Metabolism of 6-Trans Isomers of Leukotriene B4 to Dihydro Products by Human Polymorphonuclear Leukocytes*

The major dihydroxy metabolites of arachidonic acid formed by human polymorphonuclear leukocytes (PMNL) are leukotriene B, (LTB,), 6-trans-LTB,, and 12-epi-6-trans-LTB4. LTB,, and to a lesser extent its 6-trans isomers, are metabolized to 20-hydroxy prod- ucts by a hydroxylase in PMNL. We have recently reported the existence of a second pathway involving a reductase which, combined with the hydroxylase, results in the conversion of 6-trans-LTB4 to dihydro- 6-trans-LTB4. We have now investigated some of the characteristics of this novel triene reductase pathway in human PMNL and have characterized some of the products and their mechanism of formation. At low substrate concentrations, the major pathway for the initial metabolism of both 6-trans-LTB4 and 12-epi-6-trans-LTB4 is reduction of the conjugated triene chromophore to give dihydro products with sin- gle absorption maxima at about 230 nm. Dihydro-6-trans-LTB, is rapidly converted to its 20-hydroxy me- tabolite by LTB, 20-hydroxylase. However, 20-hy- droxy-6-trans-LTB, is not a substrate for the reductase. Neither 12-epi-6-trans-LTB4 nor its dihydro me- tabolite, 5,12-dihydroxy-7,9,14-eicosatrienoic increased be- tween 15 and 30 min. These results are consistent with the formation of metabolite IIb from metabolite IIIb, analogous to the situation for the metabolites of 6-trans-LTB,.

Metabolism of LTB, to 20-hydroxy-LTB4 appears to result in considerable losses of both chemotactic (15) and proaggregatory (17,18) activities with respect to PMNL. The 20hydroxy metabolite is also much less active than LTB, in causing degranulation of PMNL (5, 18). However, there are conflicting reports suggesting that 20-hydroxy-LTB, is as active as LTB, as a chemotactic agent (19) and that it has about the same affinity for LTB, binding sites in human PMNL as LTB, itself (20). In the guinea pig lung, 20-hydroxy-LTB, clearly appears to be at least as active as LTB, in contracting parenchymal strips (14,18). However, the mechanism of action of LTB, on guinea pig lung differs from that on PMNL in that it is mediated by release of thromboxane A, (21). w-Carboxy-LTB, is much less potent than its 20hydroxy precursor (5, 18,22).
We have recently observed a second pathway for the metabolism of dihydroxyeicosanoids related to LTB,, involving reduction of one of the three conjugated double bonds (23). We found that human PMNL convert 6-trans-LTB4 to a 20hydroxy product which has a single Amax at 231 nm in its UV spectrum, indicating that there are only two conjugated double bonds between the 5-and 12-hydroxyl groups (23). This product is presumably formed by the combined actions of LTB, 20-hydroxylase and a reductase.
In order to investigate the reductase reaction in more detail, it would be desirable to use a substrate such as 12-epi-6-trans-LTB,, which is not readily metabolized by the w-oxidation pathway (9). We have now shown that this isomer of LTB, is metabolized primarily to a dihydro product by human PMNL. We have investigated the mechanism of formation of this product and have examined some of the characteristics of the reductase pathway.
Preparation of Human PMNL-PMNL were prepared as described previously (9) by treatment of blood with Dextran T-500 (Pharmacia LKB Biotechnology Inc.) to remove red blood cells. Mononuclear cells were removed by centrifugation over Ficoll-Paque (Pharmacia LKB Biotechnology Inc.). Any red blood cells remaining in the pellet were lysed by treatment with 0.135 M NH4C1. After washing with 0.15 M NaC1, the cells were resuspended in Dulbecco's phosphate-buffered saline, containing 137 mM NaC1, 2.7 mM KC], 1.5 mM KH,PO,, 8.1 mM Na2HP0,, 0.5 mM MgC12, and 0.9 mM CaCI,.
