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Hemoglobin A1c in the Diagnosis and Management of Diabetes

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Hemoglobin A1c in the Diagnosis and Management of Diabetes DCCT UKPDS Hemoglobin A1c in the Diagnosis and Management of A1c Assay Diabetes Standardization Ignoring Biological Diabetes Variation in A1c Monitoring Non-enzymatic Biochemistry Separation Science ObjectivesObjectives 1.1. DescribeDescribe whatwhat clinicalclinical laboratorieslaboratories actuallyactually measuremeasure asas HbA1cHbA1c 2.2. ShowShow thatthat HbA1cHbA1c andand meanmean bloodblood glucoseglucose areare notnot interchangeableinterchangeable estimatesestimates ofof glycemicglycemic controlcontrol 3.3. ExplainExplain howhow biologicalbiological variationvariation inin HbA1cHbA1c complicatescomplicates newnew guidelinesguidelines forfor thethe diagnosisdiagnosis andand managementmanagement ofof diabetesdiabetes Main Take-home Message MBG = A1c The quantitative relationship between blood glucose MBG A1c concentration and hemoglobin A1c is not the same in all people globins (A415 nm) Globin Association is Dynamic MW 15258 15867 31125 62249 + → + ↔ Monomers Dimers Tetramer Higher Hemoglobin Concentration Higher pH Oxygenation Known Glucose Binding Sites 1 Order of affinity 8 Val1 40 17 Lys66 Lys61 61 Lys17 Val1 Lys40 Lys8 V --N=CH+ H2O 2 ׀ Non-enzymatic HCOH Labile A1c ׀ Hb glycation HOCH (aldimine/Schiff base) ׀ HCOH ׀ 1 HCOH ׀ id ap R CH2OH S V--NH2 HC=O lo w V--NH--CH2 ׀ ׀ HCOH 2 C=O ׀ ׀ HOCH HOCH ׀ + ׀ HCOH + HCOH ׀ ׀ HCOH MaillardMaillard HCOH ׀ ׀ CH2OH ReactionReaction CH2OH Glucose Stable A1c globin (ketoamine/Amadori) -NH Role of lysine V- 2 Schiff base and -amino K N=CH + H2O ׀ groups HCOH Slo ׀ 1 w id HOCH V--NH2 2 ׀ ap R HCOH ׀ V--NH2 HCOH K --NH--CH2 ׀ 2 ׀ HC=O CH2OH C=O ׀ ׀ K-NH2 HCOH HOCH ׀ ׀ ++ HOCH HCOH ׀ ׀ HCOH HCOH ׀ ׀ HCOH CH2OH 2 ׀ CH2OH Amadori globin Glucose Discovery of Hemoglobin Heterogeneity 1950 1960 1970 1980 1990 2000 2010 2020 Fast Hb Sickle Hemoglobin Heterogeneity in Normal Blood IRC-50 (Bio-Rex) Fast Hb Fast Hb cation exchange Morrison M, Cook JL. Chromatographic Kunkel HG, Wallenius G. New fractionation of normal adult hemoglobin in normal adult blood. oxyhemoglobin. Science 122:920-1,1955 Science 122:288, 1955 Identification of Hemoglobin A1c 1950 1960 1970 1980 1990 2000 2010 2020 Fast Hb Sickle AIc IRC-50 (Bio-Rex) AI Fast Hb cation exchange AII Morrison M, Cook JL. Chromatographic Allen DW, Schroeder, W.A. and fractionation of normal adult Balog, J. J Am Chem Soc 80:1628- oxyhemoglobin. Science 122:920-1,1955 34, 1958 Identification of Hemoglobin A1c 1950 1960 1970 1980 1990 2000 2010 2020 Fast Hb Sickle AIc AIa AI A AIb AIc II Hemoglobin A Heterogeneity I Allen DW, Schroeder, W.A. and Peaks designated a, b, c Balog, J. J Am Chem Soc 80:1628- in order of appearance 34, 1958 Identification of Hemoglobin A1c 1950 1960 1970 1980 1990 2000 2010 2020 Fast Hb Sickle AIc AIa “The designations, AI, AII and others, are given for A AIc Ib convenience of reference in this discussion. It is not our purpose to propose a system of nomenclature” Hemoglobin A Heterogeneity I Allen DW, Schroeder, W.A. and Peaks designated a, b, c Balog, J. J Am Chem Soc 80:1628- in order of appearance 34, 1958 Identification of Hemoglobin A1c 1950 1960 1970 1980 1990 2000 2010 2020 Fast Hb Sickle AIc Nomenclature Problem