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Evaluation of hemoglobin AIc as a measure of diabetic control Thompson, Katherine Hirsch 1977

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The Evaluation of Hemoglobin Ale as a Measure of Diabetic Control by Katherine Hirsch Thompson B.A., Pomona College, 1968 A Thesis Submitted in P a r t i a l Fulfillment of the Requirements for the Degree of Master of Science i n the Division of HUMAN NUTRITION SCHOOL OF HOME ECONOMICS We accept t h i s thesis as conforming to the reguired standard, THE UNIVERSITY OF BRITISH COLUMBIA July, 1977 (c^) Katherine Hirsch Thompson, 1977 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the requirements fo r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e fo r reference and study. I f u r t h e r agree t h a t permiss ion fo r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . It i s understood that copying or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l ga in s h a l l not be a l lowed without my w r i t t e n p e r m i s s i o n . Department of Ucryr>C PfOTn&t^tiZS The U n i v e r s i t y o f B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 ftBSTBACT Diabetic i n d i v i d u a l s have been found to have consistently higher l e v e l s of a minor hemoglobin component, HbAIc, than non-diabetic i n d i v i d u a l s . Previous investigators have suggested that v a r i a t i o n in these high levels of HbAIc may be a r e f l e c t i o n of the degree of diabetic hyperglycemia, of hypertriglyceridemia in diabetes, and of diabetic c o n t r o l . To date, evaluation of HbAIc as a c l i n i c a l l y useful parameter has been hampered by the complexity of the method of measurement, the inconsistency i n ranges of normal values reported, and the lack of a broad data base for comparison with new r e s u l t s . This investigation began with a c r i t i c a l appraisal of the methods currently i n use for measurement of HbAIc, followed by a s i m p l i f i c a t i o n and standardization of the J assay. Then the levels of HbAIc in 16 non-diabetics and 47 diabetics were determined and the mean values for these 2 groups compared. The relationships between HbAIc leve l s i n the diabetics and selected c l i n i c a l data |fasting blood sugar, 24-hour urinary sugar, age,duration of i l l n e s s , dietary record, i n s u l i n dosage, and family history of diabetes) were examined. F i n a l l y , the degree of diabetic control i n each of the diabetic patients was estimated by the attending physician on a scale of 1 to 5 (1=very good, 2=good, 3=fair, 4=poor, 5=very poor) and was compared with the HbAIc measurement. Results of the investigation have shown that the i i i chromatographic measurement of HbAIc i s unusually sensitive to the pH of developers used and also somewhat variable with respect to the length of storage time and the o p t i c a l density at which samples are read. The comparison of mean values of HbAIc for diabetics and non-diabetics has confirmed the approximately twofold higher concentration of HbAIc in diabetics. S i g n i f i c a n t correlations were found between HbAIc and fasting blood sugar (r = .442), f a t content of diet (r=-. 300) , family history of diabetes (r=-.312) and degree of diabetic control (r=.529). Thus, HbAIc values tend to be higher in patients whose fa s t i n g blood sugar i s high, whose diet contains r e l a t i v e l y l i t t l e f a t , whose r e l a t i v e s are diabetic and /or whose diabetic control i s poor. Correlations between HbAIc and duration of diabetes, HbAIc and i n s u l i n dosage or HbAIc and 24-hour urinary sugar were not s t a t i s t i c a l l y s i g n i f i c a n t (r=-.131, r=-.264, r=„067, re s p e c t i v e l y ) . The HbAIc l e v e l appears to be an accurate r e f l e c t i o n of fasting blood sugar l e v e l s averaged over a prolonged period of time (r=.587). In conclusion, HbAIc leve l s were found to provide an objective measure of diabetic control. The improved assay method makes i t a p r a c t i c a l and valuable tool for the c l i n i c i a n as well as the investigator. Measurement of HbAIc le v e l s in diabetics presents a considerable advantage over currently a v a i l a b l e measures of diabetic control in assessing the long-term effectiveness of diabetic management. iv TABLE OF CONTENTS Abstract , i i Table of Contents ......................................... i v L i s t of Tables , v i L i s t of Figures ........................................... v i i Acknowledgements ................... ... ................... . v i i i CHAPTER I INTRODUCTION 1 II LITERATURE REVIEW 4 A. Hemoglobin Ale ............................... 4 B. Diabetic Control 13 C. Glycoproteins in Diabetes 18 D. Diet and Course of Diabetes , 19 III' MATERIALS AND SETHODS 2 3 A. Subjects 2 3 B. Preparation of Hemolysates 21 C. Preparation of Chromatographic Columns 25 D. Developers 26 E. Operation of Columns 26 F. Spectrophotometry ............................ 27 G. C l i n i c a l Data 28 H. Diabetic Control 2 8 I. S t a t i s t i c a l Treatment ........................ 29 IV' RESULTS . . . 30 V DISCUSSION 44 A. Standardization of Method 44 B. Increase of HbAIc i n Diabetic Patients ....... 46 V C. P o s i t i v e C o r r e l a t i o n between HbAIc and D i a b e t i c C o n t r o l ............................. , 46 D. Advantages of HbAIc as a Measure of D i a b e t i c C o n t r o l ............................. 49 E. Negative C o r r e l a t i o n between HbAIc and D i e t a r y Fat 50 VI SDHHABY AND RECOMMENDATIONS 51 A. Summary 51 B. Recommendations .............................. 53 REFERENCES CITED - . 55 v i LIST 0? TABLES Table Page I Assessment c r i t e r i a of diabetic control 14 II HbAIc values at 552 mu and 410 mu .................. 32 III Change in HbAIc values during storage of r e d - c e l l hemolysates at -20°C 33 IV Percent HfcAIc; i n non-diabetics and diabetics 3 5,36 V Selected c l i n i c a l data of diabetic patients ........ 37,38 VI Correlation analysis of HbAIc with selected c l i n i c a l data ............... 40 VII Protein, f a t and carbohydrate intake of diabetic patients as percent of t o t a l calories 42 v i i LIST OF FIGURES Figure Page 1. Glycosylation of HbAII to form HbAIc 6 2., Chromatography of human hemoglobin on Bio-Eex-70 cation exchange resin 31 3. Correlation of the degree of diabetic control with %HbAIc ................ ............................ 41 vi i i ACKNOWLEDGEMENTS I would l i k e to thank my advisor. Dr. Joseph Leichter, f o r his d i r e c t i o n and assistance duing the course of t h i s study. My thanks are due to Dr. Wah Jun Tze for permission to study the records of patients under his care as well as f o r his h e l p f u l suggestions and cooperation i n the experimental portion of t h i s study, and to Dr. Nancy Schwartz f o r advice and c r i t i c i s m i n preparation of the thesis. I am also grat e f u l to B i l l Thompson for advice and assistance in computer programming and s t a t i s t i c a l analysis of the r e s u l t s . 1 CHAPTER I INTRODUCTION One of the most pressing needs in c l i n i c a l management of diabetes mellitus i s a r e l i a b l e method of monitoring the degree of diabetic c o n t r o l [ 1 - 3 ] . A generally accepted d e f i n i t i o n of diabetic control i s the closeness of approach to normal metabolic hoaeostasis with p a r t i c u l a r reference to carbohydrate metabolism i n diabetics [ 4 , 5 ' ] . . Continuous maintenance of near-normal blood sugar would be considered excellent control in a diabetic patient. The importance of diabetic control in the development of the secondary complications of diabetes mellitus, including retinopathy, nephropathy, neuropathy and macroangiopathy, i s a matter of much concern to patients who have diabetes, t h e i r f a m i l i e s , physicians and medical investigators. Currently available parameters of diabetic control f a l l far short of what i s needed to determine the r e l a t i o n between control and complications, or even to assess accurately the degree of control for purposes of optimizing diabetic management [ 4 ] . The measurement of Hemoglobin Ale (HbAIc) l e v e l s i s p o t e n t i a l l y a valuable t o o l i n the assessment of diabetic control. HbAIc i s a minor hemoglobin component of normal adult erythrocytes whose concentration i s elevated approximately twofold i n patients with diabetes mellitus [ 6 - 1 1 ] . I t i s one of three negatively charged minor hemoglobin components (HbAIa,b,c) that are eluted by cation exchange resin chromatography before 2 the main hemoglobin A peak (HbAII) [12-15], HbAII (frequently referred to i n the l i t e r a t u r e as HbA) constitutes the major component of normal human hemoglobin, the remainder being composed of HbAIa, HbAIb, HbAIc, HbA2 and HbF [ 13]. Studies thus far reported suggest that HbAIc l e v e l s are c l o s e l y correlated with plasma glucose lev e l s , both i n humans and i n experimental animals [16-18]. HbAIc also appears to be correlated with t r i g l y c e r i d e l e v e l s and urinary sugars [17,19]. Elucidation of the structure of HbAIc has shown that i t i s i d e n t i c a l to the major hemoglobin component, HbAII , except for the presence of a hexose group linked to the ti-ter minal amino acid (valine) of the beta chains via a Sch i f f base [20,21], Biosynthetic studies of the glycohemoglobin have shown that the glycosylation occurs nonenzymatically throughout the l i f e t i m e of the red c e l l as a postsynthetic modification of HbAII [22]., HbAIa and HbAIb apppear to be intermediates i n t h i s process [23 ]. Development of a technique of measurement of HbAIc f o r c l i n i c a l use has been hampered by the lack of agreement i n results obtained by only s l i g h t l y d i f f e r e n t procedures and by the need f o r a broader data base from which to evaluate r e s u l t s . Normal values f o r HbAIc le v e l s reported in the l i t e r a t u r e