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A study of the effect of glycogen on the oxidation of butyrate by rat liver slices

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A study of the effect of glycogen on the oxidation of butyrate by rat liver slices

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Content A STUDY OF THE EFFECT OF GLYCOGEN ON THE OXIDATION OF BUTYRATE BY RAT LIVER SLICES A Dissertation Presented to the Faculty of the Department of Biochemistry University of Southern Califomla School of Medicine In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy by Blanche Gauthier Bobbitt May 1941 This dissertation, written by under the guidance of h.BT. Faculty Committee on Studies, and approved by all its members, has # j been presented to and accepted by the Council fV Z ^ j on Graduate Study and Research, in partial fu l- ) « / V fillment of requirements for the degree of D O C T O R - O F F H I L O S O P H Y Secretary D a te .J.uae_..124l....... Committee on Studies TABLE OF CONTENTS FOREWORD......................................... 1 HISTORICAL DISCUSSION ........................... 4 EXPERIMENTAL P A R T ............................... 15 Experimental animals . . ................. . . 15 Procedures............................... 15 Warburg manometrlc method .......... 15 Calibration of apparatus 16 Solutions............... 18 Preparation of tissue slices ........ 19 Respiration experiments.................... 23 Micro-determination of acetone bodies ..... 26 Solutions ....... 26 Determination of acetoacetlc acid ...... 26 Determination of beta-hydroxybutyric acid • • 28 Determination of residual butyric acid ..... 29 Results .......... .......... ..... 34 Effect of fasting.......................... 34 Effect of glycogen ............... 36 Effect of butyrate plus glycogen ........... 38 Effect of butyrate plus glycogen on total ketone production .......................... 46 Quantitative studies on the changes in butyrate with and without glycogen............... • 42 ill DISCUSSION............, ......................... 47 CONCLUSIONS............... 55 BIBLIOGBAFHY ................................... 56 APPENDIX..................... 60 FOREWORD Ketosis, or the accumulation of ketone bodies, has been observed to occur In cases of faulty fat metabolism associated with Insufficient carbohydrate of either exogenous or endogenous, source or with impaired ability to oxidize glucose. The ketone bodies, namely acetoacetlc acid, beta-hydroxybutyric acid, and acetone are incomplete oxidation products of fatty acids. Their existence was established in studies on diabetic urine during the last century by Peters, Gerhardt, Kulz, and Stadelmann. Knoop (1904-05) showed that the even-chained fatly acids were partly responsible for the formation of ketone bodies and later determined that certain amino acids are also ketogenic. The close correlation between fat and carbohydrate metabolism is further evident upon the administration of insulin to diabetics, which results in both improved carbohydrate utilization and cessation of ketosds. Since the oxidation of carbohydrate prevents ketosis, the mechanism of this reaction has been widely investigated. Two schools of thought have developed! the one proposing antlketogenesls, the other, ketolysis. The proponents of antiketogenesis believe that ketosis is prevented when carbohydrate Is metabolized because carbohydrate exerts a fat-sparing action, hence la metabolized in preference to fat* Under such conditions, ketogenesis, i*e., the formation of ketone bodies, is suppressed* Adherents to the theory of ketolysis affirm that carbohydrate prevents ketosis by combining with the ketone bodies to form compounds which are then easily oxidized* ‘ J?he experimental work of Voit (1891), Geelmuyden (1904), and Magnus-Levy (1908) have been of great value in the develop ment of this controversy* Researches of Zeller (1914), Woodyatt (1921) and Shaffer (1922) on quantitative aspects of ketogenic- ketolytic ratios have been of great practical importance* Evidence accumulated by Embden and Oppenhelmer (1912, 1913) and by Mir sky and Broh-Kahn (1937) has been interpreted as upholding the antlketogenesis theory* On the other hand, the results of Shaffer (1921), Beuel, Gulick, and Butts (1932), Deuel, Hallman, and Murray (1938), Shapiro (1935), Butts, Dunn, and Hallman (1935-36), and Butts, Blunden, and Dunn (1937) (1,2) have contributed to the evidence in favor of ketolysis. Since the even-chained fatty acids may be ketogenic in the absence of carbohydrate, the four carbon acid, butyric, is convenient to use in ketosis studies* Heretofore, no quantita tive study has been reported on ketone body formation from butyric acid with a simultaneous determination of residual butyric acid in the presence and absence of carbohydrate* 3 Since the liver is the chief site of formation of ketone bodies (Himwich 1931, Chaikoff 1928-29, Mirsky 1936, Greenberg 1936), liver tissue is very satisfactory to use in studying ketosis produced by the addition of butyrate in the absence and in the presence of added glycogen (Quastel and Wheatley, 1933, 1934). It is the purpose of this dissertation to study such a problem employing the Warburg (1926) technique with liver slices. Not only have some of the experiments of Quastel and Wheatley (1933, 1934, 1935) been repeated using liver slices with butyrate in the presence and absence of glycogen, but also the ketone bodies formed have been separated according to the micro-acetone determination of Edson (1935) employing Rupp*s titration (1906, 1907), and then the residual butyric acid has been finally determined, thereby giving information as to whether more butyric acid is oxidized when carbohydrate is present. HISTORICAL DISCUSSION If fats are oxidized when carbohydrate metabolism is normal, the end products are carbon dioxide and water. But if the carbohydrate metabolism is disturbed, as in diabetes or even in fasting, the fats are oxidized partially to the acetone bodies. The presence of the ketone bodies in diabetic urine was observed in the 19th century, Peters reported the presence of acetone in 1857; Gerhardt later discovered aceto- acetic acid (1865); and finally Stadelmann (1883) and Kulz (1884) identified beta-hydroxybutyric acid, Opie (1900-1901) confirmed the fact that acetone bodies are present in diabetic urine. Hirschfield (1895) noted that ketosis occurs in carbo hydrate starvation and disappears upon the administration of this foodstuff. The fundamental work of Magnus-Levy (1908) demonstrated that when beta-oxybutyric acid was fed to phlorhizinlzed dogs, it was excreted in the urine; Neubauer (1910) also found when the ketone bodies were administered to diabetics, that an excretion of these substances occurred in the urine. Knoop (1904-05) proved that the precursors of the acetone bodies are the even-chained fatty acids according to experiments in which he fed the phenyl derivatives of the lower fatty acids. From these observations he advanced his famous theory of beta oxidation. Dakin supported Knoop*s theory in work published in 1909. Liver perfusion experiments of Embden 5 and Marx (1908) were also in accord with the theory of beta oxidation. That beta oxidation Is not the only mechanism of fatty acid breakdown, however, became evident from various studies made by Olutterbuck, Verkade, Deuel, Quastel, and others* Clutterbuck and Raper (1925) reported gamma and delta oxidation occurred on oxidizing the longer chain fatty acids by hydrogen peroxide. In 1935 Butts, Cutler, Hallman, and Deuel added evidence in support of .both beta and delta oxidation from feeding experiments using the sodium salts of the fatty acids which, when fed to fasting rats, caused the excretion of twice the amount of acetone bodies when caprylic acid was fed than when butyric, caproic, or acetoacetic was administered. Morehouse and Deuel (1940) further substantiated delta oxidation in results obtained by feeding the alpha-beta and the beta- gamma dideuterocaproic acids. Jowett and Quastel (1935), as a result of studies with liver slices, postulated the theory of multiple alternate oxidation. Deuel and coworkers (1936) demonstrated that a ketonuria of greater magnitude obtained after feeding the ethyl esters of caprylic or caproic acids than after isomolecular amounts of caproic or butyric acids. Also a greater ketonuria obtained after the administration of lauric than after caprylic acids. These data were interpreted to support the theory of multiple alternate oxidation. 