Hemoglobin A1c in the Diagnosis and Management of Diabetes

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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”

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)

Fast Hb AIc and DCCT Sickle AIc Diabetes Feasibility Complications

Cation Exchange HPLC

Boronate Affinity Chromatography

Immunoaffinity Doesn’t measure Antibodies made from labile A1c synthetic N-terminal beta globin peptides or purified A1c chromatography fractions What do these assays measure?

Whatever is present in Cation Exchange A1c the A1c peak; modified Val1 HPLC

Whatever is present in the bound fraction; Boronate Affinity vicinal diols on any Chromatography amino group

Whatever the antibody Immunoaffinity recognizes National Glycohemoglobin Standardization Program 1950 1960 1970 1980 1990 2000 2010 2020

Fast Hb AIc and DCCT NGSP Sickle AIc Diabetes Feasibility Complications DCCT Bio-Rex 70 Cation Exchange Reference Method

Cation Exchange HPLC A 1c Boronate Affinity Chromatography

Immunoaffinity National Glycohemoglobin Standardization Program 1950 1960 1970 1980 1990 2000 2010 2020

Fast Hb AIc and DCCT NGSP Sickle AIc Diabetes Feasibility Complications Children’s Hospital Studies CIEF for hemoglobinopathies 1950 1960 1970 1980 1990 2000 2010 2020

Fast Hb AIc and DCCT NGSP Sickle AIc Diabetes Feasibility Complications

Capillary Electrophoresis cIEF – SickleSickle Cell Trait 0.08 A0 Capillary Capillary S Inlet 50 µm i.d. Outlet 0.06 27 cm long Detector

4 Sample 4

0.04 Injection Injection A

A Anode Cathode 0.02 Buffer Autosampler Buffer A2 F A1c ++ High Voltage -- 0.00 Power Supply 5 6 7 8 9 10 11 12 13 14 15 Minutes

A1c

Hempe JM, Craver RD. Quantification of hemoglobin variants by capillary isoelectric focusing. Clin Chem 40:2288-95, 1994 Children’s Hospital Studies CIEF for hemoglobinopathies 1950 1960 1970 1980 1990A1c 2000 2010 2020

Fast Hb AIc and DCCT NGSP Sickle AIc Diabetes Feasibility Complications cIEFcIEF CorrelationCorrelation cIEF ReferenceReference Range 25 45 cIEF cIEF Normal subjects Age >1 y 20 6 h incubation 18 h incubation n 124 ) ) Mean 6.8% 30 SD 0.8 Range 5.3 - 8.4% cIEF

cIEF 15 ( ( 1c 1c 1c Correlation study A A 10 15 n 139 Mean 12.7%

Hb Mean 12.7% Hb % % 5 y =1.07x + 1.28 y =1.33x - 1.40 r2=0.94, n=40 r2=0.95, n=21 0 0 Number of ObservationsNumber 0 5 10 15 20 5 10152025 of ObservationsNumber 4 6 8 1012141618202224 % Hb A1c (HPLC - Overnight incubation) % Hb A1c vs DCCT Assay

Hempe JM, Vargas A, Craver RD. In: Petersen JR, Mohammad AA, eds. Clinical and Forensic Applications of Capillary Electrophoresis. Totowa, NJ: Humana Press, 2001:145-63 Hemoglobin Glycation Index CIEF for hemoglobinopathies 1950 1960 1970 1980 1990A1c 2000 2010 2020

Fast Hb AIc and DCCT NGSP Sickle AIc Diabetes Feasibility Complications

Comparison of A1c and MBG in Children

A1c and Hemoglobin Glycation Index CIEF for hemoglobinopathies 1950 1960 1970 1980 1990A1c 2000 2010 2020

Fast Hb AIc and DCCT NGSP Sickle AIc Diabetes Feasibility Complications

Wide population Comparison of variation A1c and MBG in Children 7-point profile sets A1c and The DCCT Research Group. Diabetes Control and Complications Trial (DCCT): results of feasibility study. Diabetes Care 10 (1):1-19, 1987 2525 ChildrenChildren’’ss HospitalHospital AA 2020 Study:Study: 1515 2.3 years 1010 2.3 years 55 128128 childrenchildren 2525 TypeType 11 diabetesdiabetes 2020 BB % HbA1c % HbA1c HbA1cHbA1c levelslevels areare 1515 High HGP Moderate influencedinfluenced bbyy factorsfactors inin 1010 HGP addition to glucose 55 Low HGP addition to glucose Three individual patients Hemoglobin Glycation 00 100100 200200 300300 400400 Hemoglobin Glycation MeanMean BloodBlood GlucoseGlucose (mg/dl)(mg/dl) PhenotypesPhenotypes Hempe et al. J Diab Comp 16:313,2002 HemoglobinHemoglobin GlycationGlycation IndexIndex HGIHGI == ObservedObserved -- PredictedPredicted HbA1cHbA1c PredictedPredicted HbA1cHbA1c == (MBG(MBG ×× slope)slope) ++ interceptintercept 3030 2525 HGI>0HGI>0 2020 HGI=0HGI=0 1515 % HbA1c % HbA1c 1010 HGI<0HGI<0 55

