Use of Theoretical Women and Model-Based Compartmental Analysis to Evaluate the Impact of Vitamin A Intake with or without a Daily Vitamin A Supplement on Vitamin A Total Body Stores and Balance During Lactation.

BACKGROUND: Inadequate vitamin A (VA) intake is common among lactating women in many communities worldwide, but high-dose VA supplementation for postpartum women is not recommended by the World Health Organization as an effective intervention. OBJECTIVES: To simulate the impact of VA intake via diet and daily VA supplements on VA total body stores (TBS) and balance in theoretical lactating women with low/moderate TBS. METHODS: We studied 6 theoretical subjects with assigned values for TBS from 219-624 µmol. Using Simulation, Analysis, and Modeling software and a previously published compartmental model for whole-body VA metabolism, we simulated TBS over 6 mo of established lactation for each subject under 4 conditions: 1) prelactation VA intake was increased to maintain VA balance (LSS); 2) prelactation VA intake was maintained (NLSS); 3) VA intake was the same as 2) but a daily VA supplement (2.8 µmol/d) was added (NLSS+S); and 4) VA intake was as 1) and the daily VA supplement was included (LSS+S). RESULTS: To compensate for the loss of VA via milk while VA balance was maintained (LSS) over 6 mo of lactation, VA intake had to increase by 0.8-1.87 µmol/d (n = 6) compared with NLSS. Over 6 mo of NLSS treatment, VA balance was negative (geometric mean, -0.77 µmol/d) compared with LSS, whereas balance was positive under NLSS+S and LSS+S conditions (0.75 and 1.5 µmol/d, respectively). For LSS, the proportion of total VA disposal was 37% via breastmilk, 32% from VA stores, and 32% from nonstorage tissues. CONCLUSIONS: Adding a daily VA supplement (2.8 µmol/d) to the diet of lactating women with suboptimal VA intake may effectively counterbalance the negative VA balance resulting from the output of VA via breastmilk and thus benefit both mother and infant by maintaining or increasing VA stores and breastmilk VA concentration.

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.

Use of Theoretical Subjects to Develop a Method for Assessing Equivalence of Dietary Vitamin A in a Mixed Diet.

BACKGROUND: Although the vitamin A (VA) equivalency of provitamin A carotenoids from single foods or capsules has been studied using several approaches, there is currently no reliable method to determine VA equivalency for mixed diets. OBJECTIVES: To reach the objective of identifying a method to determine the VA equivalency of provitamin A carotenoids in mixed diets, we tested a new approach using preformed VA as proxy for provitamin A. METHODS: We studied 6 theoretical subjects who were assigned physiologically plausible values for dietary VA intake, retinol kinetic parameters, plasma retinol pool size, and VA total body stores. Using features in the Simulation, Analysis and Modeling software, we specified that subjects ingested a tracer dose of stable isotope-labeled VA on day 0 followed by 0-µg supplemental VA or 200, 400, 800, 1200, 1600, and 2000 µg VA daily from day 14 to day 28; we assigned VA absorption to be 75%. For each supplement level, we simulated plasma retinol specific activity (SAp) over time and calculated the mean decrease in SAp relative to 0 µg. Group mean data were fitted to a regression equation to calculate predicted VA equivalency at each supplement level on day 28. RESULTS: For each subject, higher VA supplement loads resulted in lower SAp, with the magnitude of the decrease differing among subjects. The mean predicted amount of absorbed VA was within 25% of individual subjects' assigned amount for 4 of the 6 subjects, and the mean ratio of predicted to assigned amount of absorbed VA over all supplement loads ranged from 0.60 to 1.50, with an overall mean ratio of 1.0. CONCLUSIONS: Results for preformed VA suggest that this protocol may be useful for determining VA equivalency of provitamin A carotenoids in free-living subjects if mixed diets with known provitamin A content were substituted for the VA supplements.

Kinetics of omega-3 fatty acid transfer to milk differs between fatty acids and stage of lactation in dairy cows.