vary from 3.1 to 5.3% [7,8,10,24] and values for diabetics range from 6 to 12% and higher for those i n ketoacidosis [ 7,8, 11, 10,25]. A study of 5 patients by Koenig et a l . [ 8 ] , comparing glucose and HbAIc l e v e l s before and during control of their diabetes, i s 3 p r o v o c a t i v e . In these a d u l t d i a b e t i c s r e s t o r a t i o n of good c o n t r o l by d i e t and e x e r c i s e was accompanied by reduced HbAIc l e v e l s w i t h i n one or two months of reduced sugar l e v e l s i n u r i n e and plasma. I t would appear t h a t HbAIc l e v e l s could p o s s i b l y be an i n t e g r a t e d measure of the degree of d i a b e t i c c o n t r o l over the p r e v i o u s weeks and months, and as such would avoid the p i t f a l l s of blood sugar determinations which may vary widely w i t h i n a s h o r t p e r i o d of time, and of u r i n a r y sugar l e v e l s , which may n o t be comparable between p a t i e n t s and a l s o f l u c t u a t e c o n s i d e r a b l y [1.3... The purpose o f t h i s study was t h r e e f o l d : 1) t o evaluate the procedure f o r d e t e r m i n a t i o n of HbAIc and t o develop a c o n s i s t e n t method of a n a l y s i s for c l i n i c a l use, then t c compare HbAIc l e v e l s i n d i a b e t i c s with those i n n o n - d i a b e t i c s , 2) to examine , w i t h i n the d i a b e t i c p o p u l a t i o n , the r e l a t i o n s h i p s between HbAIc l e v e l s and other standard parameters of d i a b e t i c c o n t r o l , such as f a s t i n g blood glucose, u r i n a r y glucose l e v e l s , presence of secondary c o m p l i c a t i o n s or a s s o c i a t e d c o n d i t i o n s , and composition of d i e t , and 3) to examine the r e l a t i o n s h i p between HbAIc and an o b j e c t i v e o v e r a l l measure of the degree of d i a b e t i c c o n t r o l used, by the M e t a b o l i c I n v e s t i g a t i o n U n i t a t C h i l d r e n *s H o s p i t a l . a CHAPTER II LITERATURE REVIEW A. Hemoglobin Ale Hemoglobin Ale (HbAIc) was discovered as one of several minor components present in normal, r e c r y s t a l l i z e d human hemoglobin, which could be i s o l a t e d by column chromatography [15]. The presence of these minor components, comprising roughly 10% of t o t a l heme proteins, was disturbing i n that homogeneity of hemoglobin samples could no longer be assured simply by r e c r y s t a l l i z a t i o n . A method was devised to i s o l a t e the minor components on synthetic cation exchange res i n IRC-50, so that a homogeneous major component could be obtained. Clegg and Schroeder [12] subsequently used t h i s method to study the v a r i a t i o n in lev e l s of the minor components in adult human hemoglobin of normal and phenylketonuric i n d i v i d u a l s . They reasoned that whatever the source and function of these minor components, any stress to which an i n d i v i d u a l was subjected might be expected to aff e c t the l e v e l s of these components. However, they noted no s i g n i f i c a n t differences in Hbftlc l e v e l s i n normal and phenylketonuric i n d i v i d u a l s . Subsequently, extensive biochemical studies concerning the nature of HbAIc were conducted by Holmquist and Schroeder [21,22], with res u l t i n g elucidation of the structure. Hemoglobin Ale was shown to be a Schiff base condensation product between one molecule of HbAII and one molecule of a 5 ketone or aldehyde (Figure 1) . The S c h i f f base {alpha-hydroxyaldimine linkage) s h i f t s to a beta-ketoamine linkage (Amadori rearrangement) which i s stable and the reaction i s r e l a t i v e l y i r r e v e r s i b l e [26], Holmguist and Schroeder £22] also showed that the minor hemoglobin component i s synthesized i n v i t r o concurrently with HbAII and i s a normal constituent of a l l red c e l l s , i r r e s p e c t i v e of t h e i r physiological age. The sole difference between the major hemoglobin component and HbAIc was found to be the presence of a blocked N-terminal amino acid (valine) i n the minor component. Bookchin and Gallop [20] further demonstrated that the aldehyde or ketone group was i n fact a hexose molecule, guite probably linked to both beta chains of the hemoglobin tetramer, thus giving the structure as studies that HbAIc has, as the amino terminus of the beta-chain, 1 deoxy, l-(N-valyl) fructose which can be produced i n v i t r o when phosphorylated glucose or mannose are introduced into HbAII solution. fiahbar [28] was the f i r s t investigator to conclusively l i n k HbAIc to diabetes mellitus., A twofold increase i n HbAIc was observed i n diabetics, as compared to normal adults. This work was extended fey Bahfcar et a l . , [6] and T r i v e l l i et a l . . [7] to include a larger sample of both juvenile and adult diabetics, with f a i r l y consistent r e s u l t s : diabetics appeared to have roughly twice the l e v e l s of miner components as normals. In t h i s series of adult diabetics, most of whom had cardiovascular disease, no difference in l e v e l s of HbAIc was found between HbAIc. Koenig et a l . [27] concluded from t h e i r 0 0 C H 0 (J-chain of C H = N - C H 2 - C - N - C H 2 - - - C H 2 - N - C H 2 - G - N - C H 2 - - -, ' * hemoglobin ' AmnHnri ' (CHOH), - •(CHOH), A m a q o r i • C=0 I ^ I rearrangement j CH 2 OH CH ?OH (CHOH) . I glucose Schiff's base CH o0H or 2 mannose FIGURE 1: GLYCOSYLATION OF HbAII TO FORM HbAIc (7l 7 those who had recent cardiac or cerebral i n f a r c t i o n s and thase who had not. An interpretation of no r e l a t i o n s h i p between HbAIc and diabetic complications was unfortunately inferred from these r e s u l t s . The HbAIc values obtained for 7 diabetic patients with ketoacidosis and f o r 6 diabetics with intercurrent i l l n e s s were higher (13.3% and 14.3%, respectively) as compared to 11.1% f o r the t o t a l diabetic population of 75; however, t h i s was not considered s i g n i f i c a n t . The method of separation of the minor hemoglobin components remained a f a i r l y tedious and time-consuming one, despite modifications, and interest i n the topic waned for several years. Animal studies by Koenig and Cerami [1.6] paved the way f o r the expanding c l i n i c a l and research i n t e r e s t i n HbAIc which i s continuing to the present. In adult diabetic mica, the formation of increased amounts of HbAIc, i n analogy to the human si t u a t i o n , appeared to be the d i r e c t r e s u l t of the diabetic state of the animal. The biosynthesis of the glycohemoglobin occurred as a postsynthetic modification of HbAII throughout the l i f e of the red c e l l . In the diabetic mice, which had normal amounts of HbAIc at weaning, the increase of HbAIc occurred approximately 4 weeks af t e r the onset of r i s i n g plasma glucose levels. By i n j e c t i n g radiolabelled hemoglobin in t o age-matched diabetic db/db and wild-type +/• mice, Koenig and Cerami [ 1 6 ] were also able to demonstrate the difference in rate of synthesis of the glycohemoglobin i n normal and diabetic mice. HbAIc synthesis was shown to occur 2.7 times fa s t e r in diabetic than wild-type mice, even i f the red c e l l s in the diabetic 8 originated in a wild-type mouse. This result indicated conclusively that rate of synthesis and l e v e l of HbAIc were related to the genotype and le v e l s of carbohydrate metabolites of the rec i p i e n t mouse and not with those same parameters of the donor i n d i v i d u a l . Koenig et al.£l7] extended these studies to include a variety of phenotypically diabetic mice [C57BL/KsJ-db/db, C57Bl/KsJ-ob/ob, C57BL/6J-db/db, and alloxan- and streptozotocin-treated mice); i n a l l cases, the HbAIc leve l s were found to be elevated approximately twofold over l e v e l s i n normal wild-type mice., To determine whether the HbAIc concentration correlated with plasma glucose concentration, the data from C57BL/KsJ- db/db mice and mice with chemically-induced diabetes were divided into low and high plasma glucose l e v e l groups, (n=12 for each group) and the mean HbAIc l e v e l s for each group were determined. Although the mean blood glucose concentrations for the two groups were s i g n i f i c a n t l y d i f f e r e n t (p<.01), the mean HbAIc le v e l s for the two groups were not. However, these r e s u l t s cannot be construed as conclusive evidence that HbAIc i s not correlated with blood glucose concentrations, since both groups showed ex t r a o r d i n a r i l y high glucose l e v e l s (470+0.22 mg/100ml compared with 632±3mg/100ml), i n no way comparable to the human diabetic s i t u a t i o n . The data from C57BL/KsJ-db/db mice were also divided into 2 groups: obese and noh- obese, with a s t a t i s t i c a l l y s i g n i f i c a n t difference i n mean body weights (p<.001) for the two groups. However, once again, the HbAIc l e v e l s were not s i g n i f i c a n t l y d i f f e r e n t . On 9 the other hand, i n the temporary diabetes of the C57B1/6J- db/db mice, in which remission of diabetic symptoms i s spontaneous, HbAIc l e v e l s were seen to d i r e c t l y p a r a l l e l the r i s e and f a l l of plasma glucose lev e l s , with a four-week lag period for the HbAIc le v e l s , HbAIc appeared to be an accurate c o r r o l l a r y indicator of the onset and remission of diabetic symptomatology. Koenig et a l . [ 9 ] also attempted to correlate HbAIc leve l s with other changes in glycoprotein l e v e l s , such as glomerular basement membrane thickening. Measurements of muscle c a p i l l a r y basement membrane thickness varied from 612+141 to 2975±830 A i n the 21 diabetics studied. The HbAIc l e v e l s in these same patients varied from 4.49 to 10.84% of t o t a l hemoglobin . A correlation c o e f f i c i e n t of r=0.15 was obtained, but was not s t a t i s t i c a l l y s i g n i f i c a n t |p>0.05). The short time horizon (a few months) of t h i s study renders the re s u l t s questionable, however. D i s t i n c t i v e changes i n basement membrane thickening may take many years, as noted by the investigators [ 9 ] . Nonetheless, in t h i s same series of experiments [32], investigators were able to demonstrate that HbAIc le v e l s correlated p o s i t i v e l y with glucose tolerance test response |r=0.82, p<0.001, n=22 diabetic patients); or, more s p e c i f i c a l l y , with the area under the curve of a glucose tolerance test . The question had arisen of whether HbAIc le v e l s might be an inherited component of the diabetic syndrome, rather than a quantitative r e f l e c t i o n of disordered carbohydrate metabolism. 