https://doi.org/10.1007/s13300-023-01372-x
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1H NMR Lipidomic Analysis
1H NMR lipidomic analysis was performed as previously stated [12]. Lipophilic extracts were obtained from two 100-μL aliquots of freshly thawed plasma using the BUME method [22] with slight modifications. BUME was optimized for batch extractions with diisopropyl ether (DIPE) replacing heptane as the organic solvent. This procedure was performed with a BRAVO liquid-handling robot which can extract 96 samples at once. The upper lipophilic phase was completely dried in Speedvac until evaporation of organic solvents and frozen at − 80 °C until 1H NMR analysis. Lipid extracts were reconstituted in a solution of CDCl3/CD3OD/D2O (16:7:1, v/v/v) containing tetramethylsilane (TMS) at 1.18 mM as a chemical shift reference and transferred into 5-mm NMR glass tubes. 1H NMR spectra were measured at 600.20 MHz using an Avance III-600 Bruker spectrometer. A 90° pulse with water presaturation sequence (zgpr) was used. Quantification of lipid signals in 1H NMR spectra was carried out with LipSpin [23], in-house software based on Matlab (MATLAB. version 7.10.0 (R2010a); Natick, Massachusetts: The MathWorks Inc.; 2010.). Resonance assignments were based on values in the literature [24].
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1H NMR-Based Lipidomic Analysis in Relation to Cardiometabolic Traits
Whereas no differences were observed concerning age (Fig. 1), several between-gender differences were found: higher levels of 1H NMR triglycerides and ω-9 fatty acids, and lower levels of glycerophospholipids, phosphatidylcholine, sphingomyelin and ω-3 fatty acids were observed among male participants (p < 0.05; Table S3 in the supplementary material). Only minor differences were found regarding hypertension and smoking habit, whereas those on statins showed lower levels of linoleic and ω-6 fatty acids, and higher levels of sphingomyelin and arachidonic acid–eicosapentaenoic acid (ARA-EPA) (p < 0.05, Table S3). Among other adipose-related variables, FLI showed the strongest correlations with most of the 1H NMR-related lipidomic parameters, especially with 1H NMR triglycerides and ω-9 fatty acids (rs > 0.4 for both; Fig. 1). Finally, white blood cell count (as a marker of an inflammation-related variable) was directly associated with 1H NMR triglycerides, saturated fatty acids and ARA-EPA (rs = 0.2–0.3).
Associations between NMR lipidomics and clinical and laboratory parameters. Numbers in cells indicate Spearman’s R correlation index and the colour of each cell indicates the strength of association of that index according to the colour code shown in the right column. ACR albumin-to-creatinine ratio, ALAT alanine aminotransferase, BMI body mass index, eGDR estimated glucose disposal rate, eGFR estimated glomerular filtration rate, FLI fatty liver index, SBP systolic blood pressure, WBC white blood cells, WC waist circumference
Regarding T1D-specific risk factors, there were minor differences regarding diabetes duration and retinopathy status. However, HbA1c showed direct relationships with 1H NMR triglycerides, esterified cholesterol, ω-6 and ω-9 fatty acids, and ARA-EPA (p < 0.05 for all comparisons, Fig. 1). Further, only weak direct correlations were found with ACR (as a marker of diabetic kidney disease; rs = 0.1–0.2); and eGDR, as a marker of insulin sensitivity, was strongly and inversely associated with 1H NMR triglycerides (rs = − 0.415).
1H NMR-Based Lipidomic Analysis and Preclinical Carotid Atherosclerosis
Only levels of sphingomyelin were inversely associated with the presence of at least one carotid plaque (p < 0.05, Table 2), which maintained the statistical significance after adjusting for confounders such as age, sex, presence of hypertension, statin use, mean HbA1c in the last 5 years and diabetes duration (for 0.1 mmol/L increase, OR 0.50 [0.28–0.86]; p = 0.013; Table 3). When other variables associated with a higher plaque burden were assessed (presence of ≥ 3 plaques), inverse associations were found with esterified and free cholesterol, linoleic acid and ω-6 fatty acids (p < 0.05 for all comparisons; Table 2), which remained statistically associated in fully adjusted models (model 3; OR 0.055 (0.006–0.51), 0.009 (0.0–0.60), 0.17 (0.03–0.93) and 0.27 (0.07–0.97), for esterified cholesterol, free cholesterol, linoleic acid and ω-6 fatty acids, respectively; p < 0.05 for all; Table 3).
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