First-pass metabolism of midazolam by the human intestine

The in vivo intestinal metabolism of the CYP3A probe midazolam to its principal metabolite, 1'-hydroxymidazolam, was investigated during surgery in 10 liver transplant recipients. After removal of the diseased liver, five subjects received 2 mg midazolam intraduodenally, and the other five received 1 mg midazolam intravenously. Simultaneous arterial and hepatic portal venous blood samples were collected during the anhepatic phase; collection of arterial samples continued after reperfusion of the donor liver. Midazolam, 1'-hydroxymidazolam, and 1'-hydroxymidazolam glucuronide were measured in plasma. A mass balance approach that considered the net change in midazolam (intravenously) or midazolam and 1'-hydroxymidazolam (intraduodenally) concentrations across the splanchnic vascular bed during the anhepatic phase was used to quantitate the intestinal extraction of midazolam after each route of administration. For the intraduodenal group, the mean fraction of the absorbed midazolam dose that was metabolized on transit through the intestinal mucosa was 0.43 +/- 0.18. For the intravenous group, the mean fraction of midazolam extracted from arterial blood and metabolized during each passage through the splanchnic vascular bed was 0.08 +/- 0.11. Although there was significant intersubject variability, the mean intravenous and intraduodenal extraction fractions were statistically different (p = 0.009). Collectively, these results show that the small intestine contributes significantly to the first-pass oxidative metabolism of midazolam catalyzed by mucosal CYP3A4 and suggest that significant first-pass metabolism may be a general phenomenon for all high-turnover CYP3A4 substrates.     

Effect of hypothyroidism on glucose transport and metabolism in rat small intestine

A cell line (SMKT-R3) established from human renal cell carcinoma was characterized for the presence of sulfolipids and glycolipid sulfotransferases. Sulfolipids were found to constitute a large part of the acidic glycolipid fraction in SMKT-R3 cells. These findings were confirmed by metabolic labelling with 35S-sulfate. These sulfolipids were expressed at the surface of SMKT-R3 cells as ascertained by cytofluorometry using a monoclonal antibody directed to sulfolipids. Furthermore, markedly high activity levels of glycolipid sulfotransferases were observed in SMKT-R3 cells compared with other cell lines. These results suggest that the increased synthesis of sulfolipids in renal cell carcinoma tissue (Sakakibara et al., 1989. Cancer Res., 49, 335-339) is due to the elevation of the sulfotransferase activities of renal carcinoma cells themselves.     

Thiamine diphosphatase in rat small intestine

TDPase is located mostly in the proximal portion of the small intestine and its activity, like that of ALPase, decreased markedly in thiamine deficiency. The decreased enzyme activities were restored after thiamine or vitamin D3. Kinetic and other studies of the purified enzyme indicated the identity of the two enzymes.     

Vitamin B2 metabolism in the isolated perfused rat liver

We note the existence of a "partially cis-acting" regulatory protein of bacteriophage lambda: the product of the phage Q gene. We suggest that there may be a complete spectrum from "all cis" to "all trans" for such regulatory proteins. This behavior might arise because a DNA-binding protein either acts at a nearby (cis) site soon after synthesis or becomes "lost" for its trans activity on another genome through nonspecific interactions with DNA. Our proposed explanation provides one evolutionary basis for the linkage of genes for regulatory proteins and the sites at which such proteins act; it also suggests a possible rationale for the "metabolic instability" of certain regulatory proteins.     

First-pass metabolism of ethanol is predominantly gastric

Oral consumption of alcohol results in much lower blood alcohol concentrations (BACs) than does the same dose administered intravenously, suggesting significant first-pass metabolism (FPM). The questions remain, however, (1) whether this difference truly represents FPM or simply reflects slower absorption of alcohol, and (2) if there is FPM, is it mainly of gastric or hepatic origin. To study this, rats were given the same dose alcohol (1 g/kg) by either intragastric intubation or by intravenous, intraportal, and intraduodenal infusions at a rate that mimicked the loss of alcohol from the stomach. Higher BAC levels after intravenous than intragastric alcohol indicated true FPM. Higher levels after intraportal or intraduodenal infusions (in fact, comparable to those obtained with the intravenous route) demonstrated negligible FPM when the route of delivery bypassed the stomach, yet included the liver. Furthermore, rats that had developed portosystemic shunts after ligation of the portal ven exhibited blood alcohol curves and FPM equivalent to those of sham-operated controls, indicating that FPM is not dependent on first-pass flow through the liver, but reflects gastric metabolism. The absence of significant hepatic FPM is attributable to the saturation of hepatic alcohol dehydrogenase by recirculating alcohol, resulting in no appreciable increase in metabolism secondary to newly absorbed alcohol. Finally, the in vivo gastric metabolism of alcohol in pylorus-ligated rats was demonstrated by significantly lower BACs when alcohol was administered intragastrically than when an amount identical to that lost from the ligated stomach was given intraportally. Thus, the lower BACs with oral as opposed to intravenous alcohol are not simply a consequence of slow absorption, but result from FPM occurring predominantly in the stomach.     