10 A study by T a t t e r s a l l et a l . [29] was p a r t i c u l a r l y enlightening in t h i s regard. In t h i s study, the mean values of proportions of H'bAI (a*b+.c) (equivalent to (HbAI(a + b) and HbAIc together) i n 16 sets of i d e n t i c a l twins discordant for diabetes were compared to those i n 10 sets of i d e n t i c a l twins concordant f o r diabetes. In the twins discordant for diabetes, mean values f o r HbAI(a+b+c) d i f f e r e d s i g n i f i c a n t l y i n the two members of the pair: 10.4±0.74% for the diabetic twins, and 6.85±0.33 for the non-diabetic twins. By contrast, no marked differences i n those same le v e l s were noted between members of twin pairs concordant for juvenile onset diabetes, with the ove r a l l mean l e v e l of HbAI(a*b+c) observed being 11.4%. The mean value for unaffected twins of discordant pairs was not s i g n i f i c a n t l y d i f f e r e n t from that of controls. The l e v e l of HbAI(a+b+c) in diabetic twins was not correlated with blood sugar measured at the time samples were taken f o r the hemoglobin studies. In addition, they discovered that storage of normal blood to which sugar had been added to achieve a concentration of 1400 mg% for 14 days did not re s u l t in elevation of HbAI(a+b+c) l e v e l s , as measured p e r i o d i c a l l y during that time. Altogether the study l e f t no doubt that HbAI(a>b+c) l e v e l s did i n some way r e f l e c t the presence of the diabetic syndrome, not just hyperglycemia, and were not simply a concomitant r e f l e c t i o n of inherited genetic predisposition towards development of diabetes. The f i r s t longitudinal study of the re l a t i o n s h i p of HbAIc to diabetic control was that of Koenig et a l . [ 8 ] . Five diabetic patients known to be i n poor control were hospitalized for up to 11 2 months, during which time t h e i r diabetes was brought under control by c a r e f u l regulation of diet and exercise, and both glucose l e v e l s and HbAIc l e v e l s were measured p e r i o d i c a l l y . In a l l cases, reduced fasting blood sugar l e v e l s , glucose lev e l s before and af t e r meals, and urinary sugar l e v e l s were pa r a l l e l e d by reduced HbAIc l e v e l s . The temporal r e l a t i o n between urinary sugar levels and HbAIc l e v e l s demonstrated c l e a r l y the several week lag period between improved carbohydrate metabolism i n the patient and reduced glycohemoglobin l e v e l s . A s i g n i f i c a n t c o r r e l a t i o n {r=0.81, p<.01) was found between HbAIc l e v e l s and "glucose brackets" (summations of blood glucose l e v e l s one hour pre- and post-prandially f o r a l l meals taken) f o r t h i s group of patients. Three additional patients have since been studied [18], with e s s e n t i a l l y the same r e s u l t s . Paulsen and Koury [11] corroborated these r e s u l t s i n juvenile d i a b e t i c s , measuring HbAIc le v e l s in 4 children who were a c i d o t i c p r i o r to i n s u l i n treatment, and were then followed up as t h e i r condition improved. Their HbAIc le v e l s f e l l from an average of 13.5% to 10.8% within 1 to 7 months, with no s i g n i f i c a n t change noted between HbAIc l e v e l s during ketoacidosis and those measured 4 to 10 days after the st a r t of i n s u l i n therapy. Once again, HbAIc concentration appeared to represent an integrated measure over time of the ove r a l l state of carbohydrate metafoolism of the patient, over the previous weeks and months. A reduction in HbAIc was found to be contingent upon the synthesis of new red c e l l s i n an environment l e s s favorable to formation of the HbAIc modification, though 12 recent studies of Peterson et a l . [18] have shown that glycosylation i s at le a s t p a r t i a l l y reversible i n vivo. The studies of Fitzgibbons et al.[24] demonstrated conclusively that, i n f a c t , the HbAIc concentration i n older red c e l l s was s i g n i f i c a n t l y higher than that i n young c e l l s . In th e i r experiments, erythrocyte f r a c t i o n s were obtained by centrifugation i n isopycnic concentrations of dextran and were then separately analyzed . for minor and major hemoglobin components. HbAIc l e v e l s i n 15 controls and 14 diabetics were determined, with the o v e r a l l mean for controls being 4.6±0.9% HbAIc, and for diabetics, 8.6±3.1%. In the 10% youngest RBC1s for controls, HbAIc constituted 3.1+0.8% and for diabetics, 5. 1±2.1%, whereas i n the 10% oldest BBC's, HbAIc constituted 6.0±1.1% i n the controls and 10.1+3.7% in diabetics. These r e s u l t s are well worth considering i n interpreting the r e s u l t s of Schwartz et al.£25], who studied HbAIc l e v e l s i n normal, gestationally diabetic, and diabe t i c women. Of pa r t i c u l a r i n t e r e s t was the finding that pregnant diabetic women had remarkably d i f f e r e n t HbAIc l e v e l s from nonpregnant diabetic women (8.46% i n the former group as compared to 12.77% in the l a t t e r ) . This could quite possibly be due to the greatly increased percentage of new red blood c e l l s in pregnant women, rather than to some 'protective mechanism* or vastly improved dietary control, as postulated by the authors., In addition, they found that the mean HbAIc l e v e l f o r gestationally diabetic women was unusually high (8.77%), while non-diabetic pregnant obese women were no d i f f e r e n t from normals in t h i s respect 13 (6.97% average in this study). B. Diabetic Control One of the key problems i n treatment of diabetic patients today i s determination of diabetic control [30]. As stated by Knowles [ 2 ] : "The most doubtful measurement of a l l i s that of c o n t r o l . Herein, a l l studies f a l t e r , f or i t i s impossible to determine with certainty the chemical state of patients during t h e i r day-to-day l i f e and a c t i v i t y . " T y p i c a l l y , diabetic control i s monitored by periodic determinations of plasma glucose and urine reducing sugar concentrations [31]. Examples of the c r i t e r i a used f o r d i f f e r e n t i a t i n g diabetic patients into excellent, good, f a i r , and poor categories are presented i n Table I [3,32,33,34,35], A c r i t i c a l study by Malone et a l . [ 3] of such c r i t e r i a indicated a considerable degree of inconsistency. In a study of 220 children attending diabetic camp, investigators found that 50% of random urinary sugar values determined by the children d i f f e r e d from those obtained by laboratory technicians on the same specimens using the same t e s t methods (1- and 2-drop C l i n i t e s t ) . Only 54 children successfully completed a 2 4-hour urine c o l l e c t i o n . Of these, 18 could be c l a s s i f i e d as being i n good control according to urinary sugar lev e l s (less than 25 gm/ 24 hr), 11 of these 18 were also in good control according to fasting plasma glucose, and of these 11, 7 had poor control TABLE I Assessment c r i t e r i a of diabetic control (modifier! from reference 3). CRITERIA CATEGORIES E x c e l l e n t Good F a i r P o o r Percent of tests for fastin g blood glucose which were <100 mg/100 ml 100 85 70 <70 Percent of tests for 2-hour postprandial which were <150 mg/100 ml 100 85 70 <70 Percent of urine specimens free of sugar 100 75 50 <50 Urine glucose (gm/24 hr) 0-25 25-50 MOO Urine acetone (mg/100 ml) 0-10 • >30 15 applying the same c r i t e r i a to a plasma glucose measured l a t e r i n the day. The investigators concluded that d e f i n i t i o n of good or poor diabetic control s o l e l y on the basis of these c r i t e r i a was an "exercise in mass delusion" £30 ]. In the experience of Drash [ 5 ] , the majority of patients evaluated on an in-patient basis had periods of good, f a i r and poor control during any given 24-hour period. They also found a high degree of u n r e l i a b i l i t y and v a r i a b i l i t y over time i n patients' records of reducing sugar concentration i n random urine samples. A lack of co r r e l a t i o n between the apparent degree of control as determined by urinary glucose and that obtainable from records of fasting and post-prandial glucose i s also related to variable renal threshold l e v e l s f o r glucose i n dif f e r e n t patients £36], The considerable f l u c t u a t i o n (as much as 10 times normal v a r i a b i l i t y ) which has been observed in diabetic patients compared to normals in leve l s of plasma glucose, l a c t a t e , pyruvate, ketone bodies, alanine, free f a t t y acids, i n s u l i n , glucagon and growth hormone i s i n i t s e l f i n d i c a t i v e of derangement of the carbohydrate metabolism i n t h i s disease and may, i n fact, be more important in terms of long-term prognosis than the absolute values of these metabolites [ 1, 37,38]. Two p r i n c i p a l strategies have been used i n attempts to circumvent the aforementioned d i f f i c u l t i e s : 1) use of numerical data for c r i t e r i a of diabetic control i n place of d i v i s i o n s i n t o categories of good, poor, etc., and 2) use of some combination 16 of available diagnostic c r i t e r i a to obtain an assessment of control. An example of the former are the studies by Caird [39], who used 'glycosuria percentage', the percentage of c l i n i c attendances at which a random urine te s t showed glycosuria