M. Walser and S. N, Stein (Proc, Soc. Exp. Biol. Med. 82, 774 (1953) ) have used a similar helium method f o r determining the body volume

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1 14. BODY COHPOSl T i O N A#D 'SLEEP T l H E NDUCED THROUGH THE USE OF ANESTHETCS LOUS FElHSlElW U. S. DEPTi O F AGRCULTURE *... ~ *. We are interested i n determining the canposition of the l i v i n g animal without injury e i t h e r t o the animal o r t o the experiment of which the animal i s 8 p a r t. This paper w i l l b r i e f l y describe some of the methods beused a t present by d i f f e r e n t workers i n the f i e l d. n addition we w i l l review our work with the use of anesthetics as a means of determining the body fat composition of s w i n e. Methods used have ncluded t h e estimation of body density and total body water. Archimedes' principle has formed the basis f o r calcul a t i n g body density needed for the determination of body volume. Under water the baly loses an amount equivalent t o the weight of displaced f l u i d. Unfortunately a number of d i f f i c u l t i e s hinder the use of t h i s r e l a t i v e l y simple method. t is cumbersome and time-consuming because a determination of the amount of air i n the lungs and respiratory passages must be made a t the time of underwater weighing. This has prevented the use of t h i s method f o r t h e l i v i n g animal. However, t h e method has found wide use i n studying the comgosition of carcasses. Body volume determination has been W e i n other ways however. R. J. Wedgwood am8 R, W, Newman (Am. J. Phys, Anthropol. &, 26 (1953)) used air-displacement instead of water-displacement. They used an airt i g h t compartment t o enclose the subject and by successively increasing and decreasing by known amounts the air within found the pressure changes due t o these changes electrical3y end by calibration using cyclinders of known volume were able t o calculate the subject's body volume. W, E. S i r i approach. He used a calculation from the closed chamber, with (Fed. h o c. 12, 133 (1953) ) used s t i l l another gas d i l u t i o n T r i n c l p l e, obtaining a body volume change in concentration of helium in3ected i n t o a and without the subject i n the chamber. M. Walser and S. N, Stein (Proc, Soc. Exp. Biol. Med. 82, 774 (1953) ) have used a similar helium method f o r determining the body volume of i n t a c t a n i m a l s. An important p a r t o f t h e l i v i w organism i s the quantity of body water present. The preponderant single molecular element mahing, up t h e t i s s u e i s water. Lif'e with i t s great mass of chemical reactions depends on the presence of water. By mews of water the body transports most of i t s enzymes, substrates, metabolites, gases, electrolytes, antibodies and other ccolponents moved froaa c e l l t o c e l l. Body water measurement has been t r i e d by three methods: desiccation procedures, body specific gravity measurements, and d i l u t i o n techniques.

