6I8.36:6I (Received February 2, 1934.)

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183 6I8.36:6I2.352. 14 THE AUTOLYSIS OF THE PLACENTAL GLYCOGEN. BY ANNA DAVY AND A. ST G. HUGGETT. (From the Department of Physiology of the School of Medicine, The University of Leeds.

1 183 6I8.36:6I THE AUTOLYSIS OF THE PLACENTAL GLYCOGEN. BY ANNA DAVY AND A. ST G. HUGGETT. (From the Department of Physiology of the School of Medicine, The University of Leeds.) (Received February 2, 1934.) CLAUDE BERNARD [1859] showed by the iodine test that the rabbit's placenta contains glycogen. He assumed that it performs a glycogenic function for the fotus until the fetal liver is able to carry out that function for itself. Lochhead and Cramer [1908] supported this theory, showing that the glycogen is maximal in the placenta at the 21st-22nd day of pregnancy and diminishes from then until full term, during which period the glycogen is rapidly increasing in the foetal liver. Harada [1916] stated that the placenta produces 40 mg. of lactic acid per 100 g. of tissue per day after removal from the uterus. Ishikawa [1928] determined the changes in glucose during autolysis of minced placentm from full-term rabbits. He reported an increase in the glucose but no change in the lactic acid. No glycogen estimations were made. d'piane [1926] perfused human placent% with glucose and found glycogen was formed. Huggett [1929] found that the glycogen in the placenta of the rabbit was verystable despite metabolic and pharmacological experiments which in the maternal liver altered the percentage present and despite the fact that the glycogen was mainly present in the decidual (maternal) tissues of the placenta. The placental glycogen therefore appears to resemble muscle glycogen in its metabolism, being much less easily affected than the liver glycogen by starvation, carbohydrate feeding, adrenaline and insulin injections. It is in fact only altered to any appreciable degree by an excessive increase in endogenous katabolism. In the liver glycogen on autolysis yields glucose; in muscle on hydrolysis it yields lactic acid [Parnas and Wagner, 1914]. It became therefore of interest to determine quantitatively the products of autolysis of the placental glycogen. The results obtained indicate a very small formation of lactic acid confirming Harada [1916], and that the glycogen present

2 184 A. DAVY AND A. ST G. HUGGETT. yields nothing but carbohydrate, of which approximately two-thirds is glucose and one-third polysaccharide. In other words, the autolytic changes in placental glycogen resemble those in the liver rather than those in muscle. The placenta therefore probably has a similar glycogenic function as Bernard suggested, and the glycogen in it does not serve as a store of energy for the placenta itself as the glycogen in muscle does for the muscle. METHODS. The placental glycogen in the rabbit is maximal at the 21st-22nd day of pregnancy and is present almost entirely in the maternal (decidual) portion of the placenta. Consequently, the rabbits were killed at the 21st day from being served with the buck and the maternal part of the placenta separated from the foetal. The determinations were carried out on the former, though in certain experiments the effects of autolysis were determined on the whole placenta, that is, on the maternal in the presence of the foetal portion. The placenta was obtained from the rabbit by the technique described by Huggett [1929], and after removing blood with filter paper, weighed and ground up with sand as quickly as possible. The pulp so formed was divided into two parts, A and B. Portion A was mixed with three times its volume of absolute alcohol for estimation of glycogen, reducing sugar and lactic acid content. Portion B was mixed with an equal volume of 0 9 p.c. sodium chloride, a little toluene added and the whole incubated at 370 C. for 24 hours. After incubation it was treated with ice-cold absolute alcohol and the same estimations were made as on portion A. Determination of glucose and lactic acid. In order to extract these, following the method of Anderson and Macleod [1930], three volumes of absolute alcohol were used for the first extract, 75 p.c. alcohol for the second and third. The mixed extracts were evaporated, the glucose precipitated with copper sulphate and lime and then released with sulphuretted hydrogen. The glucose was determined by the method of Hanes [1929, modification of Hagedorn and Jensen's method]. The lactic acid was determined by the method of Fried emann, Cotonio and Shaffer [1927]. Determination of glycogen. PflIuger's method was used but was modified by adopting the neutralization of the potash hydrolysate with glacial acetic acid as advocated by Evans, Tsai and Young [1931]. Further, 75 p.c. alcohol was used instead of 66 p.c. to precipitate the glycogen. Apart from these modifications the estimation was performed

