Abstract
Background: Vitamin A (VA; retinol) supplementation is used to reduce child mortality in countries with high rates of malnutrition. Existing research suggests that neonates (<1 mo old) may have a limited capacity to store VA in organs other than the liver; however, knowledge about VA distribution and kinetics in individual, nonhepatic organs is limited.
Objective: We examined retinol uptake and turnover in nonhepatic organs, including skin, brain, and adipose tissue, in neonatal rats without and after VA supplementation.
Design: Sprague-Dawley neonatal rats (n = 104) were nursed by mothers fed a VA-marginal diet (0.35 mg retinol/kg diet) and treated on postnatal day 4 with an oral dose of either VA (6 μg retinyl palmitate/g body weight) or canola oil (control), both containing 1.8 μCi of [3H]retinol. Subsequently, pups (n = 4 · group−1 · time−1) were killed at 13 different times from 30 min to 24 d after dosing. The fractional and absolute transfer of chylomicron retinyl esters (CM-REs), retinol bound to retinol-binding protein (RBP-ROH), and total retinol were estimated in WinSAAM software.
Results: VA supplementation redirected the flow of CM-REs from peripheral to central organs and accumulated mainly in the liver. The RBP-ROH released from the liver was acquired mainly by the peripheral tissues but not retained efficiently, causing repeated recycling of retinol between plasma and tissues (541 compared with 5 times in the supplemented group and control group, respectively) and its rapid turnover in all organs, except the brain and white adipose tissue. Retinol stores in the liver lasted for ∼2 wk before being gradually transferred to other organs.
Conclusions: VA supplementation administered in a single high dose during the first month after birth is readily acquired but not retained efficiently in peripheral tissues of neonatal rats, suggesting that a more frequent, lower-dose supplementation may be necessary to maintain steady VA concentrations in rapidly developing neonatal tissues.
Keywords: chylomicron, extrahepatic, growth and development, mathematical modeling, neonate, retinyl esters, vitamin A deficiency, vitamin A metabolism, vitamin A supplementation
INTRODUCTION
The prevalence of vitamin A (VA4; retinol) deficiency in low- and middle-income countries has declined by ∼10% since 1991; however, in sub-Saharan Africa and south Asia, VA deficiency remains very common, affecting 1 in every 2 children (1). VA deficiency causes visual impairments that can lead to permanent blindness and is associated with an increased risk of mortality from infectious diseases (2). Neonates (<1 mo old) are particularly susceptible to these ailments because of low VA reserves and an immature immune system (3, 4). Consequently, mortality rates are highest during the neonatal period with prematurity and infections as the main cause (5, 6). To address this problem, WHO recommended periodic high-dose VA supplementation (≥50,000 IU depending on age) as a means to increase the body’s VA stores and prevent infections or reduce their severity (7, 8). This treatment proved to be effective in mitigating child mortality; however, it did not show a similar benefit in infants <6 mo old for reasons that remain unresolved (9–11).
To better understand the neonatal response to acute VA supplementation, we have previously used the neonatal rat model to examine VA storage and metabolism in multiple organs that cannot be sampled in humans because of ethical reasons. The preliminary findings of these studies indicate that, compared with adult rats in a similar VA status, the extrahepatic tissues in neonates store relatively little VA (13% compared with 44% in adult rats) (12), despite a higher proportion of retinyl esters (REs) being acquired by these tissues (40% compared with 25%) (13). VA supplementation increased the concentration of retinol in most extrahepatic organs, but only transiently (for ∼1 d) (12), suggesting that the extrahepatic tissues in neonates may have a limited retinol storage capacity.
To further explore this hypothesis, we have used model-based compartmental analysis of tracer kinetic data to examine VA metabolism in different neonatal organs with a focus on tissues that are relatively unexplored from the perspective of VA research, such as the brain, skin, white adipose tissue (WAT), and brown adipose tissue (BAT). Specifically, we have estimated the transfer of retinol, in fractional and absolute terms, between plasma and organs with distinct kinetic profiles generated for the 2 main molecular forms of VA in rat plasma: REs present in postprandial chylomicrons (CM-REs) and retinol bound to retinol-binding protein (RBP-ROH) (14, 15). Our results provide insight into the organ-specific response to acute VA supplementation and the scientific basis to help inform the most efficient treatment of VA deficiency in neonates.
METHODS
Rats and diet
Rats were housed and fed according to a protocol reported previously (12, 16). Briefly, eleven pregnant Sprague-Dawley (Charles River Laboratories) rats were fed a VA-marginal (0.35 mg retinol equivalents/kg) AIN-93G purified diet (Research Diets) (17) to render their pups into a state similar to that of low-birth-weight infants in regions with a high prevalence of VA deficiency. All animal procedures were approved by the Institutional Animal Care and Use Committee of the Pennsylvania State University.