2 141. While desiccation has given us valuable information, it i s obviously not one that can be-employed c l i n i c a l l y. n 1941 Behnke (Harvey Lecture, series 37, (l ) ) announced h i s findings using t h e specific gravity method on young men. H i s r e s u l t s showed t h a t i n normal subjects a determination of the body specific gravity enabled one t o estimate the body fat content. The primary variable t h a t determined body density was f a t with i t s low specific gravity of.92. A t t h i s time Behnke f o d a t e d h i s concept of the lean body mass, consisting of a fat-free portion of e s s e n t i a l l y constant gross composition and of a variable quantity of f a t. A t t h i s time he was not primarily interested i n determining values f o r t o t a l body water i n h i s subjects but he could have done so, f o r body water and body fat bear a reciprocal relationship t o each other; t h a t is, t h e less t h e fat content, t h e greater i s the percentage of water contained i n t h e body. The t h i r d method has been a d i l u t i o n method. An i d e a l material must be safe and give an accurate measurement of the t o t d body water. t must not be toxic t o t h e organism and must equilibrate rapidly with body water. t must not be selectively stored or excreted and should not be metabolized. ts concentration i n urine or plasma must lend i t s e l f t o ready measurement. Many substances have been employed and among the substances used have been potassium, thiourea, sulfanilamide, tritium, antipyrine and deuterium oxide. Antipyrine v a s first t r i e d f o r t h i s purpose i n 1949 by S o b e m and h i s associates (J. M o l. Chem. 31 (1949) ) These observers showed t h a t after injection antipyrine was distributed uniformly throughout the body water and t h a t i t s concentration i n body fluids could be determined accurately. Compared values f o r body water obtained with the use of antipyrine wfth those obtained from specific gravity measurements have given a satisfactory correlation between t h e two sets of values. S t i l l, there are c e r t a i n disadvantages inherent i n the use of antipyrine: t equilibrates only slowly with abnormal body fluids, such as ascites; it is selectively bound by protein and i s metabolized a t a rate which varies between subjects. However, by suitable corrections an estimate of body water can be made. =, Calculation of body f a t using the specific gravity method involves t h e assumption of a specific gravity of 1.99 for lean body mass while calculation of body f a t from t o t a l body water involves t h e assumption t h a t the lean body mass i s 73% water. These are not independent assumptions, however. t may well be t h a t they a r e the same thing, namely that 27$ Of mixed body s o l i d dissolved and suspended i n water give a substance with a specific gravity of W e must have an independent method f o r determining body f a t i n t h e l i v i n g organism. Behnke and co-workers attempted t o use such an independent t e s t when they made use of t h e r e l a t i v e s o l u b i l i t y of nitrogen i n b o w fat. Nitroaen is quite soluble i n f a t s and when body t i s s u e s a r e saturated with t h i s gas a t i t s p a r t i a l pressure i n t h e atmosphere, the amount Of nitrogen present in t h e body is dependent t o a large extent upon t h e amount of body fat, Subjects were placed i n an atmosphere of pure oxygen and all nitrogen subsequently eliminated from the body was collected. The weight of body f a t was calculated from the volume of nitrogen obtained from t h i s washout process (corrected for that dissolved i n non-lipid tissues) when -

3 142..,., t h i s was divided by the s o l u b i l i t y of gas per gram of f a t. There a r e errors in t h i s determination. t is d i f f i c u l t t o correct f o r pulmonary nitrogen, the presence of nitrogen i n the i n t e s t i n a l gases, and absorption of nitrogen through t h e skin. t takes 12 hours t o perform the washout maw procedure. With the thought that many of t h e d i f f i c u l t i e s and errors of the nitrogen elimination methcd could be avoided if a nontoxic, i n e r t foreign gas with a much greater fat: water s o l u b i l i t y r a t i o were employed, cyclopropane has been t r i e d. Cyclopropane is highly fat soluble and has been used as an anesthetic f o r years. Lesser, Blumberg and Steele (Am. J. Physiol 169, 545 (1953) ) have reported on this reseerch using r a t s. They found that 9$ of the cyclopropane in the body i s present i n the fat a t equilibrium, making the cyclopropane method r e l a t i v e l y independent of the nonlipid substances of the body. However, the s o l u b i l i t y of a l l gases varies with temperature and the mean temperature of the body fat must be estimated in each animal. An e r r o r of 1 t o 2 in estimation of mean body f a t temperature results in an e r r o r of l e s s than 4% i n the t o t a l body fat calculation. Values f o r t o t a l body fat by the cyclopropane method were found t o approximate closely the weight of t o t a l ether extractable material. The concept of t h e b a n body mss has proved usef'ul c l i n i c a l l y. t has led t o the thought that dosages of drugs should be made on the basis of the lean body mass and not on body weight. t is erroneous t o assume t h a t fat is metabolically inactive but it is probable t h a t the lean body is metabolically more active. Researches on the mechanism of thiopental detoxication have l e d t o the conclusion that i t s short duration of action is due t o its localizat i o n i n the fat depots of the body rather than in i t s rapid destruction. Following t h e intravenous injectlon of thiopental in the rat,shideman and associates (Jour. Pharm. Exper. Therap. 17, 368 (1953) ) found that drug equij.ibrium occurs between the blood, brain and l i v e r within one minute but between blood and fat u n t i l one t o two hours. When equilibrium i s ob,cained, t h e tlssue/blood r a t i o s axe appraximately.6, 2.5, and 7. mspectively, for brain, U v e r and fat. The r a t e of metabolism of theopental wa6 only 1%per hour. - H e m and Wood (Proc. SOC. Exper. B i o l. Med. 8, 318 (1952) ) pericn;lmed a s e r i e s of experiments t o see w h a t the i n f l u e z e of body fat was on the duration of thiopental. anesthesia. n an earlier paper Brodie and U8-cistes (J. Pharm. Exp. Therap. 98, 85 (195) ) had found that fat t i s s u e had a high a f f i n i t y f o r t h i o p e n t a. Herman and Wood found that the duration of thiopental anesthesia was more than doubled i n the lean r a t s compared t o the f a t rats, The fat rats had 13.5$ body fat and the lean rats 8-55 body fat. The fat rats s l e p t 23.5 minutes and t h e lean rats 52.6 minutes. The t h e from loss of righting r e f l e x u n t i l regaining of righting m f h x was taken as the duration of anesthesia. The amount of body fat w&s e a t h a t e d on the shaved, eviscerated animal by the specific gravity method. De Boer (Anesthesiology 2, 375 (1947) ) found t h a t s e m i - s t m a t i o n prolonged the sleeping time of dogs under thiopental anesthesia: the animal sad, of course, less body fat af'ter semi-starvation.