of 2% or more to Benedict's test or C l i n i t e s t . . Szabo et a l . [40] used numerical values for fasting blood sugar, weight, ketonuria and glycosuria, with no attempt to combine these variables into one f a c t o r . Examples of studies which u t i l i z e a combination of variables to obtain an o v e r a l l estimate of diabetic control include those of Keiding et a l . [41] and C o l l y e r and Hazlett [42]. Unfortunately, both of these methods rely on a f a i r degree of subjective evaluation., None of these methods can be considered as a r e l i a b l e , objective measure of control such as i s needed for optimal diabetic management and for inv e s t i g a t i v e purposes. Caird et a l . [43] have pointed out that the measures of control most l i k e l y to be relevant i n the assessment of the relationship between complications and degree of co n t r o l are those which take into account both the duration and the degree of deviation from continuous metabolic normality rather than those which only assess the s i t u a t i o n at one point i n time. However, they did not consider i t possible to measure a metabolic abnormality continuously for periods of years. The assessment of diabetic control i s central to the continuing controversy over the importance of control in 17 delaying or preventing the development of the secondary complications of diabetes. Secondary complications of diabetes, including retinopathy, neuropathy, nephropathy and atherosclerosis, account for the major morbidity and mortality associated with diabetes mellitus, es p e c i a l l y of the juvenile-onset diabetes [44,45,46]. Though i n s u l i n therapy has vastly improved the outlook f o r diabetics [47], the mean duration of l i f e after onset of diabetes i s s t i l l only 18.2 years, with vascular disease accounting for 77% of the deaths [48], Advocates of * t i g h t * diabetic control as a means of delaying the onset and reducing the severity of the secondary complications of diabetes contend that s u f f i c i e n t studies have been done to show a d e f i n i t e p o s i t i v e c o r r e l a t i o n between degree of diabetic control and improved prognosis for juvenile as well as adult diabetics [49,50,51], Others disagree, contending that regardless of the degree of control, secondary complications do develop to a greater or lesser degree, and that even on " f r e e " , i . e . , u n r e s t r i c t e d and unprescribed, diets, there i s very l i t t l e difference in the time of onset or the eventual severity of these complications [52,53,54]. In some studies [ 55,56,57 ], concerned s p e c i f i c a l l y with the r e l a t i o n s h i p between development of retinopathy and/or nephropathy, progression of these microvascular changes seemed d e f i n i t e l y to be worse under poor control; however, at best they show an association between poor control and poor prognosis, and vice versa: no cause and effect r e l a t i o n s h i p can be construed from such data. Current attempts to r e l a t e development of complications to diabetic control leave 18 unexplained those exceptional cases of apparently good control who develop severe, early complications and, conversely, of apparent poor control who escape these complications [58]. C. Glycoproteins i n Diabetes As HbAIc i s a glycoprotein, i t i s of interest to consider known variations i n other glycoproteins in diabetes. That there are increased amounts of various glycoproteins i n vascular membranes and in plasma of diabetics has been amply demonstrated [59,60]. The glycosylation appears to be insulin-independent and highly dependent upon substrate concentration [60]. Decreased u t i l i z a t i o n of insulin-requiring g l u c o s e - u t i l i z i n g pathways due to in s u l i n - d e f i c i e n c y i n diabetes mellitus may, i n fa c t , lead to o v e r - u t i l i z a t i o n of insulin-independent glucose-u t i l i z i n g pathways, such as glycoprotein synthesis £61]. The enzymes required f o r protein glycosylation are f a i r l y non-s p e c i f i c glucosyltransferases which function subsequent to protein synthesis [62]; rate of glycoprotein formation being dependent on at least one known substrate: glucose-6-phosphate [63]. , Presence of a divalent cation, optimally Mn(II) , i s also required [62]., In the diabetic state, especially i n poorly-controlled diabetes, an enhanced a v a i l a b i l i t y of glucose-6-phosphate would be expected due to increased gluconeogenesis. This same substrate appears to be one of key importance i n the synthesis of HbAIc [23]., Studies of the glucosyltransferase which i s involved i n the assembly of the hydroxylysine-linked disaccharide units of the glomerular basement membrane have 19 shown that the a c t i v i t y of t h i s enzyme i s elevated i n alloxan-diabetic r a t s . a c t i v i t y can be brought back to normal by administration of i n s u l i n [64]. Such a l t e r a t i o n s i n the biochemical composition of blood and tissues can be expected to r e s u l t i n altered physical properties of b i o l o g i c a l structures [65]. Most c h a r a c t e r i s t i c and consistent among the various lesions of diabetic microangiopathy i s an increased permeability of the basemant membrane [ 66 ]. D. Diet and Course of Diabetes Changes or differences in the proportions of f a t , protein and carbohydrate in diabetic diets have been shown to a f f e c t the frequency and severity of diabetic secondary complications, especially of large vessel disease, but also to a lesser extent in small blood vessel complications (microangiopathy) [ 67,68,69,70]. In a study by Albrink et a l . [69], the serum l i p i d records of 139 patients seen between 1931 and 1961 i n New Haven, Connecticut were reviewed., During t h i s time period, dietary recommendations had undergone a s i g n i f i c a n t change; from high f a t , low carbohydrate in the 1930*s to l i b e r a l i z e d carbohydrate and low f a t i n the l a t e 40*s and 50*s. This change in diet was accompanied by r i s i n g t r i g l y c e r i d e l e v e l s and increased freguency of large vessel disease. Serum cholesterol levels were unchanged, with no apparent r e l a t i o n to either small or 20 large vessel disease. Peterson et a l . £19] found an inverse co r r e l a t i o n between serum t r i g l y c e r i d e concentration and degree of diabetic control. Improved control was accompanied by decreased t r i g l y c e r i d e concentrations. HbAIc leve l s were also measured and correlated p o s i t i v e l y with t r i g l y c e r i d e concentration. Not a l l studies are i n agreement with the i m p l i c i t recommendation of higher f a t , more r e s t r i c t e d carbohydrate diets fo r diabetic patients. In a study by Van Eck £71], introduction of a low f a t diet to 12 hyperlipemic diabetics, a l l of whom had retinopathy, was accompanied by regression of r e t i n a l exudates. However, i t should be noted that the revised d i e t was also c a l o r i e - r e s t r i c t e d : almost a l l of the subjects l o s t weight during the study. In the epidemiological studies of Mixi et a l . £57 ] and TsuTjx""l'and «ada £72], lower rates of atherosclerosis and las s severe microangiopathy were credited to the low f a t , high carbohydrate diets of Asian peoples. A survey by Eudnick and Anderson £73] of diabetics i n Hiroshima, Japan likewise revealed a lack of atherosclerotic complications, despite what would be considered i n the West as poor diabetic management. Retinopathy, neuropathy and nephropathy were a l l present in roughly the same proportions as in western countries, though severe retinopathy was very rare. Again, the low f a t , high carbohydrate diet was suggested as a possible causative f a c t o r . Another group of diabetics noted for r e l a t i v e absence of 21 vascular disease are Yemenite Jews [ 7 4 ] . at f i r s t , t h i s was thought also to he due to adherence to a diet low i n f a t and high i n carbohydrates; however, a more thorough investigation of the s i t u a t i o n revealed that the type of carbohydrate consumed was a more prominent factor [75,76,77,78], Cohen [75 ] noted a sharp r i s e in prevalence of diabetes among Yemenite Jews 25 years following t h e i r immigration to I s r a e l , and subsequently discovered that the only s i g n i f i c a n t change i n t h e i r dietary patterns was replacement of starch with sucrose, the percentage of c a l o r i e s consumed as carbohydrates and as f a t remaining unchanged [ 7 6 ] , The r e s u l t s of interchanging starch with sucrose in an experimental situation [77,78] were increased serum ch o l e s t e r o l and decreased glucose tolerance. The r e l a t i o n of f a t to secondary complications seems tenuous, i n view of these findings. Realtive absence of severe secondary complications in Asian diabetics could conceivable also be due to low sugar consumption. As pointed out by West [ 7 0 ] , the regular association between increasing affluence, increasing sugar intake, increasing f a t intake, increasing adiposity and decreasing physical a c t i v i t y make int e r p r e t a t i o n of epidemiological evidence regarding fat intake and course or onset of diabetes d i f f i c u l t . . The single most regular association i s between increased adiposity and increased prevalence of both diabetes mellitus and atherosclerosis. In juvenile diabetics, in whom adiposity i s rarely a problem, r e s t r i c t i o n of f a t intake in an e f f o r t to maintain low serum cholesterol l e v e l s may res u l t in a poor tradeoff: that of 22 increased t r i g l y c e r i d e l e v e l s with increased carbohydrate intake. Albrink and Man [79] have suggested that adaptation to low carbohydrate, high f a t diets may be d i s t i n c t l y advantageous to diabetic i n d i v i d u a l s . 