Structural characterization of DNA amplicons by ATR-FTIR spectroscopy as a guide for screening metainflammatory disorders in blood plasma.

Attenuated total reflectance (ATR) Fourier transform infrared (FTIR) spectroscopy is a promising rapid, reagent-free, and low-cost technique considered for clinical translation. It allows to characterize biofluids proteome, lipidome, and metabolome at once. Metainflammatory disorders share a constellation of chronic systemic inflammation, oxidative stress, aberrant adipogenesis, and hypoxia, that significantly increased cardiovascular and cancer risk. As a result, these patients have elevated concentration of cfDNA in the bloodstream. Considering this, DNA amplicons were analyzed by ATR-FTIR at 3 concentrations with 1:100 dilution: (IU/mL): 718, 7.18, and 0.0718. The generated IR spectrum was used as a guide for variable selection. The main peaks in the biofingerprint (1800-900 cm-1) give important information about the base, base-sugar, phosphate, and sugar-phosphate transitions of DNA. To validate our method of selecting variables in blood plasma, 38 control subjects and 12 with metabolic syndrome were used. Using the wavenumbers of the peaks in the biofingerprint of the DNA amplicons, was generated a discriminant analysis model with Mahalanobis distance in blood plasma, and 100 % discrimination accuracy was obtained. In addition, the interval 1475-1188 cm-1 showed the greatest sensitivity to variation in the concentration of DNA amplicons, so curve fitting with Gaussian funcion was performed, obtaining adjusted-R2 of 0.993. PCA with Mahalanobis distance in the interval 1475-1188 cm-1 obtained an accuracy of 96 % and PLS-DA modeling in the interval 1475-1088 cm-1 obtained AUC = 0.991 with sensitivity of 95 % and specificity of 100 %. Therefore, ATR-FTIR spectroscopy with variable selection guided by DNA IR peaks is a promising and efficient method to be applied in metainflammatory disorders.

Detection of metabolic syndrome with ATR-FTIR spectroscopy and chemometrics in blood plasma.

Metabolic Syndrome (MetS) is a constellation of 3 or more risk factor (abdominal obesity, high triglycerides, low HDL-c, high blood pressure, and elevated blood glucose) for atherosclerotic cardiovascular disease. Considering these systemic metabolic changes in the biochemical pathways of all biomolecules, Attenuated Total Reflection-Fourier Transform Infrared (ATR-FTIR) spectroscopy is a rapid, low-cost, and reagent-free alternative technique capable of identifying spectral biomarkers that differentiate subjects with MetS from control. In this study, plasma samples from 74 subjects (14 MetS, 60 control) were analyzed on the ATR-FTIR spectrophotometer. The objective was to differentiate subjects with MetS from control with supervised chemometrics modeling (Orthogonal Partial Least Squares-Discriminant Analysis, OPLS-DA). Additionally, the inflammatory status of subjects with MetS and control (supervised by C-reactive protein - CRP, leptin, and cell-free DNA - cfDNA) was verified. The OPLS-DA model achieved 100% sensitivity and specificity in cross-validation. For 1 latent variable (93.4% of variance), RMSECV < 0.002, PRESS CV < 0.0001, and R2 > 0.9999 was obtained. Significant spectrochemical differences (p < 0.05) were found between MetS and control subjects in the following biomolecular regions (cm-1): 1717-1703 [ν(CO) and δ(NH)], 1166-1137 [ν(C-OH) + ν(CO) and ν(CC) + Î´(OH) + ν(CO)], 1113-1040 [ν(PO2-) and ν(C-OH)], and 1027-1008 [ν(CO) and v(CH2OH)]. In the OPLS-DA model loadings, amide I [1720-1600 cm-1, ν(CO)] and amide II [1570-1480 cm-1, δ(NH) + ν(CH)] had significantly greater weight than all other regions. There was a significant difference in inflammatory status between MetS patient and control (p < 0.05 for CRP and leptin, and p < 0.01 for cfDNA).