3 ~~~~~~~~~~~~~~~~~~~~~~~~~~~I GLYCOGEN IN PLACENTAL AUTOLYSIS. 185 throughout in centrifuge tubes as in the previous determinations of placental glycogen [Huggett, 1929]. Determination of total carbohydrate. This was done when necessary on the ground-up placenta before and after autolysis and also on the residues after extraction with 75 p.c. alcohol and removal therefore of sugar. The tissue or residue was hydrolysed for 5 hours with hydrochloric acid under a reflux condenser. The solution was neutralized with sodium carbonate, filtered and the proteins precipitated with zinc hydroxide. The glucose formed was estimated by the same method as in the alcoholic extracts. These procedures are summarized in Table I. TABLE I. Pulped placenta Portion A Portion B Extracted at once Mixed with 09 p.c. NaCl, autolysed 24 hours, and then extracted With 75 p.c. alcohol three times Extract Residue Evaporated Re-dissolved in water Portion C Portion D Hydrolysed with Precipitated Hydrolysed with with KOH acid CuS04 -CaO Glycogen Total cartohydrate determined determined Preci1Atate Centrifugate Suspended H20 Lactic acid H2S passed determined Glucose determined The difference between portions C and D is a measure of the carbohydrates of size intermediate between glycogen and sugars. Experimental. In the previous determinations the total carbohydrate content of the residue after triple extraction with alcohol (portion D of Table I) was not estimated. The results expressed in Tables II and IVR-VI therefore show the glucose and glycogen quantities as percentages of the original placental weight and the quantity of carbohydrate recovered as a percentage of the initial glycogen. Table III shows that the total carbohydrate content of

4 186 A. DAVY AND A. ST G. HUGGETT. the placenta is unchanged on autolysis, for the estimations on portion D show that there is an appreciable quantity of carbohydrate of molecular size intermediate between glycogen and sugars and that this is equivalent to the unrecovered portion of Tables II and IV-VI. Experiments (a), (b), (c), etc., indicate separate experiments on different placentae from different animals. Experiments (i), (ii), (iii), etc., indicate separate experiments on the same animal. All the analyses have been carried out in duplicate and the figures given represent the average of the duplicate results; they agreed to within 2 p.c. in the glucose and glycogen estimations and to within 6 p.c. in those of lactic acid. Toluene was added to the autolysate in all experiments, since a preliminary test revealed that even in 24 hours bacterial action reduced the glucose yield appreciably. Unless otherwise stated only the maternal decidual portion of the placenta was used for autolysis. Remarks Decidual portion: Aerobic autolysis TABLE II. Decidual portion: *0) gas bubbled through autolysate Decidual portion: Anaerobic autolysis N. gas f (a) 4 50 bubbled during autolysis (b) 4-67 Whole placenta: Aerobic autolysis with (a) 1-74 fluoride (b) 1-76 Effect of autolysis (aerobic and anaerobic) upon the glycogen, glucose and lactic acid content of the placenta. Percentages of the placenta A- Before incubation After incubation Lactic Glycogen Glucose acid {(a) (b) (c) Lactic Glycogen Glucose acid 0* (a) * (b) (c) Percentage of total initial carbohydrate recovered * * * * *5 0* * * * '98 0*12 66*0 (c) 2-87 * In these three experiments carbon dioxide was collected in soda-lime tubes, none was detected throughout autolysis, despite acidification afterwards with tartaric acid in the third experiment. A. Autolysis, aerobic and anaerobic. Portion B (Table I) suspended in saline was incubated for 24 hours at 370 C. In the first set of experiments autolysis proceeded alone; in the second set oxygen was bubbled through the whole time and the carbon dioxide formed was trapped in weighed soda-lime tubes; while in the third set nitrogen was bubbled through instead of oxygen to give anaerobic autolysis. In all these the results obtained were essentially the same; lactic acid was practically unchanged, glycogen disappeared almost entirely and

GLYCOGEN IN PLACENTAL AUTOLYSIS. glucose was formed to the extent of approximately 50-60 p.c. of the original glycogen.

