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Circulating cell-free DNA (cf-DNA) has emerged as a promising biomarker of ageing, tissue damage and cellular stress. However, less is known about health behaviours, ageing phenotypes and metabolic processes that lead to elevated cf-DNA levels. We sought to analyse the relationship of circulating cf-DNA level to age, sex, smoking, physical activity, vegetable consumption, ageing phenotypes (physical functioning, the number of diseases, frailty) and an extensive panel of biomarkers including blood and urine metabolites and inflammatory markers in three human cohorts (N = 5385; 17–82 years). The relationships were assessed using correlation statistics, and linear and penalised regressions (the Lasso), also stratified by sex.cf-DNA levels were significantly higher in men than in women, and especially in middle-aged men and women who smoke, and in older more frail individuals. Correlation statistics of biomarker data showed that cf-DNA level was higher with elevated inflammation (C-reactive protein, interleukin-6), and higher levels of homocysteine, and proportion of red blood cells and lower levels of ascorbic acid. Inflammation (C-reactive protein, glycoprotein acetylation), amino acids (isoleucine, leucine, tyrosine), and ketogenesis (3-hydroxybutyrate) were included in the cf-DNA level-related biomarker profiles in at least two of the cohorts.In conclusion, circulating cf-DNA level is different by sex, and related to health behaviour, health decline and metabolic processes common in health and disease. These results can inform future studies where epidemiological and biological pathways of cf-DNA are to be analysed in details, and for studies evaluating cf-DNA as a potential clinical marker.

[Abstract] Introduction: Immunosenescence and inflammaging have been implicated in the pathophysiology of frailty. Torquetenovirus (TTV), a single-stranded DNA anellovirus, the major component of the human blood virome, shows an increased replication rate with advancing age. An elevated TTV viremia has been associated with an impaired immune function and an increased risk of mortality in the older population. The objective of this study was to analyze the relation between TTV viremia, physical frailty, and cognitive impairment. Methods: TTV viremia was measured in 1,131 nonfrail, 45 physically frail, and 113 cognitively impaired older adults recruited in the MARK-AGE study (overall mean age 64.7 ± 5.9 years), and then the results were checked in two other independent cohorts from Spain and Portugal, including 126 frail, 252 prefrail, and 141 nonfrail individuals (overall mean age: 77.5 ± 8.3 years). Results: TTV viremia ≥4log was associated with physical frailty (OR: 4.69; 95% CI: 2.06-10.67, p < 0.0001) and cognitive impairment (OR: 3.49, 95% CI: 2.14-5.69, p < 0.0001) in the MARK-AGE population. The association between TTV DNA load and frailty status was confirmed in the Spanish cohort, while a slight association with cognitive impairment was observed (OR: 1.33; 95% CI: 1.000-1.773), only in the unadjusted model. No association between TTV load and frailty or cognitive impairment was found in the Portuguese sample, although a negative association between TTV viremia and MMSE score was observed in Spanish and Portuguese females. Conclusions: These findings demonstrate an association between TTV viremia and physical frailty, while the association with cognitive impairment was observed only in the younger population from the MARK-AGE study. Further research is necessary to clarify TTV's clinical relevance in the onset and progression of frailty and cognitive decline in older individuals. Ministerio de Ciencia e Innovación (España); PID2020-113788RB-I00 Xunta de Galicia; ED431B 2022/16 Xunta de Galicia; ED431F 2017/09

Aging and age-related diseases have been linked to microbial dysbiosis with changes in blood bacterial DNA concentration. This condition may promote chronic low-grade inflammation, which can be further aggravated by antioxidant nutrient deficiency. Low plasma carotenoids are associated with an increased risk of inflammation and cellular damage and predict mortality. However, no evidence is yet available on the relationship between antioxidants and the blood bacterial DNA (BB-DNA). Therefore, this study aimed to compare BB-DNA from (a) GO (nonagenarian offspring), (b) age-matched controls (Randomly recruited Age-Stratified Individuals from the General population [RASIG]), and (c) spouses of GO (SGO) recruited in the MARK-AGE project, as well as to investigate the association between BB-DNA, behavior habits, Charlson Comorbidity Index (CCI), leucocyte subsets, and the circulating levels of some antioxidants and oxidative stress markers. BB-DNA was higher in RASIG than GO and SGO, whereas GO and SGO participants showed similar values. BB-DNA increased in smokers and males with CCI ≥ 2 compared with those with CCI ≤ 1 within RASIG. Moreover, BB-DNA was positively associated with lymphocyte, neutrophil, and monocyte counts, but not with self-reported dietary habits. Higher quartiles of BB-DNA were associated with low lutein and zeaxanthin and elevated malondialdehyde plasma concentrations in RASIG. BB-DNA was also positively correlated with nitric oxide levels. Herein, we provide evidence of a reduced BB-DNA in individuals from long-living families and their spouses, suggesting a decreased microbial dysbiosis and bacterial systemic translocation. BB-DNA was also associated with smoking, CCI, leukocyte subsets, and some redox biomarkers in older participants.

