A global perspective on the status of clinical metabolomics in laboratory medicine - a survey by the IFCC metabolomics working group.

OBJECTIVES: Metabolomics aims for comprehensive characterization and measurement of small molecule metabolites (<1700 Da) in complex biological matrices. This study sought to assess the current understanding and usage of metabolomics in laboratory medicine globally and evaluate the perception of its promise and future implementation. METHODS: A survey was conducted by the IFCC metabolomics working group that queried 400 professionals from 79 countries. Participants provided insights into their experience levels, knowledge, and usage of metabolomics approaches, along with detailing the applications and methodologies employed. RESULTS: Findings revealed a varying level of experience among respondents, with varying degrees of familiarity and utilization of metabolomics techniques. Targeted approaches dominated the field, particularly liquid chromatography coupled to a triple quadrupole mass spectrometer, with untargeted methods also receiving significant usage. Applications spanned clinical research, epidemiological studies, clinical diagnostics, patient monitoring, and prognostics across various medical domains, including metabolic diseases, endocrinology, oncology, cardiometabolic risk, neurodegeneration and clinical toxicology. CONCLUSIONS: Despite optimism for the future of clinical metabolomics, challenges such as technical complexity, standardization issues, and financial constraints remain significant hurdles. The study underscores the promising yet intricate landscape of metabolomics in clinical practice, emphasizing the need for continued efforts to overcome barriers and realize its full potential in patient care and precision medicine.

A High-Throughput NMR Method for Lipoprotein-X Quantification.

Lipoprotein X (LP-X) is an abnormal cholesterol-rich lipoprotein particle that accumulates in patients with cholestatic liver disease and familial lecithin-cholesterol acyltransferase deficiency (FLD). Because there are no high-throughput diagnostic tests for its detection, a proton nuclear magnetic resonance (NMR) spectroscopy-based method was developed for use on a clinical NMR analyzer commonly used for the quantification of lipoproteins and other cardiovascular biomarkers. The LP-X assay was linear from 89 to 1615 mg/dL (cholesterol units) and had a functional sensitivity of 44 mg/dL. The intra-assay coefficient of variation (CV) varied between 1.8 and 11.8%, depending on the value of LP-X, whereas the inter-assay CV varied between 1.5 and 15.4%. The assay showed no interference with bilirubin levels up to 317 mg/dL and was also unaffected by hemolysis for hemoglobin values up to 216 mg/dL. Samples were stable when stored for up to 6 days at 4 °C but were not stable when frozen. In a large general population cohort (n = 277,000), LP-X was detected in only 50 subjects. The majority of LP-X positive cases had liver disease (64%), and in seven cases, had genetic FLD (14%). In summary, we describe a new NMR-based assay for LP-X, which can be readily implemented for routine clinical laboratory testing.

The Inflammation Biomarker GlycA Reflects Plasma N-Glycan Branching.

BACKGROUND: GlycA is a nuclear magnetic resonance (NMR) signal in plasma that correlates with inflammation and cardiovascular outcomes in large data sets. The signal is thought to originate from N-acetylglucosamine (GlcNAc) residues of branched plasma N-glycans, though direct experimental evidence is limited. Trace element concentrations affect plasma glycosylation patterns and may thereby also influence GlycA. METHODS: NMR GlycA signal was measured in plasma samples from 87 individuals and correlated with MALDI-MS N-glycomics and trace element analysis. We further evaluated the genetic association with GlycA at rs13107325, a single nucleotide polymorphism resulting in a missense variant within SLC39A8, a manganese transporter that influences N-glycan branching, both in our samples and existing genome-wide association studies data from 22 835 participants in the Women's Health Study (WHS). RESULTS: GlycA signal was correlated with both N-glycan branching (r2 ranging from 0.125-0.265; all P < 0.001) and copper concentration (r2 = 0.348, P < 0.0001). In addition, GlycA levels were associated with rs13107325 genotype in the WHS (ß [standard error of the mean] = -4.66 [1.2674], P = 0.0002). CONCLUSIONS: These results provide the first direct experimental evidence linking the GlycA NMR signal to N-glycan branching commonly associated with acute phase reactive proteins involved in inflammation.

Lipoprotein Z, a hepatotoxic lipoprotein, predicts outcome in alcohol-associated hepatitis.

