The Pennsylvania State University. The Graduate School. College of Health and Human Development VITAMIN A METABOLISM AND KINETICS

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1 The Pennsylvania State University The Graduate School College of Health and Human Development VITAMIN A METABOLISM AND KINETICS DURING THE NEONATAL PERIOD: STUDIES IN THE RAT MODEL A Dissertation in Nutritional Sciences by Joanna K. Hodges 2016 Joanna K. Hodges Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy December 2016

2 The dissertation of Joanna K. Hodges was reviewed and approved* by the following: A. Catharine Ross Professor of Nutritional Sciences and Physiology Dissertation Adviser Chair of Committee Michael H. Green Professor of Nutritional Sciences and Physiology Head of the Department of Nutritional Sciences Gregory C. Shearer Associate Professor of Nutritional Sciences and Physiology Kevin J. Harvatine Associate Professor of Nutritional Physiology *Signatures are on file in the Graduate School. ii

3 ABSTRACT Background: Vitamin A (VA, retinol) supplementation is recommended for children older than 6 mo of age in countries with high rates of malnutrition. However, the distribution and retention of VA in body tissues has not been extensively explored. Objective: This study was conducted to determine the uptake, retention and distribution of retinol, without and after supplementation with VA, in plasma and tissues in neonatal rats raised under VA-marginal conditions. Methods: Sprague-Dawley neonatal rats (n = 104, 63 males) nursed by mothers fed a VAmarginal diet (0.35 mg retinol equivalents/kg diet) were randomized and treated on postnatal day (P) 4 with an oral dose of either VA (6 μg retinyl palmitate/g body weight) or canola oil as control, both containing 1.8 μci of [ 3 H]retinol. Pups (n = 4/group/time) were euthanized at 13 times from 30 min to 24 d after dosing. The total retinol (ROH) concentration and mass was determined by ultra-performance liquid chromatography in all collected organs. The fractional and absolute (expressed in nmol/d) transfer of ROH between plasma and organs was estimated in Windows version of Simulation, Analysis and Modeling (WinSAAM) software, with distinct kinetic profiles generated for the chylomicron retinyl esters (CM- REs) and retinol bound to retinol-binding protein (RBP-ROH). Retinol concentrations and kinetic parameters determined in the present study were compared with those obtained from the previous experiment, in which neonatal rats were supplemented with VA mixed with retinoic acid (VARA). Results: In the control group, plasma VA was marginal (0.8 µmol/l), whereas liver VA was deficient (<70 nmol/g). Nonetheless, the liver contained most (~76%) of the whole-body VA iii

4 mass, while extrahepatic, non-digestive organs together contained ~13%. White adipose tissue (WAT), which was nearly absent prior to P12, contained only ~1% of the total VA mass in the body. In VA-supplemented neonates, the mean total retinol concentrations in all organs were significantly greater than in control pups. However, this increase lasted only ~1 d in most extrahepatic tissues, with the exception of WAT, where the increase lasted for 18 d. The subsequent kinetic analysis of tracer data revealed that VA supplementation redirected the flow of CM-REs away from the peripheral and toward the central organs, such as the liver, lungs and brain. Vitamin A supplementation also resulted in a greater fractional release of RBP-ROH from the liver, which was acquired mainly by the peripheral tissues, but not retained efficiently. This was evidenced by the higher turnover of RBP-derived ROH, especially in the carcass (composed largely of bones and muscles), and a substantially greater recycling number of ROH between plasma and organs in the supplemented group compared to control group (541 vs. 5 times before irreversible disposal). The turnover rate of RBP-derived ROH in WAT and brain was not higher after supplementation suggesting a greater retention capacity for ROH in these organs. Furthermore, based on the trend observed in tracer levels of various organs, ROH stores in the liver were gradually transferred to other organs starting at 2 weeks after supplementation. Finally, the comparison of results from the VA and the VARA study demonstrated that supplementation with VARA increased the fractional uptake of CM-REs into the intestines, lungs and carcass to a greater extent than did supplementation with VA and attenuated the fractional turnover as well as the recycling number of ROH from 288 to 100 times before irreversible disposal. Nevertheless, the supplementation with VA resulted in a greater accumulation of ROH in the liver, which may become available to other organs during the course of development. iv

5 Conclusions: Vitamin A administered in a single, large dose during the first few days after birth to neonatal rats raised under VA-marginal conditions accumulates mainly in the liver. The extrahepatic organs in neonates store relatively little VA and the scarcity of adipose tissue may predispose them to a low VA status. Vitamin A present in the supplement is readily acquired by all organs, but recycles repeatedly back to plasma most likely due to high expression of the stimulated by retinoic acid 6 receptor in the extrahepatic organs in neonates, a bidirectional ROH transporter induced by VA during development. Presence of retinoic acid in the VA supplement improves ROH tissue retention and attenuates recycling, most likely due to the synergistic effect of VA and retinoic acid on the activity of lecithinretinol acyltransferase, an enzyme responsible for ROH storage. Given the transient effect of large-dose VA supplementation on the extrahepatic tissue ROH contents in neonates, a more frequent, lower-dose supplementation, along with other nutrition interventions, may be necessary to maintain adequate VA levels in the rapidly developing neonatal organs. v

6 TABLE OF CONTENTS List of Figures...viii List of Tables...x List of Abbreviations...xi Acknowledgements...xiii Chapter 1. INTRODUCTION Public health relevance statement Vitamin A metabolism: an overview Mathematical modeling of vitamin A kinetics...5 Chapter 2. OBJECTIVES AND HYPOTHESES...13 Chapter 3. VITAMIN A DISTRIBUTION IN THE BODY DURING THE NEONATAL PERIOD Materials and methods Animals and diet Dose preparation and delivery Tissue collection Analyses of total retinol and retinyl esters Statistical analysis Results Animal and relative organ growth Digestive organs Plasma Liver, lungs and kidneys Extrahepatic organs and the remaining carcass Discussion and conclusions Vitamin A distribution in the body in control rats The effect of vitamin A supplementation...36 Chapter 4. VITAMIN A KINETICS IN PLASMA AND ORGANS IN NEONATAL RATS Methods Dose tracer content Tracer analysis Kinetic analysis Model structure and kinetic parameters Statistical analysis...44 vi

7 4.2 Results Mean organ tracer levels Tracer response in digestive organs Tracer response in plasma Tracer response in the liver, lungs and kidneys Tracer response in extrahepatic organs and the remaining carcass Plasma kinetic parameters Plasma transfer rates Organ kinetic parameters Percent uptake of retinol from plasma to organs Organ transfer rates Discussion and conclusions...63 Chapter 5. COMPARISON OF VITAMIN A VS. VARA EFFECTS ON RETINOL CONCENTRATIONS AND KINETICS IN PLASMA AND ORGANS IN NEONATAL RATS Methods Differences in study design between VA and VARA experiments Statistical analysis Results Comparison of vitamin A concentrations in plasma, liver and lungs Comparison of the tracer response profiles in plasma and organs Comparison of the plasma kinetic parameters Comparison of the organ kinetic parameters Comparison of the percent uptake of retinol from plasma to organs Discussion and conclusions...85 Chapter 6. SUMMARY AND FUTURE DIRECTIONS Summary Future directions...91 References...93 Appendix A: Pilot test of extraction efficiency of the lipid-rich tissues Appendix B: Comparison of results from the current and the follow-up experiment Appendix C: WinSAAM input files vii

8 LIST OF FIGURES Figure 1. Sample collection timeline...18 Figure 2. Neonatal rat body weights from P4 to P Figure 3. Organ weights as a fraction of total body weight over time in control and VAsupplemented neonatal rats between P4 and P Figure 4. Stomach and intestine total (unesterified + esterified) ROH concentration and mass in control and VA-supplemented neonatal rats between P4 and P Figure 5. Plasma total (unesterified + esterified) ROH concentration, RE concentration and ROH mass in control and VA-supplemented neonatal rats between P4 and P Figure 6. Liver, lung, and kidney total (unesterified + esterified) ROH concentration and mass in control and VA-supplemented neonatal rats between P4 and P Figure 7. Brain, white adipose tissue, brown adipose tissue, and skin total ROH concentration and mass in control and VA-supplemented neonatal rats between P4 and P Figure 8. Carcass total (unesterified + esterified) ROH concentration and mass in control and VA-supplemented neonatal rats between P4 and P Figure 9. Mean organ total (unesterified + esterified) ROH concentration and mass in control and VA-supplemented rats between P4 and P8 (0-4 d after VA supplementation)...34 Figure 10. Plasma model of VA kinetics in neonatal rats dosed orally with [ 3 H]ROH on P Figure 11. Organ models of VA kinetics in neonatal rats dosed orally with [ 3 H]ROH on P Figure 12. Mean organ fraction of ingested [ 3 H]ROH dose between d 0 and 24 after dosing in control and VA-supplemented rats dosed on P Figure 13. Stomach and intestine fraction of ingested [ 3 H]ROH dose in control and VAsupplemented rats from 0 to 24 d after dosing on P Figure 14. Plasma fraction of ingested [ 3 H]ROH dose and fraction of ingested dose as CM-REs, RBP-ROH and their sum (total ROH) in control and VAsupplemented rats from 0 to 24 d after dosing on P viii

9 Figure 15. Liver fraction of ingested [ 3 H]ROH dose and fraction of ingested dose as CM-, RBP-derived ROH and their sum (total ROH) in control and VA-supplemented rats from 0 to 24 d after dosing on P Figure 16. Lung fraction of ingested [ 3 H]ROH dose and fraction of ingested dose as CM-, RBP-derived ROH and their sum (total ROH) in control and VA-supplemented rats from 0 to 24 d after dosing on P Figure 17. Kidney fraction of ingested [ 3 H]ROH dose and fraction of ingested dose as CM-, RBP-derived ROH and their sum (total ROH) in control and VAsupplemented rats from 0 to 24 d after dosing on P Figure 18. Fraction of ingested [ 3 H]ROH dose in the brain, brown adipose tissue, skin, and carcass in control and VA-supplemented rats from 0 to 24 d after dosing on P Figure 19. Fraction of ingested [ 3 H]ROH dose as CM-, RBP-derived retinol and their sum (total ROH) in the brain and brown adipose tissue in control and VAsupplemented rats from 0 to 8 d after dosing on P Figure 20. Fraction of ingested [ 3 H]ROH dose as CM-, RBP-derived retinol and their sum (total ROH) in the skin and carcass in control and VA-supplemented rats from 0 to 2 d after dosing on P Figure 21. White adipose tissue fraction of ingested [ 3 H]ROH dose in control and VAsupplemented rats from 0 to 24 d after dosing on P Figure 22. Mean total (unesterified + esterified) ROH concentration in the liver, lungs, and plasma in control, VA- and VARA-supplemented neonatal rats on d 1 after dosing on P4 in control, VA- and VARA-supplemented neonatal rats...72 Figure 23. Plasma, liver and lung total (unesterified + esterified) ROH concentration in control, VA- and VARA-supplemented neonatal rats from 0 to 14 d after dosing on P Figure 24. Stomach and intestine fraction of ingested [ 3 H]ROH dose in control, VA- and VARA-supplemented neonatal rats from 0 to 14 d after dosing on P Figure 25. Plasma fraction of ingested [ 3 H]ROH dose in control, VA- and VARAsupplemented neonatal rats from 0 to 14 d after dosing on P Figure 26. Liver, lung and kidney fraction of ingested [ 3 H]ROH dose in control, VA- and VARA-supplemented neonatal rats from 0 to 14 d after dosing on P Figure 27. Carcass fraction of ingested [ 3 H]ROH dose in control, VA- and VARAsupplemented neonatal rats from 0 to 14 d after dosing on P ix

10 LIST OF TABLES Table 1. Sample size...15 Table 2. Organ weight as percent of total body weight in control and VA-supplemented rats between P4 and P Table 3. The effect of supplementation on organ VA concentrations in neonatal rats dosed with 200 IU of VA on P Table 4. Plasma fractional transfer coefficients in control and VA-supplemented neonatal rats dosed orally with [ 3 H]ROH on P Table 5. Plasma kinetic parameters in control and VA-supplemented neonatal rats dosed orally with [ 3 H]ROH on P Table 6. Fractional uptake of CM-REs and RBP-ROH from plasma to organs in control and VA-supplemented neonatal rats dosed orally with [ 3 H]ROH on P Table 7. Fractional turnover of CM-REs and RBP-ROH from organs in control and VAsupplemented neonatal rats dosed orally with [ 3 H]ROH on P Table 8. Percent uptake of CM-REs, RBP-ROH and total ROH from plasma to organs in control and VA-supplemented neonatal rats dosed orally with [ 3 H]ROH on P Table 9. Uptake rates of CM-REs, RBP-ROH and total ROH from plasma to organs and turnover rates of total ROH in organs in control and VA-supplemented neonatal rats dosed orally with [ 3 H]ROH on P Table 10. Plasma fractional transfer coefficients in VA- and VARA-supplemented neonatal rats dosed orally with [ 3 H]ROH on P Table 11. Plasma kinetic parameters in VA- and VARA-supplemented neonatal rats dosed orally with [ 3 H]ROH on P Table 12. Fractional uptake of CM-REs and RBP-ROH from plasma to organs in VA- and VARA-supplemented neonatal rats dosed orally with [ 3 H]ROH on P Table 13. Fractional turnover of CM-REs and RBP-ROH from organs in VA- and VARAsupplemented neonatal rats dosed orally with [ 3 H]ROH on P Table 14. Percent uptake of CM-REs and RBP-ROH from plasma to organs in VA- and VARA-supplemented neonatal rats dosed orally with [ 3 H]ROH on P x

11 LIST OF ABBREVIATIONS BAT: Brown adipose tissue CMs: Chylomicrons CM-REs: Chylomicron retinyl esters CYP: Cytochrome P450 D: Day DPM: Disintegrations per minute FCR: Fractional catabolic rate FOD: Fraction of dose H: Hour LPL: Lipoprotein lipase LRAT: Lecithin retinol acyltransferase MO: Month P: Postnatal day RBP: Retinol-binding protein RBP-ROH: Retinol-binding protein-bound retinol REs: Retinyl esters ROH: Retinol STRA6: Stimulated by retinoic acid 6 UPLC: Ultra-performance liquid chromatography xi

12 VA: Vitamin A VARA: Vitamin A combined with retinoic acid WAT: White adipose tissue WHO: World Health Organization WinSAAM: Windows version of the Simulation, Analysis and Modeling program xii

13 ACKNOWLEDGEMENTS I would like to thank Dr. Ross for giving me the opportunity to join her laboratory in 2013, a decision that changed the course of my academic career and enabled me to evolve as a researcher in molecular nutrition. I am also thankful to Dr. Ross for inspiring me as a teacher, scientist and simply a wonderful person, and for all the effort she made to secure the funding which made my research possible. I would like to thank Drs. Green and Green for patience teaching me the basics of mathematical modeling, for invaluable insights on physiology and biochemistry, for thorough revision of my writing, and for delicious dinners in a friendly atmosphere. My sincere appreciation also goes to my committee members, Dr. Shearer and Dr. Harvatine, for their feedback and helpful suggestions and to the entire Ross lab team for the welcoming work environment filled with countless acts of kindness, gratitude and exemplary work ethic. I am in tremendous debt to my newly wedded husband, Jay Garth Hodges, who propelled me with boundless affection, enormous enthusiasm and gave me the prospect of a happy ever after; and to my family for raising me, enabling me to fly across the ocean and spread my wings as a scholar in America and for supporting me financially and emotionally throughout this journey, particularly, to my mom Grażyna, brother Sylwester, dad Zdzisław, uncle and aunt Juliusz and Iwona for love, warmth, security, tears and laughter, the in-person and the intellectual faraway excursions that will remain in my memory forever. Finally, I am thankful to all the other people whose names are not listed for their contributions and the positive impact they had on my life. xiii

