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From 2013.igem.org

Contents 1 Biomarkers 1.1 TMAO 1.2 IMA 1.3 BNP

Biomarkers

TMAO

Trimethylamine N-oxide (TMAO) is a relatively common diet metabolite in animals. It originates from the degradation of choline, present in the phosphatidylcholine (lecithin) from foods like eggs, milk, liver, red meat, poultry, shellfish and fish. Choline and other trimethylamine-containing species (for example, betaine) are degraded by intestinal microbes, forming the gas trimethylamine (TMA). This molecule is then absorbed and metabolized in the liver by flavin monooxygenases (FMO), forming TMAO (Wang et al., 2011).

A study published in 2013 (Tang et al., 2013) associated TMAO levels in blood with heart disease, and pointed that gut flora has an important role in forming this molecule in humans. The demonstrated relationship between TMAO levels and future cardiac events like heart attack, stroke, and death could be established even in cases with no prior evidence of cardiac disease shown by the traditional methods.

TMAO also alters cholesterol deposition and removal from endothelial cells. Dietary TMAO aggravate the development of atherosclerotic lesions in apolipoprotein E null (apoE−/−) mice without significant alterations in plasma cholesterol, triglycerides, lipoproteins, glucose levels, and hepatic triglyceride content. But the precise molecular mechanisms in which TMAO mediates its proatherosclerotic effect are currently unknown (Koeth et al., 2013). Given this evidence, TMAO could act as good biomarker for prognosis of cardiovascular risk, although more studies are still needed to validate TMAO testing as clinical tool for preventing cardiovascular diseases.

With the aim of developing an innovative prognostic test for acute coronary syndrome, The Cardbio project included TMAO detection. This biomarker emerged in recent, good quality and strong evidence-based research and seems like a good candidate for early detection of atherosclerotic plaque formation.

Curiously, TMAO also arouses as an interesting material that Illinois team chose to work with, as you can check in their wiki. They thought a way to reduce the metabolism of L-carnitine in digestion system in order to prevent cardiovascular disease. While Illinois team use TMAO as core of a preventive mechanism counter cardiovascular disease, we thought a prognosis mechanism using the same active as a biomarker.

References

• Tang WH, Wang Z, Levison BS, Koeth RA, Britt EB, Fu X, Wu Y, Hazen SL. Intestinal microbial metabolism of phosphatidylcholine and cardiovascular risk. N Engl J Med. 2013 Apr 25;368(17):1575-84. doi: 10.1056/NEJMoa1109400.
• Koeth RA, Wang Z, Levison BS, Buffa JA, Org E, Sheehy BT, Britt EB, Fu X, Wu Y, Li L, Smith JD, DiDonato JA, Chen J, Li H, Wu GD, Lewis JD, Warrier M, Brown JM, Krauss RM, Tang WH, Bushman FD, Lusis AJ, Hazen SL.Intestinal microbiota metabolism of L-carnitine, a nutrient in red meat, promotes atherosclerosis. Nat Med. 2013 May;19(5):576-85. doi: 10.1038/nm.3145. Epub 2013 Apr 7.
• Bennett BJ, de Aguiar Vallim TQ, Wang Z, Shih DM, Meng Y, Gregory J, Allayee H, Lee R, Graham M, Crooke R, Edwards PA, Hazen SL, Lusis AJ. Trimethylamine-N-oxide, a metabolite associated with atherosclerosis, exhibits complex genetic and dietary regulation. Cell Metab. 2013 Jan 8;17(1):49-60. doi: 10.1016/j.cmet.2012.12.011.
• Wang Z, Klipfell E, Bennett BJ, Koeth R, Levison BS,Dugar B, Feldstein AE, Britt EB, Fu X, Chung YM, Wu Y, Schauer P, Smith JD, Allayee H, Tang WH, DiDonato JA, Lusis AJ, Hazen SL. Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease. Nature. 2011 Apr 7;472(7341):57-63. doi: 10.1038/nature09922.
• Mayr M. Recent Highlights of Metabolomics in Cardiovascular Research. Circulation: Cardiovascular Genetics. 2011; 4: 463-464

IMA

One of the main aspects of acute coronary syndrome is myocardial ischemia. It occurs when blood flow to heart muscle is decreased by a partial or complete blockage of coronary arteries, reducing oxygen supply. If the ischemia is detected early, it can be reversed with no myocardial permanent impairement. However, if it is prolonged, there will be cellular necrosis and myocardial infarction. Currently, the only strategy for detecting ischemia is to detect ST segment changes on the electrocardiogram (ECG) but with only around 50% sensitivity. There is, therefore, a need for an early diagnosis for myocardial ischemia so it can be treated in time.

