Sex Differences in Branched-chain Amino Acid and Tryptophan Metabolism and Pathogenesis of Youth-onset Type 2 Diabetes.

OBJECTIVES: Insulin resistance is associated with elevations in plasma branched-chain amino acids (BCAAs). BCAAs compete with aromatic amino acids including tryptophan for uptake into ß cells. To explore relationships between BCAAs and tryptophan metabolism, adiposity, and glucose tolerance, we compared urine metabolites in overweight/obese youth with type 2 diabetes (T2D) with those in nondiabetic overweight/obese and lean youth. METHODS: Metabolites were measured in 24-hour and first-morning urine samples of 56 nondiabetic adolescents with overweight/obesity, 42 adolescents with T2D, and 43 lean controls, aged 12 to 21 years. Group differences were assessed by Kruskal Wallis or ANOVA. RESULTS: Groups were comparable for age, pubertal status, and ethnicity. Youth with T2D were predominantly female and had highest percent body fat. BCAAs, branched-chain ketoacids (BCKAs), tryptophan, and kynurenine were higher in urine of subjects with T2D. There were no differences between lean controls and nondiabetic youth with overweight/obesity. T2D was associated with diversion of tryptophan from the serotonin to the kynurenine pathway, with higher urinary kynurenine/serotonin ratio and lower serotonin/tryptophan and 5-HIAA/kynurenine ratios. Urinary BCAAs, BCKAs, tryptophan, and ratios reflecting diversion to the kynurenine pathway correlated positively with metrics of body fat and hemoglobin A1c. Increases in these metabolites in the obese T2D group were more pronounced and statistically significant only in adolescent girls. CONCLUSION: Increases in urinary BCAAs and BCKAs in adolescent females with T2D are accompanied by diversion of tryptophan metabolism from the serotonin to the kynurenine pathway. These adaptations associate with higher risks of T2D in obese adolescent females than adolescent males.

Cheaper, faster, simpler trypsin digestion for high-throughput targeted protein quantification.

Introduction: LC-MS-based methods for protein quantification have a stigma of being relatively expensive and low-throughput. This is partly due to the cost and speed of trypsin digestion, which has primarily focused on advancements in research-based biomarker discovery applications that rely on protein/peptide identifications rather than clinical biomarker quantification. However, there is a need for simple, fast, and reproducibly efficient surrogate peptide recovery in clinical biomarker quantification. Methods: Multiple methodologies were evaluated to enhance tryptic digestion for the analysis of thyroglobulin, a prototypical serum protein biomarker. The main criteria for assessment were the yield and speed of formation of surrogate peptides. Various factors such as different additives, types of trypsin, microwave- and pressure-assisted systems, and enzyme concentration were considered as key variables, in addition to digestion time. Results: It was observed that digestion additives/denaturants had a significant impact on the speed and yield of digestion for each surrogate peptide. Increasing the concentration of trypsin alone was found to accelerate digestions appreciably for most surrogate peptides, without affecting the yield. However, the use of sequencing-grade trypsins and microwave/pressure-assisted systems did not offer significant advantages over the use of 'standard-grade' TPCK-treated trypsin in combination with a conventional incubator, once digestion time and additive had been optimized. Conclusion: We have dispelled the notion that trypsin digestion is inherently slow and expensive for targeted quantification of serum proteins. Additionally, we have established a groundwork for experimentation that can pave the way for the creation of efficient trypsin digestion protocols, aiming to optimize yield, speed, and cost. It is our hope that these advancements will promote the wider incorporation of such assays in clinical laboratories.

Evaluation of a Compact, Portable Centrifuge for Separating Microvolume Blood Samples at the Point of Collection.