23 CHAPTER III MATERIALS AND METHODS A. Subjects Sixteen normal and 49 diab e t i c patients were studied. Age of the non-diabetics varied from 4 to 24 years, and that of the diabetics from 9 months to 2 5 years. A l l diabetics were insulin-dependent, i . e . juvenile-onset diabetics, varying in duration from 3 months to 16 years.. Symptoms prior to diagnosis in almost a l l cases included polydypsia, polyphagia, polyuria and recent weight loss; upon examination, sugar was detected i n the urine. Ketoacidosis was rare. Five of the 49 had associated conditions unrelated to diabetes: L.D. had Addison's disease, T.C. was also e p i l e p t i c , H.M. had hypertriglyceridemia, N.R. was hypothyroid, and D.C. suffered from rheumatoid a r t h r i t i s . Another 5 of the diabetic patients had recognizable complications of diabetas: Z.C.""and B.D. both had mild retinopathy, L.T. had moderate retinopathy, P.S. had been treated for necrobiosis c u t i s , and H. D. had nevus of thigh. In addition^ 3 were in probable early stages of developing diabetic retinopathy: patients C.C, H. >3. and U.D.. Fourteen of the 49 had a family history of diabetes. Family history was coded: yes=2, no=1, for purposes of s t a t i s t i c a l analysis. The mother of M.K., the father of N.R. and a brother of S.E. were diabetic. K.S. and K.E. were 2 of 3 diabetic t r i p l e t s studied. The younger s i s t e r , K.p. was 24 also d i a b e t i c . A l l other diabetic r e l a t i v e s were farther removed i n the family history; e.g., maternal or paternal great aunt or uncle or great grandparents. MD. was an unusual neonatal diabetic who was l a t e r found to be among the 1-2% who are not transient diabetics. At the time the sample was col l e c t e d for HbAIc assay, he was s t i l l being undertreated with i n s u l i n , i n an t i c i p a t i o n of remission of diabetic symptomatology. The majority of normals were obtained through the Student Health Services at U.B.C. A l l of the remaining samples were obtained from the Metabolic Investigation Unit at Children*s Hospital. B. Preparation of Hemolysates Six ml venous blood samples were collected i n EDTA treated tubes. Blood samples were centrifuged f o r 15 minutes at 2500 BPM. Plasma was removed and red c e l l s were treated with 3.2% sodium c i t r a t e dihydrate (0.5 ml of c i t r a t e solution to 3 ml of red blood c e l l s ) . The c e l l s were separated from c i t r a t e by centrifugation at 2500 BPM for 15 minutes, then washed twice with 0.9% saline solution (2500 RPM for 15 minutes each time). Red c e l l s were hemolyzed i n d i s t i l l e d water and toluene (1 volume of water to 0.4 volumes toluene), then centrifuged at 16,000 RPM for 1 hour. The middle layer of oxyhemoglobin was then c a r e f u l l y removed and, i f not completely clear and ruby red i n colour, was centrifuged at 2500 RPM 30 minutes a d d i t i o n a l l y to remove any remaining c e l l u l a r debris. The hemoglobin was 25 then dialyzed for at least 12 hours at 0-5° C against 50 times the volume of the developer that was to be used f o r subsequent chromatography, Storage of hemolysates frozen at -20° C was found to be s a t i s f a c t o r y for up to 2 weeks; past t h i s time, increases of HbAI(a*b) would tend to d i s t o r t the res u l t s (see Results). C. Preparation of Chr o matographic Columns Bio-Rex 70 synthetic cation exchange r e s i n (200 mesh, BLo-Rad Laboratories) was prepared according to the method of Himmelhoch [80].., A s l u r r y of resin i n d i s t i l l e d water (100 ml t o t a l volume) was poured into a Buchner funnel f i t t e d with a coarse f r i t t e d glass dish and then the re s i n was washed with a series of washes (100 mis. each) under vacuum as follows: 1) 0.5 H NaOH-0.5 M NaCl, 2) water, 3) 0.5 H HCl, 4) water, 5) 0.5 H NaOH-0.5 R NaCl, 6) water u n t i l excess a l k a l i had been removed (pHydrion paper te s t neutral) . , The r e s i n was then transferred to a large container, diluted with about 10 volumes of water and allowed to sediment by gravity alone repeatedly u n t i l the supernatant f r a c t i o n was no longer hazy. The i n i t i a l process of washing can be ca r r i e d out without a Buchner funnel, though i t i s fa r slower by gravity sedimentation alone. The elimination of f i n e s was found to be absolutely e s s e n t i a l to good chromatographic separation of hemoglobin components. Following t h i s step, the resi n was allowed to e q u i l i b r a t e for 4-6 days i n Developer No., 6 at 0-5° C, then packed into 20x350 mm glass columns to uniform heights 26 of 16 cm, under gravity flow, in as few steps as possible. Further e g u i l i b r a t i o n with 1600-2000 ml of Developer No.6 at room temperature over a period of a week was necessary before columns could be considered ready to use. The f i n a l e q u i l i b r a t i o n of the column was checked by pH meter (pH = 6.74±.02). Columns were never allowed to run dry. D« Developers The developers used were those described by T r i v e l l i et a l . [ 7 ] : Developer No. 6 containing 18.37 g of monosodium phosphate monohydrate, 4.74 g of disodium phosphate and 2.60 g of potassium cyanide in 4 l i t e r s of water, pH 6.74±0.02; and phosphate buffer containing 6.52 g of anhydrous disodium phosphate and 14.35 g of monosodium phosphate monohydrate in 1 l i t e r of water, pH 6.42. Since chromatographic separation of hemoglobin components was found to be sensitive both to pH and i o n i c strength, i t was e s s e n t i a l that the pH of Developer No. 6 be kept constant and within the l i m i t s noted above. For smaller columns, more accurate control of pH would be necessary i n order to obtain consistent results {A. Cerami, personal communication). E. Operation cf Columns Prior to s t a r t i n g a chromatogram, the top cm of the re s i n in a column was s t i r r e d and allowed to s e t t l e . One ml of the hemoglobin to be chromatographed was mixed with 2 ml of Developer No. 6 and then layered c a r e f u l l y onto the column. 27 The sample was allowed to flow i n under gravity and washed twice with 2 ml of developer.., Subsequently, columns were kept f i l l e d with the appropriate buffer. I n i t i a l l y , a l l fr a c t i o n s were collected by fr a c t i o n c o l l e c t o r ; 5 ml per f r a c t i o n u n t i l a l l of the HbAIc had been eluted, then 12 ml per fr a c t i o n following the change to the stronger phosphate buffer, which was used to elute the remainder of the hemoglobin A. For routine operation, i t was found that 1 sample i n each set of samples could be colle c t e d by f r a c t i o n c o l l e c t o r , to ensure the consistency of the spectra ; the remaining 3-4 samples i n a set could then be collected i n pooled fractions by volumetric f l a s k . Following each run, columns were re-equilibrated with at l e a s t 400 ml of Developer No. 6. Spectrophotometry The o p t i c a l density of each fraction was read in a Coleman-Hitachi spectrophotometer at 410mu and the re s u l t s plotted against m i l l i l i t e r s of e f f l u e n t . The fra c t i o n s representing HbAIa+b, HbAIc and HbAII (the major component) were then separately pooled i n volumetric flasks, and these samples were also read at 410mu. From the pooled f r a c t i o n s , the percentages of HbAIa+b, and HbAIc as percentage of t o t a l hemoglobin eluted could be calculated, as described by T r i v e l l i et a l . [ 7 ] . When more than one sample was chromatographed on the same day, inspection of the chromatographic spectrum enabled the separation of the several hemoglobin components i n volumetric flasks without a f r a c t i o n c o l l e c t o r . 28 G. C l i n i c a l Data In 32 of the 49 diabetic patients, blood was drawn f o r fasting blood sugar determination on the same day as for HbAIc determination. In 39 of the 49 diabetics, a 24-hour urine was co l l e c t e d f o r glucose analysis within 1 week of c o l l e c t i o n of a blood sample for HbAIc. Glucose determination was by AEA-100 bichrbmatic semiautomatic analyzer (polarographic oxygen electrode technique) [81] for both blood and urinary glucose. For a l l the diabetic patients, complete records of nutrient intake and i n s u l i n dosage were available. In most charts, past records of more than 3 determinations of f a s t i n g blood sugar and of 24-hour urinary glucose were available from which to calculate a mean fasti n g blood sugar and mean 24-hour urinary glucose. Some t r i g l y c e r i d e l e v e l s were also available; however, a l l except those of H.H. were well within the normal range. H. Diabetic Control An estimate of diabetic control, on a scale of 1-5 (1=very good, 2=good, 3=fair, 4=poor, and 5=very poor) was assessed by Dr., Tze , the attending physician at Children's Hospital, based on personal knowledge of the patients and review of the following chemical parameters: recent urine testing for reducing sugars, 24-hour block urine glucose determination, fasting blood sugar, c l i n i c a l symptoms, and range of 24-hour urine glucose and fasting blood glucose over the duration of each patient's i l l n e s s . 