4 With t h i s background before you, s h a l l now attempt t o explain our research and findings. W e were interested i n determining fat d i r e c t l y i n the l i v i n g i n t a c t mat animal. The method had t o be quick and as accurate as possible yet done with as l i t t l e e f f o r t as possible. Anesthesia appeared t o be a lead. W e would only need t o i n j e c t t h e sleep-causing chemical and count t h e minutes of sluniber. No blood samples would have t o be taken and no blood samples would have t o be -lysed. The l i t e r a t u r e of anesthesia showed that thiopental and p e n t o t h a behaved differently. Thiopental is a thiobarbituate. We looked around f o r other anesthesia e found kemithal. t is a causing chemicals containing the thio-group. W thiobarbiturate with a d i f f e r e n t pair of side chains. using both chemicals on swine. W e s e t up a program Our program was as follows: We took three sets of animals, two e called the f i r s t two groups groups of f i v e each and one group of three. W A and B and the t h i r d group X. Group A were t o be our lean animals with group B our so-called e t r i e d everyf a t animals. The X animals were our e a e r i m e n t a l animals. W thing new on the X animals. We wanted t o be sure that a t the end of t h e experiment, at slaughter time, we would have f i v e animals lean and f i v e anbals fat. The regular diet was fed t o the B group while the A group had a,n alfalfa diluted regular d i e t. Each grayp was fed from self-feeders as a group: no individual animal. was fed away from h i s group's feeding. A t three o'clock the day before anestheeia, t h e animal on trial was placed by itself i n a pen with zero food but was allowed water t o drink. A t approximately 8:3 8:45 a.m. the animal had e i t h e r kemithal or thiopental solution used on it as reagent. Time was noted a t t h i s point and a&ain when the animal went t o the f l o o r loelng i t s a b i l i t y t o stand. When t h e animal began t o struggle we noted the time period f o r the animal t o stand on the f r o n t two feet. We called t h e period between collapsing t o t h e f l o o r e do not want t o say and standing on the f r o n t two f e e t our ''sleep" time. W that t h e animal was surgically asleep throughout t h i s period called "sleep" time. Actually t h e nn9mnlwas not asleep throughout t h e pericd but we could obtain i n using these two time refldings a d e f i n i t e physiological state of i n a b i l i t y t o stand on t h e part of the animal. Each animal was allowed t o determine h i s own a b i l i t y t o r i g h t himself. - A growth-weight curve was obtained on each animal i n order t o be c e r t a i n t h a t each animal was growing normally because of t h e frequent "sleep" times. Durinqs the growth period from weaning t o f i n a l slaughter each a n i d was given kemithal and thiopental many times. Each animal promptly went back t o eating nmediately after righting itself a f t e r anesthesia. e have had t o l e a r n how t o r e We have had very many trials. W s t r a i n growing animals f o r intravenous, intraperitoneal and r e c t a l anesthesia. We had t o determine the Bmourrt of kemithal and thiopental t o use per pound of body weight 8 that once we standardized on er d e f i n i t e value we would continue t o use it throughout t h e remaining growth period. For swine we have used with slight modification t h e method of given by Alcorn of owa (see E, R. Frank, Veterinary Surgery PJottqe, Burgess Publishing C o ~ ~ a n yminneapolis,, Minnesota, 1944) A res-int

