Ion Fast Atom Bombardment Mass Spectrometry Research Articles

Δ4-3-oxosteroid-5β-reductase deficiency: Responses to oral bile acid therapy and long-term outcomes.

BACKGROUNDDisorders of primary bile acid synthesis may be life-threatening if undiagnosed, or not treated with primary bile acid replacement therapy. To date, there are few reports on the management and follow-up of patients with Δ4-3-oxosteroid 5β-reductase (AKR1D1) deficiency. We hypothesized that a retrospective analysis of the responses to oral bile acid replacement therapy with chenodeoxycholic acid (CDCA) in patients with this bile acid synthesis disorder will increase our understanding of the disease progression and permit evaluation of this treatment regimen as an alternative to the Food and Drug Administration (FDA) approved drug cholic acid, which is currently unavailable in China.AIMTo evaluate the therapeutic responses of patients with AKR1D1 deficiency to oral bile acid therapy, specifically CDCA.METHODSTwelve patients with AKR1D1 deficiency, confirmed by fast atom bombardment ionization-mass spectrometry analysis of urine and by gene sequencing for mutations in AKR1D1, were treated with differing doses of CDCA or ursodeoxycholic acid (UDCA). The clinical and biochemical responses to therapy were monitored over a period ranging 0.5-6.4 years. Dose adjustment, to optimize the therapeutic dose, was based on changes in serum biochemistry parameters, notably liver function tests, and suppression of the urinary levels of atypical hepatotoxic 3-oxo-Δ4-bile acids measured by mass spectrometry.RESULTSPhysical examination, serum biochemistry parameters, and sonographic findings improved in all 12 patients during bile acid therapy, except one who underwent liver transplantation. Urine bile acid analysis confirmed a significant reduction in atypical hepatotoxic 3-oxo-Δ4 bile acids concomitant with clinical and biochemical improvements in those patients treated with CDCA. UDCA was ineffective in down-regulating endogenous bile acid synthesis as evidenced from the inability to suppress the urinary excretion of atypical 3-oxo-Δ4-bile acids. The dose of CDCA required for optimal clinical and biochemical responses varied from 5.5-10 mg/kg per day among patients based on maximum suppression of the atypical bile acids and improvement in serum biochemistry parameters, and careful titration of the dose was necessary to avoid side effects from CDCA.CONCLUSIONThe primary bile acid CDCA is effective in treating AKR1D1 deficiency but the therapeutic dose requires individualized optimization. UDCA is not recommended for long-term management.

Bile Acid Synthesis Disorders in Arabs: A 10-year Screening Study.

Early diagnosis of bile acid synthesis disorders (BASDs) is important because, untreated, these conditions can be fatal. Our objectives were to screen children with cholestasis or unexplained liver disease for BASD and in those with confirmed BASD to evaluate the effectiveness of cholic acid therapy. A routine serum total bile acid measurement was performed on children with cholestasis, liver cirrhosis, and liver failure. Patients were screened for BASD by fast atom bombardment ionization-mass spectrometry (FAB-MS) analysis of urine, and molecular analysis confirmed diagnosis. Treatment response to oral cholic acid (10-15 mg/kg bw/day) was assessed from liver function tests and fat-soluble vitamin levels. FAB-MS analysis of urine was used to monitor compliance and biochemical response. Between 2007 and 2016, 626 patients were evaluated; 450 with infantile cholestasis. Fifteen cases of BASD were diagnosed: 12 presented with infantile cholestasis (2.7%, 7 boys), an 8-year-old boy presented with cirrhosis, and two 18-month-old boys presented with hepatomegaly and rickets. Eleven were caused by 3β-hydroxy-Δ-C27-steroid oxidoreductase dehydrogenase deficiency, 3 from Δ-3-oxosteroid 5β-reductase deficiency, and 1 had Zellweger spectrum disorder. In all but 1, serum total bile acids were normal or low. With cholic acid therapy, 10 are alive and healthy with their native liver. Liver failure developed in 3 infants despite therapy; 2 died and 1 underwent liver transplantation. BASDs are rare but treatable causes of metabolic liver disease in Saudi Arabia. BASD should be considered in infants with cholestasis and low or normal serum total bile acid concentrations.

