If you don't remember your password, you can reset it by entering your email address and clicking the Reset Password button. You will then receive an email that contains a secure link for resetting your password
If the address matches a valid account an email will be sent to __email__ with instructions for resetting your password
A molecular mechanism underlying the albumin-cobalt binding assay.
•
Free fatty acid binding interferes with Co(II) binding to albumin.
•
Ischemia-modified albumin corresponds to albumin with increased FFA bound.
•
Increased FFA levels are sufficient to explain high ACB readings.
•
Clinical data are consistent with FFA as the trigger for high ACB/high IMA levels.
Abstract
Myocardial ischemia is difficult to diagnose effectively with still few well-defined biochemical markers for identification in advance, or in the absence of myocardial necrosis. “Ischemia-modified albumin” (IMA), a form of albumin displaying reduced cobalt-binding affinity, is significantly elevated in ischemic patients, and the albumin cobalt-binding (ACB) assay can measure its level indirectly. Elucidating the molecular mechanism underlying the identity of IMA and the ACB assay hinges on understanding metal-binding properties of albumin. Albumin binds most metal ions and harbours four primary metal binding sites: site A, site B, the N-terminal site (NTS), and the free thiol at Cys34. Previous efforts to clarify the identity of IMA and the causes for its reduced cobalt-binding capacity were focused on the NTS site, but the degree of N-terminal modification could not be correlated to the presence of ischemia. More recent work suggested that Co2+ ions as used in the ACB assay bind preferentially to site B, then to site A, and finally to the NTS. This insight paved the way for a new consistent molecular basis of the ACB assay: albumin is also the main plasma carrier for free fatty acids (FFAs), and binding of a fatty acid to the high-affinity site FA2 results in conformational changes in albumin which prevent metal binding at site A and partially at site B. Thus, this review advances the hypothesis that high IMA levels in myocardial ischemia and many other conditions originate from high plasma FFA levels hampering the binding of Co2+ to sites A and/or B. This is supported by biophysical studies and the co-association of a range of pathological conditions with positive ACB assays and high plasma FFA levels.
Myocardial ischemia occurs due to restricted blood supply to the muscular tissue of the heart (myocardium) resulting in insufficient oxygen supply. The main cause of this can be the partial or complete blockage of a coronary artery, and a critical depletion of myocardial oxygen leads to cell death, or infarction. Diagnosis of myocardial ischemia typically includes exercise-electrocardiography stress tests, coronary angiography, and imaging stress-echo tests [
I. Committee on standardization of markers of cardiac damage of the, future biomarkers for detection of ischemia and risk stratification in acute coronary syndrome.
], there are still few well-defined biochemical markers for identification of myocardial ischemia in advance, or in the absence of myocardial necrosis. One of these biomarkers is based on albumin, the most abundant protein in blood plasma. So-called “ischemia-modified albumin” (IMA) is found to be significantly elevated in ischemic patients [
I. Committee on standardization of markers of cardiac damage of the, future biomarkers for detection of ischemia and risk stratification in acute coronary syndrome.
]. IMA is solely characterised by its reduced cobalt-binding affinity, which can be measured indirectly by the Food and Drug Administration-approved albumin cobalt-binding (ACB) assay [
In the commercially available ACB test, cobalt(II) chloride (approximately 1.5 mol equivalents per albumin molecule) is added to a serum sample, to allow albumin-cobalt binding. Dithiothreitol (DTT), a metal chelator that forms a coloured complex with Co2+, is then added. The resulting ill-defined brown DTT-Co2+ product is measured by absorption spectrophotometry at 470 nm and compared to a serum-cobalt blank without DTT present. The reduced cobalt-binding capacity of IMA leaves more unbound Co2+ to complex with DTT, resulting in higher absorbance readings [
]. The ACB test has an excellent negative predictive value, i.e. low IMA readings correspond well to the absence of myocardial ischemia. However, a severe shortcoming is the high incidence of false positives, i.e. high readings in the absence of ischemia.