Persons with Down syndrome (DS) undergo a premature ageing with early onset of age-related diseases. The main endpoint of this study was the identification of blood circulating microRNAs (c-miRs) signatures characterizing DS ageing process. A discovery phase based on array was performed in plasma samples obtained from 3 young (31±2 years-old) and 3 elderly DS persons (66±2 years-old). Then, a validation phase was carried out for relevant miRs by RT-qPCR in an enlarged cohort of 43 DS individuals (from 19 up to 68 years-old). A group of 30 non-trisomic subjects, as representative of physiological ageing, was compared. In particular miR-628-5p, miR-152-3p, miR-28-5p, and let-7d-5p showed a lower level in younger DS persons (age ≤ 50 years) respect to the age-matched controls. Among those, miR-28-5p and let-7d-5p were found significantly decreased in physiological ageing ( oldest group ), thus they emerged as possible biomarkers of premature ageing in DS. Moreover, measuring blood levels of beta amyloid peptides, Aβ-42 was assessed at the lowest levels in physiological ageing and correlated with miR-28-5p and let-7d-5p in DS, while Aβ-40 correlated with miR-628-5p in the same cohort. New perspectives in terms of biomarkers are discussed.

Ageing leaves characteristic traces in the DNA methylation make-up of the genome. However, the importance of DNA methylation in ageing remains unclear. The study of subtelomeric regions could give promising insights into this issue. Previously reported associations between susceptibility to age-related diseases and epigenetic instability at subtelomeres suggest that the DNA methylation profile of subtelomeres undergoes remodelling during ageing. In the present work, this hypothesis has been tested in the context of the European large-scale project MARK-AGE. In this cross-sectional study, we profiled the DNA methylation of chromosomes 5 and 21 subtelomeres, in more than 2000 age-stratified women and men recruited in eight European countries. The study included individuals from the general population as well as the offspring of nonagenarians and Down syndrome subjects, who served as putative models of delayed and accelerated ageing, respectively. Significant linear changes of subtelomeric DNA methylation with increasing age were detected in the general population, indicating that subtelomeric DNA methylation changes are typical signs of ageing. Data also show that, compared to the general population, the dynamics of age-related DNA methylation changes are attenuated in the offspring of centenarian, while they accelerate in Down syndrome individuals. This result suggests that subtelomeric DNA methylation changes reflect the rate of ageing progression. We next attempted to trace the age-related changes of subtelomeric methylation back to the influence of diverse variables associated with methylation variations in the population, including demographics, dietary/health habits and clinical parameters. Results indicate that the effects of age on subtelomeric DNA methylation are mostly independent of all other variables evaluated. Supplementary Information The online version contains supplementary material available at 10.1007/s11357-021-00347-9.

A redox steady state is important in maintaining vital cellular functions and is therefore homeostatically controlled by a number of antioxidative agents, the most important of which are enzymes. Oxidative Stress (OS) is associated with (or/and caused by) excessive production of damaging reactive oxygen and/or nitrogen species (ROS, RNS), which play a role in many pathologies. Because OS is a risk factor for many diseases, much effort (and money) is devoted to early diagnosis and treatment of OS. The desired benefit of the “identify (OS) and treat (by low molecular weight antioxidants, LMWA)” approach is to enable selective treatment of patients under OS. The present work aims at gaining understanding of the benefit of the antioxidants based on interrelationship between the concentration of different OS biomarkers and LMWA. Both the concentrations of a variety of biomarkers and of LMWA were previously determined and some analyses have been published by the MARK-AGE team. For the sake of simplicity, we assume that the concentration of an OS biomarker is a linear function of the concentration of a LMWA (if the association is due to causal relationship). A negative slope of this dependence (and sign of the correlation coefficient) can be intuitively expected for an antioxidant, a positive slope indicates that the LMWA is pro-oxidative, whereas extrapolation of the OS biomarker to [LMWA] = 0 is an approximation of the concentration of the OS biomarker in the absence of the LMWA. Using this strategy, we studied the effects of 12 LMWA (including tocopherols, carotenoids and ascorbic acid) on the OS status, as observed with 8 biomarkers of oxidative damage (including malondialdehyde, protein carbonyls, 3-nitrotyrosine). The results of this communication show that in a cross-sectional study the LMWA contribute little to the redox state and that different “antioxidants” are very different, so that single LMWA treatment of OS is not scientifically justified assuming our simple model. In view of the difficulty of quantitating the OS and the very different effects of various LMWA, the use of the “identify and treat” approach is questionable.

The analysis of copper (Cu) and zinc (Zn) along with their major serum carriers, albumin (Alb) and ceruloplasmin (Cp), could provide information on the capacity of humans to maintain homeostasis of metals (metallostasis). However, their relationship with aging, sex, body mass index, as well as with nutritional and inflammatory markers was never investigated in a large-scale study. Here, we report results from the European large-scale cross-sectional study MARK-AGE in which Cu, Zn, Alb, Cp, as well as nutritional and inflammatory parameters were determined in 2424 age-stratified participants (35–75 years), including the general population (RASIG), nonagenarian offspring (GO), a well-studied genetic model of longevity, and spouses of GO (SGO). In RASIG, Cu to Zn ratio and Cp to Alb ratio were higher in women than in men. Both ratios increased with aging because Cu and Cp increased and Alb and Zn decreased. Cu, Zn, Alb, and Cp were found associated with several inflammatory as well as nutritional biomarkers. GO showed higher Zn levels and higher Zn to Alb ratio compared to RASIG, but we did not observe significant differences with SGO, likely as a consequence of the low sample size of SGO and the shared environment. Our results show that aging, sex, body mass index, and GO status are characterized by different levels of Cu, Zn, and their serum carrier proteins. These data and their relationship with inflammatory biomarkers support the concept that loss of metallostasis is a characteristic of inflammaging.

