The Role of Intestinal Flora in Biotin Deficiency in Conventional and Germ-Free Mice Fed a Purified Biotin-Deficient Diet without Supplementation

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1 J. Clin. Biochem. Nutr., 17, , 1994 The Role of Intestinal Flora in Biotin Deficiency in Conventional and Germ-Free Mice Fed a Purified Biotin-Deficient Diet without Supplementation with Egg White Masamichi IKEDA,* Midori IWAI, Hideko SATO, and Bunsaku SAKAKISARA Faculty of Nutrition, Kobe-Gakuin University, Nishi-ku, Kobe , Japan (Received May 17, 1994) Summary To investigate the influence of intestinal flora upon the effects of biotin deficiency, we provided germ-free and conventional mice with a purified biotin-deficient diet without egg white for 20 days. Some of the biotin-deficient germ-free mice exhibited alopecia, while conventional mice on the same diet did not. Biotin levels decreased significantly in tissues of conventional and germ-free mice fed a biotin-deficient diet except in the heart of the conventional mice. Pyruvate carboxylase activity in the liver and kidney of germ-free mice fed the deficient diet also decreased significantly, while the activity in the liver of conventional mice on the same diet showed only a slight decrease. However, no significant differences in body or tissue weights were observed between germ-free and conventional mice during the short experimental period. Thus, conventional mice fed a biotin-deficient diet showed latent biotin deficiency, while germ-free animals fed the same diet exhibited severe biotin deficiency. Enteral microbial synthesis of biotin may have made some contribution to the observed amelioration of the effects of dietary biotin deficiency. Key Words: biotin deficiency, germ-free mice, biotin, pyruvate carboxylase, intestinal flora Biotin is an essential vitamin for animals, serving as a coenzyme in several carboxylation reactions in the metabolism of carbohydrates, amino acids, fats, purines, and nucleic acids [1]. It has not been easy to produce biotin deficiency * To whom correspondence should be addressed. 103

2 104 M. IKEDA et al. in animals fed a biotin-deficient diet without addition of egg white or avidin, because of the biotin production by gut microflora. Therefore, previous studies of biotin metabolism have used animals fed a biotin-deficient diet containing egg white or those given such a diet and kept from coprophagy or treated with antibiotics. In the present report, we describe the influence of intestinal microflora on the effects of biotin deficiency in conventional and germ-free mice fed a biotin-deficient diet containing no egg white or avidin. MATERIALS AND METHODS Animals and diet. Male IQI/Jic[Gf] germ-free and conventional mice 4 weeks of age and weighing about 15 g were used. The animals were maintained in stainless steel cages with wire screen bottoms (three or four mice per cage) in a Trexler-type flexible film isolator. Germ-free mice bred in our laboratory were maintained in a standard germ-free state. The germ-free status of mice was verified at the beginning and the end of the experiment by the routine procedure [2, 3]. Conventional mice were conventionalized from the germ-free mice by being given their feces suspension in physiological saline solution as drinking water. Animals were divided into two germ-free and two conventional groups, one each of which served as a control, whereas the other served as the biotin-deficient group. Mice were fed control or biotin-deficient diet for 20 days ad libitum under a controlled temperature of 22±2 C and relative humidity of 50± 10%. Lighting was regulated automatically to provide constant periods of alternating light and darkness (the light was on from 5:00 to 20:00). The composition of the control diet used in this experiment is given in Table 1. The biotin-deficient diet had the same composition as the control except that no biotin was added. Both diets were irradiated with 5 mega rad of y-rays. Conventional and germ-free mice fed the biotin-deficient diet were placed in metabolism cages from the 17th to the 20th day, during which time feed intake was measured, and feces and urine were collected. After 20 days, the animals were sacrificed by exsanguination under light anesthesia. Liver, kidney, Table 1. Composition of experimental diet (%). aavicelrph -101, mean particle size of 40,u m (Oriental Yeast Co., Ltd., Tokyo). bthe vitamins supplied (mg/kg diet): thiamine hydrochloride, 60; riboflavin, 30; pyridoxine hydrochloride, 40; nicotinic acid, 150; Ca-pantothenate, 300; folic acid, 10; p-aminobenzoic acid, 25; biotin, l; vitamin B12, 0.415; inositol, 1,000; retinyl acetate, 20,000 IU; ergocalciferol, 6; 2-methyl-naphthoquinone, 6; tocopherol acetate, 500; ascorbic acid, 4,000. CAIN-76TM J. Clin. Biochem. Nutr.

