Quantification of pepsinogen C and prostaglandin D synthase in breast cyst fluid and their potential utility for cyst type classification.

OBJECTIVE: To quantify pepsinogen C (PEPC) and prostaglandin D synthase (PGDS) in breast cyst fluid and examine if these two parameters can be used for breast cyst type classification. DESIGN AND METHODS: We quantified PEPC and PGDS in 92 and 50 breast cyst fluids, respectively, using previously established immunofluorometric procedures. We then examined if the levels of PEPC or PGDS correlate with the type of cyst or with other clinicopathological variables. RESULTS: Quantitative analysis of the breast cyst fluids indicated that PEPC is present in all cyst fluids at various concentrations ranging from 3 to 31,000 ng/mL. PGDS positivity was confined to 30% of the cyst fluids. PEPC and PGDS levels were correlated with the breast cyst fluid cation ratio and were associated with the type of the cyst. Increased PEPC levels in breast cyst fluids were significantly correlated with a > or = 1.5 K+/Na+ ratio and were associated with the secretory/apocrine type of cyst (Type I) (p = 0.011). Immunoreactive PGDS levels were highly correlated with a low cation ratio and were associated with the transudative/flattened type of breast cyst (Type II) (p = 0.0003). A weak association was observed between PEPC levels in breast cyst fluid and menopausal status (p = 0.093). No significant associations were observed for either PEPC or PGDS concentration in breast cyst fluid and number of cysts, recurrence of the disease, family history of breast cancer, number of children, abortion, and breast feeding. CONCLUSIONS: Quantification of PEPC and PGDS in breast cyst fluid may be useful in the subclassification of cyst type in patients with gross cystic disease.

A stable isotope dilution mass spectrometric assay for the major urinary metabolite of PGD2.

1. A sensitive and specific negative ion chemical ionization mass spectrometric assay for the major urinary metabolite of PGD2 has been developed employing a chemically synthesized [18(0)4]-labelled internal standard. 2. The finding that increased urinary excretion of this metabolite occurs in a number of clinical situations suggests that the assay may prove to be a valuable tool to explore the role of PGD2 in the pathophysiology of human disease.

Thermospray liquid chromatography/mass spectrometry of prostaglandin methyl ester derivatives: application to the determination of prostaglandins E2 and D2 in rat gastric mucosa.

Methyl ester derivatives of 6-keto prostaglandin (PG) F1 alpha, PGF 2 alpha, PGE2 and PGD2 as well as methyl oximes of 6-keto PGF1 alpha, PGE2 and PGD2 have been analysed by thermospray high-performance liquid chromatography mass spectrometry. These compounds are eluted in a gradient of ammonium acetate buffer-acetonitrile at pH 3.4 using a 5 micron ODS-2 reversed-phase column. The mass spectral patterns of the PG derivatives are discussed relative to those of non-derivatized PGs. In general, the methyl ester derivatives show better characteristics than the non-derivatized PGs for the analysis of these compounds in a biological matrix, both from a chromatographic as well as from a mass spectrometric standpoint. Detection limits between 100 and 600 pg on-column can be achieved. Response curves in the selected ion monitoring mode for PGF2 alpha, PGE2 and PGD2 are linear in the range from 100 pg to 10 ng. The technique has been applied to the analysis of PGE2 and PGD2 in rat gastric mucosa.

Prostaglandin D2 and histamine during the immediate and the late-phase components of allergic cutaneous responses.

With a skin blister technique in which the mediators generated by the trauma of forming the blister are allowed to subside, we have collected human interstitial skin fluid during the course of allergic reactions to ragweed, and measured levels of histamine and prostaglandin D2 (PGD2). Of 18 ragweed-allergic individuals tested, 11 developed both an immediate and a late-phase reaction (LPR) with fivefold-elevated levels of histamine (40 ng/ml) at 30 minutes and a peak level of PGD2 (6.5 ng/ml) later at 2 1/2 hours after ragweed challenge. The other seven allergic individuals had immediate reactions without an LPR lesion and demonstrated somewhat smaller elevations of histamine (25 ng/ml) but much lower levels of PGD2 (1.6 ng/ml; p less than 0.05). The time course of appearance of these mediators was identical in both groups of patients. The fluids from unchallenged blisters of allergic and nonallergic patients and the fluids of nonallergic patients challenged with ragweed had similar levels of histamine, at the lower limit of detection, and undetectable PGD2 levels. The peak levels of PGD2 in allergic individuals correlated with the size of the LPR lesion (p less than 0.05). These data suggest that the LPR involves the secondary elaboration of mediators different from mediators responsible for the immediate manifestations of the allergic skin reaction.