5 GLYCOGEN IN PLACENTAL AUTOLYSIS. glucose was formed to the extent of approximately p.c. of the original glycogen. No carbon dioxide was formed (except in one experiment, which was not confirmed on repetition); this was despite acidification with tartaric acid. The result is to be expected, since autolytic changes in general are accomplished by hydrolysis and not by oxidation. The results on whole placentee show a slightly higher percentage of recovered carbohydrate and a lower initial percentage of glycogen. The latter is due to the fact that the maternal portion of the placenta alone contains the glycogen, and this is mixed with the foetal portion in the autolysis. The higher recovery of carbohydrate is apparently due to the dilution of the autolysate. This is confirmed by the effects of autolysis seen when maternal placente are diluted (Table IV). In all these experiments no determination was made of the total carbohydrate content of the placental residue after the alcoholic extraction of the glucose and lactic acid, that is, portion D in Table I. The residue was used solely for glycogen estimation (portion C in Table I.) In view of the constant loss of carbohydrate which formed approximately one-half to one-third of the total initial carbohydrate present, determinations were made on the total carbohydrate in the placenta before and after hydrolysis (Table III) and on the residue (portion D) after alcoholic extraction of the autolysate. TABLE III. Percentage recovery of initial carbohydrate on autolysis and determination of total carbohydrate. Percentage of total Percentage carbohydrate present of initial, &- 5A carbohydrate Before autolysis After autolysis recovered (a) (b) (c) The total carbohydrate content of the placenta is therefore practically unchanged by autolysis. On determining the total carbohydrate present in the residue (portion D) it was found to exceed the glycogen very much and to form 35 p.c. of the glucose formed in the case of the whole placenta and 50 p.c. of the glucose formed where only the maternal (decidual) portions were hydrolysed. These figures mean that the carbohydrate lost and not detected as glycogen or glucose is present as an intermediate carbohydrate product, probably a polysaccharide. The question was further investigated by taking the residue containing glycogen, after extraction of the sugars with p.c. alcohol, and further extracting it with 66 p.c. alcohol, this

6 188 A. DAVY AND A. ST G. HUGGETT.. yielded a milky solution which on acid hydrolysis gave a reducing sugar. The milky solution so obtained also formed a slight precipitate on saturation with ammonium sulphate but gave no significant colour with iodine. It seems permissible on this evidence to conclude that the residue contains glycogen together with intermediate products of its hydrolysis resembling dextrin. To determine exactly the kind of dextrin would require work with larger amounts of placenta than are at our disposal at the moment. In all probability it is a mixture of dextrins that is formed and not a single polysaccharide. B. The effect ofreaction upon placental autolysis was investigated under the following headings: (a) Change of reaction in the unbuffered solution. This was very slight, only altering from about ph 7415 to 7-4 (determined colorimetrically). (b) Autolysis when buffered at ph 5 0, 7 0 and 9*0. Buffering was maintained with the universal buffer of Prideaux and Ward [1924] except in one experiment where the buffer used was ammonium acetate. (c) Dilution of the autolysate. This was accomplished by using saline and no buffer solutions. TABLE IV. Effect of buffering and of dilution of autolysate on carbohydrate autolysis in the decidual portion of the placenta. Percentages of the placenta (decidual) Percen tial rdrate Before incubation After incubation of ini -, --,. carbo Remarks Glycog( )n Glucose Glycogen Glucose recov4 Autolysis at ph 7 0: Ammonium acetate buffer (a) 3 30 Universal phosphate p b) 4-01 buffer (c) 44 (d) hours' autolysis at ph 5-0, 7 0 and 9 0: (e) (i) 4.55 (ii) 4.55 (iii) 4.55 Effect of dilution of autolysate: (f) (i) With 2 vol. of saline * (ii) With 4 vol. of saline * (g) (i) With 1 vol. of saline * * * (ii) With 4 vol. of saline * The glycogen estimations here were accidentally lost Ltage Bred Solutions buffered at ph 7 0 yield about p.c. more glucose than in the unbuffered state. This is probably not due to the phosphate (universal) buffer used reacting with the carbohydrate, as occurs in