6 MacKay, Barnes, Carne, and Wick (1940) believe that the increased ketonuria which results after the administration of the long chain fatty acids as compared with that after butyric acid may be interpreted solely on the basis of the theory of beta oxidation* It is postulated that the acetic acid which is set free by successive beta oxidation® recombines to form acetoacetlc and beta-hydroxybutyric acids; this theory is based on a ketogenic action of acetic acid when fed to phlorhizinized dogs or to fasted rats* But such a formation of acetic acid and its recombination to form ketone bodies is probably not the case as shown by work of Stadie, Zapp, and Lukens (1941), Toenniessen and Brinkmann (1938), and Deuel, Hallman, Butts, and Murray (1936)* Stadie, Zapp, and Lukens used liver slices from depancreatized cats. After equilibrating the tissue in vitro, ketone bodies were produced by partial oxidation of fatty acids but no formation of acetic or other steam-volatile acids was found* Toenniessen and Brinkmann (1938) could not demonstrate acetic acid formation in the perfused livers of rabbits, although they stated that they believed large amounts to be formed by the catabolism of fatty acids* Finally the work of Deuel, Hallman, Butts, and Murray (1936) with the ethyl esters of the fatty acids gives no support to the oxidation of fatty acids to acetic because the same amount of ketosis resulted from administering caproic as from butyric; it was not until caprylic acid was given that an 7 increased ketosis occurred* Verkade and van der Lee (1934, 1935) suggested that fatty acids may undergo omega oxidation* This was based on the recovery of small amounts of dicarboxylic acids from the urine of human subjects after triglycerides composed of the fatty acids from eight to twelve carbon atoms were fed* Although no single oxidation theory has yet been accepted, it is, however, well agreed that the liver Is the chief site of breakdown of fat, since liver fat has a higher iodine value and phospholipid content than fat stored else where in the body* Hence it is believed that the liver desaturates fat (Leathes and Wedell, 1909, Schoenheimer and Hlttenberg, 1936) and such desaturated phospholipids are transported to the tissues for oxidation. Oxidation may also occur in the liver* As to the role of carbohydrate in fatty acid oxidation, the literature is still inconclusive* If carbohydrate metabolism can prevent ketosis, does it do so by sparing fat (antlketogenesls) or by combining in some way with the fat to make the latter more readily oxidizable (ketolysis)? Liver perfusion experiments of Embden and Marx (1908) were interpreted as indicating that carbohydrates remove fat from the oxidative processes, therefore are antiketogenic* But Magnus-Levy (1908) showed that when only a fraction of the oxidized fat is replaced by carbohydrate, the ketonuria resulting from carbohydrate fasting is overcome, Geelmuyden’s idea (1904) was that some oxidized product of glucose, such as glycuronic acid, methyl glyoxal, lactic or pyruvic acids, combines with the acetone bodies to cause their oxidation. Evidence that such condensation products of acetone bodies with glucose intermediates may occur has been cited by Henze (1931) and by Stohr and Henze (1932) in feeding experiments with “ketol," a product of aldol condensation of acetoacetic acid and methyl glyoxal followed by decarboxylation, Ketol was found to increase the blood sugar level and the liver glycogen, although it did not apparently form muscle glycogen. Zeller (1914) concluded from results obtained with high fat, low carbohydrate and low protein diets that one molecule of neutral fat is oxidized by one triose molecule, or one molecule of glucose can oxidize two molecules of fatty acid. Woodyatt (1921) and Shaffer (1922) in their studies of ketogenic- ketolytic ratios confirmed Zeller*s conclusion. Probably the greatest support for the catalytic role of glucose in the oxidation of ketone bodies is found in the in vitro results of Shaffer (1921). When glucose and aceto acetic acid were combined in alkaline solutions, the addition of hydrogen peroxide caused the acetoacetic acid to disappear at a rapid rate, increasing with the amount of glucose, with the alkalinity, and with the temperature. In the absence of glucose, however, the acetoacetic acid was oxidized only very 9 slowly* Fructose and glycerol were likewise ketolytic but lactic acid was not. Since the rate of the reaction seemed to be determined by the conversion of glucose by the alkali into a derivative which was then oxidized, Shaffer concluded that some intermediate oxidation product of glucose combined with acetoacetic acid, the new compound then being oxidized* Deuel, Gullck, and Butts (1932) reported on the varying ketolytic ability of carbohydrates, and Shapiro (1935) showed that only glucose formers can lessen ketonuria when ketogenlc acids are administered* Butts, Dunn, and Hallman (1935-36) and Butts, Blunden, and Dunn (1937) (1, 2) published evidence also in support of ketolysis from results with various anino acids and their glycogenic action* But in 1937 Mirsky and Broh-Kahn announced that glucose is not ketolytic because they found no difference in the rate of disappearance of beta- hydroxybutyric acid injected in the blood of nephrectomized rabbits with ample carbohydrate reserves as compared with fasted animals* Results from this laboratory (Blunden, Hallman, Morehouse, and Deuel, 1940), however, in rats administered 1-beta-hydroxybutyrate showed that the rate of disappearance was significantly lower in experiments carried on for 75 and 150 minute intervals than for animals receiving glucose. Thus, Deuel concluded that the "rate 10 of disappearance of beta-hydroxybutyrate from the tissues is accelerated when glucose is present.” Further, in a study of the comparative action of the fat-sparing substances, glucose and alcohol, on exogenous and endogenous ketonuria, Deuel, Hallman, and Murray (1938) demonstrated that only glucose caused a decrease in acetonuria. Glucose brought about an almost complete abolition of the endogenous ketonuria in fasting rats previously fed a high fat, low protein diet, while ethyl alcohol was entirely ineffective. Calculations showed that the extent of fat oxidation was practically identical in the fasting rats and in those receiving sufficient glucose to overcome the endogenous ketonuria. Also, the amount of glucose which was ketolytic amounted to only a little more than one per cent of the total fat oxidation of the day. Hence, Deuel concluded that "ketolysis rather than antiketogenesis is the primary mechanism whereby the metabolism of carbohydrate brings about the abolition of ketonuria.n Another method of approach to the antiketogenesis- ketolysis controversy is via the tissue slice method (Warburg, 1926). The results obtained by this procedure show considerably higher and more uniform respiration rates than when hashed tissues are employed or where perfusion of the excised organ is used. This would indicate that the respiration of the 11 intact organ is in the main preserved in the sections anl also that less variable figures obtain with tissue slice studies than with perfusion experiments* Warburg (1926) has compared respiration under these various procedures as follows: Authors Experimental Arrangement Liver Carcinoma % S Barcroft and Perfusion with blood 1*5 - 15 Shore from living animal (cat) Usui Intact liver lobe 2.8 in vitro (mouse) Russell and Film of tissue pulp 5 - 4 1*2-6 Gye (mouse) Minaml Sections of tissue 9 >13 7 -11 (rat) Outstanding work in the application of tissue slice technique to fat metabolism has been done by Quastel, Wheatley, and Jowett* In 1933 Quastel and Wheatley found that fatty acids are oxidized at considerable rates by liver slices, giving acetoacetic add as one of the products* In agreement with results obtained by perfusion experiments, the fatty acids with an even number of carbon atoms' produced the acetoacetic acid. They found that glucose did not remove acetoacetic acid formed from butyric but glycogen did, although in the latter case the respiration of the tissues was also reduced* The next year (1934), however, these 12 workers reported their Inability to repeat the experiments showing the inhibitory action of glycogen on acetoacetic acid production. These workers noted that the nutritional state of the animals used was a very Important factor in the yield of acetoacetic acid. In a study of the relation of ascorbic acid to fatty acid oxidation (1934) they found that ascorbic acid causes a significant Increase In acetoacetic production of scorbutic guinea pig liver slices in media containing butyrate or crotonate. In 1935 Jowett and Quastel reported further on the rates of oxidation of butyric# crotonic, and dl-beta-hydroxybutyric acids and showed the importance of optimum salt concentration# pH# and nutritional state, Also in the same year these men published their classical quantitative studies on nThe Oxidation of Normal Saturated Patty Acids in the Presence of Liver Slices" in which they put forward the hypothesis of multiple alternate oxidation. Still a third paper in the same year gave evidence that the kidney# spleen# and testis produce small amounts of acetoacetic acid from fatty adds but the brain gives no measurable quantity. They also showed that kidney# spleen# testis and liver destroy acetoacetic acid aerobically# the kidney removing it also anaerobically, Purther studies on acetoacetic acid breakdown appeared in November 1935 and it was noted that the rate of beta-hydroxybutyric acid 13 production from acetoacetic acid aerobically was Increased in the presence of cyanide and still further increased If glucose or glucose and