Ischemia – modified albumin by albumin cobalt binding test: a false myth or reality

Objectives

Laboratory medicine is one of the most dynamic areas of medicine. Every day, new assays or biomarkers are introduced to the clinical laboratory environment. Of course, the remarkable increase in the discovery of new biomarkers requires a serious assessment before clinical application. However, because of the commercial incentives at least partly, some of these biomarkers have been launched without sufficient evidence for their analytical and clinical performances. With additional effects such as lower cost, easy application everywhere, and inaccurately interpreted literature data, they can spread rapidly. However, clinical practice reveals their clinical uselessness soon. Therefore, those assays could be used in laboratory medicine, especially in research, although not for clinical purposes, for years. There is a proverb in Turkish: “A madman throws a stone into a well, but 40 smart people cannot get it out”. Thus, studies with insufficient assays still continue. In this opinion paper, we aimed to discuss the dark side of ischemia-modified albumin (IMA), a commonly used biomarker.

Content

Ischemia-modified albumin is a relatively newly proposed biomarker by Bar-Or, et al. [1]. Bar-Or, et al. initially observed in vitro reduction in the cobalt(II) binding capacity of human serum albumin (HSA) in ischemic conditions such as myocardial infarction and they developed an assay based on the cobalt binding capacity of HSA [2]. They based the assay on the capacity of the N-terminal of HSA to bind transition metal ions such as cobalt, nickel, copper, zinc, etc. Normally, the N-terminal of HSA is known to be a binding site for those metal ions. The authors postulated that because of the ischemia or ischemia-reperfusion, the molecular structure of the N-terminal of HSA is changed and therefore it cannot bind metal ions. This is the principal basis of the albumin cobalt binding (ACB) assay for IMA. However, structural modifications in the N-terminal of albumin could not be proven after myocardial ischemia. The majority of patients with myocardial ischemia and high IMA had wild-type albumin sequence [3].

ACB is a colorimetric assay performed in patients’ sera. In addition to patient serum, the assay mixture contains a known amount of cobalt chloride and a cobalt binding agent, dithiothreitol (DTT). After incubation for cobalt binding to albumin for 10 min, DTT is added to form brown color between cobalt and DTT, and then sodium chloride solution is added to the reaction mixture to stop this reaction. The final brown color is read at 470 nm against a serum-cobalt blank, a kind of sample blank, and the result is reported as an absorbance unit (ABSU).

