Volume 41, Issue 9

Research into diabetes mellitus and the clinical practice of diabetology are a fascinating illustration how the accumulation of knowledge leads to changing scientific theories and paradigms. The pivotal point in the history of diabetes, the discovery of insulin and its introduction to treatment, was one of the most spectacular events in medicine. Later, diabetes care provided, among other developments, models for multidisciplinary approach to treatment and the blueprint for community-based epidemiological studies. The history of diabetology also illustrates the role of chemistry, and later laboratory medicine, in the management of disease. This article is an introduction to the special issue of Clinical Chemistry and Laboratory Medicine, focused on diabetes mellitus and its complications.

The incidence of type 2 diabetes mellitus (T2D) and obesity is increasing rapidly worldwide, reaching epidemic proportions. Insulin resistance is a key feature in both conditions and plays an important pathophysiological role. Over the last two decades a central role in the origin of insulin resistance has emerged for defects in the intracellular insulin signaling cascade leading to glucose uptake. Herein, we will 1) review insulin signaling pathways leading to glucose uptake, 2) review mouse models of insulin resistance that demonstrate the pathophysiologic importance of specific defects of these pathways and 3) discuss the molecular basis for insulin resistance in some human disease states known to be associated with insulin resistance. Finally, we will briefly mention some novel treatment targets for T2D stemming from this knowledge.

Diabesity, that is to say, obesity-dependent diabetes, has emerged as a major public health problem. Though diabesity is basically explained by insulin resistance and pancreatic β cell dysfunction, new paradigms have evolved to explain these alterations in the context of the modern epidemics of obesity and diabetes. Among these, the association of inflammation with obesity is an important component of the common soil from which diabetes and cardiovascular diseases (CVD) derive. This Review presents our epidemiological findings, based primarily on the ARIC Study, in the context of the other epidemiological studies supporting the inflammatory nature of diabetes, CVD and the metabolic syndrome. We also review the characteristics of the innate immune system, including the molecular interface of innate immunity with metabolism, components of which are responsible for the presence of a state of mild, chronic and systemic inflammation related to diabesity. Finally, we present obesity as an inflammatory condition, obesitis , and propose a conceptual framework that integrates the epidemiological findings with new provocative basic science results.

The insulin-sensitising adipose hormone adiponectin is reduced in type 2 diabetic patients. We assessed the relationships between plasma adiponectin and chronic hyperglycaemia. Adiponectin levels and glycated haemoglobin (HbA 1c ) were measured at enrolment and after 90 days in 16 patients with type 2 diabetes aged (mean ± SEM) 63.0±0.6 years, with body mass index (BMI) 30.2±0.5 kg/m 2 and HbA 1c concentration 7.4± 0.1%, who did not modify their hypoglycaemic treatment during the observation period. Furthermore, plasma adiponectin was measured in 29 adult patients with type 1 diabetes and compared with 29 control subjects matched for sex, age, BMI, waist circumference and bioimpedance-estimated fat mass. In type 2 diabetic patients at enrolment, adiponectin concentration correlated with BMI (r = −0.46; p < 0.05), but not with HbA 1c . During the prospective observation, variations of adiponectin showed a significant correlation with variations of BMI (r = −0.47; p < 0.01), but not with variations of HbA 1c concentration. These results were confirmed by multivariate analysis after adjustment for sex and age. Adiponectin levels in type 1 diabetic patients (380.8±13.7 ng/ml in women, 192.5±13.9 ng/ml in men) were significantly (p < 0.05) higher than in control subjects (277.6±11.0 ng/ml in women, 102.7±5.1 ng/ml in men); plasma adiponectin correlated significantly with BMI and waist circumference, but not with HbA 1c . In conclusion, the reduction of plasma adiponectin levels in type 2 diabetic patients does not appear to be determined by chronic hyperglycaemia. Adiponectin levels are increased in type 1 diabetes, but this phenomenon is not attributable to differences in nutritional status or body composition.l21

