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Background: Quinoa is a pseudocereal with relatively high content of proteins and minerals that also contains mineral inhibitors such as phytate. The aim of the present study was to evaluate lactic acid fermentation and dry roasting on the nutritional quality and sensory attributes of quinoa. Various processes were evaluated, and quinoa grains were dry-roasted, milled, and fermented, either with or without the addition of wheat phytase or activated quinoa phytase (added as back-slop starter), for 10 hr. In other processes, raw quinoa flour was fermented for 10 hr or 4 hr and dry-roasted. Hedonic sensory evaluation was then performed to evaluate the acceptability of the fermented flours prepared as porridges., Results: The combined dry roasting and fermentation processes significantly ( p  < .05) degraded phytate between 30% and 73% from initial content. The most effective process was fermentation of raw quinoa flour followed by dry roasting, which improved the estimated zinc and iron bioavailability. Particularly, estimated zinc bioavailability improved from low (Phy:Zn 25.4, Phy·Zn:Ca 295) to moderate (Phy:Zn 7.14, Phy·Zn:Ca 81.5). Phytate degradation was mainly attributed to the activation of endogenous phytase during fermentation. Dry roasting was effective in improving the sensory attributes of the fermented quinoa flour. Porridge made with raw quinoa flour fermented for 4 hr and dry-roasted was more favorable to overall acceptability than that which was fermented for 10 hr and dry-roasted., Conclusion: Fermentation of quinoa flour for 4 hr followed by dry roasting was successful in improving both nutritional and sensory attributes of the final product., Competing Interests: The authors declare no conflict of interest., (© 2019 The Authors. Food Science & Nutrition published by Wiley Periodicals, Inc.)

Background: Pseudocereals are nutrient-rich grains with high mineral content but also phytate content. Phytate is a mineral absorption inhibitor. The study's aim was to evaluate phytate degradation during spontaneous fermentation and during Lactobacillus plantarum 299v® fermentation of quinoa, canihua, and amaranth grains and flours. It also aimed to evaluate the accessibility of iron, zinc, and calcium and to estimate their bioavailability before and after the fermentation of flours with starter culture. Lactic acid, pH, phytate, and mineral content were analyzed during fermentation., Results: Higher phytate degradation was found during the fermentation of flours (64-93%) than during that of grains (12-51%). Results suggest that phytate degradation was mainly due to endogenous phytase activity in different pseudocereals rather than the phytase produced by added microorganisms. The addition of Lactobacillus plantarum 299v® resulted in a higher level of lactic acid (76.8-82.4 g kg -1 DM) during fermentation, and a relatively quicker reduction in pH to 4 than in spontaneous fermentation. Mineral accessibility was increased (1.7-4.6-fold) and phytate : mineral molar ratios were reduced (1.5-4.2-fold) in agreement with phytate degradation (1.8-4.2-fold) in fermented flours. The reduced molar ratios were still above the threshold value for the improved estimated mineral bioavailability of mainly iron., Conclusion: Fermentation proved to be effective for degrading phytate in pseudocereal flours, but less so in grains. Fermentation with Lactobacillus plantarum 299v® improved mineral accessibility and estimated bioavailability in flours. © 2019 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry., (© 2019 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.)

Women with previously diagnosed gestational diabetes mellitus (GDM) are at increased risk of type-2-diabetes mellitus (T2D). We aimed to establish links between glucose tolerance (GT) and serum fatty acid (FA) profile in the transition from GDM to T2D. Six years after GDM, 221 women were grouped as having normal GT (NGT), impaired GT (IGT), or T2D based on oral GT test results. Fasting serum FAs were profiled, anthropometric measures taken, and dietary intake determined. Linoleic acid (LA) was significantly higher in NGT women ( p < 0.001) compared with IGT and T2D, and emerged as a strong predictor of low glucose and insulin levels, independently of BMI. Self-reported vegetable oil consumption correlated with LA serum levels and glucose levels. Delta-6-, delta-9-, and stearoyl-CoA-desaturase activities were associated with decreased GT, and delta-5-desaturase activities with increased GT. In a subgroup of women at high risk of diabetes, low LA and high palmitic acid levels were seen in those that developed T2D, with no differences in other FAs or metabolic measurements. Results suggest that proportions of LA and palmitic acid are of particular interest in the transition from GDM to T2D. Interconversions between individual FAs regulated by desaturases appear to be relevant to glucose metabolism.

