A modified relative dose-response assay employing 3,4-didehydroretinol (vitamin A2) in rats

3,4-Didehydroretinol (vitamin A2, DR, dehydroretinol), a naturally occurring analogue of retinol (vitamin A1, R), is active in vision, growth and cellular differentiation but is converted to retinol in very small amounts, if at all. When vitamin A-depleted rats were given 500 micrograms of R acetate, a naturally occurring mixture of 480 micrograms DR ester and 20 micrograms R ester or 500 micrograms DR acetate orally in corn oil, serum levels of all administered retinoids peaked between 3.5 and 5 h and then declined. When an oral dose of 600 micrograms DR/kg body wt was administered to rats with various liver reserves of vitamin A, the serum ratio of DR to R at 3.5 h was inversely related to the liver reserves of vitamin A below approximately 2 micrograms/g liver. Because the administration of DR does not affect serum R values, a single blood sample taken at 3.5 h might provide information analogous to that obtained from two blood samples in the conventional relative dose-response method.     

Use of 3,4-didehydroretinol to assess vitamin A status in rats

The acetate and palmitate esters of 3,4-didehydroretinol (DR; vitamin A2 alcohol) have been synthesized and characterized. When administered orally in corn oil to female rats, DR was present in the serum as the alcohol, but primarily as esters in the liver. As total stores of retinol (R; vitamin A1 alcohol) decrease, the ratio of DR to R in the serum markedly increases. The ratio of DR to R in serum was greater than 0.27 at liver vitamin A1 palmitate values of less than 3 micrograms/g, 0.05-0.14 at 3-19 micrograms/g, and less than 0.04 at greater than or equal to 20 micrograms/g. Thus, the ratio of DR to R in the serum at a suitable interval after the administration of dehydroretinyl acetate may aid in assessing marginal vitamin A status. DR was not demonstrably converted to R in these studies.     

The reproducibility of the modified relative dose response (MRDR) assay in healthy individuals over time and its comparison with conjunctival impression cytology (CIC).

The modified relative dose response (MRDR) assay is a minimally invasive method of detecting marginal vitamin A status. In the present study, the MRDR assay was performed four times in six well-nourished adults and in one male five-year-old child over a seven-month period. In all instances, assay ratios of dehydroretinol (DR) to retinol (R) were less than 0.03, the tentative cutoff value of 'normal' for the MRDR assay in humans. The mean ratio for all time points for all individuals was 0.015 +/- 0.005, with a mean coefficient of variation of 27 +/- 13%. In a related experiment, both MRDR and conjunctival impression cytology (CIC) tests were conducted on nine adults and the same male child. All DR/R ratios were less than or equal to 0.021, and all CIC specimens were normal. Thus, the DR/R ratio in well-nourished individuals, although showing some variation, does not oscillate between abnormal and normal responses. Furthermore, the DR/R ratios and CIC patterns were fully concordant.     

Concentration of vitamin A in the liver of foetuses and infants dying of various causes in Brasilia, Brazil

The vitamin A concentration in liver samples taken at autopsy from the central portion of the right lobe of 57 infants 0-1 year old was determined by a dual spectrophotometric and colorimetric assay. Death was due to respiratory disease (30%), complications of premature birth (16%), infections (14%), hemorrhage (14%), pneumonia (10%), cerebral edema (7%), and miscellaneous causes (9%). Gross malnutrition was noted in only 2 of these children. The median vitamin A concentration was 61 micrograms of retinol/g liver, with a range of 6-293 micrograms/g. The percent distribution of liver vitamin A levels in micrograms/g was: less than 5 (0%); 5-10 (7%); 10-20 (5%); 20-40 (16%); 40-80 (42%); 80-120 (14%); greater than 120 (16%). The mean liver level in 9 stillborn full-term infants (60 micrograms/g) was markedly lower than in 7 stillborn premature infants (125 micrograms/g). The median value for 22 infants from indigent families (54 micrograms/g) was lower than that of 35 infants from non-indigent families (65 micrograms/g). By applying the criteria that liver reserves of vitamin A less than or equal to 5 micrograms retinol/g of liver indicate a high risk of vitamin A deficiency and those less than 20 micrograms retinol/g of liver denote an inadequate reserve, no infant was at high risk but 12% had insufficient reserves.     

Effects of acute starvation on vitamin A status in rats.

