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Aggregatibacter actinomycetemcomitans belongs to the HACEK group of fastidious Gram-negative organisms, a recognized cause of infective endocarditis. A. actinomycetemcomitans is also implicated in periodontitis, with rapid progress in adolescents. We recently demonstrated that the major outer membrane protein, OmpA1 was critical for serum survival of the A. actinomycetemcomitans serotype a model strain, D7SS, and that the paralogue, OmpA2 could operate as a functional homologue to OmpA1 in mediating serum resistance. In the present work, an essentially serum-sensitive ompA1 ompA2 double mutant A. actinomycetemcomitans strain derivative was exploited to elucidate if A. actinomycetemcomitans OMVs can contribute to bacterial serum resistance. Indeed, supplementation of OMVs resulted in a dose-dependent increase of the survival of the serum-sensitive strain in incubations in 50% normal human serum (NHS). Whereas neither OmpA1 nor OmpA2 was required for the OMV-mediated serum protection, OMVs and LPS from an A. actinomycetemcomitans strain lacking the LPS O-antigen polysaccharide part were significantly impaired in protecting D7SS ompA1 ompA2. Our results using a complement system screen assay support a model where A. actinomycetemcomitans OMVs can act as a decoy, which can trigger complement activation in an LPS-dependent manner, and consume complement components to protect serum-susceptible bacterial cells.

Inflammation and infection postpartum threaten the mother and her infant. Human milk provides a defense for the infant, but inflammatory complications like mastitis may lead to the cessation of breastfeeding. Antisecretory factor (AF) has a role in the regulation of secretory processes and inflammation. The objective of the study was to describe AF-levels in plasma and breast milk, and in relation to breast complications. Breastfeeding mothers (n = 95) were consecutively recruited at a Well Baby Clinic in Ume&aring, Sweden. At inclusion four weeks postpartum, samples of venous blood (10 mL) and breast milk (10 mL) were collected. Active AF was analyzed with ELISA using a monoclonal antibody mAb43, and was detected in all samples of plasma and breast milk with a positive correlation (Spearman coefficient = 0.40, p &lt, 0.001, Pearson correlation = 0.34, p &lt, 0.01). High AF-levels in plasma correlated with high AF-levels in breast milk. The results suggest a co-regulation between active AF in plasma and breastmilk, and/or a local regulation of AF in the breast. Further studies are needed to determine the pathways for the activation of AF-levels in breast milk and plasma.

Objective To describe clinical chemistry and weight changes after short-term food or sleep deprivation or multiple deprivations during civilian survival training. Methods Data from one baseline-controlled two-period crossover study designed to compare sleep deprivation for up to 50 hours with food deprivation for up to 66 hours (n = 12) and data from regular multiple-deprivations survival training comparing participants (n = 33) with nondeprived instructors (n = 10). Results Food deprivation was associated with decreased body weight, blood glucose, serum triglycerides, sodium, chloride, and urine pH, and there were increases in blood and urine ketones and serum free fatty acids. Sleep deprivation was associated with a minor decrease in hemoglobin and erythrocyte particle count and volume fraction and an increase in leukocytes. Conclusions The clinical chemistry and body weight changes associated with food deprivation were qualitatively similar to those observed in fasting obese patients but developed quicker in the survival training setting. Sleep deprivation had few effects on the clinical chemistry profile except for hematological variables. Physicians evaluating clinical chemistry data from patients subjected to short-term food or sleep deprivation should take the physiological state into account in their assessment.

This study assessed whether the persistence of Lactobacillus reuteri DSM 17938 and ATCC PTA 5289 in saliva could delay the regrowth of mutans streptococci (MS) after a full-mouth disinfection with chlorhexidine (CHX). A randomised, double-blind, placebo-controlled study with a 6-week intervention period and 3- and 6-month follow-up was performed. 62 healthy subjects with moderate to high counts of MS were randomly assigned to a test group (n = 32) or a placebo group (n = 30). Before onset of the intervention, subjects received two sessions of professional cleaning, flossing, and application of CHX varnish and rinsed their mouth with a CHX solution between the sessions (2 days). Thereafter, the test group used probiotic lozenges (2/day) containing L. reuteri (DSM 17938 and ATCC PTA 5289; 1 × 108 CFU of each strain), and the placebo group used identical lozenges lacking the lactobacilli. Saliva samples were collected and cultured onto selective media, and isolates of L. reuteri as well as DNA directly extracted from saliva were tested by polymerase chain reaction (PCR) with specific primers. Presence of salivary MS was analysed with a chair-side test. L. reuteri was frequently detected by culture during the intervention period but in only 3 test group subjects at follow-ups. Regrowth of MS statistically significantly differed depending on the presence or absence of L. reuteri DSM 17938 detected by PCR. We conclude that cultivable L. reuteri strains may only sporadically be confirmed after termination of the intervention, but subjects with PCR-detected L. reuteri demonstrated slower regrowth of MS.

Aggregatibacter actinomycetemcomitans is an oral and systemic pathogen associated with aggressive forms of periodontitis and with endocarditis. Outer membrane vesicles (OMVs) released by this species have been demonstrated to deliver effector proteins such as cytolethal distending toxin (CDT) and leukotoxin (LtxA) into human host cells and to act as triggers of innate immunity upon carriage of NOD1-and NOD2-active pathogen-associated molecular patterns (PAMPs). To improve our understanding of the pathogenicity-associated functions that A. actinomycetemcomitans exports via OMVs, we studied the proteome of density gradient-purified OMVs from a rough-colony type clinical isolate, strain 173 (serotype e) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). This analysis yielded the identification of 151 proteins, which were found in at least three out of four independent experiments. Data are available via ProteomeXchange with identifier PXD002509. Through this study, we not only confirmed the vesicle-associated release of LtxA, and the presence of proteins, which are known to act as immunoreactive antigens in the human host, but we also identified numerous additional putative virulence-related proteins in the A. actinomycetemcomitans OMV proteome. The known and putative functions of these proteins include immune evasion, drug targeting, and iron/nutrient acquisition. In summary, our findings are consistent with an OMV-associated proteome that exhibits several offensive and defensive functions, and they provide a comprehensive basis to further disclose roles of A. actinomycetemcomitans OMVs in periodontal and systemic disease.