Incubation Conditions-Human PMNL (lo7 cells/ml) were preincubated for 2 min at 37 "C and then incubated with the two 6-trans isomers of LTB, (0.4 PM for 30 min, unless otherwise indicated). For preparative experiments, the incubations were terminated by centrifugation at 4 "C at 400 X g for 10 min. The pellet was then washed by recentrifugation in an amount of ethanol sufficient to give a final concentration of 15% when combined with the first supernatant. For analytical experiments, when it was desired to measure the amounts of products formed, the incubations (1 ml) were terminated by the addition of methanol (1 ml).
Purification of Products from Preparative Experiments-The incubation medium was extracted using cartridges (Waters CIS Sep-Paks) of ODS silica (9,28). After loading the sample onto the cartridge, it was washed with 15% ethanol in water (20 ml), water (20 ml), and petroleum ether (20 ml). Eicosanoids were then eluted with redistilled methyl formate (Aldrich) (10 ml). The material in the residue obtained after evaporation of the methyl formate was purified by RP-HPLC, which was carried out using a Waters solvent delivery system, a Berthold LB 505 radioactivity monitor, and a Waters model 490 UV detector. The stationary phase was a column (250 X 4.6 mm) of ODS silica (5 pm Ultrasphere, Beckman Instruments). The mobile phase was a gradient between water/acetic acid (1000.05) and acetonitrile/acetic acid (100:0.05) as follows: 0 min, 25% acetonitrile; 40 min, 25% acetonitrile; 45 min, 40% acetonitrile. The flow rate was 1.5 ml/min. The materials in fractions of interest were methylated and in some cases hydrogenated and then further purified by a second chromatography using methanol/water as the mobile phase. Analysis of Metabolites by RP-HPLC-After termination of the incubations, [I-"Clprostaglandin Fz,, (2000 dpm) was added to each of the samples as an internal standard to monitor recovery. The concentration of ethanol in the mixtures was adjusted to 15% by the addition of water (4.7 ml). The sample was centrifuged and the supernatant acidified to pH 3 and pumped onto an ODs-silica precolumn (Pierce Chemical Co.) connected to a 6-port switching valve (29). After washing with 2.5 mM phosphoric acid in 15% aqueous methanol, the material retained by the precolumn was analyzed on a Waters Novapak C,, column (3.9 X 150 mm) using a mobile phase consisting of a gradient between water/acetic acid (100:0.02) and acetonitrile/acetic acid (100:0.02) as follows: 0 min, 23% acetonitrile; 15 min, 23% acetonitrile; 20 min, 38% acetonitrile. The flow rate was 2 ml/min. The products were detected by separately monitoring radioactivity due to I4C and 3H as well as UV absorbance at 235 and 280 nm. The radioactivity data was integrated using a Berthold model 510 data system.

Identification of Products
High Pressure Liquid Chromatography-The metabolites formed when 6-trans-LTB, (0.4 gM) was incubated with human PMNL were analyzed by RP-HPLC (Fig. lA). The major products were metabolite Ia (retention time (tR), 18.2 min), metabolite IIa (tK, 23.4 min), and metabolite IIIa (tR, 66.8 min). Each of these metabolites was converted to its methyl ester and further purified by RP-HPLC either before or after hydrogenation using water/methanol as the mobile phase. We previously identified metabolite Ia as 20-hydroxy-6-tran.s-LTB, (9) and metabolite IIa as a 5,12,20-trihydroxy product containing a conjugated diene structure (23). Metabolite IIIa is the only product with a retention time longer than that of 6-trans-LTB,, suggesting that it is not the product of an woxidation reaction. Its slightly longer retention time would be consistent with the presence of fewer double bonds than are present in 6-trans-LTB4.