Maintenance of vitamin A stores in the body is dependent on a number of basic metabolic processes. These processes, such as protein and carbohydrate metabolism, are disrupted in acute starvation, and, as a result, alterations in vitamin A status may result. We investigated this possibility in 8-week-old Sprague-Dawley male rats. The rats were starved for 24, 48, and 72 hr but had free access to water. At 24 hours of starvation, the plasma retinol concentration was depressed, but not significantly so. After 48 and 72 hours of starvation, however, the plasma retinol concentration decreased to less than half of the control values (61 +/? 4 vs 124 +/? 12 nmol/dl at 72 hours, mean +/? SEM, (p less than 0.005). The hepatic retinoid (retinyl esters + retinol) concentration (nmol/g liver) was increased at 24 and 48 hours of starvation compared to controls (p less than 0.05), and by 72 hours the concentration was 56% greater in starved rats than in fed controls (p less than 0.001). The total hepatic retinoid content (mumol/total liver) was decreased moderately at all periods of starvation compared to controls (p less than 0.05). In both starved and fed animals, the total hepatic content per 100 g body weight, a measure of total vitamin A reserves, was statistically the same. These results demonstrate that acute starvation in rats alters the vitamin A equilibrium between the plasma and hepatic stores without affecting the overall vitamin A reserves.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of acute starvation on vitamin A status in rats

Maintenance of vitamin A stores in the body is dependent on a number of basic metabolic processes. These processes, such as protein and carbohydrate metabolism, are disrupted in acute starvation, and, as a result, alterations in vitamin A status may result. We investigated this possibility in 8-week-old Sprague-Dawley male rats. The rats were starved for 24, 48, and 72 hr but had free access to water. At 24 hours of starvation, the plasma retinol concentration was depressed, but not significantly so. After 48 and 72 hours of starvation, however, the plasma retinol concentration decreased to less than half of the control values (61 +/- 4 vs 124 +/- 12 nmol/dl at 72 hours, mean +/- SEM, (p less than 0.005). The hepatic retinoid (retinyl esters + retinol) concentration (nmol/g liver) was increased at 24 and 48 hours of starvation compared to controls (p less than 0.05), and by 72 hours the concentration was 56% greater in starved rats than in fed controls (p less than 0.001). The total hepatic retinoid content (mumol/total liver) was decreased moderately at all periods of starvation compared to controls (p less than 0.05). In both starved and fed animals, the total hepatic content per 100 g body weight, a measure of total vitamin A reserves, was statistically the same. These results demonstrate that acute starvation in rats alters the vitamin A equilibrium between the plasma and hepatic stores without affecting the overall vitamin A reserves.(ABSTRACT TRUNCATED AT 250 WORDS)     

Factors influencing the level and interanimal variability of retinyl ester hydrolase activity in rat liver

Retinyl ester hydrolase activity was studied in 25 7-wk-old rats that had been previously fed no vitamin A for 1 wk and then were fed 0, 5, 24, 60 or 240 micrograms retinol/d for 2 wk. This treatment produced rats with vitamin A reserves from depleted to normal (0.03 to 184 micrograms/g) and serum retinol concentrations of 4-67 micrograms/dl. Hydrolase activity in liver homogenates was optimal when assayed with 275 mM CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate), 2 mg/ml Triton X-100 and 5 mM retinyl palmitate at pH 7.0. By use of these concentrations and conditions, retinyl ester hydrolase activity displayed relatively low interanimal variability (fivefold). Furthermore, activity was independent of serum or liver vitamin A concentrations. When CHAPS in the assay was replaced with equimolar sodium cholate, or with reduced concentrations of CHAPS and retinyl palmitate, retinyl ester hydrolase activity was more variable, i.e., 37- and 23-fold, respectively. Furthermore, in the latter case, hydrolase activity was threefold higher in rats with liver vitamin A reserves less than 10 micrograms/g. Thus, the type and concentration of bile salt used to assay retinyl ester hydrolase affect its interanimal variability. Furthermore, hydrolase activity measured in reduced concentrations of substrate and detergent is elevated in rats with low liver reserves of vitamin A.     

Metabolism, plasma transport and biliary excretion of radioactive vitamin A and its metabolites as a function of liver reserves of vitamin A in the rat

