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A calorimetric method to assess endogenous metabolism and its application to the study of bovine sperm
Journal of Biochemical and Biophysical Methods, 7 (1983) 199-210
199
Elsevier
A calorimetric method to assess endogenous metabolism and its application to the study of bovine sperm * Philip B. Inskeep and Roy H. Hammerstedt Biochemistry Program, 406 Althouse Laboratory, Pennsylvania State University, Universi(v Park, PA 16802, U.S.A. (Received 20 November 1982) (Accepted 22 November 1982)
Summa~ Calorimetry was used to assess the importance of endogenous metabolism towards total ATP synthesis in bovine sperm in the presence of extracellular glucose. Sperm were incubated in the calorimeter with D-[U-14C]glucose without or with electron transport inhibitors, rotenone and antimycin A. Steady-state heat production during the incubations was measured for 30 rain, the incubations were terminated, and the cell suspensions removed for analysis of radioactive glucose and its metabolic end-products. Heat production (mean_+ S.E.) associated with the metabolism of glucose was calculated, from enthalpies of formation of glucose and its end-products, as -412_+ 34 m J/h/108 cells in control incubations and -263_+ 18 m J / h / 1 0 s cells in incubations with electron transport inhibitors. Measured heat production was - 4 5 5 + 3 6 and -263_+ 17 mJ / h / 1 0 s cells, respectively. Thus, heat production by endogenous pathways, the difference between measured total heat production and calculated exogenous heat production, was -43_+ 14 m J / h / 1 0 s cells for control cells and about - 6 m J / h / 1 0 s cells for inhibited cells. The ratio of heat produced per mol of ATP synthesized is similar for all ATP-producing pathways. Therefore, about 10% of total ATP synthesis in control cells and less than 2% in inhibited cells is provided by endogenous pathways when extracellular glucose is present. Key words: calorimetry; endogenous metabolism; sperm; ATP synthesis: glucose; bovine sperm.
Introduction The utilization of endogenous reserves, stored compounds such as glycogen and lipid which can be readily mobilized to satisfy cellular energy requirements via * Approved as Paper No. 6536 by the Pennsylvania Agricultural Experiment Station. Portions of this work were taken from a dissertation presented by P.B.I. in partial fulfillment for the requirements for the Ph.D. in the Biochemistry Program, The Pennsylvania State University. Preliminary results were presented at the Division of the Biological Chemistry Meeting of the American Chemical Society, August-September 1981. Abbreviations: Anti A, antimycin A; NDC, endo-norbornene-cis-5,6-dicarboxylic acid; Rote, rotenone. 0165-022X/83/$03.00 © 1983 Elsevier Science Publishers B.V.
200 ATP-yielding pathways, is a process which provides an advantage for survival of cells when extracellular substrates are depleted. However, the importance to the cell of such reserves when extracellular substrates are abundant is unclear. A conference addressing this problem was held 20 years ago [1] and, although some quantitative assessments for endogenous metabolism of microorganisms were attempted, the lack of appropriate model systems and methodologies limited the conclusions that could be drawn. Herein we describe experiments where bovine sperm, a mammalian cell system possessing simple metabolic properties [2], and a unique combination of calorimetry, carbon balance, and calculations from thermodynamic constants, have been used to quantitatively assess the importance of endogenous metabolism in bovine sperm when extracellular substrates are present. In addition, the effect of respiratory inhibitors on endogenous metabolism has been established.
Instrumentation and Methods
Materials D-[U)4C]Glucose, [1,4-14C]maleic anhydride, and [2,3-14C]maleic anhydride were purchased from Amersham, Inc. (Arlington Heights, Ill.). All biochemicals, enzymes, and ion-exchange resins were purchased from Sigma (St. Louis, Mo.). [5,614C]endo-norbornene-cis-5,6-dicarboxylic acid and [7,8-14C]endo-norbornene-c/s5,6-dicarboxylic acid were synthesized [3], recrystallized and assayed for purity by thin-layer chromatography in two solvent systems (toluene/ethyl formate/formic acid, 55 : 44 : 11, and benzene/methanol/acetic acid, 90 : 16 : 8). Greater than 99% of radioactivity co-chromatographed with authentic NDC. The buffer used for sperm preparation and in all sperm incubations contained 30 mM endo-norbornene-5,6-c/s-dicarboxylic acid, 2 mM MgC1 z, 4 mM KC1, 2 mM sodium phosphate, 107 mM NaCI, 1 mM NH4C1, 0.003% (w/v) streptomycin sulfate, and 0.005% (w/v) penicillin G at pH 7.2 as described by Hammerstedt [4]. The major buffer component, NDC, was chosen because it had been established that it is not metabolized by bacteria [5]; this conclusion was extended to sperm cells (see below).
Calorimetry The calorimeter (Microcalorimeters, Inc., St. Paul, Minn.; see Lovrien et al. [6] for a description of a similar instrument) is a differential heat conduction instrument with five glass vessels, one of which serves as a reference for the other four. Each vessel (Fig. 1) is divided into two separate compartments with a total capacity of 3.0 ml. The vessel faces have finely ground surfaces, which comprise about 60% of the total vessel surface area, and are covered by Seebeck sensors. Heat produced within the vessels passes through the sensors to large aluminum blocks on both sides of each vessel. The vessels, sensors, and heat sinks are all mounted on a drum which can be rotated 360°; thus, contents initially kept in the separated compartments of
201
~~.~--~ SINKl ~ ¢ ~
KEEBLOCK
Fig. I. Schematic representation of the calorimeter used in these experiments. Only one incubation vessel is shown. Five such vessels are mounted in the calorimeter keel block which is contained in an aluminum drum (14 cm diameter × 28 cm length). The drum is mounted inside two insulating chambers on a shaft which can be rotated by means of a crank on the outside of the outer insulating chamber. When the drum is so rotated contents of the two chambers in each vessel are mixed.
the calorimeter vessels can be mixed together by rotating the drum. The calorimeter was housed in a constant-temperature box and isothermal conditions within the calorimeter were maintained to 30 + 0.05°C with Peltier pumps. The calorimeter is capable of detecting a heat flow of less than 88 m J / m i n with a minimum signal to noise ratio of 20. Electrical calibration with 511 ohm resistors produced a non-linear curve in the range of 44-1750 m J / h (Fig. 2). An alternate method of calibration utilizing invertase-catalyzed hydrolysis of sucrose ( A H = - 1 4 . 0 m J / # m o l sucrose hydrolyzed, [7]) resulted in an identical calibration curve. Daily calibrations verified that the calibration curve was consistently reproducible. The following protocol was used for all incubations in the calorimeter: (1) The large compartment of each calorimeter vessel was loaded with 1.0 ml of oxygen-saturated buffer containing isotopically labeled glucose without or with electron transport inhibitors, flushed with H20-saturated oxygen, and sealed. With this procedure, gas-liquid equilibration within the vessels and thermal re-equilibration of the calorimeter were achieved within one hour. (2) After thermal equilibration of the calorimeter was established, sperm (0.5 ml) were loaded into the small compartments of the calorimeter vessels, and the vessels were resealed. Re-equilibration of the calorimeter was complete within 20 min after reseating the vessels. (Separate experiments (data not presented) established that sufficient oxygen was present to maintain sperm respiration beyond the termination point of the incubation and that oxygen consumption by the sperm was linear throughout the incubation.) (3) After re-equilibration was achieved, the contents of both compartments in
202
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Fig. 2. Electrical calibration curve for the calorimeter. Electric heaters (511 12 resistors mounted in calorimeter vessel stoppers) were placed in each sample vessel and current applied to each heater by a d.c. power supply was measured, The deflection of the chart recorder pen, after steady-state heat production was observed (about 5 min), was recorded for each heater at each heat output; Heat Production (m J / h ) = 3.59 m J / ( h x m A 2 x ohm)×Current (mA)2 × Resistance of heater (12). Data presented represent the composite of all calibrations performed during the 4-week period of the experiments.