Torquetenovirus (TTV) viremia has been associated with increased mortality risk in the elderly population. This work aims to investigate TTV viremia as a potential biomarker of immunosenescence. We compared levels of circulating TTV in 1813 participants of the MARK-AGE project, including human models of delayed (offspring of centenarians [GO]) and premature (Down syndrome [DS]) immunosenescence. The TTV load was positively associated with age, cytomegalovirus (CMV) antibody levels, and the Cu/Zn ratio and negatively associated with platelets, total cholesterol, and total IgM. TTV viremia was highest in DS and lowest in GO, with intermediate levels in the SGO (spouses of GO) and RASIG (Randomly Recruited Age-Stratified Individuals From The General Population) populations. In the RASIG population, TTV DNA loads showed a slight negative association with CD3+T-cells and CD4+T-cells. Finally, males with ≥4log TTV copies/mL had a higher risk of having a CD4/CD8 ratio

Frailty among elderly people leads to an increased risk for negative health outcomes. To prevent frailty, we need a better understanding of the underlying mechanisms and early detection of individuals at risk. Both may be served by identifying candidate (bio)markers, i.e. biomarkers and markers, for the physical, cognitive, and psychological frailty domains. We used univariate (Rank-ANOVA) and multivariate (elastic net) approaches on the RASIG study population (age range: 35–74 years, n = 2220) of the MARK-AGE study to study up to 331 (bio)markers between individuals with and without frailty for each domain. Biomarkers and markers identified by both approaches were studied further regarding their association with frailty using logistic regression. Univariately, we found lower levels of antioxidants, including β-cryptoxanthin and zeaxanthin, in those who were physically, cognitively or psychologically frail. Additionally, self-reported health was worse in these three frail groups. Multivariately, we observed lower levels of β-cryptoxanthin and zeaxanthin in the cognitively frail. Levels of these carotenoids were inversely associated with the risk of being cognitively frail after adjusting for confounders. Antioxidants and self-reported health are potential (bio)markers to detect persons at risk of becoming frail. The biomarkers identified may indicate the involvement of inflammation in frailty, especially for physical and cognitive frailty.

Recently, Weber et al. published a thorough investigation of the age-dependency of oxidative stress (OS) determined by the steady state concentrations of different compounds - oxidation products and antioxidants - that are in common use as biomarkers of OS in 2207 healthy individuals of the cross-sectional MARK-AGE Project. The correlations among biomarkers were significant but weak. These findings may indicate different manifestations of OS and must further be evaluated. Here, we report a refined analysis of OS based on the above-mentioned original data. We show that malondialdehyde (MDA) appears to be sensitive to both gender and age. It is significantly lower and shows a greater age-dependence in women than in men. The age-dependency of MDA in women arises in a stepwise fashion. The age-dependent slope of the steady state concentration is maximal at the age between 50 and 55 years, indicating that it may be attributed to the change of metabolism in the post-menopause. Interestingly, total glutathione (GSH) decreased with age simultaneously with the increase in MDA.Different biomarkers yield different gender- and age-dependencies. Unlike the concentration of MDA, the concentrations of the other two oxidation products, i.e. protein carbonyls and 3-nitrotyrosine were similar in men and women and appeared to be independent of age in the healthy study population. The analyzed antioxidants exhibited different gender- and age-dependencies. In conclusion, it appears that all the biomarkers assessed here reflect different types of OS and that MDA and GSH reflect the same type of OS. Keywords: Oxidative stress, Age-dependency, Gender-associated differences, Biomarkers

Overfeeding with a high-fat and/or high-carbohydrate (CHO) diet is known to increase plasma concentrations of endotoxin (lipopolysaccharide [LPS]) that may lead to metabolic disturbances like insulin resistance. The impact of CHO quality (i.e., the glycemic index [GI]) independent of fat intake on metabolic endotoxemia remains unclear. In the present study, the effects of changes in energy balance and GI on plasma endotoxin were studied.Fifteen healthy young men overconsumed diets containing 65% CHO and 20% fat for 1 week (OF; +50% of energy requirement) followed by 3 weeks of caloric restriction (CR; -50% of energy requirement) and were then randomized to 2 weeks hypercaloric refeeding (RF, +50% of energy requirement) with either a low- or high-GI (40 vs 74) diet.During OF, subjects gained 1.9 ± 0.7 kg body weight (+0.6 ± 0.8% fat mass) followed by a weight loss of 6.1 ± 0.8 kg (-2.0 ± 0.6% fat mass) and weight regain of 4.0 ± 0.6 kg (0.9 ± 0.8% fat mass). Fasting insulin and homeostasis model assessment-insulin resistance (HOMAA hypercaloric diet (OF and RF) increased plasma endotoxin irrespective of GI, whereas a negative energy balance did not reduce endotoxemia. Impaired insulin sensitivity with hypercaloric refeeding on a high-GI diet was not explained by metabolic endotoxemia.

