Determination of 15-keto-13,14-dihydro-metabolites of PGE2 and PGF2α in plasma using high performance liquid chromatography and gas chromatography-mass spectrometry

A method is described for the measurement of 15-keto-13,14-dihydrometabolites of PGE₂ and PGF_2α in peripheral human plasma. This involves purification by high performance liquid chromatography followed by determination of levels by combined gas chromatography-mass spectrometry using tetradeuterated analogs of the metabolites as internal standards. The levels of these metabolites in plasma are considered to be a more reasonable index of the entry of PGE₂ and PGF_2α into peripheral blood than are the levels of the corresponding primary prostaglandins. The endogenous levels of 15-keto-13,14-dihydro-PGE₂ and 15-keto-13,14-dihydro-PGF_2α found in peripheral plasma are 33 ± 10 pg/ml (SD; n=6) and 40 ± 16 pg/ml (SD; n=6), respectively.     

Plasma concentrations of 13,14-dihydro-15-keto-prostaglandin E2 in rabbits bearing the VX2 carcinoma: Effects of hydrocortisone and indomethacin

In rabbits bearing the prostaglandin-producing VX₂ carcinoma, the plasma concentration of 13,14-dihydro-15-keto-PGE₂ (PGE₂-M) was elevated within one week after tumor implantation and preceded the development of hypercalcemia. Both the rate of rise and magnitude of the increase were greater for the metabolite than for PGE₂; at the time of peak hypercalcemia (about 4 to 5 weeks after tumor implantation), the increase over basal in plasma PGE₂-M was about 75 fold whereas it was previously shown that the increase in PGE₂ was less than 2 fold. Indomethacin, which inhibits PGE₂ synthesis in VX₂ cells in culture, lowered in parallel plasma calcium and PGE₂-M in tumor-bearing rabbits. Administration of hydrocortisone to rabbits bearing the VX₂ tumor prevented the development of hypercalcemia when given at the time of tumor implantation and reversed the elevated plasma calcium in previously untreated animals; the steroid hormone also lowered plasma concentrations of PGE₂-M. These findings are consistent with our hypothesis that the hypercalcemic syndrome in VX₂ tumor-bearing rabbits is due to the secretion of PGE₂ by the tumor.     

Effects of hydrocortisone on the hypercalcemia and plasma levels of 13,14-dihydro-15-keto-prostaglandin E2 in mice bearing the HSDM1 fibrosarcoma

In mice bearing the prostaglandin-producing HSDM₁ fibrosarcoma, the plasma concentration of 13,14-dihydro-15-keto-PGE₂ was elevated before the development of hypercalcemia, and the magnitude of the rise was greater than that of PGE₂. When hydrocortisone, which inhibits synthesis of PGE₂ by HSDM₁ cells in culture, was administered to tumor-bearing mice, the steroid hormone prevented the rises in plasma PGE₂ metabolite and calcium concentrations. At the dose levels used, hydrocortisone did not inhibit the calcium-mobilizing action of parathyroid hormone $\text{in vivo}$ or the bone resorption-stimulating activity of $\text{PGE}{}_2\text{in vitro}$. These findings are consistent with our hypothesis that the hypercalcemic syndrome in HSDM₁ tumor-bearing mice is due to the secretion of PGE₂ by the tumor.     

Comparison of the inhibitory effect of E-prostaglandins in human and rabbit platelet-rich plasma and washed platelets

1. The anti-aggregatory potency of a number of E-type PGs was compared in human and rabbit platelet-rich plasma (PRP) and washed platelets. The potency of 13,14-dihydro-PGE₁ and 5,6-dihydro-PGE₃ is significantly higher in human than in rabbit washed platelets, while the potency of 15-keto-13,14-dihydro-PGE₁ is higher in rabbit.2. The potency of PGEs in rabbit PRP is very similar to that of washed platelets, with the exception of 1a,1b-dihomo-PGE₂, which is of a significantly lower potency in PRR.3. In human, 5,6-trans-PGE₂, PGE₃, and 15-keto-13,14-dihydro-PGE₁ are more potent in PRP than in washed platelets.4. The results indicate that the potency of E-type PGs in human and rabbit platelets is different and plasma can essentially influence the anti-aggregatory effect of PGEs; plasma can either decrease or increase potency.     