A similar pattern of products was observed after incubation of 12-epi-6-truns-LTB4 with human PMNL (Fig. 1B). In this case, however, the major product was metabolite IIIb ( tR, 82.1 rnin), which had a retention time longer than that of the substrate and was therefore unlikely to be an w-oxidation product. When rechromatographed using a mobile phase consisting of methanol/water/acetic acid (62:38:0.05) at a flow rate of 1.5 ml/min, metabolite IIIb was observed as a single peak of radioactivity and UV absorbance at 235 nm, with a retention time of 36.5 min. We detected much smaller amounts of metabolites Ib ( t R , 22 min) and IIb (tR, 27.5 min), which, by analogy with the metabolites of 6-trans-LTB4, were probably w-oxidation products. Metabolite Ib, which was a relatively minor product under the reaction conditions employed, was not homogeneous and appeared to consist of at least two components. Since these products absorb at 280 nm, they are unlikely to be dihydro metabolites, but may be Whydroxy and w-carboxy metabolites of 12-epi-6-trans-LTB4. Because of the small amounts of these products, they were not further characterized.
UV Absorption Spectra-Metabolite Ia, which was derived from g-trans-LTB,, has a UV spectrum typical of leukotrienes, with three absorption maxima a t 259, 269, and 280 nm. Metabolite IIa, on the other hand, has only one absorption maximum, at 232 nm. Metabolite IIIa, the minor product formed from 6-trans-LTB4, also has a single absorption maximum in its UV spectrum, at 231 nm. This suggests that IIIa is a dihydro metabolite of 6-trum-LTB4, containing two con-  jugated double bonds between the 5-and 12-hydroxyl groups. The major metabolite of 12-epi-6-trans-LTB4 (metabolite IIIb) has a UV spectrum similar to that of metabolite IIIa, with an absorption maximum at 231 nm (Fig. 2). Metabolite IIb has a similar UV spectrum, with a maximum at 232 nm. These results suggest that both metabolites IIb and IIIb have two conjugated double bonds and are formed by the reduction of the triene chromophore of 12-epi-6-trans-LTB4.
Gus Chromatography-Muss Spectrometry-The two major metabolites of 6-trans-LTB, (metabolites Ia and IIa) were tentatively identified on the bases of both their UV and mass spectra (9,23). However, the number and positions of the double bonds in the 20-hydroxy metabolite of 6-truns-LTB4 exhibiting maximal UV absorbance at 232 nm could not be determined, because it had been hydrogenated prior to analysis by GC-MS. After hydrogenation, the methyl ester, trimethylsilyl ether derivatives of metabolites Ia and IIa, had identical retention times and mass spectra (23).
The methyl ester, trimethylsilyl ether derivative of the major product (metabolite IIIb) formed from 12-epi-6-trans-LTB, has a mass spectrum ( . This mass spectrum resembles that of the corresponding derivative of 12-epi-6-trans-LTB, (Fig. 3B), except that comparable fragment ions containing carbons 6-11 occur at masses 2 units higher in the mass spectrum of metabolite IIIb. These results, combined with the UV spectrum of metabolite IIIb, clearly indicate that one of the three conjugated double bonds of 12-epi-6-trans-LTB, has been reduced to give a product with two conjugated double bonds. These two double bonds could theoretically be in the 6 and 8, 7 and 9, or 8 and 10 positions. The two ions at m/z 269 and 279 may be due to fragmentations on either side of the conjugated double bonds, as shown in Fig. 3A, suggesting that they are present in the 7 and 9 positions. The structure of the dihydro metabolite of 12-epi-6-truns-LTB4 would therefore be 5,12-dihydroxy-7,9,14-eicosatrienoic acid.
Secondly, the ions at m/z 272 and 285 in the mass spectrum shown in Fig. 3C are consistent with the presence of a 7,9conjugated diene structure in heptadeutero-dihydro-12-epi-6trans-LTB,. By comparison with the mass spectrum of the corresponding unlabeled compound (Fig. 3A), it can be concluded that the fragment ion at mlz 272 contains 3 deuterium atoms, whereas that at m/z 285 contains 6 deuterium atoms, as would be predicted if they arose via fragmentation on either side of the 7,g-diene. The mass spectrum shown in Fig. 3C thus provides further evidence that the conjugated double bonds are in the 7 and 9 positions.

