Methionine oxidation by hydrogen peroxide in peptides and proteins: A theoretical and Raman spectroscopy study.

The oxidation of proteins results in their deterioration via the oxidation of reactive amino acids. Oxidation of the amino acid, methionine plays an important role during biological conditions of oxidative stress, and equally a role in protein stability. In this study the oxidation of the methionine residue using the tripeptide GlyMetGly with respect to hydrogen peroxide has been studied using both Raman spectroscopy and DFT calculations. Spectral modifications following the formation of methionine sulfoxide are shown with the appearance of the SO vibration whilst there is also the modification of the CS vibrations at approximately 700 cm-1. The changes in the intensity of the CS stretching band were used to calculate the kinetic rate constant as 7.9 ±â€¯0.6 × 10-3 dm3 mol-1 s-1. The energy barrier for the reaction. is determined both experimentally and using DFT calculations. The reaction of the dairy protein beta-lactoglobulin with hydrogen peroxide is equally studied using the same technique. The solvent accessible surface area of the methionine residues within the protein were also determined and a comparison of the reaction rate constant and the energy barriers of reaction for the oxidation of the tripeptide and for the protein respectively thus, provides information about the role of the protein environment in the oxidation process.

Cholestyramine treatment of healthy humans rapidly induces transient hypertriglyceridemia when treatment is initiated.

Bile acid (BA) production in mice is regulated by hepatic farnesoid X receptors and by intestinal fibroblast growth factor (FGF)-15 (in humans, FGF-19), a suppressor of BA synthesis that also reduces serum triglycerides and glucose. Cholestyramine treatment reduces FGF-19 and induces BA synthesis, whereas plasma triglycerides may increase from unclear reasons. We explored whether FGF-19 may suppress BA synthesis and plasma triglycerides in humans by modulation of FGF-19 levels through long-term cholestyramine treatment at increasing doses. In a second acute experiment, metabolic responses from 1 day of cholestyramine treatment were monitored. Long-term treatment reduced serum FGF-19 by >90%; BA synthesis increased up to 17-fold, whereas serum BAs, triglycerides, glucose, and insulin were stable. After long-term treatment, serum BAs and FGF-19 displayed rebound increases above baseline levels, and BA and cholesterol syntheses normalized after 1 wk without rebound reductions. Acute cholestyramine treatment decreased FGF-19 by 95% overnight and serum BAs by 60%, while BA synthesis increased fourfold and triglycerides doubled. The results support that FGF-19 represses BA synthesis but not serum triglycerides. However, after cessation of both long-term and 1-day cholestyramine treatment, circulating FGF-19 levels were normalized within 2 days, whereas BA synthesis remained significantly induced in both situations, indicating that also other mechanisms than the FGF-19 pathway are responsible for stimulation of BA synthesis elicited by cholestyramine. Several of the responses during cholestyramine treatment persisted at least 6 days after treatment, highlighting the importance of removing such treatment well before evaluating dynamics of the enterohepatic circulation in humans.

Protein reactivity with singlet oxygen: Influence of the solvent exposure of the reactive amino acid residues.

The singlet oxygen quenching rate constants were measured for three model proteins, bovine serum albumin, ß-lactoglobulin and lysozyme. The results were analyzed by comparing them with the corresponding singlet oxygen quenching rate constants for a series of tripeptides with the basic formula GlyAAGly where the central amino acid (AA) was the oxidizable amino acid, tryptophan, tyrosine, methionine and histidine. It was found that the reaction rate constant in proteins can be satisfactorily modelled by the sum of the individual contributions of the oxidizable AA residues corrected for the solvent accessible surface area (SASA) effects. The best results were obtained when the SASA of the AA residues were determined by averaging over molecular dynamics simulated trajectories of the proteins. The limits of this geometrical correction of the AA residue reactivity are also discussed.

IgM-antibodies against phosphorylcholine in mothers and normal or low birth weight term newborn infants.

OBJECTIVE: To determine levels of athero-protective IgM antibodies against phosphorylcholine in mothers and term-born normal or low birth weight infants. APPROACH: Twenty three mother-infant pairs were studied, of whom 16 infants were within the normal weight range for gestational age (NGA; 3652[504] g) and 7 were small for gestational age (SGA; birth weight: 2715[255] g), the latter <2SD below the Swedish reference data mean for normal fetal growth. All infants were born at term (mean ± SD 40.5 ± 1.1 weeks). Serum was available from 6 mothers with SGA and 14 with NGA infants. Participating mothers were aged 34.0 ± 3.9 years (no difference between groups). Fourteen neonates were boys and seven were girls. Levels of anti-PC IgM were determined by ELISA. RESULTS: Neonatal IgM anti-PC levels were low (undetectable in 8 infants out of which 3 were SGA) with a median of 76[range 0-2.51] U/ml. Maternal IgM anti-PC levels were significantly higher (median 7198[range: 25.32-656.0]) U/ml) and the proportion of mothers in highest quartile (>75th percentile) was larger in mothers of NGA-infants (43%) vs. those of SGA-infants (0%, p = 0.032). CONCLUSIONS: IgM anti-PC levels are low at birth, which suggests that these antibodies do not play a "housekeeping" role in immune function during fetal life/development, but arise predominately on exposure to external antigens after birth. Furthermore, low maternal IgM anti-PC levels may play a role in placental insufficiency, contributing to poor fetal growth and a small-for-date baby. This preliminary observation may have implications for the future risk of atherosclerosis/cardiovascular disease development in pregnant women and their offspring.

