Microsomal fatty acid desaturase activities in vitamin A-deficient rat liver

The activity of microsomal fatty acid delta 9-desaturase was significantly higher in liver microsomes of vitamin A-deficient rats as compared with their controls. Feeding of vitamin A-supplemented control diet to the deficient rats restored the delta 9-desaturase activity to the control values. The activity of delta 6-desaturase was not affected by vitamin A deficiency.     

Metabolism and biological potency of 5,6-monoepoxyvitamin A aldehyde in the rat

1. The metabolism of 5,6-monoepoxyvitamin A aldehyde in the rat was found to be identical with that of vitamin A aldehyde. It promptly alleviated all the symptoms of vitamin A deficiency and promoted the growth of the vitamin A-deficient rats. 2. When administered orally, 5,6-monoepoxyvitamin A aldehyde was reduced to the corresponding alcohol in the intestine and esterified before being transported to the liver for storage. 3. 5,6-Monoepoxyvitamin A aldehyde was not converted into the furanoid form, 5,8-monoepoxyvitamin A aldehyde, during passage through the stomach. 4. Intraperitoneal administration of 5,6-monoepoxyvitamin A aldehyde led to the accumulation of 5,6-monoepoxyvitamin A in the liver and other tissues. Subcutaneous administration of this compound alleviated all the symptoms of vitamin A deficiency. 5. The small intestine is the major, if not the only, site for the metabolic reduction of 5,6-monoepoxyvitamin A aldehyde and its subsequent esterification. 6. It was demonstrated that the rat possesses the necessary enzymes for the reduction and oxidation of 5,6-monoepoxyvitamin A aldehyde to the corresponding alcohol and acid as well as the esterification of 5,6-monoepoxyvitamin A alcohol to its palmitate. These metabolic conversions were shown to be as efficient as those of vitamin A aldehyde and alcohol. 7. 5,6-Monoepoxyvitamin A aldehyde possesses a biological potency 108% that of all-trans vitamin A acetate. 8. A new visual pigment with λ_max. 480mμ, along with natural rhodopsin, was isolated from the retinas of rats maintained on 5,6-monoepoxyvitamin A aldehyde. 9. Oral administration of 5,8-monoepoxyvitamin A aldehyde to vitamin A-deficient rats led to the accumulation of 5,8-monoepoxyvitamin A in the liver and other tissues. Enzymic reduction and oxidation of 5,8-monoepoxyvitamin A aldehyde to its alcohol and acid, as well as the esterification of the alcohol, were demonstrated.     

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.     

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.     

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.     

Effect of retinol deficiency on the activity and solubility of various enzymes of the endoplasmic reticulum membranes in the rat liver

Solubilization by sodium deoxycholate and trypsin of some metabolic enzymes of unrelated compounds associated with endoplasmic reticulum membranes was carried out. The effects of urea, butanol and detergents on the retinol content in the membranes were studied. It was shown that retinol deficiency causes changes in the interactions of NADH-arylesterase with microsomal membrane components that are manifested in the decrease of the activating effect of butanol and low detergent concentrations on the NADH-reductase activity as well as in the increase in the damaging effect of urea and high detergent concentrations on the enzyme activity. Under conditions of retinol deficiency, the degree of solubilization of NADH-reductase, hydroxylase and arylesterase in the presence of sodium deoxycholate is enhanced. After treatment of liver microsomes of retinol-deficient animals with trypsin or with a trypsin-sodium cholate mixture, the content of these enzymes in the supernatant becomes much greater than that in liver microsomes of vitamin A-deficient rats. It is assumed that retinol deficiency causes of weakening of hydrophobic interactions within the membrane as well as partial translocation of the enzymes from the hydrophobic to the hydrophilic layer.     

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.     

Alterations in surface carbohydrates and in some functional properties of liver lysosomal membrane in vitamin A-deficient rat

A significant decrease in total carbohydrates and particularly in mannose, galactose and sialic acid has been observed in vitamin A-deficient rat liver lysosomal membrane. These alterations adversely affect the membrane permeability and structure-linked latency of the lysosomal enzymes.Significant reduction in the pH-dependent in vitro binding of the lysosomal arylsulfatase B to the highly purified membrane has been observed in vitamin A deficiency. This is attributed to the decrease in electro-negativity, mainly due to the observed reduction in negatively-charged sialic acid residues on the outer side of the membrane.Similar reduction in sialic acid content on the inner side of the membrane affects the microenvironment in the lysosomes. Intralysosomal pH, measured by computing the proteolytic activity of lysed lysosomes and of phagolysosomes, endocytosed with denatured ¹³¹I-labelled human serum albumin, is slightly but consistently higher in vitamin A-deficient groups compared to that in control one. This is reflected in the low rate of degradation of the entrapped proteins in vitamin A deficiency.The possible physiological significance of the observations is discussed with special reference to the loss of surface carbohydrates, particularly sialic acid, in vitamin A-deficient rats.     

