Browsing by Subject "amino acids"

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  • Woiwode, Ulrich; Ferri, Martina; Maier, Norbert M.; Lindner, Wolfgang; Lämmerhofer, Michael (2018)
    Abstract A cardinal requirement for effective 2D-HPLC separations is sufficient complementarity in the retention profiles of first and second dimension separations. It is shown that retention and enantioselectivity of chiral selectors derived from cinchona alkaloids can be conveniently modulated by structural variation of the carbamate residue of the quinine/quinidine carbamate ligand of such chiral stationary phases (CSP). A variety of aliphatic and aromatic residues have been tested in comparison to non-carbamoylated quinine CSP. Various measures of orthogonality have been utilized to derive the CSP that is most complementary to the tert-butylcarbamoylated quinine CSP (tBuCQN CSP), which is commercially available as Chiralpak QN-AX column. It turned out that O-9-(2,6-diisopropylphenylcarbamoyl)-modified quinine is most promising in this respect. Its implementation as a complementary CSP for the separation of amino acids derivatized with Sanger’s reagent (2,4-dinitrophenylated amino acids) in the first dimension combined with a tBuCQN CSP in the second dimension revealed successful enantiomer separations in a comprehensive chiral×chiral 2D-HPLC setup. However, the degree of complementarity could be greatly enhanced when simultaneously the absolute configurations were exchanged from quinine to quinidine in the chiral selector of the first dimension separation resulting in opposite elution orders of the enantiomers in the two dimensions. The advantage of such a chiral×chiral over achiral×chiral 2D-HPLC setup, amongst others, is the perfect compatibility of the mobile phase because in both dimensions the identical eluent can be used.
  • Sillanpää, Meri (Helsingin yliopisto, 2021)
    The literature study of this thesis focuses on the different analytical methods used to analyse amino acids in food and beverage samples. Amino acids are essential organic molecules and their concentrations in foods and beverages constitute, inter alia, the product’s nutritional value, quality, freshness, and flavour. Amino acid analysis of foodstuff has various applications, which exploit several analytical methods. These reviewed methods are founded on academic articles published during the past two decades. This literature review discusses the different sample matrixes, sample preparation methods, ways to derivate analytes, and different separation and detection methods utilized in the recent amino acid studies. The experimental part of this thesis was a modification of L-asparagine and L-aspartic acid test (L-Asp/L-AspAc) in Thermo Fisher Scientific Oy industrial R&D laboratory. An enzymatic photometric method is used to determine L-Asp/L-AspAc amino acids in food samples. The modification process entailed pre-testing of several candidate methods, from which the most suitable one was selected. The feasibility of the chosen test was affirmed before verification and validation of the modified test.
  • Rashid, Fatimah Azzahra Ahmad; Scafaro, Andrew P.; Asao, Shinichi; Fenske, Ricarda; Dewar, Roderick; Masle, Josette; Taylor, Nicolas L.; Atkin, Owen K. (2020)
    Leaf respiration in the dark (R-dark) is often measured at a single time during the day, with hot-acclimation lowering R-dark at a common measuring temperature. However, it is unclear whether the diel cycle influences the extent of thermal acclimation of R-dark, or how temperature and time of day interact to influence respiratory metabolites. To examine these issues, we grew rice under 25 degrees C : 20 degrees C, 30 degrees C : 25 degrees C and 40 degrees C : 35 degrees C day : night cycles, measuring R-dark and changes in metabolites at five time points spanning a single 24-h period. R-dark differed among the treatments and with time of day. However, there was no significant interaction between time and growth temperature, indicating that the diel cycle does not alter thermal acclimation of R-dark. Amino acids were highly responsive to the diel cycle and growth temperature, and many were negatively correlated with carbohydrates and with organic acids of the tricarboxylic acid (TCA) cycle. Organic TCA intermediates were significantly altered by the diel cycle irrespective of growth temperature, which we attributed to light-dependent regulatory control of TCA enzyme activities. Collectively, our study shows that environmental disruption of the balance between respiratory substrate supply and demand is corrected for by shifts in TCA-dependent metabolites.
