Effect of abomasal emptying rate on the apparent efficiency of colostral immunoglobulin G absorption in neonatal Holstein-Friesian calves.

Inadequate absorption of colostral IgG in calves increases the risk of morbidity and death and is an important source of economic loss to the dairy industry. We hypothesized that an increased rate of abomasal emptying in colostrum-fed calves would be associated with an increased apparent efficiency of absorption (AEA) of colostral IgG. This is because an increase in abomasal emptying rate causes IgG to reach the site of absorption in the small intestine earlier and at a higher luminal concentration. The main objective was, therefore, to determine the association between the AEA of colostral IgG and abomasal emptying rate in neonatal calves. Twenty-four neonatal Holstein-Friesian calves were randomly assigned to one of the following treatments: control, 2 mL of 0.9% NaCl intramuscularly; erythromycin, 8.8 mg/kg of body weight intramuscularly; ivermectin, 200 µg/kg intravenously; and gentamicin, 6.6 mg/kg intramuscularly. These treatments were selected because we have previously demonstrated that erythromycin and ivermectin increase, and gentamicin decreases, the rate of abomasal emptying in milk-fed calves. Calves were fed 3 L of pooled cow colostrum containing acetaminophen (50mg/kg of body weight) by oroesophageal intubation at 1h of age and 30 min after each treatment was administered. Jugular venous blood samples were obtained periodically after the start of feeding. Abomasal emptying rate was assessed by the time to maximal plasma acetaminophen concentration. Erythromycin increased and gentamicin decreased the abomasal emptying rate and AEA of colostral IgG compared with control, respectively, whereas ivermectin had no effect. Using data from all 24 calves, the AEA of colostral IgG was linearly and negatively associated with abomasal emptying rate (R(2)=0.22). We conclude that the abomasal emptying rate is an important determinant of the AEA of colostral IgG. Identifying a non-antimicrobial method for increasing abomasal emptying rate will provide a practical and effective method for facilitating transfer of passive immunity in colostrum-fed dairy calves.

Effectiveness of modified yeast cell wall extracts to reduce aflatoxin B1 absorption in dairy ewes.

This study investigated the effect of a modified yeast cell wall extract preparation (YCW) on the excretion of aflatoxin B1 (AFB1) and aflatoxin M1 (AFM1) in feces, urine, and milk of dairy ewes fed an aflatoxin-contaminated diet. Sixteen ewes in mid-lactation were assigned to 4 treatment groups: control, AF (60 µg of AFB1/kg of feed), YCW (2 g/kg of feed), and AF+YCW. The trial consisted of a short-term (3-d) exposure period followed by a long-term (21-d) exposure period. At the end of each exposure period, milk, urine, and feces were collected over 72 h. The treatments did not affect feed intake, milk production, milk composition, or body weight. The presence of AFM1 was detected in all matrices, whereas AFB1 was only present in feces. Daily excretion was higher following long-term exposure and reached 26.9 µg of AFB1/d in feces, 37.2 µg of AFM1/d in feces, and 10.7 µg of AFM1/d in urine. Supplementation with YCW was effective in increasing aflatoxin excretion in feces in the long-term exposure (up to 156% increase). The effect was accompanied by a trend of decreasing urinary excretion of AFM1. In contrast, the addition of YCW to the contaminated diet did not affect the transfer of aflatoxins from feed to milk under the present experimental conditions with low-producing ewes. The transfer rates of AFM1 in milk ranged from 0.24 to 0.54%. In conclusion, feed supplementation with YCW reduced the absorption of AFB1 and increased the elimination of AFB1 and AFM1 in ewe feces. Yeast cell wall extract could be used to protect ruminants from chronic exposure to aflatoxins present in feeds.

Integration of physicochemical and pharmacokinetic parameters in lead optimization: a physiological pharmacokinetic model based approach.