Time Courses for the Formation of Metabolites from 6-Trans
Isomers of LTB, The two 6-trans isomers of LTB, (0.4 PM) were incubated with human PMNL (lo7 cells/ml) for various times and the products analyzed by RP-HPLC. The time course for the formation of metabolite IIa from 6-trans-LTB4 shows that there is an initial lag phase in the formation of this product (Fig. 44). This was not true for either metabolite l a or IIIa. Initially, the amount of metabolite IIIa exceeded that of metabolite IIa, but the concentration of the former product reached a maximum and then declined, so that after 30 min, only a relatively small amount was present. Unlike the situation with metabolite IIIa, the amount of metabolite Ia in the incubation mixture increased steadily as a function of time. These results suggest that the precursor of the dihydro-20hydroxy product IIa is dihydro-6-trans-LTB, (metabolite IIIa) rather than 2O-hydroxy-6-trans-LTB4 (metabolite Ia). We did not have sufficient amounts of tritium-labeled metabolite IIIa to test this hypothesis directly. However, we found that 20hydroxy-6-trans-LTB4 was not converted to any detectable dihydro metabolites by PMNL (data not shown). These results suggest that although dihydro products are good substrates for LTB4 20-hydroxylase, 20-hydroxy products are not metabolized by the triene reductase.
The time course for the formation of products from 12-epig-trans-LTB, (Fig. 4B) bears some resemblance to that shown in Fig. 4A for 6-trans-LTB4, except that there are considerable quantitative differences. Metabolite IIb, like dihydro-20-h~droxy-6-trans-LTB4, is formed only after a lag phase. This product was barely detectable after 4 min, but after 30 min, its concentration was over 40% that of dihydro-12-epi-6trans-LTB, (metabolite IIIb), which was the major product at all time points investigated. The amount of the latter product increased up to 15 min, after which time it did not change.

Effects of LTB, on the Metabolism of 12-Epi-6-trans-LTB4
by PMNL LTB, is converted very rapidly to 20-hydroxy-LTB4 by human PMNL, and we have been unable to detect substantial amounts of dihydro metabolites of this substance in incubations with these cells. Another approach to investigating a possible interaction of LTB, with the triene reductase in PMNL would be to examine its effects on the metabolism of 6-trans isomers of LTB,. We incubated PMNL with 12-epi-6-tran~-[~H]LTB, (0.4 PM) in the presence of LTB, (2 PM) and measured the amounts of products formed after different times (Fig. 4C). The concentration of LTB, used in this experiment was higher than that of 12-epi-6-trans-LTB, because of the rapid metabolism of the former compound by LTB, 20-hydroxylase under these conditions (9). LTB, inhibited the formation of metabolites Ib, IIb, and IIIb from 12epi-6-trar~-[~H]LTB, by over 90% for the first 8 min of the reaction (Fig. 4C, inset). Between 15 and 30 min, by which time most of the LTB, had been metabolized, there was a rapid increase in the amount of dihydro-12-epi-6-trans-LTB4, so that by 30 min, the amount of this product was nearly 70% of that in control incubations (Fig. 4B) carried out in the absence of LTB,. The formation of w-oxidation products from 12-epi-6-trans-LTB, was inhibited even more by LTB, than the formation of dihydro-12-epi-6-trans-LTB,. Metabolites Ib and IIb could not be detected after a 4-min incubation. After 30 min, LTB, inhibited the formation of metabolite Ib by 85% and that of metabolite IIb by 98%. It is not surprising that the formation of metabolite IIb was inhibited more than that of the other products, since it requires the actions of both LTB, 20-hydroxylase and the triene reductase.  1 (A) and 2 PM (B). The samples were centrifuged and analyzed by precolumn extraction/RP-HPLC using a Pierce precolumn and a Waters Novapak C1, column (3.9 X 150 mm). The mobile phase was a gradient between water/acetic acid (1000.02) and acetonitrile/acetic acid (1000.02) as follows: 0 min, 23% acetonitrile; 15 min, 23% acetonitrile; 20 min, 38% acetonitrile. The flow rate was 2 ml/min. [I-"C]Prostaglandin F,, (2000 dpm) was added as an internal standard (is.) in order to monitor recovery. 30 min, and the products were analyzed by RP-HPLC. It is apparent that altering the substrate concentration has a dramatic effect on the distribution of metabolites formed from 6-trans-LTB4 (Fig. 5). At a concentration of 0.1 PM, 62% of 6-trans-LTB4 was initially metabolized by the reductase pathway (i.e. the sum of metabolites IIa and IIIa) after 30 min, compared to only 20% initially metabolized by the hydroxylase pathway (i.e. metabolite Ia) (Fig. 5A). When the substrate concentration was raised to 2 pM, however, only 6% was initially metabolized by the reductase, whereas the proportion metabolized by the hydroxylase rose to 38% (Fig. 5B). These calculations are based on the assumption that the initial reaction in the formation of dihydro-20-hydroxy-6-trans-LTB, was catalyzed by the reductase.
