The A1c Blood Test: An Illustration of Principles from General and

Research: Science and Education edited by

Products of Chemistry

George B. Kauffman California State University Fresno, CA 93740

The A1c Blood Test: An Illustration of Principles from General and Organic Chemistry

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Robert C. Kerber Department of Chemistry, SUNY at Stony Brook, Long Island, NY 11794-3400; [email protected]

Many, if not most, of the college students who take chemistry courses are motivated by chemistry’s importance in biology and medicine. Effective teaching can take advantage of such interests by relating chemical principles to medical practices. This helps to maintain student interest and attention. Diabetes has become increasingly prevalent in developed countries in recent decades. The Centers for Disease Control estimates the U.S. diabetic population at over 20 million, about 7% of the total population (1). The A1c blood test is the principal means used to assess long-term control of blood glucose concentration, and the CDC recommends at least semiannual A1c blood testing for all diabetic patients. The utility of this test derives from basic principles of chemical equilibrium and kinetics, and its results directly correlate with complications of diabetes that arise from spontaneous interactions of functional groups in carbohydrates and proteins. The A1c test therefore provides useful and relevant illustrations of general principles in introductory chemistry courses, and the related chemistry provides interesting material involving organic reactions. Glucose Glucose (blood sugar) provides a textbook example of the famous Paracelsian principle that All substances are poisons; there is none which is not a poison. The right dose differentiates a poison from a remedy…. Paracelsus (1493–1541)

A healthy person maintains a blood glucose concentration of 90 ± 20 mg兾dL (5 ± 1 mM) through feedback mechanisms involving peptide hormones: insulin, which reduces the concentration, and glucagon and epinephrine, which raise it. If the concentration of glucose in blood falls below 50 mg兾dL, hypoglycemia, which can lead to coma and death, results. On the other hand, diabetic patients suffer from hyperglycemia owing to insulin deficiency or insulin resistance. The excess glucose is not immediately toxic, but its high concentration results in slow reactions in which it becomes bonded to various proteins throughout the body (vide infra). Affected organs include the blood vessels, leading to coronary heart disease; eyes, leading to cataracts and retinopathy; kidneys, leading to nephropathy; and nerves, leading to neuropathy and limb necrosis (2). Maintenance of a stable glucose concentration is a remarkable feat, given that an average person processes about 160 grams of glucose per day, 120 grams in the brain alone. About 20 grams of free glucose is available in body fluids at www.JCE.DivCHED.org

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a given time, and an additional 180–200 grams is stored as glycogen (polyglucose) in cells (3). Instantaneous blood glucose concentrations can readily be measured in a droplet of blood using ingenious devices that rely on glucose oxidase-catalyzed oxidation of glucose to gluconic acid and hydrogen peroxide,

followed by colorimetric determination of the peroxide concentration,1

where HRP is horseradish peroxidase. However, the blood glucose concentration changes rapidly and frequently, rising upon food consumption and falling with physical activity, and it would be necessary to monitor the instantaneous concentration almost continually to ascertain a meaningful average glucose concentration. This is clearly inconvenient and impractical. Instead, the A1c test has been developed to provide a measure of blood glucose concentration averaged over a period of several weeks. Maintaining a low average glucose concentration over the long term is a high priority, as the damaging effects of excess glucose, owing to its binding to proteins (glycation), can be severe. The A1c test assays glycation of a specific protein, hemoglobin (Hb), as a measure of overall protein glycation. There are many clinical methods of measuring hemoglobin glycation, which is reported as percent of total hemoglobin bearing glycosyl groups. The methods vary in specificity for the Hb A1c isomer. One group of methods (cation-exchange chromatography, agar gel electrophoresis) is based on the reduced positive charge of the glycated protein, and the other group (boronate affinity chromatography, immunoassay) is based on structural differences. The reference method, used since 1978, is a HPLC cation exchange method. The clinical goals and outcomes defined by the 1983–1993 Diabetes Control and Complications Trial were based on the results from use of that reference method, and

Vol. 84 No. 9 September 2007

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Journal of Chemical Education