7 GLYCOGEN IN PLACENTAL A UTOLYSIS. 189 muscle metabolism, since it was obtained with the ammonium acetate buffer. It may possibly be due to the maintenance of constancy of reaction; but even in unbuffered autolysis the reaction does not materially alter. Where the medium was buffered to an acid, reaction glucose formation is markedly inhibited, and yet glycogen is broken down if anything more rapidly. In the alkaline buffer the glucose formation is only a little less pronounced than at ph 7 0. Dilution of the autolysate without buffering yields rather more glucose. This result is in line with the results of autolysis of whole placentse where more glucose was obtained than when maternal placentae alone were autolysed. C. Effect of time on the products of autolysis. Autolysis was allowed to proceed for periods varying in different experiments between 4 hours and TABLE V. Effect of time of autolysis upon the glycogenolysis of the decidual portion. Percentages in the placenta Percentage --A- 5 of initial Percentage Before incubation After incubation glycogen of initial G( recovered carbohydrate Glycogen Glucose Glycogen Glucose as glucose recovered Autolysis in unbuffered solutions: (a) (i) 12 hours (ii) 48 hours (b) 70 hours O *3 Autolysis when buffered at ph 7 0: (a) 4 hours (b) 8 hours (c) 12 hours 5.9 0* (e) 24 hours hours both in buffered and unbuffered solutions. The results are exemplified by Fig. 1. The percentage of glucose formed from glycogen increases as autolysis proceeds. The formation of glucose takes place more slowly than the breakdown of glycogen. When equilibrium was reached in each case not more than p.c. of the glycogen originally present was recovered in the form of glucose. These results emphasize the need for a rapid removal of the placenta after killing, since autolysis proceeds rapidly in the early stages. D. The autolytic enzyme. The properties of the autolytic enzyme are important in comparing the functions of glycogen in the placenta and in muscle. Meyerhof [1921] showed that fluoride inhibited lactic acid formation in muscle, and Evans [1922] showed the same thing for the glycolysis in blood. Since lactic acid formation is a relatively unimportant

8 190 A. DAVY AND A. ST G. HUGGETT. feature in the glycogenolysis of the placenta, we should not expect fluoride to affect materially the breakdown of glycogen. This is illustrated as the case by the result in Table II. There was no material alteration in the glycogen breakdown or lactic acid formation although rather more glucose was formed. This might be due to some inhibition of glycolysis. The second point about the enzyme is the fact noted above, that when autolysis is allowed to proceed, equilibrium is reached in about 24 hours \ 40~~~~~~~~~~~~~~~~~~~ \000z x - 20 _ / Hours Fig. 1. Effect of time on the autolysis of placental tissue. )- Percentage of initial glycogen recovered as glycogen. x -*- x Percentage of initial glycogen recovered as glucose. 0-0 Percentage of initial glycogen and glucose recovered as glycogen and glucose. The third point of interest is the origin of the enzyme. Is it resident in the placental tissues or does it reach it via the maternal blood from some organ such as the pancreas? In an attempt to solve this point the placenta was rendered blood-free by the vivi-perfusion method of Bogniard and Whipple [1932]. The rabbits were then killed. The placente thus obtained contained no maternal blood, and, as the maternal portion only was used, contained no foetal blood. The essence of the method is to allow Ringer-Locke solution to run in at the jugular vein and simultaneously blood to run out at the carotid artery. Adrenaline was injected towards the end of the experiment

9 GLYCOGEN IN PLACENTAL AUTOLYSIS. 191 to increase the blood-pressure and so to promote blood flow. This is allowable, since it has been shown [Huggett, 1929] that adrenaline has no effect on the placental glycogen. In about 30 mi. the fluid leaving the carotid was colourless. Table VI shows that the quantity of carbohydrate recovered is the same whether whole placentae or only maternal placente are used, and is TABLE VI. Effect of removal of maternal blood before killing on the autolysis of the maternal portion and the whole placenta. Percentage Before incubation After incubation of initial, carbohydrate Glycogen Glucose Glycogen Glucose recovered Maternal (decidual) portion of the placent.e: (a) Almost blood-free (b) Blood-free Whole placentas: Blood-free the same when the blood is washed out as when it is not. In other words, the amylase is seated within the decidual cells of the placenta, and is not merely in the maternal blood. DIsCUSSION. The products of autolysis of the placental glycogen are entirely carbohydrates, the only lactic acid produced in 24 hours being traces of the order described by Harada [1916]. The carbohydrates formed fall into two classes, soluble and insoluble in 66 p.c. alcohol. The former includes the reducing sugars of which the chief appears to be glucose. The only osazone obtained was glucosazone. There was no sign of maltosazone or of the osazone of Barbour's trisaccharide [1929]. There was, however, a small quantity of something else present, since estimations on two samples of the reducing power after hydrolysis of the alcohol-soluble fraction with acid gave higher results than when no such hydrolysis was carried out. This small quantity would, however, only account for 4 p.c. of the original glycogen. The alcohol-insoluble portion contained only a trace of glycogen but sufficient extra carbohydrate to account for the remaining glycogen. This appears to be a polysaccharide of molecular size less than that of glycogen but yielding no significant colour with iodine. Aerobic and anaerobic autolysis yield the same results and no carbon dioxide formation in either case. Increased acidity up to ph 5-0 does not inhibit glycogen breakdown, but it does decrease the formation of glucose.

