Complexity of fatty acid distribution inside human macrophages on single cell level using Raman micro-spectroscopy

Abstract

Macrophages are phagocytic cells which are involved in the non-specific immune defense. Lipid uptake and storage behavior of macrophages also play a key role in the development of atherosclerotic lesions within walls of blood vessels. The allocation of exogenous lipids such as fatty acids in the blood stream dictates the accumulation and quantity of lipids within macrophages. In case of an overexposure, macrophages transform into foam cells because of the large amount of lipid droplets in the cytoplasm. Raman micro-spectroscopy is a powerful tool for studying single cells due to the combination of microscopic imaging with spectral information. With a spatial resolution restricted by the diffraction limit, it is possible to visualize lipid droplets within macrophages. With stable isotopic labeling of fatty acids with deuterium, the uptake and storage of exogenously provided fatty acids can be investigated. In this study, we present the results of time-dependent Raman spectroscopic imaging of single THP-1 macrophages incubated with deuterated arachidonic acid. The polyunsaturated fatty acid plays an important role in the cellular signaling pathway as being the precursor of icosanoids. We show that arachidonic acid is stored in lipid droplets but foam cell formation is less pronounced as with other fatty acids. The storage efficiency in lipid droplets is lower than in cells incubated with deuterated palmitic acid. We validate our results with gas chromatography and gain information on the relative content of arachidonic acid and its metabolites in treated macrophages. These analyses also provide evidence that significant amounts of the intracellular arachidonic acid is elongated to adrenic acid but is not metabolized any further. The co-supplementation of deuterated arachidonic acid and deuterated palmitic acid leads to a non-homogenous storage pattern in lipid droplets within single cells.

Lipid droplet functions beyond energy storage

“…This repertoire is sure to expand over the next few years with the development of increasingly sophisticated lipidomics methods as well as imaging techniques based on Raman and mass spectrometry [27-34]. In many cell types, including adipocytes and hepatocytes, the majority of lipids in the droplet core correspond to triglycerides and sterol esters with a range of different FA side chains.…”

D38-cholesterol as a Raman active probe for imaging intracellular cholesterol storage

“…A similar shift has previously been reported for the deuterated fatty acid D6-arachidonic acid. 26 Note that besides a significant reduction of the CH band, the spectral profile of the CH stretches of D38-cholesterol is markedly different from the spectral bandshape of natural cholesterol in this range. The contribution of the symmetric CH 2 stretching mode at 2845 cm −1 in D38-cholesterol is relatively weak, whereas the strongest contribution in this range is found at 2925 cm −1 .…”

“…In this regard, probes based on the substitution of hydrogen by deuterium are particularly attractive, as the chemical properties of deuterated isotopologs closely resemble those of their endogenous counterparts. Deuterium-based Raman labels have been successfully used in nonlinear optical imaging applications to help increase specificity to water dynamics, 21 selected phospholipids, 22,23 neutral lipids, [24][25][26] and protein metabolism. 27,28 Labels based on stable isotopes, including deuterium, have found widespread use in clinical applications.…”

New Gateways to the Platinum Group Metal-Catalyzed Direct Deuterium-Labeling Method Utilizing Hydrogen as a Catalyst Activator

“…[49][50][51][52][53][54][55][56][57][58][59][60][61][62][63][64][65][66][67][68] Although Pt/C has been traditionally used in D 2 O for the preparation of deuterium-labeled saturated fatty acids, harsh hydrothermal reaction conditions and repetitive deuteration processes are required, and the D contents and scope of the substrates were unsatisfactory using previously reported methods. 69) Since the deuteration of alkanes, such Various fatty acids are effectively deuterium-labeled in the presence of Pt/C in an i-PrOD-d 8 /D 2 O mixed solvent (Chart 13).…”