BACKGROUND: The increased demand for decentralized blood sample collection presents numerous operational challenges for diagnostics providers. Sample degradation including sample hemolysis due to time, temperature, and handling between collection and laboratory analysis leads to limited test menus and unreliable results. Here we introduce the lightweight, portable Labcorp TrueSpin™ for rapid point-of-care blood separation using commercially available microvolume blood collection tubes. The TrueSpin is a class I FDA-registered device designed for untrained users. The centrifuge runs on AA batteries and separates a blood sample in 5 minutes. METHODS: Here we describe a series of studies evaluating sample quality and analyte stability in serum samples collected into gel microtubes and processed using the TrueSpin. Hemolysis, residual red blood cell concentration, sample volume, and serum-based chemistry analyte stability were evaluated. RESULTS: No significant difference was seen in hemolysis or residual red blood cell concentration in serum samples prepared by TrueSpin compared to the reference method. Additionally, capillary and venous blood samples separated using the TrueSpin and exposed to International Safe Transit Association 3A-simulated shipping conditions were shown to yield acceptable sample volume and quality for laboratory analysis. Finally, we show that many common serum-based chemistry analytes have limited (< 1 day) stability if uncentrifuged but improve to ≥ 3-day stability following TrueSpin separation and refrigerated or room temperature storage. CONCLUSIONS: These findings suggest that the TrueSpin is a simple and effective solution for remote sample separation and may enable broader test menus and increased test result reliability for decentralized sample collection pursuits.

Maximizing Microsampling: Measurement of Comprehensive Metabolic and Lipid Panels Using a Novel Capillary Blood Collection Device.

BACKGROUND: Demand continues to grow for patient-centric sampling solutions that enable collection of small volumes of blood outside of healthcare facilities. Various technologies have been developed to facilitate sample collection but gaps in knowledge remain, preventing these technologies from replacing standard venipuncture. METHODS: A novel blood collection device, Touch Activated Phlebotomy (TAP) II® from YourBio Health, and standard fingerstick collection using a BD Microtainer® were utilized to collect capillary serum samples. Measurements of a comprehensive metabolic and lipid panels were measured on these samples and compared to results from venous serum samples that were collected in parallel. Hemolysis was used to assess sample quality. Sample volumes obtained from self-collected TAP II samples were also determined. RESULTS: Correlation of capillary serum with respect to venous serum was demonstrated (R > 0.9) for professionally collected TAP II samples, self-collected TAP II samples, and professionally collected fingerstick samples for alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen, cholesterol, high-density lipoprotein, total bilirubin, and triglycerides. Results for creatinine demonstrated acceptable correlation, however, a consistent negative bias was observed. Biases (with unacceptable correlations) were also observed for measurements of carbon dioxide and potassium. Correlative results for albumin were not consistently acceptable across the collection techniques utilized while the remaining analytes tested did not demonstrate acceptable correlations under any condition. Correlation results, however, would improve with a wider distribution of analyte concentrations. CONCLUSIONS: Collections of small volumes of liquid blood continue to show potential as a patient-centric solution.

The pipetting Olympics: Propagating proper pipetting a priori in clinical LC-MS/MS analysis.

Introduction: Engaging pipetting events were developed to assess and challenge technicians' practical sample handling using matrices common to the clinical laboratory. As correct pipetting stands as a prerequisite for accurate clinical laboratory testing, this helped to understand sources of imprecision and bias attributed to the underlying step of aspirating and dispensing patient samples and internal standard in clinical LC-MS/MS assays while highlighting the importance for the clinical laboratory to evaluate this source of variability on an on-going basis and mitigate its impact. Methods: The events involved pipetting water, methanol, serum, and whole blood. Gravimetric analysis was used to determine the exact volumetric delivery of each matrix using two different techniques. Imprecision and bias were calculated based on the volume derived from the mass and density of each matrix, using literature values for each matrix type. Results: Low imprecision and bias were observed when pipetting water, as in common commercial pipetting assessment programs. Significantly increased imprecision and bias were observed in more applicable matrices (i.e., serum, whole blood, and methanol), indicating that water-based pipetting proficiency assessment leads to a false sense of technical ability. Additionally, the events within illuminated areas for training, leading to improved imprecision and bias. It was shown that pre-rinsing (aspirating and dispensing matrix three times to coat the tip) improved bias, particularly for delivery of methanol and whole blood. Conclusions: Precise and accurate pipetting within the clinical laboratory should not be taken for granted, nor implicitly inferred from proficiency assessment using aqueous solutions. The engaging and collegial events fostered training opportunities. Assay-specific patient sample delivery considerations (pipets and matrices) can inform the practicality of these events - the Pipetting Olympics - and drive improvements within the laboratory.

Pre-analytical conditions influencing analysis of folate in dried plasma microsamples.

