Fatty Acid-Specific Fluorescent Probes and Their Use in Resolving Mixtures of Unbound Free Fatty Acids in Equilibrium with Albumin

Abstract

We report the first measurements to profile mixtures of unbound free fatty acids. Measurements utilized fluorescent probes with distinctly different response profiles for different free fatty acids (FFA). These probes were constructed by labeling site-specific mutants of the rat intestinal fatty acid binding protein (rI-FABP) with acrylodan. The probes were produced and screened by high throughput methods and from more than 30,000 such probes we selected 6 that together have sufficient specificity and sensitivity to resolve the profile of unbound FFA (FFA u ) in mixtures of different FFA u . We developed analytical methods to determine the FFA u profile from the fluorescence (ratio) response of the different probes and used these methods to determine FFA u profiles for mixtures of arachidonate, linoleate, oleate, palmitate and stearate in equilibrium with bovine serum albumin (BSA). Measurements were performed using mixtures with a range of total FFA u concentrations, including 0.9 nM, which is similar to normal plasma levels. We also measured single FFA binding isotherms for BSA and found that binding was well described by 6-7 sites with the same binding constants (K d ). The K d values for the FFA (4 to 38 nM) were inversely related to the aqueous solubility of the FFA. We constructed a model with these parameters to predict the FFA u profile in equilibrium with BSA and found excellent agreement between the profiles measured using the FFA probes and those calculated with this model. These results should lead to a better understanding of albumin's role in buffering FFA u and to profiling FFA u in intra and extracellular biological fluids. Keywordsunbound free fatty acids; fatty acid binding proteins; fluorescent probes; site-specific mutants; high throughput screening; fatty acid profiling; binding affinities; bovine serum albumin Metabolomics is advancing rapidly as a result of new technologies and the expanding interest in systems biology (1). This advance is being driven by the recognition that physiologic phenotype is essentially a reflection of the metabolic profile, and therefore, metabolic profiling should provide an accurate representation of the states of health and disease. The activity of a given metabolite is frequently dictated by its solubility as a "free" or unbound molecule in aqueous bodily fluids. For many metabolites the unbound concentration represents a small fraction of the total, with most of the total metabolite bound in carrier complexes. Total metabolite concentrations are typically measured, but it is the unbound metabolite that interacts

Is Membrane Transport of FFA Mediated by Lipid, Protein, or Both?

“…Fourth, FFA transport properties of the adipocyte plasma membrane are distinct from lipid vesicles. In addition to the characteristics described above, which do not occur in lipid vesicles, adipocyte rate constants are ~100-fold slower than in lipid vesicles (21). Last, we observed no effect of reagents that have been shown to modulate uptake either at the metabolic level (insulin, triacsin C) or through inhibition (phloretin, DIDS, and proteases), which suggests that the effect of these reagents on measurements of uptake are at a different step than is revealed by measurements of [FFA i ].…”

Fluorescence Sensor for the Quantification of Unbound Bilirubin Concentrations

“…These results indicate that BL22P1B11-Rh is relatively insensitive to the other metabolites commonly found in adult blood. For example, the B f probes are not sensitive to FFA u (see online Supplemental Data and Supplemental Table 2), whose concentrations are normally about 1 nmol/L but in pathologic conditions can increase greatly in adults and neonates (16,(22)(23)(24). BL22P1B11-Rh is also largely insensitive to ibuprofen and indomethacin, drugs commonly used to treat ne- onates (see online Supplemental Data and Supplemental Table 3).…”

“…FABPs are small, 15-kDa water-soluble proteins that bind longchain free fatty acids (FFAs) with high affinity and are found in the cytosol of most cells (15 ). The method used to generate, purify, and characterize the B f probes is essentially that described previously for unbound FFA (FFA u ) probes (16 ) and is described in detail in Supplemental Data, which accompanies the online version of this article at http://www.clinchem.org/content/vol58/issue5.…”

“…orescence measurements using a FFA u meter (16 ) modified for bilirubin by replacing the emission filters with ones centered at 525 and 580 nm (B f meter). Measurements were at 22°C, with excitation at 375 nm, and emission intensities were measured at 525 and 575 nm [I(525) and I(575), respectively] for the SpexFluoroLog or at 525 and 580 nm for the B f meter.…”

Unbound Free Fatty Acids from Preterm Infants Treated with Intralipid Decouples Unbound from Total Bilirubin Potentially Making Phototherapy Ineffective

“…FFA u and B f were measured using fluorescently labeled mutants of fatty acid binding proteins (probes) [6,7,8]. The B f probe binds unconjugated bilirubin with high affinity (K d = 16 n M ), but is poorly sensitive to FFA (K d >3,000 n M ), conjugated bilirubin (K d >300 n M ), bilirubin photoisomers, bilirubin oxidation products, ibuprofen, and indomethacin [6].…”