Liver microsomal membrane fluidity and lipid characteristics in vitamin A-deficient rats.

Rat liver microsomes (microsomal fraction) were isolated from vitamin A-deficient and -sufficient rats and analysed for membrane lipid characteristics. Membrane fluidity was found to be significantly decreased in microsomes from the vitamin A-deficient rats, but not in liposomes prepared from lipid extracts. Microsomes from vitamin A-deficient animals showed a significant decrease in C18:2, omega 6 and an increase in C22:5, omega 6 fatty acids.     

Effect of excess and deficiency of vitamin A on the utilization of FFA by liver and skeletal muscle.

Fatty acid metabolism in liver and skeletal muscle has been studied in rats treated with high doses of vitamin A and in those made vitamin A-deficient. Ingestion of 30,000 IU of vitamin A for two days resulted in increased incorporation of palmitate-1-14C into triglycerides but not into phospholipids. Accumulation of hepatic triglycerides was observed in vitamin A-fed rats. Deficiency of vitamin A did not cause any change in the triglyceride or phospholipid content of the liver. The rate of hepatic fatty acid oxidation and ketogenesis was markedly increased in vitamin A-fed rats. The experimental evidence indicated that vitamin A may have a stimulatory effect on these processes apart from that exerted by the high plasma FFA level in vitamin A-fed rats. Oxidation of palmitate-1-14C into C32 by skeletal muscle (latissimus dorsi) was also increased as a result of vitamin A administration. Vitamin A deficiency did not cause any change in fatty acid oxidation by liver and skeletal muscle. Hepatic palmitoyl-CoA synthetase activity was decreased in vitamin A-deficient rats. The results presented suggest that vitamin A may be required for the uptake and utilization of fatty acids by liver and akeletal muscle.     

Vitamin A deficiency causes oxidative damage to liver mitochondria in rats

Mitochondrial damage in rat liver induced by chronic vitamin A-deficiency was studied using three different groups of rats: (i) control rats, (ii) rats fed a vitamin A-free diet until 50 d after birth and (iii) vitamin A-deficient rats re-fed a control diet for 30 d. No statistical difference in body weight and food intake was found between control and vitamin A-deficient rats. Liver GSH concentration was similar in both groups. However, in vitamin A-deficient rats, the mitochondrial GSH/GSSG ratio was significantly lower and the levels of malondialdehyde (MDA) and 8-oxo-7, 8-dihydro-2'-deoxyguanosine (oxo8dG) were higher when compared to control rats. These values were partially restored in re-fed rats. The mitochondrial membrane potential of vitamin A-deficient rats was significantly lower than in control rats and returned to normal levels in restored vitamin A rats. Two populations of mitochondria were found in vitamin A-deficient rats according to the composition of membrane lipids. One population showed a similar pattern to the control mitochondria and the second population had a higher membrane lipid content. This report emphasizes the protective role of vitamin A in liver mitochondria under physiological circumstances.     

Modification of Sertoli cell functions in vitamin A-deficient rats

FSH binding and cAMP responses to FSH in Sertoli cell-enriched testes were not affected by the vitamin A (retinol) status of the animals. These results indicate that changes in Sertoli cell functions during vitamin A deficiency are independent of FSH-Sertoli cell interactions. Concentrations of serum androgen binding protein (ABP) in vitamin A-deficient rats were consistently higher than those of control animals throughout the study period. The accumulation of testicular fluid after efferent duct ligation, an indication of Sertoli cell secretory function, was normal in vitamin A-deficient rats at least until 70 days of age, but declined thereafter. ABP concentrations in seminiferous tubular fluid of vitamin A-deficient rats increased transitorily during the 70-80-day age period but returned to normal by 90 days. The increment of ABP in seminiferous tubular fluid after efferent duct ligation, and ABP concentrations in interstitial fluid were consistently lower in vitamin A-deficient rats. The higher serum ABP in vitamin A-deficient rats therefore cannot be explained by an increase in the permeability of Sertoli-cell tight junctions or basement membrane.     