Atypical clinical presentation and successful treatment with oral cholic acid of a child with defective bile acid synthesis due to a novel mutation in the HSD3B7 gene.

We report definitive diagnosis and effective treatment with oral cholic acid in one Italian male child affected by 3β-hydroxy-Δ5-C27-steroid dehydrogenase (3β-HSD) deficiency. He presented with failure to thrive, hepatomegaly and multiple cystic images in kidneys; no biochemical evidence of cholestasis. Large amounts of bile acid metabolites was detected in urine by fast atom bombardment ionization mass spectrometry (FAB-MS). HSDH3B7 gene analysis identified one mutation in intron 4, at nucleotide 432, G>A substitution that has never been reported before.The replacement therapy with oral cholic acid started early after the diagnosis and is still ongoing. Three years later hepatomegaly is no longer evident, liver function is normal and the child is growing regularly. In our experience, clinical features of 3β-HSD deficiency can be very poor and even cholestasis can lack at diagnosis. Early replacement therapy with cholic acid is safe and leads to clinical and biochemical control of the disease.

Characterization of DHDP, a novel aldose reductase inhibitor isolated from Lysimachia christinae

The aldose reductase (AR) inhibitory effect of Lysimachia christinae was investigated as a functional food ingredient and a new AR inhibitor, DHDP, was isolated from L. christinae, guided by an affinity-based ultrafiltration-HPLC assay. The chemical structure of DHDP was determined to be 1,5-di-hydroxy-1,5-di-[(E)-3-(4-hydroxyphenyl)-2-propenoic]-3-pentanonyl ester by one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR), fast atom bombardment ionization mass spectrometry (FAB-MS), and ultraviolet (UV) and (IR) infrared spectroscopy. In addition, the AR inhibitory activity and mechanism of DHDP were characterized. In vitro and in silico results demonstrated that DHDP binds to the AR active site rapidly, blocking it and competitively preventing substrate access and product formation (IC50, 194.67μM; Ki, 29.68μM). Ex vivo experiments indicated that DHDP can penetrate the eye lens and successfully inhibit AR under hyperglycemic conditions, thus preventing the accumulation of sorbitol and subsequent osmotic stress.

Immunoregulation of Cellular Life Span

Our current studies focus on the molecular changes induced by aging. During aging, changes in proteins occur that alter their function and render them immunogenic. These "neoantigens" are recognized by physiologic autoantibodies. Physiologic autoantibodies and their corresponding antigens offer therapeutic strategies for disease intervention through the innate immune response. Early studies done in the 1970s showed humans and animals to have physiologic antibodies that bind to a neoantigen called senescent cell antigen (SCA), which appears on senescent and damaged cells and initiates their removal by macrophages. These studies led to the discovery that oxidation can generate a new antigen in situ. Oxidation accelerated aging of red cells, generated SCA and IgG binding, and triggered removal of red cells by macrophages. Since then, a number of laboratories have found that oxidation can generate other neoantigens. For example, oxidized LDL (OxLDL) induces antibodies that can modify the natural progression of atherosclerosis. Apoptotic cells express oxidatively modified moieties on their surfaces that are involved in macrophage recognition and phagocytosis. Physiologic autoantibodies were used to isolate SCA from brain tissue. HPLC and fast atom bombardment ionization mass spectrometry (FAB-MS) of the isolated antigen suggested that the aging antigen is a subset of band 3, a family of proteins also called anion exchange proteins (AE1-3). FAB-MS results indicate that residues matching all three band 3 isoforms (AE1, AE2, and AE3) are detected in aging antigen fractions. Among the fragments identified with FAB-MS was a sequence corresponding to an aging epitope, human band 3 sequence LFKPPKYHPDVPYVKR, residue 812-830 in AE1; HHPDVTYVK, residue 1144-1152 in AE2; or HHPEQPYVTK, residue 1135-1144 in AE3. A residue that is close to that region was identified in mouse AE1 ASGPGAAAQIQEVK, residue 762-775. The potential for altering the natural progression of diseases using select peptide-defined epitopes within or overlapping the aging antigenic site (547-553 and 824-829) is discussed using the innate immune response to band 3 in malaria as an example.

Analyses of anandamide and endocannabinoid-like compounds using collision-induced dissociation in fast atom bombardment ionization-mass spectrometry and gas chromatography/chemical ionization-mass spectrometry.