Density heterogeneity of human lung mast cells.

Suspensions of enzymatically dispersed human lung parenchymal mast cells were fractionated according to density by flotation through discontinuous Percoll gradients and examined for their responsiveness to release stimulants and pharmacologic agonists. Mast cells localized to all six density fractions (I-VI) examined: densities varied from specific gravities of 1.053 gm/ml to 1.123 gm/ml. Most (67%) lung mast cells localized to fractions III and IV, corresponding to specific gravities of 1.077 to 1.088 gm/ml, respectively. Histamine content increased with density from 2.7 +/- 0.3 pg per cell in fraction 1 to 4.8 +/- 0.7 pg per cell in fraction VI (mean +/- SEM; n = 19). Fraction III was least responsive to high concentrations of anti-IgE than to any other fractions and, along with fraction IV, the most responsive to ionophore A23187. All fractions released the arachidonate mediators prostaglandin D2 and leukotriene C4 in response to anti-IgE. In four of eight lungs tested, formyl methionine peptide (10(-6) to 10(-4) mol/L) weakly elicited histamine release (3% to 6%) in fractions I and II cells. Compound 48/80 (0.1 to 10 micrograms/ml; n = 3) failed to induce histamine release in any fractions. The cyclic adenosine monophosphate-active drugs, isoproterenol (10(-4) mol/L), dibutyryl cyclic adenosine monophosphate (3 mmol/L), and isobutylmethylxanthine (3 X 10(-4) mol/L) inhibited anti-IgE-induced histamine release from all fractions equivalently. Dimaprit (3 X 10(-5) mol/L) and cromolyn sodium (10(-5) -3 x 10(-3) mol/L) failed to significantly inhibit any fraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of prostaglandin D2 as the major eicosanoid from liver endothelial and Kupffer cells.

The capacity of freshly isolated endothelial, Kupffer and parenchymal rat liver cells to produce eicosanoids from [1-14C]arachidonic acid was investigated in order to determine the relative importance of these cells to total liver eicosanoid production. Based upon the total formation of [1-14C]arachidonate metabolites in the liver, it can be calculated that Kupffer and endothelial cells are responsible for 65 and 23%, respectively, of the total amount of eicosanoids produced by the liver. Consequently, parenchymal liver cells, representing 92.5% of the total liver mass, contribute only 12% to the total liver production of eicosanoids. The main product of Kupffer cells was prostaglandin D2 (PGD2), representing 55% of the total amount of eicosanoids produced. Liver endothelial cells produced about 4-times less eicosanoids (per mg cell protein) than Kupffer cells, and PGD2 was also the main product of these cells (44%). The production of eicosanoids by parenchymal cells was lower by a factor of 180 (per mg cell protein) than that in Kupffer cells. Besides the ability to form eicosanoids from added 14C-labeled arachidonic acid, Kupffer and endothelial liver cells were also able to produce significant amounts of PGD2 (the main liver prostaglandin) from endogenous arachidonic acid, as determined by a radioimmunoassay. It is concluded that inside the liver, Kupffer cells together with endothelial cells are of major importance in the production of eicosanoids, while the parenchymal cells may be considered metabolic target cells for these products, as indicated by the finding that the major liver prostaglandin, PGD2, could stimulate the glucose output in isolated parenchymal cells.

Increased level of salivary prostaglandins in patients with major depression.

We quantified the amounts of salivary prostaglandin (PG) D2, PGE2, and PGF2 alpha by radioimmunoassay in 32 patients with major depressive disorder, 16 patients with minor depressive disorder, 24 patients with neurotic disorders (panic, generalized anxiety, phobic, somatization, and obsessive compulsive), and 28 healthy controls. In the saliva of patients with major depressive disorder, the concentrations of immunoreactive PGs (PGD2, 385 +/- 71 pg/ml; PGE2, 498 +/- 105 pg/ml; PGF2 alpha, 444 +/- 100 pg/ml) were significantly higher than those of the healthy controls (PGD2, 129 +/- 18 pg/ml; PGE2, 207 +/- 25 pg/ml; PGF2 alpha, 164 +/- 17 pg/ml). On the other hand, the salivary concentrations of immunoreactive PGs from patients with minor depressive disorder or neurotic disorders were comparable to those of the controls. These results suggest that the level of salivary PGs may be an indicator of major depressive disorder.

The role of mediators in the response of the canine peripheral lung to 1 ppm ozone.

