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Papers by Lee Hagey

Journal of Lipid Research, 1996

... (M-90-90-145-C15-C1&17). bubble pressure method using an apparatus constructed by Karol J... more ... (M-90-90-145-C15-C1&17). bubble pressure method using an apparatus constructed by Karol J. Mysels. This method determines changes in surface tension under dynamic conditions, and its prin-ciples (30) and application to bile acids (31) have been reported previously. ...

Journal of Chemical Ecology, Jun 1, 2003

The Giant panda communicates with conspecifics by depositing a mixture of volatile compounds (cal... more The Giant panda communicates with conspecifics by depositing a mixture of volatile compounds (called scent marks) on trees and rocks. Using mass spectrometry, we identified 951 chemical components from scent glands, urine, vaginal secretions, and scent marks made by pandas. The scent marks of the two genders contained a similar array of chemicals but varied in concentration; specifically, males possessed a significantly greater amount of short chain fatty acids (F(1, 29) = 18.4, P = 0.002). Using stepwise discriminate analysis on the relative proportions of a subset of these chemicals, it was possible to classify gender (94% for males and females) and individuality (81% for males and 91% for females) from scent marks. The power to identify individual males was reduced due to the relatedness of two subjects. By cracking the identity code of Giant panda communication, we show insights into how these animals can match individuals with unique chemical profiles. Since radiocollaring is currently banned in China, the techniques described in this paper give field biologists a new means to identify and track pandas in the wild.

Gastroenterology, 2013

BACKGROUND AND AIM: Endoscopy is the first level procedure in the diagnosis of gastrointestinal (... more BACKGROUND AND AIM: Endoscopy is the first level procedure in the diagnosis of gastrointestinal (GI) masses. However gastro-duodenal or colonic lesions which mainly involve submucosa or subserosa as well as small bowel's lesions may be difficult to diagnose. The present study aimed to evaluate the role of ultrasound (US)-guided percutaneous biopsy in diagnosing GI tract lesions. PATIENTS AND METHODS: 114 patients (63 male 51 female patients, age range 55-80 years, median age 68 years) underwent US-guided biopsy of GI lesions from 2000 to 2012. In 36 patients the lesion was in the small bowel, thus endoscopically inaccessible; in 50 patients previous histology of endoscopic biopsies was repeatedly negative; in 18 patients, stenosis did not allow bioptic sampling and in 10 patients age, severe cardiological disease and sepsis prevented endoscopy. We used multi-frequency convex probes with side adapters and 18G cutting needle for histological sampling. After biopsy, patients were monitored by measuring blood pressure and pulse rate during the following 3-4 hours to evaluate possible complications. Histology was compared with postsurgery histological evaluation in 73 cases. In the remaining, histology of US-guided biopsy was the only clue to diagnosis. RESULTS: Biopsy specimens were taken from stomach in 38 cases, small bowel in 36 cases and colon-sigma in 40 cases. Final diagnosis was malignant lesions in all but three cases. One case was intestinal endometriosis, the second was bowel duplication, whereas in the third case it was a false-negative result, the correct diagnosis being gastric adenocarcinoma. Sensitivity, specificity, positive predictive value, negative predictive value and diagnostic accuracy of biopsy were respectively 99%, 100%, 100%, 66%, 99%. We observed no mortality and one complication: A case of melena due to bleeding from gastric gastrointestinal stromal tumor that was effectively controlled by positioning of metal clips. CONCLUSION: US-guided percutaneous biopsy of GI tract lesions may be an alternative, safe and effective procedure for diagnosis of GI tract lesions in those cases where conventional diagnostic approach is not feasible or successful.

Nuclear receptor, Jan 30, 2005

The nuclear hormone receptor (NR) superfamily complement in humans is composed of 48 genes with d... more The nuclear hormone receptor (NR) superfamily complement in humans is composed of 48 genes with diverse roles in metabolic homeostasis, development, and detoxification. In general, NRs are strongly conserved between vertebrate species, and few examples of molecular adaptation (positive selection) within this superfamily have been demonstrated. Previous studies utilizing two-species comparisons reveal strong purifying (negative) selection of most NR genes, with two possible exceptions being the ligand-binding domains (LBDs) of the pregnane X receptor (PXR, NR1I2) and the constitutive androstane receptor (CAR, NR1I3), two proteins involved in the regulation of toxic compound metabolism and elimination. The aim of this study was to apply detailed phylogenetic analysis using maximum likelihood methods to the entire complement of genes in the vertebrate NR superfamily. Analyses were carried out both across all vertebrates and limited to mammals and also separately for the two major domai...

Cell Reports, 2012

Genotype-phenotype mapping is hampered by countless genomic changes between species. We introduce... more Genotype-phenotype mapping is hampered by countless genomic changes between species. We introduce a computational ''forward genomics'' strategy that-given only an independently lost phenotype and whole genomes-matches genomic and phenotypic loss patterns to associate specific genomic regions with this phenotype. We conducted genome-wide screens for two metabolic phenotypes. First, our approach correctly matches the inactivated Gulo gene exactly with the species that lost the ability to synthesize vitamin C. Second, we attribute naturally low biliary phospholipid levels in guinea pigs and horses to the inactivated phospholipid transporter Abcb4. Human ABCB4 mutations also result in low phospholipid levels but lead to severe liver disease, suggesting compensatory mechanisms in guinea pig and horse. Our simulation studies, counts of independent changes in existing phenotype surveys, and the forthcoming availability of many new genomes all suggest that forward genomics can be applied to many phenotypes, including those relevant for human evolution and disease.