The utility of the collision-induced dissociation (CID) of two different forms of precursor cations generated by the fast atom bombardment (FAB) ionization of N-arachidonylethanolamine (anandamide) and a series of endocannabinoid-like compounds, such as N-oleoylethanolamine, N-palmitoylethanolamine, N-stearoylethanolamine, N-linoleoylethanolamine, N-oleoylpropanolamine, and N-palmitoylpropanolamine, as a method of providing general information on their characterizations was examined. The CID spectra of lithium-adduct [M+Li]+ ions of the amines with unsaturated hydrocarbon chains were rich in structurally informative charge-site-remote (CSR) fragmentation patterns that provide information on the locations of double bonds in hydrocarbon chains. On the other hand, the CID reactions of [M+H]+ ions produced acylium ions that are derived from the cleavage of amide bonds, thus providing information on the size of the hydrocarbon chains, although CSR fragmentations were not observed. These compounds without derivatization were analyzed using gas chromatography/chemical ionization-mass spectrometry (GC/CI-MS) with a polyethylene glycol phased column with fused silica capillary pre-tubing. Identifiable molecular-related [M+H]+ ions were observed.

Mapping of senescent cell antigen on brain anion exchanger protein (AE) isoforms using HPLC and fast atom bombardment ionization mass spectrometry (FAB-MS).

Molecular recognition of senescent cells involves oxidation of a crucial membrane protein leading to generation of a neoantigen, called 'senescent cell antigen' (SCA), and binding of physiologic autoantibodies. These IgG autoantibodies trigger macrophage removal of the cell prior to its lysis at a time when anion transport has decreased but the membrane is still grossly intact. The neoantigen SCA is generated by oxidation of a major anion transport protein called band 3 or anion exchange protein. In this study, we use IgG physiologic autoantibodies from senescent red cells to isolate SCA from brain, and HPLC and fast atom bombardment ionization mass spectrometry (FAB-MS) to compare brain SCA to band 3. HPLC peptide maps of band 3 and SCA showed substantial homology, suggesting that SCA is a subset of band 3, and includes an estimated >/=45% of the band 3 molecule. FAB-MS results indicate that residues matching all three band 3 isoforms (AE1, AE2 and AE3) are detected in SCA fractions. These findings suggest that other isoforms of band 3 may undergo the same aging changes that AE1 on red blood cells undergoes to generate SCA. This provides confirmation that SCA is on non-erythroid cell types. Implications of these studies to the generation of neoantigens by oxidation and their recognition by autoantibodies to them are discussed.

Isolation and identification of indigestible pyroglutamyl peptides in an enzymatic hydrolysate of wheat gluten prepared on an industrial scale.

An enzymatic hydrolysate of wheat gluten was further digested in vitro with porcine pepsin and pancreatin to obtain an indigestible peptide. Indigestible pyroglutamyl peptide was isolated from the digest by strong cation-exchange, size-exclusion, and reversed-phase chromatographies. The pyroglutamyl peptide was digested with pyroglutamate aminopeptidase, and the digest was reacted with phenyl isothiocyanate. The resultant phenylthiocarbamyl (PTC) peptides were purified by reversed-phase HPLC by using binary gradient elution with ammonium acetate buffer, pH 6.0, and acetonitrile. The PTC peptides were analyzed with an automatic peptide sequencer on the basis of the Edman degradation method with a modified program. Some pyroglutamyl peptides were also analyzed by fast-atom bombardment ionization mass spectrometry without the pyroglutamate amino peptidase digestion. Consequently, pyroGlu-Asn-Pro-Gln, pyroGlu-Gln-Gln-Pro-Gln, pyroGlu-Gln-Pro-Gln, pyroGlu-Gln-Pro-Gly-Gln-Gly-Gln, pyroGlu-Gln, pyroGlu-Gln-Pro, pyroGlu-Ile-Pro-Gln, pyroGlu-Ile-Pro, pyroGlu-Gln-Pro-Leu, pyroGlu-Gln-Phe-Pro-Gln, pyroGlu-Ser-Phe-Pro-Gln, pyroGlu-Phe-Pro-Gln, and pyroGlu-Gln-Pro-Pro-Phe-Ser were identified.

