Biphasic kinetics of quaternary ammonium glucuronide formation from amitriptyline and diphenhydramine in human liver microsomes.

The tricyclic antidepressant amitriptyline and the H1-receptor antagonist diphenhydramine are conjugated in human liver microsomes fortified with UDP-glucuronic acid at their tertiary amino groups with the formation of quaternary ammonium glucuronides. The kinetics of the reactions were found to be biphasic with apparent KM1 and KM2 values of 1.4 microM and 311 microM for amitriptyline and 2.6 microM and 1180 microM for diphenhydramine in four liver samples. Vmax1 values varied between 2 and 17 pmol-mg protein-1.min-1 for the two substrates and Vmax2 values between 80 and 740 pmol-mg protein-1.min-1. A close correlation existed between amitriptyline and diphenhydramine glucuronidation rates in microsomes from seven livers at concentrations corresponding to 10-40% of KM2. At low concentrations, diphenhydramine competitively inhibited the glucuronidation of amitriptyline. Vmax/K(M) values of the high-affinity UDP-glucuronosyltransferase(s) (UGTs) exceed those of the low-affinity enzyme(s) severalfold, such that the former should make the major contribution to N-glucuronidation of the drugs at therapeutic concentrations in vivo.

Renal clearance of pyridostigmine in myasthenic patients and volunteers under the influence of ranitidine and pirenzepine.

1. To assess the contribution of tubular secretion to the renal excretion of pyridostigmine, and its modification by other cationic drugs, six volunteers were given single oral doses of 60-mg pyridostigmine bromide with and without co-administration of ranitidine or pirenzepine. Renal clearances were determined by h.p.l.c. analysis of pyridostigmine and enzymic measurement of endogenous creatinine in plasma and urine. 2. In patients with myasthenia receiving continuous pyridostigmine therapy, renal clearance values were obtained in the same manner with and without ranitidine (10 patients) or pirenzepine (nine patients) co-medication. 3. Pyridostigmine was not bound to plasma proteins. Its renal clearance averaged 6.65 ml/min per kg (350% of the creatinine clearance) in all subjects, 74% being due to net tubular secretion. 4. Mean values for pyridostigmine renal clearance and for clearance by secretion were decreased in the presence of pirenzepine, but plasma concentrations were not affected significantly. Ranitidine caused a small non-significant decrease of the pyridostigmine clearance in patients. 5. Pyridostigmine had a higher elimination (2 h-1) than the absorption rate constant (0.23 h-1) when administered orally as a non-retarded preparation. 6. The renal clearance of creatinine was slightly increased by pyridostigmine in volunteers and slightly decreased by pirenzepine in the total group of subjects.

Clinical manifestations. Failure to thrive in the elderly.

1. Failure to thrive (FTT) is a diagnosis used with increasing frequency in the acute care setting. The purpose of this study was to determine how the diagnosis of FTT was being applied to the geriatric population. 2. FTT was used in the study population to describe the general deterioration seen in patients with chronic or incurable illnesses. Many patients were near the terminal stage. The diagnosis was not used specifically for the geriatric population; there was a lack of specificity in the diagnosis and treatment of FTT. 3. Common complaints of patients diagnosed as FTT included ADL changes, weight loss, and anorexia. Nursing care can significantly contribute to the management of symptoms and the prevention of complications.

Stereoselective inhibition of nortriptyline hydroxylation in man by quinidine.

1. Four volunteers phenotyped as extensive metabolizers of sparteine took 25 mg nortriptyline hydrochloride and collected urine for 72-80 h. Total free and conjugated 10-hydroxynortriptyline (10-OH-NT) accounted for 54-58% of the dose and it was reduced to 25-40% when 50 mg quinidine sulphate was ingested on the first and second day. 2. Of the four isomers of 10-OH-NT, (-)-E-10-OH-NT was selectively decreased in quantity by quinidine coadministration, while the (+)-isomer and (-)- and (+)-Z-10-OH-NT were found in unchanged or slightly increased quantities. The contribution of (-)-E-10-OH-NT to total E-10-OH-NT and the E-/Z-ratio in total 10-OH-NT were significantly reduced. 3. The quantity of the phenol, 2-hydroxynortriptyline in urine was decreased by quinidine; the relative amounts of metabolites with a primary amino group were not affected. 4. Liver microsomes from a donor in which cytochrome P450IID6 was shown to be present by in vitro phenotyping metabolized NT to E-10-OH-NT containing 86% of the (-)-isomer. Quinidine reduced the hydroxylation rate in (-)-E-10-position much more than that in (+)-E-10-position. 5. Since quinidine selectively impairs the function of cytochrome P450IID6, it is concluded that this isoform catalyses NT hydroxylation predominantly in (-)-E-10- and in 2-position.

