BIOLOGY OF REPRODUCTION (2012) 87(1):18, 1–6 Published online before print 2 May 2012. DOI 10.1095/biolreprod.112.100776
Plasma Clearance and Half-Life of Prostaglandin F2alpha: A Comparison Between Mares and Heifers1 Hemanta K. Shrestha,3,4 Mohd A. Beg,5 Ronald R. Burnette,6 and O.J. Ginther2,4,5 4
Eutheria Foundation, Cross Plains, Wisconsin Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin–Madison, Madison, Wisconsin 6 Pharmaceutical Sciences Division, School of Pharmacy, University of Wisconsin–Madison, Madison, Wisconsin 5
Horses are about five times more sensitive to the luteolytic effect of prostaglandin F2alpha (PGF) than cattle, as indicated by a recommended clinical dose of 5 mg in horses and 25 mg in cattle. Novel evaluations of the PGF plasma disappearance curves were made in mares and in heifers, and the two species were compared. Mares and heifers (n ¼ 5) of similar body weight were injected (Min 0) intravenously with PGF (5 mg per animal). Blood was sampled every 10 sec until Min 3, every 30 sec until Min 5, every 10 min until Min 60, and every 30 min until Min 240. The mean PGF concentration was greater (P , 0.05) in mares than in heifers at Min 1 through Min 60 and at Mins 180 and 240. The mean time to maximum PGF concentration was not different between mares (42.0 6 8.6 sec) and heifers (35.0 6 2.9 sec). The apparent plasma clearance, distribution halflife, elimination half-life, and maximum plasma PGF concentration were 3.3 6 0.5 L h1 kg1, 94.2 6 15.9 sec, 25.9 6 5.0 min, and 249.1 6 36.8 ng/ml, respectively, in mares and 15.4 6 2.3 L h1 kg1, 29.2 6 3.9 sec, 9.0 6 0.9 min, and 51.4 6 22.6 ng/ml, respectively, in heifers. Plasma clearance was about five times less (P , 0.0005), maximum plasma PGF concentration was five times greater (P , 0.002), and the distribution half-life and elimination half-life were about three times longer (P , 0.005) in mares than in heifers. The fivefold greater plasma clearance of PGF in heifers than in mares corresponds to the recommended fivefold greater clinical dose of PGF in cattle and supported the hypothesis that the metabolic clearance of PGF is slower in mares than heifers. half-life, heifers, mares, PGF2a, plasma clearance
INTRODUCTION The prostaglandins (PGs), thromboxanes, and prostacyclins are collectively termed prostanoids and are 20-carbon molecules derived from arachidonic acid [1, 2]. The PGs are ubiquitously produced in the body and are potent mediators for many physiological and pathological processes (e.g., smooth 1
Supported by the Eutheria Foundation, Cross Plains, WI (Projects P1HS-08 and B2-HS-08). 2 Correspondence: O.J. Ginther, Department of Pathobiological Sciences, School of Veterinary Medicine, 1656 Linden Dr., University of Wisconsin–Madison, Madison, WI 53706. E-mail:
[email protected] 3 Current address: Wisconsin National Primate Research Center, University of Wisconsin–Madison, Madison, WI 53715. Received: 21 March 2012. First decision: 19 April 2012. Accepted: 26 April 2012. Ó 2012 by the Society for the Study of Reproduction, Inc. eISSN: 1529-7268 http://www.biolreprod.org ISSN: 0006-3363
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muscle contraction, vasodilation and vasoconstriction, induction of pain and fever). The PGs play important roles in the regulation of female reproductive functions during the estrous cycle, ovulation, pregnancy, and parturition. The induction of luteolysis is a well-known effect of PGF2a (PGF). Based on the assay of a PGF metabolite (PGFM), PGF is secreted in pulses from the endometrium in many animal species, including cattle [3, 4], sheep [5, 6], and horses [7, 8]. The minimal effective systemic dose of PGF that induces corpus luteum (CL) regression with one treatment varies greatly among farm animal species. Mares are more sensitive to the luteolytic effect of PGF than cattle and sheep. The minimal effective dose of PGF in mares was calculated to be approximately 8 lg/kg body weight (total dose 1.25 mg [9, 10]). In sheep, the minimal effective systemic dose is 144 lg/kg body weight (6 mg total dose [11]), and in cattle, the dose is about 40 lg/kg body weight (15–25 mg total dose [12]). Thus, mares are about 18 