Responsiveness of the lamb ductus arteriosus to prostaglandins and their metabolites

The relative potencies of the prostaglandins A₁, A₂, E₁, E₂, F_2α and their 15-keto-, 15-keto-13,14-dihydro-, and 13,14-dihydro-metabolites were investigated on isolated lamb ductus arteriosus preparations contracted by exposure to elevated PO₂. All the prostaglandins (except PGF_2α and its 15-keto-metabolites) relaxed the tissue. However, only PGE₁, E₂, and their 13,14-dihydro-metabolites, were effective at concentrations below 10⁻⁸ M. Therefore, events that alter metabolism of circulating PGs in the perinatal period may have significant effects on the relative patency or closure of the ductus arteriosus.     

Chemical instability of 15-keto-13,14-dihydro-PGE2: The reason for low assay reliability

The spontaneous degradation of 15-keto-13,14-dihydro-PGE₂ was studied under various conditions. In aqueous media, dehydration rapidly occurred, particularly at high or very low pH. The acid catalyzed dehydration product was identified as 15-keto-13,14-dihydro-PGA₂. This compound was also formed at alkaline pH, however, at higher pH a bicyclic compound, 11-deoxy-13,14-dihydro-15-keto-11,16-cyclo-PGE₂, was formed in addition (cf. Fig. 1). The amounts of the latter compound increased with time and with increasing pH.In samples containing albumin, 15-keto-dihydro-PGE₂ was degraded even more rapidly than in buffer of the same pH. In addition to the formation of the dehydration products, a considerable part of the metabolite was bound to albumin to yield water soluble adducts. A similar binding occurred to a low molecular weight fraction of plasma. The compound that was bound was 15-keto-dihydro-PGA₂, whereas both 15-keto-dihydro-PGE₂ and the bicyclic product were relatively inert in this respect.Due to its chemical stability, the bicyclic degradation product, 11-deoxy-13,14-dihydro-15-keto-11,16-cyclo-PGE₂, is suggested as a suitable target for measurements instead of the labile parent compound, 15-keto-dihydro-PGE₂, or the reactive dehydration product, 15-keto-dihydro-PGA₂.     

Determination of cyclooxygenase products and prostaglandin metabolites using high-pressure liquid chromatography and radioimmunoassay.

A method is described for measurement of the cyclooxygenase products, thromboxane,prostacyclin, and prostaglandins (PG), and several prostaglandin metabolites. The procedure involves separation of the compounds by high-pressure liquid chromatography combined with identification and estimation by serologic analysis. These combined procedures have been used to identify and estimate five such products, PGE₂, PGE₁ PGF2α, PGF_1α, and 6-keto-PGF_1α, in the culture fluids of dog kidney cells stimulated by a tumor-promoting phorbol diester. The prostaglandin metabolites, 13,14-dihydro-15-keto-PGE₂, 13,14-dihydro-15-keto-PF2_2α, 13,14-dihydro-PGE₂, and 13,14-dihydro-PGF_2α, were not found in these culture fluids.     

Uterine vein prostaglandin levels in late pregnant dogs

We studied the uterine venous plasma concentrations of prostaglandins E₂, F_2α, 15 keto 13,14 dihydro E₂ and 15 keto 13,14 dihydro F_2α in late pregnant dogs in order to evaluate the rates of production and metabolism of prostaglandin E₂ and F_2α in pregnancy $\text{in vivo}$. We used a very specific and sensitive gas chromatography-mass spectrometry assay to measure these prostaglandins. The uterine venous concentrations of prostaglandin E₂ and 15 keto 13,14 dihydro E₂ were 1.35±.27 ng/ml and 1.89±.37 ng/ml, respectively; however, we could not find any prostaglandin F_2α and very little of its plasma metabolite in uterine venous plasma. Since uterine microsomes can generate prostaglandin F_2α and E₂ from endoperoxides, prostaglandin F_2α production $\text{in vivo}$ must be regulated through an enzymatic step after endoperoxide formation. Prostaglandin E₂ is produced by pregnant canine uterus in quantities high enough to have a biological effect in late pregnancy; however, prostaglandin F_2α does not appear to play a role at this stage of pregnancy.     