Correction for 'Formation of reactive aldehydes (MDA, HHE, HNE) during the digestion of cod liver oil: comparison of human and porcine in vitro digestion models' by Cecilia Tullberg et al., Food Funct., 2016, 7, 1401-1412.

Scope: What effect does replacing chicken or pork with herring as the main dietary source of protein have on the human plasma metabolome?, Method and Results: A randomised crossover trial with 15 healthy obese men and women (age 24-70 years). Subjects were randomly assigned to four weeks of herring diet or a reference diet of chicken and lean pork, five meals per week, followed by a washout and the other intervention arm. Fasting blood serum metabolites were analysed at 0, 2 and 4 weeks for eleven subjects with available samples, using GC-MS based metabolomics. The herring diet decreased plasma citrate, fumarate, isocitrate, glycolate, oxalate, agmatine and methyhistidine and increased asparagine, ornithine, glutamine and the hexosamine glucosamine. Modelling found that the tricarboxylic acid cycle, glyoxylate, and arginine metabolism were affected by the intervention. The effect on arginine metabolism was supported by an increase in blood nitric oxide in males on the herring diet., Conclusion: The results suggest that eating herring instead of chicken and lean pork leads to important metabolic effects, particularly on energy and amino acid metabolism. Our findings support the hypothesis that there are metabolic effects of herring intake unrelated to the long chain n-3 polyunsaturated fatty acid content., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In this work, we investigated lipid oxidation of cod liver oil during gastrointestinal (GI) digestion using two types of in vitro digestion models. In the first type of model, we used human GI juices, while we used digestive enzymes and bile from porcine origin in the second type of model. Human and porcine models were matched with respect to factors important for lipolysis, using a standardized digestion protocol. The digests were analysed for reactive oxidation products: malondialdehyde (MDA), 4-hydroxy-trans-2-nonenal (HNE), and 4-hydroxy-trans-2-hexenal (HHE) by liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry (LC/APCI-MS), and for free fatty acids (FFA) obtained during the digestion by gas chromatography-mass spectrometry (GC-MS). The formation of the oxidation products MDA, HHE, and HNE was low during the gastric digestion, however, it increased during the duodenal digestion. The formation of the oxidation products reached higher levels when digestive juices of human origin were used (60 μM of MDA, 9.8 μM of HHE, and 0.36 μM of HNE) [corrected] compared to when using enzymes and bile of porcine origin (0.96, and 1.6 μM of MDA; 0.16, and 0.23 μM of HHE; 0.026, [corrected] and 0.005 μM of HNE, respectively, in porcine models I and II). In all models, FFA release was only detected during the intestinal step, and reached up to 31% of total fatty acids (FA). The findings in this work may be of importance when designing oxidation oriented lipid digestion studies.

Determination of microalgaes' fatty acid content is often done with chloroform and methanol according to the Bligh and Dyer extraction, though faster methods exist. A number of comparisons between the Bligh and Dyer and faster methods have resulted in contradicting data, possibly due to differences in algae used and the different versions of the Bligh and Dyer method applied. Here, various forms of direct-transesterification (D-TE) and two-step transesterification (2-TE), including three versions developed in our lab, are compared with the original Bligh and Dyer (Can J Biochem Physiol 37: 911-917, 1959) extraction and two modifications thereof (Lee et al. J AOAC Int 79:487-492, 1996, and our own acidified version) on microalgae with different cell walls: Isochrysis galbana, Nannochloropsis oculata, and Phaeodactylum tricornutum. In total, fatty acid extracts from 11 methods were separated and quantified by gas chromatography with mass spectrometry. Results show that, for N. oculata and P. tricornutum, methods based on chloroform-methanol underestimated the fatty acid content compared with the 2-TE and D-TE methods, which gave similar results. Moreover, D-TE methods are faster than chloroform-methanol methods and use chemicals that are less toxic. Of the D-TE methods, the ones using hydrochloric acid and sulfuric acid recovered the most fatty acids, while boron trifluoride recovered slightly less. The main qualitative difference between the fatty acids recovered was that the chloroform-methanol methods recovered less saturated fatty acids in P. tricornutum.