14 Chapter 1. INTRODUCTION 1.1 Public health relevance statement Vitamin A (VA, retinol) is essential for the proliferation and differentiation of many cell types and, therefore, development of an entire organism (3-5). Deficiency of VA in infants leads to weakened immune system, reduced growth, xerophthalmia, blindness and ultimately death (6). During the past three decades, the prevalence of VA deficiency in lowand middle-income countries declined from 39% to 29%. However, the prevalence has not changed in sub-saharan Africa and South Asia, where one in every two preschool-age children suffers from VA deficiency and its associated symptoms (7). Many of these children become permanently blind and die within a year of losing sight (8). Given the rapid growth that occurs during the neonatal period, it is reasonable to postulate that neonates have an increased need for VA. However, humans, like many other mammals, are born with very low VA reserves, even when their mothers are well-nourished (9). This is due mainly to the low transplacental transfer of VA, which is sufficient to sustain embryonic development but insufficient to build any surplus. As a result, the levels of retinol in neonatal plasma and liver are about half of those usually observed in mothers (<0.7 µm and <10 µg/g, respectively) (10, 11), levels that would indicate deficiency if observed in older age groups. The main source of VA for a newborn is the mother s milk, especially the colostrum, which contains approximately 3 times more retinyl esters than the mature milk (9). Still, in areas of poverty and malnutrition, such as certain parts of Africa and South-East Asia, even the mother s milk is inadequate to meet the child s nutritional needs (12). 1

15 In view of this problem, the World Health Organization has initiated numerous clinical trials, many of them still ongoing, to examine the effect of large dose VA supplementation (50,000 IU/2.5 kg body weight) on infant survival (13). A meta-analysis of randomized clinical trials, in which VA was given to 6-59-mo old children, revealed a 23-34% reduction in child mortality (14-16). On the other hand, VA supplementation administered to newborns within the first few days of life had no significant overall effect on mortality outcomes in infants < 6 mo of age, despite several studies conducted in this age group (17, 18). The exact fate of VA administered to newborns remains, to a large extent, poorly understood. 1.2 Vitamin A metabolism: an overview Several comprehensive reviews on VA metabolism have been published to date, including a book chapter by Ross (4) in Modern Nutrition in Health and Disease, another one by Ross and Harrison (1) in Handbook of Vitamins, and a recent update on VA metabolism by Blaner et al. (19) in Nutrients. Rather than reiterating these sources, the purpose of this section is to provide a brief overview of the metabolic pathways, enzymes and ligands that process VA, with emphasis on their role in young infants. Vitamin A is a group of fat-soluble compounds that exhibit the biological activity of retinol (ROH), the alcohol form of VA, and are indispensable for normal vision, immune response, cell proliferation and differentiation as well as growth and development of an entire organism (3-5). The metabolism of VA, especially the digestive-absorptive phase, is closely related to that of dietary lipids and occurs both in the lumen as well as inside the enterocytes. Digestion of ROH requires bile acids to break up large fat globules into 2

16 micelles, and the enzymatic activity of retinyl ester hydrolases to cleave the fatty acid moiety away from retinyl esters (REs) and convert them into molecules of ROH (1). The primary enzymes involved in ROH hydrolysis are the pancreatic triglyceride lipase, pancreatic lipase-related protein 2, both present in the intestinal lumen, and phospholipase B located on the brush border of enterocytes (19). The unesterified ROH, together with bile acids and other products of partial lipid digestion, is absorbed across the microvilli surface into the enterocytes in the proximal portion of the small intestine. The absorption of preformed VA is very efficient. In isotope kinetic studies, approximately 70% to 90% of a physiological dose of VA was absorbed (20). Once inside the enterocytes, most of ROH is esterified by lecithin retinol acyltransferase (LRAT) and incorporated into nascent chylomicrons for secretion into the lymphatic system, which merges with general circulation. Blood-borne chylomicrons containing REs and other dietary lipids, such as triglycerides, phospholipids, fat-soluble vitamins, free and esterified cholesterol, travel to the heart first, followed by the lungs, liver and other extravascular tissues. The extrahepatic organs, such as skeletal muscle, heart, lungs and kidneys, synthesize lipoprotein lipase (LPL), which resides on the luminal surface of the capillary endothelial cells and is responsible for the hydrolysis of REs and uptake of ROH into cells (21, 22). Approximately 25-33% of REs is processed this way and deposited in the extrahepatic organs (19). The remainder is taken up by the liver together with the entire chylomicron remnant by a process called sieving, in which the remnant passes through the fenestrations present in endothelium into the space of Disse (located between the endothelium and the hepatocytes). Once inside, the remnant is taken up into hepatocytes by one of two possible receptor-mediated pathways: endocytosis by low-density lipoprotein 3

17 (LDL) receptor or LDL receptor-related protein 1. Emerging evidence also points to the existence of a third uptake mechanism, which involves scavenger receptor class B member 1, a receptor for high-density lipoprotein present on the surface of hepatocytes (19). Liver is the major site of ROH metabolism and storage in humans and rodents (23). Once in the liver, the ROH obtained by hydrolysis of REs can follow different metabolic pathways. It can be oxidized to retinal and subsequently to retinoic acid, the principal hormonal metabolite of VA and a regulator of many transcription and independent events, including growth and differentiation of different cell types during early development (24). Retinol can also be re-esterified and stored in lipid droplets within hepatic stellate cells, which function as a storage site for REs and contain up to 90% of the total ROH stored in the liver (25). Finally, ROH can be transferred from stellate cells to hepatocytes and complexed with retinol-binding protein (RBP), a 21.5 kda-protein that binds ROH in a 1:1 molar ratio for secretion into the circulation (26). In bloodstream, the RBP-ROH complex associates with another larger protein, transthyretin, also in a 1:1 ratio, which prevents it from being filtered in the kidneys prior to its delivery to the extrahepatic tissues. Apart from the liver, a few other organs, including white adipose tissue, kidneys, spleen, lungs, brain, stomach, skeletal muscle and testes, secrete RBP-ROH into plasma (27). In fact, RBP secreted into plasma is the vehicle through which extrahepatic tissues exchange ROH between each other at least 12 to 13 times before irreversible disposal in adult rats (25). This phenomenon, called recycling, is particularly useful during times of VA deficiency, when hepatic stores of ROH become depleted, and will be described in more detail in the following section. Other extrahepatic organs, such as pancreas, lungs, kidneys and intestines are also capable of storing VA for a prolonged time due to presence of cells similar to stellate 4

18 cells in these organs (25, 28). Finally, almost every cell in the body uses all-trans retinoic acid to regulate its growth, proliferation, differentiation, apoptosis and other processes related to bone development, hematopoiesis, immune response and reproductive function (1, 24). The excretion of VA occurs by urine and feces and requires the oxidation of RA by CYP26 or its conjugation with glucuronic acid, which can be added to ROH directly or to RA and its inactive metabolites. These degradation mechanisms as well as the esterification of ROH for storage in tissues keep ROH plasma level within the narrow range of µm (29). The normal tissue levels are much more dispersed and reflect recent dietary intake. For example, concentration of ROH in the liver can vary between 20 and 300 µg ROH/g of tissue and still be considered adequate (1, 4). 1.3 Mathematical modeling of vitamin A kinetics Mathematical models in biology are used to discover the qualitative and quantitative principles underlying functioning of complex biological systems. The goal of these models is to provide a mathematical description of many processes occurring simultaneously at multiple body sites and to predict the outcome of these processes under different treatment conditions (30). Compartmental models are particularly useful for studying complex living organisms, because they describe biological systems in terms of a finite number of homogenous states and processes, called compartments, which interact by means of material exchange. The compartments frequently correspond to individual organs in the body (20). Therefore, compartmental models often capture both the structure and kinetics of the system at the same time. 5

19 Compartmental analysis also allows for describing the continuum of metabolic processes without having to collect samples at impossibly small time intervals. This particular aspect has been especially useful for research in nutrition. For example, in 1970s and 80s, Foster et al. (31) and Wastney et al. (32) used compartmental analysis to characterize the short-term kinetics of zinc during the early absorptive phase in humans with several samples collected during the first few hours after dose administration. Compartmental analysis resolves the sampling site problem as well as the sampling time issue by making predictions about body sites that are impossible to access without invasive surgery. For example, in 1974, Massin et al. (33) applied compartmental analysis to measure the rate of calcium accretion and resorption from bones under different pathological conditions without sampling any bones directly. In addition to these studies, which were conducted on humans, many other ones used animal models to provide invaluable insights about the metabolism of several other nutrients, including iron, vitamin B6, glucose and cholesterol (34). The process of mathematical modeling involves two phases: (1) collecting data from an appropriately designed experiment and (2) analyzing data to extract the kinetic information. During the experimental phase, a small amount of tracer, usually a radioactive or stable isotope of the compound of interest (tracee), is introduced into the system, either orally or intravenously. After the tracer enters the system and mixes thoroughly with the tracee, data on the tracer level can be collected at appropriately spaced sampling times from plasma and accessible organs. Ideally, the tracer should satisfy the following conditions: (1) it should be easily detectable; (2) it should follow the same kinetics as the tracee; and (3) it should not perturb the kinetics of the system (35). If these conditions are met, the information 6

20 about the kinetics of the tracee can be derived from the kinetics of the tracer (30). In other words, an ideal tracer enables the modeler to see the tracee by looking at the behavior of the tracer. In the analytical phase of mathematical modeling, the researcher develops a mathematical description of the system by conceptualizing it as a series of interconnected compartments, based on her knowledge about the system biochemical, physiological and anatomical characteristics. The compartment connections represent the flow of tracee between compartments or out of the system. This flow, in turn, is the unknown in a mathematical problem, which consists of a series of ordinary, first-order, linear differential equations. The equations are set up and solved by the modeling software thus proving a quantitative representation of system kinetics based on data obtained from the experiment and a priori knowledge about the system (36). There are two main approaches to mathematical modeling of biological systems: the empirical approach and the model-based compartmental analysis. The empirical approach involves fitting the tracer response curve (plot of tracer level vs. time) to a multi-exponential equation and calculating the kinetic parameters based on the coefficients and constants obtained by solving this equation (36). The main use of this approach is to calculate kinetic parameters such as the absorption rate or the utilization rate of a nutrient. However, the representation of system kinetics derived from this method is somewhat arbitrary, as the number and type of coefficients and constants do not necessarily reflect the anatomical and physiological properties of the system. The model-based compartmental analysis, on the other hand, is more physiologically-relevant, because it conceptualizes the system as 7

21 compartments that correspond to the actual tissues and organs in the body or physical spaces, where nutrient particles behave in a similar manner (30). The model-based compartmental analysis is more informative to a biologist than the empirical analysis. However, it is also more demanding, because it requires the modeler to visualize the system structure and interconnections prior to solving the mathematical model of the system. If the hypothetical system structure is inconsistent with the empirical data, the researcher has to eliminate or add compartment or change their interconnections without violating any of the physiological constraints of the system. Only after this step is completed, the modeler can proceed to solve the model and calculate its kinetic parameters, a task which in itself may be quite intricate and time-consuming. After specifying the system structure and interconnections, the investigator must convert the observed data, often expressed in disintegrations per minute (dpm) if a radioactive isotope was used as the tracer, into a format that can be read by the modeling software. In VA research, the program of choice is the Windows version of Simulation, Analysis and Modeling (WinSAAM) created in 1962 by Mones Berman (35). This program uses data expressed as the fraction of dose recovered from a compartment (often plasma or an organ), which can be obtained by multiplying the concentration (dpm per ml or dmp per g) of tracer measured in that compartment by the volume or mass of the compartment (ml or g) (37). Data expressed as fraction of dose are entered into the program as a series of values, each corresponding to the sampling time at which it was observed. In addition, WinSAAM requires entry of the initial estimates of fractional transfer coefficients and their approximate ranges (minima and maxima) based on prior knowledge about the system. The fractional transfer coefficients are denoted as L(I,J)s and represent the fraction of tracer in 8

22 compartment (J) transferred to compartment (I) or out of the system (0) per unit of time. The number and type of L(I,J)s correspond to the connections between compartments or the flow of material out of the system. The researcher also has to specify the compartment to which the tracer was delivered or injected at the beginning of the experiment (time 0) and the initial condition, that is, the fraction of dose in that compartment at time 0. Once the WinSAAM input file is set up, the analytical phase of modeling can begin. This phase involves adjusting the model parameters in a step-wise manner to obtain a tracer response curve (continuous plot of fraction of dose over time) that best fits to the data observed in the experiment. This step can take up to several hours or even days depending on the complexity of the system and the accuracy of the initial model. Once a satisfactory fit is found, the program performs an iterative computation, in which the sum of squared residuals is minimized through the application of weighted nonlinear regression analysis. This analysis takes into account the weight assigned to data observed at different sampling times and the uniqueness of the resulting parameters (30). The computation generates a complete set of final estimates of the L(I,J)s as well as their statistical uncertainty. The L(I,J)s, in turn, can be used to calculate more complex kinetic parameters such as the mass of a nutrient in a specific compartment, transit time (the average time a nutrient spends in a compartment during a single transit) or the system residence time (the average time a nutrient spends in the system before irreversible disposal). Finally, the L(I,J)s obtained from different experimental conditions can be compared statistically to predict the system s response to various treatments or to make hypothetical statements about various regulatory mechanisms involved in maintaining the system homeostasis (35). 9

23 The most relevant aspect of model-based approach for my research is its ability to characterize VA kinetics on the anatomical, physiological and molecular level. Specifically, the model-based analysis has the capacity to lump processes occurring in spatial and temporal proximity into individual compartments, which reflect the anatomy of the body as well as the physiological and biochemical characteristics of the individual organs. For example, the model developed by Tan et al. (38) of VA kinetics in plasma of neonatal rats consisted of 8 compartments representing organs such as liver, lungs, intestines, kidneys and other non-hepatic tissues. This model allowed for distinguishing between the VA taken up by the liver vs. other organs. The model-based analysis can also separate compounds with distinct kinetic behavior in a single organ, such as the chylomicron REs and RBP-bound ROH in plasma, into individual subcompartments. This particular feature is especially useful when exploring differences among organs in terms of their uptake of the two main molecular forms of VA: REs and ROH. Besides representing the system anatomical structure and molecular function, the model-based analysis can provide quantitative and predictive information about the system under different treatment conditions, a feature that is very useful for my research. In my study, I described VA kinetics without and after VA supplementation and the model-based analysis enabled me to compare the uptake and metabolism of VA under these two conditions. The previous compartmental model of data from neonatal rats compared the kinetics of VA under control conditions and after supplementation with VA mixed with retinoic acid (VARA) and showed that the recycling number of ROH (the number of times a molecule of ROH returns to plasma after being taken up by organs) is considerably lower in the VARA-treated neonates (38). Finally, the compartmental model of data from adult 10