Fatty acids (FA) differ in their transfer efficiencies and metabolic partitioning and lactating cows provide a robust model to investigate kinetics of FA transport. The objective was to compare kinetics of n-3 polyunsaturated FA (PUFA) trafficking through plasma and into milk. In the first experiment, ten ruminally cannulated multiparous Holstein cows were used in a crossover design with 7 d periods. Cows were milked at 6 h intervals and abomasal treatments provided a single dose of 80.1 g of α-linolenic acid as free FA (ALA-FFA) or 45.5 g EPA and 32.9 g DHA (LCn3-FFA). Transfer of n-3 PUFA to milk was nearly 50% higher for ALA-FFA than LCn3-FFA (48.2 and 32.7% of the bolus) and fit a bi-exponential model. Rapid transport of n-3 PUFA, assumed to be directly through chylomicrons, was nearly twice as high in ALA-FFA than LCn3-FFA and the subsequent slow transport, assumed to be indirect transfer through tissue recycling, was over 2.5-fold higher in LCn3-FFA than in ALA-FFA. Plasma analysis revealed LCn3-FFA enriched phospholipids and cholesterol esters, which had a slow clearance. In the second experiment, 4 cows received a bolus of a mixture of ALA, EPA, and DHA prepartum while not lactating and around d 10, 55, and 225 of lactation. Transfer of ALA to milk did not differ between stages of lactation, but DHA was lower in early compared to mid and late lactation. In conclusion, dietary ALA is rapidly and efficiently transferred to milk in cows while EPA and DHA are rapidly incorporated into plasma or tissue fractions not available to the mammary gland. This demonstrates clear differences in trafficking and partitioning of n-3 PUFA that ultimately impact tissue and organelle enrichment with implications for effective doses.

Compartmental Modeling of Vitamin A Stable Isotope Data from Milk or Plasma Provides Comparable Predictions of Vitamin A Stores in Theoretical Lactating Women.

BACKGROUND: Previous compartmental models describing and quantifying whole-body vitamin A (VA) metabolism have been developed from plasma retinol kinetic data after human subjects ingest stable isotope-labeled VA. For humans, models based on data obtained from other sampling sites (e.g., excreta or milk) have not been proposed. OBJECTIVES: Our objective was to determine whether comparable model predictions of VA total body stores (TBS) in theoretical lactating women were obtained using tracer data from only retinol in plasma or VA in milk. METHODS: We used Simulation, Analysis and Modeling software to simulate values for TBS and the coefficients used in the retinol isotope dilution (RID) equation TBS = FaS/SAp (Fa, fraction of dose in stores; S, retinol specific activity (SA) in plasma/SA in stores; SAp, specific activity in plasma). We compared individual subject predictions of TBS and FaS based on modeling only plasma or only milk tracer data to previous results ("assigned values") for 12 theoretical lactating women when modeling was done based on tracer data for chylomicron retinyl esters, plasma retinol, and milk VA. RESULTS: For subjects with a wide range of TBS, model-predicted TBS based on only plasma data were comparable with assigned values (range: 94%-106%). Using only milk data, predictions ranged from 72% to 178%, but when VA intake was included in modeling, predictions were improved (97%-102%). Similar results were obtained for simulated FaS. CONCLUSIONS: If confirmed in free-living lactating women, results indicate that, similar to models based on serial plasma sampling, a model for whole-body VA kinetics, including predictions of TBS and FaS, can be identified based on tracer data for VA in milk when VA intake is included as a modeling constraint. Milk data have not been previously used for compartmental modeling of VA in humans.

Direct Use of Plasma or Milk Vitamin A Specific Activity Data to Model Retinol Kinetics and Predict Vitamin A Stores in Theoretical Lactating Women.

