PROSTAGLANDINS
FLUORESCENT DERIVATIVES OF PROSTAGLANDINS AND THROMBOXANES FOR LIQUID CHROMATOGRAPHY John Turk, Stephen J. Weiss, J.E.
Davis, and Philip Needleman
Division of Laboratory Medicine (Department of Pathology) and Department of Pharmacology, Barnes Hospital and the Washington University School of Medicine, 4960 Audubon, St. Louis, MO Abstract Fluorescent esters of the prostablandins D2, E2, F2a, and 6-keto-Floe and of thromboxane 82 have been prepared using the reagent 4-bromomethyl-7-methoxycoumarin. All of these derivatives can be separated in a single run either by thin-layer or highperformance liquid chromatography (TLC or HPLC). As little as 20 ng of PGE2 can be detected after derivatization and HPLC analysis. Identification of thromboxane B2 produced by human platelets and of 6-keto-PG Fla produced by bovine aortic microsomes has been achieved with this method.
INTRODUCTION The identification and quantitation of prostaglandins and thromboxanes has been approached in a number of ways. Bioassay is quite sensitive and has played an important role in the discovery of previously unidentified species (1,2). Such methods may, however, fail to distinguish between chemical species with overlapping pharmacologic profiles and fail to detect species with transient lifetimes whose end-products are biologically inactive. Radioimmunoassay (3,4) and mass spectrometric (5,6,7) methods both offer great specificity in species distinction as well as sensitivity in the low picogram range. Neither is well suited to the analysis of multiple components in a single run, however.
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Chromatographic methods offer the ability to perform simultaneous analyses of multiple known species and potentially to disclose the existence of previously unrecognized species. Suitable detecti;;v;ytems must be coupled with the chromatographic procedures, . Radiochemical methods have been used with thin-layer chromatography (8), and this has yielded an excellent qualitative tool but one which is not readily quantitatable. High performance liquid chromatographic (HPLC) separation with radiochemical tracers marking peak position has been described, but this involves eluant collection and subsequent analysis by mass spectrometric methods for quantitation (9). Refractive index monitoring has also been used in conjunction with HPLC (lo), but the sensitivity of this method is insufficient for detection of the small amounts of prostaglandins generated by biologi gl systems. F One approach to the problem,kf detection is to form derivatives of the prostaglandins which contain easily detectable chemical groups. Pentafluorobenzyloxine derivatives of prostaglandins containing a carbonyl group can be detected with electron capture gas chromatographic techniques in the low picogram range (11). Non-carbonyl containing prostaglandins (e.g., PGFpo()must be handled differently, however. An elegant gas chromatographic system has recently been described employing glass capillary columns and flame-ionization detection (12). Separation of all primary transformation products of arachidonic acid is achieved with a minimum detection limit of about 20 ng. This procedure requires 3 derivatization steps, however, and 2 separable isomers are generated for each carbonylcontaining species. Derivatization with chromophores absorbing strongly in the ultraviolet (UV) spectrum has also been described (11,13), allowing UV detection to be used in conjunction with HPLC. Both p-nitrobenzyloxime and p-bromophenacyl ester derivatives have been examined, and such derivatives of a number of prostaglandin species can be separated by reversed-phase HPLC. Minimum detection limits range around 50 ng. HPLC conditions for separation of such derivatives of 6keto-PGF1 and thromboxane B2 from the more classical prostaglandins were not described, however.
