Cross-Validation of Metabolic Phenotypes in SARS-CoV-2 Infected Subpopulations Using Targeted Liquid Chromatography-Mass Spectrometry (LC-MS).

To ensure biological validity in metabolic phenotyping, findings must be replicated in independent sample sets. Targeted workflows have long been heralded as ideal platforms for such validation due to their robust quantitative capability. We evaluated the capability of liquid chromatography-mass spectrometry (LC-MS) assays targeting organic acids and bile acids to validate metabolic phenotypes of SARS-CoV-2 infection. Two independent sample sets were collected: (1) Australia: plasma, SARS-CoV-2 positive (n = 20), noninfected healthy controls (n = 22) and COVID-19 disease-like symptoms but negative for SARS-CoV-2 infection (n = 22). (2) Spain: serum, SARS-CoV-2 positive (n = 33) and noninfected healthy controls (n = 39). Multivariate modeling using orthogonal projections to latent structures discriminant analyses (OPLS-DA) classified healthy controls from SARS-CoV-2 positive (Australia; R2 = 0.17, ROC-AUC = 1; Spain R2 = 0.20, ROC-AUC = 1). Univariate analyses revealed 23 significantly different (p < 0.05) metabolites between healthy controls and SARS-CoV-2 positive individuals across both cohorts. Significant metabolites revealed consistent perturbations in cellular energy metabolism (pyruvic acid, and 2-oxoglutaric acid), oxidative stress (lactic acid, 2-hydroxybutyric acid), hypoxia (2-hydroxyglutaric acid, 5-aminolevulinic acid), liver activity (primary bile acids), and host-gut microbial cometabolism (hippuric acid, phenylpropionic acid, indole-3-propionic acid). These data support targeted LC-MS metabolic phenotyping workflows for biological validation in independent sample sets.

Exploration of Human Serum Lipoprotein Supramolecular Phospholipids Using Statistical Heterospectroscopy in n-Dimensions (SHY-n): Identification of Potential Cardiovascular Risk Biomarkers Related to SARS-CoV-2 Infection.

SARS-CoV-2 infection causes a significant reduction in lipoprotein-bound serum phospholipids give rise to supramolecular phospholipid composite (SPC) signals observed in diffusion and relaxation edited 1H NMR spectra. To characterize the chemical structural components and compartmental location of SPC and to understand further its possible diagnostic properties, we applied a Statistical HeterospectroscopY in n-dimensions (SHY-n) approach. This involved statistically linking a series of orthogonal measurements made on the same samples, using independent analytical techniques and instruments, to identify the major individual phospholipid components giving rise to the SPC signals. Thus, an integrated model for SARS-CoV-2 positive and control adults is presented that relates three identified diagnostic subregions of the SPC signal envelope (SPC1, SPC2, and SPC3) generated using diffusion and relaxation edited (DIRE) NMR spectroscopy to lipoprotein and lipid measurements obtained by in vitro diagnostic NMR spectroscopy and ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The SPC signals were then correlated sequentially with (a) total phospholipids in lipoprotein subfractions; (b) apolipoproteins B100, A1, and A2 in different lipoproteins and subcompartments; and (c) MS-measured total serum phosphatidylcholines present in the NMR detection range (i.e., PCs: 16.0,18.2; 18.0,18.1; 18.2,18.2; 16.0,18.1; 16.0,20.4; 18.0,18.2; 18.1,18.2), lysophosphatidylcholines (LPCs: 16.0 and 18.2), and sphingomyelin (SM 22.1). The SPC3/SPC2 ratio correlated strongly (r = 0.86) with the apolipoprotein B100/A1 ratio, a well-established marker of cardiovascular disease risk that is markedly elevated during acute SARS-CoV-2 infection. These data indicate the considerable potential of using a serum SPC measurement as a metric of cardiovascular risk based on a single NMR experiment. This is of specific interest in relation to understanding the potential for increased cardiovascular risk in COVID-19 patients and risk persistence in post-acute COVID-19 syndrome (PACS).

Diagnostic Potential of the Plasma Lipidome in Infectious Disease: Application to Acute SARS-CoV-2 Infection.

