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.     

Increased methionine synthetase activity in zinc-deficient rat liver

To study the effect of zinc deficiency on folate metabolism, three groups of male Sprague-Dawley rats (zinc deficient (ZD), restricted-fed (RF + Zn), and ad libitum-fed control (control] were given a semipurified 25% egg white protein diet. The ZD group received less than 10.3 nmol zinc/g of diet, while the RF + Zn and control groups were given 1620 nmol zinc/g of diet. After 6-7 weeks of feeding, severe zinc deficiency developed in ZD rats. Hepatic methionine synthetase activity was increased in the ZD group compared to both the RF + Zn and control groups, but hepatic 5,10-CH2-H4folate reductase activity was similar in all groups. This increased methionine synthetase activity found in zinc-deficient rats might induce secondary alterations in folate metabolism. These changes include significantly lowered plasma folate levels, decreased 5-CH3-H4folate in liver, and increased rates of histidine and formate oxidation. The latter two findings suggest that the available non-5-CH3-H4folate is increased in zinc deficiency.     

The effect of methionine on the metabolism of serine and glycine in vitamin B-12-deficient rats.

The effect of methionine supplementation on glycine and serine metabolism was studied in vitamin B-12-deficient rats which received only 0.2% methionine in the diet. In the perfused liver, incorporation of the C-2 of glycine to the C-3 of serine was increased by addition of methionine to the perfusate. The oxidation of [1-14C]glycine to 14CO2 was however depressed. Unlike methionine, glycine did not have any significant effect on the liver folate coenzyme distribution. Oxidation of [3-14C]serine to 14CO2 both in vivo and in perfused liver was increased by methionine. A major portion of the C-3 radioactivity however was recovered in glucose. Data presented indicate that the rate of oxidation of [2-14C]histidine to 14CO2 is a more sensitive indicator of folate deficiency than the rate of oxidation of [3-14C]serine to 14CO2 although both are presumably tetrahydrofolate dependent.     

Vitamin A deficiency induces prooxidant environment and inflammation in rat aorta

We evaluated whether nutritional vitamin A deficiency generates oxidative stress and inflammation in aorta. Wistar male rats (21 days old) were given free access to a control (8 mg retinol as retinyl palmitate/kg) or a vitamin A- deficient diet for three months. One group of deficient animals was fed with the control diet fifteen days before sacrifice. Thiobarbituric acid-reactive substances (TBARS) and nitrite concentration where both analyzed in serum and aorta. Aorta Copper-Zinc Superoxide dismutase (CuZnSOD), Glutathion peroxidase (GPx) and Catalase (CAT) activities were measured. In addition, binding activity of the nuclear factor- kB (NF-kB), inducible and endothelial Nitric Oxide synthase (iNOS and eNOS, respectively) and Ciclooxygenase-2 (COX-2) expressions were determinated in aorta. Rats fed the vitamin A- deficient diet were characterized by sub-clinical plasma retinol concentration and showed increased serum and aorta concentrations of TBARS compared to controls. Lower than control activities of CuZnSOD, GPx, and CAT were observed in aorta of the vitamin A- deficient group. The binding activity of NF- kB was higher in vitamin A- deficient animals than controls. In addition, NO production evaluated as nitrite concentration increased in aorta and serum, associated with a higher expression of iNOS, eNOS and COX-2 in aorta of vitamin A-deficient rats. The incorporation of vitamin A into the diet of vitamin A-deficient rats reverted the changes observed in TBARS level, CuZnSOD and GPx activities, nitrite concentration and also, iNOS, eNOS and COX-2 expression. Prooxidant environment and inflammation are induced by vitamin A deficiency in rat aorta.     

Modification of hepatic folate metabolism in rats fed excess retinol

Feeding rats a diet containing 1000 IU of retinol/g diet enhances the folate-dependent oxidation to CO2 of formate and histidine. The activity of hepatic methylenetetrahydrofolate reductase, which plays a critical role in the regulation of liver folate metabolism, is suppressed in these animals, resulting in decreased 5-methyltetrahydrofolate synthesis. This ensures a greater concentration of hepatic tetrahydrofolate, the coenzyme on which formate and histidine oxidation depend, but also compromises the level of S-adenosylmethionine in the liver.     