Groups of 7-12 weanling Sprague-Dawley male rats were fed graded daily doses of vitamin A (5-176 micrograms retinol) for 7 or 12 weeks. Final mean liver concentrations of vitamin A, which ranged from 0.4 to 331 micrograms retinol per gram, depended both on the daily dose given and on the length of the feeding period. The mean serum retinol concentration was 24 micrograms/dl at the lowest liver vitamin A concentration, approached a plateau of 40 micrograms/dl at a liver concentration of 5-10 micrograms/g, and then very slowly increased with higher levels of vitamin A in the liver. Seven days after the oral administration of a standard dose (4.6 microCi) of 11,12-[3H2]retinyl acetate, during which period rats were fed the customary vitamin A-containing diet, bile was collected via bile duct cannulae for 1-4 hours, and then the livers and serum were extracted and analyzed. The key relationships defined were: 1) that the mean ratio of specific activities of retinol in serum to that in liver was 0.65 +/- 0.05 (SEM) (range: 0.46-0.81) at daily retinol intakes of 8-176 micrograms/day, 2) that the ratio did not vary systematically with vitamin A intake or liver reserves and 3) that the mean excretion rate of vitamin A metabolites in the bile was invariant at 0.28 microgram retinol metabolites per milliliter of bile up to a liver vitamin A concentration of 32 micrograms retinol per gram, but then increased rapidly by eightfold to a maximal rate of 2.4 micrograms retinol metabolites per milliliter of bile at a liver vitamin A value of 140 micrograms retinol per gram.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vitamin A intake and status influence retinol balance, utilization and dynamics in rats

To study effects of vitamin A status on retinol dynamics, male rats were fed purified diets varying in vitamin A concentration. Group 1 rats had marginal liver vitamin A levels (approximately 500 nmol) and were in a slight positive vitamin A balance; Group 2 had similar liver levels but were in a slight negative balance; Group 3 had lower liver levels (approximately 370 nmol) and were in a slight negative balance; Group 4 had depleted liver reserves (<10 nmol) and were in vitamin A balance. [3H]Retinol-labeled plasma was injected intravenously, and serial plasma samples were collected for 41 d while rats (six per group) consumed approximately 50 nmol retinol/d (Group 1) or -25 nmol/d (Groups 2-4). Plasma retinol was normal in Groups 1-3 (1.9-2.0 micromol/L) and lower in Group 4 (0.96 micromol/L). Plasma tracer data were fit to a three-compartment model. The central plasma retinol compartment (transit time, 1.5-1.7 h) exchanged with a fast turning-over extravascular vitamin A pool (transit time, 3-4.5 h; -40 nmol) and with a larger, slow turning-over extravascular pool (transit time, 5.5-10 d) that was the site of irreversible utilization of vitamin A. Irreversible utilization was 36 nmol/d (Group 1), 29 nmol/d (Groups 2 and 3) and 20 nmol/d (Group 4). The data indicate that in rats with low or marginal vitamin A status, vitamin A intake, vitamin A reserves and plasma retinol concentration all influence vitamin A utilization and other aspects of retinol dynamics.     

Relative amount and ester composition of vitamin A in rat hepatocytes as a function of the method of cell preparation and of total liver stores

The aim of this study was to determine the cellular distribution of vitamin A in the liver of adult, well-nourished Sprague-Dawley rats with different stores of this vitamin. Three groups of three rats each were fed AIN-76 diet. Each group received an oral supplement of either 0, 2, or 12 mg of retinol. Retinol and retinyl esters were measured in the whole liver, in hepatocytes isolated either by gravity sedimentation or by centrifugal elutriation, and in the supernatant solution recovered after hepatocyte sedimentation. Dietary supplementation with vitamin A increased the liver stores from 138 to 390 micrograms/g. A mean of 5% of the liver vitamin A was found in hepatocytes isolated by centrifugal elutriation. This percentage tended to decrease as liver vitamin A increased. In contrast, a mean of 38% of total liver vitamin A was found in hepatocytes isolated by gravity sedimentation over a threefold range of liver reserves. Whereas the retinol concentration in hepatocytes remained constant, the percentage of total vitamin A as retinyl esters, which accounted for more than 96% of the total vitamin A, increased as liver stores increased. The retinyl ester composition was similar in all three groups, major esters being palmitate (76-82%), stearate (9-12%), oleate (5-7%), and linoleate (3-4%). Over a threefold range of liver vitamin A concentrations in well-nourished rats, therefore, we estimate that only 10-20% of the total vitamin A in liver is present in the parenchymal cells.     

Liver concentrations of vitamin A and carotenoids, as a function of age and other parameters, of American children who died of various causes

To assess the age-dependent vitamin A status of children, liver samples taken at autopsy from 170 American children 0-15 yr of age were analyzed for vitamin A and carotenoids. The median liver vitamin A concentration at birth was low (11 micrograms retinol/g), remained constant to 3 mo, rapidly increased to 4 yr (130 micrograms/g) and then remained constant into adolescence. In contrast the vitamin A status of premature infants deteriorated after birth. Of infants less than 3 mo, approximately one-fourth and two-thirds showed liver vitamin A concentrations less than or equal to 5 micrograms retinol/g and less than or equal to 20 micrograms/g, respectively. On the other hand, essentially all infants greater than or equal to 6 months showed an adequate vitamin A status, defined as liver stores greater than 20 micrograms retinol/g liver. Liver carotenoid concentrations did not meaningfully correlate with age or with vitamin A concentrations. Parameters that did not significantly affect the vitamin A concentration were: 1) height and weight in infants less than 1 mo, except in the highest weight-height groups, 2) sex, although values of females were slightly higher than males, and 3) causes of death.     

