1953 - blaxter - heat tissue combustion

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Vol. 7 Excretion of B-vitamins by the refected rat 83Krehl, W. A., Teply, L. J., Sarma, P. S. & Elvehjem, C. A. (1945).Science, 101,489.Lampitt, L. H., uller, C. H. F. & Goldenberg, N. (1947). . SOC. h . d.,Lond., 66, 17.Levy, J. & Jacquot, R.(1948). C.R. Acad. Sci., Paris , 227,371.Luckey,T. ., Briggs, G. M. Jr., Moore, P. R., Elvehjem, C. A. &Hart, E. B. (1945). . iol. Chem.Mawson, E. H. & Thompson, S. Y. (1948).Biochem.J. 43,2.Mickelsen, O., Doeden, D. & Keys, A. (1945).Fed. Proc. 4,98.Mitchell, H. .& Isbell, E. R. (1942)) niv. Texas PubZ. no. 4237,p. 37.Neilands, J. B. & Strong, F. M. (1948). rch. Biochem. 19, 87.Olson, 0. .,Fager, E. E. C., Burris, R. H. & Elvehjem, C. A. (1948). . biol. Chem. 174,319.Rabinowitz,J. C. & Snell, E. E. (1947). nal. Chem. 19, 77.Roberts, E.C. & Snell, E. E. (1946). . b i d . Chem. 163, 99.Slater, E. C. & Morell, D. B. (1946).Biochem. J. 40, 644.Sreenivasan, A., Harper, A. E. & Elvehjem, C. A. (1948).J. biol. Chem. 177,117.Teply, L. . & Elvehjem, C. A. (1945). . b i d . Chem. 157, 03.

161,395.

The Heat of Combustion of the Tissues of Cattle in Relation totheir Chemical CompositionBY K. L. BLAXTER AN D J. A. F. ROOK

Hannah Da iry Research Institute, Kirk hill, Ay(Received 26January 1952)

The estimation of energy retention by cattle from the results of simultaneousdeterminations of balances of carbon and of nitrogen is based on the assumptionthat fat and protein of constant chemical composition and calorific value are the onlymaterials retained. The values employed for this purpose are usually those givenby Armsby (1917):

Nitrogen retention x 6.25 =protein retention; protein retention x 0.5254= carbonretention in protein; non-protein carbon retention x 1.31=fat stored; protein stored(8)x 5-7 =calories stored as protein (Cal.) ; fat stored (g)x 9.5 =calories stored asfat (Cal.).

The factors for protein are derived from Kohler's (1900-1) studies on the ele-mentary composition of fat-free muscles of cattle. These results with those of otherinvestigators are summarized in Table I . Kohler's (1900-1) results with horsemuscles, which are not included, gave lower values for nitrogen and for calories

Table I . Carbon and nitrogen content and the calorific value of f a t -and ash-free mammalian musclesComposition on an ash-free basis

hI %Carbon NitrogenInvestigator* Date (%) (% I Cal./kg53'40 16.30 5656.916.36 5640.9Rubner (1885)Stohmann & Langbein (1891) 52.02 16.15 -rgutinsky (1894) 52'33Kohler (I900-I) 52'69 16-57 5700'8

* Quoted in References, p. 90.6-2


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84 K. L. BLAXTERND J. A. F. ROOK I953tha n those q uote d in th e table, an d he a ttributed these low values to the presence ofglycogen. Th e carbon content of fat used i n the com putation appears to be derivedfrom th e studies of Schulze &Reineke (1867). Th ese showed variations f rom 76.50%carbon for beef fat to 77.62% carbon for hum an depot fat . T h e heat of combustionof fat, 9.5 Cal./g appears to come from a compilation of early German results byAtwater (1895) and by Fr ies (1907), th e values for cattle showing a variation of fr om9357 to 9686 Cal./kg. So m e of these analyses may b e suspect. As Fries (1907)pointed out, very few of the published calorific values determined up to that timeagreed within 1 %, even when crystalline simple organic compounds were analysed.

Ap art fro m analytical errors in th e determination of these factors for pu re proteinand fat, their use in calculation of energy storage within the animal body gives riseto two possible sources of error . Firstly, any variation in th e composition of proteinand of fat is no t satisfactorily allowed for. I n human beings, Cathcart & Cuthber t son(1931) and C uthber tson (1933) have shown that the calorific value of fat can varyfrom 8880 to 9523 Cal./kg depend ing on the source of the material. Su ch variationsare very significant in te rm s of energy storage. Secondly, errors arise as a result ofthe assumption that energy is stored only as fat or protein. It is generally believedthat the main interpretational error attached to the computation of energy balancefrom the carbon and nitrogen balance is due to the assumption that all the non-protein carbon represen ts fat. O ne gram carbon stored as glycogen would representapproximately 9.2 Cal. and as fat 12.4 Cal. T h e maximum overestimation thereforewould be 30%. I n the growing animal it is do ubtfu l whether glycogen representsmore than 2 yo of the body-weight an d the actual error is probably an overestimationof energy storage by 0.6%. A fur the r interpretational error, which does not appearto have been considered, is the possibility that, in certain instances, phospholipinmay account for a large fraction of the lipid stored. No published figures are availablefor the calorific value of phospholipins which, since they contain nitrogen, wouldbe estimated partly as protein and partly as fat.

