Search

Summary: Recent studies have shown that elevated concentrations of unconjugated bilirubin (UCB) may be a protective host factor against the development of noncommunicable diseases (NCDs), whereas low levels of UCB are associated with the opposite effect. The results of this European study, in which 2,489 samples were tested for their UCB concentration using high-performance liquid chromatography (HPLC) and additional data from the MARK-AGE database were used for analysis, provide further evidence that elevated UCB concentrations are linked to a lower risk of developing NCDs and may act as a predictive marker of biological aging as individuals with elevated UCB concentrations showed favorable outcomes in metabolic health and oxidative-stress-related biomarkers. These findings underline the significance of studying individuals with moderate hyperbilirubinemia and investigate UCB routinely, also in the setting of aging, since this condition affects millions of people worldwide but has been underrepresented in clinical research and practice until now.

Despite powerful DNA repair systems, oxidative damage/modification to DNA is an inevitable side effect of metabolism, ionizing radiation, lifestyle habits, inflammatory pathologies such as type-2 diabetes or metabolic syndrome, cancer and natural aging.One of the most common oxidative DNA modifications is 8-OHdG (8‑hydroxy-2′-deoxyguanosine), which is the most widely used marker in research and clinical diagnostics. 8-OHdG is easily and specifically detectable in various samples such as urine, plasma, cells and tissues via a large variety of methods like ELISA, HPLC, chromatographic methods, and immunochemistry.Formed by oxidation of guanine and being representative for the degree of DNA damage, 8-OHdG can be also used as biomarker for risk assessment of various cancers as well as degenerative diseases.Here, we present a highly specific, self-developed 8-OHdG antibody in successful comparison to a commercially one, tested in cells (FF95, HCT116, and HT22) and intestinal tissue, focusing on automatized evaluation via fluorescence/confocal microscopy.

Abstract Skeletal muscle aging is characterized by the loss of muscle mass, strength and function, mainly attributed to the atrophy of glycolytic fibers. Underlying mechanisms driving the skeletal muscle functional impairment are yet to be elucidated. To unbiasedly uncover its molecular mechanisms, we recurred to gene expression and metabolite profiling in a glycolytic muscle, Extensor digitorum longus (EDL), from young and aged C57BL/6JRj mice. Employing multi-omics approaches we found that the main age-related changes are connected to mitochondria, exhibiting a downregulation in mitochondrial processes. Consistent is the altered mitochondrial morphology. We further compared our mouse EDL aging signature with human data from the GTEx database, reinforcing the idea that our model may recapitulate muscle loss in humans. We are able to show that age-related mitochondrial downregulation is likely to be detrimental, as gene expression signatures from commonly used lifespan extending interventions displayed the opposite direction compared to our EDL aging signature.

The aim of this review is to provide a comprehensive and simple graphical overview on carotenoids. The review describes in four detailed figures the course of carotenoids from food to skin. Differences in chemical structures of the six most prominent carotenoids, namely, α-carotene, β-carotene, lutein, zeaxanthin, lycopene and β-cryptoxanthin, and their seasonal variations in plasma concentrations resulting from different availability of fruits and vegetables are highlighted. Furthermore, factors affecting carotenoid status, sites of storage and how and where they can be assessed (dietary intake and in vivo) are discussed. The route of carotenoid transport after ingestion, transport across the enterocyte and receptors involved in carotenoid uptake in peripheral cells are shown followed by the routes of carotenoid delivery to the skin and reasons for variations in skin carotenoid status are visualized.

Despite beneficial cardiovascular effects, substantial long-term modulation of food pattern could only be achieved in a limited number of participants. The impact of attitude towards healthy nutrition (ATHN) on successful modulation of dietary behavior is unclear, especially in the elderly. We aimed to analyze whether the personal ATHN influences 12-month adherence to two different dietary intervention regimes within a 36-month randomized controlled trial. Methods: 502 subjects were randomized to an intervention group (IG; dietary pattern focused on high intake of unsaturated fatty acids (UFA), plant protein and fiber) or control group (CG; dietary recommendation in accordance with the German Society of Nutrition) within a 36-month dietary intervention trial. Sum scores for effectiveness, appreciation and practice of healthy nutrition were assessed using ATHN questionnaire during the trial (n = 344). Linear regression models were used to investigate associations between ATHN and dietary patterns at baseline and at month 12. Results: Retirement, higher education level, age and lower body mass index (BMI) were associated with higher ATHN sum scores. ATHN was similar in CG and IG. Higher baseline intake of polyunsaturated fatty acids (PUFA) and fiber as well as lower intake in saturated fatty acids (SFA) were associated with higher scores in practice in both groups. The intervention resulted in a stronger increase of UFA, protein and fiber in the IG after 12 months, while intake of SFA declined (p < 0.01). Higher scores in appreciation were significantly associated with higher intake of fiber and lower intake of SFA in the CG at month 12, whereas no associations between ATHN and macronutrient intake were observed in the IG after 12 months. Conclusions: While ATHN appeared to play a role in general dietary behavior, ATHN did not affect the success of the specific dietary intervention in the IG at month 12. Thus, the dietary intervention achieved a substantial modification of dietary pattern in the IG and was effective to override the impact of the individual ATHN on dietary behavior.

Transportation noise is a ubiquitous urban exposure. In 2018, the World Health Organization concluded that chronic exposure to road traffic noise is a risk factor for ischemic heart disease. In contrast, they concluded that the quality of evidence for a link to other diseases was very low to moderate. Since then, several studies on the impact of noise on various diseases have been published. Also, studies investigating the mechanistic pathways underlying noise-induced health effects are emerging. We review the current evidence regarding effects of noise on health and the related disease-mechanisms. Several high-quality cohort studies consistently found road traffic noise to be associated with a higher risk of ischemic heart disease, heart failure, diabetes, and all-cause mortality. Furthermore, recent studies have indicated that road traffic and railway noise may increase the risk of diseases not commonly investigated in an environmental noise context, including breast cancer, dementia, and tinnitus. The harmful effects of noise are related to activation of a physiological stress response and nighttime sleep disturbance. Oxidative stress and inflammation downstream of stress hormone signaling and dysregulated circadian rhythms are identified as major disease-relevant pathomechanistic drivers. We discuss the role of reactive oxygen species and present results from antioxidant interventions. Lastly, we provide an overview of oxidative stress markers and adverse redox processes reported for noise-exposed animals and humans. This position paper summarizes all available epidemiological, clinical, and preclinical evidence of transportation noise as an important environmental risk factor for public health and discusses its implications on the population level.

Abstract Selenium homeostasis depends on hepatic biosynthesis of selenoprotein P (SELENOP) and SELENOP-mediated transport from the liver to e.g. the brain. In addition, the liver maintains copper homeostasis. Selenium and copper metabolism are inversely regulated, as increasing copper and decreasing selenium levels are observed in blood during aging and inflammation. Here we show that copper treatment increased intracellular selenium and SELENOP in hepatocytes and decreased extracellular SELENOP levels. Hepatic accumulation of copper is a characteristic of Wilson’s disease. Accordingly, SELENOP levels were low in serum of Wilson’s disease patients and Wilson’s rats. Mechanistically, drugs targeting protein transport in the Golgi complex mimicked some of the effects observed, indicating a disrupting effect of excessive copper on intracellular SELENOP transport resulting in its accumulation in the late Golgi. Our data suggest that hepatic copper levels determine SELENOP release from the liver and may affect selenium transport to peripheral organs such as the brain.

Mitochondria play multifaceted roles in cellular function, and impairments across domains of mitochondrial biology are known to promote cellular integrated stress response (ISR) pathways as well as systemic metabolic adaptations. However, the temporal dynamics of specific mitochondrial ISR related to physiological variations in tissue-specific energy demands remains unknown. Here, we conducted a comprehensive 24-hour muscle and plasma profiling of male and female mice with ectopic mitochondrial respiratory uncoupling in skeletal muscle (mUcp1-transgenic, TG). TG mice are characterized by increased muscle ISR, elevated oxidative stress defense, and increased secretion of FGF21 and GDF15 as ISR-induced myokines. We observed a temporal signature of both cell-autonomous and systemic ISR in the context of endocrine myokine signaling and cellular redox balance, but not of ferroptotic signature which was also increased in TG muscle. We show a progressive increase of muscle ISR on transcriptional level during the active phase (night time), with a subsequent peak in circulating FGF21 and GDF15 in the early resting phase. Moreover, we found highest levels of muscle oxidative defense (GPX and NQO1 activity) between the late active to early resting phase, which could aim to counteract excessive iron-dependent lipid peroxidation and ferroptosis in muscle of TG mice. These findings highlight the temporal dynamics of cell-autonomous and endocrine ISR signaling under skeletal muscle mitochondrial uncoupling, emphasizing the importance of considering such dissociation in translational strategies and sample collection for diagnostic biomarker analysis.

