gatitos propionil coa y biotina

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Biotin Deficiency in the Cat and the Effecton H epatic Propionyl CoA CarboxylaseCLAUDIA J. CAREY AND JAMES G. MORRISAnim al S cien ce D ep artm en t, U niversity o f C alifo rn ia ,Dav is , C alifo rn ia 95616

ABSTRACT Biotin deficiency was produced in growing kittens by feeding 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, theyexhibited derm al lesions characterized by alopecia, scaly derm atitis andachrom otrichia, which increased in severity with the deficiency. Fem alesdeveloped accum ulations of dried salivary, nasal and lacrym al secretions inthe facial region although a male did not. There was a loss of body weightin all cats as the deficiency progressed. H epatic propionyl C oA carboxylaseactivities w ere m easured on biopsy sam ples of liver during biotin deficiencyand after biotin supplem entation. In the deficient state, activities were 4%and 24% of that following biotin supplem entation. Propionyl carboxylaseactivity in the liver of the cat was com parable to that reported in the rat andchick in the deficient and norm al states. Subcutaneous injection of 0.25 m gbiotin every other day while continuing to receive the egg white diet,caused remission of clinical signs, a body weight gain and increased foodintake. J. Nutr. 107: 330-334, 1977.INDEXING KEY WORDS biotin cat propionyl CoA carboxylase

U nequivocal evidence of a requirem entfor biotin by the cat has not been reported(1) although parenteral biotin has beenused on an emperical basis to treat m iliaryeczema and general dermatitis in the cat(2). Biotin deficiency has been producedand described in a number of species including rats, m ice, poultry, swine, dogs,monkeys and man (3). It has been previously reported that the activity of biotind ependent prop ionyl-C oA carboxylase ( E C6.4.1.3) is reduced in biotin deficiency inthe rat and chick (4-8) and has been suggested by M istry et al. (9) as in indicatorof biotin deficiency. The objectives of thisstudy were to produce and characterizethe clinical signs of biotin deficiency in thecat and to measure the effect of the deficiency on hepatic propionyl carboxylaseactivity.

MAT ERIA LS AND METHODSA deficiency of biotin was produced intwo experim ents by a diet which contained

dried, raw , chicken egg white. Avidin, aprotein of the egg white binds biotin andrenders it unavailable (10). In experim ent1, a com mercial egg white preparation wasused w hereas in experim ent 2, lyophylizcdeg g w hites w ere prepared in the lab oratory.Sulfonamide was included in the diet tosu ppress intestinal synthe sis of biotin (11 ).The sem ipurified diets (table 1) consisted of 32% protein concentrate, 37.7%sucrose, 25% fat, equal parts of anim al andvegetable plus a complete mineral andvitamin mixture with the exception ofbiotin. A ll diets were fed ad libitum . Forgroups 1, 2, and 3, the protein concentratewas vitamin free casein, while for group 4the protein concentrate w as 13.5% vitam infree casein and 18.5% dried egg white.B iotin, at the level of 5 mg/kg of diet wasadded to the ration of group 1. Succinyl-sulfathiazole replaced an equal weight ofsucrose in groups 3, 4 and 5. In experim ent

Received for publication June 7, 1976.33 0


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PROPIONYL CARBOXYLASE IN BIOTIN DEFICIENCY 33 1TABLE 1

D iets u se d in th e bio tin stitd y

IngredientsCasein"Vitaminree"1EggwhiteSucrose4Animal

fat5VegetablefatMineralmix7Vitaminmix*Cholinechloride8Succinylsulfathiazole8Biotin'1Control32.037.712.5i12.54.(U30.31.00.3J**2Minusbiotin32.037.730.33Sulfon-amide32.035.730.32.04Egg

