Effects of prostaglandin E2, 16,16-dimethyl prostaglandin E2 and a prostaglandin endoperoxide analogue (U-46619) on gastric secretory volume, [H+] and mucus synthesis and secretion in the rat

The effects of orally administered prostaglandin E2, 16,16-dimethyl prostaglandin E2 and U-46619, an analogue of the prostaglandin endoperoxide PGH2, on gastric secretory volume, acid and mucus were studied in the rat. All of the compounds significantly increased the volume of gastric secretion, mucus secretion, measured as N-acetylneuraminic acid and mucus synthesis measured as the incorporation of [3H]-glucosamine into mucosal glycoprotein; however, only PGE2 and 16,16-dimethyl PGE2 inhibited acid secretion. U-46619, 1.5 mg/kg provided significant protection against ethanol-induced gastric ulcers, an effect that has been previously shown for the other two compounds. These studies provide additional evidence that prostaglandin induced mucosal protection may be related to an effect on mucus and on stimulation of nonparietal cell gastric secretion. Further study of these parameters may be important in the development of antiulcer drugs for long term clinical use.     

Potentiation of the gastro-protective effect of sulglicotide in the presence of cimetidine in the rat

The effects of sulglicotide, alone or combined with cimetidine, have been investigated on mucosal lesions induced in rats by pylorus ligation. In the same animals, the measurement of acid and pepsin output and of soluble and barrier mucus has been performed. Dose-dependent sulglicotide prevented the development of mucosal lesions and its protective effect was achieved without significant modifications in gastric acid secretion. The secretion of pepsin and of mucus was markedly inhibited at every dosage of the compound. Neither the damage to gastric mucosa nor the secretion of acid, pepsin and mucus were affected by cimetidine. The combination of the highest doses of both compounds resulted in a synergistic gastro-protective effect, not dependent on a synergistic effect on the reduction in acid secretion.     

Partial characterization of the gastrointestinal weight changes produced in the female rat by 16,16-dimethylprostaglandin E2

Oral and subcutaneous administration of 16,16-dimethylprostaglandin E2 (16,16-dimethyl PGE2) resulted in an increase in the dry weight of the stomach and small intestine of the female rat. This weight response was rapid, controlled rather than continuously progressing, dose dependent and reversible. The dry weight of the colon also increased but this was not studied in detail. Two-day treatment with 16,16-dimethyl PGE2 caused an increase in the incorporation of 3H-thymidine into the duodenum, jejunum and colon suggesting an increase in cell number. Incorporation into the stomach and ileum was not changed. The number of goblet cells per crypt was increased by prostaglandin treatment in all parts of the small intestine. Since these are mucus producing cells, the small intestine may have increased in cell number and mucus production. Both anti-secretory and cytoprotective doses of 16,16-dimethyl PGE2 caused weight increases in the stomach and small intestine. However, the weight gain by itself was not sufficient to protect the stomach or small intestine from necrotic agents after the prostaglandin was discontinued.     

Effects of prostaglandin E2, 16,16-dimethyl prostaglandin E2 and a prostaglandin endoperoxide analogue (U-46619) on gastric secretory volume, [H] and mucus synthesis and secretion in the rat

The effects of orally administered prostaglandin E₂, 16,16-dimethyl prostaglandin E₂ and U-46619, an analogue of the prostaglandin endoperoxide PGH₂, on gastric secretory volume, acid and mucus were studied in the rat. All of the compounds significantly increased the volume of gastric secretion, mucus secretion, measured as N-acetylneuraminic acid and mucus synthesis measured as the incorporation of [³H]-glucosamine into mucosal glycoprotein; however, only PGE₂ and 16,16-dimethyl PGE₂ inhibited acid secretion. U-46619, 1.5 mg/kg provided significant protection against ethanol-induced gastric ulcers, an effect that has been previously shown for the other two compounds. These studies provide additional evidence that prostaglandin induced mucosal protection may by related to an effect on mucus and on stimulation of nonparietal cell gastric secretion. Further study of these parameters may be important in the development of antiulcer drugs for long term clinical use.     

