Circadian rhythm and time-of-day-effects of (anti)oxidant biomarkers for epidemiological studies.

Various biomarkers of oxidative stress and redox status have been used in a number of clinical and epidemiological studies related to diseases and conditions that involve disturbances of the redox balance. However, a comprehensive study of diurnal variations of a set of biomarkers has not been conducted so far. Therefore, the aim of this study is to investigate circadian rhythm and time-of-day-effects of a set of frequently used biomarkers of oxidative stress, redox and antioxidant status in serum/plasma. These biomarkers include Reactive Oxygen Metabolites (ROM), Biological Antioxidant Potency (BAP), Total Thiols in Proteins (TTP), high-sensitive C-Reactive Protein (CRP) and Uric Acid (UA). During a 24-hr study, blood sampling was conducted 6 times at 4-hr intervals. The presence of circadian rhythm was analyzed with CircWave analysis, whereas the effect of time was analyzed with Repeated Measures ANOVA (RM-ANOVA). Thereby, the main focus was on the time points in working hours (8, 12 and 16 hr), which are used frequently in practice. Of all investigated biomarkers, only TTP in males demonstrated statistically significant circadian rhythm (p = 0.040). A statistically significant effect between all six time points with RM-ANOVA was observed for ROM, TTP and UA in both genders, and for BAP in females only. No statistically significant differences were observed between the time points 8 hr and 12 hr for any of the biomarkers that were assessed in our study. In conclusion, diurnal variations in some of the studied biomarkers that we demonstrate here should be taken into account when designing and conducting clinical and epidemiological studies. It is advised to standardize the time of sampling with a preference in the morning hours.

Protein and calorie restriction may improve outcomes in living kidney donors and kidney transplant recipients.

Previously, we and others showed that dietary restriction protects against renal ischemia-reperfusion injury in animals. However, clinical translation of preoperative diets is scarce, and in the setting of kidney transplantation these data are lacking. In this pilot study, we investigated the effects of five days of a preoperative protein and caloric dietary restriction (PCR) diet in living kidney donors on the perioperative effects in donors, recipients and transplanted kidneys. Thirty-five kidney donors were randomized into either the PCR, 30% calorie and 80% protein reduction, or control group without restrictions. Adherence to the diet and kidney function in donors and their kidney recipients were analyzed. Perioperative kidney biopsies were taken in a selected group of transplanted kidneys for gene expression analysis. All donors adhered to the diet. From postoperative day 2 up until month 1, kidney function of donors was significantly better in the PCR-group. PCR-donor kidney recipients showed significantly improved kidney function and lower incidence of slow graft function and acute rejection. PCR inhibited cellular immune response pathways and activated stress-resistance signaling. These observations are the first to show that preoperative dietary restriction induces postoperative recovery benefits in humans and may be beneficial in clinical settings involving ischemia-reperfusion injury.

Very long-term stability of lipid biomarkers in human serum.

The assessment of the stability of biomarkers is very important for epidemiological studies. In this study, the stability of five lipid parameters (total cholesterol, triglycerides, HDL- and LDL-cholesterol and free fatty acids) has been tested in 16 human serum samples after storage at -80 °C up at time points 0, 1, 8 and 13 y. The majority of the lipid biomarkers were stable during storage conditions, except for cholesterol. The correlations between the samples were very good at time points. Therefore, long-term storage of human serum samples allows lipid biomarker determination, provided that the samples are stored at -80 °C.

The association between exposure to different aspects of shift work and metabolic risk factors in health care workers, and the role of chronotype.

