Richard H. Moseley

Downregulated in adenoma gene encodes a chloride transporter defective in congenital chloride diarrhea

Congenital chloride diarrhea (CLD) is a recessively inherited disorder characterized by massive loss of chloride in stool. We previously identified mutations in the downregulated in adenoma (DRA) gene in patients with CLD and demonstrated that DRA encodes an intestine-specific sulfate transporter. To determine whether DRA is an intestinal chloride transporter and how mutations affect transport, Xenopus oocytes were injected with wild-type and mutagenized DRA cRNA and uptake of Cl- and SO2-4 was assayed. Both Cl- and SO2-4 were transported by wild-type DRA and an outwardly directed pH gradient stimulated Cl- uptake, consistent with Cl-/OH- exchange. Among three mutants, C307W transported both anions as effectively as wild-type, whereas transport activity was lost in V317del and the double mutant identified in 32 of 32 Finnish CLD patients. We conclude that DRA is a chloride transporter defective in CLD and that V317del is a functional mutation and C307W a silent polymorphism.Congenital chloride diarrhea (CLD) is a recessively inherited disorder characterized by massive loss of chloride in stool. We previously identified mutations in the downregulated in adenoma ( DRA) gene in patients with CLD and demonstrated that DRA encodes an intestine-specific sulfate transporter. To determine whether DRA is an intestinal chloride transporter and how mutations affect transport, Xenopus oocytes were injected with wild-type and mutagenized DRA cRNA and uptake of Cl- and[Formula: see text] was assayed. Both Cl- and[Formula: see text] were transported by wild-type DRA and an outwardly directed pH gradient stimulated Cl- uptake, consistent with Cl-/OH-exchange. Among three mutants, C307W transported both anions as effectively as wild-type, whereas transport activity was lost in V317del and the double mutant identified in 32 of 32 Finnish CLD patients. We conclude that DRA is a chloride transporter defective in CLD and that V317del is a functional mutation and C307W a silent polymorphism.

Prospective multicenter study of eligibility for antiviral therapy among 4,084 U.S. veterans with chronic hepatitis C virus infection

BACKGROUND:Many veterans may not be candidates for hepatitis C virus (HCV) treatment due to contraindications to therapy. The aims of this study were to determine the proportion of HCV-infected veterans who were eligible for interferon alfa and ribavirin therapy and to evaluate barriers to HCV treatment.METHODS:We prospectively enrolled 4,084 veterans who were referred for HCV treatment over a 1-yr period at 24 Veterans Affairs (VA) Medical Centers. Treatment candidacy was assessed using standardized criteria and the opinion of the treating clinician.RESULTS:Overall, 32.2% (95% CI, 30.8–33.7%) were candidates for HCV treatment according to standardized criteria, whereas 40.7% (95% CI, 39.2–42.3%) were candidates in the opinion of the treating clinician. Multivariable analysis identified ongoing substance abuse (OR = 17.68; 95% CI, 12.24–25.53), comorbid medical disease (OR = 9.62; 95% CI, 6.85–13.50), psychiatric disease (OR = 9.45; 95% CI, 6.70–13.32), and advanced liver disease (OR = 8.43; 95% CI, 4.42–16.06) as the strongest predictors of not being a treatment candidate. Among patients who were considered treatment candidates, 76.2% (95% CI, 74.0–78.3%) agreed to be treated and multivariable analysis showed that persons ≥50 yr of age (OR = 1.37; 95% CI, 1.07–1.76) and those with >50 lifetime sexual partners (OR = 1.44; 95% CI, 1.08–1.93) were more likely to decline treatment.CONCLUSIONS:The majority of veteran patients are not suitable candidates for HCV treatment because of substance abuse, psychiatric disease, and comorbid medical disease, and many who are candidates decline therapy. Multidisciplinary collaboration is needed to overcome barriers to HCV therapy in this population.

Sepsis and cholestasis

Sepsis-associated cholestasis should always be considered as part of the differential diagnosis of jaundice in the hospitalized or critically ill patient. The development of a disproportionate elevation of serum bilirubin in comparison with serum alkaline phosphatase and serum aminotransferases should be considered an early warning sign of an underlying infection, even in the absence of fever,leukocytosis, or other signs or symptoms. Prompt recognition and appropriate medical and surgical intervention may reduce morbidity and mortality.

Black patients with chronic hepatitis C have a lower sustained viral response rate than non-Blacks with genotype 1, but the same with genotypes 2/3, and this is not explained by more frequent dose reductions of interferon and ribavirin*.