Introduction: Determination of folate insufficiency is of considerable interest given its importance in fetal development and red blood cell formation; however, access to blood tests may be limited due to the requirement for phlebotomy as well as controlled temperature shipping of blood specimens to laboratories for testing due to the inherent instability of folate and its vitamers. Methods: An LC-MS/MS test was developed and validated for the measurement of 5-methyltetrahydrofolate (5MTHF) in dried plasma specimens collected from fingerstick blood using a laminar flow blood separation device, as well as liquid venous plasma for comparison. Two pre-analytical factors investigated influencing the measurement of 5MTHF in dried plasma were hemolysis of the fingerstick blood during collection and storage/shipment of the dried plasma. Results: Although observed infrequently, hemolysis >10 % resulted in elevated 5MTHF measurements, but hemolysis >1 % resulted in elevated chloride measurements, which were necessary to normalize 5MTHF measurements for variation in volume of dried plasma specimens. Stability of 5MTHF was improved in dried plasma relative to liquid plasma at ambient temperatures, but not sufficiently to allow for uncontrolled temperature shipping despite controlling for humidity and light exposure. Shipping studies emulating ISTA procedure 7D were conducted with a reusable cold packaging solution. The packaging failed to stabilize 5MTHF in dried plasma specimens during a 2-day summer shipping evaluation, but did provide sufficient temperature control to stabilize 5MTHF during the overnight shipping evaluation. Conclusion: Our studies provide boundary conditions with respect to hemolysis, storage, and shipping for successful analysis of 5MTHF from dried plasma specimens.

Monitoring of SARS-CoV-2 antibodies using dried blood spot for at-home collection.

The utilization of vaccines to fight the spread of SARS-CoV-2 has led to a growing need for expansive serological testing. To address this, an EUA approved immunoassay for detection of antibodies to SARS-CoV-2 in venous serum samples was investigated for use with dried blood spot (DBS) samples. Results from self-collected DBS samples demonstrated a 98.1% categorical agreement to venous serum with a correlation (R) of 0.9600 while professionally collected DBS samples demonstrated a categorical agreement of 100.0% with a correlation of 0.9888 to venous serum. Additional studies were performed to stress different aspects of at-home DBS collection, including shipping stability, effects of interferences, and other sample-specific robustness studies. These studies demonstrated a categorical agreement of at least 95.0% and a mean bias less than ± 20.0%. Furthermore, the ability to track antibody levels following vaccination with the BioNTech/Pfizer vaccine was demonstrated with serial self-collected DBS samples from pre-dose (Day 0) out to 19 weeks.

Disrupted Circadian Rhythm of Epinephrine in Males With Youth-Onset Type 2 Diabetes.

Context: Blood pressure and plasma catecholamines normally decline during sleep and rapidly increase in early morning. This is blunted in adults with type 2 diabetes (T2D). Objective: We hypothesize that increased sympatho-adrenal activity during sleep differentiates youth with T2D from nondiabetic obese youth and lean youth. Methods: Fasting spot morning and 24-hour urines were collected in obese adolescents with and without T2D, and normal-weight controls. Fractionated free urine catecholamines (epinephrine, norepinephrine, and dopamine) were measured, and the ratio of fasting spot morning to 24-hour catecholamines was calculated. Results: Urinary 24-hour catecholamine levels were comparable across the 3 groups. Fasting morning epinephrine and the ratio of fasting morning/24-hour epinephrine were higher in youth with T2D (P = 0.004 and P = 0.035, respectively). In males, the ratio of fasting morning/24-hour epinephrine was also higher in youth with T2D (P = 0.005). In females, fasting morning norepinephrine and the ratio of fasting morning/24-hour dopamine were lower in obese youth with and without T2D (P = 0.013 and P = 0.005, respectively) compared with lean youth. Systolic blood pressure was higher in diabetic participants than other groups; males trended higher than females. Conclusion: Circadian rhythm in catecholamines is disrupted in youth-onset T2D, with a blunted overnight fall in urinary epinephrine in males. Conversely, fasting morning norepinephrine and dopamine levels were lower in obese females with or without T2D. Higher nocturnal catecholamines in males with T2D might associate with, or predispose to, hypertension and cardiovascular complications. Lower catecholamine excretion in females with obesity might serve an adaptive, protective role.