Blood platelets take up glucose through insulin-independent GLUT-3 transporter. It is, however, unclear how diabetes affects further steps of glucose and glucose-derived acetyl-coenzyme A (CoA) metabolism in platelets. There is no evidence to explain whether these changes are linked to the disease-induced disturbances in platelet function. We found that activities of some key enzymes of glucose and acetyl-CoA metabolism in platelets were elevated in diabetes. Activities of hexokinase, pyruvate dehydrogenase and ATP-citrate lyase in diabetic platelets were found to be increased by 53, 56 and 88%, respectively. Accordingly, diabetes brought about 86% increase of platelet acetyl-CoA and activation of malonyl dialdehyde synthesis as well as spontaneous and thrombin-induced platelet aggregation by about 56, 50 and 15%, respectively. Significant correlations have been observed between some parameters of acetyl-CoA metabolism, platelet function and serum fructosamine in diabetic patients but not in healthy individuals. Our findings indicate that increased platelet activity in diabetic subjects may, at least in part, result from chronic hyperglycaemia-induced changes in acetyl-CoA metabolism, yielding an increase in its concentration in platelets.

Increasing evidence in both experimental and clinical studies suggests that there is a close link between hyperglycemia, oxidative stress and diabetic complications. High blood glucose level determines overproduction of reactive oxygen species (ROS) by the mitochondria electron transport chain. High reactivity of ROS determines chemical changes in virtually all cellular components, leading to DNA and protein modification and lipid peroxidation. Measurement of biomarkers such 8-hydroxy-2′deoxyguanosine (8-OHdG), isoprostanes, malondialdehyde (MDA) and nitrotyrosine is a useful tool to assess the oxidative stress of the organism. Knowledge of the mechanisms of ROS damage of is the first step for development of new therapeutic molecules and for rationalizing the use of existing drugs.

Advanced glycation irreversibly and progressively modifies proteins over time and yields the advanced glycation end-products (AGE). AGEs are thought to contribute to the development of atherosclerosis and of diabetic and uremic complications. Their inhibition has thus become a therapeutic goal. In this article, we discuss the role of various reactive carbonyl compound (RCOs) in the genesis of AGEs, postulate the existence of “carbonyl stress” in complicated diabetes and, finally, discuss therapeutic perspectives.

Advanced glycation and lipoxidation end-products (AGE/ALE) increase in tissue proteins with age and at an accelerated rate in diabetes. This Review focuses on the nature and source of AGEs/ALEs and the factors affecting their formation in tissue and plasma proteins. Lipids are identified as an important source of chemical modification of proteins in diabetes, and the role of diabetes, dyslipidemia and renal disease in formation of AGEs/ALEs is reviewed. The article concludes with a discussion of ELISA assays for AGEs/ALEs and the merits of measuring AGEs/ALEs in the clinical laboratory.

Determination of glyoxal and methylglyoxal levels in plasma is of great interest, since it allows us to evaluate oxidation processes occurring in glycated proteins. A method based on a simple derivatization procedure followed by gas chromatography/mass spectrometry (GC/MS) analysis has been developed. Ten diabetic patients were evaluated before and after improvement of glycemic control. Fasting plasma glucose, hemoglobin A 1c (HbA 1c ), advanced glycation end products (AGE), pentosidine, glyoxal and methylglyoxal levels were measured. The percentage decreases of the levels of fasting plasma glucose, HbA 1c and AGE were larger than those of pentosidine, glyoxal and methylglyoxal. These results may be explained by considering the different position of these compounds in the Maillard reaction pathways: these two sets of metabolic parameters give different pictures of patients' metabolic control. The measurement of glyoxal and methylglyoxal may be particularly important in the evaluation of the possible effect of oxidative stress. Other metabolic pathways can contribute to glyoxal production, and the observed minor decrease in these compounds can be, in principle, ascribed to such effect. However, a similar behavior of pentosidine indicates that these alternative pathways can be only partially responsible for glyoxal and methylglyoxal production.