each vessel were mixed by rotating the calorimeter drum. This started the incubation (Fig. 3).
Sperm preparation Portions of 2 ejaculates collected from the same bull within 15 min of each other i
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Fig. 3. Heat production of sperm as a function of time in the absence ( - ) and presence ( I ) of electron-transport chain inhibitors. Sperm were placed in the calorimeter vessels 25 rain prior to mixing (not shown). Thermal equilibration was achieved within 20 rain ( T = - 5 min), and the measured rate between this time and the moment of mixing ( T = 0) sperm with D-[U-~4C]glucose represents heat production of sperm in the absence of any exogenous substrate. Upon mixing, heat production rapidly increases to a new steady-state level which is maintained for the duration of the incubation. In this representative experiment, two of the four reaction tracings have been deleted for clarity.
203 were pooled, washed twice in buffer, and diluted to a known concentration, as assessed turbidimetrically [4], of about 3.5 • 108 cells/ml. This treatment resulted in a dilution of seminal plasma components of about 1000-fold [4]. Portions (0.5 ml) of these suspensions were transferred to four of the calorimeter vessels after a microscopic assessment of motility. All ejaculates used had greater than 50% motile sperm. This same sperm preparation scheme was used in experiments with buffer containing ~4C-labeled N D C except that sperm were diluted to a measured concentration near 12 • 108 cells/ml and were incubated in a respirometer. All incubations were started within 1.5 h after collection of ejaculates.
Metabolite assays Lactate, acetate and acetylcarnitine contents after 30 min of incubation were assayed in 150/~1 aliquots of the cell suspensions by ion-exchange chromatography [8]. Briefly, two sets of minicolumns were arranged in tandem so that the effluent from the first set (Dowex-50-H +) passed directly to the second set (Dowex-l-formate). Samples were applied to the top of the first column and washed with 10 ml H 2 0 . During this wash, cations (acetylcarnitine) were adsorbed to the Dowex-50-H + resin, anions (acetate, lactate and NDC) were adsorbed to the Dowex-l-formate resin, and uncharged compounds (glucose) passed through both columns and were collected in glass scintillation vials. The column sets were separated and each set was washed to elute adsorbed compounds. Acetylcarnitine was eluted from Dowex-50-H ÷ columns with 10 ml of 1 N N H 4 O H . Acetate and lactate were eluted from the Dowex-l-formate resins with 30 m M formic acid and 300 mM formic acid, respectively. In experiments with [t4C]NDC, this compound was eluted from the Dowex1-formate resin with 3 M formic acid. ~4CO 2 formation was measured in 0.5 ml portions of the sperm suspensions recovered after 30 rain of incubation in the calorimeter. Samples were transferred to glass respirometer flasks, previously loaded with 6 N K O H in the center wells and 6 N H2SO 4 in the side arms, were sealed and the acid mixed with the sperm suspensions to release ~4CO2. Separate experiments (data not presented) with sperm suspensions established that this procedure recovered 87 + 6% (S.E., n = 13) of total ~4CO2 formed by the sperm during the incubation. This factor has been used to establish the actual amount of 14CO2 produced by the sperm during the incubation in the calorimeter. All radioactive samples were assayed by liquid scintillation spectroscopy in a scintillation fluid described earlier [9].
Preparation of acid extracts of sperm suspensions Portions (0.5 ml) of sperm suspensions were added to ice-cold trichloroacetic acid, at the time that sperm were mixed with exogenous substrate ( T = 0) and at the end of the incubations in the calorimeter ( T = 30), and frozen in liquid nitrogen. Cellular debris was removed by centrifugation and these samples were assayed for adenine nucleotide levels after extraction of the trichloroacetic acid as described in an earlier report [8].
204
Incubations of sperm with 14C.labeled buffer Incubations of sperm (6.0.108 cells) from six bulls with [5,6-14 C]endo-norbor-
nene-cis-5,6-dicarboxylic acid (specific radioactivity=7.5 /~Ci/mmol) or [7,8~4C]endo-norbornene-cis-5,6-dicarboxylic acid (specific radioactivity= 4.6 /~Ci/mmol) were conducted in a differential respirometer without and with 40 mM glucose in a total volume of 1.5 ml. After 2 h, 200 /~1 of 45% trichloroacetic acid in the respirometer flask sidearms was mixed with the sperm suspensions. K O H wicks from the center wells were removed 30 min later and the amount of 14CO 2 formed was determined by liquid scintillation spectroscopy. The trichloroacetic acid was extracted from the cell suspensions as described previously. Portions (200/~1) of these samples were assayed by ion-exchange chromatography.
Incubations of sperm with exogenous [14C]glucose All other incubations were carried out in the calorimeter at 30°C for 30 min after mixing the sperm suspensions with glucose-buffer solution. Initial D-[U-lac]glucose concentration (specific radioactivity = 0.15 ~tCi//~mol) after mixing was 1.46 mM for control incubations and 3.57 mM for antimycin A/rotenone-treated incubations. For the latter incubations, antimycin A in dimethylformamide (10/xl) and rotenone in ethanol (8/d) were added to make final concentrations of 4.9 and 9.0 ~tM, respectively.
Calculations Since over 95% of glucose carbon equivalents were recovered in the form of CO 2, acetate (and acetylcarnitine), and lactate, the metabolism of glucose was divided into three operational pathways corresponding to these specific metabolic end-products. Heat released during the conversion of glucose to these end-products was calculated using standard enthalpies of reaction (Table 1). Since two of the pathways involve the formation of CO 2, we have designated CO~ as that amount of CO 2 formed from the complete oxidation of glucose (Pathway 3). It is equal to the total amount of ~4CO2 produced less the amount of 14CO2 produced concomitant with the formation of acetate and acetylcarnitine (Pathway 2). The standard A H values in Table 1 do not reflect the heat of neutralization of the buffer system by protons formed by these pathways. A titration curve of the complex buffer system, relating heat released to equivalents of acid added, was established (Fig. 4). This relationship plus the equivalents of acid released during the course of the incubations, as established by metabolite production data, was used to calculate the heat associated with buffer neutralization. This value was subtracted from total measured heat production of the sperm.