Colonisation of the gastrointestinal tract is influenced by primary microbial exposure and bioactive factors in breastmilk. The aim was to explore the prevalence of oral Candida in the first year of life in relation to selected exposures. Oral Candida was studied in 100 healthy infants at 4 and 8 weeks, 3, 6 and 12 months of age and related to delivery mode, birth weight, infant health and feeding, antibiotics, antimycotics, steroids and probiotics in mother and infant, living conditions, maternal smoking and infections The association between lactoferrin and antisecretory factor in breastmilk and maternal serum haemoglobin, transferrin, and ferritin levels in relation to oral Candida was also explored. About 11% to 15% of the infants had oral Candida at the respective age. Colonisation was fairly stable until 6 months of age. There was no conclusive impact of the investigated exposures at entry. Infants with a furry pet at home had a lower frequency of Candida at 3 months, (P0.05) whereas all but one colonised infant had older siblings at 12 months (P0.01). Lactoferrin in breastmilk was negatively associated with colonisation at 6 months of age. It is concluded that 11 to 15% had oral Candida. Exposure to furry pets and siblings impacted oral Candida.

The urinary excretion of stable metabolites of thromboxane A2, such as 11-dehydro-thromboxane B2, reflects platelet activity in vivo. Efficient sample purification is required before analysis of thromboxane metabolites, due to the presence of large amounts of interfering material in urine. Analysis by gas chromatography–mass spectrometry after extensive sample work-up procedures provides the most reliable data, but detection by enzyme immunoassay may be reliable if sample cleanup is adequate. We describe an improved immunoassay procedure for 11-dehydro-thromboxane B2, which is based on a simple one-step solid phase extraction, by using Bond-Elut Certify II columns, followed by enzyme immunoassay by using commercially available reagents. 11-Dehydro-thromboxane B2 exists in two forms, with different chemical and immunological characteristics, which are in pH-dependent equilibrium. We kept 11-dehydro-thromboxane B2 in its open ring form throughout the assay, by incubating and handling samples at pH 8.6. The extraction step achieved a recovery of 83% (95% confidence interval 74–92%), the sensitivity of the enzyme immunoassay was doubled, and the reproducibility of the assay improved under these conditions. Intra- and interassay coefficients of variation were 3 and 13.8%, respectively. A single 500-mg dose of aspirin reduced the excretion of 11-dehydro-thromboxane B2 by 77±14%, suggesting good specificity. Comparison with gas chromatography–mass spectrometry in 28 urine samples showed excellent agreement between the two methods (r2=0.94; p

In the present paper we studied the influence of albumin on the in vitro metabolism of 12-hydroxyeicosatetraenoic acid (12-HETE) and arachidonic acid in leukocytes and aspirin-treated platelets. In the presence of physiological concentrations of albumin, the metabolism of both 12-HETE and arachidonic acid was substantially altered, implicating the importance fatty acid binding proteins might have on the profile of products formed both in vitro and in vivo. The results clearly showed that albumin effectively withdraws arachidonic acid and 12-HETE from further metabolism by the leukocytes but does not influence the conversion of arachidonic acid to 12-HETE by the platelets. Thus, some of the hypotheses concerning transcellular metabolism raised from in vitro data within the eicosanoid field might have little relevance for the in vivo situation.

The effects of oxygen radicals, generated by the hypoxanthine-xanthine oxidase (XO) system, on pulmonary circulation and release of cysteinyl-containing leukotrienes (LTs) were studied in pigs after XO infusion into the right atrium. A 2.3-fold increase in pulmonary vascular resistance (PVR) (p0.05 vs. baseline) and a 2.1-fold increase in LT release (p0.05 vs. baseline) was observed. Pretreatment with indomethacin and allopurinol attenauted the vascular response (p0.01 and p0.05 vs. XO), and the LT release was inhibited by allopurinol and catalase (p0.01 and p0.02 vs. XO). We conclude that oxygen radicals stimulate lipoxygenase metabolism. This coincides with the observed increase in PVR, however, no causal relationship can be derived from the data presented.

Objectives The study was designed to compare effects of food deprivation (FD) and sleep deprivation (SD) on cognition during survival training. Methods In a cross-over design (n=12), the effects of FD (up to 66 hours followed by 500 kcal intake over 24 hours) and SD (up to 50 hours) on cognitive variables, blood glucose, and 3-OH-butyrate were studied. Results Food deprivation and SD impaired attention-dependent tasks. The FD impairment of simple reaction time was independent of blood glucose levels, which were normalized by a 500 kcal intake over 24 hours while the reaction time was not. Sleep deprivation and FD impaired maze-solving performance on all variables except rule breaks, which were significantly occurring after 50 hours of SD. Delayed word recall was impaired by SD for 50 hours. On the Balloon Analogue Risk Task, SD was associated with reduced risk-taking. In a gambling task, both SD for 50 hours and FD for 66 hours were associated with a tendency to make early choices when presented with consecutive choices, but the risk-taking was not affected. Conclusions Sleep deprivation has multiple cognitive effects, including attention, memory, visual-spatial ability, and risk-taking. Food deprivation had no affect on risk-taking, while the other tasks were affected in a way similar to SD but were less pronounced. The FD effects on cognition did not appear to depend on blood sugar levels. The need to sleep should be prioritized in survival situations to avoid cognitive impairment.