The isoelectric fractionation of hen''s-egg ovotransferrin

1. ATP sulphurylase was assayed in various organs from vitamin A-deficient and pair-fed control rats at different stages of deficiency. Activity decreased slightly in the liver and markedly in the adrenal gland. Striking differences in liver activity were observed between pair-fed control and ad libitum-fed animals. This observation suggested that diet (apart from vitamin A) strongly influenced the activity of ATP sulphurylase. 2. Total starvation caused a severe decrease in activity in liver within 48hr. This was due to a lack of protein intake. 3. By feeding groups of vitamin A-deficient and pair-fed control rats on a diet containing 80% protein, the specific activity of the liver ATP sulphurylase was maintained in the pair-fed control group at the normal level of an ad libitum-fed rat, whereas it decreased by 25% (statistically significant at P<0.01) in the deficient rat. On a 20%-protein diet, there were no significant differences between vitamin A-deficient and pair-fed control rats. These relationships held also for enzyme activity expressed per g. of liver, per total liver and per g. of DNA. There were no differences in liver protein or DNA concentration between vitamin A-deficient and control rats on either protein intake. 4. Control rats on a 20%-protein diet had liver specific enzyme activities about one-half of those in control rats on an 80%-protein diet, as well as lower liver protein concentrations. 5. It is concluded that, when the effect of protein deprivation on ATP sulphurylase is separated from the effect of vitamin A deficiency, a lowering of the enzyme activity caused by the vitamin deficiency is demonstrable.     

Vitamin A supplementation inhibits chemiluminescence and lipid peroxidation in isolated rat liver microsomes and mitochondria

In the present study we investigated if administration of vitamin A could protect rat liver microsomes and mitochondria from in vitro peroxidation. Appreciable decrease of chemiluminescence and lipid peroxidation was measured in microsomal membranes from rats receiving vitamin A, with respect to control animals. In membranes derived from control animals, the fatty acid composition was profoundly modified when subjected to in vitro peroxidation mediated by ascorbate-Fe⁺⁺, with a considerable decrease of 20:4 n6 and 22:6 n3 in mitochondria and 18:2 n6 and 20:4 n6 in microsomes. As a consequence the peroxidizability index, a parameter based on the maximal rate of oxidation of specific fatty acids was higher in supplemented animals than in control group when both kind of membranes were analyzed. These changes were less pronounced in membranes derived from rats receiving vitamin A. These results are in agreement with previous results that indicated that vitamin A may act as an antioxidant protecting membranes from deleterious effects.Abbreviations BHT butylated hydroxytoluene - BSA bovine serum albumin - CL chemiluminescence - PI peroxidizability index Member of Carrera del Investigador Científico, Consejo Nacional de Investigaciones Cientificas y Técnicas de la Republica Argentina     

The effects of vitamin A deficiency on hepatic folate metabolism in rats

The effects of severe vitamin A deficiency (liver retinol less than 2 micrograms/g) on hepatic folate metabolism in rats were studied. The oxidation of a [ring-2-14C] histidine load or a [14C]formate load to 14CO2 was significantly depressed in vitamin A-deficient rats and those given histidine also excreted more urinary formiminoglutamic acid (FiGlu) than pair-fed controls. The increase in FiGlu excretion was not due to augmented production from histidine, implicating an impairment of FiGlu catabolism. FiGlu formiminotransferase activity was unaltered in vitamin A-deficient rats, but hepatic tetrahydrofolic acid (THF) concentration was decreased by 58% in vitamin A-deficient rats given a histidine load while 5-methyl-THF concentration was increased by 39%. Formyl-THF and total folate levels were similar to controls. A redistribution of folate coenzymes was not found in vitamin A-deficient rats not force fed histidine. A 43% decrease in 10-formyl-THF dehydrogenase activity, which generates both THF and the 14CO2 from the labeled substrates, and an 81% increase in 5,10-methylene-THF reductase activity, which generates 5-methyl-THF, were found in vitamin A-deficient rats. It appears that the production of severe vitamin A deficiency results in selective changes in the activities of hepatic folate-dependent enzymes, so that when a load of a one-carbon donor is given, THF concentration decreases and metabolism of the load is impaired.     