Partial characterization of the gastrointestinal weight changes produced in the female rat by 16,16-dimethylprostaglandin E2

Oral and subcutaneous administration of 16,16-dimethylprostaglandin E₂ (16,16-dimethyl PGE₂) resulted in an increase in the dry weight of the stomach and small intestine of the female rat. This weight response was rapid, controlled rather than continuously progressing, dose dependent and reversible. The dry weight of the colon also increased but this was not studied in detail.Two-day treatment with 16,16-dimethyl PGE₂ caused an increase in the incorporation of ³H-thymidine into the duodenum, jejunum and colon suggesting an increase in cell number. Incorporation into the stomach and ileum was not changed.The number of goblet cells per crypt was increased by prostaglandin treatment in all parts of the small intestine. Since these are mucus producing cells, the small intestine may have increased in cell number and mucus production.Both anti-secretory and cytoprotective doses of 16,16-dimethyl PGE₂ caused weight increases in the stomach and small intestine. However, the weight gain by itself was not sufficient to protect the stomach or small intestine from necrotic agents after the prostaglandin was discontinued.     

Prostaglandin effect on the physical properties of gastric mucus glycoprotein and its susceptibility to pepsin

The effect of 16,16-dimethyl prostaglandin E2 (DMPGE2) on gastric mucus glycoprotein viscosity, permeability to hydrogen ion and degradation by pepsin was investigated. Preincubation with DMPGE2 produced a marked enhancement in the glycoprotein viscosity. The increase was concentration dependent and at 2.6 X 10(-5)M DMPGE2 reached a value of 178%. Permeability measurements revealed that 2.6 X 10(-7)M DMPGE2 increased the retardation ability of the glycoprotein to hydrogen ion by 10%, while 22% increase was obtained with 2.6 X 10(-4)M DMPGE2. The results of peptic activity assay showed that DMPGE2 had no inhibitory effect on the rate of glycoprotein proteolysis, and actually a small stimulatory influence was consistently observed. The results suggest that prostaglandins beneficially affect the physical properties of mucus glycoprotein which are considered to be essential for the protective function of gastric mucus.     

Effects of bile salts and prostaglandins on sodium transport in isolated rat gastric mucosa

To determine whether prostaglandins may protect against bile salt inhibition of ion transport in the stomach, gastric mucosal tissue was isolated from the rat and mounted in flux chambers. Transport of Na+ was traced with radioisotopes in the absence of bile salts and then in the presence of conjugated taurocholate or unconjugated deoxycholate at low, intermediate and high mucosal concentrations (1, 5 and 15 mmol/1). At a high (7.40) or low (3.4) mucosal pH, only the unconjugated deoxycholate inhibited active Na+ transport from mucosa to submucosa with respect to untreated controls. Inhibition of Na+ transport was apparent at a low level of deoxycholate, which also inhibited the electrical potential difference. Intermediate and high levels of deoxycholate lowered the tissue resistance. When the tissues were exposed to mucosal prostaglandin E2 or its 16,16-dimethyl analogue before and during acidified taurocholate administration, Na+ transport was not changed significantly but the electrical resistance remained high. Thus, unconjugated bile salt is more potent than conjugated bile salt in inhibiting Na+ transport and breaking the gastric mucosal barrier, and prostaglandins may afford some small protection.     

Effects of 16,16-dimethyl prostaglandin E2 and indomethacin on leukotriene B4 and inflammation in rabbit colitis

The role of increased prostaglandin production and the effects of exogenous prostaglandins on inflammation of colitis are not established. We administered intramuscular 16,16-dimethyl prostaglandin E2 (DiM-PGE2) and indomethacin to rabbits with formalin immune-complex colitis and measured leukotriene B4 (LTB4), prostaglandin E2 (PGE2) and severity of inflammation. DiM-PGE2 (100 micrograms/kg/BID) reduced LTB4 production (from 401 +/- 108 to 216 +/- 58 pg/ml) and infiltration of neutrophils, mucosal necrosis, inflammatory exudate and edema (all P less than 0.05). Other studies determined that parenteral DiM-PGE2 did not reduce the initial chemical damage induced by formalin, suggesting that cytoprotection of chemical insult was not the mechanism of suppressed inflammation in the immune colitis model. Indomethacin (10 mg/kg/d) reduced endogenous PGE2 by 80%, but did not reduce leukotriene production or inflammation. Exogenous prostaglandins cause a dose-dependent suppression of inflammation in experimental colitis, by a mechanism other than cytoprotection of chemical-induced mucosal injury.     