There have been major strides in the development of novel ways of investigating drug like properties of new chemical entities (NCE) in the last three decades. Identification of ideal properties of a clinical candidate that would give a clinical proof of concept (POC) is the most critical step in the discovery process. Besides the biological dose-response relationship, the pharmacokinetic parameters play the most vital role in influencing the therapeutic response or toxicity of NCE. While there are numerous techniques to understand various drug-like properties individually, the behavior of an NCE in a dynamic in vivo system which influences its therapeutic or toxic effects is a composite function of its various physicochemical and pharmacokinetic parameters. This implies the need to understand the collective influence of various measured parameters, and knowing how variations made in them can result in favorable pharmacodynamic or toxicokinetic properties. Understanding this behavior holds the key to a successful and time efficient lead optimization process. Physiological based pharmacokinetic models (PBPK) are of great interest in this context as they involve a natural way of integrating the individual compound property to physiological properties, providing a rational approach to predict drug like behavior (an ideal behavior identified may be addressed generally as Target Product Profile) in vivo. In the current review, various physiological pharmacokinetic models addressing absorption, tissue distribution and clearance are discussed along with their application in integrating various physicochemical and ADME parameters to predict human pharmacokinetics helping lead optimization.

Efficacy of oral cobalamin (vitamin B12) therapy.

IMPORTANCE OF THE FIELD: Cobalamin (vitamin B12) deficiency is particularly common in the elderly (> 15%). Management of cobalamin deficiency with cobalamin injections is well codified at present, but new routes of cobalamin administration (oral and nasal) are being studied, especially oral cobalamin therapy for food-cobalamin malabsorption. AREAS COVERED IN THIS REVIEW: The objective of this review is to evaluate the efficacy of oral cobalamin treatment in elderly patients. To reach this objective, PubMed data were systematically searched for English and French articles published from January 1990 to July 2008. Data from our research group on cobalamin deficiency (Groupe d'Etude des CAREnce vitamine B12 - CARE B12) were also analyzed. WHAT THE READER WILL GAIN: Three prospective randomized studies, a systematic review by the Cochrane group and five prospective cohort studies were found and provide evidence that oral cobalamin treatment may adequately treat cobalamin deficiency. The efficacy was particularly highlighted when looking at the marked improvement in serum vitamin B12 levels and hematological parameters, for example hemoglobin level, mean erythrocyte cell volume and reticulocyte count. The effect of oral cobalamin treatment in patients presenting with severe neurological manifestations has not yet been adequately documented. Oral cobalamin treatment avoids the discomfort, inconvenience and cost of monthly injections. TAKE HOME MESSAGE: Our experience and the present analysis support the use of oral cobalamin therapy in clinical practice.

[Effect of additives on absorption of Coptis chinensis total alkaloids and pharmacokinetics in mice].

OBJECTIVE: To evaluate the effect of additives on absorption of Coptis chinensis total alkaloid and their pharmacokinetics in mice. METHOD: The mice were fed with the mixture of C. chinensis total alkaloids and additives (1:1). And then the feces and orbital blood were taken to detect the content of total alkaloids by HPLC and their pharmacokinetics. RESULT: Glutin could make the absorption of jatrorrhizine, coptisine, berberine and total alkaloids increased by 30%. Tween 80 and arabic gum did not affect the absorption of berberine, but inhibit that of other alkaloids. There had no influence of lecithin on the absorption of alkaloids. The peak time of total alkaloids in blood were 2 h (Cmax 1=5.9 mg x L(-1)) and 5.0 h (Cmax 2=3.4 mg x L(-1)), respectively, AUC was 17.6 mg x h x L(-1), the elimination of Half-life t1/2 was 5.2 h. After addition of glutin, the peak time of total alkaloids in blood were 1.5 h (Cmax 1=7.6 mg x L(-1)) and 4.8 h (Cmax 2=8.5 mg x L(-1)), AUC was up to 31.1 mg x h(-1) x L(-1), the elimination of Half-life t1/2 was 6.2 h. CONCLUSION: Glutin could accelerate the mice on the absorption of C. chinensis total alkaloids, to extend the elimination half-life, increase the blood concentration and bioavailability.

Presentation of a modified dispersion model (MDM) for hepatic drug extraction and a new methodology for the prediction of the rate-limiting step in hepatic metabolic clearance.

