Danae Costopoulou

Analytical techniques for determining biotin

Biotin is a vitamin of the B-complex, which plays an important biochemical role in every living cell. In the recent years, the interest in this vitamin has been rekindled, mainly due to its association with serious human disorders, such as the inherited syndrome multiple carboxylase deficiency, which can be successfully treated with biotin administration. Diagnosis of biotin deficiency as well as monitoring of biotin levels in biological fluids of patients receiving biotin treatment is crucial. Equally important is the determination of biotin levels in pharmaceutical preparations as well as in food and food supplement products, which constitute the main source of biotin in humans. Several analytical methods for measuring biotin in various samples, e.g. human fluids, pharmaceutical formulations, food material etc., have been reported in the literature. In this review, the most representative of these methods are presented, and their characteristics are evaluated.

Levels of perfluorooctanesulfonate (PFOS) and perfluorooctanoate (PFOA) in blood samples from different groups of adults living in Greece

The production of perfluorinated compounds which finds application in a wide variety of consumer products has led to their accumulation in the environment. This fact, in combination with their chemical properties of amphiphilicity and chemical stability is the cause of their occurrence in human tissues, which according to reports is widespread, not only in occupationally exposed workers, but also in the general population. In this study, we analyzed 182 blood samples from three different groups of adults living in Greece, in order to evaluate blood levels of two environmentally most common perfluorinated compounds, perfluorooctanesulfonate (PFOS) and perfluorooctanoate (PFOA). The samples examined were from a group of individuals living in Athens, a group of inhabitants of the semi-urban and rural area of Argolida, and a group of cancer patients from the St. Savas Anticancer Hospital in Athens. The latter group was chosen in order to examine the possible relation of PFOS and PFOA levels with cancer incidence in the general population. The analytical results showed the presence of PFOS and PFOA in all samples examined, at levels similar to those reported from other European countries. There was no significant difference between the three groups however there was a significant difference between the levels of men and women in all groups examined. No correlation was found between age and PFOS and PFOA levels. This is the first study of this kind to be conducted in samples from Greece.

Levels of perfluorinated compounds in raw and cooked Mediterranean finfish and shellfish

Perfluorinated compounds (PFCs) were analyzed in several species of small Mediterranean fish and shellfish, all of which are popular in Greek diet. Analysis was conducted in raw samples and in samples cooked by the two ways preferred in Greek cuisine, i.e. fried in olive oil and grilled. PFCs above the detection limit were found in all raw samples except sardine, mussel and squid. The predominant PFC was PFOS (perfluorooctane sulfonate), the highest concentration of which was measured in picarel (20.4 ng g(-1) fresh weight). The PFOS values for the rest of the samples were between <LOD and 5.66 ng g(-1) fw. The concentrations of the detected PFCs were in most cases higher after frying or grilling, this increase being statistically significant. Based on these results, the Tolerable Daily Intake for PFOS and PFOA (perfluorooctanoic acid) through consumption of fish and seafood was well below the values proposed by EFSA.

Dioxin contamination after an accidental fire in the municipal landfill of Tagarades, Thessaloniki, Greece.

We report here a case study of dioxin contamination after a fire in the public landfill of Tagarades, which is situated southeast of Thessaloniki, Greece. The aim of this study was to determine the levels of PCDD/Fs and PCBs in food samples of meat, eggs, dairy products and vegetables, collected near the affected area, and to compare them with the usual levels found in Greek food. Soil samples were also gathered from various sites at a distance up to 5km from the landfill. Some food samples were found above the limits specified by the EU, while all food samples were above the usual levels of Greek food. The contaminated products were appropriately destroyed. The results of the soil and olive samples indicate a reverse correlation between dioxin contamination and distance from the contamination source. It should be noted that PCB levels were normal in all analyzed samples.

Determination of PCDD/F, dioxin-like PCB and PAH levels in olive and olive oil samples from areas affected by the fires in summer 2007 in Greece.

