1192

BROWNING AND DIABETIC COMPLICATIONS

NON-ENZYMATIC glycosylation (NEG) of many proteinsoccurs in diabetes mellitus, 1,2 and the amounts of

glycosylated haemoglobin and serum proteins are widelyused indices of integrated glycaemic control over the

preceding few weeks. It has also been suggested that NEGmay play a role in the long-term tissue complications ofdiabetes by such mechanisms as sulphydryl oxidation andcross-linking of proteins,3 and alterations in

immunogenicity,4 enzyme function,5 and cellular uptake anddegradation of maèromolecules. 6,7The initial step in NEG is formation of a Schiff base

between the free aldehyde group of glucose in its open-chainform and an amino group from aminoacid residues in a

protein, the N-terminal amino and e-amino groups of lysinebeing important binding sites. This rapid and reversiblereaction contrasts with subsequent slow rearrangement of the- Schiffbase to a more stable ketoamine linkage (the Amadorirearrangement). These chemical reactions have receivedmost attention in the study of NEG in the last few years,especially since it is the Amadori product that is measuredclinically in assessment of diabetic control. Interest is nowturning to a series of later dehydrations and rearrangementsinvolving the glycosylated adducts which give rise to cross-linking and appearance of brown fluorescent pigments ormelanoidins in certain proteins with a long half-life. This"browning" is the same process which has been recognised asthe discoloration of stored and heated foods since 19128 (theMaillard reaction).Collagen is the best studied protein in the context of

advanced NEG, partly because of the ease with which

collagen can be examined in skin biopsies, but also because ofits importance as a protein that is present in several tissuessubject to complication in diabetes-eg, lung (reducedelasticity9), arteries (stiffening" and accelerated atheromaI2),and basement membrane (the thickening of which has longbeen regarded as the histological hallmark of diabetic

microangiopathy13). Changes that occur in the collagen ofdiabetic patients closely resemble the changes associated withageing’""—increases in NEG, in intermolecular cross-

linking, in thermal stability, and in resistance to enzymaticdigestion, and a decrease in solubility. Many of these changeshave been reproduced by incubating collagen with glucose invitro’7, although the effects do not relate to the Amadoriproduct.18 Collagen isolated from postmortem specimens ofdura of non-diabetic subjects also displays a yellowfluorescence, the intensity of which increases linearly withage of the subjects and has the same spectral properties as

1. Brownlee M, Vlassara H, Cerami A. Nonenzymatic glycosylation and the pathogenesisof diabetic complications Ann Intern Med 1984; 101: 527-37.

2. Kennedy L, Baynes JW. Non-enzymatic glucosylation and the chronic complicationsof diabetes: An overview. Diabelologia 1984; 26: 93-98

3. Monnier VM, Stevens VJ, Cerami A. Non-enzymatic glycosylation, sulphydryloxidation, and aggregation of lens proteins in experimental sugar cataracts. J ExpMed 1979; 150: 1098-07.

4. Mangili R, Viberti GC, Vergani D. Autoantibodies to glycosylated human albumin ininsulin-dependent diabetics. Diab Res Clin Pract 1985; suppl 1: 357.

5. Eble AS, Thorpe SR, Baynes JW Nonenzymatic glucosylation and glucose-dependentcross-linking of protein. J Biol Chem 1983, 258: 9506-12.

b Williams SK, Devenny JJ, Bitensky MW Micropinocytotic Ingestions of glycosylatedalbumin by isolated microvessels Possible role in pathogenesis of diabetic

microangiopathy Proc Natl Acad Ser USA 1981, 78: 2393-977 Lorenzi M, Cagliero E, Markey B, Henriksen T, Witztum JL, Sampietro T.

