Search

Rapid and accurate serological analysis of SARS-CoV-2 antibodies is important for assessing immune protection from vaccination or infection of individuals and for projecting virus spread within a population. The quartz crystal microbalance (QCM) is a label-free flow-based sensor platform that offers an opportunity to detect the binding of a fluid-phase ligand to an immobilized target molecule in real time. A QCM-based assay was developed for the detection of SARS-CoV-2 antibody binding and evaluated for assay reproducibility. The assay was cross-compared to the Roche electrochemiluminescence assay (ECLIA) Elecsys® Anti-SARS-CoV-2 serology test kit and YHLO's chemiluminescence immunoassay (CLIA). The day-to-day reproducibility of the assay had a correlation of r2 = 0.99, p < 0.001. The assay linearity was r2 = 0.96, p < 0.001, for dilution in both serum and buffer. In the cross-comparison analysis of 119 human serum samples, 59 were positive in the Roche, 52 in the YHLO, and 48 in the QCM immunoassay. Despite differences in the detection method and antigen used for antibody capture, there was good coherence between the assays, 80–100% for positive and 96–100% for negative test results. In summation, the QCM-based SARS-CoV-2 IgG immunoassay showed high reproducibility and linearity, along with good coherence with the ELISA-based assays. Still, factors including antibody titer and antigen-binding affinity may differentially affect the various assays' responses. [ABSTRACT FROM AUTHOR]

Objective: Some patients regain weight to a variable extent from 1 year after Roux‐en‐Y gastric bypass surgery (RYGB), though rarely reaching preoperative values. The aim of the present study was to investigate whether, when, and to what extent metabolic remission occurs. Methods: Fasting metabolite and lipid profiles were determined in blood plasma collected from a nonrandomized intervention study involving 148 patients before RYGB and at 2, 12, and 60 months post RYGB. Both short‐term and long‐term alterations in metabolism were assessed. Anthropometric and clinical variables were assessed at all study visits. Results: This study found that the vast majority of changes in metabolite levels occurred during the first 2 months post RYGB. Notably, thereafter the metabolome started to return toward the presurgical state. Consequently, a close‐to‐presurgical metabolome was observed at the time when patients reached their lowest weight and glucose level. Lipids with longer acyl chains and a higher degree of unsaturation were altered more dramatically compared with shorter and more saturated lipids, suggesting a systematic and reversible lipid remodeling. Conclusions: Remission of the metabolic state was observed prior to notable weight regain. Further and more long‐term studies are required to assess whether the extent of metabolic remission predicts future weight regain and glycemic deterioration. [ABSTRACT FROM AUTHOR]

Bariatric surgery is an efficient method to induce weight loss and also, frequently, remission of type 2 diabetes (T2D). Unpaired studies have shown bariatric surgery and dietary interventions to differentially affect multiple hormonal and metabolic parameters, suggesting that bariatric surgery causes T2D remission at least partially via unique mechanisms. In the current study, plasma metabolite profiling was conducted in patients with (n = 10) and without T2D (n = 9) subjected to Roux-en-Y gastric bypass surgery (RYGB). Mixed-meal tests were conducted at baseline, after the presurgical very-low-calorie diet (VLCD) intervention, immediately after RYGB, and after a 6-week recovery period. Thereby, we could compare fasted and postprandial metabolic consequences of RYGB and VLCD in the same patients. VLCD yielded a pronounced increase in fasting acylcarnitine levels, whereas RYGB, both immediately and after a recovery period, resulted in a smaller but opposite effect. Furthermore, we observed profound changes in lipid metabolism following VLCD but not in response to RYGB. Most changes previously associated with RYGB were found to be consequences of the presurgical dietary intervention. Overall, our results question previous findings of unique metabolic effects of RYGB and suggest that the effect of RYGB on the metabolite profile is mainly attributed to caloric restriction. [ABSTRACT FROM AUTHOR]

The plasma metabolome is associated with multiple phenotypes and diseases. However, a systematic study investigating clinical determinants that control the metabolome has not yet been conducted. In the present study, therefore, we aimed to identify the major determinants of the plasma metabolite profile. We used ultra-high performance liquid chromatography (UHPLC) coupled to quadrupole time of flight mass spectrometry (QTOF-MS) to determine 106 metabolites in plasma samples from 2503 subjects in a cross-sectional study. We investigated the correlation structure of the metabolite profiles and generated uncorrelated metabolite factors using principal component analysis (PCA) and varimax rotation. Finally, we investigated associations between these factors and 34 clinical covariates. Our results suggest that liver function, followed by kidney function and insulin resistance show the strongest associations with the plasma metabolite profile. The association of specific phenotypes with several components may suggest multiple independent metabolic mechanisms, which is further supported by the composition of the associated factors. [ABSTRACT FROM AUTHOR]

