Placental transport of retinol in ewes fed high intakes of vitamin A

Twelve ewes were fed retinyl propionate equivalent to 12,000 micrograms retinol/kg body weight per day for the last two trimesters of pregnancy (group fed high vitamin A). Four ewes received a control level of 120 micrograms/kg body weight per day. Indwelling catheters were implanted surgically in fetal jugular veins and carotid arteries. Ewes or fetal lambs received [3H]retinol intravenously, and blood was sampled until parturition. The ewes fed large amounts of vitamin A maintained viable fetal lambs for 6 d less than did controls. Plasma retinyl ester concentrations were elevated in the ewes fed a high level of vitamin A but not in their fetal lambs. Rates of plasma retinol transport and clearance increased with vitamin A intake in ewes and their fetal lambs. Efficiency of placental retinol transport in the group fed high levels of vitamin A was less than one-quarter that for controls. However, placental transport rate was approximately 100 micrograms/d greater than that of controls with an equivalent amount retained by the fetoplacental unit in the group fed high vitamin A. These data indicate that placental transport of retinol is partially regulated. High maternal vitamin A intake results in high retinol transport to the fetus.     

Moderate maternal vitamin A deficiency affects perinatal organ growth and development in rats

Vitamin A deficiency during pregnancy is associated with detrimental effects in the offspring. We have developed a rat model to examine specific effects of maternal vitamin A status on perinatal growth and development. A total of 54 female rats were fed a vitamin A-free (VAF), -marginal (VAM) or -sufficient (VAS) diet from weaning until mating (at 7 weeks) and throughout pregnancy. Half of the rats in each group were injected with a single large dose of vitamin A on day 10 of pregnancy. Fetal and neonatal samples were taken on day 20 of pregnancy and the day of birth respectively. Maternal plasma retinol concentrations on day 20 and at birth were 50% and 30% lower in the VAF and VAM when compared to the VAS group. Fetal weight and survival did not differ between groups although placental:fetal ratio was higher in the VAF group than in the VAS group (0.195 (SE 0.005) v. 0.175 (SE 0.004), P < 0.05). Rats fed the VAF diet gave birth at 23.5 d, an average of 1 d later than the other groups, and had lower number of live neonates at birth. Fetal liver, heart and lung weights relative to total body weight were lower in the VAF group and had altered growth trajectories. In neonates, only the relative lung weight was reduced. In addition, an increased protein:DNA ratio indicated hypertrophy in fetal kidneys. Vitamin A injection had no additional effect on length of gestation and fetal or neonatal number. However, injection increased relative fetal organ weights in the VAF group but did not alter the effects of vitamin A deficiency in the neonate. These data suggest that chronic vitamin A deficiency during pregnancy compromises liver, heart and kidney and impairs lung growth and development during the last few days of gestation and reduces number of live neonates at birth.     

Effects of retinoic acid on the metabolism of vitamin a in rat liver

Sprague-Dawley male rats with adequate storage of vitamin A were fed vitamin A deficient diet either alone (RA-) or supplemented with retinoic acid (RA+). The rats were sacrificed at different days for the measurement of free retinol and the composition of retinyl esters in liver. At the same time the plasma retinol levels of these rats were also determined. Supplementation of retinoic acid significantly lowered the plasma retinol levels with a concomitant slower depletion of the total vitamin A in the liver of RA+ rats as compared. to RA-rats. The ratio of retinyl esters to free retinol in the liver tissue of RA- rats decreased with time, whereas in RA+ rat livers the ratio remained constant. The percentage distribution of retinyl esters in the liver tissue revealed a significant decrease in saturated retinyl esters with an increase in unsaturated retinyl esters in the RA+ group compared to RA- group. These data support the earlier findings that retinoic acid spares the utilization of retinol and suggest that it may affect the release of retinol from retinyl esters in the liver.     