The main objectives were to investigate the roles of and interplay between determinants of hepatic clearance (CL(H)) in humans, to develop a methodology and reference system for the evaluation and prediction of the rate-limiting step in CL(H), and to update the dispersion model and compare it with traditionally used liver extraction models. The new methodology enables predictions of the hepatic uptake and CL(H), dissociation, and rate-limiting step. In general, absorption, dissociation and diffusion are comparably rapid processes, and metabolism is rate-limiting. The liver appears to have a high passive uptake capacity. The Modified Dispersion Model (MDM) has a dispersion number of 0.5 and a distribution factor (df = 0.87) for the correction of a longer hepatic transit time of unbound molecules and the exclusion of the hidden unbound fraction within erythrocytes. Liver models are functionally equivalent at low CL(H), but differ for highly extracted compounds. Well-stirred and parallel-tube models demonstrate the greatest difference in performance, for example, 6- and 800,000-fold differences in the estimated in vivo intrinsic CL(H) and predicted oral bioavailability of the high CL(H) drug naloxone, respectively. The roles of and interplay between determinants of CL(H) have been further clarified and can now be better predicted. Apparent advantages with the MDM include its scientific rationale and intermediate/ balanced performance.

Comparison of intratumor and intraluminal temperatures during locoregional deep hyperthermia of pelvic tumors.

PURPOSE: To investigate whether intraluminal thermometry provides sufficient information to apply high quality deep hyperthermia in pelvic tumors. PATIENTS AND METHODS: The intratumor and intraluminal temperatures of 48 patients were analyzed per cancer type: rectum (21 male, 14 female), cervix (n=8), and bladder (n=5). Temperature-dose parameters were calculated, temperature curves within each treatment session were compared, and correlation between intratumor and intraluminal temperatures was analyzed. RESULTS: Intratumor and intraluminal temperatures at the same time points during individual treatments were highly correlated (mean correlation coefficient: 0.93). However, the quantitative level differed from 0.1 to 1.1 degrees C and the differences of the time-temperature graphs varied per tumor group. Average intratumor and intraluminal temperatures were not different in the four groups. Intratumor thermometry was found not superior over intraluminal thermometry to improve tumor temperature level and homogeneity by SAR steering. CONCLUSION: Intraluminal thermometry provides sufficient information to apply deep hyperthermia to individual patients with centrally located rectum, cervix or bladder cancer.

Functional markers of selenium status: UK Food Standards Agency workshop report.

The workshop was organised to discuss the validity and limitations of existing functional markers of Se status in human subjects and to identify future research priorities in this area. Studies presented as part of this workshop investigated: the bioavailability of Se from different dietary sources; potential functional markers of Se status; individual variation in response to Se; the effect of marginal Se status on immune function. The workshop highlighted the need to define the relationship between functional markers of Se status and health outcomes.

Nasal absorption of metoclopramide from different Carbopol 981 based formulations: In vitro, ex vivo and in vivo evaluation.

There is a need for nasal drug delivery of metoclopramide HCI (MTC) in specific patient populations where the use of commercially available intravenous and oral dosage forms may be inconvenient and/or unfeasible. In this perspective, nasal dosage forms (solution, gel and lyophilized powder) of MTC were prepared by using a mucoadhesive polymer Carbopol 981 (CRB 981). The drug release studies of formulations were performed by using a modified horizontal diffusion chamber with cellulose membrane and excised cattle nasal mucosa as diffusion barriers. After the ex vivo experiments, the morphological appearances of the nasal mucosa were analyzed with the light microscopic studies. In vivo experiments were carried on sheep model. The release of MTC from solution and powder formulations was found higher than gel formulation (p < 0.05) and no severe damage was found on the integrity of nasal mucosa after ex vivo experiments. The penetration enhancing effect of dimethyl-beta-cyclodextrin (DM-beta-CD) used in powder formulations was observed in ex vivo and in vivo experiments. In contrast to in vitro and ex vivo experiments the nasal bioavailability of gel formulation was found higher than those of the solution and powder (p < 0.05) and might represent a promising novel tool for the systemic delivery of MTC.

Coenzyme Q10 absorption and tolerance in children with Down syndrome: a dose-ranging trial.