Statistical analysis Statistical analysis was carried out using the ANOVA Procedure of the Statistical Analysis System (SAS Institute, Raleigh, N.C.). The model employed was Yig = u + animal i + inhibitorj + errorij.
205 TABLE 1 A T P YIELD A N D H E A T P R O D U C T I O N F O R D I F F E R E N T M E T A B O L I C P A T H W A Y S OPERAT I O N A L IN SPERM The first three pathways constitute the various ways exogenous glucose is metabolized by the sperm. The last pathway is the probable endogenous pathway [ 11-14]. Pathway
- mJ/bt mol a substrate
/.Lmol ATP/kt mol b substrate
Ratio mJ/ATP
Glucose ---, 2 lactate G l u c o s e + 6 02 --* 6 CO 2 + 6 H 2 0 G l u c o s e + 2 02 --, 2 acetate+ 2 CO 2 + 2 H 2 0 Lactate+ 3 02 ---, 3 CO 2 + 3 H 2 0 3-Hydroxybutyrate + 9 / 2 02 --* 4 CO 2 + 2 H 2 0 Palmitate+ 23 02 ---*16 CO 2 + 16 H 2 0
109 2820 1070 1 380 2000 10000
2 38 14 18 28 129
55 74 76 77 71 77
The values used in these calculations were A H ° at 25°C as determined for species in aqueous solution [16,17]. Due to the complexity of the thermodynamic system, and the uncertainty pertaining to actual cellular concentrations, etc., no attempt was made to calculate actual A H values for the metabolic pathways involved. Errors of less than 5% are expected because of this necessary simplifying assumption. b Values for A T P yields are based upon generally accepted metabolic pathways and stoichiometries for m a m m a l i a n cells [20]. "
Results Incubations to establish that the buffer system is metabolically inert In order to use our approach to determine the amount of endogenous metabolism
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Fig. 4 . Heat of protonation of the buffer system. Heat of protonation of the buffer was measured in the calorimeter. 1.0 ml portions of the buffer were placed in the large compartment of the calorimeter vessel and 0.5 ml portions of different concentrations of HCi were placed in the small compartment. The total a m o u n t of heat released upon mixing the contents of the two compartments was measured and a titration curve established. The biphasic nature of the resulting curve is a consequence of the presence of two buffering components.
206 TABLE 2 METABOLIC PARAMETERS OF BULL SPERM Parameter
Aerobic a
Anti A/Rote
Glucoseh Lactate Acetate Acetylcarnitine CO 2 Total heat c Exogenous heat Endogenous heat
-1.4 _+ 0.1 2.4 _+ 0.3 0.10+ 0.01 0.16 + 0.01 0.56+ 0.05 455 + 36 412 + 34 43 + 14
-2.2 + 0.1 3.9 + 0.2 0.01+ 0.01 0.07 + 0.01 0.11+ 0.01 269 + 18 263 + 17 <6
a Mean + S.E. for 18 incubations of sperm from 9 bulls. The means of each parameter for the aerobic incubations are significantly different (P < 0.05) from the corresponding means for the Anti A/Rote incubations. Sperm were prepared, incubated in the calorimeter, and the suspensions analyzed as described in the text. b /zmol/h/lO s cells. c mJ/b/10 8 cells.
occurring in s p e r m i n c u b a t e d with extracellular substrate, it was necessary to d e t e r m i n e that o n l y a single s u b s t r a t e ([lac]glucose), a n d no o t h e r c a r b o n source p r o v i d e d to the cells, was utilized. Since the only c a r b o n source other than glucose available to the s p e r m in the i n c u b a t i o n buffer was the endo-norbornene-cis-5,6-dic a r b o x y l i c acid, we synthesized N D C with ~4C i n c o r p o r a t e d in either the 5,6-ring c a r b o n s or the 7,8-carbonyl carbons. S p e r m i n c u b a t e d with [14C]NDC for 2 h w i t h o u t or with n o n - r a d i o a c t i v e glucose in the presence of l a b e l e d buffer c o n s u m e d oxygen, b u t p r o d u c e d no ~4CO 2 (limit of d e t e c t i o n was less than 0.5 n m o l e 1 4 C O 2 / h / 1 0 8 cells). In a d d i t i o n , no r a d i o a c t i v i t y (less than 0.5 n m o l e C / h / 1 0 8 cells) was recovered from acetate, acetylcarnitine, lactate or glucose fractions of s p e r m suspensions a p p l i e d to ion exchange columns, a n d 100% of r a d i o a c t i v i t y a p p l i e d to these c o l u m n s was recovered in the fraction k n o w n to c o n t a i n endonorbornene-cis-5,6-dicarboxylic acid. Thus, we c o n c l u d e that any m e t a b o l i s m of the b u f f e r was insignificant, a n d d i d not c o n t r i b u t e to heat p r o d u c t i o n d u r i n g the i n c u b a t i o n s in the calorimeter.
Metabolism of sperm when incubated with exogenous glucose Over 95% of the c a r b o n equivalents of utilized glucose was recovered in the form of lactate, acetate (including acetylcarnitine), a n d CO2 ( T a b l e 2). The heat p r o d u c e d b y the m e t a b o l i s m of exogenous substrate, c a l c u l a t e d as d e s c r i b e d above, a c c o u n t e d for 90% a n d > 98% of the total m e a s u r e d heat p r o d u c t i o n in i n c u b a t i o n s w i t h o u t a n d with a n t i m y c i n A a n d rotenone, respectively. T h e r e m a i n d e r of the total heat p r o d u c t i o n (10% a n d < 2%, respectively) can be a t t r i b u t e d to m e t a b o l i s m of e n d o g e n o u s reserves. T o t a l a d e n i n e nucleotide c o n t e n t of s p e r m (Table 3) was u n c h a n g e d d u r i n g the
207
TABLE 3 NUCLEOTIDE CONTENT AND ENERGY CHARGE OF SPERM T=0 a
ATP b ADP AMP SUM EC c
25 _+2 18 -+2 12 _+1 55 +3 0.62 _+0.03
T=20 Control
Anti A/Rote
41 +3 12 -+2 4 _+1 57 _+4 0.82 _+0.03
32 _+3 19 _-4-1 10 _+2 58 _+4 0.67 _+0.02
a Mean + S.E. for 18 incubations of sperm from 9 bulls. Means with asterisks for the control values are significantly different (P < 0.05) from the corresponding means for the T = 0 and Anti A/Rote values. Sperm were prepared, incubated in the calorimeter, inactivated with trichloroacetic acid, and analyzed as described in the text. b nmol/lO s cells. c Energy charge (EC) = (ATP + ½ ADP)/sum.
