Comparison of N-Glycopeptide to Released N-Glycan Abundances and the Influence of Glycopeptide Mass and Charge States on N-Linked Glycosylation of IgG Antibodies.

We report the comparison of mass-spectral-based abundances of tryptic glycopeptides to fluorescence abundances of released labeled glycans and the effects of mass and charge state and in-source fragmentation on glycopeptide abundances. The primary glycoforms derived from Rituximab, NISTmAb, Evolocumab, and Infliximab were high-mannose and biantennary complex galactosylated and fucosylated N-glycans. Except for Evolocumab, in-source ions derived from the loss of HexNAc or HexNAc-Hex sugars are prominent for other therapeutic IgGs. After excluding in-source fragmentation of glycopeptide ions from the results, a linear correlation was observed between fluorescently labeled N-glycan and glycopeptide abundances over a dynamic range of 500. Different charge states of human IgG-derived glycopeptides containing a wider variety of abundant attached glycans were also investigated to examine the effects of the charge state on ion abundances. These revealed a linear dependence of glycopeptide abundance on the mass of the glycan with higher charge states favoring higher-mass glycans. Findings indicate that the mass spectrometry-based bottom-up approach can provide results as accurate as those of glycan release studies while revealing the origin of each attached glycan. These site-specific relative abundances are conveniently displayed and compared using previously described glycopeptide abundance distribution spectra "GADS" representations. Mass spectrometry data are available from the MAssIVE repository (MSV000093562).

Mass Spectral Library of Acylcarnitines Derived from Human Urine.

We describe the creation of a mass spectral library of acylcarnitines and conjugated acylcarnitines from the LC-MS/MS analysis of six NIST urine reference materials. To recognize acylcarnitines, we conducted in-depth analyses of fragmentation patterns of acylcarnitines and developed a set of rules, derived from spectra in the NIST17 Tandem MS Library and those identified in urine, using the newly developed hybrid search method. Acylcarnitine tandem spectra were annotated with fragments from carnitine and acyl moieties as well as neutral loss peaks from precursors. Consensus spectra were derived from spectra having similar retention time, fragmentation pattern, and the same precursor m/z and collision energy. The library contains 157 different precursor masses, 586 unique acylcarnitines, and 4 332 acylcarnitine consensus spectra. Furthermore, from spectra that partially satisfied the fragmentation rules of acylcarnitines, we identified 125 conjugated acylcarnitines represented by 987 consensus spectra, which appear to originate from Phase II biotransformation reactions. To our knowledge, this is the first report of conjugated acylcarnitines. The mass spectra provided by this work may be useful for clinical screening of acylcarnitines as well as for studying relationships among fragmentation patterns, collision energies, structures, and retention times of acylcarnitines. Further, these methods are extensible to other classes of metabolites.

Hybrid Search: A Method for Identifying Metabolites Absent from Tandem Mass Spectrometry Libraries.

Metabolomics has a critical need for better tools for mass spectral identification. Common metabolites may be identified by searching libraries of tandem mass spectra, which offers important advantages over other approaches to identification. But tandem libraries are not nearly complete enough to represent the full molecular diversity present in complex biological samples. We present a novel hybrid search method that can help identify metabolites not in the library by similarity to compounds that are. We call it "hybrid" searching because it combines conventional, direct peak matching with the logical equivalent of neutral-loss matching. A successful hybrid search requires the library to contain "cognates" of the unknown: similar compounds with a structural difference confined to a single region of the molecule, that does not substantially alter its fragmentation behavior. We demonstrate that the hybrid search is highly likely to find similar compounds under such circumstances.

Probing the conformational behavior of a monoclonal antibody with surfactant affinity capillary electrophoresis (SurfACE).

Multiple peaks are observed for a monoclonal antibody (mAb) when analyzed by "protein MEKC" (micellar electrokinetic capillary chromatography) using SDS-containing run buffers. We present our efforts to understand the mechanism of peak formation and the factors that affect the distribution of the mAb between these peaks. We used "intrinsic" charge ladders of the mAb to determine that peak-to-peak differences in the amount of bound surfactant are comparable to the aggregation numbers of protein-bound micelles. This suggests that the peaks represent sequential unfolding intermediates formed after collisions with micelles. Since this mechanism differs from that of small-molecule MEKC, we prefer to view this technique as a variant of affinity capillary electrophoresis and call it "SurfACE." We also find that the peak distribution is highly sensitive to pH. Lower pH favors the formation of more highly bound complexes, probably through an electrostatic effect on the kinetics. If the run buffer pH is high enough, the peak distribution appears to be set during the post-injection mixing process, as the mAb encounters surfactant during its transition from the lower-pH sample environment. Analysts who wish to interpret "protein MEKC" electropherograms should take note of these effects.