Rupture of the uterus following treatment with 16-16-dimethyl E 2 prostaglandin vagitories

Rupture of the uterine body was found after induction of therapeutic abortion with vaginal suppositories containing 16,16-dimethyl prostaglandin E 2 in a 20-year-old primigravida. A short discussion is given on the cervical complications that can occur after prostaglandin induction of abortion, stating that rupture of the uterine body also can be seen. So far, no prostaglandin compound seems to avoid such complications.     

Effects of 16,16-dimethyl prostaglandin E2 and indomethacin on leukotriene B4 and inflammation in rabbit colitis

The role of increased prostaglandin production and the effects of exogenous prostaglandins on inflammation of colitis are not established. We administered intramuscular 16,16-dimethyl prostaglandin E2 (DiM-PGE2) and indomethacin to rabbits with formalin immune-complex colitis and measured leukotriene B₄ (LTB4), prostaglandin E2 (PGE2) and severity of inflammation. DiM-PGE2 (100 ug/kg/BID) reduced LTB4 production (from 401±108 to 216±58 pg/ml) and infiltration of neutrophils, mucosal necrosis, inflammatory exudate and edema (all P<0.05). Other studies determined that parenteral DiM-PGE2 did not reduce the initial chemical damage induced by formalin, suggesting that cytoprotection of chemical insult was not the mechanism of suppressed inflamation in the immune colitis model. Indomethacin (10 mg/kg/d) reduced endogenous PGE2 by 80%, but did not reduce leukotriene production or inflammation. Exogenous prostaglandins cause a dose-dependent suppression of inflammation in experimental colitis, by a mechanism other than cytoprotection of chemical-induced mucosal injury.     

Role of mucus in mucosal protection through ethanol and pepsin damage models.

Gastrointestinal mucus is considered an important part of the mucosal defence mechanism against endogenous aggressors such as acid and pepsin. The mucus gel layer, adherent to the mucosal surface creates a diffusion barrier to luminal pepsin, thus protecting the underlying epithelium from the digestion by pepsin. The mucolytic pepsin will, however, digest the mucus at its luminal surface, but that lost is normally balanced by secretion of new mucus. This dynamic balance is disrupted when the mucus is exposed to excess pepsin, which causes focal haemorrhagic damage by progressively hydrolyzing the adherent mucus. The adherent mucus gel layer cannot contribute to the protection against exogen damaging agents such as ethanol and nonsteroidal anti-inflammatory drugs, as these compounds easily penetrate the mucus barrier causing, at high concentration, epithelial exfoliation. This study describes the basic properties and characteristics of gastric mucus and compares the pepsin-induced damage with the ethanol damage model.     

Pathogenic importance of pepsin in ischemia/reperfusion-induced gastric injury

We investigated the role of pepsin in the development of ischemia/reperfusion (I/R)-induced gastric lesions in rats. Under urethane anesthesia, the pylorus was ligated, the celiac artery was clamped, and 1 ml of HCl (50-150 mM) was instilled in the stomach. Then, reperfusion was established 15 min later by removing the clamp, and 2 h later the stomach was assessed for gross mucosal damage. Pepstatin (a specific pepsin inhibitor) or pepsin was given i.g. after the pylorus was ligated while cimetidine, omeprazole, or atropine was given s.c. 30 min before the ligation. I/R produced hemorrhagic gastric injury, with a concomitant increase in the amount of pepsin secreted, and the degree of both these responses was dependent on the concentration of HCl. The formation of lesions by IR in the presence of 100 mM HCl was significantly prevented by atropine or bilateral vagotomy, but neither omeprazole nor cimetidine had any effect. Intragastric administration of pepstatin dose-dependently reduced the severity of the I/R-induced gastric lesions, the effect being significant even at 0.1 mg/kg, while that of pepsin markedly aggravated these lesions. The increased pepsin output during I/R was associated with luminal acid loss and significantly inhibited by bilateral vagotomy or pretreatment with atropine but not cimetidine or omeprazole, while pepstatin significantly inhibited the pepsin activity. In conclusion, we suggest that pepsin plays a pivotal role in the pathogenesis of I/R-induced gastric lesions, and pepsin secretion is increased during I/R, the process being associated with acid back-diffusion and mediated through a vagal-cholinergic pathway.     