During the summer of 2007, a series of massive forest fires broke out in several areas across Greece. The main sources of PCDD/Fs and dioxin-like PCBs in Greece are considered to be the uncontrolled combustion of municipal solid waste in open landfills and accidental fires in forest, rural and industrial areas. Combustion may also lead to the formation of PAHs, which are fat soluble substances of considerable toxicity. The objective of this study was to investigate PCDD/F, dioxin-like PCB and PAH contamination of olive oil produced in fire-affected areas. Olive oil is a very significant agricultural product of Greece. Samples for this study were collected from all affected oil producing areas after the fire and 1year later. PCDD/Fs, dioxin-like PCBs and PAHs were at normal levels in all samples analysed.

Perfluoroalkylated substances (PFASs) in home and commercially produced chicken eggs from the Netherlands and Greece

Dietary intake is a major route of human exposure to perfluoroalkylated substances (PFASs). However, the available information on PFAS levels in food, including chicken eggs, is limited. In the present study, home produced and commercially produced eggs (organic, battery and free range eggs) were collected from the Netherlands (n = 95) and Greece (n = 76). The egg yolks were analysed for 11 PFASs by liquid chromatography-tandem mass spectrometry using isotope dilution. PFAS levels in yolk were higher in home produced eggs from the Netherlands (median 3.1, range < LOQ - 31.2 ng g(-1)) and Greece (median 1.1, range < LOQ - 15.0 ng g(-1)) compared to the eggs collected from supermarkets. In these eggs, all PFAS levels were below the LOQ of 0.5 ng g(-1), except for a small amount of perfluorooctane sulfonate (PFOS) in 1 sample in each country (1.1 ng g(-1) and 0.9 ng g(-1) for the Netherlands and Greece respectively). PFOS was the predominant PFAS, making up on average 85% of ∑PFASs. The highest PFOS concentration was detected in a Dutch home produced egg sample (24.8 ng g(-1)). The contamination pattern was similar in both countries with the long-chain PFASs (C ≥ 8) being most frequently detected, while short-chain PFASs were rarely found. The most likely cause of the contamination of home produced eggs is ingestion of soil through pecking. Although regular consumption of home produced eggs will lead to an increased PFOS exposure, it is not expected that it will lead to exceedance of the tolerable daily intake established by EFSA.

Direct ELISA Method for the Specific Determination of Prothymosin Alpha in Human Specimens

An enzyme linked immunosorbent assay, specific for prothymosin alpha (ProT alpha) was developed using an antibody against the synthetic C-terminal peptide ProT alpha[101-109] and isolated bovine ProT alpha for the preparation of standard solutions and immunoplates. Due to the antibody used, the ELISA developed was capable of fully discriminating between ProT alpha, the naturally occuring and partially homologous peptide parathymosin alpha (ParaT alpha) and the peptide thymosin alpha1 (T alpha1), whose sequence is identical to the [1-28] sequence of ProT alpha, and its in vivo occurrence is under question. Moreover, due to its improved sensitivity, the ELISA was capable of directly determining ProT alpha concentration in human serum and tissue extracts, without any pretreatment of the samples. ProT alpha levels were directly measured in sera obtained from 48 apparently healthy individuals and 27 patients with diagnosed breast cancer and found to range from 0.67 to 2.34 microg/ml (mean value 1.27 +/- 0.49 microg/ml) and from 0.47 to 1.74 microg/ml (mean value 1.02 +/- 0.29 microg/ml), respectively. ProT alpha levels were also measured in four breast tumor and adjacent normal breast tissue extracts and found to be elevated in the tumor extracts.

PCDDs, PCDFs and PCBs in farmed fish produced in Greece: Levels and human population exposure assessment.