times more sensitive to the luteolytic effect of systemic PGF than sheep and five times more sensitive than cattle. The route for passage of PGF from the uterus to the CL differs profoundly between mares and ruminants, based on experiments that utilized unilateral and partial hysterectomy, physical stimulation of a uterine horn, intrauterine versus systemic administration of PGF, and unilateral pregnancy [13– 15]. The utero-ovarian pathway is local or unilateral in cattle and sheep and systemic or whole-body in mares. The local pathway involves close apposition of efferent uterine vessels (venous and lymphatic) with ovarian arterial vessels. In cattle, the effective luteolytic dose of PGF is approximately 20-fold greater when given systemically than when given into the uterus [12]. In ewes, a 2.0-mg dose of PGF is more effective in inducing luteolysis when injected into the uterus than when injected into the muscle [11]. In mares, no significant difference in luteolysis was found between the intrauterine and intramuscular routes for a single luteolytic injection of PGF [16]. In addition, infusion of PGF to simulate a pulse of PGFM in mares is associated with similar reduction in progesterone when a similar dose of PGF is infused by either an intravenous or an intrauterine route [17]. These results are consistent with the presence of a local utero-ovarian pathway in cattle and sheep and a systemic pathway in mares. The affinity of PGF for luteal cell membrane preparations from mares is approximately 10 times greater than that of preparations from cow CL [18]. Higher affinity of mare CL for PGF binding compared to the reported affinity in other species has been confirmed [19]. Greater affinity for binding of PGF to luteal cells may partly explain the greater sensitivity of mares to PGF for a luteolytic effect and why a local utero-ovarian pathway is not needed in mares. However, it is also possible that the metabolic clearance of PGF in mares is slower, requiring a smaller amount of PGF to induce luteolysis than in
ABSTRACT
SHRESTHA ET AL.
cattle and sheep. The biological half-life or rate of metabolic clearance of PGF in mares is unknown. The PGs are rapidly metabolized mainly in the lungs but also in other organs such as the liver and kidney. The reported percentage of PGF that is metabolized in one passage through the lungs varies considerably among species as follows: (1) guinea pigs, 90% [20]; (2) sheep, 99% [21]; (3) cattle, 65% [22]; and (4) pigs, 18% [21]. A plasma clearance of 17 L/min was reported when 15 mg PGF was infused intravenously for 30 min in heifers [23]. The biological half-life of PGs in humans is reported to be less than a minute, based on the detection of only 3% of the tritiated PGs 1.5 min after a bolus injection [24, 25]. A half-life of 1 min was reported for PGF in monkeys [26], using one monkey for each of two doses with the first sample at 30 sec. The reported studies in various species on metabolic clearance of PGF, including the plasma clearance and biological half-life, have not adequately considered the early portion of the disappearance curve. Maximum plasma PGF concentration after treatment was not determined, and a distinction between the half-life of the distribution phase and the half-life of the elimination phase was not made. Recent studies on luteolysis have shown that the hormonal responses to a bolus PGF treatment in mares [27] and heifers [28, 29] occur rapidly (within minutes). Therefore, the early portion of the disappearance curve, including the half-life of the distribution phase and maximum plasma concentration, are important considerations. The objective of the present study was to characterize maximum plasma concentration, apparent plasma clearance, distribution half-life, and elimination half-life of PGF in horses (a species with a systemic utero-ovarian pathway) and cattle (a species with a local utero-ovarian pathway). The two species were compared to determine if species differences in clearance or half-life could contribute to the greater luteolytic sensitivity to PGF in horses than in cattle. The hypothesis was that the metabolic clearance of PGF is slower in mares than in heifers.