Type 2 diabetes mellitus is a leading cause of morbidity and mortality. Cardiovascular disease (CVD) is the most prevalent complication and primarily accounts for the excess morbidity and mortality in diabetic patients, but microvascular complications, such as kidney disease and retinopathy, are frequent and contribute to the total disease burden. Lipid abnormalities in patients with type 2 diabetes are a major problem and associated with the increased risk of CVD. The most common pattern of dyslipidemia in these patients consists of elevated levels of triglycerides and low levels of high-density lipoprotein cholesterol. Low-density lipoprotein levels in these patients are often similar to that of the nondiabetic population, although there may be important qualitative differences in the pattern that contribute to the increased risk of CVD. Abnormal levels of urinary albumin occur in 30–40% of patients with type 2 diabetes and the presence of kidney disease enhances the mortality from CVD. Microalbuminuria, an early marker of diabetic nephropathy, is an independent risk factor for CVD. The increased levels of urinary albumin secretion may represent a more generalized vascular damage than renal microvascular injury alone. This Review focuses on the significance of diabetic dyslipidemia and microalbuminuria to CVD risk as well as to kidney complications. We also discuss the role of aggressive therapy to ameliorate vascular injury in the diabetic patient and reduce or prevent the cardiovascular and renal consequences of the disease.

Haemoglobin A 1c (HbA 1c ) is the pre-eminent factor for quantifying the risk of complications in patients with diabetes and for monitoring glycaemia. Intervention to lower blood glucose in the two landmark clinical trials, the UK Prospective Diabetes Study (UKPDS) and the Diabetes Control and Complications Trial (DCCT), led to a reduction in the microvascular complications of diabetes. Glycaemic status could be compared in the UKPDS and DCCT as the Bio-Rad Diamat HPLC analyser, as used in the DCCT, was introduced in 1989 for measurement of HbA 1c in the UKPDS, after liaison with the DCCT. Results from other methods used for measurement of glycated haemoglobin during the UKPDS were aligned to this method. The Bio-Rad Diamat analyser in the central laboratory for the UKPDS, reference range 4.5–6.2% HbA 1c , was certified as comparable to the DCCT by the National Glycohemoglobin Standardization Program in 1998. A median difference in HbA 1c of 0.9% was maintained over 10 years between the intensively and conventionally treated groups in the UKPDS (7.0% vs . 7.9% HbA 1c ) despite HbA 1c increasing over time. Clinical care was transferred to general practitioners after the end of the main glucose control study for post-study monitoring. Over the first 3 years of post-study monitoring, HbA 1c rose slightly in the previously intensively treated group with no appreciable increase in the conventional group, due to intensification of therapy. At near-normal HbA 1c , <6%, the updated mean value chosen to reflect glycaemic exposure throughout the UKPDS, the incidence of myocardial infarction was 2–3 times that of microvascular disease, with similar incidences for both complications at >10% updated mean HbA 1c . Relationships between the risk of complications of type 2 diabetes and updated mean HbA 1c had no observable thresholds. The UKPDS risk engine derived from the UKPDS database calculates coronary heart disease risk using HbA 1c as a continuous variable and could now replace the Framingham equations for patients with type 2 diabetes.

The Diabetes Control and Complications Trial (DCCT) and the United Kingdom Prospective Diabetes Study (UKPDS) demonstrated conclusively that risks for complications in patients with diabetes are directly related to glycemic control, as measured by glycated hemoglobin (GHb). Many diabetes organizations worldwide now recommend GHb targets in terms of DCCT/UKPDS hemoglobin A 1c (HbA 1c ). However, in 1993 there was a lack of comparability of GHb test results among methods and laboratories that represented a major obstacle to meaningful implementation of specific guidelines for diabetes care. The National Glycohemoglobin Standardization Program (NGSP) was implemented to enable laboratories to report DCCT/UKPDS-traceable GHb/HbA 1c results. The number of methods and laboratories certified by the NGSP as traceable to the DCCT has steadily increased. Proficiency testing results show marked improvement in the comparability of GHb results. By the end of 2002, 98% of surveyed laboratories (n = approx. 2000) reported GHb results as HbA 1c or equivalent compared to 50% in 1993. Ninety-seven percent of laboratories used an NGSP-certified method. For most certified methods in 2002, between-laboratory CVs were <5%. For all certified methods in 2002, the mean HbA 1c value (%) was within 0.8% HbA 1c from the NGSP target at all HbA 1c concentrations. The vast majority of laboratories in the US are now reporting results that are traceable to DCCT/UKPDS outcomes.