Breast-conservation treatment outcomes: a community hospital's experience.

In the United States, the majority of early breast cancer patients choose breast-conserving treatment in the community setting, yet there is a paucity of literature describing outcomes. In this paper, we describe our experience with breast-conserving treatment in a small community hospital. Our hospital tumor registry was used to identify breast cancer cases diagnosed at our hospital between 1997 and 2003. We limited our study to those women with initial attempts at breast-conserving surgery (BCS) who had follow-up oncology treatment at on-campus affiliated oncological services. We looked at factors that influence survival for early stage 0-II disease such as tumor and patient characteristics, completeness of local surgical tumor excision, and adjuvant treatment. We also evaluated the percentage of cases in which the initial BCS did not achieve adequate surgical oncological results and the number and type of subsequent surgeries that were required to achieve this goal. There were 185 cases with a median patient age of 55 and a median follow-up time of 53 months. Most tumors were stage 0-I (68%) or stage II (23%). A single surgery was deemed sufficient to achieve the desired oncological outcome in 54% of cases; the remaining cases (46%) required additional surgeries. A final margin of 5 mm or greater was successfully achieved in 81% of cases. Ninety-two percent of the patients underwent radiotherapy, 65% received hormonal therapy, and 49% underwent chemotherapy. One hundred and sixty one patients had successful breast-conserving surgeries (87%) and 24 patients (13%) ultimately required mastectomy. There were four loco-regional recurrences and 19 deaths during the study period. Our disease-free survival rate for early-stage cancer (stage 0-II) was 91% at 5 years. Our study shows that high-quality patient outcomes for breast-conserving treatment can be achieved in the community setting.

Hypertension and hepatic steatosis.

Hepatic steatosis (fatty liver) is increasingly recognized as a major component of the metabolic (insulin resistance) syndrome. It can progress to cirrhosis and hepatocellular carcinoma, leading to liver-related mortality. Increasing evidence shows a significant association between hepatic steatosis and hypertension; both are linked to the metabolic syndrome. This review discusses the evidence to support this association, and reviews the diagnosis and management of hepatic steatosis.

Outcome of surgical fundoplication for extraesophageal (atypical) manifestations of gastroesophageal reflux disease in adults: a systematic review.

BACKGROUND: A significant proportion of patients with gastroesophageal reflux disease (GERD) present with atypical symptoms (extraesophageal reflux; EER). The effectiveness of surgical fundoplication in treating classical reflux symptoms is well documented, but the role of surgery in alleviating EER symptoms is less clear. The aim of this study was to review the published literature to determine whether surgical fundoplication is effective in controlling EER. MATERIALS AND METHODS: A Medline, PubMed, and Cochrane database search was done to find articles on surgery for extraesophageal reflux (1991-2006). Articles on pediatric patients were excluded. The parameters looked at were patient selection, resolution of symptoms, change in the quality of life, and any adverse outcomes. RESULTS: In 25 studies, a variable proportion (15-95%) of patients with various symptoms of EOR improved after surgical fundoplication. The percentage of patients with EER responding to surgery was less than that reported for classical GERD. CONCLUSIONS: The majority of patients in most studies seem to improve symptomatically after surgery. However, a small percentage remains unchanged or worsens. The reported studies are so disparate in their methodology that firm conclusions on the role of surgery are difficult. Further studies are needed. These should be large, multicenter, prospective trials comparing medical and surgical treatment with standardized diagnostic criteria for EER. Pre- and post-treatment assessment, the type of surgery performed, and follow-up should be standardized.

Barrett's oesophagus: an audit of surveillance over a 17-year period.