Measurement of 11-ketotetranor PGF metabolites: An approach for monitoring prostaglandin F2α release in the mare

The appearance and disappearance of 15-keto-13,14-dihydro-PGF_2α and 11-ketotetranor PGF metabolites have been measured in the blood and urine of the mare following i.v. injection of prostaglandin F_2α (PGF_2α). The basal plasma concentration of the 11-ketotetranor PGF metabolites was 10-fold greater than 15-keto-13,14-dihydro PGF_2α; after injection of PGF_2α, however, 15-keto-13,14-dihydro PGF_2α increased rapidly to concentrations exceeding those of the 11-ketotetranor PGF metabolites, which also increased but to a much lesser extent. The half-life for the disappearance of 15-keto-13,14-dihydro PGF_2α was about 30-fold shorter than that of the 11-ketotetranor PGF metabolites. Similar profiles were seen in the urine, except that the concentration of the 11-ketotetranor PGF metabolites was always greater than that of the 15-keto-13,14-dihydro PGF_2α. In the mare, the main plasma metabolite of PGF_2α appears to be 15-keto-13,14-dihydro PGF_2α, whereas the 11-ketotetranor PGF metabolites predominate in the urine.Similar patterns were seen for both types of metabolite in the plasma during luteolysis and early pregnancy. Because of the differences in rates of appearance and disappearance of these metabolites, measurement of both allows the detection of peaks of PGF_2α release in samples taken less frequently than is necessary when either type is measured alone.     

Enhanced lipid peroxidation and inflammation during heat exposure in rats of different ages: Role of α-tocopherol

To investigate early events possibly related to the development of heat shock, we examined whether inflammatory-(interleukin-6, tumor necrosis factor α and 15-keto-13,14-dihydro-PGF_2α) and peroxidative-(8-iso-PGF_2α and malondialdehyde) markers are altered during acute heat exposure and aging. We also studied the relationships between inflammatory and peroxidative markers in these settings. In order to prevent these reactions developed as a consequence of the conditions mentioned above, we tested the effects of α-tocopherol. Our results demonstrated that 15-keto-13,14-dihydro-PGF_2α and malondialdehyde in the liver were altered during acute heat exposure in the young and middle-aged rats and could be predicted by changes in the levels of circulatory cytokines. Regardless of age, the supplementation with α-tocopherol prevented changes in the plasma cytokine levels and 15-keto-13,14-dihydro-PGF_2α and malondialdehyde levels in the liver, during acute heat exposure. This study notably emphasized the ability of α-tocopherol to prevent different heat induced mechanisms, involved in induction of inflammatory or peroxidative reactions.     

Synthesis of diastereomeric bis-unsaturated prostaglandins

With the report given herein all diastereomers of PGF₂, PGE₂, and PGD₂ which bear the naturally recognized 15-S hydroxylated center, whether in the natural or $\text{ent}$-prostanoic acid skeleton, have been prepared by a route involving initial introduction of the carboxyl (α) chain (1). A major advantage of the initial α-ylation⁺ route is the facile reduction of the 13,14-en-15-one system with methanolic NaBH₄ which proceeds without competing 1,4-reduction. The products are thus free of 13,14-dihydro-PG₂ contaminants (2). The initial pharmaco logical evaluation of these diastereomers will be submitted for publication in this journal (3).     

Prostaglandins and prostaglandin metabolites in human gastric juice

Human gastric juice contains higher concentrations of PG metabolites than of unmetabolized PG indicating that local metabolism might play a role in limiting the biological activity of PG in gastric mucosa and has to be considered when investigating endogenous gastric PG. A major fraction of the 15-keto-13,14-dihydro-PGE₂ (KH₂PGE₂) formed in gastric mucosa and released into the gastric lumen seems to be rapidly dehydrated to a compound co-chromatographing with KH₂PGA₂, while the amounts of the bicyclic degradation product 11-deoxy-13,14-dihydro-15-keto-11,16-cyclo-PGE₂ (11-deoxy-KH₂-cyclo-PGE₂), as measured by radioimmunoassay, in freshly extracted gastric juice are negligible. Stimulation of secretion with pentagastrin does not influence significantly the concentrations of PG and PG metabolites in human gastric juice, but total output tends to increase parallel to the increase in secretion volume. Levels of immunoreactive 6-keto-PGF_1α in human gastric juice are much lower than those of PGE₂. Since human gastric mucosa synthesizes considerable amounts of PGI₂ and 6-keto-PGF_1α in vitro, the low levels of 6-keto-PGF_1α in gastric juice might indicate that PGI₂ formed by gastric mucosa in vivo is, like PGE₂ and PGF_2α, rapidly metabolized and/or removed preferentially via the blood stream.     

Preparation of two dinor-PGI2 metabolites from 6-keto-PGF1α by mycobacterium rhodochrous

The transformation of 6-keto-PGF_1α to two prostacyclin metabolites, 2,3-dinor-6-keto-PGF_1α (I) and 2,3-dinor-6,15-diketo-13,14-dihydro-PGF_1α (II) by $\text{Mycobacterium rhodochrous}$ UC-6176 is described. The finding that the bacterium oxidized 6-keto-PGF_1α to the 6,15-diketo metabolite II shows that it contains 15-hydroxy prostaglandin dehydrogenase and Δ¹³ reductase enzyme systems.     