24 rats compared VA kinetics in animals raised under VA-deficient, -marginal and -sufficient conditions and revealed that the rate of irreversible disposal of ROH varies with VA status and that plasma ROH level is a major determinant of the disposal rate (39). The third most relevant aspect of model-based analysis for my research is its capability to quantitate the kinetics of nutrients under non-steady state conditions, that is, when the mass of the tracee in the system changes over time. This is due to a large number of data and parameters that can be handled simultaneously by the modeling software such as WinSAAM (35). For example, Tan et al. (40) successfully used model-based approach to solve a complex whole-body model of VA kinetics in growing neonatal rats. The problem of non-steady state can also be resolved outside of the modeling program by multiplying the mass of ROH in plasma, which increases with the growth of pups, by the time-invariant fractional transfer coefficients to obtain the rate of ROH transfer (expressed in nmol per d) specific to a sampling time and therefore reflective of the change over time. This particular method was previously used in the same study by Tan et al. (38) to calculate the uptake, turnover and disposal rates of ROH in continually growing rat pups. The above study by Tan et al. (38) is a pioneer project, in which the neonatal VA metabolism and kinetics were explored for the first time. The model-based compartmental analysis used in this project revealed several important aspects of VA metabolism that are unique for newborn rats. For example, the absorption of VA was found to be relatively more efficient in young compared to adult rats. The recycling of ROH between plasma and tissues was also much more extensive in neonatal rats (144 times) compared to adult rats (12-13 times). Finally, the extrahepatic tissues in neonates were more active in clearing chylomicron REs from plasma than in adult rats. In particular, compared to the adult rat extrahepatic 11

25 tissues, which took up only 25% of postprandial chylomicron REs, in neonates, these tissues took up 48%, while the remaining 52% was taken up by the liver. As a result, 51% of the whole-body VA in neonates was stored in the extrahepatic tissues, whereas only 44% was found in these tissues in adults (40). These findings raise an interesting question as to which specific non-hepatic organ stores most of the extrahepatic VA. This question as well as other objectives are addressed in chapter 2. 12

26 Chapter 2. OBJECTIVES AND HYPOTHESES In order to help guide the public heath recommendations for VA supplementation of infants, a better understanding of neonatal VA metabolism is needed. In the present study, our objective was to determine the distribution and kinetics of VA in plasma and organs in neonatal rats as a model for human infants, without and following VA supplementation at a dose equivalent, after adjustment for body weight, to that which has been given to human infants (50,000 IU/2.5 kg body weight). Previous studies conducted in our laboratory have shown that, compared to adult rats, neonates stored a greater proportion (51% vs. 44%) of the whole-body VA mass in tissues other than the liver, which is the main VA storage organ in VA-sufficient rodents (41). Moreover, a single dose of VA combined with retinoic acid (VARA) stimulated the uptake of VA by extrahepatic tissues, especially the carcass (composed largely of bones and muscles) and intestines, which together acquired nearly 75% of the recently ingested VA (42). In view of these findings, we focused here on the extrahepatic tissues, subdividing the carcass, which in our previous experiment included several organs, into its components, and determined the effect of VA supplementation, administered without RA, on retinol metabolism and kinetics in tissues that have been relatively unexplored from the perspective of VA metabolism, such as brain, skin, white adipose tissue (WAT) and brown adipose tissue (BAT). Our overarching objective consisted of the following three aims. In aim 1, we examined the concentration and mass of retinol in tissues, without and following VA supplementation, over 24 d after dose administration. Because organs grow rapidly during 13

27 the neonatal period, organ weights over time and retinol mass accumulation were also measured. In aim 2, we applied model-based compartmental analysis to quantify the fractional transfer of ROH (fraction of VA leaving one compartment and entering another/d) and the transfer rate of ROH (expressed in nmol/d) between plasma and organs in the unsupplemented (control) and VA-supplemented neonates, with distinct kinetic profiles generated for the two major molecular forms of VA in plasma: retinyl esters present in postprandial chylomicrons (CM-REs) and retinol bound to retinol-binding protein (RBP- ROH). Finally, in aim 3, we compared total ROH concentrations, tracer response profiles and kinetic parameters obtained in the current study to those from the previous experiment, in which pups were supplemented with VARA (38, 40), to determine the extent to which presence of retinoic acid in the VA supplement improves its uptake and retention in neonatal tissues. Our three aims may be summarized as follows: Aim 1. To identify the organs that store VA in neonates and to assess the relative mass and duration of these stores, particularly in the previously unexplored carcass components, such as brain, skin, and white adipose tissue (WAT) and brown adipose tissue (BAT); Aim 2. To use model-based compartmental analysis to quantify the transfer of ROH between plasma and organs in the unsupplemented (control) and VA-supplemented neonates; 14

28 Aim 3. To compare the effect of VA and VARA supplementation on ROH concentration and the transfer of CM-REs and RBP-ROH between plasma and organs in neonates. We hypothesized that a relatively larger proportion of VA would be stored extrahepatically in the rapidly growing and developing peripheral tissues, such as the skin and adipose tissue, under the unsupplemented condition and that VA supplementation would increase the proportion and mass of ROH stored in the liver and the uptake of ROH into the extrahepatic organs, such as the lungs and carcass, albeit not as effectively as VARA. Our basic approach is to randomly assign rat pups into two treatment groups, control and VA-supplemented, treat pups on postnatal day (P) 4 with an oral dose of either VA or canola oil as control, both containing the tracer, and euthanize them sequentially, in groups of 4, to collect plasma and organs at 13 time points from 30 min to 24 d after the initial treatment (Table 1). Table 1. Sample size Treatment Animals per sample collection time Sample collection times Control (canola oil) n = 4 13 VA (retinyl palmitate) 6 mg/kg (50,000 IU in infants, 200 IU in rats) n = 4 13 Total sample size N = males N = males 15

29 Chapter 3. VITAMIN A DISTRIBUTION IN THE BODY DURING THE NEONATAL PERIOD 3.1 Materials and methods Animals and diet Pregnant Sprague-Dawley rats (n = 11) were purchased from Charles River Laboratories (Kingston, NY). Upon arrival (on d 10 of pregnancy), dams were switched to a VA-marginal, AIN-93G purified diet (43) modified to contain 0.35 mg retinol equivalents/kg diet (Research Diets, New Brunswick, NJ) in order to render pups in a VAmarginal state similar to that of low birthweight infants in regions with high prevalence of VA deficiency. All dams were housed individually in a room with a 12 h light/dark cycle at 22 C with free access to food and water. All animal procedures were approved by the Institutional Animal Care and Use Committee of The Pennsylvania State University Dose preparation and delivery Vitamin A in the form of all-trans retinyl palmitate was purchased from Sigma- Aldrich (St. Louis, MO). The specific amount of VA contained in each dose was based on the international standard of 50,000 IU per 2.5 kg of body weight recommended by WHO for infants at risk of mortality in settings where VA deficiency is a public health problem (13). This standard was converted to mass units using the conversion factor of µg retinyl palmitate/iu to obtain the dose mass of 6 μg/g body weight or 21 nmol/g body weight, which is equal to 200 IU of retinol for a 10 g neonatal rat. 16

30 The amount of VA required to dose all pups was mixed with canola oil in proportions required to obtain the dose volume of 0.4 µl/g body weight + 1 µl. This more concentrated dose was chosen instead of 0.8 µl/g body weight + 1 µl used in previous experiments in order to limit the spillover from pups mouths. The oil and VA doses were stored at -20 C in vials covered with aluminum foil to protect VA from photodegradation. All dams gave birth within 2 consecutive days to a total of 116 pups that were subsequently redistributed between 11 litters to avoid any effects due to litter size differences. Approximately 10 pups from each litter were randomly assigned to the 2 treatments: control and VA-supplemented (Table 1). On P4, which marks the beginning of alveolar septation in neonatal rats (44) all pups were treated with an oral dose of either VA (6 μg retinyl palmitate/g body weight) mixed with canola oil in a ratio of 1:3 or pure canola oil as the control treatment. The dose was administered orally using a small micropipette with capillary pistons (Gilson, Middleton, WI) to limit the retention in the pipette tip. After dosing, pups muzzles were wiped with tissue paper, which was analyzed along with pipetting tips, in order to determine the amount of tracer lost during dose administration. The final mean dose corrected for losses contained 1.4 μci of [ 3 H]retinol. Immediately after dosing, each pup was returned to the mother and allowed to consume mother s milk (and diet after weaning) for the remainder of the study Tissue collection At 13 times after dose administration (Figure 1), 4 pups per group were removed from their cages, weighed, and sedated with isoflurane and, beyond 14 d of age, asphyxiated with CO2. Blood was collected from the vena cava into heparinized syringes, and the 17

31 following tissues were dissected: liver, stomach, intestines (small and large intestines with the contents), lungs, kidneys with adrenals, brain, interscapular BAT, inguinal and subcutaneous WAT (collected on and after P12 from the inguinal and scapular depots), and the remaining carcass, which also contained the eyes. Blood and liver were also collected from dams euthanized on the last day of the study. Blood samples were centrifuged at 2,000 rpm for 15 min and stored at -20 o C. Tissue samples were snap frozen in liquid nitrogen and stored at -80 o C. Figure 1. Sample collection timeline. P, postnatal day Analyses of total retinol and retinyl esters Total retinol mass (unesterified + esterified retinol) was determined by UPLC (Acquity UPLC System, Waters, Milford, MA) using an adaptation of the method described previously (40, 45). Briefly, portions of tissue (~0.2 g) were cut, weighed and homogenized in 100% ethanol with a glass homogenizer. The carcass, being a fibrous tissue, was cut into fine pieces using a multiple-blade shearers and transferred to ethanol in its entirety. Tissue homogenates (~0.2 g) and plasma aliquots (5 to 60 μl) were incubated in ethanol for 1 h or 24 h (carcass) for lipid extraction, saponified using 5, 10 or 20% potassium hydroxide to ensure a complete hydrolysis of lipids (Appendix A) and incubated at 55 o C for 30 min. 18

32 Neutral lipids were extracted by adding 4 ml of hexanes (Avantor, Centre Valley, PA) containing 0.1% of butylated hydroxytoluene/ml (Sigma-Aldrich, St. Louis, MO) and 2 ml of distilled water. The extraction step was repeated 2-3 times so as to attain at least 93% extraction efficiency, as determined in prior pilot testing of these tissues (Appendix A). After centrifugation, the upper-phase hexanes were removed, an internal standard (trimethylmethoxyphenyl-retinol) was added, the solvent was evaporated under nitrogen, and the residue was reconstituted immediately in 100 ml of methanol for injection onto the reversed-phase column of the Acquity UPLC System. To measure retinyl esters, the saponification step was omitted and the UPLC run time extended from 1 to 7 minutes. All extraction and analytical procedures were carried out under UV screened lights to protect VA from photodegradation. All results were corrected for lipid extraction efficiency Statistical analysis Values were expressed as mean ± SEM. Group means for plasma and tissue retinol mass and concentration at individual times were compared using Student s t-test with Bonferroni correction for multiple comparisons (GraphPad Prism software, version 5.0, La Jolla, CA). Changes over time were assessed using simple linear regression analysis with time as the independent variable and retinol concentration/mass as the dependent variable. A significant non-zero slope (β) indicated an increase or decrease over time. Differences with P < 5 were considered significant. To compare organs in terms of VA storage capacity, the organ retinol mass was averaged between P4 and P8, when the relative organ weights were nearly constant (Table 2). This period in rats corresponds to ~1-2.5 years of age in humans (46). The means were then compared using Wilcoxon matched-pairs signed 19

33 rank test. Total plasma volume was estimated as 3.5% of body weight based on previously published data (47). 20

34 Table 2. Organ weight as percent of total body weight in control and VA-supplemented 1 rats between P4 and P28. Day after dosing Liver (%) Lungs (%) Brain (%) WAT 2 (%) BAT (%) Kidneys (%) Skin (%) Stomach (%) Intestine (%) Carcass (%) Mean body weight (g) ± ± 3.6 ± ± 1.3 ± 17.1 ± ± ± ± ± ± ± 3.4 ± ± ± 18.1 ± ± ± ± ± ± ± ± ± ± 19.1 ± ± ± ± ± ± ± ± ± ± ± 22.8 ± ± ± ± ± ± ± ± ± ± ± 23.9 ± ± ± ± ± ± ± ± ± ± ± 22.2 ± ± ± ± ± ± ± 2.5 ± ± ± ± 17.6 ± ± ± ± ± ± ± ± 1.1 ± ± 1.3 ± 15.2 ± ± ± ± ± 1.4 Mean 4.2 ± ± ± ± ± ± 18.5 ± ± ± ± ± VA supplement was given on P4, when the mean body weight was 10.8 ± 0.1 g. Data include both control and VA-supplemented neonates, as there were no differences in organ growth due to supplementation. Each value represents the mean ± SEM of n = 8 rats. 2 No dissectible WAT was found before P12. 3 BAT, brown adipose tissue; P, postnatal day; VA, vitamin A; WAT, white adipose tissue. 21

35 3.2 Results Animal and relative organ growth Animals appeared healthy and grew rapidly, with body weights increasing from ~10 g to ~80 g, without differences due to VA supplementation (Figure 2). Figure 2. Neonatal rat body weights between P4 and P28. Inset shows the first 24 h after dosing. Each point represents the mean ± SEM of n = 4 rats. P, postnatal day; VA, vitamin A. 22

36 The ratio of organ-to-body weight was highest for the skin (0.2 ± ) and carcass (0.4 ± ). During the study, the organ-to-body weight ratio increased for the liver, intestines and carcass, and decreased for the lungs and brain. For the skin, this ratio increased prior to d 11 of the study and declined afterward (Figure 3). Figure 3. Organ weights as a fraction of total body weight over time in control and VAsupplemented rats between P4 and P28. Data include both control and VAsupplemented neonates, as there were no differences in organ growth due to VA supplementation. Carcass contained bones, muscles and connective tissue remaining after dissection of organs listed in the legend. The eyes were also contained in the carcass. No dissectible WAT was found before P12. BAT, brown adipose tissue; P, postnatal day; VA, vitamin A; WAT, white adipose tissue. 23

37 3.2.2 Digestive organs The concentration of retinol in the stomach was elevated from 30 min to 1 h (P < 1) after VA supplementation, declining to control group level at 15 h (Figure 4A). Total retinol mass in the stomach followed a similar pattern (Figure 4B). In the intestines, the concentration of retinol peaked at 1 h after dosing and remained significantly elevated for 24 h (P < 01) (Figure 4C). Total retinol mass in the intestines remained relatively constant, despite a ~900% increase in organ weight during the study (Figure 4D, inset). Figure 4. Stomach (A, B) and intestine (C, D) total (unesterified + esterified) ROH concentration and mass in control and VA-supplemented neonatal rats between P4 and P28. Insets show the first 24 h after dosing (A, C) and organ weights (B, D). Each point represents the mean ± SEM of n = 4 rats. *P < 5. P, postnatal day; VA, vitamin A. 24

38 3.2.3 Plasma In the control group, the mean concentration of retinol in plasma (0.8 ± 0.1 μmol/l) was within the marginal status of 0.7 to 1 µm (1). In the VA-supplemented neonates, plasma retinol concentration peaked at 1 h after dosing (3.6 ± 1.7 μmol/l) and returned to baseline 15 h later (Figure 5A). This increase was due mainly to retinyl esters (Figure 5B), presumably present in postprandial chylomicrons. After 15 h, plasma retinol concentration in the VA-supplemented group did not differ from that in the VA-marginal control group. Total retinol mass in plasma also did not differ, except on d 14, when there was a ~30% drop in the VA-supplemented neonates (P < 01). After d 14, the mass of retinol in plasma increased more rapidly over time than did plasma volume (Figure 5C, inset). 25