BACKGROUND: Many applications of the Simulation, Analysis and Modeling software use data on the fraction of an orally administered tracer dose (FD) in plasma; thus, researchers must scale-up measured analyte concentration to the total plasma pool. For studies in lactating women, estimating breast milk pool size is challenging. OBJECTIVES: The objectives were to determine whether the standard vitamin A modeling approach using FD data could be modified to use vitamin A specific activity in milk (SAm) and/or plasma (SAp) for compartmental analysis of vitamin A kinetics and status in theoretical lactating women. METHODS: Using 12 previously studied theoretical subjects with a wide range of assigned values for vitamin A total body stores (TBS) and the coefficient ("FaS") needed to predict TBS using a retinol isotope dilution equation, we simulated data for SAp and SAm for 49 d after oral administration of labeled vitamin A. Then we modeled datasets for SAp and SAm, as well as only SAp or SAm, incorporating a linear scaling factor to automatically convert SA to FD and including several physiologically reasonable constraints as input data. As outcomes, we compared model-predicted TBS and FaS to assigned values. RESULTS: Scaling factors effectively adjusted SA data to adequately predict vitamin A mass in plasma and breast milk pools. Data for SAp and SAm provided model predictions of TBS that were comparable to assigned values (range: 85-107%); using only SAp, ratios ranged from 92% to 108% and for SAm from 85% to 108%. Parallel results were obtained for simulated FaS. CONCLUSIONS: Results show that SA data from plasma and/or milk can be used directly for modeling vitamin A during lactation in theoretical subjects, providing accurate estimates of TBS and FaS. Results suggest that, in free-living lactating women, researchers might measure only SAp or only SAm and adequately describe whole-body vitamin A metabolism and status.

Use of Compartmental Modeling and Datasets for Theoretical Lactating Women to Determine Conditions under Which Vitamin A-Specific Activity in Breast Milk Provides Accurate Estimates of Vitamin A Total Body Stores by Retinol Isotope Dilution.

BACKGROUND: Vitamin A concentrations in breast milk are related to maternal vitamin A intake and status. OBJECTIVES: Our objective was to identify conditions under which vitamin A specific activity in breast milk (SAm) could be used instead of retinol specific activity in plasma (SAp) to predict vitamin A total body stores (TBS) by retinol isotope dilution (RID). METHODS: We used 12 previously-studied theoretical lactating women with assigned values for TBS (219-1348 µmol) and retinol kinetic parameters; we assumed subjects ingested a dose of stable isotope-labeled vitamin A. We expanded a 9-compartment steady state tracer model to include a parallel model for tracee (unlabeled retinol) and then adapted that model so vitamin A intake entered the system in 3 meals each day. Using compartmental analysis, we first simulated SAm and SAp after an overnight fast (as in actual RID experiments) and then with vitamin A intake also restricted in sequential meals on the day before sampling for RID. RESULTS: After an overnight fast, SAm at day 21 postdosing was lower than SAp. However, if vitamin A intake was also restricted in 1, 2, or 3 meals before sampling, SAm/SAp (mean ± SD) was 0.92 ± 0.042,  0.96 ± 0.016,  or 0.99 ± 0.004,  respectively; results for days 14 and 28 were similar. When either SAp or SAm was used to predict TBS by RID on day 21 after 1-d restriction, predictions for all subjects were within 25% of assigned TBS. CONCLUSIONS: Results indicate that, for theoretical lactating women with a wide range of vitamin A status, SAm will accurately predict TBS by RID at 2-4 wk postdosing if vitamin A intake is restricted for 1 d before sampling. If confirmed in community settings, results suggest that vitamin A status in lactating women can be determined without collecting blood.

Development of a Compartmental Model for Studying Vitamin A Kinetics and Status in Theoretical Lactating Women.