Abstract Background Frailty development is partly dependent on multiple factors like low levels of nutrients and high levels of oxidative stress (OS) and inflammation potentially leading to a muscle‐catabolic state. Measures of specific biomarker patterns including nutrients, OS and inflammatory biomarkers as well as muscle related biomarkers like 3‐methylhistidine (3MH) may improve evaluation of mechanisms and the complex networks leading to frailty. Methods In 220 multi‐morbid patients (≥ 60 years), classified as non‐frail (n = 104) and frail (n = 116) according to Fried's frailty criteria, we measured serum concentrations of fat‐soluble micronutrients, amino acids (AA), OS, interleukins (IL) 6 and 10, 3MH (biomarker for muscle protein turnover) and serum spectra of fatty acids (FA). We evaluated biomarker patterns by principal component analysis (PCA) and their cross‐sectional associations with frailty by multivariate logistic regression analysis. Results Two biomarker patterns [principal components (PC)] were identified by PCA. PC1 was characterized by high positive factor loadings (FL) of carotenoids, anti‐inflammatory FA and vitamin D3 together with high negative FL of pro‐inflammatory FA, IL6 and IL6/IL10, reflecting an inflammation‐related pattern. PC2 was characterized by high positive FL of AA together with high negative FL of 3MH‐based biomarkers, reflecting a muscle‐related pattern. Frail patients had significantly lower factor scores than non‐frail patients for both PC1 [median: −0.27 (interquartile range: 1.15) vs. 0.27 (1.23); P = 0.001] and PC2 [median: −0.15 (interquartile range: 1.13) vs. 0.21 (1.38); P = 0.002]. Patients with higher PC1 or PC2 factor scores were less likely to be frail [odds ratio (OR): 0.62, 95% CI: 0.46–0.83, P = 0.001 for PC1; OR: 0.64, 95% CI: 0.48–0.86, P = 0.003 for PC2] compared with patients with lower PC1 or PC2 factor scores. This indicates that increasing levels of anti‐inflammatory biomarkers and increasing levels of muscle‐anabolic biomarkers are associated with a reduced likelihood (38% and 36%, respectively) for frailty. Significant associations remained after adjusting the regression models for potential confounders. Conclusions We conclude that two specific patterns reflecting either inflammation‐related or muscle‐related biomarkers are both significantly associated with frailty among multi‐morbid patients and that these specific biomarker patterns are more informative than single biomarker analyses considering frailty identification.

This study explores age- and time-dependent variations in postprandial micronutrient absorption after a micronutrient-rich intervention meal within the Biomiel (bioavailability of micronutrients in elderly) study. Comprising 43 healthy participants, the study compares young (n = 21; mean age 26.90 years) and old (n = 22; mean age 66.77 years) men and women, analyzing baseline concentrations and six-hour postprandial dynamics of iron (Fe), copper (Cu), zinc (Zn), selenium (Se), iodine (I), free zinc (fZn), vitamin C, retinol, lycopene, β-carotene, α-tocopherol, and γ-tocopherol, along with 25(OH) vitamin D (quantified only at baseline). Methodologically, quantifications in serum or plasma were performed at baseline and also at 90, 180, 270, and 360 min postprandially. Results reveal higher baseline serum Zn and plasma lycopene concentrations in the young group, whereas Cu, Se, Cu/Zn ratio, 25(OH) vitamin D, α-tocopherol, and γ-tocopherol were higher in old participants. Postprandial variability of Zn, vitamin C, and lycopene showed a strong time-dependency. Age-related differences in postprandial metabolism were observed for Se, Cu, and I. Nevertheless, most of the variance was explained by individuality. Despite some limitations, this study provides insights into postprandial micronutrient metabolism (in serum/plasma), emphasizing the need for further research for a comprehensive understanding of this complex field. Our discoveries offer valuable insights for designing targeted interventions to address and mitigate micronutrient deficiencies in older adults, fostering optimal health and well-being across the lifespan.

Inflammatory bowel disease (IBD) is an immune-mediated gut dysfunction, which might also be associated with an inflammatory phenotype in the liver. It is known that the nutritional intake of omega-3 polyunsaturated fatty acids (n-3 PUFA) is inversely correlated to the severity and occurrence of IBD. In order to investigate whether n-3 PUFA can also reduce liver inflammation and oxidative liver damage due to colon inflammation, we explored the dextran sulfate sodium (DSS)-induced colitis model in wild-type and fat-1 mice with endogenously increased n-3 PUFA tissue content. Besides confirming previous data of alleviated DSS-induced colitis in the fat-1 mouse model, the increase of n-3 PUFA also resulted in a significant reduction of liver inflammation and oxidative damage in colitis-affected fat-1 mice as compared to wild-type littermates. This was accompanied by a remarkable increase of established inflammation-dampening n-3 PUFA oxylipins, namely docosahexaenoic acid-derived 19,20-epoxydocosapentaenoic acid and eicosapentaenoic acid-derived 15-hydroxyeicosapentaenoic acid and 17,18-epoxyeicosatetraenoic acid. Taken together, these observations demonstrate a strong inverse correlation between the anti-inflammatory lipidome derived from n-3 PUFA and the colitis-triggered inflammatory changes in the liver by reducing oxidative liver stress.

Maintenance peritoneal dialysis (PD) is commonly associated with cardiovascular diseases (CVDs), whose risk is assessed via LDL-C. Nonetheless, oxidized LDL (oxLDL), as being a key component of atherosclerotic lesions, could be also associated with atherosclerosis and related CVDs. However, its predictive value for CVDs risk assessment is subject of research studies due to the lack of specific methods to measure oxLDL status from its individual lipid/protein components. In the present study, six novel oxLDL markers, representative of certain oxidative modifications on the LDL protein and lipid components, are measured in atherosclerosis-prone PD patients (39) versus those in chronic kidney disease patients (61) under hemodialysis (HD) and healthy controls (40). LDL from serum of PD, HD and control subjects were isolated and fractionated into cholesteryl esters, triglycerides, free cholesterol, phospholipids and apolipoprotein B100 (apoB100). Subsequently the oxLDL markers cholesteryl ester hydroperoxides (-OOH), triglyceride-OOH, free cholesterol-OOH, phospholipid-OOH, apoB100 malondialdehyde and apoB100 dityrosines were measured. LDL carotenoid levels and LDL particle serum concentration were also measured. The levels of all oxLDL lipid-OOH markers were significantly elevated in PD patients versus control, while the levels of cholesteryl ester-/triglyceride-/free cholesterol-OOH were significantly elevated in PD versus HD patients, regardless of patients’ underlying medical conditions, sex, age, PD type, clinical biochemical markers and medication. It should be noted that all fractionated lipid-OOH levels were inversely correlated with LDL-P concentration, while LDL-P concentration was not correlated with LDL-C in PD patients. Moreover, LDL carotenoids were significantly lower in PD patients versus control. The increased levels of oxLDL status specific markers in both PD and HD patients (compared to control), support a potential prognostic value of oxLDL regarding CVD risk assessment in both patient groups. Lastly, the study introduces the oxLDL peroxidation markers free cholesterol-OOH and cholesteryl ester-OOH as complementary to LDL-P number, and as possible alternatives to LDL-C.

The ketogenic diet (KD) is a high-fat, low-carbohydrate diet that has been reported to have neuroprotective effects. The health effects of KD might be linked to an altered gut microbiome, which plays a major role in host health, leading to neuroprotective effects via the gut-brain axis. However, results from different studies, most often based on the 16S rRNA gene and metagenome sequencing, have been inconsistent. In this study, we assessed the effect of a 4-week KD compared to a western diet (WD) on the colonic microbiome of female C57Bl/6J mice by analyzing fecal samples using fluorescence in situ hybridization. Our results showed distinct changes in the total number of gut bacteria following the 4-week KD, in addition to changes in the composition of the microbiome. KD-fed mice showed higher absolute numbers of Actinobacteria (especially Bifidobacteria spp.) and lower absolute levels of Proteobacteria, often linked to gut inflammation, in comparison with WD-fed mice. Furthermore, an increased abundance of the typically rare genus Atopobium was observed. These changes may indicate the possible anti-inflammatory effects of the KD. However, since the overall changes in the microbiota seem low, the KD effects might be linked to the differential abundance of only a few key genera in mice.

