Gender differences in the activities of aspirin-esterases in rat tissues

The activities of aspirin (acetylsalicylic acid)-esterases were measured in several tissues (liver, kidney, adrenal glands, brain and serum) from adult male and female Wistar rats. In males, both aspirin-esterase I (assayed at pH 5.5) and II (assayed at pH 7.4) activities were higher in liver homogenates when compared to females (aspirin-esterase I: males 48.9 +/- 4.8 (N = 8) and females 29.3 +/- 4.2 (N = 8) nmol of salicylic acid formed min-1 mg protein-1; aspirin-esterase II: males 41.4 +/- 4.1 (N = 8) and females 26.1 +/- 4.5 (N = 8) nmol of salicylic acid formed min-1 mg protein-1, P < 0.001). In serum, enzyme activity was higher in females than in males (aspirin-esterase I: males 0.85 +/- 0.06 (N = 6) and females 1.18 +/- 0.11 (N = 6) nmol of salicylic acid formed min-1 mg protein-1, aspirin-esterase II: males 1.03 +/- 0.13 (N = 6) and females 1.34 +/- 0.11 (N = 6) nmol of salicylic acid formed min-1 mg protein-1, P < 0.001). In the other tissues assayed, no statistically significant difference between males and females was found. There were no statistically significant differences when the enzymes were assayed in different phases of the estrous cycle in liver and serum. These results show that the differences in aspirin-esterase activity observed between males and females are not due to the estrous cycle. The gender difference obtained in our study may indicate an involvement of gonadal hormones in the control of the hydrolysis of aspirin. This possibility is currently under investigation.     

Biphasic initial phase of platelet aggregation inhibition after oral aspirin

For one hour after the ingestion of 1 g aspirin the pharmacodynamics of acetylsalicylic acid with regard to the inhibition of platelet aggregation were studied in nine healthy male volunteers. Plasma salicylic acid (SA) and acetylsalicylic acid (ASA) levels were measured, and platelet aggregation was controlled by the collagen-induced aggregation. It took 12 - 24 minutes till the maximum of platelet aggregation inhibition was reached; maximal inhibition was only observed with ASA levels above 4.5 /microgram/ml and total ASA levels above 10 /microgram/ml. At that time already more than 50% of the total ASA were hydrolysed to minimally active SA. In spite of further increasing ASA levels inhibition of platelet aggregation decreased again. The different sensitivity of platelet- and vessel wall cyclooxygenase to aspirin does not explain our findings.     

Acetylsalicylate and salicylates in foods

Acetylsalicylic acid is effective in the prevention of cardiovascular disease. It was suggested that fruits and vegetables provide unknown amounts of acetylsalicylic acid. We could not find any acetylsalicylic acid in 30 foods using HPLC with fluorescence detection (detection limits: 0.02 mg/kg for fresh, and 0.2 mg/kg for dried products). We showed that urinary excretion of salicylates is a valid indicator for intake, and found a median salicylate excretion of 10 micromol (1.4 mg) in 24 h urine of 17 volunteers eating a variety of diets. Our data suggest that the content of (acetyl)salicylic acid of diets may be too low to affect disease risk.     

Focused antithrombotic therapy: novel anti-platelet salicylates with reduced ulcerogenic potential and higher first-pass detoxification than aspirin in rats

The use of aspirin as an anti-platelet drug is limited by its propensity to induce gastric injury and by its adverse effect on vascular prostacyclin formation. Two phenolic non-steroidal anti-inflammatory drugs (salicylic acid and diflunisal) were modified by esterification with a series of O-acyl moieties. The short-term ulcerogenic in vitro and in vivo anti-platelet properties, pharmacodynamic profiles, and extent of hepatic extraction of these phenolic esters were compared with aspirin (acetylsalicylic acid). The more lipophilic esters (longer carbon chain length in O-acyl group) show significantly less gastrotoxicity in stressed rats than does aspirin after a single oral dose. The in vitro and in vivo anti-platelet studies show that these phenolic esters inhibited (1) arachidonate-triggered human platelet aggregation and (2) thrombin-stimulated rat serum thromboxane A2 production by platelets in the clotting process almost as effectively as aspirin. The hepatic extractions of these O-acyl derivatives are significantly higher than those of aspirin. The pharmacodynamic studies show that these O-acyl derivatives of salicylic acid and diflunisal probably bind to, or combine with, the same site on the platelet cyclooxygenase as aspirin. Replacing the O-acetyl group with longer chain O-acyl moiety in this series of phenolic esters markedly reduced the potential of these agents to induce short-term gastric injury but did not lessen their activity as inhibitors of platelet aggregation. These non-acetyl salicylates may therefore represent a novel class of anti-platelet drugs with less ulcerogenic potential.     