The lack of an effect of furosemide on uterine prostaglandin metabolism in vivo

We determined the effect of 2 mg/kg intravenous furosemide on the production and metabolism of prostaglandin E₂ in the utero-placental unit of pregnant dogs. Uterine venous prostaglandins E₂ and 15-keto-13,14-dihydro E₂ were measured by gas chromatography-mass spectrometry. Even though the dose of furosemide was adequate to effect a good diuresis, neither the production nor the metabolism of prostaglandin E₂ by the uterus was altered by that dose of the drug. Using radioactive microspheres to measure hemodynamic parameters, we observed no change in uterine vascular resistance while renal vascular resistance decreased. Although the renal concentration of furosemide may be higher than the uteroplacental concentration, there is so far no evidence $\text{in vivo}$ that usual doses of furosemide enhance the production or inhibit the metabolism of prostaglandin E₂.     

Effect of PGD2, PGE2, PGF2α and PGI2 on blood pressure,heart rate and plasma catecholamine responses to spinal cord stimulation in the rat

The following experiments were designed in order to examine the inter-relationships of various prostaglandins (PG's) and the adrenergic nervous system, in conjunction with blood pressure and heart rate responses, $\text{in vivo}$. Stimulation of the entire spinal cord (50v, 0.3–3 Hz, 1.0 msec) of the pithed rat increased blood pressure, heart rate and plasma epinephrine (EPI) and norepinephrine (NE) concentration (radioenzymatic-thin layer chromatographic assay). Infusion of PGE₂(10–30 μg/kg. min, i.v.) suppressed blood pressure and heart rate responses to spinal cord stimulation while plasma EPI (but not NE) was augmented over levels found in control animals. PGI₂ (0.03–3.0 μg/kg. min, i.v.) suppressed the blood pressure response to spinal cord stimulation without any effect on heart rate or the plasma catecholamine levels. PGE₂ and PGF_2α(10–30 μg/kg. min, i.v.) did not change the blood pressure, heart rate or plasma EPI and NE responses to the spinal cord stimulation although PGF_2α disclosed an overall vasopressor effect during the pre-stimulation period. At the pre-stimulation period it was also observed that PGE₂, PGF_2α and PGI₂, had a positive chronotropic effect on the heart rate, the cardiac accelerating effect of PGE₂ was not abolished by propanolol. These $\text{in vivo}$ studies suggest that in the rat, PGE₂ and PGI₂ modulate sympathetic responses, primarily by interaction with the post-synaptic elements — PGE₂ on both blood vessels and the heart and PGI₂ by acting principally on blood vessels.     

Comparison of the effects of prostaglandins D2, F2α and E1 on spontaneous contractions of rabbit oviduct

The effects of PGD₂, PGF_2α and PGE₁ were studied on the circular muscle of post-ovulatory rabbit oviducts $\text{in vitro}$. PGE₁ inhibited spontaneous contractile activity. Lower concentrations of PGD₂ and PGF_2α were stimulatory and higher concentrations were inhibitory. Since PGD₂ may be produced in the oviduct, any hypothesis concerning the role of prostaglandins in the control of oviductal motility and ovum transport should include PGD₂ as well as PGFs and PGEs.     

Application of high performance liquid chromatography and gas chromatography-mass spectrometry to analysis of prostaglandin E1 in biological media.

A method for the quantitation of prostaglandin (PG) E₁ in biological samples of gas chromatography—mass spectrometry has been developed. PGE₁ was separated from PGE₂, 13,14-dihydro-PGE₂, and other potentially interfering prostaglandins by reversed phase high performance liquid chromatography. After conversion of PGE₁ to PGB₁ by treatment with methanolic KOH, PGB₁ was derivatized to the methyl ester trimethylsilyl ether and analyzed by selected ion monitoring using hexadeutero-PGE₁ as an internal standard. Measurable levels of PGE₁ were found in human and rat urine and in incubates of rat and rabbit renal papilla. PGE₁ excretion and production by renal slices was blocked by treatment with indomethacin. A complete mass spectrum of derivatized PGE₁ was obtained from PGE₁ generated by rabbit renal papillary slices.     

Temporal effect of prostaglandins E and F on human adrenal cAMP and cortisol output.