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Research: Science and Education

alternative methods are supposed to be calibrated to the same scale. Recommended glycated hemoglobin levels are below 7%, with intervention recommended if they exceed 8% (2c). The key point to be made in an introductory chemistry course is that knowing that the rate of glycation is directly dependent on the glucose concentration allows determination of an effective average concentration, despite wide shortterm variations. A high average glucose concentration is deduced directly from detection of large quantities of glycated hemoglobin. As pointed out by a referee, this dependence can also be presented as an application of Le Châtelier’s principle, as long as the rates are sufficient to ensure that the glycation reactions are at equilibrium. From either the kinetic or the equilibrium perspective, the dependence of the quantity of glycated hemoglobin on the average blood glucose concentration should be evident, even in the absence of detailed mechanistic modeling. Hemoglobin Hemoglobin is the protein, found in the red blood cells (erythrocytes), that carries oxygen from the lungs to the tissues of the body. Human hemoglobin is a tetrameric assemblage of four polypeptide chains, two designated α and two β, each bound to a heme unit by coordination of a histidine side chain with an iron–heme moiety. “Every milliliter of blood has approximately 5 billion erythrocytes…, and each erythrocyte is packed with 280 million molecules of hemoglobin” (4). “The concentration of hemoglobin molecules in red blood cells is so high (340 mg兾mL, 2.3 mM) that they almost could be said to be on the verge of crystallization…. The α2β2 tetramers, spheroids of axial dimensions 65 by 55 by 50 Å, are only 10 Å apart on the average” (5). Also at relatively high concentration within the erythrocyte is glucose, whose transport across the cell membrane is facilitated by transporter proteins. This maintains the intracellular glucose concentration close to that in the serum outside (6). Consequently, the erythrocyte interior provides a space where glucose and a specific protein, hemoglobin, come together in high concentrations, a favorable condition for a bimolecular reaction. Early studies of hemoglobins by ion-exchange chromatography had revealed ubiquitous minor components with reduced positive charges relative to unmodified hemoglobin. The principal component that met these criteria was designated hemoglobin A1c (7). This component constituted 5– 7% of the total hemoglobins in normal patients, but the quantity rose as high as 20% in diabetic patients. Hemoglobin A1c was eventually identified as a product of spontaneous reaction of normal hemoglobin with glucose (2). Kinetics and Mechanism of Glycation Glycation provides an example of a biologically important reaction that is not enzyme-catalyzed. Initial reaction of glucose and hemoglobin involves reversible formation of an imine:

RCH(OH)CHO + (Hb)NH2

k+1 k−1

RCH(OH)CH N(Hb) + H2O 1542


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(1)

This is followed by a less reversible, exergonic tautomerization of the imine to an aminoketone (a deoxyfructose derivative), often referred to as an Amadori rearrangement:

H+ + RCH(OH)CH N(Hb)

k+2 k−2

RC( O)CH2NH2(Hb)+

(2)

The kinetics of hemoglobin glycation has been studied both in vitro and in vivo (8–10). Results are complicated by the simultaneous reaction of the glucose at several of the accessible amine side chains in hemoglobin (there are eleven lysines and one N-terminal valine on each of the four chains of the α2β2 tetramer). Fortunately, a set of parallel secondorder reactions between a given pair of reactants shows overall second-order behavior that can be described by a composite rate constant. Individual rate constants (if needed) can then be determined from product ratios (11). The overall rate of reaction is also affected by various ligands that bind to hemoglobin, including phosphate ions (12), organic phosphates (13), and oxygen (13a, 14). Erythrocytes have an average lifespan of about 120 days in the body, so the occurrence of glycation reactions leads to a steady-state concentration of glycated hemoglobins. Their concentration is directly dependent on the average glucose concentration over the weeks prior to the determination. This averaging property is the basis for the usefulness of the A1c test as a measure of effectiveness of glucose control. Reported values for the rate and equilibrium constants for the steps in reactions 1 and 2 under physiological conditions vary widely, but the use of consensus values (k+1 = 96 × 10᎑6 L mol᎑1 s᎑1; k᎑1 = 100 × 10᎑6 s᎑1; k+2 = 14.2 × 10᎑6 s᎑1; k᎑2 = 1.7 × 10᎑6 s᎑1) has led to a biokinetic model that agrees with clinical data relating average glucose concentration to hemoglobin A1c (10). This set of rate constants may provide an interesting example for steady-state or numerical modeling in a physical chemistry course.2 A sample calculation is presented in the Supplemental Material.W The second-order kinetics that has been experimentally verified for glycation of hemoglobin is assumed to apply to other proteins as well. A high result on the A1c test indicates a high average concentration of glucose during the weeks previous to the test and therefore a proportionately high level of glycation of other proteins. Indeed, proteins with a longer residence time in the body than hemoglobin would undergo continuous glycation, rather than achieve the steady state that results from the regular turnover of hemoglobin. It is the glycation products from other proteins that are responsible for the toxic effects of excess glucose; the glycated hemoglobin that is measured in the test serves as a convenient indicator of the ongoing extent of glycation and therefore an indication of glucose toxicity. Structures of Glycation Products The N-terminal valines of the two hemoglobin β-chains are generally the most reactive glycation sites in vivo, and the hemoglobin A1c designation refers specifically to the stable product of eq 2 at these two sites. The enhanced reactivity of these sites relative to the other 46 primary amino groups of the hemoglobin tetramer probably results from their greater