pyruvate were added to the acetoaeetate- cyanide medium. In a study on the decomposition of butyric acid by liver slices Ciaranfi (1936) showed that both respiration and ketone body formation are greater in Ringer*a solution buffered by phosphate than when buffered by bicarbonate. Also on the basis of calculations of the respiratory quotients, both the oxygen consumption and to a less degree the carbon dioxide formation were increased In the presence of butyrate. The altered metabolism of fatty livers was shown by Callfano in 1937 when he produced fatty livers by phosphorus poisoning. That ketone bodies may be Intermediates not only In fat metabolism but also In carbohydrate metabolism was suggested by Krebs and Johnson (1937) from experiments on the anaerobic oxidation of pyruvic acid wherein they found that in muscle and in other tissues to a lesser extent beta-hydroxybutyric acid was the chief end product. It was not until 1938 that Gohen and Stark proved conclusively that a low liver glycogen In rats resulted In greater production of ketone bodies both In the absence and in the presence of butyrate as substrate. Leloir and Munoz (1939) obtained butyric acid oxidation in a cell-free liver brel and also 14 found that the rate of disappearance of butyric acid with liver slices was higher than that of any of the other saturated fatty acids from one to eight carbon atoms inclusive. As a result of the contributions reviewed, it seemed possible that additional data on the mechanism whereby glucose decreases ketonuria might be obtained by the employment of tissue slices. In the present investigation it was desired to determine whether the decreased ketone bodies found when liver slices are present in a butyrate medium containing glycogen as contrasted with the level when this polysaccharide is absent is to be ascribed to a suppression in their formation, which would support the antiketogenesls theory, or to an acceleration in their oxidation which would support the idea of ketolysis. By the determination of the disappearance of butyrate as well as acetone bodies, it is believed that an answer to this problem has been obtained. EXPERIMENTAL PART Experimental animals Albino rats from the stock colony of the Department of Biochemistry of the University of Southern California were employed for the experiments. Only adult males were used, four months or more of age, either well<*fed or fasted for 24, 48, or 72 hours. Before the livers were removed for sectioning, the animals were anesthetized with sodium amytal and bled from the throat. Procedures The experimental work has involved three definite procedures: (1) the msnometric method of Warburg with liver tissue slices, (2) the micro-acetone analysis of Edson employing Rupp's lodometrlc titration, and (3) the determina tion of residual butyric acid by a distillation procedure. (1) The Warburg manometrlc method The Warburg manometer described in Warburg* s ”Uber den Stoffwechsel der Tumoren” (Berlin 1926} and also in "Manometric Methods'* by Dixon (London 1934), a constant volume type of respirometer, has been used throughout this study. A bank of seven manometers was available. The water bath in which the reaction flasks, or vessels, were 16 shaken was maintained at 39°C t 0*02°. Since one end of such a manometer tube is open to the air, the manometers are very sensitive to slight barometric pressure changes, hence one of the seven was always used as a thermobarometer. The calibration of the apparatus, that is, the determination of the vessel constants, was done by liberating a known amount of carbon dioxide gas in the vessel as a result of the chemical action between sodium bicarbonate and sulfuric add after temper attire equilibrium had been attained, head perchlorate prepared according to Krebs (1930) was used as the manometer fluid. The theory of the apparatus is well discussed in Dixon's "Manometric Methods" (London 1934). It might be mentioned that the basis for calculation of the volume of gas absorbed or evolved by the surviving tissues is the vessel constant. This value represents the dimensions of a surface and actually is a factor expressed in square millimeters by which the changes in the height of the manometer fluid may be multiplied to find the volume or cubic millimeters of gas 17 concerned. It Is calculated as follows: Let x s the amount of gas In c.m.m. at H.T.P. h x the manometer reading Vgs the volume of the gas space In c.m.m. Vfr the volume of fluid added In c.m.m. T s the absolute temperature of the water bath P z the Initial pressure In the vessel p r the vapor pressure of water at temp. T Pqs the pressure of one atmosphere expressed in mm. of confining fluid For example, the lead perchlorate used had a density of 2.079, hence P0 ■ 760 x 13.59 Z7o7§-- « * . m the Bunsen absorption coefficient of the gas concerned. The total amount of gas finally present in the vessel Is the sum of the amount initially present and the amount, x, produced: iva ~ h * (va t 2 * * * and x s h . . 273 m ' x t of v0 * VF^ 0 The expression in brackets Is the vessel constant far a given gas, that is, x - h . kCOg It may be added that x is positive if gas is formed, but is negative when gas disappears. 18 When a known amount of gas, as carbon dioxide, is evolved in determining the vessel constant, the amount of carbon dioxide gas s x, the difference in manometer readings before and after evolution of the gas s h, hence kCQg • -j£- Calculation of the volume Vg may be done as follows : 0 T r ~ x 275 X - ¥F From the value V the constant for oxygen, or any other s may be determined by applying the appropriate absorption coefficient in the following formula: x s h The data for one calibration using 0.01 M sodium bicarbonate in the main part of the manometer vessel and 2/3 N sulfuric acid in the side-arm are given in Table A in the appendix. The thermobarometer is represented by the manometer number in parentheses* A summary of a series of such determinations is: made in Table B. The solutions used in the various tissue slice experiments were prepared as follows: Salt mixture. The amounts used are all calculated 19 on the basis of 0*16 M for a salt producing only two Ions; this figure Is Isotonic with serum. Stock solution for use In manometer vessels: 360 ml. 0.16 M KC1 90 ml. 0.107 M CaClg (i.e., SgfeS ) 72 ml. 0.107 M MgClg Buffer. 0.10 M NaHgPO^ adjusted to pH of 7.3 with NaOH. Indicators.. Brom thymol blue (pH 6.0 yellow 7.6 blue) B.B.H. ”4.5" (pure grey at pH of 4.5) Substrates. Sodium salts of the fatty acids 0.16 M Glycogen: 0.4 or 0.8 g. dissolved In 25 ml. 0.16 M salt. Others, jniline: 5 g. aniline hydrochloride dissolved in 25 ml. of IN NaOH, prepared fresh dally. Salt: 0.16 M NaCl The preparation of the tissue slices for the Warburg experiments requires the ability to estimate the necessary tissue slice thickness for satisfactory respiration. The following table is of Importance In this connection: (Warburg, “The Metabolism of Tumors," 1926) 20 Tissue thickness Oxygen quotient 0*21 mm* 8*8 0.30 8.8 0.31 9.4 0.50 7.8 0.95 5.8 1.24 5.9 A thickness of the tissue slices ranging between 0.2 and 0.4 mm. la generally accepted for best results. Tissues of less thickness tear apart during the shaking operation, and of greater thickness, show lowered respiration, probably due to asphyxiation of the Interior cells. Tissue thickness may be estimated as follows: Area Is measured by suspending the slices in physiological saline in a Petri dish which is superimposed upon metric ruled paper. The area of paper covered by the slice is found by counting the square millimeters covered. The tissue Is then dried to constant weight. Volume Is estimated by multiplying the dry weight by 5, since tissue is approximately one-fifth solids and four-fifths water. Thickness, then, - YQlume in cubic millimeters area In square millimeters 21 After a sufficient practice period, the thickness may be readily estimated from the translucency of the slice* For sectioning the liver tissue, an alternative procedure has been devised than the use of a straight razor as suggested by Warburg (192t>), and Dixon (1934) as well as employed by Quastel (personal communication from Professor J. S. Butts). This Involves the use of a handle (Figure 1) constructed of spring metal which holds the ordinary double-edged safety razor blade. i— f c c' Fig. 1 B and are grooves. C and C1 are rests or finger grips 23 The blade is inserted in the handle by bending the grooves B and B - * - toward each other and introducing them into the slot of the safety razor blade. The spring of the handle causes these to snap into place, thus holding the razor blade firmly, C and are rests or finger grips. The handle affords good leverage and requires effort of approximately the same value from either hand. This enables a more uniform effort in cutting than with the straight razor where the center of gravity lies near the handle. It has the further advantage that the blades can be changed for new ones when dull, by a reversal of procedure. After the vessel constants have been determined and the tissue sectioning technique acquired, the Warburg apparatus