FIG. 1. Diagram of experimental design. PGF (5 mg, i.v. bolus) was given to each mare (n ¼ 5) and heifer (n ¼ 5) 9 or 10 days after ovulation. Blood samples were collected initially at 10-sec intervals followed by 30sec, 10-min, and 30-min intervals as indicated.
Assay of PGF The plasma samples were assayed for PGF by an ELISA that was developed for fluid from luteal cavities by the first author (H.K.S.) in Dr. Milo Wiltbank’s laboratory (Department of Dairy Science, University of Wisconsin– Madison, Madison, WI). The assay was adapted in our laboratory for use in bovine and equine plasma. The PGF reference standard (catalog #16010; Cayman Chemicals, Ann Arbor, MI), the secondary antibody (rabbit anti-sheep IgG, H & L chain; catalog #402104; Calbiochem, San Diego, CA), and the primary antibody (sheep anti-prostaglandin F2a; catalog #90501; Assay Designs, Inc., Ann Arbor, MI) were commercially procured. The PGFhorseradish peroxidase (HRP) conjugate was prepared by Lori E. Anderson in the Wiltbank laboratory as previously described [32, 33]. Briefly, PGF (1 mg; Cayman Chemicals) was dissolved in 100 ll of dioxane (Fisher Scientific, Fair Lawn, NJ) before addition of tributylamine (2.5 ll; Aldrich, Milwaukee, WI) and isobutyl chloroformate (1.3 ll), then incubated at 108C 128C for 30 min. The HRP type VI (2000 U; catalog #P-8375; Sigma-Aldrich Chemical Co., St. Louis, MO) was dissolved in a mixture of dioxane (350 ll) and 0.5% sodium bicarbonate (350 ll). The PGF solution was added to the HRP solution dropwise with constant stirring and incubated at 48C for 2 h with constant stirring. To remove free PGF, the PGF-HRP solution was dialyzed against 0.1 M sodium phosphate (pH 7.0) for 40 h at 48C, with two changes of buffer. A column of Sephadex G-25 (catalog #54805 Sigma-Aldrich Chemical Co., St. Louis, MO) was saturated with 2% BSA in PBS (pH 7.0) and then rinsed with 10 column volumes of PBS. The conjugated PGF-HRP was added to the column, and 1-ml fractions were collected. Peroxidase activity in each fraction was checked by the addition of substrate solution (prepared from 0.05 M sodium acetate, 0.5 M hydrogen peroxide, and 20 mg/ml 3,3 0 ,5,5 0 -tetramethyl benzidine) to a sample from each fraction. Fractions with high peroxidase activity (dark blue color) were combined and diluted (1:50) with a mixture of equal volumes of glycerol and conjugate storage buffer (containing 0.04 M 3[N-morpholino] propane sulfonic acid, 0.12 M NaCl, 0.1 M ethylenediamine tetraacetic acid, 0.005% chlorhexidine digluconate [pH 7.2], and 0.1% gelatin) and stored at 808C. For the assay, the secondary antibody was used at a dilution of 2 lg/ml. The primary antibody and the PGF-HRP conjugate were each used at a dilution of 1:20 000. The extraction of samples and standards, preparation of buffers, and the procedure for ELISA for PGF were done as described for PGFM in our laboratory for mares [34] and cattle [29]. Serial volumes (100–7.5 ll) from pools of equine and bovine plasma from PGF-treated mares and heifers (containing high PGF concentration) were processed as for the experimental samples and resulted in displacement curves that were similar to the standard curve. The intra- and interassay coefficient of variation and sensitivity, respectively, were 6%, 13%, and 10 pg/ml for mares and 11%, 16%, and 17 pg/ml for heifers.