Characterization of the Radical Product Formed from the Reaction of Nitric Oxide with the Spin Trap 3,5-Dibromo-4-Nitrosobenzene Sulfonate

Previously, 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS) has been used in combination with electron paramagnetic resonance (EPR) spectrometry to trap nitric oxide (NO•). The reaction between DBNBS and NO• yields a radical product which gives rise to an EPR signal consisting of three lines with an AN = 0.96 mT, but the structure of this product is unknown. A two-stage high-performance liquid chromatography fractionation was performed to isolate the radical product from the other components in the DBNBS/NO• reaction mixture. The fractions containing the radical product were identified by the presence of the three-line EPR signal, and then these fractions were analyzed by negative ion fast atom bombardment–mass spectrometry (FAB–MS). Collectively, the FAB–MS data suggested that the radical product is the monosodium electrostatic complex with the dianion, bis(2,6-dibromo-4-sulfophenyl) nitroxyl. Analysis of the Gaussian and Lorentzian linewidths of the EPR signal suggested that bis(2,6-dibromo-4-sulfophenyl) nitroxyl molecules may group together to form micelles. Further studies also indicated that significant amounts of nitrogen and nitrate were produced during the reaction between DBNBS and NO•. A reaction scheme consistent with these results is presented.

Chiral recognition of cellulose tris(5-fluoro-2-methylpheylcarbamate) toward (R)- and (S)-1,1′-bi-2-naphthol detected by negative ion fast-atom bombardment mass spectrometry

Chiral recognition between cellulose tris(5-fluoro-2-methylphenylcarbamate) (MW > 120,000) as a host compound and (R,S)-1,1'-binaphthol (MW 286) as a guest compound was investigated by fast-atom bombardment mass spectrometry (FAB-MS). The use of negative ion FAB-MS made it possible to obtain a very simple mass spectrum of the guest compound with a high signal-to-noise ratio. Additionally, when nitrophenyl octyl ether, which has no OH group, was utilized as matrix, chiral recognition between the host and the guest compounds was unequivocally detected. Copyright 1999 John Wiley & Sons, Ltd.

BILE ACID SYNTHETIC DISORDER IN A SYMPTOMATIC PATIENT WITH NORMAL LIVER ENZYMES

173 The bile acid synthetic disorders (BASD) are due to inherited defects in cholate and chenodeoxycholate biosynthesis. Abnormal hepatotoxic and cholestatic bile acids result in severe liver injury, which commonly presents in the neonatal period. Patients with diagnosis in childhood typically have biochemical evidence of significant hepatic injury. We report a patient with a BASD presenting with profound malabsortion of fat soluble vitamins, but with normal hepatic aminotransferases. Case report: A 3 year old female born to non-consanguineous Liberian parents presented following a one week history of epistaxis. She was previously healthy except for rickets at age one year. She had frequent nosebleeds and easy bruisability, but no fever, diarrhea, bleeding gums or jaundice. There was no known toxic ingestion. On examination, she was a slightly pale child with height and weight both at the 75th percentile. She had hepatomegaly with a liver span of 10 cm at the right costal margin. The remainder of the physical exam was normal. The hemoglobin was 7.4 (11.5-13.5 gm/dl), platelets 553 (150-400 K/ul), PT 33.1(11.0-13.0 sec), PTT 70.0 (27.0-38.0 sec). Levels of factors VII, IX and X were low at 9%, 2% and 2% respectively. (normal - 50-150%) Factor VIII and factor XII levels were at or above normal at 231% and 87%. Warfarin level was zero. At the time of admission, total bilirubin was 0.7 (0.2-1.4 mg/dl), alkaline phosphatase 247 (145-320 U/I), ALT 39 (5-45 U/I), AST 41 (20-60 U/I) and GGT 23 (6-19 U/I) After 5 mg IV Vitamin K, PT and PTT corrected to 12.4 and 26.4 seconds. Levels of fat soluble vitamins were as follows: Vitamin E< 0.5 (3.0-12.0), 25-OH Vitamin D < 5.0 (10-55 ng/ml), Vitamin A 74(30-95 mcg/dl). Commercial choleglycine was 97 (< 68 mcg/dl). Urine was screened for abnormal bile acids by fast atom bombardment ionization mass spectrometry (FAB-MS). Results showed elevated levels of sulfate and glycosulfate conjugates of unsaturated dihydroxy- and trihydroxy-cholanoic acids, consistent with a diagnosis of 3β-hydroxy-C27-steroid dehydrogenase/isomerase deficiency. Percutaneous liver biopsy showed a moderate inflammatory infiltrate in the portal tracts and lobules, portal tracts expanded by bands of fibrosis and occasional bile plugs. Cholic acid therapy was instituted. Conclusions: Inherited defects in bile acid synthesis should be considered in patients with fat soluble vitamin deficiency. In patients with these disorders, significant hepatotoxicity from abnormal bile acids may exist even in the presence of normal liver enzymes. Maintaining a high index of suspicion for bile acid synthetic defects may result in earlier diagnosis and a better prognosis for these children.