Vitamin A status needed to maintain vitamin A concentrations in nonhepatic tissues of the pregnant rat

Vitamin A-depleted pregnant rats were fed diets containing 0, 1, 10 or 100 retinol equivalents (REq)/d during gestation. Maternal tissues of these dams, their placentas and fetuses were assayed for total vitamin A (retinol and its esters) at gestational ages 10, 13, 16 or 19 d. The vitamin A concentrations in placenta and fetuses of dams fed 10 REq/d were significantly different from those of dams fed no vitamin A. This difference was not seen in any of the six other maternal tissues assayed. We suggest that the fetus is at greater risk than other maternal tissues during severe vitamin A deprivation. For vitamin A-deprived dams, linear regression analysis indicated strong relationships between the vitamin A concentrations of nonhepatic tissues and their liver vitamin A concentrations. In contrast, among vitamin A-replete dams, these relationships were not statistically significant. An estimate was made of the minimal vitamin A status corresponding to tissue repletion by solving linear equations described by the data of the depleted dams for the vitamin A concentrations found in tissues of replete dams. This analysis revealed that a maternal vitamin A status of 3 micrograms/g liver was needed to provide the level of vitamin A found in the placenta, whole fetus and maternal tissues of vitamin A-replete dams.     

Zinc-vitamin A interaction in pregnant and fetal rats: supplemental vitamin A does not prevent zinc-deficiency-induced teratogenesis

The influence of a higher-than-normal intake of vitamin A on the detrimental effects of zinc deficiency on vitamin A metabolism was investigated in pregnant Sprague-Dawley rats. At mating, rats were fed diets containing 100 (control), 4.5, or 0.5 micrograms/g zinc combined with 4 (control) or 8 micrograms retinyl acetate/g. Low intake of zinc, but not of vitamin A, caused food intake, total body weight change, fetal weight and placental weight to be low. Incidence of teratogenic effects was more pronounced in low zinc groups than in controls. Concentrations of vitamin A in maternal plasma and liver were affected by the amount of zinc in the diet. Dietary vitamin A, however, did not affect either of these parameters. Maternal plasma zinc concentration was affected only by low dietary zinc, whereas plasma copper and iron were unaffected by the dietary treatments. Maternal liver iron was higher in zinc-deficient rats than in controls; however, maternal liver zinc and copper concentrations were not altered by dietary treatments. No significant differences in vitamin A concentration of fetal liver, fetal plasma, or placenta were seen among the groups. Fetuses from zinc-deficient dams had significantly lower levels of liver vitamin A and liver zinc than did controls. Fetal liver iron was higher in zinc-deficient fetuses than in controls, whereas fetal liver copper was not affected by dietary treatment. These data suggest that supplemental dietary vitamin A does not ameliorate the effect of zinc deficiency on vitamin A metabolism during pregnancy.     

Effects of exogenous retinol and retinoic acid on the biosynthesis of 14C-mannose labelled glycolipids and glycoproteins in rat liver

Studies were conducted to investigate the in vivo and in vitro effects of retinol and retinoic acid on the synthesis of mannolipids and mannopeptides in rat liver. The incorporation of 14C-mannose into glycolipids and glycoproteins showed a decrease in vitamin A-depleted rats as compared with vitamin A-fed rats. By means of DEAE-cellulose, silicic acid and thin-layer chromatography, the mannose-containing lipids were separated into mannosyl retinyl phosphate (MRP, Rf 0.2) and dolichyl mannosyl phosphate (DMP, Rf 0.4), respectively. A rapid increase in the synthesis of labelled MRP was observed, exhibiting a peak between 25 and 60 min after intraperitoneal administration of retinol to vitamin A-depleted rats. Similarly, administration of retinoic acid brought about elevation of 14C-mannolipid (Rf 0.2) synthesis with a peak at 60 min after injection. On the other hand, the incorporation of 14C-mannose into DMP (Rf 0.4) remained unchanged by such treatment. In vitro addition of retinyl phosphate, but not retinoyl phosphate, markedly stimulated the synthesis of 14C-mannolipid (Rf 0.2), using crude membrane of rat liver and GDP-14C-mannose as the donor. These findings strongly suggest that not only retinol but also retinoic acid plays an important biological role in mannosyl transfer reaction in rat liver. However, the molecular participation of a metabolite of retinoic acid in the formation of a mannolipid and the structure of such a metabolite remain to be established.     