39 Figure 5. Plasma total (unesterified + esterified) ROH concentration (A), RE concentration (B) and ROH mass (C) in control and VA-supplemented neonatal rats between P4 and P28. Insets show the first 24 h after dosing (A) and plasma volume (B). Each point represents the mean ± SEM of n = 4 rats (A, C) or a pooled sample of n = 4 rats euthanized at one sampling time (B). *P < 5. Dotted line represents the boundary between adequate and marginal plasma retinol concentrations (1). P, postnatal day; VA, vitamin A. 26

40 3.2.4 Liver, lungs and kidneys In the liver in control group, the mean retinol concentration (59.4 ± 1.8 nmol/g) was deficient (<70 nmol/g of tissue) (2) and it decreased steadily over time (β = -5.9 ± 1.2, P < 01). Vitamin A supplementation increased the total retinol concentration ~3-fold from the control group value at 24 h after dosing and the concentration remained elevated at most times until d 11 (P < 01) (Figure 6A). Total retinol mass in the liver increased in both groups until d 14, then declined and remained steady, despite continuous liver growth (Figure 6B). In the lungs in control group, retinol concentration increased ~4-fold from d 0 to 18 (β = 0.3 ±, P < 01). After VA supplementation, lung retinol concentration was greater than in control group, but only at 1 h (P < 1) and 24 h after dosing (P < 01), most likely due to large variation between pups within groups. After d 1, lung retinol concentration tracked similarly in both groups, with a gradual increase and a rapid decline during the last 6 days of the study (Figure 6C). The lungs also showed a progressive accumulation of retinol relative to lung weight, which lasted until d 18 and d 24 in control and VA-supplemented neonates, respectively (Figure 6D). The concentration of retinol in the kidneys of control neonates decreased by ~80% from d 0 to 8 (β = -0.2 ±, P < 01). Vitamin A supplementation resulted in a significant elevation in kidney retinol concentration from control group level, which lasted until d 2. Afterward, there were no differences due to treatment (Figure 6E). Total retinol mass in 27

41 kidneys remained at a steady level (1.8 ± 0.1 nmol) in both groups, although gradual accumulation was observed during the last 10 days of the study (Figure 6F). Figure 6. Liver (A, B), lung (C, D), and kidney (E, F) total (unesterified + esterified) ROH concentration and mass, respectively, in control and VA-supplemented neonatal rats between P4 and P28. Insets show the first 24 h after dosing (A, C, E) and organ weights (B, D, F). Each point represents the mean ± SEM of n = 4 rats. *P < 5. Dotted line represents the boundary between sufficient and deficient liver retinol concentrations (2). P, postnatal day; VA, vitamin A. 28

42 3.2.5 Extrahepatic organs and the remaining carcass In the brain, VA supplementation increased retinol concentration ~3-fold from the control group value at 8 h after dosing (P < 01), with the difference lasting until d 1 (P < 5 for all) (Figure 7A). Retinol mass in the brain increased slightly over time (Figure 7B), but the increase was insufficient to match the rapid increase in brain weight (Figure 7B, inset). In BAT, VA supplementation increased retinol concentration ~4-fold from control group value at 4 h (P < 5), and the concentration remained elevated until d 4 (P < 1 for all) (Figure 7C). Retinol did not accumulate in BAT, despite continuous tissue growth (Figure 7D). In WAT, the concentration of retinol was still significantly elevated from control group level at d 18 after dosing (P < 1) and it declined gradually over the last 10 d of the study (Figure 7E). Retinol did not accumulate in WAT, nor was there a substantial increase in WAT weight (Figure 7F). In the skin, VA supplementation increased total retinol concentration from control group level for up to 15 h after dosing (P < 01) (Figure 7G). Retinol mass in the skin increased over time ~5-fold in both groups (βcontrol = 0.5 ±, βva = 0.4 ± 0.1, P < 01 for both) (Figure 7H), in parallel to the increase in skin weight (Figure 7H, inset). 29

43 Figure 7. Brain (A, B), white adipose tissue (C, D), brown adipose tissue (E, F), skin (G, H), and carcass (I, J) total ROH concentration and mass, respectively, in control and VA-supplemented neonatal rats between P4 and P28. Insets show the first 24 h after dosing (A, C, E, G, I) and organ weights (B, D, F, H, J). Each point represents the mean ± SEM of n = 4 rats. *P < 5. BAT, brown adipose tissue; P, postnatal day; VA, vitamin A; WAT, white adipose tissue. 30

44 In carcass, the concentration of retinol was low (0.1 ± nmol/g) and it declined over time, except for d 24 in the VA-supplemented neonates, with no significant difference by treatment at any time during the study (Figure 8A). Retinol mass in the carcass was also low (0.7 ± 0.1 nmol) and declined over time, except for d 24 in the VA-supplemented group, despite a ~9-fold increase in carcass weight (Figure 8B, inset). Figure 8. Carcass total (unesterified + esterified) ROH concentration (A) and mass (B) in control and VA-supplemented neonatal rats between P4 and P28. Insets show the first 24 h after dosing (A) and carcass weight (B). Each point represents the mean ± SEM of n = 4 rats. P, postnatal day; VA, vitamin A. 31

45 3.3 Discussion and conclusions To our knowledge, this study provides novel and comprehensive information on the distribution of VA in tissues in neonatal rats raised under VA-marginal conditions. Previous studies examined the kinetics of VA administered orally to neonatal rats on P4 and showed that ~51% of the whole-body VA mass resided in the extrahepatic tissues, compared to ~44% in adult rats (40). In these experiments, however, total retinol mass in extrahepatic tissues was not measured directly but predicted using mathematical modeling based on tracer kinetics in plasma and tissues. Furthermore, several extrahepatic tissues, including skin, brain and adipose tissue, were lumped together with the carcass. Here, we analyzed these tissues individually, leaving only the skeleton, muscles and some connective tissue as the remaining carcass. Based on previous findings, we hypothesized that the extrahepatic tissues in neonates would contain a relatively higher proportion of the whole-body VA than that observed in adults and that VA supplementation would increase the proportion present in the liver. Our findings did not support these hypotheses. Instead, the results demonstrated that, despite a liver retinol concentration that would be categorized as deficient, the majority of VA reserves were still hepatic. Vitamin A supplementation had a transient effect on retinol concentrations in all extrahepatic tissues examined except WAT, but the supplementation did not affect tissue retinol distribution Vitamin A distribution in the body in control rats The liver contained ~76% of the whole-body VA mass in control, 4 to 8-d-old pups, a proportion similar to the 86% of hepatic retinol found in adult rats and humans in the VA- 32

46 adequate state (2, 48, 49). This relatively high proportion of hepatic retinol was maintained despite a liver retinol concentration that was deficient and significantly lower than that measured in adult mother rats fed the VA-marginal diet (Figure 9), a finding consistent with the low neonatal liver retinol concentration found in piglets fed a VA-free diet (~25 nmol/g) and in newborn humans (11, 50). The lungs and kidneys together stored about 5% of the whole-body VA and showed a relatively high retinol concentration consistent with the previously demonstrated role of these organs in VA storage and metabolism in rodents. Interestingly, our study showed that rats in the control group were accumulating retinol in the lungs for the 22 days of this study, despite the VA-marginal conditions. This trend may reflect the need for retinol in the lungs to support the process of secondary alveolar septation, which in rats occurs postnatally (P4 to P14), unlike in humans, in whom this process is completed during gestation (51). The remaining extrahepatic tissues each made <1% contribution to the total VA mass in the body, except the skin, digestive organs and carcass. The skin contained ~6% of the whole-body VA mass, due mostly to its large weight (~20% of the whole-body weight in 4 to 8-d-old pups) rather than high retinol concentration (~1 nmol/g). Given the large surfaceto-body weight ratio in newborns (52) and the importance of VA for the differentiation of cells in epithelial layers (4), it is possible that neonates have a greater requirement for VA to support normal skin development. Skin may also act as a reservoir of VA in neonates, in which WAT is nearly absent. The stomach and intestines each contained ~5% of the wholebody VA mass. These organs, however, were analyzed together with their contents, which contributed to the measurement. The carcass, which constituted 40% of the whole-body 33

47 Figure 9. Mean organ total (unesterified + esterified) ROH concentration (A) and mass (B) in control and VA-supplemented rats between P4 and P8 (0-4 d after VA supplementation). Each bar represents the mean ± SEM of n = 32 rats. Adult liver retinol concentration in A represents the mean ± SEM of 4 mother rats used in the study. *P < 5. BAT, brown adipose tissue; P, postnatal day; VA, vitamin A; WAT, white adipose tissue. 34

48 weight, contained only 1.2% of the whole-body retinol mass, and, together with the brain, had the lowest VA concentration (~0.1 nmol/g). Collectively, our results show that the extrahepatic, non-digestive organs in control neonates contained ~13% of the whole-body VA mass, a proportion similar to that observed under VA adequate conditions. In adult VA-sufficient rats, 14% of the whole-body VA mass was extrahepatic (compared to 44% in VA-marginal and 93% in VA-deficient adult rats) (53-55). In humans, the corresponding percentage under VA adequate conditions was estimated at 10% (56). The fact that neonates in our study stored only ~13% of retinol outside of the liver suggests that the non-hepatic tissues may not be sufficiently developed to store VA at the adult capacity. Even though the retinoid receptors, cellular retinoid-binding proteins and enzymes involved in retinol storage are expressed early in life (41), they may be rate-limiting. The low VA content in extrahepatic tissues may be attributed to the specific characteristics of the neonatal body, particularly the scarcity of WAT. White adipose tissue, which normally constitutes 5-10% of the adult rat s body weight (57), was nearly absent prior to P12 in neonatal rats and, afterward, contained only ~1% of the whole-body VA mass, compared to 10-20% in adult rats (19, 58). BAT was present from birth, but its contribution was also low (~0.5%), as was the concentration of retinol in WAT and BAT of neonates (~2 nmol/g compared to nmol/g in adult rats) (59). Such low VA storage capacity of adipose tissue may be due to the lower lipid and higher water content of neonatal tissues in general (52). 35

49 3.3.2 The effect of vitamin A supplementation Supplementation with VA resulted in a pronounced, 1- to 4-fold increase in retinol concentrations in non-digestive organs between 4 to 15 h after dosing. These elevated concentrations returned to baseline within 24 h after dosing in all organs except kidneys, liver, WAT and BAT (Table 3). In kidneys, the significant elevation lasted for 2 d, most likely due to an increased rate of retinol disposal following supplementation. A 4-d and 11- d long elevation was observed in BAT and liver, respectively. White adipose tissue was the only organ, in which the supplementation effect lasted for longer than in the liver (18 d), indicating that it may serve as a long-term VA storage depot. This finding further suggests that the scarcity of WAT in neonates may predispose them to a low VA status. The return of plasma VA concentration to a marginal level in the supplemented pups, despite a simultaneous increase in liver VA concentration, suggests that neonates may have a lower set-point for plasma retinol that is not necessarily indicative of deficiency, or that a more frequent and higher intake is needed for equilibration. The rapid (within <24 h) decline of VA concentrations in all other organs suggests that they have a relatively low VA retention capacity and utilize it quickly or release it back to plasma for transfer to the liver. This finding agrees with the early and transient (~24 h) peak in retinol concentration in the lungs, spleen and adrenal gland of neonatal (28-d-old) piglets dosed with 50,000 IU of VA (60). From these results, Riabroy and Tanumihardjo (60) concluded that extrahepatic tissues in neonatal pigs rely on recently ingested chylomicron retinyl esters as the main source of VA, making a constant supply of VA, in mother s milk or diet, necessary to maintain a normal extrahepatic VA concentration. 36

50 Table 3. The effect of supplementation on organ VA concentrations in neonatal rats dosed with 200 IU of VA on P4. Organ Time of peak VA concentration (h after dosing) Increase from control group level at peak (%) P-value Supplementation effect duration 1 (h) Plasma Carcass Stomach < 01 1 Skin < 1 15 Intestines < 5 24 Lungs < 5 24 Brain < Kidneys < 1 48 BAT < 5 96 Liver < 01 11, days WAT , days 1 Duration of significant elevation (P < 5) in organ VA concentration in the VAsupplemented group compared to the control group as determined by a Student s t-test with Bonferroni correction for multiple comparisons. 2 No dissectible WAT was found before P12. 3 BAT, brown adipose tissue; P, postnatal day; VA, vitamin A; WAT, white adipose tissue. 37

51 Vitamin A supplementation did not affect the rank order of tissues in terms of their retinol mass or concentration, except for the higher position of stomach and intestines relative to skin (retinol mass) and kidneys (retinol concentration), as is expected from the presence of the VA dose in these organs (Figure 9). This observation was similar to findings in neonatal piglets, in which the liver ranked as the highest in terms of retinol concentration, followed by the kidneys and lungs, irrespective of VA treatment (50). However, kinetic studies in rats supplemented with VARA showed a dramatic stimulatory effect of supplementation on the uptake and retention of retinyl esters by the lungs and intestines (40, 61), consistent with the previously demonstrated role of retinoic acid in upregulating genes responsible for VA storage (62-64). Therefore, VA alone may not be as effective as VARA in promoting extrahepatic retinol deposition. In conclusion, our study demonstrated that male and female Sprague-Dawley neonatal rats under VA-marginal conditions stored a majority of their VA in the liver, despite its deficient retinol concentration, an effect that may be explained by the low VA storage capacity of extrahepatic tissues. We also showed that supplementation with VA, in a dose equivalent to that which has been given to human newborns, caused a transient increase in retinol concentration in all extrahepatic organs except WAT. This may indicate that the scarcity of subcutaneous fat in neonates predisposes them to VA deficiency. These findings also suggest that a more frequent VA supplementation schedule, along with an improved dietary intake, may be necessary to meet the needs of rapidly developing neonatal tissues. More research, however, is needed to examine the link between VA storage in adipose tissue and other extrahepatic organs and the health benefits of VA supplementation. 38

52 Chapter 4. VITAMIN A KINETICS IN PLASMA AND ORGANS IN NEONATAL RATS. 4.1 Methods Dose tracer content Each dose mixture, both the control and the VA-supplemented, contained 11,12- [ 3 H]ROH (PerkinElmer, Waltham, MA) in the amount calculated to deliver ~2 μci per pup. In order to determine the tracer content of the initial dose, an aliquot of dose (3 μl) was added to 4 ml of hexanes immediately after dose preparation and left for overnight extraction. The following day, 0.5 ml of the extract was transferred to a vial containing 4 ml of scintillation fluid (ScintiVerse TM BD Cocktail, Fisher Scientific, Fair Lawn, NJ) and counted. The result showed that a 5 μl dose contained 1.8 μci of [ 3 H]ROH. During dose delivery, a small portion was left in the pipetting tip and on a pup s muzzle. The final ingested dose corrected for these losses contained 1.4 μci [ 3 H]ROH. All dose preparations were stored at -20 C in foil-wrapped vials to protect VA from photodegradation Tracer analysis Tracer analysis was conducted on the same lipid extracts as used for the analysis of ROH mass. Of the remaining 3 ml of hexane extract, 2 ml were removed and transferred into liquid scintillation vials. The solvent was evaporated under air and the residue redisolved in 4 ml of scintillation fluid (ScintiVerse TM BD Cocktail, Fisher Scientific, Fair Lawn, NJ). Tracer analysis was performed using liquid scintillation counting (LS