Iron Salts in the Post-Metallization of Mordant Dyes

Iron (II) and iron (III) salts are used as mordanting agents in lieu of chromium ions, as a source reduction approach to eliminating chromium in dye effluents following the post-metallization of acid mordant dyes. Specifically, wool fabric is dyed with Mordant Yellow 8, Yellow 30, Orange 3, Orange 6, Red 7. Red 19, Blue 1, Blue 9, Blue 13, Brown 40, Black 1, and Black 11, then treated with iron salts. The complexes are characterized by negative ion fast-atom bombardment mass spectrometry and absorp tion spectrophotometry, and the after-treated fabrics are evaluated for lightfastness and washfastness. Mordant dyes possessing either ortho, ortho'-bis(hydroxy)- phenylazopyrazole or ortho-hydroxy-phenylazonaphthol substituted chromophoric sys tems form the corresponding 1:2 metal : dye complex and undergo significant improve ments in lightfastness and washfastness following metallization.

Disulfated oligosaccharides derived from tracheobronchial mucous glycoproteins of a patient suffering from cystic fibrosis

Twenty novel disulfated oligosaccharides were purified in nanomolar quantities from tracheobronchial mucous glycoproteins from a patient with cystic fibrosis via cleavage by alkaline borohydride treatment, followed by anion-exchange chromatography, size-exclusion chromatography, and high-performance liquid chromatography (HPLC). In addition to positive ion fast-atom-bombardment mass spectrometry (FABMS), proposed structures for the resulting purified disulfated oligosaccharides were also based on carbohydrate permethylation analyses, periodate oxidation, complete sequential exoglycosidase digestion, and parallel analysis of desulfated products. Sulfate esters were found to reside on C-3 or C-6 of terminal d-galactose and on C-6 of internal d-galactose or 2-acetamido-2-deoxy- d-glucose residues. For this group of oligosaccharides, ranging in size from tri- to undeca-saccharides and possessing linear, di- and tri-antennary forms, it was also observed that sulfate esters could be located on the same or on different branches and that branched oligosaccharides can possess sulfate esters on C-3 and C-6 of different terminal galactose residues within the same structure.

Glucuronic Acid-conjugated Dihydroxy Fatty Acids in the Urine of Patients with Generalized Peroxisomal Disorders

Urine extracts from children diagnosed with generalized peroxisomal disorders were screened by continuous flow-negative ion fast atom bombardment-mass spectrometry. In 45 of 60 children with generalized peroxisomal disorders, we observed one or more intense ions (m/z 489, 505, 461, and others) that are infrequently found in children with cholestatic liver disease or normal children. Compounds giving rise to these ions were isolated using reverse phase and anion exchange chromatography. After appropriate derivatization and/or methanolysis the compounds were analyzed using capillary gas chromatography-mass spectrometry. The major compounds were found to be 12,13-dihydroxy-9-octadecenoic acid and 9,10-dihydroxy-12-octadecenoic acid, with one of the hydroxyl groups in glycosidic linkage with glucuronic acid. Minor compounds were glucuronic acid conjugates of 9,10-dihydroxy-octadecanoic acid, and 12,13-dihydroxy-6,9-, 15,16-dihydroxy-9,12-, and 9, 10-dihydroxy-12,15-octadecadienoic acids. A series of hexadecanoic, hexadecenoic, and hexadecadienoic acid glucuronides which appear to be beta-oxidation products of the C18 fatty acids were also observed, with the major species being 10, 11-dihydroxy-7-hexadecenoic acid glucuronide. In all, 16 C16 and C18 dihydroxy fatty acids were identified by gas chromatography-mass spectrometry. A series of at least 11 trihydroxy fatty acids was also observed but not fully characterized. Measurement of these compounds may prove to be useful in the diagnosis of some peroxisomal disorders.

