Survival of germfree rats without vitamin A.

Weanling, germfree rats, transferred to a conventional animal room and fed a vitamin A-deficient diet, died in 23 to 54 days. In contrast, their littermates, kept germfree and on the same diet, survived for as long as 272 days. The rats kept in germfree conditions stopped growing after 1 to 4 months but responded to supplements of retinoic acid.

The occurrence and effects of human vitamin E deficiency. A study in patients with cystic fibrosis.

The role of vitamin E in human nutrition was studied by investigation of patients with cystic fibrosis (CF) and associated pancreatic insufficiency. Vitamin E status was assessed by measurement of the plasma concentration of the principal circulating isomer, alpha-tocopherol. Results of such determinations in 52 CF patients with pancreatogenic steatorrhea revealed that all were deficient in the vitamin. The extent of decreased plasma tocopherol varied markedly but correlated with indices of intestinal malabsorption, such as the serum carotene concentration and percentage of dietary fat absorbed. Supplementation with 5-10 times the recommended daily allowance of vitamin E in a water-miscible form increased the plasma alpha-tocopherol concentrations to normal in all 19 CF patients so evaluated. Studies on the effects of vitamin E deficiency focused on possible hematologic alterations. An improved technique was developed to measure erythrocyte hemolysis in vitro in the presence of hydrogen peroxide. While erythrocyte suspensions from control subjects demonstrated resistance to hemolysis during a 3-h incubation, all samples from tocopherol-deficient CF patients showed abnormal oxidant susceptibility, evidenced by greater than 5% hemoglobin release. The degree of peroxide-induced hemolysis was related to the plasma alpha-tocopherol concentration in an inverse, sigmoidal manner. The possibility of in vivo hemolysis was assessed by measuring the survival of (51)Cr-labeled erythrocytes in 19 vitamin-E deficient patients. A moderate but statistically significant decrease in the mean (51)Cr erythrocyte half-life value was found in this group. Measurement of erythrocyte survival before and after supplementation of 6 patients with vitamin E demonstrated that the shortened erythrocyte lifespan could be corrected to normal with this treatment. Other hematologic indices in deficient subjects, however, were normal and did not change upon supplementation with vitamin E. It is concluded that CF is invariably associated with vitamin E deficiency, provided that the patient in question has pancreatic achylia and is not taking supplementary doses of tocopherol. Concomitant hematologic effects consistent with mild hemolysis, but not anemia, occur and may be reversed with vitamin E therapy. Patients with CF should be given daily doses of a water-miscible form of vitamin E to correct the deficiency.

Rat liver alpha-tocopherol binding protein.

1. The properties of rat liver cytoplasmic alpha-tocopherol binding protein have been studied. 2. The binding protein sedimented in the 3 S region of sucrose density gradients, and gel filtration indicated an approximate molecular weight of 30 500. 3. Of the tissues examined by the present assay, binding was detectable only in the liver. 4. Optimal binding was achieved by incubation at 26 degrees C for 4 h and was independent of pH between 7.4 and 9.0. 5. Pronase completely abolished binding. The binding protein was, however, almost completely resistant to trypsin, and unaffected by RNAase, DNAase, triacylglycerol lipase, and phospholipase C. 6. A variety of tocopherol analogues and other lipid-soluble compounds were tested for their ability to compete for binding. Only alpha-tocopherol and to a lesser extent alpha-tocotrienol and gamma-tocopherol exhibited competition. alpha-Tocopherol acetate, alpha-tocopherol quinone and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid had no effect on binding. 7. Tocopherol binding was reversible, and the tocopherol was not metabolized during incubation.

Are the recommended allowances for dietary antioxidants adequate?

Recommended dietary allowances are standards for maintaining health. Claims that intakes substantially above the allowances may provide protection from xenobiotics and prevent diseases, including cancer, are examined critically for five nutrients that have antioxidant potential. Major criticism is directed at the failure of epidemiologists to recognize that for many of these nutrients, metabolic differences among individuals preclude a direct relationship between dietary intake and plasma or tissue concentration. Also, the fact that no differences in disease incidence have been described within various species of animals that have markedly different metabolic patterns for some of these nutrients has not been considered. It is concluded that the experimental and epidemiological evidence to data that increased intakes of certain nutrients will have beneficial effects on human health are tenuous.

Tocopherols and polyunsaturated fatty acids in human tissues.