Improved methods are required for investigating the systemic metabolic effects of SARS-CoV-2 infection and patient stratification for precision treatment. We aimed to develop an effective method using lipid profiles for discriminating between SARS-CoV-2 infection, healthy controls, and non-SARS-CoV-2 respiratory infections. Targeted liquid chromatography-mass spectrometry lipid profiling was performed on discovery (20 SARS-CoV-2-positive; 37 healthy controls; 22 COVID-19 symptoms but SARS-CoV-2negative) and validation (312 SARS-CoV-2-positive; 100 healthy controls) cohorts. Orthogonal projection to latent structure-discriminant analysis (OPLS-DA) and Kruskal-Wallis tests were applied to establish discriminant lipids, significance, and effect size, followed by logistic regression to evaluate classification performance. OPLS-DA reported separation of SARS-CoV-2 infection from healthy controls in the discovery cohort, with an area under the curve (AUC) of 1.000. A refined panel of discriminant features consisted of six lipids from different subclasses (PE, PC, LPC, HCER, CER, and DCER). Logistic regression in the discovery cohort returned a training ROC AUC of 1.000 (sensitivity = 1.000, specificity = 1.000) and a test ROC AUC of 1.000. The validation cohort produced a training ROC AUC of 0.977 (sensitivity = 0.855, specificity = 0.948) and a test ROC AUC of 0.978 (sensitivity = 0.948, specificity = 0.922). The lipid panel was also able to differentiate SARS-CoV-2-positive individuals from SARS-CoV-2-negative individuals with COVID-19-like symptoms (specificity = 0.818). Lipid profiling and multivariate modelling revealed a signature offering mechanistic insights into SARS-CoV-2, with strong predictive power, and the potential to facilitate effective diagnosis and clinical management.

Fatty Acid-Binding Protein 5 at the Blood-Brain Barrier Regulates Endogenous Brain Docosahexaenoic Acid Levels and Cognitive Function.

Fatty acid-binding protein 5 (FABP5) at the blood-brain barrier contributes to the brain uptake of docosahexaenoic acid (DHA), a blood-derived polyunsaturated fatty acid essential for maintenance of cognitive function. Given the importance of DHA in cognition, the aim of this study was to investigate whether deletion of FABP5 results in cognitive dysfunction and whether this is associated with reduced brain endothelial cell uptake of exogenous DHA and subsequent attenuation in the brain levels of endogenous DHA. Cognitive function was assessed in male and female FABP5+/+ and FABP5-/- mice using a battery of memory paradigms. FABP5-/- mice exhibited impaired working memory and short-term memory, and these cognitive deficits were associated with a 14.7 ± 5.7% reduction in endogenous brain DHA levels. The role of FABP5 in the blood-brain barrier transport of DHA was assessed by measuring 14C-DHA uptake into brain endothelial cells and capillaries isolated from FABP5+/+ and FABP5-/- mice. In line with a crucial role of FABP5 in the brain uptake of DHA, 14C-DHA uptake into brain endothelial cells and brain capillaries of FABP5-/- mice was reduced by 48.4 ± 14.5% and 14.0 ± 4.2%, respectively, relative to those of FABP5+/+ mice. These results strongly support the hypothesis that FABP5 is essential for maintaining brain endothelial cell uptake of DHA, and that cognitive deficits observed in FABP5-/- mice are associated with reduced CNS access of DHA. SIGNIFICANCE STATEMENT: Genetic deletion of fatty acid-binding protein 5 (FABP5) in mice reduces uptake of exogenous docosahexaenoic acid (DHA) into brain endothelial cells and brain capillaries and reduces brain parenchymal levels of endogenous DHA. Therefore, FABP5 in the brain endothelial cell is a crucial contributor to the brain levels of DHA. Critically, lowered brain DHA levels in FABP5-/- mice occurred in tandem with cognitive deficits in a battery of memory paradigms. This study provides evidence of a critical role for FABP5 in the maintenance of cognitive function via regulating the brain uptake of DHA, and suggests that upregulation of FABP5 in neurodegenerative diseases, where brain DHA levels are possibly diminished (e.g., Alzheimer's disease), may provide a novel therapeutic approach for restoring cognitive function.