As can be seen, the ACB is a simple, rapid, and cheap assay and it does not require sophisticated instrumentation. Therefore it has been quickly spread worldwide for scientific research purposes. Moreover, it was approved by the United States Food and Drug Administration (FDA) for the diagnosis of myocardial ischemia in 2003. But the method is not as simple as it seems. On the contrary, it has many shortcomings. The most important one is the lack of evidence for clinical applications. Early clinical studies revealed successful results, but clinical success was quickly quenched when analytical problems were considered. Today, the ACB test for IMA is not used for clinical assessment of ischemic conditions. However, the research studies are continuing to spread. When we searched PubMed with the term “ischemia modified albumin” only, there were 280 papers published within the last five years. IMA with ACB assay has been studied not only in myocardial ischemia but also in almost every clinical condition. For example, the spectrum of studies encompasses the following clinical conditions or areas outside myocardial ischemia in alphabetical order: acne vulgaris, acute abdomen and appendicitis, alcoholic hepatitis, allergy, alopecia, Alzheimer’s disease and cognitive impairment, anemia, ankylosing spondylitis, aortic dissection, asthma, autism spectrum disorder, autoimmune gastritis, avitaminosis, Behçet’s disease, beta-thalassemia, blood transfusion, cancer (bladder, colorectal, gastric, prostatic, leukemia, etc., almost every type) and cancer therapy, carbon monoxide poisoning, celiac disease, cerebral infarction and hemorrhage (cerebrovascular accidents) and traumatic brain injury, cesarean section, cholestasis, cholecystitis, and cholecystectomy, congestive heart failure, chronic kidney disease, renal injury, end-stage renal disease and dialysis, chronic liver disease and cirrhosis, COVID-19 infection, Crimean-Congo hemorrhagic fever, cutaneous mastocytosis, dermatological diseases, diabetes mellitus and its complications, diving and decompression sickness, dyslipidemia, endometrial polyps and endometriosis, erectile dysfunction, exercise, familial Mediterranean fever, fibromyalgia, flap viability, forensic medicine, hemorrhagic shock, hernias, hidradenitis supurativa, hyperbaric oxygen therapy, hypo- and hyperthyroidism and other thyroid disorders, hypertension, infection, inflammation, infertility, intermittent claudication, intrauterine growth restriction, ionizing radiation, ischemic bowel, ischemic encephalopathy, irritable bowel syndrome and inflammatory bowel disease, lithotripsy therapy, menopouse and dysmenorrhea, mesenteric ischemia, migraine, multiple sclerosis, muscle ischemia, nasal polyps, necrotizing enterocolitis, neonatal and obstructive jaundice and phototherapy, neural tube defects, neurologic injury, obesity, insulin resistance and metabolic syndrome, ovearctive bladder, non-alcoholic fatty liver disease, obstructive sleep apnoea, organic phosphorus pesticide poisoning, oxidative stress and ischemia-reperfusion injury, ozone therapy, pleural effusions, osteoarthritis, pancreatitis, perinatal asphyxia, periodontal disease, peripheral arterial occlusive disease, phenylketonuria, pneumonia, other respiratory diseases and respiratory distress syndrome, polycytemia vera, polycystic ovary syndrome, polcystic kidney, pregnancy, delivery, hyperemesis gravidarum and preeclampsia, prematurity, prenatal screening, priapism, primary dysmenorrhea, prostatectomy, psoriasis, pulmonary embolism, pyroptosis, retinopathy, glaucome, macular degeneration, retinitis peigmentosa, and cataract, rheumatic fever, rheumatoid arthritis, sarcopenia, seizure, sepsis, sickle cell anemia, silicosis, skeletal muscle ischemia, smoking, spinal cord injury, stroke, surgery (general or orthopedic, knee, lung, intestinal, etc.), systemic lupus erythematosus, systemic sclerosis, testicular and ovarian tortion, trauma, undescended testes, urticaria, uterine artery embolisation, varicocele, vasculitis, venous thromboembolism and deep vein thrombosis, viral hepatites, vitiligo, Wilson’s disease. There are also papers on studies of analytical quality, interference, reference intervals, and studies of automotive workers, futsal players, marathon runners, etc. Meanwhile, about the half of 748 papers (n=343) on IMA is from Turkey. China and India follow Turkey with 79 and 45 papers, respectively.

Is this a normal scientific trend or spread? Or is it due to the tendency to prefer the convenience of research by being fascinated by the literature data without sufficient evidence, even though there are so many analytical and clinical problems?

The exact biochemical basis of the N-terminal modification of the albumin molecule is not clear. It is postulated that the main cause is based on free radical damage, acidosis, hypoxia, sodium, and calcium pump disruptions due to energy deficit, and free iron and copper ion exposure because of ischemia and/or ischemia-reperfusion [1]. But this postulate still needs evidence.

Clinically, the best model proposed for IMA is transient coronary artery occlusion during the percutaneous coronary intervention (PCI) [4]. There are many early studies performed on patients undergoing PCI. In 2005, we also performed a study on 21 patients (17 men, 4 women) with acute coronary syndrome treated by single-vessel PCI [5]. In this study, we collected blood specimens before, immediately after, and 6 h after the procedure of PCI. When we considered only ACB assay results or IMA values as absorbance unit (ABSU), 20 of 21 patients had increased IMA values >10% immediately after the PCI procedure concerning baseline values and remained elevated >10% in eight patients at the sixth hour of the procedure. Five of the remaining 13 patients had elevated IMA values at the sixth hour >10%, but IMA values were decreased below baseline in eight patients. These results showed that IMA by ACB test is an excellent biomarker for myocardial ischemia. However, we know that albumin concentration is an important determinant of ACB assay results. There is a warning about the results of the ACB assay being affected when serum albumin concentrations are less than lower limit or more than upper limit of reference range of albumin [6]. If the albumin concentration is lower the IMA is higher or vice versa. Theoretically, if albumin concentration is lower in the specimen, less cobalt binds to the albumin molecule, therefore a more intensive reaction between cobalt ions and DTT is expected. In our study mentioned above [5], when we adjusted IMA levels according to the formula suggested by Lippi et al. [7] ((individual serum albumin concentration/median albumin concentration of study population) x patient’s IMA value), there were no statistical differences between the groups, and the differential power of IMA for ischemia disappeared. Additionally, we found strong negative correlations between IMA levels and albumin concentrations within individual groups (the coefficients of correlations were between r=−0.705 to r=−0.757). After that, we performed a further study to clarify the relationship between albumin concentrations and IMA values [8]. In that study, we prepared a serum pool with an albumin concentration of 45 g/L and deproteinized this pool by a membrane filter (Amicon^® Ultra-4 filter; cutoff: 10,000 Da; Millipore Corp.). Then we designed two parallel studies with this protein-free serum matrix by adding the separated serum protein fraction and a commercially available human serum albumin at different albumin concentrations. We found a very strong negative correlation between IMA levels and added albumin concentrations in both pools almost close to r=1.0 in the range of 10 g/L to 60 g/L albumin concentration. In conclusion, IMA values are directly determined by albumin concentrations at the whole albumin concentration range. Another important problem we found in that study was the complex relationship between cobalt ions and DTT. The cobalt gives a strong reaction with DTT in deproteinized serum matrix but not so when water is used instead of serum matrix in reaction conditions. But, when human serum albumin (HSA) is added to this reaction mixture, the reaction color is observable. This is a very complex mystery. With all those uncertainties about the reaction between cobalt, albumin, serum matrix, and DTT, the ACB assay is used quite commonly without measuring albumin concentrations simultaneously. Also, most of the studies using ACB assay for IMA have not included albumin-adjusted IMA values. Decreased or increased serum albumin concentrations resulting from hemoconcentration, hemodilution, and various diseases or physiological conditions affect the IMA results. In total, because of the negative relationship between albumin concentration and IMA, in patients with decreased albumin concentration type 1 error (false positivity), and patients with relatively increased albumin concentration type 2 error (false negativity), may be seen. Since lower albumin levels are frequently associated with many clinical conditions, mostly type 1 error will be encountered.