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accessibility and weak basicity [pKa of 6.8 (13a, 15) as compared to about 10.5 for a lysine side chain], which leaves them partly unprotonated and nucleophilic at physiological pH. It has also been argued that the histidine residue adjacent to the N-terminus of the β-chain enhances its overall reactivity by catalyzing the Amadori rearrangement (13b, 14). Less prevalent glycated hemoglobins having carbohydrates other than glucose attached to the β-chain N-terminus have been identified (16), as have isomers of hemoglobin A1c with glucose attached to the α N-terminus and to lysine residues on either chain (17). Phosphate or organophosphate ions direct the site of glycation in favor of the preferred β-terminal valine, an effect attributed to their enhancement of the rate of the Amadori rearrangement (12, 13b). Intramolecular catalysis of Amadori rearrangement by adjacent carboxylate groups has similarly been cited in one rationalization of the enhanced reactivity of certain specific lysine residues (17b, 18). Recent advances in protein analysis by electrospray and MALDI mass spectrometry promise to accelerate the analysis of glycated hemoglobins (19). These methods of analysis show hemoglobin’s α-chain to be glycated to about two-thirds the extent of the β-chain, rather more than suggested by classical methods of analysis (20). Polyglycation of the β-chains is also evident at higher glucose concentrations (21).

The intermediate glycation products analogous to the deoxyfructosyl-lysine shown in eq 2 undergo additional slow non-enzymatic reactions, collectively referred to as Maillard reactions that result in functional degradation of the proteins. The complex products of these slow reactions are referred to as advanced glycation end products, AGEs (22). Along with the mechanisms underlying eqs 1 and 2, the chemistry underlying formation of these AGEs provides many group workshop problems useful in organic chemistry courses. One such reaction is the oxidation of the deoxyfructosyllysine to carboxymethyl-lysine and erythronic acid (23),

Advanced Glycation End Products, AGEs

catalyzed by phosphate and inhibited by metal-chelating agents; a free-radical mechanism occurring via the enediol has been suggested (23). The carboxymethyl products are not particularly toxic, and their formation may actually limit the competitive formation of more damaging byproducts (23).

The glycated hemoglobin A1c measured in the test is not toxic. But the result is a surrogate for analogous spontaneous glycations involving other proteins throughout the body.

(3)

Scheme I. Formation of glyoxal imine intermediates.

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These more toxic products are the result of formation of highly reactive α-dicarbonyl compounds (α-oxoaldehydes) or imines by a sequence of enolizations, dehydrations, and retro-aldol reactions. These reactions occur slowly in glucose solutions under physiological conditions, but more rapidly in the presence of Nα-protected lysine or proteins capable of imine formation (24). Formation of glyoxal imine intermediates is illustrated in Scheme I. These imines may undergo hydrolysis to glyoxals or they may condense directly with lysine residues from proteins via transimination reactions to form the various toxic products described below. These alternatives are not explicitly shown in Scheme I to reduce complexity. The highly electrophilic α-oxoaldehydes and imines are the direct precursors to the AGEs (25, 26). They react with particular facility with the arginine residues of proteins to form heterocyclic products, including imidazolones and pyrimidines (Scheme II) (26, 27). Since arginine residues in proteins have a high probability of occurrence in ligand and substrate recognition sites and enzyme active sites, these uncontrolled derivatization reactions often lead to functional disruption (25). Also highly damaging are reactions that result in inadvertent cross-linking of protein chains, which reduces their ability to carry out normal functions and contributes to circulation, joint, and vision problems in diabetics and the aged. One such cross-link is produced by reaction of two lysine residues from proximate chains with α-oxoaldehydes to form a bis(lysyl)imidazolium cross-link (Scheme III) (27, 28).