OBJECTIVE: Shift work has been linked to cardio-metabolic diseases, but insight into different shift work-related aspects and chronotype of shift workers and their relation with metabolic risk factors is limited. This study examined the association between current shift work status, frequency and duration of night shift work, chronotype, and metabolic risk factors in a population of health care workers. METHODS: Anthropometrics, questionnaires, and blood samples were collected from 503 shift working and 93 non-shift working health care workers employed in hospitals. Body mass index, waist circumference, cholesterol (total, HDL, LDL), triglycerides, and high-sensitivity C-reactive protein were measured. Associations of current shift work, frequency (non-night shift worker, 1-2, 3-4, ≥5 night shifts/month) and duration of night shift work (non-night shift workers, <10, 10-19, ≥20 years), and shift workers' chronotype, with metabolic risk factors were studied using linear regression analysis. RESULTS: Compared to non-shift workers, shift workers' total cholesterol level was 0.38 mmol/L lower (95%-CI = -0.73 --0.04) and LDL cholesterol was 0.34 mmol/L lower (95%-CI = -0.60 --0.08). For all other metabolic risk factors, no differences were found. The association between shift work and LDL cholesterol was especially found among shift workers working night shifts for ≥20 years (B = -0.49 (95%-CI = -0.78 --0.19)). No differences were found for night shift frequency and chronotype. CONCLUSION: In this population of health care workers employed in hospitals, no evidence for differences in metabolic risk factors was observed that could underlie a link between shift work and cardio-metabolic diseases. Further research using different aspects of shift work to study the association with metabolic risk factors is recommended.

Head skeleton malformations in zebrafish (Danio rerio) to assess adverse effects of mixtures of compounds.

The EU-EuroMix project adopted the strategy of the European Food Safety Authority (EFSA) for cumulative risk assessment, which limits the number of chemicals to consider in a mixture to those that induce a specific toxicological phenotype. These so-called cumulative assessment groups (CAGs) are refined at several levels, including the target organ and specific phenotype. Here, we explore the zebrafish embryo as a test model for quantitative evaluation in one such CAG, skeletal malformations, through exposure to test compounds 0-120 hpf and alcian blue cartilage staining at 120 hpf, focusing on the head skeleton. Reference compounds cyproconazole, flusilazole, metam, and thiram induced distinctive phenotypes in the head skeleton between the triazoles and dithiocarbamates. Of many evaluated parameters, the Meckel's-palatoquadrate (M-PQ) angle was selected for further assessment, based on the best combination of a small confidence interval, an intermediate maximal effect size and a gentle slope of the dose-response curve with cyproconazole and metam. Additional test compounds included in the CAG skeletal malformations database were tested for M-PQ effects, and this set was supplemented with compounds associated with craniofacial malformations or cleft palate to accommodate otherwise organized databases. This additional set included hexaconazole, all-trans-retinoic acid, AM580, CD3254, maneb, pyrimethanil, imidacloprid, pirimiphos-methyl, 2,4-dinitrophenol, 5-fluorouracil, 17alpha-ethynylestradiol (EE2), ethanol, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), PCB 126, methylmercury, boric acid, and MEHP. Most of these compounds produced a dose-response for M-PQ effects. Application of the assay in mixture testing was provided by combined exposure to cyproconazole and TCDD through the isobole method, supporting that in this case the combined effect can be modeled through concentration addition.

Stability of Folate and Vitamin B12 in Human Serum after Long-Term Storage: A Follow-Up after 13 Years.

In epidemiological and nutrition research, it is very important to evaluate the stability of biomarkers as function of both storage time and temperature. In this study, the stability of folate and vitamin B12 in human serum samples has been tested after long-term storage at -80°C up to 13 years. Serum samples of 16 individuals were used in this study. The concentration of folate and vitamin B12 has been determined at t=0 and at 1, 8, and 13 years after storage at -80°C. The folate concentrations in serum samples remained stable at -80°C. The concentration of vitamin B12 was decreasing during the time of the study to about 50%. The correlation of the folate and also of the vitamin B12 concentrations in the stored samples compared with the starting values was still good. Therefore, although the concentration of vitamin B12 decreased upon storage, reliable comparative analyses can still be performed.

Long-term stability of oxidative stress biomarkers in human serum.

The storage time and storage temperature might affect stability of oxidative stress biomarkers, therefore, they have to be analyzed after long-term storage of serum samples. The stability of three biomarkers reflecting oxidative stress: reactive oxygen metabolites (ROM) for hydroperoxides, total thiol levels (TTL) for the redox status and biological antioxidant potency (BAP) for the antioxidant status, was investigated at several time points during 60 months of storage at -20 and -80 °C. Biomarkers ROM and BAP showed a very good stability during storage for 60 months at both temperatures. In addition, the correlation of the data after 60 months of storage compared with the starting data was very good with correlation coefficients >0.9. The TTL assay showed good results in serum samples stored at -80 °C, but not in samples stored at -20 °C. Serum samples for analysis of the set of oxidative stress biomarkers ROM, BAP and TTL can be stored up to 60 months at -80 °C. ROM and BAP can also be stored at -20 °C during this period. The present results are very important for the biomarker-related epidemiological studies that make use of biobanks with samples stored for many years and for new project planning, including sample storage conditions.

