Food security, obesity, and meat-derived carcinogen exposure in US adults.

Risk for colorectal cancer (CRC) is increased in adults with poor diet quality, low socioeconomic status, and increased body mass index (BMI). Cooked meats contain high contents of mutagenic compounds related to CRC risk. To explore differences in meat-based carcinogen exposure, a 99-item Qualtrics survey was issued to 1648 US adults. Average monthly serving size, degree of doneness, and cooking methods of meat products were obtained. The National Cancer Institute CHARRED database was used to quantify exposure to HCAs, PAHs, and Ames Predicted and Estimated mutagenicity. Questions from validated instruments assessed food security status (FSS) and demographic variables, while height and weight were self-reported to calculate BMI. Sex, FSS and obesity status (BMI > 30 kg/m2) were compared using two-sample t-tests and multivariate regression models to determine differences in meat intake and carcinogen exposure. Statistical significance was set at P < 0.05. Eight hundred fifty-six valid responses (57.4% female and 81.8% white) were obtained. Non-obese males consumed more white meat and were exposed to greater amounts of 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline. Food insecure and obese females consumed more red meat and had higher exposure rates of several carcinogens. Pan-frying and BBQ were the primary sources of meat-derived carcinogens. Greater disparities in carcinogen exposure were observed in females regarding BMI and FSS. Public health messages to modify meat cooking methods may be most effective for CRC prevention.

Stability of the Stevia-Derived Sweetener Rebaudioside A in Solution as Affected by Ultraviolet Light Exposure.

Rebaudioside A is a natural noncaloric high-potency sweetener extracted from the leaves of Stevia rebaudiana. With rebaudioside A use increasing in foods, understanding the factors affecting its stability is necessary. This project evaluated the degradation rate constants of rebaudioside A in water, 0.1 M phosphate buffer, and 0.1 M citrate buffer at pH 3 and 7 as a function of ultraviolet (UV) light intensity (365 nm, 0 µW/cm2 for dark conditions, 27 µW/cm2 for low intensity, and 190 µW/cm2 for high intensity) at 32.5 °C. Rebaudioside A stability was adversely affected by light exposure. The pseudo-1st-order degradation rate constants increased significantly (P < 0.05) with increasing light intensity in all solutions. Under dark conditions, rebaudioside A in phosphate buffers was more susceptible to breakdown than in water and citrate buffers at both pH levels. However, exposure to UV light resulted in rebaudioside A degradation occurring approximately 10 times faster in citrate than in phosphate buffers at both pH levels. The sensitivity of rebaudioside A to UV light was greater in citrate buffers than in water or phosphate buffers. The use of light-protective packaging for beverages containing rebaudioside A will improve its stability.

Orthogonal Assessment of Biotherapeutic Glycosylation: A Case Study Correlating N-Glycan Core Afucosylation of Herceptin with Mechanism of Action.

In the development of therapeutic antibodies and biosimilars, an appropriate biopharmaceutical CMC control strategy that connects critical quality attributes with mechanism of action should enable product assessment at an early stage of development in order to mitigate risk. Here we demonstrate a new analytical workflow using trastuzumab which comprises "middle-up" analysis using a combination of IdeS and the endoglycosidases EndoS and EndoS2 to comprehensively map the glycan content. Enzymatic cleavage between the two N-acetyl glucosamine residues of the chitobiose core of N-glycans significantly simplifies the oligosaccharide component enabling facile distinction of GlcNAc from GlcNAc with core fucose. This approach facilitates quantitative determination of total Fc-glycan core-afucosylation, which was in turn correlated with receptor binding affinity by surface plasmon resonance and in vitro ADCC potency with a cell based bioassay. The strategy also quantifies Fc-glycan occupancy and the relative contribution from high mannose glycans.

A rapid approach for characterization of thiol-conjugated antibody-drug conjugates and calculation of drug-antibody ratio by liquid chromatography mass spectrometry.

We present the demonstration of a rapid "middle-up" liquid chromatography mass spectrometry (LC-MS)-based workflow for use in the characterization of thiol-conjugated maleimidocaproyl-monomethyl auristatin F (mcMMAF) and valine-citrulline-monomethyl auristatin E (vcMMAE) antibody-drug conjugates. Deconvoluted spectra were generated following a combination of deglycosylation, IdeS (immunoglobulin-degrading enzyme from Streptococcus pyogenes) digestion, and reduction steps that provide a visual representation of the product for rapid lot-to-lot comparison-a means to quickly assess the integrity of the antibody structure and the applied conjugation chemistry by mass. The relative abundance of the detected ions also offer information regarding differences in drug conjugation levels between samples, and the average drug-antibody ratio can be calculated. The approach requires little material (<100 µg) and, thus, is amenable to small-scale process development testing or as an early component of a complete characterization project facilitating informed decision making regarding which aspects of a molecule might need to be examined in more detail by orthogonal methodologies.