Intraplacental retinol distribution

With the objective of evaluating intraplacental vitamin A distribution, 234 placental samples were collected, corresponding to six samples from each of the placentas analyzed: two from the lateral maternal portion, one from the central maternal portion, two from the lateral fetal portion, and one from the central fetal portion. Samples were obtained from 39 adult puerperal mothers with low-risk pregnancies, without vitamin A deficiency or night blindness. Retinol content determination was achieved through spectrophotometry. Retinol values obtained for each region were correlated with the most probable value for each placenta (P < 0.001). Despite differences in retinol content between samples, statistical data analysis showed that intra-tissue variation had no influence on the conversion of data into information. Consequently, any portion of the placenta may be used for retinol level determination purposes, due to the correlation between all portions and the most probable value. The findings of the present study represent an advance for surveys intending to incorporate the collection and dosage of placental vitamin A levels into their analyses, thus increasing the arsenal of pre-pathological or subclinical vitamin A deficiency markers, which can allow for earlier intervention on the maternal-infant group.     

Hepatic vitamin A preloading reduces colorectal cancer metastatic multiplicity in a mouse xenograft model

Previous research in our laboratory showed that retinol inhibited all-trans retinoic acid (ATRA)-resistant human colon cancer cell invasion via a retinoic acid receptor-independent mechanism in vitro. The objective of the current study was to determine if dietary vitamin A supplementation inhibited metastasis of ATRA-resistant colon cancer cells in a nude mouse xenograft model. Female nude mice (BALB/cAnNCr-nu/nu, n = 14 per group) consumed a control diet (2,400 IU retinyl palmitate/kg diet) or a vitamin A supplemented diet (200,000 IU retinyl palmitate/kg diet) for 1 mo prior to tumor cell injection to preload the liver with vitamin A. HCT-116, ATRA-resistant, human colon cancer cells were intrasplenically injected. Mice continued to consume their respective diets for 5 wk following surgery. Consumption of supplemental vitamin A decreased hepatic metastatic multiplicity to 17% of control. Hepatic and splenic retinol and retinyl ester concentrations were significantly higher in the mice supplemented with vitamin A when compared to mice consuming the control diet. Supplemental vitamin A did not decrease body weight, feed intake, or cause toxicity. Thus, supplemental dietary vitamin A may decrease the overall number of hepatic metastasis resulting from colon cancer.     

Hepatic Vitamin A Preloading Reduces Colorectal Cancer Metastatic Multiplicity in a Mouse Xenograft Model

Previous research in our laboratory showed that retinol inhibited all-trans retinoic acid (ATRA)-resistant human colon cancer cell invasion via a retinoic acid receptor-independent mechanism in vitro. The objective of the current study was to determine if dietary vitamin A supplementation inhibited metastasis of ATRA-resistant colon cancer cells in a nude mouse xenograft model. Female nude mice (BALB/cAnNCr-nu/nu, n = 14 per group) consumed a control diet (2,400 IU retinyl palmitate/kg diet) or a vitamin A supplemented diet (200,000 IU retinyl palmitate/kg diet) for 1 mo prior to tumor cell injection to preload the liver with vitamin A. HCT-116, ATRA-resistant, human colon cancer cells were intrasplenically injected. Mice continued to consume their respective diets for 5 wk following surgery. Consumption of supplemental vitamin A decreased hepatic metastatic multiplicity to 17% of control. Hepatic and splenic retinol and retinyl ester concentrations were significantly higher in the mice supplemented with vitamin A when compared to mice consuming the control diet. Supplemental vitamin A did not decrease body weight, feed intake, or cause toxicity. Thus, supplemental dietary vitamin A may decrease the overall number of hepatic metastasis resulting from colon cancer.     

Prenatal aspects of ascorbic acid metabolism in the albino rat.