3 BIOTIN DEFICIENCY IN GERM-FREE MICE 105 lung, heart, and testis were dissected out, rinsed with physiological saline, blotted dry on filter paper, weighed, and stored at - 8O C until analysis could be performed. Assay procedures. Biotin concentrations in diets, plasma, and tissues were determined by a microbiological method using Lactobacillus plantarum ATCC 8014 as reported previously [4]; and those in feces and urine, by the isotope dilution method using [3H]biotin [5]. Tissue samples were cut into small pieces and homogenized in four volumes of cold saline with a Polytron homogenizer. Samples were acid-hydrolyzed prior to the assay according to the method of Hood [6]. Two hundred microliters of 4.0 N sulfuric acid was added to 2001u1 of plasma or the homogenate in a tube covered with aluminum foil. After the tubes had been autoclaved at 1.0 kg/cm2 pressure for 1 h, the hydrolysates were neutralized with sodium hydroxide. Pyruvate carboxylase activity was assayed by the method of Ballard and Hanson [7], and the incorporation of [14C]bicarbonate into oxaloacetate was measured. The liver and the kidney were homogenized in four volumes of 0.05 M Tris-HC1 buffer, ph 7.4, containing 5 mlvi mercaptoethanol. The resultant homogenates were centrifuged at 12,000 X g for 20 min, and the supernatants were used as the enzyme samples. Reactions were carried out in small stoppered test tubes containing the following constituents: 25 u mol of Tris-HC1 buffer, ph 7.4, 10 1u mol of sodium pyruvate, 2.5 p mol of sodium ATP, 0.75 p mot of acetyl-coa, 50 1u mol of 14C-labeled KHCO3 representing 12,000 c.p.m., 5 p mol of MgC12, and the enzyme sample in a total volume of 1 ml. After incubation for 30 min at 3TC, the reactions were stopped by the addition of 0.5 ml of 10% trichloro acetic acid. After centrifugation, any remaining CO2 was removed by bubbling unlabeled CO2 through the solution, and 1-mi aliquots of this solution were counted in a Packard 1600 TR liquid-scintillation analyzer (Packard Instrument Company, Meriden, CT). One unit of activity was defined as the amount of enzyme required to catalyze the fixation of 1,u mol of ['4C]bicarbonate per min at 37 C. Protein concentrations in the homogenates were determined by the method of Bradford [8] with a commercial dye reagent kit (Bio-Rad Laboratories, Richmond, CA). Bovine serum albumin was used as the standard. Statistical analyses. Statistical analysis was performed by the two-tailed Student's t-test, with p < 0.05 taken to indicate significance. RESULTS Body and tissue weights Twenty-five percent of the germ-free mice fed the biotin-deficient diet exhibited signs of deficiency in their skin, i.e., alopecia, whereas there were no characteristic symptoms in the conventional mice on the same diet. Body and tissue weights of germ-free and conventional mice fed the control and biotin-deficient diets for 20 days are shown in Table 2. No significant Vol. 17, No. 2, 1994

