Autoradiographic and histopathologic studies on the mode of action of an aromatic retinoid (Ro 10-9359) on chemically induced epithelial tumors in Swiss mice.

The mode of action of an aromatic analogue of retinoic acid, ethyl all-trans-9-(4-methoxy-2,3,6-trimethyl-phenyl)-3,7-dimethyl-2,4,6,8-nonatetraenoate (Ro 10-9359), a compound known to possess a considerable prophylactic and therapeutic effect on skin papillomas and carcinomas, was investigated with autoradiographic and histopathologic methods. The ip application of a single dose of 1,000 mg Ro 10-9359/kg to female Swiss mice with chemically induced skin papillomas caused a 29% regression of the mean tumor diameter after 3 days and a 51% regression after 7 days. In the tumors, the number of DNA-synthesizing cells [measured by the labeling index (LI)] and the length of the cell cycle were not affected by the retinoid; thus a mode of action at the level of cell proliferation can be excluded. In the normal skin, an increase in the LI of about 30% was observed. A small effect on the cell loss was observed; however, it was not sufficient to explain quantitatively the regression of the tumors. When measured histometrically, it appeared that the loss of the horn and the formation of necroses, 3-10 times larger than in the placebo groups, were mainly responsible for the tumor regressions caused by the retinoid. After 7 days, the proportion of stroma in the tumors was increased, and dilation of the vessels and edema in the stroma proximal to the necroses were frequent.

Histological and cytological alterations in the skin of biotin-deficient chicks.

A basal diet with practically no natural biotin was supplemented with 10, 19, 34, 62, 111 or 200 microgram of biotin per kg. Chicks receiving these graded levels of biotin were used for histological investigations of skin samples of the foot pad and the under and upper side of the middle toe. Where there was biotin deficiency, epidermal hyperplasia, papillary growth, acanthosis and hyperkeratosis were observed on the foot pad and the under side of the toe. In the stratum spinosum, cells showed big nuclei and nucleoi, and the glycogen-containing nuclear and perinuclear vacuoles had disappeared. The alterations in the skin of the upper side of the toe were similar but far less pronounced. Cell proliferation measured by the labelling index was significantly increased in all three skin samples.

An immunohistochemical study of the distribution of biotin in tissues of pigs and chickens.

An optimised indirect peroxidase-anti-peroxidase immunohistochemical technique was used to detect endogenous biotin in frozen tissue sections from biotin-supplemented and biotin-depleted pigs and chickens. A monoclonal anti-biotin antibody was used as primary antibody in this technique. Immunoreactive biotin was detected in many tissues of both species including liver, kidney, pancreas, adipose tissue, adrenal gland, testis, brain, choroid plexus, cardiac and skeletal muscle, epithelium of the respiratory and digestive systems, skin and lymphoid tissues. The specificity of immunostaining for biotin was confirmed by the finding of reduced staining intensities in tissues of biotin-depleted animals compared to those of biotin-supplemented animals. The results of this study suggest that biotin has metabolic functions in a wider range of tissues than previously known. They also indicate that endogenous tissue biotin should be considered as a source of false-positive staining when immunohistochemical or histochemical techniques which use avidin or streptavidin reagents or anti-biotin antibodies as components of the detection system, are applied to tissue sections.

Color coordinates for assessment of dietary vitamin E effects on beef color stability.

Color stability was investigated in longissimus lumborum (LL), semimembranosus (SM), and gluteus medius (GM) muscles from Holstein steers fed diets including doses of alpha-tocopheryl acetate that were 0 (EO), 250 (E250), 500 (E500), and 2,000 (E2000) mg.steer-1.d-1 for 42 or 126 d. Longissimus lumborum was aged for 14, 28, and 56 d and GM and SM were aged for 14 d. Effects of vitamin E dose on retention of redness (a*), yellowness (b*), color saturation (chroma), and proportions of redness and yellowness (hue angle) following an aging period of 14 d were E2000 > E500 = E250 > EO (P < .01). Effectiveness of dose duration on the color parameters was 126 d > 42 d (P < .01). Dietary vitamin E supplementation stabilized redness and color saturation, decreased yellowness, and extended color display life of fresh beef. A technique for estimation of color display life based on hue angle measurements of fresh beef is described. Color display life estimates based on hue angle measurements were more consistent with vitamin E supplementation effects on metmyoglobin percentage and hue angle than were estimates obtained from the metmyoglobin threshold method. Color display life across LL, SM, and GM stored until d 14 and then displayed under simulated retail conditions was extended (P < .01) 2.0 (E250) to 5.0 d (E2000). Coefficients of determination for regressions of color display life on muscle alpha-tocopherol concentration were .81, .64, and .63 in LL, SM, and GM muscles aged 14 d, respectively. Supplementation of 500 mg of alpha-tocopheryl acetate per steer daily improved (P < .01) the mean color display life of these three muscles by 2.3 d, or 100%.