53 Scintillation System, Beckman Coulter, Fullerton, CA) with each sample counted to a 1% error Kinetic analysis VA kinetics were determined using model-based compartmental analysis performed in Windows version of Simulation, Analysis, and Modeling software (WinSAAM; version 3.0.8, Kennett Square, PA). The input file for modeling plasma contained the initial estimates of fractional transfer coefficients [L(I,J)s], adapted from the previous model of VA kinetics in neonatal rats (38), and the observed data expressed as the fraction of dose (FOD; total radioactivity of plasma divided by the radioactivity of the ingested dose). Each observation was a mean of 4 pups euthanized at one sampling time and weighted by the SD. Furthermore, in order to trace the kinetics of CM-REs and RBP-ROH separately, each observed value of total ROH was defined as the sum of CM-REs and RBP-ROH using the XG function, which generated a distinct tracer response profile for CM-REs and RBP-ROH, respectively. In addition to L(I,J)s and the observed data, the organ input files contained a forcing function, which specified the amount of tracer available in plasma for uptake at any point in time, based on the solution to the plasma model. The forcing function was used to uncouple the organs from the rest of the system and model them individually given the assumption that organs derive ROH from plasma only and not from each other. Because the uptake of ROH from plasma was assumed to be unidirectional, the loss of ROH from organs included both the amount released back to plasma and the amount metabolized irreversibly. The loss of ROH from organs is, therefore, referred to as turnover throughout this dissertation. 40

54 4.1.4 Model structure and kinetic parameters The initial structure of plasma model was adapted from a previous study of VA kinetics in neonatal rats (Figure 10) (38). In order to capture the increase in tracer level of plasma observed during the last 10 d of the study (Figure 14A) and a simultaneous decrease in the tracer level of the liver (Figure 15A), compartment 7 was added to represent a portion of tracer stored in the liver for 14 d and released afterward. Figure 10. Plasma model of VA kinetics in neonatal rats dosed orally with [ 3 H]ROH on P4. Circles represent compartments and arrows represent their interconnections. Squares indicate delay components. CM-REs, chylomicron retinyl esters; DT, delay component; IC, initial condition; P, postnatal day; RBP-ROH, retinol binding protein-bound retinol; VA, vitamin A. 41

55 Structures of the organ models were also adapted from a study by Tan et al. (40) (Figure 11A, B, C). These structures fitted the observed data well, therefore, the number of compartments and parameters was left unchanged, except for adding a time interrupt at d 1, 2, 4 or 8 to capture the rapid change in the turnover rate of RBP-derived ROH in the liver, kidneys, stomach, brain, skin, BAT, and carcass. In order to calculate the turnover rate of ROH [R(0,J) = L(0,J) M(J)]; nmol/d] using the previously measured organ mass of total ROH [M(J); nmol], the organ models were simplified so that the CM-REs and RBP-ROH entered a common pool of organ total ROH, from which turnover [L(0,J)] occurred (Figure 11D). Estimating a turnover rate specific for the CM- and the RBP-derived ROH was precluded by the lack of mass data for these two entities separately. Nevertheless, the simplified models fitted the observed data equally well as the original models indicating that the CM- and RBP-derived ROH entered a common organ pool of total REs/ROH after uptake. 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 of FOD vs. time to the observed data. After finding a satisfactory fit, the final parameter values were generated through the application of a weighted nonlinear regression analysis, which minimized the residuals given the weight assigned to data, parameter constraints, and their uniqueness. The model was considered well-identified when the sum of squares from the regression analysis was less than and the fractional standard deviation of each parameter was less than

56 Figure 11. Liver (A), stomach (B) and intestine (C) models of VA kinetics in neonatal rats dosed orally with [ 3 H]ROH on postnatal day 4 and a simplified liver model (D) developed to calculate ROH turnover rate [R(I,J)]. Circles represent compartments and arrows represent their interconnections. Squares indicate compartments defined by the forcing function. Compartments 10 and 1 represent plasma CM-REs and RBP-ROH, respectively. Compartments 1X and X represent organ CM- and RBP-derived ROH, respectively. L(I,J)s represent the fractional transfer of CM-derived, RBP-derived or total ROH into or out of an organ. CM-REs, chylomicron retinyl esters; IC, initial condition; RBP-ROH, retinol binding protein-bound retinol. 43

57 4.1.5 Statistical analysis The fraction of dose (FOD) vs. time was graphed and the values at individual time points were compared with the use of Student s t-test with Bonferroni correction for multiple comparisons (GraphPad Prism software, version 5.0, La Jolla, CA). Values were expressed as mean ± SEM. The fractional transfer coefficients [L(I,J)s] were estimated in WinSAAM and expressed as the fraction of tracer in compartment (J) transferred to compartment (I) per d. The transfer rates [R(I,J)s; nmol/d] were calculated as the product of the mean VA mass in compartment J measured by UPLC during d 1 of the study [M(J); nmol] and the corresponding fractional transfer coefficient [L(I,J)] expressed as compartment fraction/d. The resulting SEMs were calculated according the error propagation formula SEM R = R FSD 2 M + FSD 2 L n. The uptake rates of total ROH were calculated as the sum of uptake rates of CM-REs and RBP-ROH and the resulting SEMs were calculated as SEM L1 +L 2 = SEM 2 L1 + SEM 2 L2. The percent uptake of ROH was calculated using the transfer rates as the basal values. All kinetic parameter values were expressed as mean ± SEM and compared statistically using Student s t-test with a t-statistic calculated for each parameter pair and evaluated for significance using the table of critical values of Student s t-distribution. A P value < 5 was considered as the level of significance. 44

58 4.2 Results Mean organ tracer levels The liver accumulated most of the tracer in both treatment groups, as evidenced by the highest mean (24-d average) fraction of dose (FOD) compared to other organs (control group: 19%, VA group: 36%) (Figure 12). The intestines and skin ranked as the second and third, although their mean FOD (~2%) was ~10-fold lower than that in the liver. The mean FOD in other extrahepatic organs was below 1%. The lowest mean FOD was found in the brain (6%). Vitamin A supplementation significantly increased the mean FOD in the liver and lungs, and decreased it in the kidneys. 45

59 Figure 12. Mean organ fraction of ingested [ 3 H]ROH dose between d 0 and 24 after dosing in control and VA-supplemented rats dosed on P4. Each bar represents the mean of n = 52 rats. Each bar represents the mean ± SEM of n = 52 rats. *P < 5. BAT, brown adipose tissue; P, postnatal day; ROH, retinol; VA, vitamin A; WAT, white adipose tissue. 46

60 4.2.2 Tracer response in digestive organs Tracer response observed in the digestive system did not differ by treatment: both groups showed a rapid loss of tracer from the stomach and the intestine and no evidence of ROH retention or recycling (Figure 13). Although the intestine FOD was higher in the VAsupplemented group compared to control group after d 2, 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. A B VA group Control group VA group Control group Figure 13. Stomach (A) and intestine (B) fraction of ingested [ 3 H]ROH dose in control and VA-supplemented rats from 0 to 24 d after dosing on P4. Insets show d 1 after dosing. Each symbol represents the mean of n = 4 rats. P, postnatal day; ROH, retinol; VA, vitamin A. 47

61 4.2.3 Tracer response in plasma In plasma, the tracer appeared within 30 min after dosing, peaked between 1-4 h and declined to a steady level within 2 d (Figure 14A). Moreover, after 14 d, there was a small increase in the plasma tracer level in both treatment groups, most likely due to a simultaneous release of RBP-ROH from the liver (Figure 15A). The CM-REs in the supplemented group (Figure 14B) peaked higher than CM-REs in control group indicating a less efficient clearance of CM-REs after supplementation (Figure 14C). A * B * VA group Control group C Figure 14. Plasma fraction of ingested [ 3 H]ROH dose (A) and fraction of ingested dose as CM-REs, RBP-ROH and their sum (total ROH) in control (B) and VAsupplemented (C) rats from 0 to 24 d after dosing on P4. Inset shows the first 2 d after dosing. Each symbol represents the mean of n = 4 rats. *P < 5. CM-REs, chylomicron retinyl esters; P, postnatal day; RBP-ROH, retinol-binding protein-bound retinol; VA, vitamin A. Total ROH RBP- ROH CM-REs Total ROH RBP- ROH CM-REs 48

62 4.2.4 Tracer response in the liver, lungs and kidneys In the liver in control pups, the total and the RBP-derived ROH increased during d 1 and remained steady afterward (Figure 15B). In the supplemented group, total ROH peaked at 15 h, due to the uptake of CM-REs (Figure 15C), declined rapidly, and increased again to a significantly higher from control level that lasted until d 14 (P < 01), indicating that ROH gradually accumulated in the liver after supplementation. A B * * * VA group Control group C Figure 15. Liver fraction of ingested [ 3 H]ROH dose (A) and fraction of ingested dose as CM-, RBP-derived ROH and their sum (total ROH) in control (B) and VAsupplemented (C) rats from 0 to 24 d after dosing on P4. Inset shows d 1 after dosing. Each symbol represents the mean of n = 4 rats. *P < 5. CM-derived ROH, chylomicron derived retinol; P, postnatal day; RBP-derived ROH, retinol-binding protein-derived retinol; VA, vitamin A. Total ROH RBP-derived ROH CM-derived ROH Total ROH RBP-derived ROH CM-derived ROH 49

63 In the lungs, FOD increased up to a maximum of ~1% and remained steady in both treatment groups (Figure 16A). The CM-derived ROH declined within 4 d after dosing in the supplemented group (Figure 16B) and within 8 h in the control group (Figure 16C). A B VA group Control group Total ROH RBP-derived ROH CM-derived ROH C Figure 16. Lung fraction of ingested [ 3 H]ROH dose (A) and fraction of ingested dose as CM-, RBP-derived ROH and their sum (total ROH) in control (B) and VA-supplemented (C) rats from 0 to 24 d after dosing on P4. Inset shows the first 2 d after dosing. Each symbol represents the mean of n = 4 rats. CMderived ROH, chylomicron derived retinol; P, postnatal day; RBP-derived ROH, retinol-binding protein-derived retinol; VA, vitamin A. Total ROH RBP-derived ROH CM-derived ROH 50

64 In the kidneys, the FOD at d 1 was significantly higher in the control group (P < 01), but declined below the supplemented group level within 8 d after dosing (Figure 17A). The CM-derived ROH in the supplemented group (Figure 17C) peaked below the CMderived ROH in the control group and declined within 8 h indicating a fractionally lower uptake and a rapid loss of CM-derived ROH after supplementation (Figure 17C). A B * * VA group Control group Total ROH RBP-derived ROH CM-derived ROH C Figure 17. Kidney fraction of ingested [ 3 H]ROH dose (A) and fraction of ingested dose as CM-, RBP-derived ROH and their sum (total ROH) in control (B) and VAsupplemented (C) rats from 0 to 24 d after dosing on P4. Inset shows the first 2 d after dosing. Each symbol represents the mean of n = 4 rats. *P < 5. CM-derived ROH, chylomicron derived retinol; P, postnatal day; RBP-derived ROH, retinol-binding protein-derived retinol; VA, vitamin A. Total ROH RBP-derived ROH CM-derived ROH 51

65 4.2.5 Tracer response in extrahepatic organs and the remaining carcass In in the remaining extrahepatic organs (brain, BAT, skin, and carcass), FOD peaked and declined within 4 to 8 d after dosing and remained at ~1% during the entire study, indicating rapid loss of tracer from these organs in both treatment groups (Figure 18). A * B VA group Control group VA group Control group C D VA group Control group VA group Control group Figure 18. Fraction of ingested [ 3 H]ROH dose in the brain (A), brown adipose tissue (B), skin (C), and carcass (D) in control and VA-supplemented rats from 0 to 24 d after dosing on P4. Insets show the first 2 d after dosing. Each symbol represents the mean of n = 4 rats. *P < 5. P, postnatal day; ROH, retinol; VA, vitamin A. 52

66 In the brain in control group, most of the tracer was delivered by RBP, whereas in the supplemented group, most was delivered by chylomicrons (Figure 19A, B). In BAT, chylomicrons delivered most of the tracer in both treatment groups (Figure 19C, D). A Total ROH RBP-derived ROH CM-derived ROH B Total ROH RBP-derived ROH CM-derived ROH C Total ROH RBP-derived ROH CM-derived ROH D Total ROH RBP-derived ROH CM-derived ROH Figure 19. Fraction of ingested [ 3 H]ROH dose as CM-, RBP-derived ROH and their sum (total ROH) in the brain (A, B), and brown adipose tissue (C, D), in control (A, C) and VA-supplemented (B, D) rats from 0 to 8 d after dosing on P4. Each symbol represents the mean of n = 4 rats. CM-derived ROH, chylomicron derived retinol; P, postnatal day; RBP-derived ROH, retinol-binding protein-derived retinol; VA, vitamin A. 53

67 In the skin and carcass, CM-derived ROH peaked and declined very rapidly in both treatment groups. Moreover, the RBP-derived ROH peaked relatively higher than the CMderived ROH suggesting RBP as the main source of ROH in the peripheral tissues (Figure 20). A Total ROH RBP-derived ROH CM-derived ROH B Total ROH RBP-derived ROH CM-derived ROH C D Total ROH RBP-derived ROH CM-derived ROH Total ROH RBP-derived ROH CM-derived ROH Figure 20. Fraction of ingested [ 3 H]ROH dose as CM-, RBP-derived ROH and their sum (total ROH) in the skin (A, B), and carcass (C, D), in control (A, C) and VAsupplemented (B, D) rats from 0 to 2 d after dosing on P4. Each symbol represents the mean of n = 4 rats. CM-derived ROH, chylomicron derived retinol; P, postnatal day; RBP-derived ROH, retinol-binding protein-derived retinol; VA, vitamin A. 54

68 In WAT, FOD appeared to be higher in the supplemented group, albeit without reaching a level of significant difference (Figure 21). VA group Control group Figure 21. White adipose tissue fraction of ingested [ 3 H]ROH dose in control and VA-supplemented rats from 0 to 24 d after dosing on P4. Each symbol represents the mean of n = 4 rats. No dissectible WAT was found before P12. 55

69 4.2.6 Plasma kinetic parameters The fractional uptake of CM-REs from plasma to organs was higher in the control group (P < 5), whereas the uptake of RBP-ROH was higher in the supplemented group (P < 01) (Table 4). The supplemented group also showed a higher fractional release and recycling of ROH from organs back to plasma (P < 01 for both), but a lower fractional irreversible loss of ROH from organs (P < 01). Table 4. Plasma kinetic parameters in control and VA-supplemented neonatal rats dosed orally with [ 3 H]ROH on P4. Parameter Parameter description L(I,J) ± SEM Control VA P-value L(2,1) Transit of ROH through the digestive system 31.4 ± ± L(15,10) Uptake of CM-REs from plasma to organs ± ± 61.1 <5 L(5,4) Release of RBP-ROH from organs to plasma 1.3 ± 2.4 ± 0.2 <01 L(6,5) Uptake of RBP-ROH from plasma to organs 16.7 ± ± 6.0 <01 L(5,6) Recycling of ROH from organs back to plasma 0.2 ± 1.1 ± <01 L(5,7) Release of RBP-ROH from liver to plasma after d ± ± 0.1 <01 L(0,6) Irreversible loss of ROH from organs 5 ± 0 02 ± 01 <01 1 CM-REs, chylomicron retinyl esters; P, postnatal day; RBP-ROH, retinol-binding protein-bound retinol; VA, vitamin A. 56