Journal of Laboratory and Clinical Medicine, 2003

Quantitation of fecal bile acid excretion can help elucidate the cause of diarrhea or steatorrhea... more Quantitation of fecal bile acid excretion can help elucidate the cause of diarrhea or steatorrhea. Fecal bile acids can be measured with gas chromatography-mass spectrometry, but this is time-consuming, expensive, and not available for clinical use. Relatively simple enzymatic methods have been described for the measurement of fecal 3␣-hydroxy bile acids, but these have not been validated in patients with gastrointestinal disease. We found that an enzymatic method yielded falsely low results in patients with malabsorption syndromes for two reasons: First, the preliminary hydrolysis step did not completely deconjugate bile acids, precluding their extraction into diethyl ether for enzymatic assay. Second, long-chain fatty acids inhibited 3␣-hydroxysteroid dehydrogenase activity. By increasing the duration of hydrolysis and the concentration of enzyme, we developed a simple, accurate, and reproducible method for measuring fecal 3␣-hydroxy bile acids that agreed well with values obtained with the use of gas chromatography-mass spectrometry (R ‫؍‬ .95), both in normal subjects and in patients with malabsorption syndromes. (J Lab Clin Med 2003;141:411-8) Abbreviations: EDTA ϭ ethylenediaminetetraacetate; GC-MS ϭ gas chromatography-mass spectrometry; NAD ϭ ␤-nicotinamide adenine dinucleotide hydrate T he cathartic effect of an excess of fecal bile acids may be an unappreciated cause of chronic diarrhea in several clinical settings, including congenital diarrhea, 1 idiopathic secretory diarrhea, 2-4 irritable bowel syndrome, 5-7 postgastrectomy or postcholecystectomy diarrhea, 8-11 Crohn's disease, 7,10 postinfectious diarrhea, 10 postvagotomy diarrhea, 11,12 microscopic colitis, 13 and diarrhea after abdominal radiation. 14,15 In addition, excessive fecal loss of bile acids can be a major factor in the pathogenesis of fat malabsorption in patients with short bowel syndrome. 16-18 Quantitation of fecal bile acid excretion can help identify the cause of diarrhea and/or steatorrhea in these clinical situations. GC-MS is considered the most accurate method for the measurement of fecal bile acids. 19 However, GC-MS analysis of fecal bile acids is time-consuming, expensive, and not available for clinical use. At least 10 enzymatic methods for measuring total fecal 3␣-hydroxy bile acids have been reported. 20-29 In general, these methods are simpler to perform than GC-MS, but some are time-consuming and complicated, requiring either corrections based on a radioactive internal standard 20,21,25 or chromatographic separation of bile acids from interfering substances. 20-22,25,28 Although these methods apparently

Developments in Primatology: Progress and Prospects

On the ventral side of the elbow of the both the slow (Nycticebus bengalensis, N. coucang) and py... more On the ventral side of the elbow of the both the slow (Nycticebus bengalensis, N. coucang) and pygmy (N. pygmaeus) lorises, one can perceive a slightly raised, fairly hair-free but barely visible swelling, termed the brachial gland (Figure 12.1). Observers of captive lorises have found that when the animal is disturbed during capture and handling, the gland secretes about 10 microliters (l) of a clear, strong-smelling liquid in the form of an apocrine sweat. Typically, male and female lorises assumed a defensive position with head bent ...

Zoological Science, 2010

Major biliary bile acids of the Medaka (Oryzias latipes): 25R-and 25S-epimers of 3α,7α,12α-trihyd... more Major biliary bile acids of the Medaka (Oryzias latipes): 25R-and 25S-epimers of 3α,7α,12α-trihydroxy-5β-cholestanoic Acid

Journal of Zoo and Wildlife Medicine, 2005

A 4.0-kg cholelith was found within the abdominal cavity of a dead wild African elephant (Loxodon... more A 4.0-kg cholelith was found within the abdominal cavity of a dead wild African elephant (Loxodonta africana) in Eritrea. Analysis of this cholelith by histochemistry, electron microscopy, electrospray mass spectroscopy, and energy-dispersive x-ray spectroscopy revealed it was composed of bile alcohols but no calcium, bilirubin, or cholesterol. Bacteria were also found in the cholelith. Similar, but smaller, bile stones have been identified previously in other wild African elephants and an excavated mammoth (Mammuthus columbi). Choleliths have been reported only once in a captive Asian elephant (Elephas maximus). Elephants, along with hyraxes (Procavia capensis) and manatees (Trichechus manatus), are unique among mammals in producing only bile alcohols and no bile acids, which may predispose them to cholelithiasis, particularly in association with bacterial infection. Dietary factors may also play an important role in cholelith formation.

