Fast atom bombardment mass spectrometry of carbohydrates contaminated with inorganic salts using a crown ether.
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Carbohydrates↗
Biomedical subjects
R Orlando
Publications and source records attributed to R Orlando.
Is canine hepatocerebellar degeneration syndrome an animal model for carbohydrate-deficient glycoprotein syndrome in humans? An example of sequencing glycoprotein glycans with mass spectrometry.
The clinical symptoms and morphologic features of canine hepatocerebellar degeneration syndrome (CHD) bear striking resemblance to those of human carbohydrate-deficient glycoprotein syndrome (CDGS). The characteristic biochemical and molecular features of human CDGS lie in the truncated carbohydrate side chains of serum transferrin and numerous other glycoproteins of affected persons. Therefore, to explore the biochemical similarities between CHD and CDGS, we compared the structures of the carbohydrate side chains of canine serum transferrin isolated from a normal and a CHD-affected dog. Because of the very small quantity of serum transferrin available from the CHD-affected dog, we used analytical procedures that minimize sample consumption. In this scheme, we used microbore liquid chromatography interfaced to electrospray tandem mass spectrometry to identify and purify the glycopeptides from the tryptic digest of canine serum transferrin. This was followed by a series of exoglycosidase digestions coupled with mass spectrometric detection to sequence the carbohydrate side chains of the glycopeptides. With these procedures we completely characterized the carbohydrate chains attached to serum transferrin isolated from both a normal and a CHD-affected dog. However, we found no discernible differences in glycosylation of the carbohydrate side chains of serum transferrin from these two animals, suggesting that the biochemical defect in puppies with CHD differs from that in children with CDGS.
Alkylation↗
Laparoscopy in chronic renal failure.
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Humans↗
A desalting approach for MALDI-MS using on-probe hydrophobic self-assembled monolayers.
One of the problems encountered in preparing samples for matrix-assisted laser desorption/ionization (MALDI) analysis is the presence of nonvolatile salts in the sample. This difficulty is often exacerbated by the necessity to prepare the sample in the appropriate sample-to-matrix ratio. This paper reports a probe surface derivatization method that greatly simplifies this sample preparation process. By constructing self-assembled monolayers of octadecyl mercaptan (C18) on the MALDI probe surface, we were able to generate a surface capable of reversibly binding polypeptides via hydrophobic interactions, which in turn, permits the analyte to be easily concentrated and desalted directly on the probe tip.
Chelating Agents↗
Risk of breast cancer in carriers of BRCA gene mutations.
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BRCA2 Protein↗
Sexual transmission of the hepatitis C virus and efficacy of prophylaxis with intramuscular immune serum globulin. A randomized controlled trial.
OBJECTIVE: To estimate the risk of sexual transmission of hepatitis C and to assess the value of prophylaxis with periodic intramuscular immune serum globulin administration. METHODS: Of 1102 steady heterosexual partners of patients with antibodies to the hepatitis C virus (HCV), 899 were enrolled in a single-blind, randomized, controlled trial. All the partners tested negative for antibodies to HCV and had normal baseline serum aminotransferase concentrations. The partners were assigned to receive 4 mL of 16% polyvalent immune serum globulin prepared from unscreened donors every 2 months (n = 450) or a placebo (n = 449). Tests for HCV infection were performed every 4 months. RESULTS: Eight hundred eighty-four partners completed the study. Seven partners became infected with HCV: 6 in the control group (incidence density, 12.00 per 1000 person-years; 95% confidence interval, 3.0 21.61) and 1 in the immune serum globulin group (incidence density, 1.98 per 1000 person-years; 95% confidence interval, 0-5.86). The risk of infection was significantly higher for partners in the control group (P = .03): for each year approximately 1% of the partners became infected. Sequence homology studies strongly suggest the sexual transmission of HCV. All immune serum globulin lots used had high enzyme-linked immunosorbent assay titers of neutralizing antibodies to HCV envelope glycoproteins and high neutralization titers in the neutralization of binding assay. CONCLUSIONS: Hepatitis C can be sexually transmitted. Immune serum globulin prepared from unscreened donors significantly reduced the risk. The treatment was safe and well tolerated. Because only immune serum globulin from unscreened donors (and not from those screened for HCV) contain anti-HCV neutralizing antibodies, hyperimmune anti-HCV immune serum globulin should be prepared from blood testing positive for antibodies to HCV, which is currently discarded.