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.     

Folate and homocysteine metabolism in copper-deficient rats.

To investigate the effect of copper deficiency on folate and homocysteine metabolism, we measured plasma, red-cell and hepatic folate, plasma homocysteine and vitamin B-12 concentrations, and hepatic methionine synthase activities in rats. Two groups of male Sprague-Dawley rats were fed semi-purified diets containing either 0. 1 mg (copper-deficient group) or 9.2 mg (control group) of copper per kg. After 6 weeks of dietary treatment, copper deficiency was established as evidenced by markedly decreased plasma and hepatic copper concentrations in rats fed the low-copper diet. Plasma, red-cell, hepatic folate, and plasma vitamin B-12 concentrations were similar in both groups, whereas plasma homocysteine concentrations in the copper-deficient group were significantly higher than in the control group (P<0.05). Copper deficiency resulted in a 21% reduction in hepatic methionine synthase activity as compared to the control group (P<0.01). This change most likely caused the increased hepatic 5-methyltetrahydrofolate and plasma homocysteine concentrations in the copper-deficient group. Our results indicate that hepatic methionine synthase may be a cuproenzyme, and plasma homocysteine concentrations are influenced by copper nutriture in rats. These data support the concept that copper deficiency can be a risk factor for cardiovascular disease.     

The effect of vitamin A deficiency on hepatic, renal and pulmonary glutathione S-transferase activities in the rat.

Feeding male weanling rats on a vitamin A-deficient diet for 6 weeks resulted in significant increases (44-57%) in glutathione S-aryl-, S-aralkyl- S-alkyl- and S-epoxidetransferase activities in the liver cytosol. Only the S-aralkyl- (27%) and S-alkyltransferase (14%) activities were significantly increased in the kidney as a result of deficiency. There was no effect on any of the pulmonary glutathione S-transferase activities. The increases in hepatic transferase activities were due primarily to increases (25-96%) in the apparent Vmax. There were no changes in the apparant Km of any of the four drug substrates employed. With 3,4-dichloronitrobenzene as the second substrate, the apparent Km for glutathione was increased by over 2-fold in vitamin A-deficient livers as compared with controls. The relationship between these results and enhanced susceptibility to chemical carcinogens in vitamin A deficiency is briefly discussed, and comparison is made between the effects of this nutritional state and pretreatment with drug inducers on the glutathione S-transferases.     

Regulation of folate-mediated one-carbon metabolism by 10-formyltetrahydrofolate dehydrogenase

10-Formyltetrahydrofolate dehydrogenase (FDH) catalyzes the NADP(+)-dependent conversion of 10-formyltetrahydrofolate to CO(2) and tetrahydrofolate (THF) and is an abundant high affinity folate-binding protein. Although several activities have been ascribed to FDH, its metabolic role in folate-mediated one-carbon metabolism is not well understood. FDH has been proposed to: 1) inhibit purine biosynthesis by depleting 10-formyl-THF pools, 2) maintain cellular folate concentrations by sequestering THF, 3) deplete the supply of folate-activated one-carbon units, and 4) stimulate the generation of THF-activated one-carbon unit synthesis by channeling folate cofactors to other folate-dependent enzymes. The metabolic functions of FDH were investigated in neuroblastoma, which do not contain detectable levels of FDH. Both low and high FDH expression reduced total cellular folate concentrations by 60%, elevated rates of folate catabolism, and depleted cellular 5-methyl-THF and S-adenosylmethionine levels. Low FDH expression increased the formyl-THF/THF ratio nearly 10-fold, whereas THF accounted for nearly 50% of total folate in neuroblastoma with high FDH expression. FDH expression did not affect the enrichment of exogenous formate into methionine, serine, or purines and did not suppress de novo purine nucleotide biosynthesis. We conclude that low FDH expression facilitates the incorporation of one-carbon units into the one-carbon pool, whereas high levels of FDH expression deplete the folate-activated one-carbon pool by catalyzing the conversion of 10-formyl-THF to THF. Furthermore, FDH does not increase cellular folate concentrations by sequestering THF in neuroblastoma nor does it inhibit or regulate de novo purine biosynthesis. FDH expression does deplete cellular 5-methyl-THF and S-adenosylmethionine levels indicating that FDH impairs the folate-dependent homocysteine remethylation cycle.     