Dose administration
On postnatal day (P) 4, which corresponds to ∼1 mo of age in humans (18), pups that were previously randomly assigned to either the VA-supplemented (n = 52; 33 males) or control (n = 52; 30 males) group received an oral dose of VA or canola oil (control), respectively. Each dose, both the control and the VA-supplemented, contained 1.8 μCi (26 ng) of 11,12-[3H]retinol (PerkinElmer) as the tracer for VA. The VA supplement consisted of all-trans retinyl palmitate (Sigma-Aldrich) in the amount of 50,000 IU/2.5 kg of body weight, as given to human infants (19), or 200 IU for a neonatal rat weighing 10 g. The proportion of labeled-to-unlabeled retinol in the supplemented dose was ∼0.05%. Doses were prepared and administered by using a method described in prior reports (12, 16).
Tissue collection
At 13 times after dosing (0.5, 1, 4, 8, and 15 h and 1, 2, 4, 8, 11, 14, 18, and 24 d), 4 pups/group were removed from cages, weighed, and sedated with isoflurane or, after P 14, asphyxiated with CO2. Blood was collected from the vena cava and the following organs were dissected: liver, stomach (including the contents), intestines (small and large including the contents), lungs, kidneys with adrenals, brain, interscapular BAT, and WAT (collected on and after P 12 from the inguinal and scapular depots); the remaining carcass was composed largely of bones, muscles, and the connective tissue as well as the eyes, spleen, and heart. Blood samples were centrifuged at 750 × g for 15 min and stored at −20°C. Tissue samples were snap-frozen in liquid nitrogen and stored at −80°C.
Tracer and retinol mass analysis
Tracer concentration and retinol mass were measured in plasma and tissue lipid extracts obtained by using a procedure described earlier (16, 20). Radioactivity analysis was performed with the use of an LS 6500 liquid-scintillation counting system (Beckman Coulter) with each sample counted to a 1% error. Total retinol (REs and unesterified retinol) mass was measured by using ultra-performance liquid chromatography (UPLC) (Acquity UPLC System; Waters).
Kinetic analysis
VA kinetics were determined by using model-based compartmental analysis in the Windows version of Simulation, Analysis, and Modeling software version 3.0.8 (21). The input file for modeling the tracer response in plasma contained the initial estimates of fractional transfer coefficients [L(I,J)s; the fraction of tracer in compartment J transferred to compartment I per day] and the observed data expressed as a fraction of the dose (total radioactivity measured in plasma and organ divided by the radioactivity of the ingested dose). Each observation was a mean of 4 pups killed at one sampling time and weighted by the SD, which was sufficient to detect differences of P < 0.05 between treatment groups for the primary outcome, irreversible loss of retinol. To generate a distinct tracer profile for CM-REs and RBP-ROH, which were not measured directly, each observed value of total retinol was defined as the sum of CM-REs and RBP-ROH.
The input files for modeling organs contained the L(I,J)s, the observed data, and a forcing function. The forcing function specified the amount of tracer available in plasma for uptake at any time during the study and was used to uncouple the organs from the rest of the system and model them individually based on the assumption that organs acquire retinol from plasma only and not from other organs. Because the uptake of retinol from plasma was assumed to be unidirectional, the loss of retinol from organs (referred to as turnover) included both the amount released back to plasma and the amount metabolized locally. Correcting the organ tracer data for the tracer present in organ’s residual plasma was previously found not to affect the values of kinetic parameters (16); therefore, all organs were modeled by using the uncorrected data.
Model structures and kinetic parameters
The initial structure of the plasma model (Figure 1) was adapted from a previous kinetic study of neonatal rats (22). To capture the increase in the fraction of tracer in plasma observed during the last 10 d of the study (and a simultaneous decline in the liver tracer fraction), compartment 7 was added to represent a portion of the tracer stored in the liver for 14 d and released afterward. The initial organ models (Figure 2A, B) were also adapted from a previous kinetic study (23). Because these models fitted the observed data well, the number of compartments and parameters in each model was left unchanged. Before calculating the retinol turnover rate [R(0,J) = L(0,J) × M(J)], organ models were simplified to merge the incoming chylomicron- and retinol-binding protein (RBP)-derived retinol into a common pool of total retinol (Figure 2C). The mass of this pool [M(J)] was previously established by UPLC analysis (12).
FIGURE 1.
Plasma model of VA kinetics in neonatal rats dosed orally with [3H]retinol on postnatal day 4. Circles represent compartments, squares represent delay components, and arrows represent their interconnections. CM-RE, chylomicron retinyl ester; DT, delay component; IC, initial condition; L, fraction; RBP-ROH, retinol bound to retinol-binding protein; VA, vitamin A.
FIGURE 2.
Liver (A) and stomach (B) models of VA kinetics in neonatal rats dosed orally with [3H]retinol on postnatal day 4 and a simplified liver model (C) developed to calculate retinol turnover rate [R(I,J)]. Circles represent compartments, squares represent components defined by the plasma forcing function, and arrows represent their interconnections. L(I,J)s represent the fractional transfer of chylomicron-derived, RBP-derived, or total retinol into or out of the organ. CM-RE, chylomicron retinyl ester; IC, initial condition; L, fraction; RBP, retinol-binding protein; RBP-ROH, retinol bound to retinol-binding protein; VA, vitamin A.