4 106 M. IKEDA et al. differences in these weights were observed between germ-free and conventional mice during the experiment. Biotin levels in tissues Biotin levels in plasma and tissues are generally used as an index of biotin nutritional status. Plasma and tissue biotin levels from the mice used in the present study are shown in Table 3. Significant decreases were observed in biotin levels in the plasma and tissues of both conventional and germ-free mice fed the biotindeficient diet, with the exception of that level in the heart of conventional mice. Biotin levels in the plasma of both groups of mice fed the experimental diet showed especially marked decreases. Although the biotin content in the livers and kidneys from germ-free mice fed the deficient diet decreased to about 20 and 27%, respectively, of those in the germ-free control group, the depletion of biotin in these organs from conventional mice fed the deficient diet was only to about 56 and 72%, respectively, of the control levels. In conventional mice fed the biotindeficient diet, the biotin content in the heart showed no decrease; whereas in germ-free mice fed the deficient diet, the content decreased significantly (p < O.OI ) to about 44% of the control level. Table 2. Body weight and tissue weights of mice fed control and biotin-deficient diets. Body weights are expressed in g. Liver, kidney, heart, lung, spleen, and testis weights are expressed as g/100 g body weight. Values are means±sd. Table 3. Biotin levels in liver, kidney, heart, lung, testis, and plasma from mice fed control and biotin-deficient diets. Biotin levels are expressed as ng/g wet weight tissue or ng/ml plasma. Values are means± SD; ap <0f15, by <0.01, Cp <0.001 compared with the respective control. J. Clin. Biochem. Nutr.

5 BIOTIN DEFICIENCY IN GERM-FREE MICE 107 Table 4. Pyruvate carboxylase activities in mice fed control and biotin-deficient diets. Activities are expressed as follows: A, mu/g wet weight; B, mu/g protein. Values are means ± SD; ap <0.05, Cp <0.001 compared with the respective control. Table 5. Biotin intake, and urinal and fecal excretion of biotin in mice fed the biotindeficient diet. The levels are expressed as ng/mouse/day. conventional group. Values are means± SD; Cp <0.001 compared with Pyruvate carboxylase activity Pyruvate carboxylase activity in the liver and kidney are known to decrease with the progress of biotin deficiency [9]. The activities of this enzyme in conventional and germ-free mice fed the control and deficient diets are shown in Table 4. Pyruvate carboxylase activity in the liver and kidney of germ-free mice fed the deficient diet decreased significantly (p < and p<0.05) to about 20 and 50%, respectively, of the control values. Conventional mice fed the deficient diet showed a slight decrease in pyruvate carboxylase activity in the liver, but not in the kidney. Biotin excretion The results of biotin balance in conventional and germ-free mice fed the biotin-deficient diet are shown in Table 5. The biotin-deficient diet contained 7.80± 0.82,u g/kg diet, as determined microbiologically. Urinary biotin excretion was slightly higher in the deficient conventional mice than in the germ-free mice on the same diet. Fecal biotin excretion was much higher in the conventional mice on the deficient diet than in the deficient germ-free mice. DISCUSSION It has not been easy to produce biotin deficiency in animals fed a purified biotin-deficient diet without inclusion of egg white because of the production of biotin by gut microflora. To hasten the onset of biotin deficiency, dietary supplementation with egg white containing avidin, prevention of coprophagy, or Vol. 17, No. 2, 1994

6 108 M. IKEDA et al. antibiotics have been used. However, egg white contains constituents other than avidin that could be involved in growth depression such as ovoinhibitor proteins and ovomucoids, which act as trypsin inhibitors [10]. Prevention of coprophagy by the use of tail cups can reduce weight gain and alter the composition of the microflora [ 11 ]. Antibiotics usually cause only temporary declines in the intestinal microflora followed by repopulation with resistant species. Such procedures, therefore, hardly represent ideal methods by which to produce experimental biotin deficiency. The deficiency brought about through such methods raises questions as to whether it represents true biotin deficiency, or is artifactually tainted due to the experimental procedure used. The best way would be to compare biotin deficiency between conventional and germ-free animals fed a purified biotin-deficient diet without inclusion of egg white. Study of biotin has been conducted with germ-free rats fed a purified biotindeficient diet without inclusion of egg white. Cherruau et al. [12] found that biotin deficiency was easily induced in germ-free rats fed such a purified biotindeficient diet without inclusion of avidin for days. It has been reported that biotin deficiency is induced more easily in mice than in rats [13, 14]. However, no detailed analysis of the influence of intestinal microflora on the effects of biotin deficiency in germ-free mice for a short term has heretofore been reported. The purpose of the present study was to investigate on a short-term basis the influence of intestinal microflora on the effects of biotin deficiency in conventional and germ-free mice fed a biotin-deficient diet containing no egg white. In the present study, some germ-free mice fed the biotin-deficient diet exhibited skin signs of deficiency, whereas there were no such characteristic signs in the conventional mice. No significant differences in body or tissue weights were observed between germ-free and conventional mice during the experiment. It was reported that the body weight and tissue weights in rats decreased significantly after 50 to 60 days under conditions comparable to those of this experiment [12], suggesting that in mice fed a biotin-deficient diet, growth retardation may take place gradually. This may explain why no reduction in body weight was observed over the short experimental period of the present study. The decreases in biotin levels were more evident in germ-free than in conventional mice fed the biotindeficient diet. Biotin levels in the heart of conventional mice fed the deficient diet did not decrease. The reason for this is unclear, but it is possible that, as the heart is one of the most important organs, that biotin reserves may be diverted there during the early stages of biotin deficiency. Cherruau et al. [12] reported that biotin deficiency was easily induced in germ-free rats fed such a purified biotindeficient diet without inclusion of avidin, and this phenomenon was presumed to be closely related to the germ-free state. Biotin deficiency in the deficient germ-free mice in the present study occurred to the extent that some animals exhibited alopecia. It is possible that during a period of more than 20 days, most mice fed the biotin-deficient diet would have exhibited alopecia. McCormick [15] reported that biotin was partially oxidized in the liver of J. Clin. Biochem. Nutr.

