Topical nitrates and the higher doses of botulinum toxin for chronic anal fissure.

BACKGROUND/AIMS: Combined BT-A (botulinum toxin A) therapy and local application of nitrates can be more effective than BT-A alone for chronic anal fissure treatment, but so far the optimal dose of BT-A is not known. The aim of our study was to learn if BT-A doses higher than those used so far could change the outcome of fissure treatment. METHODOLOGY: We enrolled 14 consecutive patients suffering from idiopathic chronic anal fissure who did not respond to previous local treatment of nitric oxide donor and subsequent BT-A therapy (25 U of Botox). They were offered a local nitroglycerin treatment. In failure cases patients received the greater doses of BT-A (50 U of Botox). RESULTS: In all 11 patients with chronic anal fissure who applied nitroglycerin after BT-A injection, an effect on the internal anal sphincter relaxation was observed but fissure healing after topical nitroglycerin occurred only in 1 case. Of 13 patients with chronic anal fissure who received 50 U of BT-A no healing was reported in 6 cases. One male from this group received a greater dose (100 U of Botox) and then the fissure healed. CONCLUSIONS: The effect of topical nitrates on internal anal sphincter relaxation after botulinum toxin injection is not the last line for nonsurgical treatment of chronic anal fissure. Always we ought to consider using the next greater dose of BT-A before surgical treatment.

Inhibition of nucleation and crystal growth of calcium carbonate by human lithostathine.

Pancreatic juice is naturally supersaturated in calcium and bicarbonate ions. A mechanism controlling CaCO3 crystal formation and growth is therefore necessary to prevent duct clogging. The present study shows that lithostathine, a glycoprotein present in human pancreatic juice at a concentration in the range of 10 mumol/L, could be involved in such a control. Lithostathine in concentrations greater than 1.5 mumol/L significantly delayed crystal nucleation and inhibited growth of preformed CaCO3 crystals from supersaturated solutions. Adsorption of lithostathine on crystals was shown by immunodetection. Albumin also adsorbed on CaCO3 crystals, but neither albumin nor other pancreatic secretory proteins inhibited crystal nucleation or growth. Lithostathine adsorbed to sites specifically inhibiting crystal growth with a dissociation constant (Kd) = 0.9 x 10(-6) mol/L. The glycosylated amino-terminal undecapeptide generated by limited trypsin hydrolysis inhibited CaCO3 crystal growth with a Kd = 3.0 x 10(-6) mol/L, similar to that of lithostathine. On the contrary, the carboxy-terminal polypeptide was inactive. A synthetic undecapeptide identical to the N-terminal end but not glycosylated was equally active. The activity disappeared upon digestion of the undecapeptide with V8 protease. The N-terminal undecapeptide of lithostathine is therefore essential to the inhibitory activity of the protein on CaCO3 crystal growth.

Immunoreactive forms of pancreatic stone protein in six mammalian species.

Secretory forms of the pancreatic stone protein (PSP S, Mr 17, 500-22,000) have been purified from human pancreatic juice. PSP S are inhibitors of CaCO3 crystal growth. The presence of similar proteins in bovine, canine, monkey, porcine, and rat pancreatic secretion was investigated in terms of biological role and immunological relationship. Pancreatic proteins were analyzed by electrophoretic separation and by subsequent immunoblotting with a rabbit polyclonal antibody against human PSP. A single immunoreactive form was detected in dog, pig, and rat (Mr 17,000), and two distinct immunoreactive forms were observed in cow and monkey (Mr 15,000 and 17,000). Inhibition of CaCO3 crystal growth was demonstrated in dog and rat. Further kinetic studies of the inhibition process in the rat showed that PSP S binds to the crystal surface according to a Langmuir adsorption isotherm with a dissociation constant (Kd) of 1.5 x 10(-6) M. These results suggest that proteins homologous to human PSP S are present in other mammalian species and may act as stabilizers of Ca(2+)-supersaturated pancreatic juice.

N-terminal sequence extension in the glycosylated forms of human pancreatic stone protein. The 5-oxoproline N-terminal chain is O-glycosylated on the 5th amino acid residue.