white13.518.5235.730.32.0532%Egghite32.035.730.32.0

* P rovided in diet (m g/kg) : D L-o-tocopherol, 159 ; m enaquinone, 15 ; thiam in-H Cl, 4.4 ; riboflavin, 4.4 ;p yrid oxin e, 2 .2 ; n ico tin ic ac id, 4 4 ; c ya no co ba la min (0 .1% B u tritu rate), 5 0 ; fo lie a cid , 1 0 ;m yo -in osito l, 22 2 ;calcium pantothenate, 5.5; retinyl acetate, 27,770 IU; cholecalciferol, 1112 IU. ** 5 m g/kg diet. ' Nutritional Biochem ical Corp., C leveland, O hio (biotin 1.2 /ig/kg). 2 Dried w hipping grade egg w hite, H en-ningsen Foods, W hite Plains, N . Y. * Fresh eggs were separated and lyophylized. 4Spreckles SugarC om pany, S an F rancisco, C alifornia. 5 R endered turkey fat w ith 0.01% butylated hydroxy anis le added.6 C risco, P rocter & G am ble, C incinnati, O hio. ' H egsted's S alt M ix, N utritional B iochem ical C orp., C leveland, Ohio. 8 Nutritional Biochemical Corp., C leveland, Ohio. -Biotin, N utritional BiochemicalC orp ., C lev ela nd, O hio .2, 32% dried, raw egg white constitutedthe protein source for group 5.Experiment 1. Eight female weanlingkittens (m ean body w eight of 1388 93 g)approximately 8 weeks of age, housed in45 X 60 X 91 cm stainless steel cages wererandom ly allocated to the four treatm ents.The kittens were vaccinated w ith felinepanleucopenia vaccine ^ and treated withan an thelmin tic .- 'A ll kittens were given the diet eitherun til clinical signs o f b iotin deficien cy w ereapparent, or for approxim ately 6 months.If the deficiency was not apparent by thistime, the cats were reallocated to a dietcontaining a higher percentage of eggwhite. Body weights were to be recordeda t w eekly in te rv al s.R em issio n o f sig ns fo llowin g a dm in istration of biotin while the dietary treatmentremained unchanged, was to be taken asc on firm ato ry e vid en ce o f a n u nc omplic ate db io ti n de fi ci ency .Experiment 2. Four weanling kittens(three females and one male) w ith a meanbody weight of 1225 34 g were giventhe same inoculation and anthelm intictreatm ent as in experim ent 1. Two kittens,one male (number 31) and one female( num ber 37 ), were fed diets based entirelyon egg white as the protein source to produce the deficiency. The other tw o kittens,

fed commercial cat food 3 ad libitum , actedas controls. Body weight and food intakewere measured on a weekly basis.A fter clinical signs of the deficie ncy w ereapparent, a biopsy sample of liver was tobe taken under general anesthesia and assayed for propionyl carboxylase activity.The kittens were repleted by parenteralbiotin injection s (0.2 5 m g on alternate d ays)then another biopsy sample of the livertaken and assayed for propionyl carboxylase activity. T he tw o kitten s receiving thecommercial cat diet were also biopsiedfor control enzym e activity values.Surgical procedure. Kittens were fastedfor 18 hours, tranquilized with acetylpro-mazine 4 and general anaesthesia was induced with halothane. A laparotomy wasthen performed under sterile conditions,the liver exposed and a sample removedfrom the right medial lobe. To prevent excessive hemorrage, the liver, at the site ofsam ple rem oval, w as co vered w ith celluloseabsorbable hemostat.5 Recovery was uneventful.Enzym e p rep ara tio n. T issu e samp les w ereblotted and weighed immediately after

1P elocine, N orden L abora tories. L incoln. N ebraska ." Plp er az in e n di pn te .n C arnation C om pany. Los A njreles, C alifornia.4 A cepronm zine A yerst. Los A nge les. C alifornia.= SurRieel Johnson & Johnson. Menlo Park, California.


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33 2 CLAUDIA J. CAREY AND JAMES G. MORRISremoval, then immersed in ice cold homogenizing medium (10% w/v) containing 0.02 M Tris -HC1buffer pH 7.4, 0.25M sucrose,7 0.001 M reduced glutathione 8(GSH), and 0.001 M EDTA9 after them ethod of Chiang and M istry (8). Following homogenation for 3 minutes in a Pot-ter-Elvehjem glass hom ogenizer, the liverhomogenate was diluted (1:4) and frozenin acetone and dry ice. Samples were keptfro ze n a t 15n til e nz ym e a ctiv itie s w eredetermined.Enzyme assay. Propionyl carboxylasefrom the liver homogenate was measuredby the radioactive labeled "CO2 fixationmethod of Lane and Halenz (12). The reaction m ixture contained, in /m oles/m l;Tris-HCl buffer pH 8.5, 50; KH14CO 3 specific activity of 973 5 cpm />mole KHCO3)107.5; ATP,8 2; MgCl2, 2; GSH,8 2.5; andn-propionyl CoA,8 0.5. A 0.45 ml aliquotof liver hom ogenate was added to 1.5 m l ofmedium and incubated in a shaking waterbath at 37 or 20 m inutes. Tw enty percentperchloric acid was added to stop the reaction and precipitate the protein. The un-reacted bicarbonate in the supernatant w asrem oved by this acidification and aliquotsof the bicarbonate free supernatant addedto a scintillation mixture " to make 15 m la nd co unted in a scintillation co unter.12

