Biotin Deficiency in the Cat and the Effect on Hepatic Propionyl CoA Carboxylase

CLAUDIA J. CAREY ANDJAMES G. MORRIS Animal Science Department, University of California, Davis, California 95616 ABSTRACT Biotin deficiency was produced in growing kittens by feed ing a diet containing dried, raw egg white. After receiving either an 18.5% egg white diet for 25 weeks, or a 32% egg white diet for 12 weeks, they exhibited dermal lesions characterized by alopecia, scaly dermatitis and achromotrichia, which increased in severity with the deficiency. Females developed accumulations of dried salivary, nasal and lacrymal secretions in the facial region although a male did not. There was a loss of body weight in all cats as the deficiency progressed. Hepatic propionyl CoA carboxylase activities were measured on biopsy samples of liver during biotin deficiency and after biotin supplementation. In the deficient state, activities were 4% and 24% of that following biotin supplementation. Propionyl carboxylase activity in the liver of the cat was comparable to that reported in the rat and chick in the deficient and normal states. Subcutaneous injection of 0.25 mg biotin every other day while continuing to receive the egg white diet, caused remission of clinical signs, a body weight gain and increased food intake. J. Nutr. 107: 330-334, 1977. INDEXING KEY WORDS biotin cat propionyl CoA carboxylase Unequivocal evidence of a requirement for biotin by the cat has not been reported (1) although parenteral biotin has been used on an emperical basis to treat miliary eczema and general dermatitis in the cat (2). Biotin deficiency has been produced and described in a number of species in cluding rats, mice, poultry, swine, dogs, monkeys and man (3). It has been previ ously reported that the activity of biotin dependent propionyl-CoA carboxylase ( EC 6.4.1.3) is reduced in biotin deficiency in the rat and chick (4-8) and has been sug gested by Mistry et al. (9) as in indicator of biotin deficiency. The objectives of this study were to produce and characterize the clinical signs of biotin deficiency in the cat and to measure the effect of the de ficiency on hepatic propionyl carboxylase activity.
MATERIALS AND METHODS

Downloaded from jn.nutrition.org by guest on June 6, 2011

dried, raw, chicken egg white. Avidin, a protein of the egg white binds biotin and renders it unavailable (10). In experiment 1, a commercial egg white preparation was used whereas in experiment 2, lyophylizcd egg whites were prepared in the laboratory. Sulfonamide was included in the diet to suppress intestinal synthesis of biotin (11). The semipurified diets (table 1) con sisted of 32% protein concentrate, 37.7% sucrose, 25% fat, equal parts of animal and vegetable plus a complete mineral and vitamin mixture with the exception of biotin. All diets were fed ad libitum. For groups 1, 2, and 3, the protein concentrate was vitamin free casein, while for group 4 the protein concentrate was 13.5% vitamin free casein and 18.5% dried egg white. Biotin, at the level of 5 mg/kg of diet was added to the ration of group 1. Succinylsulfathiazole replaced an equal weight of sucrose in groups 3, 4 and 5. In experiment
Received for publication June 7, 1976.

A deficiency of biotin was produced in two experiments by a diet which contained

330

PROPIONYL

CARBOXYLASE

IN BIOTIN

DEFICIENCY

331

TABLE 1 Diets used in the biotin stitdy

IngredientsCasein free"1Egg "Vitamin whiteSucrose4Animal

white13.518.5235.730.32.0532%Egg white32.035.730.32.0

fat5Vegetable fatMineral mix7Vitamin 30.31.00.3J**2Minusbiotin32.037.730.33Sulfon-amide32.035.730.32.04Egg mix*Choline chloride8Succinylsulfathiazole8Biotin'1Control32.037.712.5i12.54.(U