Journal of Lipid Research, 2007

The major bile acids present in the gallbladder bile of the common Australian wombat (Vombatus ur... more The major bile acids present in the gallbladder bile of the common Australian wombat (Vombatus ursinus) were isolated by preparative HPLC and identified by NMR as the taurine N-acylamidates of chenodeoxycholic acid (CDCA) and 15a-hydroxylithocholic acid (3a,15a-dihydroxy-5b-cholan-24-oic acid). Taurine-conjugated CDCA constituted 78% of biliary bile acids, and (taurine-conjugated) 15a-hydroxylithocholic acid constituted 11%. Proof of structure of the latter compound was obtained by its synthesis from CDCA via a # 14 intermediate. The synthesis of its C-15 epimer, 15b-hydroxylithocholic acid (3a,15bdihydroxy-5b-cholan-24-oic acid), is also reported. The taurine conjugate of 15a-hydroxylithocholic acid was synthesized and shown to have chromatographic and spectroscopic properties identical to those of the compound isolated from bile. It is likely that 15a-hydroxylithocholic acid is synthesized in the wombat hepatocyte by 15a-hydroxylation of lithocholic acid that was formed by bacterial 7adehydroxylation of CDCA in the distal intestine. Thus, the wombat appears to use 15a-hydroxylation as a novel detoxification mechanism for lithocholic acid.-Kakiyama,

Journal of Hepatology, 2002

Hepatology, 2013

The sea lamprey (Petromyzon marinus) is a genetically programmed animal model for biliary atresia... more The sea lamprey (Petromyzon marinus) is a genetically programmed animal model for biliary atresia as it loses its bile ducts and gallbladder during metamorphosis. However, in contrast to patients with biliary atresia or other forms of cholestasis who develop progressive disease, the post-metamorphosis lampreys grow normally to adult size. To understand how the adult lamprey thrives without the ability to secrete bile, we examined bile salt homeostasis in larval and adult lampreys. Adult livers were severely cholestatic with levels of bile salts >1 mM, but no evidence of necrosis, fibrosis, or inflammation. Interestingly, both larvae and adults had normal plasma levels (~10 μM) of bile salts. In larvae, petromyzonol sulfate (PZS) was the predominant bile salt, whereas the major bile salts in adult liver were sulfated C27 bile alcohols. Cytotoxicity assays revealed that PZS was highly toxic. Pharmacokinetic studies in free-swimming adults revealed that ~35% of intravenously injected bromosulfophthalein (BSP) was eliminated over a 72 hr period. Collection of urine and feces demonstrated that both endogenous and exogenous organic anions, including biliverdin, bile salts and BSP, were predominantly excreted via the kidney with minor amounts also detected in feces. Gene expression analysis detected marked up-regulation of orthologs of known organic anion and bile salt transporters in the kidney with lesser effects in the intestine and gills in adults compared to larvae. These findings indicate that adult lampreys tolerate cholestasis by altering hepatic bile salt composition, while maintaining normal plasma bile salt levels predominantly through renal excretion of bile products. Therefore, we conclude that strategies to accelerate renal excretion of bile salt and other toxins should be beneficial for patients with cholestasis.

Hepatology, 1988

The effect of side chain length on bile acid conjugation by human and rat liver fractions was exa... more The effect of side chain length on bile acid conjugation by human and rat liver fractions was examined. The rate of conjugation with glucuronic acid, sulfate and coenzyme A of several natural (C24) bile acids was compared with that of their corresponding nor-bile acids. The rate of coenzyme A ester formation by nor-bile acids was much lower than that of the natural bile acids. In human liver microsomes, the rate of coenzyme A formation was less than 8% of the rate for the corresponding C24 bile acid. Rat liver microsomes formed the coenzyme A ester of nor-bile acids less than 20% of the rate of their corresponding C24 homologs. Glucuronidation rates were greater than sulfation rates in both species. With human liver microsomes, nor-bile acids were glucuronidated more rapidly than their corresponding C24 homologs, whereas with rat liver microsomes the reverse was true. Purified 3 alpha-OH androgen UDP-glucuronyltransferase catalyzed the glucuronidation of both nor-bile acids and bile acids. Human liver cytosol sulfated nor-bile acids more slowly than the corresponding bile acids. Rat liver cytosol, however, sulfated nor-bile acids more rapidly than the corresponding bile acids. The highest rate was seen with lithocholylglycine. The results indicate that the novel biotransformation of nor-bile acids seen in vivo--sulfation and glucuronidation rather than amidation--is most likely explained as a consequent of defective amidation, to which the rate of coenzyme A formation contributes. Thus, side chain and nuclear structures as well as species differences in conjugating enzyme activity are determinants of the pattern of bile acid biotransformation by the mammalian liver.

Hepatology, 1989

Canalicular transport of bilirubin diglucuronide, dibromosulfophthalein and several glutathione c... more Canalicular transport of bilirubin diglucuronide, dibromosulfophthalein and several glutathione conjugates is deficient in mutant TR- rats. In contrast, transport of cholyltaurine (taurocholate), a conjugated bile acid, is normal. Previous studies using normal rats have shown that C23 nor-dihydroxy bile acids are conjugated with sulfate or glucuronide during hepatic transport in contrast to the natural C24 bile acids, which are amidated with glycine or taurine. Studies were performed to test the hypothesis that (a) in the TR- rat, nordeoxycholate would be conjugated with glucuronate or sulfate just as in the normal rat, and (b) that such conjugates would have defective biliary secretion. [C23-14C]Nordeoxycholate was administered intravenously to bile fistula rats (TR- and normal), and the biliary recovery of metabolites was assessed by chromatography and mass spectrometry. In both groups of rats, the major biotransformation product of nordeoxycholate was the side chain (23-ester) glucuronide. Conjugation on the nucleus with sulfate and glucuronide at the 3-position (ethereal linkage) also occurred, as well as amidation at the C23 carboxylic acid group. In the mutant rats, biliary secretion of the 3-sulfate and 3-glucuronide conjugates was less than 10% and 1%, respectively, of that of normal rats, whereas biliary secretion of the 23-ester glucuronide and the 23-taurine amidate, as well as unchanged nordeoxycholate, was not decreased. Canalicular secretion of nor-bile acid 3-ether glucuronides and 3-sulfates appears to involve the "bilirubin transport system," which is deficient in mutant rats. Canalicular secretion of unconjugated, amidated or esterified nordeoxycholate is mediated via another pathway, probably the "bile acid transport system."(ABSTRACT TRUNCATED AT 250 WORDS)