BACKGROUND AND AIMS: Lipoprotein Z (LP-Z) is an abnormal free cholesterol (FC)-enriched LDL-like particle discovered from patients with cholestatic liver disease. This study aims to define the diagnostic value of LP-Z in alcohol-associated hepatitis (AH) and interrogate the biology behind its formation. APPROACH AND RESULTS: We measured serum levels of LP-Z using nuclear magnetic resonance spectroscopy, a well-established clinical assay. Serum levels of LP-Z were significantly elevated in four AH cohorts compared with control groups, including heavy drinkers and patients with cirrhosis. We defined a Z-index, calculated by the ratio of LP-Z to total apolipoprotein B-containing lipoproteins, representing the degree of deviation from normal VLDL metabolism. A high Z-index was associated with 90-day mortality independent from the Model for End-Stage Liver Disease (MELD) and provided added prognosticative value. Both a Z-index ≤ 0.6 and a decline of Z-index by ≥0.1 in 2 weeks predicted 90-day survival. RNA-sequencing analyses of liver tissues demonstrated an inverse association in the expression of enzymes responsible for the extrahepatic conversion of VLDL to LDL and AH disease severity, which was further confirmed by the measurement of serum enzyme activity. To evaluate whether the FC in LP-Z could contribute to the pathogenesis of AH, we found significantly altered FC levels in liver explant of patients with AH. Furthermore, FC in reconstituted LP-Z particles caused direct toxicity to human hepatocytes in a concentration-dependent manner, supporting a pathogenic role of FC in LP-Z. CONCLUSIONS: Impaired lipoprotein metabolism in AH leads to the accumulation of LP-Z in the circulation, which is hepatotoxic from excessive FC. A Z-index ≤ 0.6 predicts 90-day survival independent from conventional biomarkers for disease prognostication.

High-Density Lipoprotein and Long-Term Incidence and Progression of Aortic Valve Calcification: The Multi-Ethnic Study of Atherosclerosis.

BACKGROUND: Aortic valve calcification (AVC) shares pathological features with atherosclerosis. Lipoprotein components have been detected in aortic valve tissue, including HDL (high-density lipoprotein). HDL measures have inverse associations with cardiovascular disease, but relationships with long-term AVC progression are unclear. We investigated associations of HDL cholesterol, HDL-particle number and size, apoC3-defined HDL subtypes, and, secondarily, CETP (cholesteryl ester transfer protein) mass and activity, with long-term incidence and progression of AVC. METHODS: We used linear mixed-effects models to evaluate the associations of baseline HDL indices with AVC. AVC was quantified by Agatston scoring of up to 3 serial computed tomography scans over a median of 8.9 (maximum 11.2) years of follow-up in the Multi-Ethnic Study of Atherosclerosis (n=6784). RESULTS: After adjustment, higher concentrations of HDL-C (high-density lipoprotein cholesterol), HDL-P (HDL particles), large HDL-P, and apoC3-lacking HDL-C were significantly associated with lower incidence/progression of AVC. Neither small or medium HDL-P nor apoC3-containing HDL-C was significantly associated with AVC incidence/progression. When included together, a significant association was observed only for HDL-C, but not for HDL-P. Secondary analyses showed an inverse relationship between CETP mass, but not activity, and AVC incidence/progression. In exploratory assessments, inverse associations for HDL-C, HDL-P, large HDL-P, and apoC3-lacking HDL with AVC incidence/progression were more pronounced for older, male, and White participants. ApoC3-containing HDL-C only showed a positive association with AVC in these subgroups. CONCLUSIONS: In a multiethnic population, HDL-C, HDL-P, large HDL-P, and apoC3-lacking HDL-C were inversely associated with long-term incidence and progression of AVC. Further investigation of HDL composition and mechanisms could be useful in understanding pathways that slow AVC.

Associations Between High-Density Lipoprotein Particles and Ischemic Events by Vascular Domain, Sex, and Ethnicity: A Pooled Cohort Analysis.