MATERIALS AND METHODS Animals and Treatment The mares were nonlactating mixed breeds of large ponies and apparent pony-horse crosses and were 7 to 17 yr of age. The dairy heifers (Holsteins) were 18 to 24 mo of age. The management of animals, including housing and feeding and the ultrasonographic monitoring for ovulation detection, has been described and was similar between mares [30] and heifers [28]. Animals with docile temperament were selected and were accustomed to the handling procedures. The experiment was done during October in the northern temperate zone. Animals were handled according to the United States Department of Agriculture Guide for Care and Use of Agricultural Animals in Agricultural Research and Teaching. Mares (n ¼ 5) and heifers (n ¼ 5) were selected so that body weight was similar between species (mares, 474.8 6 11.4 kg; heifers, 474.4 6 11.6 kg). Dinoprost tromethamine (Lutalyse; Pfizer Animal Health, New York, NY) was used for PGF treatment. According to the manufacturer, the product contains the naturally occurring PGF (dinoprost) as the tromethamine salt. A 5-mg i.v. dose of PGF was used. This dose is luteolytic after a single treatment in mares but not in heifers. The experiment was done during diestrus 9 or 10 days after ovulation. On the day of the experiment, an indwelling catheter (14 gauge; Angiocath; Becton Dickinson, Sandy, UT) was placed into the left jugular vein as described [31]. The PGF dose of 5 mg (1 ml Lutalyse) was administered as a rapid bolus injection into the right jugular vein at least 30 min after catheterization of the left jugular vein. Blood samples were collected from the indwelling catheter just before PGF treatment (Min 0), followed by every 10 sec until Min 3, every 30 sec until Min 5, at Min 10, every 10 min until Min 60, and every 30 min until Min 240 (Fig. 1). The blood samples were collected into heparinized tubes and immediately placed in ice water for approximately 10 min, then centrifuged
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(2000 3 g for 10 min). The plasma was decanted and stored at 208C until assayed.
PLASMA CLEARANCE OF PGF2alpha IN MARES AND HEIFERS
FIG. 2. Mean 6 SEM concentrations of PGF in jugular plasma of mares and heifers treated with 5 mg PGF (1 ml lutalyse, i.v.; Min 0 ¼ just before treatment). The mean of maximum PGF concentration in individuals was greater (P , 0.002) in mares (249.1 6 36.8 ng/ml) than in heifers (51.4 6 22.6 ng/ml). Probabilities for a main effect of species (S) and time (T) and their interaction (ST) are shown. An asterisk (*) on the x-axis indicates a difference (P , 0.05) between the two species within a time.
Plasma clearance of PGF and first-pass metabolism in lungs were not measured separately. Therefore, plasma clearance was termed apparent plasma clearance and was determined for each animal as follows: dose of PGF (5 mg ¼ 5 000 000 ng) divided by area under curve (AUC; [ng/ml] 3 min) for plasma PGF concentration from Min 0 to the time of a return to the basal concentration. The basal PGF concentration was defined as the mean plasma PGF concentration at Min 0 (mare ¼ 81.0 6 67.4 pg/ml; heifer ¼ 71.8 6 26.2 pg/ ml). The variation in basal PGF concentrations at Min 0 likely reflects differences among animals in the location of the blood sample relative to endogenous PGF pulses [34]. The AUC was calculated using the trapezoid rule, with the minimum concentration in each individual animal as the base. The individual disappearance curve for plasma PGF was described by the twocompartment bi-exponential model: Ct ¼ aeat þ bebt, where Ct is the PGF concentration at time t [35]. The projected initial (Min 0) plasma concentration of PGF is the sum of coefficients a and b. For mares, PGF concentrations from the maximum until 60 min were used, and for heifers concentrations from the maximum until 40 min were used. The discrepancy in the length of time was necessary because mean PGF concentration plateaued after Min 40 in heifers (Fig. 2). The PGF concentrations were log (natural) transformed. The distribution half-life was defined as t1/2(a) ¼ 0.693/a, where a is the slope during distribution phase and 0.693 is the natural log of 2. The slope (b) for the elimination phase was calculated using a mono-exponential curve and included data from Min 10 until Min 60 for mares and from Min 5 until Min 40 for heifers. The elimination half-life was defined as t1/2(b) ¼ 0.693/b, where b is the slope during the elimination phase.