is ready to be used for respiration experiments. Tissue slices not exceeding 20 mg. dry weight were used in this study. The slices prepared from rat liver immediately after excision of the liver were bathed in 0.9 per cent sodium chloride and then Immersed in the manometer vessels. Usually three slices were used in each vessel so that average thickness in different vessels would be approximately the same. The medium in which the slices were immersed consisted of the followings Salt mixture (K, Ca, Mg) 0.3 ml. Phosphate buffer 0.6 Sodium chloride 0.16 M 2.1 24 When additional metabolites were added, as sodium butyrate or glycogen, they replaced a similar volume or sodium chloride solution. Into the inner cup of each manometer vessel was inserted a small roll of filter paper moistened with 0,2 ml, of 2 N sodium hydroxide to absorb carbon dioxide evolved during respiration of the tissues. The vessels were attached to the manometers and gassed for 5 minutes with a mixture of 95 per cent oxygen and 5 per cent carbon dioxide. The manometers with vessels attached were then transferred to the water bath, in which the vessels were shaken usually for 2 hours. Ten to twenty minutes were allowed for temperature equilibrium, after which the readings of oxygen uptake were taken every thirty minutes. The difference between the second and third readings was always used for calculation of the oxygen quotient. At the end of the two hour period the slices were removed from the vessels, washed in distilled water, dried at about 105° 0, and weighed to constant weight. The pH of each vessel was checked. The filter paper absorbers were removed from the Inner cups of the vessels, 0,1 ml, of 50 per cent sulfuric acid delivered into each cup, and the cups finally dried out with filter paper. Acetoacetic acid was then determined either manome trie ally or by the micro-acetone method of Edson (1935), described later. The manometrle estimation of acetoacetic acid as 25 developed by Quastel and Wheatley (1935) was accomplished as follows* The residual solution (3.0 ml*) after removal of tissue slices was acidified by adding 0*3 ml* of 1*0 N acetic acid, which brought the pH approximately to 4*5, as checked by an indicator* Into tbs vessel side arm was run 0*2 ml* of a solution of aniline hydrochloride, prepared fresh each day* The vessels, with carbon dioxide as gas phase, were shaken 10 to 15 minutes in the water bath before the manometer readings were taken* As soon as temperature equilibrium was apparent, the aniline solution was tipped into the vessel mixture to decompose acetoacetlc acid* This reaction was first found by Wohl (1901, 1907), used by Ostern (1933) and by Krebs (1933) in manometrlc determination of oxalacetlc acid, and applied by Quastel and Wheatley (1933) to acetoacetlc acid determinations in tissue slice studies* According to Wohl: COOH.CO.CHg.COOH ♦ NHg.CgHg CgHg.NH.OO.C.O.CHg ♦ HgO ♦ COg oxalacetlc aniline pyruvanillde The above reaction is probably analogous with acetoacetlc acid, that is, 1 mol carbon dioxide is evolved for each mol acetoacetlc add, the evolution being considered complete in 45 to 55 minutes* A typical experiment is given in detail in the appendix, Table C* 26 The various quotients used to express the results of the respiration experiments are the following: Qqo = cubic millimeters of oxygen consumed per milligram of dry tissue per hour ^Ac - cul3ic millimeters of carbon dioxide equivalent to acetoacetlc acid formed per milligram of dry tissue per hour qq s cubic millimeters of carbon dioxide equivalent to beta-hydroxybutyric acid farmed per milligram of dry tissue per hour «Ket * oum of «Ac “ld ®B-OH (2) Mloro-determinatlon of acetone bodies* Edson (1935) has combined the Van Slyke (1917) method of determining acetone bodies with Rupp’s lodometrlc titration (1906, 1907) for the mercury* Solutions: 20$ copper sulfate 10$ calcium hydroxide suspension Denlges reagent: 10$ mercuric sulfate in 50 vol. % sulfuric acid made as follows: 75 gm. red mercuric oxide dissolved In 1 liter 4 N sulfuric; 500 ml. sulfuric sp. g. 1*835 diluted to 1 liter* Combine 1 liter 50 $ sulfuric with 3*5 liters mercuric sulfate and 10 liters water* 1 N hydrochloric add 35$ formaldehyde 3$ potassium iodide 7*5 N sodium hydroxide glacial acetic acid 0*01 H iodine 0.01 H thiosulfate 1$ starch solution 5$ potassium dlchromate Determination of acetoacetlc acid: After removal of the tissue slices and excess sodium 27 hydroxide from the Warburg vessels, there is run Into each vessel 0.5 ml, each of 20 per cent copper sulfate and 10 per cent calcium hydroxide suspension. The solution is transferred to a 25 ml, Erlenmeyer flask, after which the Warburg vessel is rinsed twice with 5,0 ml, of distilled water. The contents of each Brlenmeyer flask are made up to 15 ml. volume and allowed to stand 20 minutes before filtering, 12,5 ml, of the filtrate is pipetted into a flat bottomed Pyrex glass flask provided with ground glass connection for a condenser, 4,5 ml, of Deniges combined reagent is added to each flask and the mixture refluxed for 30 minutes. 2 ml, distilled water is washed down each condenser, then the acetone-mercury precipitate Is filtered with suction on a sintered glass funnel (#4 porosity). The flask Is rinsed with two 5 ml. portions- of water which are also us;ed to wash tbe precipitate. The filtrate is saved for the determination of beta-hydroxybutyric acid. The precipitate is dissolved on the funnel in 10 ml, warm N hydrochloric acid. The mercuric chloride solution Is run into a filter flask by suction. The filter is washed well with water. To the mercuric chloride solution are added in the following order: 0,5 ml, 35 per cent formaldehyde, 5,0 ml, 3 per cent potassium iodide, and 2,0 ml. 7,5 N sodium hydroxide. The solution is allowed to stand for at 28 least 2 minutes, then is shaken. Finally are added 1.0 ml. glacial acetic acid and 10.0 ml. 0.01 N iodine solution. The excess iodine is titrated with 0.01 N thlosulfate. According to Edson (1935), taking the 12.5 ml. aliquot into account, 1.0 ml. 0.01 N iodine equals 0.138 mg. acetoacetlc acid or 30.2 micro liters of carbon dioxide. The principle of this iodine titration (Rupp 1906, 1907) is as follows. Metallic mercury will precipitate from alkaline solution by means of formaldehyde, then may be changed to mercuric iodide by means of excess n/10 iodine, the unchanged iodine being titrated with thiosulfate. To change any combined mercury in alkaline solution, potassium iodide, then alkali are used. The resulting mercury iodide- potassium iodide is reduced immediately by formaldehyde at ordinary temperature. Formaldehyde does not interfere with the iodine reaction if the solution has previously been made acid, hence the use of the glacial acetic add. The calculation according to Rupp is: 1 Hg ♦ 2 I * 2 K I — * K2HgI4 200.4 g. Hg s 2 I; 0.01002 g. Hg a 1 ml. n/10 I. Determination of beta-hydroxybutyric acid: The filtrate from the acetoacetlc acid precipitation is returned to the flat-bottomed flask and brought to boiling. 1.0 ml. of 5 per cent potassium dicbromate is 29 added through the top of the condenser and boiling is continued for 90 minutes, after which time 5*0 ml* of water . t is run down the condenser, then the acetone-mercury precipitate is filtered by gravity on a sintered glass funnel (#3 porosity). The flask is thoroughly rinsed and the washings run through the funnel until the filtrate appears to be free of dlchromate as it drops into the filter flask. The filtrate is saved for determination of residual butyric acid. The precipitate is dissolved in 10.0 nil. of warm N hydrochloric acid and treated as described above for the acetone-mercury precipitate due to acetoacetlc acid. Considering the original 12.5 ml. aliquot, 1.0 ml. of 0.01 N iodine is equivalent to 0.185 mg. of beta-hydroxybutyric acid or 39.9 micro liters of carbon dioxide. (3) Determination of residual butyric acid The filtrate obtained after separating the acetone- mercury precipitate due to beta-hydroxybutyric acid is made up to approximately 250 ml. volume and distilled in an all-glass distillation apparatus consisting of a Kjeldahl flask, connecting tube with Hopkins* trap, and a condenser fitted into a filter flask. The last nemed la always filled to the same level with distillate, which represents a volume of 185 ml. The distillate is immediately titrated with 0.01 N sodium hydroxide using phenolphthalein as indicator. 30 A similar procedure has been used by Deuel, Hallman, Greeley, Butts, and Halliday (1940) in distilling acetoacetate and acetone from hashed tissues of the rat* The apparatus used was the same except in the determination of butyric acid, a Hopkins* trap was found necessary to hold back the sulfuric acid vapors* The method for butyric acid distillation was developed on the basis that butyric acid is readily distillable with steam* The validity of the method has been tested with known amounts of butyric acid, recoveries of which are reported below* The tltratable acid obtained in the distillate is concluded to be butyric acid because it may be qualitatively recognized by odor and was never quantitatively recovered unless butyrate had been used as substrate in the tissue slice media* Five titrations using 0*01 N sodium hydroxide and phenolphthaleln were made on distillates from water (3) and from water plus; 1*0 ml. of 60 per cent sulfuric acid (2)* The readings weres 1. 