192 A. DAVY AND A. ST G. HUGGETT. Increased alkalinity up to ph 9*0 has little or no effect on either process. Phosphate and fluoride ions have no gross effect on placental autolysis.

10 192 A. DAVY AND A. ST G. HUGGETT. Increased alkalinity up to ph 9*0 has little or no effect on either process. Phosphate and fluoride ions have no gross effect on placental autolysis. This compares with muscle autolysis [Meyerhof, 1921]. Sahyun and Alsberg [1931] have observed that in hydrolysis of pure liver glycogen with takadiastase equilibrium is reached when only 52 p.c. of the glycogen broken down is present as reducing sugars. They state that in addition to the monosaccharide formed a polysaccharide is present. In this respect therefore there appears to be some parallel between the autolytic hydrolysis of glycogen in the placenta and the hydrolysis by takadiastase of glycogen in the liver. Lochhead and Cra m e r [1908] extracted with glycerol from the placenta an enzyme which converted pure glycogen into glucose. The glycogen used had not, however, been prepared from the placenta. These experiments therefore confirm Claude Bernard's original view that the placenta serves as a store of carbohydrate for the faetus, and they offer no evidence of parallelism of function between muscle and placental glycogen. Taken in conjunction with previous experiments [Huggett, 1929] the decidual glycogen appears to be nearly independent of the maternal metabolism, to be remarkably stable and to be controlled mainly by the fwetal demands although present in the maternal (decidual) tissues of the placenta. SUMMARY. 1. Autolysis of minced rabbit's placenta results in disappearance of the glycogen and its replacement by approximately two-thirds of glucose and one-third of a dextrin-like polysaccharide. 2. No carbon dioxide and only traces of lactic acid were found. 3. The same result was obtained with whole placente and decidual portions only, and was unaltered by the absence of oxygen, by fluoride or phosphate, or by previous removal of maternal blood. 4. Alteration in reaction did not alter the disappearance of glycogen, but more glucose was formed at ph 5 0 than at ph 7 0. Our thanks are due to Dr Arthur Wormall of this department for many helpful suggestions. We are indebted to the Government Grant Committee for agrant to one ofus (A. St G. H.) which has partly covered the expenses incurred.

11 GLYCOGEN IN PLACENTAL AUTOLYSIS. 193 REFERENCES. Anderson, J. A. and Macleod, J. J. R. (1930). Biochem. J. 24, Barbour, A. D. (1929). J. biol. Chem. 85, 29. Bernard, C. (1859). C. R. Acad. Sci. Pari8, 48, 77 and 673. Bogniard, R. P. and Whipple, G. H. (1932). J. exp. Med. 55, 654. Evans, C. L. (1922). J. Physiol. 56, 146. Evans, C. L., Tsai, C. and Young, F. G. (1931). Ibid. 73, 67. Friedemann, T. E., Cotonio, M. and Shaffer, P. A. (1927). J. biol. Chem. 73, 333. Hanes, C. S. (1929). Biochem. J. 23, 99. Harada, T. (1916). Acta Sch. med. Univ. Kioto, 1, 291. Holmes, E. G. and Gerard, R. W. (1929). Biochem. J. 23, 738. Huggett, A. St G. (1929). J. Physiol. 67, 360. Ishikaw a, E. (1928). Biochem. Z. 195, 469. Lochhead, J. and Cramer, W. (1908). Proc. Roy. Soc. B, 80, 265. Meyerhof, 0. (1921). Pflugers Arch. 188, 114. Parnas, J. K. and Wagner, R. (1914). Biochem. Z. 61, 387. d'piane, G. (1926). Ginec. nod. 5, 328. (Quoted from Needham, J., Chemical Embryology.) Prideaux, E. B. R. and Ward, A. T. (1924). J. chem. Soc., Lond., 125, 426. Sahyun, M. and Alsberg, C. L. (1931). J. biol. Chem. 93, 235. Simpson, W. W. and Macleod, J. J. R. (1927). J. Physiol. 64, 253.

6I2.744.I5: e3. sufficiently high'. There exists in such cases a certain concentration of the. by direct analysis.

6I2.744.I5: e3. sufficiently high'. There exists in such cases a certain concentration of the. by direct analysis. 194 THE DIFFUSION OF ACTATE INTO AND FROM MUSCE. BY S. C. DEVADATTA. 6I2.744.I5:547.472e3 (From the Department of Physiology, Edinburgh University.) CERTAIN constituents of the voluntary muscles of the