Chemical treatments for reducing the yellow discoloration of channel catfish (Ictalurus punctatus) fillets.

The effect of chemical pretreatments on the color and carotenoid content of yellow discolored channel catfish fillets was studied. The color and carotenoid content of the fillets were analyzed by the L*a*b* color system and high-performance liquid chromatography, respectively. Untreated fillets turned more yellow and darker after 12-d storage. Sodium bicarbonate had a beneficial effect on reducing the degree of yellowness, but the fillets still turned darker after storage. Sodium bisulfite gave the best results with fillets becoming less yellow and brighter after storage. Ascorbic acid, butylated hydroxyanisole, citric acid, and sodium metabisulfite were not successful at reducing the discoloration. The sum of carotenoid contents of untreated fillets decreased significantly (P < 0.05) during storage as compared to the fresh fillets. However, the sum of the carotenoid contents of fillets treated by various chemicals was not significantly (P > 0.05) different from the fresh or untreated fillets. The appearance of catfish fillets may be improved during storage by pretreating with sodium bisulfite.

Physical and chemical stability of tagatose powder.

Tagatose is a reduced-calorie monosaccharide that displays prebiotic properties. Water can interact with powdered tagatose to varying extents, depending upon the storage environment. Adsorbed water can impact the stability of tagatose, altering its functionality and usability as an ingredient. The objective of this study was to evaluate the physical and chemical stability of bulk tagatose powder as a function of relative humidity (RH) and temperature. Powdered tagatose was stored in desiccators at 20, 30, and 40 °C and 33% to 85% RH. Moisture contents (MC), physical characteristics, tagatose degradation profiles, and browning kinetics were monitored for 12 mo. The critical RH associated with deliquescence (RH0) was approximately 85% at 20 °C. MC values below RH0 were all less than 2% (wb). The MC at 85% RH ranged from 55% to 80% (wb), increasing as temperature decreased. At 33% RH and 20 °C tagatose remained a free flowing powder. As either temperature or RH increased, varying degrees of physical caking occurred. At 85% RH, tagatose deliquesced at all temperatures. Browning occurred in all samples at 40 °C. Despite physical caking and browning, measurable tagatose degradation was only observed in the deliquesced sample at 85% RH and 40 °C, where 20% loss occurred in 6 mo. Although extreme RHs and temperatures are required for tagatose degradation to occur, intermediate RHs and temperatures promote physical caking and deliquescence, which create handling problems during product formulation. The exposure of tagatose to elevated relative humidities and temperatures should be avoided to maintain its physical and chemical quality.

Tagatose stability in milk and diet lemonade.

UNLABELLED: The monosaccharide tagatose has been shown to behave physiologically as a prebiotic. To provide its healthful prebiotic effect to consumers, tagatose must not break down during food processing and storage. The objective of this study was to evaluate the storage and thermal stabilities of tagatose in milk and lemonade. Tagatose (0.9% to 1.5%) was added to commercially available shelf stable milk and diet lemonade. Samples were stored at 20, 40, 61, and 81 °C. Tagatose loss was monitored chromatographically. Pseudo-first-order rate constants for tagatose degradation were determined along with the reaction's activation energy. No tagatose degradation was observed in lemonade at temperatures equal to or less than 61 °C. Degradation occurred faster in milk because of its higher pH in comparison to lemonade and its dairy proteins enabling the Maillard reaction. The activation energy for tagatose degradation in milk was 24.6 kcal/mol. Using this activation energy, it was estimated that less than 0.1% tagatose would be lost during pasteurization and less than 4% would be lost during storage at 25 °C for 6 mo. Although tagatose degradation occurs in beverages, the extent of its loss during pasteurization and storage would be very low. Tagatose can be formulated into beverages with minimal concern about its degradation and the subsequent loss of prebiotic activity. PRACTICAL APPLICATION: Tagatose can be incorporated into beverages as a prebiotic to improve the healthful characteristics of the product without significant degradation.

Thermal stability of tagatose in solution.