0*54 ml 2. 0.48 3. 0.58 4. 0.50 5. 0.57 Average 0.53 Five recoveries of butyric acid were made by distilling 31 the following mixture si 1* Water, 1.0 ml* sulfuric, 0.2 ml. butyrate (0.16 M) 3.64 ml. 2. Same as No. 1 with tissue medium 3.59 3. Water, 4.5 ml. Benig^s, 0.2 ml. butyrate, tissue medium 3.67 4. Same as No. 3 with glycogen 5. Same as No. 3 with 1.0 ml. dichr ornate 3.69 3.65 Average 3.65 Corrected for water blank 3.12 The average recovery after correction for water blank Is approximately 97.5 per cent. Finally, five recoveries of butyric acid were made on the filtrates obtained after precipitation of the acetone bodies. Typical tissue media were made up, the first three with glycogen, the last two without glycogen. After copper- lime precipitation, a 12.5 ml. aliquot was used for the micro-acetone procedure, followed exactly as previously described. 1. 3.07 ml. 2. 2.98 3. 3.00 4. 3.04 5. 2.97 Average 3.01 Calculation, of the recovery factor 32 3.01 - 0.53 « 2.48 2.48 x = 2.98 o Recovery factor = s 93.1 per cent Validity of Edson*s micro-acetone determination of acetone bodies was checked by usdng a solution of beta- hydroxybutyric acid (0.55 mg. in 5.0 ml.), the source of which was the calcium-zinc salt of 1-beta-hydroxybutyric acid prepared according to the ^procedure described by v Blunden (1938). The recoveries were as follows: 1. 0.56 mg. 2. 0.53 3. 0.55 4. 0.56 These results compare favorably with Edson's recoveries which ranged from 95 per cent to 110 per cent in 6 trials. The recovery values tend to be high because Edson used Van Slyke's factor of 8.45 g. of mercury precipitate equals 1 g. of beta-hydroxybutyrate. An experimental study made in Deuel's laboratory with both the 1- and the dl-beta-hydroxy- butyrate shows that this value is too low and that the factor 9.51 is more nearly accurate (Blunden, Hallman, 33 Morehouse, and Beuel, 1940), Jowett and Quastel (1935) also reported a higher value of 9.85 following experiments using presumably the dl-beta-hydroxybutyrate. EXPERIMENTAL RESULTS Effect of fasting A study on the effect of fasting on acetoacetlc acid content with and without butyrate substrate was made with rat liver slices from 10 animals that had been fasted 24, 48, or 72 hours. A tabulation of these results Is made In Table I with a comparison of values obtained with unfasted rats. Because consistently higher values of acetoacetlc acid quotients maintained with the 48 hour fasted animals, such nutritional states were employed in the remaining experiments. Since the spontaneous acetoacetlc acid content of unfasted rat liver is generally quite high, fasting brought about much higher quantities, an advantage when changes in amounts of ketone bodies are to be studied. Further, the 48 hour fasting period was chosen because not such wide-spread changes in the general condition throughout the animal body might be expected to occur as after longer fasts. TABLE I THE EFFECT OF FASTING ON THE Q0g AND Q^ OF THE LIVER SLICES OF MALE RATS Unfasted Fasted 24 hours Fasted 48 hours Fasted 72 hours Butyric-ConC• 0 0.01M 0 0.01H 0 0.011 0 0.01M Quotient ^02 Qac $02 §AC Q°2 Qac Q02 $AC Qp2 Qac $02 ^AC Q02 $AC QG2 Qac 9.1 8.9 1.14 1.13 10.0 9.9 4.34 4.21 8.0 8.5 1.99 1.96 10.0 5.58 9.9 5.34 8.2 8.5 2.64 2.32 12.4 5.16 7.1 7.0 7.4 2.19 1.95 2.15 10.6 5.59 12.6 6.25 11.9 6.12 5.3 5.6 1.19 1.08 7.5 8.8 3.33 4.47 8.5 0 , 11.7 7.1 1.46 1.79 10.2 4.48 8.0 7.9 1.77 1.55 9.7 3.38 9.5 2.66 7.9 1.28 7.8 4.38 7.6 1.08 9.3 2.96 7.3 3.37 10.9 7.37 8.0 1.12 7.8 2.77 8.6 4.04 11.0 6.72 8.2 3.48 7.0 2.14 8.8 2.14 13.7 5.32 8.2 2.14 13.9 6.04 9.2 2.04 13.9 6.15 7.4 1.12 8.9 3.68 8.3 1.32 10.0 5.46 8.4 2.51 12.3 5.89 Grouped experiments are on the liver slioes of the same rat. 36 Effect of glycogen. Using the livers from unfasted and from 48 hour fasted animals, the effect of the addition of gLycogen to the tissue medium was studied. The quotients found are listed In Table II. Because greater lowering of the acetoacetlc acid quotients occurred when the glycogen concentration was one per cent of the medium, this amount was used In later experiments. Seventeen rats were used. TABLE II THE EFFECT OF GLYCOGEN ON THE %z AND QAC OF THE LITER SLICES OF MALE RATS Unfasted Fasted 48 hours fionc. in per oent 0 0*5 1.0 0 1.0 Quotient O j > Ao O2 Ac °2 Ac 02 Ao 02 Ac 6.6 1.99 7.6 1.49 7.4 0 7.9 2.79 8.2 0.77 8.3 7.9 1.82 9.0 0 7.4 8.3 2.82 8.1 0.80 6.9 1.77 8.6 1.64 7.0 3.05 6.0 2.41 6.3 1.25 7.4 1.54 7.6 2.78 10.2 1.31 10.6 1.30 7.2 1.49 8.3 1.40 10.4 1.09 12.8 0.52 7.1 1.09 9.3 0 9.9 0.30 7.9 0.15 10.3 1.23 10.3 0.55 9.3 1.30 9.0 0.21 7.92 3.05 8.21 0.19 8.5 1.14 9.6 0. 23 7.76 2.65 10.75 0.95 8.5 0.56 8.37 1.50 8.5 1.69 8.42 0 7.9? 1.86 13.68 1.80 7.73 1.47 10.22 0.29 10.39 1.85 12.12 1.82 8.52 1.42 9.46 0.38 10.63 2.02 10.0 1.79 8.68 1.41 8.84 1.12 8.6 1.64 8.88 2.14 9.34 0.49 7.4 1.54 8.2 0.77 8.3 2.82 8.1 0.80 8.0 2.41 7.6 2.78 7.59 2.07 7.48 1.26 9.03 2.20 8.02 1.72 6.91 2.72 7.56 2.04 38 Effect of butyrate plus glycogen Since glycogen was found usually to lower the endogenous acetoacetlc acid content, it was decided to study the effect on exogenous ketosis, employing butyrate as substrate* The values from these experiments are reported in Table III. TABLE III THE EFFECT OF BUTYRATE WITH AND WITHOUT GLYCOGEN ON THE Q0j> AND Qag OF THE LIVER SLICES OF MALE RATS FASTED 48 HOURS Substrate 0 Glycogen 1% Butyrate O.OIM Glycogen 1$ + Butyrate O.OIM Quotient Og Ao 0 < > Ao ___Sa..... Ao O2 Ac 7.9 2.79 8.2 0.77 11.8 5.87 7.4 8.3 2.8 2 8.1 0.80 7.0 3.05 8.0 2.41 13.5 , 5.06 7.6 2.78 10.9 4.61 7.59 2.07 7.48 1.26 13.21 5.86 10.79 3.88 13.76 5.36 12.26 4.37 9.02 2.20 8.02 1.72 13.22 6.57 13.64 4.98 15.57 6.68 12.48 5.77 6.91 2.72 7.56 2.04 13.85 7.17 12.31 4.88 13.32 6.38 12.52 5.44 40 Effect of butyrate plus glycogen on total ketone production The presence of glycogen having been found so often to lower the acetoacetlc acid content, it seemed necessary to determine whether such an effect were at the expense of the beta-hydroxybutyric acid. The micro-acetone determina tion of Edson was used in order to recover the ketone bodies separately, the sum of such recoveries being total ketones found. The quotients obtained in these experiments are shown in Table IV, T A B L E T 17 EFFECTS OF BUTYRATE AND GLYCOGEN ON TOTAL KETONE PRODUCTION OF LITER SLICES OF MALE RATS FASTED 48 HOURS Substrate_________ 0 Glycogen 1%____ Butyrate O.OIM Glycogen lf> f Butyrate Q«0 1 2 1 itient °2 Ao B-OH Ket °2 Ac B-OH Ket °2 Ao B-OH Ket °s Ao B-OH Ket i * No. 1 7.02 2.65 1.64 4.29 6.20 3.18 2.65 5.83 10.28 11.89 8.54 9.08 4.54 2.16 13.08 11.24 10.54 9.84 0.34 0.14 0.15 0.29 0.49 0.43 2 7.64 5.07 2.20 7.27 7.17 4.28 1.89 6.17 12.02 13.70 12.54 9.01 3.45 4.13 15.99 13.14 14.22 13.09 11.05 12.08 3.91 3.92 14.96* 16.00 3 6.19 3.07 0.90 3.97 5.22 3.18 0.75 3.93 9.64 13.94 6.13 8.63 3.38 2.92 9.51 11.55 12.18 9.66 10.01 18.75 3.07 0.79 13.08* 19.54 4 6.76 0.68 1.39 2.07 8.33 1.13 1.18 2.31 10.33 15.12 9.66 9.66 3.37 1.77 13.03 11.43 12.38 11.13 6.70 6.11 1.94 1.57 8.64 7.68 5 7.57 0.98 0.99 1.87 10.95 5.21? 12.42 5.59 2.41 8.00 6.83 1.2 3 ? 14.33 7.13 3.02 10.15 11.35 6.05 2.52 8.57 6 6.58 1.16 , 2.50 3.66 12.12 6.23 3.20 9.43 12.68 5.59 2.76 8.35 8.88 0.96 2.25 3.21 13.48 6.81 3.75 10.56 12.86 6.17 2.60 8.77 7 7.04 1.88 1.50 3.38 12.61 6.09 2.85 8.94 12.82 4.91 2.68 7.59 7.13 0.77 2.86 3.63 11.85 6.15 2.66 8.81 13.25 2.29 8 7.57 0.78 2.21 2.99 10.73 5.85 2.29 8.14 11.13 7.37 2.23 9.60** 7.49 0.81 2.07 2.88 11.37 5.64 3.94 9.58 13.29 5.36 5.55 10.91 9 7.73 0.94 1.51 2.45 10.34 5.21 2.64 7.85 10.75 5.06 2.14 7.20 8.51 0.95 1.48 2.43 11.46 5.26 2.90 8.16 12.93 5.54 2.08 7.62 1 0 9.07 2.01 0.96 2.97 10.01 5.01 2.>91 7.92 11.26 4.35 1.78 6.13 8.44 2.34 1.15 3.49 11.42 5.31 2.94 8.25 11.52 4.38 1.71 6.09 11 7.70 0.87 0.74 1.61 7.33 3.98 1.05 5.03 9.73 4.20 0.92 5.12 8.26 0.86 0.64 1.50 10.22 5.73 1.26 6.99 11.30 4.24 0.79 5.03 12 6.46 0.75 2.85 3.60 10.49 3.21 1.43 4.64 10.94 3.49 1.43 4.92 7.18 1.15 1.87 3.02 13.22 6.08 3.17 9.25 11.48 5.62 1.23 6.85 13 9.29 1.14 2.18 3.32 11.51 4.76 1.39 6.15 12.29 3.45 1.29 4.74 8.96 0.91 2.25 3.16 14.27 3.53 2.78 6.31 14.10 3.42 1.66 5.08 *?atty liver **Diarrhoea TABLE 17 (cont.) 14 8.38 0.75 2.15 2.90 7*73 1.15 0.61 1.76 7.73 0.79 1.89 2.68 7.47 1.28 0.38 1.66 15 7.10 1.53 1.47 3.00 7.54 0.65 0.68 1.3 3 7.50 1.52 1.44 2.96 8.39 1.06 0.78 1.84 16 6.96 1.27 1.17 2.44 7.33 0.74 0.58 1.32 7.13 1.43 1.04 2.47 8.27 0.85 0.68 1.53 17 7.43 1.27 1.56 2.83 8.32 1.22 1.08 2.30 8.32 1.32 1.57 2.89 9.87 1.34 0.81 2.15 18 7.39 0.83 1.26 2.09 7.33 1.33 1.06 2.39 8.47 0.79 1.19 1.98 8.58 1.50 1.01 2.51 19 7.14 0,88 1.24 2.12 7.19 1.24 0.93 2.17 7.43 0.89 1.44 2.33 10.53 1.50 1.09 2.59 20 8.37 3.29 2.08 5.37 8.75 1.92 1.88 3.80 9.86 3.31 1.72 5.03 9.45 1.27 1.48 2.75 21 7.98 0.83 1.71 2.54 9.67 1.01 1.45 2.46 7.33 0.96 2.45 3.41 9.63 1.49 0.97 2.46 22 7.01 0.84 1.17 2.01 7.68 1.00 0.83 1.83 7.85 0.82 1.52 2.34 8.48 1.31 0.96 2.27 23 6.51 0.82 0.95 1.77 7.72 0.77 1.06 1.83 7.98 1.12 1.45 2.57 8.15 0.97 1.00 1.97 11.37 5.37 2.57 7.94 9,77 4.30 2.08 6.38 11.17 4.02 1.76 5.78 11.82 3.14 1.43 4,57 12.43 3.44 1.84. 