Empiricism in Microsampling: Utilizing a Novel Lateral Flow Device and Intrinsic Normalization to Provide Accurate and Precise Clinical Analysis from a Finger Stick.

BACKGROUND: Phlebotomy plays a key role in clinical laboratory medicine but poses certain challenges for the patient and the laboratory. Dried blood spots simplify collection and stabilize specimens effectively, but clinical reference intervals are based primarily on serum or plasma. We evaluated use of dried separated blood plasma specimens to simplify plasma sample collection via finger stick; however, this sampling technique posed substantial analytical challenges. We discuss herein our efforts to overcome these challenges and provide accurate and precise clinical measurements. METHODS: Microsamples of whole blood were collected via finger stick using a collection device employing laminar-flow separation of cellular blood and plasma fractions with subsequent desiccation. Samples were analyzed on modern autoanalyzers with FDA-approved reagent and calibration systems, as well as commercially available reagents with laboratory-developed assay parameters. Measured analyte concentrations from extracted dried plasma samples were normalized to a coextracted endogenous analyte, chloride. RESULTS: Chloride normalization reduced variability incurred through extraction and undefined plasma volume. Excellent correlation of normalized measurements from dried finger-stick samples (whole blood and plasma) versus matched venous samples facilitated developing mathematical transformations to provide concordance between specimen types. Independent end-to-end performance verification yielded mean biases <3% for the 5 analytes evaluated relative to venous drawn samples analyzed on FDA-approved measurement systems. CONCLUSION: Challenges inherent with this microsampling technique and alternate sample matrix were obviated through capabilities of modern autoanalyzers and implementation of chloride normalization. These results demonstrate that self-collected microsamples from a finger stick can give results concordant with those of venous samples.

Quality over quantity: A qualitative, targeted bottom-up proteomics approach to genotyping apolipoprotein L1.

A targeted, bottom-up proteomic assay was developed for the qualitative detection of apolipoprotein L1 (ApoL1) protein variants (G0, G1, and G2) in blood plasma for identification of high and low renal risk genotypes. Following trypsin digestion of liquid or dry plasma, surrogate peptides specific to each ApoL1 variant were detected by liquid chromatography-tandem mass spectrometry along with two surrogate peptides common among all variants which served as internal (positive) controls to verify correct sample processing. Using isotopically labeled peptide internal standards, the presence or absence of each surrogate peptide was determined using multiple objective metrics including: 1) retention time confirmation relative to its internal standard, 2) comparison of the internal standard-normalized response relative to pre-established thresholds for confident detection, and 3) ion ratio monitoring. Based on the pattern of variant-specific surrogate peptides detected, the genotype was accurately inferred. The final, optimized method was fully validated for liquid plasma specimens, as well as dry plasma specimens collected on a laminar flow blood separation device. For both specimen types, the latter which can be self-collected for remote or in-home sampling, the assay was shown to be reproducible, interference-free with the exception of gross hemolysis, and accurate relative to Sanger sequencing (100% agreement). This targeted, qualitative bottom-up proteomic assay for detection of ApoL1 variants in blood provides a high-throughput, cost-effective alternative to molecular methods and has potential implications to improve organ allocation by facilitating screening kidney donors for high-risk ApoL1 genotypes, but could be applicable for genotyping other clinically relevant blood proteins variants.

More sensitivity is always better: Measuring sub-clinical levels of serum thyroglobulin on a µLC-MS/MS system.