First-pass metabolism of ethinyl estradiol in dogs and rats

The contraceptive steroid ethinyl estradiol was extensively metabolized when given orally in solution to dogs. It was thought at first that metabolism occurred exclusively in the liver. However, use of standard equations to predict the oral bioavailability of drugs known to be metabolized by hepatic first pass resulted in significantly higher values than those obtained experimentally. To rationalize the data and to determine whether ethinyl estradiol also is metabolized in the gut wall during absorption, metabolism in rats was studied. The drug was administered in solution intraduodenally, intraportally, and intravenously as a bolus by first-order infusion. The results indicate that, in rats, 40% of the drug is metabolized by the gut wall and 79% of the drug in the portal blood is metabolized by the liver intraduodenal administration.     

beta-Glucosidase activity in the rat small intestine toward quercetin monoglucosides

In order to evaluate the positional specificity for a glucoside group in the hydrolysis of flavonoid glucosides in the rat small intestine, beta-glucosidase activity was measured with the quercetin monoglucosides, quercetin-3-O-beta-D-glucopyranoside (Q3G), quercetin-4'-O-beta-D-glucopyranoside (Q4'G) and quercetin-7-O-beta-D-glucopyranoside (Q7G), as well as with quercetin-3-O-rutinoside (rutin) and p-nitrophenyl-beta-D-glucopyranoside (NPG) by using the HPLC technique. Enzymes were prepared from rat small intestinal mucosa of the duodenum, jejunum and ileum, among which the enzyme activity of the jejunum was highest for all the glycosides tested. Q4'G was the richest substrate for a beta-glucosidase solution among these glycosides, while rutin and NPG were both poor substrates. This suggests that dietary flavonoid glucosides are primarily hydrolyzed and liberated aglycones in the jejunum.     

Action of levorin on glucose transport in the rat small intestine

The technique of accumulating preparation of the mucosa and "turned out sac" was used to show that levorin, a polyenic antibiotic in a concentration of 10(-6) M, lowered the transport rate and accumulation of glucose by the epithelial cells of the rat thin intestine under conditions of oxygenation. Suppression of the glucose transport in the first stages resulted in partial inhibition of the transmembrane transfer. It is suggested that levorin suppression of the glucose transport through the erythrocyte apical membrane in the thin intestine is associated with a decrease in the electrochemical gradient of Na+.     

Influence of acetate on glucose metabolism in the perfused hind-quarter of the rat

Single unit activity was recorded in the cat vagus in order to detect possible receptors firing in response to changing lung CO2 concentration. The cats were ventilated at a high rate (60-120 breaths per min) and inspired CO2 concentration was altered between 0 and 8% in a step-like fashion, each phase consisting of about 10 breaths. Thus the effects of changing intrapulmonary CO2 concentration could be differentiated from the effects of stretch of lung tissue. Activity was recorded in 7 cats from 120 units firing in phase with ventilation. Many receptors showed some CO2 sensitivity, but no fiber was found discharging in response to CO2 exclusively. The results provide no evidence for the occurence of specific CO2 receptors in the feline lung with vagal afferents functionally similar to those reported for the avian lung.     

Mechanism of nitric oxide-induced contraction in the rat isolated small intestine

1. The contractile response to nitric oxide (NO) in ral ileal myenteric plexus-longitudinal muscle strips was pharmacologically analysed. 2. NO (10(-7) M) induced only contraction while 10(-6) M NO induced contraction followed by relaxation. Methylene blue (up to 10(-4) M) did not affect the NO-induced contractions but significantly reduced the relaxation evoked by 10(-6) M NO. Administration of 8-bromo-cyclic GMP (10(-6)-10(-4) M) only induced relaxation. 3. Sodium nitroprusside (SNP; 10(-7)-10(-5) M) induced concentration-dependent contractions per se; the contractile response to NO, administered within 10 min after SNP, was concentration-dependently reduced. The guanosine 3':5'-cyclic monophosphate (cyclic GMP) content of the tissues was not increased during contractions with 10(-8) M NO and 10(-6) M SNP; it was increased by a factor of 2 during contraction with 10(-7) M NO, and by a factor of 12 during relaxation with 3 x 10(-6) M NO. 4. The NO-induced contractions were not affected by ryanodine (3 x 10(-5) M) but were concentration-dependently reduced by nifedipine (10(-8)-10(-7) M) and apamin (3 x 10(-9)-3 x 10(-8) M). 5. These results suggest that cyclic GMP is not involved in the NO-induced contraction in the rat small intestine. The NO-induced contraction is related to extracellular Ca2+ influx through L-type Ca2+ channels, that might be activated in response to the closure of Ca(2+)-dependent K+ channels.     

