Deconjugation Activity Research Articles

Transfer of [ 3H]estrone-[ 35S]sulfate across guinea pig fetal membranes

The possible role of fetal membrane deconjugating activity in the movement of a charged steroid conjugate between fetal and maternal compartments was investigated. The ability of amnion and chorion laeve to transfer [ 3H]estrone-[ 35S]sulfate was assessed in both orientations of guinea pig tissue at 45 days and near parturition. While early amnion was impermeable, late tissue transferred approximately 50% (w/w) of the substrate in a bidirectional process that was non-saturable and independent of either deconjugation or ATP. Transfer across early chorion was similar to late amnion. Saturation curves from each tissue were superimposable, as were those of the time course. Transfer across both early and late chorion proceeded in the absence of deconjugation, with no effect of tissue orientation or ATP depletion. However, late chorion exhibited a decrease in estrone-sulfate transfer, as verified by concentration dependency and time course analyses, though transport across the tissue remained non-saturable. The results in amnion were congruous with the presence and absence of tight junctions in the epithelium of early and late tissue, respectively. However, sulfoconjugate transfer across early chorion proceeded in the presence of a paracellular barrier, suggesting specialized regulation of the transport process which extended late into gestation.

Transfer of steroidal and nonsteroidal compounds across guinea pig fetal membranes.

Transfer of steroidal and nonsteroidal compounds across guinea pig amnion and chorion laeve was investigated as a function of stage of gestation, tissue orientation, steroid specificity, and molecular size. Each fetal membrane was examined at early and late stages of gestation, before and after pubic symphysis relaxation. Early amnion was impermeable to macromolecules and small charged molecules while [3H]estrone and [3H]pregnenolone were transferred, the latter depending on tissue orientation and involving conjugation at the basolateral interface. After symphysis dilation, amnion transferred all substrates tested with the exception of BSA; the molecular weight cutoff was approximately 5,000. Unlike amnion, early chorion transferred both free and conjugated steroids as well as inorganic sulfate. Transfer of estrone involved conjugation and depended on tissue orientation. Transfer of [3H]estrone-sulfate, [3H]estrone-glucuronide, and [3H]pregnenolone-sulfate was similar despite selective deconjugating activity toward estrone-sulfate. Near term, chorion was impermeable to inorganic sulfate and transfer of estrone-glucuronide depended on tissue orientation, involving deconjugation in the maternal to fetal direction. At no stage of gestation did chorion transfer macromolecules. These results suggest that the transfer of free and conjugated steroids across fetal membranes is differentially regulated by tissue, its stage of development, and direction of transfer.

Cholylglycine hydrolase and 7α-dehydroxylase: optimum assay conditions in vitro and caecal enzyme activities ex vivo

Increasing evidence implicates deoxycholic acid (DCA) in the pathogenesis of cholesterol-rich gallbladder stones. However, relatively little is known about the activities of the two intestinal bacterial enzymes (cholylglycine hydrolase and cholic acid 7α-dehydroxylase) responsible for the deconjugation and subsequent dehydroxylation of conjugated cholic acid (CA), to form DCA. We, therefore, established optimal reaction conditions for measuring the activities of these two enzymes in vitro, and applied these conditions to the determination of the enzymes in caecal aspirates from six subjects undergoing clinically-indicated colonoscopy. With respect to cholylglycine hydrolase activity: zero order kinetics were found over 20 min at 37°C (pH optimum 4.0), with K m and V max values of 1.66 mmol/l and 0.90 mmol CA min −1 mg prot −1, respectively. For cholic acid 7α-dehydroxylation: zero order kinetics were found over 7.5 min at 37°C, under anaerobic conditions (pH optimum 8.0), with K m and V max values of 5.23×10 −8 mol/l and 1.88×10 −7 mol DCA min −1 mg prot −1, respectively. Applying these reaction conditions to the caecal aspirates, endogenous cholylglycine hydrolase activities ranged from 0.49 to 2.43 units (mg protein −1 min −1) and CA 7α-dehydroxylase activities from 1.75 to 5.82×10 −7 units (mg protein −1 min −1). This study is unique in assaying quantitatively both the deconjugation and dehydroxylation enzyme activities in human caecal samples — an essential first step to further studies of intestinal bacterial enzymes in the pathogenesis of cholesterol gallstone disease.

Relationships Among Bile Tolerance, Bile Salt Deconjugation, and Assimilation of Cholesterol by Lactobacillus acidophilus

The relationships among growth in the presence of bile, deconjugation of sodium taurocholate, and assimilation of cholesterol by 19 cultures of Lactobacillus acidophilus were examined. Cultures of L. acidophilus were grown at 37°C in lactobacilli MRS broth supplemented with sodium thioglycollate, sodium taurocholate, and cholesterol (cholesterol phosphatidyl choline micelles). Deconjugation activity was maximum in the late exponential phase of growth, which also coincided with maximum assimilation of cholesterol. Considerable variation existed among cultures in their ability to grow in the presence of bile, to deconjugate sodium taurocholate, and to assimilate cholesterol. However, statistical analyses revealed no significant correlations.