], efforts to elucidate the molecular causes of reduced cobalt binding concentrated on this site. It was hypothesized that ischemia causes the N-terminal end of the albumin protein to undergo structural modifications, hence that IMA corresponded to N-terminally modified albumin [
]; more recently, no correlation was found between the ACB assay and an enzyme-linked immunosorbent assay that specifically detects N-terminal modification of albumin in patients with either acute coronary syndrome or non-ischemic chest pain [
Insignificant role of the N-terminal cobalt-binding site of albumin in the assessment of acute coronary syndrome: discrepancy between the albumin cobalt-binding assay and N-terminal-targeted immunoassay.
]. Similarly, patients suffering from acute-on-chronic liver failure have significantly elevated ACB assay readings, but the same proportion of N-terminally modified albumin as healthy individuals [
]. In the light of such findings, low plasma pH as a result of acidosis, and altered plasma cysteine/cystine ratio as a consequence of hypoxia or oxidative stress have also been suspected as molecular causes of reduced cobalt binding [
]. Indeed, a positive correlation has been identified between the highly elevated serum levels of free fatty acids (FFAs) in patients with acute ischemic myocardia and high levels of IMA [
Utility of serum Fatty Acid concentrations as a marker for acute myocardial infarction and their potential role in the formation of ischemia-modified albumin: a pilot study.
], we have demonstrated that the conformational changes that FFA-binding to albumin elicits in the protein is sufficient to cause reduced cobalt binding capacity [
]. This review will present essential background information on metal ion-albumin interactions and discuss the molecular basis of FFA-mediated inhibition of metal (in particular Co2+) binding. It will also provide a clinical perspective to highlight how conclusions from biochemical/bioinorganic investigations are reflected in patient data.
2. Albumin – a carrier of essential and xenobiotic metal ions in plasma
Albumin is a ∼66 kDa protein containing 585 amino acids, contributing to around 50% of the total protein concentration in blood plasma, and up to 75% of the colloidal activity [
]. Importantly, albumin also serves as an important carrier of inorganic ions, including those required for regular physiological function (Ca2+, Cu2+, Zn2+) [
Nickel(II) transport in human blood serum. Studies of nickel(II) binding to human albumin and to native-sequence peptide, and ternary-complex formation with L-histidine.
]. Before considering cobalt binding in depth, we will briefly summarise the interactions of albumin with other d-block metal ions, with the exception of Cr3+, Fe3+, and Mn2+, which are preferentially transported by transferrin, another important metal ion transporter in blood plasma. Whilst Fe3+ can, in principle, also bind to albumin, this only occurs in cases of severe iron overload [
]. Metal binding to such sites can be studied using a variety of techniques. Stability constants for the binding of d-block metals, including Zn2+, Cu2+, Ni2+ and Cd2+, were originally derived from equilibrium dialysis experiments [
The interaction of zinc, nickel and cadmium with serum albumin and histidine-rich glycoprotein assessed by equilibrium dialysis and immunoadsorbent chromatography.
Equilibrium dialysis of metal-serum albumin. I. Successive stability constants of Zn(II)-serum albumin and the Zn2+-induced cross-linking self-association.