As a dietary intervention, methionine restriction (MR) has been reported to increase longevity and improve metabolism disorders. However, the effects of MR on alleviating neurodegenerative diseases such as Alzheimer's disease (AD) are largely unexplored. Here we sought to investigate the neuroprotective effects of low methionine intake in mild cognitive impairment (MCI) patients and APP/PS1 AD model mice, and to uncover the underlying mechanisms. In a cohort composed of 45 individuals diagnosed with MCI and 61 healthy controls without cognitive impairment, methionine intake was found to be positively associated with the increased risk of MCI, where no sex differences were observed. We further conducted a 16-week MR intervention (0.17% methionine, w/w) on APP/PS1 AD model mice. Although MR reduced Aβ accumulation in the brain of both male and female APP/PS1 mice, MR improved cognitive function only in male mice, as assessed by the Morris water maze test. Consistently, MR restored synapse ultrastructure and alleviated mitochondrial dysfunction by enhancing mitochondrial biogenesis in the brain of male APP/PS1 mice. Importantly, MR effectively balanced the redox status and activated cystathionine-β-synthase (CBS)/H2S pathway in the brain of male APP/PS1 mice. Together, our study indicated that lower dietary methionine intake is associated with improved cognitive function, in which CBS/H2S pathway plays an essential role. MR could be a promising nutritional intervention for preventing AD development.

Methionine is an essential amino acid, involved in the promotion of growth, immunity, and regulation of energy metabolism. Over the decades, research has long focused on the beneficial effects of methionine supplementation, while data on positive effects of methionine restriction (MR) were first published in 1993. MR is a low-methionine dietary intervention that has been reported to ameliorate aging and aging-related health concomitants and diseases, such as obesity, type 2 diabetes, and cognitive disorders. In addition, MR seems to be an approach to prolong lifespan which has been validated extensively in various animal models, such as Caenorhabditis elegans, Drosophila, yeast, and murine models. MR appears to be associated with a reduction in oxidative stress via so far mainly undiscovered mechanisms, and these changes in redox status appear to be one of the underlying mechanisms for lifespan extension and beneficial health effects. In the present review, the association of methionine metabolism pathways with redox homeostasis is described. In addition, the effects of MR on lifespan, age-related implications, comorbidities, and diseases are discussed.

Mice with constitutive disruption of the Selenop gene have been key to delineate the importance of selenoproteins in neurobiology. However, the phenotype of this mouse model is exquisitely dependent on selenium supply and timing of selenium supplementation. Combining biochemical, histological, and behavioral methods, we tested the hypothesis that parvalbumin-expressing interneurons in the primary somatosensory cortex and hippocampus depend on dietary selenium availability in Selenop−/− mice. Selenop-deficient mice kept on adequate selenium diet (0.15 mg/kg, i.e. the recommended dietary allowance, RDA) developed ataxia, tremor, and hyperexcitability between the age of 4–5 weeks. Video-electroencephalography demonstrated epileptic seizures in Selenop−/− mice fed the RDA diet, while Selenop ± heterozygous mice behaved normally. Both neurological phenotypes, hyperexcitability/seizures and ataxia/dystonia were successfully prevented by selenium supplementation from birth or transgenic expression of human SELENOP under a hepatocyte-specific promoter. Selenium supplementation with 10 μM selenite in the drinking water on top of the RDA diet increased the activity of glutathione peroxidase in the brains of Selenop−/− mice to control levels. The effects of selenium supplementation on the neurological phenotypes were dose- and time-dependent. Selenium supplementation after weaning was apparently too late to prevent ataxia/dystonia, while selenium withdrawal from rescued Selenop−/− mice eventually resulted in ataxia. We conclude that SELENOP expression is essential for preserving interneuron survival under limiting Se supply, while SELENOP appears dispensable under sufficiently high Se status.

Glucosinolates are plant secondary metabolites found in cruciferous vegetables (Brassicaceae) that are valued for their potential health benefits. Frequently consumed representatives of these vegetables, for example, are white or red cabbage, which are typically boiled before consumption. Recently, 3-alk(en)yl-4-hydroxythiazolidine-2-thiones were identified as a class of thermal glucosinolate degradation products that are formed during the boiling of cabbage. Since these newly discovered compounds are frequently consumed, this raises questions about their potential uptake and their possible bioactive functions. Therefore, 3-allyl-4-hydroxythiazolidine-2-thione (allyl HTT) and 4-hydroxy-3-(4-(methylsulfinyl) butyl)thiazolidine-2-thione (4-MSOB HTT) as degradation products of the respective glucosinolates sinigrin and glucoraphanin were investigated. After consumption of boiled red cabbage broth, recoveries of consumed amounts of the degradation products in urine collected for 24 h were 18 ± 5% for allyl HTT and 21 ± 4% for 4-MSOB HTT (mean ± SD, n = 3). To investigate the stability of the degradation products during uptake and to elucidate the uptake mechanism, both an in vitro stomach and an in vitro intestinal model were applied. The results indicate that the uptake of allyl HTT and 4-MSOB HTT occurs by passive diffusion. Both compounds show no acute cell toxicity, no antioxidant potential, and no change in NAD(P)H dehydrogenase quinone 1 (NQO1) activity up to 100 μM. However, inhibition of glycogen synthase kinases-3 (GSK-3) in the range of 20% for allyl HTT for the isoform GSK-3β and 29% for 4-MSOB HTT for the isoform GSK-3α at a concentration of 100 μM was found. Neither health-promoting nor toxic effects of 3-alk(en)yl-4-hydroxythiazolidine-2-thiones were found in the four tested assays carried out in this study, which contrasts with the properties of other glucosinolate degradation products, such as isothiocyanates.

The steady-state redox status is physiologically important and therefore homeostatically maintained. Changes in the status result in signaling (eustress) or oxidative damage (distress). Oxidative stress (OS) is a hard-to-quantitate term that can be estimated only based on different biomarkers. Clinical application of OS, particularly for selective antioxidant treatment of people under oxidative stress, requires quantitative evaluation and is limited by the lack of universal biomarkers to describe it. Furthermore, different antioxidants have different effects on the redox state. Hence, as long as we do not have the possibility to determine and quantify OS, therapeutic interventions by the “identify-and-treat” approach cannot be assessed and are, therefore, not likely to be the basis for selective preventive measures against oxidative damage.

An inadequate selenium (Se) status can accelerate the aging process, increasing the vulnerability to age-related diseases. The study aimed to investigate plasma Se and Se species in a large population, including 2200 older adults from the general population (RASIG), 514 nonagenarian offspring (GO), and 293 GO Spouses (SGO). Plasma Se levels in women exhibit an inverted U-shaped pattern, increasing with age until the post-menopausal period and then declining. Conversely, men exhibit a linear decline in plasma Se levels with age. Subjects from Finland had the highest plasma Se values, while those from Poland had the lowest ones. Plasma Se was influenced by fish and vitamin consumption, but there were no significant differences between RASIG, GO, and SGO. Plasma Se was positively associated with albumin, HDL, total cholesterol, fibrinogen, and triglycerides and negatively associated with homocysteine. Fractionation analysis showed that Se distribution among plasma selenoproteins is affected by age, glucometabolic and inflammatory factors, and being GO or SGO. These findings show that sex-specific, nutritional, and inflammatory factors play a crucial role in the regulation of Se plasma levels throughout the aging process and that the shared environment of GO and SGO plays a role in their distinctive Se fractionation.