00 100100 200200 300300 400400 500500 600600 MeanMean BloodBlood GlucoseGlucose (mg/dl)(mg/dl) DiabetesDiabetes ControlControl andand ComplicationsComplications TrialTrial (DCCT)(DCCT)

1515 Moderate HGP AA 150150 1010 Low HGP 100100 High HGP 55 First 100 patients 5050 Frequency 1515 Frequency BB High HGP % HbA1c % HbA1c Moderate 00 1010 HGP --2.22.2 --1.21.2 --0.20.2 0.80.8 1.81.8 2.82.8 HGI 55 Low HGP Three individual patients Hypothesis: High HGP patients 0 100 200 300 400 are more susceptible to Mean Blood Glucose (mg/dl) microvascular complications n=1441 DiabetesDiabetes ControlControl andand ComplicationsComplications TrialTrial (DCCT)(DCCT) 0.35 Retinopathy 0.30 Moderate HGP 0.25 150150 0.20 Low HGP 0.15 100100 High HGP 0.10 0.05 5050 Frequency Frequency 0.10 Nephropathy 0 0.08 0 --2.22.2 --1.21.2 --0.20.2 0.80.8 1.81.8 2.82.8 0.06 HGI Risk Probability Risk Probability 0.04 Conclusion: HGI reflects biological variation in A1c and 0.02 biological variation in A1c and complications risk due to factors other than mean blood glucose 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 McCarter et al. Diabetes Care 27: 1259, YearsYears ofof FollowFollow--upup 2004 HGI and Biological Variation in A1c Study Reference Patients A1c Blood glucose Children’s JDC 2002 Type 1 CIEF MBG - 30 d Hospital children meter data DCCT Diabetes Type 1 BioRex MBG - 7 pt Care 2004 adults HPLC profile set UKPDS ADA Type 2 BioRad Fasting 2005 adults HPLC glucose DirecNet Diabetes Type 1 NGSP MBG - CGM Care 2007 children Immunoassay Children’s Pediatric Type 1 NGSP MBG – Raw Hospital Diab 2010 children Immunoassay meter data LowModerate High 300 MBG 300 250 250

200 200

150 150

100 100 12 A1C 12 11 11 10 10 9 9

%8 mg/dl 8 7 7 6 6 4 HGI 4 2 2 0 0 % -2 -2 -4 -4 Sequential Clinic Visits Soros AA, Chalew SA, McCarter RJ, Shepard R, Hempe JM. Hemoglobin glycation index: a robust measure of hemoglobin A1c bias in pediatric type 1 diabetes patients. Pediatric Diabetes 11:455-461, 2010 Hemoglobin Glycation Index CIEF for hemoglobinopathies 1950 1960 1970 1980 1990A1c 2000 2010 2020 HGI IFCC Fast Hb AIc and DCCT NGSP Sickle AIc Diabetes Feasibility Complications

Diabetes Care 30(9):2399-2400, 2007 1. The NGSP cation exchange reference A1c method is non-specific 2. The IFCC developed a mass spectrometry based reference method that quantifies N-terminal hexapeptides of  globin modified by glucose 3. IFCC standardized results were 25% lower than NGSP standardized results 4. Recommended reporting A1c in mmol A1c/mol of Hb 5. And since MBG = A1c, consider reporting A1c as “estimated average glucose” - eAG Into Estimated Average Glucose Values. RJ. Translating theA1CAssay Heine D, Schoenfeld H, J,BorgR,Zheng Nathan DM, Kuenen