] and need to be complemented by techniques that address structural features. For true transition metal ions such as Cu2+ and Co2+, electronic spectroscopic methods such as circular dichroism allow metal binding to albumin to be studied via transfer of chirality from metal-binding amino acid residues to the d-d/charge-transfer bands of complexed metal ions, providing insight into the geometry of metal-protein interactions [
]. The same ions have unpaired electrons, and can also be investigated using electron paramagnetic resonance (EPR) spectroscopy, which provides insight into the chemical environment surrounding the metal ion [
]. To obtain structural information on the binding of diamagnetic d10 ions, such as Zn2+ and Cd2+, that are largely silent in the aforementioned spectroscopies, nuclear magnetic resonance (NMR) methods have been employed, making use of either partially-characterised 1H-resonances of metal-binding residues, or NMR-active nuclei such as the 111Cd or 113Cd isotopes of cadmium [
]. Further information on the coordination mode, geometry and identification of likely donor ligands has been gained using extended X-ray absorption fine structure spectroscopy (EXAFS) [
. Site A, the multi-metal binding site (MBS) (blue); NTS/ATCUN motif (green); Cys34 (red). The precise location of site B is not yet known. The boxed labels indicate the six sub-domains of albumin. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
One of the first metal binding sites to be identified on albumin was the N-terminal binding site (NTS), which arises from the first triplet amino acid motif of human albumin: Asp1–Ala2–His3 (Figs. 1 and 2) [
]. It involves the N-terminal amino group, the N(delta) of His3, and two deprotonated backbone amide nitrogen atoms. This square planar configuration of N-donor atoms (Fig. 2) is particularly suitable for Cu2+ and Ni2+, which has led to the NTS being referred to by the acronym ‘ATCUN’, for the Amino Terminal Cu(II) and Ni(II) binding motif [
]. The ATCUN motif is present in the majority of albumins from different mammalian species, though porcine and canine albumins are notable exceptions, as they lack His3 [
]. The NTS motif is thought to have high conformational flexibility in the absence of bound metal, reflected in the crystal structures of albumin, all of which lack defined structures of the first few N-terminal residues [
]. Interestingly, the N-terminal X-X-His motif is not unique to albumin – many other proteins, such as the peptide hormone Hepcidin, can also bind Ni2+ and Cu2+ ions via an ATCUN motif [
Fig. 2Contrasting geometries of metal binding sites on albumin. Left: square planar coordination of Cu2+ or Ni2+ at the NTS site; the structure shown is derived from molecular modelling. The N-terminal amino group, two deprotonated backbone amide N atoms and the N(delta) of the imidazole ring of His3 form a square plane around the central metal ion. Right: tetrahedral coordination of Zn2+ at site A in human serum albumin (pdb 5ijf). His67 uses its N(epsilon) N atom, whilst His247 binds via N(delta). Asp249 binds in mono-dentate fashion, with the second carboxylate O at ca. 2.6 Å distance, too long for a metal-ligand bond. Typically for zinc sites in proteins, angles between ligands deviate substantially from the ideal tetrahedral angle (109.5°) and vary between 95° and 125°. Metal ions are rendered in gold, N atoms in blue, O atoms in red, carbon atoms in grey. No H atoms are shown.
Cu2+ binds preferentially to the NTS in albumin, occupying approximately 1–2% of the available NTS – equating to around 15% of total copper in blood plasma [
]. Owing to the d9 electronic configuration of Cu2+, preference to form square planar complexes, and high stability in the Irving-Williams series, Cu2+ is coordinated at the NTS with 1 pM affinity [
Nickel(II) transport in human blood serum. Studies of nickel(II) binding to human albumin and to native-sequence peptide, and ternary-complex formation with L-histidine.
] for the d10 divalent cations Zn2+ and Cd2+, site A can also bind Cu2+, Ni2+ and Co2+ – hence it is also referred to as the ‘multi-metal’ binding site [
], and so distorted trigonal bipyramidal coordination of Zn2+ was proposed, with water (or chloride) as the fifth ligand completing the inner coordination sphere. However, the recent X-ray crystallographic structures of human and equine albumins discounted participation of Asn99 and showed site A to be essentially tetrahedral (Fig. 2), with the fourth ligand being a water molecule [
] (HSAB principle) provide a good coordination environment for metal ions with a small ionic radius and moderate charge (e.g. 2+ cations). Notably though, the affinity of site A for Cu2+ is 4 orders of magnitude lower than that of the NTS [
], thus site A only becomes populated by Cu2+ when more than one molar equivalent of Cu2+ is present. Finally, the comparison of apo- and Zn-bound crystal structures of albumin has revealed high structural similarity at site A. Thus, in marked contrast to the flexible NTS, site A is essentially ‘pre-formed’ for metal binding [
]. It is important to note that site A is an inter-domain site, with His67 from domain I, and His247 and Asp249 from domain II.