The pentosidine cross-link,

similarly forms a stabilized aromatic heterocycle linking two protein chains, in this case through an arginine residue on one and a lysine on the other. The five-carbon moiety that makes up the rest of the heterocycle derives from a pentose, probably ribose (29). These protein-altering reactions occur spontaneously, without enzyme catalysis. The products result in reduction of protein activity and flexibility and hence to cell damage. These altered proteins are found in everyone, and their quantity increases with age. But uncontrolled diabetics have unusually high quantities of circulating glucose, so the quantities of AGEs formed in their bodies are significantly higher than in non-diabetics of the same age. (In this sense, diabetics age faster.)

Scheme II. Formation of heterocyclic AGEs.

Scheme III. Formation of bis(lysyl)imidazolium cross-links.

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Conclusion The hemoglobin A1c test provides a convenient measure of long-term average glucose concentration, which indicates the extent of ongoing glycation of proteins in the body. Formation of the glycation products responsible for many of the debilitating effects of diabetes is directly proportional to the average glucose concentration in the bloodstream, which can be viewed either as a consequence of their second-order reaction (first order each in glucose and in protein) or of Le Châtelier’s principle applied to the equilibria of eqs 1 and 2. The enhanced occurrence of these spontaneous reactions in uncontrolled diabetics can be used by teachers in introductory courses to illustrate the consequences of kinetic order. The spontaneous nature of these reactions makes them particularly straightforward examples of the effect of concentration on rate in medically relevant reactions. Detailed kinetic analyses may be carried out in advanced physical chemistry courses.3 The glycation reactions provide interesting examples whose mechanisms involve sequences of simple steps (e.g., imine formation, tautomerization, condensations) that can be worked out by team-learning groups in secondsemester organic chemistry courses. The biological and medical relevance of the reactions should provide immediacy and motivation to the students. W

Supplemental Material

This set of rate constants may provide an interesting example for steady-state or numerical modeling in a physical chemistry course. A sample calculation is presented in this issue of JCE Online. Notes 1. Use of a related glucometer in an error analysis laboratory exercise has been suggested by Edmiston, P. L.; Williams, T. R. An Analytical Experiment in Error Analysis: Repeated Determination of Glucose using Commercial Glucometers. J. Chem. Educ. 2002, 77, 377–379. 2. A referee reports that he “found it a somewhat daunting, but ultimately rewarding challenge to build a spreadsheet to predict the time variance (of glycated and unglycated hemoglobins), based on the rate constants provided...” 3. Adapted from ref 22.

Literature Cited 1. National Diabetes Fact Sheet, 2005. http://www.cdc.gov/diabetes/pubs/pdf/ndfs_2005.pdf (accessed Jun 2007). 2. (a) Kilpatrick, E. S. J. Clin. Pathol. 2000, 53, 335–339. (b) Pizzorno, J. E.; Murray, M. T. A Textbook of Natural Medicine. www.healthy.net/library/books/textbook/section2/glyhem.pdf (accessed Jun 2007). (c) Laboratory Medicine Practice Guidelines. www.nacb.org/lmpg/diabetes/6_diabetes_hemoglob.pdf (accessed Jun 2007). 3. Garrett, R. H.; Grisham, C. M. Biochemistry, 3rd ed.; Thomson Brooks/Cole: Belmont, CA, 2005; p 705. 4. Dickerson, R. E.; Geis, I. Hemoglobin: Structure, Function, Evolution, and Pathology; Benjamin/Cummings: Menlo Park, CA, 1983; p 21. 5. Dickerson, R. E.; Geis, I. Hemoglobin: Structure, Function, Evolution, and Pathology; Benjamin/Cummings: Menlo Park, CA, 1983; p 127.