The fetoplacental pressor effects of low-dose acetylsalicylic acid and angiotensin II in the ex vivo cotyledon model

OBJECTIVE: Our purpose was to investigate perfusion pressure changes ex vivo induced by angiotensin II on fetoplacental vasculature pretreated with low-dose acetylsalicylic acid. STUDY DESIGN: Two cotyledons from each of 12 placentas were perfused. The intervillous space of one cotyledon was infused with acetylsalicylic acid (5 x 10(-5) mol/L) similar to the serum concentration of women receiving daily low-dose aspirin therapy (60 to 81 mg). The control cotyledon was infused with an equivalent amount of normal saline solution. Two doses of angiotensin II, 1 x 10(-11.5) and 1 x 10(-10) moles, were injected as boluses into the chorionic arteries of each cotyledon. A 3 x 10(-7) mole dose of angiotensin II was also injected into the intervillous space. Statistical analysis was performed with analysis of variance, and results are expressed as mean pressure change in millimeters of mercury +/- SEM. RESULTS: Perfusion pressure response did not vary between cotyledons pretreated with acetylsalicylic acid and control cotyledons when 3 x 10(-7) moles of angiotensin II was injected into the intervillous space (8.0 +/- 1.9 mm Hg vs 9.8 +/- 1.6 mm Hg, p = 0.59). There were no differences between cotyledons in pressure response to 1 x 10(-11.5) moles of angiotensin II injected into the fetal circuit (5.9 +/- 0.8 mm Hg vs 6.7 +/- 0.9 mm Hg, p = 0.51). However, in the cotyledons pretreated with acetylsalicylic acid there was a decrease in the pressor response to 1 x 10(-10) moles of angiotensin II (14.1 +/- 1.4 mm Hg vs 21.5 +/- 3.3 mm Hg, p = 0.05). CONCLUSIONS: Low-dose aspirin infused into the intervillous space decreases vasoconstriction elicited by angiotensin II in the fetoplacental compartment. This suggests that maternal low-dose aspirin therapy has effects in the fetoplacental circulation in addition to its effects in the maternal circulation.     

The role of endogenous nitric oxide in the gastroprotective action of morphine

Morphine in a dose of 1 mg/kg s.c. decreased mucosal lesions induced by 100% ethanol or acidified aspirin by 79% and 85%, respectively, in rats. When the animals were pretreated with NG-nitro-L-arginine (40 mg/kg i.v.), the mucosal lesions were aggravated in both tests and the gastroprotective action of morphine decreased to 17% and 20%, respectively. This decrease in morphine protection was antagonized by L-arginine but not by D-arginine in the case of ethanol-induced lesions; however, L-arginine failed to restore the gastroprotective effect of morphine when the mucosal damage was induced by acidified aspirin. The protective action of either prostaglandin E2 (0.1 mg/kg orally) or cysteamine (50 mg/kg orally) was not influenced by NG-nitro-L-arginine (L-NNA). When L-NNA was given simultaneously with either indomethacin (10 mg/kg p.o.) or N-ethyl-maleimide (50 mg/kg s.c.), compounds which also reduced the gastroprotective action of morphine, almost complete inhibition of the gastroprotective action of morphine against 100% ethanol-induced lesions was observed as a result of the addition of the inhibitory activities of the latter substances. These results suggest that: (1) Endogenous nitric oxide is likely to be involved in the gastroprotective action of morphine. (2) The protective action of nitric oxide is independent of both mucosal prostaglandins and sulfhydryls.     