0.5 h incubation. However, sperm incubated in the absence of respiratory inhibitors were able to increase their energy charge (i.e. convert A M P and A D P to ATP), while sperm with electron-transport inhibitors were capable only of maintaining initial energy charge. This increase of ATP, yielding a higher steady-state energy charge, occurred within the first 5 rain of incubation (unpublished observations). This change represents a difference between the initial and final thermodynamic states of the system and is representative of other possible changes in cellular metabolite concentrations. The heat produced by these processes is not encompassed in the calculations, and is a potential source of error in this method. However, when an estimation of the a m o u n t of heat actually evolved by the process was made using the appropriate heats of reaction for A T P synthesis [10], the total a m o u n t of heat p r o d u c e d during the redistribution of adenine nucleotide s during the incubations was approximately + 0 . 5 3 m J / 1 0 s cells. This value is negligible c o m p a r e d to - 2 6 9 m J / 1 0 8 cells total heat produced by the sperm. Therefore, we concluded that formation of nmoles of intracellular metabolites can be ignored in cells that are transforming/~moles of substrates during the incubation period.
Discussion To introduce this method of studying endogenous metabolism a simple model system, m a m m a l i a n sperm, was used. Sperm provide an ideal model system since biosynthesis, growth, and most branch pathways of intermediary metabolism (such as the pentose phosphate pathway) do not occur to any significant extent in sperm [2], and the metabolism of exogenous substrates can be defined precisely using standard isotopic tracer methods. Indeed, over 95% of glucose carbon equivalents
208
utilized by sperm was recovered in our experiments as CO 2, acetate, acetylcarnitine and lactate. Calorimetry has long been accepted as a useful method for assessing overall metabolism because heat production is a byproduct of all chemical reactions. Since the amount of heat released by a specific metabolic reaction can be precisely related to the stoichiometry of the reaction (Table 1), it is possible to calculate the amount of heat produced by sperm due to the metabolism of extracellular glucose (Table 2). The total amount of heat produced by the sperm equals the heat produced by the metabolism of glucose and the heat produced by metabolism of endogenous substrates. Thus, the difference between total heat production, as measured by calorimetry, and the calculated heat production, due to the metabolism of glucose, is equal to the heat produced by the metabolism of endogenous substrates. Using this approach, we estimated that about 10% of heat produced by sperm is contributed by metabolism of endogenous substrates. In addition, endogenous metabolism was absent for sperm incubated with the electron transport chain inhibitors, antimycin A and rotenone. This observation is consistent with data [11-14] indicating that phospholipids serve as the endogenous substrate for sperm. Utilization of phospholipids as an energy source involves the fl-oxidation pathway and is dependent on a functional electron transport chain. Since ATP synthesis is a central process in the metabolism (and heat production), it is useful to convert the heats associated with metabolism to ATP production via
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AEROBIC
ANTI A/ROTE
Fig. 5. ATP synthesis of sperm via different metabolic pathways. The amount of ATP synthesized via endogenous metabolism and the metabolism of glucose was calculated as described in the text. Each bar represents the rate of ATP synthesis of sperm during the incubations and is subdivided to show the proportion of ATP furnished by different individual pathways: open area, ATP from complete oxidation of glucose to CO2; striped area, ATP from oxidation of glucose to acetate + CO2; shaded area, ATP from glucose conversion to lactate; and cross-hatched area, ATP from endogenous pathways. Endogenous metabolism in antimycin A plus rotenone-treated cells contributed less than 2% of total ATP production and is not shown in this figure.
209 the different metabolic pathways (Table 1). A T P production from the metabolism of glucose is readily calculated using well established stoichiometries for the pathways involved [21]. In order to estimate ATP production from the metabolism of endogenous reserves, however, where the precise metabolic pathways are not known with certainty, we have used an empirical relationship between heat production and ATP production for respiration-linked processes. The ratio of mJ heat produced per #mol A T P produced for m a n y common substrates degraded by respiration-linked pathways is 75 (Table 1). Since endogenous metabolism was eliminated by electron transport inhibitors and thus appears to depend on respiration, we have used this relationship to estimate that the amount of A T P produced by endogenous metabolism is 0.7 # m o l / h / 1 0 8 cells (Fig. 5). Thus, metabolism of endogenous reserves in sperm when glucose is present accounts for approximately 10% of total ATP production. Application of these methods to more complex cell types where a significant amount of the ~4C substrate is incorporated into cellular components is possible. Standard cell fractionation techniques [15] can be used to establish an accurate carbon balance between exogenous substrate and both low molecular weight endproducts and cellular macromolecules. Precise zaH ° values for low molecular weight compounds are available [ 16,17] while mean values for protein, lipid, nucleic acids or glycogen can be calculated [18-20]. Values for heat produced via the synthesis of these high molecular weight compounds from substrates provided to the cells can then be calculated. This should provide estimations of an order-of-magnitude precision. In summary, these experiments have demonstrated that the relative importance of endogenous metabolism in the presence of extracellular substrates can be precisely determined for a simple mammalian cell system. Further studies detailing the effect of overall metabolic rate and nature of exogenous substrate on the relative importance of endogenous metabolism are in progress.
Simplified description of the method and its advantages Heat production of sperm is readily measured using a heat-conduction, batch-type calorimeter. Since sperm metabolismis simple and only a few metabolic end-products are formed when sperm are incubated with radioactiveglucose, heat production due to metabolismof the glucose is readily calculated using well established thermodynamic constants. The difference between the heat production calculated for glucose utilization and the heat production measured by calorimetry is the heat production due to metabolism of endogenous substrates. Thus, calorimetry affords an easy method of assessing the relative importance of endogenous metabolism to cell bioenergetics, even though the identity of the endogenous substrates is not known with certainty.
Acknowledgements Susan Magargee provided technical assistance. Ejaculated bovine sperm was generously provided by the Dairy Breeding Research Center at the Pennsylvania State University.