Oxidative stress and antioxidants play a role in age-related diseases and in the aging process. We here present data on protein carbonyls, 3-nitrotyrosine, malondialdehyde, and cellular and plasma antioxidants (glutathione, cysteine, ascorbic acid, uric acid,α-tocopherol, and lycopene) and their relation with age in the European multicenter study MARK-AGE. To avoid confounding, only data from countries which recruited subjects from all three study groups (five of eight centers) and only participants aged ≥55 years were selected resulting in data from 1559 participants. These included subjects from (1) the general population, (2) members from long-living families, and (3) their spouses. In addition, 683 middle-aged reference participants (35–54 years) served as a control. After adjustment for age, BMI, smoking status, gender, and country, there were differences in protein carbonyls, malondialdehyde, 3-nitrotyrosine,α-tocopherol, cysteine, and glutathione between the 3 study groups. Protein carbonyls and 3-nitrotyrosine as well as cysteine, uric acid, and lycopene were identified as independent biomarkers with the highest correlation with age. Interestingly, from all antioxidants measured, only lycopene was lower in all aged groups and from the oxidative stress biomarkers, only 3-nitrotyrosine was increased in the descendants from long-living families compared to the middle-aged control group. We conclude that both lifestyle and genetics may be important contributors to redox biomarkers in an aging population.

Metallothionein (MT) family are cysteine-rich proteins that regulate zinc (Zn) homeostasis and protect against oxidative damage. Studies in transgenic mice have shown that MT favorably influence longevity, although their role in human aging is not completely understood. Within the European multicenter study MARK-AGE, we analyzed MT induction after Zn treatment in peripheral blood mononuclear cells (PBMCs) and its relation with redox biomarkers in 2,936 age-stratified subjects (35–75 years) including the general population (RASIG), centenarian offspring (GO), and their spouses (SGO). We found that the lymphocyte capability to induce MT in response to Zn is not affected by aging. However, GO participants showed lower Zn-induced MT and increased basal expression of MT1A, MT1X, and ZnT-1 genes than RASIG subjects. Moreover, Zn-induced MT levels were found to be inversely related with oxidative stress markers (plasma protein carbonyls, 3-nitrotyrosine, and malondialdehyde) in the whole population, but not in GO subjects. In conclusion, our results support the hypothesis that the response to Zn is attenuated in PBMCs of centenarian offspring compared to the general population as a consequence of a tighter control of Zn homeostasis which is likely to provide them constant protection against stress stimuli over the whole lifespan. published

Objective Only a few studies have used objective measurements to investigate the relationship between sleep quality and obesity. These studies showed controversial results. Methods Sleep efficiency was measured by Actiwatch 2 in 132 healthy students (age 23.3 ± 3.7 years, BMI 23.1 ± 4.1 kg/m2) for 12 ± 3 nights, differentiating between work and free days. Physical activity, dietary habits, and autonomic function (heart rate variability, HRV) were analyzed as potential determinants of sleep quality and its relationship with body composition. Results Sleep efficiency was 87.0% in women and 84.9% in men (P

cope Orange fruits and products thereof represent important dietary sources of carotenoids, particularly β-cryptoxanthin. Since previous studies reported a positive effect of vegetable processing on carotenoid absorption, our objective was to compare the bioavailability of β-cryptoxanthin from either fresh navel oranges (Citrus sinensis L. Osbeck) or pasteurized orange juice. Methods and results The study was designed as a randomized 2-way cross-over study. Twelve volunteers consumed two meals delivering 744 μg of β-cryptoxanthin from either fresh navel oranges or pasteurized orange juice. Eight blood samples were collected over 9.5 h after test meal consumption and analyzed using HPLC-DAD. Additionally, carotenoid bioaccessibility was assessed after in vitro digestion of the same test foods. β-cryptoxanthin bioavailability from pasteurized orange juice was 1.8-fold higher than from fresh oranges (P = 0.011). Similarly, mean absorption of the non-dose adjusted carotenoids lutein (P = 0.301), zeaxanthin (P = 0.216), and zeinoxanthin (P = 0.090) were slightly higher from orange juice, although not reaching statistical significance. The in vitro digestion revealed a 5.3-fold higher bioaccessibility of β-cryptoxanthin from orange juice. Dietary fiber contents in the test foods were inversely associated with carotenoid bioavailability. Conclusion Orange juice represents a more bioavailable source of β-cryptoxanthin than fresh oranges.

Due to the successful implementation of vaccination strategies early-life morbidity and mortality due to infectious disease has been reduced dramatically. Vaccines against tetanus and diphtheria are among the most frequently used vaccines worldwide, but various studies in different European countries have shown that protection against tetanus and particularly against diphtheria is unsatisfactory in adults and older persons. In this study we analyzed tetanus- and diphtheria-specific antibody concentrations in 2100 adults of different age from 6 selected European countries (Austria, Belgium, Germany, Greece, Italy, Poland) in order to investigate differences in the level of protection against tetanus and diphtheria across Europe. Our data reveal that tetanus- and diphtheria-specific antibody concentrations vary greatly between countries, which is also reflected in the percentage of persons with antibody concentrations below the protective level (0.1 IU/ml), which ranged from 2 to 31% percent for tetanus and 28–63% for diphtheria. In most countries, tetanus- and diphtheria-specific antibody concentrations decrease with age. This phenomenon is more pronounced in countries with generally low antibody levels, such as Italy, Poland and Greece. Interestingly, tetanus-specific antibody concentrations are generally higher in males than in females, which is probably due to vaccination during their military service or more frequent booster vaccinations after injuries, whereas no gender-related differences were found for diphtheria-specific antibodies. In conclusion, our study demonstrates that the European population is not fully protected against tetanus and diphtheria. Measures to improve protection should include a life-long perspective on vaccination, more education to increase awareness of and compliance with vaccination guidelines, and a harmonization of recommendations and incentives across Europe.