  • Wang, Qin; Wurtz, Peter; Auro, Kirsi; Morin-Papunen, Laure; Kangas, Antti J.; Soininen, Pasi; Tiainen, Mika; Tynkkynen, Tuulia; Joensuu, Anni; Havulinna, Aki S.; Aalto, Kristiina; Salmi, Marko; Blankenberg, Stefan; Zeller, Tanja; Viikari, Jorma; Kahonen, Mika; Lehtimaki, Terho; Salomaa, Veikko; Jalkanen, Sirpa; Jarvelin, Marjo-Riitta; Perola, Markus; Raitakari, Olli T.; Lawlor, Debbie A.; Kettunen, Johannes; Ala-Korpela, Mika (2016)
    Background: Hormonal contraception is commonly used worldwide, but its systemic effects across lipoprotein subclasses, fatty acids, circulating metabolites and cytokines remain poorly understood. Methods: A comprehensive molecular profile (75 metabolic measures and 37 cytokines) was measured for up to 5841 women (age range 24-49 years) from three population-based cohorts. Women using combined oral contraceptive pills (COCPs) or progestin-only contraceptives (POCs) were compared with those who did not use hormonal contraception. Metabolomics profiles were reassessed for 869 women after 6 years to uncover the metabolic effects of starting, stopping and persistently using hormonal contraception. Results: The comprehensive molecular profiling allowed multiple new findings on the metabolic associations with the use of COCPs. They were positively associated with lipoprotein subclasses, including all high-density lipoprotein (HDL) subclasses. The associations with fatty acids and amino acids were strong and variable in direction. COCP use was negatively associated with albumin and positively associated with creatinine and inflammatory markers, including glycoprotein acetyls and several growth factors and interleukins. Our findings also confirmed previous results e.g. for increased circulating triglycerides and HDL cholesterol. Starting COCPs caused similar metabolic changes to those observed cross-sectionally: the changes were maintained in consistent users and normalized in those who stopped using. In contrast, POCs were only weakly associated with metabolic and inflammatory markers. Results were consistent across all cohorts and for different COCP preparations and different types of POC delivery. Conclusions: Use of COCPs causes widespread metabolic and inflammatory effects. However, persistent use does not appear to accumulate the effects over time and the metabolic perturbations are reversed upon discontinuation. POCs have little effect on systemic metabolism and inflammation.
  • Marttinen, Eeva (Helsingfors universitet, 2010)
    Nitrogen is usually the growth limiting nutrient in boreal forest soils. Most of the nitrogen is bound to organic fraction, and low bioavailability of nitrogen delimits plant growth in boreal forest soils. Amino acids are easily available nitrogen compounds and thus they are important nitrogen sources for soil microorganisms. Almost all boreal forest trees form mycorrhizal assoociations with fungi. Mycorrhizal fungi produce wide variety of enzymes which break down organic nitrogen compounds. So far there is little knowledge of amino acid mineralization mechanisms of ectomycorrhizal fungi. L-amino acid oxidase (LAO) catalyses the mineralization of amino acids to ammonium. The ectomycorrhizal fungi Hebeloma spp. and Laccaria spp. have been shown to possess LAO enzyme activities. It has been proposed that LAO is one of the nitrogen mineralization mechanisms in ectomycorrhizal fungi, but so far no LAO genes have been described from basidiomycete fungi. In this study the first LAO gene sequences from the basidiomycete fungus Hebeloma cylindrosporum was described. The RACE-PCR -method was used to determine the 3´ and 5´ end sequences of the cDNA of the LAO1 gene. Based on the obtained sequences, primers to isolate the genomic DNA and cDNA sequences of the LAO1 gene were designed. The structure of the LAO1 gene, which is composed of five exons and four introns, was determined. Binding site of nitrogen regulating protein was found from upstream region of LAO1-gene. The partial genomic DNA sequence of gene adjacent to LAO1-gene was also measured. In the L. bicolor genome the gene preceding the LAO1 gene has been annotated as a putative pyruvate decarboxylase. In this study the partial cDNA sequence of another LAO-homolog of H. cylindrosporum was also determined. The LAO gene from another basidiomycete fungus, Laccaria bicolor, was also recognised. The gene model of LAO gene of L. bicolor was unannotated in the NCBI database. Based on the phylogenetic tree of LAO-related protein sequences, the ancestral form of LAO gene has been duplicated. This study provides molecular biological information on the catabolic mechanisms of amino acids in ectomycorrhizal fungi. Ammonium ions, produced by ectomycorrhizal fungi, might be a significant source of nitrogen for plants and other soil microbes. It is possible that LAO is an important factor of nitrogen cycle in soils of boreal forests.