Five tissues from three adults who died suddenly and three adult cancer victims were analyzed for alpha- and gamma-tocopherols and fatty acids. When compared with two reports in 1949 and 1958, the three presumably normal subjects had alpha-tocopherol concentrations in liver, muscle and adipose tissue in about the same range as earlier, but heart was twice as high and lung was three times higher than earlier. The content of gamma-tocopherol in all tissues was considerably higher than in 1949 or 1958. Tissues from cancer patients were no lower in alpha-tocopherol than tissues from normal subjects. Adipose tissue from three of the six subjects contained linoleic acid exceeding 15 per cent of total fatty acids. Tocopherols in these subjects, expressed on a tissue weight or tissue lipid basis, were not remarkable. The molar ratio of polyunsaturated fatty acids to alpha-tocopherol in heart and lung was calculated and its possible use in evaluating vitamin E status is discussed.

Megavitamin E supplementation in man.

To assess possible toxic and/or beneficial effects of vitamin E supplementation, a group of 28 adults voluntarily ingesting 100 to 800 IU/day of tocopherol for an average of 3 years were evaluated in this study. Half of the subjects claimed a feeling of improved health or well being, but no specific beneficial effects were noted consistently; the other half indicated no change in health status after beginning vitamin E supplements. No gross evidence of toxicity was apparent on reviewing past medical histories with the subjects. Plasma alpha-tocopherol was found to be elevated significantly in the group from 650 micrograms/100 ml (control mean) to 1,340 micrograms/100 ml; however, 25% of the values were within 2 SD of the control mean. Plasma alpha-tocopherol levels did not correlate with total daily dose but did relate to plasma triglyceride and cholesterol concentrations. Total plasma carotenoids were also significantly increased along with vitamin A levels; the former did not correlate with plasma vitamin E, whereas the latter showed a significant correlation. Laboratory screening for toxic side effects of vitamin E supplementation by performance of 20 standard clinical blood tests failed to reveal any disturbance in liver, kidney, muscle, thyroid gland, erythrocytes, leukocytes, coagulation parameters, or blood glucose. It is concluded that megavitamin E supplements in this group produced no apparent toxic side effects and that subjective claims for beneficial effects were highly variable.

Expressing dietary values for fat-soluble vitamins: changes in concepts and terminology.

The 9th ed. of the Recommended Dietary Allowances has abandoned the use of international units for expressing the food values for vitamins A, D, and E, and has set forth expressions based on absolute weights, including vitamin K. These changes will necessitate a revision in the manner in which both experimental and applied nutritionists deal with these vitamins. In this review, the background and rationale for these changes are presented in an effort to aid investigators in their transition to this new concept. Changes in the nomenclature of vitamin E are also explained.

Tissue storage of vitamins A and E in rats drinking or infused with total parenteral nutrition solutions.

The total parenteral nutrition (TPN) rat and its sham-operated control were used as a model to compare the metabolism and storage of vitamins A and E when they are administered intravenously or orally. Male Fisher rats were depleted of both vitamins for several months with a diet free of vitamins A or E, but containing retinoic acid for growth. TPN solutions containing aqueous dispersions of retinol, retinyl palmitate and dl-alpha-tocopheryl acetate were infused at 2.3 ml/h into the jugular veins of 10 TPN rats. Eight sham-operated control rats drank similar volumes from food cups. TPN rats received 115.3 +/- 4.5 (mean +/- SEM) micrograms of retinol equivalents and 2.2 +/- 0.2 mg of alpha-tocopherol equivalents per day; controls received 146.4 +/- 16.5 micrograms and 2.1 +/- 0.3 mg, respectively. After 7 days the animals were fasted overnight and killed. Plasma levels of retinol were 27.8 +/- 1.5 micrograms/dl for TPN rats, and 27.4 +/- 1.2 for controls. Plasma alpha-tocopherol was 1909 +/- 183 micrograms/dl for TPN rats and 1063 +/- 77 for controls. The only forms of the vitamins found in plasma after overnight fasting were unesterified retinol and unesterified alpha-tocopherol. Sham-operated control rats stored amounts of vitamins A and E similar to values reported in the literature for fed animals. TPN rats stored more of both vitamins than controls in liver, heart, and spleen, but not in testes. The enhanced liver vitamin storage by TPN rats did not appear to be due to a slight increase in lipid content. The results indicate that both vitamins A and E infused in TPN solutions maintain blood levels and are stored in tissues.