TABLE 1. Apparent plasma clearance, distribution half-life, and elimination half-life of prostaglandin F2a after a single bolus intravenous injection (5 mg) in each mare and heifer (n ¼ 5/species).a
Statistical Analyses Species
The plasma PGF concentration data were not normally distributed (ShapiroWilk test) and were transformed to ranks for comparison between mares and heifers. The PGF concentrations in mares and heifers were compared by the SAS MIXED procedure (version 9.2; SAS Institute, Inc., Cary, NC), using a REPEATED statement to minimize autocorrelation between sequential measurements and with spatial power to account for uneven intervals between samples. The PGF concentrations were analyzed for the main effects of species (equine and bovine) and time and for the species-by-time interaction. When an interaction was obtained, species and time effects were further analyzed. Unpaired Student t-tests were used for comparisons between the two species for differences within a time, apparent plasma clearance and half-lives, maximum plasma PGF concentration, and time to reach the maximum concentration. Least Significant Difference test was used to locate differences between times within a species. A probability of P 0.05 was considered significant.
Mares Mare A Mare B Mare C Mare D Mare E Mean 6 SEM Heifers Heifer A Heifer B Heifer C Heifer D Heifer E Mean 6 SEM Probabilityb
RESULTS
Apparent plasma clearance (L h1 kg1)
Distribution half-life (sec)
Elimination half-life (min)
4.9 3.5 2.0 3.1 3.1 3.3 6 0.5
150.7 103.1 60.8 68.6 87.8 94.2 6 15.9
12.6 33.0 26.9 17.5 39.8 25.9 6 5.0
11.2 21.0 13.5 20.9 10.3 15.4 6 2.3 P , 0.0005
16.8 38.5 28.1 26.3 36.5 29.2 6 3.9 P , 0.002
8.8 7.9 6.8 9.5 12.3 9.0 6 0.9 P , 0.005
a
The injection was given on Day 9 or 10 after ovulation in both mares and heifers. b The probabilities for a difference between the two species are shown; comparison by unpaired t-test between the two species.
Probabilities for the results of the factorial analyses that were significant or approached significance are shown in Figure 2. Probabilities for differences in discrete end points are 3
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shown in Table 1 or in the text. The plasma concentrations of PGF in mares and heifers are shown (Fig. 2). The main effect of species and time and the interaction of species-by-time were significant. The interaction represented primarily greater (P , 0.05) concentrations of PGF in mares than in heifers at Min 1 through 60 (except at Min 10) and at Mins 180 and 210. The PGF concentration increased (P , 0.05) between Mins 0 and 1 in mares and Mins 0 and 0.5 in heifers and decreased (P , 0.05) thereafter. The maximum PGF concentration was greater (P , 0.002) in mares (249.1 6 36.8 ng/ml) than in heifers (51.4 6 22.6 ng/ml). However, the time to the maximum concentration was not different between mares (42.0 6 8.6 sec) and heifers (35.0 6 2.9 sec). The disappearance curve for plasma PGF concentration after a single i.v. injection of PGF in each mare and heifer is shown (Fig. 3). The apparent plasma clearance, distribution half-life, and elimination half-life for individuals and the mean for each species are presented in Table 1. The plasma clearance was
Disappearance Curve
SHRESTHA ET AL.