Site B
The other Cd2+ binding site (site B), which is distinct from site A and the NTS and readily identifiable using 111Cd or 113Cd NMR (Fig. 3a), appears to bind Cd2+ with similar affinity to the multi-metal binding site A [
]. In contrast, site B's affinity for zinc is markedly less than that of site A. Based on NMR data, it is likely that only one nitrogen donor ligand is involved at site B, suggesting this site to be harder (HSAB principle) than site A. The location of site B has remained elusive, but site-directed mutagenesis of His39Leu excluded His39 from involvement in either site A or B [
Fig. 3Co2+ competes with both Cd2+ and Zn2+ for albumin binding under physiological conditions (pH 7.4, 50 mM Tris-Cl, 50 mM NaCl) but not with Cu2+. (a) 111 Cd NMR spectra of Cd2BSA (1.5 mM) with increasing addition of Co2+. 111-Cd resonances corresponding to both site A and B (∼140 ppm and 35 ppm, respectively) are affected by Co2+. (b) Isothermal calorimetry experiments performed in the presence of 1 mol. equiv. of Cu2+ (●) or Zn2+ () demonstrate that addition of Zn2+ decreases albumin's affinity and capacity for Co2+-binding, while addition of Cu2+ has no significant effect.
Albumin contains 17 disulfide bonds, which contribute to the structural stability of the protein. One free thiol residue (Cys34) is located between helices 2 and 3 of subdomain IA (Fig. 1) [
]. Cys34 is not involved in any intramolecular bridging, however it often forms intermolecular disulfides with small sulfur-containing molecules such as cysteine and glutathione [
]. The restricted location of Cys34 in a crevice of albumin helps to improve its specificity for binding metal ions that favour linear coordination, including Hg2+, Au+, Ag+ and Pt2+[
Albumin is an important transporter of Ca2+ in blood plasma. Many reports suggest that this occurs in a non-specific fashion, involving various carboxylate side chains on the surface of albumin [
]. It may be significant that one of the Ca2+ sites detected by crystallography involves the key site A ligand, Asp248 (corresponding to Asp249 in human albumin) and indeed, Ca2+ ions were found to interfere with the 113Cd signals for both sites A and B of human albumin [
]. However, the affinity of albumin for Ca2+ binding is relatively weak (Kd of 0.67 mM), with only around 45% of the 2.4 mM of circulating Ca2+ bound to albumin [
]. While it is widely assumed that its binding resembles that of Ni2+ and Cu2+ (d8 and d9 metal ions, respectively, with preference for the formation of square planar/tetragonal complexes), Co2+ (d7) behaves in fact more like Zn2+ (d10), has a similar ionic radius (0.58 vs. 0.60 Å for Zn2+ [
Evidence that the principal CoII-binding site in human serum albumin is not at the N-terminus: implication on the albumin cobalt binding test for detecting myocardial ischemia.
] have since rejected this claim. Competition with Cd2+ and Cu2+ monitored by electronic absorption spectroscopy strongly suggested that sites A and B are the preferred Co2+ binding sites [
Insignificant role of the N-terminal cobalt-binding site of albumin in the assessment of acute coronary syndrome: discrepancy between the albumin cobalt-binding assay and N-terminal-targeted immunoassay.
Evidence that the principal CoII-binding site in human serum albumin is not at the N-terminus: implication on the albumin cobalt binding test for detecting myocardial ischemia.
]. Co2+ binding to sites A and B was also confirmed by 111Cd NMR spectroscopy for bovine albumin (Fig. 3a), and competition with Zn2+ was evident from ITC (Fig. 3b) [
Evidence that the principal CoII-binding site in human serum albumin is not at the N-terminus: implication on the albumin cobalt binding test for detecting myocardial ischemia.