Factors affecting the exchange of tocopherol between red blood cells and plasma.

The purpose of this study was to clarify factors that affect the equilibrium distribution of alpha-tocopherol between red blood cells and plasma. Plasma labeled with 14C-alpha-tocopherol was incubated with red cells for 4 to 6 hr and the distribution of the tocopherol determined in the two compartments (red blood cell (RBC: plasma ratio). Use of heparin as anticoagulant gave a higher RBC: plasma ratio than acid citrate dextrose. The RBC:plasma ratio was affected by the hematocrit of the incubation mixture, higher ratios being obtained with lower hematocrits. The ratio was not related to the plasma concentration of alpha-toxopherol, indicating that the red cell was not saturated up to four times normal plasma levels. Of possible clinical significance was the finding that the RBC: plasma ratio was related to the total lipid concentration of plasma. Red cell content of alpha-tocopherol decreased as plasma lipids increased, until at three times normal plasma lipid concentration the red cells had one-third or less of their normal concentration of alpha-tocopherol. The implications of this observation on the relationship between plasma and tissue aplha-tocopherol are discussed.

Simultaneous determination of alpha-tocopherol and retinol in plasma or red cells by high pressure liquid chromatography.

This paper describes a rapid, microprocedure for the simultaneous determination of alpha-tocopherol (vitamin E) and retinol (vitamin A) in plasma, and of alpha-tocopherol alone in red cells since cells do not contain retinol. A total lipid extract from 0.1 ml plasma or 0.125 ml red cells and containing internal standards of alpha-tocopheryl acetate and retinyl acetates is injected onto a high pressure liquid chromatography with a reverse phase column developed with methanol-water. An ultraviolet detector with 280-nm filter is used. The chromatogram is complete in 8 min and the alpha-tocopherol and retinol are quantitated by the peak height ratio method. Comparison of results with both plasma and red cells gave excellent agreement with conventional methods for these vitamins. The procedure should be particularly useful for clinical studies and nutrition surveys.

Plasma carotenoids in normal men after a single ingestion of vegetables or purified beta-carotene.

Changes in seven plasma carotenoids were measured in 30 men for 11 d after ingesting a single dose of pure beta-carotene or a high carotenoid vegetable. A controlled, low-carotenoid diet was fed in a crossover design. Maximum plasma concentrations of beta-carotene occurred 24-48 h after dosing with beta-carotene (12 or 30 mg) or carrots (270 g). A large intake of broccoli (600 g) or tomato juice (180 g) did not change any plasma carotenoids. We concluded that 1) normal subjects vary widely, three to fourfold, in efficiency of carotenoid absorption; 2) peak plasma response to beta-carotene in a capsule occurs at 24-48 h; 3) a large single intake of carrots produces a small increase in plasma beta-carotene but single intakes of broccoli or tomato juice do not change plasma carotenoids; and 4) plasma response to pure beta-carotene is greater than the response to a similar amount of beta-carotene in carrots.

Plasma carotenoid response to chronic intake of selected foods and beta-carotene supplements in men.

We determined serial changes in four major plasma carotenoid fractions (alpha-carotene, beta-carotene, lutein/zeaxanthin, and lycopene) in 30 men consuming defined daily doses of carotenoids from foods (broccoli, carrots, or tomato juice) or from purified beta-carotene in capsules (12 or 30 mg) for 6 wk while fed a controlled diet. Compared with baseline, beta-carotene increased in the 30- and 12-mg-capsule and carrot groups whereas alpha-carotene increased in the carrot group and lutein increased in the broccoli group. Lower lutein concentrations in recipients of beta-carotene capsules suggested an interaction between these two carotenoids. Lycopene declined in all groups except the tomato-juice group. Total carotenoid concentration changes only reflected the large increases in beta-carotene concentrations and not the smaller changes observed in other individual carotenoids. Overall, purified beta-carotene produced a greater plasma response than did similar quantities of carotenoids from foods sources. However, some foods increased plasma concentrations of certain carotenoids.

Lipofuscin accumulation resulting from senescence and vitamin E deficiency: spectral properties and tissue distribution.