Background: Frailty is gaining importance in cardiothoracic surgery and is a risk factor for adverse outcomes and mortality. Various frailty scores have since been developed, but there is no consensus which to use for cardiac surgery. Methods: In an all-comer prospective study of patients presenting for cardiac surgery, we assessed frailty and analyzed complication rates in hospital and 1-year mortality, as well as laboratory markers before and after surgery. Results: 246 included patients were analyzed. A total of 16 patients (6.5%) were frail, and 130 patients (52.85%) were pre-frail, summarized in the frail group (FRAIL) and compared to the non-frail patients (NON-FRAIL). The mean age was 66.5 ± 9.05 years, 21.14% female. The in-hospital mortality rate was 4.88% and the 1-year mortality rate was 6.1%. FRAIL patients stayed longer in hospital (FRAIL 15.53 ± 8.5 days vs. NON-FRAIL 13.71 ± 8.94 days, p = 0.004) and in intensive/intermediate care units (ITS/IMC) (FRAIL 5.4 ± 4.33 days vs. NON-FRAIL 4.86 ± 4.78 days, p = 0.014). The 6 min walk (6 MW) (317.92 ± 94.17 m vs. 387.08 ± 93.43 m, p = 0.006), mini mental status (MMS) (25.72 ± 4.36 vs. 27.71 ± 1.9, p = 0.048) and clinical frail scale (3.65 ± 1.32 vs. 2.82 ± 0.86, p = 0.005) scores differed between patients who died within the first year after surgery compared to those who survived this period. In-hospital stay correlated with timed up-and-go (TUG) (TAU: 0.094, p = 0.037), Barthel index (TAU-0.114, p = 0.032), hand grip strength (TAU-0.173, p < 0.001), and EuroSCORE II (TAU 0.119, p = 0.008). ICU/IMC stay duration correlated with TUG (TAU 0.186, p < 0.001), 6 MW (TAU-0.149, p = 0.002), and hand grip strength (TAU-0.22, p < 0.001). FRAIL patients had post-operatively altered levels of plasma-redox-biomarkers and fat-soluble micronutrients. Conclusions: frailty parameters with the highest predictive value as well as ease of use could be added to the EuroSCORE.

The assessment of dietary carotenoids via blood measurements has been widely used as a marker for fruit and vegetable consumption. In the present study, modern, non-invasive approaches to assess dietary carotenoids, such as skin measurements and an app-based short dietary record (ASDR), were compared with conventional methods such as plasma status and handwritten 3-day dietary records. In an 8-week observational study, 21 healthy participants aged 50–65 years recorded their daily consumption of carotenoid-rich fruits and vegetables via a specially developed ASDR. Anthropometry, blood samplings and assessment of skin carotenoids via Raman and reflection spectroscopy were performed at baseline, after four weeks and at the end of the study. App-based intake data showed good correlations with plasma α-carotene (r = 0.74, p < 0.0001), β-carotene (r = 0.71, p < 0.0001), and total plasma carotenoids (r = 0.65, p < 0.0001); weak correlations with plasma lutein/zeaxanthin and β-cryptoxanthin (both r = 0.34, p < 0.05); and no correlation with plasma lycopene. Skin measurements via reflection and Raman spectroscopy correlated well with total plasma carotenoids (r = 0.81 and 0.72, respectively; both p < 0.0001), α-carotene (r = 0.75–0.62, p < 0.0001), and β-carotene (r = 0.79–0.71, p < 0.0001); moderately with plasma lutein/zeaxanthin (both r = 0.51, p < 0.0001); weakly with plasma β-cryptoxanthin (r = 0.40–0.31, p < 0.05); and showed no correlation with plasma lycopene. Skin measurements could provide a more convenient and noninvasive approach of estimating a person’s fruit and vegetable consumption compared to traditional methods, especially in studies that do not intend blood sampling. ASDR records might function as a suitable, convenient tool for dietary assessment in nutritional intervention studies.

The influence of nutritional factors on frailty syndrome is still poorly understood. Thus, we aimed to confirm cross-sectional associations of diet-related blood biomarker patterns with frailty and pre-frailty statuses in 1271 older adults from four European cohorts. Principal component analysis (PCA) was performed based on plasma levels of α-carotene, β-carotene, lycopene, lutein + zeaxanthin, β-cryptoxanthin, α-tocopherol, γ-tocopherol and retinol. Cross-sectional associations between biomarker patterns and frailty status, according to Fried’s frailty criteria, were assessed by using general linear models and multinomial logistic regression models as appropriate with adjustments for the main potential confounders. Robust subjects had higher concentrations of total carotenoids, β-carotene and β-cryptoxanthin than frail and pre-frail subjects and had higher lutein + zeaxanthin concentrations than frail subjects. No associations between 25-Hydroxyvitamin D3 and frailty status were observed. Two distinct biomarker patterns were identified in the PCA results. The principal component 1 (PC1) pattern was characterized by overall higher plasma levels of carotenoids, tocopherols and retinol, and the PC2 pattern was characterized by higher loadings for tocopherols, retinol and lycopene together and lower loadings for other carotenoids. Analyses revealed inverse associations between PC1 and prevalent frailty. Compared to participants in the lowest quartile of PC1, those in the highest quartile were less likely to be frail (odds ratio: 0.45, 95% CI: 0.25–0.80, p = 0.006). In addition, those in the highest quartile of PC2 showed higher odds for prevalent frailty (2.48, 1.28–4.80, p = 0.007) than those in the lowest quartile. Our findings strengthen the results from the first phase of the FRAILOMIC project, indicating carotenoids are suitable components for future biomarker-based frailty indices.

Abstract Background A poor fat‐soluble micronutrient (FMN) and a high oxidative stress status are associated with frailty. Our aim was to determine the cross‐sectional association of FMNs and oxidative stress biomarkers [protein carbonyls (PrCarb) and 3‐nitrotyrosine] with the frailty status in participants older than 65 years. Methods Plasma levels of vitamins A (retinol), D3, E (α‐tocopherol and γ‐tocopherol) and carotenoids (α‐carotene and β‐carotene, lycopene, lutein/zeaxanthin, and β‐cryptoxanthin), PrCarb, and 3‐nitrotyrosine were measured in 1450 individuals of the FRAILOMIC initiative. Participants were classified into robust, pre‐frail, and frail using Fried's frailty criteria. Associations between biomarkers and frailty status were assessed by general linear and logistic regression models, both adjusted for cohort, season of blood sampling, gender, age, height, weight, and smoking. Results Robust participants had significantly higher vitamin D3 and lutein/zeaxanthin concentrations than pre‐frail and frail subjects; had significantly higher γ‐tocopherol, α‐carotene, β‐carotene, lycopene, and β‐cryptoxanthin concentrations than frail subjects, and had significantly lower PrCarb concentrations than frail participants in multivariate linear models. Frail subjects were more likely to be in the lowest than in the highest tertile for vitamin D3 (adjusted odds ratio: 2.15; 95% confidence interval: 1.42–3.26), α‐tocopherol (2.12; 1.39–3.24), α‐carotene (1.69; 1.00–2.88), β‐carotene (1.84; 1.13–2.99), lycopene (1.94; 1.24–3.05), lutein/zeaxanthin (3.60; 2.34–5.53), and β‐cryptoxanthin (3.02; 1.95–4.69) and were more likely to be in the highest than in the lowest tertile for PrCarb (2.86; 1.82–4.49) than robust subjects in multivariate regression models. Conclusions Our study indicates that both low FMN and high PrCarb concentrations are associated with pre‐frailty and frailty.

Background Degenerative aortic valve (AoV) disease and resulting aortic stenosis are major clinical health problems. Murine models of valve disease are rare, resulting in a translational knowledge gap on underlying mechanisms, functional consequences, and potential therapies. Naïve New Zealand obese (NZO) mice were recently found to have a dramatic decline of left ventricular (LV) function at early age. Therefore, we aimed to identify the underlying cause of reduced LV function in NZO mice. Methods and Results Cardiac function and pulmonary hemodynamics of NZO and age‐matched C57BL/6J mice were monitored by serial echocardiographic examinations. AoVs in NZO mice demonstrated extensive thickening, asymmetric aortic leaflet formation, and cartilaginous transformation of the valvular stroma. Doppler echocardiography of the aorta revealed increased peak velocity profiles, holodiastolic flow reversal, and dilatation of the ascending aorta, consistent with aortic stenosis and regurgitation. Compensated LV hypertrophy deteriorated to decompensated LV failure and remodeling, as indicated by increased LV mass, interstitial fibrosis, and inflammatory cell infiltration. Elevated LV pressures in NZO mice were associated with lung congestion and cor pulmonale, evident as right ventricular dilatation, decreased right ventricular function, and increased mean right ventricular systolic pressure, indicative for the development of pulmonary hypertension and ultimately right ventricular failure. Conclusions NZO mice demonstrate as a novel murine model to spontaneously develop degenerative AoV disease, aortic stenosis, and the associated end organ damages of both ventricles and the lung. Closely mimicking the clinical scenario of degenerative AoV disease, the model may facilitate a better mechanistic understanding and testing of novel treatment strategies in degenerative AoV disease.

