Combined LDI/SAT test to evaluate intestinal lactose digestion and mucosa permeability.

BACKGROUND: Intestinal mucosal damage causes impaired digestive capacity and increased mucosal permeability. Quantification of damage can be used to improve treatment options. Currently, the Lactose Digestion Index (LDI) and the Sugar Absorption Test (SAT) are used for evaluation. The investigation studied whether both tests could be combined to provide a useful multifunctional test and whether measurements in blood (LDI) could be replaced by measurements in urine. MATERIALS AND METHODS: The LDI (25 g 13C-lactose, 0.5 g 2H-glucose), the SAT (5 g lactulose, 1 g L-rhamnose) and the LDI/SAT combination test were performed in seven lactose-digesting and eight lactose-maldigesting adults. Plasma glucose 13C-enrichment was determined by gas-chromatography/combustion/isotope ratio mass-spectrometry (GC/C/IRMS), 2H enrichment determined by gas-chromatography/mass-spectrometry (GC/MS) and urinary sugars by gas-chromatography (GC). RESULTS: The results of the separate LDI test were not different from those of the LDI/SAT in the lactose-digester group (0.82 +/- 0.06 vs. 0.81 +/- 0.09), nor in the lactose-maldigester group (0.36 +/- 0.12 vs. 0.35 +/- 0.06). A significant correlation was found between the 10-h urinary-lactose/lactulose ratio and the LDI (R2 = 0.71, P < 0.01). There were no differences in the lactulose/L-rhamnose ratio between lactose-digesters and lactose-maldigesters using both the SAT and LDI/SAT tests. CONCLUSION: The LDI/SAT test is a reliable method of measuring digestion and permeability simultaneously. The 10-h period urinary lactose/lactulose excretion ratio following lactose consumption reflects lactose digestive capacity.

Gall bladder dysmotility: a risk factor for gall stone formation in hypertriglyceridaemia and reversal on triglyceride lowering therapy by bezafibrate and fish oil.

BACKGROUND AND AIM: The aim of this study was to unravel the mechanisms responsible for the increased risk of gall stone disease in hypertriglyceridaemia (HTG) and to compare the effects of triglyceride lowering therapy by bezafibrate and fish oil on determinants of cholelithiasis (biliary lipid composition and gall bladder motility) in HTG patients. PATIENTS AND METHODS: Gall bladder motility (ultrasonography) was studied postprandially and during infusion of cholecystokinin (CCK). Determinants of cholelithiasis and serum lipids were compared between nine HTG patients and 10 age, sex, and body mass index matched normolipidaemic controls. The effects of bezafibrate and fish oil in HTG patients were studied in a randomised cross over trial. RESULTS: HTG patients showed 14-fold higher serum triglyceride (TG) levels than controls. Biliary lipid composition, fasting gall bladder volumes, and CCK levels did not differ between HTG patients and controls. Gall bladder emptying was reduced in HTG patients compared with controls during CCK infusion (-22%) as well as in response to a meal (-37%; both p<0.001). Postprandial CCK levels were significantly higher in HTG patients. Both bezafibrate and fish oil reduced serum TG levels (-68% and -51% v baseline, respectively; both p<0.01). Fasting CCK levels were not affected whereas CCK induced gall bladder emptying increased during bezafibrate (+29%; p<0.001) and tended to increase on fish oil therapy (+13%; p=0.07). Postprandial gall bladder motility improved on bezafibrate and fish oil (+47 and +25% v baseline, respectively; both p<0.02) at least partly due to increased gall bladder sensitivity to CCK (both p<0.05 v baseline). Bezafibrate but not fish oil increased the molar ratio of cholesterol to bile acids (+40%; p

Extracorporal albumin dialysis (MARS) improves cholestasis and normalizes low apo A-I levels in a patient with benign recurrent intrahepatic cholestasis (BRIC).

The familial cholestatic diseases Benign Recurrent Intrahepatic Cholestasis (BRIC) and Progessive Familial Intrahepatic Cholestasis type 1 (PFIC1) are characterized by intermittent or permanently elevated plasma bile salt levels, therapy-resistant extreme pruritus and peculiar biochemical abnormalities including low apolipoprotein apo A-I. Previously, symptomatic improvement has been demonstrated in BRIC patients after extracorporal albumin dialysis (MARS). We hypothesized that MARS improves cholestasis, induces changes in the bile salt profile and normalizes apo A-I serum levels in BRIC. A 17-year-old-female patient with BRIC experienced an episode of cholestasis lasting for more than 6 months with extreme pruritus and diarrhoea not responding to standard therapy. During a period of five days the patient was treated 3 x 8 h with MARS. The procedures were well tolerated and resulted in reduction of plasma bile salts by 58%. The plasma bile salt profile changed into a more hydrophilic composition after MARS. Diarrhoea discontinued and the pruritus improved significantly from 9 to 4 on a subjective scale. These effects lasted 4 months until a relapse occurred. Low plasma apo A-I levels (0.52 g/l) normalized after MARS (0.98 g/l). The procedures were well tolerated. Fatigue was noted as the only transient side-effect. In conclusion, MARS may induce a long-term symptomatic improvement and decrease of cholestatic markers in BRIC. Further studies evaluating efficacy and mechanism of MARS in patients with BRIC are needed.