Negative-ion fast atom bombardment mass spectrometry of sulfonylurea herbicides

Seven sulfonylurea herbicides were studied using negative-ion fast atom bombardment mass spectrometry (FAB-MS). The spectra were characteristic of the structure of the examined herbicides with fragmentation at the sulfonylurea bridge representing both aromatic moieties. The fragmentation pattern was established based on the FAB- B/E mass spectrometry/mass spectrometry linked-scan spectra. Background-subtracted negative ion FAB-MS spectra of the herbicides exhibit [M‐1] ‐ peaks and their fragmentation products. Application of triethylenetetramine as a FAB matrix resulted in enhanced fragmentation. The 3-nitrobenzyl alcohol FAB spectra typically exhibited high-abundance [M‐1] ‐ peaks and fewer fragment

Reply To: Δ4-3-Oxosteroid 5β-reductase deficiency and neonatal hemochromatosis

Dr. Clayton's letter raises pertinent points with regard to the interpretation of the finding of increased urinary excretion of Δ4-3- oxo bile acids. These metabolites can be detected in a variety of severe cholestatic liver disorders. The interpretation of their presence in liver failure is much more complex. Currently the finding of high proportions of Δ4-3-oxo bile acids and markedly reduced or absent primary bile acids is used as supportive evidence of a quantitative defect in Δ4-3-oxosteroid 5β-reductase enzyme activity. This pattern of urinary bile acid metabolites is not necessarily a simple reflection of liver dysfunction. Fast atom bombardment ionization-mass spectrometry analyses of urine from many patients with liver failure, including four with the clinical diagnosis of neonatal hemochromatosis, did not reveal this bile acid profile. An additional five patients have been identified who had a clinical diagnosis of neonatal hemochromatosis, and had a bile acid metabolite pattern similar to the two patients that we described.1Shneider BL Setchell KDR Whitington PF Neilson KA Suchy FJ. Δ4-3-Oxosteroid 5β-reductase deficiency causing neonatal liver failure and hemochromatosis.J PEDIATR. 1994; 124: 234-238Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar Definitive diagnosis of a primary genetic deficiency of the enzyme is hampered by the fact that it is not expressed in fibroblasts. When possible we have attempted to obtain additional evidence for an enzyme defect by immunoblotting for the 38 kd protein with monoclonal antibodies against the enzyme.2Russell DW Setchell KDR Bile acid biosynthesis.Biochemistry. 1992; 31: 4737-4749Crossref PubMed Scopus (644) Google Scholar As pointed out by Dr. Clayton, the finding of an absence of immunodetectable protein may be caused by a generalized destruction of hepatic proteins. The finding of a truncated protein in our first patient is intriguing.1Shneider BL Setchell KDR Whitington PF Neilson KA Suchy FJ. Δ4-3-Oxosteroid 5β-reductase deficiency causing neonatal liver failure and hemochromatosis.J PEDIATR. 1994; 124: 234-238Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar This feature is more consistent with a primary defect, rather than simple nonspecific loss of proteins as a result of hepatocellular injury. At present, confirmatory evidence of a primary enzyme defect can best be established from the observation that an interruption of primary bile acid therapy in these patients, after liver function has returned to normal, results in a reemergence of Δ4-3-oxo bile acids as major metabolites in urine. A human complementary DNA encoding 5β-reductase was recently sequenced, and molecular genetic techniques should soon be available to aid in clarifying the role of a primary genetic deficiency of this enzyme in the pathogenesis of liver failure.3Kondo K-H Kai M-H Setoguchi Y et al.Cloning and expression of cDNA of human Δ4-3-oxosteroid 5β-reductase and substrate specificity of the expressed enzyme.Eur J Biochem. 1994; 219: 357-363Crossref PubMed Scopus (121) Google Scholar The role of biliary excretion of iron has been persistently underestimated and poorly studied, especially in the newborn period. Examination of the studies of Hultcrantz et al.4Hultcrantz R Angelin B Bjorn-Rasmussen E Ewerth S Einarsson K Biliary excretion of iron and ferritin in idiopathic hemochromatosis.Gastroenterology. 1989; 96: 1539-1545PubMed Google Scholar indicates that normal biliary excretion amounts to between 0.4 and 2.3 mg of iron/day, in the range of normal uptake of iron. We agree that the finding of 5β-reductase deficiency should not be the end of the diagnostic evaluation in an infant with neonatal liver failure, but it should be part of the evaluation. Only through systematic evaluation of infants with neonatal liver failure will we determine the significance of this finding, in particular its relationship to the development of neonatal hemochromatosis. Unfortunately, at this time pediatric hepatologists are faced with very difficult therapeutic decisions. More exact molecular probes may simplify those decisions in the future. 9/35/58922