Effect of vitamin A deficiency on hepatic and intestinal drug metabolizing enzymes in rats

Feeding of vitamin A-deficient diet to male weanling rats for 10 weeks caused significant reduction in the hepatic cytochrome P-450, cytochrome b5, aminopyrine N-demethylase and arylhydrocarbon hydroxylase activities. Contrary to this, the levels of these Phase I enzymes were found to be significantly elevated in all the 3 portions (proximal, middle and distal) of the intestine in deficient animals as compared to corresponding pair-fed controls. Of the Phase II enzymes studied, UDP-glucuronyltransferase showed a significant decrease whereas glutathione S-transferase showed a significant increase in vitamin A-deficient rat liver and small intestine. The study suggests that vitamin A deficiency causes an imbalance between the Phase I and phase II drug metabolizing enzyme systems which may decrease the capacity of the organism to withstand the neoplastic effects of chemical carcinogens in vitamin A deficiency.     

Role of vitamin A in sulphate metabolism: Studies on the enzymic activation of sulphate by various tissue extracts of normal and vitamin A-deficient rats

1. The enzymic activation of sulphate by various tissue extracts of vitamin A-deficient rats and their pair-fed controls was studied. 2. Vitamin A deficiency does not impair the enzymic activity in liver, colon and brain. However, a significant decrease in activity was observed in epiphyseal cartilage.     

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.     

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.     

L-gulonolactone oxidase activity and vitamin C status in riboflavin-deficient rats

The effect of riboflavin deficiency on the activity of L-gulonolactone oxidase [L-gulono-γ-lactone : oxygen 2-oxidoreductase, EC 1.1.3.8] and on vitamin C status was studied. A marked decrease in the specific activity of L-gulonolactone oxidase was observed in the liver microsomes isolated from riboflavin-deficient rats: the specific activity was approx. one-third of that in the microsomes isolated from control rats. The L-ascorbic acid content in the liver of the riboflavin-deficient rats was approx. one-half of that in the liver of the control rats. It seems that the rate of production of L-ascorbic acid in the riboflavin-deficient rats is limited by the decreased level of L-gulonolactone oxidase activity. Immunotitration using rabbit antiserum directed to L-gulonolactone oxidase revealed that a substantial amount of an inactive form of this enzyme is present in the liver microsomes of the riboflavin-deficient rats. L-Gulonolactone oxidase activity in the microsomes of these rats increased by approx. 35% upon addition of FAD, but it was slightly decreased by the addition of FMN or riboflavin. These results indicate that the liver microsomes of the riboflavin-deficient rats contain a protein which exhibits L-gulonolactone oxidase activity upon addition of FAD.     

Tissue distribution and subcellular localization of retinol-binding protein in normal and vitamin A-deficient rats.

Levels of retinol-binding (RBP), the plasma transport protein for vitamin A, were measured by radioimmunoassay in sera and in a large number of tissues from both normal and vitamin A-deficient rats. The tissues included liver, kidney, fat, muscle, brain, eye, salivary gland, thymus, lung, heart, intestine, spleen, adrenal, testes, thyroid, and red blood cells. The RBP levels in tissues other than serum, liver, and kidneys varied from 12 mug/g of tissue for normal spleen to an undetectable level in red blood cells. Much of the RBP in the tissues with low levels may have been due to residual serum in the samples. In general, except for liver, RBP levels were lower in tissues from vitamin A-deficient rats than in those from normal rats. In normal rats, the liver, kidney, and serum levels were 30 plus or minus 4 (mean plus orminus SEM), 151 plus or minus 22, and 44 plus or minus 3 mug/g, respectively. In vitamin A-deficient rats, the liver RBP level was about three times the normal level whereas the kidney and serum levels were about one-fifth the normal values. When normal liver homogenates were fractionated by centrifugation, 67% of the RBP was recovered in the microsomal fraction and only 9% was found in the soluble 105,000 g supernate. In contrast, 76% of the RBP in homogenates of normal kidneys was in the soluble fraction. Similar results were obtained with deficient livers and kidneys. Incubation with deoxycholate released the liver RBP into the soluble fraction. RBP is produced in the liver and removed from the blood by the kidneys. The levels of RBP in normal and deficient liver, serum, and kidney appear to reflect the relative rates of RBP secretion and turnover.     