Measurement of parameters of cholic acid kinetics in plasma using a microscale stable isotope dilution technique: application to rodents and humans.

A stable isotope dilution method is described that allows measurement of cholic acid (CA) kinetics, that is, pool size, fractional turnover rate (FTR), and synthesis rate in mice, rats, and humans. Decay of administered [2,2,4,4-2H4]CA enrichment was measured in time in 50-microl plasma samples by gas-liquid chromatography/electron capture negative chemical ionization-mass spectrometry, applying the pentafluorobenzyl-trimethylsilyl derivative. The kinetic data expressed species-dependent differences. The CA pool sizes were 16.8 +/- 2.1, 10.6 +/- 1.2, and 2.4 +/- 0.7 micromol/100 g body weight for mice, rats, and humans, respectively. The FTR values were 0.44 +/- 0.03, 0.88 +/- 0.10, and 0.46 +/- 0.14 per day for mice, rats, and humans. The corresponding synthesis rates were 7.3 +/- 1.6, 9.3 +/- 0.1, and 1.0 +/- 0.2 micromol/100 g body weight per day. The human data agreed well with literature data obtained by conventional isotope dilution techniques. For rats and mice these are the first reported isotope dilution data. The method was validated by confirmation of isotopic equilibrium between biliary CA and plasma CA in the rat. Its applicability was demonstrated by the observation of increased CA FTR and CA synthesis rate in rats fed cholestyramine, which is known to increase fecal bile acid excretion. The presented stable isotope dilution method enables the determination of CA kinetic parameters in small plasma samples. The method can be applied in unanesthetized rodents with an intact enterohepatic circulation and may also be valuable when studying the development of human neonatal bile acid kinetics.

13C-carbohydrate breath tests: impact of physical activity on the rate-limiting step in lactose utilization.

BACKGROUND: 13CO2 breath tests can be used to monitor carbohydrate digestion in the small intestine. However, after ingestion of 13C-substrates, 13CO2 excretion in breath originates from two sources: a digestive/oxidative fraction, derived from the small intestine, and a fermentation fraction, derived from undigested substrate spill-over in the colon. In this study, the determinants of the digestive/oxidative fraction were analysed in order to improve the sensitivity/specificity of the 13C-carbohydrate breath tests. METHODS: 13C-carbohydrate breath tests were performed in healthy adults using 13C-lactose, pre-digested 13C-lactose, 13C-glucose, and 13C-galactose as substrates. The effect of exercise (bicycling, 50 W), increasing the metabolism of digested/absorbed substrate, on the outcome of the test was analysed. RESULTS: In rest, no difference was observed in the 4-h cumulative percentage dose recovered in breath (4-h cPDR) after administration of glucose, pre-digested lactose, and lactose, which were 20.3 +/- 4.5%, 19.2 +/- 5.5%, and 19.9 +/- 4.9%, respectively. The 13CO2 excretion rate after 13C-galactose consumption was significantly slower than after 13C-glucose consumption. Exercise increased 4-h cPDR of 13C-glucose significantly: 76.0 +/- 1.0% vs. 22.7 +/- 2.3%. This effect was also observed using 13C-lactose as substrate: 66.1 +/- 6.2% vs. 19.6 +/- 3.9%. One subject had non-symptomatic lactose maldigestion indicated by a positive H2 breath test. The 13CO2 breath test of this subject in rest was indistinguishable from that of the others (4-h cPDR 16.6 vs. 19.6 +/- 3.9%), whereas the test was clearly indicative during exercise (4-h cPDR 20.5 vs. 66.1 +/- 6.2%). CONCLUSION: In healthy volunteers in rest, glucose oxidation is the rate-limiting step in lactose conversion into 13CO2. Increase of metabolism (for instance, by exercise) can shift this step to intestinal hydrolysis of lactose, making the 13C-lactose breath test more sensitive.