* Provided in diet (mg/kg) : DL-o-tocopherol, 159 ; menaquinone, 15 ; thiamin-HCl, 4.4 ; riboflavin, 4.4 ; pyridoxine, 2.2 ; nicotinic acid, 44 ; cyanocobalamin (0.1% Bu triturate), 50 ; folie acid, 10 ; myo-inositol, 222 ; calcium pantothenate, 5.5; retinyl acetate, 27,770 IU; cholecalciferol, 1112 IU. ** 5 mg/kg diet. ' Nu tritional Biochemical Corp., Cleveland, Ohio (biotin 1.2 /ig/kg). 2Dried whipping grade egg white, Henningsen Foods, White Plains, N. Y. * Fresh eggs were separated and lyophylized. 4Spreckles Sugar Company, San Francisco, California. 5 Rendered turkey fat with 0.01% butylated hydroxy anisleadded. 6 Crisco, Procter & Gamble, Cincinnati, Ohio. ' Hegsted's Salt Mix, Nutritional Biochemical Corp., Cleve land, Ohio. 8 Nutritional Biochemical Corp., Cleveland, Ohio. o-Biotin, Nutritional Biochemical Corp., Cleveland, Ohio.

Downloaded from jn.nutrition.org by guest on June 6, 2011

2, 32% dried, raw egg white constituted the protein source for group 5. Experiment 1. Eight female weanling kittens (mean body weight of 1388 g) 93 approximately 8 weeks of age, housed in 45 X 60 X 91 cm stainless steel cages were randomly allocated to the four treatments. The kittens were vaccinated with feline panleucopenia vaccine ^ and treated with an anthelmintic.-' All kittens were given the diet either until clinical signs of biotin deficiency were apparent, or for approximately 6 months. If the deficiency was not apparent by this time, the cats were reallocated to a diet containing a higher percentage of egg white. Body weights were to be recorded at weekly intervals. Remission of signs following administra tion of biotin while the dietary treatment remained unchanged, was to be taken as confirmatory evidence of an uncomplicated biotin deficiency. Experiment 2. Four weanling kittens (three females and one male) with a mean body weight of 1225 g were given 34 the same inoculation and anthelmintic treatment as in experiment 1. Two kittens, one male (number 31) and one female ( number 37 ), were fed diets based entirely on egg white as the protein source to pro duce the deficiency. The other two kittens,

fed commercial cat food 3 ad libitum, acted as controls. Body weight and food intake were measured on a weekly basis. After clinical signs of the deficiency were apparent, a biopsy sample of liver was to be taken under general anesthesia and as sayed for propionyl carboxylase activity. The kittens were repleted by parenteral biotin injections (0.25 mg on alternate days) then another biopsy sample of the liver taken and assayed for propionyl carbox ylase activity. The two kittens receiving the commercial cat diet were also biopsied for control enzyme activity values. Surgical procedure. Kittens were fasted for 18 hours, tranquilized with acetylpromazine 4 and general anaesthesia was in duced with halothane. A laparotomy was then performed under sterile conditions, the liver exposed and a sample removed from the right medial lobe. To prevent ex cessive hemorrage, the liver, at the site of sample removal, was covered with cellulose absorbable hemostat.5 Recovery was un eventful. Enzyme preparation. Tissue samples were blotted and weighed immediately after
1Pelocine, Norden Laboratories. Lincoln. Nebraska. "Plperazine ndipnte. n Carnation Company. Los Anjreles, California. 4 Acepronmzine Ayerst. Los Angeles. California. = SurRieel Johnson & Johnson. Menlo Park, Cali fornia.

332

CLAUDIA

J. CAREY AND JAMES G. MORRIS

removal, then immersed in ice cold ho mogenizing medium (10% w/v) contain ing 0.02 M Tris -HC1 uffer pH 7.4, 0.25 b M sucrose,7 0.001 M reduced glutathione 8 (GSH), and 0.001 M EDTA9 after the method of Chiang and Mistry (8). Follow ing homogenation for 3 minutes in a Potter-Elvehjem glass homogenizer, the liver homogenate was diluted (1:4) and frozen in acetone and dry ice. Samples were kept frozen at 15 enzyme activities were until determined. Enzyme assay. Propionyl carboxylase from the liver homogenate was measured by the radioactive labeled "CO2 fixation method of Lane and Halenz (12). The re action mixture contained, in /moles/ml; Tris-HCl buffer pH 8.5, 50; KH14CO3 spe cific activity of 9735 cpm/>mole KHCO3)10 7.5; ATP,8 2; MgCl2, 2; GSH,8 2.5; and n-propionyl CoA,8 0.5. A 0.45 ml aliquot of liver homogenate was added to 1.5 ml of medium and incubated in a shaking water bath at 37 20 minutes. Twenty percent for perchloric acid was added to stop the re action and precipitate the protein. The unreacted bicarbonate in the supernatant was removed by this acidification and aliquots of the bicarbonate free supernatant added to a scintillation mixture " to make 15 ml and counted in a scintillation counter.12