The Addition of Ultrasound Arterial Examination to Upper Extremity Vein Mapping before Hemodialysis Access.

BACKGROUND: Routine upper extremity vein mapping by ultrasound (Ven-US) is recommended by current National Kidney Foundation/Kidney Disease Outcomes Quality Initiative guidelines before arteriovenous fistula (AVF) creation. However, the impact of concomitant arterial US (Art-US) examination is not clear. METHODS: The Ven-US protocol at our institution was modified to include Art-US starting January 2013. Therefore, retrospective review of patients who received Ven-US with Art-US between January 2013 and July 2014 was performed. The Art-US component included distal brachial and radial artery diameters, level of brachial bifurcation, and Doppler Allen's test. A plan for hemodialysis (HD) access was proposed by 2 attending vascular surgeons (VS1 and VS2) and based on a set of criteria for fistula creation (CFC) using Ven-US findings alone. The Art-US findings were subsequently reviewed, and the plan was changed based on either vascular surgeon judgment (VS1 and VS2) or predetermined arterial anatomic criteria (CFC). RESULTS: In total, 163 patients (326 arms) were included. The mean age was 53 years, most patients were male (60%), and most were HD dependent at the time of US evaluation (67%). The initial plan based on Ven-US was: 17-19% radiocephalic (RC) AVF, 33-48% brachiocephalic AVF, 20-27% brachiobasilic AVF, and 14-23% grafts. The Art-US revealed 159 radial arteries (49%) with diameter <2 mm, 16 brachial arteries (5%) with high bifurcation, 93 (29%) incomplete palmar arches, and 7 arms (2%) with arterial waveform blunting. Review of Art-US findings resulted in an overall change to the operative plan from 4% to 12% of patients. Those with an initially planned RC AVF were more likely to have a change in operative approach (21-57%) compared with all other types of planned access (1-3%, P < 0.001). CONCLUSIONS: Preoperative Art-US may significantly change the operative plan, particularly when planning a RC AVF, and should be performed before HD access surgery at the wrist.

Increased use of brachiocephalic arteriovenous fistulas improves functional primary patency.

OBJECTIVE: As vascular surgeons strive to meet the Fistula First Initiative, some authors have observed a decrease in arteriovenous fistula (AVF) maturation rates in association with an increase in AVF creation. In May 2012, we adopted a practice change in an attempt to maintain the same high level of AVF creation while leading to a decrease in fistula failures. METHODS: A retrospective study was conducted of all dialysis access procedures performed by a single vascular surgeon before (period 1; before May 1, 2012) and after (period 2; after May 1, 2012) the change in practice pattern. The adopted change included favoring the brachiocephalic location unless the patient was an ideal anatomic candidate for a radiocephalic AVF, creating a larger and standardized arteriotomy, and using a large venous footplate whenever possible. The main outcome measure was primary functional patency at 1 year. Secondary outcome measures included primary patency at 1 year, time to maturation, type of fistula created, steal syndrome, and tunneled hemodialysis catheter infections. RESULTS: Of 213 vascular access procedures performed, 191 (90%) were AVFs. There was no difference in use of AVFs between period 1 (93% AVFs) and period 2 (88% AVFs; P = .2). Use of brachiocephalic AVFs increased from 38% in period 1 to 56% in period 2 (P = .01), with a corresponding trend toward a decrease in radiocephalic AVFs in period 2 (36% in period 1 to 27% in period 2; P = .2). Primary functional patency at 1 year was 47% in period 1 and 63% in period 2 (P = .03). Primary patency at 1 year was 51% in period 1 and 70% in period 2 (P = .001). Time to reach functional maturation was decreased in period 2 (median, 76 vs 82.5 days; P = .046). There was no difference in steal syndrome (P = 1.0), and the incidence of hemodialysis catheter infections was lower in period 2 (0 vs 7 [7%]; P = .006). CONCLUSIONS: Increasing brachiocephalic AVF creation and reducing our reliance on radiocephalic AVFs resulted in a significant increase in primary functional patency at 1 year. This was achieved while maintaining the same high percentage of fistulas, a lower rate of central catheter infections, and the same low incidence of steal syndrome.