Small intestinal dysmotility following abdominal irradiation in the rat small intestine

Abdominal symptoms such as diarrhoea, abdominal cramps and vomiting are common during and after abdominal radiotherapy for gynaecological and pelvic malignancy. It has recently been recognized that small intestinal dysmotility may contribute to these symptoms but the underlying mechanisms are unclear in part because of the technical difficulties inherent in performing studies in irradiated small intestine. The aim of the current study was to evaluate small intestinal motor activity using perfused micromanometric techniques in 6-8-cm segments of ileum during arterial perfusion with isotonic oxygenated fluorocarbon solution. Intestinal segments from six rats were studied 4 days after treatment with 10 Gy abdominal irradiation. Ileal segments from nine nonirradiated animals acted as controls. For each experiment the total number of pressure waves, high-amplitude (> 20 mmHg, long-duration > 6 sec) pressure waves, and long (> 20 associated) bursts of pressure waves were determined. Irradiation had no effect on the overall number of pressure waves, but increased high-amplitude long-duration (HALD) pressure waves (248 vs 7, P < 0.01). In control animals HALD waves were localized to a single recording site but after radiotherapy 74% of HALD waves were temporally associated with similar pressure waves in other manometric channels. Forty-seven per cent of associated HALD waves migrated aborally. Retrograde migration of HALD waves was seen in five segments following irradiation. Irradiation abolished bursts of > 20 pressure waves.     

Enhancement of Ca++ uptake by lactose in the rat small intestine

In previous experiments, lactose was shown to increase the absorption of Ca++ by the small intestine of the rat and other mammals. To further investigate the mechanism of the lactose effect, Ca++ uptake was studied in everted gut sac preparations. Gut sacs from the rat ileum were preincubated with or without lactose for 45 minutes, and then the tissue uptake of 45Ca over the first 3 minutes was measured in the presence or absence of lactose. The presence of 160 mM lactose increased the initial rate of Ca++ uptake in the first minute by 64% compared to the NaCl control. The lactose effect was dependent on the presence of lactose in the preincubation medium only and not on the presence of lactose during the measurement of Ca++ uptake. Lactose increased Ca++ absorption when the Ca++ concentrations ranged from 0.1 to 10 mM. However, the magnitude of the enhancement was dependent on the lactose concentration and was reduced below 160 mM lactose. When Ca++ and lactose uptake during a 45 minute period was measured in parallel experiments, no evidence for the co-transport of lactose and Ca++ into the tissue was found. These and other data indicated that lactose is not interacting directly with Ca++ in solution but is interacting with the absorptive cells of the intestine to increase their permeability to Ca++.     

Oxyntomodulin reduces hydromineral transport through rat small intestine

Glicentin (GLIC) and oxyntomodulin (OXM) are released from the ileum and colon during digestion. Both hormones reduce fluid and proton secretion in the stomach. The luminal concentration of sodium and chloride underlying the nutrient absorption, the effect of OXM on electrolyte transport through the small intestine, was assessed in vivo using ligated loops and in vitro using Ussing chambers. In vivo, a zero transport state, estimated by the net water, chloride, and sodium fluxes, was observed when an 80 mM NaCl normoosmolar solution (274 mosm) was administered intraluminally. Active secretion was observed with hyperosmotic challenge (474 mosm). The amplitude of this active secretion increased 2.5- to 3-fold when an electrogenic challenge (NaCl 40 mM) was substituted to the hyperosmotic one. OXM (800 fmol/ml plasma) did not modify the basal transport in the duodenum or in the jejunum (t = 45 min). When active secretion was induced by the hyperosmotic challenge, OXM (200 fmol/ml plasma) had no effect on duodenal or jejunal transport (t = 50 min). When active secretion was induced by an electrogenic challenge, OXM (300 fmol/ml plasma) preferentially reduced the hydromineral transport in jejunum. In vitro, OXM also induced a reduction in the ion transport towards the jejunal lumen (EC50 = 20 pM), the amplitude of which depended upon the integrity of the tetrodotoxin-sensitive neurons. In conclusion, OXM was able to reduce the large secretion induced in rat jejunum in vivo by an electrogenic gradient. In vitro, the antisecretory effect of OXM was partly mediated by the neurons present in the intrajejunal wall.     