The effects of various substrate concentrations on the formation of products from 6-trans-LTB4 and 12-epi-6-trans-LTB, are shown in Fig. 6. It should be noted that the scale for the ordinate, which shows the amounts of products formed, is different for Fig. 6A (6-trans-LTB4) and Fig. 6B (12-epi-6trans-LTB,). The formation of trihydroxy trienes from these two substrates is nearly linear with the substrate concentration up to at least 2 p~, suggesting that the o-oxidation reaction has a fairly high Vmax. The rate of formation of woxidation products from 6-trans-LTB4 is about four times greater than from 12-epi-6-truns-LTB4.
The formation of reductase products is affected quite differently from that of hydroxylase products as the substrate concentration is raised. At low substrate concentrations (0.1 pM), the major pathway for the initial metabolism of both 6trans-LTB, and l2-epi-6-trans-LTB4 is the formation of dihydro products. However, the amounts of these products reach maximal levels at substrate concentrations between 0.4 and 0.8 p~ and then decline at higher concentrations. These results suggest that both the K,,, and the V,,, of the reductase pathway are substantially lower than those of LTB, 20hydroxylase.
Effects of Concentration of PMNL on the Metabolism of 12-Epi-6-trans-LTB4 Various concentrations of PMNL were incubated with 12epi-6-trans-LTB4 (0.4 p~) for 30 min, and the amounts of products formed were analyzed by HPLC. At all cell concentrations, the reductase product, metabolite IIIb, was formed to the greatest extent (Fig. 7). As the concentration of PMNL was raised, the amounts of dihydro-12-epi-6-trans-LTB4 (IIIb) and 20-hydroxy-l2-epi-6-trans-LTB4 (Ib) first increased in a nearly linear fashion and then reached nearly maximal levels due to limitations in substrate availability. The pattern for the formation of the combined reductase/ hydroxylase product (IIb) was quite different. At low concentrations of PMNL, metabolite IIb was only a minor product,  whereas at higher concentrations, quite substantial amounts were formed. This would suggest that there is no coupling between the reductase and hydroxylase reactions. The dihydro product is probably first formed by the PMNL and then released into the medium, where it can be converted by a second cell to the dihydro-20-hydroxy product. Thus, the formation of metabolite IIb requires two subsequent interactions of substrate with PMNL, whereas the formation of the other products requires only one such interaction.

DISCUSSION
There are two major pathways for the metabolism of 6trans isomers of LTB, by human PMNL: formation of dihydro products by a triene reductase, and o-oxidation (Fig. 8). The w-hydroxylation of these products is presumably catalyzed by LTB, 20-hydroxylase, since LTB, strongly inhibits this process (Fig. 4C). Both 6-truns-LTB4 and dihydro-6-trans-LTB4 are relatively good substrates for the hydroxylase (although not nearly as good as LTB, itself), and we have identified 20hydroxy products derived from both of these substances by UV spectrophotometry and GC-MS. On the other hand, 12epi-6-trans-LTB, and its dihydro metabolite are much poorer substrates for LTB, 20-hydroxylase. We have detected Woxidized metabolites of these substances by HPLC, but have not yet isolated sufficient quantities for analysis by GC-MS.