BACKGROUND: High-density lipoprotein (HDL) cholesterol concentration (HDL-C) is an established atheroprotective marker, in particular for coronary artery disease; however, HDL particle concentration (HDL-P) may better predict risk. The associations of HDL-C and HDL-P with ischemic stroke and myocardial infarction (MI) among women and Blacks have not been well studied. We hypothesized that HDL-P would consistently be associated with MI and stroke among women and Blacks compared with HDL-C. METHODS: We analyzed individual-level participant data in a pooled cohort of 4 large population studies without baseline atherosclerotic cardiovascular disease: DHS (Dallas Heart Study; n=2535), ARIC (Atherosclerosis Risk in Communities; n=1595), MESA (Multi-Ethnic Study of Atherosclerosis; n=6632), and PREVEND (Prevention of Renal and Vascular Endstage Disease; n=5022). HDL markers were analyzed in adjusted Cox proportional hazard models for MI and ischemic stroke. RESULTS: In the overall population (n=15 784), HDL-P was inversely associated with the combined outcome of MI and ischemic stroke, adjusted for cardiometabolic risk factors (hazard ratio [HR] for quartile 4 [Q4] versus quartile 1 [Q1], 0.64 [95% CI, 0.52-0.78]), as was HDL-C (HR for Q4 versus Q1, 0.76 [95% CI, 0.61-0.94]). Adjustment for HDL-C did not attenuate the inverse relationship between HDL-P and atherosclerotic cardiovascular disease, whereas adjustment for HDL-P attenuated all associations between HDL-C and events. HDL-P was inversely associated with the individual end points of MI and ischemic stroke in the overall population, including in women. HDL-P was inversely associated with MI among White participants but not among Black participants (HR for Q4 versus Q1 for Whites, 0.49 [95% CI, 0.35-0.69]; for Blacks, 1.22 [95% CI, 0.76-1.98]; Pinteraction=0.001). Similarly, HDL-C was inversely associated with MI among White participants (HR for Q4 versus Q1, 0.53 [95% CI, 0.36-0.78]) but had a weak direct association with MI among Black participants (HR for Q4 versus Q1, 1.75 [95% CI, 1.08-2.83]; Pinteraction<0.0001). CONCLUSIONS: Compared with HDL-C, HDL-P was consistently associated with MI and ischemic stroke in the overall population. Differential associations of both HDL-C and HDL-P for MI by Black ethnicity suggest that atherosclerotic cardiovascular disease risk may differ by vascular domain and ethnicity. Future studies should examine individual outcomes separately.

Association of beta-hydroxybutyrate with development of heart failure: Sex differences in a Dutch population cohort.

BACKGROUND: In the failing heart, energy metabolism is shifted towards increased ketone body oxidation. Nevertheless, the association of beta-hydroxybutyrate (ß-OHB) with development of heart failure (HF) remains unclear. We investigated the association between plasma ß-OHB and the risk of HF in a prospective population-based cohort. DESIGN: Plasma ß-OHB concentrations were measured in 6134 participants of the PREVEND study. Risk of incident HF with reduced (HFrEF) or preserved (HFpEF) ejection fraction was estimated using multivariable-adjusted Cox regression models. RESULTS: During median follow-up for 8.2 years, 227 subjects were diagnosed with HF (137 with HFrEF; 90 with HFpEF). Cox regression analyses revealed a significant association of higher ß-OHB concentrations with incident HF (HR per 1 standard deviation increase, 1.40 (95% CI: 1.21-1.63; P < .001), which was largely attributable to HFrEF. In women, the hazard ratio (HR) for HFrEF per 1 standard deviation increase in ß-OHB was 1.73 (95% confidence interval (CI): 1.17-2.56, P = .005) in age, BMI, type 2 diabetes, hypertension, myocardial infarction, smoking, alcohol consumption, total cholesterol, HDL-C, triglycerides, glucose, eGFR and UAE adjusted analysis. In men, in the same fully adjusted analysis, the HR was 1.14 (CI: 0.86-1.53, P = .36) (P < .01 for sex interaction). In N-terminal pro-brain natriuretic peptide (NT-proBNP)-stratified analysis, the age-adjusted association with HF was significant in women with higher NT-proBNP levels (P = .008). CONCLUSIONS: This prospective study suggests that high plasma concentrations of ß-OHB are associated with an increased risk of HFrEF, particularly in women. The mechanisms responsible for the sex differences of this association warrant further study.

A new phenotypic classification system for dyslipidemias based on the standard lipid panel.

BACKGROUND: Dyslipoproteinemias can be classified by their distinct lipoprotein patterns, which helps determine atherosclerotic cardiovascular disease (ASCVD) risk and directs lipid management but this has required advanced laboratory testing. OBJECTIVE: To develop a new algorithm for classifying lipoprotein disorders that only relies on the standard lipid panel. METHODS: Lipid thresholds for defining the different lipoprotein phenotypes were derived for Non-High-Density Lipoprotein-Cholesterol (NonHDL-C) and Triglycerides (TG) to be concordant when possible with the current US Multi-Society guidelines for blood cholesterol management. RESULTS: The new classification method categorizes patients into all the classical Fredrickson-like phenotypes except for Type III dysbetalipoproteinemia. In addition, a new hypolipidemic phenotype (Type VI) due to genetic mutations in apoB-metabolism is described. The validity of the new algorithm was confirmed by lipid analysis by NMR (N = 11,365) and by concordance with classification by agarose gel electrophoresis/beta-quantification (N = 5504). Furthermore, based on the Atherosclerosis Risk in Communities (ARIC) cohort (N = 14,742), the lipoprotein phenotypes differ in their association with ASCVD (TypeV>IIb > IVb > IIa > IVa > normolipidemic) and can be used prognostically as risk enhancer conditions in the management of patients. CONCLUSIONS: We describe a clinically useful lipoprotein phenotyping system that is only dependent upon the standard lipid panel. It, therefore, can be easily implemented for increasing compliance with current guidelines and for improving the care of patients at risk for ASCVD.