BACKGROUND: Low vitamin A status and suboptimal milk vitamin A concentrations are problems in many populations worldwide. However, limited research has been done on whole-body vitamin A kinetics in women of reproductive age, especially during lactation. OBJECTIVES: Goals were to develop compartmental models describing retinol kinetics in theoretical nonlactating (NL) and lactating (L) women and to determine whether the retinol isotope dilution (RID) method accurately predicted vitamin A total body stores (TBS) in the groups and individuals. METHODS: We adapted 12 previously-used theoretical females with assigned values for retinol kinetic parameters and TBS (225-1348 µmol); subjects were NL or L (nursing one 3- to 6-mo-old infant) during 49-d kinetic studies after isotope dosing. We used an established compartmental model, adding a compartment for chylomicrons and, for L, another for mammary gland milk with inputs from holo-retinol-binding protein and chylomicron retinyl esters and output to milk. Using compartmental analysis, we simulated tracer responses in compartments of interest and calculated TBS using the RID equation TBS =   FaS/SAp [Fa, fraction of dose in stores; S, retinol specific activity in plasma/specific activity in stores; SAp, specific activity of retinol in plasma]. RESULTS: Models for both groups were well identified. Simulated plasma tracer responses were similar for NL and L, with L always below NL; milk tracer paralleled plasma from 10 d postdosing. Geometric mean FaS ratios (L/NL) were ∼0.75 during days 2-30. Using appropriate group FaS, RID provided accurate TBS predictions for >80% of NL and L subjects after day 18 when CV% for FaS was ∼10%. CONCLUSIONS: These new physiologically-based models for vitamin A kinetics may be useful for future research in women of reproductive age. Results indicate that, in groups like these, RID to assess an individual's vitamin A status should be done at 21-28 d after isotope dosing.

Does the Amount of Stable Isotope Dose Influence Retinol Kinetic Responses and Predictions of Vitamin A Total Body Stores by the Retinol Isotope Dilution Method in Theoretical Children and Adults?

BACKGROUND: To minimize both cost and perturbations to the vitamin A system, investigators limit the amount of stable isotope administered when estimating vitamin A total body stores (TBS) by retinol isotope dilution (RID). OBJECTIVES: We hypothesized that reasonable increases in the mass of stable isotope administered to theoretical subjects would have only transient impacts on vitamin A kinetics and minimal effects on RID-predicted TBS. METHODS: We adapted previously used theoretical subjects (3 children, 3 adults) with low, moderate, or high assigned TBS and applied compartmental analysis to solve a steady state model for tracer and tracee using assigned values for retinol kinetic parameters and plasma retinol. To follow retinol trafficking when increasing amounts of stable isotope were administered [1.39-7 (children) and 2.8-14 µmol retinol (adults)], we added assumptions to an established compartmental model so that plasma retinol homeostasis was maintained. Using model-simulated data, we plotted retinol kinetics versus time and applied the RID equation TBS = FaS/SAp [Fa, fraction of dose in stores; S, retinol specific activity (SA) in plasma/SA in stores; SAp, SA in plasma] to calculate vitamin A stores. RESULTS: The model predicted that increasing the stable isotope dose caused transient early increases in hepatocyte total retinol; increases in plasma tracer were accompanied by decreases in tracee to maintain plasma retinol homeostasis. Despite changes in kinetic responses, RID accurately predicted assigned TBS (98-105%) at all loads for all theoretical subjects from 1 to 28 d postdosing. CONCLUSIONS: Results indicate that, compared with doses of 1.4-3.5 µmol used in recent RID field studies, doubling the stable isotope dose should not affect the accuracy of TBS predictions, thus allowing for experiments of longer duration when including a super-subject design (Ford et al., J Nutr 2020;150:411-8) and/or studying retinol kinetics.

The Use of Datasets for Theoretical Subjects to Validate Vitamin A-Related Methods and Experimental Designs.

We review recent work in which model-based compartmental analysis has been applied to data for theoretical human subjects in order to study questions related to vitamin A kinetics and metabolism. Using model simulations in this way, one can validate experimental designs, evaluate or improve vitamin A assessment methods, study the influence of perturbations on assessment methods, and/or advance information related to retinol kinetics. We also provide some information on the rationale for assigning physiologically appropriate values for specified characteristics [e.g., plasma retinol concentration, vitamin A total body stores (TBS), vitamin A intake] to hypothetical individuals, and in addition, we outline how one might first select an appropriate compartmental model for whole-body vitamin A metabolism and then specify physiologically reasonable values for the associated retinol kinetic parameters. In the studies discussed here, the Simulation, Analysis, and Modeling software was used to simulate responses in key model compartments for hypothetical subjects so that model predictions could be compared to assigned values or projected outcomes. For example, in the case of the retinol isotope dilution (RID) method that is used to assess vitamin A status, application of this approach has provided a way to evaluate the accuracy of TBS predictions under different steady state and non-steady state conditions, thus increasing confidence about the validity of RID results obtained in the field. Although datasets for theoretical subjects have been used to evaluate protocols in pharmacokinetics, to our knowledge, other nutrition researchers have not previously used approaches such as those described here. Our results to date indicate that this strategy has the potential to provide useful information related not only to vitamin A but perhaps to other nutrients as well.