Diffusion coefficient in native mucus gel of rat small intestine.

The diffusion coefficients of [3H] water, urea, benzoic acid, antipyrine, aminopyrine, alpha-methyl-glucoside, L-phenylalanine and of hydrogen ions were measured at 38 degrees C in native mucus gel from rat small intestine. The diffusion in the gel was reduced to 37-53% (for hydrogen ions to 7%) compared with buffer solution. In addition, the buffering capacity of the gel retarded the permeation of hydrogen ions before a steady state flux was attained. A model calculation revealed that in the preparation a gel layer of 80 microns thickness represents 23% of the total permeation resistance for substances with high epithelial permeability. The aqueous part of the pre-epithelial diffusion resistance amounts to 77% of the total resistance.

Effect of villosity and distension on the absorptive and secretory flux in the small intestine.

The effects of villosity and distension on the absorptive and secretory flux in the small intestine were investigated theoretically in a simplified model. In the case of low epithelial permeability, villosity increases both fluxes by surface enlargement, but in the case of high epithelial permeability, this occurred only if the intervillous spaces are very narrow. Otherwise, the flux is reduced due to the intervillous diffusion resistance, which is more effective than the enlargement of the surface area in that case. Distension increases the fluxes due to the additional surface exposed, by opening the intervillous spaces. In the case of low epithelial permeability this increase exceeds that expected from the enlargement of the smooth inner cylindrical surface area. In the case of high epithelial permeability, however, the increase of the fluxes exceeds surface enlargement only in the first phase, just after opening the intervillous spaces. Otherwise, the increase of the flux is less, since the hindrance by the intervillous diffusion resistance is more effective than the increase of the smooth inner cylindrical surface area. In the intervillous spaces the concentration gradient is non-linear with the steepest slope at the entrance due to the permeation through the lateral surfaces of the villi. The gradient approaches linearity in the center of broad intervillous spaces and becomes steeper when the width decreases and the epithelial permeability increases. In rat small intestine broad intervillous spaces are formed at the front sides of the trapezoidal villi by the predominant circular distension. The diffusion resistance in these spaces and the increase of the supravillous diffusion resistance weaken the increase of the absorptive and secretory flux by distension, especially in the case of high epithelial permeability.

Influence of distension on absorption and villous structure in rat jejunum.

In urethan-anesthetized rats the appearance rates of urea (U), antipyrine (A), and alpha-methyl-D-glucoside (MG) in the venous blood of perfused jejunal segment were measured in the undistended state and after elevation of the intraluminal pressure up to 10 cmH2O. Serosal and inner cylindrical surface area of the jejunal segment were enlarged by maximally 100 and 150%, respectively. The absorption rates, however, increased only by 34 (U), 28 (A), and 26% (MG). The increase of the supravillous diffusion resistance contributed only partially to this effect. The "cylindrical" permeability coefficient (Pcyl, average permeability coefficient related to inner cylindrical area, neglecting villous structure) decreased by 39, 57, and 50%, respectively. Due to circular stretching, broad intervillous spaces were formed that covered finally approximately 40% of the mucosal surface area. The additional intervillous diffusion resistance in these spaces was more effective than the absorption through the lateral surface of the villi. Thus the overall permeability of the mucosa, i.e., Pcyl, was reduced. Calculations based on a simplified model of the mucosa confirmed the experimental results.

Closed rat jejunal segment in situ: role of pre-epithelial diffusion resistance (unstirred layer) in the absorption process and model analysis.

After intraluminal injection of 0.5 ml buffer solution into closed jejunal segments (length, 3-5 cm) of anesthetized rats the appearance rates of a series of labeled substances in jejunal venous blood were measured for 30 min in situ (initial concentration, 0.02-10 mmol/l or 1 GBq/l tritiated water). The appearance rates quickly rose to a maximum and then declined almost exponentially. Model analysis of the descending of branch of the curves by two one-compartment models (perfect luminal mixing, radial diffusion without convection) revealed a relative pre-epithelial diffusion resistance of nearly 100% for benzoic acid, salicylic acid, L-lysine (0.02 and 1 mmol/l), alpha-methyl-D-glucoside, and L-phenylalanine; 80% to 95% for aniline and butanol; 50% to 80% for benzyl alcohol, theophylline, aminopyrine, antipyrine, dodecanol, and D-galactose; approximately 40% for tritiated water; approximately 30% for L-lysine (10 mmol/l); 10% to 20% for urea and benzylamine; and approximately 4% for erythritol. The shape of the curves was well described by a two-compartment model (intestinal lumen and "tissue", radial luminal diffusion without convection); the variability of the data, however, prevented closer analysis of the parameters of this model. Since pre-epithelial diffusion (unstirred layer) in the closed jejunal segment without peristalsis is the rate limiting step in the absorption process of highly permeant substances, information on intestinal epithelium can be obtained only with poorly permeant substances.