Polyunsaturated fatty acids (PUFAs), especially the n-3 series, are known for their protective effects. Considering that cardiovascular diseases are risk factors for dementia, which is common at aging, the aim of this study was to evaluate whether fatty acid status in the elderly was associated with cognitive function and cardiovascular risk. Forty-five elderly persons (age ≥60 years) were included and divided into two groups based on their Mini-Mental Status Examination score adjusted for educational level: the case group (n = 12) and the control group (n = 33). Serum fatty acid composition, homocysteine (Hcy), hs-CRP, lipid profile and different cognitive domains were evaluated. The case group, characterized by reduced cognitive performance, showed higher levels of 14:0, 16:0, 16:1n-7 fatty acids and lower levels of 22:0, 24:1n-9, 22:6n-3 (DHA) and total PUFAs compared to the control group (p < 0.05). The n-6/n-3 ratio was elevated in both study groups, whereas alterations in Hcy, hs-CRP and lipid profile were observed in the case group. Cognitive function was positively associated with the 24:1n-9, DHA and total n-3 PUFAs, while 14:0, 16:0 and 16:1n-7 fatty acids, the n-6/n-3 ratio and Hcy were inversely associated. In addition, n-3 PUFAs, particularly DHA, were inversely associated with cardiovascular risk, assessed by Hcy levels in the elderly.

A low folate or low thiamine status may be associated with the risk of preterm delivery, small for gestational age (SGA) offspring and adverse pregnancy outcomes.5-Methyltetrahydrofolate (5MTHF) and thiamine diphosphate (TDP) were measured directly in cord-blood erythrocytes (CBEs) of early preterm (n=26;32 weeks gestational age; including 50% multiple births), late preterm (n=38; 32 to37 weeks; including 24% multiple births) and term newborns (n=60, 37-42 weeks) via high-performance liquid chromatography and fluorescence detection. Associations between 5MTHF and TDP with gestational age, newborn anthropometrics (birth weight, newborn's length and head circumference) and risk of being SGA were explored.Group comparison as well as multivariate linear regression analysis of cord-blood vitamins revealed that 5MTHF was significantly lower in late preterms compared with terms but did not differ between singletons and multiples. TDP tended to be higher in preterms than in terms and lower in multiples than in singletons in both early and late preterms. Multivariate analysis on birth outcomes showed that 5MTHF was significantly positively associated with gestational age, birth weight and newborn's length. 5MTHF, increasing gestational age and parity were associated with a significantly reduced risk for being SGA, while TDP, multiple births and gender were not associated with the risk for being SGA.Higher CBE concentrations of 5MTHF were associated with improved birth outcomes. Lower TDP concentrations were observed in multiple births. Future studies evaluating cord-blood vitamin concentrations and their associations with birth outcomes should additionally include dietary intakes and maternal blood concentrations at different stages of pregnancy.

Metabolism is essential to understand human health. To characterize human metabolism, a high-resolution read-out of the metabolic status under various physiological conditions, either in health or disease, is needed. Metabolomics offers an unprecedented approach for generating system-specific biochemical definitions of a human phenotype through the capture of a variety of metabolites in a single measurement. The emergence of large cohorts in clinical studies increases the demand of technologies able to analyze a large number of measurements, in an automated fashion, in the most robust way. NMR is an established metabolomics tool for obtaining metabolic phenotypes. Here, we describe the analysis of NMR-based urinary profiles for metabolic studies, challenged to a large human study (3007 samples). This method includes the acquisition of nuclear Overhauser effect spectroscopy one-dimensional and J-resolved two-dimensional (J-Res-2D) 1H NMR spectra obtained on a 600 MHz spectrometer, equipped with a 120 μL flow probe, coupled to a flow-injection analysis system, in full automation under the control of a sampler manager. Samples were acquired at a throughput of ∼20 (or 40 when J-Res-2D is included) min/sample. The associated technical analysis error over the full series of analysis is 12%, which demonstrates the robustness of the method. With the aim to describe an overall metabolomics workflow, the quantification of 36 metabolites, mainly related to central carbon metabolism and gut microbial host cometabolism, was obtained, as well as multivariate data analysis of the full spectral profiles. The metabolic read-outs generated using our analytical workflow can therefore be considered for further pathway modeling and/or biological interpretation.

Aging is associated with alterations in the content and patterns of DNA methylation virtually throughout the entire human lifespan. Reasons for these variations are not well understood. However, several lines of evidence suggest that the epigenetic instability in aging may be traced back to the alteration of the expression of DNA methyltransferases. Here, the association of the expression of DNA methyltransferases DNMT1 and DNMT3B with age has been analysed in the context of the MARK-AGE study, a large-scale cross-sectional study of the European general population. Using peripheral blood mononuclear cells, we assessed the variation of DNMT1 and DNMT3B gene expression in more than two thousand age-stratified women and men (35-75 years) recruited across eight European countries. Significant age-related changes were detected for both transcripts. The level of DNMT1 gradually dropped with aging but this was only observed up to the age of 64 years. By contrast, the expression of DNMT3B decreased linearly with increasing age and this association was particularly evident in females. We next attempted to trace the age-related changes of both transcripts to the influence of different variables that have an impact on changes of their expression in the population, including demographics, dietary and health habits, and clinical parameters. Our results indicate that age affects the expression of DNMT1 and DNMT3B as an almost independent variable in respect of all other variables evaluated. published