  • Soronen, Päivi (Helsingin yliopisto, 2019)
    Nitrogen (N) availability often limits plant growth in the boreal forest ecosystem. There has been a lack of reliable method to study soil N supply as in traditionally used potassium chloride (KCl) extraction sampling and sample preparation disturb soil structure and stimulate N mineralization, leading to the overestimation of inorganic N forms ammonium (NH4+) and nitrate (NO3-) and underestimation of organic N forms such as amino acids. Diffusion-based microdialysis technique for the sampling of soil diffusive N fluxes gives an opportunity to study soil N supply at a scale that is relevant for plant N uptake, as microdialysis probe has a membrane that reminds the plant fine root in its scale and also, to some extent, in its function. During sampling, the movement of water inside the microdialysis probe induces diffusive flux of solutes across the membrane surface along the concentration gradient. The aim of this study was to test the performance of microdialysis technique at different soil moisture content levels and its capability to monitor temporal changes in diffusive N fluxes in laboratory experiments (ex situ). Soil fine-scale N dynamics were further studied by comparing the diffusive N fluxes in the field (in situ) in boreal forest soil to multiple factors that are thought to affect forest soil N availability. In this study, soil diffusive NH4+, NO3- and amino acid N fluxes were sampled ex situ from sieved soils taken from three different sites – clear-cut, spruce stand (MT spruce) and pine stand (VT pine) in Lapinjärvi, Finland in November 2017. In ex situ microdialysis experiments, the diffusive N fluxes were observed at three different soil moisture content levels and after N addition. In situ microdialysis sampling was run at the logging residue experiment of the Lapinjärvi clear-cut site and at the MT spruce site in June 2018 and at the pine logging residue experiment in Kiikala, Finland in September 2018. The results from the in situ microdialysis were compared with soil moisture content, pH, C-to-N ratio and temperature as well as with the net N mineralization and net nitrification rates, microbial biomass C and N contents and the concentrations of volatile monoterpenes and condensed tannins, factors that are assumed to affect N availability in forest soil. Nitrogen fluxes sampled ex situ showed that the total amino acid flux in the soil taken from the clear-cut site was only half of that in the MT spruce soil whereas NO3- flux was two times higher at the clear-cut site than at the MT spruce site. MT spruce soil with a moisture content of 60 % water-holding capacity (WHC) had significantly higher NH4+ flux than the same soil in its field moisture content (44 % WHC). Nitrogen pulse was detected in all soil samples as increased NH4+ flux after the N addition, followed by a subsequent decrease near to the initial level. In situ microdialysis sampling showed that the total amino acid fluxes were 5–15 nmol N cm-2 h-1 and they dominated the total diffusive N fluxes in Lapinjärvi and Kiikala. On average, the smallest share of the total free amino acids (54 %) was observed at the control plots of the logging residue experiment in Lapinjärvi. No correlation between the KCl-extractable NH4+-N concentration and the diffusive NH4+ flux was found, but instead the KCl-extractable NH4+-N concentration showed a significant positive correlation with the diffusive fluxes of both total free amino acid N and nitrate. Moreover, the diffusive NH4+ flux correlated positively with the net N mineralization rate. In general, ex situ microdialysis sampling showed 2–10 times higher amino acid fluxes and 10–20 times higher ammonium fluxes than the in situ microdialysis that reflects the effect of sampling, sample storage and preparation. The effect of soil moisture on the diffusive N fluxes could be further studied in laboratory experiments and in situ. The results of this study showed that the diffusive fluxes of different N forms are decoupled from the bulk soil concentrations. Moreover, microdialysis could be possibly used to quantify the transformation processes of N compounds in soil. These results increase the evidence that microdialysis has potential to detect temporal changes in N fluxes and possibly give new information about the ongoing processes at soil microsites.