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6. Garrett, R. H.; Grisham, C. M. Biochemistry, 3rd ed.; Thomson Brooks/Cole: Belmont, CA, 2005; pp 287–289. 7. Bunn, H. F.; Haney, D. N.; Kamin, S.; Gabbay, K. H.; Gallop, P. M. J. Clin. Invest. 1976, 57, 1652–1659. 8. Higgins, P. J.; Bunn, H. F. J. Biol. Chem. 1981, 256, 5204– 5208. 9. Svendsen, P. A.; Christiansen, J. S.; Søegaard, U.; Nerup, J. Diabetologia 1981, 21, 549–553. 10. Mortensen, H. B.; Vølund, A. Scand. Lab. Invest. 1988, 48, 595–602. 11. Frost, A. A.; Pearson, R. G. Kinetics and Mechanism; John Wiley & Sons, Inc.: New York, 1953; pp 151–152. 12. (a) Watkins, N. G.; Neglia-Fisher, C. I.; Dyer, D. G.; Thorpe, S. R.; Baynes, J. W. J. Biol. Chem. 1987, 262, 7207–7212. (b) Kunika, K.; Itakura, M.; Yamashita, K. Life Sci. 1989, 45, 623–630. 13. (a) Lowrey, C. H.; Lyness, S. J.; Soeldner, J. S. J. Biol. Chem. 1985, 260, 11611–11618. (b) Gil, H.; Peña, M.; Vasquez, B.; Uzcategui, J. J. Phys. Org. Chem. 2002, 15, 820–825. 14. Bai, Y.; Ueno, H.; Manning, J. M. J. Protein Chem. 1989, 8, 299–315. 15. Garner, M. H.; Bogardt, R. A., Jr.; Gurd, F. R. N. J. Biol. Chem. 1975, 250I, 4398–4404. 16. Garrick, L. M.; McDonald, M. J.; Shapiro, R.; Bleichman, M.; McManus, M.; Bunn, H. F. Eur. J. Biochem. 1980, 106, 356–359. 17. (a) Bunn, H. F.; Shapiro, R.; McManus, M.; Garrick. L; McDonald, M. J.; Gallop, P. M.; Gabbay, K. H. J. Biol. Chem. 1979, 254, 3892–3898. (b) Shapiro, R.; McManus, M. J.; Zalut, C.; Bunn, H. F. J. Biol. Chem. 1980, 255, 3120–3127. (c) Neglia, C. I.; Cohen, H. J.; Garber, A. R.; Thorpe, S. R.; Baynes, J. W. J. Biol. Chem. 1985, 260, 5406–5410. 18. Acharya, A. S.; Roy, R. P.; Dorai, B. J. Protein Chem. 1991, 10, 345–358. 19. Miedema, K. Clinical Chemistry 1997, 43, 705–707. 20. (a) Roberts, N. B.; Green, B. N.; Morris, M. Clinical Chemistry 1997, 43, 771–778. (b) Lapolla, A.; Tubaro, M.; Reitano, R.; Arico, N. C.; Ragazzi, E.; Seraglia, R.; Vogliardi, S.; Traldi, P.; Fedele, D. Diabetologia 2004, 47, 1712–1715. 21. Peterson, K. P.; Pavlovich, J. G.; Goldstein, D.; Little, R.; England, J.; Peterson, C. M. Clinical Chemistry 1998, 44, 1951– 1958. 22. Singh, R.; Barden, A.; Mori, T.; Beilin, L. Diabetologia 2001, 44, 129–146. 23. Ahmed, M. U.; Thorpe, S. R.; Baynes, J. W. J. Biol. Chem. 1986, 261, 4889–4894. 24. Thornalley, P. J.; Langborg, A.; Minhas, H. S. Biochem. J. 1999, 344, 109–116. 25. (a) Thornalley, P. J. Ann. N. Y. Acad. Sci. 2005, 1043, 111– 117. (b) Niwa, T. J. Chrom. B: Biomed. Sci. Appl. 1999, 731, 23–36. 26. Oya, T.; Hattori, N.; Mizuno, Y.; Miyata, S.; Maeda, S.; Osawa, T.; Uchida, K. J. Biol. Chem. 1999, 274, 18492–18502. 27. Thornalley, P. J.; Battah, S.; Ahmed, N.; Karachalias, N.; Agalou, S.; Babaei-Jadidi, R.; Dawnay, A. Biochem. J. 2003, 375, 581–592. 28. Brinkmann, E.; Wells-Knecht, K. J.; Thorpe, S. R.; Baynes, J. W. J. Chem. Soc., Perkins Trans I 1995, 2817–2818. 29. (a) Grandhee, S.; Monnier, V. M. J. Biol. Chem. 1991, 266, 11649–11653. (b) Biemel, K. M.; Reihl, O.; Conrad, J.; Lederer, M. O. J. Biol. Chem. 2001, 276, 23405–23412.


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The A1c Blood Test: An Illustration of Principles from General and

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