Although liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays for thyroglobulin (Tg) are resistant to autoantibody (TgAb) interference, recent studies have demonstrated approximately 40% of TgAb-positive individuals with recurrent thyroid cancer have Tg concentrations below the lower limit of quantification (LLOQ) of the LC-MS/MS assays described to date (i.e., <0.5 ng/mL), resulting in false-negative findings during post-thyroidectomy monitoring. To reduce false negative results due to insufficient analytical sensitivity, a new Tg assay was developed on a commercially available LC-MS/MS system operating at microliter/minute flow-rates (i.e., µLC-MS/MS) to maximize the analytical sensitivity and achieve a LLOQ of 0.02 ng/mL. When applied to the measurement of TgAb-negative and TgAb-positive patient serum samples previously measuring below the LLOQ of current immunometric and LC-MS/MS assays (LLOQ, 0.1-0.2 ng/mL), concentrations were measurable by µLC-MS/MS in 66% and 44% of samples, respectively - possibly explaining the persistence of TgAb in those patients. Patients with low Tg concentrations measured by µLC-MS/MS (<0.1 ng/mL) also exhibited elevation in their Tg concentrations upon hormone stimulation, indicating the detected Tg was produced from remnant thyroid tissue and would be suitable as a tissue biomarker. Forty-eight TgAb-positive patient specimens with undetectable Tg by both conventional LC-MS/MS (<0.15 ng/mL) and immunometric (<0.1 ng/mL) assays demonstrated measureable Tg concentrations by the new µLC-MS/MS assay in approximately one-third of the specimens, despite all patients being disease free at the time of collection, suggesting interference-free monitoring of low Tg levels may be feasible prior to the on-set of recurrent disease using a sensitive LC-MS/MS assay.

Large-volume injection gas chromatography-vacuum ultraviolet spectroscopy for the qualitative and quantitative analysis of fatty acids in blood plasma.

Qualitative and quantitative determination of fatty acids in plasma is of extreme importance as these are indicators of metabolic diseases. In this work, a sensitive and rugged method for detecting and quantifying fatty acids (as fatty acid methyl ester derivatives, FAMEs) in blood plasma was developed. The use of large-volume injection (LVI) gas chromatography-vacuum ultraviolet spectroscopy (GC-VUV) for analysis of fatty acids in blood plasma allowed the injection of higher sample volumes to accommodate sufficient analyte on-column for necessary detection ranges with a run time of 45 min. Calibration curves exhibited consistent linearity and reproducibility and were used along with internal standards for the quantification of 11 saturated and 21 unsaturated fatty acids. Intra-day and inter-day (n = 6) CVs had an average of 5 and 6%, respectively, and recoveries an average of 105%. The concentrations of EPA, DHA, and AA, as well as the omega-3 index and omega-6/omega-3 ratio, were calculated and compared with clinically actionable measurement ranges. Due to the use of LVI, the more volatile analyte (C8:0) was lost and therefore impossible to quantify. The volatility cutoff was determined to be the C10:0 analyte with a molecular weight of 186.295 g/mol.

A novel NMR-based assay to measure circulating concentrations of branched-chain amino acids: Elevation in subjects with type 2 diabetes mellitus and association with carotid intima media thickness.

OBJECTIVES: Plasma branched-chain amino acid (BCAA) levels, measured on nuclear magnetic resonance (NMR) metabolomics research platforms or by mass spectrometry, have been shown to be associated with type 2 diabetes mellitus (T2DM) and cardiovascular disease (CVD). We developed a new test for quantification of BCAA on a clinical NMR analyzer and used this test to determine the clinical correlates of BCAA in 2 independent cohorts. DESIGN AND METHODS: The performance of the NMR-based BCAA assay was evaluated. A method comparison study was performed with mass spectrometry (LC-MS/MS). Plasma BCAA were measured in the Insulin Resistance Atherosclerosis Study (IRAS, n = 1209; 376 T2DM subjects) and in a Groningen cohort (n = 123; 67 T2DM subjects). In addition, carotid intima media thickness (cIMT) was measured successfully in 119 subjects from the Groningen cohort. RESULTS: NMR-based BCAA assay results were linear over a range of concentrations. Coefficients of variation for inter- and intra-assay precision ranged from 1.8-6.0, 1.7-5.4, 4.4-9.1, and 8.8-21.3%, for total BCAA, valine, leucine, and isoleucine, respectively. BCAA quantified from the same samples using NMR and LC-MS/MS were highly correlated (R2 = 0.97, 0.95 and 0.90 for valine, leucine and isoleucine). In both cohorts total and individual BCAA were elevated in T2DM (P = 0.01 to ≤0.001). Moreover, cIMT was associated with BCAA independent of age, sex, T2DM and metabolic syndrome (MetS) categorization or alternatively of individual MetS components. CONCLUSIONS: BCAA levels, measured by NMR in the clinical laboratory, are elevated in T2DM and may be associated with cIMT, a proxy of subclinical atherosclerosis.

Absolute Protein Quantification by Mass Spectrometry: Not as Simple as Advertised.

