Irina Shalaurova

Purpose: To investigate associations between lipid oxidation biomarkers (oxylipins), antioxidant micronutrients, lipoprotein particles, and apolipoproteins in multiple sclerosis (MS). Methods: Blood and neurological assessments were collected from 30 healthy controls, 68 relapsing remitting MS subjects, and 37 progressive MS subjects. Hydroxy (H) and hydroperoxy lipid peroxidation products of the polyunsaturated fatty acids (PUFAs) arachidonic (20:4, ω-6), linoleic (octadecadienoic acid or ODE, 18:2, ω-6), eicosapentaenoic (20:5, ω-3), and α-linolenic (18:3, ω-3) acids were measured using liquid chromatography–mass spectrometry. Antioxidant micronutrients, including β-cryptoxanthin and lutein/zeaxanthin, were quantified by high-performance liquid chromatography. Lipoprotein and metabolite profiles were obtained using nuclear magnetic resonance spectroscopy. Regression models were adjusted for age, sex, body mass index, and disease status. Results: The 9-hydroxy octadecadienoic acid to 13-hydroxy octadecadienoic acid ratio (9-HODE/13-HODE ratio), which reflects autoxidative versus enzymatic oxidation, was associated with MS status (p = 0.002) and disability on the Expanded Disability Status Scale (p = 0.004). Lutein/zeaxanthin (p = 0.023) and β-cryptoxanthin (p = 0.028) were negatively associated with the 9-HODE/13-HODE ratio. Apolipoprotein-CII, a marker of liver-X-receptor (LXR) signaling, was associated with 9-HODE/13-HODE ratio and other oxylipins. Octadecadienoic fatty acid-derived oxylipins were negatively associated with LC3A, a mitophagy marker, and positively correlated with 7-ketocholesterol, a cholesterol autoxidation product. Conclusions: Autoxidation of PUFAs is associated with greater disability in MS. Higher β-cryptoxanthin and lutein/zeaxanthin were associated with reduced auto-oxidation. Lipid peroxidation shows associations with LXR signaling, mitophagy, inflammation, and cholesterol autoxidation.

BACKGROUND: Genome- and epigenome-wide association studies have associated variants and methylation status of CPT1a (carnitine palmitoyltransferase 1a) to reductions in VLDL (very low-density lipoprotein) cholesterol and triglyceride levels. The objective of this study was to determine the mechanisms by which CPT1a-dependent mitochondrial fatty acid oxidation influences hepatic and lipoprotein metabolism. METHODS: Eight-week-old male and female Cpt1a -floxed mice ( Cpt1a ^fl/fl ) and Cpt1a -floxed mice expressing the human apo B ₁₀₀ transgene ( Cpt1a ^fl/fl /B100 ^Tg ) were administered control adeno-associated virus or adeno-associated virus encoding Cre-recombinase under control of a liver-specific promoter (TBG-Cre [thyroxin-binding globulin]). Control and liver-specific knockout mice were placed on a low-fat control or western-type diet (42% kcal fat, 0.2% cholesterol) for 16 weeks. Livers were collected and used for histological and lipid analysis, while gene and protein expression were measured by bulk RNA-sequencing and immunoblotting, respectively. Lipoprotein composition in plasma was determined by size exclusion chromatography and nuclear magnetic resonance. Rates of VLDL-triglyceride secretion were quantified after lipase inhibition with poloxamer 407. Liquid and gas chromatography–mass spectrometry were used to measure bile acid species and fecal neutral sterols, respectively. RESULTS: We report significant associations between the presence of CPT1a SNPs (single nucleotide polymorphisms) and reductions in plasma cholesterol, as well as positive associations between hepatic Cpt1a expression and plasma cholesterol levels across inbred mouse strains. Mechanistic studies show that both wild-type and human apo B ₁₀₀ (apoB)–transgenic mice with liver-specific deletion of Cpt1a (liver-specific knockout) display lower circulating apoB levels consistent with reduced LDL (low-density lipoprotein)-cholesterol and LDL particle number. Despite a reduction in steady-state plasma lipids, VLDL-triglyceride and VLDL cholesterol secretion rates are increased, suggesting accelerated clearance of apoB-LPs (apoB-containing lipoproteins) in liver-specific knockout mice. Mechanistic approaches show greater PPARα (peroxisome proliferator–activated receptor α) signaling which favors enhanced lipoprotein lipase–mediated metabolism of apoB-LPs, including increases in apo AIV and apo CII and reductions in apo CIII and Angptl3 (angiopoietin-like 3). CONCLUSIONS: These studies provide mechanistic insight linking genetic variants and methylation status of CPT1a to reductions in circulating apoB-LPs in humans.