210
References 1 Whipple, H., ed. (1963) Ann. N.Y. Acad. Sci. 102. 2 Hammerstedt, R.H. (1981) in Reproductive Processes and Contraception (McKerns, K.W., ed.), pp. 305-318. Plenum Press, New York. 3 Fieser, L.F. and Williamson, K.L. (1975) Organic Experiments, 3rd ed., pp. 118-122. D.C. Heath and Co., Lexington, Mass. 4 Hammerstedt, R.H. (1975) Biol. Reprod. 13, 389-396. 5 Mallette, M.F. (1967) J. Bacteriol. 94, 283-290. 6 Lovrien, R., Jorgenson, G., Ma, M.K. and Sund, W.E. (1980) Biotech. Bioeng. 22, 1249-1269. 7 Bauer, C.R. and Gemmill, C.L. (1952) Arch. Bioc. Biop. 35, 110-116. 8 Inskeep, P.B. and Hammerstedt, R.H. (1982) Biol. Reprod. 27, 735-743. 9 Hiipakka, R.A. and Hammerstedt, R.H. (1978) Biol. Reprod. 19, 368-379. 10 Alberty, R.A. (1969) J. Biol. Chem. 244, 3290-3302. 11 Lardy, H.A. and Phillips, P.H. (1941) Am. J. Physiol. 133, 602-609. 12 Lardy, H.A. and Phillips, P.H. (1941) Am. J. Physiol. 134, 542-548. 13 Hartree, E.F. and Mann, T. (1959) Biochem. J. 71,423-434. 14 Hartree, E.F. and Mann, T. (1961) Biochem. J. 80, 464-476. 15 Munro, H.N. and Fleck, A. (1966) in Methods of Biochemical Analysis, Vol. 1 (David Glick, ed.), pp. 287-297. Interscience Publishers, Inc., New York. 16 Kharasch, M.S. (1929) Bur. Stds. J. Res. 2, 359-430. 17 Wilhoit, R.C. (1969) in Biochemical Microcalorimetry (Brown, H.D., ed.), pp. 305-318. Academic Press, New York. 18 Erickson, L.E. (1978) Biotech. Bioeng. 20, 1623-1638. 19 Erickson, L.E., Minkevich, I.G. and Eroshin, V.K. (1978) Biotech. Bioeng. 20, 1595-1621. 20 Crabtree, B. and Taylor, D.J. (1979) in Biochemical Thermodynamics (Jones, M.N., ed.), pp. 333-378. Elsevier Scientific Pub., Amsterdam. 21 Dagley, S. and Nicholson, D.E. (1970) An Introduction to Metabolic Pathways, Wiley and Sons, New York.
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Elsevier
A calorimetric method to assess endogenous metabolism and its application to the study of bovine sperm * Philip B. Inskeep and Roy H. Hammerstedt Biochemistry Program, 406 Althouse Laboratory, Pennsylvania State University, Universi(v Park, PA 16802, U.S.A. (Received 20 November 1982) (Accepted 22 November 1982)
Summa~ Calorimetry was used to assess the importance of endogenous metabolism towards total ATP synthesis in bovine sperm in the presence of extracellular glucose. Sperm were incubated in the calorimeter with D-[U-14C]glucose without or with electron transport inhibitors, rotenone and antimycin A. Steady-state heat production during the incubations was measured for 30 rain, the incubations were terminated, and the cell suspensions removed for analysis of radioactive glucose and its metabolic end-products. Heat production (mean_+ S.E.) associated with the metabolism of glucose was calculated, from enthalpies of formation of glucose and its end-products, as -412_+ 34 m J/h/108 cells in control incubations and -263_+ 18 m J / h / 1 0 s cells in incubations with electron transport inhibitors. Measured heat production was - 4 5 5 + 3 6 and -263_+ 17 mJ / h / 1 0 s cells, respectively. Thus, heat production by endogenous pathways, the difference between measured total heat production and calculated exogenous heat production, was -43_+ 14 m J / h / 1 0 s cells for control cells and about - 6 m J / h / 1 0 s cells for inhibited cells. The ratio of heat produced per mol of ATP synthesized is similar for all ATP-producing pathways. Therefore, about 10% of total ATP synthesis in control cells and less than 2% in inhibited cells is provided by endogenous pathways when extracellular glucose is present. Key words: calorimetry; endogenous metabolism; sperm; ATP synthesis: glucose; bovine sperm.
Introduction The utilization of endogenous reserves, stored compounds such as glycogen and lipid which can be readily mobilized to satisfy cellular energy requirements via * Approved as Paper No. 6536 by the Pennsylvania Agricultural Experiment Station. Portions of this work were taken from a dissertation presented by P.B.I. in partial fulfillment for the requirements for the Ph.D. in the Biochemistry Program, The Pennsylvania State University. Preliminary results were presented at the Division of the Biological Chemistry Meeting of the American Chemical Society, August-September 1981. Abbreviations: Anti A, antimycin A; NDC, endo-norbornene-cis-5,6-dicarboxylic acid; Rote, rotenone. 0165-022X/83/$03.00 © 1983 Elsevier Science Publishers B.V.
200 ATP-yielding pathways, is a process which provides an advantage for survival of cells when extracellular substrates are depleted. However, the importance to the cell of such reserves when extracellular substrates are abundant is unclear. A conference addressing this problem was held 20 years ago [1] and, although some quantitative assessments for endogenous metabolism of microorganisms were attempted, the lack of appropriate model systems and methodologies limited the conclusions that could be drawn. Herein we describe experiments where bovine sperm, a mammalian cell system possessing simple metabolic properties [2], and a unique combination of calorimetry, carbon balance, and calculations from thermodynamic constants, have been used to quantitatively assess the importance of endogenous metabolism in bovine sperm when extracellular substrates are present. In addition, the effect of respiratory inhibitors on endogenous metabolism has been established.
Instrumentation and Methods
Materials D-[U)4C]Glucose, [1,4-14C]maleic anhydride, and [2,3-14C]maleic anhydride were purchased from Amersham, Inc. (Arlington Heights, Ill.). All biochemicals, enzymes, and ion-exchange resins were purchased from Sigma (St. Louis, Mo.). [5,614C]endo-norbornene-cis-5,6-dicarboxylic acid and [7,8-14C]endo-norbornene-c/s5,6-dicarboxylic acid were synthesized [3], recrystallized and assayed for purity by thin-layer chromatography in two solvent systems (toluene/ethyl formate/formic acid, 55 : 44 : 11, and benzene/methanol/acetic acid, 90 : 16 : 8). Greater than 99% of radioactivity co-chromatographed with authentic NDC. The buffer used for sperm preparation and in all sperm incubations contained 30 mM endo-norbornene-5,6-c/s-dicarboxylic acid, 2 mM MgC1 z, 4 mM KC1, 2 mM sodium phosphate, 107 mM NaCI, 1 mM NH4C1, 0.003% (w/v) streptomycin sulfate, and 0.005% (w/v) penicillin G at pH 7.2 as described by Hammerstedt [4]. The major buffer component, NDC, was chosen because it had been established that it is not metabolized by bacteria [5]; this conclusion was extended to sperm cells (see below).
Calorimetry The calorimeter (Microcalorimeters, Inc., St. Paul, Minn.; see Lovrien et al. [6] for a description of a similar instrument) is a differential heat conduction instrument with five glass vessels, one of which serves as a reference for the other four. Each vessel (Fig. 1) is divided into two separate compartments with a total capacity of 3.0 ml. The vessel faces have finely ground surfaces, which comprise about 60% of the total vessel surface area, and are covered by Seebeck sensors. Heat produced within the vessels passes through the sensors to large aluminum blocks on both sides of each vessel. The vessels, sensors, and heat sinks are all mounted on a drum which can be rotated 360°; thus, contents initially kept in the separated compartments of
201
~~.~--~ SINKl ~ ¢ ~
KEEBLOCK
Fig. I. Schematic representation of the calorimeter used in these experiments. Only one incubation vessel is shown. Five such vessels are mounted in the calorimeter keel block which is contained in an aluminum drum (14 cm diameter × 28 cm length). The drum is mounted inside two insulating chambers on a shaft which can be rotated by means of a crank on the outside of the outer insulating chamber. When the drum is so rotated contents of the two chambers in each vessel are mixed.