N-Alkylnicotinium halides: a class of cationic matrix additives for enhancing the sensitivity in negative ion fast-atom bombardment mass spectrometry of polyanionic analytes

The addition of some surfactants to the fast-atom bombardment (FAB) matrix previously has been demonstrated to enhance analyte signals in fast-atom bombardment mass spectrometry. In particular, cationic surfactants appear to enhance the negative ion FAB detectability of analytes that exist as anionic species in the matrix solution. It has been proposed that the charged surfactant concentrates the oppositely charged analyte near the surface, which results in larger signals for the analyte. Cationic surfactants that contain a fixed positive charge and an additional basic site were prepared with different hydrophobic moieties and were evaluated for their effectiveness as FAB matrix additives. The compound N-octylnicotinium bromide (ONBr) is shown to improve greatly the analyte-related signals in negative ion fast-atom bombardment mass spectrometry for a variety of polyanionic analytes, relative to other surfactants (e.g., cetylpyridinium salts). This surfactant not only enhances detectability, but also simplifies the pseudomolecular ion region of the resulting spectra by reducing or eliminating metal cation adduct peaks. The simple mechanism of enhancement via surface activity is evaluated, and alternative mechanisms are considered. It is clearly shown that ONBr, as a FAB matrix additive, will allow mass spectrometry to be used for the analysis of anionic compounds that normally exhibit very low responses.

A new cause of progressive intrahepatic cholestasis: 3 β-Hydroxy-C 27-steroid dehydrogenase/isomerase deficiency

There have been a few reports of infants with severe neonatal cholestasis related to a defect in primary bile acid synthesis. To assess the importance of such deficiency among children with progressive intrahepatic cholestasis (Byler disease), screening for inborn errors in bile acid synthesis was performed by fast atom bombardment ionization-mass spectrometry of urine samples from 30 affected children. Bile acid analysis revealed a specific fast atom bombardment ionization-mass spectrometry profile for 3 beta-hydroxy-C27 steroid dehydrogenase/isomerase deficiency in five children who had jaundice, hepatosplenomegaly, and fatty stools beginning at ages ranging from 4 to 46 months. None of them had pruritus. Liver function tests showed persistently normal serum gamma-glutamyltransferase activity, low serum cholesterol and vitamin E levels, normal serum bile acid concentrations despite raised serum bilirubin levels, and decreased prothrombin time and clotting factor V. In four of the cases a similar disease was observed in siblings. Liver function returned to normal after oral ursodeoxycholic acid therapy. We conclude that 3 beta-hydroxy-C27-steroid dehydrogenase/isomerase deficiency should be considered when idiopathic cholestatic liver disease with clinical features akin to Byler disease is characterized by the association of normal serum gamma-glutamyltransferase activity, normal serum bile acid concentration, absence of pruritus, and a return to normal liver function during ursodeoxycholic acid therapy. Early identification of these children is essential because they benefit from bile acid therapy and might thus avoid the need for liver transplantation.