The effect of protaglandin F2a upon early gravidity (author's transl)

4 women were administered prostaglangin F2alpha, produced by the Upjohn firm, during the first trimester of pregnancy in order to induce abortion. The preparation was administered by intrauterine injection in a dose of 5 mg. The subjects were observed radioimmunologically by means of radio receptors in order to monitor the levels of human chorionic gonadotropin, prolactin, and progesterone. Observations were made at the beginning of the treatment, and after 2, 6, and 24 hours. A rapid increase in the level of progesterone was noted, accompanied by an increase in the level of prolactin. Results appeared to be in agreement with previous studies in concluding that the action of prostaglandins does not depend primarily on the luteolytic effect.     

Clinical pharmacokinetics of oxaprozin

Oxaprozin is a nonsteroidal anti-inflammatory drug which reaches peak plasma concentrations 2 to 6 hours after oral administration. Oxaprozin binds extensively, in a concentration-dependent manner, to plasma albumin. The area under the plasma concentration-time curve (AUC) of oxaprozin is linearly proportional to the dose for oral doses up to 1200 mg. At doses greater than 1200 mg there is an increase in the unbound fraction of drug, leading to an increased clearance and volume of distribution (Vd) of total oxaprozin. Accumulation of the drug at steady state is between 40 and 58% lower than predicted by single dose data. After administration of multiple doses, the apparent oral clearance (CL/F) and Vd of total oxaprozin increased while those of the unbound drug decreased significantly. Substantial concentrations of oxaprozin are attained in synovial fluid, which is a proposed site of action for nonsteroidal anti-inflammatory drugs. Relationships between total plasma, unbound plasma and synovial concentrations, and therapeutic and toxicological effects have yet to be established. Oxaprozin is eliminated following biotransformation to glucuroconjugated metabolites which are excreted in urine and bile, with little drug being eliminated unchanged. Two hydroxylated metabolites have been shown to possess anti-inflammatory activity. Hepatic disease and rheumatoid arthritis do not significantly alter the disposition of oxaprozin. Patients with renal impairment demonstrate an increase in unbound plasma concentrations of oxaprozin. A significant drug interaction has been demonstrated between oxaprozin and aspirin (acetylsalicylic acid).     

Potential for cross-contamination from use of a needleless injector

In this study the severity of aspirin-induced gastric mucosal damage was investigated in rats with obstructive cholestasis. Cholestasis was induced by ligation and resection of the bile duct under general anesthesia. Two weeks after operation, the rats were fasted for 24 hours. Aspirin was administered orally in doses of 0, 128, 192, 266 and 335 mg/kg, and the animals were killed four hours after dosing. The dose of 266 mg/kg was chosen for a study of the time-dependency; other groups of animals were killed at time intervals of one, three, five, seven and nine hours after aspirin administration. The results showed that aspirin induces more severe gastric damage in bile duct resected rats compared with sham-operated and control animals. Salicylate levels of serums were also measured but there was no significant difference in serum salicylate levels between bile duct resected, sham-operated and control rats. It can be concluded that cholestasis can potentiate aspirin-induced gastric damage in rats.     

Cholecystokinin cholecystography

Copper sulfate, given orally in an emetic dose, decreased the absorption of therapeutic doses of acetylsalicylic acid or lithium carbonate, more than was expected judging from the recovery of salicylic acid in the vomit. Ten volunteers participated.     

A dual effect of 1-methyl-4-phenylpyridinium (MPP+)-analogs on the respiratory chain of bovine heart mitochondria

Various gastrointestinal functions such as mucosal blood flow and mucus secretion can be influenced immunologically. Rats were systemically sensitized with 4-hydroxy-3-iodo-5-nitro-phenylacetic acid (NIP), a synthetic antigen. Mucosal release of gastrin, prostaglandin F2 alpha, 6-keto-prostaglandin F1 alpha, and leukotriene C4 was measured after intragastric or in vitro antigen challenge. Gastric protection from ethanol was determined. In sensitized rats, intragastric antigen challenge increased release of gastrin from the antral mucosa ex vivo and tended to increase release of prostaglandin F2 alpha. Likewise, antral mucosa of sensitized rats released significantly more gastrin and prostaglandin F2 alpha during in vitro antigen challenge than during incubation in the absence of antigen. Release of 6-keto-prostaglandin F1 alpha and leukotriene C4 was not affected by the immunologic reaction. Topical antigen challenge in sensitized rats reduced gastric mucosal damage caused by ethanol by 50%. The immunologically induced gastroprotection was significantly attenuated by pretreatment with indomethacin. The findings show that specific antigen challenge renders the gastric mucosa more resistant against the injurious effect of ethanol indicating that the stomach is a target organ of immunological reactions. As gastrin and prostaglandins exert potent protective effects, release of these mediators may contribute to the protective response to gastric mucosal immune activation.     