Deconjugating activity for sulfoconjugated dopamine in homogenates of organs from dogs

To clarify the possibility that sulfoconjugated dopamine (DA) may play a physiological role by being converted to active free DA, we examined the deconjugating activity in homogenates of organs from dogs. Each tissue homogenate was incubated with sulfoconjugated DA, and the deconjugating activity of the organs was compared. The kidney and liver exhibited the highest deconjugating activities. In contrast, the intestine and heart showed lower arylsulfatase activities, and almost no activity was found in the brain or skeletal muscle. Moreover, in the heart, the deconjugating activity for sulfoconjugated DA was higher in the atrium than the ventricle. These results indicate that sulfoconjugated DA is converted to active free DA in homogenates of organs from dogs and that the deconjugating activity varies between different parts of an organ. Sulfoconjugated DA must be looked upon as a possible precursor or reservoir for the production of active free DA.

Metabolism of Ecdysteroids During the Embryogenesis ofManduca Sexta

Abstract The combination of ion suppression, reverse phase high pressure liquid chromatography (IS-RPHPLC) and radioimmunoassay (RIA) employing two relatively non-specific, complimentary antisera was used to identify and quantify ecdysteroids of divergent polarities during embryogenesis of the tobacco hornworm, Manduca sexta. Newly layed eggs (0–1 hr) contained high levels (>30 μg/g) of a maternally derived, polar conjugate of 26-hydroxy-ecdysone (26E) but less than 0.6 and 0.2 μg/g of the polar conjugates of ecdysone (E) and 20-hydroxyecdysone (20E), respectively. The only free ecdysteroid detected was 26E (0.4 μg/g). Between 4 and 12 hr after oviposition, marked deconjugation activity occurred as titers of free 26E increased to 14 μg/g while levels of conjugated 26E fell to 19 μg/g. By this time there were only negligible levels of free E (less than 0.1 μg/g). After 12 hr, the deconjugation of the maternal polar 26E-26-phosphate conjugate slowed and levels of free 26E fell as it was metabolized. At 24 hr, low titers of free 20,26-dihydroxyecdysone (20,26E, 0.4 μg/g) and a β-D-glucose conjugate of 26E (0.8 μg/g) were detected, presumably formed by embryonic 20-monooxygenase and uridine diphospho-α-D-glucose (UDPG) glucosyltransferase activities, respectively. About the time of the deposition of the first larval cuticle (66–72 hr), these 26E metabolites reached high concentrations, (5 μg/g free 20,26E and 7 μg/g 26E-glucose conjugate) while free 26E fell to 2 μg/g. In newly hatched larvae, the 26E-glucose conjugate was the major detectable ecdysteroid. At no time during embryogenesis did the concentrations of free E and 20E exceed 0.5 μg/g and 0.3 μg/g, respectively.

Small-bowel bacterial overgrowth in the postgastrectomy syndrome.

Jejunal flora, bile acid deconjugation, and breath hydrogen (H2) and methane (CH4) excretion were studied in 22 Billroth II (BII)-operated patients with chronic postprandial symptoms, dumping (9), vomiting (7), pain (10), and diarrhoea (14). Sixteen were below 90% of desirable weight. Two control groups were included, one comprising 5 symptom-free, BII-operated volunteers and another comprising 12 healthy, unoperated volunteers. The numbers of bacteria recovered from jejunal secretions in the postgastrectomy patients did not differ significantly from those recovered in the symptom-free BII-operated controls but were significantly lower in the unoperated controls. Production of fermentation gas in anaerobic media supplemented with carbohydrates occurred in 17 of 22 postgastrectomy patients and in 4 of 5 BII-operated controls but in none of the unoperated controls. Bacterial bile acid deconjugating activity did not differ significantly between the postgastrectomy patients and the BII-operated controls but was significantly lower in the unoperated controls. Breath H2 excretion after glucose ingestion was significantly higher in the postgastrectomy patients than in both the BII-operated and the unoperated controls. The addition of pectin or guar gum to the glucose meal largely prevented postprandial symptoms and breath hydrogen excretion. Six out of 12 postgastrectomy patients treated with metronidazole recorded symptomatic effects, mainly on diarrhoea. Our findings indicate that jejunal bacterial overgrowth may be a major cause of the symptoms in some postgastrectomy patients. The tests available for demonstration of small-bowel bacterial overgrowth, perhaps with the exception of the glucose H2 breath test, did not differentiate satisfactorily between symptom-producing and non-symptom-producing abnormal jejunal flora. Thus these tests may seem to have a limited practical diagnostic value in such patients.

Influence of three antimicrobial agents--penicillin, metronidazole, and doxycyclin--on the intestinal microflora of healthy humans.

Antimicrobial drugs may affect the normal gut microflora in a potentially harmful manner. The purpose of the present study was to ascertain whether any of several tests could detect drug influence on the intestinal flora of healthy subjects. Jejunal secretions and feces were cultivated aerobically and anaerobically, with measurement of fermentation gas production in tubes supplemented with glucose or lactulose; bacterial bile acid deconjugation was measured with the 14C-GCA test; and pulmonary H2 and CH4 excretion were measured with gas-solid chromatography after lactulose and glucose ingestion in 18 healthy subjects before and after peroral treatment with either penicillin, metronidazole, or doxycyclin. Bacterial numbers and fermentation gas production were unchanged after treatment, as was the bacterial bile acid deconjugating activity. Pulmonary H2 excretion after lactulose ingestion was significantly reduced after penicillin and metronidazole but not after doxycyclin treatment. Pulmonary CH4 disappeared after metronidazole but remained unchanged after penicillin and doxycyclin treatment. It is concluded that pulmonary H2 and CH4 measurement after lactulose ingestion may serve as a sensitive determinant for drug effects on the normal gut microflora.