Interaction of human endothelial cells with elevated glucose concentrations andnative and glycosylated low density lipoproteins Diabetologia 1984, 26: 218-22

8 Maillard LC Action des acides amines stir les sucres; formation des mélanoidines parvoie méthodique CR Acad Sci (Paris) 1912, 154: 66-68

9 Schuyler MR, Niewoehner DE, Inkly SR, Kohn R Abnormal lung elasticity in

juvenile diabetes mellitus Am Rev Resp Dis 1976; 113: 37-4110. Rosenbloom AL, Silverstein JH, Lezotte DC, Richardson K, McCallum M. Limited

|oint mobility in childhood diabetes mellitus indicates increased risk formicrovascular disease. N Engl J Med 1981, 305: 191-94

collagen incubated in vitro with glucose. 16 The fluorescenceof collagen from diabetics corresponds to that of non-diabetics twice their age." In all probability this prematureageing of diabetic collagen is due to advanced glycosylationreactions and cross-linking, but the chemical nature of theseevents is unclear. Pongor and co-workers 19 proposed a

structure for one of these links: after acid hydrolysis ofbrowned polylysine and browned serum albumin, a

chromophore was released and was identified as

2-(2-furoyl)-4(5)-(2-furanyl)-lH-imidazole (FFI). This novelcompound suggests that protein cross-links could be formedby a precursor of FFI in which two glucose molecules(perhaps from the Amadori product) and two amino groupsfrom lysine residues condense to form the tricyclic conjugateprecursor of FFI.The relation between collagen browning and diabetic

complications has now been further explored by Monnierand colleagues.20 Collagen was isolated from skin biopsies of41 insulin-dependent diabetics and the age-correctedfluorescence was related to the severity of various

complications. This measure of browning was shown to behigher in patients with worse retinopathy, arterial stiffness(aortic pulse-wave velocity), and joint stiffness than in thoseof similar age and duration of diabetes who had lesser degreesof or no such complications. The trend was not significant fornephropathy, but there was a correlation between collagenfluorescence and blood pressure. It is possible that arterialstiffness due to advanced NEG in diabetics may cause orexacerbate an increase in blood pressure, which is itself a riskfactor for diabetic retinopathy.2I But the exact role of

browning in the aetiology of diabetic complications isunknown. Is it simply a marker for the most certain of thecausative factors-the duration and severity of

hyperglycaemia?21Two features of browning that are of relevance to diabetic

complications, however, are its irreversibility and the factthat soluble proteins such as albumin and IgG can becovalently trapped by browned proteins, at least in vitro.23This binding process will presumably progress

independently of the presence of glucose and may be a

mechanism whereby immunoglobulins and albumin are

11. Pillsbury HC, Hung L, Kyte MC, Freis ED. Arterial pulse waves and velocity andsystolic time intervals in diabetic children. Am Heart J 1974; 87: 783-90

12. Jarrett RJ, Keen H, Chakrabarti R. Diabetes, hyperglycaemia and arterial disease In:Keen H, Jarrett J, eds. Complications of Diabetes. 2nd ed. London Edward Arnold,1982: 179-203.

13 Williamson JR, Kilo C. Capillary basement membranes in diabetes. Diabetes 1983; 32(suppl 2): 96-100.

14. Schneider SL, Kohn RR. Effects of age and diabetes mellitus on the solubility andnonenzymatic glucosylation of human skin collagen. J Clin Invest 1981, 67:1630-35.

15 Yue DK, McLennan S, Delbridge L, Handlesman DJ, Reeve T, Turtle JR Thethermal stability of collagen in diabetic rats: Correlation with severity of diabetesand non-enzymatic glycosylation. Diabetologia 1983; 24: 282-85.

16. Monnier VM, Kohn RR, Cerami A. Accelerated age-related browning of humancollagen in diabetes mellitus. Proc Natl Acad Sci USA 1984; 81: 583-87.

17. Kohn RR, Cerami A, Monnier VM. Collagen ageing in vitro by nonenzymaticglycosylation and browning. Diabetes 1984; 33: 57-59.

18. Lyons TJ, Kennedy L Effect of in vitro non-enzymatic glycosylation of human skincollagen on susceptibility to collagenase digestion. Europ J Clin Invest. 1985, 15:128-31.

19. Pongor S, Ulrich PC, Bencsath FA, Cerami A. Ageing of proteins Isolation andidentification of a fluorescent chromophore from the reaction of polypeptides withglucose Proc Natl Acad Sci USA 1984, 81: 2684-88.

20. Monnier VM, Vishwanath V, Frank KE, Elmets CA, Dauchot P, Kohn RR. Relationbetween complications oftype 1 diabetes mellitus and collagen-linked fluorescenceN Engl J Med 1986; 314: 403-08.

