Defining HbA1c: the indispensable decision to approach measurement standardization
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1 Defining HbA1c: the indispensable decision to approach measurement standardization Jan-Olof Jeppsson IFCC WG for HbA1c standardization Clinical Chemistry, Malmö University Hospital Sweden 6 November, 2007 J.O. Jeppsson 1
2 Overview Biochemical background Purification of primary calibrators IFCC reference method Validation of the reference method J.O. Jeppsson 2
3 Electrofocusing of hemoglobins HbS HbA3 HbA1c HbAo HbF Intmed methb HbA2 J.O. Jeppsson 3
4 Contents of the HbA1c peak at ion exchange chromatography Glc α β Glc Carb carb α β α β J.O. Jeppsson 4
5 BioRex 70 Ion Exchange Chromatography NGSP/DCCT HbA1c HbA0 HbA1c HbA0 Goldstein et al in Clark WL,Larner J, Pohl SL eds Methods of Diabetes Research Vol 2; 1986:
6 Mono S Ion Exchange Chromatography of HbA1c HbA0 HbA1c HbF HbA1d HbA1a+b _α-chain, Jeppsson JO et al Clin Chem 1986;35: J.O. Jeppsson 6
7 IFCC WG-HbA1c Definition of analyte Development of primary calibrators Establishment of a validated reference method Developing a network of reference laboratories worldwide J.O. Jeppsson 7
8 Glycation sites of Hemoglobin β-chains: Yellow α-chains: Blue N-terminal Valine: red Lysine green Glutathione HbA 3 or HbA 1d HOOCCHCH 2 CH 2 CONHCHCONHCH 2 COOH NH 2 CH 2 SH J.O. Jeppsson 8
9 Preparation of calibrators Hemoglobin Cation exchange chromatography SP Sepharose HPLC, ph 6.2 HbA1c (crude) HbA0 (crude) Affinity chromatography Glycogel Boronate, ph 8.0 Cation exchange chromatography SP Sepharose HPLC, ph 6.2 HbA1c (pure) HbA0 (pure) J.O. Jeppsson 9 Finke A et al Clin Chem Lab Med 1998:36(5);
10 ESI/MS of HbA1c Da Non-glycated α-chain Da Mono-glycated β-chain J.O. Jeppsson 10
11 ESI/MS of HbA Da Non-glycated α-chain Da Non-glycated β-chain J.O. Jeppsson 11
12 Standard addition experiment with HPLC-ESI/MS 0.08 response(hba 0 ) / response(hba 1c ) Equals: 2.55 % HbA y= x R^2 = Fig. 13 Ratio: [HbA 0 ] / [HbA 1c ] J.O. Jeppsson 12
13 β-chains: Chains: Yellow α-chains: Chains: Blue N-terminal terminal Valine: Red Hexapeptide:Green HbA1c is the stable adduct of glucose to the N-terminal amino acid, valine of the β chain of hemoglobin J.O. Jeppsson 13
14 VHLTPE E Proteolytic Cleavage of β-chain by Endoproteinase Glu-C KSAVTALWGKVNVDEVNVDE VGGE ALGRLLVVYPWTQRFFE SFGDLSTPDAVMGNPKVKAHGKKVLGAFS KVLGAFS DGLAHLDNLKGTFATLSE LHCDKLHVDPE NFRLLGNVLVCVLAHHFGKE FTPPVQAAYQKVVAGVANALAHKYH J.O. Jeppsson 14
15 The Analytical Challenge Proteolytic cleavage of β-chain (146 amino acids) HbAo-peptide Glu-C Val His Leu Thr Pro Glu Glu Lys Ser... HbA1c-peptide Glc Val His Leu Thr Pro Glu Glu Lys Ser... J.O. Jeppsson 15
16 Photometric detection of peptides HbAo Hexapeptide HbA1c Hexapeptide C18 Chromatography J.O. Jeppsson 16
17 Flow Chart for Reference Methods Hemolysate Enzymatic Cleavage HPLC-Capillary Capillary Electrophoresis HPLC-Mass Spectrometry Approved by IFCC 2001 J.O. Jeppsson 17
18 ESI mass spectra of nonglycated and glycated βn-terminal hexapeptides Kobold U et al Clin Chem 1977: 43(10); J.O. Jeppsson 18