Effect of graded biotin levels in the diet on liver pyruvate carboxylase of chicks fed ad libitum and after starvation.

Decreasing levels of biotin in the diet from 200 mug/kg to 0 mug/kg reduced the pyruvate carboxylase activity per g liver and in the whole liver of chicks to 17% of the normal activity. Oral supplementation with 3 mg biotin per chick increased the enzyme activity within 24 hours back to normal. In animals well supplemented with biotin (200 mug/kg diet) 24 hours of starvation increased the per g liver pyruvate carboxylase activity. When the pyruvate carboxylase activities were related to the whole liver, no effect of starvation was found.

Autoradiographic localisation of 3H-biotin in chick tissues.

In an autoradiographic study with a 243 days' exposure time of the autoradiographs, the tissue distribution of 3H-labelled biotin was investigated. Label being always related to cytoplasma, no label was found in the nucleus in cells suitable for such an observation. High labelling was found in differentiated cells, e.g. in hepatocytes, proximal tubular cells of kidney, villous epithelia of small intestine, fat cells, epithelial cells of proventriculus, and uropygial gland and in ependymal cells of the plexus chorioideus. Undifferentiated and rapidly proliferating tissue compartments were only weakly labelled as for example undifferentiated kidney cortex, bone marrow, lymphatic tissue. Organs engaged in fat synthesis showed intensive labelling. In these organs as well as in liver and kidney the accumulation of label is in agreement with the known biochemical function. The accumulation of label, e.g. in osteoblasts or in the ependymal cells of the plexus chorioideus, gives evidence that biotin is an essential vitamin not only for the liver and the kidney.

Biotin deficiency in chicks fed a wheat-based diet.

A wheat-based diet produced severe biotin deficiency symptoms appearing at the age of ten to fourteen days and becoming very severe in the third and fourth week (group 1). Biotin supplementation with 50 mug/kg (group 2) reduced the symptoms almost completely, but did not restore completely growth compared to chicks receiving the diet supplemented with 300 mug biotin/kg compared to chicks receiving the diet supplemented with 300 mug biotin/kg (group 3). The plasma level of biotin was about, or lower than, 100 ng/100 ml plasma in groups 1 and 2, indicating biotin deficiency. In group 3, plasma biotin was above 200 ng/100 ml. Liver biotin, after two weeks, was low in group 1 (less than 600 ng/g), medium in group 2 (1000 to 1500 ng/g) and in group 3 above 2000 ng/g. Plasma and liver biotin levels are found to be suitable parameters for diagnosis of subclinical biotin deficiency in chicks.

Relationships between vitamin A and vitamin E in the chick.

Plasma levels of vitamins A and E were analysed during the dietary administration of two levels of vitamin A (10 000 or 50 000 IU/kg) in combination with four levels of supplemental vitamin E (0, 50, 100, or 150 mg/kg) and with or without a supplement of oil to the diet. Tocopherol levels in plasma were markedly decreased by the higher vitamin A supplementation. In contrast, the various vitamin E intakes had no influence on plasma retinol levels. The addition of oil to the diet did not affect this interaction. The absorption, distribution and elimination of labeled 3H-dl-alpha-tocopheryl acetate after an oral or intravenous administration, in combination with a high oral dose of vitamin A (100 000 IU/chick), were studied. The high oral single dose of vitamin A reduced the levels of radioactivity in all the analysed tissues and organs, when both vitamins were administered orally. However, vitamin A did not affect distribution and elimination of radioactivity, when an interaction in the gastro-intestinal tract was avoided by different routes of administration.

Stereological composition of the liver of biotin deficient and control chicks.