]. It is important to note however, that even though Co2+ and Zn2+may be regarded as metal ions with similar properties, the apparent binding constant for Co2+ binding to its strongest site on bovine albumin (log Kapp = 4.6 ± 0.3 × 104 M−1; Fig. 5b; [
]. Most importantly, this weaker than anticipated binding of Co2+ to the NTS means that the initially proposed molecular basis of the ACB assay to assess the likelihood of myocardial infarction required revision, since the original studies assumed that Co2+ binds exclusively to the NTS [
]. FFAs are the main source of energy for heart and skeletal muscle. Disturbances of the levels and/or distribution of fatty acids in the body have been linked to a spectrum of pathological disorders, including diabetes, cardiovascular and neurological diseases, and cancer [
]. Owing to the abundance of albumin in plasma, and the importance of fatty acids in metabolism and disease progression, binding of FFAs to albumin has been studied intensively in the past four decades [
Up to seven medium-to-long chain (C10-C18) fatty acid binding sites (FA1-7) have been identified on albumin, spread over the three domains (see Table 1 and Fig. 4) [
]. The binding affinities depend on both the site and the FFA chain length. Four additional binding locations have been described for short-to-medium chain fatty acids [
], however for the purposes of this review we will focus on FA1-7 (Fig. 4). In a normal physiological state, albumin circulates with between 0.1–2 equivalents of FFAs bound, however it pathologically can bind in excess of 6 equivalents [
]. The seven identified binding sites can be broadly split into two categories: the high-affinity sites (FA2, FA4 and FA5) and the low-affinity sites (FA1, FA3, FA6 and FA7) [
]. The high-affinity site FA2 is close to metal-binding site A and therefore of particular interest. This relatively hydrophobic site is, like metal site A, an inter-domain site and is located between sub-domains IA and IIA (Fig. 4) [
]. Compared to FFA-free albumin, accommodation of a fatty acid molecule in site FA2 requires a change in the mutual arrangement of domains I and II. While short-chain FFAs (<C8) were originally thought to be too short to successfully dock in the FA2 site [
], more recent 1H and 111Cd NMR studies indicated that octanoate can bind to this site. Molecular modelling suggested that the half-pocket in domain II is sufficient to accommodate octanoate, and therefore does not require the domain-domain movement [
Table 1The location and characteristics of fatty acid binding sites FA1-7 of albumin. Particular attention is drawn to binding site FA2, since occupation of this site by FFAs causes an allosteric switch in metal binding at site A, owing to its close proximity; both are located between subdomains IA and IIA.
. Site A and FA2 are both located between subdomains IA-IIA. The inter-domain nature and the proximity of FA2 to site A allows for the allosteric switching of metal ion binding
], this is not the case when FA2 is occupied by a longer chain FFA (e.g. myristic acid, C14), as the distance between the metal-coordinating residues is too large after the conformational change [
]. This crucial discovery suggested that FFA and zinc concentration(s) in blood plasma may be correlated through an allosteric mechanism based on albumin [
]. Competition experiments monitored by ITC demonstrated that the zinc-binding capacity of both bovine and human albumin for site A was dramatically reduced [
]. Five equivalents of myristate were sufficient to completely inhibit Zn2+coordination to site A in bovine albumin (Fig. 5a), with site B also affected more or less severely [
]. Indeed, the data in Fig. 5a indicate that the largest effect is seen between 0 and 2 molar equivalents. The downstream implications of this allosteric switch for the fate of plasma zinc are discussed elsewhere [
Fig. 5Isothermal titration calorimetry experiments demonstrate the mutual modulation of metal and fatty acid binding to bovine albumin. The presence of the C14:0 fatty acid myristate (○, 0 mol. equiv.; ●, 1 mol. equiv.; ▽, 3 mol. equiv.; and ★ 5 mol. equiv.) affects the binding capacity of albumin for Zn2+ (a) and Co2+ (b) under near-physiological conditions (pH 7.4, 50 mM Tris-Cl, 50 mM NaCl). Co2+ binding to albumin is not only weaker than that of Zn2+, but the effect of FFAs on Zn2+ binding is also much more pronounced than that of Co2+. (c) The presence of 1 mol. equiv. of Zn2+ () or Co2+ (●) affects the energetics of fatty acid binding relative to the metal-free experiment (○), likely due to the need to remove the metal before the FFA can bind. Notably, again the effect for Zn2+ is larger than that for Co2+.