The corrected fluorescence emission spectra and tissue distributions of the autofluorescent pigments which accumulate during normal aging and as a consequence of vitamin E deficiency were studied in albino rats. In the retinal pigment epithelium, both the age-related pigment (lipofuscin) and the pigment related to vitamin E deficiency had essentially identical emission spectra. Peak emission occurred from 590 to 650 nm. Young animals which had been kept on a vitamin E deficient diet for 17 weeks after weaning showed significant accumulations of autofluorescent pigment in uterus, duodenum, and retinal pigment epithelium, but not in the spinal cord or inferior olivary nucleus of the brain. Old animals (96 weeks) fed a commercial diet with adequate vitamin E had accumulated lipofuscin in the retinal pigment epithelium, spinal cord gray matter, and inferior olivary nucleus, but not in the duodenum or uterus. Thus, while the auto-fluorescent pigments related to aging and vitamin E deficiency have similar properties, their tissue distributions are quite different.

Effects of moderate-intensity light on vitamin A-deficient rat retinas.

The effects of moderate-intensity light (150 to 200 ft-cd) on retinal structure were compared between retinol-adequate and retinol-deficient rats after 1 to 6 days of light exposure during the 12 hr light phase of the cycle. Both damage to the outer segments and loss of photoreceptor cells were accelerated in retinol-adequate rats. Outer segments in retinas of retinol-adequate rats showed an abnormal staining pattern and disruption of disc structure in the distal portion about 2 days before those of retinol-deficient rats. After 4 days of exposure 24% of the photoreceptor cells had degenerated in the retinol-adequate retinas, but only 6% in the retinol-deficient retinas. By 6 days 65% and 41% of the photoreceptors had degenerated in the retinol-adequate and retinol-deficient retinas, respectively. Thus light exposure induced more rapid degeneration of photoreceptor cells in rats receiving adequate retinol than in those deficient in this vitamin.

Deficiencies of vitamins E and A in the rat. Retinal damage and lipofuscin accumulation.

The interrelationships of vitamins E and A in maintaining various structural components of the retina were studied in four groups of weanling female rats fed purified diets adequate or deficient in each vitamin: +E, +A; -E, +A; +E, -A; and -E, -A. Groups deficient in retinol (-A) were supplemented with retinoic acid. After 14, 21, and 35 weeks, the retinas were examined histologically and ultrastructurally. At 35 weeks, the doubly deficient rats (-E, -A) had lost 92% of their rod nuclei, whereas rats deficient in vitamins A (+E, -A) or E (-E, +A) alone had lost only 34% and 20%, respectively. Vitamin E deficiency resulted in extensive lipofuscin deposits in the retinal pigment epithelium as early as 21 weeks, but the presence of vitamin A doubled the number of lipofuscin granules (-E, +A vs. -E, -A) and induced an even greater increase in their autofluorescence. Another clear influence of vitamin A was seen when +E, +A retinas autofluoresced not only much more than +E, -A retinas, which had similar numbers of granules, but also more than -E, -A retinas, which had about twice as many lipofuscin granules. In the retina, unlike the uterus, the lipofuscin-specific autofluorescence and lipofuscin granule number were not proportional. Moreover, the numbers of granules were influenced by both vitamins E and A, whereas the intensity of lipofuscin-specific autofluorescence was determined almost exclusively by vitamin A. Probably the accelerated loss of photoreceptor cells in -E, -A retinas resulted from both oxidation of membranes and oxidation of retinal vitamin A stores in the absence of vitamin E protection.

Intrinsic, light-independent, regional differences in photoreceptor cell degeneration in vitamin A-deficient rat retinas.

The retinas of retinol-deficient rats reared in darkness or cyclic light were examined to determine whether regional degeneration of photoreceptor cells was induced by environmental lighting or resulted from intrinsic differences between ocular hemispheres. Weanling male Sprague-Dawley rats were fed a retinol-adequate or retinol-deficient diet and were reared in either cyclic light or darkness through 29 weeks. The nasal retinal quadrants were examined by light microscopy and the number of photoreceptor nuclei was counted in a 630 micron segment beginning 200 micron from the optic nerve. Retinol-deficient rats reared in darkness for 29 weeks lost 24% of their photoreceptors in the inferior nasal quadrant but only 11% in the superior nasal quadrant. Deficient rats reared in cyclic light for 29 weeks lost 39% of their photoreceptor cells in the inferior nasal quadrant and only 16% in the superior nasal quadrant. Photoreceptor cells degenerated faster in the inferior nasal quadrant than in the superior nasal quadrant in darkness or cyclic light. These results indicate that regional differences in rate of photoreceptor cell loss in retinol-deficient rats are not induced by the lighting conditions but occur as a result of intrinsic differences between the ocular hemispheres.