We tested the hypothesis that the in vivo response of the canine peripheral lung to 1 ppm ozone is mediated, in part, by histamine and cyclooxygenase and lipoxygenase products of arachidonic acid metabolism. Ozone was delivered for 5 min to lobar segments through a wedged bronchoscope and resulted in a mean (+/- 1 SE) increase in collateral system resistance (Rcs) of 220.7 +/- 13.8% immediately after exposure. Four 5-min exposures of ozone to the same segments over a 3-h period yielded reproducible Rcs responses, i.e., tolerance to the exposure regimen was not exhibited. Analyses of bronchoalveolar lavage fluid obtained from the isolated segment 1 min after a single exposure to ozone indicated significant increases, compared with control, in mean concentrations of PGD2 (135.3 +/- 33.3 pg/ml versus 47.8 +/- 16.0; p less than 0.025) and histamine (1.43 +/- 0.19 ng/ml versus 1.18 +/- 0.17; p less than 0.05). Additionally, a molecule that exhibited high reactivity with LTB4 antibody was found in greater concentrations in ozone-exposed segments compared to controls (821.5 +/- 206.7 pg/ml versus 437.5 +/- 78.8; p less than 0.05). In contrast, the concentration of TxB2 was not significantly greater in ozone-exposed segments compared to controls (37.2 +/- 6.6 pg/ml versus 33.7 +/- 10.3; p less than 0.05). Cyclooxygenase inhibition (indomethacin, 5 mg/kg, IV) significantly inhibited the Rcs response by 32% (p less than 0.05) and histamine H1-receptor blockade (chlorpheniramine maleate, 5 mg/kg, IV) reduced the response by 30% (p less than 0.05). However, blockade of thromboxane synthetase (UK-37,248, 3 mg/kg, IV) had no significant effect on the ozone-induced response.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of short-term systemic glucocorticoid treatment on human nasal mediator release after antigen challenge.

The effect of systemic glucocorticoid treatment on early- and late-phase nasal allergic reactions after allergen challenge was determined in a double-blind, cross-over study in 13 allergic individuals. The subjects were pretreated for 2 d before challenge with 60 mg prednisone per day or a matching placebo. A previously described model using repeated nasal lavages for measuring mediator release in vivo was utilized. Symptom scores obtained repeatedly before, during, and after the challenge and the number and timing of sneezes were recorded. The mediators measured were histamine. N-alpha-p-tosyl-L-arginine methyl ester (TAME)-esterase activity, kinins, PGD2, and LTC4/D4. Albumin was also measured as a marker of plasma transudation. Blood samples were taken for determination of total number of white blood cells, differential count, and total blood histamine content. No effect of steroid therapy was found on the appearance of symptoms or any of the mediators, except a reduction in kinins, in the early phase of the allergic reaction. However, in the late phase, the prednisone reduced the number of sneezes (P less than 0.01), as well as the level of histamine (P less than 0.05), TAME-esterase activity (P less than 0.05), kinins (P less than 0.05), and albumin (P less than 0.05). Only low levels of leukotrienes were found in the late phase, but the quantities of these mediators seemed to be decreased by the glucocorticoid treatment (P = 0.06). PGD2 did not increase during the LPR and thus was not affected by glucocorticosteroids. The immediate response to a second challenge 11 h after the first was also evaluated. Whereas the appearance of mediators was enhanced over the initial response to the same challenge dose in placebo-treated subjects, this enhancement was abrogated after prednisone treatment. As this dose of drug is known to be clinically effective in treating hay fever, the present study confirms the earlier findings of others that short-term systemic glucocorticoid treatment inhibits the late phase but not the immediate phase of antigen challenge. Furthermore, secondary enhancement of immediate responses is inhibited. This study shows that glucocorticoids inhibit the generation or release of inflammatory mediators during the late reaction and the physiologic response.

Determination of 9 alpha, 11 beta-prostaglandin F2 by stereospecific antibody in various rat tissues.

