19

THE LANCET

Glycosylation and DiseaseWHEN abnormally high concentrations of

haemoglobin A1c were reported in subjects withdiabetes mellitus the practical implications were

exciting.’ Because the increase resulted from non-

enzymatic glycosylation, the HbA,1c value was deemedto reflect the average plasma glucose concentrationduring the life-span of the red cell. Subsequently itemerged that many other circulatory and tissue

proteins undergo glycosylation proportional to theexisting glucose concentrations.2 Thus glycosylation ofother proteins might provide evidence of pre-existingglucose concentrations over a different time-scale

according to the half-life of the proteins concerned, andmight be of particular value when shorter-term

therapeutic changes needed evaluation, as for examplein diabetic pregnancy.3 In view of the ubiquitousnature of glycosylation the possibility arose thatexcessive glycosylation might alter function and

perhaps provide the link between longstanding raisedglucose concentration and the development of diabeticcomplications. To what extent have initial

expectations been realised?Numerous methodological difficulties in the

measurement of HbA1 have been identified andcircumvented. Concentrations ofHbA,a and HbAjb arenot much increased in subjects with glucoseintolerance, and the increases in HbA1c during periodsof poor glucose control are reflected proportionately intotal HbA,-an observation that has allowed

development of more rapid and cheaper methodssuitable for clinical use.4,s They include small columnchromatography, colorimetric procedures, agar gelelectrophoresis, high performance liquidchromatography, and isoelectric focusing. The last isthe most rapid procedure and thus best suited to

providing same-day results for the clinic.Immunoassay and affinity gel separation procedures

1 Editorial. Glycosylated haemoglobins and disease. Lancet 1977, ii: 22-23.2 McFarland KF, Catalano EW, Day JF, Thorpe SR, Baynes JW. Nonenzymatic

glycosylation of serum proteins in diabetes mellitus. Diabetes 1979; 28: 1011-133 Kennedy L, Mehl TD, Riley WJ, Merimee TJ. Non-enzymatically glycosylated serum

protein in diabetes mellitus: an index of short-term glycaemia. Diabetologia 1981;21: 94-98

4 Kynoch PAM, Lehmann H. Rapid estimation (2½ hours) of glycosylated haemoglobinfor routine purposes. Lancet 1977, ii: 16.

5 Welch SG, Boucher BJ. A rapid micro-scale method for the measurement of

haemoglobin A1(a+b+c). Diabetologia 1978; 14: 209-11.

have also been described but have been less fullyevaluated. (Mayer and Freedman6 provide a goodreview of methodology.) All methods have drawbacks,but the common aim should be a coefficient of variationof less than 6%:7 column procedures require closeattention to temperature and osmolality; isoelectric

focusing requires expensive equipment and high-doseaspirin causes interference. After early enthusiasmsome doubts were raised when HbA1 concentrationsseemed to change as a result of quite short-termchanges of glucose concentrations,8-Io ° but these

changes proved to be due to the formation of labileSchiff bases, distinct from the stable ketoamine. Theselabile components can be eliminated either byincubation of red cells for 11 hours in isotonic saline" 11

or by more rapid incubation in acid medium.I2,13Provided that labile components have been removedthe clinician can be confident that HbA, reflects

average glucose levels over the lifespan of the redcells; 14-17 indeed nomograms have been prepared forestimation of average glucose concentrationstherefrom.18 HbA, is more informative in the clinicthan occasional blood and urine results, which aresubject to timing and other biases, 19 and large numbersof samples can now be examined quickly and cheaply.As with all investigations, the results must be

interpreted cautiously. High HbA, values have beenreported in the presence of iron-deficiency anaemia, 20perhaps owing to the high proportion of old red cells,and the converse is observed with haemolysis.2I Highvalues are also observed in the presence of abnormal

haemoglobins. In uraemia a raised HbA, may have

6. Mayer TK, Freedman ZR. Protein glycosylation in diabetes mellitus: a review oflaboratory measurements and of their clinical utility. Clin Chim Acta 1983; 127:147-84.

7. Peterson CM, Jovanovic L, Raskin P, Goldstein DE. A comparative evaluation ofglycosylated haemoglobin assays: feasibility of references and standards.

Diabetologia 1984; 26: 214-17.8. Svendsen PA, Christiansen JS, Welinder B, Nerup J. Fast glycosylation of

haemoglobin Lancet 1979; i; 6039. Widness JA, Rogler-Brown TL, McCormick KL, et al. Rapid fluctuations in

glycohemoglobin (hemoglobin A1c) related to acute changes in glucose. J Lab ClinMed 1980; 95: 386-95.