Removal of moderately oxidized proteins is mainly carried out by the proteasome, while highly modified proteins are no longer degradable. However, in the case of proteins modified by nitration of tyrosine residues to 3-nitrotyrosine (NO2Y), the role of the proteasome remains to be established. For this purpose, degradation assays and mass spectrometry analyses were performed using isolated proteasome and purified fractions of native cytochrome c (Cyt c) and tyrosine nitrated proteoforms (NO2Y74-Cyt c and NO2Y97-Cyt c). While Cyt c treated under mild conditions with hydrogen peroxide was preferentially degraded by the proteasome, NO2Y74- and NO2Y97-Cyt c species did not show an increased degradation rate with respect to native Cyt c. Peptide mapping analysis confirmed a decreased chymotrypsin-like cleavage at C-terminal of NO2Y sites within the protein, with respect to unmodified Y residues. Additionally, studies with the proteasome substrate suc-LLVY-AMC (Y-AMC) and its NO2Y-containing analog, suc-LLVNO2Y-AMC (NO2Y-AMC) were performed, both using isolated 20S-proteasome and astrocytoma cell lysates as the proteasomal source. Comparisons of both substrates showed a significantly decreased proteasome activity towards NO2Y-AMC. Moreover, NO2Y-AMC, but not Y-AMC degradation rates, were largely diminished by increasing the reaction pH, suggesting an inhibitory influence of the additional negative charge contained in NO2Y-AMC secondary to nitration. The mechanism of slowing of proteasome activity in NO2Y-contaning peptides was further substantiated in studies using the phenylalanine and nitro-phenylalanine peptide analog substrates. Finally, degradation rates of Y-AMC and NO2Y-AMC with proteinase K were the same, demonstrating the selective inability of the proteasome to readily cleave at nitrotyrosine sites. Altogether, data indicate that the proteasome has a decreased capability to cleave at C-terminal of NO2Y residues in proteins with respect to the unmodified residues, making this a possible factor that decreases the turnover of oxidized proteins, if they are not unfolded, and facilitating the accumulation of nitrated proteins.

The presence of circulating microbiome in blood has been reported in both physiological and pathological conditions, although its origins, identities and function remain to be elucidated. This study aimed to investigate the presence of blood microbiome by quantitative real-time PCRs targeting the 16S rRNA gene. To our knowledge, this is the first study in which the circulating microbiome has been analyzed in such a large sample of individuals since the study was carried out on 1285 Randomly recruited Age-Stratified Individuals from the General population (RASIG). The samples came from several different European countries recruited within the EU Project MARK-AGE in which a series of clinical biochemical parameters were determined. The results obtained reveal an association between microbial DNA copy number and geographic origin. By contrast, no gender and age-related difference emerged, thus demonstrating the role of the environment in influencing the above levels independent of age and gender at least until the age of 75. In addition, a significant positive association was found with Free Fatty Acids (FFA) levels, leukocyte count, insulin, and glucose levels. Since these factors play an essential role in both health and disease conditions, their association with the extent of the blood microbiome leads us to consider the blood microbiome as a potential biomarker of human health.

When the amount of reactive oxygen species produced by human metabolism cannot be balanced by antioxidants, this phenomenon is commonly referred to as oxidative stress. It is hypothesised that diets with high amounts of plant food products may have a beneficial impact on oxidative stress status. However, few studies have examined whether a vegan diet is associated with lower oxidative stress compared to an omnivorous diet. The present cross-sectional study aimed to compare the levels of five oxidative stress biomarkers in vegans and omnivores. Data of 36 vegans and 36 omnivores from Germany and of 21 vegans and 18 omnivores from Finland were analysed. HPLC coupled with mass spectrometry or fluorescence detection and ELISA methods were used to measure the oxidative stress biomarkers malondialdehyde (MDA), protein carbonyls and 3-nitrotyrosine in plasma and 8-hydroxy-2′-deoxyguanosine (8-OHdG) and 8-iso-prostaglandin F2α (8-iso-PGF2α) in 24 h urine. Analyses of variance and covariance, considering potential confounders, were used. Vegans and omnivores showed no differences in MDA and protein carbonyl concentrations. In Finnish but not in German vegans, the concentrations of 3-nitrotyrosine were lower compared to those in omnivores (p = 0.047). In Germany, vegans showed lower excretion levels of 8-iso-PGF2α than omnivores (p = 0.002) and with a trend also of 8-OHdG (p = 0.05). The sensitivity analysis suggests lower 8-iso-PGF2α excretion levels in women compared to men, independently of the dietary group. The present study contributes to expanding our knowledge of the relationship between diet and oxidative stress and showed that 3-nitrotyrosine, 8-OHdG and 8-iso-PGF2α tended to be lower in vegans. Furthermore, studies are recommended to validate the present findings.

Background: The number of people above the age of 60 years is raising world-wide being associated with an increase in the prevalence of aging-associated impairments and even diseases. Recent studies suggest that aging is associated with alterations in bacterial endotoxin levels and that these changes may add to low-grade inflammation, the so-called ‘inflammaging’, and aging-associated liver degeneration. However, mechanisms involved, and especially, the interaction of intestinal microbiota and barrier in the development of aging-associated inflammation and liver degeneration have not been fully understood. Objective: The aim of the present study was to determine if intestinal microbiota composition changes with age and if these alterations are associated with changes of markers of intestinal barrier function and the development of inflammation and liver degeneration. Methods: Blood, liver, small and large intestinal tissue of male 2-, 15-, 24- and 30-months old C57BL/6 mice fed standard chow were obtained. Intestinal microbiota composition, expression levels of antimicrobial peptides in small intestine and markers of intestinal barrier function were measured. Furthermore, indices of liver damage, inflammation and expression levels of lipopolysaccharide binding protein (Lbp) as well as of toll-like receptors (Tlr) 1-9 in liver tissue were assessed. Results: Pairwise comparisons of the microbial community in the small intestine showed differences between 2- and 24-, 15- and 24-, as well as 15- and 30-months old animals while Shannon’s diversity, species richness and evenness indexes did not differ in both small and large intestine, respectively, between age groups. Concentrations of nitric oxide were significantly lower in small intestine of 15-, 24- and 30-months old mice compared to 2-months old mice while mRNA expression of the antimicrobial peptides defensin alpha 1 and lysozyme 1 was unchanged. In contrast, in liver tissue, older age of animals was associated with increasing inflammation and the development of fibrosis in 24- and 30-months old mice. Numbers of inflammatory foci and neutrophils in livers of 24- and 30-months old mice were significantly higher compared to 2-months old mice. These alterations were also associated with higher endotoxin levels in plasma as well as an increased mRNA expression of Lbp and Tlr1, Tlr2, Tlr4, Tlr6 and Tlr9 in livers in older mice. Conclusion: Despite no consistent and robust changes of microbiota composition in small and/or large intestine of mice of different age were observed, our data suggest that alterations of markers of intestinal barrier function in small intestine are associated with an induction of several Tlrs and beginning hepatic inflammation in older mice and increase with age.

Abstract Background There is a growing interest in the role of inflammageing for chronic disease development. Cytokines are potent soluble immune mediators that can be used as target biomarkers of inflammageing; however, their measurement in human samples has been challenging. This study aimed to assess the reliability of a pro- and anti-inflammatory cytokine panel in a sample of healthy people measured with a novel electrochemiluminescent multiplex immunoassay platform (Meso Scale Discovery, MSD), and to characterize their associations with metabolic and inflammatory phenotypes. Results Overall, the majority of cytokines were above the limit of detection (in at least 85.3% of the samples). Cytokines IL-6, IL-8, TNF-α, IL-10, IL-13, and IFN-γ showed overall good to fair reliability (ICC > 0.40), whereas IL-1β, IL-2, IL-4, and IL-12p70 showed poor reliability (ICC

Abstract Echocardiography is the most commonly applied technique for non-invasive assessment of cardiac function in small animals. Manual tracing of endocardial borders is time consuming and varies with operator experience. Therefore, we aimed to evaluate a novel automated two-dimensional software algorithm (Auto2DE) for small animals and compare it to the standard use of manual 2D-echocardiographic assessment (2DE). We hypothesized that novel Auto2DE will provide rapid and robust data sets, which are in agreement with manually assessed data of animals. 2DE and Auto2DE were carried out using a high-resolution imaging-system for small animals. First, validation cohorts of mouse and rat cine loops were used to compare Auto2DE against 2DE. These data were stratified for image quality by a blinded expert in small animal imaging. Second, we evaluated 2DE and Auto2DE in four mouse models and four rat models with different cardiac pathologies. Automated assessment of LV function by 2DE was faster than conventional 2DE analysis and independent of operator experience levels. The accuracy of Auto2DE-assessed data in healthy mice was dependent on cine loop quality, with excellent agreement between Auto2DE and 2DE in cine loops with adequate quality. Auto2DE allowed for valid detection of impaired cardiac function in animal models with pronounced cardiac phenotypes, but yielded poor performance in diabetic animal models independent of image quality. Auto2DE represents a novel automated analysis tool for rapid assessment of LV function, which is suitable for data acquisition in studies with good and very good echocardiographic image quality, but presents systematic problems in specific pathologies.