Priming with Retinoic Acid, an Active Metabolite of Vitamin A, Increases Vitamin A Uptake in the Small Intestine of Neonatal Rats.

Given that combined vitamin A (VA) and retinoic acid (RA) supplementation stimulated the intestinal uptake of plasma retinyl esters in neonatal rats, we administrated an RA dose as a pretreatment before VA supplementation to investigate the distinct effect of RA on intestinal VA kinetics. On postnatal days (P) 2 and 3, half of the pups received an oral dose of RA (RA group), while the remaining received canola oil as the control (CN). On P4, after receiving an oral dose of 3H-labeled VA, pups were euthanized at selected times (n = 4-6/treatment/time) and intestine was collected. In both CN and RA groups, intestinal VA mass increased dramatically after VA supplementation; however, RA-pretreated pups had relatively higher VA levels from 10 h and accumulated 30% more VA over the 30-h study. Labeled VA rapidly peaked in the intestine of CN pups and then declined from 13 h, while a continuous increase was observed in the RA group, with a second peak at 10 h and nearly twice the accumulation of 3H-labeled VA compared to CN. Our findings indicate that RA pretreatment may stimulate the influx of supplemental VA into the intestine, and the increased VA accumulation suggests a potential VA storage capacity in neonatal intestine.

Influence of Vitamin A Status on the Choice of Sampling Time for Application of the Retinol Isotope Dilution Method in Theoretical Children.

BACKGROUND: Vitamin A status may influence the choice of a blood sampling time for applying the retinol isotope dilution (RID) equation to predict vitamin A total body stores (TBS) in children. OBJECTIVES: We aimed to identify time(s) after administration of labeled vitamin A that provide accurate estimates of TBS in theoretical children with low or high TBS. METHODS: We postulated 2- to 5-y-old children (12/group) with low (<200 µmol) or high TBS (≥700 µmol) and used compartmental analysis to simulate individual subject values for the RID equation TBS =   FaS/SAp (Fa, fraction of dose in stores; S, retinol specific activity in plasma/in stores; SAp, retinol specific activity in plasma). Using individual SAp and group geometric mean FaS values from 1-28 d, we calculated individual and group mean TBS and compared them to assigned values. RESULTS: Mean TBS was accurately predicted for both groups at all times. For individuals, predicted and assigned TBS were closest when the CV% for FaS was low [12-14%; 4-13 d (low), 12-28 d (high)]. The mean percentage error for TBS was <10% from 2-19 d (low) and 7-28 d (high). Predicted TBS was within 25% of assigned TBS for ≥80% of children from 3-23 d (low) and 9-28 d (high). Within groups, RID tended to overestimate lower TBS and underestimate higher TBS. CONCLUSIONS: Using a good estimate for FaS, accurate RID predictions of TBS for individuals will be obtained at many times. If vitamin A status is low, results indicate that early sampling (e.g., 4-13 d) is optimal; if vitamin A status is high, sampling at 12-28 d is indicated. When vitamin A status is unknown, sampling at 14 d is recommended, or a super-subject design can be used to obtain the group mean FaS at various times for RID prediction of TBS in individuals.

Pregnancy and Lactation Alter Vitamin A Metabolism and Kinetics in Rats under Vitamin A-Adequate Dietary Conditions.