In view of the recent finding that prostaglandin D2 is stereospecifically converted to 9 alpha, 11 beta-prostaglandin F2, an isomer of prostaglandin F2 alpha, a highly specific and sensitive radioimmunoassay for 9 alpha, 11 beta-prostaglandin F2 was developed and applied to determine the content of this prostaglandin in various rat tissues. Antisera against 9 alpha, 11 beta-prostaglandin F2 were raised in rabbits immunized with the bovine serum albumin conjugate, and [3H]9 alpha, 11 beta-prostaglandin F2 was enzymatically prepared from [3H]prostaglandin D2. The assay detected 9 alpha, 11 beta-prostaglandin F2 over the range of 20 pg to 1 ng, and the antiserum showed less than 0.04% cross-reaction with prostaglandin F2 alpha, prostaglandin F2 beta and 9 beta, 11 beta-prostaglandin F2. To avoid postmortem changes, tissues were frozen in liquid nitrogen immediately after removal. The basal level of 9 alpha, 11 beta-prostaglandin F2 was hardly detectable in various tissues of the rat examined, including spleen, lung, liver and brain; although it was found to be 0.31 +/- 0.06 ng/g wet weight in the small intestine. During convulsion induced by pentylenetetrazole, enormous amounts of prostaglandin D2 (ca. 180 ng/g wet weight) and prostaglandin F2 alpha (ca. 70 ng/g) were produced in the brain; however, 9 alpha, 11 beta-prostaglandin F2 was detected neither there nor in the blood. This result demonstrates that the conversion to 9 alpha, 11 beta-prostaglandin F2 is a minor pathway, if one at all, of prostaglandin D2 metabolism in the rat brain.

Predominance of prostaglandin D2 and I2 in the rat gastric mucosa--analysis by high-performance liquid chromatography.

We have developed a method for measuring prostaglandins (PGs) in rat gastric mucosa by high-performance liquid chromatography (HPLC). The levels of PGD2 and 6-keto-PGF1 alpha, a degradation product of PGI2, were five times higher than those of PGE2 and PGF2 alpha. Oral administration of indomethacin (6 mg/kg body weight) completely abolished the synthesis of all detectable PGs uniformly. These results suggest that endogenous PGs, especially PGD2 and I2, play some roles in the function of the gastric mucosa.

Decreased levels of prostaglandins I2 and E2 in acute gastric mucosal lesions induced by hypoxia.

We have observed that the contents of prostaglandin (PG) D2 and 6-keto-PGF1 alpha were five times higher than those of PGE2 and PGF2 alpha in rat gastric mucosa. In order to elucidate the role of PGs in the function of gastric mucosa, we studied the effect of hypoxia on the levels of PGs in relation to the degree of gastric mucosal lesions. 6-Keto-PGF1 alpha levels were significantly decreased only by severe and long-term hypoxia (10% O2, 18 hours) when severe ulcerative lesions were observed. PGE2 levels were significantly decreased even by mild and short-term hypoxia (13% O2, 4 hours) when slight ulcerative lesions were observed. PGF2 alpha and PGD2 levels were significantly decreased by mild and short-term hypoxia; however, there was no significant difference from the control group under severe and long-term hypoxia. These results suggest that each of the PGs plays a different role in the pathogenesis of acute gastric mucosal lesions induced by hypoxia.

Metabolism of prostaglandin D2 in isolated rat lung: the stereospecific formation of 9 alpha,11 beta-prostaglandin F2 from prostaglandin D2.

The metabolic transformation of exogenous prostaglandin D2 was investigated in isolated perfused rat lung. Dose-dependent formation (2-150 ng) of 9 alpha,11 beta-prostaglandin F2, corresponding to about 0.1% of the perfused dose of prostaglandin D2, was observed by specific radioimmunoassay both in the perfusate and in lung tissue after a 5-min perfusion. To investigate the reason for this low conversion ratio, we analyzed the metabolites of tritium-labeled 9 alpha,11 beta-prostaglandin F2 and prostaglandin D2 by boric acid-impregnated TLC and HPLC. By 5 min after the start of perfusion, 9 alpha,11 beta-prostaglandin F2 disappeared completely from the perfusate and the major product formed remained unchanged during the remainder of the 30-min perfusion. The major product was separated by TLC and identified as 13,14-dihydro-15-keto-9 alpha,11 beta-prostaglandin F2 by GC/MS. In contrast, pulmonary breakdown of prostaglandin D2 was slow and two major metabolites in the perfusate increased with time, each representing 56% and 11% of the total radioactivity at the end of the perfusion. The major product (56%) was identified as 13,14-dihydro-15-ketoprostaglandin D2 and the minor one (11%) was tentatively identified as 13,14-dihydro-15-keto-9 alpha,11 beta-prostaglandin F2 based on the results from radioimmunoassays, TLC, HPLC, and the time course of pulmonary breakdown. These results demonstrate that the metabolism of prostaglandin D2 in rat lung involves at least two pathways, one by 15-hydroxyprostaglandin dehydrogenase and the other by 11-ketoreductase, and that the 9 alpha,11 beta-prostaglandin F2 formed is rapidly metabolized to 13,14-dihydro-15-keto-9 alpha,11 beta-prostaglandin F2.