The extended lipid panel assay: a clinically-deployed high-throughput nuclear magnetic resonance method for the simultaneous measurement of lipids and Apolipoprotein B.

BACKGROUND: Standard lipid panel assays employing chemical/enzymatic methods measure total cholesterol (TC), triglycerides (TG), and high-density lipoprotein cholesterol (HDL-C), from which are calculated estimates of low-density lipoprotein cholesterol (LDL-C). These lipid measures are used universally to guide management of atherosclerotic cardiovascular disease risk. Apolipoprotein B (apoB) is generally acknowledged to be superior to LDL-C for lipid-lowering therapeutic decision-making, but apoB immunoassays are performed relatively infrequently due to the added analytic cost. The aim of this study was to develop and validate the performance of a rapid, high-throughput, reagent-less assay producing an "Extended Lipid Panel" (ELP) that includes apoB, using the Vantera® nuclear magnetic resonance (NMR) analyzer platform already deployed clinically for lipoprotein particle and other testing. METHODS: Partial least squares regression models, using as input a defined region of proton NMR spectra of plasma or serum, were created to simultaneously quantify TC, TG, HDL-C, and apoB. Large training sets (n > ~ 1000) of patient sera analyzed independently for lipids and apoB by chemical methods were employed to ensure prediction models reflect the wide lipid compositional diversity of the population. The analytical performance of the NMR ELP assay was comprehensively evaluated. RESULTS: Excellent agreement was demonstrated between chemically-measured and ELP assay values of TC, TG, HDL-C and apoB with correlation coefficients ranging from 0.980 to 0.997. Within-run precision studies measured using low, medium, and high level serum pools gave coefficients of variation for the 4 analytes ranging from 1.0 to 3.8% for the low, 1.0 to 1.7% for the medium, and 0.9 to 1.3% for the high pools. Corresponding values for within-lab precision over 20 days were 1.4 to 3.6%, 1.2 to 2.3%, and 1.0 to 1.9%, respectively. Independent testing at three sites over 5 days produced highly consistent assay results. No major interference was observed from 38 endogenous or exogenous substances tested. CONCLUSIONS: Extensive assay performance evaluations validate that the NMR ELP assay is efficient, robust, and substantially equivalent to standard chemistry assays for the clinical measurement of lipids and apoB. Routine reporting of apoB alongside standard lipid measures could facilitate more widespread utilization of apoB for clinical decision-making.

GlycA and hsCRP are independent and additive predictors of future cardiovascular events among patients undergoing angiography: The intermountain heart collaborative study.

BACKGROUND: GlycA is an inflammatory marker that is raised in patients with cardiometabolic diseases and associated with cardiovascular (CV) events. We sought to determine if GlycA adds independent value to hsCRP for CV risk prediction. METHODS: Patients in the Intermountain Heart Collaborative Study who underwent coronary angiography and had plasma GlycA and hsCRP levels were studied (n = 2996). Patients were followed for 7.0 ±â€¯2.8 years. GlycA and hsCRP were moderately correlated (r = 0.46, P < .0001). GlycA and hsCRP concentrations were stratified into high and low categories by their median values. Multivariable cox hazard regression was utilized to determine the associations of GlycA quartiles, as well as high and low categories of GlycA and hsCRP, with major adverse cardiovascular events (MACE) defined as the composite of death, myocardial infarction (MI), heart failure (HF) hospitalization, and stroke. RESULTS: The highest GlycA quartile was associated with future MACE [HR: 1.43; 95% CI: 1.22-1.69; P < .0001]. Patients with high GlycA and high hsCRP had more diabetes, hyperlipidemia, hypertension, HF, renal failure and MI, but not coronary artery disease. High GlycA and hsCRP (H/H) versus low GlycA and hsCRP (L/L) was associated with MACE, death and HF hospitalization, but not MI or stroke. Combined MACE rates were 33.5%, 41.3%, 35.7% and 49.1% for L/L, L/H, H/L and H/H categories of GlycA/hsCRP, respectively (P-trend < .0001). The interaction between GlycA and hsCRP was significant for the outcome of death (P = .03). CONCLUSION: In this study, levels of GlycA and hsCRP were independent and additive markers of risk for MACE, death and HF hospitalization.