The pancreatic stone protein isolated from human calculi (PSP) derives from the immunoreactive protein forms detected in human pancreatic juice (PSP S2-5) through the tryptic cleavage of the Arg-11-Ile-12 bond. Among the eleven amino acids of the PSP S2-5 N-terminal extension Z-E-A-Q-T-E-L-P-Q-A-R, the first residue is an oxoproline and the fifth, a threonine, bears the single carbohydrate chain of the protein molecules. Variations in the glycan chain composition account for the differences in the Mr of PSP S2-5. The PSP S2-5 forms are very soluble in aqueous solutions between the pH values 5.0-9.0, whereas the proteolysated form is scarcely soluble.

Characterization in rat pancreatic juice of a protein homologous to the human pancreatic stone protein.

1. The pancreatic stone protein (PSP, Mr 15,000) which has been discovered in human calculi derives from the native glycosylated forms of the protein (Mrs 17,500-22,000) which are present in human pancreatic juice through tryptic cleavage of the Arg 11-Ile 12 bond. 2. In the present study, a homologous native form of the protein (Mr 17,000) was purified from rat pancreatic juice. 3. Its N-terminal amino acid sequence was found to display a high degree of homology with that of the human native protein forms, apart from the fact that it was not glycosylated. 4. In rat as in human, tryptic cleavage of the Arg 11-Ile 12 bond transforms a soluble protein into one which is practically insoluble at neutral pH.

Protein content of precipitates present in pancreatic juice of alcoholic subjects and patients with chronic calcifying pancreatitis.

Chronic calcifying pancreatitis is characterized by the formation of intraductal protein plugs or precipitates and calcified stones in ducts. Similar precipitates may be collected by endoscopic retrograde catheterization of the main pancreatic duct. They are present in the pancreatic juice of alcoholic subjects and patients with chronic calcifying pancreatitis. Protein analysis of these precipitates was performed to try to elucidate the mechanism of stone formation. Two protein fraction were separated by extraction of precipitates. One fraction was easily soluble in saline and contained a small amount of most of the proteins of pancreatic juice. The other fraction was soluble in citrate or ethylenediaminetetraacetate and contained a few proteins with close isoelectric points and identical molecular weight (13,500). These proteins showed immunological identity with the "stone protein" isolated from human pancreatic calculi. Our data demonstrate that the major citrate-soluble protein of precipitates in pancreatic juice is identical with "stone protein". They are strongly support the concept that this protein is the organic matrix of pancreatic stones. Different mechanisms are proposed to explain the phenomenon of protein precipitation that frequently occurs in alcoholic subjects and patients with chronic calcifying pancreatitis.

Physiological conditions for the study of basal and meal stimulated exocrine pancreatic secretion in the dog. Absence of feedback inhibition of basal secretion.

Pancreatic secretion has been studied in dogs in basal and postprandial conditions, as nearly physiological as possible. When pancreatic juice was excluded from the duodenum pancreatic secretion was not raised, compared with secretion during the return of juice to the duodenum. In fact, in seven mongrels, returning pancreatic juice led a transient rise in pancreatic secretion. This was not seen in five beagles. These results indicate that dogs do not manifest the feedback control of pancreatic secretion by pancreatic juice observed in other species. Pancreatic secretory activity was determined in 10 dogs after stimulation by food. The highest secretion rates occurred during the initial 60 min. The maximal secretion of protein occurred before the maximal secretion of fluid and bicarbonate. The effect of the meal diminished slowly during the subsequent minutes but did not reach basal levels after 2 h. In physiological conditions, maximal pancreatic secretion of fluid and bicarbonate was about one-fifth and of protein was almost one-seventh of the maximal secretory capacity obtained with secretin and cholecystokinin, respectively. Potential specific activity of trypsinogen was unchanged during the different experimental conditions. Trypsinogen output represented a constant average of 20% of protein output. The interindividual variability of pancreatic secretion rates was reduced when outputs were expressed per kilogram of body weight. In general, a significant positive correlation was found between body weight and the secretory outputs. No differences were observed in the response of mongrel and beagle dogs to a meal.