Testicular atrophy, zinc concentration, and angiotensin-converting enzyme activity in the testes of vitamin a-deficient rats

Angiotensin-converting enzyme (ACE) as a part of the renin angiotensin system (RES) regulates blood pressure and fluid and electrolyte homeostasis, and the enzyme is considered to have a function in reproduction. Reduced enzyme activities have been observed in atrophied testes as a results of zinc and pituitary deficiencies. Vitamin A deficiency causes atrophy of testes. The present study was conducted on three groups of male, 3-wk-old, Wistar rats. After 54 d of the experimental period, testicular weights of the vitamin A-deficient rats (Agroup, allowed free access to vitamin Adeficient diet) was significantly lower than its pair-fed, PF (given restricted amount control diet) and A+ (allowed free access to control diet) groups. Zinc concentrations and both soluble and particulate ACE activities in the testes of vitamin A-deficient rats (Agroup) were significantly lower than the other two groups. No significant differences were observed regarding zinc concentration, particulate ACE, and total ACE activities in the testes of PF and A+ groups. Vitamin A deficiency did not significantly affect the enzyme activity in the lung. From the observations of the present study, we speculate that testicular atrophy in vitamin A deficiency may have resulted from lower zinc concentration and decreased ACE activity in that organ.     

Effect of Folate Deficiency on Local Cerebral Glucose Utilization in the Rat

There is considerable debate on the role of folate in CNS function. Recent work indicates that folate deficiency may affect CNS serotonin metabolism, and clinical studies describe many consequences of such a deficiency. On the other hand some workers maintain that folate deficiency alone causes CNS abnormalities. We maintained rats, through dietary deprivation, at folate levels below 4 ng/ml for more than 6 weeks and showed that at that time both their liver and brain folate levels were significantly reduced. We then studied their local cerebral glucose utilization (LCGU) using the [14C]deoxyglucose technique. This method assesses cerebral function by measuring regional metabolic activity. We also determined LCGU in rats given the same diet but replenished with folate (folate control) and in others given free access to commercially available food (normal controls). Our results show that this degree of folate deficiency has no effect on cerebral function. This contrasts with the focal suppression of LCGU we previously reported in a model of vitamin B12 deficiency.     

Studies on the production and assessment of experimental histidinemia in the rat.

Intraperitoneal administration to rats of D- or DL-alpha-hydrazinoimidazolylpropionic acid was found to produce a substantial inactivation of hepatic histidine ammonia-lyase (EC 4.3.1.3) in vivo. Proportional to this loss in enzyme activity was an impairment of the ability of treated rats to oxidize L-[ring-2-14C] histidine to 14CO2. Rats in which hepatic histidine ammonia-lyase activity was either depressed by DL-hydrazinoimidazolylproprionic acid injection or elevated by feeding a high protein diet displayed proportionately altered rates of 3H2O release into plasma water following L-[3-3H] histidine administration. Plasma L-histidine clearance following loading with this amino acid was similarly affected by these treatments. Administration of DL-alphal-hydrazinoimisazolylproprionic acid to rats was also found to inactivate non-specifically pyridoxal 5-phosphate enzymes in vivo; pyridoxine injection was found to reverse the DL-alpha-hydrazinoimidazolylproprionic acid-induced inactivation of hepatic aspartate aminotransferase (EC 2.6.1.1) in vivo, but not that of hepatic histidine ammonia-lyase. These findings demonstrate that histidine ammonia-lyase is the rate-limiting factor in L-histidine degradation in the rat. The potential usefulness of DL-hydrazinoimidazolylproprionic acid in the production of an animal model for histidinemia (hereditary histidine ammonia-lyase deficiency) is discussed.     

