Influence of whey protein hydrolysis in combination with dextran glycation on immunoglobulin E binding capacity with blood sera obtained from patients with a cow milk protein allergy.

Food protein allergies are a major global concern. Hydrolysis of food proteins reduces their allergenicity, but another novel approach is the covalent attachment of polysaccharides to proteins via the Maillard reaction (i.e., glycation), which blocks some IgE binding epitopes on the protein allergen. We wanted to examine whether enzymatic hydrolysis, combined with glycation, could further reduce IgE binding for people with a cow milk protein allergy. Whey protein isolate (WPI) was hydrolyzed by immobilized trypsin and chymotrypsin to degree of hydrolysis (DH) values of 17 to 27%. Immobilized enzymes were used to avoid heat-treating the hydrolysate (to inactivate the enzymes, because heating could also affect the IgE binding ability of the protein). The resultant whey protein isolate hydrolysates (WPIH) were then glycated with 10-kDa dextran (DX) in aqueous solutions held at 62°C for 24 h. We analyzed the molar mass (MW) of WPIH samples and their corresponding glycates (WPIH-DX) using size-exclusion chromatography with multi-angle laser light scattering. We obtained blood sera from 8 patients who had been diagnosed with a cow milk protein allergy, and we used a composite serum for IgE binding analysis. The average MW values of samples WPIH-1 to WPIH-3 decreased from 11.15, 9.46, and 7.57 kDa with increasing DH values of 18.7, 22.5, and 27.1%. Glycation significantly reduced the high bitterness of the WPIH samples, as assessed by a trained sensory panel. The WPIH-DX glycates had significantly reduced WPI-specific IgE binding capacity compared to WPI or unglycated WPIH; we found an almost 99% reduction in IgE binding for the WPIH-DX glycate made from WPIH with a DH value of 27.1%. Hydrolysis of WPI followed by glycation with DX via the Maillard reaction significantly decreased the allergenicity of whey proteins.

Glycation of whey protein with dextrans of different molar mass: Effect on immunoglobulin E-binding capacity with blood sera obtained from patients with cow milk protein allergy.

A growing concern around the world is the number of people who are suffering from food protein allergies. One potential approach to decrease protein allergenicity is to block IgE-binding epitopes of the protein allergen by attachment of polysaccharides via the Maillard reaction (i.e., glycation). Protein glycation has been extensively studied to modify various functional properties. We wanted to examine whether glycates could reduce IgE binding in patients with cow milk protein allergy and to explore how the size (molar mass; MW) of the polysaccharide affects this IgE-binding capacity. Glycation was performed using the initial step of the Maillard reaction performed in aqueous solutions. The specific goal of this study was to reduce the IgE-binding capacity of whey protein isolate (WPI) through glycation with dextran (DX). Blood sera were obtained from 8 patients who had been diagnosed with cow milk protein allergy, and a composite sera sample was used for IgE-binding analysis by the ImmunoCap (Phadia, Uppsala, Sweden) method. The WPI was glycated with DX of MW ranging from 1 to 2,000 kDa, and the MW of purified glycates was determined using size-exclusion chromatography coupled with multiangle laser light scattering. The WPI to DX molar ratios in the glycates made from DX that had MW values of 1, 3.5, 10 (G10), 150, 500, and 2,000 kDa were 1:4, 1:3, 1:2, 1:1.5, 1:1, and 1:1, respectively. With the increase in the MW of DX, there was an increase in the MW values of the corresponding glycates but a decrease in the number of bound DX. The WPI-DX glycates had lower whey protein IgE-binding capacity than native WPI, with the lowest IgE-binding capacity obtained in the G10 glycate. The DX binding ratios and morphology results from atomic force microscopy images suggested that glycation of WPI with small-MW DX resulted in extensive protein surface coverage, probably due to the attachment of up to 4 DX molecules per whey protein. The lower IgE binding of the G10 glycate was likely due to greater steric hindrance (or a physical barrier) at the surface of the protein. In summary, our results demonstrate that glycating WPI with DX via Maillard reaction can potentially be used to decrease the allergenicity of whey protein.

Properties of acid gels made from sodium caseinate-maltodextrin conjugates prepared by a wet heating method.