During the modeling phase, the fractional transfer coefficients were adjusted in a step-wise manner to obtain the best fit of the model-calculated plot to the observed data. After a satisfactory fit was found, the final parameter values were generated through a weighted nonlinear regression analysis, which minimized the residuals given the weight assigned to data, parameter constraints, and their uniqueness. The parameters were considered well identified if the sum of squares from regression analysis was <10−5 and the parameter fractional SD was <0.5.
Statistical analysis
The fraction of dose versus time was plotted, and the values at individual time points were compared with the use of Student’s t test with Bonferroni’s correction for multiple comparisons (GraphPad Prism version 5.0). The fractional transfer coefficients [L(I,J)s] were expressed as the fraction of tracer in compartment J transferred to compartment I per day. The absolute transfer rates [R(I,J)s; in nanomoles per day] were calculated as the product of the mean mass of retinol in compartment J during day 1 of the study [M(J); in nanomoles] and the corresponding fractional transfer coefficient [L(I,J)]. The resulting SEMs were calculated according to the error propagation formula
 |
where FSD is the fractional standard deviation.
All kinetic parameters were expressed as means ± SEMs and compared statistically with the use of Student’s t test, with the t statistic evaluated for significance by using the table of critical values of t distribution. A P value < 0.05 was considered the level of significance.
RESULTS
Mean organ tracer levels
The liver accumulated most of the tracer in both treatment groups (Figure 3). The intestines and skin ranked second and third, but there was an ∼10-fold difference between their mean fraction of dose (∼2%) and that of the liver. The mean fraction of dose in other extrahepatic organs was <1%. The lowest value was found in the brain (0.06%). VA supplementation significantly increased the fraction of tracer in the liver and lungs and decreased it in the kidneys.
FIGURE 3.
Mean organ fraction of the ingested [3H]retinol dose between days 0 and 24 after dosing in control and VA-supplemented rats dosed on postnatal day 4. The inset shows the nonhepatic tissues. Bars represent the means ± SEMs, n = 52 rats. *P < 0.05 (Student’s t test). BAT, brown adipose tissue; VA, vitamin A; WAT, white adipose tissue.
Tracer response in digestive organs
The tracer response in the digestive system did not differ by treatment: both groups showed a rapid loss of tracer from the stomach and intestine and no evidence of retinol retention or recycling (Figure 4). Although the fraction of tracer in the intestines after day 4 of the study was higher in the VA-supplemented group than in the control group, the difference was not significant. The absorption efficiency, based on the fraction of tracer lost during digestion [L(0,2)] was 99% in the control group and 88% in the supplemented group.
FIGURE 4.
Stomach (A) and intestine (B) fraction of ingested [3H]retinol dose in control and VA-supplemented rats from 0 to 24 d after dosing on postnatal day 4. Insets show the first 24 h after dosing. Each symbol represents the mean of 4 rats. There were no significant differences between groups. VA, vitamin A.
Tracer response in plasma
The tracer appeared in plasma within 30 min after dosing, peaked between 1 and 4 h, and declined to a steady concentration within 2 d, indicating a thorough mixing of the tracer with the endogenous retinol (Figure 5A). The CM-REs in the supplemented group peaked higher than in the control group, indicating less efficient clearance of CM-REs from plasma after supplementation (Figure 5B, C). After 14 d, there was a small increase in plasma tracer in both treatment groups, most likely due to a simultaneous release of RBP-ROH from the liver (Figure 6A).
FIGURE 5.
Plasma fraction of ingested [3H]retinol dose (A) and fraction of ingested dose as CM-REs, RBP-ROH, and total ROH in control (B) and VA-supplemented (C) rats from 0 to 24 d after dosing on postnatal day 4. The inset shows the first 48 h after dosing. Tracer profiles for CM-REs and RBP-ROH were generated by the model based on the assumption that total ROH is the sum of CM- and RBP-ROH. Each symbol represents the mean of 4 rats. *P < 0.05 (Student’s t test). CM-RE, chylomicron retinyl ester; RBP-ROH, retinol bound to retinol-binding protein; ROH, retinol; VA, vitamin A.
FIGURE 6.
Liver (A–C), lung (D–F), and kidney (G–I) fraction of ingested [3H]retinol dose and fraction of ingested dose as CM-derived, RBP-derived, and total ROH in control and VA-supplemented rats from 0 to 24 d after dosing on postnatal day 4. Insets show the first 48 h after dosing. Tracer profiles for CM- and RBP-derived ROH were generated by the model based on the assumption that total ROH is the sum of CM- and RBP-ROH. Each symbol represents the mean of 4 rats. *P < 0.05 (Student’s t test). CM, chylomicron; RBP, retinol-binding protein; RBP-ROH, retinol bound to retinol-binding protein; ROH, retinol; VA, vitamin A.
Tracer response in the liver, lungs, and kidneys
The total and RBP-derived retinol in the liver in the control group increased on day 1 and remained steady afterward (Figure 6A, B). In the supplemented group, liver total retinol peaked at 15 h due to the uptake of CM-REs, declined rapidly, and increased again to an amount significantly higher than in the control group. This increase lasted until day 14 (P < 0.001) and declined afterward (Figure 6C). In the lungs, total retinol increased up to a maximum of ∼1% and remained steady in both treatment groups (Figure 6D). The chylomicron-derived retinol declined within 4 d in the supplemented group and within 8 h in the control group (Figure 6E, F). In the kidneys, total retinol was significantly higher in the control group on day 1 (P < 0.001), but declined below the supplemented-group amount on day 14 after dosing (Figure 6G). The chylomicron-derived retinol in the supplemented group peaked below the control group amount and declined within 8 h (Figure 6H, I).
