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Conjugation and Other Methods in Polymeric Vaccines
Published in Mesut Karahan, Synthetic Peptide Vaccine Models, 2021
Chromatography is the general name of the methods for separating components in complex solutions. Amino acid analysis is used for differentiation of amino acids and peptides, determination of amino acid composition of proteins, analysis of peptide sequences, and diagnosis of diseases belonging to amino acid metabolism. According to this information high performance liquid chromatography (HPLC) is a chromatographic technique that is used to separate, identify, and quantify components of a mixture, for example the separation of chemical compounds or identification of constituents of a biological sample; a typical HPLC system contains a stationary phase, a mobile phase of varying polarity, and an ultraviolet detector (Blum 2014). This technique is commonly used because it is sensitive, easily adaptable to quantitative determinations, non-volatile (suitable for decomposition of heat-degradable compounds), and has applicability with the most commonly used substances (amino acids, peptides, drugs, pesticides) (Mauldin et al. 2006; Fornaguera and Solans 2018) (Figure 7.1).
Hereditary and Metabolic Diseases of the Central Nervous System in Adults
Published in Philip B. Gorelick, Fernando D. Testai, Graeme J. Hankey, Joanna M. Wardlaw, Hankey's Clinical Neurology, 2020
Amino acid analysis: Elevated plasma and urine homocysteine and methionine levels.Decreased levels of cysteine.
Serologic Evaluation Using Monoclonal and Polyclonal Antibodies — Their Diagnostic and Prognostic Usefulness
Published in John T. Kemshead, Pediatric Tumors: Immunological and Molecular Markers, 2020
The polyamines, putrescine and spermidine, were evaluated as tumor markers for diagnosis and monitoring of medulloblastomas. Strictly speaking they are not tumor markers, but are metabolic products of nucleic acids reflecting cellular growth and proliferation. The assay technique uses chromatography and amino acid analysis. Approximately 30–40% of tumors have raised levels (> 184 and 150 pmol/ml for putrescine and spermidine, respectively, at diagnosis). Marton et al. found that 15/16 patients had a raised level correlating with response to therapy.49
Laboratory testing for mitochondrial diseases: biomarkers for diagnosis and follow-up
Published in Critical Reviews in Clinical Laboratory Sciences, 2023
Abraham J. Paredes-Fuentes, Clara Oliva, Roser Urreizti, Delia Yubero, Rafael Artuch
Quantitative analyses of amino acids can be conducted by reversed-phase high-performance liquid chromatography (HPLC), ion exchange chromatography, GC–MS, or ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS). In the past decades, ion exchange chromatography has been the most widely employed method (reference method), but its long analysis time (e.g. 2–3 h per sample), the need for large sample volumes and the presence of some interfering compounds that react with ninhydrin are reasons why many laboratories are transitioning to the use of UPLC–MS/MS. These methods require a simple sample pretreatment step that uses 6-aminoquinolyl-N-hydroxysuccinimildyl carbamate (or similar compound) as a derivatization reagent and allow quantification of 42 amino acids and related compounds of clinical interest on 2 μL of sample in a 9 min run [109]. Moreover, validated, commercially available amino acid analysis kits can be used for selective, accurate, and fully quantitative amino acid analyses.
Integration of stool microbiota, proteome and amino acid profiles to discriminate patients with adenomas and colorectal cancer
Published in Gut Microbes, 2022
Sofie Bosch, Animesh Acharjee, Mohammed Nabil Quraishi, Irene V Bijnsdorp, Patricia Rojas, Abdellatif Bakkali, Erwin EW Jansen, Pieter Stokkers, Johan Kuijvenhoven, Thang V Pham, Andrew D Beggs, Connie R Jimenez, Eduard A Struys, Georgios V Gkoutos, Tim GJ de Meij, Nanne KH de Boer
By means of standard operating procedure, targeted amino acid analysis was performed on fecal samples using a targeted High Performance Liquid Chromatography (HPLC) technique, specifically amino acid analysis (AAA).39 The 500 mg fecal subsample and 1000 µL distilled water were mixed by vortex for one minute to homogenize the samples. The samples were then recoded and investigated by an independent laboratory researcher, blinded for the diagnosis. To prevent potential bias by differences in fecal water content, samples were frozen at minus 30 degrees and subsequently freeze-dried for 24 hours (Christ Alpha 2–4). Depending on the fecal consistency of the sample, the residual after freeze drying was approximately 30–70 mg. Consistently maintaining a feces-water ratio of 20 mg:1 mL this residual was mixed with distilled water. This mixture was again vigorously homogenized using vortex. For the analysis of the amino acid profile, 400 µL of the mixture was pipetted into a filter and centrifuged for 20 minutes at 14.000 g (Hettig Zentrifugen Mikro 2 R). Subsequently, the supernatant was mixed with an internal standard solution with a one-to-one ratio. This final mixture was centrifuged for 10 minutes and filtered (Whatman) into compatible containers for the final amino acid analyses (Biochrome 30). Amino acids were separated by ion-exchange chromatography and detected by UV-absorbance after post-column derivatization with ninhydrin.
Transcerebral exchange kinetics of large neutral amino acids during acute inspiratory hypoxia in humans
Published in Scandinavian Journal of Clinical and Laboratory Investigation, 2019
Rasmus H. Dahl, Ronan M. G. Berg, Sarah Taudorf, Damian M. Bailey, Carsten Lundby, Mette Christensen, Fin S. Larsen, Kirsten Møller
We measured all LNAAs with the exception of tryptophan (AAAs: phenylalanine and tyrosine; BCAAs: valine, leucine, and isoleucine; others: histidine and methionine). Plasma was isolated immediately after sampling by centrifugation at 3000 g for 10 min. at 4 °C, and stored at −20 °C until analysis. On the day of amino acid analysis, plasma was thawed and deproteinised using sulfosalicylic acid (containing norleucine as internal standard). Amino acid analysis was performed by high performance liquid chromatography (HPLC) with fluorometric detection, utilizing post-column derivatization of free amino acids with o-phthalaldehyde (OPA). Chromatographic separation was performed by ion-exchange chromatography, using a lithium amino acid analysis HPLC-column (4.0 × 100 mm, 5 µm, Pickering Laboratories). The HPLC system consisted of a Waters 510 dual piston pump system controller, a Waters 420 fluorescence detector and a Waters 717 plus autosampler all controlled by a Waters Empower software system (Milford, MA) for chromatographic analysis.