Covalent attachment of polysaccharides to proteins (conjugation) via the Maillard reaction has been extensively studied. Conjugation can lead to a significant improvement in protein functionality (e.g., solubility, emulsification, and heat stability). Caseins have previously been successfully conjugated with maltodextrin (Md), but the effect on the detailed acid gelation properties has not been examined. We studied the effect of conjugating sodium caseinate (NaCN) with 3 different sized Md samples via the Maillard reaction in aqueous solutions. The Md samples had dextrose equivalents of 4 to 7, 9 to 12, and 20 to 23 for Md40, Md100, and Md200, respectively. The conjugation reaction was performed in mixtures with 5% NaCN and 5% Md, which were heated at 90°C for 10 h. The degree of conjugation was estimated from the reduction in free amino groups as well as color changes. Sodium dodecyl sulfate-PAGE analysis was performed to confirm conjugation by employing staining of both protein and carbohydrate bands. The molar mass of samples was determined by size-exclusion chromatography coupled with multi-angle laser light scattering. After the conjugation reaction, samples were then gelled by the addition of 0.63% (wt/vol) glucono-δ-lactone at 30°C, such that samples reached pH 4.6 after about 13 h. The rheological properties of samples during acidification was monitored by small-strain dynamic oscillatory rheology. The microstructure of acid gels at pH 4.6 was examined by fluorescence microscopy. Conjugation resulted in a loss of 10.8, 8.8, and 11.9% of the available amino groups in the protein for the NaCN-Md40 conjugates (C40), NaCN-Md100 conjugates (C100), and NaCN-Md100 conjugates (C200), respectively. With a decrease in the size of the type of Md, an increase occurred in the molar mass of the resultant conjugate. The weight average molar masses of NaCN-Md samples were 340, 368, and 425 kDa for the conjugates C40, C100, and C200, respectively. Addition of Md to NaCN dispersion resulted in slightly shorter acid gelation times even without the conjugation reaction. The storage modulus (G') of acid gels was significantly lower in conjugated samples compared with the corresponding (unreacted) mixtures of Md and NaCN. The sample with the lowest G' value at pH 4.6 was the C40 conjugate. Fluorescence microscopy showed that gels made by conjugates had slightly larger pores. These results indicate that conjugation of casein modified its acid gelation properties, presumably by the Md polysaccharide moiety hindering aggregation and rearrangements of the casein network.

Dietary flavonoid fisetin increases abundance of high-molecular-mass hyaluronan conferring resistance to prostate oncogenesis.

We and others have shown previously that fisetin, a plant flavonoid, has therapeutic potential against many cancer types. Here, we examined the probable mechanism of its action in prostate cancer (PCa) using a global metabolomics approach. HPLC-ESI-MS analysis of tumor xenografts from fisetin-treated animals identified several metabolic targets with hyaluronan (HA) as the most affected. Efficacy of fisetin on HA was then evaluated in vitro and also in vivo in the transgenic TRAMP mouse model of PCa. Size exclusion chromatography-multiangle laser light scattering (SEC-MALS) was performed to analyze the molar mass (Mw) distribution of HA. Fisetin treatment downregulated intracellular and secreted HA levels both in vitro and in vivo Fisetin inhibited HA synthesis and degradation enzymes, which led to cessation of HA synthesis and also repressed the degradation of the available high-molecular-mass (HMM)-HA. SEC-MALS analysis of intact HA fragment size revealed that cells and animals have more abundance of HMM-HA and less of low-molecular-mass (LMM)-HA upon fisetin treatment. Elevated HA levels have been shown to be associated with disease progression in certain cancer types. Biological responses triggered by HA mainly depend on the HA polymer length where HMM-HA represses mitogenic signaling and has anti-inflammatory properties whereas LMM-HA promotes proliferation and inflammation. Similarly, Mw analysis of secreted HA fragment size revealed less HMM-HA is secreted that allowed more HMM-HA to be retained within the cells and tissues. Our findings establish that fisetin is an effective, non-toxic, potent HA synthesis inhibitor, which increases abundance of antiangiogenic HMM-HA and could be used for the management of PCa.

Effects of stabilizing or increasing respiratory motor outputs on obstructive sleep apnea.

To determine how the obstructive sleep apnea (OSA) patient's pathophysiological traits predict the success of the treatment aimed at stabilization or increase in respiratory motor outputs, we studied 26 newly diagnosed OSA patients [apnea-hypopnea index (AHI) 42 ± 5 events/h with 92% of apneas obstructive] who were treated with O2 supplementation, an isocapnic rebreathing system in which CO2 was added only during hyperpnea to prevent transient hypocapnia, and a continuous rebreathing system. We also measured each patient's controller gain below eupnea [change in minute volume/change in end-tidal Pco2 (ΔVe/ΔPetCO2)], CO2 reserve (eupnea-apnea threshold PetCO2), and plant gain (ΔPetCO2/ΔVe), as well as passive upper airway closing pressure (Pcrit). With isocapnic rebreathing, 14/26 reduced their AHI to 31 ± 6% of control (P < 0.01) (responder); 12/26 did not show significant change (nonresponder). The responders vs. nonresponders had a greater controller gain (6.5 ± 1.7 vs. 2.1 ± 0.2 l·min(-1)·mmHg(-1), P < 0.01) and a smaller CO2 reserve (1.9 ± 0.3 vs. 4.3 ± 0.4 mmHg, P < 0.01) with no differences in Pcrit (-0.1 ± 1.2 vs. 0.2 ± 0.9 cmH2O, P > 0.05). Hypercapnic rebreathing (+4.2 ± 1 mmHg PetCO2) reduced AHI to 15 ± 4% of control (P < 0.001) in 17/21 subjects with a wide range of CO2 reserve. Hyperoxia (SaO2 ∼95-98%) reduced AHI to 36 ± 11% of control in 7/19 OSA patients tested. We concluded that stabilizing central respiratory motor output via prevention of transient hypocapnia prevents most OSA in selected patients with a high chemosensitivity and a collapsible upper airway, whereas increasing respiratory motor output via moderate hypercapnia eliminates OSA in most patients with a wider range of chemosensitivity and CO2 reserve. Reducing chemosensitivity via hyperoxia had a limited and unpredictable effect on OSA.