CGM + SMBG AG (mg/dl) G(gd)=2. b1 46.7 AG (mg/dl) =28.7xHbA1c– r 2 = 0.84,p<0.0001 HbA1c (%) Diabetes Care Average of4different assays 31:1-6, 2008 people the sameway inall A1c isrelated toMBG Underlying assumption: Diabetes Care 31:1-6, 2008 Diabetologia 51:904, 2008

eAG does not improve patient understanding of diabetes management

Brick, J. C. et al. (2009) A Randomized Comparison of the Terms Estimated Average Glucose Versus Hemoglobin A1C. Diabetes Educ. 35: 596-602. Hempe, J.M., Soros, A.A. & Chalew, S.A. Diabetes Care 33:1449, 2010

eAG = 28.7 A1c - 46.7 A1c 6% = eAG of 126 250 eAG>MBG MBG overestimates eAG MBG 225 225 10 eAG

175 Failure to reach 8 A1c (%) 175 target MBG Exceed target MBG/risk for mg/dl 150 7 150 hypoglycemia

125 MBG (mg/dl) 125 6

100 100 5

75 75 4 Low Moderate High Low Moderate High HGI Group HGI Group 225 MBG 10 A1c 200 9

175 Failure to reach 8 A1c (%) target MBG Exceed target 150 MBG/risk for 7 hypoglycemia

MBG (mg/dl) 125 6

100 5

75 4 American Diabetes Association. Standards of medical careLow in diabetes--2010.Moderate DiabetesHigh Care 2010;33 Suppl 1:S11-S61. HGI Group 225 MBG 10 A1c 200 9

175 Failure to reach 8 A1c (%) target MBG Exceed target 150 MBG/risk for 7 hypoglycemia

MBG (mg/dl) 125 6

100 5

75 4 Low Moderate High HGI Group American Diabetes Association. Standards of medical care in diabetes--2010. Diabetes Care 2010;33 Suppl 1:S11-S61. American Diabetes Association. Standards of medical care in diabetes--2010. Diabetes Care 2010;33 Suppl 1:S11-S61. Diabetes Care 32 (7):1327-1334, 2009

Non-proliferative retinopathy graded by fundus photography (n=28,000 from 9 countries) Diabetes Diabetes Diabetes A1c A1c Both Diabetes Both

Diabetes NG Diabetes FPG NG OGTT

van 't Riet E.; et al. Relationship between A1C and glucose levels in the general Dutch population: the new Hoorn study. Diabetes Care 33 (1):61-66, 2010. Diabetes Diabetes Diabetes A1c A1c Both Diabetes Both

Diabetes NG Diabetes FPG NG OGTT

van 't Riet E.; et al. Relationship between A1C and glucose levels in the general Dutch population: the new Hoorn study. Diabetes Care 33 (1):61-66, 2010. onic Chr

Acute

Hyperglycemia

Hypoglycemia Microvascular and Macrovascular Disease Coma and Death Metformin + sulfonylureas Insulin Hypoglycemia

Currie CJ, et al. Survival as a function of HbA1c in people with type 2 diabetes: a retrospective cohort study. The Lancet 2010;In Press, Corrected Proof. Diabetes Care 33:1025-1027, 2010 Translational Research Program

Hemoglobin Analysis Development of multidimensional hemoglobin separation techniques Identification of chemical modifiers Clinical Research Basic Research Prospective Analysis of Mechanisms of Mouse Diabetes Existing Non-enzymatic Model of Study Clinical Trial Hemoglobin Hemoglobin Data Glycation Glycation

Best way to use HGI association with Quantitative in vitro In vivo assessment HGI and clinical phenotype bioassay of non- of factors that hemoglobin and outcome in large enzymatic influence non- assays to populations hemoglobin enzymatic improve patient glycation hemoglobin care glycation ObjectivesObjectives

1.1. MBGMBG  A1cA1c 2.2. ConsiderConsider bothboth MBGMBG andand A1cA1c forfor moremore personalizedpersonalized patientpatient carecare 3.3. EvenEven expertexpert committeecommittee recommendationsrecommendations shouldshould bebe takentaken withwith aa graingrain ofof saltsalt

-Ind

S S It Takes a Long Time to Change Course

Funding Children’s Hospital RIC ADA, JDRF, P & C Carroll Foundation Collaborators Stuart Chalew, M.D. Bob McCarter, Sc.D. Alfonso Vargas, M.D. Ricardo Gomez, M.D. Randal Craver, M.D. Arlette Soros, M.D. Students Amanda McGeehee Rachel Shepard Allyson Bazarsky Thanks Children’s Hospital Lab Endocrine Nurses "An important scientific innovation rarely makes its way by gradually winning over and converting its opponents. What does happen is that its opponents gradually die out and that the growing generation is familiar with the idea from the beginning."

Max Planck 1858-1947