Hepatology, 2006

Mrp4 is a member of the multidrug resistance-associated gene family that is expressed on the baso... more Mrp4 is a member of the multidrug resistance-associated gene family that is expressed on the basolateral membrane of hepatocytes and undergoes adaptive upregulation in response to cholestatic injury or bile acid feeding. However, the relative importance of Mrp4 in a protective adaptive response to cholestatic injury is not known. To address this issue, common bile duct ligation (CBDL) was performed in wild-type and Mrp4-/- mice and animals followed for 7 days. Histological analysis and serum aminotransferase levels revealed more severe liver injury in the absence of Mrp4 expression. Western analyses revealed that Mrp4, but not Mrp3, was significantly increased after CBDL in wild-type mice. Serum bile acid levels were significantly lower in Mrp4-/- mice than in wild-type CBDL mice, whereas serum bilirubin levels were the same, suggesting that Mrp4 was required to effectively extrude bile acids from the cholestatic liver. Mrp3 and Ostalpha-Ostbeta were upregulated in Mrp4-/- mice but were unable to compensate for the loss of Mrp4. High-performance liquid chromatography analysis on liver extracts revealed that taurine tetrahydroxy bile acid/beta-muricholic acid ratios were increased twofold in Mrp4-/- mice. In conclusion, hepatic Mrp4 plays a unique and essential protective role in the adaptive response to obstructive cholestatic liver injury.

Hepatology, 2003

Experiments were performed to test whether conjugated bile acid administration would decrease bac... more Experiments were performed to test whether conjugated bile acid administration would decrease bacterial overgrowth, bacterial translocation, and endotoxemia in ascitic cirrhotic rats. Cholylsarcosine, a deconjugation-dehydroxylation resistant and cholylglycine, a deconjugation-dehydroxylation susceptible bile acid were used. Rats with CCl(4)-induced cirrhosis and ascites were fed cholylsarcosine, cholylglycine (both at 70 mg/kg/d), or placebo for 2 weeks. Healthy rats, as controls, were treated similarly. In cirrhotic rats receiving placebo, bile secretion from an acute biliary fistula was lower than in healthy rats (27.2 +/- 6.5 vs. 53.0 +/- 3.1 microL/kg/min; mean +/- SE, P<.05). The administration of conjugated bile acids to cirrhotic rats normalized bile secretion (cholylsarcosine, 51.8 +/- 6.29; cholylglycine, 52.72 +/- 8.9 microL/kg/min). Total ileal bacterial content was 6-fold higher in ascitic cirrhotic rats than in healthy rats. Conjugated bile acid administration reduced bacterial content to normal levels. Bacterial translocation was less in cirrhotic animals receiving conjugated bile acids (cholylsarcosine, 33%; cholylglycine, 26%) than in animals receiving placebo (66%). Endotoxemia was decreased in cirrhotic rats by conjugated bile acid feeding (cholylsarcosine, 0.098 +/- 0.002; cholylglycine 0.101 +/- 0.007 EU/mL) compared with placebo (0.282 +/- 0.124, P…

Hepatology, 2005

Experiments were performed in 2 volunteers to define the biotransformation and physiological prop... more Experiments were performed in 2 volunteers to define the biotransformation and physiological properties of norursodeoxycholic acid (norUDCA), the C 23 (C 24-nor) homolog of UDCA. To complement the in vivo studies, the biotransformation of norUDCA ex vivo using precision-cut human liver slices was also characterized. In the human studies, both a tracer dose given intravenously and a physiological dose (7.9 mmol, 3.0 g) given orally were excreted equally in bile and urine. By chromatography and mass spectrometry, the dominant biotransformation product of norUDCA in bile and urine was the C-23 ester glucuronide. Little N-acyl amidation (with glycine or taurine) occurred. The oral dose induced a sustained bicarbonate-rich hypercholeresis, with total bile flow averaging 20 L/kg/min, a rate extrapolating to 2 L/d. The increased bile flow was attributed to cholehepatic shunting of norUDCA as well to the lack of micelles in bile. Phospholipid and cholesterol secretion relative to bile acid secretion decreased during secretion of norUDCA and its metabolites, presumably also because of the absence of micelles in canalicular bile. When incubated with human liver slices, norUDCA was glucuronidated, whereas UDCA was conjugated with glycine or taurine. In conclusion, in humans, norUDCA is glucuronidated rather than amidated. In humans, but not animals, there is considerable renal elimination of the C-23 ester glucuronide, the dominant metabolite. NorUDCA ingestion induces a bicarbonaterich hypercholeresis and evokes less phospholipid and cholesterol secretion into bile than UDCA. Molecules that undergo cholehepatic shunting should be powerful choleretics in

Journal of Lipid Research, 1996

... (M-90-90-145-C15-C1&17). bubble pressure method using an apparatus constructed by Karol J... more ... (M-90-90-145-C15-C1&17). bubble pressure method using an apparatus constructed by Karol J. Mysels. This method determines changes in surface tension under dynamic conditions, and its prin-ciples (30) and application to bile acids (31) have been reported previously. ...