29 S t a t i s t i c a l Treatment S i g n i f i c a n c e of the d i f f e r e n c e between mean HbAIc values f o r d i a b e t i c s and n o n - d i a b e t i c s was determined by Student's t -t e s t . C o r r e l a t i o n c o e f f i c i e n t s and p a r t i a l c o r r e l a t i o n c o e f f i c i e n t s were determined a c c o r d i n g to computer program UBC TRIP. 30 CHAPTER IV RESULTS Representative p r o f i l e s of the minor and major hemoglobin components of normal and diabetic human bloods are given i n Figure 2. The p r o f i l e s are s i m i l a r when o p t i c a l density i s read at 552 mu rather than 410 mu, although a higher concentration of hemoglobin must be applied to obtain the same peak heights [82 ]. Amberlite IRC-50 resin may be used i n place of the Bio-Rex 70 [83]; however, i t appears that the HbAI (a*b) and HbAIc are l e s s c l e a r l y separated. Variation i n pH of the developers used results i n a di f f e r e n t p r o f i l e [13 ]. Table II shows the r e s u l t s of assays read both at 410 mu and 552 mu. The fact that the r e s u l t s are not i d e n t i c a l can be explained on the basis of much greater v a r i a b i l i t y of readings on the Coleman- Hitachi spectrophotometer in t h i s range of o p t i c a l densities; however, the consistently higher values calculated would seem to i n d i c a t e that t h i s i s partly a function of the wavelength used. Since a higher concentration of hemoglobin would be required to obtain r e l i a b l e o p t i c a l density readings at 552 mu, i t was decided to use o p t i c a l density 410 mu throughout the study. The e f f e c t of storage on HbAIc i s shown i n Table I I I . Storage at -20°C for several weeks resulted i n d i s t o r t i o n s of the HbAI(a+b)/HbAIc r a t i o towards higher values as well as a general increase i n the l e v e l s observed i n most cases. The T A B L E I I Rb Ale values at 552 my. and 410.mu. £amp_le o ^ i K 41.0 my. ^HbATc 0._D._ 552 mji %HbA.l£ 1 . 152 3. 1 .020 3.7 2 . 148 5.4 .018 5.7 3 .116 6.3 .019 14. 6 4 . 153 6.2 .024 10.4 5 . 164 6.9 .024 9.6 6 .253 7.3 .026 7. 9 MeantS.D. 5.9±1.5 8.7±3.8 HbAIc was calculated as percentage of t o t a l hemoglobin eluted. Values measured at 552 mji were s i g n i f i c a n t l y higher than those at 410 nyi (p < .05, Student's t-test; n = 6) . TABLE H I The e f f e c t o f p r o l o n g e d s t o r a g e a t -20°c of d i a l y z e d r e d c e l l h e m o l y s a t e s on the s t a b i l i t y o f t h e he m o g l o b i n components, d e m o n s t r a b l e by Bio-Rex 70 c h r o m o t o g r a p h y . minor 22l2AIia.£b)_ £HbAIc iHbACia*bJ_/HbAIc week 1 2 3 a 1 2 3 H 1 2 3 a 1 2. 3 2. 5 2.8 3.2 7.9 7. 9 7.9 8. 5 .29 .32 . 35 . 38 2 1.8 2. 8 2. 9 2.8 6.1 7. 5 7.6 5.8 . 30 . 37 . 38 • " 8 3 2.6 3. 1 5. 1 5.2 10.7 10. 6 12.7 1 2. 9 • 2 4 .29 .40 .40 U 2.4 2. 8 3.5 2.8 9.5 10. 4 11.0 10.8 . 25 . 27 .32 . 26 34 separation of HiAI (a+b) and HbAIc was l e s s d i s t i n c t after 3-4 weeks of storage and elution of HbAII was followed b j yet another minor component which could be eluted only with great d i f f i c u l t y . Comparison of HbAIc values in diabetics and non-diabetics i s presented i n Table IV (mean values: non-diabetics = 4.2+1.2; diabetics = 8.3+1.6, n1=16, n2=47). , The mean HbAIc values f o r the two groups »ere found to be s i g n i f i c a n t l y d i f f e r e n t (t=12.83, P«<3.0000 01) and compare favorably with the results of Fitzgibbons et a l . [24] {mean values: non-diabetics = 4.6±1.8; diabetics = 8.6+3.1; n1=18, n2=14). A variety of selected c l i n i c a l data on the patients studied i s presented i n Table V. Selection was based on usefulness i n assessing the degree of diabetic control i n each of these patients, as well as for purposes of comparison with previous HbAIc investigations. Bange of current f a s t i n g blood sugar was from 51 mg/100ml to 390 mg/100ml (mean, 192 mg/100ml), and of current urinary sugars from 0.08 gm/24hours to 221.1gm/24hours (mean 63.1 gm/24hours) . Current f a s t i n g blood sugars correlated poorly with current urinary sugars - (r=0.205,p>0.10) , as did mean fasting blood sugars with mean urinary sugars {r=-0.029, p>0.25). Current fasting blood sugars were highly correlated with mean fa s t i n g blood sugars (r=0.588, p<0.0005), as were current urinary sugars with mean urinary sugars (r=0. 683, p<0.0005), perhaps i n d i c a t i n g a ce r t a i n consistency of response on the part of any one patient to the i l l n e s s . TABLE IV Percent HbAIc in non-diabetics and diabetics. Non-diabetics: Mean±S.D. Diabetics: i n j e c t Age Sex 3bAT4a±bj_i!l_ SH 1 <25 F 1.6 3. 1 SH2 <25 F 2.0 5.4 s i n <25 F 1.9 6.3 SH4 <25 F 1.2 4.2 SH5 <2S F 1. 3 3.5 SH6 <25 F 2.5 4 . 2 LR 24 F 1.3 3.7 CG <12 M 1.5 3. 5 SL 23 F 1.9 3.6 AD 23 F 2.1 4. 1 LP <12 M 1.7 4. 2 A V <12 M 1.9 4.5 DA <12 M 2.2 5 . 2 RB <12 M 2.5 5. 1 AK 40 F 2. 1 4.3 GB 12 F 1 . 4 4.3 1 . 8 ± 0 . 8 4 . 2+1 . 2 BT 12 2 1.6 7. 0 PS 12 F 1 .6 7 . 0 LA 18 M 2. 1 8 . 6 LD 16 M 2 . 0 8 . 3 ZC 22 M 1.9 6. 0 SR 12 M 2.3 9 . 0 LT 20 M 1.6 8 . 0 TC 15 F 1.9 6. 5 BC 8 F 2.6 7. 2 HM 20 F 2.7 8. 8 HD 16 M 2.5 8. 3 CC 16 M 3.2 9.6 PT 20 F 2.5 7. 9 SD 22 F 2.1 5. 7 SJ 22 F 1.9 8. 2 HW 23 M 1.9 6. 4 KD 3 F 1. 8 7. 3 BP 13 F 1.5 7. 0 MK 18 F 2.6 10. 7 MD <1 M 4.1 13. 1 NR 13 M 2.0 8. 8 DC 13 F 3.2 10. 1 FA 13 F 2.0 8. 7 MM 15 M 3.0 10. 0 BD 14 F 2.3 8. 3 continued TABLE IV continued Subject Age Sex. HbATla + bLl^L HbAIcJSl. RK 14 F 2.2 7. 0 SE 16 F 1.8 8. 6 PE 16 F 2.1 10. 4 FM 15 F 2.8 11. 3 HD 15 M 2.9 11. 1 SM 9 F 3.0 9. 7 KS 17 F 2.3 10. 5 KE 17 F 2.8 8. 4 KO 15 F 3.0 8.7 WD 15 M 1.8 7. 2 KC 6 M 2.1 8. 4 MJ 9 F 2.3 7. 9 KW 15 M 1.8 6. 1 MT 1 1 F 2.6 7. 2 PD 18 2.9 7. 8 WE 12 M 1.6 5. 8 BS 14 F 2.6 10. 7 TG 13 M 2.4 9. 5 EP 14 M 1.8 7. 6 DR 18 M 2.3 7. 3 PP 10 F 2.0 7. 6 KR 13 M 2.4 6. 9 Mean±S.D. 2.3±0.5 8.3±1.6 HbAI(a + b) and HbAIc were calculated as the % of t o t a l hemoglobin eluted. Non-diabetic and diabetic mean HbAIc values w e r e s i g n i g i c a n t l y d i f f e r e n t from each other (p << .000001, Student's t-test; n1=16, n2=47). 37 Selected c l i n i c a l data of diabetic pa tients. J2BJECT HbSIC J i t s DTP. i t s . c a ^ i o o a l H E A N CUR PRNT aZ2!ibc n TK u DIABR-IC QQNTPOL i S s i s i i x I s c o s B i f ig>( BT 7.0 12 7.5 251 27.6 24. 4 62 2 SR 9.0 12 4 51 272 9.3 20.6 60 3 convulsions PS 9.9 15 12 190 72 3 necrobiosis cutis LT 8.0 20 8 238 221. 1 169.3 i o o 4 •oderate retinopathy Lt 8.6 18 .1 173 130.1 47.2 3 4 4 TC 6. 5 15 10 64 2. 5 epilepsy LD 8.3 16 13 217 13.0 71.1 66 1 Addison's disease BC 7.2 8 7.5 99 127 37. 9 31.4 44 1 ZC 6.0 22 11 + 208 72.0 60 2 nild retinopathy HH 8.8 20 15 252 392 11.6 46 3 hypertricjlycerideaia HD 8.3 16 7 160. 0 131.3 68 3 nems of thiqh CC 9.6 16 '•5 173.3 87.1 55 4 PT 7.9 20 12 274 243 26. 8 28.6 62 1 SD 5.7 22 6 69 119 4. 9 11.3 66 0. 5 SJ 8. 2 22 12 209 219 91.2 144.4 46 2. 5 BW 6.3 23 16 83 163 21.3 99.4 33 2 BT 8.0 21 16 72 122 84 1 KG 7. 1 25 4 • 55 113 54.0 62 1 K D 7.3 3 0.5 180 31.7 2. 5 BP 7.0 13 7 261 250 18. H 11.3 72 2 nK 10.7 18 5 253 211 141.5 112. 1 72 4 BD 13. 1 <1 <1 • 390 434 2. 4 5 N R 8.8 13 5 • 271) 308 3 2 2. 5 hypothyroid DC 10. 1 13 5.5 216 332 19.5 : 51.6 80 3 rheumatoid a r t h r i t i s FA 8.7 13 2 301 2"40 40.6 62.3 60 2 HM 10.0 15 10 217 189 141.2 74.0 82 3 con t inuGr] 38 T A B L E V c o n t i n u e d IBJF.CT H b A I c D u n . cnn tEMT rt FA N I.:ISIM,TV DIA 3 F T pun.' DDS F.5 SQHIES! S i t s E a Z l Q O E i BO fl. 3 111 6.5 386 223 6 1. 14 9 2 . 9 59 3 RK 7 . 0 IU 7 186 216 7 0 . 14 1143. 6 76 2 . 5 SE 8 . 6 16 14 • 182 221 3 6 . 0 09 2 PE 1 0 . « 16 3 273 257 3 0 . 0 7 6.9 52 2 . 5 FN 11.3 15 12 77 270 1 . 1 2 1 . 6 146 1 OD 1 1 . 1 15 7.5 323 214.0 5 0 . 6 115 2 . 5 SM 9.7 9 1 1149 175 3 6 . 9 1 0 0 . 2 2 2 2 . 5 KS 1 0 . 5 17 5 • 2H6 216 5 0.5 52 2 KE 8.14 17 2 88 225 0 . 0 8 50 2 . 5 KO 8.7 15 14.5 231 21B 2 5 . 1 17.7 U6 2 SD 7 . 2 15 3 239 1 2 1 . 3 8 1 . f i 50 2 KC 8.14 6 1 98 5 . 3 1 HJ 7 . 9 9 1 26 1 KS 6 . 1 15 - 3 222 2 3 . 5 7 2 . 0 714 1 . 5 HT 7. 2 11 3 192 2 1 . 0 1.5 PD 7 . 8 ia 5 2U2 9 8 . 7 62 2 . 5 BE 5 . 8 12 6.5 137 5 1 . 1 7 0 . 0 38 1 . 5 BS 10.7 1« 2 235 5 6 . 6 2 . 5 TG 9.5 13 10 • 7 5.7 32 2 . 5 EP 7 . 6 11 10 1 2 1 . 1 HO 2 DR 7. 3. 18 10 1 2 3 . 3 97.11 67 2 PP 7 . 6 10 1 126 1 KB 6^ 2 13 1 122 1 Means 8 . U 1 5 . 0 7.3 192 229 6 3 . 1 6 7 . 0 55 A5SQCXAT^n CONDITIONS • i l d r e t i n o p a t h y k e t o a c i d o s i s b l u r r e d v i s i o n i d u r a t i o n o f d i a b e t e s ' f a m i l y h i s t o r v of d i a b e t e s J f a s t i n q b l o o d s u q a r * o r i n a r y suq,u s 1 u n i t = .0^5.11 fnq p u r e c r y s t a l l i n e z i n c i n s u l i n S e l e c t i o n w a s b a s e d o n u s e f u l n e s s i n a s s n r , s i n q d e q r c e o f d i a b r > t i c c o n t r o l . " * " i n t h ? f a n i l y h i s t o r y c o l u i c n i n d i c a t e s t h a t a t l e a s t o n e rnerohor o f t h e f a m i l y ( c o n s a n q u i n i t y 1/R o r g r e a t e r ) h a s b e e n d i a q n o s e d a s d i a he t i c . rtea n f a s t i n q b l o o d s u g a r i n d u°an u r i n a r y s u g a r uoc» o b t a i n e d b y a v e r a q i n q the? r e s p e c t i v e v a l u e s o v . ? r t h e d u r a t i o n D f t h e i l l r . p s s . B l ^ n k s p a c e s i n t h e a b o v e i n d i c a t e t h o s e c a s e s w i t h l e s s t h a n t h r e e t e s t r e s u l t s f o r f a s t i n q b l o o d s u q . i r o r u r i n a r y s u g a r . T o t a l u n i t s o f i n s u l i n i s t h e sum o f l o n q - a c t i n q a n d s h o r t - a c t i n q t y p e s o f i n s u l i n , m e a s u r e d a s i n t e r n a t i o n a l u n i t s . D i a b e t i c c o n t r o l w a s e s t i m a t e d by t h e a t t e n d i n q p h y s i c i a n on a s c i l e o f 1 t o 5 ( 1 = v c c y g o o d , 2 = q o o d , 3 = f a i r , H= p o o r , 5 = v e r y p o o r ) . 39 In Table VI, the co r r e l a t i o n analysis of these variabLes with HbAIc i s presented. Both current f a s t i n g blood sugar and mean fast i n g blood sugar are strongly correlated with Hbfilc. The p a r t i a l c o r r e l a t i o n of Hbaic with mean fast i n g blood sugars {eliminating interdependence of mean fasting blood sugar and current fasting blood sugar) remains highly s i g n i f i c a n t (r partial=0.484, p<0.0025). The corr e l a t i o n with family h i s t o r y of diabetes i s also moderately s i g n i f i c a n t (r=.312,P<.05).„ Neither urinary sugar nor mean urinary sugar were correlated with HbAIc to a s i g n i f i c a n t degree. In Figure 3, the regression curve of percentage HbAIc vs. the diabetic control estimate on a scale of 1 to 5 (1=very good, 2=godd, 3=fair, 4=poor, and 5=very poor) i s plotted according to the equation 6.4*0.87x, obtained from the regression analysis.. The c o r r e l a t i o n between diabetic control and HbAIc concentration i s highly s i g n i f i c a n t (r=.529, p<. 