70 Vitamin A supplementation decreased the fraction of plasma ROH utilized irreversibly per day and the transit time of ROH in plasma resulting in a much higher recycling number of ROH between plasma and organs in the supplemented group compared to control group (Table 5). The transit time of ROH in organs was higher in the control group. Table 5. Plasma kinetic parameters in control and VA-supplemented neonatal rats dosed orally with [ 3 H]ROH on P4. Parameter Parameter description Control VA Plasma residence time T(5,5) Average time a molecule of retinol spends in plasma over multiple transits Plasma transit time Average time a molecule of retinol t(5) spends in plasma during a single transit Tissue transit time Average time a molecule of retinol t(6) spends in organs during a single transit Fractional catabolic rate Fraction of plasma retinol utilized FCR(5,5) irreversibly per day Average number of times a molecule of Recycling number v(5) retinol recycles through plasma before irreversible disposal 1 P, postnatal day; VA, vitamin A. 8.2 h 4.0 d 1.4 h 11 min 3.3 d 22 h Plasma transfer rates The transfer rates (expressed in nmol/d) of CM-REs, RBP-ROH and total ROH from plasma to organs were all significantly higher in the supplemented group. The transfer rate of CM-REs was ~50-fold higher (345.3 ± vs. 7.3 ± 4.3 nmol/d; P < 5), the transfer rate of RBP-ROH was ~10-fold higher (59.1 ± 3.9 vs. 5.8 ± 0.5 nmol/d; P < 01), and the transfer rate of total ROH was ~30-fold higher (404.4 ± vs ± 4.4 nmol/d; P < 5) after supplementation. 57

71 4.2.8 Organ kinetic parameters After supplementation, the fractional uptake of CM-REs was significantly higher in the lungs, liver (P < 01 for both) and brain (P < 1), and significantly lower in other organs (P < 5 for skin; P < 01 for other) (Table 6). The fractional uptake of RBP- ROH was higher in the lungs, carcass, liver, kidneys (P < 01 for all) and skin (P < 1), 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 (P < 1). Table 6. Fractional uptake of CM-REs and RBP-ROH from plasma to organs in control and VA-supplemented neonatal rats dosed orally with [ 3 H]ROH on P4. Uptake of CM-REs Uptake of RBP-ROH (fraction of plasma REs transferred/d) (fraction of plasma ROH transferred/d) Organ L(I,J) ± SEM L(I,J) ± SEM P-value P-value Control VA Control VA Stomach ± ± Intestines 44.3 ± ± 2.9 <01 3 ± 0 4 ± Liver ± ± 14.4 < ± ± 0.4 <01 Lungs 3.6 ± ± 1.1 < ± ± 0.3 <01 Kidneys 33.9 ± ± 0.4 < ± ± 0.5 <01 BAT 8.8 ± ± 0.1 < ± 0.3 ± WAT ± ± Brain 0.3 ± ± 0.1 <1 0.5 ± 0.2 ± 0.1 <1 Skin ± ± 7.1 <5 4.6 ± ± 10.2 <1 Carcass 87.3 ± ± 4.0 < ± ± 3.3 <01 1 Stomach model did not include a CM-RE uptake component. 2 No dissectible WAT was found before P12. 3 BAT, brown adipose tissue; CM-REs, chylomicron retinyl esters; P, postnatal day; RBP- ROH, retinol-binding protein-bound retinol; VA, vitamin A; WAT, white adipose tissue. 58

72 After supplementation, the fractional turnover of CM-derived ROH was significantly higher in the lungs, kidneys, liver (P < 01 for all) and brain (P < 1), with lungs showing the largest, ~60-fold difference, and significantly lower in the intestines (P < 01) and BAT (P < 1) (Table 7). The fractional turnover of RBP-derived ROH was higher in the carcass, lungs, kidneys (P < 01 for all), skin and liver (P < 1), with carcass showing the largest, ~30-fold difference, and significantly lower in the brain (P < 1). Table 7. Fractional turnover of CM- and RBP-derived ROH in organs in control and VAsupplemented neonatal rats dosed orally with [ 3 H]ROH on P4. Turnover 2 of CM-derived ROH (fraction of organ ROH transferred/d) Turnover 2 of RBP-derived ROH (fraction of organ ROH transferred/d) Organ L(I,J) ± SEM L(I,J) ± SEM Control VA P-value Control VA P-value Stomach 6.8 ± ± ± ± Intestines 0.3 ± 0.2 ± <01 ± ± 1.00 Liver 0.4 ± ± 0.1 < ± 0.3 ± <1 Lungs 0.6 ± ± 3.9 < ± 1.8 ± 0.2 <01 Kidneys 2.1 ± ± 3.6 < ± ± 0.7 <01 BAT 1.4 ± ± <1 1.6 ± ± WAT ± 0.1 ± Brain 0.6 ± ± 0.1 < ± ± 2.0 <1 Skin ± ± ± ± 2.2 <1 Carcass 33.4 ± ± ± ± 10.4 <01 1 No dissectible WAT was found before P12. 2 Turnover included ROH release to plasma and irreversible loss due to metabolism. 3 BAT, brown adipose tissue; CM-REs, chylomicron retinyl esters; P, postnatal day; RBP- ROH, retinol-binding protein-bound retinol; VA, vitamin A; WAT, white adipose tissue. 59

73 4.2.9 Percent uptake of retinol from plasma to organs Vitamin A supplementation redirected the flow of CM-REs away from the peripheral tissues and toward the liver. In the control group, the highest percent of plasma CM-REs was taken up by the skin, followed by the carcass and liver (Table 8). However, the skin and carcass also showed the highest fractional turnover of CM-derived ROH (Table 7). In the supplemented group, these proportions were reversed toward a greater CM-REs percent uptake into the liver, which also showed a greater retention CM-derived ROH compared to skin and carcass (Table 7). The highest percent of plasma RBP-ROH was taken up by the liver in the control group and the carcass in the supplemented group (Table 8). The highest percent of total ROH was taken up by the skin in the control group and the liver in the supplemented group. Table 8. Percent uptake of CM-REs, RBP-ROH, and total ROH from plasma to organs in control and VA-supplemented neonatal rats dosed orally with [ 3 H]ROH on P4. Organ CM-REs uptake (%) RBP-ROH uptake (%) Total ROH uptake (%) Control VA Control VA Control VA Stomach Intestines Liver Lungs Kidneys BAT WAT Brain Skin Carcass Stomach model did not include a CM-RE uptake component. 2 No dissectible WAT was found before P12. 3 BAT, brown adipose tissue; CM-REs, chylomicron retinyl esters; P, postnatal day; RBP-ROH, retinol-binding protein-bound retinol; VA, vitamin A; WAT, white adipose tissue. 60

74 Organ transfer rates After supplementation, the uptake rate (in nmol/d) of CM-REs was significantly higher in all the examined organs (P < 1 for liver; P < 5 for other), with the largest, ~500-fold difference observed in the lungs (Table 9). The uptake rate of RBP-ROH was higher in the carcass, liver, lungs, kidneys (P < 01 for all), and skin (P < 1), with carcass showing the largest, 50-fold difference. Brain was the only organ, in which the uptake rate of RBP-ROH was significantly lower after supplementation (P < 5). The uptake rate of total ROH was higher in all the examined organs (P < 01 for carcass; P < 1 for lungs, skin and kidneys; P < 5 for other), except the stomach and WAT, with the largest difference observed in the lung, liver (~60-fold) and carcass (~50-fold). After supplementation, the ROH turnover rate was significantly higher in all the examined organs (P < 01 for carcass, skin, liver and intestines; P < 1 for lungs, BAT and kidneys), except the brain, WAT and stomach, with the largest, ~90-fold difference observed in the carcass. 61

75 Table 9. Uptake rate of CM-REs, RBP-ROH and total ROH from plasma to organs and turnover rate of total ROH in organs in control and VA-supplemented neonatal rats dosed orally with [ 3 H]ROH on P4. Organ Uptake rate 1 of CM-REs (nmol/d) Uptake rate 1 of RBP-ROH (nmol/d) R(I,J) ± SEM R(I,J) ± SEM P-value Control VA Control VA P-value Stomach ± ± Intestines 0.3 ± ± 4.7 <5 1 ± 0 2 ± 0 5 Liver 0.7 ± ± 69.1 <1 3.0 ± ± 0.4 <01 Lungs 2 ± ± 3.6 <5 0.2 ± 1.8 ± 0.2 <01 Kidneys 0.2 ± ± 1.1 <5 1.3 ± ± 0.3 <01 BAT 0.1 ± 3.7 ± 1.3 <5 0.1 ± 0.1 ± 0.37 WAT ± 0 5 ± Brain 02± ± 0.3 <5 0.2 ± 0.1 ± <5 Skin 3.1 ± ± 24.6 <5 1.6 ± ± 4.6 <1 Carcass 0.6 ± ± 8.4 <5 2.3 ± ± 6.4 <01 Uptake rate 1 of total ROH (nmol/d) Turnover rate 1,2 of total ROH (nmol/d) Organ R(I,J) ± SEM R(I,J) ± SEM Control VA P-value Control VA P-value Stomach 3.3 ± ± ± ± Intestines 0.3 ± ± 4.7 <5 0.7 ± 1.6 ± <01 Liver 3.8 ± ± 69.1 <5 4.4 ± ± 13.6 <01 Lungs 0.2 ± 12.4 ± 3.6 <1 0.1 ± 1.6 ± 0.5 <1 Kidneys 1.5 ± ± 1.1 <1 2.8 ± ± 0.4 <1 BAT 0.2 ± 3.8 ± 1.3 <5 0.2 ± 0.5 ± 0.1 <1 WAT 2 ± 0 5 ± ± 0 0 ± Brain 0.2 ± 0.9 ± 0.3 <5 0.2 ± 0.2 ± 0.91 Skin 4.7 ± ± 25.1 <1 2.3 ± ± 4.1 <01 Carcass 2.9 ± ± 10.6 < ± ± 22.6 <01 1 Transfer rates were calculated as the product of total ROH mass measured by UPLC in compartment, from which the tracer was leaving (compartment J) and the corresponding fractional transfer coefficient [L(I,J)]. 2 Turnover rate included ROH release to plasma and irreversible loss due to metabolism. 3 Stomach model did not include a CM-RE uptake component. 4 No dissectible WAT was found before P12. 5 Turnover rate calculated using retinol mass measured on d 1 after dosing due to the presence of VA supplement in the digestive organs at earlier time points (Figure 4). 6 BAT, brown adipose tissue; CM-REs, chylomicron-associated retinyl esters; P, postnatal day; RBP-ROH, retinol-binding protein-bound retinol; VA, vitamin A; WAT, white adipose tissue. 62

76 4.3 Discussion and conclusions Despite the importance of VA for development, knowledge about its uptake and retention in rapidly developing neonatal tissues is limited. Here, we quantified the uptake of CM-REs, RBP-ROH and total ROH into organs in neonatal rats raised under VA-marginal conditions, without and after VA supplementation with 200 IU of retinyl acetate. The transfer of ROH from plasma to organs was estimated using model-based compartmental analysis in WinSAAM with the use of forcing function to uncouple the organs from the rest of the system and model them individually. Our findings demonstrated that VA supplementation redirected the flow of CM-REs away from the peripheral tissues (skin and carcass) and toward the liver for subsequent greater fractional release as RBP-ROH and re-delivery 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 after supplementation. 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. After supplementation, the fractional uptake of CM-REs was also significantly higher in the lungs and brain and significantly lower in BAT and intestines. However, unlike the peripheral tissues, BAT and intestines did not compensate with a higher fractional uptake of RBP-ROH. We have also shown that VA supplementation resulted in a much higher (>100-fold) recycling number of ROH between plasma and organs due to a lower fraction of plasma ROH metabolized irreversibly and a higher fraction returning to plasma after being taken up by organs. Retinol turnover was particularly high in the carcass, but no different from control 63

77 group in the brain or WAT, consistently with our findings in Aim 1. Finally, in both treatment groups, we have observed a gradual loss of tracer from the liver during the last 10 d of the study and a progressive accumulation of tracer in plasma and the extrahepatic tissues during the same period. Our finding that VA supplementation redirected the flow of CM-REs from peripheral tissues to the liver may have a two-fold explanation: (1) the capacity of peripheral tissues to take up a large amount of CM-REs may be limited and/or saturated by supplementation; (2) the capacity of the liver to take up a large amount of CM-REs may be relatively high compared to that of other organs. The first explanation (limited uptake of CM-REs by peripheral tissues) is consistent with the rapid loss of CM-derived ROH from nearly all extrahepatic organs and the substantially higher recycling number of RBP-ROH between plasma and organs in the VAsupplemented group compared to control group (541 vs. 5 times before irreversible disposal). The first explanation is also consistent with the relatively low LRAT expression (65) and ROH concentration (59, 66, 67) in the lungs, skin and adipose tissue during the neonatal period. Both the activity of LRAT in lungs and the uptake of CM-REs in the lungs and other extrahepatic tissues have been previously shown to be upregulated by retinoic acid (40, 68). Therefore, it is reasonable to postulate that, under VA-marginal conditions, the activity of LRAT in the extrahepatic tissues in neonates is low, which limits the ROH retention capacity of these tissues and causes a large release of ROH back to the circulation for later uptake into the liver. 64

78 Another possible mechanism behind the limited capacity of peripheral tissues to retain ROH may be a relatively high activity of Stimulated by retinoic acid 6 (STRA6), a transmembrane receptor for RBP and a mediator of ROH uptake (19). STRA6 acts as a bidirectional transporter, importing ROH into the cell when the intracellular level is low and exporting ROH when there is excess (69, 70). This function of STRA6 is particularly important during embryonic development to maintain retinoid homeostasis in developing tissues. Dietary VA excess during this period was found to upregulate STRA6 expression specifically in the developing embryo but not in the placenta suggesting that STRA6 serves a specific purpose of protecting the embryo from toxic effects of excessive maternal retinoids (71). Previous studies have also found that STRA6 expression was elevated in LRATdeficient mice, in which the intracellular ROH levels were high due to the inability to esterify ROH (19, 72). Therefore, the high expression of STRA6 during the neonatal period, particularly in tissues that continue to differentiate postnatally, such as the skin and lungs, may be a remnant from the gestational period and may protect the still immature organs from excessive VA intake by the mother or in the post-weaning diet. Of note is the fact that, in a systematic comparison of STRA6 activity in different organs, the highest level was found in the undifferentiated keratinocytes in the basal epidermis (73). In these cells, the activity of STRA6 was even higher than that in placenta and cells of the retinal pigment epithelium. Moreover, STRA6 activity in the skin was shown to be induced by VA and retinoic acid (74) and to contribute to VA-mediated epidermal differentiation (69, 75). In our study, the fractional uptake of RBP-ROH was particularly high in the skin and carcass. In the supplemented group, these tissues together acquired 89% 65

79 of plasma RBP-ROH. This finding may be related to the relatively high number of undifferentiated cells in the neonatal skin and carcass. The second explanation (enhanced CM-REs uptake into the liver) is consistent with the relative level of lipoprotein lipase (LPL) activity in different organs during development. LPL is responsible for the margination of chylomicrons at the interior side of tissue capillaries and the hydrolysis of their contents, which include retinyl esters in their four most abundant forms: retinyl palmitate, retinyl oleate, retinyl stearate, and retinyl linoleate (19). In a series of in vitro experiments, Blaner et al. (21) demonstrated that triglycerides are the preferred substrate for LPL, however, when the majority of triglycerides are hydrolyzed (~75%), retinyl ester hydrolysis proceeds. Moreover, several in vivo studies found that increased LPL expression is associated with increased CM-REs uptake into the heart, skeletal muscle and adipose tissue, but not the lungs or kidneys (19), and that a partial knockout of LPL in mice leads to a delayed RE clearance from plasma (76). These findings indicate that LPL expression and activity correlates with a tissue capacity to acquire CM- REs. LPL is usually considered an extrahepatic enzyme highly expressed in tissues that require a steady supply of fatty acids, such as heart, skeletal muscle, adipose tissue and mammary gland during lactation (77). However, in newborn rats, LPL is actively synthesized by hepatocytes at a rate 7-fold higher than in adult rats and its activity reaches peak at around P7 (78). 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 in the peripheral tissues, which are low in glycogen and fat stores during development (78). Presence of LPL in the neonatal liver may also enhance the uptake of RE by other 66

