Tricarboxylic Acid Metabolism Studies in the Ovary Throughout the Menstrual Cycle. S. J. Behrman, M.D., M.R.C.O.G., and Gregory S. Duboff, M.S., D.Sc.

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1 Tricarboxylic Acid Metabolism Stdies in the Ovary Throghot the Menstral Cycle S. J. Behrman, M.D., M.R.C.O.G., and Gregory S. Dboff, M.S., D.Sc. THE VARIOUS ENDOCRINE CHANGEs dring the hman menstral cycle have been intensively stdied. The significant role of the ovary dring the menstral cycle is evident from its demonstrated prodction of estrogens and progesterone nder the inflence of the gonadotrophins. Yet very little is known of the metabolic activity of the ovary, at a celllar level, dring the menstral cycle, and of the manner in which the varios hormones, both endogenos and exogenos, affect these metabolic activities. Three basic qestions therefore need clarification: ( 1) What, if at all existent, are the actal oxidative and glycolytic metabolic pathways operative within the ovary? (2) What contribtion to the changes in the menstral cycle and ovlation do the basic metabolic activities within the ovary make? ( 3) What are the interrelationships of the ovarian and gonadotrophic hormones associated with the menstral cycle and the fndamental ovarian celllar metabolism? As sch basic information has direct application to the pathways of hormone tilization dring the menstral cycle and the phenomenon of ovlation, a possible explanation for the mechanism of ovlation and contribtions of clinical vale in the management of disorders of the menstral cycle cold be obtained. This is the ltimate aim of this preliminary investigation. From the Deparbnent of Obstetrics and Gynecology, University of Michigan School of Medicine, Ann Arbor, Michigan. This investigation has been spported by a grant from the Josiah Macy, Jr. Fondation (Project #579). This paper was presented at the Fifteenth Annal Meeting of the American Society for the Stdy of Sterility, Atlantic City, N.J., April 3-5,

2 Vol. 11, No.2, 196 OVARIAN METABOLISM STUDIES All living cells reqire energy for two reasons: first, to maintain themselves in the living state, and, second, to do work, sch as secretion, absorption, bilding p and breaking down cell material, and movement. This necessary energy is obtained from the degradation of food elements in three wellrecognized stages. Stage I is the oxidative-enzymatic degradation of foodstffs into carbohydrates, fatty acids, and amino acids. Stage II is the formation of phosphate esters of the energy-rich phosphate bonds at the celllar level. Stage III is the release of this energy by means of energy-transport mechanisms, e.g., hydrogen, high-energy phosphates, etc., which at the biomoleclar level are dependent on TPN and DPN, and which in trn appear to be dependent pon the steroids, e.g., estrogen. 7 9 Finally, this special form of chemical energy is stored in the pyrophosphate bonds of Acetoacetate Fat Glcose t Pyrvate! Jill Lactate CO2 4 lo lo H 2 Acetyl-co-A Cltrate/alacetate H v "\. Cis Aconitate H2 Malate H 2 t \..,....., H I so Citrate \c H2 H2 +-+I Fme rate Oxal sccinate H 2 Sccinate \ C2 d.keto Gltarate 211 Fig. 1. Kreb's cycle.