RESULTS AND DISCUSSIONExperiment 1. The mean regression co

efficient of body weight (g) with respectto time (weeks) in groups 1 to 4 up to 25w eeks w as 59.9 , 33 .7, 49.8, and 59 .0 g/w eek ,respectively. A t 25 weeks, accumulateddried salivary, nasal and lacrymal secretions were observed in the facial region ofboth cats from group 4; this condition became more pronounced with time. In thesam e cats, a lopecia beg an at the extrem itiesand progressed over the whole body. Thealopecia w as accom panied by achrom otri-c hia and scaly derm atitis. C oin cidental w iththese changes, there was a progressive lossof body w eight; the m ean linear regressioncoefficient of body weight on time was88g/week. In the term inal stages of thedeficiency, both cats had a foul smellingdiarrhea and were euthanized at 245 and282 days due to their extreme emaciatedand anore xic state.As no clinical signs were apparent in the

cats fed the minus biotin diet ( group 2 ) at25 weeks, their diet was changed by replacing the 32% casein with 32% eggwhite.13 All other ingredients were keptconstant. There was almost an imm ediatebody w eight loss, the linear regression coefficient of body weight on time was-43.5 g/week from the 30th to 40th week.Four to five weeks follow ing this dietarychange, both cats exhibited the sam e signsas the cats in group 4, nam ely, accum ulateddried salivary, nasal and lacrymal secretions, alopecia, achrom otrichia, scale derm atitis and diarrhea. Hem oglobin concentrations and packed cell volume wereperiodically monitored and at all timeswere in the normal range. Subcutaneousinjection of 0.25 m g of o-biotin 14 was commenced at 46 weeks while they continuedto receive the 32% egg white diet. Therewas an immediate response to the therapyas evidenced by the gain in body weight(linear regression coefficient of +36 g/w eek) and a progressive rem ission of signs.In neither group 2 nor 4 were abnormalities of gait observed, as has been reported in rats, m ice, swine, m onkeys, andman (3). However, other symptoms ofbiotin deficiency were sim ilar to those reported in these species. None of the signsof biotin deficiency described in groups 2and 4 were exhibited by the cats with thesulfonamide treatment (group 3) duringthe 46 weeks of trial. This indicates, thateither the "vitam in free" casein containedadequate biotin for the cat, or the sulfona-m ides did not com pletely inhibit intestinalm icrobial synthesis of biotin, or there w asa com bination of both factors.Experiment 2. The mean regression coefficient of body weight on tim e was +30.0g/week for the two cats during the first 12weeks of consuming the 32% egg whitediet ( group 5 ). Mean ( SE) food intakewas 209 29 g/week for the male and193 17 g/week for the female cat. A lopec ia and g eneralized scaly derm atitis w ereevident on the male cat at 8 weeks and in-"Trizma Base, Sigma, St. Louis, Missouri.' Malllnckrodt Chemical Works, Los Angeles, California.8 S igm a. S t. L ouis, M issouri.Eastm an Kodak Co., Rochester, New York.10N ew England Nuclear, G ardena, C alifornia.11Aquasol, New England Nuclear, lot #617.12P ackard 300 3 S cintillation c ounter.ls Hennlngsen Foods, White Plains, New York.14Nutritional Biochemical Corp., Cleveland, Ohio.