Photodynamic therapy (PDT) is an important clinical approach for cancer treatment. It involves the administration of a photosensitizer, followed by its activation with light and induction of cell death. The underlying mechanism is an increased production of reactive oxygen species (ROS) leading to oxidative stress, which is followed by cell death. However, effectiveness of PDT is limited due to an initiation of endogenous rescue response systems like heme oxygenase-1 (HO-1) in tumor cells. In recent years, consuming of antioxidant supplements has become widespread, but the effect of exogenously applied antioxidants on cancer therapy outcome remains unclear. Thus, this study was aimed to investigate if exogenous antioxidants might decrease ROS-induced cytotoxicity in photodynamic treatment. Lycopene, β-carotene, vitamin C, N-acetylcysteine, trolox, and N-tert-butyl-α-phenylnitrone in different doses were administered to human melanoma cells prior exposure to photodynamic treatment. Supplementation with vitamin C resulted in a significant decrease of the cell death rate, whereas the other tested antioxidants had no effect on cell viability and oxidative stress markers. The simultaneous application of vitamin C with the HO-1 activity inhibitor zinc protoporphyrine IX (ZnPPIX) caused a considerable decrease of photodynamic treatment-induced cytotoxicity compared to ZnPPIX alone. It can be summarized that exogenously applied antioxidants do not have a leading role in the protective response against photodynamic treatment. However, further studies are necessary to investigate more antioxidants and other substances, which might affect the outcome of photodynamic treatment in cancer therapy.

Oxidative stress is an inevitable process in the nucleus, especially in antitumor chemotherapy, and adaptation by defense mechanisms seems to be one element in the development of long-term resistance to many chemotherapeutic drugs. In this study, a potential chromatin repair mechanism during oxidative stress was investigated in HT22 cells. The 20S proteasome has been shown to be largely responsible for the degradation of oxidatively modified histone proteins in the nucleus. Poly(ADP-ribosyl)ation reactions also play an important role in DNA repair as a consequence of oxidative damage and single-strand breaks. Such a reaction may occur also with the 20S proteasome-with a known increase in enzymatic activity-and also with histones-reducing their proteolytic susceptibility as shown for the first time here. After hydrogen peroxide treatment of HT22 cells, degradation of the model peptide substrate suc-LLVY-MCA and degradation of oxidized histones by nuclear proteasome increased. During the removal of protein carbonyls, single-strand breaks and 8-hydroxy-2'-deoxyguanosine, proteasome, and poly(ADP-ribose) polymerase-1 enzymes were shown to play tightly interacting roles. Our results following the repair of oxidative damage show the proteolytic activation of proteasome concerning poly(ADP-ribosyl)ation together with a decline in poly(ADP-ribosyl)ation of oxidized histones, leading to a selective recognition of oxidatively modified histones. (C) 2009 Elsevier Inc. All rights reserved.

Several studies have demonstrated that proteasome activity decreases whereas protein oxidation increases with aging in various tissues. However, no studies are available correlating both parameters directly comparing different tissues of one organism. Therefore, we determined whether there is an age-related change in proteasome activity and protein oxidation in heart, lung, liver, kidney and skeletal muscle samples of 6-, 10-, 18- and 26-month-old rats. There was a significant age-related increase in protein carbonyls at 18 and 26 months compared to young rats. Thereby, protein carbonyl formation was rather due to a general than a specific protein carbonylation as shown by immunblot studies. The highest increase in protein carbonyl formation was found in liver, lung and kidney samples. Proteasome activity decreased significantly with age in lung and liver samples. Proteasome activity in liver and lung decreased by factor five compared to young rats. Strong correlations between proteasome activity and protein oxidation were found in liver and lung, whereas in other tissues only a trend was found. These results demonstrate that the increase in protein oxidation and the decline in proteasome activity are correlating. Further studies are needed to determine the mechanisms which cause organ-specific aging-rates and their consequences.

Free radical attack on beta-carotene results in the formation of high amounts of carotene breakdown products (CBPs) having biological activities. As several of the CBPs are reactive aldehydes, it has to be considered that these compounds are able to modify proteins. Therefore, the aim of the study was to investigate whether CBP-modification of proteins is leading to damaged proteins recognized and degraded by the proteasomal system. We used the model proteins tau and ferritin to test whether CBPs will modify them and whether such modifications lead to enhanced proteasomal degradation. To modify proteins, we used crude CBPs as a mixture obtained after hypochloric acid derived BC degradation, as well as several single compounds, as apo8'-carotenal, retinal, or beta-ionone. The majority of the CBPs found in our reaction mixture are well known metabolites as described earlier after BC degradation using different oxidants. CBPs are able to modify proteins, and in in vitro studies, we were able to demonstrate that the 20S proteasome is able to recognize and degrade CBP-modified proteins preferentially. In testing the proteolytic response of HT22 cells toward CBPs, we could demonstrate an enhanced protein turnover, which is sensitive to lactacystin. Interestingly, the proteasomal activity is resistant to treatment with CBP. On the other hand, we were able to demonstrate that supraphysiological levels of CBPs might lead to the formation of protein-CBP-adducts that are able to inhibit the proteasome. Therefore, the removal of CBP-modified proteins seems to be catalyzed by the proteasomal system and is effective, if the formation of CBPs is not overwhelming and leading to protein aggregates.