Circulating concentrations of vitamin D, 25(OH)D, and 1,25(OH)2D are lower in obese than lean individuals, but little is known about the adipose tissue content of these molecules. The aim of this study was to explore the possibility to use time-of-flight secondary ion mass spectrometry (TOF-SIMS) to measure vitamin D and its metabolites in fat tissue in obese and lean subjects. Abdominal subcutaneous adipose tissue (SAT) biopsies were obtained from three lean and three obese women, and paired biopsies SAT and visceral adipose tissue (VAT) were obtained from three obese subjects during gastric bypass surgery. TOF-SIMS was used to measure vitamin D3, 25(OH)D3, and 1,25(OH)2D3 in adipose tissue. We found that vitamin D3, 25(OH)D3, and 1,25(OH)2D3 in adipose tissue can be measured with TOF-SIMS. In adipose tissue, vitamin D3 and its metabolites were located in adipocyte lipid droplets. The content of vitamin D3 (P=0.006) and 25(OH)D3 (P=0.018) were lower in SAT in obese compared with lean women. TOF-SIMS has the potential to semi-quantitatively measure vitamin D metabolites in adipose tissue, and offers a possibility to compare vitamin D levels in different depots and groups of individuals. It also gives the opportunity to explore the localization of vitamin D metabolites at a cellular level., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Diet is a significant modifiable risk factor for cardiovascular disease and high fish intake has been associated with vascular health in population studies. However, intervention studies have been inconclusive. In this study, male low-density lipoprotein receptor-deficient mice were given 16-week high fat/high sucrose diets, supplemented with either minced herring fillets or minced beef. The diets were matched in total fat and cholesterol content; taurine content and fatty acid composition was analysed. Body weights were recorded throughout the study; plasma lipids were analysed at week 8 and 16. Body composition and adipocyte size were evaluated at study end. Atherosclerosis was evaluated at week 12 (ultrasound) and at termination (en face histology). Herring-fed mice had a higher proportion of long-chain n-3 polyunsaturated fatty acids in the hepatic triacylglycerides (TAG) and phospholipid fractions. The herring-fed mice had increased body weight (P=0.007), and reduced epididymal adipocyte size (P=0.009), despite similar food intake and body composition as the beef-fed mice. The herring-fed mice had lower plasma TAG and very-low-density lipoprotein (VLDL)-cholesterol concentrations throughout the study (TAG; P=0.0012 and 0.004, VLDL-cholesterol; P=0.006 and 0.041, week 8 and 16, respectively). At week 16, the herring-fed had higher plasma concentrations of HDL-cholesterol (P=0.004) and less atherosclerotic lesions in the aortic arch (P=0.007) compared with the beef-fed mice. In conclusion, dietary herring in comparison to beef markedly improved vascular health in this mouse model, suggesting that herring provides an added value beyond its content of macronutrients.

