Lipidome of Activated Hepatic Stellate Cells Figure 4. HSCs contain a metabolically homogenous population of LDs. A. Freshly isolated HSCs were cultured for 6 days and additionally incubated with 25 mM deuterated arachidonic acid (AA-d8) for another 24 h. After fixation confocal Raman microspectroscopy was performed as described. Raman images in the 1595 cm21 (RE) and 2180–2280 cm21 (AA-d8) regions are shown in arbitrary units for LD enriched sites perinuclearly (upper panels) and at the cell extension (from a different cell; lower panels). B. To determine metabolic activity of HSC LDs, freshly isolated HSCs were cultured for 4 days and subsequently incubated for 5 h with 25 mM Bodipy C-12. After fixation cells were analyzed by fluorescence microscopy. doi:10.1371/journal.pone.0034945.g004 HSC by HPLC-MS. We observed that 15–20% of the neutral lipids consisted of RE in quiescent HSCs (Fig. 5A). The main RE species was retinyl palmitate (6868%), followed by retinyl stearate (1764%). The remainder being retinyl linoleate and retinyl oleate (763% and 963%, respectively; n = 3). In accordance with the Raman experiments, a strong decline in the RE amount (mainly retinyl palmitate) was seen on day 4 after plating, and this decline continued upon further activation (Fig. 5B). However, activated cells had a somewhat increased level of TAG and a 2–3 fold higher level of cholesterol esters (Fig. 5B). Surprisingly, in activated HSCs TAG species with relatively high m/z values between 900–1050 were much more enriched, than species with lower m/z values like TAG (18:2,18:2,18:2), (Figs. 5B, 6A and 6B). Further analysis by MS/MS of the TAG species that were elevated in activated HSCs showed that most of the increased high m/z species had one or more long chain polyunsaturated fatty acids (PUFA) incorporated i.e. fatty acids of 20 and 22 carbon chain length and 4 or more double bonds. The most predominant PUFA was docosapentae- noic acid (22:5). Quantification of the TAG species containing two or three PUFAs (TAG-PUFAs) revealed that they were hardly detectable in quiescent HSC (Fig. 6C), but increased more than 10-fold upon activation. Especially TAG species with three PUFA moieties were specific for activated HSC at day 7 (Fig. 6C). After prolonged culture for one month, no TAG-PUFAs were present any more (data not shown). HPLC-MS analysis of the phospholipid fractions of HSCs at day 0, 4 and 7 revealed no increase in PUFA species in phosphatidylcholine (PC), the most abundant phospholipid, upon activation (Fig. 6B). For a detailed PC-PUFA analysis see Fig. S3. Similar results were observed for phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylserine (PS) species (not PLoS ONE | www.plosone.org 5 April 2012 | Volume 7 | Issue 4 | e34945