Johannes Cremers

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.

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 (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.

Fatty acids of erythrocyte membrane in acute pancreatitis patients

AIM To evaluate changes in the fatty acid composition of erythrocyte membrane phospholipids during severe and mild acute pancreatitis (AP) of alcoholic and nonalcoholic etiology. METHODS All consecutive patients with a diagnosis of AP and onset of the disease within the last 72 h admitted to the Hospital of Lithuanian University of Health Sciences between June and December 2007 were included. According to the Acute Physiology and Chronic Health Evaluation (APACHE II) scale, the patients were subdivided into the mild (APACHE II score < 7, n = 22) and severe (APACHE II score ≥ 7, n = 17) AP groups. Healthy individuals (n = 26) were enrolled as controls. Blood samples were collected from patients on admission to the hospital. Fatty acids (FAs) were extracted from erythrocyte phospholipids and expressed as percentages of the total FAs present in the chromatogram. The concentrations of superoxide dismutase and glutathione peroxidase were measured in erythrocytes. RESULTS We found an increase in the percentages of saturated and monounsaturated FAs, a decrease in the percentages of total polyunsaturated FAs (PUFAs) and n-3 PUFAs in erythrocyte membrane phospholipids of AP patients compared with healthy controls. Palmitic (C16:0), palmitoleic (C16:1n7cis), arachidonic (C20:4n6), docosahexaenoic (DHA, C22:6n3), and docosapentaenoic (DPA, C22:5n3) acids were the major contributing factors. A decrease in the peroxidation and unsaturation indexes in AP patients as well as the severe and mild AP groups as compared with controls was observed. The concentrations of antioxidant enzymes in the mild AP group were lower than in the control group. In severe AP of nonalcoholic etiology, the percentages of arachidic (C20:0) and arachidonic (C20:4n6) acids were decreased as compared with the control group. The patients with mild AP of nonalcoholic etiology had the increased percentages of total saturated FAs and gama linoleic acid (C18:3n6) and the decreased percentages of elaidic (C18:1n9t), eicosapentaenoic acid (EPA, C20:5n3), DPA (C22:5n3), DHA (C22:6n3) as well as total and n-3 PUFAs in erythrocyte membrane phospholipids. CONCLUSION The composition of FAs in erythrocyte membranes is altered during AP. These changes are likely to be associated with alcohol consumption, inflammatory processes, and oxidative stress.

The Sensory Difference Threshold of Menthol Odor in Flavored Tobacco Determined by Combining Sensory and Chemical Analysis

Abstract Cigarettes are an often-used consumer product, and flavor is an important determinant of their product appeal. Cigarettes with strong nontobacco flavors are popular among young people, and may facilitate smoking initiation. Discriminating flavors in tobacco is important for regulation purposes, for instance to set upper limits to the levels of important flavor additives. We provide a simple and fast method to determine the human odor difference threshold for flavor additives in a tobacco matrix, using a combination of chemical and sensory analysis. For an example, the human difference threshold for menthol odor, one of the most frequently used tobacco flavors, was determined. A consumer panel consisting of 20 women compared different concentrations of menthol-flavored tobacco to unflavored cigarette tobacco using the 2-alternative forced choice method. Components contributing to menthol odor were quantified using headspace GC-MS. The sensory difference threshold of menthol odor corresponded to a mixture of 43 (37–50)% menthol-flavored tobacco, containing 1.8 (1.6–2.1) mg menthol, 2.7 (2.3–3.1) µg menthone, and 1.0 (0.9–1.2) µg neomenthyl acetate per gram of tobacco. Such a method is important in the context of the European Tobacco Product Directive, and the US Food and Drug Administration Tobacco Control Act, that both prohibit cigarettes and roll-your-own tobacco with a characterizing flavor other than tobacco. Our method can also be adapted for matrices other than tobacco, such as food.

Identification of flavour additives in tobacco products to develop a flavour library

Objectives This study combines chemical analysis and flavour descriptions of flavour additives used in tobacco products, and provides a starting point to build an extensive library of flavour components, useful for product surveillance. Methods Headspace gas chromatography-mass spectrometry (GC-MS) was used to compare 22 commercially available tobacco products (cigarettes and roll-your-own) expected to have a characterising flavour and 6 commercially available products not expected to have a characterising flavour with 5 reference products (natural tobacco leaves and research cigarettes containing no flavour additives). The flavour components naturally present in the reference products were excluded from components present in commercially available products containing flavour additives. A description of the remaining flavour additives was used for categorisation. Results GC-MS measurements of the 33 tobacco products resulted in an overview of 186 chemical compounds. Of these, 144 were solely present in commercially available products. These 144 flavour additives were described using 62 different flavour descriptors extracted from flavour databases, which were categorised into eight groups largely based on the definition of characterising flavours from the European Tobacco Product Directive: fruit, spice, herb, alcohol, menthol, sweet, floral and miscellaneous. Conclusions We developed a method to identify and describe flavour additives in tobacco products. Flavour additives consist of single flavour compounds or mixtures of multiple flavour compounds, and different combinations of flavour compounds can cause a certain flavour. A flavour library helps to detect flavour additives that are characteristic for a certain flavour, and thus can be useful for regulation of flavours in tobacco and related products.

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.

Simple and Fast Determination of Ammonia in Tobacco

The presence of ammonia in tobacco is an important factor for the absorption of nicotine, and for product taste. The determination of ammonia in tobacco is usually performed by ion chromatography with conductivity detection devices. Here a new method is presented to measure the concentration of ammonia in tobacco based on an automated enzymatic method. This method is easy to perform and can be used on routine clinical analyzers. The enzymatic ammonia determination showed an intra-assay and inter-assay variation of 4-7 and 5-8%, respectively as determined with 3 brands of cigarettes. A comparison with the established HPLC-IC method gave similar results with respect to both concentrations in cigarettes and the reproducibility of the method. In one working day 50-60 samples of cigarettes or tobacco can be processed and analyzed.

Sensory analysis of characterising flavours: evaluating tobacco product odours using an expert panel

Objectives Tobacco flavours are an important regulatory concept in several jurisdictions, for example in the USA, Canada and Europe. The European Tobacco Products Directive 2014/40/EU prohibits cigarettes and roll-your-own tobacco having a characterising flavour. This directive defines characterising flavour as ‘a clearly noticeable smell or taste other than one of tobacco […]’. To distinguish between products with and without a characterising flavour, we trained an expert panel to identify characterising flavours by smelling. Methods An expert panel (n=18) evaluated the smell of 20 tobacco products using self-defined odour attributes, following Quantitative Descriptive Analysis. The panel was trained during 14 attribute training, consensus training and performance monitoring sessions. Products were assessed during six test sessions. Principal component analysis, hierarchical clustering (four and six clusters) and Hotelling’s T-tests (95% and 99% CIs) were used to determine differences and similarities between tobacco products based on odour attributes. Results The final attribute list contained 13 odour descriptors. Panel performance was sufficient after 14 training sessions. Products marketed as unflavoured that formed a cluster were considered reference products. A four-cluster method distinguished cherry-flavoured, vanilla-flavoured and menthol-flavoured products from reference products. Six clusters subdivided reference products into tobacco leaves, roll-your-own and commercial products. Conclusions An expert panel was successfully trained to assess characterising odours in cigarettes and roll-your-own tobacco. This method could be applied to other product types such as e-cigarettes. Regulatory decisions on the choice of reference products and significance level are needed which directly influences the products being assessed as having a characterising odour.