3 212 BEHRMAN & DUBOFF Fertility & Sterility adenosinetriphosphate (A TP) and is released when this bond is hydrolyzed, i.e., the Kreb' s cycle (Fig. 1). On a biomoleclar level, therefore, the chemical reactions that lead to the release of energy may be divided into two grops. The first comprises reactions in which the available material is incompletely brned, the end prodcts being, apart from carbon dioxide and water, one of three sbstances: acetic acid in the form of acetocoenzyme A (Co A), alpha-ketogltaric acid, or oxalacetic acid. The first of these contribtes the greater amont of energy. Alpha-ketogltaric acid arises from several amino acids, sch as gltamic acid, histidine, arginine, proline, and hydroxyproline; whereas oxalacetic acid arises from aspartic acid and from the benzene ring of tyrosine and phenylalanine. These Kreb's cycle intermediates are metabolically closely interrelated, staging the third level of the energy release by way of the tricarboxylic-acid cycle, which represents a common terminal pathway of oxidation of all foodstffs. Ths, the Kreb's cycle describes how one aceticacid eqivalent is brned completely to carbon dioxide and water, a series of di- and tricarboxylic acids appearing as intermediate stages. The prpose of this paper, therefore, is to report the activity of the tricarboxylic acid cycle of Kreb dring varying phases throghot the normal menstral cycle. According to the wealth of pblished material, organ perfsion has been a rewarding approach to the stdy of the metabolic fnctions of endocrine glands. However, the extension of this general technic to the ovary does not lend itself readily to a stdy of the problems otlined above. Accordingly, we ndertook a stdy of the metabolic activities within homogenates of the hman ovary obtained immediately after srgical removal. METHODS AND MATERIALS The procedres sed in this stdy were derived from methods developed in other laboratories (Frohman et al., 3 Dajani,2 and Marshall and Rand4 ) and modified in sch a manner as to be adapted to working with ovarian tisses. Ovaries were obtained from patients. Of these, 12 were normally menstrating women who had had tboplasties, a myomectomy, and an appendectomy. The day of the cycle was calclated from the first day of the last period and confirmed by histologic examination of the endometrim obtained from D & C or endometrial biopsy. For specimens were obtained from wedge resections for polycystic ovarian disease, 3 from postmenopasal hysterectomies, and 1 from a 6-week postpartm sterilization.

4 Vol. 11, No. 2, 196 OVARIAN METABOLISM STUDIES 213 Samples of ovary were prepared as follows: Sections of ovary obtained from the operating room were removed as qickly as possible and placed in dry ice to sspend all celllar activity. The tisses were then thawed ot in a soltion of polyvinylperrilidinone-scrose-salt soltion, blotted, and weighed. They were then minced into small pieces within the soltion and homogenized gently with a glass homogenizer. This procedre was merely to free as mch of the cells as possible from the stroma. The whole was then centrifged at a low speed to precipitate the tisse debris. The spernatant material was recentrifged at a higher speed to sediment the cells ot of the soltion. The sediment was washed twice with five-times normal (hypertonic) saline. The cells were then resspended in Kreb's-Ringer's soltion containing antibiotics. An aliqot of this was added to a sbstrate consisting of sodim acetate, A TP and DPN in a flask, and the air replaced by 95 per cent oxygen with 5 per cent C2. This soltion was then incbated at 37o to 3 C. for 4 hors and shaken freqently. Another aliqot, which was not activated, was sed as a control. Both flasks were removed from the incbator and placed immediately in a deep freeze at -65 C. to terminate enzyme activity. The soltions were then permitted to thaw ot and were frther homogenized vigorosly in a Vertis homogenizer, following which eight parts of acetone were added, the ph adjsted to between 2 to 3, and the soltions were allowed to stand in the refrigerator overnight for extraction of the organic acids. The contents in the flasks were then transferred to large centrifge bottles and centrifged at high speed to precipitate the acetone-insolble material. The spernatant flids were transferred to rond-bottom flasks and evaporated down to abot cc. on a rotary film evaporator. The concentrated soltion was treated with ether to remove fats, and the ether extract was discarded. The remaining aqeos soltion was recentrifged to remove any solid particles, after which it was evaporated to dryness. The dry reside was taken p in per cent tertiary amyl alcohol in chloroform, and sitable aliqots sed for the next phase in the procedre. This consisted of a colmn chromatographic technic, employing hydrated silicic acid. Complete separation of the citric-acid cycle intermediates, as well as celiain other related acids from the tisse extracts was achieved, as shown in Fig. 2. The validity of the chromatographic fractionation was frther demonstrated by recovery stdies of the acids added to tisse homogenates, as well as from pre sol-