Dihydro-6-trans-LTB4 (metabolite IIIa) is rapidly converted to its 20-hydroxy derivative, and we have not yet conclusively identified the former by GC-MS. However, this product has a Amax at 231 nm as would be expected for a dihydro product with two conjugated double bonds. Its retention time upon analysis by RP-HPLC, which was slightly longer than that of 6-trans-LTB4, would also be consistent with this structure. In order to investigate the formation of non-w-hydroxylated dihydro metabolite from 6-trans isomers of LTB, in more detail, we incubated PMNL with 12-epi-6trans-LTB,, since the latter is not a very good substrate for LTB, 20-hydroxylase. The major product (metabolite IIIb) formed from this substrate under most conditions was identified on the basis of its retention time, UV spectrum, and mass spectrum as dihydro-12-epi-6-truns-LTB4.
There are three possible positions for the conjugated double bonds in dihydro-12-epi-6-truns-LTB4: 6 and 8, 7 and 9, or 8 and 10. If these double bonds were present in the 6-and 8positions, an intense ion would be expected in the mass spectrum at m/z 255 (CH = CH-CH = CH-CH(OSiMe,)-(CH2),-C02Me), due to cleavage between carbons 9 and 10. This is the most abundant ion above m/z 100 in the mass spectrum of the methyl ester, trimethylsilyl ether derivative of 5-hydroxy-6,8,11,14-eicosatetraenoic acid (30). No such ion is present in the mass spectrum of dihydro-12-epi-6-truns-LTB,, suggesting that the conjugated double bonds are not in the 6-and 8-positions.
Comparison of the mass spectra of the unlabeled and deuterium-labeled dihydro metabolites of 12-epi-6-truns-LTB4 suggests that the two conjugated double bonds are in the 7and 9-positions. The fragment ion at m/z 279 in the mass spectrum of the unlabeled metabolite probably arose due to cleavage between carbons 6 and 7, on the carboxyl side of the conjugated diene structure, and therefore consists of C7-Czo (Fig. 3A). This is supported by the presence of an analogous fragment ion in the mass spectrum of the corresponding deuterium-labeled metabolite at m/z 285 (Fig. 3C). There are no significant ions in the mass spectra of unlabeled and deuterium-labeled 12-epi-6-truns-LTB4 (Figs. 3, B and D ) which correspond to the above ions. The ions at m / z 282 (Fig.  3B) and 288 (Fig. 30) in the mass spectra of the latter compounds do not have the appropriate masses and were presumably formed due to subsequent cleavages between carbons 4 and 5 and carbons 12 and 13. The ions at m/z 269 ( Fig. 3A) and 272 (Fig. 3C) in the mass spectra of the unlabeled and deuterium-labeled dihydro metabolites may be due to cleavage between carbons 10 and 11 on the alkyl side of the conjugated diene structure. However, it must be borne in mind that analogous ions are present at m/z 267 (Fig. 3B) and m / z 270 (Fig. 3 0 ) in the mass spectra of unlabeled and deuterium-labeled 12-epi-6-trans-LTB4. From the above considerations, the mass spectral evidence would strongly suggest that 12-epi-6-truns-LTB4 is metabolized to 5,12-dihydroxy-7,9,14-eicosatrienoic acid by human PMNL.
The reduction of 5,12-dihydroxy trienes could occur by a number of different mechanisms. In most cases involving the enzymatic reduction of carbon-carbon double bonds, the double bond is activated by conjugation with an oxo group. For example, prostaglandins are biologically inactivated by conversion to 13,14-dihydro-15-0~0 metabolites by the successive actions of 15-hydroxyprostaglandin dehydrogenase and prostaglandin A13-reductase. Only 15-oxoprostaglandins, and not unmetabolized 15-hydroxyprostaglandins, are substrates for the reductase (31). Similarly, steroid 5a-reductases catalyze the reduction of the 4,5-double bond, which is conjugated with the 3-oxo group of steroids. The reductases involved in fatty acid chain elongation and P-oxidation also catalyze the reduction of double bonds which are activated by conjugation with the oxo groups of CoA esters. One of these enzymes catalyzes the reduction of a 2,4-dienoyl-CoA derived from linoleic acid to a 3-enoyl-CoA metabolite (ie. reduction is coupled to migration of one of the double bonds, as appears to occur with 6-trans isomers of LTB,) (32).