Transfer of ascorbic acid and/or its derivatives from maternal blood into the fetus was studied during the last week of gestation in the rat. Rats were injected intravenously with [1-14C]-ascorbic acid and the rate of transfer estimated by the concentration and content of label present in placentas and fetuses. At all times studied the concentration of label in the placenta was greater than in the fetus. The highest capacity of the placenta to concentrate label was found on day 15 decreasing to a low at day 19 and again increasing up to day 21. While in the fetuses, the concentration of label per gram of tissue remained remarkably constant throughout the study. The quantity of labeled compounds transferred into the fetus per gram of placental tissue increased between day 15 and day 21 of gestation.     

Retinoic acid supplementation of a vitamin A-deficient diet inhibits retinoid loss from hamster liver and serum pools

These studies were performed to follow a spectrum of relevant parameters in male Syrian golden hamsters fed either a vitamin A-deficient diet or the same diet supplemented with retinoic acid at 3 micrograms/g diet. Body weight and life span were not affected by the vitamin A-deficient diet until after 6-7 wk. Squamous metaplastic lesions of the Formalin-fixed tracheas were not generally observed in the hamsters fed the deficient diet until 6-7 wk, at which time blood retinol and liver retinyl palmitate levels had also decreased. Blood glucose levels remained normal (90 mg/dl) until about 7 wk but declined to about 40% of normal at 9 and 10 wk. Dietary retinoic acid supplementation of the vitamin-deficient diet (3 micrograms/g diet) inhibited the loss of retinol from blood and of retinyl palmitate from the liver so that these compounds were still present at 10 wk, but were not detectable in hamsters fed the vitamin A-deficient diet without retinoic acid.     

Expression of retinoic acid nuclear receptors and tissue transglutaminase is altered in various tissues of rats fed a vitamin A-deficient diet.

The effects of vitamin A nutritional status on the levels of expression of retinoic acid nuclear receptors (RAR), and the retinoic acid-responsive gene, tissue transglutaminase, were determined in rats. Weanling male Sprague-Dawley rats fed a vitamin A-deficient diet for approximately 7 wk developed vitamin A deficiency, as confirmed by the depletion of liver retinol and retinyl palmitate. Controls were fed the same diet supplemented with 24 mg/kg retinyl acetate. The levels of expression of RAR beta mRNA were approximately 80% lower in bladder, brain, liver, lung and trachea and those of RAR gamma mRNA were approximately 50% lower in bladder, lung and trachea of rats fed the vitamin A-deficient diet than in controls. The levels of expression of RAR alpha mRNA were approximately 90% lower in brain and approximately 30% greater in liver, kidney, intestine and lung of rats fed the vitamin A-deficient diet. Vitamin A deficiency also resulted in reduced expression of tissue transglutaminase in the bladder, lungs and trachea, which paralleled the effects observed for RAR beta and RAR gamma. When vitamin A-deficient rats were subsequently fed a retinol-deficient diet supplemented with retinoic acid for 4 wk, the expression of RAR (beta and gamma) and tissue transglutaminase returned to the control levels. These results indicate that vitamin A nutritional status in rats influences the expression of both RAR and tissue transglutaminase in certain tissues.     

An interaction between zinc and vitamin A in pregnant and fetal rats

A possible interaction between zinc and vitamin A metabolism was studied in pregnant rats. Rats were depleted of vitamin A by feeding retinoic acid during growth. At mating, they were fed diets containing 100, 9, or 0.5 microgram/g zinc and were given orally 400, 8, or 0 microgram/kg body weight/day of retinyl palmitate. Low intake of zinc, but not of vitamin A, caused maternal body weight gain, placental weight, and fetal weight all to be low. The number of implantation sites affected and the proportion of fetuses malformed were dependent on intake of both zinc and vitamin A, and there was a significant interaction between these nutrients in regard to both of these parameters. In maternal as well as in fetal animals, plasma and liver zinc concentrations were low in groups fed low levels of zinc, but not in those given low vitamin A. Liver vitamin A values were affected by vitamin A intake but not by dietary zinc concentration. However, plasma vitamin A concentration in both maternal and fetal animals was significantly reduced by low intake of either zinc or vitamin A. There was a significant interaction between these two nutrients in regard to plasma vitamin A. These data indicate an interaction between zinc and vitamin A metabolism possibly at the level of vitamin A mobilization from the liver.     