Downloaded from www.biolreprod.org. FIG. 3. Individual animal (A, B, C, D, E) plasma disappearance curves of PGF from maximum to Min 20 for mares and heifers. The concentrations are from the jugular plasma after treatment with 5 mg PGF (1 ml Lutalyse, i.v.; Min 0 ¼ just before treatment). The solid line represents a bi-exponential curve calculated for actual plasma PGF concentrations (solid dots). The plasma PGF concentration is in log scale. Note the more rapid steepness of initial decline in plasma PGF concentration (distribution phase) in heifers than in mares.
similar to our use of the term distribution half-life. Previous studies have been limited also by infrequent or delayed sampling and small numbers of animals. The current study is novel in that it directly compared the PGF disappearance curve between two species and had an adequate number of animals for consideration of variability among animals. In each species, the apparent plasma clearance and distribution half-life were more than doubled between the individuals with the lowest and highest values. The distribution half-life of about 1.5 min (94 sec) in mares is apparently more than the commonly reported biological half-
smaller and the distribution half-life and the elimination halflife were longer in mares than in heifers. DISCUSSION To our knowledge, this is the first report on the elimination half-life for PGF in any species. Previous reports on the biological half-life of PGF did not distinguish between distribution half-life and elimination half-life. However, based on inspection of the reported and current profiles, the terms half-life and biological half-life in previous reports seem 4
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PLASMA CLEARANCE OF PGF2alpha IN MARES AND HEIFERS
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individuals was about five times greater in mares than in heifers. The greater maximum plasma concentrations apparently resulted from a lower percentage of PGF metabolized in a single passage through lungs. The reported clearance in one passage through the lungs in heifers is 65% [22] but is unknown in mares. A comparison is needed on the difference between the two species on the efficiency of PGF metabolism by the lung. Metabolism of prostaglandins is most rapid and efficient in the lungs. Lungs receive approximately 100% of cardiac output (from the right side) and hence all the blood passes through the lungs during each circulation, unlike for other organs such as the kidney or liver [40]. The rate of any enzymatic reaction is expected to have an upper limit as the concentration of the substrate is increased; the enzyme has a limited capacity [41]. At a higher concentration of a drug (substrate), the rate of metabolism approaches an upper limit, and the process is said to be capacity limited or to show saturability. The capacitylimited state or saturability is important for the action of PGF. Reaching the capacity-limited state would allow PGF to have a spillover effect and reach the target tissues or organs at a more effective concentration. Although PGF is rapidly and efficiently metabolized in the body, its enzymatic conversion by PGDH may reach a capacity-limited state or saturation, and more PGF in its active form may escape metabolism and become available to the tissues for an effective role (e.g., luteolysis). In this regard, research is needed for PGF half-life with different doses. The affinity of equine luteal cell membrane preparations for PGF is approximately 10 times greater than for bovine luteal cell membrane preparations [18]. Higher affinity of mare luteal cell membrane preparations for PGF binding, compared to reported affinity in other species, has been confirmed [19]. The reported greater affinity of mare CL to binding of PGF compared to other species and the current results of slower metabolic clearance in mares than in heifers likely account for the greater sensitivity of mare CL to the luteolytic effect of PGF. In regard to PGF sensitivity, the luteolytic dose of PGF may induce side effects such as sweating, excitement, diarrhea, or colic in mares [42] but not in cattle. The functional significance of the maximum PGF concentration during the distribution phase after a bolus of PGF on induction of luteolysis is not known. However, a bolus PGF treatment induces acute hormonal responses in mares and heifers. In mares, progesterone increases within 2 min, LH increases within 5 min, and cortisol and FSH increase within 10 min after a bolus treatment with PGF (2.5 mg intravenous [27]). In heifers, progesterone increases within 4 min and cortisol and prolactin increase after 6 min of a bolus dose of PGF (4 mg intrauterine [29]). These acute hormonal responses to a bolus of PGF are nonphysiological reactions to an overdose of PGF [27, 43] and seem to be associated with the PGF profile concentrations during the distribution phase. In conclusion, the apparent plasma clearance of PGF was about five times less and the distribution and elimination halflives were about three times longer in mares than in heifers of similar body weight when injected intravenously with a similar bolus dose of PGF. The results indicated that the reported greater luteal sensitivity of mares to PGF compared to heifers is at least partly attributable to slower metabolic clearance of PGF in mares, resulting in greater maximum and greater profile concentration of PGF during the early portion of the plasma PGF disappearance curve. The hypothesis that metabolic clearance of PGF is slower in mares than in heifers was supported.