Serum albumin is the most abundant protein in human blood. It is responsible for binding, transporting and distributing a number of small molecules and metallic ions, such as Fe2+, Ni2+, Cd2+ e Co2+. Studies conducted by Bar-Or et al., revealed that albumin extracted from patients with ischemia in cardiac tissues presented reduced cobalt binding. This reduction was considered to be likely caused due to the loss of two aminoacids in the albumin N-terminal – Asp1 e Ala2 – which constitutes an important binding site for metals in the protein (Sadler et al., 1994) and are known for being particularly susceptible to degradation, comparing to other N-terminal residues in other species (Chan et al., 1995). Recent studies (Oh et al., 2012; Lu et al., 2012) suggests, however, that the reduced albumin affinity for cobalt occurs not by the N-terminal damage, but by the binding of free fatty acids, which are increased in ischemic cases (Apple et al., 2004), in this portion of the protein, obstructing the cobalt binding site. Therefore, IMA detection could be a measure of free fatty acids in blood, which have been recently pointed as good biomarkers for prognosis of acute coronary syndrome (Breitling et al., 2011).

A colorimetric test for ischemia modified albumin (IMA) was developed (Bar-Or et al., 2000), based on the measure of free cobalt after the addition of patient sera with ischemia suspicion. Studies comparing the clinical use of this cobalt binding assay (CBA) with other biomarkers point to a high sensibility for ischemia detection, but with low specificity (Bhagavan et al., 2003; Christenson et al., 2001). This assay has a high negative predictive value and can be used in initial triage in clinic, and was approved by FDA (Foods and Drugs Administration) for detection/exclusion of acute myocardial infarction in 2003. Therefore, the Cardbio project chose IMA as a biomarker to be detected by its construction. Associated with other more specific biomarkers, IMA detection can improve the sensibility of our test and it also accomplishes our goal to detect the early alterations caused by acute coronary syndrome instead of diagnosing late events that cannot be reversed.

References

• Bar–Or D, Lau E, Rao N, Bampos N, Winkler JV, Curtis CG. Reduction in the cobalt binding capacity of human albumin with myocardial ischemia. Ann Emerg Med 1999;34(4 Suppl):S56.
• Bar–Or D, Lau E, Rao N, Bampos N, Winkler JV, Curtis CG. Characterization of the Co2+ and Ni2+ binding amino-acid residues of the N-terminus of human albumin. Eur J Biochem 2001;268:42-7
• Sadler PJ, Tucker A, Viles JH. Involvement of a lysine residue in the N-terminal Ni21 and u21 binding site of serum albumins. Comparison with Co21, Cd21, Al31. Eur J Biochem 1994;220:193–200.
• Chan B, Dodsworth N, Woodrow J, Tucker A, Harris R. Site-specific N-terminal auto-degradation of human serum albumin. Eur J Biochem 1995;227:524–8.
• Bar-Or, D., Lau, E. & Winkler, J.V. (2000) A novel assay for cobalt-albumin binding and its potential as a marker for myocardial ischemia - a preliminary report. J. Emerg Med. 19,311-315.
• Bhagavan, N. V.; Lai, E. M.; Rios, P. A.; Yang, J. S.; Ortega-Lopez, A. M.; Shinoda, H.; Honda, S. A. A.; Rios, C. N.; Sugiyama, C. E.; Ha, C. E. Evaluation of human serum albumin cobalt binding assay for the assessment of myocardial ischemia and myocardial infarction. Clin. Chem. 2003, 49, 581−585.
• Christenson, R. H.; Duh, S. H.; Sanhai, W. R.; Wu, A. H. B.; Holtman, V.; Painter, P.; Branham, E.; Apple, F. S.; Murakami, M.; Morris, D. L. Characteristics of an albumin cobalt binding test for assessment of acute coronary syndrome patients: A multicentre study. Clin. Chem. 2001, 47, 464−470.
• Oh BJ, Seo MH, Kim HS. 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. Biomarkers. 2012 Aug;17(5):394-401
• Lu J, Stewart AJ, Sadler PJ, Pinheiro TJ, Blindauer CA. Allosteric inhibition of cobalt binding to albumin by fatty acids: implications for the detection of myocardialischemia. J Med Chem. 2012 May 10;55(9):4425-30
• Apple, F. S.; Kleinfeld, A. M.; Adams, J., III. Unbound free fatty acid concentrations are increased in cardiac ischemia. Clin. Proteomics J. 2004, 1, 41−44.
• Breitling, L. P.; Rothenbacher, D.; Grandi, N. C.; Marz, W.; Brenner, H. Prognostic usefulness of free fatty acids in patients with stable coronary heart disease. Am. J. Cardiol. 2011, 108, 508−513.
• David C. Gaze. Ischemia Modified Albumin: A Novel Biomarker for the Detection of Cardiac Ischemia. Drug Metab. Pharmacokinet. 24 (4): 333–341 (2009).