Vitamin E deficiency and the retina: photoreceptor and pigment epithelial changes.

To investigate the role of normal vitamin E levels and the interrelationships between vitamin E and A in maintaining the visual cells of the retina, weanling female Sprague-Dawley rats were fed vitamin E-free diets differing tenfold in their vitamin A content (0.8 and 8.0 mg of retinol per kilogram of diet). Rats on vitamin E-free diets with the higher vitamin A level exhibited marked disruption of photoreceptor outer segment membranes and a fivefold increase in the number of lipofuscin granules in the pigment epithelial cells which ingest these membranes. Rats on vitamin E-free diets with the lower vitamin A level showed the same retinal damages plus significant loss of photoreceptor cells compared to age-matched rats on control diets. Rods and cones were involved equally, and their pattern of loss was not like that found in vitamin A deficiency. Normal levels of vitamin E probably protect photoreceptor membranes from oxidative damage and retard the accumulation of their remnants and other products of lipid breakdown in the pigment epithelium. The vitamin A status of rats has a significant influence on the extent of damage induced by vitamin E deficiency.

Deficiencies of vitamins E and A in the rat: lipofuscin accumulation in the choroid.

The effects of vitamin E and A deficiencies on the formation of lipofuscin in the melanocytes and fibroblasts of the choroidal stroma and in the endothelial cells of the choriocapillaris were studied. Weanling female albino rats (Sprague-Dawley) were divided into three groups and fed purified diets adequate or deficient in vitamins E and A: +E, +A; -E, -A. After 35 weeks, vitamin E deficient rats (-E, +A) exhibited increased lipofuscin-specific autofluorescence in the choroid compared to the controls (+E, +A). By electron microscopy and morphometric methods the choroidal stroma of vitamin E deficient rats displayed an increase in lipofuscin content as measured by the number of lipofuscin granules and their size, if compared with the controls. However, in vitamin E deficiency, only animals with a supply of vitamin A (-E, +A) showed higher amounts of lipofuscin in the choroidal stroma; animals deficient in both vitamins (-E, -A) stayed on the same level as the controls (+E, +A). In the endothelial cells of the choriocapillaris, on the other hand, no significant increases in lipofuscin content were observed in either group of vitamin E deficient animals (-E, +A), (-E, -A). Apparently vitamin E deficiency affects the choroid by increasing lipofuscin formation only in the melanocytes and fibroblasts. Vitamin A appears to play a role in lipofuscin formation.

Structural and biochemical changes in vitamin A--deficient rat retinas.

The levels of rhodopsin and opsin were investigated in relation to the maintenance of retinal structure in retinas of vitamin A--deficient rats in low levels of cyclic illumination (1.5 to 2 foot-candles). Rhodopsin levels decreased in the deficient retinas to approximately 20% of control at 9 weeks, and this level was retained through 39 weeks on the deficient diet. Opsin levels decreased at a slower rate but reached about 20% of control levels at 32 weeks. Despite the decrease in rhodopsin levels, obvious deterioration of disc structure was not observed until 16 weeks of deficiency, when opsin levels had already decreased to 60% to 70% of control. The structural disruption of photoreceptor outer segments was localized initially in discs of the distal third. Rod cell degeneration preceded cone cell degeneration in vitamin A--deficient retinas. Most of the rods and cones persisted in the posterior retina at 23 weeks on the deficient diet; however, by 40 weeks, only 11% of the rod nuclei remained. In contrast, about 63% of the cone nuclei were present at 40 weeks of deficiency. The photoreceptor cells were affected by the deficiency to a greater extent in the inferior hemisphere than in the superior hemisphere of the eye.

Ineffectiveness of testosterone and FSH in maintaining the vitamin A deficient rat testis.

The combined administration of follicle-stimulating hormone and testosterone to sexually mature male rats whose testes were in the early stages of damage from retinol deficiency did not prevent the continued degeneration of the germinal epithelium. It is concluded that the action of retinol on the testis is probably not mediated through, nor influenced by, the pituitary or steroid hormones.