Characterisation of capillary ionic liquid columns for gas chromatography-mass spectrometry analysis of fatty acid methyl esters.

Due to their distinct chemical properties, the application of ionic liquid (IL) compounds as gas chromatography (GC) stationary phases offer unique GC separation especially in the analysis of geometric and positional fatty acid methyl ester (FAME) isomers. Elution behaviour of FAME on several commercialised IL capillary columns including phosphonium based SLB-IL59, SLB-IL60, SLB-IL61 and SLB-IL76 and imidazolium based SLB-IL82, SLB-IL100, and SLB-IL111 as well as a general purpose column SLB-5ms, were evaluated in gas chromatography-mass spectrometry (GC-MS) analysis. The phases were further characterised by using a linear solvation energy relationship (LSER) approach according to the equivalent chain length (ECL) index of FAME. Among all tested IL columns, elution temperatures of saturated FAME increased as their McReynolds' polarity value decreased, except for IL60. ECL values increased markedly as the stationary phase polarity increased, particularly for the polyunsaturated FAME. The LSER study indicated a lowest l/e value at 0.864 for IL111, displaying phase selectivity towards unsaturated FAME, with higher peak capacity within a carbon number isomer group. s and e descriptors calculated from LSER were validated by excellent correlation with dipole moments and lowest unoccupied molecular orbital (LUMO) energies, with R(2) values of 0.99 and 0.92 respectively, calculated using GAUSSIAN.

Profiling of soil fatty acids using comprehensive two-dimensional gas chromatography with mass spectrometry detection.

Profiling of phospholipid fatty acids (PLFA) represents a challenging goal for distinguishing the diversity of microbial communities and biomass in the complex and heterogeneous soil ecosystem. Comprehensive two-dimensional gas chromatography (GC×GC) coupled with simultaneous flame ionisation and mass spectrometry detection was applied as a culture-independent method for PLFA profiling of microbial classification in forest soil. A number of column sets were evaluated for the GC×GC separation of fatty acid methyl esters (FAME). Due to better isomeric separation and compound patterns on the 2D contour plot, an apolar-polar column combination was selected for soil microbial PLFA characterisation. A comprehensive view of PLFA composition with carbon chain length varying from 12 to 20 was observed in forest soil samples, with the commonly reported bacterial FAME of iso-/anteiso-, methyl-branched-, cyclopropyl-, and hydroxyl-substituted FA identified by their mass spectral and retention time according to authentic standards. Notably, some uncommon oxygenated FAME were found in high abundance and were further characterised by GC×GC coupled with high resolution mass spectrometry. This tentatively revealed geometric pairs of methyl 9,10-epoxyoctadecanoate isomers.

Integrated multidimensional and comprehensive 2D GC analysis of fatty acid methyl esters.

Fatty acid methyl ester (FAME) profiling in complex fish oil and milk fat samples was studied using integrated comprehensive 2D GC (GC × GC) and multidimensional GC (MDGC). Using GC × GC, FAME compounds--cis- and trans-isomers, and essential fatty acid isomers--ranging from C18 to C22 in fish oil and C18 in milk fat were clearly displayed in contour plot format according to structural properties and patterns, further identified based on authentic standards. Incompletely resolved regions were subjected to MDGC, with Cn (n = 18, 20) zones transferred to a (2)D column. Elution behavior of C18 FAME on various (2)D column phases (ionic liquids IL111, IL100, IL76, and modified PEG) was evaluated. Individual isolated Cn zones demonstrated about four-fold increased peak capacities. The IL100 provided superior separation, good peak shape, and utilization of elution space. For milk fat-derived FAME, the (2)D chromatogram revealed at least three peaks corresponding to C18:1, more than six peaks for cis/trans-C18:2 isomers, and two peaks for C18:3. More than 17 peaks were obtained for the C20 region of fish oil-derived FAMEs using MDGC, compared with ten peaks using GC × GC. The MDGC strategy is useful for improved FAME isomer separation and confirmation.