5.28 11.42 2.92 1.07 3.99 11.37 4.69 2.01 6.70 11.90 4.26 1.64 5.90 12.86 4,77 2.18 6.95 11.43 4.34 1.77 6.11 9.78 2.38 1.36 3.74 9.09 2.04 1.11 3.15 12.40 6.67 1.83 8.50 11.13 3.24 1.52 4.76 13.18 5.40- 3.41 8.81 13.16 4.05 2.84 6.89 12.32 5,91 1.97 7.88 11.53 5.27 2.78 8.05 13.12 6.33 2.99 9.32 11.09 4.62 2.43 7.05 42 Quantitative studies on the changes in butyrate with and without glycogen Although in the majority of experiments a marked decrease in the quantity of ketone bodies was noted in the presence of glycogen, it may be argued that this results from a suppression in the transformation of butyric acid to these compounds because of the preferential oxidation of the glycogen. Under such, conditions, therefore, the butyrate remaining in the medium should be greater when i glycogen Isb in the substrate than when it is absent. In order to determine this fact, the butyrate remaining in the tissue medium at the end of the respiration experiments was determined after acetoacetate and beta- hydroxybutyrate had been quantitatively removed and determined. The speed of disappearance of butyrate is calculated from the difference between the quantity of the original butyrate added and the sum of the butyrate recovered at the end plus the amount of acetoacetate and beta-hydroxybutyrate (calculated as butyrate) also present. Because the latter compounds are formed when butyrate is absent from the medium, correction la made for the amounts of these which were found in the control tests. These results are summarised in Table V, 43 It is also recognized that the decreased ketone body recovery in the glycogen-butyrate medium may partly result from the decrease in the endogenous content. In Table V the corrections are based on control experiments where the tissues were suspended in the basal salt solution without either butyrate or glycogen. TABLE 7 THE COMPARATIVE ACETOACETATE AND HYDROXYBUTYRATE EEC0VERY ABB BUTYRATE OXIDATION OP LIVER SLICES IK SUBSTRATES OP BUTYRATE ALONE OR BUTYRATE ANB GLYCOGEN Expt. NO. Butyrate added Butyrate and & tones reooyerec Total //gms Butyric oxidized beyond aoetone bodies Total Total per mg tissue ml N/1001 p gras NaOH Aeetoaeetic as butyric B*!QH butyric as o • H U £ J B Butyrio as such «1. corrected 1^grams /ul. corrected 1 ^ grams ml N/100 1 NaOH f^gma 5 B 2*20 2818 126.6 497.6 42.6 167.5 1.99(2.14) 1883.2. 2548.3 269.7 25.7 BG 3,20 2818 99.7 391.7 31.5 123.9 1.90(2.04) 1795.2 2310.8 507.2 45.7 BG 3.20 2818 143.6 564.2 44.4 174.4 1.61(1.73) 1522.4 2261.0 557.0 38.2 6 B 3.20 2818 101.3 398.2 16.0 63.2 1.96(2.11) 1856.8 2318.2 509.8 52.0 B 3.20 2818 103.5 406.8 24.7 96.9 2.01(2.16) 1900.8 2404.5 413.5 45.9 BG 3.20 2818 87.9 345.4 7.4 29.0 1.98(2.13) 1874.4 2248.8 569.2 58.6 BG 3.20 2818 97.1 381.6 4.2 16.4 1.94(2.08) 1830.4 2228.4 589.6 62.1 7 B 3.20 2818 75.4 296.2 10.6 41.6 2.02(2.17) 1909.6 2247.4 570.6 72.2 B 3.20 2818 75.4 296.1 7.5 29.4 2.04(2.19) 1927.2 2252.7 565.3 72.3 BG 3.20 2818 54.6 214.5 7.6 29.9 1.88(2.02) 1777.6 2022.0 796.0 104.7 *8 B 3.20 2818 87.9 345.3 2.6 10.3 1.98(2.13) 1874.4 2230.0 588.0 67.6 B 3.20 2818 83.2 327.2 31.0 121.7 2.02(2.17) 1909.6 2358.5 459.5 53.4 BG 3.20 2818 131.4 516.4 1.8 7.1 1.75(1.88) 1654.4 2177.9 640.1 64.0 BG 3.20 2818 93.0 365.6 69.6 273.4 1.68(1.81) 1592.8 2231.8 586.2 57.5 9 B 3.20 2818 105.9 416.2 28.3 111.1 2.22(2.39) 2103.2 2630.5 187.5 15.1 B 3.20 2818 107.1 421.1 34.7 136.4 2.11(2.27) 1997.6 2555.1 262.9 21.2 BG 3.20 2818 105.5 414.5 16.4 64.4 1.97(2.12) 1865.6 2344.5 473.5 36.9 BG 3,20 2818 108.0 404.9 13.0 51.0 1.94(2.09) 1839.2 2295.1 522.9 46.7 10 B 3.20 2818 87.7 344.8 57.4 225.4 1.96(2.11) 1856.8 2427.0 391.0 25.2 B 3.20 2818 88.9 349.3 53.4 210.8 1.98(2.13) 1874.4 2434.5 383.5 27.0 BG 3.20 2818 61.6 242.2 20.4 80.4 1.80(1.94) 1707.2 2029.8 788.2 55.5 BG 3.20 2818 62.9 247.3 18.6 73.1 1.78(1.92) 1689.6 2010.0 808.0 56.5 TABLE 7 (eont.) 11 B 3.20 2818 112.3 441.4 13.0 50.9 2.00(2.15) 1892.0 2384.3 433.7 24.1 B 3.20 2818 155.8 612.4 18.2 71.7 1.84(1.98) 1742.4 2426.5 391.5 24,5 BG 3.20 2818 108.2 425.3 7.4 29.3 1.62(1.74} 1531.2 1985.8 832.2 51.4 BG 3.20 2818 111.5 438.4 3.3 13.0 1.72(1.85) 1628.0 2079.4 738.6 44.8 12 B 3.20 2818 56.0 220.0 -23.1 -90.8 1.86(2.00) 1760.0 1889.2 928.2 74.9 B 3.20 2818 160.1 629.2 25.3 99.4 1.78(1.92) 1689.6 2418.2 399.8 25.6 BG 3.20 2818 68.6 269.6 -25.1 -98.6 1.71(1.84) 1619.2 1790.2 1027.8 76.1 BG 3.20 2818 129.8 510.1 -31.4 -123.4 1.67(1.80) 1584.0 1970.7 847.3 61.0 13 B 3.20 2818 111.4 437.8 -24.7 -97.1 1.73(1.86) 1636.8 1977.5 840.5 56.4 B 3.20 2818 67.8 266.4 15.1 59.3 1.64(1.76) 1548.8 1874.5 943.5 69.9 BG 3.20 2818 70.0 275.1 -26.8 -105.3 1.60(1.72) 1513.6 1683.4 1134.6 78.8 BG 3.20 2818 65.8 258.6 -15.3 -60.1 1.60(1.72) 1513.6 1712.1 1105.9 80.7 *Diarrhoea Calculations made as follows: Acetoacetic ^AG o ^ ain©d“SV»Q^Q of tissue alone x mgs. tissue x 2 hrs. = ^lCOg Hydrosybutyrate Qb-oh ottained-av.Qb_gh of tissue alone x mgs. tissue x 2 hrs. = /*lC0g Micrograms /<ie02 % 22 400 000 "re 108 11 01 10 bod7 as ^utyrate Butyric as such ml. 0.01N KaOH cor. for 93$ recovery 1 ml. 0.01N Ha0H^:880 /*g Butyric B = butyrate in substrate (0.2 ml. of 0.16 1} BG = butyrate and glyoogen in substrate In the following series of tests, recorded In Table VI, the corrections on the butyrate experiments are based on values obtained as above, while the corrections in the butyrate-glycogen tests were obtained from values found for liver slices in the salt solution containing glycogen. TABLE 71 THE COMPARATIVE ACETOACETATE AND HYDROXYBUTYRATE RECOVERY AND BUTYRATE OXIDATION OF LIVER SLICES IN SUBSTRATE OF BUTYRATE ALONE, OF GLYCOGEN ALONE, OR OF BUTYRATE AND GLYCOGEN Expt. Butyrate added Butyrate and ketones recovered Total Butyric oxidized No. ml N/lOOl ^ugma|Acetoacetic as butyric(B-OH butyric as butyrio Butyric as such p gms beyond acetone NaOH j i i . % corrected Jy«graras| y<l. corrected I ^tgrams ml N/lOO NaOH y^gms bodies Total Total per mg tissue 14 B 3.20 2818 130.6 513.2 15.6 61.3 1.93(2.08) 1830.4 2404.9 413.1 29.1 BG 3.20 2818 78.8 309.7 40.4 158.7 1.85(1.99) 1751.2 2219.6 598.4 46.8 15 B 3.20 2818 68.0 267.2 8.2 3 2.2 1.89(2.03) 1786.4 2085.8 732.2 53.8 BG 3.20 2818 53.4 209.9 8.2 32.2 1.89(2.03) 1786.4 2028.5 789.5 67.5 16 B 3.20 2818 61.0 239.7 21.6 84.9 1.90(2.04) 1795.2 2119.8 698.2 47.8 BG 3.20 2818 63.6 249.9 13.2 51.9 1.73(1.86) 1636.8 1938.6 879.4 58.6 17 B 3.20 2818 109.2 429.2 14.5 57.0 1.76(1.89) 1663.2 2149.4 668.6 41.5 BG 3.20 2818 88.2 346.6 20.7 81.4 1.71(1.84) 1619.2 2047.2 770.8 52.1 18 B 3.20 2818 118.0 463.7 28.6 112.4 1.92(2.06) 1812.8 2388.9 429.1 28.8 BG 3.20 2818 36.2 142.3 19.0 74.7 1.86(2.00) 1760.0 1977.0 841.0 64.7 19 B 3.20 2818 40.5 159.2 0.5 2.0 2.31(2.48) 2182.4 2343.6 474.4 35.1 BG 3.20 2818 15.1 59.3 2.3 9.0 2.27(2.44) 2147.2 2215.5 602.5 53.3 20 B 3.20 2818 64.7 252.3 -1.3 -5.1 2.08(2.24) 1971.2 2218.4 599.6 62.5 BG 3.20 2818 -1.5 -5.9 -4.6 -18.1 1.93(2.08) 1830.4 1806.4 1011.6 82.9 fABLET VI (cont.) 21 B 3.20 2818 73.8 290.0 21.8 85.7 1.98(2.13} 1874.4 2250.1 567.9 69.2 BG 3.20 2818 48.7 191.4 28,4 111.6 1.82(1.96} 1724.8 2027.8 790.2 90.8 22 B 3.20 2818 79.2 311.2 9.8 38.5 1.98(2.13) 1874.4 2224.1 593.9 76.1 BG 3.20 2818 79.7 313.2 36.5 143.4 . 1.97(2.12) 1865.6 2322.2 495.8 51.1 23 B 3.20 2818 90.0 353.7 30.1 118.3 2.02(2.17) 1909.6 2381.6 436.4 52.0 BG 3.20 2818 79.5 312.4 29.7 116.7 1.92(2.06) 1812.8 2241.9 576.1 54.4 Calculations: Same as for Table V, except butyrate quotients corrected by quotients from tissues alone; quotients for butyrate with glycogen corrected by quotients from tissue plus glycogen. Exp. 1 14 15 16 17 18 19 20 21 22 23 TABLE VII SUMMARY TABLE OF COMPARATIVE ACETOACETATE AND HYLROXYBUTYRATE RECOVERY AND OF BUTYRATE OXIDATION OF LIVER SLICES IN BUTYRATE ALONE OR WITH GLYCOGEN Acetone bodies recovered per 10 mg. dry tissue per Hour Butyrate oxidized per 10 mg. dry tissue per hour Acetoacetate Hydroxybutyrate Total B BG B BG B BG B BG 361.3 242.2 43.2 124.1 404.5 366.3’ 290.8 467.9 196.4 179.5 23.7 27.5 220.1 207.0. 538.2 675.0 164.2 166.7 58.2 34.6 222.4 201.3 478.4 586.6 266.5 234.3 35.4 55.0 301.9 289.3 415.2 521.1 311.1 109.4 75.4 57.4 386.5 166.8 287.9 646.7 118.0 52.5 . 1.5 8.0 119.5 60.5 351.5 533.2 262.9 -4.8 -5.3 -14.8 257.6 -19.6 624.6 829.1 353.8 220.1 104.6 128.3 458.4 348.4 692.5 908.3 399.0 322.9 49.4 147.8 448.4 470,7 761.4 511.1 420.9 294.6 140.6 110.0 561.7 404.6 519.5 543.5 338.1 251.5 . 