Controlled studies of coenzyme Q(10) dosing and tolerance have been reported in adults, but not in pediatric patients. This study compares low- and high-dose coenzyme Q(10) (LiQ-NOL syrup) absorption and tolerance in children with Down syndrome. After a 1-month low-dose (1.0 mg/kg/day) run-in period, all participants received high-dose coenzyme Q(10) (10.0 mg/kg/day) for two additional months (in randomized sequence as one daily dose or split into two daily doses). Chemistry profiles and complete blood counts were determined just before and at the study completion. Plasma coenzyme Q(10) concentrations were determined initially and at each study visit. Parents reported adverse events and study drug evaluations using standardized forms. Most of the 16 children who completed this study tolerated high-dose coenzyme Q(10) well. Uncooperative behavior resulted in premature withdrawal of two participants, and may have been treatment-related. Pre- and posttreatment laboratory test changes were considered to be clinically nonsignificant. Study results indicate that high-dose coenzyme Q(10) (10 mg/kg/day) is well-absorbed and well-tolerated by most children with Down syndrome, and appears to provide plasma concentrations which are comparable to previous adult studies administering much higher coenzyme Q(10) dosages.

Chlorogenic acid is absorbed in its intact form in the stomach of rats.

The bioavailability of chlorogenic acid, a major polyphenol of the human diet that is particularly abundant in coffee and various fruits, was explored in rats. To identify the form under which it is absorbed through the gut mucosa and the site of absorption along the gastrointestinal tract, rats were fed a diet supplemented with chlorogenic acid (0.25%, wt:wt). Chlorogenic acid and its metabolites were estimated in the stomach, small intestine and cecal contents as well as in bladder urine and plasma by HPLC with coulometric detection at several time points (1.5, 3, 4.5, and 7 h) after the beginning of the meal. Minor hydrolysis of chlorogenic acid (<1%) occurred in the stomach and small intestine contents, whereas 15-32% of ingested chlorogenic acid was hydrolyzed into caffeic acid in the cecum. Chlorogenic acid and caffeic acid appeared early (at 1.5 h) in plasma and urine, suggesting an absorption of chlorogenic acid into the upper part of the gastrointestinal tract. Gastric absorption of chlorogenic acid was further examined by infusing chlorogenic acid in the ligated stomach of food-deprived rats. After 30 min of infusion, intact chlorogenic acid was found in the gastric vein and aorta. No other metabolites could be detected by HPLC-electrospray ionization-MS-MS. These results show for the first time that chlorogenic acid is quickly absorbed in the rat stomach in its intact form.

Absorption and elimination of formate following oral administration of calcium formate in female human subjects.

Calcium formate is a water-soluble salt of an essential mineral nutrient with potential for use as a dietary calcium supplement. Formate ion is a product of endogenous and xenobiotic metabolism, but sustained high plasma formate concentrations (such as occur in cases of methanol poisoning) are toxic to the retina and optic nerve. Humans and primates have reduced capacity for formate oxidation compared with rodents and dogs and are thus more sensitive to methanol (and formate) intoxication. To assess the potential for accumulation of formate ion upon repeated administration of calcium formate as a potential dietary calcium supplement, we measured plasma concentrations of formate in 14 adult human subjects before and after oral administration of a single large dose of calcium formate (3900 mg; ca. 3-6 times the anticipated dose for calcium supplementation). Plasma formate concentrations increased briskly from 0.024 +/- 0.008 mM (endogenous formate) to reach C(max) (0.50 +/- 0.04 mM) at 60 min postdose and then declined with a half-life of 59 +/- 7 min. By 225 min postdose, plasma formate concentration had returned to baseline. With such a short half-life, repeated use of calcium formate as a dietary supplement, even three times daily, should not lead to progressive accumulation of formate. These findings are discussed in light of the production of formate by endogenous and xenobiotic metabolism and the kinetics of formate during methanol poisoning.

The effect of delta G on the transport and oral absorption of macromolecules.