Background/Objectives: The metabolic vulnerability index (MVX)—a composite biomarker reflecting metabolic malnutrition and inflammation—is associated with increased mortality risk, but its association across different levels of chronic disease burden has not been explored. We aimed to examine the associations of MVX and its subcomponents (Inflammation Vulnerability Index, IVX and Metabolic Malnutrition Index, MMX) with all-cause mortality according to multimorbidity status. Methods: In the PREVEND study, which included 6054 participants (mean age 54 years; 49.5% male), MVX was calculated using six plasma biomarkers measured simultaneously via nuclear magnetic resonance spectroscopy. Hazard ratios (HRs) with 95% confidence intervals (CIs) were estimated. Results: During a median follow-up of 14.0 years, 911 deaths were recorded. In analyses adjusted for several established risk factors, the HRs (95% CIs) of mortality per 1 standard deviation increment in MVX were 1.32 (1.13–1.54; p < 0.001), 1.23 (1.08–1.40; p = 0.002), and 1.29 (1.16–1.43; p < 0.001) for individuals with no disease, one disease, and multimorbidity, respectively. The corresponding HRs (95% CIs) were 1.22 (1.05–1.42; p = 0.010), 1.17 (1.03–1.34; p = 0.015), and 1.25 (1.13–1.38; p < 0.001) for IVX and 1.29 (1.11–1.48; p = 0.001), 1.16 (1.02–1.31; p = 0.032), and 1.14 (1.03–1.25; p = 0.004) for MMX. The ratio of HRs showed no statistical evidence that sex modified the associations of MVX, IVX, and MMX with mortality in each multimorbidity category. However, the associations appeared stronger in males with chronic disease and in females without chronic conditions, suggesting possible sex-related trends. Conclusions: MVX, IVX, and MMX are independent risk indicators of all-cause mortality regardless of the burden of chronic disease, with MVX showing the strongest associations across different multimorbidity statuses. MMX should be interpreted as a proxy for metabolic malnutrition rather than a direct nutritional assessment tool.

Purpose: Ketone bodies could be useful biomarkers in multiple sclerosis (MS) because the pathophysiological processes underlying MS disease progression induce metabolic stress. The purpose was to assess the relationships of ketone bodies with biomarkers of metabolic, inflammatory, and oxidative stress in MS. Methods: Blood samples and neurological assessments were obtained from 153 healthy controls (HC), 187 relapsing-remitting (RRMS), and 91 progressive MS (PMS) patients. AcAc, BHB, and acetone were measured using proton nuclear magnetic resonance spectroscopy. Indices of inflammatory vulnerability (IVX), metabolic malnutrition (MMX), and metabolic vulnerability (MVX) were computed from the NMR profiles. Cholesterol, apolipoprotein, lipid peroxidation, and antioxidant profiles were obtained. Regression analysis adjusted for age, sex, body mass index, and HC, RRMS, or PMS disease status. Results: AcAc and BHB levels were greater in MS compared to HC. BHB and ketone bodies were positively associated with disability on the MS Severity Scale and ambulation time. BHB was positively associated with IVX, MMX, and MVX. AcAc was positively associated with MMX and negatively associated with IVX and MVX. Total ketone body concentration was positively associated with MMX and MVX. BHB and AcAc levels were negatively associated with the amino acids alanine, valine, and leucine. Conclusions: Ketone bodies are associated with inflammatory vulnerability, metabolic vulnerability, and ambulatory disability measures in MS.