An experimental and theoretical study of the amino acid side chain Raman bands in proteins.

The Raman spectra of a series of tripeptides with the basic formula GlyAAGly where the central amino acid (AA) was tryptophan, tyrosine, phenylalanine, glycine, methionine, histidine, lysine and leucine were measured in H2O. The theoretical Raman spectra obtained using density functional theory (DFT) calculations at the B3LYP/6-311+G(2df,2pd) level of theory allows a precise attribution of the vibrational bands. The experimental results show that there is a blue shift in the frequencies of several bands of the amino acid side chains in tripeptides compared to free amino acids, especially in the case of AAs containing aromatic rings. On the other hand, a very good agreement was found between the Raman bands of AA residues in tripeptides and those measured on three model proteins: bovine serum albumin, ß-lactoglobulin and lysozyme. The present analysis contributes to an unambiguous interpretation of the protein Raman spectra that is useful in monitoring the biological reactions involving AA side chains alteration.

Circulating proprotein convertase subtilisin kexin type 9 has a diurnal rhythm synchronous with cholesterol synthesis and is reduced by fasting in humans.

OBJECTIVE: To gain insight into the function of proprotein convertase subtilisin kexin type 9 (PCSK9) in humans by establishing whether circulating levels are influenced by diurnal, dietary, and hormonal changes. METHODS AND RESULTS: We monitored circulating PCSK9 in a set of dynamic human experiments and could show that serum PCSK9 levels display a diurnal rhythm that closely parallels that of cholesterol synthesis, measured as serum lathosterol. In contrast to these marked diurnal changes in cholesterol metabolism, serum low-density lipoprotein (LDL) cholesterol levels remained stable during the diurnal cycle. Depletion of liver cholesterol by treatment with the bile acid-binding resin, cholestyramine, abolished the diurnal rhythms of both PCSK9 and lathosterol. Fasting (>18 hours) strongly reduced circulating PCSK9 and lathosterol levels, whereas serum LDL levels remained unchanged. Growth hormone, known to be increased during fasting in humans, reduced circulating PCSK9 in parallel to LDL cholesterol levels. CONCLUSIONS: Throughout the day, and in response to fasting and cholesterol depletion, circulating PCSK9 displays marked variation, presumably related to oscillations in hepatic cholesterol that modify its activity in parallel with cholesterol synthesis. In addition to this sterol-mediated regulation, additional effects on LDL receptors may be mediated by hormones directly influencing PCSK9.

Low levels of antibodies against phosphorylcholine in Alzheimer's disease.

Phosphorylcholine (PC) may play an important role in the atherogenic and pro-inflammatory effects of oxidized low density lipoproteins. We recently demonstrated that low levels of IgM antibodies against PC (anti-PC) are associated with development of myocardial infarction and stroke. We here evaluate the association between anti-PC and dementia and Alzheimer's disease (AD). We conducted a nested case-control study of 182 incident dementia cases (serum collected before onset of dementia) matched to 366 controls and a case-control study of 97 prevalent dementia cases (serum collected after dementia onset) matched to 205 controls. Controls were matched on gender and age at blood draw (+/- 1 year). Participants were from the Swedish Twin Registry. Anti-PC levels were measured by ELISA. The odds ratio (OR) of dementia was modeled using conditional logistic regression. Patients with dementia had significantly lower mean anti-PC levels than controls (39.1 versus 49.5 U/ml). The likelihood of having dementia or AD was doubled for individuals with the lowest 25% anti-PC levels (OR=2.04 and 2.70, respectively). The results were similar after adjustments for potential confounders. There was no association between anti-PC levels and incident dementia. Low levels of atheroprotective anti-PC could play a role in AD and dementia. Potential mechanisms include decreased anti-inflammatory potential and effects on the vasculature. Further attention is merited to elucidate the role of anti-PC in AD development and the usefulness of anti-PC as a part of risk prediction, prognosis, diagnosis, or treatment.

Flexible strategy for immobilizing redox-active compounds using in situ generation of diazonium salts. Investigations of the blocking and catalytic properties of the layers.