In vivo production of thromboxane (TX) A2 and the cysteinyl-containing leukotrienes (LT) C4, D4, and E4 in correlation to airway responses was studied. Bronchial provocation with specific allergen in atopic asthmatics was followed by a significant increase in urinary concentration of immunoreactive LTE4 (34 +/- 6 before versus 56 +/- 7 ng/mmol creatinine after allergen challenge; n = 5) and 11-dehydro-TXB2 (164 +/- 29 versus 238 +/- 25 ng/mmol creatinine). In the presence of the leukotriene-antagonist ICI-204,219, which significantly increased the PD20 for allergen, the increment in urinary excretion of LTE4 was even higher (60 +/- 8 versus 288 +/- 128 ng/mmol creatinine; n = 5). In contrast, provocation with histamine (n = 5) did not provoke release of leukotrienes or thromboxane, nor was inhalation of LTD4 (n = 7) associated with increased urinary concentration of 11-dehydro-TXB2. Furthermore, bronchoconstriction induced by inhalation of lysine-aspirin in aspirin-sensitive asthmatics (n = 4) was followed by increased levels of LTE4 in the urine, whereas the levels of 11-dehydro-TXB2 remained the same. Finally, the basal levels of LTE4 in the urine of nine aspirin-sensitive asthmatics were elevated as compared with 15 other asthmatics (112 +/- 54 versus 38 +/- 20 ng/mmol creatinine; p less than 0.001). The findings support that the cysteinyl-leukotrienes are potential mediators of allergen-induced asthma and that the release of LTE4 and 11-dehydro-TXB2 into the urine appeared to be a direct and dose-dependent effect of the antigen-antibody reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

15(S)-Hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE) was by far the most abundant metabolite of arachidonic acid in chopped human bronchi, as identified by reverse phase HPLC, uv spectrometry, and GC/MS. The quantitation of monohydroxyeicosatetraenoic acids (mono-HETEs) was performed by the use of 16(S)-hydroxy-9(Z),12(Z),14(E)-heneicosatrienoic acid as internal standard. Thus, significant amounts of 15-HETE were obtained in incubations of bronchi in buffer alone, but the addition of exogenous arachidonic acid (3-100 microM), dose-dependently increased the formation, with maximal levels reached at around 10 min. In contrast, challenge with ionophore A23187 or anti-human IgE did not stimulate the production of 15-HETE in the bronchi. Nordihydroguaiaretic acid inhibited the production of 15-HETE, whereas indomethacin did not. Small amounts of 8,15-diHETEs were detected in incubations with exogenous 15H(P)ETE. Lipoxins were however not detected under any of the incubation conditions used. Furthermore, removal of the airway epithelium substantially diminished the production of 15-HETE in the bronchi. Finally, bronchi were obtained from three patients with asthma, and the amounts of 15-HETE in these specimens were significantly higher than those found in tissues from nonasthmatics. Also, in peripheral lung parenchyma and pulmonary blood vessels 15-HETE was the major mono-HETE after stimulation with arachidonic acid but the levels were about 10 times lower than in the bronchi. As another difference, challenge of the parenchyma with the ionophore A23187 made 5-HETE the predominant mono-HETE. Taken together, airway epithelium appears to be the major source of 15-HETE in the human lung and the findings in specimens of asthmatics raise the possibility that 15-HETE somehow is involved in airway inflammation.

Exogenous [3H]leukotriene B4 (LTB4) was converted into several polar and non-polar metabolites in the chopped human lung. One of the major metabolites was identified as 5(S),12-dihydroxy-6,8,14-eicosatrienoic acid (10,11-dihydro-LTB4) by means of co-chromatography with authentic standards, ultraviolet spectrometry and gas chromatography-mass spectrometry. Analysis on chiral straight phase HPLC revealed the presence of both the 12(S) and 12(R) epimers of dihydro-LTB4. Dihydro-LTB4 was also formed from endogenously generated LTB4 in ionophore A23187 stimulated incubations. The dihydro metabolites were approximately 100 times less potent than LTB4 in causing guinea pig lung strip contraction and leukocyte-dependent inflammation in the hamster cheek pouch in vivo.

12-Hydroxyeicosatetraenoic acid (12-HETE) is one of the major metabolites formed from arachidonic acid in platelets. We have recently shown that the in vitro metabolism of 12-HETE by human leukocytes, with and without stimulation, is effectively inhibited by the addition of physiological concentrations of albumin, probably by sequestration of the compound. In the present paper, we have studied the in vivo metabolism of 12-HETE in the rabbit, using either [1- 14 C]- or [ 14 C(U)]12-HETE. Distribution of radioactivity was followed in urine, plasma, and bile, as well as in a number of tissues. In most of the tissues examined, the hydrophilic radioactivity constituted more than 50% of the total radioactivity after 20 min. When the lipophilic fraction was analyzed, around 15% of the radioactivity was shown to be unesterified 12-HETE, and only a very minor part could be detected as metabolites. The dominating lipophilic compound in the circulation after i.v. administration of radiolabeled 12-HETE was at all time points (1–60 min.) the parent compound, as analyzed by HPTLC and HPLC. A comparison of the plasma metabolite profiles obtained when [1- 14 C]- and [ 14 C(U)]12-HETE were used displayed almost identical patterns, thus indicating that β-oxidized metabolites either were not formed or were rapidly removed from the circulation. The appearance of large amounts of water-soluble radioactivity with time supported the latter conclusion. Several minor metabolites were seen that chromatographed in the dihydroxy acid region as judged by HPLC and TLC. The major one of these compounds represented about 10% of the lipophilic plasma radioactivity after 60 min., while unmetabolized 12-HETE at this stage still represented about 30%. The metabolite had a polarity similar to 12,20-dihydroxyeicosatetraenoic acid; however, when chromatographed together, these two compounds separated, indicating a different structure of the metabolite. Our findings are in agreement with in vitro data concerning the protective effect of albumin on the metabolism of 12-HETE and is the first extensive metabolic study of 12-HETE in vivo covering all metabolic possibilities involving the carbon skeleton.