Lipid profile and effect of statin treatment in pooled phase II and phase III baricitinib studies.

OBJECTIVES: Lipid profiles are altered by active disease in patients with rheumatoid arthritis (RA) and may be further modified by treatment with Janus kinase inhibitors and other disease-modifying antirheumatic drugs. METHODS: Lipid data were analysed from phase II and III studies of 4 mg (n=997) and 2 mg (n=479) oral baricitinib administered once daily in patients with moderate-to-severe active RA. Lipoprotein particle size and number and GlycA were evaluated with nuclear magnetic resonance in one phase III study. The effect of statin therapy on lipid levels was evaluated in patients on statins at baseline and in patients who initiated statins during the study. RESULTS: Treatment with baricitinib was associated with increased levels of total cholesterol, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) and triglycerides, but no significant change in LDL-C:HDL-C ratio. Lipid levels plateaued after 12 weeks of treatment. Baricitinib treatment increased large LDL and decreased small, dense LDL particle numbers and GlycA. Lipid changes from baseline were not significantly different between baseline statin users and non-users. In patients who initiated statin therapy during the study, LDL-C, triglycerides (baricitinib 4 mg only) and apolipoprotein B decreased to pre-baricitinib levels; HDL-C and apolipoprotein A-I levels remained elevated. CONCLUSIONS: Baricitinib was associated with increased LDL-C, HDL-C and triglyceride levels, but did not alter the LDL-C:HDL-C ratio. Evaluation of cardiovascular event rates during long-term treatment is warranted to further characterise these findings and their possible clinical implications. TRIAL REGISTRATION NUMBER: NCT00902486, NCT01469013, NCT01185353, NCT01721044, NCT01721057, NCT01711359, NCT01710358, NCT01885078.

Comparability of Lipoprotein Particle Number Concentrations Across ES-DMA, NMR, LC-MS/MS, Immunonephelometry, and VAP: In Search of a Candidate Reference Measurement Procedure for apoB and non-HDL-P Standardization.

BACKGROUND: Despite the usefulness of standard lipid parameters for cardiovascular disease risk assessment, undiagnosed residual risk remains high. Advanced lipoprotein testing (ALT) was developed to provide physicians with more predictive diagnostic tools. ALT methods separate and/or measure lipoproteins according to different parameters such as size, density, charge, or content, and equivalence of results across methods has not been demonstrated. METHODS: Through a split-sample study, 25 clinical specimens (CSs) were assayed in 10 laboratories before and after freezing using the major ALT methods for non-HDL particles (non-HDL-P) or apolipoprotein B-100 (apoB-100) measurements with the intent to assess their comparability in the current state of the art. RESULTS: The overall relative standard deviation (CV) of non-HDL-P and apoB-100 concentrations measured by electrospray differential mobility analysis, nuclear magnetic resonance, immunonephelometry, LC-MS/MS, and vertical autoprofile in the 25 frozen CSs was 14.1%. Within-method comparability was heterogeneous, and CV among 4 different LC-MS/MS methods was 11.4% for apoB-100. No significant effect of freezing and thawing was observed. CONCLUSIONS: This study demonstrates that ALT methods do not yet provide equivalent results for the measurement of non-HDL-P and apoB-100. The better agreement between methods harmonized to the WHO/IFCC reference material suggests that standardizing ALT methods by use of a common commutable calibrator will improve cross-platform comparability. This study provides further evidence that LC-MS/MS is the most suitable candidate reference measurement procedure to standardize apoB-100 measurement, as it would provide results with SI traceability. The absence of freezing and thawing effect suggests that frozen serum pools could be used as secondary reference materials.

Obesity is associated with an altered HDL subspecies profile among adolescents with metabolic disease.

We aimed to determine the risk factors associated with the depletion of large HDL particles and enrichment of small HDL particles observed in adolescents with T2D. Four groups of adolescents were recruited: 1) lean insulin-sensitive (L-IS), normal BMI and no insulin resistance; 2) lean insulin-resistant (L-IR), normal BMI but insulin resistance (fasting insulin levels ≥ 25 mU/ml and homeostatic model assessment of insulin resistance ≥ 6); 3) obese insulin-sensitive (O-IS), BMI ≥ 95th percentile and no insulin resistance; and 4) obese insulin-resistant (O-IR), BMI ≥ 95th percentile and insulin resistance. Plasma was separated by using gel-filtration chromatography to assess the HDL subspecies profile and compared with that of obese adolescents with T2D (O-T2D). Large HDL subspecies were significantly lower across groups from L-IS > L-IR > O-IS > O-IR > O-T2D (P < 0.0001); small HDL particles were higher from L-IS to O-T2D (P < 0.0001); and medium-sized particles did not differ across groups. The contributions of obesity, insulin resistance, and diabetes to HDL subspecies profile were between 23% and 28%, 1% and 10%, and 4% and 9%, respectively. Obesity is the major risk factor associated with the altered HDL subspecies profile previously reported in adolescents with T2D, with smaller contributions from insulin resistance and diabetes.