BACKGROUND: Vitamin A (VA) plays critical roles in prenatal and postnatal development; however, limited information is available regarding maternal VA metabolism during pregnancy and lactation. OBJECTIVES: We investigated the impact of pregnancy and lactation on VA metabolism and kinetics in rats, hypothesizing that changes in physiological status would naturally perturb whole-body VA kinetics. METHODS: Eight-week old female rats (n = 10) fed an AIN-93G diet received an oral tracer dose of 3H-labeled retinol to initiate the kinetic study. On d 21 after dosing, six female rats were mated. Serial blood samples were collected from each female rat at selected times after dose administration until d 14 of lactation. Model-based compartmental analysis was applied to the plasma tracer data to develop VA kinetic models. RESULTS: Our compartmental model revealed that pregnancy resulted in a gradual increase in hepatic VA mobilization, presumably to support different stages of fetal development. Additionally, the model indicates that during lactation, VA derived from dietary intake was the primary source of VA delivered to the mammary gland for milk VA secretion. CONCLUSION: During pregnancy and lactation in rats with an adequate VA intake and previous VA storage, the internal redistribution of VA and increased uptake from diet supported the maintenance of VA homeostasis.

Use of Model-Based Compartmental Analysis and Theoretical Data to Further Explore Choice of Sampling Time for Assessing Vitamin A Status in Groups and Individual Human Subjects by the Retinol Isotope Dilution Method.

BACKGROUND: An optimal blood sampling time for application of the retinol isotope dilution (RID) method for predicting vitamin A total body stores (TBS) (i.e., vitamin A status) has not been established. OBJECTIVES: Objectives were to identify sampling times that provide accurate estimates of TBS by RID in groups and individuals by applying compartmental modeling to data for theoretical adults and children. METHODS: We selected previously generated hypothetical adults and children (20 per group) that had a wide range of assigned values for TBS and vitamin A kinetic parameters. We used the Simulation, Analysis and Modeling software to simulate individual kinetic responses; then we calculated geometric mean values for the RID equation coefficients and each individual's plasma retinol specific activity at various times, using those values to predict group mean and individual subject TBS. Predicted values for TBS were compared with assigned values. RESULTS: Accurate estimates of group mean TBS were obtained at all sampling times from 1 to 30 d in both adults and children. For individuals, correlations between RID-predicted TBS and assigned values increased with time in the adults (R2 = 0.80 at day 14, 0.96 at day 21, and 0.99 at day 28); a similar trend was observed for the children, with R2 = 0.82 at day 7 and increasing to 0.97 at days 21 and 28 (P < 0.001 for all comparisons). CONCLUSIONS: Although no single, unique time provided the most accurate prediction of TBS for all individuals within these groups, applying the RID method at 21 or 28 d yielded predictions that were within 25% of assigned values for 90% or 95% of adults, respectively; corresponding values for children were 80% from 10 to 20 d, and 85% at 21 and 28 d. For most subjects, early times (<14 d for adults and <10 d for children) provided less accurate predictions.

Development of a Compartmental Model to Investigate the Influence of Inflammation on Predictions of Vitamin A Total Body Stores by Retinol Isotope Dilution in Theoretical Humans.

BACKGROUND: Inflammation, both acute and chronic, is associated with reductions in the synthesis of retinol-binding protein (RBP) and the concentration of retinol in plasma. Consequently, it is currently recommended that the retinol isotope dilution (RID) method not be used to estimate vitamin A total body stores (TBS) in subjects with inflammation. OBJECTIVES: To apply compartmental analysis to study the effects of inflammation on hepatic apo-RBP input, plasma retinol pool size, and RID-predicted TBS in theoretical subjects with known steady state values for these parameters. METHODS: We selected 6 previously generated hypothetical subjects who ingested a dose of stable isotope-labeled vitamin A (day 0). Starting with each subject's published steady state model for retinol tracer kinetics, we developed a parallel model for unlabeled retinol and RBP that incorporated links between these entities and tied liver retinol secretion to RBP availability. Then we perturbed the steady state model by initiating chronic or acute inflammation on day 0 or acute inflammation on day 3 or 9 and simulating results for RBP, plasma retinol, and TBS. RESULTS: Chronic inflammation led to substantial reductions in RID-predicted TBS for at least 2 weeks after tracer administration. In contrast, acute inflammation induced on day 0 or 3 resulted in less dramatic impacts on TBS (37% or 20% reduction, respectively, from steady state levels, with levels rebounding by 14 days). When inflammation was induced 9 days after administration of the tracer, the effects on predicted TBS were minimal. Overall, for acute inflammation initiated at 0, 3, or 9 days, accurate predictions of TBS were obtained by 2 weeks. CONCLUSIONS: Compartmental analysis can be applied in the novel way described here to study the influence of perturbations such as inflammation on predictions of vitamin A status using RID. Such an approach has potential value for studying other perturbations and different nutrients.