Programmed Effects in Neurobehavior and Antioxidative Physiology in Zebrafish Embryonically Exposed to Cadmium: Observations and Hypothesized Adverse Outcome Pathway Framework.

Non-communicable diseases (NCDs) are a major cause of premature mortality. Recent studies show that predispositions for NCDs may arise from early-life exposure to low concentrations of environmental contaminants. This developmental origins of health and disease (DOHaD) paradigm suggests that programming of an embryo can be disrupted, changing the homeostatic set point of biological functions. Epigenetic alterations are a possible underlying mechanism. Here, we investigated the DOHaD paradigm by exposing zebrafish to subtoxic concentrations of the ubiquitous contaminant cadmium during embryogenesis, followed by growth under normal conditions. Prolonged behavioral responses to physical stress and altered antioxidative physiology were observed approximately ten weeks after termination of embryonal exposure, at concentrations that were 50-3200-fold below the direct embryotoxic concentration, and interpreted as altered developmental programming. Literature was explored for possible mechanistic pathways that link embryonic subtoxic cadmium to the observed apical phenotypes, more specifically, the probability of molecular mechanisms induced by cadmium exposure leading to altered DNA methylation and subsequently to the observed apical phenotypes. This was done using the adverse outcome pathway model framework, and assessing key event relationship plausibility by tailored Bradford-Hill analysis. Thus, cadmium interaction with thiols appeared to be the major contributor to late-life effects. Cadmium-thiol interactions may lead to depletion of the methyl donor S-adenosyl-methionine, resulting in methylome alterations, and may, additionally, result in oxidative stress, which may lead to DNA oxidation, and subsequently altered DNA methyltransferase activity. In this way, DNA methylation may be affected at a critical developmental stage, causing the observed apical phenotypes.

Tissue-Specific Effects of Vitamin E Supplementation.

A multivitamin and mineral supplementation study of 6 weeks was conducted with male and female mice. The control group received a standard dose of vitamins and minerals of 1× the Recommended Daily Intake (RDI), whereas a second group received 3× RDI. A third group received a high dose of vitamin E (25× RDI), close to the upper limit of toxicity (UL), but still recommended and considered to be harmless and beneficial. The high dose of vitamin E caused a number of beneficial, but also adverse effects. Different biomarkers of tissue toxicity, oxidative stress related processes and inflammation were determined. These biomarkers did not change in plasma and erythrocytes to a large extent. In the liver of male mice, some beneficial effects were observed by a lower concentration of several biomarkers of inflammation. However, in the kidney of male mice, a number of biomarkers increased substantially with the higher dose of vitamin E, indicating tissue toxicity and an increased level of inflammation. Since this dose of vitamin E, which is lower than the UL, cause some adverse effects, even after a short exposure period, further studies are required to reconsider the UL for vitamin E.

Short-Term Preoperative Calorie and Protein Restriction Is Feasible in Healthy Kidney Donors and Morbidly Obese Patients Scheduled for Surgery.

INTRODUCTION: Surgery-induced oxidative stress increases the risk of perioperative complications and delay in postoperative recovery. In mice, short-term preoperative dietary and protein restriction protect against oxidative stress. We investigated the feasibility of a calorie- and protein-restricted diet in two patient populations. METHODS: In this pilot study, 30 live kidney donors and 38 morbidly obese patients awaiting surgery were randomized into three groups: a restricted diet group, who received a synthetic liquid diet with 30% fewer calories and 80% less protein for five consecutive days; a group who received a synthetic diet containing the daily energy requirements (DER); and a control group. Feasibility was assessed using self-reported discomfort, body weight changes, and metabolic parameters in blood samples. RESULTS: Twenty patients (71%) complied with the restricted and 13 (65%) with the DER-diet. In total, 68% of the patients reported minor discomfort that resolved after normal eating resumed. The mean weight loss on the restricted diet was significantly greater (2.4 kg) than in the control group (0 kg, p = 0.002), but not in the DER-diet (1.5 kg). The restricted diet significantly reduced levels of serum urea and plasma prealbumin (PAB) and retinol binding protein (RBP). CONCLUSIONS: A short-term preoperative calorie- and protein-restricted diet is feasible in kidney donors and morbidly obese patients. Compliance is high and can be objectively measured via changes in urea, PAB, and RBP levels. These results demonstrate that this diet can be used to study the effects of dietary restriction on surgery-induced oxidative stress in a clinical setting.