Tagatose, a monosaccharide similar to fructose, has been shown to behave as a prebiotic. To deliver this prebiotic benefit, tagatose must not degrade during the processing of foods and beverages. The objective of this study was to evaluate the thermal stability of tagatose in solutions. Tagatose solutions were prepared in 0.02 and 0.1 M phosphate and citrate buffers at pHs 3 and 7, which were then held at 60, 70, and 80 degrees C. Pseudo-1st-order rate constants for tagatose degradation were determined. In citrate and phosphate buffers at pH 3, minimal tagatose was lost and slight browning was observed. At pH 7, tagatose degradation rates were enhanced. Degradation was faster in phosphate buffer than citrate buffer. Higher buffer concentrations also increased the degradation rate constants. Enhanced browning accompanied tagatose degradation in all buffer solutions at pH 7. Using the activation energies for tagatose degradation, less than 0.5% and 0.02% tagatose would be lost under basic vat and HTST pasteurization conditions, respectively. Although tagatose does breakdown at elevated temperatures, the amount of tagatose lost during typical thermal processing conditions would be virtually negligible. Practical Application: Tagatose degradation occurs minimally during pasteurization, which may allow for its incorporation into beverages as a prebiotic.

Effect of eculizumab on haemolysis-associated nitric oxide depletion, dyspnoea, and measures of pulmonary hypertension in patients with paroxysmal nocturnal haemoglobinuria.

Pulmonary hypertension (PH) is a common complication of haemolytic anaemia. Intravascular haemolysis leads to nitric oxide (NO) depletion, endothelial and smooth muscle dysregulation, and vasculopathy, characterized by progressive hypertension. PH has been reported in patients with paroxysmal nocturnal haemoglobinuria (PNH), a life-threatening haemolytic disease. We explored the relationship between haemolysis, systemic NO, arginine catabolism and measures of PH in 73 PNH patients enrolled in the placebo-controlled TRIUMPH (Transfusion Reduction Efficacy and Safety Clinical Investigation Using Eculizumab in Paroxysmal Nocturnal Haemoglobinuria) study. At baseline, intravascular haemolysis was associated with elevated NO consumption (P < 0.0001) and arginase-1 release (P < 0.0001). Almost half of the patients in the trial had elevated levels (> or =160 pg/ml) of N-terminal pro-brain natriuretic peptide (NT-proBNP), a marker of pulmonary vascular resistance and right ventricular dysfunction previously shown to indicate PH. Eculizumab treatment significantly reduced haemolysis (P < 0.001), NO depletion (P < 0.001), vasomotor tone (P < 0.05), dyspnoea (P = 0.006) and resulted in a 50% reduction in the proportion of patients with elevated NT-proBNP (P < 0.001) within 2 weeks of treatment. Importantly, the significant improvements in dyspnoea and NT-proBNP levels occurred without significant changes in anaemia. These data demonstrated that intravascular haemolysis in PNH produces a state of NO catabolism leading to signs of PH, including elevated NT pro-BNP and dyspnoea that are significantly improved by treatment with eculizumab.

Discovery and development of the complement inhibitor eculizumab for the treatment of paroxysmal nocturnal hemoglobinuria.

The complement system provides critical immunoprotective and immunoregulatory functions but uncontrolled complement activation can lead to severe pathology. In the rare hemolytic disease paroxysmal nocturnal hemoglobinuria (PNH), somatic mutations result in a deficiency of glycosylphosphatidylinositol-linked surface proteins, including the terminal complement inhibitor CD59, on hematopoietic stem cells. In a dysfunctional bone marrow background, these mutated progenitor blood cells expand and populate the periphery. Deficiency of CD59 on PNH red blood cells results in chronic complement-mediated intravascular hemolysis, a process central to the morbidity and mortality of PNH. A recently developed, humanized monoclonal antibody directed against complement component C5, eculizumab (Soliris; Alexion Pharmaceuticals Inc., Cheshire, CT, USA), blocks the proinflammatory and cytolytic effects of terminal complement activation. The recent approval of eculizumab as a first-in-class complement inhibitor for the treatment of PNH validates the concept of complement inhibition as an effective therapy and provides rationale for investigation of other indications in which complement plays a role.

Effect of the complement inhibitor eculizumab on thromboembolism in patients with paroxysmal nocturnal hemoglobinuria.