Tissue prostaglandin levels in familial adenomatous polyposis patients treated with sulindac

BACKGROUND: Recent work has demonstrated a correlation between frequency of aspirin ingestion and colorectal cancer prevention. Sulindac, another nonsteroidal anti-inflammatory drug (NSAID), has been shown to cause polyp regression and a fall in cell proliferation in patients with familial adenomatous polyposis, who are destined to develop colorectal cancer unless the colon is removed. However, the mode of action of NSAIDs in colorectal carcinogenesis prevention remains to be determined, although a prostaglandin-mediated mechanism seems likely. METHODS: Rectal or duodenal biopsies from 20 patients with familial adenomatous polyposis, who had been randomized to sulindac or placebo, were analyzed for prostaglandin (PG) E2 and F2 alpha levels before and after treatment. RESULTS: A significant fall in prostaglandin E2 and F2 alpha levels was seen in patients who were on sulindac; this correlated with a visual improvement in number and size of polyps in the same patients (P = 0.0096; PGE2, P = 0.036; PGF2 alpha, Spearman's rank correlation). CONCLUSIONS: Nonsteroidal anti-inflammatory drugs may prevent colorectal cancer by their inhibition of prostaglandin synthesis. Prostaglandins may be implicated in carcinogenesis through an increase in cell proliferation, through immunosuppression, by increasing neovascularization, or via a mutagenic effect.     

Aspirin inhibition and acetylation of the plant cytochrome P450, allene oxide synthase, resembles that of animal prostaglandin endoperoxide H synthase

The enzymatic reactions leading to octadecanoid lipid signaling intermediates in plants are similar to those of animals and are inhibited by nonsteroidal anti-inflammatory drugs (NSAIDs) such as salicylic acid and aspirin. In animals, NSAIDs inhibit the cyclooxygenase (COX) activity of prostaglandin endoperoxide H synthase, which ultimately blocks the formation of prostaglandins. In plants, NSAIDs block the formation of 12-oxo-phytodienoic acid and jasmonates, which are the equivalent signaling compounds. In this study we show that NSAIDs act as competitive inhibitors of allene oxide synthase (AOS), the cytochrome P450 that initiates plant oxylipin synthesis. We also show that aspirin causes the time-dependent inhibition and acetylation of AOS, which leads the irreversible inactivation of this enzyme. This inhibition and acetylation superficially resembles that observed for the inactivation of COX in animals. In AOS, aspirin acetylates three serine residues near the C-terminal region that appear to be highly conserved among AOS sequences from other plants but are not conserved among "classical" type P450s. The role of these serine residues is unclear. Unlike animal COX, where acetylation of a single serine residue within the substrate channel leads to inactivation of prostaglandin endoperoxide H synthase, the three serine residues in AOS are not thought to line the putative substrate channel. Thus, inhibition by aspirin may be by a different mechanism. It is possible that aspirin and related NSAIDs could inhibit other P450s that have motifs similar to AOS and consequently serve as potential biochemical targets for this class of drugs.     

Combination of two doses of acetyl salicylic acid: experimental study of arterial thrombosis

The antithrombotic effect of high dose acetylsalicylic acid is well known, and recently, in vitro studies hinted the potent thrombotic effect of ultra-low dose of acetylsalicylic acid (<1mg/day) showing a significant decrease in bleeding time. In this study, we investigated the effect of a combination between a high and an ultra-low dosage (100 mg/kg+ 10(-30) mg/kg) on an arterial thrombosis induced by a laser beam. We used an intravital microscopic technique, allowing to evaluate (anti)-thromboembolic events at previously determined locations of microvasculature. Thrombus formation was induced by argon-laser shot. The instrumental test setup was completed with a video system, to select mesenteric arterioles with the same diameter (between 15 and 25 microm). The changes in platelet aggregability were determined by Cardinal and Flower method, and the concentration of acetylsalicylic acid in the plasma was measured by high pressure liquid chromatography. Antithrombotic effect of high dose (100 mg/kg) acetylsalicylic acid was confirmed in all results obtained. Asa injected at ultra-low dose (10(-30) mg/kg) had a potent thrombotic properties and decreased significantly the bleeding time. The subcutaneous administration of the combination of the two doses permitted to come back to the control values, and the bleeding time was shortened compared to control group.     