The $\text{in vitro}$ modulating effects of the E and F series prostaglandins upon the cAMP and cortisol output of normal human adrenal dice were evaluated with time and compared to the effects of ACTH. PGE₁ and PGE₂ significantly increased human adrenal cAMP levels; cortisol output increased in a dose related manner. Although the cortisol levels produced by E prostaglandins and ACTH were quantitatively similar, on a molar basis ACTH was greater than 10⁶ fold more effective. PGE_1α, PGF_2α, PGF_1β and PGF_2β depressed adrenal cAMP, except PGF_2α and PGF_2β at 100 μg/ml. PGF_1α and PGF_2α depressed cortisol levels at all doses. Similarly, PGF_1β and PGF_2β also depressed cortisol output, except PGF_2β at 100 μg/ml which was stimulatory. In both series of prostaglandins the temporal responses were dose related in regard to the cyclic nucleotide and steroid alterations. The findings demonstrate the E and F series prostaglandins act antagonistically in respect to cAMP and cortisol output. In addition, as the cAMP level and cortisol output are not always correlated, it appears that these prostaglandin mediated events are separable. The relationship between adrenal prostaglandins and cyclic nucleotides, therefore, invites a more sophisticated second messenger concept in terms of adrenocortical function, than currently proposed.     

Effects of 19-hydroxy-prostaglandins on oviductal and uterine motility

The effects of 19-hydroxy-prostaglandins (19-OH-PGs) were tested $\text{in}$$\text{vivo}$ on the rabbit oviduct and uterus and on the rhesus monkey ($\text{Macaca}$$\text{mulatta}$) uterus. The 19-OH-PGEs suppressed spontaneous oviductal and uterine activity in the rabbit. The qualitative effect on the rabbit oviduct of 19-OH-PGEs was similar to that of PGE₂. However, the typical response of the rabbit uterus to PGE₂ was an increase in muscle activity. With regard to the rabbit oviduct, 19(R)-OH-PGE₂ was as potent as PGE₂, but 19(S)-OH-PGE₂ was approximately $\text{1}\text{2}$ as potent as PGE₂. Based on the dose of 19-OH-PGEs usually required to cause a minimal suppression and the dose of PGE₂ required to cause a minimal stimulation of rabbit uterine activity, 19(R)-OH-PGE₂ was twice as potent as PGE₂ while 19(S)-OH-PGE₂ was $\text{1}\text{2}$ as potent as PGE₂. Stimulatory effects on the rabbit oviduct and uterus were observed following administration of 19-OH-PGEs and PGF_2α. The potency on the rabbit oviduct of 19(S)-OH-PGF_2α was about $\text{1}\text{5}$ to $\text{1}\text{10}$ that of PGF_2α; the potency of 19(R)-OH-PGF_2α was about $\text{1}\text{10}$ to $\text{1}\text{20}$ that of PGF_2α. Both 19-OH-PGFs were approximately $\text{1}\text{5}$ to $\text{1}\text{10}$ as potent as PGF_2α on the rabbit uterus. At the doses tested 19-OH-PGFs were inactive on the monkey uterus. Thus, these compounds are at least $\text{1}\text{5}$ as active as PGF_2α. In contrast, 19(R)-OH-PGE₂ had approximately the same potency as PGE₂ in stimulating monkey uterine activity; but 19(S)-OH-PGE₂ was approximately $\text{1}\text{3}$ as potent as PGE₂.     

Prostaglandin removal and metabolism by isolated perfused rat lung

We have investigated the mechanism(s) involved in the removal of prostaglandins (PG) from the pulmonary circulation by the lung. Unidirectional fluxes of PG from the circulation into the lung are measured in an isolated perfused rat lung preparation. Evidence is presented which suggests that a transport system for PG exists in lung tissue. This transport system is responsible for the removal of some PG from the circulation by the lung. PGE₁ and PGF_2α are substrates for this system, whereas PGB₁, PGA₁, and 15-keto-PGF_2α are not. Since PGA₁ is a substrate for the intracellular PG dehydrogenase, the selectivity of the lung's metabolism system for circulating PG is probably due to the selectivity of the transport system for PG. It is shown that the percentage of the pulmonary arterial concentration (C_A) of PGE₁ or PGF_2α that is metabolized on passage through the pulmonary circulation decreases rapidly as C_A increases. When the lungs were perfused with PGE₁ (PGF_2α), the metabolites detected in the venous effluent were 15-keto-PGE₁ (PGF_2α) and 15-keto-13,14-dihydro-PGE₁ (PGF_2α). The time course pattern of the appearance of metabolites in the venous effluent after the initiation of a constant C_A, and the relative concentrations of the metabolites in the venous effluent, were examined as a function of C_A.