10. Goldstein DE, Peth SB, England JD, Hess RL, Da Costa J. Effects of acute changes inblood glucose on HbA1c. Diabetes 1980; 29: 623-27.

11. Bolli G, Compagnucci P, Cartechini MG, De Feo P, Santeusanio F, Brunetti P.Analysis of short-term changes in reversibly and irreversibly glycosylatedhaemoglobin A(: relevance to diabetes mellitus. Diabetologia 1981, 21: 70-72.

12. Nathan DM, Avezzano ES, Palmer JL. A rapid chemical means for removing labileglycohemoglobin. Diabetes 1981; 30: 700-01.

13. Bisse E, Berger W, Fluckiger R. Elimination of labile glycohemoglobin during samplehemolysis at pH 5. Diabetes 1982; 31: 630-33.

14. Ditzel J, Kjaergaard J-J. Haemoglobin A1c concentrations after initial insulin

treatment for newly discovered diabetes. Br Med J 1978; 1: 741-42.15. Gabbay KH, Hasty K, Breslow JL, Ellison RC, Bunn F, Gallop PM. Glycosylated

hemoglobins and long-term blood glucose control in diabetes mellitus. J ClinEndocrinol Metab 1977; 44: 859-64.

16. Borsey DQ, Fraser DM, Gray RS, Elton RA, Smith AF, Clarke BF. Glycosylatedhemoglobin and its temporal relationship to plasma glucose in non-insulin

dependent (type 2) diabetes mellitus. Metabolism 1982; 31: 362-64.17. Koenig RJ, Peterson CM, Jones RL, Saudek C, Lehrman M, Cerami A. Correlation of

glucose regulation and hemoglobin A1c in diabetes mellitus. N Engl J Med 1976;295: 417-20.

18. Welborn TA, Knuiman M, Davis RE, Stanton K, McCann V, Constable I Applyingthe correlation between glycosylated haemoglobin and plasma glucose levels.

Diabetologia 1983; 24: 461-62.19. Nathan DM, Singer DE, Hurxthal K, Goodson JD The clinical information value of

the glycosylated hemoglobin assay. N Engl J Med 1984; 310: 341-46.20. Brooks AP, Metcalfe J, Day JL, Edwards M. Iron deficiency and glycosylated

haemoglobin A1. Lancet 1980; ii. 141.21. Panzer S, Kronik G, Lechner K, Bettelheim P, Neumann E, Dudczak R Glycosylated

hemoglobins (GHb). an index of red cell survival. Blood 1982; 59: 1348-50.

20

several causes;22 2 the change seems to be due

predominantly to the increase in HbA1a and Alb thoughcoincidental alteration in red cell kinetics may alter

HbA1c as well. Some care may also be required inpregnancy, when HbA tends to decrease a little.23Heavily jaundiced or lactescenr4 serum may give riseto falsely high values (but washing of red cells withsaline, as for removal of labile Schiff components, willexclude these artifacts).Measurement of HbA, as a screening procedure for

abnormal glucose tolerance seemed at one time anattractive possibility, but the false-negative rate is toohigh-normal HbA1 values in 16-65% of subjects withabnormal glucose tolerance.6 The detection of anassociation between high HbA, levels in the firsttrimester of pregnancy and the prevalence of

congenital abnormalities is of interest. The higher riskof congenital abnormality in babies born to suchwomen is probably due to glucose abnormalities at thetime of conception and during the first trimester.25Planning of conception to coincide with the bestpossible glucose tolerance, as indicated by HbArconcentrations, might reduce this risk. In research, theability to determine average glucose control reasonablyaccurately over several weeks from a single estimationallows sensitive quantification of the effects ofintervention.Concurrent with the investigations into

measurements of HbA, interest has grown in the

glycosylation of other circulating proteins. Again therehave been methodological difficulties but largebatches can now be handled by a modification of theDolhofer and Wieland26 procedure. Estimation of totalcirculating glycosylated proteins and possiblyindividual proteins such as albumin may provideinformation complementary to that yielded by HbA, 2’The short half-life of these proteins (2-3 weeks) allowsestimates of the effect of changes of glucose control overa different time-scale. This may be of particular valuein pregnancy, where the effects of therapeutic changesmay need evaluation fortnightly or weekly. Estimationof proteins could also have methodological advantagesin that samples can be stored and batched, and themethods are more readily standardised than those forHbA,.What of the relation between glycosylation and

diabetic complications? One suggestion is that Hb

glycosylation might alter oxygen affinity and thus

22. Fluckiger R, Harmon W, Meier W, Loo S, Gabbay KH. Hemoglobin carbamylation inuremia. N Engl J Med 1981; 304: 823-27.

23. Hanson U, Hagenfedt L, Hagenfeldt K. Glycosylated hemoglobins in normal

pregnancy sequential changes and relation to birth weight. Obstet Gynecol 1983; 62:741-43.