T o provide fu rth er information on th e accuracy of prediction of energy storagefrom Armsby's (1917) factors, an examination of the carbon and nitrogen contentsan d energy value of body tissues of cattle was und ertake n. Co mp arable analyses ofisolated tissue components were also made, and the results used to illustrate thepossibility of error.Materials METHODS

Samples of nine muscles, of five brains and of five livers were analysed. They wereobtained from freshly killed animals and were dried at 100' before analysis. Fivesamples of depot fa t were dried i n vacuo over calcium chloride. Details of thepreparation of brain phospholipins, liver glycogen and myosin are given later.Chemical methods

Nitrogen was determined by macro-Kjeldahl analysis. Carbon was determinedby combustion of the sample in a stream of oxygen in a tu be packed with copper oxideand lead chromate, using th e precautions listed by Pregl (1924). T h e carbon dioxide


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VOl. 7 Factors for calculation of energy storage 85was absorbed in solid sodium hydroxide dispersed with pumice stone. Ash wasdetermined by incineration at 550'.Calorimetric methods

Calorific values were determined in a standard pattern of bomb calorimeter. Thewater equivalent of the bomb was determined by use of benzoic acid (thermochemicalstandard grade, British Drug Houses Ltd.). The Regnault-Pfaundler cooling cor-rection was used, and a correction was inserted for the heats of formation and ofsolution of the higher oxides of nitrogen. Sulphur was determined by analysis of thewashings of the bomb. No allowance was made for possible impurities in the oxygensupply, but care was taken to make all determinations at the same oxygen pressure.The increase in temperature of the calorimeter was measured by a thermistor circuitcalibrated to give a range from 15 o 20, and to read to the nearest 0-001'. Detailsof this instrument and instructions for its calibration and use are given by Beakley(1951),o whom we are indebted for assistance. The thermistor circuit was calibratedusing a long-stem mercury-in-glass thermometer with an N.P.L. certificate, readingto the nearest O.OI', over the range 15-20'.Preparation of myosin, f a t , phospholipins and glycogen

Myosin. Muscle, freed from visible fat, was homogenized in a KC1-NaHCO, buffer,and the protein in the extract was precipitated by dilution in 20 vol. water (Edsall,1930). The precipitate was redissolved in the buffer and again precipitated bydilution. It was finally dried with acetone and then by desiccation in vacuo overCaC1,. All precipitations were carried out at 3'.

Fat . Perinephric fat was extracted with hot ethanol, the extract was dissolved in etherand impurities were precipitated with acetone. Th e fat was dried over CaCl, in vacuo.

Glycogen. Glycogen was prepared from cow liver using the method of Bell &Young (1934).Repeated precipitations of the glycogen with ethanol were employed.

Phospholipins. Two preparations were made from the brains of two cows. Thefirst method employed extraction with hot ethanol, and the other the chlorofonn-methanol extraction described by Folch, Ascoli, Lees, Meath & le Baron (1951).The crude extracts were purified by repeated precipitation from ether with acetone,and finally dried in vacuo over CaCl,. The preparation was thought to be largelylecithins with some slight admixture of cephalins.

RESULTSThe analyses of the tissues are expressed on an ash-free basis, and are given inTable 2. From the carbon and nitrogen content of the tissues the calorific value wascalculated using Armsby's (1917)actors, with the results presented in Fig. I. InTable 3 the mean calculated values have been compared with the mean observedvalues. It will be noted that Armsby's factors gave a significant overestimation ofthe calorific value of muscle tissue. This may be due to several causes. The fat andprotein present in the sample may have had on average a lower calorific value thanthe preparations used by Armsby (1917)s the basis for the derivation of his factors.


