Quantitative determination of low density lipoprotein oxidation by FTIR and chemometric analysis

Abstract

This study was conducted to develop a quantitative FTIR spectroscopy method to measure LDL lipid oxidation products and determine the effect of oxidation on LDL lipid and protein. In vitro LDL oxidation at 37 degrees C for 1 h produced a range of conjugated diene (CD) (0.14-0.26 mM/mg protein) and carbonyl contents (0.9-3.8 microg/g protein) that were used to produce calibration sets. Spectra were collected from the calibration set and partial least squares regression was used to develop calibration models from spectral regions 4000-650, 3750-3000, 1720-1500, and 1180-935 cm(-1) to predict CD and carbonyl contents. The optimal models were selected based on their standard error of prediction (SEP), and the selected models were performance-tested with an additional set of LDL spectra. The best models for CD prediction were derived from spectral regions 4000-650 and 1180-935 cm(-1) with the lowest SEP of 0.013 and 0.013 mM/mg protein, respectively. The peaks at 1745 (cholesterol and TAG ester C=O stretch), 1710 (carbonyl C-O stretch), and 1621 cm(-1) (peptide C=O stretch) positively correlated with LDL oxidation. FTIR and chemometrics revealed protein conformational changes during LDL oxidation and provided a simple technique that has potential for rapidly observing structural changes in human LDL during oxidation and for measuring primary and secondary oxidation products.

Rhodobacter megalophilus sp. nov., a phototroph from the Indian Himalayas possessing a wide temperature range for growth

“…S2(a) Supplementary Fig. S2b) was due to N-H stretching of peptide linkages of the polypeptide chain of lipoproteins (Lam et al, 2004). Furthermore, the high intensities of peaks observed in strain JA194 T ( Supplementary Fig.…”

Fourier transform infrared spectroscopy evaluation of low density lipoprotein oxidation in the presence of quercetin, catechin, and α‐tocopherol

“…Blood samples from five fasting human males were obtained in vacutainers containing 1 mg/mL EDTA and centrifuged for 10 min at 2000 × g and 4°C to separate the plasma. Human LDL were isolated from the fresh human plasma by sequential floating ultracentrifugation, according to the method described by Lam et al (9). The protein content of LDL was determined according to Lowry et al (10) with BSA as a standard.…”

“…Prior to oxidation, all LDL samples were diluted to a final concentration of 50 µg of protein/mL with EDTA-free phosphate buffer (pH 7.4). The in vitro oxidation of LDL was performed in the absence (control) or presence of 0.5 µM quercetin, 2.0 µM catechin, and 0.05 µM α-tocopherol (in ethanol) per mg LDL protein, as previously described (9). The concentrations of quercetin, catechin, and α-tocopherol used in this study corresponded to those reported in human plasma (4,11,12) and will from here on also be referred to as their physiological concentrations.…”

“…This technique is rapid, requires only a small sample, and provides information on functional group composition. We have demonstrated that FTIR can be used to quantify LDL lipid primary and secondary oxidation products as well as changes in LDL protein conformations (9). Studies that simultaneously measure the primary and secondary oxidation products of lipoprotein lipids and of apoB-100 conformational changes and their interrelation are rare.…”

“…In the present study, we used the FTIR techniques developed in our laboratories (9) in combination with band-fitting procedures to simultaneously evaluate the production of primary and secondary oxidation products and apoB-100 modifications in the presence of the antioxidants quercetin, catechin, and α-tocopherol during LDL oxidation. Integrated intensities of the fitted component of LDL spectral bands were measured and analyzed to obtain the relative concentrations of the LDL lipid components and protein conformational structures.…”