Additional heterogeneity of normal hemoglobin Clegg MD, Schroeder WA. J Am Chem Soc 1959;81:6065-9. Characterization of Hemoglobin A1c 1950 1960 1970 1980 1990 2000 2010 2020 Fast Hb Sickle AIc Chromatography of Paper electrophoresis of tryptic peptides chromatography fractions AIcT-1 Holmquist, W. R. & Schroeder, W. A. A new N-terminal blocking group Fraction 5 of hemoglobin AIc, but not AII, involving a Schiff base in hemoglobin contained chemically modified N-terminal AIc. Biochemistry 5: 2489-2503, 1966 beta globin peptides Characterization of Hemoglobin A1c 1950 1960 1970 1980 1990 2000 2010 2020 Fast Hb Sickle AIc Mass Spectrometry Globin chains dimers -+ Bookchin, R. M. & Gallop, P. M. “The studies described in the present Structure of hemoglobin AIc: nature of report indicate that a hexose is linked to the N-terminal beta chain blocking group. the N-terminal of the AIc chain - the possible attachment of other structures to Biochem Biophys. Res Commun. 32: 86- the hexose has not been excluded…” 93, 1968 Hemoglobin A1c Elevated in Diabetes 1950 1960 1970 1980 1990 2000 2010 2020 Fast Hb AIc and Sickle AIc Diabetes Fast Hb Diabetic Normal Fast Hb Higher in Fetal People with Diabetes Rahbar S. An abnormal hemoglobin in L.A. Trivelli et al. Hemoglobin components red cells of diabetics. Clinica Chimica in patients with diabetes mellitus. N Engl J Acta 22:296-8, 1968 Med 284 (7):353-357, 1971 Hemoglobin A1c Elevated in Diabetes 1950 1960 1970 1980 1990 2000 2010 2020 Fast Hb AIc and Sickle AIc Diabetes AIc correlated with blood glucose Fast Hb Higher in People with Diabetes Koenig RJ et al. Correlation of L.A. Trivelli et al. Hemoglobin components glucose regulation and hemoglobin in patients with diabetes mellitus. N Engl J AIc in diabetes mellitus. NEJM Med 284 (7):353-357, 1971 1976;295:417-20. Hemoglobin A1c Elevated in Diabetes 1950 1960 1970 1980 1990 2000 2010 2020 Fast Hb AIc and Sickle AIc Diabetes AssessingAssessing GlycemiaGlycemia 700 Type I Diabetes 600 AIc 500 400 correlated 300 with blood 200 glucose 100 Blood Glucose (mg/dl) Blood Glucose (mg/dl) 0 10 20 30 40 50 60 70 Time (days) Blood glucose variation Koenig RJ et al. Correlation of glucose regulation and hemoglobin within individuals AIc in diabetes mellitus. NEJM 1976;295:417-20. Hemoglobin A1c Elevated in Diabetes 1950 1960 1970 1980 1990 2000 2010 2020 Fast Hb AIc and Sickle AIc Diabetes AssessingAssessing GlycemiaGlycemia 700 Type I Diabetes 600 500 400 300 200 100 Blood Glucose (mg/dl) Blood Glucose (mg/dl) 0 10 20 30 40 50 60 70 Time (days) Blood glucose variation R. J. Jarrett; et al. "Instant" blood sugar measurement using Dextrostix and a within individuals reflectance meter. Diabetes. 19 (10):724-726, 1970 Characterization of Hemoglobin A1c 1950 1960 1970 1980 1990 2000 2010 2020 Fast Hb AIc and Sickle AIc Diabetes Gas Chromatography Two-step model of IS Of CHO hemoglobin glycation glu Labile A1c Stable A1c man glu ? man Bunn HF, et al. Further identification of the nature and linkage of the carbohydrate in hemoglobin A1c. H. F. Bunn; et al. The glycosylation of Biochem Biophys Res Commun hemoglobin: relevance to diabetes 1975;67:103-9 mellitus. Science 200 (7):21-27, 1978 Diabetes Control and Complications Trial 1950 1960 1970 1980 1990 2000 2010 2020 Fast Hb AIc and DCCT Sickle AIc Diabetes Labile fraction removed by Bio-Rex 70 Two-step model of RBC hemoglobin glycation