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A study has been made of the acid pyrophosphatase, carbamoylphosphate ornithinecarbamoyl(CPOC) transferase, invertase, and malic dehydrogenase activities of Saccharomyces cerevisiae grown in a defined medium containing either an optimal or a suboptimal concentration of d -biotin; the effect of supplementing the biotin-deficient medium with l -aspartic acid and/or oleic acid was also examined. Acid pyrophosphatase, invertase, and malic dehydrogenase activities were frequently higher in yeast grown under conditions of varying biotin deficiency than in biotin-optimal yeast. It is concluded that synthesis of these enzymes is not specifically biotin dependent and that the different activities in biotin-deficient yeast are probably the result of an alteration in the normal induction and repression mechanisms and possibly also of a decreased synthesis of repressor substances. CPOC transferase activity was always low in yeast grown in biotin-deficient media as compared with biotin-optimal yeast, and it is suggested that synthesis of this enzyme may be specifically biotin dependent. Yeast grown in biotin-deficient medium, with or without oleic acid, was shown to be more fragile than yeast grown in biotin-optimal medium or in biotin-deficient medium supplemented with aspartic acid; these observations are discussed in relation to changes in the cell wall metabolism of yeast accompanying biotin deficiency.

Incorporation of gluconeogenic precursors such as l -alanine-U- 14 C, l -aspartate-U- 14 C, succinate-1,4- 14 C, NaH 14 CO 3 and glycerol-U- 14 C into blood glucose and liver glycogen was studied in control and pair-fed biotin-deficient rats. It was observed that only glycerol-U- 14 C was utilized equally efficiently by control and deficient animals for glucose synthesis. A study of the levels of gluconeogenic intermediates, as well as the levels of reduced and oxidized forms of pyridine nucleotides in livers of fasted control and deficient animals, indicated two sites of control of gluconeogenesis in biotin deficiency: at the pyruvate carboxylase step and at the glyceraldehyde-3-phosphate dehydrogenase step. Under these conditions, abnormally large amounts of lactate were excreted in the urine of biotin-deficient animals.

The cells of biotin-restricted yeasts contain 5-aminoimidazole (or its riboside), inosine, and deamido-DPN. In addition to these substances, hypoxanthine and nicotinic acid have been reported in the medium, but are not detectable within the cells. If the biotin-restricted yeasts are supplied with aspartate or biotin, the amount of these materials is markedly decreased.

The effect of biotin deficiency on the in vivo oxidation of glucose-C 14 labeled in various positions has been studied in the rat. In all cases there is more C 14 O 2 output by the biotin-deficient animal as compared with controls. Incorporation of glucose-C 14 into liver glycogen is also reduced markedly in biotin deficiency. These findings are in agreement with the role of biotin in CO 2 fixation reactions.

The interrelationship between insulin deficiency and biotin deficiency and its relation to carbohydrate metabolism has been studied. Severe biotin-deficient animals show no change in blood sugar levels. Administration of equal amount of anti-insulin serum increased the blood sugar levels to the same extent in normal and biotin-deficient animals, indicating insulin levels were unaltered in biotin deficiency. Oxidation of C 14 -glucose to CO 2 is also unaltered. C 14 O 2 incorporation into glucose is slightly decreased in biotin-deficient animals. Anti-insulin serum treated biotin-deficient animals show three- to fourfold increase in C 14 O 2 incorporation into glucose. Incorporation of 2-C 14 -acetate and 2-C 14 -propionate into glucose, glycogen, and CO 2 , and C 14 O 2 into glucose and glycogen is unaltered in biotin-deficient animals. However, there is a significant decrease in the incorporation of these substrates into long-chain fatty acids.

A comparison of the metabolism of glucose in vitro by adipose tissue from normal, biotin-deficient, and biotin-restored rats has been made. Insulin enhances the uptake of glucose by tissue from all three types of animals. In the biotin-deficient animal, 41% of the glucose is converted to lactic acid as compared to 7% in the normal. A corresponding decrease in the net CO 2 production is observed in the biotin-deficient animal signifying a lowered fatty acid synthesis. A single injection of 300 μg. biotin results in a rapid restoration of the metabolic pattern toward the normal.