496.0 622.2 DISCUSSION The present experiments offer cogent proof that the presence of carbohydrate accelerates the rate at which butyric acid disappears from a medium In which liver slices are suspended. Thus It was shown In every case that the total butyrate (as acetoacetate, hydroxybutyrate, and unchanged butyrate) remaining in the medium at the end of the tests was invariably less, if glycogen had been added to the butyrate substrate. In a series of ten tests in which slices from the same liver were placed In respirometers containing respectively phosphate buffer solution alone, plus glycogen, plus butyrate, and plus butyrate and glycogen, it was found that the average butyrate which disappears beyond the ketone body stage after the addition of this component alone was 496.0 micrograms per 10 milligrams dry liver per hour, while the level found when glycogen as well as butyrate was added amounted to approximately 622.2 micrograms per 10 milligrams per hour, which corresponds with an increased rate of disappearance of butyric acid of 25.4 per cent over the basal level. Although a considerable variability obtains in the basal rate of disappearance of butyrate in different livers, the comparative results with and without glycogen were all 48 carried out simultaneously on slices from the same livers. Despite the considerable differences obtained, the results are significant. This is shown by the fact that in 9 out of 10 cases the basal rate of butyrate disappearance does not equal the level found when glycogen and butyrate were both present in the medium. In addition to the greater rate of butyrate disappear ance exhibited by the sections treated with glycogen, there is also a marked decrease in the level of total ketone bodies which remain at the end of the test. Although the decrease is not always noted in both of the separate fractions (acetoacetate and hydroxybutyrate), it was found that the total ketones in all cases except experiment No. 22, Table VII, were lower than those found in the vessel with butyrate but without glycogen. The average total ketone bodies in the liver slices immersed in butyrate were 338.1 micrograms per 10 milligrams of dry liver tissue, while the mean for those tests with slices of corresponding livers to which glycogen had been added had been decreased to 251.5 micrograms. The decreased amount of the ketone bodies with the addition of glycogen must be due to a more rapid disappear ance of these constituents. If It were due to a suppression in the formation from exogenous butyric acid, then the butyric acid left at the end of the tests In those cases 49 should be Increased. Actually, the total butyrate which still remained unchanged at the conclusion of the experiments was usually slightly less In the glycogen-butyrate medium than In the tests vihere butyrate alone was used. Another possible reason which may be advanced to explain the lowered amount of ketone bodies in the carbo- hydrate-supplemented media is that a decrease in the endogenous ketosia takes place. Since less ketone bodies would then result spontaneously, a lowered content of the acetone bodies might be found even though the rate of production from butyric acid were unaltered. However, in both groups of experiments a correction has been made for the rate of the endogenous content by subtracting from the butyrate and butyrate-glycogen tests, the values of ketone bodies found respectively with liver slices in the basal medium alone and with glycogen. The validity of the experiments reported here is also indicated by the fact that the results with butyrate closely approximate those of Quastel both in regards to oxygen and acetoacetlc acid quotients. The close comparison is evident in Table VIII which follows. TABLE VIII THE VALUES FOE Q02 ^ $AG OBTAINED WITH LIVER SLICES OF RATS IMMERSED IN VARIOUS CONCENTRATIONS OF BUTTRATE COMPARED WITH RESULTS REPORTED BY JOWETT AND QUASTEL (1935). Biochem. J. Z& 2159 (1935) Rat liver. Oxygen Glycerophosphate Buffer Sodixun phosphate Buffer Jowett and Quastel Fatty Aoid Cone. H 0 . * 0.01 0.02. 9.1, 8.9 10.0, 9.9 12.0, 9.9 5.3, 5.6 7.5, 8.8 7.8, 8.1 7.6, 8.0 9.3, 7.8 10.7, 9.6 1.14, 1.13 4.34, 4.21 4.39, 3.99 1.19, 1.08 3.33, 4.47 4.38, 4.37 1.08, 1.12 2.96, 2.77 4.02, 3.34 0.01 Butyrie QOj ‘ AC 9.77(P. 2165) 9.43(P.2166) 0.96(P.2165) 1.00(P.2166) 12.74(P.2165) 12.34(P.2166) 4.55(P.2165) 4.13(P.2166) 3.42-5.68(9- exp.) (P.2165) 51 The results also are supported by the experiments of Quastel (1935), where it was demonstrated that the addition of glyco gen decreases the aeetoacetic acid quotient although he also found decreased oxygen quotients (from 9*9 to 7*0)• However, an attempt to repeat this work (1934) was unsuccessful. Several reasons may be suggested for this difficulty. The animals used were presumably unfasted, hence the liver glycogen was probably normal. There was then no need of more glycogen. In fact, excessive glycogen might have interferred with the oxidative process, a suggestion which might also explain the decrease in oxygen quotients of approximately 30 per cent. Differences in oxygen quotients, of course, occur with different tissue slices, but 30 per cent is a very significant figure. Further, no mention is made as to the method of preparing the glycogen solution. In order to be certain that changes in osmotic pressure would be minimized, in this study the glycogen was prepared in 0.16 M sodium chloride. Also, glycogen equivalent to one per cent of the tissue medium was used instead of 0.5 per cent (Quastel, 1933), and was prepared 48 hours before use and refrigerated. Finally, to show real differences In ketone body content, the 48 hour fasted livers which exhibit greater spontaneous ketosia would be preferable. Therefore, in the remainder of the experiments reported here the livers 52 from 48 hour fasted animals were used. Consideration of Table IV shows that on the whole the oxygen quotients were comparable In the presence and absence of glycogen. The respiration, therefore, was not appreciably altered, but the acetoacetic acid quotient was. Further support of the work is to be found in the fact that the results on the ketolytic effect of carbohy drate also agree with those reported by Cohen and Stark (1938). However, these investigators failed to report their oxygen quotients which are necessary to evaluate the validity of the experiments. Lowered ketone body content may originate as readily with a poorly metabolizing or oxygen-starved tissue as by a ketolytic effect. Moreover, for a complete solution of the problem, a simultaneous determination of butyrate is also necessary, a procedure not carried out by any of the other investigators. The variability in the amount of butyric acid presum ably completely oxidized by the tissue from one liver as compared with that of other livers indicates that the metabolic processes Involved are specific for the individual liver. In other words, each liver Is a law unto itself. Hence, this type of data is not subject to statistical treatment. Calcula tion by tha Fisher "t" (1934) method gives a value of 1.35, which is not significant. Nutritional condition is probably 53 involved, and the storage of fat and other substances as well as glycogen are important factors. Some evidence for altered metabolism may be seen from experiments Nos. 2 and 3, in Table IV, in which very fatty livers were employed. The increase in ketosis when glycogen was added to butyrate is apparent from the extremely high total ketone quotients found, 16.00 and 19.54. Respiration studies with tissue from fatty livers seem certainly worthy of some future investigation. As to the method of analyzing for residual butyric acid, further comments should be made. The acid obtained by distilling the filtrate remaining after precipitation of the ketone substances beyond any question was butyric. It is true that such acids as lactic might be thought to interfere. If this were the case, then the distillates obtained from tissue media to which no substrates were added should show a titration value much higher than that for distilled water. This, however, was not the case. No tltratable acid was found to be present in the tissue medium which lacked butyrate. In fact, the titration readings from the media in which tissue had metabolized without added substrate or with glycogen were always comparable with those from an equal volume of distilled water. Also, when the ketone quotients from added butyrate were low, more residual butyric acid was found. However, to make corrections that would be beyond any criticism, the 54 titration values of the tissue media with added glycogen were always subtracted from the corresponding values of media with added butyrate and glycogen, while those of media without any substrate were subtracted from the corresponding values for media with added butyrate. Prom such calculations! the disappearance of more butyric acid occurred when glycogen had been added to the medium. The fact that no volatile acid is found in the liver tissue when butyrate is absent indicates the improbability that butyric acid is an Intermediate in the normal oxidation of tax. If the ketone bodies originate by multiple alternate oxidation, there would be no necessity for butyrate to be postulated as an intermediate. The explanation of the increased disappearance of butyric acid in the presence of glycogen can only be made on the basis of ketolysis. Glycogen usually decreased the endogenous ketone body content, true, but it also decreased the exogenous. This must have occurred by oxidizing butyric acid beyond the ketone body stage. Hence evidence is hereby added to that already accumulated which supports the theory that “ketolysis; rather than antiketogenesis is the primary mechanism” whereby carbohydrates effect a decrease in the ketone body content of liver tissue. CONCLUSIONS 1* The effects of fasting on the acetoacetate content of liver tissue from male rats have been investigated by use of the Warburg technique. The highest and least variable values were reached after a fasting period of 48 hours. 