Many candidate biomarkers of human ageing have been proposed in the scientific literature but in all cases their variability in cross-sectional studies is considerable, and therefore no single measurement has proven to serve a useful marker to determine, on its own, biological age. A plausible reason for this is the intrinsic multi-causal and multi-system nature of the ageing process. The recently completed MARK-AGE study was a large-scale integrated project supported by the European Commission. The major aim of this project was to conduct a population study comprising about 3200 subjects in order to identify a set of biomarkers of ageing which, as a combination of parameters with appropriate weighting, would measure biological age better than any marker in isolation. (C) 2015 The Authors. Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Only a few studies have used objective measurements to investigate the relationship between sleep quality and obesity. These studies showed controversial results.Sleep efficiency was measured by Actiwatch 2 in 132 healthy students (age 23.3 ± 3.7 years, BMI 23.1 ± 4.1 kg/m(2) ) for 12 ± 3 nights, differentiating between work and free days. Physical activity, dietary habits, and autonomic function (heart rate variability, HRV) were analyzed as potential determinants of sleep quality and its relationship with body composition.Sleep efficiency was 87.0% in women and 84.9% in men (P 0.05) and was higher at free days when compared to work days in women (P 0.05). Lower sleep efficiency was associated with a higher fat mass. This was true for sleep efficiency on work days in women [fat mass index (FMI): r = -0.35, P 0.01] and for free days in men (FMI: r = -0.37, P 0.05). Poor sleep efficiency was associated with less physical activity (r = 0.29, P 0.05) and impaired HRV in women (r = 0.60, P 0.05) and with a higher fat intake in men (r = -0.39, P 0.05).Poor sleep efficiency was associated with higher fat mass. The relationship between sleep quality and fat mass differs between work and free days and may be explained by physical activity and autonomic function in women and dietary habits in men.

Within the MARK-AGE project, a population study (3337 subjects) was conducted to identify a set of biomarkers of ageing which, as a combination of parameters with appropriate weighting, would measure biological age better than any single marker. The MARK-AGE project involves 14 European countries and a total of 26 research centres. In such a study, standard operating procedures (SOPs) are an essential task, which are binding for all MARK-AGE Beneficiaries. The SOPs cover all aspects of subject's recruitment, collection, shipment and distribution of biological samples (blood and its components, buccal mucosa cells or BMC and urine) as well as the anthropometric measurements and questionnaires. published

Objective. Cognitive impairment reduces quality of life and is related to vascular and neurodegenerative disorders. However, there is also a close relationship between these diseases and oxidative stress. Thus, the purpose of this study was to assess whether inflammation and oxidative damage are associated with low cognitive performance in the elderly with different housing conditions.Methods. The study groups consisted of 32 institutionalized and 25 noninstitutionalized Brazilian elderly subjects. Oxidative damage, inflammation markers, and cognitive function were evaluated.Results. The results demonstrated pronounced oxidative stress in the institutionalized elderly group, which also had a lower antioxidant status compared to noninstitutionalized subjects. High levels of proinflammatory cytokines were also observed in the institutionalized elderly. Furthermore, the raised levels of inflammatory markers were correlated with increased oxidative stress, and both were associated with low cognitive performance. However, based on multiple linear regression analysis, oxidative stress appears to be the main factor responsible for the cognitive decline.Conclusions. The findings suggest that individuals with lower antioxidant status are more vulnerable to oxidative stress, which is associated with cognitive function, leading to reduced life quality and expectancy.

In two work packages of the MARK-AGE project, 37 immunological and physiological biomarkers were measured in 3637 serum, plasma or blood samples in five batches during a period of 4 years.The quality of the serum and plasma samples was very good as judged by the low number of biomarker measurements (only 0.2%) that were rejected because of a high hemolysis, icteria or lipemia of the samples.Using quality control samples, day-to-day and batch variations were determined. The mean inter-assay variation of the five batches were all below 8%, with an average inter-assay coefficient of variation of all biomarkers of 4.0%. Also the precision of the measurements was very good, because all measurements were between 90% and 115% of the defined target values.A possible mix-up of samples was determined by comparison of the extreme testosterone levels of men and women. It was concluded that 3% of the sample identification could be mixed-up.Considering the complex procedure from collection to analysis, including preparation, handling, shipment and storage, of the samples in the MARK-AGE project, both the quality of the samples and the quality of the measurements are very good. published

3-Nitrotyrosine (3NT) is known as an important indicator of nitrosative stress and has been linked to various diseases. Our aim was to develop an indirect ELISA (enzyme-linked immunosorbent assay) method suitable for the detection of protein-bound 3NT in clinical plasma and serum samples. Nitrated protein standards and reduced protein standards were prepared. Limit of detection was determined for standards; recovery and reproducibility were determined for human plasma samples. The limit of detection for this method is 1.82±0.56 pmol/mg protein. Mean recovery of standards was 95%. 3NT concentration in plasma samples of obese and normal weight subjects was determined to be between 2 pmol/mg and 19 pmol/mg. No time-consuming sample preparation or expensive laboratory equipment is required, and applied antibodies are commercially available. Sensitivity, rapid analysis time, possibilities of high throughput applications and small sample volumes make this ELISA attractive for use in clinical laboratories.

Photodynamic therapy (PDT) is a potential tool in cancer treatment. Today this therapy is established among others for the treatment of nonmelanoma skin cancer. However, the more dangerous skin cancer--the melanoma--still has to be removed by surgery. Therefore, we investigated the effects of PDT and additional administration of heme oxygenase I (HO-I) and poly(ADP-ribose) polymerase (PARP) inhibitors on the treatment of melanoma cells in comparison to nonmalignant keratinocytes. Therefore, cocultures were established with WM451LU melanoma cells and HaCaT keratinocytes. In the coculture some 65% melanoma cells and 35% HaCaT cells were present before PDT, whereas after PDT the proportion was 41% melanoma cells and 59% HaCaT cells. Combination of both inhibitors improves these results to only 16% melanoma cells and 84% HaCaT cells. PDT is, therefore, a potent skin cancer treatment, which might also be interesting for melanoma treatment. The cytotoxic effects of PDT are largely mediated by ROS. Addition of HO-I and PARP inhibitors could improve the efficiency of photodynamic treatment.