GlycA, a novel biomarker of systemic inflammation and cardiovascular disease risk.

BACKGROUND: GlycA is a novel spectroscopic marker of systemic inflammation with low intra-individual variability and other attributes favoring its clinical use in patients with chronic inflammatory and autoimmune diseases. GlycA is unique in its composite nature, reflecting both increased glycan complexity and circulating acute phase protein levels during local and systemic inflammation. Recent studies of GlycA from cross-sectional, observational and interventional studies have been highly informative, demonstrating that GlycA is elevated in acute and chronic inflammation, predicts death in healthy individuals and is associated with disease severity in patients with chronic inflammatory diseases such as rheumatoid arthritis, psoriasis and lupus. Moreover, following treatment with biological therapy in psoriasis, reduction in skin disease severity was accompanied by a decrease in GlycA levels and improvement in vascular inflammation. CONCLUSIONS: Collectively, these findings suggest GlycA is a marker that tracks systemic inflammation and subclinical vascular inflammation. However, larger prospective studies and randomized trials are necessary in order to assess the impact of novel therapies on GlycA in patients with chronic inflammatory conditions, which may be concomitant with cardiovascular benefits.

The effects of basal insulin peglispro vs. insulin glargine on lipoprotein particles by NMR and liver fat content by MRI in patients with diabetes.

BACKGROUND: In Phase 2/3 studies of basal insulin peglispro (BIL) compared to insulin glargine, patients with type 1 or type 2 diabetes previously treated with insulin and randomized to BIL had an increase in serum triglycerides (TGs). To further understand lipoprotein changes, a lipid substudy which included liver fat content was designed to assess relationships among the measured variables for each diabetes cohort and compare the hepato-preferential insulin BIL to glargine. METHODS: In three cohorts of patients with diabetes (type 1, type 2 insulin naïve, and type 2 previously on insulin; n = 652), liver fat content (LFC) was determined by magnetic resonance imaging (MRI) and blood lipids were analyzed by nuclear magnetic resonance (NMR) spectroscopy at baseline, 26 and 52 weeks of treatment. Apolipoproteins, adiponectin, and other lipid parameters were also measured. Descriptive statistics were done, as well as correlation analyses to look for relationships among LFC and lipoproteins or other lipid measures. RESULTS: In patients with type 1 diabetes treated with BIL, but not glargine, small LDL and medium and large VLDL subclass concentrations increased from baseline. In patients with type 2 diabetes previously on insulin and treated with BIL, large VLDL concentration increased from baseline. In insulin naïve patients with type 2 diabetes treated with BIL, there were very few changes, while in those treated with glargine, small LDL and large VLDL decreased from baseline. Baseline LFC correlated significantly in one or more cohorts with baseline large VLDL, small LDL, VLDL size, and Apo C3. Changes in LFC by treatment showed generally weak correlations with lipoprotein changes, except for positive correlations with large VLDL and VLDL size. Adiponectin was higher in patients with type 1 diabetes compared to patients with type 2 diabetes, but decreased with treatment with both BIL and glargine. CONCLUSIONS: The lipoprotein changes were in line with the observed changes in serum TGs; i.e., the cohorts experiencing increased TGs and LFC with BIL treatment had decreased LDL size and increased VLDL size. These data and analyses add to the currently available information on the metabolic effects of insulins in a very carefully characterized cohort of patients with diabetes. Clinicaltrials.gov registration numbers and dates NCT01481779 (2011), NCT01435616 (2011), NCT01454284 (2011), NCT01582451 (2012).

Does a lack of physical activity explain the rheumatoid arthritis lipid profile?