Lecithin:retinol acyltransferase from mouse and rat liver. CDNA cloning and liver-specific regulation by dietary vitamin a and retinoic acid

Lecithin:retinol acyltransferase (LRAT), present in microsomes, catalyzes the transfer of the sn-1 fatty acid of phosphatidylcholine to retinol bound to a cellular retinol-binding protein. In the present study we have cloned mouse and rat liver LRAT cDNA and tested the hypothesis that LRAT mRNA, like LRAT activity, is regulated physiologically in a liver-specific manner. The nucleotide sequences of mouse and rat liver LRAT cDNA each encode a 231-amino acid protein with 94% similarity between these species, and approximately 80% similarity to a cDNA for LRAT from human retinal pigment epithelium. Expression of rat LRAT cDNA in HEK293T cells resulted in functional retinol esterification and storage. RNA from several rat tissues hybridized with liver LRAT cDNA. However, LRAT mRNA was virtually absent from the liver of vitamin A-deficient animals, while being unaffected in intestine and testis. LRAT mRNA was rapidly induced by retinoic acid (RA) in liver of vitamin A-deficient mice and rats (P < 0.01). LRAT mRNA and enzymatic activity were well correlated in the same livers of rats treated with exogenous RA (r = 0.895, P < 0.0001), and in a dietary study that encompassed a broad range of vitamin A exposure (r = 0.799, P < 0.0001). Liver total retinol of <100 nmol/g was associated with low LRAT expression (<33% of control).We propose that RA, derived exogenously or from metabolism, serves as an important signal of vitamin A status. The constitutive expression of liver LRAT during retinoid sufficiency would serve to divert retinol into storage pools, while the curtailment of LRAT expression in retinoid deficiency would maintain retinol for secretion and delivery to peripheral tissues.     

Change in the metabolism of 7,12-dimethylbenz(a)anthracene under the influence of vitamin A]

The in vitro metabolism of 7,12-dimethylbenz(a)anthracene in its incubation with the liver microsomes, the liver and mammary gland homogenates of rats, kept on vitamin A-enriched diet, was studied. Vitamin A inhibited the formation of lipophilic metabolites and increased the generation of water-soluble metabolites. The amount of lipophilic metabolites extracted from the microsomes and the liver and mammary gland homogenates were decreased by a factor of 2.2 and 5, respectively. The amount of unmetabolized DMBA in the liver microsomes was the same in control and experimental animals.     

Vitamin A deficiency leads to severe functional disturbance of the intestinal epithelium enzymes associated with diarrhoea and increased bacterial translocation in gnotobiotic rats

A disturbance of the integrity of the intestinal epithelium with an increased risk for bacterial translocation is one of the suggested factors underlying the increased incidence of infections and septicaemia during vitamin A deficiency. In the present study the effects of vitamin A deficiency on the enzymic activity of enterocytes in response to bacterial colonization with a non-pathogenic Escherichia coli strain were studied in monocolonized and conventional Wistar rats. The monocolonized, but not the conventional, vitamin A-deficient rats had markedly reduced weight compared to their pair-fed controls and presented neurological symptoms, such as hind leg weakness, tremor and slow gait. Moreover, only in the monocolonized vitamin A-deficient rats were severe diarrhoea and bacterial translocation to extraintestinal sites-mainly kidneys-detected. Measurements of enterocyte brush-border enzyme activities revealed that lactase, sucrase, gamma-glutamyltranspeptidase (GGT) and dipeptidyl peptidase IV (DPP IV) were significantly reduced in the monocolonized vitamin A-deficient rats compared to the pair-fed controls, indicating a severe functional disturbance of the enterocytes. In conventional vitamin A-deficient rats only sucrase activity was markedly lower than in the respective controls. Our observation, that the deficient vitamin A status led to a strong reduction of enterocyte enzymic activities, associated with diarrhoea and increased bacterial translocation, mainly in the gnotobiotic rats, suggests that the composition of the bacterial flora, i.e. the colonization state, has a strong influence on triggering the severity of the functional disturbances of the intestinal epithelium, and adds to the clinical manifestations of vitamin A deficiency.     

Changes in rat liver mitochondrial lipids in vitamin A deficiency

The alterations in the lipid profiles of rat liver mitochondria due to vitamin A deficiency were studied. The amount of total lipids and phospholipids were decreased with a concomitant increase in triglycerides and cholesterol levels in mitochondria, isolated from vitamin A-deficient animals. Of particular significance was the observation that the content of lysolecithin, a potent cytolytic agent, was increased. An analysis of individual fatty acids showed that the percentage of polyunsaturated fatty acids was decreased significantly in vitamin A deficiency. Further, mitochondria from vitamin A-deficient animals, when incubated in 0.1 M Tris-HCl buffer (pH 7.4)in vitro, produced increased amounts of malondialdehyde and lipofuchsin pigments indicating increased susceptibility of the mitochondrial membrane to peroxidative damage. These results suggest a possible role of vitamin A in the prevention of the decomposition of structural lipids.     

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.