Adult↗
A new strategy for MALDI on magnetic sector mass spectrometers with point detectors.
We have developed a new strategy to perform matrix-assisted laser desorption/ionization (MALDI) on magnetic sector instruments equipped with nonintegrating point detectors. This approach is based on using liquid matrices to provide very long-lasting analyte ion currents and to overcome the inability of these instruments to simultaneously detect ions over a range of mass-to-charge values. Here, we demonstrate some of the capabilities of MALDI on a magnetic sector for analyzing biological samples. These capabilities include the ability to perform MS/MS experiments, to attain resolutions over 8000 (full width at half-maximum), and to determine relative molecular masses with < 20 ppm error for a single measurement and < 2 ppm error for the average of five measurements. All three of these experiments can be performed on the same 10 pmol sample loading. We also compare the results of MALDI on a magnetic sector instrument with point detection with the reported capabilities of MALDI on time-of-flight-based mass analyzers and with fast atom bombardment on sector-based instruments. This work demonstrates that researchers with magnetic sector-based instruments can perform MALDI experiments with a relatively simple and inexpensive upgrade to their existing equipment.
Angiotensinogen↗
Identification of the glycosylation site and glycan structures of recombinant endopolygalacturonase II by mass spectrometry.
A series of mass spectrometric experiments was performed to characterize the carbohydrate chains attached to endopolygalacturonase II (EPG-II) overexpressed in Aspergillus niger. First, an aliquot of trypsin-digested EPG-II was analyzed by capillary high-performance liquid chromatography (HPLC) coupled with electrospray ionization (ESI) mass spectrometry (HPLC/MS). The ESI mass spectrometer was operated in the tandem mass spectrometric (MS/MS) mode and utilized precursor ion scanning of the HexNAc+ oxonium ion at m/z 204 to selectively detect glycopeptides eluting from the HPLC. Aliquots of the fraction identified by HPLC/MS/MS to contain a glycopeptide were then sequentially digested with Aspergillus saitoi alpha-1-2-mannosidase and peptide N:glycosidase F (N-glycanase), followed by matrix-assisted laser-desorption/ionization mass spectrometric analysis to provide structural information from the carbohydrate chain. The masses of the carbohydrate chains in the glycopeptides were thus determined and subsequently used to search the Complex Carbohydrate Structure Database for the probable structures of the glycans. This analysis determined that recombinant EPG-II has a single N-glycosylation site, the carbohydrate chain at this site is heterogeneous, and the glycan structure is of the high-mannose type. No sites of O-glycosylation were detected in EPG-II.
Amino Acid Sequence↗
Laparoscopy in the critically ill.
BACKGROUND: Laparoscopy was evaluated in critically ill patients with suspected acute cholecystitis, mesenteric ischemia, or gastrointestinal perforation. We studied laparoscopy to assess its utility, accuracy, and effect on cardiopulmonary stability. METHODS: Twenty-six surgical ICU patients with possible abdominal sepsis underwent laparoscopy. Nineteen were post cardiac surgery; the remainder had other diagnoses. Video laparoscopy was performed with hemodynamic monitoring and inotropic support as needed. Eight patients had bedside laparoscopy. RESULTS: Fifteen patients had suspected acute cholecystitis. Laparoscopy was positive in 10; four had open cholecystectomy, four laparoscopic cholecystectomy, and two tube cholecystostomy. Nine patients had suspected mesenteric ischemia; laparoscopy was positive in five, revealing cirrhosis in two and ischemic bowel in three. Two patients had suspected perforated viscus with colonic perforation in one and one false negative. There were no adverse hemodynamic events. CONCLUSIONS: Laparoscopy can be performed safely in critically ill patients. It is useful in patients with acute cholecystitis and in patients who are post cardiac surgery with refractory lactic acidosis in whom a diagnosis of mesenteric ischemia is considered.
Acute Disease↗
Prevalence of hepatitis C virus genotypes in southern Italy.