In an ultrastructural and stereological study the livers of 28-day-old chicks, fed either a biotin-deficient or a supplemented diet, were investigated. The biotin-deficient chicks showed the known dermal lesions and were significantly retarded in growth. About 30% less hepatocytic nuclei per unit volume liver were found in the deficient chicks than in the controls. In biotin deficiency mainly the rough endoplasmic reticulum (RER) and the mitochondria were affected. RER was diminished in volume and surface density, whereas in mitochondria only the surface densities of the outer and inner membrane were reduced. The reduction of RER is in agreement with the known reduction of protein synthesis in biotin deficiency.

Phagocytosis of alpha-tocopheryl nicotinate by the reticuloendothelial system: a microautoradiographic study.

The cellular distribution of labelled alpha-tocopheryl nicotinate after intravenous administration to normal rats was examined by light microscopic autoradiography. The compound was found to have accumulated in cells of the reticuloendothelial system.

The bioavailability of supplemental biotin in cattle.

A trial using 12 yearling heifers was carried out to test whether biotin metabolism and bioavailability are influenced by continuous dietary supplementation with biotin. Six of these heifers received no biotin supplementation (controls), while six received a daily dietary supplement of 20 mg biotin over the whole experimental period of four months. During each of three test periods (on days 14 and 21, 56 and 63, and 118 and 124), single test dosages of 40 mg (oral) and 5 mg (intravenous) biotin were given to each animal in a crossover test design. Blood samples were collected up to 72 h after each of these single doses, and at approximately two-weekly intervals for the assessment of baseline values. Serum biotin levels were determined by an ELSA test. Areas under the curves (AUC) were calculated as the target parameter for the assessment of the bioavailability of orally administered biotin. Serum biotin baseline levels were 300-800 ng/l in the controls and 3000-8000 ng/l in the supplemented animals. In both groups, AUC values in the first test period (days 14 and 21) were significantly higher than in subsequent periods. However, the biotin supplementation showed no significant effect. There was no significant difference in elimination half-lives between groups with and without biotin supplementation. The range was 5-18 h. Furthermore, there was no significant difference in the bioavailability of biotin between the test periods or between the biotin-supplemented and unsupplemented animals. Overall bioavailability was 48%.

Biotin kinetics in serum of cattle after intravenous and oral dosing.

Single oral (p.o.) or intravenous (i.v.) doses of biotin were given to four cattle (400-450 kg body weight) in two consecutive tests two weeks apart. Dosages were p.o. 20, 40, 80 or 160 and i.v. 5, 10, 20, 40 mg biotin per 300 kg body weight. A three-compartment model was used to describe the course of serum concentrations with time. After i.v. administration, terminal half-lives of about 8 h were found. Areas under the curves were linearly related to both the p.o. and the i.v. doses. The systemically available fraction of the p.o. dose was 50 to 60%. On the basis of kinetic parameters, the biotin uptake via the feed was estimated to be 2.5 mg/day, which was about half of that estimated to be in the hay consumed. The data suggest that there was no relevant ruminal synthesis of biotin.

Effects of dietary retinol on hepatic retinol storage and on plasma and tissue alpha-tocopherol in pigs.

A feeding experiment was carried out with pigs to investigate the effects of graded dietary retinol supplements on hepatic and plasma retinol and on alpha-tocopherol in plasma and selected tissues. Four groups of twelve weanling pigs each with a mean body weight of 7 kg were fed a complete ration containing 54 IU vitamin E/kg and supplemented with 5, 10, 20 or 40 x 10(3) IU retinol/kg for Treatments 1 to 4, respectively. The feed was fed in restricted amounts for 150 days when the pigs were slaughtered at 105 kg body weight. Overall daily body weight gain and feed conversion ratio were 660 g and 2.49, respectively, without treatment differences (p less than 0.01). Hepatic retinol concentration (y, IU/g fresh weight) was linearly related to dietary retinol concentration (x, IU/kg feed) as expressed by y = -88.9 + 0.077 x [r2 = 0.94]. Total hepatic retinol (y, IU) and total retinol intake (x, IU) were linearly related as expressed by y = -194.9 x 10(3) + 0.4585 x [r2 = 0.98]. Hepatic retinol retention relative to intake ranged from 30.0% to 44.9%. Plasma retinol concentration was not affected by dietary retinol (p less than 0.01). Plasma alpha-tocopherol levels remained on a plateau for 42 days in all treatments. After 150 days plasma levels exceeded those at 42 days in Treatments 1 to 3, but not in Treatment 4. The effect of retinol on tissue alpha-tocopherol varied with tissues. While there was no effect on M. Longissimus and backfat, alpha-tocopherol levels in heart and liver showed an inverse relationship with dietary retinol.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of vitamin E on mammary and blood leukocyte function, with emphasis on chemotaxis, in periparturient dairy cows.

