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(1) What is Glycation?
Glycation refers to non enzymatic addition of hexoses to proteins which leads to formation of reversible and irreversible glycation products. Where as glycosylation refers to non-enzymatic addition of glucose to proteins. Effects of glycation of haemoglobin has been mentioned, but multiple proteins in body are altered in the same way often with disturbed function e.g. Albumin, lens protein, collegen, lipo proteins.
Glycated Haemoglobin:- Haemoglobin A has been observed to be glycated post translationally arid non enzymatically at the terminal valine of the beta chain producing Hb Ale. There are two minor products of glycation of haemoglobin, these are haemoglobin Ala, and haemoglobin Alb. Together these three are considered as glyc atedhaemoglobin.
Glycated Protein:- Plasma protein are also glycated similarly and serum fructosamine measurement represents the glycated component of all the plasma protein.
(2) Pathogenesis
Hyperglycemia causes excessive amounts of irreversible advanced glycation end products (AGEs) to accumulate on long life extra cellular matrix proteins and probably also on DNA in tissues that develop diabetic complications. AGEs begin to accumulate when chemically reversible early glycation products form. These inturn results in formation of Schiff base adducts at a rate proportional to glucose concentration and duration of exposure. The Schiff base adducts quickly rearrange to form more stable glycated haemoglobin like Amadori products, which leads equilibrium over a period of several weeks. Both types of early glycation products are totally reversible. In contrast irreversible AGEs accumulate over a period of years, as early glycation products undergo a slow complex series of chemical rearrangements. One of the two classes of AGEs is formed by the condensation of two Amadori products and resembles the heterocyclic imidazole derivative, 2 furoyl -4(5)-(2-furanyl) 1 - imidazole. The second class of AGEs is formed by reaction of an Amadori product with Amadori derived fragmentation products, resulting in formation of heterocyclinc pyrrole based structure. Arginine and lysine contributes to the formation of these products.
Accumulation of AGEs contributes to pathogenesis of lesions by cross linking of extra cellular protein, altered cell matrix interaction and modification of DNA structure and function. Cross linking of collagen fibrils by non enzymatic glycation alters the physical properties of extra cellular matrix, resulting in change in the organization of the intra cellular act in cytoskeleton resulting in decrease flexibility and permeability of tissues and reduced turnover.
(3) Role in diagnosis of long term control
Monitoring of metabolic balance in diabetes involves the assay of cumulative markers of protein glycation. These are
(a) Haemoglobin lAc. The percentage of haemoglobin A that is glycated is directly proportional to the time that the red cells have been exposed to glucose and glucose concentration. Measurement of glycated haemoglobin fraction gives an integrated picture of the mean blood glucose level during half the average life span of RBC, i.e. 60 days. Normal value is upto 6 gm/dl.
(b) Fructosamine - Serum fructosamine measurement represents the glycated component of all the plasma proteins. These proteins have variety of half lives in circulation but albumin with half life of 8 days is dominant. Measurement of fructosamine have not been widely accepted as albumin half life may change substantially in varied conditions.
(c) Haemoglobin AGE- Assessment of long term control of glucose by haemoglobin AGEe measurement have shown superior results then haemoglobin Ale. Hb-AGE presents at high level in red cells of diabetic patients and differs from glucose derived Amadori product haemoglobin Ale in being chemically irreversible and thus persisting for the circulating life of RBCs.
(d) Glycation index of Hair-Kobayashi-(7) al proposed glycation index of hair for non invasive estimation of diabetic control. Glycation index A(390)/A(412) is based on the ratio of glycation protein to cystine induced coloration. This index for back and scalp hairs from hyper cholesterolimic mice with hyperglycemia, diabetic rats and diabetic patients gave significantly higher values (2.0-2.6 fold) than those of normal subjects (P.). The glycation indices (mean+or-SD) of hairs from diabetic and non-diabetic subjects were 3.00+or- 0.96 and 1.51 +or-0.45 respectively. These indices correlated well with the level of haemoglobin Ale.
Advantage of Hb Ale measurement
(1) It is very useful for assessment of long term blood sugar control. Frequent measurement of blood sugar is not necessary.
(2) blood glucose values can be manipulated by patients by taking extra insulin or oral hypoglycemic agents or missing meal on the day of test. So that patient can bring out good results. While glycated Hb is unaffected by time, type of sample venous or capillary, fed or fasting state.