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Fluorescence detection is an appealing possibility since it offers sensitivity comparable or superior to UV detection in most instances, and in view of recent advances in detector technology (14,15) may shortly offer much greater sensitivity than that presently available. In addition, fluorescent derivatives can be visualized on thin-layer chromatograms in small amounts, providing an inexpensive tool for qualitative analysis. Application of fluorescence detection to prostaglandins has been limited by the lack of native fluorescence and the absence of functional groups in the native structure which react readily with conventional fluorescent derivatizing reagents such as fluorescamine, ophthaldehyde, or dansyl chloride. Recently, however, Dunges (16) has described the use of 4-bromomethyl-7-methoxycoumarin (Mmc-Br) to form fluorescent esters of carboxylic acids in a single-step preparation. The present report describes the use of Mmc-Br to form fluorescent esters of PGD2, PGE2, thromboxane B2, PGF2% , and 6keto-PGFI< as well as HPLC conditions for the separation of these derivatives and an internal standard. Application of these methods to the detection of thromboxane B2 and 6-keto-PGFId derived from biological sources is also described. MATERIALS Mmc-Br (4-bromomethyl-7-methoxycoumarin) was obtained from Regis Chemical (Morton Grove, Ill.). Prostaglandin and thromboxane standards were generously donated by Dr. J.Pike of the Upjohn Co. (Kalamazoo, Mich.). Imidazole and indomethacin were obtained from NuCheck Prep. (Elysian, Minn.), and (l-l4C) arachidonic acid (197 microcuries per mg) was obtained from Amersham-Searle Corp. (Arlington Heights, Ill.). Sodium borohydride, sodium sulfate, potassium carbonate, and all solvents (acetic acid, acetone, benzene, chloroform, dioxane, ethanol, ethyl acetate, hexanes, iso-octane, methanol, and petroleum ether) were obtained from Fisher Chemical (St. Louis, MO.). Silica Gel G and F254 TLC plates were obtained from Brinkman Instruments (Westbury, N.Y.).A thermostated water bath and portable UV light source (348 nm) were obtained from Scientific Products (St. Louis, MO.). Reacti-vials (teflon capped, screw top, conical reaction vials) with volumes of 0.3 or 1.0 ml and compatible magnetic stirring bars were obtained from Pierce (Rockford, Ill.). The mc.del 8500 high pressure liquid chromatography system was a prc_iuct of Varian (Palo Alto, Calif.), as was the prototype flow-through fluorometer, which was provided on loan from that company. The Aminco-Bowman spectrophotofluorometer was a product of American Instruments (Silver Spring, Md.). The model 930 thin-layer radiochromatogram scanner was a product of Vanguard Systems (Dobbs Ferry, NY). Bovine aortas were obtained from a local slaughter house.
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METHODS Extraction conditions: Biological mixtures containing prostagland=ere centrifuged for 10 min. at 5000 g to remove particulate matter and were then extracted with an equal volume of petroleum ether, which was discarded. From this point on handling of prostaglandin-containing aqueous solutions derived from bioloqical or chemical sources was identical. The solution was adjusted to pH 3.0 with 1N HCl and extracted 3 times with eaual volumes of ethyl acetate. The pooled extracts were washed once with one-sixth volume of water to remove residual HCl, and the aqueous phase was discarded. The organic phase was dried over sodium sulfate, concentrated to dryness under nitrogen, and taken up in acetone. Reaction conditions: Material to be derivatized was introduced into a 0.3 ml (or 1.0 ml) reacti-vial as an acetone solution and concentrated to dryness under nitrogen. At least a 3-fold molar excess of Mmc-Br was then introduced as an acetone solution (2 mg/ml). A stirring bar and about 25 mg of potassium carbonate were then added, and the vials were capped and placed in a 67O C water bath for 10 min. with magnetic stirring. The vials were then placed on ice for about 5 min. (to reduce solvent loss on opening), and an aliquot of the reaction solution was immediately subjected to chromatography. Thin-layer chromatography: TLC was performed on Silica Gel G or Silica Gel F254 plates in the following solvent systems (proportions expressed as volume to volume): chloroform 100, methanol 7 (system A); benzene 17, ethyl acetate 1 (system B, reference 