Fish is among the essential components of Mediterranean diet and has beneficial effects on human health. Farmed fish is an affordable alternative to wild fish and a significant food export product for Greece. Published studies worldwide have reported significant levels of environmental pollutants in fish tissues. Especially for PCDDs/Fs and PCBs, the studies suggest that the most important contribution to human dietary intake is from fish and seafood. In the present study, we investigate the levels of PCDDs/Fs, dioxin-like and non dioxin-like PCBs in the most common farmed fish species produced in Greece i.e. sea bass, sea bream and rainbow trout. These species are widely consumed in Greece and are also exported to many countries worldwide. The mean levels found were WHO-PCDD/F-TEQ: 0.22 pg g(-1) wet weight (w.w.), WHO-PCDD/F-PCB-TEQ: 0.88 pg g(-1) w.w. for sea bream, WHO-PCDD/F-TEQ: 0.13 pg g(-1) w.w., WHO-PCDD/F-PCB-TEQ: 0.68 pg g(-1) w.w. for sea bass and WHO-PCDD/F-TEQ: 0.10 pg g(-1) w.w., WHO-PCDD/F-PCB-TEQ: 0.43 pg g(-1) w.w. for rainbow trout. For non dioxin-like PCBs, mean sum values found were 8.02 ng g(-1) w.w. for sea bream, 5.24 ng g(-1) w.w. for sea bass and 2.90 ng g(-1) w.w. for rainbow trout. All concentrations found were far below maximum levels set by the European Union and in the same range as wild-caught fish also presented for comparison. Daily intake from the consumption of farmed fish species examined is calculated at 1.3 pg WHO-TEQ kg(-1) b.w., which is at the lowest end of TDI values proposed by the WHO.

Determination of perfluoroalkylated substances (PFASs) in drinking water from the Netherlands and Greece

In the present study 11 perfluoroalkylated substances (PFASs) were analysed in drinking tap water samples from the Netherlands (n = 37) and from Greece (n = 43) by applying LC-MS/MS and isotope dilution. PFASs concentrations above the limit of quantification, LOQ (0.6 ng/l) were detected in 20.9% of the samples from Greece. Total PFAS concentrations ranged between <LOQ and 5.9 ng/l, with the highest concentrations noted for the three Aegean islands Mykonos, Kalymnos and Syros and for the town Tripoli in the Peloponnese. In the Dutch situation, total PFASs concentrations above the LOQ were detected in 48.6% of the samples, varying from <LOQ to 54 ng/l. The highest concentrations were detected around Amsterdam (including Schiphol airport) and more generally, PFASs were detected in the drinking water from the western part of the Netherlands. This seems attributable to the source, which is purified surface water in this area. Short-chain PFASs and especially perfluoropentanoic acid (PFPeA), perfluorohexanoic acid (PFHxA), perfluoroheptanoic acid (PFHpA), perfluorooctanoic acid (PFOA), perfluorobutane sulfonate (PFBS), and perfluorohexane sulfonate (PFHxS) were detected most frequently, whereas long-chain PFASs (C > 8) were only rarely detected. In the drinking water samples from the eastern part of the Netherlands, where drinking water is sourced from groundwater reservoirs, no PFASs were detected. This demonstrates that exposure to PFASs through drinking water in the Netherlands is dependent on the source. Additionally, five samples of bottled water from each country were analysed in the current study, with all of them originating from ground wells. In these samples, all PFASs were below the LOQ.

Infant dietary exposure to dioxins and dioxin-like compounds in Greece.

The dietary exposure of infants to polychlorinated dibenzo dioxins and furans (PCDD/Fs) and dioxin like polychlorinated biphenyls (dl-PCBs) is an issue of great social impact. We investigated for the first time the dietary intake of these compounds in infants living in Greece. We included in our study two age groups: 0-6 months, when infants are fed exclusively by human milk and/or formula milk, and 6 to 12 months, when solid food is introduced to nutrition. We took into consideration analytical results for PCDD/Fs and dl-PCBs concentrations in the most popular infant formulae in the Greek market, previous data for mother milk concentrations of PCDD/Fs and dl-PCBs from Greece, and finally analytical data for fat-containing food products from the Greek market. In the first study group, it was found than in infants exclusively fed by breast milk, the calculated sum of PCDD/Fs and dioxin-like PCBs (60.3-80.4 TEQ pg/kg body weight) was significantly higher than that of infants that consume a combination of human milk and formula (31.2-41.6 TEQ pg/kg body weight). In the second study group, separate daily intake estimations were performed for babies receiving human milk (estimated total daily intake 19.76-24.95 TEQ pg/kg body weight) and formula milk (estimated total daily intake 1.60-2.24 TEQ pg/kg body weight). The risks of this exposure should not be overestimated because nursing is restricted to a limited period of human life and besides, the potential consumption of higher levels of dioxin-like compounds is fully compensated by the significant benefits of breast-feeding.