Henri Joyeux

Prognosis of patients with nonmalignant chronic intestinal failure receiving long-term home parenteral nutrition

BACKGROUND/AIMS Long-term survival of patients with intestinal failure requiring home parenteral nutrition (HPN) has been only partly shown. Therefore, we described the survival of these patients and explored prognosis factors. METHODS Two hundred seventeen noncancer non-acquired immunodeficiency syndrome adult patients presenting with chronic intestinal failure enrolled from January 1980 to December 1989 in approved HPN programs in Belgium and France; prognosis factors of survival were explored using multivariate analysis. Data were updated in March 1991; not one of the patients was lost to follow-up. RESULTS Seventy-three patients died during the survey, and the mortality rate related to HPN complications accounted for 11% of deaths. Probabilities of survival at 1, 3, and 5 years were 91%, 70%, and 62%, respectively. Three independent variables were associated with a decreased risk of death: age of patients younger than 40 years, start of HPN after 1987, and absence of chronic intestinal obstruction. In patients younger than 60 years of age included after 1983 with a very short bowel, who could represent suitable candidates for small bowel transplantation, the 2-year survival rate was 90%, a prognosis that compared favorably with recent reports of survival after small bowel transplantation. CONCLUSIONS HPN prognosis compares favorably with recent reports of survival after small bowel transplantation.

Intestinal adaptation in patients with short bowel syndrome. Measurement by calcium absorption.

Functional adaptation of remaining intestine was evaluated in 30 patients with extensive small bowel resection. Calcium and xylose absorption tests were compared. Calcium absorption was measured by a double- radiotracer technique. Serum xylosemia was measured 2 hr afterd-xylose ingestion. Patients were divided into two groups according to the time interval between surgery and evaluation: less (group I) or more (group II) than two years. A statistically significant correlation was found between xylosemia and remaining small bowel length (r=0.71; P<0.001) and between calcium absorption and remaining small bowel length (r=0.75; P<0.001) in group I. A significant correlation was also observed between calcium absorption and time after surgery (r=0.65; P=0.001) but not for xylose absorption. Calcium absorption value was significantly increased in group II patients compared with group I patients matched for remaining small bowel length (36.2±12.5% vs 14.5±9.1%; P < 0.001) while no difference was observed between the two groups concerning xylose absorption. These data indicate that intestinal calcium absorption continues to increase for more than two years after a major bowel resection in man. The intestine does not seem to recover all its functions at the same time.

Characterisation of a newly isolated Caco-2 clone (TC-7), as a model of transport processes and biotransformation of drugs

Abstract Three clones isolated from early (clone PD-7 and PF-11) and late (TC-7) passages of the parental Caco-2 epithelial cell line, were characterized for their ability to transport and metabolize endogenous compounds as well as xenobiotics. All three clones were able to form a homogeneous well-differentiated epithelial monolayer as demonstrated by the presence of microvilli at the apical pole of the cells and a high transepithelial electrical resistance. These cell monolayers were further characterized for their ability to transport different probes such as mannitol for the paracellular route, testosterone for passive diffusion and taurocholic acid for the presence of active biliary acids transporters. Only small differences were observed between the parental cell line Caco-2 and the different clones in terms of transepithelial electrical resistance, mannitol paracellular transport and testosterone passive diffusion. However, large differences were observed in the active transport of taurocholic acid with V max / K m values of 0.037, 0.048, 0.060 and 0.178 for Caco-2 parental cell line, clones PD-7, PF-11 and TC-7, respectively. Among transport processes, clones were also characterized for the expression of various enzyme systems involved in the biotransformation of endogenous compounds and xenobiotics, such as cytochromes P450 and UDP-glucuronosyltransferases. All cell types expressed cytochrome P450IA1, as demonstrated by the O-deethylation of 7-ethoxyresorufin. However, a 3 day β-naphthoflavone pretreatment induced 10.1 ± 3.0− and 10.4 ± 5.9− fold increases in 7-ethoxyresorufin O-deethylation in Caco-2 cells and PF-11 clone, respectively, while 24.7 ± 9.6− and 22.7 ± 8.1− fold increases were observed for PD-7 and TC-7 clones, respectively. These two clones also exhibited a much higher catalytic activity towards 1-naphthol, a substrate for UDP-glucuronosyltransferases. Since the intestinal epithelium plays an important role in the rate of absorption of intact drugs following their oral administration, both transport and metabolic characteristics make the Caco-2/TC-7 clone a suitable in vitro model for studying the intestinal disposition of drugs.