Tracer response in extrahepatic organs
Total retinol in the remaining extrahepatic organs (brain, BAT, skin, and carcass) peaked and declined within 4–8 d after dosing and remained at ∼1% during the entire study, indicating a rapid loss of tracer from these organs (Figure 7A, D, G, J). In the brain, most of the tracer in the control group was delivered by RBP, whereas in the supplemented group, most was delivered by chylomicrons (Figure 7B, C). In BAT, chylomicrons delivered most of the tracer in both treatment groups (Figure 7E, F). In the skin and carcass, the chylomicron-derived retinol peaked and declined very rapidly, below the peak in RBP-derived retinol, suggesting RBP as the main source of retinol in these tissues (Figure 7H, I, K, L). In WAT, total retinol appeared to be higher in the supplemented group, but the difference was not statistically significant (Figure 8).
FIGURE 7.
Brain (A–C), BAT (D–F), skin (G–I), and carcass (J–L) fraction of ingested [3H]retinol dose and fraction of ingested dose as CM-derived, RBP-derived, and total ROH in control and VA-supplemented rats from 0 to 24 d after dosing on postnatal day 4. Insets show the first 48 h after dosing. Tracer profiles for CM- and RBP-derived ROH were generated by the model based on the assumption that total ROH is the sum of CM- and RBP-ROH. The carcass contained bones, muscles, and connective tissue remaining after dissection of other organs. Each symbol represents the mean of 4 rats. *P < 0.05 (Student’s t test). BAT, brown adipose tissue; CM, chylomicron; RBP, retinol-binding protein; ROH, retinol; VA, vitamin A.
FIGURE 8.
WAT fraction of ingested [3H]retinol dose in control and VA-supplemented rats from 0 to 24 d after dosing on postnatal day 4. No dissectible WAT was found before postnatal day 12. Each symbol represents the mean of 4 rats. There were no significant differences between groups. VA, vitamin A; WAT, white adipose tissue.
Plasma kinetic parameters
The fractional uptake of CM-REs from plasma to organs [L(15,10)] was higher in the control group, whereas the uptake of RBP-ROH [L(6,5)] was higher in the supplemented group (Table 1). The supplemented group also showed a higher fractional release [L(5,4)] and recycling [L(5,6)] but a lower fractional, irreversible loss of retinol from organs [L(0,6)]. VA supplementation decreased the fraction of plasma retinol used irreversibly per day and the transit time of retinol in plasma, resulting in a much higher recycling number of retinol between plasma and organs in the supplemented than in the control group (Table 2). The transit time of retinol in organs was higher in the control group.
TABLE 1.
Plasma kinetic parameters in control and VA-supplemented neonatal rats dosed orally with [3H]retinol on postnatal day 41
| L(I,J) ± SEM |
||||
| Parameter | Parameter description | Control | VA | P2 |
| L(2,1) | Transit of retinol through the digestive system | 31.4 ± 2.4 | 32.3 ± 2.1 | 0.97 |
| L(15,10) | Uptake of CM-REs from plasma to organs | 1067.3 ± 227.9 | 492.1 ± 61.1 | <0.05 |
| L(5,4) | Release of RBP-ROH from organs to plasma | 1.3 ± 0.0 | 2.4 ± 0.2 | <0.001 |
| L(6,5) | Uptake of RBP-ROH from plasma to organs | 16.7 ± 1.2 | 135.4 ± 6.0 | <0.001 |
| L(5,6) | Recycling of retinol from organs back to plasma | 0.2 ± 0.0 | 1.1 ± 0.0 | <0.001 |
| L(5,7) | Release of RBP-ROH from the liver to plasma after 14 d | 0.9 ± 0.1 | 2.1 ± 0.1 | <0.001 |
| L(0,6) | Irreversible loss of retinol from organs | 0.05 ± 0.00 | 0.002 ± 0.001 | <0.001 |
CM-RE, chylomicron retinyl ester; L, fraction; RBP-ROH, retinol bound to retinol-binding protein; VA, vitamin A.
Parameters were compared by using Student’s t test.
TABLE 2.
Plasma kinetic parameters in control and VA-supplemented neonatal rats dosed orally with [3H]retinol on postnatal day 41
| Parameter | Parameter description | Control | VA |
| Plasma residence time | Average time a molecule of retinol spends in plasma over multiple transits | 8.2 h | 4.0 d |
| Plasma transit time | Average time a molecule of retinol spends in plasma during a single transit | 1.4 h | 11 min |
| Organ transit time | Average time a molecule of retinol spends in organs during a single transit | 3.3 d | 22 h |
| Fractional catabolic rate | Fraction of plasma retinol used irreversibly per day | 2.9 | 0.2 |
| Recycling number | Average number of times a molecule of retinol recycles through plasma before irreversible disposal | 5 | 541 |
VA, vitamin A.