5 rope i s fastened around the snout and attached t o a post approximately one foot above t h e ground. A hobble is made of a piece of pipe about one and one-fourth inches i n diameter and sixteen inches long, having a strong ring two inches i n diameter welded on each end of t h e pipe. A strong rope runs through each r i n g and is attached t o each of the rear l e g s. The opposite ends of t h e rope are attached t o a t h i r d ring which i s i n t u r n fastened t o a block and tackle. The latter i s fastened t o a post a t a point s i x f e e t off t h e ground. As the block and tackle i s tightened the animal is stretched out and stands on his f r o n t t w o feet. n t h i s position the animal can easily be given t h e anesthesia producing chemical. As soon as t h e animal begins t o collapse on h i s f r o n t feet t h e block and tackle is released and the animal i s then completely freed of h i s r e s t r a i n t. This restraining set-up has allowed two men t o handle swine of varying s i z e s and weights easily and has made it possible t o i n j e c t accurately the drug without danger of breaking t h e needle or syringe. Our results were approximately as follows: Figure 1 shows the relationship between carcass s p e c i f i c gravity and percent carcass fat obtained by physical analysis. Figures 2 and 3 show the relationship between carcass specific gravity and the "sleep1' times obtained by using Kemithal and Thiopental respectively. Our follow-up experiments t h i s year were conducted a l i t t l e differently. We took a group of pigs a f t e r weaning and fed a l l animals t h e regular d i e t. This time we used only thiopental as our anesthesia-producing drug. As the animals g r e w both thiopental and antipyrine were injected intraperitoneally and t h e "sleep" time and body water determined. Periodic a l l y one animal was sacrificed and the specific gravity of t h e carcass determined. n addition the body of t h i s anwl was analyzed chemically. want t o present t o you a p i c t u r e of one animal as t h e animal grew from 59.4 t o 141 pounds. n figure 4 you w i l l note t h s t as t h e a n a l weight and percent body fat increase the animal "sleeps" less and less. n conclusion we have here another approach t o the possible solution of determining quickly, accurately and d i r e c t l y t h e body f a t Naturally t h e r e are percentage of growing s w i n e who are i n experiments. unknowns about t h i s procedure but w h a t we do know leads us t o believe t h a t the method is a valid one and is one of the few approaches available f o r determining the fat content of the l i v i n g animal.

6 1115. A.98- er 4 b L k o*ooo $ Carcass Fat (Physical Analysls) h er 4 td L o.geo a Q, :1.2- b u (d i r 6 7 t 9 8 Remithel * S l e e p * Tlme-minutes i b a) u i oeor Thiopental n S l e e p * Tlme-mlnutes 18

7 so time-minutes 18 2 Anlmal g h 1 P 4 Y x-x x welght vs. s l e e p t i n e Body r a t VS. c (ThloDental used) s l e e p time