BACKGROUND: In rheumatoid arthritis (RA), cardiovascular risk is associated with paradoxical reductions in total cholesterol, low density lipoprotein-cholesterol (LDL-C), and high density lipoprotein-cholesterol (HDL-C). Concentrations of small LDL (LDL-P) and HDL (HDL-P) particles are also reduced with increased inflammation and disease activity in RA patients. Here we sought to identify which measure(s) of inflammation, disease activity and cardiometabolic risk contribute most to the RA-associated lipoprotein profile. METHODS: NMR lipoprotein measurements were obtained for individuals with RA (n = 50) and age-, gender-, and body mass index (BMI)-matched controls (n = 39). Groups were compared using 39 matched pairs with 11 additional subjects used in RA only analyses. Among RA patients, relationships were determined for lipoprotein parameters with measures of disease activity, disability, pain, inflammation, body composition, insulin sensitivity and exercise. Percentage of time spent in basal activity (<1 metabolic equivalent) and exercise (≥3 metabolic equivalents) were objectively-determined. RESULTS: Subjects with RA had fewer total and small LDL-P as well as larger LDL and HDL size (P < 0.05). Among RA patients, pain and disability were associated with fewer small HDL-P (P < 0.05), while interleukin (IL)-6, IL-18, and TNF-α were associated with LDL size (P < 0.05). BMI, waist circumference, abdominal visceral adiposity and insulin resistance were associated with more total and small LDL-P, fewer large HDL-P, and a reduction in HDL size (P < 0.05). Most similar to the RA lipoprotein profile, more basal activity (minimal physical activity) and less exercise time were associated with fewer small LDL-P and total and small HDL-P (P < 0.05). CONCLUSIONS: The RA-associated lipoprotein profile is associated with a lack of physical activity. As this was a cross-sectional investigation and not an intervention and was performed from 2008-13, this study was not registered in clinicaltrials.gov.

Steatohepatitis and liver fibrosis are predicted by the characteristics of very low density lipoprotein in nonalcoholic fatty liver disease.

BACKGROUND & AIMS: A major challenge in the management of nonalcoholic fatty liver disease (NAFLD) is to identify patients with nonalcoholic steatohepatitis (NASH) and early liver fibrosis. The progression of NAFLD is accompanied by distinctive changes in very low density lipoprotein (VLDL), a lipoprotein particle produced exclusively in the liver. Herein, we sought to determine the characteristics of VLDL profiles associated with NASH and liver fibrosis. METHODS: We evaluated VLDL profiles of 128 patients from a single centre NAFLD registry, and examined VLDL size, total and subclass VLDL concentrations in relation to NAFLD activity score (NAS), steatohepatitis and liver fibrosis as determined by liver biopsy. RESULTS: A near linear relationship was observed between mean VLDL particle size and NAFLD activity score (NAS). In multivariate models, VLDL particle size was significantly associated with both NAS and NASH, after adjustment for BMI and diabetes. A decrease in small VLDL particle concentration was associated with more advanced liver fibrosis. In receiver operative characteristic analyses, mean VLDL size performed similarly to cytokeratin 18 in predicting NASH, whereas small VLDL particle concentration had similar performance to NAFLD fibrosis score in predicting stage 2 or above liver fibrosis. CONCLUSIONS: The increase in mean VLDL size in NASH and decrease in small VLDL particle concentration in liver fibrosis likely reflect changes in the number and state of hepatocytes associated with NASH and fibrosis. In addition to its value in risk stratification of cardiovascular diseases, circulating VLDL profile may provide information for the staging of NAFLD disease severity.

Differences in GlycA and lipoprotein particle parameters may help distinguish acute kawasaki disease from other febrile illnesses in children.

BACKGROUND: Glycosylation patterns of serum proteins, such as α1-acid glycoprotein, are modified during an acute phase reaction. The response of acute Kawasaki disease (KD) patients to IVIG treatment has been linked to sialic acid levels on native IgG, suggesting that protein glycosylation patterns vary during the immune response in acute KD. Additionally, the distribution and function of lipoprotein particles are altered during inflammation. Therefore, the aim of this study was to explore the potential for GlycA, a marker of protein glycosylation, and the lipoprotein particle profile to distinguish pediatric patients with acute KD from those with other febrile illnesses. METHODS: Nuclear magnetic resonance was used to quantify GlycA and lipoprotein particle classes and subclasses in pediatric subjects with acute KD (n = 75), post-treatment subacute (n = 36) and convalescent (n = 63) KD, as well as febrile controls (n = 48), and age-similar healthy controls (n = 48). RESULTS: GlycA was elevated in acute KD subjects compared to febrile controls with bacterial or viral infections, IVIG-treated subacute and convalescent KD subjects, and healthy children (P <0.0001). Acute KD subjects had increased total and small low density lipoprotein particle numbers (LDL-P) (P <0.0001) and decreased total high density lipoprotein particle number (HDL-P) (P <0.0001) compared to febrile controls. Consequently, the ratio of LDL-P to HDL-P was higher in acute KD subjects than all groups tested (P <0.0001). While GlycA, CRP, erythrocyte sedimentation rate, LDL-P and LDL-P/HDL-P ratio were able to distinguish patients with KD from those with other febrile illnesses (AUC = 0.789-0.884), the combinations of GlycA and LDL-P (AUC = 0.909) or GlycA and the LDL-P/HDL-P ratio (AUC = 0.910) were best at discerning KD in patients 6-10 days after illness onset. CONCLUSIONS: High levels of GlycA confirm enhanced protein glycosylation as part of the acute phase response in KD patients. When combined with common laboratory tests and clinical characteristics, GlycA and NMR-measured lipoprotein particle parameters may be useful for distinguishing acute KD from bacterial or viral illnesses in pediatric patients.