Organ kinetic parameters
After supplementation, the fractional uptake of CM-REs was significantly higher in the liver, lungs, and brain and significantly lower in other organs (Table 3). The fractional uptake of RBP-ROH was higher in the liver, lungs, kidneys, skin, and carcass, with the largest, ∼40-fold difference observed in the carcass. Brain was the only organ with a significantly lower fractional uptake of RBP-ROH in the supplemented group. After supplementation, the fractional turnover of chylomicron-derived retinol was significantly higher in the liver, lungs, kidneys, and brain with the lungs showing the largest, ∼60-fold difference and significantly lower in the intestines and BAT (Table 4). The fractional turnover of RBP-derived retinol was higher in the liver, lungs, kidneys, skin, and carcass with the carcass showing the largest, ∼30-fold difference and significantly lower in the brain.
TABLE 3.
Fractional uptake of CM-REs and RBP-ROH from plasma to organs of control and VA-supplemented neonatal rats dosed orally with [3H]retinol on postnatal day 41
| Uptake of CM-REs (fraction of plasma REs transferred/d) |
Uptake of RBP-ROH (fraction of plasma retinol transferred/d) |
|||||
| L(I,J) ± SEM |
L(I,J) ± SEM |
|||||
| Organ | Control | VA | P2 | Control | VA | P2 |
| Stomach3 | — | — | — | 7.5 ± 1.9 | 10.0 ± 9.7 | 0.98 |
| Intestines | 44.3 ± 1.2 | 19.1 ± 2.9 | <0.001 | 0.03 ± 0.00 | 0.04 ± 0.01 | 0.20 |
| Liver | 107.3 ± 10.2 | 304.7 ± 14.4 | <0.001 | 8.7 ± 0.6 | 17.0 ± 0.4 | <0.001 |
| Lungs | 3.6 ± 0.2 | 15.0 ± 1.1 | <0.001 | 0.4 ± 0.1 | 4.2 ± 0.3 | <0.001 |
| Kidneys | 33.9 ± 3.4 | 4.8 ± 0.4 | <0.001 | 3.6 ± 0.6 | 6.9 ± 0.5 | <0.001 |
| BAT | 8.8 ± 0.2 | 5.3 ± 0.1 | <0.001 | 0.3 ± 0.0 | 0.3 ± 0.1 | 0.82 |
| WAT4 | — | — | — | 0.05 ± 0.01 | 0.11 ± 0.04 | 0.22 |
| Brain | 0.3 ± 0.1 | 1.2 ± 0.1 | <0.01 | 0.5 ± 0.0 | 0.2 ± 0.1 | <0.01 |
| Skin | 454.7 ± 144.4 | 103.2 ± 7.1 | <0.05 | 4.6 ± 0.4 | 44.2 ± 10.2 | <0.01 |
| Carcass | 87.3 ± 5.3 | 32. 0 ± 4.0 | <0.001 | 6.6 ± 0.5 | 270.5 ± 3.3 | <0.001 |
BAT, brown adipose tissue; CM-RE, chylomicron retinyl ester; L, fraction; RBP-ROH, retinol bound to retinol-binding protein; RE, retinyl ester; VA, vitamin A; WAT, white adipose tissue.
Parameters were compared by using Student’s t test.
The stomach model did not include a CM-RE uptake component.
No dissectible WAT was found before postnatal day 12.
TABLE 4.
Fractional turnover of chylomicron- and RBP-derived retinol in organs of control and VA-supplemented neonatal rats dosed orally with [3H]retinol on postnatal day 41
| Turnover2 of chylomicron-derived retinol (fraction of organ retinol transferred/d) |
Turnover2 of RBP-derived retinol (fraction of organ retinol transferred/d) |
|||||
| L(I,J) ± SEM |
L(I,J) ± SEM |
|||||
| Organ | Control | VA | P3 | Control | VA | P3 |
| Stomach | 6.8 ± 0.2 | 6.2 ± 0.9 | 0.52 | 19.1 ± 4.9 | 105.8 ± 110.1 | 0.45 |
| Intestines | 0.3 ± 0.0 | 0.2 ± 0.0 | <0.001 | 0.0 ± 0.0 | 0.0 ± 0.0 | 1.00 |
| Liver | 0.4 ± 0.1 | 1.4 ± 0.1 | <0.001 | 0.2 ± 0.0 | 0.3 ± 0.0 | <0.01 |
| Lungs | 0.6 ± 0.2 | 34.4 ± 3.9 | <0.001 | 0.2 ± 0.0 | 1.8 ± 0.2 | <0.001 |
| Kidneys | 2.1 ± 0.5 | 21.1 ± 3.6 | <0.001 | 5.2 ± 0.9 | 10.3 ± 0.7 | <0.001 |
| BAT | 1.4 ± 0.1 | 0.9 ± 0.0 | <0.01 | 1.6 ± 0.2 | 1.7 ± 0.4 | 0.87 |
| WAT4 | — | — | — | 0.1 ± 0.0 | 0.1 ± 0.1 | 0.58 |
| Brain | 0.6 ± 0.2 | 1.4 ± 0.1 | <0.01 | 11.6 ± 1.1 | 3.4 ± 2.0 | <0.01 |
| Skin | 153.5 ± 62.5 | 48.1 ± 11.7 | 0.12 | 1.1 ± 0.1 | 9.6 ± 2.2 | <0.01 |
| Carcass | 33.4 ± 4.3 | 48.7 ± 18.2 | 0.43 | 4.1 ± 0.3 | 142.8 ± 10.4 | <0.001 |
BAT, brown adipose tissue; L, fraction; RBP, retinol-binding protein; VA, vitamin A; WAT, white adipose tissue.