Functionally null mutations in patients with the cblG-variant form of methionine synthase deficiency

A column-switching liquid chromatographic method is described for the simultaneous determination of aspirin and salicylic acid in human plasma. Blood samples are taken into chilled tubes containing a fluoride anticoagulant, and the plasma is isolated by centrifugation. Following a simple acidification step, a 200 microL aliquot of the sample is injected directly onto the HPLC system. The C-18 extraction column is washed with acidified water for 2 min, after which time the compounds are removed by back-flushing directly onto the analytical column (C-8 Nucleosil, 5 microns, 250 mm x 4.6 mm). The flow rate through both columns is 1 mL/min, and the analytes are quantified by measurement of their UV absorbance at 225 nm. The mobile phase is a mixture of water-methanol-acetonitrile-orthophosphoric acid (650:200:150:1 v/v/v/v). The method is linear in the concentration ranges 0.10-5.00 micrograms/mL for aspirin and 0.25-15.00 micrograms/mL for salicylic acid. Both compounds have a limit of quantitation of 0.10 microgram/mL and a limit of detection of 0.04 microgram/mL. Extensive stability tests have been carried out, and validation studies reveal the method to be reproducible and repeatable. Excellent recoveries from plasma obviate the need for an internal standard. The procedure is easier to execute and requires less sample handling than methods currently described in the literature. It has been successfully applied to the investigation of the levels of aspirin and salicylic acid in a healthy, nonfasting volunteer following a 600 mg oral dose of aspirin.     

Effects of captopril related to increased levels of prostacyclin and angiotensin-(1-7) in essential hypertension

OBJECTIVE: To evaluate the contribution of angiotensin-(1-7) [Ang-(1-7)] and prostaglandins to the acute and long-term antihypertensive actions of captopril in mild-to-moderate essential hypertensive patients. DESIGN AND METHODS: Blood pressure, cardiac rate and the plasma concentrations of angiotensin I (Ang I), angiotensin II (Ang II), Ang-(1-7), prostaglandin E2 and 6-keto prostaglandin F1 alpha (the breakdown product of prostacyclin) were determined in the peripheral venous blood of 24 essential hypertensive subjects before and 3 h after administration of 50 mg captopril. Eleven of 24 patients completed a 6-month treatment period with captopril monotherapy (50 mg twice a day). The hemodynamic and hormonal response produced by a last 50 mg dose of captopril was determined once again in the 11 subjects who maintained blood pressure control with captopril monotherapy for 6 months. RESULTS: The fall in blood pressure produced 3 h after drug intake was comparable for the first and the last 50 mg captopril dose. Although the first response to captopril increased plasma levels of Ang I only, the response to the last dose of the drug (6 months after) caused significantly higher levels of Ang I and Ang-(1-7). Neither acute nor chronic therapy with captopril had a significant effect on plasma concentrations of Ang II. Although plasma levels of prostaglandin E2 and 6-keto prostaglandin F1 alpha were not modified by a first exposure to captopril, the concentrations of 6-keto prostaglandin F1 alpha but not prostaglandin E2 rose significantly in subjects treated with the inhibitor for 6 months. A negative correlation was also demonstrated between diastolic blood pressure and plasma Ang-(1-7) levels in the 11 essential hypertensive subjects in whom blood pressure was controlled with captopril monotherapy. CONCLUSIONS: Inhibition of angiotensin converting enzyme with captopril had a significant effect on blood pressure that was not directly accounted for by a suppression of plasma Ang II levels. Continuous therapy with captopril unmasked a contribution of Ang-(1-7) and prostacyclin to the antihypertensive actions of this drug.     