OBJECTIVE: To audit whether our patients with Barrett's oesophagus (BO) enter into our endoscopic surveillance programme and whether they continue with it after entry. We have determined the incidence of oesophageal adenocarcinoma among our surveyed patients. DESIGN: We retrospectively audited prospectively collected data from our BO surveillance programme over the years 1987-2003. SETTING: An inner city teaching hospital. RESULTS: During these years, 466 patients with BO were diagnosed (392 long segment, >or=3 cm), 29 had oesophageal adenocarcinoma at diagnosis, 232 [195 with intestinal metaplasia (IM) on biopsy] had at least one follow-up endoscopy, and 205 have not been re-endoscoped. In 27 out of 205 no IM was present. Of the remaining 178 out of 205 with IM, 30 were within 2 years of diagnosis and 148 have not been re-endoscoped for the following reasons: age (51), non-attendance (35), not referred back by general practitioner (30), non-oesophageal cancer (14), severe concurrent illness (12), death (three), refused follow-up (two), left the area (one). The 195 patients with IM who entered endoscopic surveillance consisted of 108 men and 87 women (aged 62.9 years, range 31-96), were followed for a total of 1068 patient-years (average 5.5 years), and had 556 endoscopies (average 2.9 per patient). Ninety-seven out of 195 patients remain under active endoscopic surveillance but 98 discontinued for the following reasons: age (31), non attendance (21), death (21 including one from oesophageal adenocarcinoma), refused follow up (seven), concurrent illness (six), left the area (four), no IM on repeat biopsies (three). Of the 195 patients with IM, four developed low-grade dysplasia, two high-grade dysplasia and four adenocarcinoma (incidence 0.37%); 178 out of 195 have been maintained on proton pump inhibitor (PPI) therapy. CONCLUSIONS: The majority of patients with BO either do not enter or do not continue in an endoscopic surveillance programme. This needs to be acknowledged when the workload and cost of BO surveillance programmes are considered. The incidence of adenocarcinoma was low compared with many published series, and we speculate whether this is the result of maintenance PPI therapy.

Factors relating to compliance with a gluten-free diet in patients with coeliac disease: comparison of white Caucasian and South Asian patients.

BACKGROUND & AIMS: To identify factors relating to compliance with a gluten-free diet amongst white Caucasian and South Asians with coeliac disease. METHODS: Cross-sectional survey, with case note review of 130 adult patients with coeliac disease (90 white Caucasian and 40 South Asians). RESULTS: 87 (66.9%) of the 130 questionnaires were returned; whites: 73.3%, South Asians: 52.5% (P = 0.02). White Caucasians' assessment of their own strictness to the gluten-free diet correlated with small bowel histological recovery (OR 10.00, 95% CI 3.2-33.06) and negative endomysial antibodies (OR 34.94, CI 6.58-185.40). This was not seen in the South Asian patients. Amongst the white coeliacs, factors correlating with compliance with a gluten-free diet were: Coeliac Society membership, understanding food labelling, obtaining sufficient gluten-free products, explanation by a physician, and regular dietetic follow-up. These factors were not identified amongst the South Asians, who were less likely to attend dietetic clinics, join the Coeliac Society and be satisfied with information provided by doctors and dieticians. CONCLUSIONS: In contrast to the South Asians, factors were identified which related to compliance with a gluten-free diet amongst white Caucasian coeliac patients. This study has shown that the treatment approach to ethnic minorities with coeliac disease must be improved.

Disproportionation Kinetics of Hypoiodous Acid As Catalyzed and Suppressed by Acetic Acid-Acetate Buffer.

The kinetics of the disproportionation of hypoiodous acid to give iodine and iodate ion (5HOI right harpoon over left harpoon 2I(2) + IO(3)(-) + H(+) + 2H(2)O) are investigated in aqueous acetic acid-sodium acetate buffer. The rate of iodine formation is followed photometrically at -log [H(+)] = 3.50, 4.00, 4.50, and 5.00, &mgr; = 0.50 M (NaClO(4)), and 25.0 degrees C. Both catalytic and inhibitory buffer effects are observed. The first process is proposed to be a disproportionation of iodine(I) to give HOIO and I(-); the iodide then reacts with HOI to give I(2). The reactive species (acetato-O)iodine(I), CH(3)CO(2)I, is postulated to increase the rate by assisting in the formation of I(2)O, a steady-state species that hydrolyzes to give HOIO and I(2). Inhibition is postulated to result from the formation of the stable ion bis(acetato-O)iodate(I), (CH(3)CO(2))(2)I(-), as buffer concentration is increased. This species is observed spectrophotometrically with a UV absorption shoulder (lambda = 266 nm; epsilon = 530 M(-)(1) cm(-)(1)). The second process is proposed to be a disproportionation of HOIO to give IO(3)(-) and I(2). Above 1 M total buffer, the reaction becomes reversible with less than 90% I(2) formation. Rate and equilibrium constants are resolved and reported for the proposed mechanism.