A Compartmental Model Describing the Kinetics of ß-Carotene and ß-Carotene-Derived Retinol in Healthy Older Adults.

BACKGROUND: Descriptive and quantitative information on ß-carotene whole-body kinetics in humans is limited. OBJECTIVES: Our objective was to advance the development of a physiologically based, working hypothesis compartmental model describing the metabolism of ß-carotene and ß-carotene-derived retinol. METHODS: We used model-based compartmental analysis (Simulation, Analysis and Modeling software) to analyze previously published data on plasma kinetics of [2H8]ß-carotene, [2H4]ß-carotene-derived retinol, and [2H8]retinyl acetate-derived retinol in healthy, older US adults (3 female, 2 male; 50-68 y); subjects were studied for 56 d after consuming doses of 11 µmol [2H8]ß-carotene and, 3 d later, 9 µmol [2H8]retinyl acetate in oil. RESULTS: We developed a complex model for labeled ß-carotene and ß-carotene-derived retinol, as well as preformed vitamin A, using simulations to augment observed data during model calibration. The model predicts that mean (range) ß-carotene absorption (bioavailability) was 9.5% (5.2-14%) and bioefficacy was 7.3% (3.6-14%). Of the absorbed ß-carotene, 41% (25-58%) was packaged intact in chylomicrons and the balance was converted to retinol, with 58% (42-75%) transported as retinyl esters in chylomicrons and 0-2% by retinol-binding protein. Most (95%) chylomicron ß-carotene was cleared by the liver. Later data revealed differences in the metabolism of retinyl acetate- versus ß-carotene-derived retinol; data required that both ß-carotene and derived retinol be recycled from extrahepatic tissues (e.g. adipose) in HDL. Of total bioconversion [73% (47-99%)], 82% occurred in the intestine, 17% in the liver, and 0.83% in other tissues. CONCLUSIONS: Our model advances knowledge about whole-body ß-carotene metabolism in healthy adults, including the kinetics of transport in all lipoprotein species, and suggests hypotheses to be tested in future studies, such as the possibility that retinol derived from hepatic conversion over a long period of time might contribute to plasma retinol homeostasis and total body vitamin A stores.

Are Fatty Acids Gluconeogenic Precursors?

It is widely accepted that the tricarboxylic acid (TCA) cycle is a critical partner for gluconeogenesis (GNG) in hepatocytes. Although researchers in the 1950s showed, using radiolabeled long-chain fatty acids, that acetate derived from fatty acid ß-oxidation contributes carbon to glucose, fatty acids are not included on lists of gluconeogenic precursors in many textbooks of biochemistry and nutritional biochemistry. Here, by following the flow of carbon atoms through the mitochondrial TCA cycle and into cytosolic GNG, it is shown that carbons in acetyl-CoA derived from fatty acid ß-oxidation will be found in glucose. Specifically, it is evident that, after the condensation of acetyl-CoA and oxaloacetate (OAA) to make citrate at the start of the TCA cycle, the 2 carbons lost from the cycle as carbon dioxide come from OAA, not acetyl-CoA. Carbons from acetyl-CoA are retained as the cycle progresses toward malate, and when malate exits the mitochondrion for GNG, carbons that originated in acetyl-CoA and OAA are found to contribute equally to glucose. With influx of other critical precursors into the TCA cycle and efflux of malate into the cytosol for GNG, the TCA cycle is in balance. During fasting-induced GNG, there is a net gain of glucose in glucogenic cells; however, the fact that there is no net gain in the TCA cycle is irrelevant as far as precursors are concerned. Given the physiological importance of fat as a source of reserve energy, and knowing that some cell types rely on glucose as their primary supplier of energy, a role for fatty acids in glucose production aligns both with intuition and with evidence provided by a careful look at the biochemistry and older isotope studies. Hopefully, subsequent editions of textbooks will list fatty acids among the gluconeogenic precursors.