HCV is ubiquitous. In 50% of all cases it causes chronic hepatitis that often evolves into liver cirrhosis and hepatocellular carcinoma. Recently HCV has been classified in 5 genotypes by Okamoto. The purpose of this study is to evaluate the prevalence of 5 genotypes in Campania, a region of southern Italy, where the prevalence of anti-HCV antibodies ranges from 0.87 to 4%, and to evaluate the correlation between the HCV genotypes and the severity of histological damage. One-hundred- and-thirty-five anti-HCV positive patients were enrolled and tested by PCR to identify HCV-RNA. One-hundred-and-twenty-four patients resulted HCV-RNA positive. Genotyping was performed as described by Okamoto et al. with minor modifications of the specific primer to type III proposed by Silini et al. Eight patients were negative for all genotypes. Eight patients were positive for type I(1a), 61 for type II(1b), 39 for type III(2a), 11 for type IV(2b) and 1 for type V(3a). In 4 cases two different genotypes were present in the same sample [II(1b)-IV(2b), III(2a)-II(1b) twice, III (2a)-IV(2b)]. Histological evaluation of liver damage showed: CPH (22 cases), minimal CAH (56), severe CAH (31) and liver cirrhosis (15). There was no statistically significant correlation between the 5 genotypes and the severity of histological damage. Data on the prevalence of genotype II (1b) in Italy are similar to those reported for other European countries. The prevalence of genotypes in southern Italy is similar to that reported in the population of northern Italy.
Adult↗
The effect of age on plasma MEGX concentrations.
AIMS: The study was designed to assess the effect age on the monoethylglycinexylidide (MEGX) liver function test. METHODS: Plasma MEGX concentration was measured in 16 young and 17 elderly healthy volunteers 10, 15, 30, 45 and 60 min after intravenous injection of 1 mg kg(-1) lignocaine. RESULTS: Lower MEGX concentrations were noted in elderly subjects. The difference between young and old individuals became increasingly significant as the sampling time was increased, mainly because of a progressive decrease in intersubject variability within each of the two study groups. Linear regression analysis consistently showed that the strength of the relationships between MEGX concentration and age increased with increased sampling time and was maximal at 60 min (r = -0.70; P < 0.0001; reduction in MEGX formation (95% CI) 0.51 (0.33-0.69) ng ml(-1) per year). CONCLUSIONS: These data indicate that the rate of MEGX production declines significantly with age. Therefore, when the results of the MEGX test are to be compared in different study groups, subjects should be strictly matched for age.
Adolescent↗
Long-term interferon-alpha therapy does not affect sex hormones in males with chronic hepatitis C.
The effects of interferon-alpha (IFN-alpha), given at a dosage of 6 MU thrice weekly for 12 months, on gonadal function were investigated in 18 males affected by chronic hepatitis C. Periodically, all patients were clinically monitored and questioned about sexual function. Gonadotropin and serum androgen concentrations (follicle-stimulating hormone, luteinizing hormone, total testosterone, free testosterone, androstenedione, dehydroepiandrosterone, dehydroepiandrosterone sulfate, and sex hormone binding globulin) were tested every 3 months. Ten of 18 patients (55%) responded to IFN-alpha therapy. Serum total testosterone and sex hormone binding globulin values decreased slightly at the third month of treatment, then returned to baseline values. Serum free testosterone and other sex hormones remained essentially unchanged during IFN-alpha therapy. Four patients (22.2%) complained of sexual dysfunction (impaired libido, erectile failure, and impaired ejaculation), which was unrelated to any significant hormonal change and resolved after IFN therapy was stopped. Serum sex hormones values did not differ between responders and nonresponders to IFN-alpha. This study indicates that 12 months treatment with 6 MU of IFN-alpha thrice weekly does not significantly affect gonadal function in men with chronic hepatitis C. The sexual dysfunction observed could be ascribed to such other side effects of IFN as asthenia, fatigue, or anxiety, or it could have a psychologic basis.
Adolescent↗
The structures of arabinoxyloglucans produced by solanaceous plants.