Vitamin A deficiency alters splenic dendritic cell subsets and increases CD8Gr-1 memory T lymphocytes in C57BL/6J mice

Vitamin A-deficient populations have impaired T cell-dependent antibody responses. Dendritic cells (DCs) are the most proficient antigen-presenting cells to naïve T cells. In the mouse, CD11b⁺ myeloid DCs stimulate T helper (Th) 2 antibody immune responses, while CD8α⁺ lymphoid DCs stimulate Th1 cell-mediated immune responses. Therefore, we hypothesized that vitamin A-deficient animals would have decreased numbers of myeloid DCs and unaffected numbers of lymphoid DCs. We performed dietary depletion of vitamin A in C57BL/6 J male and female mice and used multicolor flow cytometry to quantify immune cell populations of the spleen, with particular focus on DC subpopulations. We show that vitamin A-depleted animals have increased polymorphonuclear neutrophils, lymphoid DCs, and memory CD8⁺ T cells and decreased CD4⁺ T lymphocytes. Therefore, vitamin A deficiency alters splenic DC subpopulations, which may contribute to skewed immune responses of vitamin A-deficient populations.     

Free Radical-Induced Liver Injury. I. Effects of Dietary Vitamin E Deficiency on Triacylglycerol Level and its Fatty Acid Profile in Rat Liver

Effects of dietary vitamin E deficiency on the fatty acid compositions of total lipids and phospholipids were studied in several tissues of rats fed a vitamin E-deficient diet for 4, 6, and 9 months. No significant differences were observed between the vitamin E deficiency and controls except in the fatty acid profiles of liver total lipids. Triacylglycerol (TAG) accumulation was found in the liver of rats fed a vitamin E-deficient diet. The levels of TAG-palmitate and -oleate increased particularly in the liver from such animals. The fatty acid compositions of hepatic phospholipids were not affected by the diet. Increased TAG observed in the liver of rats fed a vitamin E-deficient diet was restored to normal when the diet was supplemented with 20 mg α-tocopheryl acetate/kg diet. These findings indicate that dietary vitamin E deficiency causes TAG accumulation in the liver and that the antioxidant, vitamin E, is capable of preventing free radical-induced liver injury.     

Effects of selenium deficiency on fatty acid metabolism in rats fed fish oil-enriched diets.

The hepatic fatty acid metabolism was investigated in rats stressed by selenium deficiency and enhanced fish oil intake. Changes in the composition of lipids, peroxides, and fatty acids were studied in the liver of rats fed either a Sedeficient (8 microg Se/kg) or a Se-adequate (300 microg Se/kg) diet, both rich in n-3 fatty acid-containing fish oil (100 g/kg diet) and vitamin E (146 mg alpha-tocopherol/kg diet). The two diets were identical except for their Se content. Se deficiency led to a decrease in hair coat density and quality as well as to changes in liver lipids, individual lipid fractions and phospholipid fatty acid composition of the liver. The low Se status did reduce total and reduced glutathione in the liver but did not affect the hepatic malondialdehyde level. In liver phospholipids (PL), Se deficiency significantly reduced levels of palmitic acid [16:0], fatty acids of the n-3 series such as DHA [22:6 n-3], and other long-chain polyunsaturates C-20-C-22, but increased n-6 fatty acids such as linoleic acid (LA) [18:2 n-6]. Thus, the conversion of LA to arachidonic acid was reduced and the ratio of n-6/n-3 fatty acids was increased. As in liver PL, an increase in the n-6/n-3 ratio was also observed in the mucosal total fatty acids of the small intestine. These results suggest that in rats with adequate vitamin E and enhanced fish oil intake, Se deficiency affects the lipid concentration and fatty acid composition in the liver. The changes may be related to the decreased levels of selenoenzymes with antioxidative functions. Possible effects of Se on absorption, storage and desaturation of fatty acids were also discussed.     

Biosynthetic studies on mannolipids and mannoproteins of normal and vitamin A-depleted hamster livers.

The incorporation of [1-14C]mannose into hamster liver glycolipids and glycoproteins was studied in normal and vitamin A-depleted hamsters. Severly (25% weight loss) and mildly (no weight loss) deficient animals were compared to vitamin A-fed controls. The incorporation of [14C]mannose into glycolipids and glycoproteins decreased in mild and severe vitamin A deficiency by 63-90% compared to vitamin A-fed animals. These results were essentially the same whether expressed per g of wet liver, per DNA or per protein. The size of the pools of mannose, glucose and galactose and their specific radioactivity in liver were determined by gas-liquid chromatography of the boronates of the hexitols (Eisenberg, Jr, F. (1972) Methods Enzymol. XXVIIIB, 168-178) in normal and vitamin A-deficient conditions. It was found that the amount of free hexoses per g of liver was very similar in normal and vitamin A-deficient conditions. The specific radioactivities for mannose and glucose were greater in vitamin A deficiency, thus excluding the possibility that the observed severe decrease in glycopeptide and glycolipid synthesis is a reflection of a similar decrease in the specific radioactivity of the precursor pools. Quantitation of mannose in glycoprotein showed a 79% decrease in vitamin A deficiency. Specific radioactivity of mannose in glycoproteins, 20 min after injection of the label, was 187 dpm/mug of mannose in the normal and 48 kpm/mug of mannose in the vitamin A-deficient livers. It is concluded that vitamin A is necessary for the biosynthesis of liver mannose-containing glycoproteins and glycolipids.     

