Chylomicrons against light scattering: The battle for characterization

Abstract

Chylomicrons (CMs) are lipoprotein particles circulating in blood and transporting dietary lipids. Optically speaking, CMs are small compared to the wavelength of visible light and widely distributed by the size and refractive index (RI). Consequently, intensity of light scattered by the CMs scales with up to the sixth power of their size, hampering simultaneous analysis of 60 and 600 nm CMs. We present an accurate method for quantitative characterization of large-size CM subpopulation by the distributions over size and RI. For the first time the CM characteristics have been determined at a single particle level based on angle-resolved light-scattering measurements. We applied the developed method to 2 key processes relating to CM metabolism, namely in vivo dynamics of CMs in blood plasma after a meal and in vitro lipolysis of CMs by the lipoprotein lipase in postheparin plasma. We have observed the substantial variations in CM concentration, size and RI distributions. This opens the way for a multitude of medical applications involving screening of CM metabolism, which we exemplified by revealing large differences in CM characteristics after a 12-hour fast between a healthy volunteer and a patient with atherosclerosis.

Refractive index to evaluate staining specificity of extracellular vesicles by flow cytometry

“…Furthermore, for the RI > 1.42 lipoprotein population >300 nm, we found an RI of 1.51, which was also found by others in a recent study [26]. Altogether, we can conclude that our RI values for EVs and lipoproteins are in agreement with literature, suggesting that the inaccuracies in the determined RI are not substantial.…”

“…Here, we used a threshold at RI = 1.42 to discriminate the two populations with different RI present in plasma and platelet concentrate (Figure 1(b)). This threshold is based on the facts that (i) lipoproteins consist of lipids and proteins which both have an RI > 1.44 [24,26,27] and (ii) EVs are reported to have an RI < 1.40 [13,14]. Because EVs may differ in composition, for example due to the presence of RNA, some EVs may have an RI > 1.42.…”

“…Flow-SR can, for instance, be used as a diagnostic tool to check lipoprotein metabolism (e.g. monitor lipoprotein concentrations after a meal), or to study their involvement in pathogenesis of, for example, atherosclerosis [26]. Flow-SR, thereby, is applicable to all flow cytometers, but the particle size range to which it can be applied will differ per instrument as a result of the detection limit and the wavelength used [12].…”

Single‐Particle Characterization by Elastic Light Scattering

“…The same characterization method was used for blood microparticles (extracellular vesicles), [ 11 ] also combining the standard LSP measurement with side‐ and forward‐scattering signals. [ 93,214,215 ] Resulting characterization uncertainties significantly vary over the sample. The median size uncertainties (1 SD) were 8, 19, and 5 nm for 0.4 and 1 µm beads and near‐spherical blood microparticles larger than 0.4 µm, respectively.…”

“…Similarly, obtained uncertainties (or other quality‐of‐fit parameters) has been used to discard a part of the processed particles, as belonging to an alternative (more complicated) model. [ 11,64,215 ] Finally, the most rigorous approach is to try a regression with different models and choose the most suitable one for a specific particle. Examples include using the F‐test and the Bayesian information criterion for nested (spheres and spheroids [ 200 ] ) and non‐nested models (spheres and bispheres [ 93,214 ] ), respectively.…”