Another problem with the ACB assay is the presence of other transition metal ions in biological fluids. In 2008, we reported a study on the effects of calcium(II), magnesium(II), copper (II), and iron(II) ions on IMA and we found that ACB assay is not affected by calcium(II), magnesium(II), and iron(II) ions, but copper(II) ions [9]. Moreover, in addition to N-terminal, the albumin molecule has three more metal binding sites [6] and this molecular feature may also interfere with the ACB assay.

Other deficiencies of IMA and ACB assay

In humans, the half-life of the albumin molecule is three weeks. If IMA is a reality, presumably, N-terminal modification has a similar half-life. But IMA rapidly returns to baseline values within 24 h. This is an inexplicable contradiction. A possible explanation for this contradiction would be the rapid clearance of IMA, but there is not any evidence for this explanation.

There are manual or automatized ACB assays or some modifications of it but none of them are standardized. As there is neither a reference material nor a reference method. The results are reported as absorbance units (ABSU) or arbitrary units (kU/L). There are commercially available immunoassays with the ELISA technique which are used for research but the characteristics of their calibrators are unclear. Neither the ACB assay nor immunoassays are traceable. Additionally, there is not a strong correlation between ACB assays and immunoassays (essentially ELISA assays). In conclusion, the results are significantly different and are not comparable or transferable, hence there is a harmonization problem for IMA, too.

The color reagent in the ACB assay is DTT solution. DTT is a general sulfhydryl (-SH) reducing reagent at the same time. It can react with disulfide groups and reduce the SH groups in both albumin molecules and other proteins in the serum, even free disulfide groups in the serum matrix such as oxidized glutathione as well. Those non-specific reactions interfere with the color reaction between cobalt and DTT.

Finally, IMA does not shows any correlation with other cardiac markers such as cardiac troponins, creatine kinase MB isoenzyme, and myoglobin [5]. The strongest correlation is between IMA and albumin. This is an expected finding because the ACB assay essentially measures albumin concentration. It is interesting that despite all these analytical and clinical problems, the ACB assay was cleared by FDA for the detection of myocardial ischemia in 2003.

There are few systematic reviews or meta-analyses evaluating the clinical application of IMA and these papers conclude with positive evaluations. Positive evaluations are inevitable because these meta-analyses review only the clinical side of ACB assay without considering the technical problems of it. Of these, the newest one is partly opposite to the prior meta-analyses [10]. Even this meta-analysis concludes with positive evaluations of IMA such as area under the curve value of 0.750 for the diagnosis of acute coronary syndrome with a pooled diagnostic odds ratio of 3.72. However, the authors added that because of the high heterogeneity between the clinical studies the accuracy of these findings could not be ascertained.

Summary and outlook

In conclusion, there is insufficient evidence for the existence of IMA as a biomarker. The ACB assay essentially measures serum albumin concentration. There are many other interfering factors with the ACB assay. Therefore, the measurement of IMA in any pathological condition is a useless effort.

As a final word, I would like to end my article with the aphorisms of Heraclitus: “Panta rhei” (everything flows), “no man ever bath in the same river twice”.