Closure of the ductus arteriosus in premature infants by inhibition of prostaglandin synthesis

Inhibition of prostaglandin synthesis constricts the ductus arteriosus in fetal lambs in utero. We administered the inhibitors, aspirin or indomethacin to 18 premature infants with patent ductus arteriosus, and assessed the effects clinically and by echocardiography (left atrial/aortic-root ratio). After aspirin (20 mg per kilogram, every six hours for four doses) the ductus closed permanently in one infant within 24 hours; in another, constriction occurred with clinical improvement, and the third did not respond. In five infants given 0.3 mg per kilogram of indomethacin, complete closure occurred within one day; two of them, who received three doses had an elevated serum creatinine for one week. In one infant the ductus reopened, requiring a second dose of indomethacin 11 days after the first. Ten infants received 0.1 mg per kilogram of indomethacin, and closure occurred within 24 to 30 hours in eight. One had a soft murmur for four days, and one did not respond to two doses of indomethacin. A murmur reappeared after three to seven days in three infants but only one required further treatment. In infants receiving a single dose of 0.3 mg per kilogram, or one or more doses of 0.1 mg per kilogram, renal function was unaltered.     

Effect of pharmacological doses of ascorbic acid on copper acetate-induced ovulation in the rabbit

The effect of pharmacological doses of ascorbic acid on copper aceta te-induced ovulation was investigated in 18 rabbits. Ovulation was induced by iv administration of copper acetate (.3 mg/kg body weight) and laparotomies were performed 24 hours later. Ascorbic acid (100 mg/kg, iv) was administered at the same time as the copper acetate or at varying intervals thereafter. Those receiving ascorbic acid 5 hours aft er copper acetate had significantly (p less than .05) fewer corpora lutea, which indicates that ascorbic acid had an inhibitory effect on ovulation most marked 5 hours after the copper acetate. It is suggested that ascorbic acid acts by inhibiting synthesis of prostaglandins involved in the process of ovulation.     

Stimulation of prostaglandin biosynthesis by vasoactive substances in methylcholanthrene-transformed mouse BALB/3T3

Prostaglandins E2 and F2alpha are present in the culture medium of methylcholanthrene-transformed mouse BALB/3T3 cells. The production of these prostaglandins is stimulated when the cells are incubated in the presence of serum, arachidonic acid, thrombin, and bradykinin, or if they are mechanically manipulated. Whereas the appearance of prostaglandins resulting from the latter four treatments is complete in several minutes, in the presence of serum the prostaglandin levels are still increasing ever after 2 hours. Stimulation by all of these treatments is additive. Indomethacin inhibits these stimulations, suggesting that the production of prostaglandins results from de novo biosynthesis.     

Different effects of indomethacin and nabumetone on prostaglandin-mediated gastric responses to central vagal activation in rats

Intracisternal injection of a stable thyrotropin-releasing hormone (TRH) analog increases gastric prostaglandins release and mucosal resistance to injury through central vagal pathways. The effects of two nonsteroidal anti-inflammatory drugs, indomethacin (INDO) and nabumetone on intracisternal injection of various doses of TRH-induced gastric acid secretion and changes in mucosal resistance were investigated in urethane-anesthetized rats. Doses of INDO (5 mg/kg) and nabumetone (13.75 mg/kg) producing similar acute anti-inflammatory response in the carrageenin-induced paw edema were injected i.p. in all studies. INDO potentiated the acid secretion induced by intracisternal injection of TRH at 25, 50 and 200 ng by 5.1-, 1.9- and 1.4-fold, respectively, whereas nabumetone did not modify the secretory response to TRH. Moderate erosions were observed in 100% of rats treated with the combination of INDO and TRH (200 ng) whereas no erosions were observed when TRH or INDO were given alone or TRH in combination with nabumetone. TRH at 7 ng reduced mucosal damage induced by intragastric administration of ethanol (60%, 1 ml/kg) by 63%. The mucosal protective action of TRH was abolished by INDO but not altered by nabumetone pretreatment. These data indicate that at comparable anti-inflammatory doses, nabumetone, unlike INDO, neither blocks the protection against ethanol injury induced by low doses of TRH injected intracisternally nor potentiates the gastric acid secretion or lesions induced by higher dose of TRH. We speculate that these differences reflect reduced inhibition of gastric prostaglandins by nabumetone.