Metabolic syndrome is a significant worldwide public health challenge and is inextricably linked to adverse renal and cardiovascular outcomes. The inhibition of the transient receptor potential cation channel subfamily C member 6 (TRPC6) has been found to ameliorate renal outcomes in the unilateral ureteral obstruction (UUO) of accelerated renal fibrosis. Therefore, the pharmacological inhibition of TPRC6 could be a promising therapeutic intervention in the progressive tubulo-interstitial fibrosis in hypertension and metabolic syndrome. In the present study, we hypothesized that the novel selective TRPC6 inhibitor SH045 (larixyl N-methylcarbamate) ameliorates UUO-accelerated renal fibrosis in a New Zealand obese (NZO) mouse model, which is a polygenic model of metabolic syndrome. The in vivo inhibition of TRPC6 by SH045 markedly decreased the mRNA expression of pro-fibrotic markers (Col1α1, Col3α1, Col4α1, Acta2, Ccn2, Fn1) and chemokines (Cxcl1, Ccl5, Ccr2) in UUO kidneys of NZO mice compared to kidneys of vehicle-treated animals. Renal expressions of intercellular adhesion molecule 1 (ICAM-1) and α-smooth muscle actin (α-SMA) were diminished in SH045- versus vehicle-treated UUO mice. Furthermore, renal inflammatory cell infiltration (F4/80+ and CD4+) and tubulointerstitial fibrosis (Sirius red and fibronectin staining) were ameliorated in SH045-treated NZO mice. We conclude that the pharmacological inhibition of TRPC6 might be a promising antifibrotic therapeutic method to treat progressive tubulo-interstitial fibrosis in hypertension and metabolic syndrome.

Obesity has been linked to lower concentrations of fat-soluble micronutrients and higher concentrations of oxidative stress markers as well as an altered metabolism of branched chain amino acids and phospholipids. In the context of morbid obesity, the aim of this study was to investigate whether and to which extent plasma status of micronutrients, amino acids, phospholipids and oxidative stress differs between morbidly obese (n = 23) and non-obese patients (n = 13). In addition to plasma, malondialdehyde, retinol, cholesterol and triglycerides were assessed in visceral and subcutaneous adipose tissue in both groups. Plasma γ-tocopherol was significantly lower (p < 0.011) in the obese group while other fat-soluble micronutrients showed no statistically significant differences between both groups. Branched-chain amino acids (all p < 0.008) and lysine (p < 0.006) were significantly higher in morbidly obese patients compared to the control group. Malondialdehyde concentrations in both visceral (p < 0.016) and subcutaneous (p < 0.002) adipose tissue were significantly higher in the morbidly obese group while plasma markers of oxidative stress showed no significant differences between both groups. Significantly lower plasma concentrations of phosphatidylcholine, phosphatidylethanolamine, lyso-phosphatidylethanolamine (all p < 0.05) and their corresponding ether-linked analogs were observed, which were all reduced in obese participants compared to the control group. Pre-operative assessment of micronutrients in patients undergoing bariatric surgery is recommended for early identification of patients who might be at higher risk to develop a severe micronutrient deficiency post-surgery. Assessment of plasma BCAAs and phospholipids in obese patients might help to differentiate between metabolic healthy patients and those with metabolic disorders.

Overnutrition contributes to insulin resistance, obesity and metabolic stress, initiating a loss of functional beta-cells and diabetes development. Whether these damaging effects are amplified in advanced age is barely investigated. Therefore, New Zealand Obese (NZO) mice, a well-established model for the investigation of human obesity-associated type 2 diabetes, were fed a metabolically challenging diet with a high-fat, carbohydrate restricted period followed by a carbohydrate intervention in young as well as advanced age. Interestingly, while young NZO mice developed massive hyperglycemia in response to carbohydrate feeding, leading to beta-cell dysfunction and cell death, aged counterparts compensated the increased insulin demand by persistent beta-cell function and beta-cell mass expansion. Beta-cell loss in young NZO islets was linked to increased expression of thioredoxin-interacting protein (TXNIP), presumably initiating an apoptosis-signaling cascade via caspase-3 activation. In contrast, islets of aged NZOs exhibited a sustained redox balance without changes in TXNIP expression, associated with higher proliferative potential by cell cycle activation. These findings support the relevance of a maintained proliferative potential and redox homeostasis for preserving islet functionality under metabolic stress, with the peculiarity that this adaptive response emerged with advanced age in diabetes-prone NZO mice.

Neurodegenerative diseases (NDD) such as Alzheimer's (AD) and Parkinson's disease (PD) are distinct clinical entities, however, the aggregation of key neuronal proteins, presumably leading to neuronal demise appears to represent a common mechanism. It has become evident, that advanced glycation end products (AGEs) trigger the accumulation of such modified proteins, which eventually contributes to pathological aspect of NDDs. Increased levels of AGEs are found in amyloid plaques in AD brains and in both advanced and early PD (incidental Lewy body disease). The molecular mechanisms by which AGE dependent modifications may modulate the susceptibility towards NDDs, however, remain enigmatic and it is unclear, whether AGEs may serve as biomarker of NDD. In the present study, we examined AGEs (CML: Carboxymethyllysine and CEL: Carboxyethyllysine), markers of oxidative stress and micronutrients in the plasma of PD and AD patients and controls. As compared to healthy controls, AD females displayed lower levels of CEL while higher levels of CML were found in AD and PD patients. A somewhat similar pattern was observed for protein carbonyls (PC), revealing lower values exclusively in AD females, whereas AD males displayed significantly higher values compared to healthy controls and PD. Sex-specific differences were also observed for other relevant markers such as malondialdehyde, 3-nitrotyrosine, γ -tocopherols, retinol, plasma proteins and α-carotene, while α-tocopherols, β-carotene, lutein/zeaxanthin, β-cryptoxanthin and lycopene showed no relevant association. Taken together, our study suggests yet unappreciated differences of the distribution of AGEs among the sexes in NDD. We therefore suggest to make a clear distinction between sexes when analyzing oxidative (AGEs)-related stress and carbonyl-related stress and vitamins.

Regular consumption of fruits and vegetables, which is related to high plasma levels of lipid-soluble micronutrients such as carotenoids and tocopherols, is linked to lower incidences of various age-related diseases. Differences in lipid-soluble micronutrient blood concentrations seem to be associated with age. Our retrospective analysis included men and women aged 22–37 and 60–85 years from the Berlin Aging Study II. Participants with simultaneously available plasma samples and dietary data were included (n = 1973). Differences between young and old groups were found for plasma lycopene, α-carotene, α-tocopherol, β-cryptoxanthin (only in women), and γ-tocopherol (only in men). β-Carotene, retinol and lutein/zeaxanthin did not differ between young and old participants regardless of the sex. We found significant associations for lycopene, α-carotene (both inverse), α-tocopherol, γ-tocopherol, and β-carotene (all positive) with age. Adjusting for BMI, smoking status, season, cholesterol and dietary intake confirmed these associations, except for β-carotene. These micronutrients are important antioxidants and associated with lower incidence of age-related diseases, therefore it is important to understand the underlying mechanisms in order to implement dietary strategies for the prevention of age-related diseases. To explain the lower lycopene and α-carotene concentration in older subjects, bioavailability studies in older participants are necessary.

Adipogenesis is a fundamental process of white adipose tissue function, supporting lipid storage and release, while avoiding its spillover and ectopic accumulation in tissues and organs. During aging adipogenesis is impaired and among other factors, oxidative stress contributes to this process. Adipogenesis requires functional and dynamic mitochondria; however, this organelle itself becomes dysfunctional during aging and accounts for most of reactive oxygen species (ROS) production. Here, we evaluated whether oxidative stress impairs adipogenesis through functional impairment of mitodynamics by utilizing hyperoxia as a continuous source of oxidative stress while maintaining cellular viability. This negatively impacted mitochondrial function, including respiration and dynamics and ultimately blocked adipogenesis. Interestingly, this state was reversible by using the antidiabetic drug, Rosiglitazone, which reduced oxidative stress, restored mitochondrial dynamics and respiration and augmented adipogenesis. Moreover, in vitro results were in agreement with in vivo models of oxidative stress and aging, in which mice depleted of the superoxide dismutase enzyme 1 (SOD1) and old wild-type C57BL/6JRj mice demonstrated the same trend of adipogenic potential. Importantly, in humans the results follow the same pattern, showing a downregulation of adipogenic markers during aging. Since the levels of oxidative stress and peripheral insulin resistance increase with age, while adipogenesis decreases during aging, our model helps to understand a possible way to overcome physiologically low, steady stress conditions and restore adipogenesis, avoiding accumulation of deleterious hypertrophic adipocytes in favor of beneficial hyperplasia.