80 mechanisms, such as the receptor-mediated endocytosis, by tethering the incoming CM particles to the surface of capillary endothelium (71, 79). In our study, a higher fractional uptake of CM-REs after supplementation was found not only in the liver (~2-fold difference) but also in the lungs and brain (~3-fold difference). LPL activity in the lungs is high at birth, during the synthesis of phosphatidylcholine, the chief component of lung surfactant, the declines to about 50% of adult value, and then peaks again around P15 (80, 81), during the final stage of alveolar septation in rats (44). In the brain, LPL activity follows a different developmental pattern. It is low at birth but peaks at P4 (82), concurrently with the supplement delivery in our experiment, remains elevated until about P15 enabling rapid fatty acid deposition in the developing brain (83-85), and declines at weaning. Moreover, the cerebral LPL is especially active in the hippocampus, the learning center of the brain, where it has been previously shown to regulate the levels of vitamin E, another lipid-soluble vitamin (45). Interestingly, after birth, the activity of LPL in BAT is about twice as high as it is in the liver (81). However, unlike in other tissues, its activity in BAT remains constant between birth and P10 and declines sharply afterward (81). Similarly, LPL activity in WAT shows no change between the time of adipose tissue development (around P10) and 21 months (83). Moreover, a treatment of BAT and WAT with retinoic acid showed no effect on LPL expression in these tissues (86) and VA supplementation during suckling even reduced LPL expression in WAT at weaning (87). These findings suggest that the reason why VA supplementation had no effect on the fractional uptake of CM-REs into BAT was that LPL in this organ was already functioning at its maximum capacity. Whether VA or retinoic acid can upregulate LPL activity in other tissues is unknown at present. However, retinoic acid 67

81 is an agonist of PPARα (88), which in turn can induce the activity of LPL in the liver specifically (89), suggesting that the regulatory effect of VA on LPL in this organ is possible. As for the changes observed in organ tracer levels after d 14 of our study, we speculate that they may reflect the specific pattern of LPL and STRA6 expression over time in different parts of the body. During the second half of the sucking period (~P10-P21), LPL activity declines in the liver and increases in other tissues, such as the heart, skeletal muscle and kidneys (81, 83), reaching adult levels by weaning (90). Meanwhile, the expression of STRA6 decreases ~2-fold in the lungs (65) and other extrahepatic tissues (91). This timing overlaps with a progressive decline in the liver tracer observed after d 14 in our study and a simultaneous accumulation of tracer in plasma and the extrahepatic tissues. It is, therefore, possible that changes in the distribution of retinoid homeostatic genes, such as LPL and STRA6, during development correspond to changes in the distribution of VA stores in the neonatal body. In conclusion, our results in Aim 2 showed that a high dose of VA (200 IU) ingested on P4 by neonatal rats raised under VA-marginal conditions was acquired mainly by the liver and stored for about 2 weeks before being gradually transferred to other tissues. With respect to the two main molecular forms of ROH, the uptake of postprandial CM-REs was significantly higher in the supplemented group lungs, brain and liver, and the uptake of RBP- ROH was particularly high in the peripheral tissues, such as the skin, bone and muscle. However, the RBP-ROH acquired by the peripheral tissues was not retained efficiently, resulting in a repeated recycling of ROH between plasma and tissues. Given low ROH retention capacity of the extrahepatic tissues in neonates, an infrequent high-dose VA 68

82 supplementation may not be an optimal strategy to maintain the extrahepatic tissue ROH at a steady level. 69

83 Chapter 5. COMPARISON OF VITAMIN A VS. VARA EFFECTS ON RETINOL CONCENTRATIONS AND KINETICS IN PLASMA AND ORGANS IN NEONATAL RATS. 5.1 Methods Differences in study design between VA and VARA experiments The previous (VARA) and the current (VA) study of VA metabolism in neonatal rats followed the same protocol except for the following differences: (1) in the VARA study, pups were dosed with VA combined with one-tenth the amount of retinoic acid, whereas, in the VA study, pups received VA by itself; (2) the VARA study lasted for 14 days, whereas the VA study lasted for 24 d; (3) in addition to the VA study sample collection times, in the VARA study, samples were collected at 2.5, 6, 11 h and 6 d but not at 30 min after dosing; (3) in the VARA study, n = 3 pups were killed per group per sampling time, whereas n = 4 pups were killed per group per time in the VA study; (4) in the VARA study, the carcass contained muscles and bones as well as skin, BAT, WAT, and brain, tissues that were dissected in the VA study leaving only muscles and bones as the remaining carcass; (5) in the VARA study, ROH mass was measured only in plasma, liver and lungs, whereas all the collected organs were analyzed for ROH mass in the VA study; (6) in the VARA study, total ROH in plasma was not separated into CM-REs and RBP-ROH prior to ROH mass analysis, therefore precluding the calculation of transfer rates (nmol/d), which are based on the mass of plasma CM-REs and RBP-ROH. 70

84 5.1.2 Statistical analysis Prior to comparison, models of ROH kinetics from the VA study were truncated by removing data for d 18 and 24. The parameter L(5,7), which in the VA study represented the release of RBP-ROH from liver after d 14, was also removed and the remaining parameters were recalculated based on the truncated data. In order to match the carcass from the VARA, data for the skin, BAT, WAT, brain and the remaining carcass from the VA study were summed up and the SEMs were calculated based on the error propagation formula SEM 1+ +n = SEM SEM2 n. Finally, ROH mass data for the control group in the VA and the VARA study (ROH concentration and fraction of dose at each sampling time) were pooled together to create a single comparison standard for both studies. Fraction of dose (FOD) vs. time and the mean total ROH concentration in plasma, liver and lungs were graphed and the values for control, VA- and VARA-supplemented neonates were compared using a two-way ANOVA with Bonferroni correction for multiple comparisons. Values were expressed as mean ± SEM (concentration) or mean (fraction of dose). The fractional transfer coefficients [L(I,J)s] were estimated in WinSAAM and expressed as the fraction of tracer in compartment (J) transferred to compartment (I) per d. The percent uptake of ROH into organs was calculated using the fractional transfer coefficients of CM-REs and RBP-ROH as the basal values. All kinetic parameters were expressed as mean ± SEM and compared statistically using Student s t-test with a t-statistic calculated for each parameter pair and evaluated for significance using the table of critical values of Student s t-distribution. Differences with P < 5 were considered significant. 71

85 5.2 Results Comparison of vitamin A concentrations in plasma, liver and lungs During day 1 of the study, the mean total ROH concentration was significantly higher in the liver in the VA-supplemented pups compared to control (P < 01) and VARAsupplemented ones (P < 01) and in the VARA-supplemented pups compared to controls (P < 5) (Figure 22). Figure 22. Mean total (unesterified + esterified) ROH concentration in the liver, lungs, and plasma (μmol/l) in control, VA- and VARA-supplemented neonatal rats during d 1 after dosing on P4. Each bar represents the mean ± SEM of n = 42 (control), n = 24 (VA), or n = 18 (VARA) rats. P < 5: # VA/VARA vs. control, *VA vs. VARA. P, postnatal day; ROH, retinol; VA, vitamin A, VARA, VA combined with retinoic acid. 72

86 During the study, total ROH concentration in plasma was significantly higher in both the VA- and the VARA-supplemented group compared to controls at 1 and 4 h after dosing (VA vs. control: P < 1 for both; VA vs. VARA: P < 1 at 1 h and P < 5 at 4 h) (Figure 23A). In the liver, total ROH concentration was significantly higher in the VAsupplemented pups compared to control and VARA-supplemented pups at all sampling times from 4 h to 14 d (VA vs. control: P < 1 for all; VA vs. VARA: P < 5 at 8 d and P < 1 for all other), except d 1 after dosing when it was higher only from the control group (Figure 23B). In the VARA-supplemented neonates, liver ROH concentration was higher compared to control pups only at 1 and 2 d after dosing (P < 01 for both). In the lungs, total ROH concentration was higher in the VARA-supplemented pups compared to control and VA-supplemented ones at 8 and 24 h after dosing (P < 01 for both) and compared only to control pups from 4 to 24 h after dosing (P < 01 for all). In the VA-supplemented neonates, lung ROH concentration was higher compared to control and VARA-supplemented pups during the last 6 days of the study (VA vs. other: P < 5 at 8 and 11 d; P < 01 at 14 d) and compared only to control pups at all sampling times after 8 h, except 4 d after dosing (P < 5 at 1 d; P < 1 for all other) (Figure 23C). 73

87 74 Figure 23. Plasma (A), liver (B), and lung (C) total (unesterified + esterified) ROH concentration in control, VA- and VARAsupplemented neonatal rats from 0 to 14 d after dosing on P4. Insets show the first 24 h after dosing. Each point represents the mean ± SEM of n = 4 (VA study) or n = 3 (VARA study) rats. P < 5: *supplemented vs. control, # VA vs. other: ^VARA vs. other, VA vs. control. P, postnatal day; ROH, retinol; VA, vitamin A, VARA, VA combined with retinoic acid.

88 5.2.2 Comparison of the tracer response profiles in plasma and organs There were no significant differences in the tracer response observed in the digestive organs between the VA- and the VARA-supplemented pups (Figure 24). In the intestines, the dose appeared to peak higher and decline more gradually in the VA study, both in control and supplemented neonates, however, these differences did not reach significance (Figure 24C, D). Figure 24. Stomach (A) and intestine (B) fraction of ingested [ 3 H]ROH dose in control, VA- and VARA-supplemented neonatal rats from 0 to 14 d after dosing on P4. Each symbol represents the mean of n = 4 (VA study) or n = 3 (VARA study) rats. P, postnatal day; ROH, retinol; VA, vitamin A, VARA, VA combined with retinoic acid. 75

89 In plasma, tracer response was also similar in the two studies, both without and after supplementation (Figure 25). Figure 25. Plasma fraction of ingested [ 3 H]ROH dose in control, VA- and VARAsupplemented neonatal rats from 0 to 14 d after dosing on P4. Each symbol represents the mean of n = 4 (VA study) or n = 3 (VARA study) rats. P, postnatal day; ROH, retinol; VA, vitamin A, VARA, VA combined with retinoic acid. 76

90 In the liver, the VA-supplemented pups showed a significantly higher FOD compared to VARA-supplemented ones at 11 and 14 d after dosing (P < 1 for both) (Figure 26A, B). In the kidneys, FOD was also significantly higher at these time points in the VA- compared to VARA-supplemented pups (d 11: P < 1; d 14: P < 01) (Figure 26E, F). In the lungs, differences were observed between the control groups at 2 and 14 d after dosing (P < 1 for both) most likely due to procedural differences between studies, such as sampling of a different lung lobe prior to tissue analysis (Figure 26C, D). 77

91 Figure 26. Liver (A), lung (B), and kidney (C) fraction of ingested [ 3 H]ROH dose in control, VA- and VARA-supplemented neonatal rats from 0 to 14 d after dosing on P4. Each symbol represents the mean of n = 4 (VA study) or n = 3 (VARA study) rats. *P < 5. P, postnatal day; ROH, retinol; VA, vitamin A, VARA, VA combined with retinoic acid. 78

92 In the carcass, the VARA-supplemented pups showed a significantly higher FOD compared to VA-supplemented ones at 1 h after dosing (P < 1) (Figure 27). A significant difference was also observed between the control groups at 4 h after dosing (P < 01). Figure 27. Carcass fraction of ingested [ 3 H]ROH dose in control, VA- and VARAsupplemented neonatal rats from 0 to 14 d after dosing on P4. Each symbol represents the mean of n = 4 (VA study) or n = 3 (VARA study) rats. *P < 5. P, postnatal day; ROH, retinol; VA, vitamin A, VARA, VA combined with retinoic acid. 79

93 5.2.3 Comparison of the plasma kinetic parameters The statistical comparison of plasma kinetic parameters revealed that there was no difference between the VA and the VARA supplementation in terms of their effect on the fractional uptake of CM-REs or RBP-ROH from plasma to organs or the irreversible loss of ROH from organs (Table 10). However, the VARA supplementation resulted in a significantly greater fractional release of RBP-ROH from organs (P < 01) and a significantly lower recycling of RBP-derived ROH from organs back to plasma (P < 5). Table 10. Plasma fractional transfer coefficients in VA- and VARA-supplemented neonatal rats dosed orally with [ 3 H]ROH on P4. Parameter Parameter description L(I,J) ± SEM VA VARA P-value L(2,1) Transit of ROH through the digestive system 31.9 ± ± L(15,10) Uptake of CM-REs from plasma to organs ± ± L(5,4) Release of RBP-ROH from organs to plasma 4.0 ± ± 0.1 <01 L(6,5) Uptake of RBP-ROH from plasma to organs ± ± L(5,6) Recycling of RBP-ROH from organs back to plasma 1.2 ± ± <5 L(0,6) Irreversible loss of ROH from organs 0 ± 0 1 ± CM-REs, chylomicron retinyl esters; P, postnatal day; RBP-ROH, retinol-binding protein-bound retinol; VA, vitamin A; VARA, vitamin A combined with retinoic acid. 80

94 The VARA-supplemented pups also showed a higher fractional catabolic rate of plasma ROH and a higher transit time of ROH in plasma, therefore, a lower recycling number of ROH between plasma and organs compared to VA-supplemented pups (Table 11). Table 11. Plasma kinetic parameters in VA- and VARA-supplemented neonatal rats dosed orally with [ 3 H]ROH on P4. Parameter Parameter description VA VARA Plasma residence time Average time a molecule of retinol spends in plasma over multiple transits Plasma transit Average time a molecule of retinol spends time in plasma during a single transit Tissue transit Average time a molecule of retinol spends time in organs during a single transit Fractional Fraction of plasma retinol utilized catabolic rate irreversibly per day Recycling Average number of times a molecule of number retinol recycles through plasma before irreversible disposal 1 P, postnatal day; VA, vitamin A h h 6.0 min 8.4 min 20.4 h 29 h

95 5.2.4 Comparison of the organ kinetic parameters The fractional uptake of CM-REs into the intestines, lungs and carcass was significantly higher in the VARA-supplemented pups, whereas the uptake into the liver and kidneys was higher in the VA-supplemented ones (P < 01 for all) (Table 12). The fractional uptake of RBP-ROH into the intestines and liver was significantly higher in the VARA-supplemented pups (P < 01 for both), whereas the uptake into the lungs, kidneys and carcass was higher in the VA-supplemented ones (P < 01 for all). Table 12. Fractional uptake of CM-REs and RBP-ROH from plasma to organs in VAand VARA-supplemented neonatal rats dosed orally with [ 3 H]ROH on P4. Uptake of CM-REs Uptake of RBP-ROH (fraction of plasma REs transferred/d) (fraction of plasma ROH transferred/d) Organ L(I,J) ± SEM L(I,J) ± SEM P-value VA VARA VA VARA P-value Stomach ± ± Intestines 19.1 ± ± 14.8 <01 4 ± ± 0.3 <01 Liver ± ± 3.7 < ± ± 1.0 <01 Lungs 15.0 ± ± 0.3 < ± ± <01 Kidneys 4.9 ± ± 0.1 < ± ± 0.1 <01 Carcass ± ± 6.3 < ± ± 0.1 <01 1 Stomach model did not include a CM-RE uptake component. 2 CM-REs, chylomicron retinyl esters; P, postnatal day; RBP-ROH, retinol-binding protein-bound retinol; VA, vitamin A; VARA, vitamin A combined with retinoic acid. 82