A versatile two-step method is developed to covalently immobilize redox-active molecules onto carbon surfaces. First, a robust anchoring platform is grafted onto surfaces by electrochemical reduction of aryl diazonium salts in situ generated. Depending on the nature of the layer termini, -COOH or -NH(2), a further chemical coupling involving ferrocenemethylamine or ferrocene carboxylic acid derivatives leads to the covalent binding of ferrocene centers. The chemical strategy using acyl chloride activation is efficient and flexible, since it can be applied either to surface-reactive end groups or to reactive species in solution. Cyclic voltammetry analyses point to the covalent binding of ferrocene units restricted to the upper layers of the underlying aryl films, while AFM measurements show a lost of compactness of the layers after the chemical attachment of ferrocene centers. The preparation conditions of the anchoring layers were found to determine the interfacial properties of the resulted ferrocenyl-modified electrodes. The ferrocene units promoted effective redox mediation providing that the free redox probes are adequately chosen (i.e., vs size/formal potential) and the underlying layers exhibit strong blocking properties. For anchoring films with weaker blocking effect, the coexistence of two distinct phenomena, redox mediation and ET at pinholes could be evidenced.

Low levels of IgM antibodies against phosphorylcholine-A potential risk marker for ischemic stroke in men.

BACKGROUND: Natural antibodies specific for phosphorylcholine (anti-PC) have been implicated as protective factors in atherosclerosis. We herein determined the relationship between IgM anti-PC and incidence of cardiovascular disease (CVD). METHODS: We studied 349 incident cases (200 men) of first events of CVD (coronary heart disease (CHD; n=203 or ischemic stroke; n=146) and 693 age- and sex-matched controls identified through 12 years of follow-up (1991-2003) of subjects from the cardiovascular cohort within the Malmö Diet and Cancer Study. Relative risks (RR) of CVD with 95% confidence intervals (CI) of incident CVD with adjustments for age, smoking, total cholesterol and blood pressure were determined. Anti-PC-levels were measured using ELISA (Athera CVDefine). RESULTS: As determined using Athera CVDefine, significant associations were attained with values of anti-PC below 17U/ml (corresponding to the lowest 9th percentile), which remained after taking confounders into account (RR: 1.79, 95% CI: 1.09-2.94, p=0.021). If men were studied separately, significance was evident at values below 17U/ml (RR: 2.01, 95% CI: 1.11-3.67, p=0.022), which was not the case among women. Furthermore, values below 17U/ml were also associated with ischemic stroke (RR=3.67, 95% CI: 1.34-10.1, p=0.01), but not with CHD. CONCLUSION: Low IgM anti-PC could be a novel risk marker for development of ischemic stroke in men. Further studies are needed to establish gender and subgroup differences.

Gluten-free vegan diet induces decreased LDL and oxidized LDL levels and raised atheroprotective natural antibodies against phosphorylcholine in patients with rheumatoid arthritis: a randomized study.

INTRODUCTION: The purpose of this study was to investigate the effects of vegan diet in patients with rheumatoid arthritis (RA) on blood lipids oxidized low-density lipoprotein (oxLDL) and natural atheroprotective antibodies against phosphorylcholine (anti-PCs). METHODS: Sixty-six patients with active RA were randomly assigned to either a vegan diet free of gluten (38 patients) or a well-balanced non-vegan diet (28 patients) for 1 year. Thirty patients in the vegan group completed more than 3 months on the diet regimen. Blood lipids were analyzed by routine methods, and oxLDL and anti-PCs were analyzed by enzyme-linked immunosorbent assay. Data and serum samples were obtained at baseline and after 3 and 12 months. RESULTS: Mean ages were 50.0 years for the vegan group and 50.8 years for controls. Gluten-free vegan diet induced lower body mass index (BMI) and low-density lipoprotein (LDL) and higher anti-PC IgM than control diet (p < 0.005). In the vegan group, BMI, LDL, and cholesterol decreased after both 3 and 12 months (p < 0.01) and oxLDL after 3 months (p = 0.021) and trendwise after 12 months (p = 0.090). Triglycerides and high-density lipoprotein did not change. IgA anti-PC levels increased after 3 months (p = 0.027) and IgM anti-PC levels increased trendwise after 12 months (p = 0.057). There was no difference in IgG anti-PC levels. In the control diet group, IgM anti-PC levels decreased both after 3 and 12 months (p < 0.01). When separating vegan patients into clinical responders and non-responders at 12 months, the effects on oxLDL and anti-PC IgA were seen only in responders (p < 0.05). CONCLUSION: A gluten-free vegan diet in RA induces changes that are potentially atheroprotective and anti-inflammatory, including decreased LDL and oxLDL levels and raised anti-PC IgM and IgA levels.