Differential ferritin expression is associated with iron deficiency in coeliac disease.

BACKGROUND: Sixty percent of people with coeliac disease (CD) are iron deficient. Many, however, remain iron replete despite the disease. AIMS: (i) To characterize the changes in duodenal iron transport proteins in CD with and without iron deficiency. (ii) To examine if iron-activated gut lymphocytes can inhibit iron export in an enterocyte cell model. METHODS: Endoscopic duodenal biopsies were collected from patients with normal duodenum with and without iron deficiency anaemia and untreated CD sufferers with iron deficiency (n=10 each group). mRNA expression of iron transport proteins was determined by quantitative real time PCR. Protein localization and expression was determined from histology sections in patients with normal duodenum (n=20), and patients with untreated CD with and without iron deficiency (n=20). In addition, CaCo2 cells were cocultured with iron-activated lymphocytes 55Fe was used to determine the effect on CaCo2 cell iron transport. RESULTS: The expression of divalent metal transporter 1 and ferroportin was increased in CD with or without iron deficiency. Ferritin expression was increased in CD but only in those with associated iron deficiency. TNF-alpha produced by activated lymphocytes inhibited iron export from CaCo2 cells. CONCLUSION: Increased enterocyte ferritin expression may promote iron deficiency in CD and this effect seems to be dependent upon TNF-alpha expression in gut lymphocytes.

Endoscopic surveillance for Barrett's oesophagus: the patients' perspective.

OBJECTIVE: Barrett's oesophagus (BO) is associated with the development of oesophageal adenocarcinoma and endoscopic surveillance is commonly practised. In view of the clinical, psychological and economic implications of BO, we have studied patients' attitudes towards surveillance, their levels of anxiety and quality of life. METHODS: Patients with BO undergoing endoscopic surveillance were asked to complete a questionnaire,including seven questions on their experience of surveillance, the Hospital Anxiety and Depression Scale,the Trust in Physician Scale (TIPS) and the Short Form-36 quality of life questionnaire. RESULTS: Of the 250 patients invited, 178 responded (71%).One hundred and fifty-one (60%) completed the questionnaire sufficiently for analysis [median age 66 (range 41-79) years, 101 male]. Twenty-nine percent of patients had received too little information concerning surveillance of BO and 22% no information at all. Information was deemed difficult to understand by 24%. Hospital Anxiety and Depression Scale anxiety score: 14%abnormal, 25% borderline. Quality of life was lower than the general population in seven of eight Short Form-36 parameters. Increasing TIPS score correlated with having received (r= 0.33, P <0.001) and understood (r = 0.2, P= 0.037) BO information, and negatively with the belief that endoscopic surveillance reduced oesophageal adenocarcinoma risk (r= - 0.19, P =0.025). Increasing TIPS score was associated with less anxiety (P= 0.015) and depression (P =0.001), and better quality of life. CONCLUSION: Patients undergoing endoscopic surveillance for BO suffer anxiety and have impaired quality of life. As trust in their physicians is correlated with receiving sufficient BO information, we surmise that ensuring BO patients understand their condition is likely to improve quality of life, anxiety and depression.

Intrinsic charge ladders of a monoclonal antibody in hydroxypropylcellulose-coated capillaries.

Capillary zone electrophoresis (CZE) has been used to resolve the charge heterogeneity of an intact ( approximately 150 kDa) monoclonal IgG antibody (mAb). Although this microheterogeneity can also be observed by isoelectric focusing, CZE allows the net charge of each variant to be measured as a function of pH and other solution conditions. Separation was achieved in both borate and Tris run buffers using capillaries that had been statically coated with hydroxypropylcellulose (HPC). The HPC coating makes inadvertent chromatographic retention of the mAb undetectably small and decreases electroosmotic flow (EOF) to approximately 10(-5) cm(2) V(-1) s(-1), with reasonable stability over dozens of runs under the conditions tested (pH 8.5 and 9.0 for each buffer). We also describe a novel means of measuring small, positive EOF coefficients and larger, negative net mobilities in the same run. This allows determination of accurate electrophoretic mobilities despite variations in EOF. The resolved mAb charge variants (which most likely result from deamidation or partial truncation) constitute what we call an "intrinsic" charge ladder. As with conventional charge ladders formed by deliberate modification of a homogeneous protein, net charge is obtained by extrapolating a plot of electrophoretic mobility versus (assumed) incremental charge difference. At a given pH, the mAb is more negatively charged in borate than in Tris, reflecting specific binding of the B(OH)(4)(-) anion. We also report hydrodynamic radii calculated from the slopes of these plots.