16); chloroform 90, methanol 8, acetic acid 1.0, water 0.8 (system C, reference 17); benzene 90, dioxane 45, acetic acid 4.5 (system D, reference 18); or the organic phase of ethyl acetate 110, aceticacid,20, is&octane 50,-water.100 (system E, reference 19). Spots were identified with a portable UV lamp or by staining with iodine vapor. High performance liquid chromatography: HPLC of prostaglandincontaining mixtures was performed with a programmed gradient of 2 solvent systems: chloroform 35, iso-octane 65, methanol 1.0 (system F) and chloroform 100, methanol 1.35 (system G). The Mmc derivative of acetic acid was eluted in hexanes 100, methanol 1.0 (system H). The flow rate was 100 ml per hr., resulting in a column pressure of about 110 atmospheres (1620 psi). Sample volumes of 20 ul were applied to the Varian CN-10 Micropak column with a loop injector. Eluant was monitored in a prototype Varian Fluorichrom.flowthrough fluorometer with a deuterium arc-lamplight source. Excitation filters employed were Varian 7-54 (325 nm band) and Varian 7-60 (360 nm band), and the emission filter was Varian 3-74 (385 nm cutoff). Chart speed on the Varian model 9176 recorder was 0.5 cm per min. Solvent program I was employed to display multiple prostaglandin species: l)lOO% F, 14 min., 2)increase G 10% per min., 4 min.,
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3)60% F and 40% 6, 8 min., 4)increase G 10% per min., 6 min., 5) 100% G, 8 min. To display only the more polar prostaglandins the accelerated program II was employed: 1)80% F and 20% G, 8 min., 2)increase G 10% per min., 8 min., 3)100% G, 2 min. Preparation of standards: Derivatives of the following standard compounds were prepared under reaction conditions described above: aceticacid,'PGD2, PGE2, PGF2ti , 6-keto-PGFlH , and thromboxane B2. The derivative of acetic acid, 4-methyl-7-methoxycoumarin acetate (Mmc-AC) was purified by seven recrystallizations from ethanol at which point it exhibited a melting point (165-167OC, dec.) and migration coefficient in system B (Rf. 0.37) which corresponded to literature values (16). Derivatives of the standard prostaglandins (Nmc-PG) were purified by preparative TLC in system A. Silica gel containing the adducts was scraped from the plates and extracted 4 times with the solvent system chloroform 4, methanol 1. Solvent was evaporated under nitrogen, and the residue was dissolved in sufficient chloroform so that a 2 ul aliquot produced an HPLC chromatogram with a peak height of about 50% full scale at attenuation 5, gain 10, recorder span 10 millivolts. Reduction of 6-keto-PGFI, was performed by dissolving 750 ug of the compound in 10 ml of methanol and gradually adding 10 mg of sodium borohydride over 30 min. while stirring at 4OC. Thirty ml of water was then added. The solution was extracted as described above, and the material so recovered was derivatized under standard conditions. The solution from the derivatization vial was then subjected to preparative TLC in system A. A faint band of fluorescence corresponding to Mmc-6-keto-PGF o( (Rf0.18) was observed, but the major band wasmorepolar (RfO.1 I!i ) and was assumed to represent Mmc6-hydroxy-PGFlc( . This material was recovered from the TLC plate as described above. Reaction kinetics: Several vials with identical concentrations of PGmlO ug/ml) and Mmc-Br (2 mg/ml) were prepared and reacted under standard conditions except that the incubation period was varied from D to 40 min. Aliquots (20 ul) of reaction solution were injected into the HPLC system at the end of the incubation period and peak heights were compared at the various time points. Extent of reaction: A comparison was made between the peak height produced by a 20 ul aliquot of a 1.0 mg/ml acetone solution of standard MC-AC and that produced by a 20 ul aliquot of a solution containing acetic acid (0.18 mg/ml) and Mmc-Br (2 mg/ml) that had been reacted under standard conditions. Extent of reaction with prostaglandins was evaluated by TLC of multiple aliquots (10 to 250 ul) from a reaction solution of PGE2 (650 us/ml) and In separate lanes of the same TLC plate standard Mmc-Br (2 mg/ml). PGE was applied in amounts from 1 to 60 ug. The plate was develope Z in system C which clearly separated PGE2 (Rf0.32) from MmcPGE2 (Rf0.55) and spots were located by fluorescence and iodine staining.