Gastroduodenal mucus bicarbonate barrier: protection against acid and pepsin

Secretion of bicarbonate into the adherent layer of mucus gel creates a pH gradient with a near-neutral pH at the epithelial surfaces in stomach and duodenum, providing the first line of mucosal protection against luminal acid. The continuous adherent mucus layer is also a barrier to luminal pepsin, thereby protecting the underlying mucosa from proteolytic digestion. In this article we review the present state of the gastroduodenal mucus bicarbonate barrier two decades after the first supporting experimental evidence appeared. The primary function of the adherent mucus gel layer is a structural one to create a stable, unstirred layer to support surface neutralization of acid and act as a protective physical barrier against luminal pepsin. Therefore, the emphasis on mucus in this review is on the form and role of the adherent mucus gel layer. The primary function of the mucosal bicarbonate secretion is to neutralize acid diffusing into the mucus gel layer and to be quantitatively sufficient to maintain a near-neutral pH at the mucus-mucosal surface interface. The emphasis on mucosal bicarbonate in this review is on the mechanisms and control of its secretion and the establishment of a surface pH gradient. Evidence suggests that under normal physiological conditions, the mucus bicarbonate barrier is sufficient for protection of the gastric mucosa against acid and pepsin and is even more so for the duodenum. acid-base transporters; cystic fibrosis transmembrane conductance regulator channel; surface pH gradient; mucus gels; trefoil peptides     

Correlation of gastric mucosal damage with sialic acid profile in rats: effect of hydrochloric acid, pepsin and hypertonic saline

Sialic acids occupy terminal positions on gastric mucus glycoprotein where they contribute to the high viscosity of mucin. Desialylation of mucus may lead to degradation of the mucus and eventually to the breakdown of the gastric mucus barrier. The effect of a variety of damaging agents (0.1 M HCl, 2 mg ml(-1) pepsin and 2 M NaCl) on sialic acid profile was determined in pylorus-ligated rats. The relationship between sialic acid, galactose, pyruvate and the extent of gastric mucosal damage were studied. Instillation of pepsin significantly increased total sialic acid, galactose and macroscopic mucosal lesions in the stomach. Instillation of 0.1 M HCl reduced the total sialic acid but this decrease was not significant. Acidity led to a significant increase in the amount of free sialic acid in the gastric instillates and the macroscopic lesions induced by acid was not significantly different from the control animals (0.15 M NaCl). 2 M NaCl induced the macroscopic lesions in the stomach and also free sialic acid in the instillates. Pepsin potentiates the action of 2 M NaCl. In all the agents examined with the exception of acid, it was observed that an increase in free sialic acid and galactose was accompanied by gastric mucosal erosion and elevation of pyruvate concentration. It is concluded that gastric acidity alone is not inherently damaging and that resistance of gastric mucosa to destructive agents may be dependent on the integrity of the sialic acids.     

Effects of 16, 16-dimethyl prostaglandin E2 on bile-induced histamine release and permeability alterations in canine oxyntic mucosa.

Damage to the stomach results in excessive movement of hydrogen ion (H+) out of the lumen, and increased movement of sodium (Na+) and potassium (K+) into the lumen. Histamine liberation during damage probably adds to the destruction by increased capillary permeability and formation of edema. Previous reports have shown that the synthetic prostaglandin analogue 16,16-dimethyl prostaglandin E2 (Dm PGE2) protects dog gastric mucosa from aspirin- and ethanol-induced gastric mucosa damage. The effects of dm PGE2 on bile salt (sodium taurocholate) induced injury has not been investigated. Using the canine Heidenhain pouch, the present study examined the action of dm PGE2 on gastric mucosal damage induced by 5 mM sodium taurocholate in 100 mM HCl. Bile salt damaged the pouch mucosa as evidenced by an increased loss of H+, and increased net fluxes of both Na+ and K+. There was also an increase in the histamine content of the fluid irrigating the Heidenhain pouch. Intravenous injection of dm PGE2 in the doses 0.1 and 1.0 microgram/kg 1/2 h before administration of the sodium taurocholate in HCl significantly reduced the net loss of H+ and the gain of Na+, K+, and histamine. It is concluded the dm PGE2 effectively protects the canine gastric mucosa from the damaging effects of bile salt and that the mechanism of dm PGE2 protection of canine oxyntic mucosa may be mediated in part via inhibition of the gastric mucosal release of histamine.     