cats fed the minus biotin diet ( group 2 ) at 25 weeks, their diet was changed by re placing the 32% casein with 32% egg white.13 All other ingredients were kept constant. There was almost an immediate body weight loss, the linear regression co efficient of body weight on time was -43.5 g/week from the 30th to 40th week. Four to five weeks following this dietary change, both cats exhibited the same signs as the cats in group 4, namely, accumulated dried salivary, nasal and lacrymal secre tions, alopecia, achromotrichia, scale der matitis and diarrhea. Hemoglobin concen trations and packed cell volume were periodically monitored and at all times were in the normal range. Subcutaneous injection of 0.25 mg of o-biotin 14was com menced at 46 weeks while they continued to receive the 32% egg white diet. There was an immediate response to the therapy as evidenced by the gain in body weight (linear regression coefficient of +36 g/ week) and a progressive remission of signs. In neither group 2 nor 4 were abnor malities of gait observed, as has been re ported in rats, mice, swine, monkeys, and man (3). However, other symptoms of biotin deficiency were similar to those re ported in these species. None of the signs of biotin deficiency described in groups 2 and 4 were exhibited by the cats with the RESULTS AND DISCUSSION sulfonamide treatment (group 3) during Experiment 1. The mean regression co the 46 the "vitamin free" casein contained either weeks of trial. This indicates, that efficient of body weight (g) with respect adequate biotin for the cat, or the sulfonato time (weeks) in groups 1 to 4 up to 25 weeks was 59.9, 33.7, 49.8, and 59.0 g/week, mides did not completely inhibit intestinal respectively. At 25 weeks, accumulated microbial synthesis of biotin, or there was of both factors. dried salivary, nasal and lacrymal secre a combination 2. The mean regression co Experiment tions were observed in the facial region of efficient of body weight on time was +30.0 both cats from group 4; this condition be g/week for the two cats during the first 12 came more pronounced with time. In the weeks of consuming the 32% egg white same cats, alopecia began at the extremities diet ( group 5 ). Mean ( SE) food intake and progressed over the whole body. The was 209 g/week for the male and 29 alopecia was accompanied by achromotri193 17 g/week for the female cat. Alo chia and scaly dermatitis. Coincidental with pecia and generalized scaly dermatitis were these changes, there was a progressive loss evident on the male cat at 8 weeks and inof body weight; the mean linear regression coefficient of body weight on time was "Trizma Base, Sigma, St. Louis, Missouri. 88g/week. In the terminal stages of the ' Malllnckrodt Chemical Works, Los Angeles, Cali deficiency, both cats had a foul smelling fornia. 8 Sigma. St. Louis, Missouri. Eastman Kodak Co., Rochester, New York. diarrhea and were euthanized at 245 and 10New England Nuclear, Gardena, California. 11Aquasol, New England Nuclear, lot #617. 282 days due to their extreme emaciated 12Packard 3003 Scintillation counter. and anorexic state. ls Hennlngsen Foods, White Plains, New York. 14Nutritional Biochemical Corp., Cleveland, Ohio. As no clinical signs were apparent in the

