total TAG and ca. 80% of total PC after a 3 h incubation period at 10 degrees C. Further, the 1,2-DAG branch point into TAG or PC was polarised towards TAG synthesis (6:1) via the MAG pathway but more evenly distributed between TAG and PC (1:1) via the G3P pathway. Effect of long-chain saturated, monounsaturated and polyunsaturated fatty acids on the synthesized TAG/PC ratio was assessed by individually exchanging 16:0, 18:1n-9 or 18:2n-6, for 16:0+18:2n-6, in mucosal solutions. TAG synthesis was influenced considerably more than PC synthesis, via either pathway, by exogenous fatty acids utilised. 18:1n-9 significantly stimulated TAG synthesis via the MAG pathway yielding a TAG/PC ratio of 12:1. Alternatively, 18:2n-6 stimulated TAG synthesis the most via the G3P pathway (TAG/PC=4:1). 16:0 significantly attenuated TAG synthesis via either pathway. Micellar fatty acid species also significantly affected intestinal active transport mechanisms as shown by decreasing transepithelial potential (TEP) and short-circuit current (SSC) with increasing fatty acid unsaturation. The epithelial integrity was, however, not compromised after 3 h of exposure to any of the fatty acids. The implications of these findings on dietary fatty acid composition and enterocytic lipid droplet accumulation are discussed.

Fatty acid utilisation and metabolism in caecal enterocytes of rainbow trout (Oncorhynchus mykiss) fed dietary fish or copepod oil.

A combined fatty acid metabolism assay was employed to determine fatty acid uptake and relative utilisation in enterocytes isolated from the pyloric caeca of rainbow trout. In addition, the effect of a diet high in long-chain monoenoic fatty alcohols present as wax esters in oil derived from Calanus finmarchicus, compared to a standard fish oil diet, on caecal enterocyte fatty acid metabolism was investigated. The diets were fed for 8 weeks before caecal enterocytes from each dietary group were isolated and incubated with [1-14C]fatty acids: 16:0, 18:1n-9, 18:2n-6, 18:3n-3, 20:1n-9, 20:4n-6, 20:5n-3, and 22:6n-3. Uptake was measured over 2 h with relative utilisation of different [1-14C]fatty acids calculated as a percentage of uptake. Differences in uptake were observed, with 18:1n-9 and 18:2n-6 showing the highest rates. Esterification into cellular lipids was highest with 16:0 and C18 fatty acids, accounting for over one-third of total uptake, through predominant incorporation in triacylglycerol (TAG). The overall utilisation of fatty acids in phospholipid synthesis was low, but highest with 16:0, the most prevalent fatty acid recovered in intracellular phosphatidylcholine (PC) and phosphatidylinositol (PI), although exported PC exhibited higher proportions of C20/C22 polyunsaturated fatty acids (PUFA). Other than 16:0, incorporation into PC and PI was highest with C20/C22 PUFA and 20:4n-6 respectively. Recovery of labelled 18:1n-9 in exported TAG was 3-fold greater than any other fatty acid which could be due to multiple esterification on the glycerol 'backbone' and/or increased export. Approximately 20-40% of fatty acids taken up were beta-oxidised, and was highest with 20:4n-6. Oxidation of 20:5n-3 and 22:6n-3 was also surprisingly high, although 22:6n-3 oxidation was mainly attributed to retroconversion to 20:5n-3. Metabolic modification of fatty acids by elongation-desaturation was generally low at <10% of [1-14C]fatty acid uptake. Dietary copepod oil had generally little effect on fatty acid metabolism in enterocytes, although it stimulated the elongation and desaturation of 16:0 and elongation of 18:1n-9, with radioactivity recovered in longer n-9 monoenes. The monoenoic fatty acid, 20:1n-9, abundant in copepod oil as the homologous alcohol, was poorly utilised with 80% of uptake remaining unesterified in the enterocyte. However, the fatty acid composition of pyloric caeca was not influenced by dietary copepod oil.

Enzyme activities of intestinal triacylglycerol and phosphatidylcholine biosynthesis in Atlantic salmon (Salmo salar L.).

The substitution of fish oil with plant-derived oil in diets for carnivorous fish, such as Atlantic salmon, has previously revealed the potentially deleterious supranuclear accumulation of lipid droplets in intestinal cells (enterocytes) which may compromise gut integrity, and consequently, fish health. This suggests that unfamiliar dietary lipid sources may have a significant impact on intestinal lipid metabolism, however, the mode of lipid resynthesis is largely unknown in teleost fish intestine. The present study aimed at characterising three key lipogenic enzymes involved in the biosynthesis of triacylglycerol (TAG) and phosphatidylcholine (PC) in Atlantic salmon enterocytes: monoacylglycerol acyltransferase (MGAT), diacylglycerol acyltransferase (DGAT), and diacylglycerol cholinephosphotransferase (CPT). Furthermore, to investigate the dietary effect of plant oils on these enzymes, two experimental groups of fish were fed a diet with either capelin (fish oil) or vegetable oil (rapeseed oil:palm oil:linseed oil, 55:30:15 w/w) as the lipid source. The monoacylglycerol (MAG) pathway was highly active in the intestinal mucosa of Atlantic salmon as demonstrated by MGAT activity (7 nmol [1-(14)C]palmitoyl-CoA incorporated min(-1) mg protein(-1)) and DGAT activity (4 nmol [1-(14)C]palmitoyl-CoA incorporated min(-1) mg protein(-1)), with MGAT appearing to also provide adequate production of sn-1,2-diacylglycerol for potential utilisation in PC synthesis via CPT activity (0.4 nmol CDP-[(14)C]choline incorporated min(-1) mg protein(-1)). Both DGAT and CPT specific activity values were comparable to reported mammalian equivalents, although MGAT activity was lower. Nevertheless, MGAT appeared not to be the rate-limiting step in salmon intestinal TAG synthesis. The homology between piscine and mammalian enzymes was established by similar stimulation and inhibition profiles by a variety of tested cofactors and isomeric substrates. The low dietary n-3/n-6 PUFA ratio presented in the vegetable oil diet did not significantly affect the activities of MGAT, DGAT, or CPT under optimised assay conditions, or in vivo intestinal mucosa lipid class composition, when compared to a standard fish oil diet.