OBJECTIVE: To determine the effect of vitamin E supplementation on the immune system of dairy cows. DESIGN: The following immune parameters were followed: production of chemotactic factors and superoxide by mammary macrophages and chemotactic responsiveness of blood neutrophils. ANIMALS: 16 healthy Holstein dairy cows. PROCEDURE: Dairy cows were assigned to 1 of 2 experimental groups: control (no vitamin E supplementation) and vitamin E supplemented. Supplementation of vitamin E started 4 weeks before and continued up to 8 weeks after parturition, and included oral supplementation of vitamin E at the rate of 3,000 IU/cow/d. In addition, the same group of cows received 1 injection of vitamin E (5,000 IU) 1 week prior to the expected date of parturition. Blood samples were collected weekly throughout the experimental period. RESULTS: Vitamin E supplementation enhanced by 30 to 83% (P < 0.05) chemotactic responsiveness of blood neutrophils beginning 2 weeks before to 4 weeks after parturition, compared with controls. There were no differences in production of superoxide or chemotactic factors by mammary macrophages between control and vitamin E-supplemented cows. CONCLUSIONS: Vitamin E supplementation prevents the periparturient inhibition of neutrophil chemotaxis. It is unlikely that vitamin E affects directly the function of mammary macrophages.

Available biotin content of various feed ingredients.

The availability to the chick of the microbiologically determined biotin in various feed ingredients was assayed using a standard curve methodology. The chicks were grown up to 28 days of age. Ingredients of the standard diet were partially replaced by the test ingredient. Each test mixture was also supplemented with 200 micrograms biotin/kg and fed to a control group. The percentage of growth of the test chicks compared with the controls proved to be a suitable parameter for the assessment of biotin availability. Availability of biotin was expressed in percentage of the biotin determined microbiologically. In the tested cereals availability was distinctly low, whereas in some other feed ingredients the availability was reduced less.

Incompatibility between lasalocid and chloramphenicol in broiler chicks after a long-term simultaneous administration.

Two growth experiments were conducted to evaluate in broiler chicks the compatibility between lasalocid medication in the feed (at 90 or 125 ppm) and a long-term administration of chloramphenicol either via the feed (500 ppm) or via the drinking water (500 mg/liter). The simultaneous administration of lasalocid and chloramphenicol generally caused severe growth depression, decreased feed intake and impaired feed conversion. Several chicks showed evident symptoms of intoxication, such as ataxia, leg weakness and paralysis. The development and frequency of these symptoms were dependent on the dosage of lasalocid and on the duration of the simultaneous administration. Biochemical examinations (Experiment 2) revealed in the affected chicks significant changes in several parameters, in particular a markedly increased activity of creatine kinase and GOT in the plasma. It confirmed that the observed leg weakness and paralysis were caused by myodegeneration.

Clinical study on the effect of biotin on skin conditions in dogs.

In a collaborative study with small-animal veterinary surgeons, dogs with fur and skin conditions were treated with biotin (approximately 5 mg biotin/10 kg body weight/day) for 3 to 5 weeks. In total 119 cases could be treated which were reported to show symptoms such as dull coat, brittle hair, loss of hair, scaly skin, pruritus or dermatitis. Cases requiring other treatments with e.g. glucocorticoids, were excluded from the study. In 60% of the cases all symptoms were reported to be cured after the biotin treatment and in a further 31% an improvement was noted; in only 9% no effect was recorded. All breeds responded but to a variable extent: e.g. in Poodles the response was lower (no response in 6 out of 11 cases) than in Alsatians where all improved and 14 out of 29 were completely cured. The results confirm the favourable effect of biotin for treatment of fur and skin conditions in dogs.