The metabolism of arachidonic acid by washed human platelets was investigated. [1-14C]Arachidonic acid was extensively converted to [1-14C]12-hydroxyeicosatetraenoic acid. In addition, several minor labeled products were formed with a considerably lower specific activity, indicating their preferential formation from endogenous substrate. These were dihydroxy metabolites of arachidonic acid with conjugated triene structures and were identified as 14,15-dihydroxyeicosatetraenoic acid (three isomers) and 8,15-dihydroxyeicosatetraenoic acid (three isomers). The identification was based on comparison with reference compounds with respect to chromatographic properties, characteristic UV spectra, and mass spectrometry of several derivatives. Bradykinin (10(-8)-10(-5) M) was found to enhance the formation of all these compounds. In addition, the monohydroxy acid fraction was found to contain 15-hydroxyeicosatetraenoic acid. The present investigation thus demonstrates the occurrence of a 15-lipoxygenase in human platelets in addition to the previously known 12-lipoxygenase.

Conjugates of prostaglandins and thromboxanes with tritium labeled amino acids were prepared and employed as labeled ligands in prostaglandin and thromboxane radioimmunoassays. Assays for PGF2 alpha, 15-keto-13, 14-dihydro-PGF2 alpha, TXB2 and 15-keto-13,14-dihydro-TXB2 were evaluated in comparative studies using either these heterologous ligands or the corresponding homologous tritiated eicosanoid as tracers. Binding properties for the respective antibodies were found to be similar using either tracer. Three biological studies were also conducted, viz. study of the release of TXB2 during collagen induced platelet aggregation, of 15-keto-13,14-dihydro-TXB2 during guinea pig pulmonary anaphylaxis, and of PGF2 alpha (measured as 15-keto-13,14-dihydro-PGF2 alpha in peripheral plasma) during bovine luteolysis. The analyses gave comparable results using either the heterologous or the homologous assay. Thus, this type of labeled prostanoid conjugates may serve as a convenient alternative to homologous tracers in radioimmunoassay. Heterologous tracers may even in certain cases provide the only simple solution to the problem of preparing a labeled ligand of high specific activity.

The pattern of metabolites appearing in the circulation after intravenous injection of [9β-3H]prostaglandin F2α was investigated in the human. Analysis of profiles of products was performed by two-dimensional TLC and autoradiography. Identification of labeled metabolites was accomplished by comparing their chromatographic behaviour with reference compounds in several chromatographic systems. After injection of [9β-3H] prostaglandin F2α the initially formed metabolite was 15-keto-13,14-dihydroprostaglandin F2α. However, this compound only dominated the spectrum of metabolites during the first few minutes, and several more polar products soon appeared. About 20 min after the injection the most prominent metabolite was 5α,7α-dihydroxy-11-ketotetranorprostane-1,16-dioic acid, which remained the dominating plasma compound and was also the major metabolite in urine. Several other highly oxidized products were also identified in plasma. Also these metabolites appeared later and remained longer in the circulation than the initially formed 15-ketodihydro metabolite. Our findings suggested that the more degraded metabolites might serve as more reliable plasma parameters for monitoring prostaglandin production than the traditional parameter, 15-keto-dihydroprostaglandin F2α. This hypothesis was supported by radioimmunoassay of metabolite levels in plasma appearing after either exogenous (intravenous administration) or endogenous prostaglandin F2α (late human pregnancy and parturition). In all cases studied, the tetranor metabolites remained elevated in the circulation for several hours, in contrast to their precursor, 15-ketodihydroprostaglandin F2α, which disappeared rapidly.

Prostaglandin F2 alpha (PGF2 alpha), prostaglandin E2 (PGE2) and thromboxane B2 (TXB2) levels were determined by radioimmunoassay (RIA) in knee joint fluid in two groups of patients with classical or definite rheumatoid arthritis (RA). One group had no treatment for one week prior to the study. The second group consisted of patients who developed acute exudative arthritis despite the use of various non-steroidal anti-inflammatory drugs (NSAID). The main metabolites of PGF2 alpha in plasma and urine were measured in the untreated group. The conclusion to be drawn from this study are: 1) Treatment with NSAID effectively lowers PGF2 alpha and PGE2 and TXB2 levels in joint fluid. 2) Signs and symptoms of arthritis may persist in spite of lowered levels of PGF2 alpha and PGE2 in the joint fluid. 3) The main metabolites of PGF2 alpha in plasma and urine are above the low-normal limit in untreated patients. 4) The ratio TXB2/PG was low in untreated patients, suggesting that the thromboxane pathway is quantitatively minor in rheumatoid joint inflammation.