life of PGF (;1 min) in other species (see Introduction). However, the distribution half-life in heifers was only about half of a minute (29 sec). The reported [23] clearance of PGF in heifers is 17 L/min when 15 mg PGF was infused intravenously for 30 min (0.5 mg/min). For comparison, when the apparent plasma clearance (15.4 L h1 kg1) for our results was converted to liters per minute, the clearance was seven times greater (121.8 L/min) in the current study than in the reported study [23]. The difference may represent infusion in the reported study versus bolus injection in the present study. The rapid metabolism of PGF after a bolus treatment is consistent with an increase in plasma PGFM concentration within 2 min after bolus treatment with 0.5 mg PGF in mares [17] and in heifers [29]. The hypothesis that the metabolic clearance of PGF is slower in mares than in heifers was supported. The slower metabolic clearance of PGF in mares was indicated by a smaller apparent plasma clearance, longer distribution half-life, and longer elimination half-life than in heifers. The plasma clearance was about fivefold greater in heifers than in mares. In this regard, the manufacturer-recommended clinical dose of PGF for inducing luteolysis is fivefold greater in cattle (25 mg) than in horses (5 mg). The difference in apparent plasma clearance occurred with the same dose of PGF (5 mg per animal) in each species, and body weights were similar. The threefold greater distribution half-life and elimination half-life and the smaller plasma clearance in mares compared to that in heifers indicated that a greater portion of the injected PGF remained in the circulation longer in mares than in heifers. This was well reflected in the plasma PGF concentration profiles of the two species (Fig. 2). The PGF concentrations were greater in mares than in heifers from 1 min after PGF injection until 60 min. In addition, the mean of the maximum PGF concentration in individual animals was greater in mares than in heifers. Thus, the greater luteolytic sensitivity to PGF in mares than in heifers, as indicated by the smaller PGF dose requirements, was represented by a larger amount of PGF for a longer period in the circulation and therefore longer exposure at the cellular level in target tissues. The metabolism of PGs is mediated by NAD þ-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH), which catalyzes the initial rate-limiting step in the conversion of PGs to their inactive 15-keto metabolites [36, 37]. The PGDH is ubiquitously expressed in mammalian tissues and can be found in vascular beds, including those in lungs and kidneys [38, 39]. A species difference in the activity of PGDH may be responsible for the considerable variation in the reported percentages of PGF that is metabolized in one passage through the lungs (see Introduction). Although PGDH activity apparently has not been reported for either horses or cattle, a slower metabolic clearance of PGF in mares in the current study likely represents less PGDH activity in mares than in heifers. In a two-compartment model [35], the steep decline in PGF concentration during the distribution phase occurs during the distribution of PGF by the central compartment (blood vessels) to the peripheral compartment (tissues). The contribution from elimination of a drug during this phase is usually limited. In the present study, both mares and heifers were similar in body weight and presumably had similar blood volume, which is reported to be about 8% of the body weight [40]. When each of the two species was treated with the same dose of PGF (5 mg), a similar maximum plasma PGF concentration was expected. Although the time to reach the maximum plasma PGF concentration (,1 min after treatment) did not differ between the two species, the mean maximum PGF concentration among
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ACKNOWLEDGMENT The authors thank M.C. Wiltbank, University of Wisconsin, for providing PGF-HRP conjugate, P. Crump for statistical advice, Pfizer Animal Health for a gift of Lutalyse, and M.A. Siddiqui for technical assistance.
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