2. For sectioning liver tissue an alternative procedure has been devised which involves the use of a handle constructed of spring metal which holds the ordinary double-edged safety razor blade. 3. & quantitative study has been made of the effect of glycogen on the oxidation of butyrate by liver slices. Butyrate has been recovered as acetoacetlc.jrcid, beta- hydroxybutyric, acid (both according to the method of Edson) , and as the residual, unchanged acid. 4. ^A^method for the determination of residual butyric acid in tissue media has been developed, a recovery value of better than 93 per cent having bean shown. 5. Experiments have been reported which show that glycogen added to tissue media lowers both the endogenous and the exogenous content of rat liver sections. 6. Evidence has been presented which supports the theory that the effect of carbohydrate on fat oxidation is ketolytic. BIBLIOGRAPHY 1. Blunden, H., Proe. Soc. Exper. Biol* and Med., 38: 466, 1938. 2. Blunden, H., L.F. Hallman, M.G. Morehouse, and H.J. Deuel, Jr., J. Biol. Chem.. 135: 757, 1940. 3. Bobbitt>, B.Gr. , and H. J. Deuel, Jr. , Am. J. Physiol. , 131: 521, 1940. ~ 4. Butts, J.S., H. Blxmden, and M.S. Dunn, J. Biol. Ohem., 119: 247, 1937. (1) 5. Butts, J.S. , H. Blunden, and M.S. Dunn, J. Biol. 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Dixon, Malcolm, Manometrlc Methods, London, 1934. 20. Bdson, N.L. , Biochem. J., 29s 2082, 1935. 21. Bnbden, 8., Centralbl f. Physiol., 18: 832, 1905* 22. Embden, 8., and Marx, Beitr. chem. Physiol, u. Path., 11: 318, 1908. 23. Embden, 8., and M. Oppenheimer, Blochem. Z., 45: 186, 1912. 24. Bnbden, 8., and M. Oppenheimer, Biochem. Z. , 55: 337, 1913. 25. Bnbden, 8., H. Salomon, and F. Schmidt, Biochem. Z., 55: 301, 1913. 26. Fisher, R.A., Statistical Methods for Research Workers, Edinburgh, 1934. 27. Qeelmuyden, H.C., Ztschr. f. Physiol. Chem., 41: 135, 1904. 28. 8erhardt, C., W. Med. Presse, 28: 672, 1865. 29. Oreenberg, M.M., J. Biol. Chem., 112: 431, 1936. 30. Henze, M., Ztschr. f. Physiol. Chem., 195: 248, 1931. 31. Himwlch, H.E., W. Coldfarb, and A. Weller, J. Biol. Chem.,, 93: 337, 1931. 32. Hirschfield, F., Z. klin. Med., 28: 176, 1895. (1) 33. Hirschfield, F., Z. kiln. Med., 31: 22, 1895. (2) 34. Jowett, M., and J.H. Quastel, Blochem. J., 29: 2143, 2159, and 2181, 1935. “ 35. Knoop, F., Beitr. chem. Physiol, u. Path., 6: 150, 1904-05. 36. Krebs, H.A., Biochem. Z. , 220: 250, 1930. 37. Krebs, H.A., Ztschr. f. Physiol. Chem., 218: 156, 1933. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 58 Krebs, H.A., and W.A. Johnson, Blochem. J., 31i 645, 1937. Kul2 , E., 2. Biol., 20: 165, 1884. Leathes, J.B., and E. Wedell, j. Physiol., 38: xxxvili, 1909. “ Lelolr, L.F., and J.M. Munoz, Blochem. J., 33: 734, 1939. MacKay, E.M., H.H. Barnes, H.O. Carne, and A.N. Wick, J. Biol. Ohem., 135: 157, 1940. Magnua-Levy, A., Ergebn. d. Inn. Med. u. Kinderh., 1: 353, 372, 374, and 384, 19057 Mirsky, I.A., Am. J. Physiol», 15: 424, 1905-06. Mirsky, I.A., Am. J. Physiol., 116: 322, 1936. Mirsky, I.A., and H.H. Broh-Kahn, Am. J. Physiol., 119: 734, 1937. Morehouse, M.S., and H.J. Beuel, Jr., Proc. Soc. Exper. Biol, and Med., 45: 96, 1940. Neubauer, 0., Yerhandl. d. deufc. Gong. Inn. Med., 27: 566, 19101 Opie, E.L., J. Exper. Med., 5: 397, 1900-1901. Ostern, P., Ztschr. f. Physiol. Chem., 218: 160, 1933. Peters, Prager, Vlertel .jahrsschrlft, 55: 81, 1857. Quastel, J.H., and A.H.M. Wheatley, Biochem. J., 27: 1753, 1933. Quastel, J.H., and A.H.M. Wheatley, Blochem. J., 28: 1014, 1934. Quastel, J.H., and A.H.M. Wheatley, Biochem. J., 29: 2773, 1935. Rupp, Ber. deutsch. chem. Ges., 39 : 3702, 1906. Rupp, Ber. deutsch. chem. Ges., 40: 3276, 1907. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 5? Schoenhelraer, R., and B. Rittenberg, J. Biol. Chem., 113: 505, 1936. Shaffer, P.A., J. Biol. Chem., 47: 433 and 449, 1921. Shaffer, P.A., J. Biol. Chem., 54; 399, 1922. Shapiro, X., J. Biol. Chem., 108: 373, 1935. St&delmann, E., Arch. Exper• Path. Pharmakol., 17: 49, 1883. Stadie, W.C., J.A. Zapp, Jr., and F.B.W. Lukens, J. Biol. Chem., 137: 75, 1941. ~ Stohr, R., and M. Henze, Ztschr. f. Physiol. Chem., 206: 1; 212: 111, 19321 Toeniessen, E., and E. Brinkmann, Z. Physiol. Chem., 252: 169, 1938. ~ Van Slyke, B., J. Biol. Chem., 32: 455, 1917• ¥erkade, P.E., and J. van der Lee, Biochem. J., 28: 31, 1934. " Verkade, P.E., and J. van der Lee, Z. Physiol. Chem., 237: 186, 1935. Voit, Carl, Ztschr. f. Biol., 28: 245, 1891. Warburg, Otto, tJber den Stoffwechsel der Tumoren, Berlin, 1926. Wohl, T J . Metarb, Ber. chem. Gee., 34: 1139, 1901. Wohl, XJ. Metarb, Ber. chem. Ges., 40: 2282 and 2308, 1907. Woodyatfe, R.T., Arch. Int. Med., 28: 125, 1921. Zeller, H., Arch, f. d. ges. Physiol., 17: 213, 1914. TABLE A. DATA FOR ONE DETERMINATION OF VESSEL CONSTANTS Manometer . #1 : - #2... #3_ #4 #5 _ #6 __ (#7) 0•OlMNaHCO^-ml. 2.0 2.0 2.0 2.0 2.0 2.0 h2o 1.1 1.1 1.1 1.1 1.1 . 1 3.1 2/3NH2SO^-ml. 0.1 0.1 0.1 0.1 0.1 0.1 0.1 3.2 3.2 3.2 3.2 3.2 3.2 3.2 Readings 42.8 61.8 76.0 65.5 69.2 62.2 92.0 36.0 58.9 76.3 65.4 69.9 64.O 94*3 34*8 58.2 76.1 65.5 69.4 63.9 94.9 34-9 58.7 76.8 65.9 69.8 64.2 95.4 35.6 59.7 77.9 67.2 70.7 65.1 96.7 Acid tipped in Readings 252.0 248.3 280.0 266.3 250.8 248.2 111.5 249.0 246.9 279.6 264.8 249.8 247.2 113.2 248.9 246.8 279.0 264.3 249.3 246.9 113.8 Difference 213.3 187.1 201.1 197.1 178.6 181.8 17.1 Diff. cor. 196.2 170.0 I84.O 180.0 161.5 164.7 TABLE B SUMMARY OF A SERIES OF DETERMINATIONS OF VESSEL CONSTANTS Manometer #1 #2 #3 • #4 #5 #6 #7 Differences, cor. 195.0 170.6 I64.4 176.1 194*1 182.9 160.9 158.3 175.7 195*3 170.1 184.4 175.1 195*0 171.0 I84.O 163.4 176.3 170.6 183.8 163.3 182.9 185.4 163.1 198.6 172.5 183.6 162.9 197.2 172.6 181.7 163.5 176.2 198.8 169.6 159.1 158.8 172.0 196.9 161.8 158.4 198.1 168.9 180.8 161.1 160.8 174-6 168.6 181.2 159.1 • 160.0 172,3 196.2 170.0 184.0 180.0 161.5. 164.7 Average 196.5 . 169.7 182.9 182.9 161.8 160.8 174*8 kC02 ^F ~ 3200 2.269 2.627 2.438 2.438 2.756 2.773 2.551 kQ2 VF = 3200 1.955 2.292 2.102 2.102 2.420 2.437 2.215 kC02 VF = 3500 2.268 • 2.606 2.416 2.416 2.733 2.750 2.529 TABLE C TYPICAL RESPIRATION EXPERIMENT Rat #7909, 300 gms., fasted 48 hours. 39°C * o.02° Manometer #1 #2 #3 (#4) #5 #6 #7 Flask: Salt Mixture 0.3 0.3 0.3 0.3 0.3 0.3 0.3 Buffer 0.6 0.6 0.6 0.6 0.6 0.6 0.6 Glycogen 1.9 1.9 1.9 Butyrate, 0.16M 0.2 0.2 0.2 0.2 NaCI, 0.16M 2.1 0.2 1.9 2.1 1.9 Inner oup: NaOH, 2N 0.2 0.2 0.2 0.2 0.2 0.2 0.2 Liver Slices +3 +3 +2 *3 +3 43 In bath 5:35 Readings 5:50 285.9 278.0 224.6 232.7 221.8 224.6 264.3 6:20 259.6 256.0 188.0 224.8 185.9 193.4 232.1 6:50 238.9 238.0 157.5 221.0 157.9 168.1 205.6 7:20 218.9 219.8 128.4 217.2 132.1 141.9 180.3 Tissue out 7:35 pH checked. Alkali washed out. 0.3ml. of l.GN acetic acid added to flask. Change in pH checked. 0.2ml aniline run into side arm. V™ = 3500. Flasks returned 8:10 Readings 8:25 70.0 58.3 63.9 77.8 65.2 43.6 69.9 8:28 65.0 58.6 62.8 76.7 65.9 41.8 70.9 8:31 62.8 59.8 62.9 76.8 66.8 41.0 72.0 8:34 62.2 61.0 63.7 77.8 67.1 41.0 73.6 8:37 63.8 62% 6 64.8 79.2 67.7 41.0 74.8 Aniline tipped in 8:40 Readings 9:20 89.8 83.8 118.5 91.9 114.8 82.1 115.5 9:25 91.2 83.8 118.9 92.2 114.7 81.9 115.8 9:30 92.9 84.7 119.8 92.9 114.7 82.1 116.9 'TABLE G (oont.) Tissue wt. mg* A \ A hA -oor. °2 ko -Vp = 3200 A Q°2 ^ hG02 A ha02-oor. w » = 3500 qac 8.7 8.7 8.5 20.7 18.0 30.5 16.9 14.2 26.7 1.955 2.292 2.102 7.59 7.48 13.21 29.1 22.1 55.0 15.4 8.4 41.3 2.268 2.606 2.416 2.07 1.26 5.86 3.8 13.7 8.5 28.0 24.2 2.42 13.76 47.0 33.3 2.733 5.36 9.7 25.3 21.5 2.437 10.79 41.1 27.4 2.75 3.88 8.2 26.5 22.7 2.215 12.26 42.1 28.4 • 2.529 4.37 UMI Number: DP21531 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted.. In. the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, . a note will indicate the deletion. UMI DP21531 Published by ProQuest LLC (2014). Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. 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Creator Bobbitt, Blanche Gauthier (author)

Core Title A study of the effect of glycogen on the oxidation of butyrate by rat liver slices

School School of Medicine

Degree Doctor of Philosophy

Degree Program Biochemistry

Degree Conferral Date 1941-05

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag chemistry, biochemistry,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Deuel, H.J. (committee chair), [illegible] (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-604885

Unique identifier UC11354912

Identifier DP21531.pdf (filename),usctheses-c17-604885 (legacy record id)

Legacy Identifier DP21531.pdf

Dmrecord 604885

Document Type Dissertation

Rights Bobbitt, B. G.

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus, Los Angeles, California 90089, USA