Summary.  In previous hepatitis C virus (HCV) treatment studies, Black patients not only had a lower sustained viral response (SVR) rate to interferon and ribavirin (RBV) than non‐Black patients but also a higher frequency of HCV genotype 1 (GT‐1) infection. The aim of this community‐based study was to determine whether Black patients have a lower SVR rate independent of genotype. We prospectively enrolled 785 patients (24.8% Black, 71.5% White, 3.7% others) who received interferon alpha‐2b 3 MU three times weekly + RBV 1000–1200 mg/day for 24 weeks (GT‐2/3) or 48 weeks (GT‐1). Black patients were more commonly infected with GT‐1 (86.8%vs 64.8%, P < 0.001) and less frequently had an SVR compared with non‐Black patients (8.4%vs 21.6%, P < 0.001). Within GT‐1, Black patients had a lower SVR rate than non‐Black patients (6.1%vs 14.1%, P = 0.004) but not within GT‐2/3 (50.0%vs 36.5%, P = 0.47). Black patients had lower baseline haemoglobin levels (14.8 vs 15.3 g/dL, P < 0.001) and neutrophil counts (2900 vs 4100/mm3, P < 0.001) and required more frequent dose reductions of RBV (29.8%vs 18.5%, P < 0.001) and interferon (4.7%vs 1.6%, P = 0.012). However, dose reductions were not associated with lower SVR rates while early treatment discontinuations were (2.9%vs 25.7%, P < 0.001). Independent predictors of SVR were GT‐1 [odds ratio (OR) 0.33; 95% confidence interval (CI) 0.20–0.55; P < 0.001], Black race (OR 0.45; 95% CI 0.22–0.93; P = 0.030), and advanced fibrosis, stages 3 + 4 (OR 0.53; 95% CI 0.31–0.92; P = 0.023). In conclusion, Black patients infected with HCV GT‐1 (but not GT‐2/3) have a lower SVR rate than non‐Black patients. This is not explained by their lower baseline haemoglobin levels and neutrophil counts that lead to higher rates of ribavirin and interferon dose reductions.

Trans fat feeding results in higher serum alanine aminotransferase and increased insulin resistance compared with a standard murine high-fat diet

Diets high in trans fats are associated with an increased risk of cardiovascular disease and components of the metabolic syndrome. The influence of these toxic fatty acids on the development of nonalcoholic fatty liver disease has not been significantly examined. Therefore, we sought to compare the effect of a murine diet high in trans fat to a standard high-fat diet that is devoid of trans fats but high in saturated fats. Male AKR/J mice were fed a calorically identical trans fat diet or standard high-fat diet for 10 days, 4 wk, and 8 wk. Serum alanine aminotransferase (ALT), lipid, insulin, and leptin levels were determined and the quantitative insulin-sensitivity check index (QUICKI) was calculated as a measure of insulin resistance. Additionally, hepatic triglyceride content and gene expression of several proinflammatory genes were assessed. By 8 wk, trans fat-fed mice exhibited higher ALT values than standard high-fat-fed mice (126 +/- 16 vs. 71 +/- 7 U/l, P < 0.02) despite similar hepatic triglyceride content at each time point. Trans fat-fed mice also had increased insulin resistance compared with high-fat-fed mice at 4 and 8 wk with significantly higher insulin levels and lower QUICKI values. Additionally, hepatic interleukin-1beta (IL-1beta) gene expression was 3.6-fold higher at 4 wk (P < 0.05) and 5-fold higher at 8 wk (P < 0.05) in trans fat-fed mice compared with standard high-fat-fed mice. Trans fat feeding results in higher ALT values, increased insulin resistance, and elevated IL-1beta levels compared with standard high-fat feeding.

A familial form of incomplete septal cirrhosis

The clinical features and hepatic histology of a disorder resembling idiopathic portal hypertension and nodular regenerative hyperplasia but most consistent with incomplete septal cirrhosis, occurring in four family members, are described. This represents the first description of the familial occurrence of this entity. Features common to incomplete septal cirrhosis and the noncirrhotic nodular conditions of the liver that may present with complications of portal hypertension are discussed.

Ursodeoxycholate stimulates Na+-H+ exchange in rat liver basolateral plasma membrane vesicles.