The leukotrienes (LT's) are a group of arachidonic acid derivatives implicated as mediators of allergic bronchoconstriction and acute inflammation. Tracheal spirals and strips of lung parenchyma from guinea pigs were used under non-flow conditions to characterize the contractions caused by LTA4, LTB4 and LTC4. Cumulative administrations of leukotrienes desensitized the lung strip, whereas non-cumulative dose-response relationships for the leukotrienes and histamine were reasonably parallel. Half maximal contractions of the lung strips were obtained at a final bath concentration of 1 nM for LTC4 and 300 nM for LTA4 or LTB4, as compared with 6 000 nM for histamine. In the trachea, LTC4 was approximately 100 times more potent than LTA4 and histamine. Leukotrienes B4 and C4, but not acetylcholine or histamine, elicited release of the bronchoconstrictive thromboxane A2 from the lung under non-flow conditions. Indomethacin blocked the contractile response to LTB4, whereas the contractile effect of LTC4 remained unaltered. The beta-adrenoceptor agonist isoproterenol and the LTC4 antagonist FPL 55712 attenuated the contraction, but not the release of thromboxane A2, induced by LTC4. Changing to a perifusion technique rendered the lung strips less sensitive to the direct action of LTC4, and released thromboxane A2 now contributed significantly to the contractile response. In addition, the perifusion experiments indicated that LTB4 released histamine as well. We conclude that the chemoattractant LTB4 is an indirectly acting bronchoconstrictor, whereas the slow reacting substance LTC4 contracts the airway muscle by a predominantly direct mechanism. The exquisite bronchoconstrictive activity of LTC4 may be unrelated to its ability to induce formation of thromboxane A2.

Both vasopressin and PGF2 alpha are effective uterine stimulants in the non-pregnant human uterus, especially around the onset of menstruation. In order to clarify the relationship of these hormones to menstrual pain, plasma concentrations of vasopressin and two prostaglandin metabolites (15-keto-13,14-dihydro-PGF2 alpha and 11-ketotetranor PGF metabolites) were measured in serial blood samples taken premenstrually and during menstruation. Five women with premenstrual pain gave 7-9 blood samples at intervals of 30 minutes on the day preceding the onset of menstruation. From 5 women with severe primary dysmenorrhea a corresponding series of blood samples were taken during the first day of menstruation. Two groups of 5 women with no symptoms served as controls, either premenstrually or during menstruation. In the women with premenstrual pain the vasopressin concentrations were significantly higher than in the corresponding control group. Even higher and markedly fluctuating vasopressin levels were found in the women with dysmenorrhea who, in general, had more intense pain than the women with premenstrual symptoms. In the group with dysmenorrhea there was also a significant rise in plasma concentration of the PG metabolites. No such increase was seen in the group with premenstrual pain. It is concluded that the pathophysiology of premenstrual pain could imply increased vasopressin secretion. The more severe pain in primary dysmenorrhea seems to be the result of a combined effect of vasopressin and PGF2 alpha.

A radioimmunoassay was developed for a mono-O-methyl derivative of thromboxane B2. The antibodies showed high specificity for this compound and cross reacted only 1.2% with thromboxane B2 and less than 0.1% with prostaglandins and prostaglandin metabolites. The method had a sensitivity of 7 picog. The radioimmunoassay was employed in studies where thromboxane A2 was generated in human platelets and immediately converted into mono-O-methyl thromboxane B2 by treatment of the sample with a large volume of methanol. In some of the experiments, thromboxane B2 was simultaneously measured by a separate radioimmunoassay. Using these two assays it was demonstrated that thromboxane A2 could be detected only during the earlier stages of the platelet aggregation, whereas thromboxane B2 rapidly reached a constant level. In a separate experiment, the half-life of thromboxane A2 in buffer was found to be 32.5±2.5 (S.D.) sec at 37°C; the compound was more stable at lower temperatures. The t 1 2 for thromboxane A2 was also considerably longer in plasma.

Leg lymph was collected from pentobarbital anaesthetized rabbits before and after scalding injury of the paw (75 degrees C for 20 s), and the contents of cyclic AMP (cAMP), prostaglandin E2 (PGE2) and PGF2 alpha and thromboxane B2 (TXB2) in lymph were determined. After injury lymph flow increased about four times. The maximal rate of flow was found between 30 and 60 min after scalding. The efflux of cAMP and immunoreactive iPGE2, iPGF2 alpha and iTXB2 also increased. The maximum values were detected at approximately 0-30, 30-60, 30-60 and 180-240 min, respectively, after the injury. The output of cAMP, iPGE2 and iPGF2 alpha and iTXB2 in lymph of the contralateral non-scalded paw remained low throughout the experiments. When rabbits were injected with indomethacin (2.5 mg/kg) or diclofenac sodium (2.5 mg/kg) immediately after the scalding injury, the efflux of cAMP, iPGE2 and iPGF2 alpha were low. Lymph flow was markedly reduced after treatment with diclofenac sodium; treatment with indomethacin did not significantly affect lymph flow. The results suggest a prostaglandin-dependent formation of cAMP following scalding injury which may be related to the initial responses to scalding.

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.