Hemolysis and hemoglobinemia contribute to serious clinical sequelae in hemolytic disorders. In paroxysmal nocturnal hemoglobinuria (PNH) patients, hemolysis can contribute to thromboembolism (TE), the most feared complication in PNH, and the leading cause of disease-related deaths. We evaluated whether long-term treatment with the complement inhibitor eculizumab reduces the rate of TE in patients with PNH. Clinical trial participants included all patients in the 3 eculizumab PNH clinical studies, which recruited patients between 2002 and 2005 (n = 195); patients from these studies continued treatment in the current multinational open-label extension study. Thromboembolism rate with eculizumab treatment was compared with the pretreatment rate in the same patients. The TE event rate with eculizumab treatment was 1.07 events/100 patient-years compared with 7.37 events/100 patient-years (P < .001) prior to eculizumab treatment (relative reduction, 85%; absolute reduction, 6.3 TE events/100 patient-years). With equalization of the duration of exposure before and during treatment for each patient, TE events were reduced from 39 events before eculizumab to 3 events during eculizumab (P < .001). The TE event rate in antithrombotic-treated patients (n = 103) was reduced from 10.61 to 0.62 events/100 patient-years with eculizumab treatment (P < .001). These results show that eculizumab treatment reduces the risk of clinical thromboembolism in patients with PNH. This study is registered at http://clinicaltrials.gov (study ID no. NCT00122317).

The clinical sequelae of intravascular hemolysis and extracellular plasma hemoglobin: a novel mechanism of human disease.

CONTEXT: The efficient sequestration of hemoglobin by the red blood cell membrane and the presence of multiple hemoglobin clearance mechanisms suggest a critical need to prevent the buildup of this molecule in the plasma. A growing list of clinical manifestations attributed to hemoglobin release in a variety of acquired and iatrogenic hemolytic disorders suggests that hemolysis and hemoglobinemia should be considered as a novel mechanism of human disease. EVIDENCE ACQUISITION: Pertinent scientific literature databases and references were searched through October 2004 using terms that encompassed various aspects of hemolysis, hemoglobin preparations, clinical symptoms associated with plasma hemoglobin, nitric oxide in hemolysis, anemia, pulmonary hypertension, paroxysmal nocturnal hemoglobinuria, and sickle-cell disease. EVIDENCE SYNTHESIS: Hemoglobin is released into the plasma from the erythrocyte during intravascular hemolysis in hereditary, acquired, and iatrogenic hemolytic conditions. When the capacity of protective hemoglobin-scavenging mechanisms has been saturated, levels of cell-free hemoglobin increase in the plasma, resulting in the consumption of nitric oxide and clinical sequelae. Nitric oxide plays a major role in vascular homeostasis and has been shown to be a critical regulator of basal and stress-mediated smooth muscle relaxation and vasomotor tone, endothelial adhesion molecule expression, and platelet activation and aggregation. Thus, clinical consequences of excessive cell-free plasma hemoglobin levels during intravascular hemolysis or the administration of hemoglobin preparations include dystonias involving the gastrointestinal, cardiovascular, pulmonary, and urogenital systems, as well as clotting disorders. Many of the clinical sequelae of intravascular hemolysis in a prototypic hemolytic disease, paroxysmal nocturnal hemoglobinuria, are readily explained by hemoglobin-mediated nitric oxide scavenging. CONCLUSION: A growing body of evidence supports the existence of a novel mechanism of human disease, namely, hemolysis-associated smooth muscle dystonia, vasculopathy, and endothelial dysfunction.

Preliminary report of the effects of complement suppression with pexelizumab on neurocognitive decline after coronary artery bypass graft surgery.

BACKGROUND AND PURPOSE: Pharmacological modulation of complement activation recently has been postulated as a therapeutic target in the treatment of neurological injury. We hypothesized that pexelizumab, a humanized scFv monoclonal antibody directed against the C5 complement component, would limit deficits in patients undergoing coronary artery bypass graft surgery with cardiopulmonary bypass. METHODS: The Phase II Pexelizumab study was a 914-patient, double-blind, placebo-controlled, 65-center study of patients undergoing coronary artery bypass graft surgery. Patients were randomized to pexelizumab bolus, bolus plus infusion, or placebo. Neurological and neurocognitive functions were assessed as secondary endpoints at baseline and on postoperative days (POD) 4 and 30. Cognitive deficits were assessed with multivariable tests accounting for baseline cognition, age, diabetes, years of education, sex, elevated creatinine, history of myocardial infarction, neurological disorder or congestive heart failure, and cardiopulmonary bypass time. RESULTS: Pexelizumab had no statistically significant effect on the primary composite endpoint or on overall cognition. When domain specific effects were examined, a decline of at least 10% in the visuo-spatial domain was observed on POD 4 in 56% of patients receiving placebo compared with 40% receiving pexelizumab by bolus and infusion (P=0.003). Similarly, on POD 30, a 10% decline was present in 21% of patients in the placebo group versus only 12% of the drug bolus plus infusion group (P=0.016). No differences were seen between treatment groups in any of the other domains. CONCLUSIONS: Pexelizumab administration for 24 hours perioperatively had no effect on global measures of cognition but may reduce dysfunction in the visuo-spatial domain.