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Linearity: The relationship between the concentration of prostaolandin introduced into the reaction vial and the heiqht (or area) of the HPLC peak produced by a standard aliquot of the reaction ’ solution was evaluated as follows. Aliquots (10 ul) from acetone solutions of PGE2 or PGF2& varying in concentration from 10 ng/ul to 10 ug/ul were added to vials containing 90 ul of Mmc-Br in acetone (2 mg/ml). After incubation under standard conditions, a 20 ul aliquot of the reaction solution from each vial was subjected to HPLC and the areas of the resultant peaks were compared. Biological preparations: Washed platelets were prepared from the blood of healthy human volunteers-as previously described (20). Platelet suspensions (4.5 to 5.5 x 105 per ul) were incubated at37OC with continuous stirring in a Payton dual-channel aggregometer. The suspensions (2 ml) were treated with arachidonic acid alone (final concentration i750 ng/ml platelt suspension) or with arachidonic acid after a 10 min. incubation with imidazole (final concentration 300 ug/ml) or after a 30 min. incubation with‘indomethacin After 10 min. exposure to arachidonic (final concentration 10 ug/ml). acid (only indomethacin-treated platelets failed to aggregate during this period) the suspensions were subjected to the standard extraction and derivatization procedures described above. Final reaction volume was 200 ul. Aliquots of 20 ul were subjected to HPLC. Bovine aortic microsomes were prepared by the method of Gryglewski et al (21), and 15-hydroperoxy-arachidonic acid was prepared by the method of Hamberg ard Samuelsson (22). PGH2 was prepared as previously described (23,24). A 0.2 ml aliquot of the microsome preparation (protein concentration 5 mg/ml) was diluted in 0.8 ml of 100 mM potassium phosphate buffer (pH7.8). PGH (4 ug) was then added alone or after a 10 min. incubation at 37OC o $ the microsomes with 5 ug of 15-hydroperoxy-arachidonic acid. After the addition of PGH2, the mixture was incubated at 370C for 10 min. and was then subjected to the standard extraction and derivatization procedures. Final reaction volume was 200 ul. Aliquots of 20 ul Similar experiments were performed using were subjected to HPLC. (l-14C)-PGH prepared from (1-14C)-arachidonic acid. About 200 ng (50,000 cpm ? of the radio-labelled substrate was substituted for Conditions unlabelled PGH2 in the experiments described above. were otherwise identical except that the entire extract of the incubation medium was subjected directly to TLC in system E. RESULTS Rapid formation of fluorescent adducts from the non-fluorescent carboxyjic acid starting materials was observed on TLC. Migration coefficients are listed in Table 1. Clear separation of all prostaglandins from their Mmc derivatives was obtained in system C,.with the Flmc derivatives exhibiting larger migration coefficients. The order of migration of the Nnc derivatives paralleled that of the
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Table I .
TLC Migration Coefficients of Mmc-PG Adducts.
Mmc Adduct
-A
B
0.56
___
___
___
E2
0.42
0.55
0.30
0.25
TB2
0.32
0.48
0.44
0.33
F26
0.23
0.38
0.22
0.15
6KF
0.18
0.56
0.22
0.10
6HF
0.10
0.16
0.11
0.06
D2
D
&
About 300 ng of each species was applied to the plate. See Methods for solvent systems. D2 is PGD2; E2 is PGE2; TB2 is thromboxane B2; F@ is PGF2% ; 6KF is 6-keto-PGFld: 6HF is 6-hydroxy-PGFld . Dash means Rf was not determined.
Table 2.
Relationship of Amount of Prostaglandin Derivatized to Fluorescent Peak Area. Concentration in Reaction Vial (us/ml)
Compound
Amount Applied to Column (ng)
Pea Area (mm1 x attenuation)
E2
1000
2 x 104
7.77 2 0.41 x 104
E2
100
2 x 103
7.81 + 0.62 x lo3
FPM
100
2 x 103
7.90 + 0.51 x 103
F26
20
4 x 102
1.61 2 0.15 x lo3
E2
10
2 x 102
8.15 t 0.90 x lo2
Fzo(
4
8 x lo1
3.22 ~0.50
E2
1
2 x 101
8.00 + 2.10 x lo1
x lo2
Aliquots (20 ul) of reaction solution were eluted from the HPLC column with 60% F and 40% G. Standard deviations are indicated ~I=&I). Abbreviations as in Table 1. See Methods for further
.