96 The fractional turnover of both the CM- and the RBP-derived ROH was significantly higher in the intestines in the VARA-supplemented group (Table 13). In the liver, the CMderived ROH turnover was higher in the VA-supplemented group, whereas the RBP-derived ROH turnover was higher in the VARA-supplemented group. The fractional turnover of both the CM- and the RBP-derived ROH in all other organs was significantly greater in the VAsupplemented group (P < 01 for all). Table 13. Fractional turnover of CM- and RBP-derived ROH from organs in VA- and VARA-supplemented neonatal rats dosed orally with [ 3 H]ROH on P4. Turnover of CM-derived ROH (fraction of organ ROH transferred/d) Turnover of RBP-derived ROH (fraction of organ ROH transferred/d) Organ L(I,J) ± SEM L(I,J) ± SEM VA VARA P-value VA VARA P-value Stomach 6.2 ± ± ± ± Intestines 0.2 ± 7.9 ± 0.3 <01 ± 0.7 ± <01 Liver 1.4 ± ± < ± 0.4 ± <01 Lungs 37.4 ± ± < ± ± <01 Kidneys 21.2 ± ± < ± ± 0.1 <01 Carcass 88.7 ± ± 0.1 < ± ± <01 1 CM-REs, chylomicron retinyl esters; P, postnatal day; RBP-ROH, retinol-binding protein-bound retinol; VA, vitamin A; VARA, vitamin A combined with retinoic acid. 83

97 5.2.5 Comparison of the percent uptake of retinol from plasma to organs The percent uptake of CM-REs was highest in the liver in the VA-supplemented pups and in the carcass in the VARA-treated ones (Table 14). The opposite was found for the RBP-ROH. The percent uptake of RBP-ROH was highest in the carcass in the VA group and in the liver in the VARA group. Table 14. Percent uptake of CM-REs and RBP-ROH from plasma to organs in VA- and VARA-supplemented neonatal rats dosed orally with [ 3 H]ROH on P4. Organ CM-REs uptake (%) RBP-ROH uptake (%) VA VARA VA VARA Stomach Intestines Liver Lungs Kidneys Carcass Stomach model did not include a CM-RE uptake component. 2 CM-REs, chylomicron retinyl esters; P, postnatal day; RBP-ROH, retinol-binding protein-bound retinol; VA, vitamin A; VARA, vitamin A combined with retinoic acid. 84

98 5.3 Discussion and conclusions Previous studies have shown that an oral supplementation of neonatal rats with VA and a small proportion (1:10 ratio) of retinoic acid (VARA) increased the uptake of VA into the extrahepatic tissues. However, in randomized controlled trials of VA supplementation in infants, the supplement consists of VA alone. In order to determine whether VARA supplementation offers additional benefits in terms of improved VA tissue uptake and retention, in Aim 3, we have compared these two forms of supplementation in terms of their effect on organ ROH concentrations and the fractional transfer of ROH between plasma and organs in neonatal rats raised under VA-marginal conditions. Our results demonstrated that the recycling number of ROH between plasma and organs was higher in the VA- vs. VARA-supplemented neonates due to a lower fractional catabolic rate of plasma ROH in the VA-treated pups. In particular, VA supplementation resulted in a much higher fractional turnover of both the CM- and the RBP-derived ROH in the kidneys, lungs and carcass, but not in the liver and intestines, where it was higher in the VARA-supplemented pups. We have also found that the fractional uptake of CM-REs into the intestines, carcass and lungs was significantly higher in the VARA-supplemented pups, whereas the uptake into the liver and kidneys was higher in the VA-treated ones, as was their liver ROH concentration throughout the study. Finally, in the liver, lungs and carcass, in both the VA- and the VARA-supplemented pups, there was a reciprocal relationship between the uptake of the two forms of ROH: a significantly higher fractional uptake of CM-REs in the VA vs. VARA group coincided with a significantly lower fractional uptake of RBP-ROH and vice versa. This relationship was present in all the examined organs except the kidneys and intestines. 85

99 Our finding that the recycling number of ROH between plasma and organs was lower after supplementation with VARA may be attributed to the synergistic effect of VA and retinoic acid on the activity of LRAT, a phenomenon previously demonstrated and replicated in both the neonatal and the adult rats (61, 92-94). The upregulation of LRAT by VARA is particularly prominent in the neonatal lungs, liver and kidneys (65), however, given presence of this enzyme in other organs, such as intestines, skin, adipose tissue and, to a lower extent, skeletal muscle (95, 96), it is possible that this effect occurs in other tissues. The primary role of LRAT is to esterify ROH for long-term storage. Therefore, its induction by VARA may lead to a greater tissue retention and lower recycling number of ROH, which would explain our findings in the VARA- vs. VA-treated animals. Regarding the more potent effect of VARA vs. VA on the uptake of CM-REs into the lungs, intestines and carcass but not into the liver, we propose the following mechanistic explanation. First, LRAT activity in the lungs may be more responsive to VARA than its activity in the liver. This is consistent with the previous finding that retinoic acid treatment increased LRAT activity in both the lungs and the liver but to a different extent: the increase in the liver was less than 2-fold, whereas the increase in the lungs was 6- to 9-fold (68). The above speculation is also consistent with results of a study that compared the residence time of ROH in various organs with or without retinoic acid supplementation. This study found the strongest effect of retinoic acid on ROH residence in the lungs, which showed a 75-fold higher value after supplementation compared to 14-fold difference in the liver (97). The effect of VARA in the lungs, intestines and carcass may be further enhanced by the activity of STRA6, a cell membrane transporter for RBP-ROH. STRA6 is highly expressed in all three organs, especially during development (65, 73, 98), but is absent from 86

100 the liver (69). Moreover, it can cooperate with LRAT in the formation of REs (19), and its expression in the neonatal lungs has been shown to increase in response to oral supplementation with VARA (65, 98). The differential effect of VARA vs. VA on the uptake of CM-REs into the lungs and liver may also result from the induction of CYP26A1 and CYP26B1 in the liver and lungs, respectively. Both the CYP26A1 and the CYP26B1 metabolize retinoic acid irreversibly converting it to its inactive metabolites (99). However, the CYP26A1 resides mainly in the liver and is highly responsive to retinoic acid. In fact, it shows the largest fold increase in expression after retinoic acid treatment compared to other VA-metabolizing enzymes (63, 99). In contrast, the CYP26B1 is more highly expressed in the lungs compared to liver and is less responsive to retinoic acid (98). Given this difference in tissue distribution and retinoic acid sensitivity of the two CYP26 variants, it is possible that a large dose of VA combined with retinoic acid induces its own catabolism to a greater extent in the liver than in the lungs resulting in a lower accumulation of hepatic ROH. This speculation concurs with our finding of the higher liver ROH concentration in the VA- vs. VARA-supplemented pups and the higher fractional turnover of RBP-derived ROH in the liver vs. lungs in the VARA-treated pups. With respect to the higher uptake and concentration of ROH in the kidneys in the VA-supplemented neonates, we speculate that it may be correlated with the greater recycling of ROH between plasma and organs observed in this group. Although the liver is the main site of RBP synthesis, approximately 50% of the circulating ROH is sequestered in the kidneys, filtered out into urine, reabsorbed and released back into the circulation after binding to RBP synthesized in the cells of the proximal convoluted tubules (23). Moreover, 87

101 kidneys have a considerable storage capacity for REs, similarly as WAT, even in the LRATdeficient animal model (96). Nevertheless, LRAT expression in the kidneys is relatively lower than in the liver or lungs (95), which may explain the significantly higher turnover of ROH observed in the kidneys of the VA- vs. VARA-supplemented neonates. Lastly, the reciprocal relationship between the CM-RE and the RBP-ROH uptake into organs may be the consequence of a highly regulated activity of several enzymes, which respond in an organ-specific manner and ensure that a relatively high uptake of CM-REs is followed by a relatively low uptake of RBP-ROH and vice versa. The absence of this relationship in the intestines and kidneys may be related to the constitutive high expression of LRAT in the intestines (95, 100, 101) and the narrow range of its expression in the kidneys (65). In conclusion, in Aim 3, we found that, in neonatal rats raised under VA-marginal conditions, VARA supplementation administered on P4 increased the fractional uptake of CM-REs into the intestines, lungs and carcass to a greater extent than did supplementation with VA. VARA supplementation also attenuated the frequent recycling of ROH between plasma and organs observed in the VA-supplemented animals by decreasing the turnover of ROH in the kidneys, lungs and carcass. However, the supplementation with VA resulted in a higher concentration of ROH in the liver during the entire study as well as in the lungs during the last 6 days of the study. Given the improved ROH retention in several extrahepatic organs after oral supplementation with VARA, a small amount of retinoic acid combined with VA may serve as a targeted treatment of low VA levels in organs that develop in late gestation and postnatally, such as the lungs and intestines. 88

102 Chapter 6. SUMMARY AND FUTURE DIRECTIONS 6.1 Summary In the present study, we have determined the distribution of ROH in the body and the transfer of ROH between plasma and organs in neonatal rats raised under VA-marginal conditions, without and after supplementation on P4 with VA in the amount equivalent to that which is given to newborns as part of randomized controlled trials conducted in lowincome countries. Our results demonstrated that most of the VA present in the body of a neonatal rat accumulated in the liver, irrespective of the treatment, even when the liver ROH concentration was deficient, as was found in control animals. Moreover, irrespective of the treatment, both the mean ROH mass and the mean tracer level in the liver remained considerably (>10-fold) higher than in other organs. We have also found that the ingestion of a single large dose of VA (200 IU) on P4 resulted in a transient increase in ROH concentrations in the extrahepatic organs most likely due to their limited ROH storage capacity. This was evidenced by the high loss of tracer from nearly all non-hepatic organs, a finding which may be explained by a relatively low expression of LRAT and high expression of STRA6 in these tissues during the neonatal period. The kinetic analysis of tracer data obtained from our experiment further revealed that the uptake of postprandial CM-REs after supplementation was significantly higher in the liver, lungs and brain, possibly due to a relatively high expression of LPL in these organs during development. Moreover, the RBP-ROH released from organs, such as the liver and kidneys, after supplementation was acquired primarily by the peripheral tissues, such as the 89

103 skin, bone and muscle, but the increased uptake was not matched by an increased storage capacity resulting in a higher turnover rate of ROH and a substantially higher ROH recycling number in the VA-supplemented compared to control neonates. The only two organs in which ROH turnover was not higher after supplementation were the WAT and brain suggesting a greater ROH retention capacity of these organs. Retinol stores in the liver lasted for about 2 weeks after supplementation and were gradually transferred to other organs afterward, as evidenced by a decrease in the liver tracer level after d 14 of the study and a simultaneous increase in the tracer level in plasma and almost all non-hepatic organs. Finally, the comparison of results from the VA and the VARA study demonstrated that the supplementation with VARA was more potent at increasing the fractional uptake of CM-REs into the intestines, lungs and carcass and that the VARA-supplemented pups showed a lower fractional turnover of ROH in the lungs, kidneys and carcass and a less frequent ROH recycling between plasma and tissues compared to the VA-supplemented pups. Nevertheless, the supplementation with VA resulted in a greater uptake and accumulation of ROH in the liver, which may become available to other tissues during the course of development. Given the transient effect of VA supplementation on tissue ROH contents in neonates, a more frequent, smaller-dose supplementation, along with other nutrition interventions, may be necessary to prevent ROH levels in the extrahepatic tissues from falling to low values. 6.2 Future directions Our study has several limitations that can inform future research in this area. First, the plasma levels of labeled REs and RBP-retinol were not assessed directly. The kinetics of 90

104 CM- and RBP-derived ROH in organs were determined based on the assumption that the observed total labeled ROH in plasma is the sum of the labeled REs and the labeled RBP- ROH. In order to validate our findings, we conducted a follow-up experiment focused on the early post-absorptive phase (first 30 h after supplementation), in which the total labeled ROH in plasma was separated into the labeled REs and RBP-ROH prior to measurement. The tracer response profiles of CM- and RBP-derived ROH generated in this analysis were similar to those obtained from the current experiment (Appendix B), which lends further confidence in the accuracy of our model. However, the follow-up experiment was performed only on the samples of brain tissue. Future studies could measure the labeled REs and RBP- ROH in other organs to determine if there are any discrepancies between the model-predicted and the observed values. Second, the analysis of whole bodies of pups killed at 1 h after dosing showed that, on average, 65% of the ingested tracer was recovered from the whole body. The remaining 35% was either lost during sample processing or unable to be recovered. In order to assess the proportion of tracer lost during processing, we have summed up the tracer recovered from all the organs (including the remaining carcass) collected at 1 h after dosing and compared it to the tracer recovered from the whole-body of pups killed at the same time point. The calculation revealed that the recovery from all organs summed together equaled 53% vs. 65% from the whole-body analysis. We have also compared the uptake rate of total ROH derived from the plasma model to that from the individual organs summed together and found a close match between these values (control group plasma vs. summed organs: 13.1 ± 4.4 nmol/d vs nmol/d; VA group plasma vs. summed organs: ± nmol/d vs nmol/d). Based on these findings, we conclude that the loss of tracer due 91

105 processing was minimal and that most of the tracer was lost due to the conversion of labeled ROH to its non-extractable metabolites during the early post-prandial phase. In order to address this problem, future studies should measure other substrates of ROH metabolism to identify them and determine their role in the neonatal VA metabolism. Third, in our study we were not able to elucidate the specific mechanisms responsible for the uptake and storage of ROH in neonatal tissues. Given the organ-specific and timedependent effect of VA supplementation on ROH stores in a neonatal body, future studies could delve deeper into these mechanisms and analyze the expression of retinoid homeostatic genes, such as STRA6, LPL, LRAT and CYP26, in the previously-unexplored neonatal organs, such as the skin, brain, adipose tissue and muscle, over the course of development. Finally, our study demonstrated that a single high-dose VA supplementation was not optimal for establishing adequate extrahepatic stores of ROH and, instead, resulted in the repeated recycling of ROH between plasma and tissues in neonatal rats. Based on these findings, future research may determine if a smaller dose of VA delivered more frequently is more effective in maintaining the extrahepatic ROH stores and able to improve some of the negative health outcomes observed in newborns subject to VA deficiency. 92

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115 APPENDIX A Pilot test of extraction efficiency of the lipid-rich tissues Organ KOH concentration Number of extractions Extraction efficiency (%) (%) Stomach Intestines Brain Skin WAT Carcass KOH, potassium hydroxide; WAT, white adipose tissue. 102

116 APPENDIX B Comparison of results from the current and the follow-up experiment Fraction of ingested [ 3 H]ROH dose as CM- and RBP-derived ROH and their sum (total ROH) in the brain in VA-supplemented rats from 0 to 30 h after dosing on P4. Panel A shows the tracer response profiles of CM- and RBP-derived ROH estimated by the model based on a direct measurement of labeled RE and RBP-ROH in plasma. Panel B shows the corresponding profiles estimated by the model based on a function, which defined total ROH in plasma as the sum of labeled RE and RBP-ROH. Each symbol represents the mean of n = 2-4 rats. CM-derived ROH, chylomicron derived retinol; P, postnatal day; RBP-derived ROH, retinol-binding proteinderived retinol; VA, vitamin A. 103