NMR Spectroscopy-Based Lipoprotein and Glycoprotein Biomarkers Differentiate Acute and Chronic Inflammation in Diverse Healthy and Disease Population Cohorts

Abstract

Understanding the distribution and variation in NMR-based inflammatory markers is crucial to the evaluation of their clinical utility in disease prognosis and diagnosis. We applied high-resolution ¹H NMR spectroscopy of blood plasma and serum to measure the acute phase reactive glycoprotein signals (GlycA and GlycB) and the subregions of the lipoprotein-based Supramolecular Phospholipid Composite signals (SPC₁, SPC₂, and SPC₃) in a large multicohort population study. A total of 5702 samples were studied to determine the signal variations in a range of chronic and acute inflammatory conditions. We found that while GlycA and GlycB were increased in inflammation, the SPC regions behaved independently of Glyc signals, with SPC₂ and SPC₃ being reduced in chronic inflammation in comparison to healthy controls (p-value SPC₂ = 2.9 × 10^-10, p-value SPC₃ = 2.2 × 10^-3) and SPC₁ (p-value = 0.29) being unchanged. SPC₁ was decreased in acute inflammation, indicating a link to the immune response (p-value = 2.5 × 10^-11). These findings confirm the independent biological relevance of all three SPC subregions and contraindicate the use of aggregate SPC values as general inflammatory markers.

Keywords: C-reactive protein; NMR spectroscopy; SARS-CoV-2; SPC; Supramolecular Phospholipid Composite signals; acute inflammation; blood plasma metabolic phenotyping; cardiovascular disease risk; chronic inflammation; diabetes; glycoproteins; inflammation.

1

(A) Overview of the blood lipoproteins. Very low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), and the two subfractions visible using the DIRE experiment, the low-density lipoprotein (LDL) and high-density lipoprotein (HDL). (B) The segments of the composite SPC peak, SPC₁, SPC₂, and SPC₃ in the DIRE NMR spectrum with the associated lipoprotein subfractions from which the peak arises from shown.

2

(A) PCA scores plot and the associated loadings of the variables of healthy individuals (blue square), those with chronic inflammation (cardiovascular disease, yellow circle), and individuals with acute inflammation (acute SARS-CoV-2 infection, red triangle). (B) Box plots of healthy individuals (blue), those with chronic inflammation (yellow), and those with acute inflammation (red) for SPC₁, SPC₂, SPC₃, GlycA, GlycB, and SPC/Glyc. Adjusted p-values of all plots can be found inTables S4–S9.

3

(A) Box plot of healthy individuals (blue), those with chronic inflammation (yellow), and those with acute inflammation (red) for the SPC₃/SPC₂ ratio.

4

Box plots showing changes in the inflammatory markers for the healthy participants and nonhealthy participants of the population cohort at time point 1 and time point 2, collected 5 years apart, showing no significant difference between the time points and across the age ranges. Criteria of healthy participants of the BHAS study have been described previously. ,

5

(A) OPLS-DA scores of healthy participants (blue) and patients with cardiovascular disease (red) (R²X = 0.48, AUROC = 0.77, CV-AUROC = 0.77) and (B) −log10 p-values of the inflammatory markers. Red is higher in cardiovascular disease, and blue is lower in cardiovascular disease in comparison to healthy individuals. (C) OPLS-DA of healthy participants (blue) and patients with type 2 diabetes (red) (R²X = 0.46, AUROC = 0.84, CV-AUROC = 0.83) and (D) −log10 p-values of the inflammatory markers. All p-values for both models can be found in Tables S10 and S11.