The fermented cereal-based gruel togwa is used as weaning food for children in Tanzania. Togwa is rich in minerals but these are often not available for uptake in the human intestine due to natural inhibitors, such as phytate (IP(6)). The yeasts Pichia kudriavzevii TY13, Hanseniaspora guilliermondii TY14 and TY20, isolated from Tanzanian togwa, and selected for high phytase activity in complex yeast medium YPD, were now studied regarding their ability to degrade IP(6) in maize-based model togwa. A modified constitutively high-phytase producing Saccharomyces cerevisiae BY80 and commercial Aspergillus ficuum phytase were included for comparison. In addition, a strain of Lactobacillus plantarum was included in the model-togwa set-up. All yeasts in the study grew and reached final cell density 1.5-2 log units higher than the start value. S. cerevisiae BY80 degraded 85% of the IP(6) in 48 h; the same degradation level as with A. ficuum phytase (89%). Of the togwa-isolated yeasts, P. kudriavzevii TY13 and H. guilliermondii TY14 showed strong phytate degradation in the model-togwa; 95% or more of the initial IP(6) was degraded after 48 h. This corresponds to a remaining level of 0.4 and 0.3μmol IP(6)/g dw. Co-inoculation with L. plantarum did not increase IP(6) degradation. Moreover, fermentation with P. kudriavzevii TY13 yielded a successive increase in inorganic phosphate (P(i)), from 0.7 to 5.4 mM, suggesting a phytase production in TY13 which is fairly insensitive to P(i) repression. The study shows that phytate in a model togwa is available for yeast phytase enzymes, and addresses the importance of strain selection for effectively degrading the phytate. Certain yeasts originating from togwa seem to have developed a natural high phytase production, and P. kudriavzevii TY13 and H. guilliermondii TY14 seem particularly well adapted to phytate degradation in togwa, and is our choice for further studies and strain improvement., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Celiac disease is caused by an inappropriate immune response to incompletely digested gluten proteins. We investigated whether synthetic peptides with high affinity to wheat gliadin could be selected with a phage display technique and whether complexes between such peptides and gliadin could sustain gastric and pancreatic digestion. Two synthetic peptides, P61 and P64, were selected because of their high affinity to immobilized gliadin. They were allowed to form complexes with gliadin, whereafter the complexes were subjected to in vitro digestion with gastric and pancreatic enzymes. The digestion products were analyzed with Western blot and RP HPLC. The results showed that both peptides formed stable complexes with intact gliadin and that complexes between gliadin and peptide P64 partly resisted gastrointestinal digestion. The two peptides reduced the binding of serum anti-gliadin IgA antibodies by 12%, and 11.5%, respectively, and the binding of anti-gliadin antibodies of the IgG isotype by 13% and 10%. Thus peptides produced by a phage display technique could interact stably with gliadin partly masking epitopes for antibody binding. A combination of peptides of this kind may be used to block gliadin-immune system interactions., (Copyright © 2011 International Union of Biochemistry and Molecular Biology, Inc.)

The growing awareness of the relationship between diet and health has led to an increasing demand for food products that support health above and beyond providing basic nutrition. Probiotics are live organisms present in foods, which yield health benefits related to their interactions with the gastrointestinal tract. Phytases are a subgroup of phosphatases that catalyse the desphosphorylation of phytate, which reduces its negative impact on mineral bioavailability, and generates lower inositol phosphates. The aims of this investigation were to (i) study the ability of the probiotic candidate Bifidobacterium pseudocatenulatum to degrade phytate in synthetic medium, to (ii) identify the lower inositol phosphates generated, to (iii) study its survival under conditions mimicking gastrointestinal passage and finally to (iv) assess adhesion of the bacteria to Caco-2 cells. The first steps of InsP(6) degradation by B. pseudocatenulatum phytate-degrading enzyme/s were preferentially initiated at the DL-6-position and 5-position of the myo-inositol ring. It suggests that the main InsP(6) degradation pathway by B. pseudocatenulatum by sequential removal of phosphate groups was D/L-Ins(1,2,3,4,5)P(5) or D/L-Ins(1,2,3,4,6)P(5); D/L-Ins(1,2,3,4)P(4); to finally Ins(1,2,3)P(3) and D/L-Ins(1,2,4)P(3)/D/L-Ins(1,3,4)P(3). This human strain also showed a notable tolerance to bile as well as a selective adhesion capacity (adhesion to control surfaces was zero), to human intestinal Caco-2 cells comparable to the commercial probiotic B. lactis. The phytate-degrading activity constitutes a novel metabolic trait which could contribute to the improvement of mineral absorption in the intestine as a nutritional probiotic feature with potential trophic effect in human gut.

Using a combination of High-Performance Ion Chromatography analysis and kinetic studies, the pathway of myo-inositol hexakisphosphate dephosphorylation by a phytase from a Malaysian waste-water bacterium was established. The data demonstrate that the phytase preferably dephosphorylates myo-inositol hexakisphosphate in a stereospecific way by sequential removal of phosphate groups via D-I(1,2,3,4,5)P(5), D-I(2,3,4,5)P(4), D-I(2,3,4)P(3), D-I(2,3)P(2) to finally I(2)P. It was estimated that more than 90% of phytate hydrolysis occurs via D-I(1,2,3,4,5)P(5). Thus, the phytase from the Malaysian waste-water bacterium has to be considered a 6-phytase (E.C. 3.1.3.26). A second pathway of minor importance could be proposed which is in accordance with the results obtained from analysis of the dephosphorylation products formed by the action of the phytase under investigation on myo-inositol hexakisphosphate. It proceeds via D/L-I(1,2,4,5,6)P(5), D/L-I(1,2,4,5)P(4), D/L-I(1,2,4)P(3), D/L-I(2,4)P(2) to finally I(2)P.