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native species in all systems. Clear separation of the Mmc derivatives of PGD2, PGE2, PGF2cr , 6-keto-PGFIoc , and thromboxane B2 was obtained in system A, as shown in Figure 1. As little as 40 ng of PGE could be visually detected after derivatization and TLC on pla zes with fluorescent indicator. About 5 ug of PGE2 was required to produce a visible stain with iodine vapor. Determination of excitation and emission spectra of dilute solutions (10 ug/ml or less) of standard Mmc-PGE2 in solvent F revealed an excitation maximum near 340 nm and an emission maximum near 405 nm. Kinetic analyses under standard reaction conditions revealed that the fluorescence generated from the reaction of Mmc-Br and PGE2 increased linearly for about 5 min. At this point maximal fluorescence was achieved, which was stable for reaction times up to 40 min. (see Methods section). Evidence that the reaction is quantitative is provided by the fact that reaction mixtures containing acetic acid generated peaks on HPLC which represented 102% (S.D. t 6%,n = 4) of the fluorescence predicted from that generated 'by pure Mmc-Ac. In addition, no unreacted PGE2 was detectable on TLC when the plates were loaded with a sufficient volume of reaction solution to produce a visible iodine stain for PGE2 had as much as 4% of the starting material failed to react (see Methods section). The reaction is relatively free cf major fluorescent by-products. Unreacted Mmc-Br and its decomposition products elute shortly after injection, and minor by-productsare observed eluting near Mmc-PGD2 and Mmc-6-keto-PGFI, as seen in Figure 2. The magnitude of the fluorescent signal approximates a linear function of the concentration of carboxylic acid initially added to the reaction vial as seen in Table 2. This relationship extends from the limit of sensitivity (20 ng of PGE2 derivatized and applied to the column) through at least 3 orders of magnitude. Equimolar amounts of PGE2 and PGF2* produce equivalent fluorescent peak areas (Table 2). HPLC separation of the Mmc adducts of the following species was achieved in a single 40 min. run by means of solvent program I: PGD2, PGE2, PGF20( , 6-keto-PGFI, , 6-hydroxy-PGFI, , and thromboxane Figure 3 is the chromatogram of a crude reaction mixture of !&se compounds. In the absence of thromboxane B2 or PGF20( isocratic resolution of the remaining species can be achieved in the solvent system chloroform 40, hexanes 60, methanol 2.0. Run time can be considerably shortened by omitting Mmc-6-hydroxy-PGFId (internal standard) and/or sacrificing resolution of the less polar species to obtain more rapid appearance of the more polar species as in solvent program II. Thromboxane B synthesis by human platelets as detected by the methods descn ?Ied here is demonstrated in Figure 4. Panel 4a is the chromatogram of a reaction mixture of standards. Panel 4d is the chromatogram of a derivatized extract from platelets
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6Ht 'F' 'Tb ' b, 6KF *?nlx*E2 Figure 1. Separation of standard Mmc-PG adducts in System A applied as a mixture in the central lane and separately in the other lanes. Abbreviation and TLC conditions as in Table 1.
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0
IO
20
30
time (mid
Figure 2. HPLC of crude reaction mixture. Mmc-Br (2 mg/ml) and 6-keto PGF1, ( 600 ug/ml) were reacted under standard conditions. An aliquot (20 ul) of the crude reaction solution was eluted from the HPLC column with the solvent 60% F and 40% G. Instrument settings: attenuation 100, gain 10, mv span 10.
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I I
6KF
\n 6HF
0
IO
20
30
40
time (mid Figure 3. HPLC separation of Mmc adducts of multiple species. An aliquot (20 ul) was injected from a reaction mixture (1.0 ml) of Mmc-Br (2 mg/ml) and about 150 ug D2, 200 ug E2, 300 ug TB2, 150 ug F2ti , and 300 ug 6KF. Standard Mmc-6HF (1 ul) was also injected, and the sample was eluted with solvent program I. Abbreviations as in Table 1. Peak identity confirmed by spiking with standards. Settings: attenuation 200, gain 10, mv span 10. See Methods.
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C 6KF
Figure 4. Thromboxane B2 production by human platelets documented by HPLC. a) Elution of standards by solvent program I. Other panels are from derivatized extracts of incubation medium co-injected with standard Mmc-6HF. b) Platelets aggregated with arachidonic acid after exposure to imidazole. c) Platelets treated with arachidonic acid after exposure to indomethacin. d) Platelets aggregated with arachidonic acid. e) Material from d) spiked with standard Mmc-TB2. Abbreviations as in Table 1. See Methods for further detail. Expected elution times of Mmc-E2 and Mmc-TB2 are indicated by vertical lines. Settings: gain 10, attenuation 5, mv span 10.