Role of central/peripheral chemoreceptors and their interdependence in the pathophysiology of sleep apnea.

Unstable periodic breathing with intermittent ventilatory overshoots and undershoots commonly occurs in chronic heart failure, in hypoxia, with chronic opioid use and in certain types of obstructive sleep apnea. Sleep promotes breathing instability because it unmasks a highly sensitive dependence of the respiratory control system on chemoreceptor input, because transient cortical arousals promote ventilatory overshoots and also because upper airway dilator muscle tonicity is reduced and airway collapsibility enhanced. We will present data in support of the premise that carotid chemoreceptors are essential in the pathogenesis of apnea and periodicity; however it is the hyperadditive influence of peripheral chemoreceptor sensory input on central chemosensitivity that accounts for apnea and periodic breathing. This chemoreceptor interdependence also provides a significant portion of the normal drive to breathe in normoxia (i.e. eupnea) and in acute hypoxia. Finally, we discuss the effects of preventing transient hypocapnia (via selective increases in FICO(2)) on centrally mediated types of periodic breathing and even some varieties of cyclical obstructive sleep apnea.

Mercury distribution and lipid oxidation in fish muscle: effects of washing and isoelectric protein precipitation.

Nearly all the mercury (Hg) in whole muscle from whitefish (Coregonus clupeaformis) and walleye (Sander vitreus) was present as methyl mercury (MeHg). The Hg content in whole muscle from whitefish and walleye was 0.04-0.09 and 0.14-0.81 ppm, respectively. The myofibril fraction contained approximately three-fourths of the Hg in whitefish and walleye whole muscle. The sarcoplasmic protein fraction (e.g., press juice) was the next most abundant source of Hg. Isolated myosin, triacylglycerols, and cellular membranes contained the least Hg. Protein isolates prepared by pH shifting in the presence of citric acid did not decrease Hg levels. Addition of cysteine during washing decreased the Hg content in washed muscle probably through the interaction of the sulfhydryl group in cysteine with MeHg. Primary and secondary lipid oxidation products were lower during 2 °C storage in isolates prepared by pH shifting compared to those of washed or unwashed mince from whole muscle. This was attributed to removing some of the cellular membranes by pH shifting. Washing the mince accelerated lipid peroxide formation but decreased secondary lipid oxidation products compared to that of the unwashed mince. This suggested that there was a lipid hydroperoxide generating system that was active upon dilution of aqueous antioxidants and pro-oxidants.

The heterogeneity of obstructive sleep apnea (predominant obstructive vs pure obstructive apnea).

STUDY OBJECTIVES: To compare the breathing instability and upper airway collapsibility between patients with pure OSA (i.e. 100% of apneas are obstructive) and patients with predominant OSA (i.e., coexisting obstructive and central apneas). DESIGN: A cross-sectional study with data scored by a fellow being blinded to the subjects' classification. The results were compared between the 2 groups with unpaired student t-test. SETTING AND INTERVENTIONS: Standard polysomnography technique was used to document sleep-wake state. Ventilator in pressure support mode was used to introduce hypocapnic apnea during CO(2) reserve measurement. CPAP with both positive and negative pressures was used to produce obstructive apnea during upper airway collapsibility measurement. PARTICIPANTS: 21 patients with OSA: 12 with coexisting central/mixed apneas and hypopneas (28% ± 6% of total), and 9 had pure OSA. MEASUREMENTS: The upper airway collapsibility was measured by assessing the critical closing pressure (Pcrit). Breathing stability was assessed by measuring CO(2) reserve (i.e., ΔPCO(2) [eupnea-apnea threshold]) during NREM sleep. RESULTS: There was no difference in Pcrit between the 2 groups (pure OSA vs. predominant OSA: 2.0 ± 0.4 vs. 2.7 ± 0.4 cm H(2)O, P = 0.27); but the CO(2) reserve was significantly smaller in predominant OSA group (1.6 ± 0.7 mm Hg) than the pure OSA group (3.8 ± 0.6 mm Hg) (P = 0.02). CONCLUSIONS: The present data indicate that breathing stability rather than upper airway collapsibility distinguishes OSA patients with a combination of obstructive and central events from those with pure OSA.