Several structural features, most notably the presence of alpha-L-Araf-(1-->2)-alpha-D-Xylp side chains, distinguish the arabinoxyloglucans (AXGs) produced by solanaceous plants from the xyloglucans produced by other dicotyledonous plants. However, previous studies did not establish the exact order of attachment of the various side chains along the backbone of these AXGs. Therefore, oligosaccharide subunits of the AXGs secreted by suspension-cultured tobacco and tomato cells were generated by treatment of the isolated AXGs with a fungal endo-beta-(1-->4)-D-glucanase (EG). The oligosaccharides were reduced with sodium borohydride to the corresponding oligoglycosyl alditol derivatives and purified by a combination of gel-permeation chromatography, reversed-phase HPLC, and HPAE chromatography. The isolated oligoglycosyl alditols were chemically characterized by NMR spectroscopy, matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDITOFMS), fast-atom bombardment mass spectrometry (FABMS), FABMS/MS, and glycosyl-linkage analysis. The results confirmed that the AXGs from these species are composed of a (1-->4)-linked beta-D-Glcp backbone substituted at O-6 with various side chains. Both tobacco and tomato AXG contain alpha-D-Xylp and alpha-L-Araf-(1-->2)-alpha-D-Xylp side chains. However, oligosaccharide fragments of tomato AXG were also shown to contain beta-D-Galp-(1-->2)-alpha-D-Xylp and beta-Araf-(1-->3)-alpha-L-Araf-(1-->2)-alpha-D-Xylp side chains that are not present in the tobacco AXG. This is the first report of beta-Araf residues in a xyloglucan. The primary structures of 20 oligosaccharides generated by EG-treatment of tobacco AXG were determined. The generation of such a large number of oligosaccharides is due in part to the presence of O-acetyl substituents at O-6 of many of the backbone beta-D-Glcp residues of tobacco AXG. The presence of either an O-acetyl or a glycosidic substituent at O-6 of a beta-D-Glc p residue in the AXG backbone protects the glycosidic bond of this residue from cleavage by the EG. Removal of the O-acetyl substituents prior to EG-treatment of the AXG-results in oligosacharide fragments that are smaller than those produced by EG-treatment of the O-acetylated AXG. Therefore, analysis of the complex mixture of oligosaccharides obtained by EG treatment of native tobacco AXGs provides information regarding the distribution of AXG side chains that would be lost if the AXG is de-O-acetylated prior to EG-treatment. Furthermore, the large library of oligosaccharide fragments generated by this approach revealed additional correlations between the structural features of AXGs and diagnosis chemical shift effects in their 1H NMR spectra.
Arabinose↗
Simplifying the exoglycosidase digestion/MALDI-MS procedures for sequencing N-linked carbohydrate side chains.
Exoglycosidase digestion coupled with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is an effective technique for sequencing the N-linked carbohydrate side chains of a glycoprotein. However, the buffers currently used in the enzymatic procedures are detrimental to MALDI-MS, and thus desalting is required before the digestion products can be analyzed. We demonstrate that a 25 mM ammonium acetate solution adjusted to the proper pH can replace the normal exoglycosidase digestion buffers. The use of these ammonium acetate solutions permits direct MALDI-MS analysis of the digestion mixture without desalting. More importantly, we show that many of the commonly used exoglycosidases retain both their activity and their specificity under these conditions.
Carbohydrate Sequence↗
A new matrix for matrix-assisted laser desorption/ionization on magnetic sector instruments with point detectors.
A new matrix consisting of a binary mixture of alpha-cyano-4-hydroxycinnamic acid and 3-amino-quinoline has been developed specifically for matrix-assisted laser desorption/ionization (MALDI) analysis on magnetic sector mass spectrometers with point detectors. The major benefit of this matrix is that it is a viscous liquid with a self-healing surface. These properties provide very long-lasting and reasonably constant ion currents from pmol quantities of analyte, allowing MALDI-magnetic sector spectra to be obtained even though a non-integrating point detector is used. The applicability of this matrix for biological samples is demonstrated by analyzing a mixture of complex N-linked oligosaccharides with the new liquid matrix and with several traditional solid MALDI matrices both on magnetic sector and time-of-flight instruments.
Indicators and Reagents↗
Identifying the glycosylation sites and site-specific carbohydrate heterogeneity of glycoproteins by matrix-assisted laser desorption/ ionization mass spectrometry.