The role of histidine in the anemia of folate deficiency

The amino acid histidine is metabolized to glutamic acid in mammalian tissue. Formiminoglutamic acid (FIGLU) is an intermediary in this reaction, and tetrahydrofolic acid is the coenzyme that converts it to glutamic acid. A test for folate deficiency concerns the measurement of urinary FIGLU excretion after a histidine load. It was observed that folate-deficient individuals receiving the histidine for the FIGLU test made hematological response that alleviated the anemia associated with this deficiency. This was unusual in that a biochemical test to determine the deficiency results in a beneficial effect for one aspect of the deficiency. The studies reported in this paper give a metabolic explanation for this phenomenon. Urine was collected for 24 hr from 25 folate-deficient subjects, 10 vitamin B(12)-deficient subjects, and 15 normal controls. Urinary excretion of histidine was a mean of 203 mg with a range of 130-360 mg for the folate-deficient subjects; 51.5 mg with a range of 30-76.6 mg for normal subjects; and 60.0 mg with a range of 32.3-93.0 mg for the vitamin B(12)-deficient subjects. All the folate-deficient subjects subsequently made a hematological response to the histidine administered for the FIGLU test. No hematological response was observed in the vitamin B(12)-deficient individuals. When folic acid was given to folate-deficient subjects who received no histidine, urinary histidine levels returned to normal levels rapidly and this was followed by a hematological response. Others have shown that volunteers fed a histidine-free diet developed anemia. In normal subjects, histidine is excreted much more in the urine than other essential amino acids are. Hemoglobin protein contains 10% histidine. Under normal conditions, dietary histidine can supply sufficient histidine to prevent anemia. When the dietary intake is diminished or the urinary excretion is greatly increased, anemia results. It is concluded that folate deficiency causes histidine depletion through increased urinary excretion of this amino acid. Feeding histidine replenishes tissue levels of histidine, resulting in hemoglobin regeneration. Folic acid administration results in return of histidine to normal urinary levels. Thus, a combination of folic acid histidine would be beneficial for folate deficient individuals.     

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.     

Dietary nickel and folic acid interact to affect folate and methionine metabolism in the rat

A previous experiment using rats indicated that dietary nickel (Ni), folic acid, and their interaction affected variables associated with one-carbon metabolism. That study used diets that produced only mild folate deficiency. Thus, an experiment was performed to determine the effect of a severe folate deficiency on nickel deprivation in rats. A 2×2 factorially arranged experiment used groups of six weanling Sprague-Dawley rats. Dietary variables were nickel, as NiCl₂·6H₂O, 0 or 1 μg/g and folic acid, 0 or 4 mg/kg. All diets contained 10 g succinylsulfathiazole/kg to suppress microbial folate synthesis. The basal diet contained <20 ng Ni/g. After 50 d, an interaction between nickel and folate affected the urinary excretion of formiminoglutamic acid (FIGLU) and the liver concentration of S-adenosylmethionine (SAM). Because of this, it is proposed that the physiological function of nickel is related to the common metabolism shared by SAM and FIGLU. Possibly the physiological function of nickel could be related to the tissue concentration of 5-methyltetrahydrofolate (MTHF) or tetrahydrofolate (THF).     

Metabolic responses of folic acid and related compounds to thyroxine in rats

This study deals with the effects of thyroidectomy and feeding thyroid powder on histidine and folic acid metabolism. Normal rats maintained on a soy protein diet, low in methionine but supplemented with vitamin B-12, oxidize approx. 10% of an injected dose of [2-¹⁴C]histidine in 3 h and excrete low levels of formiminoglutamic acid. Addition of methionine increases histidine oxidation to approx. 20%. The feeding of thyroid powder or the injection of high levels of thyroxine decreases histidine oxidation and increases formiminoglutamic acid excretion. Surgical thyroidectomy at weaning increases histidine oxidation to approx. 45% and, thus, resembles the effect of methionine in promoting histidine oxidation and decreasing formiminoglutamic acid excretion. The feeding of methionine to the thyroidectomized animal further increases histidine oxidation to 65%. The distribution of folate forms in the liver was determined by column chromatography following administration of a dose of tritiated folic acid. In the normal animal, tetrahydrofolate accounts for 38% of the total folate present. The feeding of methionine increases this to 48%, which is consistent with the observed increase in histidine metabolism. Thyroidectomy increases the percentage of tetrahydrofolate to 63% and the feeding of methionine further increases it to 68%. The percentage of tetrahydrofolate relative to total folate is in proportion to the observed rate of histidine metabolism. The action of thyroidectomy in increasing histidine oxidation may be accounted for by its effect in increasing the proportion of tetrahydrofolate.     