Interrelationships between the development of the gastric cytoprotective effects of prostacyclin, atropine, cimetidine and the gastric mucosal superoxide dismutase activity in rats

Gastric mucosal damage was produced by the intragastric administration of 96% ethanol or 0.6 M HCl. The cytoprotective doses of prostacyclin (PGI2) (5 micrograms/kg), atropine (0.025 mg/kg) or cimetidine (2.5 mg/kg) were given intraperitoneally 30 min before the administration of the necrotizing agents. The animals were killed 1 hr later. The number and severity of gastric mucosal lesions (ulcer) were recorded. At the time of the sacrifice of the animals, superoxide dismutase (SOD) was prepared from the gastric fundic mucosa and its activity was measured. It was found that PGI2 (5 micrograms/kg), atropine (0.025 mg/kg) and cimetidine (2.5 mg/kg) significantly decreased the number and severity of gastric mucosal lesions (ulcers) produced by the intragastric administration of 96% ethanol a 0.6 M HCl, PGI2, atropine, cimetidine, given in cytoprotective doses, significantly mounted the ethanol-induced increase of gastric mucosal SOD activity; PGI2, atropine, cimetidine, given them in cytoprotective doses significantly shunted the HCl-induced decrease of gastric mucosal SOD activity. It has been concluded that; chemically different cytoprotective agents (PGI2, atropine, cimetidine) give rise to similar tendencies in the changes of gastric mucosal SOD activity; both the significant decrease (in the ethanol-model) and the significant increase (in the HCl-model) of this enzyme seem to be involved in the development of gastric mucosal protection by PGI2, atropine and cimetidine.     

In vitro electrophysiological effects of cimetidine, prostaglandin E2 and acetylcholine on the rabbit gastric mucosa

The electrophysiological effects of cimetidine, cytoprotective dose of prostaglandin E2 (PGE2) and acetylcholine were determined in parallel in Ussing-chambered rabbit fundic and antral mucosal preparations. In the fundic mucosal preparations both cimetidine and PGE2 caused an increase in transmucosal potential difference (PD) and in short-circuit current (ISC); the transepithelial resistance (Rt) was essentially unchanged. Addition of acetylcholine to the pretreated fundic preparations produced further gradual increases in PD and ISC; cimetidine pretreatment delayed this effect of acetylcholine. In contrast to fundic mucosa, cimetidine did not cause any electrical change of the antral preparation but decreases in PD, Rt and ISC were detected after the addition of PGE2. Acetylcholine produced a rapid initial PD elevation followed by a PD drop of both antral tissues independent of pretreatment. These findings suggest that both cimetidine and PGE2 generated electrical hyperpolarisation of rabbit fundic mucosa. These changes may be favourable for mucosal protection. No "beneficial" electrical changes were detected on the antral mucosa after administration of cimetidine and PGE2. Acetylcholine increased the effects of other stimuli on the fundic mucosa. In the rabbit antral mucosa acetylcholine generated biphasic changes of electrical properties.     

Roles of endogenous prostaglandins and nitric oxide in gastroduodenal ulcerogenic responses induced in rats by hypothermic stress.

We examined the roles of endogenous prostaglandins (PGs) and nitric oxide (NO) in the gastroduodenal ulcerogenic responses to hypothermic stress (28 approximately 30 degrees C) in anesthetized rats. Lowering body temperature provoked damage in the gastroduodenal mucosa, with an increase of gastric acid secretion and motility. These responses were completely abolished by bilateral vagotomy or atropine, while 16,16-dimethyl PGE2 decreased the mucosal ulcerogenic response with no effect on acid secretion. The non-selective COX inhibitors, indomethacin or aspirin, worsened these lesions with enhancement of gastric motility and no effect on acid secretion, while the selective COX-2 inhibitor NS-398 did not affect any of these responses. On the other hand, the non-selective NOS inhibitor L-NAME but not aminoguanidine (a relatively selective inhibitor of iNOS), significantly potentiated the acid secretory and mucosal ulcerogenic responses in the stomach but reduced the duodenal damage in response to hypothermia, the effects being antagonized by co-administration of L-arginine. Hypothermia itself decreased duodenal HCO3- secretion under both basal and mucosal acidification-stimulated conditions. Both indomethacin and aspirin further decreased the HCO3- response to the mucosal acidification, while L-NAME significantly increased the HCO3- secretion even under hypothermic conditions, similar to 16,16-dimethyl PGE2. These results suggest that 1) hypothermic stress caused an increase of acid secretion and motility as well as a decrease of duodenal HCO3-secretion, resulting in damage in both the stomach and duodenum, 2) the COX-1 but not COX-2 inhibition worsened these lesions by enhancing gastric motility and further decreasing duodenal HCO3- response, 3) the cNOS but not iNOS inhibition worsened gastric lesions by increasing acid secretion but decreased duodenal damage by increasing HCO3- secretion. Thus, it is assumed that the gastroduodenal ulcerogenic and functional responses to hypothermic stress are modified by cNOS/NO as well as COX-1/PGs.     