Turnover included retinol released back to plasma and irreversible loss due to metabolism.
Parameters were compared by using Student’s t test.
No dissectible WAT was found before postnatal day 12.
Organ transfer rates
After supplementation, the absolute uptake (uptake rate expressed in nmol/d) of CM-REs was significantly higher in all organs with the largest, ∼500-fold difference observed in the lungs (Table 5). The uptake rate of RBP-ROH was higher in the liver, lungs, kidneys, skin, and carcass with the carcass showing the largest, 50-fold difference. The brain was the only organ in which the uptake rate of RBP-ROH was significantly lower after supplementation. The uptake rate of total retinol was higher in all organs, except the stomach and WAT, with the largest difference observed in the liver, lungs (∼60-fold), and carcass (∼50-fold) (Table 6). After supplementation, the turnover rate of total retinol was significantly higher in all organs, except the stomach, WAT, and brain, with the largest, ∼90-fold difference observed in the carcass.
TABLE 5.
Uptake rate of CM-REs and RBP-ROH from plasma to organs of control and VA-supplemented neonatal rats dosed orally with [3H]retinol on postnatal day 41
| Uptake rate of CM-REs, nmol/d |
Uptake rate of RBP-ROH, nmol/d |
|||||
| R(I,J) ± SEM |
R(I,J) ± SEM |
|||||
| Organ | Control | VA | P2 | Control | VA | P2 |
| Stomach3 | — | — | — | 2.7 ± 0.7 | 4.4 ± 4.2 | 0.71 |
| Intestines | 0.3 ± 0.2 | 13.4 ± 4.7 | <0.05 | 0.01 ± 0.00 | 0.02 ± 0.00 | 0.05 |
| Liver | 0.7 ± 0.4 | 213.8 ± 69.1 | <0.01 | 3.0 ± 0.2 | 7.4 ± 0.4 | <0.001 |
| Lungs | 0.02 ± 0.01 | 10.5 ± 3.6 | <0.05 | 0.2 ± 0.0 | 1.8 ± 0.2 | <0.001 |
| Kidneys | 0.2 ± 0.1 | 3.4 ± 1.1 | <0.05 | 1.3 ± 0.2 | 3.0 ± 0.3 | <0.001 |
| BAT | 0.1 ± 0.0 | 3.7 ± 1.3 | <0.05 | 0.1 ± 0.0 | 0.1 ± 0.0 | 0.37 |
| WAT4 | — | — | — | 0.02 ± 0.00 | 0.05 ± 0.02 | 0.19 |
| Brain | 0.00 ± 0.00 | 0.8 ± 0.3 | <0.05 | 0.2 ± 0.0 | 0.1 ± 0.0 | <0.05 |
| Skin | 3.1 ± 2.0 | 72.4 ± 24.6 | <0.05 | 1.6 ± 0.2 | 19.3 ± 4.6 | <0.01 |
| Carcass | 0.6 ± 0.3 | 22.5 ± 8.4 | <0.05 | 2.3 ± 0.2 | 118.1 ± 6.4 | <0.001 |
BAT, brown adipose tissue; CM-RE, chylomicron-associated retinyl ester; R, rate; RBP-ROH, retinol bound to retinol-binding protein; VA, vitamin A; WAT, white adipose tissue.
Parameters were compared by using Student’s t test.
The stomach model did not include a CM-RE uptake component.
No dissectible WAT was found before postnatal day 12.
TABLE 6.
Transfer rate of total retinol between plasma and organs of control and VA-supplemented neonatal rats dosed orally with [3H]retinol on postnatal day 41
| Uptake rate of total retinol, nmol/d |
Turnover rate2 of total retinol, nmol/d |
|||||
| R(I,J) ± SEM |
R(I,J) ± SEM |
|||||
| Organ | Control | VA | P3 | Control | VA | P3 |
| Stomach | 3.3 ± 0.8 | 4.4 ± 4.2 | 0.71 | 10.4 ± 1.0 | 13.5 ± 1.84 | 0.18 |
| Intestines | 0.3 ± 0.2 | 13.4 ± 4.7 | <0.05 | 0.7 ± 0.0 | 1.6 ± 0.14 | <0.001 |
| Liver | 3.8 ± 0.5 | 221.2 ± 69.1 | <0.05 | 4.4 ± 0.4 | 84.6 ± 13.6 | <0.001 |
| Lungs | 0.2 ± 0.0 | 12.4 ± 3.6 | <0.01 | 0.1 ± 0.0 | 1.6 ± 0.5 | <0.01 |
| Kidneys | 1.5 ± 0.2 | 6.4 ± 1.1 | <0.01 | 2.8 ± 0.3 | 4.5 ± 0.4 | <0.01 |
| BAT | 0.2 ± 0.0 | 3.8 ± 1.3 | <0.05 | 0.2 ± 0.0 | 0.5 ± 0.1 | <0.01 |
| WAT | 0.02 ± 0.00 | 0.05 ± 0.02 | 0.19 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.43 |
| Brain | 0.2 ± 0.0 | 0.9 ± 0.3 | <0.05 | 0.2 ± 0.0 | 0.2 ± 0.0 | 0.91 |
| Skin | 4.7 ± 2.1 | 91.7 ± 25.1 | <0.01 | 2.3 ± 0.4 | 41.1 ± 4.1 | <0.001 |
| Carcass | 2.9 ± 0.4 | 140.7 ± 10.6 | <0.001 | 1.2 ± 0.2 | 113.4 ± 22.6 | <0.001 |
BAT, brown adipose tissue; R, rate; VA, vitamin A; WAT, white adipose tissue.