Retinyl ester (vitamin A ester) and carotenoid composition in human liver

Surgical liver biopsy samples from seven diseased and five healthy human subjects, 3-33 years of age, were analyzed by reversed-phase high-pressure liquid chromatography for retinol (vitamin A alcohol), retinyl esters (vitamin A esters), and carotenoids. Total liver vitamin A values ranged from 7.8 to 2860 nmol/g liver (2.2 to 817 micrograms/g). As a percentage, liver retinol decreased with increasing liver reserves of vitamin A. Retinyl palmitate was the predominant vitamin A ester (57 to 83 mole%) in all samples, with retinyl stearate (5.5 to 11.4%), oleate (4.9 to 17.2%), and myristate plus palmitoleate (pair not resolved; 3.3 to 11.9%) next most common. Lesser amounts of retinyl linoleate, linolenate, and arachidonate were found. Normal livers had significant amounts of several carotenoids: lutein (0.2 to 16.2 nmol/g), lycopene (10.2 to 55.1 nmol/g), alpha-carotene (3.0 to 7.3 nmol/g), and beta carotene (5.8 to 25 nmol/g). Total carotenoid values ranged from 26.5 to 67 nmol/g in normal liver samples. There was no correlation between liver vitamin A and individual or total carotenoids in normal livers.     

Modification of vitamin A metabolism in rats fed a copper-deficient diet

The liver is the main storage site of vitamin A and copper. Inverse relationships between copper and vitamin A liver concentrations have been suggested. We have investigated the consequences of a copper-deficient diet on liver and blood vitamin A storage in Wistar rats. Animals were fed either a copper-deficient diet for 45 days from weaning, or an identical diet containing adequate amounts of copper. Concentrations of vitamin A were determined by isocratic high performance liquid chromatography using UV detection. We have observed in the liver of the rats fed a copper-deficient diet a significantly higher mean level of retinyl esters (148 +/- 37 micrograms/g of liver) and retinol (3.3 +/- 1.4 micrograms/g of liver) compared to the mean concentration of the retinyl esters (53 +/- 8.5 micrograms/g of liver) (p less than 0.01) and retinol (1.4 +/- 0.5 micrograms/g of liver) (p less than 0.01) in controls. Opposite results were observed in the serum of the group fed a copper-deficient diet as these rats had a significantly lower level of retinol (22 +/- 4 micrograms/100 ml) compared to the mean concentration in the controls (64 +/- 20 micrograms/100 ml) (p less than 0.01). These findings suggest that a copper-deficient diet may cause defective transport of vitamin A from liver to blood. This experimental model may be useful to further investigate unusual liver vitamin A and copper concentrations observed in children during various hepatobiliary diseases.     

Vitamin A relative dose response test: validation by intravenous injection in children with liver disease

The relative dose response (RDR) test has been proposed as a tool for estimation of total body stores of vitamin A. In this study we have validated a variation of this test in children with or without liver disease. Administration of vitamin A was done by intravenous injection of 1000 micrograms of retinyl palmitate. We conclude that the RDR test by intravenous injection performed after 5 h is a reliable and sensitive indicator of vitamin A status. Values found were greater than 20% when liver vitamin A concentration is less than 20 micrograms/g liver and less than 10% when liver vitamin A concentration is greater than 20 micrograms/g liver. We also conclude that the RDR test can be applied to evaluate the efficiency of vitamin A therapy and we confirm that plasma retinol levels should not be used to screen vitamin A status.     

A marginal vitamin A status alters the distribution of vitamin A among parenchymal and stellate cells in rat liver

The aim of this study was to determine whether the distribution of vitamin A between parenchymal and stellate cells in rats with low vitamin A liver reserves is different from that observed in rats with adequate reserves. Retinol and retinyl esters were quantitated in parenchymal and stellate cells of the liver of three groups (n = 3) of adult Sprague-Dawley rats with mean total liver vitamin A reserves of 1.2, 14.5 and 28.9 micrograms retinol/g fresh liver for groups 1, 2 and 3, respectively. The amount of vitamin A per 10(6) cells was similar in parenchymal and stellate cells of group 1, whereas in groups 2 and 3, a much higher amount was found in stellate cells (11-fold and 27-fold higher, respectively). In group 1, 83% of the liver vitamin A was present in parenchymal cells. As liver stores rose, however, a progressively greater amount was stored in stellate cells. In group 3, 82% of the liver vitamin A was found in stellate cells. In groups 2 and 3, 87-91% of the vitamin A was found as retinyl ester, whereas in group 1, 52% was found as retinol. The retinyl ester composition was similar in parenchymal and stellate cells, major esters being palmitate (84%) and stearate (10%), with smaller amounts of oleate (2.5%) and other esters. Thus, in rats with low liver vitamin A reserves, most of the liver vitamin A is found in parenchymal cells.     