5 U4 s I L I c I c A c I SEQUENCE OF ELUTION r<:.. ::> AMYL-ALCOHOL CHLOROFORM SOLUTION I< -:::: CITRATE r-- - ISOCITRATE r-- -MALATE- -.!.CON I TATE- -«-KETO- GLUTARATE - -SUCCINATE-- BEHRMAN & DUBOFF PERCENT RECOVERIES OF ORGANIC ACID FROM MIX TURES OF PURE SOLUTIONS AND FROM ACIDS ADDED TO OVARIAN TISSUE HOMOGENATES zo ADDED GAMMA PURE TO EQUIVALENT SOLUTIONS HOMOGENATES MEAN* RANGE MEAN* RANGE!.ACETATE 99.7 BS ,-55, Z. PYRUVATE XALACETATE o-az. o 4. FUMARATE n. o-9. o Fertility & Sterility c L M N LACTATE FUMARATE r-- - OXALACETATE r- -- PYRUVATE r-- -- ACETATE / 5. LACTATE 97. s b. SUCCIXATE CJ CI-KETO- GLUTARATE Z..97., ACO:"o:'ITATE t MALATE Z lo.isoci'frate 95. H ( CITRATE.). I OZ AVERAGE OF THREE DETERMINATIONS t THE SUM OF CIS AND TRANS FORMS ZOO GAMMA CITRIC ACID \ Fig. 2. Recovery of citric-acid cycle intermediates from ovarian tisse homogenates by chromatographic fractionation. tions. Fractions collected from the chromatographic colmn were titrated against a dilte sodim hydroxide soltion and the concentrations of the specific organic acids calclated from the alkaline normality eqivalent. The reslts are tablated in Table 1 for the recoveries of the mixtres of pre soltions and from acids added to ovarian tisse sspensions.

TABLE 1. 215 OVARIAN METABOLISM STUDIES Vol. 11, No. 2, 196 Ovarian-tisse Concentrations of Kreb's Cycle Acids (J.tg./Gm.

6 TABLE OVARIAN METABOLISM STUDIES Vol. 11, No. 2, 196 Ovarian-tisse Concentrations of Kreb's Cycle Acids (J.tg./Gm.) Day of menstral cycle 3 5 Acetate Pyrvate Oxalacetate Fmarate Sccinate A-Ketogltarate Aconitate Malate Isocitrate Citrate Lactic ' RESULTS Figre 3 shows the reslts of the potential cell activity of the Kreb' s acid cycle in terms of total organic acids determined on 12 patients throghot the normal menstral cycle. There is a significant increase in oxidative activity (with increased energy otpt), beginning abot the thirteenth day with an apparentpeak arond Days to 16 and followed by a gradal decrease TOTAL ACIDS I DAYS Fig. 3. OF MENSTRUAL CYCLE Total organic acids recovered from ovarian tisse homogenates in 12 patients.

Fertility & Sterility BEHRMAN & DUBOFF 216 to preovlatory levels. This marked increase of metabolic activity closely corresponds to the estimated time of ovlation.

7 Fertility & Sterility BEHRMAN & DUBOFF 216 to preovlatory levels. This marked increase of metabolic activity closely corresponds to the estimated time of ovlation. Since we are primarily interested in the tricarboxylic acids, we plotted these hydroxy acids as their mono-, di-, and tri-forms (Fig. 4). This gives frther evidence that the agmentation of energy activity is reflected in the over-all increase of the several species of the Kreb's cycle intermediates. A frther analysis of the individal acids (Table 1) confirms the fact that the greatest increase in the concentration of energy-rich phosphate esters occrs arond the time of ovlation. In addition to the Kreb' s cycle intermediates, the related lactic acid vales were also determined. These are graphically shown in Fig. 5. The significance of this will be dealt with later. Figre 6 shows sperimposed on the crve established for normally menstrating women, estimations of the total Kreb' s cycle acids in 4 cases of polycystic ovary, 3 cases of postmenopasal women ( 3, 15, and 1 years), and 1 case of postpartm ovary. It is evident that none of these showed the marked degree of oxidative activity demonstrated by the ovaries from normono-, Dl-, AND TRICARBOXYLIC ACIDS MON-1... TRI-... II) c -- 2 "' w..j /6 II) m wl a: :ac,.."' :1 DAYS OF MENSTRUAL CYCLE Fig. 4. Total acids plotted in their mono-, di- and tri-forms.

a:: ACI OS OVARIAN DISEASE B 3 CASES OF POSTMENOPAUSAL OVARIES I CASE OF 6 WKS POSTPARTUM OVARY E <I)... <( 1&1.