Crucially, although the binding preferences of Zn2+ and Co2+ are not identical, the presence of myristate also clearly reduced the binding capacity of bovine albumin for Co2+ (Fig. 5b) [
]. This is in agreement with the fact that Co2+ does not bind preferentially to site A, but site B (which in BSA is affected by FFA binding, but less severely) [
], and can also bind to the NTS motif which is not expected to be adversely affected by the presence of FFA. Similarly to Zn2+, it appears that the bound metal ion must first be removed from site A before fatty acid binding can occur at FA2, identified by a reduction in the exothermicity of the FFA-binding reaction (Fig. 5c) [
]. The number of apparent Co2+ binding sites in these experiments was in broad agreement with other experimental data, although we note that the selected experimental conditions did not allow to fully saturate all three binding sites. The apparent number of binding sites in the absence of myristate amounted to 2.4, and reduced to ca. 1.3 sites, implying that (at least) one binding site became non-functional (Fig. 6a). This is consistent with an inhibition of cobalt-binding to site A, as a result of FFA binding to the nearby FA2 site [
]. Most importantly, increasing the levels of FFA in a mock ACB assay is sufficient to lead to increased formation of the Co-DTT product, with concomitant higher absorbance readings (Fig. 6b). The magnitude of the changes in absorbance at 470 nm is broadly in line with effects seen in clinical studies [
]. We next explore whether this molecular mechanism may be reflected in clinical data.
Fig. 6Increasing FFA (myristate, C14:0) decreases the total Co2+ binding capacity of BSA, (a) reflected in the number of apparent binding sites of albumin for Co2+ (No data for 4 mol. eq. Myr). (b) In turn, this affects the formation of the Co-DTT complex as part of the ACB assay (b), used for the detection of myocardial ischemia.
As indicated previously, the diagnostic specificity of the ACB assay is very low, resulting in a high proportion of false positives, i.e. high readings despite the absence of ischemia [
Utility of serum Fatty Acid concentrations as a marker for acute myocardial infarction and their potential role in the formation of ischemia-modified albumin: a pilot study.
Ischemia-modified albumin levels in patients with end-stage renal disease patients on hemodialysis: does albumin analysis method affect albumin-adjusted ischemia-modified albumin levels?.
]. These conditions, therefore, should have a common feature that can explain elevated IMA levels, and we propose that this common feature is elevated plasma FFAs. The latter have been shown to independently influence the ACB assay to the same extent as ACS and other conditions [
Utility of serum Fatty Acid concentrations as a marker for acute myocardial infarction and their potential role in the formation of ischemia-modified albumin: a pilot study.
]. Together with the biochemical and biophysical studies detailed in Section 3, it is compelling to suggest that IMA corresponds to albumins in which FA2 is occupied. To further explore this hypothesis, Table 2 compiles selected conditions which are positive for the ACB assay and reports quantitative data on serum FFAs drawn from the literature. Cobalt binding to albumin is both specific and proportional to the total serum albumin concentration, and so many studies adjust for the total albumin level [
Table 2Selected conditions associated with a positive ischemia modified albumin (IMA) test and increased free fatty acids (FFAs) with the corresponding IMA and FFA levels.
Utility of serum Fatty Acid concentrations as a marker for acute myocardial infarction and their potential role in the formation of ischemia-modified albumin: a pilot study.