The profiles of circulating metabolites of prostaglandin F2 alpha were investigated in a number of species, viz. rat, rabbit, guinea pig, cattle and sheep. The aim of the study was to identify in each animal major plasma metabolites that outlast the initially formed, short-lived 15-keto-13, 14-dihydroprostaglandin F2 alpha and might thus serve as better parameters for monitoring prostaglandin production in vivo. Tritium-labeled prostaglandin F2 alpha was injected intravenously and frequent blood samples were collected. The metabolic profiles at different stages were visualized using two-dimensional thin-layer chromatography and autoradiography. Identification of circulating products was achieved by comparison with reference compounds using several chromatographic methods, and by gas chromatography-mass spectrometry in cases where larger amounts of the prostaglandin had been administered. In the rabbit a similar study was also done with tritium-labeled prostaglandin E2. Certain species differences were seen in the removal of labeled compounds from the circulation, the elimination being most efficient in the guinea pig. Further differences were seen in the profiles of circulating prostaglandin metabolites. The first appearing major prostaglandin F2 alpha metabolite was always 15-ketodihydroprostaglandin F2 alpha. However, this compound was later replaced in the circulation by a number of more degraded products, the profiles of which were relatively typical for each species. Thus, in cattle, rat and guinea pig, the earliest-formed metabolites, 15-ketodihydroprostaglandin F2 alpha and its tetranor counterpart, 5 alpha, 7 alpha-dihydroxy-11-ketotetranorprostanoic acid, remained comparatively prominent plasma products, whereas highly polar dicarboxylic acids rapidly dominated the metabolite spectrum in the ovine and lapine circulation. These differences were further supported by separate kinetic experiments, using unlabeled prostaglandin F2 alpha and radioimmunological determination of formed products. These latter experiments also demonstrated further pronounced species differences in the rat of elimination of the different prostaglandin metabolites. A considerable interconversion between prostaglandin E and F compounds was also demonstrated in some species. In conclusion, the traditional prostaglandin parameters in plasma, the 15-ketodihydrometabolites, were found not to be the best parameters in all species. It is suggested that species differences in prostaglandin metabolism are taken ito consideration when the optimal analytical protocol is sought for future biological studies. Some alternatives are suggested in the present paper.

Many difficulties encountered in prostaglandin and thromboxane assay can be overcome by metabolic consideration of which compound is the best target for measurement. The following requirements must be fulfilled: the chosen compound should represent a constant and preferably major fraction of the studied pathway; it should not be formed as an artifact during collection and processing of the samples; its half-life in the biological material under study should be long; and it must be chemically stable. Our knowledge of prostaglandin metabolism now enables us to solve most assay problems for prostaglandins of the E and F type and, at least in some cases, also compounds of the D type. In general, either the 15-ketodihydro metabolites or the beta-oxidized end products thereof should be monitored; either as such or as chemically stable degradation products. In the thromboxane area, most assays have so far been directed at TxB2. The present study, however, shows that this compound is a highly unsuitable target for monitoring, since it may be formed in large amounts during sample collection. Metabolic studies indicate that an early metabolite, 11-dehydro-TxB2, is a better alternative. Quantification of this product however presents certain problems: it occurs in two different forms and the equilibrium is highly dependent on for example pH. A recently developed radioimmunoassay was employed in kinetic studies on TxB2 metabolism in the human and confirmed that 11-dehydro-TxB2 gives a more reliable picture of events than its parent compound, TXB2.

The prostaglandins, thromboxanes and leukotrienes constitute a rapidly growing family of compounds, all of which are oxygenated derivatives of certain polyunsaturated fatty acids, such as arachidonic acid. Most of these metabolites are biologically very potent substances, displaying a wide variety of actions in many different biological systems. The prostaglandins were originally discovered in the reproductive tract, though it is now known that most cell types in the body possess the enzymes necessary for their biosynthesis. Due to the ubiquitous occurrence of the prostaglandins and related substances, as well as their strong and diversified biological effects, they are believed to participate in numerous biological processes, physiological as well as pathological. In certain cases such a participation has also been proved: in the reproductive tract, prostaglandins are known to be involved in such events as pregnancy, parturition, dysmenorrhea and luteolysis.

In order to identify suitable parameters for measurement of thromboxane production in vivo the metabolism of TXB2 was studied in the human. [3H8]-TXB2 was given intravenously to a healthy human volunteer. Blood samples were collected for 50 min after the injection, and urine was collected for 24 hours. The urinary and blood metabolic profiles were visualized by the use of two-dimensional TLC and autoradiography. Identification of metabolites was achieved with GC/MS and in some cases by cochromatography with reference compounds in TLC and GC. In blood, unmetabolized TXB2 was the dominating compound during the first 30 min. Three less polar metabolites appeared, two of which were identified as 11-dehydro-TXB2 and 11, 15-didehydro-13, 14-dihydro-TXB2, respectively. The third compound was tentatively identified as 15-dehydro-13, 14-dihydro-TXB2. Since 11-dehydro-TXB2 was one of the major metabolites in blood as well as urine, it was deemed suitable as target for measurement of thromboxane production in vivo . The advantages of 11-dehydro-TXB2 over its parent compound, TXB2, were demonstrated in experiments where unlabeled TXB2 was injected i.v. to a human volunteer, and the blood and urinary levels of both compounds were then followed by radioimmunoassay. Measured levels of 11-dehydro-TXB2 were found to give a more reliable picture of metabolic events than TXB2, the latter compound to a large extent reflecting technical difficulties during blood sample collection.

The metabolism of tritium labeled 15-methyl-PGF2α, administered intra-amniotically, was studied in seven cases of mid-trimester abortion. The disappearance of the compound from the amniotic sac was a very slow process. A metabolite, dinor-15-methyl-PGF2α, was found in small amounts in the amniotic fluid and also in extracts of placenta and fetal liver, lung, and kidney. Plasma levels of 15-methyl-PGF2α in the maternal circulation were determined.

A method was developed for quantitative determination of endogenous production of prostaglandin (PG)E1, PGE2 and PGE3 by Rana temporaria lung, heart and urinary bladder homogenates, since these tissues contain the precursors, 8,11,14-eicosatrienoic, arachidonic and 5,8,11,14,17-eicosapentaenoic acids. Following homogenization and shaking at 22°C for 30 min, media were extracted by XAD-2, treated with sodium hydroxide in order to convert PGE compounds into PGB compounds, purified by thin-layer chromatography, and analyzed by high-performance liquid chromatography with homo-PGE1 as an internal standard. The ratio of prostaglandins E1, E2 and E3 compared to the ratio of fatty acid precursors in tissue suggested that the tissue content of precursor is not the only factor determining the type of prostaglandin synthesized.