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86 K. L. BLAXTERND J. A. F. ROOKTable 2 . Elementary analyses and calori$c values of bovine tissues,expressed on a dr y, ash-free basis

I953

Sample no.I23456789

1 0I 1I 213141516I7I 8192021222324

Brain

Liver

Tissue Carbon (%)Muscle 55'52

53'6255'3052'2457'4753-6953'7753-3853.3661-8363.5161-2462-5963.9656.0055'4455'2357'5857'63746674'99Samplesdeterioratedbefore analysis

Fat

Nitrogen ( yo ) Cal./kg14'05 58761.5'42 565913.80 582315.10 573I1-36 653914'32 574514'60 59441402 579114-70 57969.08 72447.88 7228-41 74349.22 72029'45 706412-81 606911-46 637411.17 63289'73 604811.81 60970'27 93290.71 91710.13 94640'08 93830.07 941

Table 3 . Prediction of calorific value of tissues from their carbonand nitrogen content, using Armsby's (1917) actorsObserved Calculatedvalue value (B- ) withA B standard error

Muscle 5878.2 6010.9 132'7f43'9*Tissue (Cal-ikg) (Cal./k!) (Cal./kg)Brain 7229'0 7332.6 103.6f 13.0Fat 9250.0 9254 '5 4'5 *57.1All tissues 136.7f 5.3*'Liver 6183.2 6413.2 230'Of 135.2

* Significant at woz> P>o-or. ** Significant at P


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VOl. 7 Factors f o r calculation of energy storage 87

/I I I I 15000 6000 7000 8000 9000 10,OOO

C a l c u l a t e d v a l u e (Cal./kg)Fig. I . The accuracy of prediction of the calorific values of tissues of cattle from theircarbon and nitrogen content using Armsby's (1917)actors.

Table 4. Elementary analyses an d calorific values of purified tissue constituentsexpressed on a dr y, ash-free basisTissue Nitrogen Carbonconstituent (% I (70) Cal./kg

Fat (depot) 0 '00 76.5 I 9398Glycogen 1.70 45'27 4117Phospholipin I 1-97 - 7866

2 1-97 62.90 8137Myosin 1 5 '5 0 57'0.5 5949The values for glycogen and for the mixed phospholipins of brain are, to our

knowledge, the only published observations on the calorific value of these substances.The values are shown in Fig. 2 together with limited analytical data on other purifiedtissue components as summarized by Fries (1907). The values for non-proteinnitrogen-containing substances are all above the line that relates the calorific valueof tissues solely to their fat and protein content according to the Armsby factors.The values for phospholipin, for glycogen and for succinic acid lie below the line.The deviation of phospholipin is small, and the error introduced by nitrogenousextractives would be in the reverse direction to that noted in Table 3. It may beconcluded, therefore, that the presence of glycogen and of other carbohydratederivatives probably accounts for the major part of the discrepancy. In any event,Fig. 3 suggests that the discrepancy that arises from the application of Armsby'sfactors to analytical data for whole tissues is due to the assumption that nitrogen


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11,000 r10.000 -

m: 000 -3000 -0 Succinic acid20001000

--0 10 20 30 40 50

Nitrogen(%)Fig. 2. The calorific values of constituents of pure tissues of cattle in relation to their nitrogen content.

0-0, alues of Armsby (1917);0 ,values of Fries (1907);A, alues obtained in present work.

m

6750 -6500 -6250 -6000 -5750 -5500 -5250 I I I ' I I I I I I I I I I I I I I I I1 2 3 4 5 6 7 8 9 1 01 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2 2 3

Nitrogen (%)Fig. 3. The relation between the calorific values of dry, ash-free tissues of cattle

and their nitrogen content.


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VOl. 7 Factors fo r calculation of energy storage 89retention represents retention of a mixture of purified protein and triglyceride fat,rather than of a highly complex system of nitrogen-containing and nitrogen-freesubstances.

To make allowance for the discrepancies that arise from Armsby's assumptions,an empirical approach was adopted. The calorific value of materials containing nonitrogen and containing 16.0yo nitrogen was evaluated by statistical analysis of thedata in Table 2. The calorific value of the ash-free dry matter of the tissues wasrelated to their nitrogen content and the results are shown graphically in Fig. 3.

Cal.=9367- 52.9 N, (1 )he equation was:where Cal.=Calories/kg dry, ash-free tissue and N = he nitrogen content expressedas a percentage.

The regression was very highly significant (P


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90 K. L. BLAXTERND J . A. F. ROOK I953organic compounds that are deposited along with protein when an animal retainsnitrogen. This process assumes that such a deposition is of constant composition,which is certainly not correct. The same criticism is true of other factors used in carbonand nitrogen balance methods, and an empirical measure using material obtainedfrom animals in a normal state of nutrition may possibly minimize this error inpractical experiments.It may be suggested therefore that the factors summarized in Table 5 should beused for the computation of the results of carbon and nitrogen balances rather thanthose of Armsby.