Importance of the paracellular pathway for the transport of a new bisphosphonate using the human CACO-2 monolayers model

The transport of a new bisphosphonate, Tiludronate, was investigated on the human adenocarcinoma cell line, CACO-2. Experiments were performed 7-16 days after cells achieved confluence, conditions under which they form well-differentiated monolayers joined by tight junctions. Tiludronate transport rate across CACO-2 monolayers was independent of the temperature (4 degrees versus 37 degrees), of the polarity of the cell membrane (apical-to-basolateral versus basolateral-to-apical), and of the presence of metabolic poisons (sodium azide). Its transport was enhanced by either the presence of EGTA in the incubation buffer, i.e. when extracellular Ca2+ concentration was reduced, or by the pretreatment of monolayers with EGTA, i.e. when the intercellular spaces and the tight junctions were widened. Based on these different observations, we could suggest that Tiludronate mainly used the paracellular pathway to cross the intestinal epithelium. An increase in the Tiludronate permeability coefficient was also observed following treatment of cells with high Tiludronate concentrations, as a consequence of the direct effect of this compound on the extracellular Ca2+ ions. Hence, for high drug concentrations, i.e. 20 mM, we observed a decrease in free extracellular Ca2+ concentration, an increase in the transepithelial electrical resistance and an increase in the transport of [14C]polyethyleneglycol ([14C]PEG400), a probe for the paracellular pathway. The results indicate that Tiludronate is transported across CACO-2 monolayers by the paracellular route. Moreover, it can affect its own transport by its concentration-dependent effect on tight junction widening.

Inhibition of cytochrome P-450p (P450IIIA1) gene expression during liver regeneration from two-thirds hepatectomy in the rat.

Regenerating liver from partial hepatectomy (HPX) is known to exhibit a strong and transient deficiency in both spectrally detectable microsomal cytochrome P-450 (P-450) and related monooxygenase activities. Male Wistar rats (250-300 g) were HPX or sham operated and liver was excised at different times after operation. The time course of accumulation of five different forms of P-450 (including P-450b/e, P-450c, P-450d, P-450p and P-450UT-A) was determined in the regenerating liver, by Western blots developed with specific antibodies. With the exception of P-450c, whose level was not affected, the accumulation of other forms strongly decreased during the first 24 hr after HPX. For P-450b/e and P-450d, 80% of initial level was restored at 96 hr, whereas for P-450p and P-450UT-A, two major forms in control rat liver, the accumulation was only 20-25% of the initial, 1 week after HPX. No significant decrease was observed in sham operated animals. Plasmid pDex 12 containing a cDNA insert coding for P-450p was used to further investigate the effects of HPX on P-450p mRNA level and gene transcription. Northern blot analysis of RNA from regenerating liver (cDNA insert of pDex 12 being used as a probe) demonstrated that P-450p mRNA level decreased strongly to a minimum 12 hr after operation. This was correlated with a strong and transient decrease in P-450p gene transcription determined from nuclear run on experiments, the time course of which, however, did not account for the early decrease in mRNA level. We conclude that P-450p deficiency in the regenerating liver results from a combination of transient inhibition of gene transcription and early increase of mRNA degradation. Time course and amplitude of the decrease in P-450 UT-A accumulation suggest an inhibition of gene transcription as observed with P-450p.

Bisphosphonates increase tight junction permeability in the human intestinal epithelial (Caco-2) model