7 BIOTIN DEFICIENCY IN GERM-FREE MICE 109 rats and partially excreted in urine in a matter of hours. Biotin metabolites containing ureide groups were found to be excreted in urine and feces, but some of these metabolites did not respond to the biotin assay utilizing L. planturum that was used. Therefore, we employed an isotope-dilution assay in the present study; and our results suggest that biotin excretion into urine and feces of mice is dependent, at least partly, on the presence of intestinal flora. Fecal biotin excretion levels were much higher in the conventional than in the germ-free mice, which may be due to the production of biotin by intestinal microflora. Biotin balance in deficient germ-free mice in the present study showed a negative imbalance of ng/day/mouse, as was shown in Table 5. Thus, germ-free mice fed the deficient diet lost a total of 3,046 ng of biotin during the 20 days of the experiment, which would result in biotin deficiency. Therefore, the total biotin content in mice before the experiment affects the level of biotin deficiency, in accordance with the previous observation reported by Cherruau et al. [12] that the earlier the deprivation, the earlier the deficiency manifests itself with more severe symptoms. On the other hand, the conventional mice excreted more biotin in feces and urine than the germ-free mice, and showed no characteristic signs of biotin deficiency despite significant decreases in tissue biotin levels. These findings indicate that intestinal microflora contribute to the biotin supply in conventional mice but that the amount is not enough to completely ameliorate biotin deficiency. Coates et al. [16] reported that biotin was synthesized by microbial action in the gut of conventional birds but that the birds derived little benefit from the synthesized biotin. Subsequently, Bitsch et al. [17] and Rader et al. [18, 19] also reported that enteral microbial synthesis is not adequate to cover the biotin requirement of rats. These findings are in accordance with those of the present study that enteral microbial synthesis of biotin is not sufficient for the biotin requirement of mice fed a purified biotin-deficient diet. It is known that the composition of gastrointestinal microflora is influenced by the type of diet [20-22]. Thus, with different diet compositions, amounts of biotin sufficient for the requirement of mice may be produced by intestinal microflora. REFERENCES 1. Lynen, F. (1967): The role of biotin-dependent carboxylations in biosynthetic reactions. Biochem. J., 102, Committee for Standardization of Sterility Test for Germfree Animals in Japan (1972): Recommended requirement for sterility test for germ-free animals in Japan. Exp. Anim., 21, Maejima, K., and Nomura, T. (1975): An experience of application of sterility test of germfree mice and rats recommended by JEARA. Exp. Anim., 24, Wright, L.D., and Skeggs, H.R. (1944): Determination of biotin with Lactobacillus ayabinosus. PYoc. Soc. Exp. Biol. Med., 56, Dakshinamuruti, K., and Allan, L. (1979): Isotope dilution assay for biotin: Use of Vol. 17, No. 2, 1994

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