Progesterone and the main plasma metabolite of PGF2alpha, 15-keto-13, 14-dihydro-PGF2alpha, were determined at hourly intervals in the peripheral circulation during luteolysis in two heifers. The prostaglandin release was found to occur during 2-3 days as rapid pulses with a duration of 1-5 hours prior to and during luteolysis, which was indicated by decreasing levels of progesterone.

[9β-3H]Prostaglandin F2α was incubated with the particle free fraction of a homogenate of guinea pig lung. One less polar metabolite appeared, which was assigned thje strcuture 9α.11α-dihydroxy-15-ketoprost-5-enoic acid. The identification was based on gas chromatographic and mass spectrometric analyses of the trimethylsilyl ether, acetate, methoxime-trimethlsily ether and methoxime-acetate derivatives of the methyl ester. The position of the double bond was established by oxidative ozonolysis of the methlester and acetate followed by gas chromatogralphymass spectrometry.

SUMMARY 1. The preparation of tritium-labeled prostaglandin F1, is described. 2. After administration to rats, about 30 % of the radioactivity was recovered in urine and about 8% in feces. 3. The labeled products in urine consisted almost exclusively of more polar metabolites. 4. The main labeled urinary metabolite has been shown to be 2,3-dinorprostaglandin Fi,. 10 MINUTES AcknowledgmentsThe authors are greatly indebted to Dr. R. Ryhage for the mass spectrometric analyses. The technical assistance of Miss C. Carlon and Miss U. Hedin is gratefully 0 acknowledged. REFERENCES FIG. 4. Gas-liquid chromatography of the hydrogenated tri- acetate of Metabolite II. Material (1.2 X 106 d.p.m. of 3H) ob- 1. ~NGG~RD, E., AND SAMUELSSON, B., J. Biol. Chem., 239, 4097 tained from Fractions 12 to 20 in the experiment described in Fig. (1964). 1 was treated with diazomethane, hydrogenated (PtOz in ethanol), 2. SAMUELSSON

Incubation of 8,11,14-eicosatrienoic acid with homogenates of sheep vesicular gland yielded a new product, which has been identified as 9α, 15-dihydroxy-11-ketoprost-13-enoic acid. The identification was based on mass spectrometry of the methyl ester and the methoxime derivative, and on infrared spectroscopy and ultraviolet spectroscopy, before and after treatment with alkali. Reduction of the isolated compound with borodeuteride yielded prostaglandin F1α containing deuterium in the ring, as established by mass spectrometry of a derivative. Further proof for the location of the keto group in the isomer was obtained through biosynthesis from [9-3H,3-14C]- and [11-3H,2-14C]8,11,14-eicosatrienoic acids. Incubation with [13D-3H,3-14C]8,11, 14-eicosatrienoic acid revealed that the 13d-hydrogen atom is retained during the formation of the isomer, which is also the case with prostaglandin E1. The mechanism of the conversion of 8,11,14-eicosatrienoic acid into prostaglandins is discussed. The identification of 9α,15-dihydroxy-11-ketoprost-13-enoic acid and the finding that 3H at C-9 is retained during its formation further support the involvement of an endoperoxide in the biosynthesis of prostaglandins.

Laboratory experimentation was conducted on the metabolism of PG(prostaglandin)F2alpha in a female subject. Laboratory preparation of the chemicals involved is described in detail. Tritium-labelled PGF2alpha was administered intravenously in a female subject and collection of venous blood from the contralateral arm was started simultaneously and continued for 10 minutes after completion of the injection. This collected blood was submitted to a number of laboratory analytic procedures in order to follow the initial transformations through PGF2alpha metabolism. 2 radioactive products--1 major metabolite and 1 minor metabolite--appeared in the blood and were identified. The structures of these 2 metabolites are analyzed and reported. Although the pathways in the formation of these 2 metabolites have not been studied in humans, the 1st step is believed to be enzymatic oxidation of the allylic alcohol group at C-15, reduction of the Delta 13 double bond, and subsequent reduction of the oxo group at C-15.

[9β-3H]Prostaglandin F2α was injected intravenously into female subjects and the structures of metabolites appearing in the urine were determined. Apart from the earlier identified main urinary metabolite, 5α,7α-dihydroxy-11-ketotetranorprosta-1,16-dioic acid and its δ-lactone, the following metabolites were found: 7α,9α,18-trihydroxy-13-ketodinorprost-3-enoic acid, 7α,9α-dihydroxy-13-ketodinorprost-3-en-1,18-dioic acid, 7α,9α-dihydroxy-13-keto-(dinor, ω-dinor)prost-3-en-1,16-dioic acid, the γ-lactone of 7α,9α,13-trihydroxy-(dinor,ω-dinor)prost-3-en-1,16-dioic acid, 5α,7α,11-trihydroxytetranorprosta-1,16-dioic acid and 5α,7α,16-trihydroxy-11-ketotetranorprostanoic acid. The two latter metabolites were also present as δ-lactone derivatives.

[9β- 3 H]Prostaglandin F 1α was injected subcutaneously to male guinea pigs. Six metabolites were isolated from urine; three C 18 compounds: 7α,9α-dihydroxy-13-ketodinorprostanoic acid, 7α,9α,13-trihydroxy-dinorprost-11-enoic acid (dinorprostaglandin F 1α ) and 7α,9α,13-trihydroxy-dinorprostanoic acid (dihydro-dinorprostaglandin F 1α ); and three C 16 compounds: 5α,7α-dihydroxy-11-ketotetranorprostanoic acid, 5α,7α,11-trihydroxy-tetranorprost-9-enoic acid (tetranorprostaglandin F 1α ) and 5α,7α 11-trihydroxy-tetranorprostanoic acid (dihydro-tetranorprostaglandin F 1α ). Identification was based on gas chromatography-mass spectrometry of several derivatives, as well as comparison with reference compounds.