Metabolism and effect of 7-oxo-lithocholic acid 3-sulfate on bile flow and biliary lipid secretion in rats

Significant amounts of oxo and sulfated bile acids are excreted in the urine of patients with cholestatic liver diseases, but these bile acid species are found only in traces in bile. Several possible explanations for this finding include poor canalicular secretion, rapid biotransformation by oxidoreduction and extrahepatic sulfation of oxo bile acids. To assess the physiological actions and hepatic metabolism of oxo and sulfated bile acids, we synthesized 7-oxo-lithocholic acid 3-sulfate, examined the effect of intravenous infusion of this bile acid (5.0 μmol/min/kg body wt) on bile flow and biliary lipid secretion in Sprague-Dawley rats with a bile fistula and compared this effect with that of the unconjugated analog, 7-oxo-lithocholic acid, and the primary bile acid conjugate, chenodeoxycholic acid 3-sulfate. Bile flow and bile acid secretion increased immediately after infusion of all three bile acids. The highly polar 7-oxo-lithocholic acid 3-sulfate significantly reduced the biliary secretion of phospholipids (p < 0.05) and cholesterol (p < 0.05), but by contrast the more hydrophobic analog, 7-oxo-lithocholic acid, caused a significant increase in the biliary phospholipids (p < 0.01) and cholesterol (p < 0.01) secretion. Infusion of chenodeoxycholic acid 3-sulfate also resulted in an increase in secretion of biliary cholesterol and phospholipids, but the magnitude of the change was less than that induced by 7-oxo-lithocholic acid infusion. Biliary lipid secretion was inversely related to the polarity of the infused bile acid. Negative ion fast atom bombardment—mass spectrometry and gas chromatography—mass spectrometry analyses indicated that the major bile acids excreted in the bile after infusion of 7-oxo-lithocholic acid were taurine and glycine conjugates of the parent compound and the corresponding conjugates of its metabolite, ursodeoxycholate, whereas chenodeoxycholate and its conjugates were minor metabolites. By contrast, 7-oxo-lithocholic acid 3-sulfate and chenodeoxycholic acid 3-sulfate were only partially amidated with taurine and glycine and did not undergo further metabolic transformation of the steroid nucleus. These data demonstrate that sulfation of 7-oxo-lithocholic acid significantly alters its physiological properties and hinders biotransformation, including oxidoreduction, hydroxylation and amidation. The findings that the sulfate conjugates of 7-oxo-lithocholic acid and chenodeoxycholic acid are efficiently and preferentially secreted into bile, and not into urine, would strongly indicate that the bile acid sulfates, found in relatively high proportions in the urine of patients with cholestasis, are of extrahepatic and most probably of renal origin.

Detection of ubiquinone in parasitic and free-living protozoa, including species devoid of mitochondria

Ubiquinone (coenzyme Q, CoQ) was analyzed and individual homologues quantified in 11 species of parasitic and freeliving protozoa by a combination of thin-layer chromatography and high performance liquid chromatography. Fast atom bombardment ionization-mass spectrometry was used for the first time to confirm the identity of the fractionated CoQ homologues and proved to be a fast, gentle and convenient method for ubiquinone identification. Ubiquinone was detected in all organisms including those devoid of identifiable mitochondria. However, significantly lower levels of CoQ were present in those organisms lacking this respiratory organelle (5- to 50-fold lower in Entamoeba histolytica (CoQ 9) and 15- to 350-fold for Giardia lamblia (CoQ 9) and Tritrichomonas foetus (CoQ 10). Coenzyme Q 9 was the predominant homologue in promastigotes of Leishmania donovani and Leishmania major. Lower amounts of CoQ 8 and CoQ 10 were also detected in L. donovani and CoQ 8 in L. major. Comparison of the in vitro cultivated promastigote and amastigote forms of Leishmania pifanoi and Leishmania amazonensis revealed CoQ 9 to be the sole detectable ubiquinone homologue in the amastigote (macrophage) stage, whereas CoQ 8 and CoQ 10 were also present in the promastigotes (life cycle stage found in the insect gut) of L. pifanoi, and CoQ 7 and CoQ 8 in promastigotes of L. amazonensis. Interestingly, the total amounts of CoQ were similar in both forms of these organisms. The free-living ciliates, Tetrahymena thermophia and Paramecium tetraurelia contained CoQ 8 as the predominant ubiquinone species and this homologue was also present in the isolated cilia from P. tetraurelia. The marine ciliate, Parauronema acutum contained CoQ 7 as well as CoQ 8. Comparison of xenosome-containing P. acutum with organisms lacking the symbiont revealed that twice the level of CoQ 8 was present in cells grown with this cytoplasmic gram-negative bacterium. Results suggest that CoQ is ubiquitous amongst the protozoa, regardless of the presence of mitochondria, and may function in alternative roles to that of mitochondrial electron transport chain component.