21. Knowler WC, Bennett PH, Ballintine EJ. Increased incidence of retinopathy in

diabetics with elevated blood pressure: A six-year follow-up study in Pima IndiansN Engl J Med 1980, 302: 645-50.

22. Tchobroutsky G. Relation of diabetic control to development of microvascularcomplications Diabetologia 1978; 15: 143-52

23. Brownlee M, Pongor S, Cerami A Covalent attachment of soluble proteins by

nonenzymatically glycosylated collagen Role in the in situ formation of immuno

complexes J Exp Med 1983, 138: 1739-44

1193

deposited in diabetic basement membrane.24 Low-densitylipoprotein is also trapped by NEG products on collagen,25and this may be important in the development of

atherosclerosis in diabetes.Control of glycaemia in diabetic rats corrects NEG of

haemoglobin and of liver and kidney tissue, but not the NEGof tail collagen, nor its increased thermal stability.26 Inhuman diabetes, evidence is accumulating that, on the onehand, progress of early microangiopathic complications suchas background retinopathy may be at least slowed by a year ortwo of strict metabolic control27,28 but, conversely, advanceddisease in organs such as the kidney remains largelyunaltered.29 The detailed biochemistry of browning in themany long-lived body proteins, the links between this processand the pathogenesis of different diabetic complications, andthe relation of browning to their reversibility, if any, are allurgent topics for research in the next few years.

ULTRAFILTRATION AND HAEMOFILTRATIONFOR REFRACTORY CONGESTIVE CARDIACFAILURE

MOST patients with congestive heart failure andsevere underlying heart disease respond to treatmentwith diuretics, vasodilators, inotropic agents,angiotensin-converting-enzyme inhibitors, bedrest, anddiet. Symptoms are relieved and life may be prolonged.1However, some patients with pulmonary and peripheraloedema are resistant to conventional - therapy andphysical methods for the removal of oedema fluid may beconsidered. This is not a new idea. Southey’s tubes,venesection, and both thoracocentesis and pericardio-centesis have been used in the past. The value of

peritoneal dialysis with hypertonic solutions was

described in 1968, and appeared to initiate clinical

improvement.2 Nevertheless, although the technique issimple and cheap, it was never widely used becauseefficiency of fluid removal is poor, diaphragmaticsplinting is deleterious in the presence of impairedventilatory function, and there is a real risk of peritonitis.Haemodialysis to remove larger volumes of extracellularfluid may relieve symptoms, but is often limited byadverse haemodynamic effects.Use of ultrafiltration and haemofiltration has now

been advocated as a method of managing refractoryheart failure. The terminology of these techniques isconfusing, and they may be employed together. Haemo-dialysis equipment with a semipermeable membrane isused for ultrafiltration; diffusion of solutes into thedialysate is modified by application of hydrostaticpressure. Hydrostatic pressure is also used for

haemofiltration, but in this technique a compact, highlypermeable membrane produces an ultrafiltrate of blood

24. Miller K, Michael AF Immunopathology of renal extracellular membranes indiabetes: Specificity of tubular basement membrane immunofluorescence. Diabetes1976; 25: 701-08.

25. Brownlee M, Vlassara H, Cerami A. Non-enzymatic glycosylation products on collagencovalently trap low-density lipoprotein. Diabetes 1985; 34: 938-41.

26 McLennan S, Yue DK, Marsh M, et al The prevention and reversibility of tissue non-enzymatic glycosylation in diabetes. Diab Med 1986, 3: 141-46

27. Lauritzen T, Frost-Larsen K, Larsen H-W, Deckert T, and the Steno Study Group.Two-year experience with continuous subcutaneous insulin infusion in relation toretinopathy and neuropathy. Diabetes 1985; 34 (suppl 3). 74-79.

28 Kroc Collaborative Study Group. The Kroc study patients at two years: A report onfurther retinal changes. Diabetes 1985, 34 (suppl 1). 37A.

29 Viberti GC, Bilous RW, Mackintosh D, Bending JJ, Keen H. Long-term correction of

hyperglycaemia and progression of renal failure in insulin-dependent diabetes. BrMed J 1983; 286: 598-602.