Relationship between antipyrine absorption and blood flow rate in rat jejunum, ileum, and colon.

The appearance rate of antipyrine in intestinal venous blood was measured in anesthetized rats during perfusion (0.2 ml/min) of a buffered solution with 1 mmol/l labeled antipyrine through a jejunal, ileal, or colonic segment (length: 2-5 cm). When the blood flow rate was increased from 0.9-1.2 to 1.6-2.0 ml min-1 g-1 by raising the systemic blood pressure from 80 to 130 mm Hg, the absorption of antipyrine increased only in the colon. Stepwise reduction of the blood flow rate from 1.4-1.7 to 0.2-0.3 or stepwise raise from 0.2-0.3 to 1.4 ml min-1 g-1 by constriction or release of the mesenteric artery decreased or increased the absorption rate of antipyrine. The relation between absorption and flow rate can be described by curves which ascend at low and level off into a horizontal section at high flow rates. At the same blood flow rate the regional absorption rate decreased in the order jejunum, ileum, and colon with the largest step between ileum and colon. Model analysis yielded the following results for jejunum, ileum, and colon, respectively: permeability-surface area product 0.083, 0.074, and 0.037 ml min-1 g-1; fraction of absorptive site blood flow rate 0.24, 0.19, 0.08. The differences can be attributed mainly to the change of the surface area from jejunum to ileum and colon.

Influence of dietary fiber and intraluminal pressure on absorption and pre-epithelial diffusion resistance (unstirred layer) in rat jejunum in situ.

The appearance rates of antipyrine, benzoic acid, benzylamine, urea, and alpha-methyl-D-glucoside (MG) in jejunal venous blood of anesthetized rats were measured with and without dietary fibers methylcellulose, carboxymethylcellulose sodium, guaran, and sodium alginate in the luminal solution. Raising the concentration of methylcellulose from 0 to 17.5 g/l resulted in an exponential increase in the viscosity of the solution to 98 cSt, a linear decrease of the diffusion coefficient for antipyrine by 28%, and an increase in antipyrine absorption in the perfused jejunal segment by 23%. The simultaneous increase in intraluminal pressure and radius resulted in a linear relation between absorption rate and apparent mucosal surface area. Similar results were obtained by raising intraluminal pressure directly using a carbohydrate-free perfusion solution. In the perfused rat jejunum, the effect of increased pre-epithelial diffusion resistance (i.e. reduced diffusion coefficient and lengthened diffusion distance) induced by methylcellulose on absorption was overcome by the effect of the enlarged apparent mucosal surface area. Preperfusion of a substrate-free, guaran containing solution followed by perfusion with a guaran-free solution containing antipyrine and MG retarded the increase in the appearance rate of these substrates due to the additional viscous guaran layer left after preperfusion. Constant distension of the intestinal wall was achieved by injecting 0.5 ml of the solution into a closed jejunal segment. Addition of the carbohydrates to the injection solution (approx. 100 cSt viscosity) resulted in a 3% to 20% reduction in the diffusion coefficients and in the absorption of antipyrine, benzoic acid, and MG. Diffusion coefficients for urea and benzylamine were reduced by 5% to 12%; absorption varied in the range of the control (-22% to +43%). Model analysis revealed that, in the closed jejunal segment of the rat, the limiting step in the absorption process of antipyrine, benzoic acid, and MG was pre-epithelial diffusion resistance; the reduction of absorption, therefore, corresponded roughly to that of the diffusion coefficient. In the case of urea and benzylamine, pre-epithelial diffusion resistance was only 20% of the total permeation resistance: the influence of the polymers on absorption, therefore, was not always significant.

In vivo studies of mucosal-serosal transfer in rat jejunum.