8 LACTIC 1&1 ::» Cl) Cl) t= <( z TOTAL ACIDS- ii: LACTIC ACID""""' E <I)... <( 1&..J > Cl) Ill 1&1 a:: "'... :I DAYS OF MENSTRUAL CYCLE Fig. 5. Lactic acid recovery in ovarian tisse homogenates compared with total organic acids. TOTAL..,!!! A 4 CASES OF POLYCYSTIC z C... :! a:: ACI OS OVARIAN DISEASE B 3 CASES OF POSTMENOPAUSAL OVARIES I CASE OF 6 WKS POSTPARTUM OVARY E <I)... <( 1&1..J Ill a:: 1&1 "' i DAYS OF MENSTRUAL CYCLE Fig. 6. Total organic acids obtained dring normal menstral cycle compared with those obtained from postmenopasal, polycystic and postpartm ovaries.

9 21 BEHRMAN & DUBOFF Fertility & Sterility mally menstrating women. In fact, none of these cases showed levels exceeding that fond in the preovlatory phase. In order to eliminate the possibility of artefacts as a case of the foregoing observations, a nmber of chromatographic fractionations of mixtres with known concentrations of the organic acids were performed, and the recovery of acids added to different specimens of ovaries that were regarded as relatively normal. The virtally insignificant vales obtained for the several experiments with cell sspensions withot sbmission to incbation and activation (in which the yields of organic acids were from less than 1 JLg. to a maximm of 5 JLg. for any of the organic acids) is an index of the absence of any significant celllar damage dring the maniplation of the ovarian tisse in preparing it for analysis. The identity of the acids obtained by colmn chromatography was frther confirmed by the ltraviolet absorption spectrophotometric procedre with the se of one-tenth normal hydrochloric acid as the solvent. DISCUSSION Althogh the experiments appear to offer a means for measring the prodcts of the tricarboxylic-acid cycle in the ovary, the interpretation of these findings presents several difficlties. An important stride toward or goal has been made, bt no knowledge of the relative importance of enzymatic activity within the tricarboxylic system of enzymes has as yet been established. Also, there are well..;recognized alternate pathways of oxidative and glycolytic metabolism in animal tisses. A failre to demonstrate increased concentrations of tricarboxylic intermediates in the aberrant ovaries (polycystic ovarian disease, postmenopasal ovary, etc.), as seen at the time of ovlation in the normal ovary, may possibly be de to a blocking or inhibiting effect of one or more of the necessary enzymes reqired for transference of the energy from one member to the next in the.kreb's cycle. We have not as yet explored the importance of the alternate pathways that may be involved. Stdy of the alternate pathways is the sbject of work now in progress. Nevertheless, the importance and exact role of the many metabolic pathways in the activity of the ovarian gland mst be established in order to nderstand the complexity of the interaction of endocrine sbstances that inflence the ovary dring its reprodctive activity. Review of the literatre reveals no precedence of investigations in the area of intermediate metabolism in tisses per se, or as it pertains to the problems nder consideration.