Turnover rate included retinol released back to plasma and irreversible loss due to metabolism.
Parameters were compared by using Student’s t test.
Turnover rate calculated by using retinol mass measured on day 1 after dosing because of the presence of the VA supplement in digestive organs at earlier time points.
Percentage of uptake of retinol from plasma to organs
VA supplementation redirected the flow of CM-REs away from peripheral tissues and toward the liver, lungs, and brain (Table 7). In the control group, the highest percentage of plasma CM-REs was taken up by the skin followed by the liver and carcass. In the supplemented group, these proportions were reversed toward the highest CM-RE uptake by the liver. The highest percentage of plasma RBP-ROH was taken up by the liver in the control group and the carcass in the supplemented group. The highest percentage of total retinol was taken up by the skin in the control group and the liver in the supplemented group.
TABLE 7.
Percentage of uptake of CM-REs, RBP-ROH, and total retinol from plasma to organs of control and VA-supplemented neonatal rats dosed orally with [3H]retinol on postnatal day 41
| CM-RE uptake, % |
RBP-ROH uptake, % |
Total retinol uptake, % |
||||
| Organ | Control | VA | Control | VA | Control | VA |
| Stomach2 | — | — | 23.2 | 2.8 | 16.3 | 0.9 |
| Intestines | 6.1 | 3.9 | 0.1 | 0.01 | 1.8 | 2.7 |
| Liver | 12.1 | 62.8 | 27.0 | 4.8 | 23.0 | 44.7 |
| Lungs | 0.8 | 3.1 | 1.2 | 1.2 | 1.2 | 2.5 |
| Kidneys | 4.0 | 1.0 | 11.2 | 2.0 | 9.1 | 1.3 |
| BAT | 2.0 | 1.1 | 0.9 | 0.1 | 1.2 | 0.8 |
| WAT3 | — | — | 0.2 | 0.03 | 0.1 | 0.0 |
| Brain | 0.04 | 0.2 | 1.5 | 0.1 | 1.2 | 0.2 |
| Skin | 62.7 | 21.3 | 14.3 | 12.5 | 28.5 | 18.5 |
| Carcass | 12.1 | 6.6 | 20.4 | 76.5 | 17.6 | 28.4 |
BAT, brown adipose tissue; CM-RE, chylomicron retinyl ester; RBP-ROH, retinol bound to retinol-binding protein; VA, vitamin A; WAT, white adipose tissue.
The stomach model did not include a CM-RE uptake component.
No dissectible WAT was found before postnatal day 12.
DISCUSSION
Despite the importance of VA for development, knowledge about its uptake and retention in rapidly developing neonatal tissues is limited. Here, we quantified the transfer of CM-REs, RBP-ROH, and total retinol in and out of organs in neonatal rats raised under VA-marginal conditions without and after VA supplementation. Our findings demonstrated that VA supplementation redirected the flow of CM-REs away from peripheral tissues (the skin and carcass) and toward the liver for subsequent greater fractional release as RBP-ROH and redelivery to peripheral tissues. This was evidenced by a higher fractional uptake of CM-REs into the liver, which took up the highest proportion (63%) of CM-REs, and a higher fractional uptake of RBP-ROH into the carcass, which took up the highest proportion (76%) of RBP-ROH. The reverse pattern was observed in control neonates, with most CM-REs (63%) acquired by the skin and the highest proportion of RBP-ROH (29%) acquired by the liver. We also found that VA supplementation resulted in a higher retinol turnover in most tissues and a much greater (>100-fold) retinol recycling number between plasma and organs. Retinol turnover was particularly high in the carcass but no different from the control group turnover in the brain and WAT, suggesting greater retinol retention in these tissues. Finally, we have observed a gradual loss of tracer from the liver in both treatment groups during the last 10 d of the study and a simultaneous accumulation of tracer in plasma and the extrahepatic organs.