Na+:H+ and Cl-:HCO3- exchange are localized, respectively, to basolateral (blLPM) and canalicular (cLPM) rat liver plasma membranes. To determine whether these exchangers play a role in bile formation, we examined the effect of a choleretic agent, ursodeoxycholate (UDCA), on these exchange mechanisms. 22Na (1 mM) and 36Cl (5 mM) uptake was determined using outwardly directed H+ and HCO3- gradients, respectively. Preincubation of blLPM vesicles with UDCA (0-500 microM) resulted in a concentration-dependent increase in initial rates of amiloride-sensitive pH-driven Na+ uptake, with a maximal effect at 200 microM. UDCA (200 microM) increased Vmax from 23 +/- 2 (control) to 37 +/- 7 nmol/min per mg protein; apparent Km for Na+ was unchanged. Preincubation with tauroursodeoxycholate (200 microM), taurocholate (10-200 microM) or cholate, chenodeoxycholate, or deoxycholate (200 microM) had no effect on pH-driven Na+ uptake. UDCA (200 microM) had no effect on either membrane lipid fluidity, assessed by steady-state fluorescence polarization using the probes 1,6-diphenyl-1,3,5-hexatriene, 12-(9-anthroyloxy) stearic acid, and 2-(9-anthroyloxy) stearic acid (2-AS), or Na+,K+-ATPase activity in blLPM vesicles. In cLPM vesicles, UDCA (0-500 microM) had no stimulatory effect on initial rates of HCO3(-)-driven Cl- uptake. Enhanced basolateral Na+:H+ exchange activity, leading to intracellular HCO3- concentrations above equilibrium, may account for the bicarbonate-rich choleresis after UDCA infusion.

Thiamine Transport by Basolateral Rat Liver Plasma Membrane Vesicles

Hepatic thiamine transport is thought to be a saturable, Na(+)- and energy-dependent process. However, the transport of this organic cation has not been examined in experimental models that allow direct characterization of carrier-mediated processes. Recently, a sinusoidal organic cation/H+ antiport was identified, using N1-methylnicotinamide as a marker. To determine whether thiamine is a substrate for this antiport, the characteristics of thiamine uptake were examined in rat liver basolateral membrane vesicles. An inwardly directed Na+ gradient had no effect on thiamine uptake as compared with an identical K+ gradient. An outwardly directed H+ gradient stimulated thiamine uptake as compared with pH-equilibrated conditions, and H(+)-dependent uptake was not the result of an H+ diffusion potential. Identical pH gradients stimulated uptake under voltage-clamped conditions, consistent with electroneutral thiamine/H+ exchange. Unlabeled intravesicular thiamine trans-stimulated [3H]thiamine uptake. Choline and imipramine cis-inhibited thiamine/H+ exchange; a series of other organic cations and thiamine analogues had no effect. Carrier-mediated [3H]thiamine uptake showed two saturable systems. In conclusion, a thiamine/H+ antiport is present on the sinusoidal membrane, distinct from Na+/H+ and NMN+/H+ exchange.

Hepatic uptake of γ-butyrobetaine, a precursor of carnitine biosynthesis, in rats

Gamma-butyrobetaine (GBB) is a precursor in the biosynthesis of carnitine, which plays an important role in the beta-oxidation of fatty acids, and is converted to carnitine by gamma-butyrobetaine dioxygenase (BBD) predominantly in liver. We investigated the molecular mechanism of hepatic uptake of GBB in rat hepatocytes. Cellular localization of rat Octn2 (rOctn2:Slc22A5) was studied by Western blot analysis. Uptake of deuterated GBB (d(3)-GBB) was examined in HEK293 cells expressing rOctn2 (HEK293/rOctn2) and freshly isolated rat hepatocytes. d(3)-GBB was quantified by use of liquid chromatography-tandem mass spectrometry. Western blot analysis demonstrated an expression of OCTN2 protein in hepatic basolateral membrane but not in bile canalicular membrane fraction. Furthermore, we found that d(3)-GBB was taken up by rOctn2 in an Na(+)-dependent manner with K(m) value of 13 microM. The apparent K(m) value for d(3)-GBB transport in freshly isolated rat hepatocytes was 9 microM. d(3)-GBB uptake by the rat hepatocytes was inhibited by gamma-aminobutyric acid (GABA) to 30% of the control, whereas it was inhibited by carnitine to 62% of the control, even at 500 microM. Furthermore, d(3)-GBB uptake by rat hepatocytes was decreased by 45% with rat Gat2 (Slc6A13, a major liver GABA transporter) silenced by the microRNA method. Accordingly, the present study clearly demonstrates that GBB is taken up by hepatocytes for carnitine biosynthesis not only via Octn2 but also via the GABA transporter, possibly Gat2.

Needle in a Haystack

A 50-year-old woman reported multiple, loose bowel movements associated with mild, cramping abdominal pain. She had been well until 2 months earlier, when her bowel habits changed from one formed stool per day to frequent loose stools of moderate volume.