Utility of serum Fatty Acid concentrations as a marker for acute myocardial infarction and their potential role in the formation of ischemia-modified albumin: a pilot study.
Elevated serum and cerebrospinal fluid free fatty acid levels are associated with unfavorable functional outcome in subjects with acute ischemic stroke.
Hormone, metabolic peptide, and nutrient levels in the earliest phases of rheumatoid arthritis-contribution of free fatty acids to an increased cardiovascular risk during very early disease.
Serum fatty acid profiles and potential biomarkers of ankylosing spondylitis determined by gas chromatography-mass spectrometry and multivariate statistical analysis.
Ischemia-modified albumin levels in patients with end-stage renal disease patients on hemodialysis: does albumin analysis method affect albumin-adjusted ischemia-modified albumin levels?.
Evaluation of oxidative stress in women with polycystic ovarian syndrome as represented by serum ischemia modified albumin and its correlation with testosterone and insulin resistance.
Utility of serum Fatty Acid concentrations as a marker for acute myocardial infarction and their potential role in the formation of ischemia-modified albumin: a pilot study.
]. The pain and the stress associated with such syndromes is thought to trigger a sympathetic discharge, with the release of catecholamines which activate hormone-sensitive tissue lipase – the enzyme which hydrolyses triglycerides and hence liberates FFAs into the circulation [
]. This leads to elevated serum free fatty acid concentrations within 1–2 hours from the onset of ACS, and the degree of increase in FFA concentration has been positively associated with serious ventricular arrhythmias [
Utility of serum Fatty Acid concentrations as a marker for acute myocardial infarction and their potential role in the formation of ischemia-modified albumin: a pilot study.
]. In addition, the IMA levels detected via the ACB assay increase within minutes of the onset of ischemia, stay high for 6 to 12 hours before returning to normal level within 24 hours. This correlates to similar changes in FFA levels, which return to normal after 24 to 48 hours after myocardial ischemia [
], but is in contrast to explanations invoking N-terminally modified albumin, as albumin has a half-life of ca. 20 days and so IMA should be detected for several days following ischemia [
]. In fatty liver disease for example, there is insulin resistance which causes a withdrawal of the inhibition of dephosphorylation of hormone-sensitive lipase activity to reduce fat hydrolysis [
]. Similarly, chronic kidney disease is associated with raised FFA concentrations arising from TNF-α-induced adipose tissue lipolysis as a consequence of systemic inflammation [
]. It is therefore consistent with our hypothesis that those disease states are associated with positive ACB assays. Further conditions with positive ACB readings include diabetes [
Fasting serum triglycerides, free fatty acids, and malondialdehyde are increased in preeclampsia, are positively correlated, and decrease within 48 hours post partum.
Evaluation of oxidative stress in women with polycystic ovarian syndrome as represented by serum ischemia modified albumin and its correlation with testosterone and insulin resistance.
]) no variation in FFA levels compared to healthy controls have been found. Yet some specific long-chain FFAs were measured at higher concentrations and their increased binding affinity for albumin may explain the observed changes in albumin metal-binding capacities [
]), however they were not included in our analysis as “IMA levels” were measured with an immunoassay (see next section) instead of the ACB assay.
5. Proposed alternatives to the ACB assay
An enzyme-linked immunosorbent assay has been developed as an alternative to the ACB assay to specifically detect N-terminal modification of albumin. However, no correlation has been found between the results of this assay and IMA levels measured via the ACB assay in patients with either acute coronary syndrome or non-ischemic chest pain [
Insignificant role of the N-terminal cobalt-binding site of albumin in the assessment of acute coronary syndrome: discrepancy between the albumin cobalt-binding assay and N-terminal-targeted immunoassay.
]. This is consistent with metal binding sites A and B playing a more important role in cobalt binding than the N-terminus.