The antioxidative effect of herring (Clupea harengus) light muscle press juice (PJ) against hemoglobin-(Hb-) mediated oxidation of washed cod mince during ice storage was tested. The PJ was fractionated into low-molecular-weight (LMW; <1 [corrected] kDa) and high-molecular-weight (HMW; >1, >3.5, and > 50 kDa) fractions; it was preheated (10 min, 100 degrees C) and tested with or without removing heat coagulated proteins. Its antioxidative effect was compared with that given by endogenous levels of two tentative antioxidant candidates: ascorbic acid and uric acid. Oxidation was followed by determining rancid odor, peroxide value, and redness. Whole herring PJ and the LMW-PJ fraction significantly (p < 0.001) extended the oxidation lag phase of controls, from 2 up to 8 and 7 days, respectively. The HWM-PJ fractions were significantly (p < 0.05) less efficient than the whole and LMW-PJ samples, giving only 3.5-4.5 days of lag phase. Heat-treated PJ, with and without the heat-coagulated proteins, gave 7 and 5 days of oxidation lag phase, respectively. Heating different batches of the LMW-PJ fraction grouped the results into two categories: one where heating almost fully destroyed the antioxidative activity (fractions prepared from spring-caught herring) and another where heating had no or a minor effect (fractions prepared from fall-caught herring). The spring LMW-PJ had low ascorbic acid levels (18-42.6 microM), and 50-100% were destroyed by the heating. In fall LMW-PJ, the levels were 76.2-137.6 microM, and only 43-51% were destroyed. Ascorbic acid fortification of heated spring LMW-PJ to reach the levels found in the corresponding unheated spring LMW-PJ sample and the heated fall LMW-PJ gave back most of the antioxidative activity, which proved an important role of ascorbic acid for the antioxidative activity of LMW-herring PJ. This conclusion is drawn despite the fact that pure solutions with endogenous levels of ascorbic acid (giving 8.4-19.6 microM in final model) only very slightly delayed Hb-mediated oxidation of the washed cod mince.

Using a combination of high-performance ion chromatography analysis and kinetic studies, the pathway of myo-inositol hexakisphosphate dephosphorylation by the beta-propeller phytase of Shewanella oneidensis was established, which was then compared with that of Bacillus subtilis 168, Bacillus amyloliquefaciens ATCC 15841, and B. amyloliquefaciens 45 beta-propeller phytases. The data demonstrate that all of these beta-propeller phytases dephosphorylate myo-inositol hexakisphosphate in a stereospecific way by sequential removal of phosphate groups via d-Ins(1,2,4,5,6)P5, Ins(2,4,5,6)P4 to finally Ins(2,4,6)P3. Thus, the beta-propeller phytases prefer the hydrolysis of every second phosphate over that of adjacent ones. This finding does not support previous phytate degradation models proposed by J. Kerovuo, J. Rouvinen, and F. Hatzack (2000. Biochem. J. 352: 623-628) and R. Greiner, A. Farouk, M. Larsson Alminger, and N.G. Carlsson (2002. Can. J. Microbiol. 48: 986-994), but seems to fit with the structural model given by S. Shin, N.C. Ha, B.C. Oh, T.K. Oh, and B.H. Oh (2001. Structure, 9: 851-858).