Vitamin A Absorption Efficiency Determined by Compartmental Analysis of Postprandial Plasma Retinyl Ester Kinetics in Theoretical Humans.

BACKGROUND: Better methods are needed for determining vitamin A absorption efficiency in humans to support development of dietary recommendations and to improve the accuracy of predictions of vitamin A status. OBJECTIVES: We developed and evaluated a method for estimating vitamin A absorption efficiency based on compartmental modeling of theoretical data on postprandial plasma retinyl ester (RE) kinetics. METHODS: We generated data on plasma RE and retinol kinetics (30 min to 8 h or 56 d, respectively) after oral administration of labeled vitamin A for 12 theoretical adults with a range of values assigned for vitamin A absorption (55-90%); we modeled all data to obtain best-fit values for absorption and other parameters using Simulation, Analysis, and Modeling software. We then modeled RE data only (16 or 10 samples), with or without added random error, and compared assigned to predicted absorption values. We also compared assigned values to areas under RE response curves (RE AUCs). RESULTS: We confirmed that a unique value for vitamin A absorption cannot be identified by modeling plasma retinol tracer kinetics. However, when RE data were modeled, predicted vitamin A absorptions were within 1% of assigned values using data without error and within 12% when 5% error was included. When the sample number was reduced, predictions were still within 13% for 10 of the 12 subjects and within 23% overall. Assigned values for absorption were not correlated with RE AUC (P = 0.21). CONCLUSIONS: We describe a feasible and accurate method for determining vitamin A absorption efficiency that is based on compartmental modeling of plasma RE kinetic data collected for 8 h after a test meal. This approach can be used in a clinical setting after fasting subjects consume a fat-containing breakfast meal with a known amount of vitamin A or a stable isotope label.

Perturbed Vitamin A Status Induced by Iron Deficiency Is Corrected by Iron Repletion in Rats with Pre-Existing Iron Deficiency.

BACKGROUND: Although iron deficiency is known to interrupt vitamin A (VA) metabolism, the ability of iron repletion to restore VA metabolism and kinetics in iron-deficient rats is not well understood. OBJECTIVES: In the present study, we examined the effects of dietary iron repletion on VA status in rats with pre-existing iron deficiency. METHODS: Weanling Sprague-Dawley rats were fed a VA-marginal diet (0.35 mg retinol/kg diet) containing either a normal concentration of iron [35 ppm, control group (CN)] or reduced iron (3 ppm, iron-deficient group, ID-); after 5 wk, 4 rats/group were killed for baseline measurements. A 3H-labeled retinol emulsion was administered intravenously to the remaining rats (n = 6, CN; n = 10, ID-) as tracer to initiate the kinetic study. On day 21 after dosing, n = 5 ID- rats were switched to the CN diet, generating an iron-repletion group (ID+). Blood samples were collected at 34 time points ≤92 d after dose administration, when all rats were killed and iron and VA status were determined. RESULTS: At baseline, ID- rats had developed iron deficiency, with a reduced plasma VA concentration (0.67 compared with 1.20 µmol/L in ID- and CN rats, respectively; P < 0.01) and a tendency toward higher liver VA (265 compared with 187 nmol in ID- and CN rats, respectively; P = 0.10). On day 92, iron deficiency persisted in ID- rats, accompanied by 2-times higher liver VA (456 nmol compared with 190 nmol in ID- and CN rats, respectively; P < 0.001) but lower plasma VA (0.64 compared with 0.94 µmol/L in ID- and CN rats, respectively; P = 0.05). ID+ rats not only recovered from iron deficiency, but also exhibited less liver VA sequestration (276 nmol) and normal plasma VA (0.91 µmol/L, not different from CN rats). CONCLUSIONS: Our results suggest that iron repletion can remove the inhibitory effect of iron deficiency on hepatic mobilization of VA and restore plasma retinol concentrations in iron-deficient rats, setting the stage for kinetic studies of VA turnover in this setting.