Transfer of vitamin A from intestine to plasma in lambs fed low and high intakes of vitamin A

Three groups of lambs were fed a low carotene diet supplemented with vitamin A proprionate equivalent to 0, 100 or 12,000 micrograms retinol per kilogram per day via rumen cannulae. These groups were mildly deficient, normal (control) and intoxicated, respectively. After 17 weeks, abomasal cannulae were implanted, [15-3H]retinol was injected into the abomasum, and jugular blood was sampled for 48 hours. After 7 days, the tracer was injected into a jugular vein, and blood was sampled for 48 hours. Rates of retinol and retinyl ester transport and retinol clearance from plasma reflected intake (P less than 0.05); clearance of retinyl esters was unaffected. Mean efficiencies of total vitamin A (retinol and retinyl ester) transfer from digestive tract to plasma were 86, 60 and 60%, whereas those of retinol were 91, 58 and 14% (P less than 0.05) for mildly deficient, control and intoxicated lambs, respectively. Thus lower transfer efficiencies were associated with higher transport rates of plasma retinol and retinyl ester and with increased clearance of retinol but not retinyl ester from plasma. These results suggest that vitamin A transfer from intestine to plasma is sensitive to vitamin A intake, and that retinol is the primary form of vitamin A affected.     

Influence of iron on vitamin A nutritional status

Iron deficiency seems to deteriorate vitamin A metabolism leading to a reduction in serum retinol and an increase in hepatic retinol and retinyl ester. These alterations probably result from an increase in retinol sequestration to the liver and/or impairment in the activity of hepatic retinyl ester hydrolases decreasing vitamin A mobilization.     

Conceptus uptake of the 106RuNO-nitro complex in relation to gestational stages

106RuNO-nitro complex was administered intravenously to pregnant rats to study uptake of the radioruthenium complex by the conceptus in relation to gestational stages. Each conceptus was sampled periodically with respect to its placenta, fetal membrane, fetal fluid and fetus. Perceptible radioactivity in the fetus was detected only in the later stage of gestation and its relative concentration, defined as the ratio radioactivity per unit weight in the body tissue at sacrifice to that in the whole-body at dosing, was very low compared with other tissues. The average number of fetuses in one litter was 13 and the transfer rate of nitro complex into the fetuses 24 hr after injection to rats on the 20th day of gestation was about 1% of initial maternal dose. The relative concentration in the placenta and fetal membrane was much higher than in the fetus and decreased with time after injection. These results indicate that the placenta and fetal membrane play significant roles as barriers to the transfer of 106RuNO-nitro complex into the fetus.     

Reproducibility of relative dose response (RDR) test and serum retinol and retinyl ester concentrations in children after a 2-week interval.

Reproducibility of the relative dose response test (RDR), a test designed to measure vitamin A status, was tested in 23 Belizean children, 5-8 years after 2-week interval during which no treatment was given.

As required for the RDR test, serum retinol concentrations were determined before and 5 hours after an oral dose of vitamin A. An RDR score > 14% was used as the criterion of inadequate vitamin A status. The HPLC method used to measure serum retinol concentrations also determined the concentrations of four retinyl esters.

The RDR test was reproducible for 17 of 23 subjects: 3 scored > 14% on both tests; 14, < 14% on both. Six subjects scored > 14% on only one test. The concordance correlation coefficient (rc) for the percent change in the two tests was 0.24; for fasting serum retinol concentration, rc = 0.81. For retinyl palmitate and stearate, the esters present in highest concentrations at 5 hours, concordance correlation coefficients were 0.75 and 0.59, respectively.

The failure of the RDR test to classify 26% of the subjects reproducibly reduces the usefulness of the test. In addition, the reproducibility of the retinyl ester concentrations in serum 5 hours after the retinyl palmitate dose and the relatively high concentrations in some subjects suggests that some individuals may not metabolize sufficient retinol in 5 hours to cause a maximal increase in serum retinol, resulting in an underestimation of deficiency in a population in which the RDR test is used.     