the calorimeter vessels can be mixed together by rotating the drum. The calorimeter was housed in a constant-temperature box and isothermal conditions within the calorimeter were maintained to 30 + 0.05°C with Peltier pumps. The calorimeter is capable of detecting a heat flow of less than 88 m J / m i n with a minimum signal to noise ratio of 20. Electrical calibration with 511 ohm resistors produced a non-linear curve in the range of 44-1750 m J / h (Fig. 2). An alternate method of calibration utilizing invertase-catalyzed hydrolysis of sucrose ( A H = - 1 4 . 0 m J / # m o l sucrose hydrolyzed, [7]) resulted in an identical calibration curve. Daily calibrations verified that the calibration curve was consistently reproducible. The following protocol was used for all incubations in the calorimeter: (1) The large compartment of each calorimeter vessel was loaded with 1.0 ml of oxygen-saturated buffer containing isotopically labeled glucose without or with electron transport inhibitors, flushed with H20-saturated oxygen, and sealed. With this procedure, gas-liquid equilibration within the vessels and thermal re-equilibration of the calorimeter were achieved within one hour. (2) After thermal equilibration of the calorimeter was established, sperm (0.5 ml) were loaded into the small compartments of the calorimeter vessels, and the vessels were resealed. Re-equilibration of the calorimeter was complete within 20 min after reseating the vessels. (Separate experiments (data not presented) established that sufficient oxygen was present to maintain sperm respiration beyond the termination point of the incubation and that oxygen consumption by the sperm was linear throughout the incubation.) (3) After re-equilibration was achieved, the contents of both compartments in
202
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Fig. 2. Electrical calibration curve for the calorimeter. Electric heaters (511 12 resistors mounted in calorimeter vessel stoppers) were placed in each sample vessel and current applied to each heater by a d.c. power supply was measured, The deflection of the chart recorder pen, after steady-state heat production was observed (about 5 min), was recorded for each heater at each heat output; Heat Production (m J / h ) = 3.59 m J / ( h x m A 2 x ohm)×Current (mA)2 × Resistance of heater (12). Data presented represent the composite of all calibrations performed during the 4-week period of the experiments.
each vessel were mixed by rotating the calorimeter drum. This started the incubation (Fig. 3).
Sperm preparation Portions of 2 ejaculates collected from the same bull within 15 min of each other i
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Fig. 3. Heat production of sperm as a function of time in the absence ( - ) and presence ( I ) of electron-transport chain inhibitors. Sperm were placed in the calorimeter vessels 25 rain prior to mixing (not shown). Thermal equilibration was achieved within 20 rain ( T = - 5 min), and the measured rate between this time and the moment of mixing ( T = 0) sperm with D-[U-~4C]glucose represents heat production of sperm in the absence of any exogenous substrate. Upon mixing, heat production rapidly increases to a new steady-state level which is maintained for the duration of the incubation. In this representative experiment, two of the four reaction tracings have been deleted for clarity.
203 were pooled, washed twice in buffer, and diluted to a known concentration, as assessed turbidimetrically [4], of about 3.5 • 108 cells/ml. This treatment resulted in a dilution of seminal plasma components of about 1000-fold [4]. Portions (0.5 ml) of these suspensions were transferred to four of the calorimeter vessels after a microscopic assessment of motility. All ejaculates used had greater than 50% motile sperm. This same sperm preparation scheme was used in experiments with buffer containing ~4C-labeled N D C except that sperm were diluted to a measured concentration near 12 • 108 cells/ml and were incubated in a respirometer. All incubations were started within 1.5 h after collection of ejaculates.
Metabolite assays Lactate, acetate and acetylcarnitine contents after 30 min of incubation were assayed in 150/~1 aliquots of the cell suspensions by ion-exchange chromatography [8]. Briefly, two sets of minicolumns were arranged in tandem so that the effluent from the first set (Dowex-50-H +) passed directly to the second set (Dowex-l-formate). Samples were applied to the top of the first column and washed with 10 ml H 2 0 . During this wash, cations (acetylcarnitine) were adsorbed to the Dowex-50-H + resin, anions (acetate, lactate and NDC) were adsorbed to the Dowex-l-formate resin, and uncharged compounds (glucose) passed through both columns and were collected in glass scintillation vials. The column sets were separated and each set was washed to elute adsorbed compounds. Acetylcarnitine was eluted from Dowex-50-H ÷ columns with 10 ml of 1 N N H 4 O H . Acetate and lactate were eluted from the Dowex-l-formate resins with 30 m M formic acid and 300 mM formic acid, respectively. In experiments with [t4C]NDC, this compound was eluted from the Dowex1-formate resin with 3 M formic acid. ~4CO 2 formation was measured in 0.5 ml portions of the sperm suspensions recovered after 30 rain of incubation in the calorimeter. Samples were transferred to glass respirometer flasks, previously loaded with 6 N K O H in the center wells and 6 N H2SO 4 in the side arms, were sealed and the acid mixed with the sperm suspensions to release ~4CO2. Separate experiments (data not presented) with sperm suspensions established that this procedure recovered 87 + 6% (S.E., n = 13) of total ~4CO2 formed by the sperm during the incubation. This factor has been used to establish the actual amount of 14CO2 produced by the sperm during the incubation in the calorimeter. All radioactive samples were assayed by liquid scintillation spectroscopy in a scintillation fluid described earlier [9].
Preparation of acid extracts of sperm suspensions Portions (0.5 ml) of sperm suspensions were added to ice-cold trichloroacetic acid, at the time that sperm were mixed with exogenous substrate ( T = 0) and at the end of the incubations in the calorimeter ( T = 30), and frozen in liquid nitrogen. Cellular debris was removed by centrifugation and these samples were assayed for adenine nucleotide levels after extraction of the trichloroacetic acid as described in an earlier report [8].
204
Incubations of sperm with 14C.labeled buffer Incubations of sperm (6.0.108 cells) from six bulls with [5,6-14 C]endo-norbor-
nene-cis-5,6-dicarboxylic acid (specific radioactivity=7.5 /~Ci/mmol) or [7,8~4C]endo-norbornene-cis-5,6-dicarboxylic acid (specific radioactivity= 4.6 /~Ci/mmol) were conducted in a differential respirometer without and with 40 mM glucose in a total volume of 1.5 ml. After 2 h, 200 /~1 of 45% trichloroacetic acid in the respirometer flask sidearms was mixed with the sperm suspensions. K O H wicks from the center wells were removed 30 min later and the amount of 14CO 2 formed was determined by liquid scintillation spectroscopy. The trichloroacetic acid was extracted from the cell suspensions as described previously. Portions (200/~1) of these samples were assayed by ion-exchange chromatography.