The investigations of the effect of biotin deficiency upon carbohydrate metabolism by washed cell suspensions of Saccharomyces cerevisiae 139 show that biotin stimulates the rate of fermentation and oxidation of carbohydrates by deficient cells. Aspartic acid partially replaces biotin in stimulating the rate of fermentation and oxidation of glucose by biotin-deficient yeast. Biotin or biotin plus aspartic acid elicits a greater response than aspartic alone in the stimulation of glucose metabolism by yeast, suggesting roles for biotin in both fermentation and oxidation other than its role in the metabolism of aspartic acid. The rate of adaptation of biotin-deficient cell suspensions of S. cerevisiae to sucrose fermentation is much more rapid in the presence of biotin, suggesting that this vitamin is concerned in the synthesis of the adaptive enzymes necessary for fermentation of this sugar. It has been found that cell-free extracts with high fermentative ability can be obtained if the synthetic medium is supplemented with 0.5% casein hydrolyzate.

Administration of l -ascorbic acid to biotin-deficient rats delayed the onset of deficiency but did not abolish all the clinical symptoms of the deficiency. Biotin-deficient animals excreted less than half the amount of ascorbic acid compared to the animals that received biotin. Similarly, liver and adrenals of the biotin-deficient animals contained less than half the total ascorbic acid as compared to the control rats. Blood serum levels of ascorbic acid also showed significant, differences between the two groups. Ascorbic acid synthesis from labeled glucose showed a marked reduction in the specific activity as well as the total counts incorporated by the biotin-deficient rat compared to the control receiving biotin. These effects are a result of an impairment in glucose utilization as well as a decrease in l -gulonolactone oxidase activity in biotin deficiency.

The effect of dietary administration of 1% orotic acid on the biotin content of liver as well as the influence of biotin deficiency on the development of fatty liver in orotic acid-treated rats have been studied. The amount of biotin in the livers of orotic acid-fed rats was lower than in the controls. In biotin-deficient rats, the development of orotic acid fatty liver was inhibited while the administration of large doses of biotin led to an increase of liver fat. The data suggest that there may be a relationship between biotin and fatty infiltration of the liver induced by orotic acid.

Actithiazic acid, an antibiotic known to interfere with the biotin metabolism of certain bacteria, did not significantly increase or decrease the degree of biotin deficiency (measured by liver and muscle total biotin content) induced in the rat by administration of raw egg white. Two biotin-like substances, “avidin uncombinable biotin” (AUB) and SC factor, have been found in the supernatant of Escherichia coli cultures as well as in the urine and stool of the rat. The “avidin uncombinable biotin” of the urine of actithiazic acid-treated rats was increased about ten times over the concentration of AUB found in the urine of untreated controls. The AUB of the stool was similarly but less consistently increased. The AUB and SC factors are normally produced in small quantities by E. coli , but under the influence of actithiazic acid this production is increased five-to tenfold. This increase of biotin vitamers was found in supernatants of cultures of crude rat stool flora and in pure cultures of a coliform organism (isolated from this rat stool) as well as in similar cultures of a pure strain of E. coli K-12. These findings suggest an enteric origin of AUB and SC. Moreover, no AUB could be detected in rat muscle or liver, as contrasted with the large amounts found in stool and urine. No significant amount of “true” biotin is elaborated into the medium by E. coli , although the organisms themselves contain considerable biotin in bound form. AUB and SC have been separated chromatographically, but their chemical identification has not been accomplished.

The lack of an effect of a biotin deficiency upon production of the carbamyl phosphate-synthesizing enzyme of Streptococcus lactis 8039 suggests that biotin is not involved directly in the production of this enzyme. The decreased biotin content of active fractions of the enzyme after each step in purification and the inability of avidin to affect the activity of the purified enzyme suggest that biotin is not a cofactor in the enzyme. Sulfate ion specifically protects the enzyme and also reactivates enzyme preparations which have been inactivated by dialysis, heat treatment, or aging. Some properties of the enzyme were determined.

The effect of ethionine on the biotin content of liver as well as the effect of biotin deficiency on the development of fatty liver and on lipogenesis in vitro in ethionine-treated rats are reported. The amount of biotin in the livers of ethionine-treated rats was lower than in the controls. In biotin-deficient rats, the development of ethionine-induced fatty liver was inhibited. The incorporation of acetate-1-C 14 into total lipids was decreased in slices from livers of biotin-deficient rats and also from livers of ethionine-treated rats fed on a biotin-free and on a biotin-supplemented diet. The data suggest that there may be a relationship between biotin and ethionine-induced fatty infiltration of the liver.