The contribution of newly synthesized cholesterol to bile salt synthesis in rats quantified by mass isotopomer distribution analysis.

A new stable isotope procedure has been developed and validated in rats, applying [1-(13)C]acetate infusion to quantify the production of bile salts from de novo synthesized cholesterol making use of the mass isotopomer distribution analysis (MIDA) principle. Ions (m/z) 458-461, 370-373 and 285-288 were monitored by GC/MS (EI-mode) for the methyl trimethylsilylether derivatives of cholate, chenodeoxycholate and beta-muricholate, respectively. Rats with intact exteriorized enterohepatic circulation and rats with chronic bile diversion were infused with [1-(13)C]acetate for up to 14 h. After 10 h of infusion the enterohepatic circulation of the intact group was interrupted to deplete the existing bile salt pool (acute bile diversion). The fractions of biliary cholesterol and individual bile salts derived from newly synthesized cholesterol were determined by MIDA at t=14 h. In rats with acute bile diversion, these fractions were 20, 25, 27 and 23% for biliary cholesterol, cholate, chenodeoxycholate and beta-muricholate, respectively. After bile diversion for 8 days to induce hepatic cholesterol and bile salt synthesis, these fractions increased significantly to 32, 47, 41 and 47%, respectively. Calculated enrichments of the acetyl-CoA precursor pools were similar for all bile salts and biliary cholesterol within the two rat groups. However, chronic enterohepatic interruption decreased the acetyl-CoA pool size almost two-fold. We conclude that MIDA is a validated new stable isotope technique for studying the synthetic pathway from acetyl-CoA to bile salts. This technique provides an important new tool for studying bile salt metabolism in humans using stable isotopes.

Non-invasive detection of low-intestinal lactase activity in children by use of a combined 13CO2/H2 breath test.

BACKGROUND: The aim of the study was to diagnose hypolactasia with a higher accuracy than with the traditional H2 breath test. METHODS: We used a combined 13C-lactose 13CO2/H2 breath test, which was performed in 33 patients in whom lactase activity was measured. RESULTS: Lactase activity was reduced in 13 cases. The sensitivity and specificity of the H2 test were 54% and 90%; those of the 13CO2 test 69% and 70%. False-negative results did not always occur in the same patients. In five of six patients with both test results abnormal, lactase activity was low. In 13 of 15 patients with both test results normal, lactase activity was normal. In 6 of 12 cases with only 1 test abnormal, lactase activity was low. CONCLUSION: The combined H2/13CO2 breath test (sensitivity, 85%; specificity, 65%) is more adequate for diagnosis of hypolactasia than the H2 breath test alone.

Differential effects of 17alpha-ethinylestradiol on the neutral and acidic pathways of bile salt synthesis in the rat.

Effects of 17alpha-ethinylestradiol (EE) on the neutral and acidic biosynthetic pathways of bile salt (BS) synthesis were evaluated in rats with an intact enterohepatic circulation and in rats with long-term bile diversion to induce BS synthesis. For this purpose, bile salt pool composition, synthesis of individual BS in vivo, hepatic activities, and expression levels of cholesterol 7alpha-hydroxylase (CYP7A), and sterol 27-hydroxylase (CYP27), as well as of other enzymes involved in BS synthesis, were analyzed in rats treated with EE (5 mg/kg, 3 days) or its vehicle. BS pool size was decreased by 27% but total BS synthesis was not affected by EE in intact rats. Synthesis of cholate was reduced by 68% in EE-treated rats, while that of chenodeoxycholate was increased by 60%. The recently identified Delta22-isomer of beta-muricholate contributed for 5.4% and 18.3 % (P < 0.01) to the pool in control and EE-treated rats, respectively, but could not be detected in bile after exhaustion of the pool. A clear reduction of BS synthesis was found in bile-diverted rats treated with EE, yet biliary BS composition was only minimally affected. Activity of CYP7A was decreased by EE in both intact and bile-diverted rats, whereas the activity of the CYP27 was not affected. Hepatic mRNA levels of CYP7A were significantly reduced by EE in bile-diverted rats only; CYP27 mRNA levels were not affected by EE. In addition, mRNA levels of sterol 12alpha-hydroxylase and lithocholate 6beta-hydroxylase were increased by bile diversion and suppressed by EE. This study shows that 17alpha-ethinylestradiol (EE)-induced intrahepatic cholestasis in rats is associated with selective inhibition of the neutral pathway of bile salt (BS) synthesis. Simultaneous impairment of other enzymes in the BS biosynthetic pathways may contribute to overall effects of EE on BS synthesis.