For the first time a dual pathway for dephosphorylation of myo-inositol hexakisphosphate by a histidine acid phytase was established. The phytate-degrading enzyme of Klebsiella terrigena degrades myo-inositol hexakisphosphate by stepwise dephosphorylation, preferably via D-Ins(1,2,4,5,6)P5, D-Ins(1,2,5,6)P4, D-Ins(1,2,6)P3, D-Ins(1,2)P2 and alternatively via D-Ins(1,2,4,5,6)P5, Ins(2,4,5,6)P4, D-Ins(2,4,5)P3, D-Ins(2,4)P2 to finally Ins(2)P. It was estimated that more than 98% of phytate hydrolysis occurs via D-Ins(1,2,4,5,6)P5. Therefore, the phytate-degrading enzyme from K. terrigena has to be considered a 3-phytase (EC 3.1.3.8). A second dual pathway of minor importance could be proposed that is in accordance with the results obtained by analysis of the dephosphorylation products formed by the action of the phytate-degrading enzyme of K. terrigena on myo-inositol hexakisphosphate. It proceeds preferably via D-Ins(1,2,3,5,6)P5, D-Ins(1,2,3,6)P4, Ins(1,2,3)P3, D-Ins(2,3)P2 and alternatively via D-Ins(1,2,3,5,6)P5, D-Ins(2,3,5,6)P4, D-Ins(2,3,5)P3, D-Ins(2,3)P2 to finally Ins(2)P. D-Ins(2,3,5,6)P4, D-Ins(2,3,5)P3, and D-Ins(2,4)P2 were reported for the first time as intermediates of enzymatic phytate dephosphorylation. A role of the phytate-degrading enzyme from K. terrigena in phytate breakdown could not be ruled out. Because of its cytoplasmatic localization and the suggestions for substrate recognition, D-Ins(1,3,4,5,6)P5 might be the natural substrate of this enzyme and, therefore, may play a role in microbial pathogenesis or cellular myo-inositol phosphate metabolism.

Using a combination of high-performance ion chromatography analysis and kinetic studies, the pathway of dephosphorylation of myo-inositol hexakisphosphate by the phytases purified from faba bean and lupine seeds, respectively, was established. The data demonstrate that the legume seed phytases under investigation dephosphorylate myo-inositol hexakisphosphate in a stereospecific way. The phytase from faba bean seeds and the phytase LP2 from lupine seeds degrade phytate by sequential removal of phosphate groups via D-Ins(1,2,3,5,6)P(5), D-Ins(1,2,5,6)P(4), D-Ins(1,2,6)P(3), and D-Ins(1,2)P(2) to finally Ins(2)P, whereas the phytases LP11 and LP12 from lupine seeds generate the final degradation product Ins(2)P via D-Ins(1,2,4,5,6)P(5), D-Ins(1,2,5,6)P(4), D-Ins(1,2,6)P(3), and D-Ins(1,2)P(2).

The pathway of dephosphorylation of myo-inositol hexakisphosphate by the phytate-degrading enzymes of Bacillus subtilis 168, Bacillus amyloliquefaciens ATCC 15841, and Bacillus amyloliquefaciens 45 was established using a combination of high-performance ion chromatography analysis and kinetic studies. The data demonstrate that all the Bacillus phytate-degrading enzymes under investigation dephosphorylate myo-inositol hexakisphosphate by sequential removal of phosphate groups via two independent routes; the routes proceed via D-Ins(1,2,4,5,6)P5 to Ins(2,4,5,6)P4 to finally Ins(2,4,6)P3 or D-Ins(2,5,6)P3 and via D-Ins(1,2,4,5,6)P5 to D-Ins(1,2,5,6)P4 to finally D-Ins(1,2,6)P3. The resulting myo-inositol trisphosphate D-Ins(1,2,6)P3 was degraded via D-Ins(2,6)P2 to finally Ins(2)P after prolonged incubation times in combination with increased enzyme concentration.

A sensitive and simple method for the simultaneous determination of nutritionally important minerals in food samples is in great demand. Ion chromatography coupled with UV-vis detection is shown to be an appropriate technique for this objective. The method is based on the formation of mineral complexes by pyridine-2,6-dicarboxylic acid in the mobile phase. The complexes are then postcolumn derivatized with 4-(2-pyridylazo)resorcinol (PAR), resulting in mineral-PAR complexes that are detected by UV-vis absorption at 500 nm. This facilitates the simultaneous separation and quantification of minerals in one chromatographic run. Within 16 min, Cu, Ni, Zn, Co, Mn, and Fe are analyzed. When a 50 microL injection volume is used, the average detection limit is 5 ppb in the injection liquid. The detection limit makes it a superior alternative to AAS and, in several applications, also an alternative to ICP-MS techniques. Different sample treatments were evaluated. The concentration of acid in the treated sample varied with the sample treatment, which may cause a limitation for the injection volume. A crucial prerequisite to achieve the reported detection limits and to obtain reliable results is to completely exclude all contamination from instruments and materials.