19 Two-dimensional separation of N-terminal hexapeptides of hemoglobin C 18 Chromatography Capillary Electrophoresis Non-glycated hexapeptide Glycated hexapeptide J.O. Jeppsson 19 Jeppsson JO et al Clin Chem Lab Med 2002;40(1):78-89
20 Calibration function for HPLC-CE y = x R 2 = (HbA1)/(HbA0).Target values J.O. Jeppsson 20
21 Possible Interferences The hexapeptide represent an unique sequence Hb S and C Acetylated and carbamylated hemoglobin Potassium cyanide Sodium azid- excluded J.O. Jeppsson 21
22 Hemoglobinopathies in the N-terminal N part Hb S Glu Val Hb C Glu Lys Hb Raleigh Val Ac-Ala Hb Okayama His Gln Hb Fukuoka His Tyr Hb Tyne Pro Ser J.O. Jeppsson 22
23 Possible Interferences The hexapeptide represent an unique sequence Hb S and C Acetylated and carbamylated hemoglobin Potassium cyanide Sodium azid- excluded J.O. Jeppsson 23
24 Comparison between a HPLC and IFCC reference method 14,00 13,00 12,00 11,00 10,00 9,00 8,00 7,00 6,00 5,00 4,00 3,00 2,00 1,00 y = 0,979x + 1,650 R 2 =0,994 0,00 0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00 9,00 10,00 11,00 12,00 13,00 % HbA1c IFCC Reference Method y=x Hoelzel W et al Clin Chem 2004:50;
25 Comparison between converted HPLC and IFCC reference method 13,00 12,00 11,00 10,00 9,00 8,00 7,00 6,00 5,00 4,00 3,00 y=x y= 0,94x y= 1,06x 2,00 1,00 0,00 0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00 9,00 10,00 11,00 12,00 13,00 IFCC Reference Method
26 Harmonization of HbA1c Designated Comparison Methods (DCM) National Glycohemoglobin Standardization Program(NGSP) for DCCT in US Japan (JDS/JSCC), a set of national calibrators and HPLC/KO500 Mono S (EQUALIS) ion exchange chromatography in Sweden J.O. Jeppsson 26
27 Comparison between the IFCC reference method and three designated comparison methods 12,00 NGSP = 0,92xIFCC + 2,15 10,00 DSM 8,00 6,00 NGSP Mono-S JDS 4,00 Mono S = 0,989xIFCC ,00 JDS = 0,927xIFCC+1,73 0,00 0,00 2,00 4,00 6,00 8,00 10,00 12,00 % HbA1c IFCC method Hoelzel et al Clin Chem 2004;50: J.O. Jeppsson 27
28 30 25 x 20 Frequency x x-2s x + 2s 5 0 2,5 2,6 2,7 2,8 2,9 3,0 3,1 3,2 3,3 3,4 3,5 3,6 3,7 3,8 3,9 4,0 4,1 4,2 % HbA1c IFCC:Tentative reference range: % (Non-diabetics-OGTT: ADA/WHO, n=120) J.O. Jeppsson 28
29 Summary I We defined the analyte HbA1c as the glucose adduct to the N- terminal amino acid, valine of the β-chain of hemoglobin Primary calibrators, pure HbA1c (monoglycated β-chain and non-glycated α-chain) and pure HbA0 (nonglycated β-chain and nonglycated α-chain) have been purified A reference method has been developed as follows: Hemoglobin is cleaved into peptides by the proteolytic enzyme endoproteinase Glu-C The resulting glycated and nonglycated N-terminal hexapeptides of the β-chain are separated from the crude peptide mixture by reversed-phase HPLC J.O. Jeppsson 29
30 Summary II The hexapeptides are quantified by mass spectrometry or capillary electrophoresis at ultraviolet detection HbA1c is determined as the ratio of the glycated and nonglycated β N-terminal hexapeptides of hemoglobin A strong correlation was obtained between IFCC-RM and all DCM methods The specific IFCC reference method gives HbA1c lower values than the DCM methods Reference range: % or mmol/mol J.O. Jeppsson 30
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