Journal of Chemical Ecology, Jun 1, 2003

The Giant panda communicates with conspecifics by depositing a mixture of volatile compounds (cal... more The Giant panda communicates with conspecifics by depositing a mixture of volatile compounds (called scent marks) on trees and rocks. Using mass spectrometry, we identified 951 chemical components from scent glands, urine, vaginal secretions, and scent marks made by pandas. The scent marks of the two genders contained a similar array of chemicals but varied in concentration; specifically, males possessed a significantly greater amount of short chain fatty acids (F(1, 29) = 18.4, P = 0.002). Using stepwise discriminate analysis on the relative proportions of a subset of these chemicals, it was possible to classify gender (94% for males and females) and individuality (81% for males and 91% for females) from scent marks. The power to identify individual males was reduced due to the relatedness of two subjects. By cracking the identity code of Giant panda communication, we show insights into how these animals can match individuals with unique chemical profiles. Since radiocollaring is currently banned in China, the techniques described in this paper give field biologists a new means to identify and track pandas in the wild.

Gastroenterology, 2013

BACKGROUND AND AIM: Endoscopy is the first level procedure in the diagnosis of gastrointestinal (... more BACKGROUND AND AIM: Endoscopy is the first level procedure in the diagnosis of gastrointestinal (GI) masses. However gastro-duodenal or colonic lesions which mainly involve submucosa or subserosa as well as small bowel's lesions may be difficult to diagnose. The present study aimed to evaluate the role of ultrasound (US)-guided percutaneous biopsy in diagnosing GI tract lesions. PATIENTS AND METHODS: 114 patients (63 male 51 female patients, age range 55-80 years, median age 68 years) underwent US-guided biopsy of GI lesions from 2000 to 2012. In 36 patients the lesion was in the small bowel, thus endoscopically inaccessible; in 50 patients previous histology of endoscopic biopsies was repeatedly negative; in 18 patients, stenosis did not allow bioptic sampling and in 10 patients age, severe cardiological disease and sepsis prevented endoscopy. We used multi-frequency convex probes with side adapters and 18G cutting needle for histological sampling. After biopsy, patients were monitored by measuring blood pressure and pulse rate during the following 3-4 hours to evaluate possible complications. Histology was compared with postsurgery histological evaluation in 73 cases. In the remaining, histology of US-guided biopsy was the only clue to diagnosis. RESULTS: Biopsy specimens were taken from stomach in 38 cases, small bowel in 36 cases and colon-sigma in 40 cases. Final diagnosis was malignant lesions in all but three cases. One case was intestinal endometriosis, the second was bowel duplication, whereas in the third case it was a false-negative result, the correct diagnosis being gastric adenocarcinoma. Sensitivity, specificity, positive predictive value, negative predictive value and diagnostic accuracy of biopsy were respectively 99%, 100%, 100%, 66%, 99%. We observed no mortality and one complication: A case of melena due to bleeding from gastric gastrointestinal stromal tumor that was effectively controlled by positioning of metal clips. CONCLUSION: US-guided percutaneous biopsy of GI tract lesions may be an alternative, safe and effective procedure for diagnosis of GI tract lesions in those cases where conventional diagnostic approach is not feasible or successful.

Nuclear receptor, Jan 30, 2005

The nuclear hormone receptor (NR) superfamily complement in humans is composed of 48 genes with d... more The nuclear hormone receptor (NR) superfamily complement in humans is composed of 48 genes with diverse roles in metabolic homeostasis, development, and detoxification. In general, NRs are strongly conserved between vertebrate species, and few examples of molecular adaptation (positive selection) within this superfamily have been demonstrated. Previous studies utilizing two-species comparisons reveal strong purifying (negative) selection of most NR genes, with two possible exceptions being the ligand-binding domains (LBDs) of the pregnane X receptor (PXR, NR1I2) and the constitutive androstane receptor (CAR, NR1I3), two proteins involved in the regulation of toxic compound metabolism and elimination. The aim of this study was to apply detailed phylogenetic analysis using maximum likelihood methods to the entire complement of genes in the vertebrate NR superfamily. Analyses were carried out both across all vertebrates and limited to mammals and also separately for the two major domai...

Cell Reports, 2012

Genotype-phenotype mapping is hampered by countless genomic changes between species. We introduce... more Genotype-phenotype mapping is hampered by countless genomic changes between species. We introduce a computational ''forward genomics'' strategy that-given only an independently lost phenotype and whole genomes-matches genomic and phenotypic loss patterns to associate specific genomic regions with this phenotype. We conducted genome-wide screens for two metabolic phenotypes. First, our approach correctly matches the inactivated Gulo gene exactly with the species that lost the ability to synthesize vitamin C. Second, we attribute naturally low biliary phospholipid levels in guinea pigs and horses to the inactivated phospholipid transporter Abcb4. Human ABCB4 mutations also result in low phospholipid levels but lead to severe liver disease, suggesting compensatory mechanisms in guinea pig and horse. Our simulation studies, counts of independent changes in existing phenotype surveys, and the forthcoming availability of many new genomes all suggest that forward genomics can be applied to many phenotypes, including those relevant for human evolution and disease.