Purpose: Metabolic vulnerabilities can exacerbate inflammatory injury and inhibit repair in multiple sclerosis (MS). The purpose was to evaluate whether blood biomarkers of inflammatory and metabolic vulnerability are associated with MS disability and neurodegeneration. Methods: Proton nuclear magnetic resonance spectra were obtained from serum samples from 153 healthy controls, 187 relapsing–remitting, and 91 progressive MS patients. The spectra were analyzed to obtain concentrations of lipoprotein sub-classes, glycated acute-phase proteins, and small-molecule metabolites, including leucine, valine, isoleucine, alanine, and citrate. Composite indices for inflammatory vulnerability, metabolic malnutrition, and metabolic vulnerability were computed. MS disability was measured on the Expanded Disability Status Scale. MRI measures of lesions and whole-brain and tissue-specific volumes were acquired. Results: Valine, leucine, isoleucine, alanine, the Inflammatory Vulnerability Index, the Metabolic Malnutrition Index, and the Metabolic Vulnerability Index differed between healthy control and MS groups in regression analyses adjusted for age, sex, and body mass index. The Expanded Disability Status Scale was associated with small HDL particle levels, inflammatory vulnerability, and metabolic vulnerability. Timed ambulation was associated with inflammatory vulnerability and metabolic vulnerability. Greater metabolic vulnerability and inflammatory vulnerability were associated with lower gray matter, deep gray matter volumes, and greater lateral ventricle volume. Conclusions: Serum-biomarker-derived indices of inflammatory and metabolic vulnerability are associated with disability and neurodegeneration in MS.

Background: Heart failure (HF) features a shift in metabolism towards enhanced utilization of ketone bodies. While elevations in plasma natriuretic peptides represent a biochemical hallmark of HF, natriuretic peptides may promote lipolysis, thereby contributing to fatty acid availability for ketogenesis. Methods: We cross-sectionally tested to what extent fasting plasma total ketone bodies (measured using nuclear magnetic resonance spectroscopy) are associated with N-terminal pro-BNP (NT-proBNP; electrochemiluminescent sandwich immunoassay) in individuals with and without HF. Results: Among 6217 participants from the Prevention of REnal and Vascular ENd-stage Disease (PREVEND) study, 203 were identified with HF. NT-proBNP was four-fold and total ketone bodies were 25% higher in HF participants (each p < 0.001). In both participants with and without HF, total ketone body levels correlated with NT-proBNP (r = 0.116 and 0.185, respectively; p < 0.001). In multivariable linear regression analysis adjusted for relevant covariates, total ketone bodies remained associated with NT-proBNP in the whole cohort (std β = 0.08, p < 0.001), without a difference in participants with and without HF (p interaction: 0.52). Conclusion: This general population-based study reveals an independent association of fasting total body ketone bodies with plasma NT-proBNP. Our findings suggest that a metabolic defense mechanism could be operative, providing the myocardium with ketone bodies to meet its energy demands.

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.

Nonalcoholic fatty liver disease (NAFLD) is associated with mitochondrial damage. Circulating mitochondrial metabolites may be elevated in NAFLD but their associations with liver damage is not known. This study aimed to assess the association of key mitochondrial metabolites with the degree of liver fibrosis in the context of NAFLD and nonalcoholic steatohepatitis (NASH). Cross-sectional analyses were performed on two cohorts of biopsy-proven NAFLD and/or NASH subjects. The association of circulating mitochondrial metabolite concentrations with liver fibrosis was assessed using linear regression analysis. In the single-center cohort of NAFLD subjects (n = 187), the mean age was 54.9 ±13.0 years, 40.1% were female and 86.1% were White. Type 2 diabetes (51.3%), hypertension (43.9%) and obesity (72.2%) were prevalent. Those with high citrate had a higher proportion of moderate/significant liver fibrosis (stage F ≥ 2) (68.4 vs. 39.6%, p = 0.001) and advanced fibrosis (stage F ≥ 3) (31.6 vs. 13.6%, p = 0.01). Citrate was associated with liver fibrosis independent of age, sex, NAFLD activity score and metabolic syndrome (per 1 SD increase: β = 0.19, 95% CI: 0.03–0.35, p = 0.02). This association was also observed in a cohort of NASH subjects (n = 176) (β = 0.21, 95% CI: 0.07–0.36, p = 0.005). The association of citrate with liver fibrosis was observed in males (p = 0.005) but not females (p = 0.41). In conclusion, circulating citrate is elevated and associated with liver fibrosis, particularly in male subjects with NAFLD and NASH. Mitochondrial function may be a target to consider for reducing the progression of liver fibrosis and NASH.