Central role of cyclooxygenase in the response of canine peripheral airways to antigen.

We studied the effects of antigen aerosol challenge on the airways of the canine peripheral lung and examined the roles of cyclooxygenase products, histamine, and cholinergic activity in the responses. One-minute deliveries of 1:10,000 or 1:100,000 concentrations of Ascaris suum antigen aerosol through a wedged bronchoscope resulted in mean maximal increases in collateral system resistance (Rcs) of 415 and 177%, respectively, after 4-8 min. Repeated antigen challenge (1:100,000) resulted in significantly decreased responsiveness to antigen after the initial exposure (P less than 0.005). Bronchoalveolar lavage fluid obtained from the isolated, challenged segment had a significant increase in mean (+/- SE) prostaglandin D2 (PGD2) concentration vs. control (222.0 +/- 65.3 vs. 72.7 +/- 19.5 pg/ml; P less than 0.05); histamine concentrations were variable and not significantly different (4.1 +/- 2.6 vs. 1.2 +/- 0.2 ng/ml; P greater than 0.05). In nine experiments, cyclooxygenase inhibition significantly attenuated the antigen-induced increase in Rcs by 53.4% (P less than 0.001), and the concentration of PGD2 in lavage fluid was reduced by 96.0% (P less than 0.01). Blockade of histamine H1-receptors (n = 8) or cholinergic receptors (n = 7) did not significantly affect the airway response (P greater than 0.05). These data indicate that the canine peripheral lung responds in a dose-dependent manner to antigen aerosol challenge and exhibits characteristics of antigen tachyphylaxis. Results also suggest that cyclooxygenase products play a central role in the acute bronchoconstrictive response of the lung periphery.

Release of prostaglandin D2 into human airways during acute antigen challenge.

Among the many possible mediators of the early asthmatic response, prostaglandin D2, a bronchoconstrictor, is the principal cyclooxygenase metabolite of arachidonic acid that is released upon the activation of mast cells and is also synthesized by human alveolar macrophages. We performed bronchoalveolar lavage in five patients with chronic stable asthma, before and up to nine minutes after local provocative challenge with Dermatophagoides pteronyssinus. The lavage fluid was analyzed for products of arachidonic acid metabolism. Prostaglandin D2 levels in all five patients rose an average of 150-fold, from less than 8 to 332 +/- 114 pg per milliliter (mean +/- SEM; P less than 0.050), after local instillation of the antigen. Levels of 15-hydroxyeicosatetraenoic acid, which may also have a role in the pulmonary allergic response, were detectable in lavage fluid before challenge and increased after provocation with the antigen in four of the five patients. The activity of beta-glucuronidase, an enzyme released by macrophages and mast cells upon stimulation, tended to increase in the lavage fluid after provocation in all patients. These studies provide evidence that the release of prostaglandin D2 into the airways is an early event after the instillation of D. pteronyssinus in patients who are sensitive to this antigen.

Basal level of prostaglandin D2 in rat brain by a solid-phase enzyme immunoassay.

A solid-phase enzyme immunoassay for prostaglandin D2 (PGD2) was developed in which PGD2 was labeled with horseradish peroxidase. After competitive binding to the immobilized antibody between enzyme-labeled and free PGD2, the activity of the enzyme bound to the antibody was assayed fluorometrically using 3-(p-hydroxyphenyl)-propionic acid and hydrogen peroxide as substrates. The procedure allowed determinations of 3-100 pg for PGD2. The IC50 value for PGD2 in the solid-phase enzyme immunoassay was about 25 pg and the sensitivity was improved about 10 times compared to those in radioimmunoassay and in solution-phase enzyme immunoassay. The solid-phase enzyme immunoassay was applied to the measurement of PGD2 content in rat brain and thereby an octadecylsilyl silica cartridge and a reversed-phase HPLC were sequentially used for sample preparations. Heads were immediately frozen in liquid nitrogen after decapitation to avoid a postmortem formation of PGD2. PGD2 contents measured by solid-phase enzyme immunoassay correlated well with the values obtained by radioimmunoassay (r = 0.966) after raising its contents by intravenous administration of PGD2. The in vivo level of PGD2 in rat brain was extremely low but determined to be 0.11 +/- 0.03 ng/g tissue (mean +/- S.E.M.) with this enzyme immunoassay. The result was equal to the value extrapolated to zero time from the postmortem change.