10 Vol. 11, No. 2, 196 OVARIAN METABOLISM STUDIES 219 There may be vale in digressing at this point to discss the celllar biochemistry that is being considered here and to reflect on the chemical principles of the Kreb's cycle system. The Kreb's cycle and associated enzymatic systems catalyze the oxidation of pyrvic acid, of fatty acids, and of certain key natral amino acids to carbon dioxide and water. Althogh each starting point may be different, nevertheless, as in the case of Rome, all the metabolic roads lead to the Kreb's cycle. A predominant featre of this cycle is that it permits small amonts of any oxidizable sbstrate to be degraded by sccessive enzymes ntil nothing is left bt carbon dioxide and water. Coincident with these varios oxidative processes by one enzyme to another, there is the tapping of oxidative energy in the form of high-energy phosphates ( P4). The primary pyrophosphoric ester ( Kreb' s cycle acid + phosphate) is formed by the operation of the reversibly redcible coenzymes TPN and DPN, which serve as transformers of oxidative energy to phosphate-bond energy. This TPN-DPN shnt is in jxtaposition to the action of the endogenos steroid hormones, and possibly to that of the gonadotrophic hormones. There is reason to believe that these hormones implement the reglation of an orderly seqence of hydrogen (energy) transport. A qestion that cold readily arise at this point might well be the reason for an increase in aerobic oxidation and glycolysis at some point in the menstral cycle. Two explanations might be sggested. First, in the repetitively recrring menstral cycle there is marked celllar growth and synthesis within the ovary associated with folliclar development, its matration, and evental ovlation, followed by regression and celllar degradation. Ths, in this alternation of synthesis and degradation, energy is consmed. Second, the particlar direction of acceleration of the tricarboxylic-acid-cycle activity may be for the prpose of frnishing energy for work, sch as is involved in contraction or in explsion of the ovm from the follicle. It is difficlt to see how bond energy cold be sed this way, as the phenomenon of contraction is generally associated with mscle strctres. To bring the process of ovlation into line with the latter concept one cold speclate, for example, that a change in biochemical architectre and environment, with release of energy, cold be responsible for the rptre of the follicle. One frther item that reqires brief discssion is the lactic acid content of the ovary throghot the menstral cycle, as indicated in Table 1 and Fig. 5. In glandlar tisse, pyrvate is oxidized as fast as it can be formed from glcose and, therefore, lactic acid does not accmlate. However, nder or

11 2 BEHRMAN &. DUBOFF Fertility & Sterility conditions of experimentation, lactic acid accmlated dring the storage (freezing) of the gland, i.e., nder anaerobic conditions. This indicates that the metabolic pathway chosen by glcose is mainly that of the tricarboxylic acid cycle, rather than the anaerobic lactic-acid pathway. SUMMARY 1. A method for the stdy of tricarboxylic-acid metabolism in the hman ovary has been developed and stdied. 2. Qantitative recovery of members of the citric-acid cycle indicates that this is one of the important sorces of energy within the ovary, and is characterized by marked agmentation of the di- and tricarboxylic acids at the time of ovlation and for a short period thereafter. 3. Random samples from postmenopasal, postpartm, and polycystic ovaries do not show this agmentation. 4. The possible implications have been briefly discssed, and areas for frther research demarcated. REFERENCES I. BcHANAN, J. M., SAKAMI, W., GRIN, S., and WILSON, D. W. ]. Biol. Chern. 157:747, 1945; 159:695, DAJANI, R. M., and ORTEN, J. M. ]. Biol. Chern. 231, FROHMAN, C. E., ORTEN,]. M., and SMITH, A. H. ]. Biol. Chern. 193:277, MARSHALL, C. S., and RANDS, R. D. ]. Am. Chern. Soc. 72:2692, MELROSE, D. R., and TERNER, C. Biochem.]. 49:1, MICHEALis, L., and SALMON, K. Physiol. 13:63-693, 193. Respiration of mammalian erythrocytes. ]. Gen. 7. TALALAY, P., and WILLIAMs-AsHMAN, H. G. Proc. Nat. Acad. Sc. 44:15, VILLEE, C. A. Estradiol-indced stimlation of citrate tilization by placenta. ]. Biol. Chern. 215:171-12, VILLEE, C. A., and HAGERMAN, D. D. Effects of estradiol on metabolism of hman placenta in vitro. ]. Biol. Chem. 5:73-2, WARBURG,. Z. Physiol. Chern. 59:112, 199.

THE EFFECT OF INTRAUTERINE PROGESTERONE TREATMENT ON THE ENDOMETRIAL PROSTAGLANDIN F CONTENT IN THE RABBIT*

FERTILITY AND STERILITY Copyright 1977 The American Fertility Society Vol. 2, No.5, May 1977 Printed in U.s.A. THE EFFECT OF INTRAUTERINE PROGESTERONE TREATMENT ON THE ENDOMETRIAL PROSTAGLANDIN F CONTENT