Abstract The human Caco-2 monolayer model was used to elucidate the potential role of bisphosphonates in the regulation of the junctional complex opening. Experiments were performed in parallel with EGTA, a potent free Ca 2+ chelator, which was already demonstrated to increase intestinal epithelium permeability both in vitro and in vivo. Following treatment of Caco-2 cells with increasing non-toxic concentrations of tiludronate, a new bisphosphonate, different phenomena could be observed such as (i) free Ca 2+ complex formation at tiludronate concentrations above 0.3 mM and a Ca 2+ IC 50 around 10–20 mM, (ii) increase in monolayer permeability, i.e., decrease in the transmembrane epithelial electrical resistance, at tiludronate concentrations above 10 mM and a TEER IC 50 around 40 mM, associated with (iii) an opening of the junctional complex (desmosomes, intermediate and tight junctions) as demonstrated by scanning electron microscopy of the cell monolayer and electron microscopy of the apical pole of cells. (iv) The direct consequence of these observations was an increase in monolayer permeability at the level of the paracellular route. Hence, the paracellular transport not only of the PEG 400 probe was increased following treatment of Caco-2 monolayers with tiludronate concentrations above 20 mM but also that of tiludronate itself at concentrations above 15 mM. These results are in agreement with a previous study (Boulenc et al., Biochem. Pharmacol. , 46 (1993) 1591–1600) which showed that tiludronate was specifically transported across Caco-2 cells via the paracellular route. Therefore, by its specific effect on free Ca 2+ concentration, tiludronate could regulate the junctional complex opening of the intestinal epithelium monolayer and enhance its own transport across the intestinal wall.

Sodium lauryl sulphate increases tiludronate paracellular transport using human epithelial caco-2 monolayers

Abstract The potential effect of the common pharmaceutical wetting agent, sodium lauryl sulphate (SLS), on the transport of the hydrophilic bisphosphonate, tiludronate, was investigated both by performing physico-chemical determinations of the SLS-tiludronate interaction and by measuring the paracellular transport of tiludronate (Boulenc et al., Biochem. Pharmacol., 46 (1993) 1591–1600) across the in vitro human intestinal epithelium model, i.e., Caco-2. SLS did not affect the contact angles determined with different liquids (glycerin, dioxane, sulphuric acid, water, mercury, heptane and decane) on tiludronate tablets. SLS influenced neither the disintegration of tiludronate tablets nor tiludronate solubility. However, both the efficiency and effectiveness of SLS, in reducing surface tension, were affected by tiludronate. Hence, the presence of 0.48 g/l tiludronate in a water solution changed the efficiency of SLS in reducing the surface tension from 1 to 0.3 g/1. Before evaluating tiludronate transport across Caco-2 monolayers, the reversible (absorption enhancement of orally administered drugs) and irreversible (cell cytotoxicity) effects of SLS on the viability of Caco-2 monolayers were investigated. Following a 1 h exposure of well-differentiated Caco-2 cells to SLS concentrations above 100 mg/l, mitochondrial dehydrogenase activity decreased in a concentration-dependent manner, cytosolic lactate dehydrogenase leakage occurred, mannitol transport was irreversibly increased and a structural separation of the tight junctions was observed by electron microscopy. SLS concentrations up to 80 mg/l did not affect mitochondrial dehydrogenase and cytosolic lactate dehydrogenase activities, while both a reversible increase in mannitol paracellular transport and tight junction opening were observed. Under these incubation conditions, tiludronate paracellular transport was increased in a concentration-dependent manner. These studies demonstrate that SLS increased tiludronate paracellular transport through its specific and transient effect on the permeability of the intercellular space.

Production of labelled 25 hydroxyvitamin D from 14C vitamin D as a marker of liver function after 65 per cent hepatectomy in dogs

Abstract In order to appreciate hepatocyte activity after 65 per cent hepatectomy, plasma 14 C 25-hydroxyvitamin D ( 14 C 25-OH-D, % total dose x litre −1 x body weight-kg-) was measured following an intraportal bolus injection of 4 μCi 14 C vitamin D. As disturbance of the spleno-pancreatic venous flow might lead to post operative hepatic atrophy, the hepatectomized dogs were separated into three groups: no shunt (group I, n=8), mesocaval (group II, n=3) or portocaval shunt (group III, n=8). As compared with five controls, the 14 C 25-OH-D level at day 6 was within the normal range (mean ± SEM=231±21) in groups I (187±28) and II (184±12) and was significantly depressed in group III (114±14, p The results were compared with post operative proaccelerin levels, which were decreased in group III (61±9%) and normal (100%) in the other groups. From the present results, it appears that 25 hydroxylation is a reliable index of liver function and that portacaval shunts impair liver activity after 65 per cent hepatectomy. The latter finding could result either from a major reduction in hepatic blood flow or suppression of such hepatotrophic factors as pancreatic hormones.