Mass spectrometric epitope mapping has become a versatile method to precisely determine a soluble antigen's partial structure that directly interacts with an antibody in solution. Typical lengths of investigated antigens have increased up to several 100 amino acids while experimentally determined epitope peptides have decreased in length to on average 10–15 amino acids. Since the early 1990s more and more sophisticated methods have been developed and have forwarded a bouquet of suitable approaches for epitope mapping with immobilized, temporarily immobilized, and free‐floating antibodies. While up to now monoclonal antibodies have been mostly used in epitope mapping experiments, the applicability of polyclonal antibodies has been proven. The antibody's resistance towards enzymatic proteolysis has been of key importance for the two mostly applied methods: epitope excision and epitope extraction. Sample consumption has dropped to low pmol amounts on both, the antigen and the antibody. While adequate in‐solution sample handling has been most important for successful epitope mapping, mass spectrometric analysis has been found the most suitable read‐out method from early on. The rapidity by which mass spectrometric epitope mapping nowadays is executed outperforms all alternative methods. Thus, it can be asserted that mass spectrometric epitope mapping has reached a state of maturity, which allows it to be used in any mass spectrometry laboratory. After 25 years of constant and steady improvements, its application to clinical samples, for example, for patient characterization and stratification, is anticipated in the near future. © 2016 Wiley Periodicals, Inc. Mass Spec Rev 37:229–241, 2018. [ABSTRACT FROM AUTHOR]

Glucagon-like peptide 1 (GLP-1), secreted from intestinal L cells, glucose dependently stimulates insulin secretion from β-cells. This glucose dependence prevents hypoglycemia, rendering GLP-1 analogs a useful and safe treatment modality in type 2 diabetes. Although the amino acid glutamine is a potent elicitor of GLP-1 secretion, the responsible mechanism remains unclear. We investigated how GLP-1 secretion is metabolically coupled in L cells (GLUTag) and in vivo in mice using the insulin-secreting cell line INS-1 832/13 as reference. A membrane-permeable glutamate analog (dimethylglutamate [DMG]), acting downstream of electrogenic transporters, elicited similar alterations in metabolism as glutamine in both cell lines. Both DMG and glutamine alone elicited GLP-1 secretion in GLUTag cells and in vivo, whereas activation of glutamate dehydrogenase (GDH) was required to stimulate insulin secretion from INS-1 832/13 cells. Pharmacological inhibition in vivo of GDH blocked secretion of GLP-1 in response to DMG. In conclusion, our results suggest that nonelectrogenic nutrient uptake and metabolism play an important role in L cell stimulus-secretion coupling. Metabolism of glutamine and related analogs by GDH in the L cell may explain why GLP-1 secretion, but not that of insulin, is activated by these secretagogues in vivo. [ABSTRACT FROM AUTHOR]

BACKGROUND: Hyperthyroidism is associated with alterations in metabolism that are currently only partially understood. The objective of the study was to investigate changes in metabolism associated with reinstatement of euthyroidism in Swedish patients. METHODS: Eighty metabolites in plasma were profiled from 10 subjects with Graves' disease (GD) at baseline and after 9 and 15 months of treatment to reinstate euthyroidism. Thyroid parameters, thyrotropin (TSH), TSH receptor antibodies, free triiodothyronine, and free thyroxine were followed. Main findings were validated in plasma from 20 subjects with GD at baseline and at three, six, and nine months. The study was conducted at the endocrinology clinic in Malmö, Sweden. RESULTS: Euthyroidism was reinstated at three months, and thyroid status did not change further during the 15-month follow-up. This was paralleled by altered levels of 9/19 detected acylcarnitines (p < 0.05 after adjustment for multiple testing). Levels of short-chain acylcarnitines were decreased, intermediate-chain acylcarnitines elevated, and long-chain acylcarnitines unaltered. CONCLUSIONS: GD and treatment of the disease is associated with pronounced acyl chain length-dependent alterations in acylcarnitine levels. These changes may be impacted by ethnicity and or dietary differences. [ABSTRACT FROM AUTHOR]

Latent autoimmune diabetes in adults (LADA) usually refers to GAD65 autoantibodies (GADAb)-positive diabetes with onset after 35 years of age and no insulin treatment within the first 6 months after diagnosis. However, it is not always easy to distinguish LADA from type 1 or type 2 diabetes. In this study, we examined whether metabolite profiling could help to distinguish LADA (n = 50) from type 1 diabetes (n = 50) and type 2 diabetes (n = 50). Of 123 identified metabolites, 99 differed between the diabetes types. However, no unique metabolite profile could be identified for any of the types. Instead, the metabolome varied along a C-peptide-driven continuum from type 1 diabetes via LADA to type 2 diabetes. LADA was more similar to type 2 diabetes than to type 1 diabetes. In a principal component analysis, LADA patients overlapping with type 1 diabetes progressed faster to insulin therapy than those overlapping with type 2 diabetes. In conclusion, we could not find any unique metabolite profile distinguishing LADA from type 1 and type 2 diabetes. Rather, LADA was metabolically an intermediate of type 1 and type 2 diabetes, with those patients closer to the former showing a faster progression to insulin therapy than those closer to the latter. [ABSTRACT FROM AUTHOR]