High-protein diet is a promising strategy for diabetes treatment supporting body weight control, improving glycaemic status, cardiovascular risk factors and reducing liver fat. Here, we investigated effects of diets high in animal (AP) or plant (PP) protein on oxidative stress and antioxidant status in individuals with type 2 diabetes (T2DM). 37 obese individuals (age 64.3 ± 1.0 years) with T2DM were randomized to an isocaloric diet (30 energy(E)% protein, 30 E% fat and 40 E% carbohydrates) rich in AP or PP for 6 weeks. Markers of oxidative and nitrosative stress and antioxidant status in plasma and nitrate/nitrite levels in urine were assessed. Gene expression in subcutaneous adipose tissue (SAT) was analysed by RNA-Seq and real-time PCR. Both AP and PP diets similarly reduced plasma levels of malondialdehyde (PAP = 0.003, PPP = 1.6 × 10−4) and protein carbonyls (PAP = 1.2 × 10−4, PPP = 3.0 × 10−5) over 6 weeks. Nitrotyrosine (NT) increased upon both AP and PP diets (PAP = 0.005, PPP = 0.004). SAT expression of genes involved in nitric oxide (NO) and oxidative stress metabolism and urine NO metabolite (nitrate/nitrite) levels were not changed upon both diets. Plasma levels of carotenoids increased upon PP diet, whereas retinol, alpha- and gamma-tocopherol slightly decreased upon both diets. AP and PP diets similarly improve oxidative stress but increase nitrosative stress markers in individuals with T2DM. Mechanisms of the NT regulation upon high-protein diets need further investigation. Keywords: Animal protein, Plant protein, Type 2 diabetes, Oxidative stress, Antioxidant status

Abstract Background The assessment of ventricular volumes using conventional echocardiography methods is limited with regards to the need of geometrical assumptions. In the present study, we aimed to evaluate a novel commercial system for three-dimensional echocardiography (3DE) in preclinical models by direct comparison with conventional 1D- and 2D-echocardiography (1DE; 2DE) and the gold-standard technique magnetic resonance imaging (MRI). Further, we provide a standard operating protocol for image acquisition and analysis with 3DE. Methods 3DE was carried out using a 30 MHz center frequency transducer coupled to a Vevo®3100 Imaging System. We evaluated under different experimental conditions: 1) in vitro phantom measurements served as controlled setting in which boundaries were clearly delineated; 2) a validation cohort composed of healthy C57BL/6 J mice and New Zealand Obese (NZO) mice was used in order to validate 3DE against cardiac MRI; 3) a standard mouse model of pressure overload induced-heart failure was investigated to estimate the value of 3DE. Results First, in vitro volumetry revealed good agreement between 3DE assessed volumes and the MRI-assessed volumes. Second, cardiac volume determination with 3DE showed smaller mean differences compared to cardiac MRI than conventional 1DE and 2DE. Third, 3DE was suitable to detect reduced ejection fractions in heart failure mice. Fourth, inter- and intra-observer variability of 3DE showed good to excellent agreement regarding absolute volumes in healthy mice, whereas agreement rates for the relative metrics ejection fraction and stroke volume demonstrated good to moderate observer variabilities. Conclusions 3DE provides a novel method for accurate volumetry in small animals without the need for spatial assumptions, demonstrating a technique for an improved analysis of ventricular function. Further validation work and highly standardized image analyses are required to increase reproducibility of this approach.

A new 2,4-dinitrophenylhydrazine (DNPH)-based photometric assay is developed for the quantification of carbonyls in protein samples from any biological source by protein carbonyl-DNPH hydrazone formation at acidic pH in the presence of denaturing urea, and subsequent hydrazone solubilization in the presence of SDS and stabilization from acid hydrolysis at pH 7.0. At this neutral (ntr) pH, interfering unreacted DNPH is uncharged and its thus increased hydrophobicity permits its 100% effective removal from the solubilizate with ethyl acetate/hexane wash. The ntrDNPH assay is more reliable and sensitive than the standard (std) DNPH photometric assay because it eliminates its main limitations: (i) interfering unreacted DNPH (pKa 1.55) that is nonspecifically bound to the TCA (pKa 0.7)-protein pellet is not effectively removed after wash with EtOH: ethyl acetate because it is positively charged, (ii) acid (TCA-induced) hydrolysis of the protein carbonyl-DNPH hydrazone, (iii) sample protein concentration re-determination, (iv) loss of sample acid (TCA)-soluble proteins, (v) DNA interference, and (vi) requires high protein quantity samples (≥ 1 mg). Considering ntrDNPH assay’s very low protein limit (1 µg), its cumulative and functional sensitivities are 2600- and 2000-fold higher than those of the stdDNPH assay, respectively. The present study elucidates the DNA interference mechanism on the stdDNPH assay, and also develops a standardized protocol for sample protein treatment and fractionation (into cytoplasmic/aqueous, membrane/lipid-bound, and histone/DNA-bound proteins; see Supplement section V) in order to ensure reproducible carbonyl determination on defined cell protein fractions, and to eliminate assay interference from protein samples containing (i) Cys sulfenic acid groups (via their neutralization with dithiothreitol), and (ii) DNA (via its removal by streptomycin sulfate precipitation). Lastly, the ntrDNPH assay determines carbonyl groups on cell wall polysaccharides, thus paving the way on studies to investigate cell walls acting as antioxidant defense in plants, fungi, bacteria and lichens. Keywords: Photometric method, 2,4-dinitrophenylhydrazine, Protein carbonyls, Cell wall polysaccharide carbonyls, Oxidative stress, DNA interference, Protein fractionation

A new fluorometric assay is presented for the ultrasensitive quantification of total protein carbonyls, and is based on their specific reaction with rhodamine B hydrazide (RBH), and the production of a protein carbonyl-RBH hydrazone the fluorescence of which (at ex/em 560/585 nm) is greatly enhanced by guanidine-HCl. Compared to the fluorescein-5-thiosemicarbazide (FTC)-based fluorometric assay, the RBH assay uses a 24-fold shorter reaction incubation time (1 h) and at least 1000-fold lower protein quantity (2.5 µg), and produces very reliable data that were verified by extensive standardization experiments. The protein carbonyl group detection sensitivity limit of the RBH assay, based on its standard curve, can be as low as 0.4 pmol, and even lower. Counting the very low protein limit of the RBH assay, its cumulative and functional sensitivity is 8500- and 800-fold higher than the corresponding ones for the FTC assay. Neither heme proteins hemoglobin and cytochrome c nor DNA interfere with the RBH assay. Keywords: Fluorometric method, Rhodamine B hydrazide, Protein carbonyls, Protein oxidation, Oxidative stress, DNA

Under oxidative stress 26S proteasomes suffer reversible disassembly into its 20S and 19S subunits, a process mediated by HSP70. This inhibits the degradation of polyubiquitinated proteins by the 26S proteasome and allows the degradation of oxidized proteins by a free 20S proteasome. Low fluxes of antimycin A-stimulated ROS production caused dimerization of mitochondrial peroxiredoxin 3 and cytosolic peroxiredoxin 2, but not peroxiredoxin overoxidation and overall oxidation of cellular protein thiols. This moderate redox imbalance was sufficient to inhibit the ATP stimulation of 26S proteasome activity. This process was dependent on reversible cysteine oxidation. Moreover, our results show that this early inhibition of ATP stimulation occurs previous to particle disassembly, indicating an intermediate step during the redox regulation of the 26S proteasome with special relevance under redox signaling rather than oxidative stress conditions.