Other studies on human serum albumin have utilised Cu2+ and Ni2+ instead of Co2+to assess reduced metal binding. In some cases, these studies were inspired by the originally proposed mechanism involving binding to the NTS [
]. Even though Cu2+ and Ni2+ do indeed preferentially bind to the N-terminus, these studies were successful in demonstrating poor binding capacity of albumin for these ions in coronary artery syndromes – similar to the ACB assay [
]. It should however be considered that site A is a potent secondary binding site for these metal ions once the NTS is saturated, as explained in Section 2 [
]. Therefore, providing that such tests employ an appropriate metal: albumin molar ratio (≥2), FFAs can affect the binding capacity of albumin for Cu2+ and Ni2+ binding to site A (and site B) like for Zn2+ or Co2+ [
]. Most recently, a 13C NMR-based protocol using 13C-methyl-labeled oleic acid (OA) as a reporter molecule has also been developed to measure the amount of long chain FFAs bound to albumin as an alternative to the ACB assay that is not dependent on total albumin concentrations [
Use of the ACB assay to measure IMA levels in multiple pathological conditions has gained traction in recent years. The diagnostic value of this test critically depends on understanding its molecular basis. In the light of compelling evidence, there is now increasing recognition of the fact that N-terminal modification is not a plausible explanation for reduced cobalt binding by albumin [
Insignificant role of the N-terminal cobalt-binding site of albumin in the assessment of acute coronary syndrome: discrepancy between the albumin cobalt-binding assay and N-terminal-targeted immunoassay.
Ischemia-modified albumin as a marker of acute coronary syndrome: The case for revising the concept of "N-terminal modification" to "fatty acid occupation" of albumin.
]. Nonetheless, the FFA-based mechanism is not yet widely accepted either, with many recent studies claiming that IMA corresponds to a marker for “oxidative stress”. In principle, an altered redox balance may well affect the ill-defined chemistry of complex formation between Co2+ and DTT, as both agents are prone to oxidation. This alternative hypothesis which does not require covalent modification of albumin may also be more compatible with the timescales of increased and returned to normal ACB readings. At present, corresponding quantitative data and experiments to demonstrate the viability of this hypothesis are scarce, and it leaves unclear the role of albumin in the readout, although the possibility of ternary complex formation was raised [
]. The correlation between ACB assay readings and FFA levels is clear (Fig. 6b), provides a coherent explanation of the chemical identity of IMA, and is consistent with all clinical observations. Serum FFA, in particular unbound FFA, concentrations are useful biomarkers for early diagnosis of ACS [
Ischemia-modified albumin as a marker of acute coronary syndrome: The case for revising the concept of "N-terminal modification" to "fatty acid occupation" of albumin.
]. A comprehensive understanding of the chemical species contributing to the overall readouts, including effects of pH and redox chemistry, should enable the design of a test with much better specificity and diagnostic value.
Conflict of interest
There are no financial or other relationships that might lead to a conflict of interest for the authors.
Acknowledgements
This work was supported by the Leverhulme Trust (grant ref. RPG-2017-214), BBSRC (grant ref. BB/J006467/1) and the British Heart Foundation (grant refs. PG/15/9/31270 and FS/15/42/31556).
References
Picano E.
Palinkas A.
Amyot R.
Diagnosis of myocardial ischemia in hypertensive patients.
I. Committee on standardization of markers of cardiac damage of the, future biomarkers for detection of ischemia and risk stratification in acute coronary syndrome.
30082-6&id=gr1.jpg){kind=link}
30082-6&id=gr2.jpg){kind=link}
) demonstrate that addition of Zn2+ decreases albumin's affinity and capacity for Co2+-binding, while addition of Cu2+ has no significant effect.30082-6&id=gr3.jpg){kind=link}
30082-6&id=gr4.jpg){kind=link}
) or Co2+ (●) affects the energetics of fatty acid binding relative to the metal-free experiment (○), likely due to the need to remove the metal before the FFA can bind. Notably, again the effect for Zn2+ is larger than that for Co2+.30082-6&id=gr5.jpg){kind=link}
30082-6&id=gr6.jpg){kind=link}