Glycogen metabolism in β-cells may affect downstream metabolic pathways controlling insulin release. We examined glycogen metabolism in human islets and in the rodent-derived INS-1 832/13 β-cells and found them to express the same isoforms of key enzymes required for glycogen metabolism. Our findings indicate that glycogenesis is insulin-independent but influenced by extracellular glucose concentrations. Levels of glycogen synthase decrease with increasing glucose concentrations, paralleling accumulation of glycogen. We did not find cAMP-elicited glycogenolysis and insulin secretion to be causally related. In conclusion, our results reveal regulated glycogen metabolism in human islets and insulin-secreting cells. Whether glycogen metabolism affects insulin secretion under physiological conditions remains to be determined. [ABSTRACT FROM AUTHOR]

To obtain insight into pH change-driven molecular dynamics, we studied the higher order structure changes of protein G'e at the molecular and amino add residue levels in solution by using nanoESI- and IM-mass spectrometry, CD spectroscopy, and protein chemical modification reactions (protein footprinting). We found a dramatic change of the overall tertiary structure of protein G'e when the pH was changed from neutral to atidic, whereas its secondary structure features remained nearly invariable. Limited proteolysis and surface-topology mapping of protein G'e by fast photochemical oxidation of proteins (FPOP) under neutral and addic conditions reveal areas where higher order conformational changes occur on the amino-acid residue level. Under neutral solution conditions, lower oxidation occurs for residues of the first linker region, whereas greater oxidative modifications occur for amino-acid residues of the IgG-binding domains I and II. We propose a dynamic model of pH-induced structural changes in which protein G'e at neutral pH adopts an overall tight conformation with all four domains packed in a firm assembly, whereas at addic pH, the three IgG-binding domains form an elongated alignment, and the N-terminal, His-tagcarrymg domain unfolds. At the same time the individual IgG-binding domains themselves seem to adopt a more compacted fold. As the secondary structure features are nearly unchanged at either pH, interchange between both conformations is highly reversible, explaining the high reconditioning power of protein G'e-based affinity chromatography columns. [ABSTRACT FROM AUTHOR]

The development and application of a miniaturized affinity system for the preparation and release of intact immune complexes are demonstrated. Antibodies were reversibly affinity-adsorbed on pipette tips containing protein G´ and protein A, respectively. Antigen proteins were digested with proteases and peptide mixtures were exposed to attached antibodies; forming antibody-epitope complexes, that is, immune complexes. Elution with millimolar indole propionic acid (IPA)-containing buffers under neutral pH conditions allowed to effectively isolate the intact immune complexes in purified form. Size exclusion chromatography was performed to determine the integrity of the antibody-epitope complexes. Mass spectrometric analysis identified the epitope peptides in the respective SEC fractions. His-tag-containing recombinant human glucose-6-phosphate isomerase in combination with an anti-His-tag monoclonal antibody was instrumental to develop the method. Application was extended to the isolation of the intact antibody-epitope complex of a recombinant human tripartite motif 21 (rhTRIM21) auto-antigen in combination with a rabbit polyclonal anti-TRIM21 antibody. Peptide chip analysis showed that antibody-epitope binding of rhTRIM21 peptide antibody complexes was not affected by the presence of IPA in the elution buffer. By contrast, protein G´ showed an ion charge structure by electrospray mass spectrometry that resembled a denatured conformation when exposed to IPA-containing buffers. The advantages of this novel isolation strategy are low sample consumption and short experimental duration in addition to the direct and robust methodology that provides easy access to intact antibody-antigen complexes under neutral pH and low salt conditions for subsequent investigations. Copyright © 2014 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Background: A multifunctional diet (MFD) combining foods and ingredients with proven functional properties, such as fatty fish and fiber-rich foods, among others, was developed and shown to markedly reduce cardiometabolic risk-associated factors., Objective: Here, we aim at examining metabolic physiological changes associated with these improvements., Methods: Adult overweight individuals without other risk factors were enrolled in an 8-week randomized controlled intervention following a parallel design, with one group ( n  = 23) following MFD and one group ( n  = 24) adhering to a control diet (CD) that followed the caloric formula (E%) advised by the Nordic Nutritional Recommendations. Plasma metabolites and lipids were profiled by gas chromatography and ultrahigh performance liquid chromatography/mass spectrometry., Results: Weight loss was similar between groups. The MFD and CD resulted in altered levels of 137 and 78 metabolites, respectively. Out of these, 83 were uniquely altered by the MFD and only 24 by the CD. The MFD-elicited alterations in lipid levels depended on carbon number and degree of unsaturation., Conclusion: An MFD elicits weight loss-independent systematic lipid remodeling, promoting increased circulating levels of long and highly unsaturated lipids., Clinical Trial Registration: https://clinicaltrials.gov/ct2/show/NCT02148653?term=NCT02148653&draw=2&rank=1, NCT02148653., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Arroyo, Ocariz, Rogova, Al-Majdoub, Björck, Tovar and Spégel.)