Effects of vitamin A deficiency on cell proliferation and morphology of trachea of the hamster

Abstract. Regulation by vitamin A of cell proliferation and differentiation of epithelial tissues is well-established. Deficiency of vitamin A in experimental animals leads to the development of hyperplasia and squamous metaplasia. The objective of the present study was to examine, for young hamsters, the effects of variable levels of the vitamin in the liver and trachea, on cell proliferation and morphology of tracheal epithelium and on body weights. Newly born litters were maintained on vitamin A-supplemented and vitamin A-deficient diets, and various parameters were examined at different ages. Retinol and retinyl palmitate levels were determined by high performance liquid chromatography. For animals on the supplemented diet, concentrations of liver retinyl palmitate and retinol increased progressively with age, reaching highest levels of approximately 84 and 1 -9 μg/g liver, respectively, at 28 d. In contrast, in animals on the vitamin A-deficient diet, the retinyl palmitate and retinol levels decreased progressively, reaching the lowest levels of approximately 0–32 and 0–09 μg/g, respectively. No significant reduction in retinol was observed in the trachea of animals maintained on the deficient diet for at least 20 d; their tracheas were depleted of retinol at 28 d. No vitamin A-associated differences were, however, observed in the labelling indices, growth fraction or in the morphology of the tracheal epithelium. Both the control and vitamin A-deficient animals gained weight progressively until 36 d of age, although the weight of animals in the latter group remained below those in the former group. These results show that mild-to-severe deficiency of vitamin A had no effects on cell proliferation or tracheal morphology of the hamster. The hyperplasia and squamous metaplasia in the trachea occurs only at an extreme vitamin A-deficiency when the tissue levels of the vitamin are depleted.     

Contribution of vitamin A deficiency to calculogenic risk factors of urine: studies in children.

Studies in experimental animals showed that vitamin A deficiency enhanced the severity of urinary calculi disease. In India, children with low socioeconomic status are the major victims of bladder stone disease, and vitamin A deficiency is also more prevalent among these children. However, no systematic study is available to correlate the vitamin A-deficient status of children with their predisposition to urinary calculi disease. Vitamin A-deficient and normal boys were the subjects of this study. Twenty-four-hour samples of urine were collected from all the children at the beginning of the study and after normalizing the vitamin A status of the deficient children. Important risk factors were estimated in urine. Plasma vitamin A levels were also measured in these children. Among the deficient group, only children with plasma vitamin A levels of 15 micrograms and lower exhibited calcium oxalate crystalluria. Most importantly, abnormal crystalluria was observed in all children whose plasma vitamin A levels were 13 micrograms/dl or less. Compared to normal children the urine of vitamin A-deficient children showed the following changes: (a) reduced concentration of crystal growth inhibitors, namely citrate and glycosaminoglycans; (b) a decline in inhibitory activity toward calcium oxalate crystal growth; and (c) enhanced excretion of high risk factors, namely calcium and oxalate. Correction of vitamin A status normalized the above abnormal properties of urine. The results of this study strongly support the hypothesis that the vitamin A-deficient state is one of the factors that can enhance the risk of urolithiasis in susceptible populations.     

Vitamin A and lipid peroxidation: effect of retinol deficiency

The effect of alimentary vitamin A deficiency on some parameters of lipid peroxidation (LPO) in young rats was studied. It was found that under vitamin A deficiency the content of diene conjugates in liver homogenates and microsomes diminishes, whereas that of malonic aldehyde in small intestinal mucosa, liver and testis homogenates is unaffected. However, the malonic aldehyde production in liver homogenates and microsomes decreases after 60 min incubation at 37 degrees C without addition of prooxidants. At the same time, enzymatic NADPH-dependent and nonenzymatic ascorbate-dependent LPO in liver microsomes of vitamin A-deficient rats does not change significantly. The decrease of LPO intensity in vitamin A-deficient animals may be due to the reduced content in liver microsomes of the main LPO substrates, i.e., arachidonic and linoleic acids, as well as to the decrease of cytochrome P-450 level in rat liver.     