The effect of retinol deficiency on the metabolism of all-trans-retinyl acetate in rat testes

Following intratesticular injection, the metabolism of all-trans-[11-3H] retinyl acetate was studied in rats fed a vitamin A-deficient diet supplemented either with retinyl palmitate (-A + RP) or retinoic acid (-A + RA). Analysis of testicular metabolites by HPLC at 6 h and 24 h demonstrated the hydrolysis of retinyl acetate to retinol, esterification of retinol to retinyl palmitate and the formation of trace amounts of retinoic acid and other metabolites in both groups of rats. Eight and nine metabolite peaks were present in the -A + RP and -A + RA groups, respectively. The HPLC profile was similar in both groups of rats but the amounts of metabolites differed.     

Effect of retinoic acid and retinyl acetate feeding upon lipid metabolism in adrenalectomized rats.

Many reports have appeared in the literature suggesting that vitamin A may exert some of its effects via changes in adrenocortical activity. A series of experiments were performed in order to assess the possible role of the adrenal gland in vitamin A-induced lipid alterations in rats. Adrenalectomized, sham-operated, and intact rats were fed retinoic acid or retinyl acetate at several levels. Either 25 or 100 retinol equivalents (RE)/g dry diet were fed to male Sprague-Dawley rats for periods of 7 or 28 days. Neither compound had an effect on the concentration of liver glycerides, phospholipids, cholesterol, or total lipids. Vitamin A, especially in the form of retinoic acid, was found to induce an elevation of plasma triglycerides. The presence of the adrenal gland was not necessary for the induction of hypertriglyceridemia nor was there any indication of increased adrenocortical output (as measured by plasma corticosterone level) as a result of vitamin A feeding. There was a reduction in circulating retinol as a result of retinoic acid feeding at either 25 or 100 RE in sham-operated and adrenalectomized rats but not in unoperated rats. These experiments demonstrate that vitamin A, especially in the form of retinoic acid, fed at as low as 25 RE/g diet to the rat can induce hypertriglyceridemia, and that the adrenal gland does not mediate this effect.     

Vitamin A in blood plasma and urine of dogs is affected by the dietary level of vitamin A

Dogs differ from other species with respect to the occurrence of a high percentage of retinyl esters in blood plasma and the excretion of substantial amounts of vitamin A in the urine. Our investigation focussed on the effects of different concentrations of vitamin A in the diet, ranging from concentrations below NRC requirements of 25 IU/kg body weight (BW) to 2400 IU/kg BW, on the levels of retinol and retinyl esters (palmitate/oleate and stearate) in canine blood plasma and urine. The plasma levels of retinyl esters paralleled the levels of vitamin A in the feed (r = 0.91; p < 0.001). The highest plasma level (12.1 +/- 0.4 mg/l) was observed at the highest level in the diet. This observation may be explained by the fact that in dogs retinyl esters are associated with lipoproteins. Even under prolonged feeding on vitamin A levels below NRC requirements, retinyl esters were still present in the plasma (2.8 +/- 0.1 mg/l). Levels of retinol were not affected (1.2 +/- 0.03 vs. 1.0 +/- 0.03 mg/l, respectively). In the urine, the concentration of retinol and retinyl palmitate/oleate increased with the first increase of vitamin A in the diet to 1.2 +/- 0.4 mg/l of total vitamin A. Urinary levels were elevated and fluctuated with up to four peaks while dietary vitamin A levels were above NRC requirements. But the amount of retinol and retinyl esters excreted did not show any dependence on the amount of vitamin A in the diet. When the amount of vitamin A in the diet was at or below requirements, only traces of retinol and retinyl esters were detected in urine. Thus, contrary to current knowledge for most other mammals, retinyl ester levels in plasma and retinol and retinyl esters in the urine of dogs proved to be clearly but differently affected by the amount of vitamin A supplied with the diet. Contrary to retinol, plasma levels of retinyl esters closely reflect the actual supply of vitamin A with the feed. The occurrence of retinol and retinyl esters in urine may, however, be due to dietary supply of vitamin A in excess of standard requirements, thereby providing a useful indicator of a dietary supply of vitamin A above requirement. The mechanism involved in the possible regulation of urinary excretion of retinol and retinyl esters remains to be elucidated.