Prostaglandin F2α, unlabeled or tritium-labeled, was administered intraamniotically to eight women in the second trimester of pregnancy. The absorption of PGF2α from amniotic fluid and the metabolism of the compound were studied in samples of blood, amniotic fluid, and urine. The radioactivity in amniotic fluid was found to disappear very slowly, and 5 to 10 hrs after the administration several other radioactive compounds appeared in the amniotic fluid. The main product was identified as 9α, 11α-dihydroxy-15- -ketoprost-5-enoic acid by gas chromatography-mass spectrometry of two derivatives. The metabolites excreted into urine were found to be identical with those obtained after intravenous injection of PGF2α. Low concentrations of PGF2α, around 0.2–0.3 nanog/ml, were found in plasma following intraamniotic administration. The metabolite was found in much higher concentrations; the maximum levels ranged between 1.5 and 14.8 nanog/ml.

The main urine metabolite of prostaglandin F2 alpha (5 alpha, 7 alpha-dihydroxy-11-ketotetranor-phostane-1, 16-dioic acid, PGF-metabolite) was determined by mass spectrometry (MS) and radioimmunoassay (RIA) in burned patients treated under routine conditions. The amount of the PGF-metabolite as determined by MS was 130 and 67 microgram/24 h (normal value 24 +/- 17 microgram/24 h) on days 3 and 5 respectively in one patient. In serial determinations using RIA the urine level of the PGF-metabolite was within normal values during the first days and rose to a broad peak 1-4 weeks after the injury. Thromboxane B2 (TXB2) was identified and quantitated in burn blister fluid. The amount of TXB2 was 1.7 ng/ml.

Fig. 1 summarizes the structures of primary PGE2 and PGF2a (upper line), their initially formed 15-ketodihydro-metabolites which appera early in blood after release (middle), and their β- and ω-oxidized metabolites, which appear later and remain longer in the circulation and also dominate the urinary profile (lower line). No single compound can be put forward as the ideal assay parameter: depending on aim and design of the study, either of these compounds may be monitored. The chemical instability of PGE compounds should however be kept in mind: generally it is safer to induce degradation by alkali treatment into a stable product prior to assay.

The effects of leucotrienes C4 (LTC4), D4 (LTD4), Prostacyclin (PGI2) and Thromboxane A2 (TXA2) were studied on superfused human cerebral artery strips. LTC4 and LTD4 neither contracted nor relaxed the strips. PGI2 caused a dose-dependent relaxation from 0.2 nmol to 0.8 nmol. When given simultaneously with a vasoconstrictor substance, PGI2 had an almost complete inhibitory effect. TXA2 caused a dose-dependent contraction from 0.03 nmol to 0.15 nmol. Also, TXA2 was more potent than 5-hydroxytryptamine (5-HT) and prostaglandin E2 (PGE2) with a contraction threshold of about 0.1 nmol. Prostaglandin E1 (PGE1), H2 (PGH2), and F2a (PGF2a) had contraction thresholds of about 1 nmol. Only a stable endoperoxide analogue (EPA), was more potent than TXA2, with a contraction threshold of 0.005 nmol. The possible role of these substances in producing ischaemic manifestations after subarachnoidal haemorrhage (SAH) is discussed.

Radioimmunoassay is based on a competition between labeled and unlabeled molecules of a certain compound for the binding sites on an antibody directed against the same compound. The presence of a large amount of unlabeled compound will lead to an extensive displacement of labeled molecules from the binding sites, thus, to a high radioactivity in the unbound fraction. The absolute amounts of the compound assayed are obtained from comparison with a standard curve with known amounts.

The effect of intrauterine iodine infusion on estrous cycle length was studied in four cows. The infusions were performed at various times of the estrous cycle: early, middle, late, and during luteolysis. Blood samples were drawn every third hour from the jugular vein. Progesterone and 15-keto-13,14-dihydroprostaglandin F(2α) (the main metabolite of PGF(2α)) were measured to monitor luteal activity and prostaglandin release. No release of prostaglandins was observed immediately following intrauterine infusion. Infusion in two cows on day 5 of the estrous cycle resulted in prostaglandin release after 54 and 69 hrs., respectively, followed by luteal regression and the occurrence of estrus at approx. five days after infusion. Infusions performed on days 11 or 12 resulted in prostaglandin release after 147 and 120 hrs., respectively, followed by luteolysis and heat after a 19 day estrous cycle. Infusion in two cows at days 16 and 17 resulted in prostaglandin release after 117 hrs. in both animals. One cycle was prolonged whereas the other cycle was normal in duration. One cow infused on day 20 following the occurrence of the first prostaglandin surge had a cycle length of 26 days, whereas another cow infused on day 20 was not affected because luteolysis was essentially complete by the time of infusion. One animal infused on day 5 did not respond to the iodine infusion. In this animal, however, the corpus luteum was not completely developed prior to the infusion. From this study it can be concluded: 1) intrauterine iodine infusions performed after the development of a progesterone secreting corpus luteum result in prostaglandin release within three to six days with the subsequent occurrence of luteolysis; 2) luteolysis w(r)as in all cases observed in connection with prostaglandin F(2α) release of the same order of magnitude and duration as during normal luteolysis. kw|Keywords|k]prostaglandin release; k]progesterone; k]cow; k]es trous cycle; k]iodine infusion

The leukotrienes and the related hydroxy acids constitute a chemically and biologically rather heterogenous group of compounds. Thus, the assay methods employed for their detection and quantification by necessity differ considerably, depending on what compound is under study.