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PROSTAGLANDINS aggregated with arachidonic acid. A peak apparently co-migrating with Mmc-thromboxane B2 is observed. Co-migration is confirmed in panel 4e, which is the chromatogram of the extract in 4d, now spiked with standard Mmc-thromboxane B2. Panel 4b is the chromatogram of a derivatized extract from platelets treated with the thromboxane-synthetase i n h i b i t o r imidazole. Thromboxane B2 synthesis is suppressed, and PGE2 appears due to the isomerization of PGH2 which occurs in the absence of i t s enzymatic conversion to thromboxane B2 (25). Panel 4c demonstrates the nearly complete suppression of p l a t e l e t prostaglandin synthesis by the cyclooxygenase i n h i b i t o r indomethacin. An identical response to these pharmacologic manipulations of p l a t e l e t suspensions has been documented in previously published t h i n - l a y e r radio-chromatograms (22). Production of the prostacyclin isomerization product 6-ketoPGFIm by bovine aortic microsomes is demonstrated in Figure 5. Elu~ion of standard Mmc-6-keto-PGFl~by solvent program I I is shown in panel 5a. Panel 5b is th~ chromatogram of a derivatized extract of the medium in which bovine aortic microsomes and PGH2 were incubated. Panel 5c demonstrates co-migration of the peak observed in 5b and Mmc-6-keto-PGFl~ when the extract is spiked with the standard material. Panel 5d demonstrates complete suppression of 6-keto-PGFl~ synthesis when the microsomes are treated with the prostacyclin-synthetase i n h i b i t o r 15-hydroperoxy-arachidonic acid (18). Confirmation of the q u a l i t a t i v e accuracy of these observations is provided in Figure 6. Panel 6a is the t h i n - l a y e r radiochromatogram of the extract of medium in which bovine aortic microsomes were incubated with (1-14C)-PGH2. Co-migration of standard 6-keto-PGFiw and the resultant radioactive peak is observed. Panel 6b again demonstrates suppression of 6-keto-PGFl~ synthesis by treatment of the microsomes with 15-hydroperoxyZarachidonic acid. DISCUSSION Dunges' recent description of 4-bromomethyl-7-methoxycoumarin (Mmc-Br) as an effective reagent for fluorescent l a b e l l i n g of carboxylic acids greatly extends the range of compounds to which fluorescence detection methods can be applied (16). The report established that the adducts are carboxylic acid esters; that the reaction is quantitative and complete in less than 40 minutes at 55~C; that the molar fluorescent y i e l d of the adduct is independent of the i d e n t i t y of the carboxylic acid; and that ~ the i n t e n s i t y of the fluorescent signal is l i n e a r l y related to the amount of carboxylic acid derivatized. The findings presented in this report indicate that these properties also obtain when prostaglandin species are the carboxylate donors in the reaction. In addition, the derivatization procedure has been simplified somewhat by the use of heated, magnetically s t i r r e d , sealed reaction v i a l s rather than a micro-refluxing apparatus as o r i g i n a l l y described. This also allows the use of a higher reaction temperature (67 o vs. 55°C) and a shorter reaction time (10 min. vs. 60 min.).
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’
’
:
d
b
4
Figure 5. Production of 6-keto-PGFl, by bovine aortic microsomes documented by HPLC. a) Elution of standard Mmc-6KF by solvent program II. Other panels are derivatized extracts of incubation media. b) Bovine aortic microsomes and PGH2. c) Material from b) spiked with standard Mmc-6KF. d) Microsomes treated with 15-hydroperoxy-arachidonic acid, then PGH2. Expected elution time of WC-6KF indicated by vertical line. Abbreviations as in Table 1. See Methods for further detail. Settings: gain 10, mv span 10, attenuation 5.
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a
b
A2
D,
E2F2a6KF
Figure 6. Production of 6-keto-PGF1& by bovine aortic microsames documented by thin-layer radiochromatograms. TLC in system E of extracts of incubation media. Dark circles indicate migration of standards applied to same plate and located by iodine staining. a) Bovine aortic microsomes and PGH2. b) Microsomes treated with 15hydroperoxy-arachidonic acid, then PGH2. Abbreviations as in Table 1. See Methods for further detail.