The relationship between the adenine nucleotide metabolism and the conversion of the xanthine oxidase enzyme system in ischemia-reperfusion of the rat small intestine

The time course of the energy metabolism after reperfusion, the relationship between the conversion of xanthine dehydrogenase to xanthine oxidase (D-to-O conversion) during ischemia, and the changes of the energy metabolism after reperfusion were studied using an ischemia-reperfusion model in the small intestine of the rat. The rat jejunum underwent an occlusion of the superior mesenteric artery and vein for either 30 minutes (group 1, n = 6) or 90 minutes (group 2, n = 6) with collateral interruption, and then it was reperfused. The contents of the adenine nucleotides in the small intestine of the rat were measured by high-performance liquid chromatography (HPLC) before ischemia, and 30, 60, and 90 minutes of ischemia, as well as 30, 60, 120, and 180 minutes after reperfusion. The recovery level of adenosine triphosphate (ATP) in group 1 (6.05 +/- 0.80 mumol/g dry weight) 30 minutes after reperfusion was significantly higher than that in group 2 (2.28 +/- 1.12 mumol/g dry weight) (P < .001). In addition, the ATP content after reperfusion in group 2 did not change from 30 to 180 minutes after reperfusion. The D-to-O conversion during ischemia in group 1 was not significantly greater than that before ischemia; however, that of group 2 did increase significantly during ischemia (P < .005). These results suggest that the tissue damage from ischemia-reperfusion injury after reperfusion under 90 minutes' ischemia is accomplished within the first 30 minutes after reperfusion. Therefore, the ATP level at 30 minutes after reperfusion may be useful for the evaluation of intestinal viability. Thus, the conversion of the xanthine oxidase enzyme system might play an important role in the expression of ischemia-reperfusion injury.     

Effect of flow on first-pass metabolism of drugs: single pass studies on 4-methylumbelliferone conjugation in the serially perfused rat intestine and liver preparations

Intracellular in vivo recordings of physiologically identified inferior colliculus central nucleus (ICc) auditory neurons (n = 71) were carried out in anesthetized guinea pigs. The neuronal membrane characteristics are described showing mainly quantitative differences with a previous report [Nelson, P.G. and Erulkar, S.D., J. Neurophysiol., 26 (1963) 908-923]. The spontaneous spike activity was consistent with the discharge pattern of most extracellularly recorded units. The action potentials showed different spike durations, short and long, and some of them exhibited hyperpolarizing post-potentials. There were also differences in firing rate. The ICc neurons exhibited irregular activity producing spike trains as well as long silent periods (without spikes). Intracellular current injection revealed membrane potential adaptation and shifts that outlasted the electrical stimuli by 20-30 ms. Both evoked synaptic potentials and the spike activity in response to click and tone-burst stimulation were analyzed. Depolarizing-hyperpolarizing synaptic potentials were found in response to contralateral and binaural sound stimulation that far outlasted the stimulus (up to 90 ms). When ipsilaterally stimulated, inhibitory responses and no-responses were also recorded. Although few cells were studied, a similar phenomenon was observed using tone-burst stimulation; moreover, a good correlation was obtained between membrane potential shifts and the triggered spikes (input-output relationship). These in vivo results demonstrate the synaptic activity underlying many of the extracellularly recorded discharge patterns. The data are consistent with the known multi-synaptic ascending pathway by which signals arrive at the ICc as well as the descending corticofugal input that may contribute to the generation of long duration post-synaptic potentials.     

Effect of oxygen shortage on the metabolism of oestrone in the hemoglobin-free perfused rat liver

Cell-associated enterotoxin B was detected in lysates of cells of Staphylococcus aureus S-6 and 4916 disrupted by sonication or lysostaphin treatment. As much as 67% of this total cell-associated toxin was surface-bound, located outside the cytoplasmic membrane, and was released during protoplasting of this organism by lysostaphin treatment in hypertonic medium. The remainder of the cell-associated toxin was termed cytoplasmic and was released during osmotic lysis of the protoplasts. Levels of cell-associated toxin as a function of the age of the cells showed a rapid increase in both surface-bound, cytoplasmic, and total cell-associated toxin levels during the period of active toxin synthesis (late exponential phase of growth). These cell-associated toxin levels then reached a peak as the culture entered stationary phase, at a time corresponding to a decrease in the rate of toxin synthesis, and decreased slowly thereafter.     

Effect of alcohol ingestion on the epithelial cell population in rat small intestine

Chronic administration of alcohol to well-nourished rats led to striking changes in the small intestinal cell population. The present experiments corroborate the view that alcohol is directly toxic to the small intestine.