8 147. MEt. AUNAN: Thank you, D r. Feinstein, A t t h i s time we w i l l c a l l on Don Kropf from Wisconsin. bb. KROPF: Speaking of sleep time an wondering what anesthetics some of the latecomers use. With regard t o these three good papers we h v e now heard have Just B few cormpent8 t h a t should like t o throw out and then we w i l l have a l i t t l e discussion. With regard t o the lean-meter, we have been investigating the p o s s i b i l i t y of using t h i s instrument on carcasses and especially we are working with t h i s point in view: f we can determine somehow t h e amount of muscling, tbe. s i z e of the eye muscle, without breaking t h e loins when you work i n packing plants the packers don't want t o break t h e loin, if possible, and so i f something like t h i s can be done it sure w i l l help u8 a l o t. -- Another c w t should l i k e t o throw out as far as t h i s carcass work i s concerned i s we were talking about changing the shape of t h i s l i t t l e disk. t 16 round and i f you t r y t o f i t it inside o f t h e carcusa, sa;y w d r k i q from the r i b toward the outer side of the carcas6 it is very difficult t o use it fn t h i s place. Perhapa a long rectangular shape disk would do t h e job nicer. A t t h i s tine if atly of you have queetions or c o ~ m n t st o d i r e c t t o Dr. Pearson, D r. Wilder, or Dr. Feinetein should l i k e t o hear them. MR. BBDENSTEN: should like t o ask D r. Feinstein whether or not he has experienced any d i f f i c u l t y with the antipyrine technic in determining body water. have p a r t i c u l a r reference t o the difficulties tfst we have experienced i n getting an extremely e r r a t i c rate of metabolism or something affecting the concentration of the antipyrine or antipyrine-like substances which show up on the o p t i c a l density. We have been able t o get absolutely no l i n e or plot on the semilogarithmic paper in order t o determine the concentration in the serum. was wondering if you had any d i f f i c u l t i e s of that kind. MR. E'ENSPEN: Probably you noticed tbat in tb tallr X said that the antipyrine -8 injected intraperitoneally in the swine. We found when doing t h i s that absorption into the cavity WLLS slower than when injected i n t o the blood stream Sad it agparently seemed t o make the determination work out a l i t t l e b e t t e r. We have never noticed d i f f i c u l t y 88 far as the antipyrine rea8lrig on the rectum was concerned. We always had enough n i t r i t e t o c a w e the reaction t o come up very quickly, within t e n minutes, t is a mstter of having the proper n i t r i t e concentration, and we take resdiags at 5, 1, l5 and 2 minute intervals j u s t t o be sure we have accurate readings. After the materia is injected i n t o the peritoneum we take the blood. on swine. MR, WEZLNmON: This is i n regard t o checks with antipyrine W e have not gotten as good checks with swine as we have

9 148. with c a t t l e, but on the 32 hogs t h a t we determined body water with antipyrine and t h a t were l a t e r ground up and analyzed f o r moisture the correlation was.73. m a t corresponds t o a correlation of about.93 with the cattle. Same of our points weren't on the l i n e as w e l l as we should like t o see them. For t h e cattle they were much larger. MR. NAUMA": Doctor, i n which one of the sleep-inducing; agents do you have the most confidence? MR. FEWSTEN: Well, with kemithal t h e animal goes out very quickly and it comes back very quickly, as though t h e animal took just a l i t t l e sleep and got back on i t s feet without any d i f f i c u l t y. With pentothal t h e animal does not go out quite as easily and it does not come back on i t s feet as n i c e l y with pentothal. We had a l i t t l e d i f f i c u l t y with kemithal i n getting a sample t h a t was consistent. That is, t h e l o t s were not consistent and we have not found out yet w h y they are not consistent. Chemically they seem t o be a l i k e but on assay procedure they don't seem t o be alike. MR. NAUMA": On t h i s point, do you f e e l t h a t the natural stimulus t o get up is s u f f i c i e n t t o avoid any great e r r o r along that line? MR. FEXNSTEN: Well, we are using a natural stimulus - hunger. Swine a r e always hungry. They have been without feed f o r 18 o r 2 hours. have seen them drag t h e i r backside which has not completely come out of slumber, so t o speak, dragging t h e whole body over t o the feed bin and start t o eat, p r a c t i c a l l y l i e i n there and eat. So there i s a stimulus there f o r t h e animal t o get up. MR. KASTELC: Would you mind indicating how much antipyrine you use when you inject t h e hogs intraperitoneally? kilos. MR. FEEJsrrEN: We are using roughly about 5 grams per 1 MR. KROPF: Are there any other questions or comments? MR. ACKER: Were the same a n h l s used i n both anesthetics i n the bottom two graphs on t h i s page? week. MR. FENS1EN: Yes, they were done a week apart - within a MR. ACKER: D i d you run correlations on t h e two anesthetics and did you get a much longer one with one than with the other? MR. FENSEN: the curves. graphs. You w i l l notice t h e different time periods on MR. A m : But the same animals you can estimate from the wondered i f you ran correlations.