Conjugated dienes in which the double bonds are not activated by conjugation with an oxo group can also be reduced by reductases. For example, sterol 14-reductase converts As,14sterols to the corresponding As-sterols (33).
There are thus two probable mechanisms for the formation of 5,12-dihydroxy-7,9,14-eicosatrienoic acid from 12-epi-6trans-LTB,. The simplest mechanism would be the direct reduction of the conjugated triene system, coupled with migration of the double bonds, to give the corresponding conjugated diene (Fig. 9). On the other hand, the 5-or the 12hydroxyl group of 12-epi-6-trans-LTB4 could first be oxidized to an oxo group by a dehydrogenase. Alternatively, and perhaps less likely, the oxo intermediate could also be formed by hydroxylation at Cs to give a geminal diol, followed by dehydration. The formation of the oxo intermediate, which would result in activation of the double bonds, would be followed by two steps of reduction, coupled with migration of the double bonds, to give the dihydro product. Although this mechanism is more complicated than the direct reduction of one of the double bonds, it is analogous to the formation of 13,14dihydroprostaglandins from prostaglandins. These products are formed by the initial actions of 15-hydroxyprostaglandin dehydrogenase and prostaglandin A13 reductase as discussed above, followed by reduction of the 15-oxo group of the 13,14dihydro-15-oxoprostaglandin to give a 13,14-dihydroprostaglandin (34).
One way to distinguish between the above two possibilities would be to incubate 12-epi-6-tr~ns-[5,6,8,9,11,12,14,15-~H] LTB, with PMNL and analyze the resulting dihydro product by GC-MS. If the triene group of the substrate were reduced directly, no deuterium atoms would be lost from the substrate. On the other hand, if there were an oxo intermediate, the deuterium atom in either the 5-position or the 12-position would be lost (Fig. 9). The results of this experiment clearly indicate that a deuterium atom was lost from the 5-position during the formation of heptadeutero-5,12-dihydroxy-7,9,14- eicosatrienoic acid, strongly suggesting the presence of an intermediate with a 5-oxo group. We have not detected any such compounds in incubations of human PMNL with either 6-trans-LTB4 or 12-epi-6-trans-LTB4. However, it is possible that such intermediates could be very short-lived and be rapidly converted to the dihydro metabolites IIIa and IIIb. There is some evidence for the formation of an oxo metabolite of LTB, by rat hepatocytes (35). Although this product was not isolated and identified, it could be an intermediate in the formation of isomers of LTB, metabolites formed by these cells (35).
Because of the limited availability of labeled and unlabeled substrates, the experiments on the effects of substrate concentration illustrated in Fig. 6, A and B , were not designed for the determination of the K, and V,,, values for the hydroxylase and reductase enzymes. However, these data suggest that the V,,, for the formation of dihydro products is considerably lower than that for the formation of w-oxidation products. It would also appear that the reductase pathway is subject to substrate inhibition, since the amounts of dihydro products formed from both 6-trans-LTB4 and 12-epi-6-trans-LTB, decrease as the substrate concentration is raised from 0.8 to 2 pM (Fig. 6). At low substrate concentrations, the reductase pathway is the major pathway for the initial metabolism of 6-trans-LTB4 and 12-epi-6-trans-LTB4, suggesting that the K, for the formation of dihydro products from these substrates is lower than that for their hydroxylation.
Since LTB, is rapidly metabolized by LTB, 20-hydroxylase and since 20-hydroxylated products are not substrates for the reductase, we have not yet been able to detect dihydro metabolites of LTB, in human PMNL. There is indirect evidence that LTB, may be a substrate for the reductase; however, LTB, strongly inhibited the conversion of 12-epi-6-trans-LTB, to both dihydro and 20-hydroxylated products by PMNL (Fig. 4C), suggesting that LTB, competitively inhibited the metabolism of this substrate by both the triene reductase and the 20-hydroxylase. An alternative explanation of these data, which cannot be excluded, is that LTB, did not inhibit the reductase directly, but rather competitively inhibited the active uptake of 12-epi-6-trans-LTB4 into the PMNL. We are currently investigating which of these hypotheses is correct.