Diurnal Variation of Hormonal and Lipid Biomarkers in a Molecular Epidemiology-Like Setting.

INTRODUCTION: Many molecular epidemiology studies focusing on high prevalent diseases, such as metabolic disorders and cancer, investigate metabolic and hormonal markers. In general, sampling for these markers can occur at any time-point during the day or after an overnight fast. However, environmental factors, such as light exposure and food intake might affect the levels of these markers, since they provide input for the internal time-keeping system. When diurnal variation is larger than the inter-individual variation, time of day should be taken into account. Importantly, heterogeneity in diurnal variation and disturbance of circadian rhythms among a study population might increasingly occur as a result of our increasing 24/7 economy and related variation in exposure to environmental factors (such as light and food). AIM: The aim of the present study was to determine whether a set of often used biomarkers shows diurnal variation in a setting resembling large molecular epidemiology studies, i.e., non-fasted and limited control possibilities for other environmental influences. RESULTS: We show that markers for which diurnal variation is not an issue are adrenocorticotropic hormone, follicle stimulating hormone, estradiol and high-density lipoprotein. For all other tested markers diurnal variation was observed in at least one gender (cholesterol, cortisol, dehydroepiandrosterone sulfate, free fatty acids, low-density lipoprotein, luteinizing hormone, prolactin, progesterone, testosterone, triglycerides, total triiodothyronine and thyroid-stimulating hormone) or could not reliably be detected (human growth hormone). DISCUSSION: Thus, studies investigating these markers should take diurnal variation into account, for which we provide some options. Furthermore, our study indicates the need for investigating diurnal variation (in literature or experimentally) before setting up studies measuring markers in routine and controlled settings, especially since time-of-day likely matters for many more markers than the ones investigated in the present study.

Quality control data of physiological and immunological biomarkers measured in serum and plasma.

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.

Long-term stability of cancer biomarkers in human serum: biomarkers of oxidative stress and redox status, homocysteine, CRP and the enzymes ALT and GGT.

AIM: Five frequently used biomarkers in cancer research and epidemiological studies were tested for their assay stability upon storage of serum for 12 months at -20 and -70/-80°C. MATERIALS & METHODS: The biomarker assays include reactive oxygen metabolites (ROM), the total thiol levels (TTL), homocysteine (HCy), C-reactive protein (HS-CRP) and two liver enzymes, alanine aminotransferase (ALT) and γ-glutamyltransferase (GGT). RESULTS: The assays for ROM, HCy, HS-CRP and GGT were stable in human serum samples at the two temperatures tested. The two other assays TTL and ALT, however, showed statistically significant differences in their stability between -20 and -80°C. CONCLUSION: Therefore, storage at -80°C is advised to maintain a reliable assay outcome when serum samples have to be stored for longer periods.

Biomarkers of oxidative stress and redox status in a short-term low-dosed multivitamin and mineral supplementation study in two human age groups.

A 60-day intervention study was conducted in which the participants took a low dose of a multivitamin and mineral supplement. The study consists of a final number of 66 volunteers (30 males and 36 females), divided into two age groups of 30-35 and 60-65 years. For 30 days they took a multivitamin and mineral supplement with 1× the recommended daily intake (RDI) followed by another 30 days with 2× the RDI. The aim of the study was to monitor oxidative stress and redox status of both young and old age groups. In serum, the expected increase of the water-soluble vitamins folate and vitamin B12 was observed with a concomitant decrease in homocysteine. Serum biomarkers of oxidative stress, the reactive oxygen metabolites, of the antioxidant status, the biological antioxidant potential did not change. However, the total thiol levels in serum, biomarker of the redox status, decreased significant, only in both groups of elderly after 60 days. In erythrocytes, there was a change in the glutathione metabolism as observed by an increase in glutathione reductase and to a lower extend in glutathione peroxidase, indicating an increase in oxidative stress in all groups. It is concluded that a low-dosed multivitamin and -mineral supplementation have different effects on the redox status in young versus old. It remained to explain why a low dose of a multivitamin and -mineral supplement cause increased oxidative stress.