24. Falko JM, O’Dorisio TM, Cataland S. Spurious elevations in glycosylated hemoglobin(HbA1) secondary to hypertriglyceridaemia. Arch Intern Med 1982; 142: 1370-71.

25. Leslie RDG, Pyke DA, John PN, White JM. Haemoglobin A1 in diabetic pregnancy.Lancet 1978; ii: 958-59

26 Dolhofer R, Wieland OH. Increased glycosylation of serum albumin in diabetesmellitus. Diabetes 1980; 29: 417-21.

27 Kennedy L, Baynes JW. Non-enzymatic glycosylation and the chronic complications ofdiabetes: an overview. Diabetologia 1984; 26: 93-97.

contribute to ischaemia.28 In vivo, however,glycosylation and oxygen affinity seem to beunrelated 29 Another is that glycosylation of low-density lipoprotein contributes to lipid abnormalitiessince glycosylated low density lipoprotein seems toreact indifferently with its receptor.10 Glycosylation ofnerve proteins31 might be important in pathogenesis ofneuropathy, and of basement membrane proteins inpathogenesis of nephropathy and other micro-

angiopathies.32 There is some evidence that increasedglycosylation of lens proteins may contribute to thedevelopment of cataract, and the glycosylation of

collagen33 to abnormalities in large blood vessels andthe diabetic-hand syndrome. The multiplicity offactors occurring during the development of compli-cations of diabetes makes any particular pathogeneticrole for glycosylation hard to disentangle. Whetherdeterioration of function is occurring because of or inparallel with excessive glycosylation is impossible tosay. Competing claims for the importance of differentpathogenetic mechanisms remain to be evaluated.Little can be concluded from a lack of, or a very weak,relation between concentrations of glycosylatedcirculatory proteins and the presence or absence ofcomplications owing to the vastly different turnovervalues between circulatory and tissue proteins. Finalconclusions must await the outcome of studies in whichindividual mechanisms can be separately modified-as, for example, with agents that might inhibit

glycosylation34 versus agents modifying sorbitolaccumulation. Glycosylation must certainly beincluded in the extensive list of ways in which

prolonged glucose intolerance might cause these

complications.

Screening for NasopharyngealCarcinoma

THE value of screening for cancer is controversial. Intheory, detection of premalignant stages followed byearlier treatment should lead to substantial reductionin mortality, but the actual results may be

disappointing. Screening for carcinoma of the cervix,for example, which began about 20 years ago with

28. McDonald JM, Bleichman M, Bunn HF, Noble RW. Functional properties of theglycosylated minor components of human adult hemoglobin. J Biol Chem 1979;254: 702-07.

29. Samaja M, Melotti D, Carenini A, Pozza G. Glycosylated haemoglobins and theoxygen affinity of whole blood. Diabetologia 1982; 23: 399-402.

30. Witzum JL, Mahoney EM, Branks MJ, Fisher M, Elam R, Steinberg D.

Nonenzymatic glycosylation of low-density lipoprotein alters its biologic activity.Diabetes 1982; 31: 283-90.

31. Vlassara H, Brownlee M, Cerami A. Excessive nonenzymatic glycosylation of

peripheral and central nervous system myelin components in diabetic rats. Diabetes1983; 32: 670-74.

32. Lubec G, Vycudilik W, Coradella H, Pollak A. Enzymatic reversibility ofnonenzymatic glycosylation of the glomerular basement membrane. Nephron 1983;33: 26-28.

33. Stevens VJ, Rouzer CA, Monnier VM, Cerami A. Diabetic cataract formation.

potential role of glycosylation of lens crystallins. Proc Natl Acad Sci USA 1978; 75:2918-22.

34 Schnider SL, Kohn RK. Effects of age and diabetes mellitus on the solubility andnonenzymatic glycosylation of human skin collagen. J Clin Invest 1981; 67:

1630-34.