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PROPIONYL CARBOX YLASE IN BIOTIN DEFICIENCY 33 3creased in severity with tim e. At 12 w eeks,the female began developing signs of thedried lacrymal, nasal, and salivary secretions which were pronounced at 18 weeks.However, the female's coat maintained ahealthy appearance throughout the trial.She continued to gain weight until 24weeks, whereas the male ceased gainingweight at 5 to 6 weeks. The mean regression coefficient of body weight over tim efor both cats from the 24th to 33rd weekwas 25 .3g /week .D uring this latter phase of the d eficiency,food intake was depressed somewhat forthe fem ale to 163.7 11 g/week and evenm ore so in the m ale to 98.8 11 g/week.Other critical signs of biotin deficiencyobserved in the male included alopecia,generaliz ed scaly derm atitis w ith achrom o-tric hia . A n a cc umula tio n o f d rie d se cre tio nsas described in experiment 1 was not asevident in the male cat. He had a noticeable loss of subcutaneous fat and becameemaciated at an earlier stage than any ofthe other cats. A lthough the coat of thefemale in experiment 2 maintained ahealthy appearance, hair loss in the facialarea first observed in the 18th week, resembled the condition of "spectacle eye"described in rats (13). This female alsoe xh ib ite d in cre asin g a cc umula tio ns o f d rie dlacrym al, nasal, and salivary secretions asthe experiment progressed. In contrast tothe m ale, subcutaneo us fat w as m aintained.Sexual differences in response to biotindeficiency have been reported to occur inother species (14). The failure of the malecat in the experiment to develop accumulations of dried secretions and a greaterdepletion of subcutaneous fat, may indicate a differential response of the felinese xe s to b io tin d efic ie nc y.Diarrhea, present in som e of the anim alsof experiment 1, was not observed at anytime in the cats of experiment 2.Administration of biotin (0.25 mg onalternate days) to both cats resulted in animm ediate increase in mean food intake to252 27.9 g/w eek. B ody w eight follow edin a sim ilar response with a mean linearregression coefficient of +42.4 g/week.Rem ission of clinical signs accom paniedthe increased food intake and w eight gain.Enzyme acti vi ty. The acti vi ty of hepati cp ro pio ny l c arb ox yla se , fo llow in g p ro lo ng ed

TABLE 2Hepatic propionyl carboxy lase ac tiv ity in b iol indeficient, supplemented and contro l cats

U nits * o f en zy mectivitynumberstatus3131

+37t37+5+9 +per

g f re shiver17.10.4850.70.813.70.4647.32.8547.51.5648.1

0.45per

m g l iv errotein0.0730.0024)*0.3080.0054)0.0130.0024)0.2960.018(4)0.213O.C074)0.216 0. 00 3 ( 2)

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

biotin deprivation was greatly depressedcompared to that after administration ofbiotin (table 2). M ean specific activities of0.07 and 0.0 1 on oleCO2 fixed/hour/m gprote in w ere m easured follow ing consum ption of the egg white diet for 30 weeks.W ith the injections of biotin, m ean specificac tivities increased to 0.31 / m oles nd 0.30/ m olesCO-, fixed/hour/m g protein. C ontrol cats biopsied at the same time hadmean specific activities of 0.22 and 0.21/tm oles CO'2 fixed/hour/m g protein . T hesevalues are comparable w ith other valuesobtained from the literature for the rat andchick as shown in table 3, but are less thanreported for the cow.The enzyme activity of the male cat inthe biotin deficient state was 24% of itsenzym e activity following biotin supplem entation. W hile in the fem ale, the activityof the enzym e following prolonged biotindeprivation was 4% of the supplementedstate. Chang and M istry (8) report thatpropionyl carboxylase activity in rats decreased to 5% of the control value afterconsuming an egg white diet for 30 days.P ro pio ny l c arb ox yla se a ctiv itie s in th e c hic kwere also reported to be reduced to 12%of the controls after they had been givena biotin deficient diet for only 20 days ( 7 ) .It has been reported that prolongeddeprivation of biotin in rats and chicks doesnot completely deplete the liver of thevitam in (15). The enzyme activities measured in the cat, follo wing prolo nged bio tindeprivation, also indicate some residualbiotin in this species.The clinical signs observed in the catfollowing long-term feeding of egg white


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3 34 CLA UDIA J. CA REY A ND JA MES G. MORR ISTA BLE 3

Hepat ic p ropionyl carboxylase act ivi ty in the l ivers o f the ca t and other ver tebratespec ies

SpeciesCatRatChickCowBiotinUnits/mg*statusrotein0.013+0.3080.015+0.480.07-0.12+0.270.001+0.1220.078+0.294+ 5.2SourceLiver

homogenateLiverhomogenateLiverm i to chondrial ac et on eowderLiverm i to chondrial ac et on eowderCellf re e e nz ymereps0-4.5%f rac ti on l iv e romogenate100,000gupernatant100,000gupernatant100,000gupernatant100,000gupernatantAcetonepowder extractReferenceThis

studyThisstudy(6)(6)(16)(16)(7)(7)(7)(7)(17)