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Fluorescent derivatives of PGD2, PGE2, PGF2, , 6-keto-PGFIoc, and thromboxane B2 are readily formed and can be separated from each other in a single one-dimensional thin-layer chromatogram. Most laboratories currently require TLC in 2 systems (C or D and E) to display all of these prostaglandin species. The minimum detection limit of 40 ng of PGE2 derivatized and applied to the plate represents an improvement of 2 orders of magnitude compared to iodine staining and of one order of magnitude compared to densitometry after charring (26). Scanning techniques do exist for -in situ quantitation of the fluorescence on TLC plates (16) but were not explored in this study. A bonded phase column was employed in the HPLC studies to avoid problems inherent in the use of adsorption columns such as difficulty in maintaining constant surface activity due to accumulation of polar contaminants from biological extracts. After prolonged use of the CN-10 Micropak column changes in peak shape and retention time do occur, but original column properties can be reestablished by a 30 min. isopropanol wash. Column performance is unaffected by the injection of large amounts of unreacted Mmc-Br present in crude reaction mixtures. HPLC resolution of the Mmc adducts of the 5 prostaglandin species listed above was achieved with this column in a single 40 min. run. In addition, the Mmc derivative of 6-hydroxy-PGFIti (which was prepared by the reduction of 6-keto-PGFId) could be separated from the derivatives of the 5 naturally occurring species and may serve as a convenient internal standard. Compounds eluting earlier than the naturally occurring species would be less likely to be useful as internal standards since that region of the chromatogram is populated by unreacted Mmc-Br, its decomposition products, and multiple unidentified species extractetl from biological materials. Clear separation of multiple species, linearity of fluorescent signal with concentration, availability of an internal standard, and the ability to detect small amounts of material make this procedure a promising one for quantitation of prostaglandins from biological sources. While rigorously quantitative studies have not yet been undertaken, definitive qualitative identification of thromboxane B2 produced by human platelets and of 6-keto-PGFIo( produced by bovine aortic microsomes has been achieved. Identification of both species rests on co-migration with standard Mmc adducts and suppression of production of the species by appropriate pharmacologic stimuli. The amount of thromboxane B2 demonstrated in Figure 4 was derived from the platelets present in 2 ml of human blood. Compared to the most effective previously described HPLC method,which involved derivatization with UV chromophores (13), the method described here offers somewhat greater sensitivity and the ability to resolve 6-keto-PGFId and thromboxane B2 from the more
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classical prostaglandins. Recently described gas chromatographic methods can resolve all of the species examined here (11,12), and the sensitivity with flame-ionization detection is equivalent to that obtained with this method. Sensitivity with electron-capture detection of pentafluorobenzyloxime derivatives is far greater, however, and extends into the low picogram range (11). The method described here does offer some advantages over gas chromatographic methods,however. First, the number of derivatization steps is reduced from 3 to 1, greatly facilitating sample handling, and the adducts can be visualized on TLC. In addition, only one adduct is formed from each species rather than two in the case of the oxime derivatives. This simplifies the appearance and interpretation of the chromatogram and reduces potential for interference from non-prostaglandin substances. Finally, fluorescent analysis is non-destructive, allowing eluant recovery for radiochemical or other analyses. Potentially useful modifications of the system include application to additional arachidonic acid metabolites, development of the capability for post-column derivatization, and augmentation of sensitivity. Arachidonic acid and relatively nonpolar metabolites such as HETE, HHT, and 15-keto-prostaglandins possibly would be more easily resolved with a reversed phase system, given the interferences present in the early region of the chromatogram in the normal phase. The general method of derivatization and detection should be readily applicable to these compounds, however. The low fluorescence of unreacted MmcBr and the rapidity of its reaction with carboxylic acids suggest that derivatization of column effluent may be feasible, provided thatthe eluting solvent does not interfere with the reaction. This would obviate pre-column sample derivatization and with stream-splitting techniques could allow recovery of unmodified sample components for bioassay or chemical analyses. Augmentation of the sensitivity of fluorescence detection by 2 to 3 orders of magnitude by replacing conventional arc-lamps with laser light sources has recently been reported (14,15). How rapidly such improvements in detector technology might become generally accessible is uncertain, however.
In summary, fluorescent esters of prostaglandins can be formed in a rapid, single-step preparation. Such adducts of PGD2, PGE2, PGF2o(,6-keto-PGFl,, and thromboxane B2 can be clearly separated from each other in a single run both by TLC and by HPLC. The minimum detectable amount of PGE2 derivatized and applied to the HPLC column is 20 ng, which approximates the sensitivity of flame-lonization detection. Qualitative identification of thromboxane B2 produced by human platelets and of 6-keto-PGFld produced by bovine aortic microsomes has been achieved using these methods.
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ACKNOWLEDGEMENTS This study was supported by grants from the National Institutes of Health (SCOR-HL-17646 and HL-20787) to Philip Needleman. We are indebted to Angela Wyche for invaluable technical assistance. REFERENCES 1.
Kulkarni, P.S., R. Roberts, and P. Needleman. Paradoxical Endogenous Synthesis of a Coronary Dilating Substance from Arachidonate. Prostaglandins 12~337-353, 1976.
2.
Bunting, S., R. Gryglewski, S. Moncada, and J. Vane. Arterial Walls Generate from Prostaglandin Endoperoxides a Substance (Prostaglandin X) which Relaxes Strips of Mesenteric and Celiac Arteries and Inhibits Platelet Aggregation. Prostaglandins -12: 897-913, 1976.
3.
Samuelsson, B., E. Granstrom, K. Green, M. Hamberg, and S. Hammarstrom. Prostaglandins. Ann. Rev. Biochem. -44:669-695, 1975.
4.