Benign recurrent intrahepatic cholestasis: altered bile acid metabolism.

Altered bile acid metabolism has been claimed to play a role in the etiology of benign recurrent intrahepatic cholestasis (BRIC). Therefore, we studied bile acid metabolism in detail in 10 patients with this syndrome. Pool sizes of both primary bile acids were estimated simultaneously, using deuterated cholic acid and chenodeoxycholic acid. The pool sizes of cholic acid and chenodeoxycholic acid, expressed in micromoles per kilogram body weight, were significantly contracted in BRIC patients during a cholestasis-free period: 8.0 +/- 4.2 and 11.7 +/- 4.7, respectively, versus 24.1 +/- 11.7 and 22.9 +/- 7.8 in controls. Fractional turnover rates (per day) for cholic acid and chenodeoxycholic acid were increased: 0.70 +/- 0.29 and 0.58 +/- 0.27, respectively, versus 0.29 +/- 0.12 and 0.23 +/- 0.10 in controls. Bile acid pool composition expressed as percentages in BRIC patients was cholic acid 34 +/- 17, chenodeoxycholic acid 38 +/- 9, deoxycholic acid 27 +/- 18, and lithocholic acid 1 +/- 1, with a glycine to taurine conjugation ratio of 6.7 +/- 4.9. Corresponding values for 32 controls were cholic acid 57 +/- 13, chenodeoxycholic acid 29 +/- 9, deoxycholic acid 14 +/- 9, and lithocholic acid less than 1, with a glycine to taurine conjugation ratio of 2.4 +/- 1.3. Fecal bile acid loss, in micromoles per kilogram body weight per day, was 11.2 +/- 9.0 in BRIC patients compared with 2.8 +/- 1.4 in controls. The serum 7 alpha-hydroxycholesterol level (nanomoles per liter) was significantly increased in BRIC patients: 326 +/- 179 versus 171 +/- 90 in controls. These results suggest that in BRIC patients spillover of bile acids into the colon occurs, which leads to increased fecal bile acid loss and a reduced bile acid pool size. Increased serum 7 alpha-hydroxycholesterol is probably indicative of an accelerated bile acid synthesis rate due to increased activity of cholesterol 7 alpha-hydroxylase, the enzyme catalyzing the first step in the major pathway of bile acid synthesis. The results of our study suggest that in BRIC patients a contracted bile acid pool increases the susceptibility of the liver for cholestatic agents.

Identification of 5-hydroxy-6-indolyl-O-sulfate in urine of patients with malignant melanoma.

It has previously been shown that enzymatically hydrolyzed urine of patients with malignant melanoma contains 5,6-dihydroxyindole (5, 6DHI ). In this study we describe the elucidation of the entire structure of urinary 5, 6DHI -conjugate. Differential hydrolysis of melanotic urine revealed that, in contrast to beta-glucuronidase, sulfatase can liberate 5, 6DHI from its conjugated form. 5, 6DHI -sulfate was synthetized by reacting 5, 6DHI with sulfur trioxide trimethylamine complex. Thin-layer chromatography (TLC) documented its close similarity to the Thorm ahlen -positive compound usually entitled "C." Gas chromatographic-mass spectrometric (GC-MS) analysis of methylated and subsequently hydrolyzed synthetic 5, 6DHI -sulfate showed that the synthetic product consisted of a mixture of 5-hydroxy-6-indolyl-O-sulfate and 6-hydroxy-5-indolyl-O-sulfate (with a certain amount of 5, 6DHI -disulfate). 5, 6DHI -sulfate was purified with use of DEAE-cellulose column chromatography from melanotic urine. Methylation of this conjugate with deuterated dimethylsulfate and subsequent GC-MS analysis of the hydrolyzed product provided evidence that 5, 6DHI from melanotic urine was almost exclusively sulfated in position 6. It was concluded (1) that 5, 6DHI is excreted as a 6-O-sulfate, and (2) that this compound is consistent with Thorm ahlen -positive compound "C".

Identification of Thormählen-positive compound "B" in urine of patients with malignant melanoma.

The identification of the entire structure of the Thormählen-positive compound B from melanotic urine is described. The compound was separated from other Thormählen-positive compounds using DEAE-cellulose column chromatography. On the basis of differential enzymic hydrolysis followed by gas chromatography-mass spectrometry analysis and comparison with synthetically prepared compounds it is possible to conclude that the Thormählen-positive compound B is a mixture of O-sulphate 5-hydroxy-6-methoxyindole and 6-hydroxy-5-methoxyindole with predominance of the latter.