  • Ylinen, Vappu; Pylkkö, Päivi; Peura, Jussi; Tuomola, Essi; Valaja, Jarmo (2018)
    The effects of low-protein diets supplemented with DL-methionine (MET) and L-histidine (HIS) on growth, pelt size and pelt quality were studied in two performance trials conducted at the Kannus Research Farm Luova Ltd, Finland. Both trials were conducted with 200 blue foxes, caged male-female pairs, initial age on average 20 weeks (trial 1) and 25 weeks (trial 2). In trial 1, diets contained digestible crude protein (DCP) 24%, 20% and 16% of metabolisable energy (ME). In trial 2, diets contained DCP 20%, 16.5% and 13% of ME. In both trials, the middle protein level was fed with or without MET and the lowest protein level was fed with MET and with or without HIS. In trial 1, blue foxes showed the greatest average daily gain (ADG) in the highest protein diet. Pelt size and pelt quality were not affected. In trial 2, blue foxes showed the greatest ADG in the low-protein groups. Pelt size and pelt quality were not affected.
  • Ketonen, Krista (Helsingfors universitet, 2014)
    Variation of the protein and amino acid content of barley, wheat and oats were studied. Diets based on grain samples of different protein content were optimized for pigs and poultry.The study went on to optimize diets for pigs and poultry with grains of different protein contents. The amino acid and raw protein analysis was undertaken on 38 grain samples. Correlations were calculated between different variables in grain samples and linear regression analysis was conducted between the protein and amino acid composition. The best estimate for amino acid concentrations of cereals was the protein content. The relative content of amino acids decreases as protein content increases and especially so in barley and wheat. Most reliable regression equations between amino acid and protein content were made for barley and wheat samples. For oat reliable regression equations could not be made. Oats also differed by other features from barley and wheat as it correlated with different variables compared to barley and wheat. Amount of needed protein concentrate levels decreased when barley and wheat protein and amino acid contents were considered in optimization. Protein concentrate levels did not decrease when used oat sample with highest protein content.
  • Taipale, Sami Johan; Kahilainen, Kimmo Kalevi; Holtgrieve, Gordon William; Peltomaa, Elina Talvikki (2018)
    The first few months of life is the most vulnerable period for fish and their optimal hatching time with zooplankton prey is favored by natural selection. Traditionally, however, prey abundance (i.e., zooplankton density) has been considered important, whereas prey nutritional composition has been largely neglected in natural settings. High-quality zooplankton, rich in both essential amino acids (EAAs) and fatty acids (FAs), are required as starting prey to initiate development and fast juvenile growth. Prey quality is dependent on environmental conditions, and, for example, eutrophication and browning are two major factors defining primary producer community structures that will directly determine the nutritional quality of the basal food sources (algae, bacteria, terrestrial matter) for zooplankton. We experimentally tested how eutrophication and browning affect the growth and survival of juvenile rainbow trout (Oncorhynchus mykiss) by changing the quality of basal resources. We fed the fish on herbivorous zooplankton (Daphnia) grown with foods of different nutritional quality (algae, bacteria, terrestrial matter), and used GC-MS, stable isotope labeling as well as bulk and compound-specific stable isotope analyses for detecting the effects of different diets on the nutritional status of fish. The content of EAAs and omega-3 (ω-3) polyunsaturated FAs (PUFAs) in basal foods and zooplankton decreased in both eutrophication and browning treatments. The decrease in ω-3 PUFA and especially docosahexaenoic acid (DHA) was reflected to fish juveniles, but they were able to compensate for low availability of EAAs in their food. Therefore, the reduced growth and survival of the juvenile fish was linked to the low availability of DHA. Fish showed very low ability to convert alpha-linolenic acid (ALA) to DHA. We conclude that eutrophication and browning decrease the availability of the originally phytoplankton-derived DHA for zooplankton and juvenile fish, suggesting bottom-up regulation of food web quality.