The measurement of glycated haemoglobin has become centrally important in the monitoring of glycaemic control in the patient with diabetes. A number of analytical techniques have been described to measure this important haemoglobin fraction, and the fraction measured depends on the technique used; this has resulted in laboratories reporting different fractions i.e. , HbA 1c , Hba 1c and total glycated haemoglobin. This paper outlines methods currently used in clinical laboratories for the measurement of this glycated fraction. It describes the principles behind these methods and goes on to describe the new IFCC reference method, which will be used in the future to standardise methods used in the clinical laboratory. Analytical goals and factors that interfere with methods are also discussed.

Diabetes mellitus is an excellent case study of the evolution, and successful application, of point-of-care testing. It offers a valuable history of the way in which technology has evolved, and continues to evolve, to meet the needs of patients whilst also providing for a more optimal delivery of care. Diabetes mellitus is also a good exemplar of where test and treatment regimes must operate in complete harmony in order to achieve the greatest benefit. Thus whilst the measurement of blood glucose is central to the screening, diagnosis and management of diabetes, it is in the latter use, largely related to supporting compliance with therapy, that point-of-care testing is of greatest relevance. In addition, there are other tests, more associated with the management of diabetes and early detection of the complications associated with diabetes, that are appropriate to the point-of-care testing modality. This Review will focus on the developments in technology and the harnessing of this innovation to support the delivery of clinical, organisational and economic benefits in the care of patients with diabetes mellitus.

Various guidelines are in use for validating blood glucose meters. There are currently no analytical guidelines for validating procedures available for non-invasive or minimally invasive blood glucose meters. As a result manufacturers of these meters need to comply with different regulations in different countries with concomitant high costs. Some of the differences between different guidelines will be discussed in this article and recommendations for improvement will be presented.

Obtaining representative physiological samples for glucose analysis remains a challenge especially when developing less invasive glucose monitoring systems for diabetic patients. In the present study the glucose content of the stratum corneum was compared with the amount of glucose obtained by short aqueous extractions from a site on the dorsal wrist, using high pressure liquid chromatography with pulsed amperometric detection. Ten successive aqueous 1-minute extractions of the site yielded a total of 60 ng cm −2 . The total glucose content of the stratum corneum of the site, determined from 30 successive tape-strippings of the site, was 360 ng cm −2 . After tape-stripping, the transcutaneous aqueous extraction rate was 86±13 ng cm −2 min −1 , compared with rates of 80–600 ng cm −2 min −1 obtained with suction effusion or microdialysis after tape-stripping. Glucose on the surface of the skin and within the stratum corneum should be considered as sources of extraneous glucose contamination during testing of less invasive glucose monitoring devices.