Delta G (DeltaG) is the biologically active fragment of Zonula Occludens Toxin (Zot), an absorption enhancer, that reversibly opens the tight junctions of epithelial and endothelial cells in the small intestine and brain. This study evaluates the possible use of DeltaG in enhancing the oral bioavailability of macromolecules using large paracellular markers as model agents. The transport of [(14)C]Inulin and [(14)C]PEG4000 was evaluated across Caco-2 cells with DeltaG (0, 100, 180 microg/ml). The apparent permeability coefficients (P(app)) were calculated. The in vitro toxicity of DeltaG (180 microg/ml) was assessed. Sprague Dawley rats were dosed intraduodenally (ID) with the following treatments: [(14)C]Inulin or [(14)C]PEG4000 (30 microci/kg) w/o DeltaG (720 microg/kg)/protease inhibitors (PI). Blood was collected and plasma was analyzed for radioactivity. DeltaG (180 microg/ml) increased [(14)C]Inulin and [(14)C]PEG4000 P(app) by 82.6 and 24.4%, respectively, without any toxicity. After ID administration with DeltaG/PI, C(max) and AUC were significantly (p < 0.05) increased for both Inulin and PEG4000. However, Inulin displayed greater enhancement ratios in vitro and in vivo. This study suggests that DeltaG may be used to enhance the oral bioavailability of macromolecules (e.g., proteins) after coadministration through modulation of paracellular transport.

Enhanced oral bioavailability of poorly absorbed drugs. I. Screening of absorption carrier for the ceftriaxone complex.

In this study, we attempted to improve the oral absorption of ceftriaxone (CTX) by using an absorption carrier and the CTX complex together. After the CTX-Ca-carrageenan gel complex was prepared, several kinds of compounds (Capmul MCM C10, Gelucire 44/14, glycyrrhizin) were screened as potential oral enhancers for our experiment and the intestinal morphologies in rats were examined. Of these compounds, the mono- and diglyceride mixtures, Capmul MCM C10 greatly enhanced the gastrointestinal absorption of CTX when this carrier was coadministered with the complex in rats. Percent bioavailability was attained in rats by the enteral route ranging from 55 to 79% when this complex was administered with various doses of Capmul MCM C10. Furthermore, the surface morphology of the complex has a highly smooth appearance as seen under scanning electron microscopy after preparation. No treatment-related signs of any damage were observed on the administrations intestinal membrane when morphologies were examined in rats after the complex and the absorption carrier were coadministered. The results of the observation suggest that Capmul MCM C10 is a promising carrier, having a good balance between bioavailability enhancing activity and safety, for the oral delivery of CTX when it is coadministered with complex.

Bioavailability and bioequivalence trials: statistics & pharmacokinetic principles.

This document has been prepared as a set of workshop material, which will be presented in the International Congress on Medical and Care Compunetics in June 2004 in Den Hague, The Netherlands. The workshop is divided into two parts and deals with the fundamentals of the bioavailability and bioequivalence studies. First part of the workshop deals with the pharmacokinetic principles, and the second part discusses the statistical approaches and the data analysis using the Statistical Analysis Software (SAS).

The effect of polyunsaturated fatty acids, including conjugated linoleic acid, on calcium absorption and bone metabolism and composition in young growing rats.

The effect of polyunsaturated fatty acids (PUFA), in particular conjugated linoleic acid (CLA), on Ca and bone metabolism is unclear. In a 2x2 factorial design study, forty male 4-week-old rats were fed a control diet containing 70 g added fat (soyabean oil (SBO; n-6 PUFA-rich diet) or menhaden oil-safflower oil (MSO; n-3 PUFA-rich diet))/kg diet with 0 or 10 g CLA/kg for 8 weeks. Ex vivo prostaglandin E2 biosynthesis by bone organ culture was significantly higher (P<0.001) in rats consuming SBO compared with MSO, irrespective of CLA. Addition of the CLA treatment to either diet further lowered (P<0.05) ex vivo prostaglandin E2 production. Neither PUFA type nor CLA altered circulating or femoral mRNA levels of osteocalcin (a marker of bone formation) or insulin-like growth factor-I (a mediator of bone metabolism). While urinary pyridinium crosslinks levels (markers of bone resorption) were unaffected by CLA irrespective of PUFA type, they were significantly higher (P<0.05) in rats consuming SBO compared with MSO irrespective of CLA. Net fractional (%) and absolute (mg) Ca absorption were significantly (P<0.01 and P<0.05 respectively) higher in CLA-supplemented than unsupplemented animals fed on the n-3 PUFA-rich diet, whereas CLA had no effect in animals fed the n-6 PUFA-rich diet. There was no effect of CLA supplementation on bone mineral mass. In conclusion, CLA supplementation over 8 weeks appeared to enhance Ca absorption in young growing rats fed an n-3 PUFA-rich diet, but had no measurable effect on bone metabolism or bone mass over this time frame.