Distribution of retinol in rat liver cells: effect of age, sex and nutritional status

1. We have recently shown that the stellate cells, under normal conditions, contain a majority (more than 80%) of the total store of retinol in liver (Blomhoff et al. 1985). 2. In the present work we have studied the role of the various liver cells in rats of different ages, sex and vitamin A status. 3. In most of these groups of rats, storage of retinol in parenchymal cells was proportional to the liver store of retinol, and less than 10% of total retinol in the liver could be recovered in the parenchymal cells. The only exception was parenchymal cells isolated from vitamin A-deficient rats. In rats containing 5 nmol retinol/g liver, about 16% of total retinol could be recovered in parenchymal cells, while in rats with only 1 nmol retinol/g liver, about 40% of total retinol could be recovered in parenchymal cells. 4. These results indicate that parenchymal cells played a minor role in liver storage of retinol, and that stellate cells stored more than 90% of liver retinol in most instances. Only in rats with a low retinol status did the percentage of retinol in parenchymal cells increase.     

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

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

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

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

Vitamin A during lactation: relationship of maternal diet to milk vitamin A content and to the vitamin A status of lactating rats and their pups

We have investigated the effects of maternal vitamin A intake during pregnancy and lactation or during lactation alone on the concentration of vitamin A in rat's milk and on vitamin A levels in plasma and liver of dams and their pups. Groups of Sprague-Dawley rats were fed diets having either a high vitamin A content [15 retinol equivalents (R.E.)/g diet] or a low vitamin A content (0.6 R.E./g) for 42 d, including 7-8 d prior to pregnancy, pregnancy, and for 14 d of lactation. The concentration of vitamin A in milk on d 14 of lactation was significantly greater on the high vitamin A diets [114 +/- 16 micrograms/dl (mean +/- SEM; n = 8) versus 52 +/- 7.3 micrograms/dl (n = 11), P less than 0.005]. However, milk vitamin A concentration on d 1 of lactation did not vary with maternal vitamin A intake during pregnancy. In a second study in which supplementation with vitamin A (30 R.E./g diet) was begun on d 1 postpartum, the milk vitamin A content increased progressively with duration of lactation. Maternal plasma vitamin A concentrations did not differ between rats fed the higher or lower vitamin A diets. However, liver vitamin A concentrations both of dams and of their 14-d-old pups were significantly higher when dams were fed the higher vitamin A diets during pregnancy and/or lactation. The results of these studies indicate that the transfer of vitamin A from mother to offspring by milk and the vitamin A status of dams and their suckling neonates is influenced by maternal vitamin A intake during lactation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Response of plasma levels of vitamin A to a dose of vitamin A as an indicator of hepatic vitamin A reserves in rats.

Rats were fed diets deficient [-A] or sufficient [+A] (3 mg retinol equivalents/kg) in vitamin A, and without [-RA] or with [+RA] (12 mg/kg) retinoic acid supplementation for up to 33 days. Rats with plasma vitamin A levels ranging from 7 to 62 micrograms/dl were studied at intervals during progressive depletion of liver stores of vitamin A (expt. 2) and when liver stores were nearly exhausted (less than 10 micrograms/g) or replete (up to 100 micrograms/g) with vitamin A (expt. 1). A dose of retinyl acetate in corn oil (20 micrograms retinol equivalents) was administered by intubation directly into the stomach. The relative dose response (RDR), expressed as a percentage and defined as the absolute magnitude of the rise in plasma vitamin A levels 5 hours after the dose of retinyl acetate, divided by the plasma level of vitamin A attained after 5 hours, was determined for each rat and correlated over a wide range of vitamin A plasma and liver levels. An RDR above 50% invariably was associated with low plasma levels (10 to 30 micrograms/dl) and low liver stores (less than 10 micrograms/g) of vitamin A, whereas an RDR of less than 40% was associated with plasma levels above 30 micrograms/dl and liver stores ranging from 3 to 100 micrograms/g.