The importance of measuring microalbuminuria is well established. However, only scanty data are available concerning the biological variability of albumin excretion in type 2 diabetic subjects. We report our experience from a large clinical trial of a new antihypertensive drug (Lercanidipine) designed to reduce albumin excretion and blood pressure in type 2 diabetic patients with hypertension and microalbuminuria. Eighty seven patients with persistent microalbuminuria were studied within 1 year of the clinical trial. The measurements were performed on blood and timed urine samples frozen at −80 °C and shipped to a central laboratory unit. Preliminary experiments were performed to assess albumin stability in urine under various conditions (4 °C, −20 °C and −80 °C), particularly with regard to the albumin/creatinine ratio. Urine samples can be stored up to 3 weeks at 4 °C or up to 2 months at −80 °C. The biological variability of the albumin excretion rate was 25.7%, while that of the albumin/creatinine ratio was 13.4%. These data are useful in defining the analytical goals of imprecision for microalbuminuria (CV = 13% for albumin, and CV = 6% for albumin/creatinine ratio). No correlation between albumin/creatinine ratio and HbA 1c was found in the cohort of 61 microalbuminuric patients who completed the trial. The results of this study confirm that the albumin/creatinine ratio is much more suitable for monitoring albumin excretion in longitudinal studies than the albumin excretion rate.

Measurement of proinsulin is an important tool in the assessment of pancreatic β cell function in patients with type 2 diabetes. The goal of this study was to perform a technical and clinical evaluation of two specific chemiluminescence assays (CLIA) for the determination of intact and total proinsulin in comparison to a radioimmunoassay (RIA) method for the measurement of total proinsulin. A total of 191 serum samples from patients with type 2 diabetes were used to perform a regression analysis. The total proinsulin CLIA showed higher proinsulin levels than the two other proinsulin assays (mean ± SD: 55.9±58.1 pmol/l, p < 0.001 in both cases). The intact proinsulin CLIA (22.5±20.9 pmol/l) gave lower values than the RIA for total proinsulin (31.9±25.4 pmol/l, p < 0.001 vs . CLIA, r = 0.948). The RIA has a 95% cross-reactivity to des31,32-proinsulin, which is secreted during the process of β cell deterioration. The intact proinsulin CLIA has virtually no crossreactivity with des31,32-proinsulin (1.4%) and is therefore more specific for intact proinsulin than the RIA. This test does not measure further degradation products, in contrast to the total proinsulin CLIA. The CLIA is, therefore, more specific for total proinsulin measurement than the RIA. Both CLIAs could be performed much faster (4 hours) than the RIA method (75 hours/4 days). In conclusion, the CLIA methods show improved qualitative outcomes, higher specificity and several technical advantages over the RIA method.

In 1997 the American Diabetes Association lowered the threshold for diagnosis of diabetes from a fasting plasma glucose concentration of 7.8 mmol/l to 7.0 mmol/l and advised that the oral glucose tolerance test no longer be used in routine clinical practice. In 1999 the World Health Organization endorsed the reduction in fasting plasma glucose threshold but recommended retaining the oral glucose tolerance test for anyone with impaired fasting glucose (6.1 mmol/l–6.9 mmol/l). This Review discusses the impact of these changes on the prevalence of diabetes and examines the implications for individuals and specific high-risk groups. The phenotype of those diagnosed with diabetes and the predictive value for the development of complications according to the different criteria are compared. It is clear that these changes in diagnostic criteria have major importance both for individuals and for resource planning at a national level.

In the new lowered diagnostic discriminator for diabetes mellitus (DM) from the American Diabetes Association (ADA), fasting peripheral venous plasma glucose (f-vPG) of 7.0 mmol/l is identical to the 99.9 centile of f-vPG (7.05 mmol/l, 95%CI: 6.91–7.20 mmol/l) in a low-risk reference population. We investigated its diagnostic concordance with other diagnostic discriminators. As no index test is available for DM we used the ADA discriminator as gold standard. We isolated a low-risk reference population (n = 424) from a randomised general population (n = 726) by ruling out of all cases with clinical and biochemical risk indicators for DM. We based our analysis on measurements traceable to primary standard concentration, a bias of <1.5% and CV% < 2.5. The distribution of the fasting capillary whole blood glucose (f-CBG; mmol/l) in the reference population was ln Gaussian with the 99.9 centile of 6.62 mmol/l (95% CI 6.47–6.77 mmol/l) and the 97.5 centile of 5.92 mmol/l (5.82–6.02 mmol/l). The 6.1 mmol/l f-CBG WHO limit corresponds approximately to the 97.6 centile, and this limit is thus not traceable to the ADA discriminator, which corresponds to f-CBG of 6.4 mmol/l. This is the case in groups only, as recalculation will introduce unpredictable errors. Thus, in our general population a varying number of subjects will be at risk of DM as a mere consequence of different limits. The f-CBG limit of 6.1 mmol/l will thus lead to 2.4% false-positive diagnoses or, in EU, to around 44 × 10 6 adults being diagnosed. The number of cases at risk of DM vary from 5.4 × 10 6 to 44 × 10 6 in EU. We conclude that application of different diagnostic limits results in highly variable number of diagnosed DM cases, and therefore one diagnostic discriminator is needed to provide reproducible diagnoses.