[A study on absorption and utilization of calcium, iron and zinc in mineral-fortified and dephytinized soy milk powder consumed by boys aged 12 to 14 years].

OBJECTIVES: In order to understand the absorption and utilization of calcium, iron and zinc in cow milk, soy milk powder and dephytinized soy milk powder in the children and adolescents, and to lay a foundation for the improvement of protein and mineral nutrition in Chinese residents with popularizing consumption of soy milk powder. METHODS: Totally, 57 boys aged 12 to 14 years were selected and divided into three groups, matched by age, hemoglobin concentration, height and weight. The boys were given 220 ml of fortified and dephytinized soy milk, soy milk or cow milk, respectively, containing calcium 270 mg, iron 4 mg and zinc 4 mg, which were labeled with stable isotopes (44)Ca, (58)Fe and (70)Zn in a single serving size, and their feces were labeled with brilliant blue and dysprosium. Feces specimens were collected for all the subjects and absorption rate of calcium, iron and zinc were measured for all the children who took fortified and dephytinized soy milk, soy milk or cow milk, respectively. Calcium in feces was measured with heat ionized mass spectrometry, and recovery of (58)Fe and (70)Zn in feces and content of dysprosium were measured with induction coupling iso-ionic mass spectrometry, and then absorption rates of iron and zinc were calculated and adjusted by the recovery rate of dysprosium. RESULTS: Iron absorption rate was (6.7 +/- 3.8)% in soy milk powder group, (15.5 +/- 9.2)% in the cow milk group and (20.6 +/- 7.3)% in dephytinized soy milk powder group, respectively. Calcium absorption rate was (43.5 +/- 10.7)%, (64.2 +/- 11.4)%, and (50.9 +/- 6.6)% in the three groups, respectively. Absorption rate of zinc was (11.3 +/- 6.5)%, (31.2 +/- 10.4)% and (20.1 +/- 7.4)%, respectively. Significant increase in absorption of calcium, iron and zinc was observed in the groups with fortified and dephytinized soy milk powder, as compared with those consuming nondephytinized soy milk powder. Absorption rate of calcium and zinc was significantly higher in the group with cow milk than that in the group with dephytinized and fortified soy milk powder, and iron bioavailability was lower in the group with cow milk than that with dephytinized soy milk powder, with no significant difference. Absorption rates of calcium, iron and zinc were higher in children of China due to their long term adaptation to dietary intake of lower mineral and protein, and higher fiber. CONCLUSIONS: Bioavailability of calcium, iron and zinc in soy milk powder could be increased by dephytinized treatment for it.

Modulation of absorption of beta-carotene and tissue accumulation of beta-carotene and vitamin A by different surfactants in rats.

BACKGROUND/AIMS: The absorption of beta-carotene is closely associated with the absorption of dietary fats in the duodenum. Aim of the study was to evaluate two different surfactants, taurocholate and Pluronic L-81, which are known to stimulate or inhibit the absorption of dietary fats, respectively with regard to the absorption of beta-carotene and tissue accumulation of beta-carotene and vitamin A. METHODS: Rats were kept on a vitamin-A- deficient diet for 4 weeks and then either kept on this diet or fed this diet enriched with beta-carotene (200 mg/kg feed) alone or in combination with taurocholate (10 g/kg) or Pluronic L-81 (5 ml/kg) for another two weeks. RESULTS: beta-carotene was not detectable in liver or plasma of rats fed the deficient diet. The supplementation of beta-carotene alone led to an increase of beta-carotene in plasma and organs (p < 0.05) and resulted in an increase of vitamin A in the liver (p < 0.01), indicating its conversion. The addition of taurocholate enhanced the absorption of beta-carotene (p < 0.01), but had little affect on the levels of total vitamin A in the liver. In contrast, Pluronic L-81 caused a reduced uptake of beta-carotene as indicated by lower concentrations in plasma and liver (p < 0.01) as well as reduced total vitamin A levels in the liver (p < 0.01) either caused by the reduced availability of beta-carotene or a reduced conversion into vitamin A. CONCLUSIONS: The study shows that surfactants can modulate beta-carotene absorption differently. The results for taurocholate confirm known observations concerning an enhanced absorption of beta-carotene. Pluronic L-81 might diminish the uptake of beta-carotene into the enterocyte, which would be in disagreement with regard to its function in the absorption of total lipids in general, or might effect the excretion into the blood by modulation chylomicron secretion.