Early effects of feeding excess vitamin A: mechanism of fatty liver production in rats

Oral administration of vitamin A (30,000 IU daily for 2 days) to young rats caused a marked increase in hepatic glycogen, cholesterol, and glycerides, while hepatic phospholipid content remained almost unaltered. In an examination of the pathogenesis of the lipid accumulation, it was found that more glucose-(14)C was incorporated into liver lipids in vitamin A-fed rats, whereas incorporation of glucose-(14)C and dl-glycine-(14)C into liver protein remained unaltered. The increase in glucose-(14)C incorporation was confined to the glyceride-glycerol portion of the lipids; incorporation into liver fatty acids was inhibited. Plasma free fatty acid concentrations were elevated. It is postulated that in the vitamin A-fed rats, increased accumulation of lipids in the liver is caused by a stimulation of fatty acid mobilization from adipose tissue and enhanced formation of glycerophosphate through glycolysis, with consequent increase in the glyceride synthesis in the liver. The weight of the adrenals was increased, whereas cholesterol concentration in the gland was decreased, after administration of vitamin A to rats. This indicates adrenocortical stimulation. Interestingly enough, vitamin A feeding did not affect either the level of liver lipids or of plasma FFA in adrenalectomized rats.     

Impaired blood clearance of bacteria and phagocytic activity in vitamin A-deficient rats

The effect of vitamin A deficiency on the functional integrity of the reticuloendothelial system and the phagocytic capacity of circulating polymorphonuclear leukocytes was evaluated in retinoate-cycled vitamin A-deficient rats under conditions such that secondary dietary imbalances were eliminated. Kinetics of blood clearance of 2 X 10(7) Escherichia coli injected intravenously was depressed within 8 days of the withdrawal of retinoic acid; all animals were profoundly affected by Day 12 of deficiency. In vitro, the phagocytic activity of polymorphonuclear leukocytes was similarly affected; by Day 12 of deficiency, phagocytic capacity in all deficient animals was less than 40% of the appropriate control values (P less than 0.01). Animals rendered vitamin A deficient by this procedure also displayed marked susceptibility to endogenous bacterial infection, as judged from the proportion of deficient rats that spontaneously developed bacteremia during the later stages of deficiency. These data together demonstrate unequivocally that reticuloendothelial and polymorphonuclear leukocytic functions are impaired in vitamin A deficiency in the absence of other dietary imbalances.     

Vitamin A and the biosynthesis of sulphated mucopolysaccharides. Experiments with rats and cultured neoplastic mast cells

1. The uptake and incorporation of [(35)S]sulphate into mucopolysaccharides by colon and duodenum in vitro are unaffected by the vitamin A status of the animals. 2. Uptake and incorporation in vivo are unaffected at 4hr. after injection of [(35)S]sulphate, but at later times are decreased in some tissues of vitamin A-deficient animals. 3. The rate of removal of (35)S from blood, its rate of appearance in urine, the plasma concentration of sulphate and the uronic acid content of several tissues are not significantly altered in vitamin A deficiency. 4. These results, and direct measurement of (35)S in mucopolysaccharides at various times after injection of [(35)S]sulphate, suggest that the synthesis of mucopolysaccharides is unaffected but that their turnover is increased in vitamin A deficiency. 5. Neither the growth rate of, nor the incorporation of [(35)S]sulphate into heparin by, P815Y and HC cultured neoplastic mast cells is decreased when the horse serum necessary for growth is treated with ultraviolet light or is replaced by serum from vitamin A-deficient rats. 6. The addition of citral is no more toxic to growth rate or to incorporation of (35)S than is the addition of vitamin A itself. 7. It is concluded that neoplastic mast cells in culture do not require vitamin A for growth or for the synthesis of heparin. 8. None of these results is compatible with the view that vitamin A or a derivative is directly involved in the biosynthesis of sulphated mucopolysaccharides.     