In the recent American Diabetes Association (ADA)/WHO recommendations, the oral glucose tolerance test (OGTT) was replaced by the measurement of a single fasting glucose concentration with a decision limit for the detection of type 2 diabetes mellitus (DM) reduced. This proposal, however, misses all cases of isolated post-prandial hyperglycaemia. Therefore, a study was undertaken to develop a post-challenge, one-sample mode of diagnosis. OGTT was performed in 240 high-risk subjects who were suspected to suffer from type 2 DM. Glucose concentrations were determined at 30 min intervals in the capillary blood, venous blood and plasma, and insulin was determined in venous plasma only. The test results were classified in non-disease and disease group according to the decision limits recommended by ADA/WHO. Furthermore, the early insulin response and an insulin sensitivity index were used to determine new cut-off values. These were identified as the concentrations demonstrating the highest diagnostic efficiency and were lower than the WHO limits. The 2 h post-load plasma concentration led to higher efficiency at a cut-off value of 9.0 mmol/l glucose (162 mg/dl) compared to concentrations of samples taken in the fasting state, at an earlier time of the OGTT, or in venous and capillary blood. Under this condition, 72 diabetic patients (35%) were detected in the study group (n = 207), whereas only 36 (17%) were found with one sample in the fasting state and 53 (26%) with two samples using the ADA/WHO criteria. Therefore, a single venous plasma sample taken after 2 h post-glucose challenge appeared to be most efficient for the early detection of DM.

The clinical laboratory plays an essential role in both the diagnosis and therapeutic monitoring of diabetes mellitus. Here, a short review of the main laboratory parameters used in the monitoring of diabetes–glucose (including point-of-care testing), HbA 1c and microalbuminuria–is presented. The impact of pre-analytical and analytical performance criteria on the clinical decision making process is discussed.

This article provides an overview of the role of metabolite toxicity, low-grade inflammation and disturbed cellular signaling in obesity, glucose intolerance and diabetes. It also highlights links between this continuum of deteriorating glucose tolerance and atherosclerosis. Obesity, diabetes mellitus, and cardiovascular disease are all related to diet and to the level of physical activity. They have reached epidemic proportions worldwide. Glucose intolerance and diabetes increase the risk of atherosclerotic events. Moreover, obesity, and glucose intolerance or diabetes, are components of the metabolic syndrome, which also imparts an increased cardiovascular risk. There is increasing recognition that common mechanisms contribute to diabetes and cardiovascular disease. Following increased calorie intake and/or decreased physical activity, fuel metabolism generates excess of ‘toxic’ metabolites, particularly glucose and fatty acids. Homeostasis is affected by the endocrine output from the adipose tissue. Reactive oxygen species are generated, creating oxidative stress, which exerts major effects on signaling pathways, further affecting cellular metabolism and triggering low-grade inflammatory reaction. This perspective on the diabetic syndrome has been reflected in the approach to its treatment, which integrates maintenance of glycemic control with primary and secondary cardiovascular prevention. Laboratory medicine should support diabetes care with an integrated package of tests which, in addition to glycemic control, enable assessment and monitoring of the risk of microvascular complications as well as cardiovascular disease.