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Unbound free fatty acid profiles in human plasma and the unexpected absence of unbound palmitoleate

2017; Elsevier BV; Volume: 58; Issue: 3 Linguagem: Inglês

10.1194/jlr.m074260

1539-7262

Andrew H. Huber, Alan M. Kleinfeld,

Lipid metabolism and biosynthesis

We determined for the first time the profiles of the nine most abundant unbound FFAs (FFAus) in human plasma. Profiles were determined for a standard reference plasma of pooled healthy adults for which the Lipid MAPSMAPS Consortium had determined the total FFA profiles. Measurements were performed by using 20 different acrylodan-labeled fatty acid binding protein mutants (probes), which have complementary specificities for the nine FFAs that comprise more than 96% of long-chain plasma FFA. The acrylodan fluorescence emission for each probe changes upon binding a FFAu. The plasma concentrations of each of the nine FFAus were determined by combining the measured fluorescence ratios of the 20 probes. The total molar FFAu concentration accounted for <10−5 of the total FFA concentration, and the mole fractions of the FFAu profiles were substantially different than the total FFA profiles. Myristic acid, for example, comprises 22% of the unbound versus 2.8% of the total. The most surprising difference is our finding of zero unbound cis-9-palmitoleic acid (POA), whereas the total POA was 7.2%. An unidentified plasma component appears to specifically prevent the release of POA. FFAus are the physiologically active FFAs, and plasma FFAu profiles may provide novel information about human health. We determined for the first time the profiles of the nine most abundant unbound FFAs (FFAus) in human plasma. Profiles were determined for a standard reference plasma of pooled healthy adults for which the Lipid MAPSMAPS Consortium had determined the total FFA profiles. Measurements were performed by using 20 different acrylodan-labeled fatty acid binding protein mutants (probes), which have complementary specificities for the nine FFAs that comprise more than 96% of long-chain plasma FFA. The acrylodan fluorescence emission for each probe changes upon binding a FFAu. The plasma concentrations of each of the nine FFAus were determined by combining the measured fluorescence ratios of the 20 probes. The total molar FFAu concentration accounted for 1 mM, and almost all FFAs are bound to serum albumin. The pool of circulating FFAs is composed of as many as 40 distinct molecular species of FFAs. Approximately 30 of these are long-chain FFAs (14–26 carbons), of which 9 FFAs (Table 1) make up >96% of the total long-chain FFAs by molarity (3.Quehenberger O. Armando A.M. Brown A.H. Milne S.B. Myers D.S. Merrill A.H. Bandyopadhyay S. Jones K.N. Kelly S. Shaner R.L. et al.Lipidomics reveals a remarkable diversity of lipids in human plasma.J. Lipid Res. 2010; 51: 3299-3305Abstract Full Text Full Text PDF PubMed Scopus (912) Google Scholar). The distribution of the different FFAs (the FFA profile) is expected to reflect the physiologic state, and changes in the profiles have been correlated with disease (4.Yli-Jama P. Meyer H.E. Ringstad J. Pedersen J.I. Serum free fatty acid pattern and risk of myocardial infarction: a case- control study.J. Intern. Med. 2002; 251: 19-28Crossref PubMed Scopus (75) Google Scholar, 5.Villa P.M. Laivuori H. Kajantie E. Kaaja R. Free fatty acid profiles in preeclampsia.Prostaglandins Leukot. Essent. Fatty Acids. 2009; 81: 17-21Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 6.Lv W. Yang T. Identification of possible biomarkers for breast cancer from free fatty acid profiles determined by GC-MS and multivariate statistical analysis.Clin. Biochem. 2012; 45: 127-133Crossref PubMed Scopus (67) Google Scholar, 7.Tomita K. Teratani T. Yokoyama H. Suzuki T. Irie R. Ebinuma H. Saito H. Hokari R. Miura S. Hibi T. Plasma free myristic acid proportion is a predictor of nonalcoholic steatohepatitis.Dig. Dis. Sci. 2011; 56: 3045-3052Crossref PubMed Scopus (29) Google Scholar, 8.Hagenfeldt L. Wahren J. Pernow B. Raf L. Uptake of individual free fatty acids by skeletal muscle and liver in man.J. Clin. Invest. 