Schizophrenia, depression and posttraumatic stress disorder (PTSD) are severe mental disorders and complicated diagnostic entities, due to their phenotypic, biological and genetic heterogeneity, unknown etiology, and poorly understood alterations in biological pathways and biological mechanisms. Disturbed homeostasis between overproduction of oxidant species, overcoming redox regulation and a lack of cellular antioxidant defenses, resulting in free radical-mediated pathology and subsequent neurotoxicity contributes to development of depression, schizophrenia and PTSD, their heterogeneous clinical presentation and resistance to treatment. Metabolomics is a discipline that combines different strategies with the aim to extract, detect, identify and quantify all metabolites that are present in a biological sample and might provide mechanistic insights into the etiology of various psychiatric disorders. Therefore, oxidative stress research combined with metabolomics might offer a novel approach in dissecting psychiatric disorders, since these data-driven but not necessarily hypothesis-driven methods might identify new targets, molecules and pathways responsible for development of schizophrenia, depression or PTSD. Findings from the oxidative research in psychiatry together with metabolomics data might facilitate development of specific and validated prognostic, therapeutic and clinical biomarkers. These methods might reveal bio-signatures of individual patients, leading to individualized treatment approach. In reviewing findings related to oxidative stress and metabolomics in selected psychiatric disorders, we have highlighted how these novel approaches might make a unique contribution to deeper understanding of psychopathological alterations underlying schizophrenia, depression and PTSD. Keywords: Schizophrenia, Depression, Posttraumatic stress disorder, Oxidative stress, Lipid peroxidation, Metabolomics, Biomarkers

Association of oxidative stress with carcinogenesis is well known, but not understood well, as is pathophysiology of oxidative stress generated during different types of anti-cancer treatments. Moreover, recent findings indicate that cancer associated lipid peroxidation might eventually help defending adjacent nonmalignant cells from cancer invasion. Therefore, untargeted metabolomics studies designed for advanced translational and clinical studies are needed to understand the existing paradoxes in oncology, including those related to controversial usage of antioxidants aiming to prevent or treat cancer. In this short review we have tried to put emphasis on the importance of pathophysiology of oxidative stress and lipid peroxidation in cancer development in relation to metabolic adaptation of particular types of cancer allowing us to conclude that adaptation to oxidative stress is one of the main driving forces of cancer pathophysiology. With the help of metabolomics many novel findings are being achieved thus encouraging further scientific breakthroughs. Combined with targeted qualitative and quantitative methods, especially immunochemistry, further research might reveal bio-signatures of individual patients and respective malignant diseases, leading to individualized treatment approach, according to the concepts of modern integrative medicine. Keywords: Carcinogenesis, Cancer, Oxidative stress, Lipid peroxidation, 4-hydroxynonenal, Glutathione, Metabolomics, Immunochemistry, Biomarkers, Omics science

Abstract Mitochondrial dysfunction is usually associated with various metabolic disorders and ageing. However, salutary effects in response to mild mitochondrial perturbations have been reported in multiple organisms, whereas molecular regulators of cell-autonomous stress responses remain elusive. We addressed this question by asking whether the nuclear factor erythroid-derived-like 2 (Nrf2), a transcription factor and master regulator of cellular redox status is involved in adaptive physiological responses including muscle mitohormesis. Using a transgenic mouse model with skeletal muscle-specific mitochondrial uncoupling and oxidative phosphorylation (OXPHOS) inefficiency (UCP1-transgenic, TG) we show that additional genetic ablation of Nrf2 abolishes an adaptive muscle NAD(P)H quinone dehydrogenase 1 (NQO1) and catalase induction. Deficiency of Nrf2 also leads to decreased mitochondrial respiratory performance although muscle functional integrity, fiber-type profile and mitochondrial biogenesis were not significantly altered. Importantly, Nrf2 ablation did not abolish the induction of key genes and proteins of muscle integrated stress response including the serine, one-carbon cycle, and glycine synthesis (SOG) pathway in TG mice while further increasing glutathione peroxidase (GPX) activity linked to increased GPX1 protein levels. Conclusively, our results tune down the functions controlled by Nrf2 in muscle mitohormesis and oxidative stress defense during mitochondrial OXPHOS inefficiency.

The production of reactive species is an inevitable by-product of metabolism and thus, life itself. Since reactive species are able to damage cellular structures, especially proteins, as the most abundant macromolecule of mammalian cells, systems are necessary which regulate and preserve a functional cellular protein pool, in a process termed “proteostasis”. Not only the mammalian protein pool is subject of a constant turnover, organelles are also degraded and rebuild. The most important systems for these removal processes are the “ubiquitin-proteasomal system” (UPS), the central proteolytic machinery of mammalian cells, mainly responsible for proteostasis, as well as the “autophagy-lysosomal system”, which mediates the turnover of organelles and large aggregates. Many age-related pathologies and the aging process itself are accompanied by a dysregulation of UPS, autophagy and the cross-talk between both systems. This review will describe the sources and effects of oxidative stress, preservation of cellular protein- and organelle-homeostasis and the effects of aging on proteostasis in mammalian cells.

The European Cooperation in Science and Technology (COST) provides an ideal framework to establish multi-disciplinary research networks. COST Action BM1203 (EU-ROS) represents a consortium of researchers from different disciplines who are dedicated to providing new insights and tools for better understanding redox biology and medicine and, in the long run, to finding new therapeutic strategies to target dysregulated redox processes in various diseases. This report highlights the major achievements of EU-ROS as well as research updates and new perspectives arising from its members. The EU-ROS consortium comprised more than 140 active members who worked together for four years on the topics briefly described below. The formation of reactive oxygen and nitrogen species (RONS) is an established hallmark of our aerobic environment and metabolism but RONS also act as messengers via redox regulation of essential cellular processes. The fact that many diseases have been found to be associated with oxidative stress established the theory of oxidative stress as a trigger of diseases that can be corrected by antioxidant therapy. However, while experimental studies support this thesis, clinical studies still generate controversial results, due to complex pathophysiology of oxidative stress in humans. For future improvement of antioxidant therapy and better understanding of redox-associated disease progression detailed knowledge on the sources and targets of RONS formation and discrimination of their detrimental or beneficial roles is required. In order to advance this important area of biology and medicine, highly synergistic approaches combining a variety of diverse and contrasting disciplines are needed.

Cardiac remodeling and contractile dysfunction are leading causes in hypertrophy-associated heart failure (HF), increasing with a population’s rising age. A hallmark of aged and diseased hearts is the accumulation of modified proteins caused by an impaired autophagy-lysosomal-pathway. Although, autophagy inducer rapamycin has been described to exert cardioprotective effects, it remains to be shown whether these effects can be attributed to improved cardiomyocyte autophagy and contractility. In vivo hypertrophy was induced by transverse aortic constriction (TAC), with mice receiving daily rapamycin injections beginning six weeks after surgery for four weeks. Echocardiographic analysis demonstrated TAC-induced HF and protein analyses showed abundance of modified proteins in TAC-hearts after 10 weeks, both reduced by rapamycin. In vitro, cardiomyocyte hypertrophy was mimicked by endothelin 1 (ET-1) and autophagy manipulated by silencing Atg5 in neonatal cardiomyocytes. ET-1 and siAtg5 decreased Atg5–Atg12 and LC3-II, increased natriuretic peptides, and decreased amplitude and early phase of contraction in cardiomyocytes, the latter two evaluated using ImageJ macro Myocyter recently developed by us. ET-1 further decreased cell contractility in control but not in siAtg5 cells. In conclusion, ET-1 decreased autophagy and cardiomyocyte contractility, in line with siAtg5-treated cells and the results of TAC-mice demonstrating a crucial role for autophagy in cardiomyocyte contractility and cardiac performance.

Liver fibrosis is a critical complication of obesity-induced fatty liver disease. Wnt1 inducible signaling pathway protein 1 (WISP1/CCN4), a novel adipokine associated with visceral obesity and insulin resistance, also contributes to lung and kidney fibrosis. The aim of the present study was to investigate the role of CCN4 in liver fibrosis in severe obesity. For this, human liver biopsies were collected from 35 severely obese humans (BMI 42.5 ± 0.7 kg/m2, age 46.7 ± 1.8 y, 25.7% males) during bariatric surgery and examined for the expression of CCN4, fibrosis, and inflammation markers. Hepatic stellate LX-2 cells were treated with human recombinant CCN4 alone or in combination with LPS or transforming growth factor beta (TGF-β) and examined for fibrosis and inflammation markers. CCN4 mRNA expression in the liver positively correlated with BMI and expression of fibrosis markers COL1A1, COL3A1, COL6A1, αSMA, TGFB1, extracellular matrix turnover enzymes TIMP1 and MMP9, and the inflammatory marker ITGAX/CD11c. In LX-2 cells, the exposure to recombinant CCN4 caused dose-dependent induction of MMP9 and MCP1. CCN4 potentiated the TGF-β-mediated induction of COL3A1, TIMP1, and MCP1 but showed no interaction with LPS treatment. Our results suggest a potential contribution of CCN4 to the early pathogenesis of obesity-associated liver fibrosis.