In anesthetized rats, the appearance rates of a series of labeled substances in jejunal venous blood (phi B) and serosal bath (phi S) were measured in vivo (intestinal blood flow rate 1.5 ml min-1 g-1) after intraluminal administration of 0.5 ml buffer solution (initial concentration 1 mmol/1 or 1 GBq/1 tritiated water) into a closed jejunal segment (length 4-5 cm). Between 32% (erythritol) and 93% (salicylic acid) of the administered activity (unchanged substance and possible metabolites) appeared in the intestinal venous blood within 60 min. The fraction recovered from the serosal bath after 15 (60) min was 11 (6)% for tritiated water, 7 (4)% for aniline, 3 (7)% for aminopyrine, 5 (4)% for butanol, 3 (3)% for benzyl alcohol, 2 (4)% for benzylamine, 1-2% for benzoic acid, theophylline, methyl-alpha-D -glucopyranoside, L-lysine, antipyrine, and urea, and less than 1% for L-phenylalanine, D-galactose, erythritol, and salicylic acid. During single pass perfusion of a jejunal segment (length 3-4 cm) the fraction of serosal transfer phi S/(phi B + phi S) was 19% for tritiated water, 4.9% for antipyrine, 0.5% for benzoic acid, and 0.08% for salicylic acid. Distension of the intestinal wall by administration of 1 ml buffer solution instead of 0.5 ml increased the appearance rate of benzoic acid and antipyrine in intestinal venous blood by a factor of 2 and serosal transfer by a factor of approximately 3.(ABSTRACT TRUNCATED AT 250 WORDS)

Subclasses of muscarinic receptors in isolated gastric mucosal cells: receptor characterization and parietal cell function.

Muscarinic receptors were characterized in isolated intact chief and parietal cell enriched cell populations from canine and guinea-pig gastric mucosa by binding of tritiated N-methylscopolamine ([3H]NMS). Antagonist and agonist binding was studied by displacement of [3H]NMS with non-radioactive atropine, pirenzepine, pilocarpine and carbachol. Model analysis points to the existence of two binding sites in each of the two cell populations. The number of binding sites per cell was 1.7-1.8 times higher in parietal than in chief cell populations. Subclasses of muscarinic receptors as characterized by pirenzepine binding were compatible with the suggested A- and C- (high and low affinity) binding sites. The observation that in canine cells GMPPNP induced a conformational change of the high affinity binding site for pirenzepine could suggest that their proportion might depend on environmental factors. Binding parameters were related to specific parietal cell function as measured by aminopyrine accumulation as index for acid secretion. The carbachol effects depended on the calcium concentration and were competitively inhibited by pirenzepine. The physiological relevance of muscarinic receptor heterogeneity in gastric mucosal cells is unknown although the data support the hypothesis that involvement of muscarinic binding sites in calcium transport mechanisms connected with parietal cell function and possible conformational changes of the binding sites might be regulatory parameters in gastric secretory processes.

Drug absorption by the rat jejunum perfused in situ. Dissociation from the pH-partition theory and role of microclimate-pH and unstirred layer.

In anaesthetized rats the rate of appearance of benzoic acid and aminopyrine in jejunal venous blood was measured; the pH of the luminal perfusion solution was varied between 4 and 10.5. The pH-absorption curves were less steep than predicted by the unmodified pH-partition theory. A reduction of the mucosal unstirred layer thickness by means of the segmented-flow technique considerably increased the absorption rate without essentially changing the shape of the pH-absorption curves. The pH at the surface of the jejunal mucosa was 6.0, 6.5, 6.6, and 8.0 for luminal solutions of pH 4.0, 6.0, 8.0, and 10.8, respectively. From the absorption data the microclimate-pH was calculated which would explain best the observed pH-absorption curves. These calculated pH-values correspond well to the values measured at the mucosal surface. Therefore, it was concluded that a microclimate-pH caused the deviation of the intestinal pH-absorption curves of benzoic acid and aminopyrine from the prediction of the unmodified pH-partition theory. The mucosal unstirred layer represented only a considerable permeation resistance and was not responsible for the deviating shape of the pH-absorption curves.

Appearance of 14C-polyethylene glycol 4000 in intestinal venous blood: influence of osmolarity and laxatives, effect on net water flux determination.

In anaesthetized rats the appearance rate of 14C-polyethylene glycol 4000 (14C-PEG 4000) was measured in the intestinal venous blood after intraluminal administration into a jejunal, ileal, and colonic segment. The absorption rate was small, especially in the colon (0.5-4.2% within 60 min in an intestinal segment of about 300 mg wet tissue weight depending on the batch of 14C-PEG 4000). The absorbed PEG in the plasma consisted mainly of molecules with lower molecular weight than 4000 which were included in the commercial batches of 14C-PEG 4000. The appearance rate of 14C-PEG decreased with time after single dose but remained constant, when a 14C-PEG solution was perfused continuously through the intestinal lumen. A hypotonic solution increased and a hypertonic one decreased slightly the absorption of PEG in the jejunum and ileum but not in the colon. The influence of bisacodyl (100 mgl(-1)) and ricinoleate (10 mmol l(-1)) on the absorption of PEG was small or absent, while deoxycholate (5 mmol 1(-1)) raised the absorption rate considerably, predominantly of the high molecular weight fraction. If in intestinal absorption studies a batch of commercial 14C-PEG 4000 with a small low molecular weight fraction is used, the error in the determination of net water flux caused by the absorption of PEG can be neglected. The influence of osmolarity and laxatives is insignificant. Bile acids increase the intestinal permeability of PEG 4000, so that the net water flux determination can be biased considerably.