Journal of Laboratory and Clinical Medicine, 2003

Quantitation of fecal bile acid excretion can help elucidate the cause of diarrhea or steatorrhea... more Quantitation of fecal bile acid excretion can help elucidate the cause of diarrhea or steatorrhea. Fecal bile acids can be measured with gas chromatography-mass spectrometry, but this is time-consuming, expensive, and not available for clinical use. Relatively simple enzymatic methods have been described for the measurement of fecal 3␣-hydroxy bile acids, but these have not been validated in patients with gastrointestinal disease. We found that an enzymatic method yielded falsely low results in patients with malabsorption syndromes for two reasons: First, the preliminary hydrolysis step did not completely deconjugate bile acids, precluding their extraction into diethyl ether for enzymatic assay. Second, long-chain fatty acids inhibited 3␣-hydroxysteroid dehydrogenase activity. By increasing the duration of hydrolysis and the concentration of enzyme, we developed a simple, accurate, and reproducible method for measuring fecal 3␣-hydroxy bile acids that agreed well with values obtained with the use of gas chromatography-mass spectrometry (R ‫؍‬ .95), both in normal subjects and in patients with malabsorption syndromes. (J Lab Clin Med 2003;141:411-8) Abbreviations: EDTA ϭ ethylenediaminetetraacetate; GC-MS ϭ gas chromatography-mass spectrometry; NAD ϭ ␤-nicotinamide adenine dinucleotide hydrate T he cathartic effect of an excess of fecal bile acids may be an unappreciated cause of chronic diarrhea in several clinical settings, including congenital diarrhea, 1 idiopathic secretory diarrhea, 2-4 irritable bowel syndrome, 5-7 postgastrectomy or postcholecystectomy diarrhea, 8-11 Crohn's disease, 7,10 postinfectious diarrhea, 10 postvagotomy diarrhea, 11,12 microscopic colitis, 13 and diarrhea after abdominal radiation. 14,15 In addition, excessive fecal loss of bile acids can be a major factor in the pathogenesis of fat malabsorption in patients with short bowel syndrome. 16-18 Quantitation of fecal bile acid excretion can help identify the cause of diarrhea and/or steatorrhea in these clinical situations. GC-MS is considered the most accurate method for the measurement of fecal bile acids. 19 However, GC-MS analysis of fecal bile acids is time-consuming, expensive, and not available for clinical use. At least 10 enzymatic methods for measuring total fecal 3␣-hydroxy bile acids have been reported. 20-29 In general, these methods are simpler to perform than GC-MS, but some are time-consuming and complicated, requiring either corrections based on a radioactive internal standard 20,21,25 or chromatographic separation of bile acids from interfering substances. 20-22,25,28 Although these methods apparently

Developments in Primatology: Progress and Prospects

On the ventral side of the elbow of the both the slow (Nycticebus bengalensis, N. coucang) and py... more On the ventral side of the elbow of the both the slow (Nycticebus bengalensis, N. coucang) and pygmy (N. pygmaeus) lorises, one can perceive a slightly raised, fairly hair-free but barely visible swelling, termed the brachial gland (Figure 12.1). Observers of captive lorises have found that when the animal is disturbed during capture and handling, the gland secretes about 10 microliters (l) of a clear, strong-smelling liquid in the form of an apocrine sweat. Typically, male and female lorises assumed a defensive position with head bent ...

Zoological Science, 2010

Major biliary bile acids of the Medaka (Oryzias latipes): 25R-and 25S-epimers of 3α,7α,12α-trihyd... more Major biliary bile acids of the Medaka (Oryzias latipes): 25R-and 25S-epimers of 3α,7α,12α-trihydroxy-5β-cholestanoic Acid

Journal of Zoo and Wildlife Medicine, 2005

A 4.0-kg cholelith was found within the abdominal cavity of a dead wild African elephant (Loxodon... more A 4.0-kg cholelith was found within the abdominal cavity of a dead wild African elephant (Loxodonta africana) in Eritrea. Analysis of this cholelith by histochemistry, electron microscopy, electrospray mass spectroscopy, and energy-dispersive x-ray spectroscopy revealed it was composed of bile alcohols but no calcium, bilirubin, or cholesterol. Bacteria were also found in the cholelith. Similar, but smaller, bile stones have been identified previously in other wild African elephants and an excavated mammoth (Mammuthus columbi). Choleliths have been reported only once in a captive Asian elephant (Elephas maximus). Elephants, along with hyraxes (Procavia capensis) and manatees (Trichechus manatus), are unique among mammals in producing only bile alcohols and no bile acids, which may predispose them to cholelithiasis, particularly in association with bacterial infection. Dietary factors may also play an important role in cholelith formation.

