Piet Beekhof

Long-term stability of parameters of antioxidant status in human serum

Abstract The antioxidant status of serum or plasma can be determined using several commercially available assays. Here, four different assays, total antioxidant status (TAS), its second-generation assay (TAS2), biological antioxidant potential (BAP), and enzymatic assay using horseradish peroxidase (EAOC), were applied on human serum samples to test the temperature stability of antioxidants, upon storage of serum for 12 months. The two or three most commonly used temperatures for storage, that is, − 20, − 70 (or − 80), and − 196°C, were selected. The general conclusion is that all assays were stable at the temperatures tested. In addition, there were almost no statistically significant differences between the samples stored at different temperatures. Only the rank order of the EAOC assay was not very good in samples stored at − 20°C. Also three components contributing to the total antioxidant capacity, uric acid, creatinine and bilirubin, showed no statistically significant differences between the temperatures. Therefore, storage at − 20°C is sufficient to maintain a proper assay outcome of most of the total antioxidant assays, although storage at − 70/80°C is to be preferred for longer storage times.

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.

Short-Term Stability of Biomarkers of Oxidative Stress and Antioxidant Status in Human Serum

The oxidation and antioxidant status of serum are often determined in serum samples which have been frozen for some time. The oxidative stress process is prone to fast alterations in the sample because of the possible instability of the reactants. Here one oxidation assay (ROM) and three antioxidant assays (FRAP, TAS, and BAP) have been tested on their performance and stability at short-time storage. The most commonly used temperatures for storage and handling of serum samples (

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.

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

Abstract 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.

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

Abstract 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.

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 Stability of Parameters of Lipid Metabolism in Frozen Human Serum: Triglycerides, Free Fatty Acids, Total-, HDL- and LDL-cholesterol, Apolipoprotein-A1 and B

Background: In large epidemiological studies it is important to test the stability of biomarkers as a function of both temperature and duration of storage. In this study the stability of seven lipid parameters have been tested in human serum samples after storage at three different temperatures up to 1 year. Methods: Serum samples of 16 human individuals were used in this study. The concentration of all parameters have been determined at T=0 and at several time points up to 1 year after storage at –20°C, –70°C and –196°C. Results: Most of the lipid biomarkers, cholesterol, triglycerides HDL- and LDL cholesterol, apolipoprotein-A1 and –B are stable on long-term storage for one year at the three temperatures tested. The levels of both HDL- and LDL cholesterol showed a small decrease for samples stored at -20°C only. The free fatty acids, however, are not stable and showed a decrease to about 80% of the starting value. The rank order between the samples, however, remained very good. Conclusions: This study shows that free fatty acids are not stable in human serum samples probably due to thawing and freezing effects, although the rank order and correlation between the samples from different time points remained the same. HDL- and LDL-cholesterol showed a very small deviation on storage at -20°C. The other lipid parameters remained perfectly stable in this study. No differences were observed between storage at -70°C or -196°C, Therefore it is advised to store serum samples at -70°C for longer periods.

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

BackgroundParaoxonase-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.ResultsIn 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.ConclusionsIt 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.

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.