GlycA: A Composite Nuclear Magnetic Resonance Biomarker of Systemic Inflammation.

BACKGROUND: Nuclear magnetic resonance (NMR) spectra of serum obtained under quantitative conditions for lipoprotein particle analyses contain additional signals that could potentially serve as useful clinical biomarkers. One of these signals that we named GlycA originates from a subset of glycan N-acetylglucosamine residues on enzymatically glycosylated acute-phase proteins. We hypothesized that the amplitude of the GlycA signal might provide a unique and convenient measure of systemic inflammation. METHODS: We developed a spectral deconvolution algorithm to quantify GlycA signal amplitudes from automated NMR LipoProfile(®) test spectra and assessed analytic precision and biological variability. Spectra of acute-phase glycoproteins and serum fractions were analyzed to probe the origins of the GlycA signal. GlycA concentrations obtained from archived NMR LipoProfile spectra of baseline plasma from 5537 participants in the Multi-Ethnic Study of Atherosclerosis (MESA) were used to assess associations with demographic and laboratory parameters including measures of inflammation. RESULTS: Major acute-phase protein contributors to the serum GlycA signal are α1-acid glycoprotein, haptoglobin, α1-antitrypsin, α1-antichymotrypsin, and transferrin. GlycA concentrations were correlated with high-sensitivity C-reactive protein (hsCRP) (r = 0.56), fibrinogen (r = 0.46), and interleukin-6 (IL-6) (r = 0.35) (all P < 0.0001). Analytic imprecision was low (intra- and interassay CVs 1.9% and 2.6%, respectively) and intraindividual variability, assessed weekly for 5 weeks in 23 healthy volunteers, was 4.3%, lower than for hsCRP (29.2%), cholesterol (5.7%), and triglycerides (18.0%). CONCLUSIONS: GlycA is a unique inflammatory biomarker with analytic and clinical attributes that may complement or provide advantages over existing clinical markers of systemic inflammation.

A novel protein glycan biomarker and LCAT activity in metabolic syndrome.

BACKGROUND: The cholesterol-esterifying enzyme, lecithin:cholesterol acyltransferase (LCAT), is instrumental in high-density lipoprotein (HDL) remodelling. LCAT may also modify oxidative and inflammatory processes, as supported by an inverse relationship with HDL antioxidative functionality and a positive relationship with high-sensitivity C-reactive protein (hsCRP). GlycA is a recently developed proton nuclear magnetic resonance (NMR) spectroscopy-measured biomarker of inflammation whose signal originates from a subset of N-acetylglucosamine residues on the most abundant glycosylated acute-phase proteins. Plasma GlycA correlates positively with hsCRP and may predict cardiovascular disease even independent of hsCRP. Here, we tested the extent to which plasma GlycA is elevated in metabolic syndrome (MetS), and determined its relationship with LCAT activity. MATERIALS AND METHODS: Plasma GlycA, hsCRP, serum amyloid A (SAA), tumour necrosis factor-α (TNF-α) and LCAT activity were measured in 58 subjects with MetS (including 46 subjects with type 2 diabetes mellitus (T2DM)) and in 45 nondiabetic subjects without MetS. RESULTS: Plasma GlycA was higher in MetS coinciding with higher hsCRP and LCAT activity (P < 0.01 for each). In all subjects combined, GlycA was correlated positively with hsCRP, SAA and LCAT activity (P < 0.001 for each), but not with TNF-α. Age- and sex-adjusted multivariable linear regression analysis revealed that GlycA was positively associated with LCAT activity (P = 0.029), independent of the presence of MetS, T2DM, hsCRP and SAA. GlycA was unrelated to diabetes status. CONCLUSION: A pro-inflammatory glycoprotein biomarker, GlycA, is higher in MetS. Higher plasma levels of this glycoprotein biomarker relate to increased LCAT activity in the setting of MetS.