A sensitive and facile method is described to identify the glycosylation sites and site-specific heterogeneity in the carbohydrate attached to glycoproteins. In this procedure, the peptide backbone of the glycoprotein is cleaved enzymatically. The resulting peptide/glycopeptide mixture is divided into three fractions. The first is analyzed directly by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), while the other two aliquots are analyzed by MALDI-MS after enzymatic release of the N-linked chains and the N- and O-linked chains. Comparison of these MALDI mass spectra provides the molecular weight of each carbohydrate side chain and of the peptide to which it was attached. This information combined with the amino acid sequence of the protein identifies the glycosylation sites, and provides information concerning site-specific oligosaccharide heterogeneity. This approach does not require time-consuming liquid chromatographic separations and can be performed on as little as 10 pmol of glycoprotein. Thus, our approach is faster and simpler than procedures currently used for glycosylation site mapping, and may offer a slight sensitivity advantage.
Animals↗
New immobilization chemistry for probe affinity mass spectrometry.
Probe affinity mass spectrometry (PAMS) is a technique that combines affinity separations directly with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). In this approach, a binding molecule, such as an antibody, lectin or receptor, is covalently attached to the surface of a MALDI probe. This permits the analyte of interest to be selectively captured and concentrated on the probe surface prior to MALDI-MS analysis. A major limitation of our initial PAMS immobilization chemistry was that it produced only a relatively small number of binding sites on the probe, as it was based on forming a single monolayer of the binding molecule. Because of this limitation, we have investigated new immobilization chemistries for PAMS that are not confined by monolayer formation and thus allow a larger number of analyte molecules to be captured by the probe. We have developed a new PAMS chemistry that first attaches very high molecular weight (approximately 500,000) dextrans to the MALDI probe, followed by immobilization of the binding molecules to the probe-bound dextrans. Because the size of each dextran molecule is significantly larger than the binding molecule, multiple binding molecules can be linked to the same dextran chain. We have demonstrated that these surfaces possess approximately 500 times more analyte binding sites than probes prepared with our original PAMS chemistry. This chemistry is applicable to any binding molecule that contains a primary amine and is suitable, therefore, for a wide range of applications.
Chromatography, Affinity↗
Effect of moderate or severe liver dysfunction on the pharmacokinetics of gamma-hydroxybutyric acid.
OBJECTIVES: To assess the effect of moderate or severe liver dysfunction on the pharmacokinetics of gamma-hydroxybutyric acid (GHB). METHODS: The absorption and disposition kinetics of GHB were studied in eight cirrhotic patients without ascites (Child's class A) and eight cirrhotic patients with ascites (Child's class C), after administration of a single oral dose of 25 mg.kg-1. The liver metabolic function of each patient was evaluated by measuring antipyrine clearance and the formation rate of the lidocaine metabolite monoethylglycinexylidide (MEGX). RESULTS: Compared to those previously determined in eight healthy control subjects given the same GHB dose, mean AUC values were double or greater in the cirrhotic patients. Accordingly, apparent oral clearance was markedly reduced (from 9.1 to 4.5 and 4.1 ml.min-1.kg-1 in nonascitic and ascitic patients, respectively). Terminal half-life (t1/2), was significantly longer in nonascitic patients than in control subjects (32 vs 22 min). A further significant prolongation of t1/2, most likely due to an increased distribution volume, was observed in patients with ascites (56 min). Nonetheless, GHB plasma concentrations fell to either undetectable or negligible levels by the end of the usual dosing intervals (6-8 h). More limited changes were noted in the absorption parameters. The peak level (Cmax) increased only in nonascitic patients, but not proportionally to the increase in AUC. The time to Cmax increased from 30 to 45 min in both cirrhotic groups. These findings are consistent with a slowed rate of GHB absorption in cirrhotic patients. Adverse effects were similar, for intensity and duration, to those recorded in healthy volunteers, i.e., mild and transient. CONCLUSIONS: Although liver cirrhosis causes significant modifications of GHB disposition kinetics, the increase in t1/2 is not such as to cause drug accumulation on repetitive dosing. However, in consideration of the higher mean plasma levels observed in cirrhotic patients, it appears wise to keep the initial GHB daily dose at the lower end of the therapeutic range and to carefully monitor the patients if upward dose adjustments are required.
Aged↗