Effect of intravenous cimetidine, ranitidine and pentagastrin and intragastric prostaglandin E1 treatments on gastric transmucosal potential difference in the rat.

Effects of intravenous cimetidine, ranitidine and intragastric prostaglandin E1 (alprostadil) treatments on the transmucosal potential difference (PD) of the stomach were compared. It was also investigated whether the above-mentioned drugs influenced the decrease of PD which followed both intragastric administration of 30% alcohol or Ca++ solution in 5 Mm final concentration and intravenous administration of pentagastrin. Both cimetidine and ranitidine treatments led to significant (p < 0.05) increase of PD, the effect of ranitidine was dose dependent. Prostaglandin E1 in a dose of 40 micrograms/kg led to significant decrease of PD (< 0.05). Both intragastric administration of prostaglandin E1 in a dose of 40 micrograms/kg and intravenous administration of ranitidine in a dose of 10 mg/kg significantly diminish the effect of Ca++ and alcohol to decrease PD. Neither prostaglandin E1, nor ranitidine pretreatment had any effect on the rapid and highly significant (p < 0.01) decrease of PD following i.v. pentagastrin administration. It is hypothesized that transmucosal PD of the stomach provides information not only on the actual condition of the mucosal barrier but on the electrophysiology of gastric secretion as well.     

Sulfation in vitro of mucus glycoprotein by submandibular salivary gland: effects of prostaglandin and acetylsalicylic acid

Enzymatic sulfation of mucus glycoprotein by rat submandibular salivary gland and the effect of prostaglandin and acetylsalicylic acid on this process were investigated in vitro. The sulfotransferase enzyme which catalyzes the transfer of sulfate ester group from 3'-phosphoadenosine-5'-phosphosulfate to submandibular gland mucus glycoprotein has been located in the detergent extracts of Golgi-rich membrane fraction of the gland. Optimum enzyme activity was obtained at pH 6.8 with 0.5% Triton X-100, 25 mM NaF and 4 mM MgCl2, using the desulfated glycoprotein. The enzyme was also capable of sulfation of the intact mucus glycoprotein, but the acceptor capacity of such glycoprotein was 68% lower. The apparent Km of the submandibular gland sulfotransferase for salivary mucus glycoprotein was 11.1 microM. The 35S-labeled glycoprotein product of the enzyme reaction gave in CsCl density gradient a 35S-labeled peak which coincided with that of the glycoprotein. This glycoprotein upon reductive beta-elimination yielded several acidic 35S-labeled oligosaccharide alditols which accounted for 75% of the 35S-labeled glycoprotein label. Based on the analytical data, the two most abundant oligosaccharides were identified as sulfated tri- and pentasaccharides. The submandibular gland sulfotransferase activity was stimulated by 16,16-dimethyl prostaglandin E2 and inhibited by acetylsalicylic acid. The rate of enhancement of the glycoprotein sulfation was proportional to the concentration of prostaglandin up to 2.10(-5) M, at which point a 31% increase in sulfation was attained. The inhibition of the glycoprotein sulfation by acetylsalicylic acid was proportional to the drug concentration up to 2.5.10(-4) M at which concentration a 48% reduction in the sulfotransferase activity occurred. The apparent Ki value for sulfation of salivary mucus glycoprotein in presence of acetylsalicylic acid was 58.9 microM. The results suggest that prostaglandins may play a role in salivary mucin sulfation and that this process is sensitive to such nonsteroidal anti-inflammatory agents as acetylsalicylic acid.