Stable isotopically labeled (SIL) tryptic peptides, cleavable SIL peptides, and a full-length SIL protein were compared for internal calibration (i.e., as internal calibrators) and external calibration (i.e., as internal standards) when quantifying three forms of unlabeled, human thyroglobulin (Tg) by bottom-up protein analysis. All SIL materials and human proteins were standardized by amino acid analysis to ensure traceability of measurements and allow confident assignment of accuracy. The three forms of human Tg quantified were (1) the primary reference material BCR457-a native protein purified from human thyroids, (2) a commercially available form also purified from human thyroids, and (3) a full-length recombinant form expressed and purified from a human embryonic kidney 293 cell-line. Collectively, the results unequivocally demonstrate the lack of commutability of tryptic and cleavable SIL peptides as internal calibrators across various bottom-up assays (i.e., denaturing/digestion conditions). Further, the results demonstrate the potential during external calibration for surrogate protein calibrators (i.e., recombinant proteins) to produce inaccurate concentration assignments of native protein analytes by bottom-up analysis due to variance in digestion efficiency, which is not alleviated by altering denaturation/digestion stringency and indicates why protein calibrators may not be commutable in bottom-up protein assays. These results have implications regarding the veracity of "absolute" protein concentration assignments by bottom-up assays using peptide calibrators, as well as protein calibrators, given that absolute accuracy was not universally observed. Nevertheless, these results support the use of recombinant SIL proteins as internal standards over SIL peptides due to their ability to better mimic the digestion of human-derived proteins and mitigate bias due to digestion-based matrix effects that were observed during external calibration.

Medication Error When Switching from Warfarin to Rivaroxaban Leading to Spontaneous Large Ecchymosis of the Abdominal and Chest Wall.

Non-vitamin K oral anticoagulant (NOAC) therapy may be inappropriate if prescription was incorrect, the patient's physiological parameters change, or interacting concomitant medications are erroneously added. The aim of this report was to illustrate inappropriate NOAC prescription in a 78-year-old woman with non-valvular atrial fibrillation and borderline renal dysfunction who was switched from warfarin to rivaroxaban and subsequently developed bruising with hemorrhagic shock and acute on chronic renal failure. Administration of 4-factor prothrombin complex concentrate effectively reversed coagulopathy and stopped bleeding. Retrospective determination of circulating plasma levels of rivaroxaban and warfarin confirmed that excessive anticoagulation was likely due to warfarin that the patient probably continued to take although rivaroxaban was initiated. Pharmacodynamic interaction between rivaroxaban and warfarin may not only be additive but synergistic. In patients at high risk of complications, judicious prescribing and dosing of NOACs, and regular monitoring of concomitant medications and renal function are highly recommended.

Targeted peptide measurements in biology and medicine: best practices for mass spectrometry-based assay development using a fit-for-purpose approach.

Adoption of targeted mass spectrometry (MS) approaches such as multiple reaction monitoring (MRM) to study biological and biomedical questions is well underway in the proteomics community. Successful application depends on the ability to generate reliable assays that uniquely and confidently identify target peptides in a sample. Unfortunately, there is a wide range of criteria being applied to say that an assay has been successfully developed. There is no consensus on what criteria are acceptable and little understanding of the impact of variable criteria on the quality of the results generated. Publications describing targeted MS assays for peptides frequently do not contain sufficient information for readers to establish confidence that the tests work as intended or to be able to apply the tests described in their own labs. Guidance must be developed so that targeted MS assays with established performance can be made widely distributed and applied by many labs worldwide. To begin to address the problems and their solutions, a workshop was held at the National Institutes of Health with representatives from the multiple communities developing and employing targeted MS assays. Participants discussed the analytical goals of their experiments and the experimental evidence needed to establish that the assays they develop work as intended and are achieving the required levels of performance. Using this "fit-for-purpose" approach, the group defined three tiers of assays distinguished by their performance and extent of analytical characterization. Computational and statistical tools useful for the analysis of targeted MS results were described. Participants also detailed the information that authors need to provide in their manuscripts to enable reviewers and readers to clearly understand what procedures were performed and to evaluate the reliability of the peptide or protein quantification measurements reported. This paper presents a summary of the meeting and recommendations.