Oxidized and cross-linked modified proteins are known to accumulate in ageing. Little is known about whether the accumulation of proteins modified by advanced glycation end products (AGEs) is due to an affected intracellular degradation. Therefore, this study was designed to determine whether the intracellular enzymes cathepsin B, cathepsin D and the 20S proteasome are able to degrade AGE-modified proteins in vitro. It shows that AGE-modified albumin is degraded by cathepsin D, while cathepsin B was less effective in the degradation of aldehyde-modified albumin and the 20S proteasome was completely unable to degrade them. Mouse primary embryonic fibroblasts isolated from a cathepsin D knockout animals were found to have an extensive intracellular AGE-accumulation, mainly in lysosomes, and a reduction of AGE-modified protein degradation compared to cells isolated from wild type animals. In summary, it can be assumed that cathepsin D plays a significant role in the removal of AGE-modified proteins.

In physiological conditions intracellular radical formation is mostly due to mitochondrial activity. This is in contrast to clinical and pathophysiological situations, where the oxidant formation is additionally driven by xenobiotics and inflammation. Oxidative damage accumulation in macromolecules especially in proteins has been considered as a cause of cellular damage and pathology impairing the clinical outcome of patients. However, up to now strategies to measure oxidative stress in clinical settings are limited. A lot of parameters and techniques are available for the determination of oxidized proteins in biological systems. Unfortunately, most of them are no reliable markers in clinical settings due to their unknown clinical relevance or the lack in clinical feasibility. Major problems are the sample availability, sample stability and cost-, time- and man-power intensive methods. The present review focuses on the measurement of protein oxidation products from a chemical, biological, and medical point of view.

Lipid peroxidation products like malondialdehyde, 4-hydroxynonenal and F2-isoprostanes are widely used as markers of oxidative stress in vitro and in vivo. This study reports the results of a multi-laboratory validation study by COST Action B35 to assess inter-laboratory and intra-laboratory variation in the measurement of lipid peroxidation. Human plasma samples were exposed to UVA irradiation at different doses (0, 15 J, 20 J), encoded and shipped to 15 laboratories, where analyses of malondialdehyde, 4-hydroxynonenal and isoprostanes were conducted. The results demonstrate a low within-day-variation and a good correlation of results observed on two different days. However, high coefficients of variation were observed between the laboratories. Malondialdehyde determined by HPLC was found to be the most sensitive and reproducible lipid peroxidation product in plasma upon UVA treatment. It is concluded that measurement of malondialdehyde by HPLC has good analytical validity for inter-laboratory studies on lipid peroxidation in human EDTA-plasma samples, although it is acknowledged that this may not translate to biological validity. © 2010 Informa UK, Ltd.

Solar ultraviolet (UV) A radiation is a well known trigger of signaling responses in human skin fibroblasts. One important consequence of this stress response is the increased expression of matrix metalloproteinase-1 (MMP-1), which causes extracellular protein degradation and thereby contributes to photoaging of human skin. In the present study we identify the proteasome as an integral part of the UVA-induced, intracellular signaling cascade in human dermal fibroblasts. UVA-induced singlet oxygen formation was accompanied by protein oxidation, the cross-linking of oxidized proteins, and an inhibition of the proteasomal system. This proteasomal inhibition subsequently led to an accumulation of c-Jun and phosphorylated c-Jun and activation of activator protein-1, i.e. transcription factors known to control MMP-1 expression. Increased transcription factor activation was also observed if the proteasome was inhibited by cross-linked proteins or lactacystin, indicating a general mechanism. Most importantly, inhibition of the proteasome was of functional relevance for UVA-induced MMP-1 expression, because overexpression of the proteasome or the protein repair enzyme methionine sulfoxide reductase prevented the UVA-induced induction of MMP-1. These studies show that an environmentally relevant stimulus can trigger a signaling pathway, which links intracellular and extracellular protein degradation. They also identify the proteasome as an integral part of the UVA stress response.

Photodynamic therapy (PDT) is a promising antitumor treatment strategy. However, effectiveness of PDT is limited due to an initiation of rescue responses in tumor cells, including the induction of heme oxygenase-1 (HO-1). Furthermore, the main sources of free radical production in PDT-induced oxidative stress are not clear. Here, human melanoma cells were loaded with the photosensitizer 5-aminolevulinic acid and exposed to non-thermal light of 420-800 nm at different doses. It was shown that inhibition of HO-1 activity by zinc protoporphyrin IX increased PDT-induced cytotoxicity in a dose-dependent manner. Interestingly, the cytotoxic effects were not diminished by the simultaneous application of the iron chelator desferrioxamine. Importantly, PDT together with non-toxic doses of hemin increased the number of dead cells. From these results can be concluded that heme but not iron act as the main source of free radicals in PDT treatment. This is supported by the fact that during PDT ferritin is readily up-regulated, able to bind excess iron formed by the HO-1 action. The combined treatment of photosensitizers with HO-1 inhibitors might increase the effectiveness of PDT in tumor treatment.