Downloaded from jn.nutrition.org by guest on June 6, 2011

PROPIONYL

CARBOXYLASE

IN BIOTIN

DEFICIENCY

333

creased in severity with time. At 12 weeks, the female began developing signs of the dried lacrymal, nasal, and salivary secre tions which were pronounced at 18 weeks. However, the female's coat maintained a healthy appearance throughout the trial. She continued to gain weight until 24 weeks, whereas the male ceased gaining weight at 5 to 6 weeks. The mean regres sion coefficient of body weight over time for both cats from the 24th to 33rd week was 25.3g/week. During this latter phase of the deficiency, food intake was depressed somewhat for the female to 163.7 11 g/week and even more so in the male to 98.8 11 g/week. Other critical signs of biotin deficiency observed in the male included alopecia, generalized scaly dermatitis with achromotrichia. An accumulation of dried secretions as described in experiment 1 was not as evident in the male cat. He had a notice able loss of subcutaneous fat and became emaciated at an earlier stage than any of the other cats. Although the coat of the female in experiment 2 maintained a healthy appearance, hair loss in the facial area first the condition the "spectacle eye" sembled observed in of 18th week, re described in rats (13). This female also exhibited increasing accumulations of dried lacrymal, nasal, and salivary secretions as the experiment progressed. In contrast to the male, subcutaneous fat was maintained. Sexual differences in response to biotin deficiency have been reported to occur in other species (14). The failure of the male cat in the experiment to develop accumu lations of dried secretions and a greater depletion of subcutaneous fat, may indi cate a differential response of the feline sexes to biotin deficiency. Diarrhea, present in some of the animals of experiment 1, was not observed at any time in the cats of experiment 2. Administration of biotin (0.25 mg on alternate days) to both cats resulted in an immediate increase in mean food intake to 252 27.9 g/week. Body weight followed in a similar response with a mean linear regression coefficient of +42.4 g/week. Remission of clinical signs accompanied the increased food intake and weight gain. Enzyme activity. The activity of hepatic propionyl carboxylase, following prolonged

TABLE 2
Hepatic propionyl carboxylase activity in biolin deficient, supplemented and control cats
activitynumber status3131 Units* of enzyme liver17.1 g fresh protein0.073 mg liver

+37t37 +5 +9 +per

(4)*0.308 0.002 0.4850.7 (4)0.013 0.005 0.813.7 (4)0.2960.018 0.002 0.4647.3 2.8547.51.5648.1 (4)0.213 (4)0.216 O.C07 0.003 (2) 0.45per

* A unit is defined as the amount of enzyme which fixes 1 /moleof COs/hour. ** Mean value and standard error with number of samples in parentheses. t Mole cat.

biotin deprivation was greatly depressed compared to that after administration of biotin (table 2). Mean specific activities of 0.07 and 0.01 onoleCO2 fixed/hour/mg protein were measured following consump tion of the egg white diet for 30 weeks. With the injections of biotin, mean specific activities increased to 0.31 /moles nd 0.30 a /molesCO-, fixed/hour/mg protein. Con trol cats biopsied at the same time had mean specific activities of 0.22 and 0.21 /tmoles CO'2 fixed/hour/mg protein. These values are comparable with other values obtained from the literature for the rat and chick as shown in table 3, but are less than reported for the cow. The enzyme activity of the male cat in the biotin deficient state was 24% of its enzyme activity following biotin supple mentation. While in the female, the activity of the enzyme following prolonged biotin deprivation was 4% of the supplemented state. Chang and Mistry (8) report that propionyl carboxylase activity in rats de creased to 5% of the control value after consuming an egg white diet for 30 days. Propionyl carboxylase activities in the chick were also reported to be reduced to 12% of the controls after they had been given a biotin deficient diet for only 20 days ( 7 ). It has been reported that prolonged deprivation of biotin in rats and chicks does not completely deplete the liver of the vitamin (15). The enzyme activities mea sured in the cat, following prolonged biotin deprivation, also indicate some residual biotin in this species. The clinical signs observed in the cat following long-term feeding of egg white

Downloaded from jn.nutrition.org by guest on June 6, 2011

334

CLAUDIA

J. CAREY AND JAMES G. MORRIS

TABLE 3 Hepatic propionyl carboxylase activity in the livers of the cat and other vertebrate species Units/mg* status protein SpeciesCatRatChickCowBiotin 0.013+ 0.3080.015+ 0.480.07-0.12+ 0.270.001+ 0.1220.078+ 0.294+ 5.2SourceLiver homogenateLiver homogenateLiver powderLiver mitochondrial acetone powderCell mitochondrial acetone preps0-4.5% free enzyme homogenate100,000 fraction liver supernatant100,000 g supernatant100,000 g supernatant100,000 g supernatantAcetone g powder extractReferenceThis studyThis study(6) '(6)(16)(16)(7)(7)(7)(7)(17)

* One unit is equivalent to 1 jamleof CO2 fixed/hour.

protein are similar to those described in other species. The remission of these signs with parenteral administration of biotin confirmed the deficiency and thereby the need for biotin by the cat. The reduced activity of the biotin dependent enzyme, propionyl carboxylase in the biotin defi ciency, substantiates a biotin requirement for the cat.
ACKNOWLEDGMENTS

8.