* O ne u nit is e qu iv ale nt to 1 jam leo f CO2 f ix ed /h ou r.

protein are sim ilar to those described inother species. T he rem ission of these signsw ith parenteral adm inistration of biotinconf irm ed the def iciency and thereby theneed for biotin by the cat. T he reducedactiv ity of the biotin dependent enz ym e,propiony l carbox y lase in the biotin def ic iency , substan tiates a bio tin requirem en tfor the cat.ACKNOWLEDGMENTS

Gratitude is ex pressed to A rt A guirre,John B ry an, and Ernest A very for theirc on ti nued as si st ance .L ITER ATUR E CIT ED

1. N ational R esearch Council (1972) N utrientrequirem ents of laboratory anim als, N o. 10,p. 4. N ational A cadem y of S ciences, W ashington, D .C .2. Joshua, J. O. (1959) The use of biotin incertain sk in diseases of the cat. V et. R ee. 71,102.3. Gy rgy ,P. (1954) B iotin. In The V itam ins,C hem istry , Phy siology , Pathology , ( S ebrell,W . H. & Harris, R . S ., eds.), V ol. I, pp. 512-618. A cadem ic Press Inc., N ew Y ork .4. Lardy , H. A . & Peanask y , R . (1953) M etabolic functions of biotin. Phy siol. R ev . 33,560-565.5. L ardy , H. A . & A dler, J. (1956) S ynthesisof succinate f rom propionate and bicarbonateby soluble enz ym es f rom liv er m itochondria.J. B iol. Chem . 219, 933-942.6. K osow , D. P. & Lane, M . D. (1961) R estoration of biotin-def iciency -induced depression of propiony l carbox ylase activ ity in v iv oand m v itro. B iochem . B iop. R es. Comm . 4,92-95.7. A rinze, J. C. & M istry , S . P. ( 1971 ) A ctiv ities of som e biotin enz ym es and certain as

pects of gluconeogenesis during biotin def iciency . Com p. B iochem . Phy siol. 38B , 285-294.8. Chiang, G. S . & M istry , S . P. (1974) A ctiv ities of py ruvate carbox y lase and propiony lCoA carbox ylase in rat tissues during biotind ef ic ie nc y and resto ratio n o f th e activ itie s af te rbiotin adm inistration. Proc. Soc. Exp. B iol.M ed. 146, 21-24.9. M istry , S . P., Dakshinam urti, K . & M odi, V . V .( 1 962 ) Im pairm ent of glucose utilization inbiotin def iciency . A rch. B iochem . B iop. 96,674-675.10. Eak in, R . E., S nell, E . E. & W illiam s, R . J.( 1941 ) T he concentration and assay ofav idin, the injury producing protein in rawegg w hite. J. B iol. Chem . 140, 535-543.11. Daft, F. S ., A shburn, L . L . & Sebrell, W . H.(1942) B iotin def iciency and other changesin rats giv en sulfanily gnanidine or succiny lsulfathiozole in purif ied diets. S cience 96,321-322.12. Lane, M . D. & Halenz , D. R . (1962) M itochondrial propiony l carbox ylase. In M ethodsof Enz ym ology , ( Colow ick , S . P. & K aplan,N . O., eds.), V ol. V , pp. 576-577. A cadem icPress Inc., N ew Y ork .13. N ielsen, E. & Elv ehjem , C. A . ( 1941 ) Cureof spectacle ey e condition in rats w ith biotinconcentrates. Proc. S oc. Exp. B iol. M ed. 48,349-352.14. Okey , P., Pencharz , R . & Lepkov sk y , S .(1950) S ex horm onal ef fects in incipientbiotin def iciency . A m . J. Phy siol. 161, 1-13.15. Dak shinam urti, K . & M istry , S . P. (1963)T issue and intracellular distribution of biotin"COOH in rats and chick s. J. B iol. Chem .238 , 294 -296 .16. K osow , D. P. & Lane, M . D. (1961) Propiony l apocarbox ylase activ ation cataly zed bycell f ree enz ym e ex tracts. B iochem . B iop. R es.Com m . 5, 191-195.17. Halenz , D. R . & L ane, M . D. (1960) Properties and purif ication of m itochondrial propiony l carbox ylase. J. B iol. Chem . 235, 878-884.

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