Journal of Lipid Research, 2007

The major bile acids present in the gallbladder bile of the common Australian wombat (Vombatus ur... more The major bile acids present in the gallbladder bile of the common Australian wombat (Vombatus ursinus) were isolated by preparative HPLC and identified by NMR as the taurine N-acylamidates of chenodeoxycholic acid (CDCA) and 15a-hydroxylithocholic acid (3a,15a-dihydroxy-5b-cholan-24-oic acid). Taurine-conjugated CDCA constituted 78% of biliary bile acids, and (taurine-conjugated) 15a-hydroxylithocholic acid constituted 11%. Proof of structure of the latter compound was obtained by its synthesis from CDCA via a # 14 intermediate. The synthesis of its C-15 epimer, 15b-hydroxylithocholic acid (3a,15bdihydroxy-5b-cholan-24-oic acid), is also reported. The taurine conjugate of 15a-hydroxylithocholic acid was synthesized and shown to have chromatographic and spectroscopic properties identical to those of the compound isolated from bile. It is likely that 15a-hydroxylithocholic acid is synthesized in the wombat hepatocyte by 15a-hydroxylation of lithocholic acid that was formed by bacterial 7adehydroxylation of CDCA in the distal intestine. Thus, the wombat appears to use 15a-hydroxylation as a novel detoxification mechanism for lithocholic acid.-Kakiyama,

Journal of Hepatology, 2002

Hepatology, 2013

The sea lamprey (Petromyzon marinus) is a genetically programmed animal model for biliary atresia... more The sea lamprey (Petromyzon marinus) is a genetically programmed animal model for biliary atresia as it loses its bile ducts and gallbladder during metamorphosis. However, in contrast to patients with biliary atresia or other forms of cholestasis who develop progressive disease, the post-metamorphosis lampreys grow normally to adult size. To understand how the adult lamprey thrives without the ability to secrete bile, we examined bile salt homeostasis in larval and adult lampreys. Adult livers were severely cholestatic with levels of bile salts >1 mM, but no evidence of necrosis, fibrosis, or inflammation. Interestingly, both larvae and adults had normal plasma levels (~10 μM) of bile salts. In larvae, petromyzonol sulfate (PZS) was the predominant bile salt, whereas the major bile salts in adult liver were sulfated C27 bile alcohols. Cytotoxicity assays revealed that PZS was highly toxic. Pharmacokinetic studies in free-swimming adults revealed that ~35% of intravenously injected bromosulfophthalein (BSP) was eliminated over a 72 hr period. Collection of urine and feces demonstrated that both endogenous and exogenous organic anions, including biliverdin, bile salts and BSP, were predominantly excreted via the kidney with minor amounts also detected in feces. Gene expression analysis detected marked up-regulation of orthologs of known organic anion and bile salt transporters in the kidney with lesser effects in the intestine and gills in adults compared to larvae. These findings indicate that adult lampreys tolerate cholestasis by altering hepatic bile salt composition, while maintaining normal plasma bile salt levels predominantly through renal excretion of bile products. Therefore, we conclude that strategies to accelerate renal excretion of bile salt and other toxins should be beneficial for patients with cholestasis.

Hepatology, 1988

The effect of side chain length on bile acid conjugation by human and rat liver fractions was exa... more The effect of side chain length on bile acid conjugation by human and rat liver fractions was examined. The rate of conjugation with glucuronic acid, sulfate and coenzyme A of several natural (C24) bile acids was compared with that of their corresponding nor-bile acids. The rate of coenzyme A ester formation by nor-bile acids was much lower than that of the natural bile acids. In human liver microsomes, the rate of coenzyme A formation was less than 8% of the rate for the corresponding C24 bile acid. Rat liver microsomes formed the coenzyme A ester of nor-bile acids less than 20% of the rate of their corresponding C24 homologs. Glucuronidation rates were greater than sulfation rates in both species. With human liver microsomes, nor-bile acids were glucuronidated more rapidly than their corresponding C24 homologs, whereas with rat liver microsomes the reverse was true. Purified 3 alpha-OH androgen UDP-glucuronyltransferase catalyzed the glucuronidation of both nor-bile acids and bile acids. Human liver cytosol sulfated nor-bile acids more slowly than the corresponding bile acids. Rat liver cytosol, however, sulfated nor-bile acids more rapidly than the corresponding bile acids. The highest rate was seen with lithocholylglycine. The results indicate that the novel biotransformation of nor-bile acids seen in vivo--sulfation and glucuronidation rather than amidation--is most likely explained as a consequent of defective amidation, to which the rate of coenzyme A formation contributes. Thus, side chain and nuclear structures as well as species differences in conjugating enzyme activity are determinants of the pattern of bile acid biotransformation by the mammalian liver.

Hepatology, 1989

Canalicular transport of bilirubin diglucuronide, dibromosulfophthalein and several glutathione c... more Canalicular transport of bilirubin diglucuronide, dibromosulfophthalein and several glutathione conjugates is deficient in mutant TR- rats. In contrast, transport of cholyltaurine (taurocholate), a conjugated bile acid, is normal. Previous studies using normal rats have shown that C23 nor-dihydroxy bile acids are conjugated with sulfate or glucuronide during hepatic transport in contrast to the natural C24 bile acids, which are amidated with glycine or taurine. Studies were performed to test the hypothesis that (a) in the TR- rat, nordeoxycholate would be conjugated with glucuronate or sulfate just as in the normal rat, and (b) that such conjugates would have defective biliary secretion. [C23-14C]Nordeoxycholate was administered intravenously to bile fistula rats (TR- and normal), and the biliary recovery of metabolites was assessed by chromatography and mass spectrometry. In both groups of rats, the major biotransformation product of nordeoxycholate was the side chain (23-ester) glucuronide. Conjugation on the nucleus with sulfate and glucuronide at the 3-position (ethereal linkage) also occurred, as well as amidation at the C23 carboxylic acid group. In the mutant rats, biliary secretion of the 3-sulfate and 3-glucuronide conjugates was less than 10% and 1%, respectively, of that of normal rats, whereas biliary secretion of the 23-ester glucuronide and the 23-taurine amidate, as well as unchanged nordeoxycholate, was not decreased. Canalicular secretion of nor-bile acid 3-ether glucuronides and 3-sulfates appears to involve the "bilirubin transport system," which is deficient in mutant rats. Canalicular secretion of unconjugated, amidated or esterified nordeoxycholate is mediated via another pathway, probably the "bile acid transport system."(ABSTRACT TRUNCATED AT 250 WORDS)