1972; 51: 2324-2330Crossref PubMed Scopus (127) Google Scholar).TABLE 1The 9 FFAs whose unbound plasma profiles were determined in this studyCommon NameAbbreviationStructuresaFatty acid chain length and double bond number.MyristicMA14:0PalmiticPA16:0cis-9-palmitoleicPOA16:1StearicSA18:0OleicOA18:1LinoleicLA18:2α-LinolenicLNA18:3 (ω-3)ArachidonicAA20:4Eicoaspentaenoic 5,8,11,14,17EPA20:5Docosahexaenoic 4,7,10,13,16,19DHA22:6These nine FFAs comprise 96% of the total FFA by molarity as determined by the Lipid MAPS Consortium (3.Quehenberger O. Armando A.M. Brown A.H. Milne S.B. Myers D.S. Merrill A.H. Bandyopadhyay S. Jones K.N. Kelly S. Shaner R.L. et al.Lipidomics reveals a remarkable diversity of lipids in human plasma.J. Lipid Res. 2010; 51: 3299-3305Abstract Full Text Full Text PDF PubMed Scopus (912) Google Scholar).a Fatty acid chain length and double bond number. Open table in a new tab These nine FFAs comprise 96% of the total FFA by molarity as determined by the Lipid MAPS Consortium (3.Quehenberger O. Armando A.M. Brown A.H. Milne S.B. Myers D.S. Merrill A.H. Bandyopadhyay S. Jones K.N. Kelly S. Shaner R.L. et al.Lipidomics reveals a remarkable diversity of lipids in human plasma.J. Lipid Res. 2010; 51: 3299-3305Abstract Full Text Full Text PDF PubMed Scopus (912) Google Scholar). Because most FFAs are bound to albumin, the total plasma FFA concentrations do not directly reflect the physiologically active FFA concentrations. The albumin-bound FFAs cannot enter cells, bind to proteins, or serve as substrates for FFA-utilizing enzymes. Only FFAs that are soluble monomers in the aqueous phase can be transported across membranes, bind to specific sites on proteins, and function in enzymatic reactions. The soluble FFAs (unbound FFA; FFAu) are a tiny fraction of the total FFAs (bound and unbound). In humans, the molar FFAu concentration is typically −Rj1QjKdj>)(Eq. 6) where for each probe j, the brackets represent αi weighted averages. The FFAuT values were first calculated for each probe by using the αi obtained from the initial FFAu profile. The average FFAuT value and SD of all probes was determined, and probes whose FFAuT deviated by ≥2 SDs were identified. In most cases, the deviation could be corrected, or if saturation occurred, a new profile was determined with the outlier probe removed. We created an "artificial NIST" plasma by combining FFA-HSA complexes of the nine FFAs in Table 1 using the ratios of the individual total FFA concentrations determined for the NIST 1950 SRM plasma by the Lipid MAPS Consortium (3.Quehenberger O. Armando A.M. Brown A.H. Milne S.B. Myers D.S. Merrill A.H. Bandyopadhyay S. Jones K.N. Kelly S. Shaner R.L. et al.Lipidomics reveals a remarkable diversity of lipids in human plasma.J. Lipid Res. 2010; 51: 3299-3305Abstract Full Text Full Text PDF PubMed Scopus (912) Google Scholar). FFA profile measurements were performed by using artificial NIST diluted 50-fold in the buffer. The ability to determine FFAu profiles accurately from the measured R values depends on the uncertainties in the measured R values. To estimate what level of uncertainty will allow an accurate determination of the FFAu profiles, we used the mole fractions, the set of αi, determined from the measured NIST FFAu. These αi were used to generate R values for each probe by solving equation 6 for R j, whereRj=FFAuTRmjQjKdj>+Ro1+FFAuTQjKdj(Eq. 7) Starting sets of ideal R values (no errors) for 20 probes were calculated for each of three total FFAu concentrations: 0.5, 1.5, and 5.0 nM, a range that is typical for different donors. For each total FFAu concentration, a random, maximum error of 1% and 2%, was applied to 20 copies of the ideal 20-probe set of R values for each FFAu total. Twenty FFAu profiles at each total FFAu concentration were then determined from each nonideal set of R values by using MLAB. The CV for each FFAu in each FFAu profile was calculated from the set of the 20 "replicate" FFAu profiles (supplemental Table S4). The HSA equilibrium dissociation constants (Kd), at 22°C, for the nine FFAus were determined from binding isotherms measured, as described previously for BSA (10.Huber A.H. Kampf J.P. Kwan T. Zhu B. Kleinfeld A.M. Fatty acid-specific fluorescent probes and their use in resolving mixtures of different unbound free fatty acids in equilibrium with albumin.Biochemistry. 