Neurological disorder and excessive accumulation of calcium in brain of clinically vitamin A-deficient rats

Three groups of rats were fed two types of synthetic diets for 52 d. The—A group was allowed free access to a vitamin A-deficient diet and showed classical signs of vitamin A deficiency. The brain was the only organ in our experiment where no significant weight difference was present among the three groups. In the brain, calcium concentration was significantly higher in the—A group when compared with the PF (Pair-fed; allowed restricted amount of control diet) and +A groups (allowed free access to control diet). In the tibia, calcium and magnesium concentrations were significantly lower in the—A group when compared with other two groups. Excessive accumulation of calcium in brain and apparently similar unbalance in bone, mineral concentration were observed in central nervous system (CNS) degenerative diseases. Our results suggest that abnormal metabolism of calcium and magnesium in some tissues and excessive accumulation of calcium in brain may be responsible for the development of neurological disorders in vitamin A-deficient rats.     

Biliary metabolites of all-trans-retinoic acid in the rat

Biliary metabolites from physiological doses of all-trans-[10-3H]retinoic acid were examined in normal and vitamin A-deficient rats. The bile from normal and vitamin A-deficient rats contained approximately 60% of the administered dose following a 24-h collection period. However, vitamin A-deficient rats show a 6-h delay in the excretion of radioactivity compared to normal rats. Retinoyl-beta-glucuronide excretion was particularly sensitive to the vitamin A status of the rats. In normal rats, retinoyl-beta-glucuronide reached a maximum concentration of 235 pmol/ml of bile 2 h following the dose and then rapidly declined. Vitamin A-deficient rats show a relatively constant concentration of this metabolite (100-150 pmol/ml of bile) over a 10-h collection period. Retinoic acid excretion was low in both normal and deficient rats. The concentration of retinotaurine, a recently identified biliary metabolite, was approximately equal to retinoyl-beta-glucuronide in normal rats and appeared in the bile 2 h later than the glucuronide.     

Vitamin A deficiency modulates iron metabolism via ineffective erythropoiesis

Vitamin A modulates inflammatory status, iron metabolism and erythropoiesis. Given that these factors modulate the expression of the hormone hepcidin (Hamp), we investigated the effect of vitamin A deficiency on molecular biomarkers of iron metabolism, the inflammatory response and the erythropoietic system. Five groups of male Wistar rats were treated: control (AIN-93G), the vitamin A-deficient (VAD) diet, the iron-deficient (FeD) diet, the vitamin A- and iron-deficient (VAFeD) diet or the diet with 12 mg atRA/kg diet replacing all-trans-retinyl palmitate by all-trans retinoic acid (atRA). Vitamin A deficiency reduced serum iron and transferrin saturation levels, increased spleen iron concentrations, reduced hepatic Hamp and kidney erythropoietin messenger RNA (mRNA) levels and up-regulated hepatic and spleen heme oxygenase-1 gene expression while reducing the liver HO-1 specific activity compared with the control. The FeD and VAFeD rats exhibited lower levels of serum iron and transferrin saturation, lower iron concentrations in tissues and lower hepatic Hamp mRNA levels compared with the control. The treatment with atRA resulted in lower serum iron and transferrin concentrations, an increased iron concentration in the liver, a decreased iron concentration in the spleen and in the gut, and decreased hepatic Hamp mRNA levels. In summary, these findings suggest that vitamin A deficiency leads to ineffective erythropoiesis by the down-regulation of renal erythropoietin expression in the kidney, resulting in erythrocyte malformation and the consequent accumulation of the heme group in the spleen. Vitamin A deficiency indirectly modulates systemic iron homeostasis by enhancing erythrophagocytosis of undifferentiated erythrocytes.     

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.