Disadvantages of Hb Ale measurement.
(1) It can not diagnose hypoglycemia or DKA
(2) It is sometimes possible to obtain normal values in patients suffering from
frequent and dangerous episodes of hypoglycemia, if these are balanced by other
episodes of excessive hyperglycemia.
(3) Values will be unduely low for a given blood glucose level if the life span of RBC
is shortened like in patients with haemolytic anaemia, chronic sepsis,
Rheumatoid arthiritis, chronic blood loss.
(4) If measured by electrophoretic method, patients who drink 30 or more units of
ethanol per week, values may be higher because acetaldehyde derived from
ethanol binds non enzymatically to side chain of Hb moves in same direction. This
error is not there in electroendosmosis method.
(4) Does glycation adds to Diabetes Mellitus complication
The magnitude and duration of target tissue exposure to abnormally high levels of blood glucose correlate closely with the extent and rate of progretion of retinopathy, nephropathy and neuropathy, although genetic determinants of tissue susceptibility and independent accelerating factors such as hypertension also influence the individual clinical course.
Many lines of evidence attest to a multifactorial pathogenesis of diabetic complications. Increased sorbitol pathway metabolism and non enzymatic glycation products have been implicated by many investigators in the pathogenesis of vascular and neural dysfunction as well as early vascular structural changes in animal models of diabetes. The tissues like retinal capillaries, renal tissue and the cardiovascular system are dependent on the integrity of their supporting framework of collagen. It is the modification of the properties by glycation that results in many complications. AGEs elicits a wide range of cell mediated responses leading to vascular dysfunction, matrix expansion and athero and glomerulosclerosis.
Sirns -TJ (1996) stressed that it is the major glycation cross link responsible for vascular stiffening Schmidt - AM et al in 1996 hypothesized that one of the mechanism underlying advanced periodontal disease in diabetes may involve oxidant stress in gingiva induced by the effect of AGEs.
In a study of Ramalho et al in 1996, there was significant differences in fluorescence levels between glycated and non glycated protein in cataractous lenses from diabetic patients in which all proteins analysed presented higher glycation levels than in non diabetic patients, This suggests increased glycation products are involved in cataract development in diabetics.
Patients with end stage renal disease on dialysis displayed high AGEs.levels in serum. Diabetics without complication showed normal values of AGEs. In the case of retinopathy the increase in AGEs was associated with seventy of retinal status. Li-YM et al (1995) suggested that antibacterial activity of lysozyme and lactoferrin is inhibited by binding of advance glycation modified protein thereby increasing susceptibility to bacterial infections in diabetic population.
How to Prevent Glycation
Aminoguanidine hydrochloride is protype compound acts by selectively blocking reactive carbonyls of AGE's precursors. Aminoguanidine effectively inhibits AGE's induced cross linking of collegen and it prevents cross link induced defects of heparin binding to collegen and fibronectin.
In rat eye aminoguanidine treatment prevents diabetes induced
increase in retinal vascular permeability and subsequent intercapillary deposition of extravasated protein. Development of retinal microaneurysms also reduced in long term diabetes.
In aortic wall of diabetic rats aminoguanidine treatment reduces AGE's content, decreases the quantity of plasma protein linked to diabetic matrix and diminishes the excessive cross linking of aortic matrix protein itself.
In diabetic kidney aminoguanidine treatment decreases glomerular basement membrane AGE's content and prevents both increased synthesis of matrix components such as laminin and reduced matrix susceptibility to degradation by proteases, because of this the pathognomonic basement membrane thickening of diabetes does not occur. Also there occurs concomitant reduction in albumin excretion.
Based on these studies aminoguanidine and its analogues have potential therapeutic role in prevention of diabetic complications.
In few studies by La Selva (1996) thiamine has shown important role in prevention of AGE's.
In one study thiamine have shown to correct delayed replication and decreasing production of lactate and advanced glycation end products to bovine retinal and human umblical vein endothelial cells cultured under high glucose concentration. In another study done in vitro thiamine pyrophosphate and pyridoxamine inhibited antigenic AGE's and were more effective than aminoguanidine suggesting that these two compounds may have novel therapeutic potential in preventing vascular complications of diabetes.
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Aminoguanidine Biochem Biophys Res-commun, 1996, 220(1): 113- 9.
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glucose control by Haemoglobin AGE measurement Lancet 1996, Feb 24, 374, 513-5.
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