BNP

BNP (Brain Natriuretic Peptide) is synthesized mainly by the ventricles, and their circulatory concentrations are significantly elevated in congestive heart failure (CHF).

The plasma concentration of BNP has been used to assist in the accurate diagnosis of heart failure in patients admitted with symptoms of decompensated heart failure (Abassi et al., 2004).

In humans, BNP is produced from proBNP, which contains 108 aminoacids and, after proteolytic processing, releases a mature molecule and a 32 aminoacid N-terminal fragment in the circulation. BNP was originally cloned from brain but is now considered a blood hormone produced mainly in the heart ventricles (Ogawa et al. 1991). It is now known that these peptides have effects such as diuresis, natriuresis, vasodilation, and act as a circulating hormone in the inhibition of aldosterone synthesis and renin secretion. Thus, BNPs seems to play an important role in the regulation of blood pressure and blood volume (Nishikimi et al., 2006).

BNP is released by injured heart in very expressive proportions. Therefore, physicians have become very interested in measuring the plasma levels of BNP as a diagnostic tool in cardiology. In fact, several studies have shown that the measurement of circulating BNP can discriminate between patients with decompensated congestive heart failure and patients with dyspnea due to noncardiac etiology (Lemos et al., 2001). Evaluation of BNP levels should not be used as an independent test, but its high sensitivity and negative predictive value may be useful to add other information to the physician in making a diagnosis of heart failure. The main strength of BNP is the excellent negative predictive value with regard to left ventricular dysfunction and heart failure, but other specific diagnostic tools are required to define the actual abnormality (Vuolteenaho et al., 2005).

The Cardbio project chose to use BNP in its construction because it is a biomarker already used with diagnostic purposes, with several commercial assays already developed for its quantitative immunodetection. These assays could be used as controls, to validate our construction. In addition to this, since BNP is a small, unstable molecule that can be underestimated by immunoassays relying on antibody recognition (Tamm et al., 2008), a synthetic biology approach could improve the heart failure diagnosis based on this biomarker.

References

• Vuolteenaho O, Ala-Kopsala M, Ruskoaho H. BNP as a biomarker in heart disease. Adv Clin Chem. 2005;40:1-36.
• Lemos, J. A., Morrow, D. A., Bentley, J. H., Omland, T., Sabatine, M. S., McCabe, C. H., Hall, C., Cannon, C. P., & Braunwald, E. (2001). “The prognosis value of B-type natriuretic peptide in patients with acute coronary syndromes.” N Engl J Med 345, 1014–1021.
• Toshio Nishikimi, Nobuyo Maeda, Hiroaki Matsuoka (2006). “The role of natriuretic peptides in cardioprotection.” Elsevier Cardiovascular Research 69 (2006) 318 – 328
• Zaid Abassi, Tony Karram, Samer Ellaham, Joseph Winaver, Aaron Hoffman (2004).”Implications of the natriuretic peptide system in the pathogenesis of heart failure: diagnostic and therapeutic importance.” Elsevier Pharmacology & Therapeutics 102 (2004) 223– 241
• Natalia N. Tamm, Karina R. Seferian, Alexander G. Semenov, Kadriya S. Mukharyamova, Ekaterina V. Koshkina, Mihail I. Krasnoselsky, Alexander B. Postnikov, Daria V. Serebryanaya, Fred S. Apple, MaryAnn M. Murakami, and Alexey G. Katrukha. Novel Immunoassay for Quantification of Brain Natriuretic Peptide and Its Precursor in Human Blood. Clinical Chemistry 54:9 1511–1518 (2008)

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