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.

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.

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.

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.