0001). The considerable variation i n dietary intake of c a l o r i e s derived from protein, fat and carbohydrate within t h i s group of juvenile diabetics i s evident i n Table VII. Percentage of cal o r i e s contributed by protein varies from 15.4 to 24.4 (mean 19.7%), the percentage of c a l o r i e s from f a t from 26.3 to 42.4 (mean 33.3%), and the percentage of ca l o r i e s from carbohydrate from 38.7 to 55.2 (mean 47.1%). Without considerably more detailed knowledge of energy needs of each patient, c a l o r i e s cannot be evaluated i n terms of under- and over-nutrition and hence are not included i n the correlation analysis. A strong 40 TABLE VI C o r r e l a t i o n a n a l y s i s of HbAIc with s e l e c t e d c l i n i c a l data. VARIABLE R F SIGNIFICANCE Age • 2 5 9 47 3 . 3 9 .05<p<.10 d u r a t i o n of diabe t e s • 131 47 0.88 n. s. f a m i l y h i s t o r y of d i a b e t e s + . 3 1 2 46 4.99 . 0 2 5 < p < . 0 5 c u r r e n t f . b . s , * + . 4 4 2 29 7.04 . 0 1 < p < . 0 2 5 mean f . b . s . + . 5 8 7 34 1 7 . 9 1 . 0 0 0 1 < p < . 0 0 0 5 c u r r e n t u . s . 2 + . 0 6 7 36 0.16 n. s. mean u.s. 0 4 8 30 0.07 n. s. i n s u l i n dose • 2 6 4 41 3.0 7 .05<p<.10 d i a b e t i c c o n t r o l ^ 5 2 9 47 1 8 . 2 9 .0000S<p<.0001 1 f a s t i n g blood sugar 2 u r i n a r y sugar 3 defined, i n Table V i y = 6 - 4 • -87x p < 0 - 0 0 2 5 F I G U R E 3: C O R R E L A T I O N O F T H E D E G R E E O F D I A B E T I C C O N T R O L W I T H °U H b A I c TABLE VII 42 Protein, fat and carbohydrate intake of diabetic patients as percent of t o t a l c a l o r i e s . SUBJECT PROTEIN FAT CARBOHYDRATE KCALORIES BT 15. 8 34. 8 49. 6 2513 PS 21.4 38.4 40.3 2672 LA 19.9 36. 1 43. 0 3085 LD 20. 9 28.8 50. 3 2504 ZC 20. 1 41.1 38. 8 3064 SR 19. 9 38.5 49.6 2386 LT 16.5 34. 8 48. 7 2663 TC 18.9 26.9 54.2 1505 BC 21. 2 29.2 49. 8 1389 HM 23. 3 26.3 49.4 1197 HD 20.6 34.5 44. 9 2629 CC 18.5 32. 3 50. 3 3087 PT 23. 9 40.7 35. 3 1303 SD 18. 1 32. 1 49. 8 2273 SJ 19. 8 37. 8 42.4 1311 HW 21 .1 39. 2 39.7 2317 KD 19.3 32.2 48. 6 1371 BP 13.0 38.0 44.0 2500 MK 20. 8 30.7 49. 2 2261 NR 22.5 35. 2 42. 4 2000 DC 20. 0 3 4.5 44. 5 1747 FA 18.8 34. 3 46.9 2319 MM 19.0 31.3 49.7 2930 BD 19.4 31.2 4 9.4 1877 RK 20. 3 37.2 42. 5 2128 SE 15. 4 33. 1 51.5 1740 PE 24 . 4 30. 1 45.5 1345 FM 22.0 30.0 48. 0 1400 DD 19.3 29.7 51.0 2879 SM 18.5 30.4 51. 1 17 39 KS 18.3 26. 5 55. 2 1356 KE 10.3 26.5 55.2 1356 KO 19. 8 26.5 5 3. 7 15 95 WT 18.9 42. 4 3 8. 7 2867 MG 19. 2 36. 4 44.4 1965 WD 18.9 33.7 47. 4 2268 MJ 20. 8 31.3 47. 9 1728 KW 22. 0 33.0 45. 0 2500 MT 17.0 34.0 49. 0 2126 PD 16.9 33.5 49. 6 1396 WE 19.7 32.5 47. 8 24 36 TG 20.5 34.6 44. 9 2500 EP 20. 0 33.0 47.0 2550 DR 20.0 33.0 47. 0 3342 MeantS.D. 19.7±1.91 33.3±4. 0 47. 1±4. 4 2139±594 Values qiven for protein, f a t and carbohydrate are the percentages of t o t a l c a l o r i e s represented by each of these dietary constituents (n = 44) . U3 negative c o r r e l a t i o n between percentage of c a l o r i e s supplied by fa t and Hb&Ic was found (r=-.300, p<.05). Neither percentage of cal o r i e s supplied by protein nor percentage of c a l o r i e s supplied by carbohydrate were s i g n i f i c a n t l y correlated with Hb&Ic {r=.192, r=.211, r e s p e c t i v e l y ) . 44 CHAPTER V DISCUSSION A. Standardization of Method A recurrent problem in evaluation of HbAIc as a c l i n i c a l l y useful parameter of diabetic control i s the v a r i a b i l i t y in normal values given by d i f f e r e n t i n v estigators. Koenig jet al.[ 9] consider 3-5% HbAIc to be normal f o r adult humans. More s p e c i f i c a l l y , Clegg and Schroeder [12] found a mean HbAIc of 4.2% i n 6 non-diabetics, and Eahbar et al.f 6 ] found a mean HbAIc of 4.6%, both of which are consistent with the' r e s u l t s of Fitzgibbons et al.[24] (4.6±1.8, n=15) and those reported herein (4.2±1.2, n=16) (Table IV). By contrast, some investigators have obtained generally higher values f o r percentage of HbAIc i n non-diabetics. Paulsen and Koury [11] found 5.2±1.0% HbAIc i n 65 non-diabetic c h i l d r e n and 5.3±0.8% HbAIc in 20 non-diabetic adults; Schwartz et al.[25] found 5.7±0.4 %HbAIc (n = 12) in normal adults; and Huismah and Horton [83] observed a range of 4.3-6.6% in 14 samples of normal adult erythrocytes. It i s worth noting that the l a t t e r studies were a l l carried out at 540-580 mu, whereas the former were read at 410-415 mu. This observation i s i n accordance with the results of our comparative measurements (see Table I I ) , though other f a c t o r s may be involved, such as differences i n storage time or 45 temperature, pH of imffers,etc. Huisman and Meyering [84] have suggested that ca l c u l a t i o n of the percentages of the d i f f e r e n t hemoglobin f r a c t i o n s may be more accurate at 4 15 mu than at 5 80 mu due to the higher molar extinction of hemoglobin at 415 mu. Not a l l investigators have calculated percentage of HbAI(a*b) separately from HbAIc. Tattersa.1.1 et a l . [ 2 9 ] collected a l l 3 minor components together in a standardized volume of eluate (250 ml). Values obtained for HbAI(a+b+c) were 6.9% in normals and 11.9% for diabetics. A s i m i l a r system was used by T r i v e l l i et a l . [7] with a mean value for non-diabetics obtained equal to 6.5±1.5%. Use of such a combined measure may be useful c l i n i c a l l y , though i t i s probably less r e l i a b l e , i n view of the r e l a t i v e i n s t a b i l i t y of HbAI(a + b) as compared to HbAIc (Table I I I ) . Huisman and Horton [83] measured changes i n HbAI(a+b)and HbAIc concentrations during i n v i t r o aging of s t e r i l e blood samples at 4,25, and 37°C. „ No s i g n i f i c a n t change in the percentages of HbAIc were observed while a d e f i n i t e increase i n HbAI(a+b) was shown, with the degree of increase mainly dependent on time of storage and not on temperature. Separation of HbAI (a + b) and HbAIc was also poorer after prolonged storage. These r e s u l t s are s i m i l a r to those obtained in our storage study (Table I I I ) , though some increase i n HbAIc over time was noted in our study. Other storage studies have shown no a l t e r a t i o n i n % HbAIc: Koenig and Cerami [16] found that storage at -20°C f o r 4 days had no s i g n i f i c a n t e f f e c t on HbAIc. Likewise, Schwartz et a l . 46 [25] compared fresh hemolysates with the same samples stored up to 6 months at -20°C and found no detectable e f f e c t on the percentage of HbAIc, though the chromatographic peak appeared broader aft e r prolonged storage. In our study, freezing and thawing i n the course of the experiment may have speeded up i n v i t r o aging. B« Increase of HbAIc in Diabetic Patients The observed elevation of %HbAIc in diabetics as compared to nohdiabetics was approximately twofold, when mean values were compared {8.6% HbAIc for diabetics, 4.2% HbAIc for nondiabetics){Table IV)., The same proportionate difference has been observed by Bahbar et a l . [ 6 ] , Paulsen and Koury [11], T r i v e l l i et a l . £7 3* Koenig et a l . [17], Fitzgibbons et a l . [24], Schwartz et a l . [25], and T a t t e r s a l l et a l . [29]. C. Positive c o r r e l a t i o n between HbAIc and diabetic control Correlation of HbAIc le v e l s with the estimated degree of diabetic control i s perhaps the most s i g n i f i c a n t r e s u l t of t h i s study. Previous in v e s t i g t o r s have indicated i n a limited study t h i s type of r e l a t i o n s h i p : Koenig et a l . [8,9] and Peterson et a l . [18,19] measured HbAIc le v e l s i n a t o t a l of 8 maturity-onset diabetics before and 1-2 months after normoglycemia had been achieved. In both of these studies, the HbAIc l e v e l s were much higher when the patients were under poor control (as shown by high urine and blood glucose levels) than when diabetic management had been improved. Paulsen and Koury [11] found 47 consistently higher HbAIc l e v e l s in keotacidotic i n s u l i n -dependent diabetics than i n those who were nonacidotic. HbAI(a+b+c) le v e l s i n the former group decreased to nonacidotic l e v e l s 1 to 2 months following improvement of diabetic c o n t r o l . Our r e s u l t s confirm these preliminary investigations and show conclusively that HbAIc can be used as a simple and direct substitute f o r a complex assessment of degree of diabetic control. In accord with t h i s positive c o r r e l a t i o n between diabe t i c control and HbAIc, HbAIc was also strongly correlated with current fa s t i n g blood sugar and with the mean of recorded fasting blood sugars for any one patient (Table VI). As noted previously, f a s t i n g blood sugars, either alone [57], or in combination with other parameters [39], are frequently used as a measure of the degree of diabetic control. Although there may be considerable fl u c t u a t i o n i n fasting blood sugar on a day-to-day basis, the mean of a l l recorded fas t i n g blood sugars since onset bf diabetes for any one person may be considered a reasonable estimate of the degree of control averaged over the entire duration of the i l l n e s s , and as such serves as a useful measure for between-patient comparisons. HbAIc was not highly correlated with levels of glucose in 24-hour urine c o l l e c t i o n s , although t h i s measure i s also sometimes used as an indicator of degree of diabetic control [85]., One possible explanation i s that 24-hour urine c o l l e c t i o n s are performed only over the weekends and hence may 48 not be a true r e f l e c t i o n of the state of carbohydrate metabolism during the remainder of the seek, when carbohydrate intake may be d i f f e r e n t . The amount of glucose s p i l l e d into the urine i n any one 24-hour period varies d i r e c t l y with the amount of carbohydrate consumed [86], and also with the renal threshold l e v e l , which may d i f f e r from patient to patient [36]. Furthermore, the c o r r e l a t i o n between degree of control as measured by f a s t i n g blood sugar and degree of control as measured by urinary glucose i s not high [3,5], Previous reports of c o r r e l a t i o n between urinary sugars and HbAIc [ 1 7 ] show a four-week lag period between a change i n urinary sugar and a change i n HbAIc levels under conditions of improved diabetic control in i n d i v i d u a l patients. Unfortunately, urinary sugars in t h i s study were not evaluated within 1 month prior to determination of HbAIc. HbAIc was not strongly correlated with duration of diabetes, age of subjects or i n s u l i n dosage {Table VI). Koenig et al.