1. Furberg CD, Yusuf S. Effect of vasodilators on survival in chronic congestiveheart failure. Am J Cardiol 1985; 55: 1110-13.

(a solution of water, electrolytes, and molecules up toMW6000) without contact with dialysate. Arterial-to-venous communication may be used, but with a bloodpump venous-to-venous link is practical. The efficacy ofthese methods for removal of excess fluid has beenshown in oedematous patients3-5 and during cardiacsurgery.6

In six patients with refractory congestive heart failure,considerable amelioration of cardiac symptoms wasobserved during two courses of ultrafiltration whichresulted in loss of an average 7-7 litres of fluid and 7-3 kgbodyweight.7 Nine similar patients lost an average of12.7 litres (range 3-7-23) with up to five courses ofultrafiltration;8 during and following the five courses,measurements of heart rate, blood pressure, cardiac

output, and left ventricular function were unchanged,but ventricular filling pressure was reduced transientlyand serum sodium was increased. Treatment waslimited by development of muscle cramps or

hypotension.8 Haemofiltration appears to have a similarbeneficial effect; loss of fluid and weight is achievedwithout haemodynamic deterioration.9-11 After physicalremoval of fluid, weight reduction appears to bemaintained for some weeks and treatment may be

repeated frequently (one patient had thirty courses overthree months with loss of 122 litres of fluid7). In

addition, patients become more responsive to standardheart-failure therapy.The poor prognosis of patients with refractory heart

failure is well known, and in one series only two of ninepatients survived for a year.8 Although most patientswho have been treated by these techniques were

unsuitable for operative intervention, in a few casesultrafiltration stabilised the patient’s condition

sufficiently for valve surgery or cardiac transplantationto be carried ouC. Physical removal of fluid will affectonly the secondary features of heart failure and not theprimary pathophysiological disorder, therefore therewas no evidence of improvement in cardiac function oroutput or of a persistent effect on filling pressure.

Ultrafiltration and to a lesser extent haemofiltrationare complex and expensive, requiring special equipmentwith highly trained medical, technical, and nursingpersonnel. In many countries, including the UK, thesefacilities are under-provided. Since the techniques donot alter prognosis, they should be used for palliationonly when definitive treatment, such as cardiac surgeryor transplantation, is contemplated.2. Cairns KB, Porter GA, Kloster FE, Bristow JD, Grinwold HE. Clinical and

hemodynamic results of peritoneal dialysis for severe cardiac failure. AmHeart J 1968; 76: 227-34.

3. Silverstein ME, Ford CA, Lysaght MJ, Henderson LW. Treatment of severefluid overload by ultrafiltration. N Engl J Med 1974; 291: 747-51.

4. Kramer P, Wigger W, Rieger J, Matthaei D, Scheler F. Arteriovenoushaemofiltration: A new and simple method for treatment of overhydratedpatients resistant to diuretics. Klin Wschr 1977; 55: 1121-22.

5. Editorial: Haemofiltration. Lancet 1983; i: 1196-97.6. Magilligan DJ. Indications for ultrafiltration in the cardiac surgical patient. J

Thorac Cardiovasc Surg 1985; 89: 183-89.7. Fauchald P, Forfang K, Amlie J. An evaluation of ultrafiltration as treatment of

therapy-resistant cardiac edema. Acta Med Scand 1986; 219: 47-51.8. Simpson IA, Rae AP, Gribben J, Boulton-Jones JM, Allison ME, Hutton I.

Ultrafiltration in the management of refractory congestive heart failure. BrHeart J 1986; 55: 344-47.

9. Blanke H, Kreuzer H, Wigger W, Scheler F. Die Behandlung der akutenLinksherzinsuffizenz anurischer patienten mit der Hamofiltration. Dtsch MedWschr 1977; 102: 1804-47.

10. Mulder AW, Flendrig JA, Fiersen H. Hemofiltratie bij ernstige, chronilschedecompensatio cordis: Cen nieuw therapentisch perspectief. Ned TijdschrGeneeskd 1983; 127: 1755-59.

11. Page E, Machecourt J, Dechelette E, Wolf JE, Bourlard P, Denis B. Traitementdes ilnsuffisances cardiaques avec oedemes refractaires par ultrafiltrationextracorporelle. Arch Mal Coer 1984; 77: 1040-45.