Hepatology, 2006

Mrp4 is a member of the multidrug resistance-associated gene family that is expressed on the baso... more Mrp4 is a member of the multidrug resistance-associated gene family that is expressed on the basolateral membrane of hepatocytes and undergoes adaptive upregulation in response to cholestatic injury or bile acid feeding. However, the relative importance of Mrp4 in a protective adaptive response to cholestatic injury is not known. To address this issue, common bile duct ligation (CBDL) was performed in wild-type and Mrp4-/- mice and animals followed for 7 days. Histological analysis and serum aminotransferase levels revealed more severe liver injury in the absence of Mrp4 expression. Western analyses revealed that Mrp4, but not Mrp3, was significantly increased after CBDL in wild-type mice. Serum bile acid levels were significantly lower in Mrp4-/- mice than in wild-type CBDL mice, whereas serum bilirubin levels were the same, suggesting that Mrp4 was required to effectively extrude bile acids from the cholestatic liver. Mrp3 and Ostalpha-Ostbeta were upregulated in Mrp4-/- mice but were unable to compensate for the loss of Mrp4. High-performance liquid chromatography analysis on liver extracts revealed that taurine tetrahydroxy bile acid/beta-muricholic acid ratios were increased twofold in Mrp4-/- mice. In conclusion, hepatic Mrp4 plays a unique and essential protective role in the adaptive response to obstructive cholestatic liver injury.

Hepatology, 2003

Experiments were performed to test whether conjugated bile acid administration would decrease bac... more Experiments were performed to test whether conjugated bile acid administration would decrease bacterial overgrowth, bacterial translocation, and endotoxemia in ascitic cirrhotic rats. Cholylsarcosine, a deconjugation-dehydroxylation resistant and cholylglycine, a deconjugation-dehydroxylation susceptible bile acid were used. Rats with CCl(4)-induced cirrhosis and ascites were fed cholylsarcosine, cholylglycine (both at 70 mg/kg/d), or placebo for 2 weeks. Healthy rats, as controls, were treated similarly. In cirrhotic rats receiving placebo, bile secretion from an acute biliary fistula was lower than in healthy rats (27.2 +/- 6.5 vs. 53.0 +/- 3.1 microL/kg/min; mean +/- SE, P<.05). The administration of conjugated bile acids to cirrhotic rats normalized bile secretion (cholylsarcosine, 51.8 +/- 6.29; cholylglycine, 52.72 +/- 8.9 microL/kg/min). Total ileal bacterial content was 6-fold higher in ascitic cirrhotic rats than in healthy rats. Conjugated bile acid administration reduced bacterial content to normal levels. Bacterial translocation was less in cirrhotic animals receiving conjugated bile acids (cholylsarcosine, 33%; cholylglycine, 26%) than in animals receiving placebo (66%). Endotoxemia was decreased in cirrhotic rats by conjugated bile acid feeding (cholylsarcosine, 0.098 +/- 0.002; cholylglycine 0.101 +/- 0.007 EU/mL) compared with placebo (0.282 +/- 0.124, P…

Hepatology, 2005

Experiments were performed in 2 volunteers to define the biotransformation and physiological prop... more Experiments were performed in 2 volunteers to define the biotransformation and physiological properties of norursodeoxycholic acid (norUDCA), the C 23 (C 24-nor) homolog of UDCA. To complement the in vivo studies, the biotransformation of norUDCA ex vivo using precision-cut human liver slices was also characterized. In the human studies, both a tracer dose given intravenously and a physiological dose (7.9 mmol, 3.0 g) given orally were excreted equally in bile and urine. By chromatography and mass spectrometry, the dominant biotransformation product of norUDCA in bile and urine was the C-23 ester glucuronide. Little N-acyl amidation (with glycine or taurine) occurred. The oral dose induced a sustained bicarbonate-rich hypercholeresis, with total bile flow averaging 20 L/kg/min, a rate extrapolating to 2 L/d. The increased bile flow was attributed to cholehepatic shunting of norUDCA as well to the lack of micelles in bile. Phospholipid and cholesterol secretion relative to bile acid secretion decreased during secretion of norUDCA and its metabolites, presumably also because of the absence of micelles in canalicular bile. When incubated with human liver slices, norUDCA was glucuronidated, whereas UDCA was conjugated with glycine or taurine. In conclusion, in humans, norUDCA is glucuronidated rather than amidated. In humans, but not animals, there is considerable renal elimination of the C-23 ester glucuronide, the dominant metabolite. NorUDCA ingestion induces a bicarbonaterich hypercholeresis and evokes less phospholipid and cholesterol secretion into bile than UDCA. Molecules that undergo cholehepatic shunting should be powerful choleretics in