Table 5 . Suggested fac tors fo r the computation of the calorie storagefrom carbon and nitrogen balancesMultiplication factors

fA

\Armsby's (1917)Computation* original SuggestedProtein stored from nitrogen retention 6.25 6-25Calories stored as protein from protein stored (Cal./kg) 5700 5322Carbon content of protein from protein (g/g) 0'5254 0. 5 120t

I 0 0- .0 0-- =1.31Fat retention from retention of non-protein carbon 74'84t =6.5Calories stored a s fat from fat retention (Cal./kg) 9500 9367

* In practice it is not necessary to compute protein retention or fat retention, the factors being com-bined and expressed in terms of nitrogen and carbon retentions alone. Th e full stages are given herefor simplicity.t Seep. 89.

I. The calorific value and carbon and nitrogen content of the tissues of cattlehave been determined.

2. Factors normally used for the prediction of calorific value from the carbonand nitrogen content of tissues have been examined and have been shown to causea small systematic error.

3. Analyses of purified fat and protein agree with those on which these factorswere based.4. It is pointed out that the error arises from the assumption that pure proteinand triglyceride fat are the only materials stored by the animal during growth orfattening. The error can be reduced by using as a base-line a statistical estimate ofthe carbon content and calorific value of a tissue containing 1 6 % nitrogen.

5. Supporting evidence is adduced from analyses of purified tissue constituentsand a set of factors is suggested.

SUMMARY

REFERENCESArgutinsky, P. (1894).PJiig. Arch. ges. Physiol. 55 , 345.Armsby, H. .(1917).he Nutrition of Farm Animals. New York: T he Macmillan Co.Atwater, W. 0. (189j). ull. U.S. ff.xp . Stas. no . 21.Atwater, W. . & Bcnedict, F. G. (1904). ubl. Cavneg. Instn. no. 42.Beakley, W. R.(19j1). . sci. Instrum. 28,176.Bell, D. . &Young, F. G. (1934). Biochem. J . 28, 882.Cathcart, E.P.& Cuthbertson, D. P. (1931). .Physiol. 72,349.


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VOl. 7 Factors fo r calculation of energy storage 9Cuthbertson, D. P. (1933). Biochem.J.27, 1099.Edsall, J. T. (1930). J . bid. Chem. 89, 289.Folch, J. , Ascoli, I. , Lees, M., Meath, J. A. & le Baron, F. N. 1951). J. bioZ. Chem. xgx, 833.Fries, J. A. (1907). Bull. US. ur . Anim. Ind. no. 94.Kohler, A. (1900-1). Hoppe-SeyZ.2. 1, 79.Lusk, G. (1931). The Elements of the Science of Nutrition. Philadelphia: W. B. Saunders Co.Pregl, F. (1924). Quantitative Organic Micro-analysis. London: J. and A. Churchill.Rubner,M. (1885). Z . BWZ. 21, 12.Schulze,E. & Reineke, A. (1867). L a d w . Ve~sStu. ,97.Stohmann& Langbein (1891). J . prukt . Chem. (N.F.) 4,364 (quoted by Armsby, 1917).

The Cholinesterase Problem in the Newborn PuppyBY R. A. McCANCE AND L. M. BROWNMedical Research Coumil Department of Experimental Medicine,University of Cambridge

(Received2 Februuy 1952)McCance, Hutchinson, Dean & Jones (1949)howed that the pseudo-cholinesteraseactivity in the serum of the puppy might increase up to twenty-five times its initialvalue in the first 3 days of life, and decrease again within about 10 ays. Th e massiverise was attributed to the absorption of intact molecules of enzyme through theintestinal wall from the mothers colostrum, which exhibited a very high cholinesteraseactivity. The present study was undertaken ( a ) to provide some information aboutthe rate of destruction of the colostrum cholinesterase by digestive enzymes of thepuppy, adult dog and pig in vitro; and ( 6 ) to find out whether the cholinesterase inthe serum was taken up by any of the puppys tissues, thus giving a clue to its function.

METHODSAnimals

Organs from adult dogs that had been killed for experimental purposes in otherDepartments were obtained immediately after death. Since neither the serum northe red cells showed any change in cholinesterase activity as a result of theseexperiments, it is unlikely that the activity of the organs was altered. Fresh pigpancreas and duodenum were obtained from the local slaughterhouse.

Most of the puppies were born in the Department. They were weighed, killed bya blow on the head, decapitated and bled into a centrifuge tube. The organs wereremoved and prepared for cholinesterase estimations, and samples of pancreas werealso taken for preparation of proteolytic enzymes and histological examination.

Digestion experimentsPreparation of proteolytic enzymes. Pancreas from pigs, adult dogs and newborn

puppies was minced or cut up finely with scissors, washed with acetone and ether,allowed to dry at room temperature and then sieved and stored. T o obtain enterokinase,the mucosa was scraped from the wall of the duodenum and dried, sieved and storedin the same way as the pancreas. A mixture of intestinal ereptic enzymes was obtained

1953 - blaxter - heat tissue combustion

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