Our finding of the redirected flow of CM-REs from peripheral tissues to the liver may have a 2-fold explanation: 1) the capacity of peripheral tissues to acquire a large amount of CM-REs may be limited and/or saturated by supplementation; 2) the capacity of the liver to acquire a large amount of CM-REs may be relatively high compared with other organs. The first explanation (limited uptake of CM-REs by peripheral tissues) is consistent with the rapid loss of chylomicron-derived retinol from the skin and carcass observed in our study (Figure 7). It is also consistent with the relatively low retinol concentration in the skin, adipose tissue, and carcass (12, 24–26) and the low expression of lecithin-retinol acyltransferase (LRAT), an enzyme responsible for retinol storage, in the lungs during the neonatal period (27). Both the activity of LRAT and the uptake of CM-REs have been shown to be upregulated by retinoic acid (23, 28). Therefore, under VA-marginal conditions, a low activity of LRAT in peripheral tissues may limit retinol retention, causing a large release of retinol back to the circulation for subsequent uptake into the liver.
The limited capacity of peripheral tissues to retain retinol may be further explained by the relatively high activity of the receptor stimulated by retinoic acid 6 (STRA6) in developing tissues (29). STRA6 acts as a bidirectional transporter, importing retinol into the cell when the intracellular concentration is low and exporting it when it is high (30, 31). This function of STRA6 is particularly important during the embryonic stage when retinoic acid must be maintained within a narrow concentration range to ensure proper tissue development. Dietary VA excess during this period was found to upregulate STRA6, specifically in the embryo but not in the placenta, suggesting that STRA6 serves a unique developmental role of protecting the embryo from toxic effects of excessive maternal retinoids (32). A high expression of STRA6 in neonates, particularly in tissues that continue to develop postnatally, may be a remnant from the gestational period that protects these tissues from excessive VA intake by the mother.
The second explanation (enhanced CM-RE uptake by the liver) is consistent with the relatively high activity of lipoprotein lipase (LPL) in the neonatal liver. LPL is responsible for the margination of chylomicrons at the interior side of tissue capillaries and the hydrolysis of their contents, which include REs (33). In a series of in vitro experiments, Blaner et al. (34) demonstrated that triglycerides are the preferred substrate for LPL; however, when the majority of triglycerides is hydrolyzed (∼75%), RE hydrolysis proceeds. Several in vivo studies also reported that an increased expression of LPL in the heart, skeletal muscle, and adipose tissue is correlated with an increased CM-RE uptake into these organs (33) and conversely that a partial knockout of LPL leads to a delayed RE clearance (35). These findings indicate that LPL expression correlates with a tissue capacity to acquire CM-REs.
LPL is considered an extrahepatic enzyme highly expressed in tissues that require a steady supply of fatty acids, such as the heart, skeletal muscle, adipose tissue, and mammary gland during lactation (36). However, in newborn rats, LPL is actively synthesized by hepatocytes at a rate 7-fold higher than in adult rats (37). The putative role of hepatic LPL is to channel triglycerides into the liver for enhanced production of VLDL and ketone bodies, which can serve as an energy source for the peripheral tissues low in glycogen and fat stores during development (37). The presence of LPL in the neonatal liver may also enhance the uptake of RE by other mechanisms, such as the receptor-mediated endocytosis, by tethering the incoming chylomicron particles to the surface of capillary endothelium (32, 38).
Regarding the trends observed in organ tracer amounts over time in our study, we speculate that they may reflect changes in the distribution of LPL and STRA6 expression during development. During the second half of the suckling period (∼P 10–∼P 21), LPL activity declines in the liver and increases in other tissues, such as the heart, skeletal muscle, and kidney, reaching adult levels at weaning (39–41). Meanwhile, the expression of STRA6 decreases in the lungs (27) and other extrahepatic tissues (27, 42). This timing overlaps with a progressive decline of the tracer in the liver and a simultaneous accumulation in plasma and several extrahepatic tissues after day 14 of our study.
In conclusion, the analysis of tracer response in neonatal organs demonstrated that a high dose of VA (200 IU) ingested on P 4 by pups raised under VA-marginal conditions accumulated mainly in the liver and was stored for ∼2 wk before being gradually transferred to other tissues. Further compartmental analysis showed that the uptake of postprandial CM-REs was particularly high in the liver, lungs, and brain, whereas the uptake of RBP-ROH was high in the peripheral tissues, such as the skin, bones, and muscles. The peripheral tissues, however, did not retain the acquired retinol, resulting in its repeated recycling between plasma and organs. Given the low retinol retention in extrahepatic tissues of neonates, an infrequent high dose of VA may not be the optimal strategy to maintain the extrahepatic retinol at a steady concentration.
Acknowledgments
We thank JB Green for editorial assistance.
The authors’ responsibilities were as follows—JKH: conducted the research, analyzed the data, and wrote the manuscript; LT: conducted the research; MHG: assisted with the kinetic analysis and interpretation; ACR: conducted the research and had primary responsibility for the final content; and all authors: designed the research and read and approved the final manuscript. None of the authors reported a conflict of interest related to the study.
Footnotes
Abbreviations used: BAT, brown adipose tissue; CM-RE, chylomicron retinyl ester; LPL, lipoprotein lipase; LRAT, lecithin-retinol acyltransferase; P, postnatal day; RBP, retinol-binding protein; RBP-ROH, retinol bound to retinol-binding protein; RE, retinyl ester; STRA6, stimulated by retinoic acid 6; UPLC, ultra-performance liquid chromatography; VA, vitamin A; WAT, white adipose tissue.
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