Long-term stability of biomarkers of the iron status in human serum and plasma.

CONTEXT: In epidemiological research, it is very important to test the stability of biomarkers as function of both storage time and temperature. OBJECTIVE: In this study, the stability of biomarkers of the iron status was tested up to 1 year of storage. MATERIALS AND METHODS: The biomarkers include total iron, unsaturated iron binding capacity, ferritin, transferrin, soluble transferrin receptor, ceruloplasmin and haptoglobin. RESULTS: The concentrations of all biomarkers tested remain constant upon storage at -20, -70 and -196 °C. CONCLUSION: All biomarkers of the iron status were stable at the temperatures tested for 1 year.

Selective induction of IL-6 by aluminum-induced oxidative stress can be prevented by selenium.

In this study the acute toxic effects of aluminum (Al) on mice have been investigated, including the interactions of Al and selenium (Se). Focus was put on the systemic effects of (co)exposure to Al and Se as a reflection of the redox status in the liver, kidney and brain. Short-term exposure (16 h) to Al resulted in an increase in the systemic inflammation parameters IL-6 and PAI-1, whereas serum levels of TNF-α remained unaffected. The different response pattern of IL-6 and TNF-α probably indicates an increased intracellular oxidative stress and altered redox status in the liver, because the selective increase in IL-6 serves as a protective intrahepatocellular process driven by oxidative stress. The intracellular glutathione concentration GSHtot decreased significantly upon Al exposure. Both the increase in IL-6 and decrease in glutathione status could be prevented by co-exposure to Se, but not the increase in PAI-1. The redox status of the kidney and brain was not markedly affected. Therefore it was concluded that short-term exposure to Al causes adverse effects on the intracellular oxidative stress processes in the liver, as reflected by the selective increase in the IL-6 concentration. This process can be restored by co-administration of the trace element Se as a part of the glutathione redox system.

Long-term (in)stability of folate and vitamin B12 in human serum.

BACKGROUND: In epidemiological research it is very important to test the stability of biomarkers as a function of both storage time and temperature. In this study the stability of both folate and vitamin B12 in human serum samples have been tested after storage at three different temperatures up to 1 year. METHODS: Serum samples of 16 individuals were used in this study. The concentration of folate and vitamin B12 has been determined at T=0 and at several time points up to 1 year after storage at -20°C, -70°C and -196°C. The statistical difference from the initial value at T=0 were determined with a t-test. RESULTS: Folate in serum samples remained stable at -70°C but was not stable during storage at -20°C. A fast decrease was observed after Day 4 which resulted in a stable level of about 60% of the original value measured at T=0 (p<0.001). The rank order of folate concentration in the samples, however, was not affected. The stability of vitamin B12 was good at all temperatures tested. CONCLUSIONS: Measurements of folate concentrations in serum stored at -20°C are not reliable. The rank order, however, was not changed. Vitamin B12 was stable at all temperatures tested. For both folate and vitamin B12 storage at -70°C is sufficient to maintain the original concentration for 1 year. Storage at -196°C in liquid nitrogen is not necessary for these nutrients.

Long term stability of paraoxonase-1 and high-density lipoprotein in human serum.

BACKGROUND: Paraoxonase-1 (PON1) is an enzyme with numerous functions and receives an increasing interest in clinical and epidemiological studies. Sometimes samples are stored for longer periods at a certain temperature. Therefore the stability of PON1 activity must be checked and retained upon storage for longer periods. RESULTS: In this study the stability of PON1 activity has been tested in human serum samples during storage up to 12 months at 3 commonly used temperatures, -20°C, -70°C and -196°C. It was found that the stability of the PON1 activity is constant during 12 months of storage at -70°C and -196°C. Storage at -20°C resulted in a small but statistically significant decrease after 6 months to about 94% of its original value. Nonetheless, the rank order between the samples at T = 0 and 12 months remained the same. The same temperature dependence was found for the associated high-density lipoprotein. CONCLUSIONS: It can be concluded that -70°C is the right temperature for storage to maintain the PON1 activity for at least one year. Storage at a lower temperature in liquid nitrogen (-196°C) is not necessary.