2006; 45: 14263-14274Crossref PubMed Scopus (35) Google Scholar). Measurements were done by titrating 600 µM fatty acid free HSA in HEPES buffer with the sodium salts of the fatty acids. The fatty acid salts were added slowly to reach concentrations from ∼90 to 3,300 µM (depending on the FFA) and incubated for 10 min, and the equilibrium FFAu concentrations were measured with ADIFAB2 (10.Huber A.H. Kampf J.P. Kwan T. Zhu B. Kleinfeld A.M. Fatty acid-specific fluorescent probes and their use in resolving mixtures of different unbound free fatty acids in equilibrium with albumin.Biochemistry. 2006; 45: 14263-14274Crossref PubMed Scopus (35) Google Scholar). Scatchard analysis revealed a single class of between five and eight binding sites and Kd values ranging from ∼5 to 50 nM, depending on the FFA (supplemental Table S5). Supplemental Table S5 also shows that results for BSA, from our earlier study (10.Huber A.H. Kampf J.P. Kwan T. Zhu B. Kleinfeld A.M. Fatty acid-specific fluorescent probes and their use in resolving mixtures of different unbound free fatty acids in equilibrium with albumin.Biochemistry. 2006; 45: 14263-14274Crossref PubMed Scopus (35) Google Scholar), are similar to those obtained in the present study for HSA. The protein components of the probes used to determine FFAu profiles are mutants of a His-tagged rI-FABP with a Glu-131 to Asp substitution and a COOH-terminal affinity tag comprising Arg-132, Gly-133, and six histidines (Fig. 1). Site selection for mutagenesis (10.Huber A.H. Kampf J.P. Kwan T. Zhu B. Kleinfeld A.M. Fatty acid-specific fluorescent probes and their use in resolving mixtures of different unbound free fatty acids in equilibrium with albumin.Biochemistry. 2006; 45: 14263-14274Crossref PubMed Scopus (35) Google Scholar) was guided by the X-ray crystallography and NMR structures for the wild-type protein (11.Sacchettini J.C. Gordon J.I. Banaszak L.J. The structure of crystalline Escherichia coli-derived rat intestinal fatty acid binding protein at 2.5 A resolution.J. Biol. Chem. 1988; 263: 5815-5819Abstract Full Text PDF PubMed Google Scholar, 12.Cistola D.P. Sacchettini J.C. Banaszak L.J. Walsh M.T. Gordon J.I. Fatty acid interactions with rat intestinal and liver fatty acid-binding proteins expressed in Escherichia coli.J. Biol. Chem. 1989; 264: 2700-2710Abstract Full Text PDF PubMed Google Scholar), as well as our previous studies of the thermodynamics of mutant FABPs (14.Richieri G.V. Low P.J. Ogata R.T. Kleinfeld A.M. Mutants of rat intestinal fatty acid binding protein illustrate the critical role played by enthalpy-entropy compensation in ligand binding.J. Biol. Chem. 1997; 272: 16737-16740Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 15.Richieri G.V. Low P.J. Ogata R.T. Kleinfeld A.M. Thermodynamics of fatty acid binding to engineered mutants of the adipocyte and intestinal fatty acid binding proteins.J. Biol. Chem. 1998; 273: 7397-7405Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). The FFAu probes consisted of between 3 and 20 substitutional mutations of the wild-type protein, modified by the His tag addition (Fig. 1). Most of the mutations were within the FFA binding pocket of the wild-type protein, and in some cases a cysteine was introduced at positions on or near the protein's α-helical region (Table 2). In the sequences presented in Table 2 and Fig. 1, amino acids are numbered from the first residue of the mature protein. The initiator methionine residue of the wild-type protein was removed by aminopeptidase activity, leaving an alanine as residue 1. Thus, counting backward and skipping zero, the initiator methionine is at residue position −1. Seventeen of the probes were labeled with acrylodan on a cysteine at positions 26, 27, or 30 (Table 2). The remaining three probes were labeled at lysine at position 27. The response profiles were calculated by using equation 3, a FFAu concentration of 1 nM for each of the nine FFAus, and the probe calibrations in supplemental Table S1. For each probe, the largest responses occurred for those FFAus for which the probe is most sensitive (supplemental Table S3). The 20 probes have complementary specificities for all nine FFAus, but no probe has absolute specificity for any single FFAu. Several probes have high degrees of specificity