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[Letter to the Editor] In vitro use of free fatty acids bound to albumin: A comparison of protocols
Long-chain free fatty acids (FFAs) are important metabolic substrates for
energy production and lipid
synthesis that are also involved in signaling processes (1,2). Two of the most common fatty acids in humans are the long-chain saturated FFA palmitate (C16:0) and the monounsat- urated oleate (C18:1). Western diets rich in fatty acids are associated with increased levels of plasma cholesterol, hepatic steatosis, and a greater risk of cardiovascular disease (3,4). High levels of circulating FFAs, in particular
of saturated FFAs, are implicated in insulin resistance and pancreatic -cell dysfunction, and are predictive of diabetes development (5–8). In vitro exposure to high levels of FFAs leads to lipotoxicity, causing cellular dysfunction and death (5,9). The low solubility of
long-chain
FFAs in aqueous solutions represents one of the major limitations for in vitro and in vivo studies. To overcome this problem, FFAs can be conjugated to albumin, allowing the preparation of solutions in the physiological concen-
tration range. The fraction of unbound FFAs accessible for cellular uptake depends on the ratio of total FFAs to albumin (10). Thus, the biological effect of FFAs can be augmented by increasing the FFA concentration or by decreasing the bovine serum albumin (BSA) concentration. The unbound FFA concentration is also determined by the relative affinities of the FFAs for albumin (10). For example, when using a total FFA concentration of 0.5 mM in the presence of 1% w/v (151 µM) BSA, corresponding to an FFA/albumin molar
Figure 1. Cell death induced by different free fatty acid (FFA)/albumin binding protocols. (A) INS-1E cells were cultured 16 h in the absence (CTL) or presence of 0.5 mM palmitate (PAL) in medium containing 1% charcoal-absorbed BSA, FFA-free BSA (1, 0.85, 0.8, 0.75%) or FFA-free BSA (0.75, 0.7, 0.67%) precomplexed to palmitate (n = 2–20). (B) INS-1E cells were cultured 16 h in the absence (CTL) or presence of 0.5 mM oleate (OLE) in medium containing 1% char- coal-absorbed BSA, 0.75% FFA-free BSA or 0.67% FFA-free BSA precomplexed to oleate (n = 7–14). (C) Human islets were cultured 72 h in the absence (CTL) or presence of 0.5 mM palmitate (PAL) in medium contain- ing 1% charcoal-absorbed BSA or FFA-free BSA (0.75, 0.7, 0.67%) precomplexed to palmitate (n = 4–11). (D) Human islets were cultured 72 h in the absence (CTL) or pres- ence of 0.5 mM oleate (OLE) in 1% char- coal-absorbed BSA or 0.67% FFA-free BSA precomplexed to oleate (n = 5–11). Results represent mean ± SEM; *P < 0.05, **P < 0.01, and ***P < 0.001 compared to CTL.
Vol. 58 | No. 5 | 2015 228
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