[9] and T r i v e l l i et a l . [ 7 ] also found no c o r r e l a t i o n between age or duration of diabetes and HbAIc levels. However, the strong c o r r e l a t i o n between HbAIc and diabetic control may obscure a weaker co r r e l a t i o n with these variables., Given a group of diabetics a l l of whom were in the same c l a s s i f i c a t i o n for degree of diabetic c o n t r o l , an increasing severity and freguency of diabetic complications would probably be noticeable with increasing duration of the disease, and hence also with increasing age. Long- term diabetics are generally those with the highest freguency of retinopathy, neuropathy and nephropathy 49 [ 45,47,87,88]. The weak negative c o r r e l a t i o n (p<.10) between i n s u l i n requirement and Hbftlc (Table VI) may be an i n d i c a t i o n that maintenance of glucose at lower l e v e l s (by higher dosage of insulin) i s advantageous i n reducing glycoprotein formation., On the other hand, i f i n s u l i n dosage i s taken as a measure of severity of diabetes {higher i n s u l i n requirement i n d i c a t i n g a more severe case of diabetes), then the negative c o r r e l a t i o n i s an i n d i c a t i o n of no r e l a t i o n between HbAIc and severity of diabetes. D. Advantages of HbAIc as a Measure of Diabetic Control HbAIc concentration i s correlated with blood glucose concentration over previous weeks and months. As a predictor of i n s u l i n requirement i t i s thus far superior to other i n d i c a t o r s of carbohydrate metabolism which can fluctuate widely within a few hours and can i n some cases be misleading. For example, i f the patient*s diet were altered p r i o r to an o f f i c e v i s i t , a f a l s e i n d i c a t i o n of diabetic control might be obtained. In addition, HbAIc measurement involves no subjective element of diabetic control assessment. Eeguiring only one blood sample every few months, HbAIc of f e r s a consistent measure of long-term blood glucose regulation. "This technic [ s i c ] may allow future d e f i n i t i v e prospective studies of the r e l a t i o n of blood glucose control to the development and progression of the various diabetic complications" [4]. 50 a further advantage of using HbAIc as an index of diabetic control i s the probable biochemical l i n k between the diabetic state and increased glycoprotein synthesis. The p o s s i b i l i t y exists for showing a cause and e f f e c t r e l a t i o n s h i p between increased glycohemoglobin and development of the microangiopathy of diabetes [73]. I f , indeed, HbAIc synthesis i s i n d i c a t i v e of these changes c h a r a c t e r i s t i c of diabetic microangiopathy, then determination of HbAIc levels could not only be used as an independent and objective index of diabetic control, but could also be considered as an early indicator of these changes, at a stage when reversion to tighter diabetic control might be of s i g n i f i c a n t therapeutic value. E. Negative c o r r e l a t i o n between HbAIc and percentage of c a l o r i e s consumed as fat The negative c o r r e l a t i o n between percent of c a l o r i e s consumed as f a t i n i n d i v i d u a l diets and l e v e l of HbAIc (r=-.300) implies that increased intake of carbohydrate and/or protein leads to increased glycohemoglobin synthesis. Modification of diabetic diets has been shown to a f f e c t the course and freguency of secondary complications [74,75,76,77]. That the differences in composition of diet might also be r e f l e c t e d i n the rate of glycoprotein synthesis has not previously been suggested. 51 CHAPTER VI SUMMARY SND RECOMMENDATIONS A., Summary The purpose of the present inves t i g a t i o n was threefold: 1) to evaluate the technique of measurement of HbAIc and to compare glycohemoglofein lev e l s in diabetics with those i n non-diabetics; 2) to examine the relat i o n s h i p between HbAIc and selected c l i n i c a l data of diabetic patients, including age, duration of diabetes, current and mean fasting blood sugar l e v e l s , i n s u l i n requirement and proportion of f a t , protein and carbohydrate in the diet, and 3) to evaluate HbAIc as an objective measure of diabetic c o n t r o l . Evaluation of the method used for determination of HbAIc has shown that the method i s sensitive to the pH of buffers used, to storaqe time of samples pr i o r to analysis and to the opt i c a l density at which HbAIc samples are read. Comparison of HbAIc l e v e l s in diabetics and non-diabetics has confirmed the nearly twofold increase in diabetic hemoglobin <p<.001) . within the diabetic population studied, a p o s i t i v e c o r r e l a t i o n was found between HbAIc l e v e l and degree of diabetic control on a scale of 1 to 5 (1=very good, 2=good, 3=fair, 4=poor, 5=very poor) (r=0.529, p<. 0001). Thus, low HbAIc le v e l s {close to normal) are associated with good diabetic control., As 5 2 an objective, one-step measure of degree of diabetic c o n t r o l , HbAIc represents a s i g n i f i c a n t advance over currently available parameters of diabetic c o n t r o l . A s i g n i f i c a n t c o r r e l a t i o n was found between HbAIc and family history of diabetes (r=.312). HbAIc levels thus tend to be higher i n patients who have diabetic r e l a t i v e s {consanguinity greater than 1/8). That HbAIc i s a r e f l e c t i o n of plasma glucose levels i s shown by the positive c o r r e l a t i o n both with current fasting blood sugar and mean fasting blood sugar, averaged over the duration of the i l l n e s s {r=.U42, r=.587, r e s p e c t i v e l y ) . Ho s i g n i f i c a n t correlations were found between HbAIc and duration of diabetes {r=-.131), urinary sugar l e v e l s <r=. 067), age of subjects {r=-.259) or i n s u l i n dosage {r=-.264). In examining the re l a t i o n s h i p of HbAIc to the type of d i e t consumed, BbAIc was found to be s i g n i f i c a n t l y negatively correlated with the percentage of c a l o r i e s consumed as fat i n the diet {r=-.300), though not s i g n i f i c a n t l y correlated with either the percentage of c a l o r i e s consumed as protein or carbohydrate. The percentage of f a t i n the d i e t may thus be of pi v o t a l importance in determination of the amount of glycohemoglobin formed. Future study of the r e l a t i o n between HbAIc and development of the secondary complications of diabetes may y i e l d a d e f i n i t i v e resolution to the current controversy over the effectiveness of good diabetic control i n delaying or a l l e v i a t i n g c h a r a c t e r i s t i c vascular le s i o n s . Increased HbAIc 53 lev e l s may also serve as an early indicator of derangement of carbohydrate metabolism of a severity which eventually r e s u l t s in increased deposition of glycoproteins i n basement membrane. B. Recommendations In order to f a c i l i t a t e future comparative studies involving HbAIc, one standardized procedure should be used f o r a l l determinations of HbAIc l e v e l s . A standard range of normal and diabetic values would then be comparable for a l l subsequent studies. A prospective study of the relationship between HbAIc and the development of secondary complication would be es p e c i a l l y valuable i n order to focus present divergent philosophies of diabetic management on a v e r i f i e d association (or lack of i t ) between degree of diabetic control and frequency and severity of diabetic complications. The a v a i l a b i l i t y of an objective, quantitative measure of diabetic control,namely HbAIc, should make such a study f e a s i b l e . Further investigations of increased glycoprotein synthesis in diabetes, both i n vivo and in v i t r o , would be worthwhile i n order to elucidate the etiology of diabe t i c vascular complications, as well as to introduce the p o s s i b i l i t y f o r therapeutic intervention {e.q., tighter diabetic control or change i n hypoglycemic agent) at an early stage. In p a r t i c u l a r , the k i n e t i c s of the glucosyl- transferases involved i n glycoprotein synthesis and the metal requirements of such 54 enzymes should receive careful attention. Moreover, the relati o n s h i p between diabetic control as measured by HbAIc lev e l s and basement membrane thickening should tie studied over a longer time period, i n order to properly evaluate the possible c o r r e l a t i o n between these two measures. In addition, the f e a s i b i l i t y of higher f a t diets f o r juvenile diabetics should be investigated, f i r s t i n animal studies, l a t e r i n controlled t r i a l s with human subjects. Closer attention to current divergent dietary practices among juvenile and adult diabetics may y i e l d valuable information with respect to the a d v i s a b i l i t y of low f a t diets for diabetics. 55 REFBHENCES CITED A l b e r t i , K.G.M.M., A. Dornhorst & A.S. Rowe (197 5) Metabolic rhythms i n normal & diabetic man. , In Contemporary Topics i n the Study of Diabetes £ Metabolic Endocrinology, (ed. Eleazar Shafrir) Academic Press, N.Y. pp. 15-54. Knowles, J r . , H.C (1970) Control of diabetes and the progression of vascular disease. In Diabetes M e l l i t u s : Theory and Practice, (ed. M. Ellenberg and H. Rifkin) McGraw-Hill Book Co, Toronto. pp.666-673. Malone, J.I., J.M. Hellrung, E..W. Molphus, A.L. Rosenbloom, A. T. Grgic S F. T. 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