10 149. MR, FENSCPEN: W e ran no correlations, but the two materials w i l l give the time even though they a r e not used i n the same proportion. n other words, we used thiopental at 12 kilograms per pound of body weight and kemithal at 2 1 kilograms of body weight. H. ACKEB: Will the two give a corresponding time on both animals or W i l l one be a longer time snd the other a shorter time? Fw, ib": The two anesthesias w i l l work a l i k e. animals sleep less and the lean animals sleep more. M3. ACKER: Did you ever The fat run the time? MR, FENSCEN: W e have the data. MR. ACKER: Are they correlated? MR. FENS!EN: Yes, they are correlated. MR, PEARSON: want t o ask D r. F'einstein a question about t h e intraperitoneal injection of antipyrine. As understand the antipyrine procedure, when you make the injection you assume an instantaneous dist r i b u t i o n throughout the body. MR, EENSTEN: You never take your blood samples i m e d i a t e l y. W e take three o r four blood saqples i n order t o get back t o zero time, and it is done the 8wap t h a t you do but injecting antipyrine i n t o the blood stream. DR, PEARSON: The point here i s t h a t you assume t h a t time you have c q l e t e d i s t r i b u t i o n throughout t h e e n t i r e body will have t o make a correction factor i f t h a t is not t r u e. n of intraperitoneal injection we have made some and our volumes lower. a t zero and you t h e case are much MR. FENSTEN: Well, the whole theory of antipyrine being diluted i n water is d e f i n i t e l y wrong because you a r e going t o have one part of the body completely different from the statement that it dissolves throughout t h e body water. f you are going t o assume t h a t it i s n ' t true, then the method is no good from the start. You are assuming that the antipyrine dissolves a f t e r it reaches the body water and the cavity i s a p a r t of it. f it i s n t t t r u e then the method i s n ' t good. MFt. WHEELER: think i f you have any more questions D r. Feinstein w i l l be glad t o t a l k t o you after t h e meeting, and should like t o t u r n it back t o Woodie. MR, A U " : And a t t h i s time will t u r n it back t o D r. Kline. t h i n k before we get up and stretch we had better have another b a l l o t t o e l e c t our conference chairman for next year. Yesterday, you w i l l r e c a l l, f i v e men were elected t o the Executive Committee: Pearson, Aunm, Henrickson, Kemp and Blumer. You will now vote for one of those men for your conference chairman. Please cast your b a l l o t s. CHARMAPJ KLNE:

11 15. (Balloting, taking of conference picture, and recess.) CHARMAN mve right along. m:now l e t ' s come t o order again, please. W e must should l i k e t o mabe t h i s one announcement before turning it back t o Woodie t o proceed and t h a t is t o s a y ' t h a t your new conference chairman for next year is D r. Pearson of Michigan State University. Now then, Woodie, wiu you proceed? M. All": At t h i s time we w i l l c a l l on D r. Zane Palmer. W e welcome you back t o the Reciprocal Meat Conference. You have been gone the past few years and it is certainly a pleasure t o have you with us again. MR. PALMER: Thank you, Woodie. Gentlemen of the Reciprocal Meat Conference, it i s definitely a pleasure to be back i n t h i s group.