Incubations of sperm with exogenous [14C]glucose All other incubations were carried out in the calorimeter at 30°C for 30 min after mixing the sperm suspensions with glucose-buffer solution. Initial D-[U-lac]glucose concentration (specific radioactivity = 0.15 ~tCi//~mol) after mixing was 1.46 mM for control incubations and 3.57 mM for antimycin A/rotenone-treated incubations. For the latter incubations, antimycin A in dimethylformamide (10/xl) and rotenone in ethanol (8/d) were added to make final concentrations of 4.9 and 9.0 ~tM, respectively.
Calculations Since over 95% of glucose carbon equivalents were recovered in the form of CO 2, acetate (and acetylcarnitine), and lactate, the metabolism of glucose was divided into three operational pathways corresponding to these specific metabolic end-products. Heat released during the conversion of glucose to these end-products was calculated using standard enthalpies of reaction (Table 1). Since two of the pathways involve the formation of CO 2, we have designated CO~ as that amount of CO 2 formed from the complete oxidation of glucose (Pathway 3). It is equal to the total amount of ~4CO2 produced less the amount of 14CO2 produced concomitant with the formation of acetate and acetylcarnitine (Pathway 2). The standard A H values in Table 1 do not reflect the heat of neutralization of the buffer system by protons formed by these pathways. A titration curve of the complex buffer system, relating heat released to equivalents of acid added, was established (Fig. 4). This relationship plus the equivalents of acid released during the course of the incubations, as established by metabolite production data, was used to calculate the heat associated with buffer neutralization. This value was subtracted from total measured heat production of the sperm.
Statistical analysis Statistical analysis was carried out using the ANOVA Procedure of the Statistical Analysis System (SAS Institute, Raleigh, N.C.). The model employed was Yig = u + animal i + inhibitorj + errorij.
205 TABLE 1 A T P YIELD A N D H E A T P R O D U C T I O N F O R D I F F E R E N T M E T A B O L I C P A T H W A Y S OPERAT I O N A L IN SPERM The first three pathways constitute the various ways exogenous glucose is metabolized by the sperm. The last pathway is the probable endogenous pathway [ 11-14]. Pathway
- mJ/bt mol a substrate
/.Lmol ATP/kt mol b substrate
Ratio mJ/ATP
Glucose ---, 2 lactate G l u c o s e + 6 02 --* 6 CO 2 + 6 H 2 0 G l u c o s e + 2 02 --, 2 acetate+ 2 CO 2 + 2 H 2 0 Lactate+ 3 02 ---, 3 CO 2 + 3 H 2 0 3-Hydroxybutyrate + 9 / 2 02 --* 4 CO 2 + 2 H 2 0 Palmitate+ 23 02 ---*16 CO 2 + 16 H 2 0
109 2820 1070 1 380 2000 10000
2 38 14 18 28 129
55 74 76 77 71 77
The values used in these calculations were A H ° at 25°C as determined for species in aqueous solution [16,17]. Due to the complexity of the thermodynamic system, and the uncertainty pertaining to actual cellular concentrations, etc., no attempt was made to calculate actual A H values for the metabolic pathways involved. Errors of less than 5% are expected because of this necessary simplifying assumption. b Values for A T P yields are based upon generally accepted metabolic pathways and stoichiometries for m a m m a l i a n cells [20]. "
Results Incubations to establish that the buffer system is metabolically inert In order to use our approach to determine the amount of endogenous metabolism
;51- I
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1.0 H+
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Fig. 4 . Heat of protonation of the buffer system. Heat of protonation of the buffer was measured in the calorimeter. 1.0 ml portions of the buffer were placed in the large compartment of the calorimeter vessel and 0.5 ml portions of different concentrations of HCi were placed in the small compartment. The total a m o u n t of heat released upon mixing the contents of the two compartments was measured and a titration curve established. The biphasic nature of the resulting curve is a consequence of the presence of two buffering components.
206 TABLE 2 METABOLIC PARAMETERS OF BULL SPERM Parameter
Aerobic a
Anti A/Rote
Glucoseh Lactate Acetate Acetylcarnitine CO 2 Total heat c Exogenous heat Endogenous heat
-1.4 _+ 0.1 2.4 _+ 0.3 0.10+ 0.01 0.16 + 0.01 0.56+ 0.05 455 + 36 412 + 34 43 + 14
-2.2 + 0.1 3.9 + 0.2 0.01+ 0.01 0.07 + 0.01 0.11+ 0.01 269 + 18 263 + 17 <6
a Mean + S.E. for 18 incubations of sperm from 9 bulls. The means of each parameter for the aerobic incubations are significantly different (P < 0.05) from the corresponding means for the Anti A/Rote incubations. Sperm were prepared, incubated in the calorimeter, and the suspensions analyzed as described in the text. b /zmol/h/lO s cells. c mJ/b/10 8 cells.
occurring in s p e r m i n c u b a t e d with extracellular substrate, it was necessary to d e t e r m i n e that o n l y a single s u b s t r a t e ([lac]glucose), a n d no o t h e r c a r b o n source p r o v i d e d to the cells, was utilized. Since the only c a r b o n source other than glucose available to the s p e r m in the i n c u b a t i o n buffer was the endo-norbornene-cis-5,6-dic a r b o x y l i c acid, we synthesized N D C with ~4C i n c o r p o r a t e d in either the 5,6-ring c a r b o n s or the 7,8-carbonyl carbons. S p e r m i n c u b a t e d with [14C]NDC for 2 h w i t h o u t or with n o n - r a d i o a c t i v e glucose in the presence of l a b e l e d buffer c o n s u m e d oxygen, b u t p r o d u c e d no ~4CO 2 (limit of d e t e c t i o n was less than 0.5 n m o l e 1 4 C O 2 / h / 1 0 8 cells). In a d d i t i o n , no r a d i o a c t i v i t y (less than 0.5 n m o l e C / h / 1 0 8 cells) was recovered from acetate, acetylcarnitine, lactate or glucose fractions of s p e r m suspensions a p p l i e d to ion exchange columns, a n d 100% of r a d i o a c t i v i t y a p p l i e d to these c o l u m n s was recovered in the fraction k n o w n to c o n t a i n endonorbornene-cis-5,6-dicarboxylic acid. Thus, we c o n c l u d e that any m e t a b o l i s m of the b u f f e r was insignificant, a n d d i d not c o n t r i b u t e to heat p r o d u c t i o n d u r i n g the i n c u b a t i o n s in the calorimeter.