Intestinal first-pass effects of estrone sulfate and estrone in the rat.

Mixtures of (14C)-estrone and (3H)-estrone-sulfate (10 micrograms and 13 micrograms, respectively) were instilled into the lumina of intact jejunal loops of rats. Estrone was faster absorbed into portal blood than was estrone-sulfate (72% vs. 17% in 30 min). In portal blood the chemical forms derived from estrone were: unchanged estrone (72%), estrone-glucuronide (14%), estrone-sulfate (5%) and estradiol (9%); no estriol was formed. The chemical forms of estrone sulfate were: estrone-sulfate (86%), estrone (6%), estrone-glucuronide (2%), estradiol (2%) and estradiol-sulfate (4%). These metabolites were also found in the lumen. It is concluded that the intestinal first-pass effects do not significantly influence the hormonal activities of the two estrogens.

Influence of blood flow on intestinal absorption of xenobiotics.

The dependence of intestinal absorption of xenobiotics on the blood flow rate increases from blood flow independent to blood flow limited absorption as the absorbability of the substances increases. Since the absorbed substances are mainly drained by the blood flowing through the subepithelial vessels, not only the total flow rate of an intestinal segment but also the intramural blood flow pattern influences the absorption rate. The villous countercurrent exchange represents an additional resistance to the absorption. In rat jejunum a time-dependent decreases of absorption complicates the analysis of experimental data.

The longitudinal intraluminal concentration gradient in the perfused rat jejunum and the appropriate mean concentration for calculation of the absorption rate.

In anaesthetized rats, jejunal segments 30 cm in length were perfused (rates:0.1, 0.2,0.5 ml/min) with solutions containing antipyrine, salicylic acid, urea, L-lysine, L- and D-phenylalanine. The intraluminal concentration was determined approx. 10, 20, and 30 cm from the inflow cannula. The intraluminal concentration gradients were almost exponential and corresponded to the gradient predicted for a laminar flow through a solute-permeable circular tube. This gradient has a slightly greater curvature than the exponential one. Theoretical considerations showed that the logarithmic mean (Co-Cz)/ln(Co/Cz) of the in- and outflow concentration is the best average value for application in non-integrated absorption equations. This is also valid for the temporal concentration gradient after single administration of a substance into an intestinal segment.

Absorption and metabolism of naphthalene and benzo(a)pyrene in the rat jejunum in situ.

Naphthalene or benzo(a)pyrene (100 nmol) was instilled into the closed rat intestinal loop in situ and the appearance of the free compound and its metabolites was determined in portal blood. Naphthalene appeared mostly unchanged in blood whereas benzo(a)pyrene was extensively metabolized by mucosal cells. The results suggest that absorption and metabolism are competing processes in the gut.

Rat jejunum perfused in situ: effect of perfusion rate and intraluminal radius on absorption rate and effective unstirred layer thickness.

In anaesthetized rats a jejunal segment was perfused in situ varying the perfusion rate (0.1, 0.2, 0.5 ml/min) in a randomized order. The intraluminal radius of the segments was small (1.7 mm) or enlarged (3.1 mm) by increasing the intraluminal pressure. The appearance rate of butanol, antipyrine, salicylic acid, D- and L-phenylalanine but not of urea in the venous blood of the jejunal segments was increased up to 35%, when the intraluminal perfusion rate was raised from 0.1 to 0.5 ml/min. Two factors contribute to this effect: the flattening of the concentration gradient down the segment and the reduction of the effective unstirred layer thickness. The length and the intraluminal radius of the perfused segments was not altered, when the perfusion rate was varied. Therefore, a change of the absorbing area did not contribute to the increase of the absorption rate induced by the increase of the perfusion rate. In the series with small intraluminal radius the experimental data corresponded to the theoretical predictions obtained for a laminar intraluminal flow. In the segments with enlarged intraluminal radius the increase of the absorption rate by raising the perfusion rate was less than expected for a laminar flow indicating that the flow might have been turbulent. The enlargement of the intraluminal radius from 1.7 to 3.1 mm increased the absorption rate up to 100%.