9.

10.

Gratitude is expressed to Art Aguirre, John Bryan, and Ernest Avery for their continued assistance.
LITERATURE CITED

11.

1. National Research Council (1972) Nutrient requirements of laboratory animals, No. 10, p. 4. National Academy of Sciences, Washing ton, D.C. 2. Joshua, J. O. (1959) The use of biotin in certain skin diseases of the cat. Vet. Ree. 71, 102. 3. Gyrgy,P. (1954) Biotin. In The Vitamins, Chemistry, Physiology, Pathology, ( Sebrell, W. H. & Harris, R. S., eds.), Vol. I, pp. 512618. Academic Press Inc., New York. 4. Lardy, H. A. & Peanasky, R. (1953) Meta bolic functions of biotin. Physiol. Rev. 33, 560-565. 5. Lardy, H. A. & Adler, J. (1956) Synthesis of succinate from propionate and bicarbonate by soluble enzymes from liver mitochondria. J. Biol. Chem. 219, 933-942. 6. Kosow, D. P. & Lane, M. D. (1961) Res toration of biotin-deficiency-induced depres sion of propionyl carboxylase activity in vivo and m vitro. Biochem. Biop. Res. Comm. 4, 92-95. 7. Arinze, J. C. & Mistry, S. P. ( 1971 ) Activi ties of some biotin enzymes and certain as

12.

13.

14. 15.

16.

17.

pects of gluconeogenesis during biotin de ficiency. Comp. Biochem. Physiol. 38B, 285294. Chiang, G. S. & Mistry, S. P. (1974) Activi ties of pyruvate carboxylase and propionyl CoA carboxylase in rat tissues during biotin deficiency and restoration of the activities after biotin administration. Proc. Soc. Exp. Biol. Med. 146, 21-24. Mistry, S. P., Dakshinamurti, K. & Modi, V. V. ( 1962 ) Impairment of glucose utilization in biotin deficiency. Arch. Biochem. Biop. 96, 674-675. Eakin, R. E., Snell, E. E. & Williams, R. J. ( 1941 ) The concentration and assay of avidin, the injury producing protein in raw egg white. J. Biol. Chem. 140, 535-543. Daft, F. S., Ashburn, L. L. & Sebrell, W. H. (1942) Biotin deficiency and other changes in rats given sulfanilygnanidine or succinyl sulfathiozole in purified diets. Science 96, 321-322. Lane, M. D. & Halenz, D. R. (1962) Mito chondrial propionyl carboxylase. In Methods of Enzymology, ( Colowick, S. P. & Kaplan, N. O., eds.), Vol. V, pp. 576-577. Academic Press Inc., New York. Nielsen, E. & Elvehjem, C. A. ( 1941 ) Cure of spectacle eye condition in rats with biotin concentrates. Proc. Soc. Exp. Biol. Med. 48, 349-352. Okey, P., Pencharz, R. & Lepkovsky, S. (1950) Sex hormonal effects in incipient biotin deficiency. Am. J. Physiol. 161, 1-13. Dakshinamurti, K. & Mistry, S. P. (1963) Tissue and intracellular distribution of biotin "COOH in rats and chicks. J. Biol. Chem. 238, 294-296. Kosow, D. P. & Lane, M. D. (1961) Pro pionyl apocarboxylase activation catalyzed by cell free enzyme extracts. Biochem. Biop. Res. Comm. 5, 191-195. Halenz, D. R. & Lane, M. D. (1960) Prop erties and purification of mitochondrial pro pionyl carboxylase. J. Biol. Chem. 235, 878884.

Downloaded from jn.nutrition.org by guest on June 6, 2011