Levine, L., R. Guttierroz-Cernosek, and H. Van Vunakis. Specific Antibodies: Reagents for Quantitative Analysis of Prostaglandins. Advances in Bioscience 9:71-83, 1973.
5.
Green, D., M. Hamberg, and B. Samuelsson. Quantitative Analysis of Prostaglandin and Thromboxanes by Mass Spectrometric Methods. Advances Prostaglandin Thromboxane Res. 1:47-59, 1976.
6.
Lawson, A. The Scope of Mass Spectroscopy in Clinical Chemistry. Clinical Chemistry 21:803-824, 1975.
7.
Kelly, R.W., and P.L. Taylor. Tert-Butyl Dimethylsilyl Ethers as Derivatives for Qualitative Analysis of Steroids and Prostaglandins by Gas Phase Methods. Anal. Chem. 48:465-467, 1976.
8.
Chasalow, F.T. and B.B. Phariss. Luteinizing Hormone Stimulation of Ovarian Prostaglandin Biosynthesis. Prostaglandins 1:107-117, 1972.
9.
Carr, K., B.J. Sweetman, and J.C. Frolich. High Performance Liquid Chromatography of Prostaglandins: Biological Applications. Prostaglandins -11:3-14, 1976.
10.
Lustgarten, R.K. High Performance Liquid Chromatographic Separation of C-15 Epimers of 15-Methyl Prostaglandin E2 Methyl Ester and 15-Methyl Prostaglandin E2. J. Pharm. Sci. -10:1533-35, 1976.
AUGUST
1978 VOL. 16 NO. 2
PROSTAGLANDINS
11.
Fitzpatrick, F.A., M.A. Wynalda, and O.G. Kaiser. Oximes for High Performance Liquid and Electron Capture Gas Chromatography of Prostaglandins and Thrombaxanes. Anal. Chem. -49:1032-35, 1977.
12.
Fitzpatrick, F.A. Separation of Prostaglandins and Thromboxanes with Glass Capillary Columns. Anal. Chem. -50:47-52, 1978.
13.
Fitzpatrick, F.A. High Performance Liquid Chromatographic and D from in vitro Determination of Prostaglandins F %4:%502, 1676. Enzyme Incubations. Anal. Chem. _*
14.
Diebold, G.N. and R.N. Zare. Laser Fluorimetry: Subpicogram Detection of Aflatoxins Using High Pressure Liquid Chromatography. Science 196:1439-41, 1977.
15.
Richardson, J.H. and M.E. Ando. Sub Part per Trillion Detection of Polycyclic Aromatic Hydrocarbons by Laser Induced Molecular Fluorescence. Anal. Chem. 49:955-959, 1977.
16.
Dunges, W. 4-Bromomethyl-7-Methoxycoumarin as a New Fluorescence Label for Fatty Acids. Anal. Chem. 49:442-445, 1977.
17.
Nugteren, D.H. and E. Hazelhof. Isolation and Properties of Intermediates in Prostaglandin Biosynthesis. Biochem. Biophys. Acta 326:448-461, 1973.
18.
Bill, T.L., J.B. Smith, and M.J. Silber. Metabolism of (14C)Arachidonic Acid by Human Platelets. Biochem. Biophys. Acta 424.303-314, 1976. -*
19.
Johnson, R.A., D.R. Morton, J.H. Kinner, R.R. Gorman, J.C. McGuire, and F.R. Sun. The Chemical Structure of Prostaglandin X (Prostacyclin). Prostaglandins -12:915-918, 1976.
20.
Minkes, M., N. Stanford, M. Chi, G. Roth, A. Raz, P. Needleman, and P. Majerus. Cyclic Adenosine 3',5'-monophosphate Inhibits the Availability of Arachidonate to Prostaglandin Synthetase in Human Platelet Suspensions. J. Clin. Invest. -59:449-454, 1977.
21.
Gryglewski, R.N., S. Bunting, S. Moncada, R.N. Flower, and J.R. Vane. Arterial Walls are Protected Against Deposition of Platelet Thrombi by a Substance (Prostaglandin X) Which They Make from Prostaglandin Endoperoxides. Prostaglandins -12:685-711, 1976.
22.
Hamberg, M. and B. Samuelsson. On the Specificity of Oxygenation of Unsaturated Fatty Acids Catalyzed by Soyabean Lipoxygenase. J. Biol. Chem. 242:5239-45, 1967.
‘AUGUST
1978 VOL. 16 NO. 2
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