incubation in Cation Labile A1c Stable A1c physiological Exchange saline >5 h A1c Highly D. E. Goldstein, et al. In: Methods in Variable Diabetes Research, edited by J. H. F. Bunn; et al. The glycosylation of Larner, S. L. Pohl, and W. L Clarke, hemoglobin: relevance to diabetes New York:Wiley, 1986, p. 475-504 mellitus. Science 200 (7):21-27, 1978 Diabetes Control and Complications Trial 1950 1960 1970 1980 1990 2000 2010 2020 Fast Hb AIc and DCCT Sickle AIc Diabetes Feasibility Labile fraction removed by Bio-Rex 70 Wide population RBC variation incubation in Cation physiological Exchange saline >5 h A 1c 7-point profile sets D. E. Goldstein, et al. In: Methods in The DCCT Research Group. Diabetes Diabetes Research, edited by J. Control and Complications Trial Larner, S. L. Pohl, and W. L Clarke, (DCCT): results of feasibility study. New York:Wiley, 1986, p. 475-504 Diabetes Care 10 (1):1-19, 1987 Diabetes Control and Complications Trial 1950 1960 1970 1980 1990 2000 2010 2020 Fast Hb AIc and DCCT Sickle AIc Diabetes Feasibility Complications Effect of Intensive Management Development of Retinopathy Primary Prevention Cohort On Hemoglobin A1c Levels EDIC The DCCT Research Group. The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. NEJM 1993;329:977-86. Commercial Glycated Hemoglobin Assays 1950 1960 1970 1980 1990 2000 2010 2020 Fast Hb AIc and DCCT Sickle AIc Diabetes Feasibility Complications Clinical Monitoring of Blood Glucose Cation Exchange HPLC Boronate Affinity Chromatography Immunoaffinity Commercial Glycated Hemoglobin Assays 1950 1960 1970 1980 1990 2000 2010 2020 Fast Hb AIc and DCCT Sickle AIc Diabetes Feasibility Complications Bio-Rex 70 Labile fraction Cation Exchange removed by RBC HPLC incubation in PBS >5 h Diabetic Boronate Affinity Chromatography Non-diabetic A 1c A1c Immunoaffinity D. E. Goldstein, et al. Methods for quantitating glycosylated hemoglobins: high performance liquid chromatography and thiobarbituric acid colorimetry. In: Methods in Diabetes Research, edited by J. Larner, S. L. Pohl, and W. L Clarke, New York:Wiley, 1986, p. 475-504 Commercial Glycated Hemoglobin Assays 1950 1960 1970 1980 1990 2000 2010 2020 Fast Hb AIc and DCCT Sickle AIc Diabetes Feasibility Complications Tosoh A1c 2.2 Diabetic Diabetic Cation Exchange Non-diabetic Good Control Poor Control HPLC A1c Labile Boronate Affinity Chromatography Immunoaffinity Khuu et al. Evaluation of a fully automated high-performance liquid chromatography assay for hemoglobin A1c. Arch Pathol Lab Med 123:763-7, 1999 Commercial Glycated Hemoglobin Assays 1950 1960 1970 1980 1990 2000 2010 2020 Fast Hb AIc and DCCT Sickle AIc Diabetes Feasibility Complications | Vicinal HO-C Cation Exchange diol | HPLC HO-C | Boronate Affinity Chromatography Immunoaffinity R. L. Garlick; et al. Characterization of glycosylated hemoglobins. Relevance to monitoring of diabetic control and analysis of other proteins. J Clin Invest 71 (5):1062-1072, 1983 Commercial Glycated Hemoglobin Assays 1950 1960 1970 1980 1990 2000 2010 2020 Fast Hb AIc and DCCT Sickle AIc Diabetes Feasibility Complications Boronate Affinity | Chromatography Vicinal Add mixture of HO-C glycated and diol non-glycated | hemoglobins Doesn’t HO-C Hemoglobins modified by vicinal measure diols bind (G, glycated) labile A1c | Non-diol hemoglobins pass through (N, non-glycated) R.
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