Composition of myelin lipids and synthesis of 3-ketodihydrosphingosine in the vitamin B 6 -deficient developing rat

Abstract— Recoverable myelin in 20-day-old, Vitamin B₆-deficient rats was reduced from that in control animals. The composition of myelin lipids from deficient animals was nearly identical to that for controls except for a striking reduction in phospholipids polyunsaturated fatty acids. Enzymatic synthesis in vitro of 3-ketodihydrosphingosine was impaired in the deficient animals; addition of the vitamin Bs cofactor to these incubation mixtures restored activity of the condensing enzyme to control levels.     

Changes in mitochondrial and microsomal lipid peroxidation and fatty acid profiles in adrenal glands, testes, and livers from alpha-tocopherol-deficient rats

Studies were done to evaluate the effects of alpha-tocopherol deficiency in rats on the fatty acid composition and sensitivity to lipid peroxidation (LP) of mitochondria and microsomes from adrenal glands, testes, and livers. In control (alpha-tocopherol-sufficient) animals, adrenal concentrations of alpha-tocopherol were approximately 10 times greater than those in livers and testes. Dietary deficiency of alpha-tocopherol for 8 weeks decreased adrenal and hepatic concentrations by 80-90% and testicular concentrations by approximately 60-70%. Incubation of testicular or hepatic mitochondria and microsomes from control rats with FeSO(4) (1.0 mM) caused a time-dependent stimulation of LP as indicated by the formation of thiobarbituric acid reactive substances (TBARS); the rate of TBARS production increased in preparations from alpha-tocopherol-deficient animals. TBARS formation was not demonstrable in adrenal mitochondria or microsomes from alpha-tocopherol sufficient rats, but reached high levels in alpha-tocopherol-deficient preparations. The fatty acid composition of mitochondria and microsomes was tissue-dependent. In particular, arachidonic acid comprised approximately 40% of the total fatty acids in adrenal membranes, but only 20-25% in testes and livers. alpha-Tocopherol deficiency increased oleic acid concentrations in adrenal and hepatic mitochondria and microsomes but not in testes. In all three tissues, linoleic acid concentrations decreased by approximately 50%, but arachidonic acid levels were unaffected by alpha-tocopherol deficiency. The results indicate a close relationship between tissue sensitivity to LP in vitro and alpha-tocopherol concentrations. Nonetheless, any oxidative stress in vivo caused by alpha-tocopherol deficiency seems to spare arachidonic acid in mitochondria and microsomes but decreases linoleic acid concentrations. It is possible that because of the important physiological functions of arachidonic acid, metabolic adaptations serve to maintain membrane content during periods of oxidative stress.     

Effects of vitamin A deficiency on cell proliferation and morphology of trachea of the hamster

Regulation by vitamin A of cell proliferation and differentiation of epithelial tissues is well-established. Deficiency of vitamin A in experimental animals leads to the development of hyperplasia and squamous metaplasia. The objective of the present study was to examine, for young hamsters, the effects of variable levels of the vitamin in the liver and trachea, on cell proliferation and morphology of tracheal epithelium and on body weights. Newly born litters were maintained on vitamin A-supplemented and vitamin A-deficient diets, and various parameters were examined at different ages. Retinol and retinyl palmitate levels were determined by high performance liquid chromatography. For animals on the supplemented diet, concentrations of liver retinyl palmitate and retinol increased progressively with age, reaching highest levels of approximately 84 and 1.9 micrograms g liver, respectively, at 28 d. In contrast, in animals on the vitamin A-deficient diet, the retinyl palmitate and retinol levels decreased progressively, reaching the lowest levels of approximately 0.32 and 0.09 micrograms/g, respectively. No significant reduction in retinol was observed in the trachea of animals maintained on the deficient diet for at least 20 d: their tracheas were depleted of retinol at 28 d. No vitamin A-associated differences were, however, observed in the labelling indices, growth fraction or in the morphology of the tracheal epithelium. Both the control and vitamin A-deficient animals gained weight progressively until 36 d of age, although the weight of animals in the latter group remained below those in the former group. These results show that mild-to-severe deficiency of vitamin A had no effects on cell proliferation or tracheal morphology of the hamster. The hyperplasia and squamous metaplasia in the trachea occurs only at an extreme vitamin A-deficiency when the tissue levels of the vitamin are depleted.