THE LABORATORY AND DIABETES RDG LESLIE, ERNESTO LOPEZ, STEPHANIE CUNNINGHAM INSTITUTE OF CELL AND MOLECULAR SCIENCE, QUEEN MARY, UNIVERSITY OF LONDON, LONDON UK.
Diabetes mellitus (DM) is a disorder characterized by increased concentrations of glucose in the circulation. The glucose level has varied over the years and can presently be determined by either a raised fasting glucose or a raised glucose following oral glucose challenge (Table 1A). Type 1 diabetes, insulin-dependent diabetes mellitus, and type 2 diabetes, noninsulin-dependent diabetes mellitus, represent two distinct diseases. Clinically, the distinction can sometimes be misleading and uncertain. The loss of insulin secretory capacity is most severe in type 1 diabetes while decreased insulin sensitivity is most severe in patients with type 2 diabetes. Secondary diabetes accounts for barely 1%–2% of all new cases. Monogeneic forms of diabetes are rare. Type 1 diabetes patients are considered insulin-dependent at diagnosis, while the type 2 diabetes patients are initially non-insulin-dependent. Ketosis-prone diabetes (KPD) and Latent Autoimmune Diabetes in Adults (LADA) are each exclusive forms of diabetes and have a different natural history from these major types. KPD is diagnosed with the categorical initial clinical feature of ketoacidosis, though subsequently insulin therapy is, for a while, not required. In contrast, LADA can be diagnosed by the co-occurrence of three traits, not one of which is categorical or exclusive to the condition. The three traits are: adult onset non-insulin requiring diabetes, an islet autoantibody such as glutamic acid decarboxylase autoantibodies (GADA) or cytoplasmic islet cell auto antibodies (ICA) and the lack of need for insulin treatment for several months postdiagnosis. LADA is the most prevalent autoimmune diabetes.
Definitions & Classification In 2000, The World Health Organization (WHO) updated the definition of DM to reflect the better understanding of ‘milder’ glucose intolerance. Table 1B indicates the criteria for impaired glucose tolerance (IGT) and impaired fasting glycaemia (IFG). Both metabolic states intermediate between normal glucose tolerance and DM. Subjects with IFG or IGT are at high risk of progression to diabetes. In certain cases, glycated haemoglobin or haemoglobin A1c (HbA1c) gives equal or almost equal sensitivity and specificity to glucose measurement. HbA1c levels of 6.0% or higher are considered abnormal by most laboratories.
Table 1B: Values for Diagnosis DM and other categories of hyperglycaemia
Diabetes Mellitus Impaired
Fasting
2-h post glucose load
>=110mg/dl
>= 200mg/dl (Venous >=180mg/dl)
Glucose <110mg/dl
Tolerance (IGT) Impaired
Fasting >=100mg/dl
>=140mg/dl
&
<
200mg/dl
(Venous >=120mg/dl & <180mg/dl) <140mg/dl (Venous <120mg/dl)
Glycaemia (IFG)
Table 1A: WHO criteria for DM 1
Symptoms + >200mg/dl at any time
2
Fasting glucose (8 h) > 126mg/dl
3
2 h postload glucose >200mg/dl (with 75g anhydrous glucose dissolved in water)
*In the absence of unequivocal hyperglycaemia, these criteria should be confirmed by repeat testing on a different day. The third measure (OGTT) is not recommended for routine clinical use.
Glycosylation Haemoglobunin HbA1c has proved of great clinical benefit, but there may be more to levels of this molecule than a simple relationship to mean blood glucose. The nonenzymatic reaction of the amino groups of amino acids, peptides or proteins with reducing sugars, results in the formation of brown complexes and protein-protein cross-links.
This nonenzymatic reaction was known as the
‘Maillard reaction’. The Amadori products, referred to as glycated products, are different from the enzymatically glycosylated proteins. These early glycation products undergo further complex reactions such as rearrangement, dehydration and condensation to become irreversibly cross-linked, heterogeneus fluorescent derivates termed advanced glycation end products (AGEs). Reducing sugars and other carbohydrates react with nucleophiles. It is now clear that dicarbonyl compounds are key intermediates in the formation of AGEs. The Amadori product can breakdown via its enol form to give highly reactive free α-dicarbonyl glyoxal compounds such as 3-deoxyglucosone, methylglyoxal and glyoxal. These molecules cross-link proteins and have been detected in vivo.
Biological implications: hemoglobin glycation and AGE formation The glycation of hemoglobin increases its oxygen affinity and makes it more susceptible to oxidation. Thus, most of the primary effects of AGEs leading to the complications of diabetes and aging are triggered by AGE formation on longlive proteins. Collagen has a very low turnover rate and is a prime target. Especially damaging are the effects of AGEs on vascular collagen, which often results in coronary disease, kidney damage, retinal pathology, poor peripheral circulation and other lesions.
Formation of advanced AGEs is thought to
constitute one pathway in the pathogenesis of microvascular complications ( i ).
Glycation of collagen, reflected in its furosine and carboxymethyllysine (CML) content and in further formation of AGEs, predicts the development and progression of diabetic retinopathy and nephropathy which are independent of HbA1c levels ( ii - iii - iv - v - vi ). A close association between diabetic retinopathy, lymphocyte CML levels ( vii ) and the increased CML levels in red blood cells of diabetic patients compared to healthy controls, suggest an important role of the intracellular AGEs levels in the pathogenesis of diabetic complications ( viii ).
Glycated haemoglobin Glycohemoglobin (Ghb or HbA1c) is a measure of long-term mean glycaemia that predicts risk of diabetic complications both in patients with type 1 and type 2 diabetes ( ix - x ). HbA1c depends on several elements such as insulin regime, enzymatic and non-enzymatic protein glycation, red cells lifespan, oxygen tension and age and genetic factors of glucose metabolism. Alone, HbA1c levels do not inform about the glycaemic control of the patients. Therefore, only HbA1c is not a reliable method for diabetes or impaired glucose tolerance diagnosis. Biological variation between individuals suggests a different susceptibility to enzymatic glycation ( xi ), that factors vary in influencing the membrane glucose uptake, variation in the glucose binding to hemoglobin ( xii - xiii ) and variation in the glycolytic enzymes activity, which might facilitate the glycation of hemoglobin or enhance its deglycation ( xiv -) may affect the accumulation of intracellular HbA1c. Reports suggest that although the interindividual variation in HbA1c unrelated to glycemia is minimal, there is substantial interindividual variability in HbA1c and that people can be classified as “low glycators” or alternatively “high glycators” ( xv - xvi - xvii ) according to the glycation rate. Furthermore, HbA1c may be misleading if the life span of the red cell is reduced or if an abnormal haemoglobin due to, for example, thalassaemia is present. However, standardistion of HbA1c means the error in that measurement is less than in the measurement of blood glucose and
potentially of oral glucose tolerance. As a result HbA1c could be incorporated into the diagnosis of diabetes, say a level of greater than 6.5% suggesting that a blood glucose screen is warranted. Blood glucose testing is currently needed before the clinician can provide a diagnosis and therapy ( xviii ).
i
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