Ethnicity, gender, and socioeconomic status as risk factors for esophagitis and Barrett's esophagus.

Barrett's esophagus is thought to be a disease occurring predominantly in White Caucasian males of higher socioeconomic status. There are no published studies simultaneously examining risk of Barrett's esophagus according to ethnicity, gender, and socioeconomic status within a single data set. The authors conducted a retrospective case-control analysis within a cross-sectional study to determine risk of Barrett's esophagus in relation to sociodemographic variables in a large United Kingdom population. All patients undergoing upper gastrointestinal endoscopy at two clinical centers between January 2000 and January 2003 were evaluated. Data on ethnicity, age, gender, socioeconomic status, and the presence of Barrett's esophagus and esophagitis at endoscopy were collected. A total of 20,310 patients were analyzed. Barrett's esophagus was more common in White Caucasians (401/14,095 (2.8%)) than in South Asians (16/5,190 (0.3%)) (adjusted odds ratio (OR)=6.03, 95% confidence interval (CI): 3.56, 10.22), as was esophagitis (2,500/14,095 (17.7%) vs. 557/5,190 (10.7%); adjusted OR=1.76, 95% CI: 1.57, 1.97). Patients with Barrett's esophagus were also more likely to be male (adjusted OR=2.70, 95% CI: 2.18, 3.35) and of higher socioeconomic status (adjusted OR=1.58, 95% CI: 1.16, 2.15 (top tertile vs. bottom tertile)). White Caucasian ethnicity, male gender, and higher socioeconomic status are independent risk factors for Barrett's esophagus.

The emerging role of the liver in iron metabolism.

Iron is essential in health and well-being and its dysregulation is a common theme in disease. Recent advances in our understanding of the molecular biology underlying hemochromatosis and anemia has provided insight into the complex mechanisms implicated in iron metabolism. The proximal small bowel is the major site of iron absorption and, it is becoming increasingly clear that the regulation of this process involves the liver and, in particular, the hepatic antimicrobial peptide hepcidin. A number of studies have shown hepcidin to have an inhibitory function at the level of small bowel iron absorption, although its exact site of action remains to be elucidated. Clearly, identifying the target of hepcidin is of importance and is likely to lead to the development of therapeutic agents in the treatment of iron disorders.

The role of hemochromatosis susceptibility gene mutations in protecting against iron deficiency in celiac disease.

BACKGROUND & AIMS: Celiac disease and hereditary hemochromatosis are common HLA-defined conditions in northwestern Europe. We sought to determine whether there is a genetic relationship between the 2 diseases and if hemochromatosis susceptibility gene (HFE) mutations are protective against iron deficiency in celiac disease. METHODS: Polymerase chain reaction amplification using sequence-specific primers capable of identifying the 2 HFE gene mutations (H63D and C282Y) and the HLA class I and II alleles was used to type 145 white patients with celiac disease and 187 matched controls. Hemoglobin and fasting serum iron levels in celiac patients were measured at diagnosis. RESULTS: HFE gene mutations, H63D or C282Y, were identified in 70 celiac patients (48.3%) and 61 controls (32.6%) (P = 0.004). The C282Y mutation was associated with HLA-A*03 and B*07 alleles in controls and with A*01, A*03, B*08, and DRB1*0301 alleles in celiac patients; the H63D mutation was associated with HLA-A*25 and DRB1*03 alleles in controls and A*29 and DRB1*03 alleles in celiac patients. At diagnosis, celiac patients with the C282Y mutation had higher mean hemoglobin and fasting serum iron levels compared with the HFE wild type (P = 0.0002 and 0.006, respectively). This was not observed with the H63D mutation. CONCLUSIONS: In celiac disease, HFE gene mutations are common and are in linkage disequilibrium with different HLA alleles compared with controls. A disease-specific haplotype that carries C282Y and DQB1*02 is suggested. We propose that HFE gene mutations provide a survival advantage by ameliorating the iron deficiency seen in celiac patients.