Metabolism of sperm when incubated with exogenous glucose Over 95% of the c a r b o n equivalents of utilized glucose was recovered in the form of lactate, acetate (including acetylcarnitine), a n d CO2 ( T a b l e 2). The heat p r o d u c e d b y the m e t a b o l i s m of exogenous substrate, c a l c u l a t e d as d e s c r i b e d above, a c c o u n t e d for 90% a n d > 98% of the total m e a s u r e d heat p r o d u c t i o n in i n c u b a t i o n s w i t h o u t a n d with a n t i m y c i n A a n d rotenone, respectively. T h e r e m a i n d e r of the total heat p r o d u c t i o n (10% a n d < 2%, respectively) can be a t t r i b u t e d to m e t a b o l i s m of e n d o g e n o u s reserves. T o t a l a d e n i n e nucleotide c o n t e n t of s p e r m (Table 3) was u n c h a n g e d d u r i n g the
207
TABLE 3 NUCLEOTIDE CONTENT AND ENERGY CHARGE OF SPERM T=0 a
ATP b ADP AMP SUM EC c
25 _+2 18 -+2 12 _+1 55 +3 0.62 _+0.03
T=20 Control
Anti A/Rote
41 +3 12 -+2 4 _+1 57 _+4 0.82 _+0.03
32 _+3 19 _-4-1 10 _+2 58 _+4 0.67 _+0.02
a Mean + S.E. for 18 incubations of sperm from 9 bulls. Means with asterisks for the control values are significantly different (P < 0.05) from the corresponding means for the T = 0 and Anti A/Rote values. Sperm were prepared, incubated in the calorimeter, inactivated with trichloroacetic acid, and analyzed as described in the text. b nmol/lO s cells. c Energy charge (EC) = (ATP + ½ ADP)/sum.
0.5 h incubation. However, sperm incubated in the absence of respiratory inhibitors were able to increase their energy charge (i.e. convert A M P and A D P to ATP), while sperm with electron-transport inhibitors were capable only of maintaining initial energy charge. This increase of ATP, yielding a higher steady-state energy charge, occurred within the first 5 rain of incubation (unpublished observations). This change represents a difference between the initial and final thermodynamic states of the system and is representative of other possible changes in cellular metabolite concentrations. The heat produced by these processes is not encompassed in the calculations, and is a potential source of error in this method. However, when an estimation of the a m o u n t of heat actually evolved by the process was made using the appropriate heats of reaction for A T P synthesis [10], the total a m o u n t of heat p r o d u c e d during the redistribution of adenine nucleotide s during the incubations was approximately + 0 . 5 3 m J / 1 0 s cells. This value is negligible c o m p a r e d to - 2 6 9 m J / 1 0 8 cells total heat produced by the sperm. Therefore, we concluded that formation of nmoles of intracellular metabolites can be ignored in cells that are transforming/~moles of substrates during the incubation period.
Discussion To introduce this method of studying endogenous metabolism a simple model system, m a m m a l i a n sperm, was used. Sperm provide an ideal model system since biosynthesis, growth, and most branch pathways of intermediary metabolism (such as the pentose phosphate pathway) do not occur to any significant extent in sperm [2], and the metabolism of exogenous substrates can be defined precisely using standard isotopic tracer methods. Indeed, over 95% of glucose carbon equivalents
208
utilized by sperm was recovered in our experiments as CO 2, acetate, acetylcarnitine and lactate. Calorimetry has long been accepted as a useful method for assessing overall metabolism because heat production is a byproduct of all chemical reactions. Since the amount of heat released by a specific metabolic reaction can be precisely related to the stoichiometry of the reaction (Table 1), it is possible to calculate the amount of heat produced by sperm due to the metabolism of extracellular glucose (Table 2). The total amount of heat produced by the sperm equals the heat produced by the metabolism of glucose and the heat produced by metabolism of endogenous substrates. Thus, the difference between total heat production, as measured by calorimetry, and the calculated heat production, due to the metabolism of glucose, is equal to the heat produced by the metabolism of endogenous substrates. Using this approach, we estimated that about 10% of heat produced by sperm is contributed by metabolism of endogenous substrates. In addition, endogenous metabolism was absent for sperm incubated with the electron transport chain inhibitors, antimycin A and rotenone. This observation is consistent with data [11-14] indicating that phospholipids serve as the endogenous substrate for sperm. Utilization of phospholipids as an energy source involves the fl-oxidation pathway and is dependent on a functional electron transport chain. Since ATP synthesis is a central process in the metabolism (and heat production), it is useful to convert the heats associated with metabolism to ATP production via
..J la.I o
"_o e, iu3 taJ o
:L
AEROBIC
ANTI A/ROTE
Fig. 5. ATP synthesis of sperm via different metabolic pathways. The amount of ATP synthesized via endogenous metabolism and the metabolism of glucose was calculated as described in the text. Each bar represents the rate of ATP synthesis of sperm during the incubations and is subdivided to show the proportion of ATP furnished by different individual pathways: open area, ATP from complete oxidation of glucose to CO2; striped area, ATP from oxidation of glucose to acetate + CO2; shaded area, ATP from glucose conversion to lactate; and cross-hatched area, ATP from endogenous pathways. Endogenous metabolism in antimycin A plus rotenone-treated cells contributed less than 2% of total ATP production and is not shown in this figure.
209 the different metabolic pathways (Table 1). A T P production from the metabolism of glucose is readily calculated using well established stoichiometries for the pathways involved [21]. In order to estimate ATP production from the metabolism of endogenous reserves, however, where the precise metabolic pathways are not known with certainty, we have used an empirical relationship between heat production and ATP production for respiration-linked processes. The ratio of mJ heat produced per #mol A T P produced for m a n y common substrates degraded by respiration-linked pathways is 75 (Table 1). Since endogenous metabolism was eliminated by electron transport inhibitors and thus appears to depend on respiration, we have used this relationship to estimate that the amount of A T P produced by endogenous metabolism is 0.7 # m o l / h / 1 0 8 cells (Fig. 5). Thus, metabolism of endogenous reserves in sperm when glucose is present accounts for approximately 10% of total ATP production. Application of these methods to more complex cell types where a significant amount of the ~4C substrate is incorporated into cellular components is possible. Standard cell fractionation techniques [15] can be used to establish an accurate carbon balance between exogenous substrate and both low molecular weight endproducts and cellular macromolecules. Precise zaH ° values for low molecular weight compounds are available [ 16,17] while mean values for protein, lipid, nucleic acids or glycogen can be calculated [18-20]. Values for heat produced via the synthesis of these high molecular weight compounds from substrates provided to the cells can then be calculated. This should provide estimations of an order-of-magnitude precision. In summary, these experiments have demonstrated that the relative importance of endogenous metabolism in the presence of extracellular substrates can be precisely determined for a simple mammalian cell system. Further studies detailing the effect of overall metabolic rate and nature of exogenous substrate on the relative importance of endogenous metabolism are in progress.
Simplified description of the method and its advantages Heat production of sperm is readily measured using a heat-conduction, batch-type calorimeter. Since sperm metabolismis simple and only a few metabolic end-products are formed when sperm are incubated with radioactiveglucose, heat production due to metabolismof the glucose is readily calculated using well established thermodynamic constants. The difference between the heat production calculated for glucose utilization and the heat production measured by calorimetry is the heat production due to metabolism of endogenous substrates. Thus, calorimetry affords an easy method of assessing the relative importance of endogenous metabolism to cell bioenergetics, even though the identity of the endogenous substrates is not known with certainty.
Acknowledgements Susan Magargee provided technical assistance. Ejaculated bovine sperm was generously provided by the Dairy Breeding Research Center at the Pennsylvania State University.
210
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