Novel role for CFTR in fluid absorption from the distal airspaces of the lung.

The active absorption of fluid from the airspaces of the lung is important for the resolution of clinical pulmonary edema. Although ENaC channels provide a major route for Na(+) absorption, the route of Cl(-) transport has been unclear. We applied a series of complementary approaches to define the role of Cl(-) transport in fluid clearance in the distal airspaces of the intact mouse lung, using wild-type and cystic fibrosis Delta F508 mice. Initial studies in wild-type mice showed marked inhibition of fluid clearance by Cl(-) channel inhibitors and Cl(-) ion substitution, providing evidence for a transcellular route for Cl(-) transport. In response to cAMP stimulation by isoproterenol, clearance was inhibited by the CFTR inhibitor glibenclamide in both wild-type mice and the normal human lung. Although isoproterenol markedly increased fluid absorption in wild-type mice, there was no effect in Delta F508 mice. Radioisotopic clearance studies done at 23 degrees C (to block active fluid absorption) showed approximately 20% clearance of (22)Na in 30 min both without and with isoproterenol. However, the clearance of (36)Cl was increased by 47% by isoproterenol in wild-type mice but was not changed in Delta F508 mice, providing independent evidence for involvement of CFTR in cAMP-stimulated Cl(-) transport. Further, CFTR played a major role in fluid clearance in a mouse model of acute volume-overload pulmonary edema. After infusion of saline (40% body weight), the lung wet-to-dry weight ratio increased by 28% in wild-type versus 64% in Delta F508 mice. These results provide direct evidence for a functionally important role for CFTR in the distal airspaces of the lung.

Investigation of the coordinated functional activities of cytochrome P450 3A4 and P-glycoprotein in limiting the absorption of xenobiotics in Caco-2 cells.

The coordination of the functional activities of intestinal CYP3A4 and P-gp in limiting the absorption of xenobiotics in Caco-2 cells was investigated. Growing Caco-2 cells were exposed to increasing concentrations of doxorubicin (1-2 microM) in plastic flasks to encourage a subpopulation of cells, that displayed an intrinsically higher multidrug resistance (mdr) phenotype than the parent cells, to survive and grow. Doxorubicin-exposed (hereinafter referred to as type I cells) and nonexposed Caco-2 cells (parent cells) on collagen-coated inserts were also treated with either 0 (control) or 0.25 microM 1alpha,25-dihydroxyvitamin D(3) to promote cellular CYP3A4 expression. Increased P-gp protein expression, as detected by Western blotting, was noted in type I cells (213 +/- 54.35%) compared to that of parent cells (100 +/- 6.05%). Furthermore, they retained significantly less [(3)H]vincristine sulphate (p < 0.05), a P-gp substrate, after efflux (272.89 +/- 11.86 fmol/mg protein) than the parent cells (381.39 +/- 61.82 fmol/mg protein). The expression of CYP3A4 in parental cells after 1alpha,25-dihydroxyvitamin D(3) treatment was quantified to be 76.2 +/- 7.6 pmol/mg protein and comparable with that found in human jejunal enterocytes (70.0 +/- 20.0 pmol/mg protein). Type I cells, however, expressed a very low quantity of CYP3A4 both before and after the treatment that was beyond the minimum detection limit of Western blotting. Functionally, the rates of 1-hydroxylation of midazolam by CYP3A for both cell types ranged from 257.0 +/- 20.0 to 1057.0 +/- 46.0 pmol/min/mg protein. Type I cells, although having a higher P-gp expression and activity comparatively, metabolized midazolam less extensively than the parent cells. The results suggested that there were noncoordinated functional activities of intestinal CYP3A4 and P-gp in Caco-2 cells, although they both functioned independently to minimize intestinal epithelial absorption of xenobiotics.