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In developing countries, diabetes prevalence is rising dramatically due to rapid urbanisation, growth and ageing of the population (Mbanya et al. 2010)), as well as by eating behaviours, food supply and fast food facilities in urban areas. In sub-Saharan Africa, type 2 diabetes accounts for >90% of the cases and population prevalence proportions range from 1 to 12%. Besides infectious conditions, timely diagnosis and screening for non-communicable diseases deserve attention, as both have an important impact on local economies and population morbidity and mortality. Primary prevention and treatment can reduce the incidence of both diabetes and related diseases such as retinopathy, cataract, atherosclerosis, nephropathy and diabetic neuropathy (Hall et al. 2011).
Recently, an International Expert Committee with members of the American Diabetes Association, the European Association for the Study of Diabetes and the International Diabetes Federation revised the diagnostic criteria for diabetes mellitus. Currently, diagnosis of diabetes is based on plasma glucose concentrations [either a fasting plasma glucose (FPG) concentration ≥126 mg/dl (7.0 mm), a random plasma glucose concentration ≥200 mg/dl (11.1 mm) or a 2-h plasma glucose value ≥ 200 mg/dl in the 75 g oral glucose tolerance test (OGTT)] or a haemoglobin (Hb) A1c ≥48 mmol/mol (The International Expert Committee 2009). Venous blood glucose remains a widely used tool for diagnosing and monitoring of patients with diabetes, but this analysis is subject to pre-analytical variation (pre-analytical conditions for blood prelevations: fasting; pre-analytical conditions of transport: an expected loss of glucose of ±10 mg/dl/h) (Janssen & Delanghe 2010). Although HbA1c is now considered as a good diagnostic tool with high specificity and a low sensitivity (Zemlin et al. 2011), various haemoglobinopathies (e.g. thalassaemias) factors that impact red blood cell survival and red blood cell age, uraemia, hyperbilirubinaemia and iron deficiency may influence the results (Herman & Cohen 2012). The cost of analysis limits the use of HbA1c in certain regions of sub-Saharan Africa. In view of the particular problems of diabetes diagnosis in Africa, there is a call for an affordable method, which is adapted to regional hospitals in developing countries, requiring only simple equipment and chemicals.
Serum fructosamine assays measure the glycation of all serum proteins and reflect glycaemia over ±2 weeks (Mosca et al. 1987; Lindsey et al. 2004). This analyte presents several advantages to traditional methods (FPG and OGTT) including a greater pre-analytical stability and a correct interpretation in patients with haemoglobinopathies or anaemia (Schnedl et al. 2000; Narbonne et al. 2001). Although multiple studies have demonstrated a significant correlation between fructosamine and HbA1c, differences between fructosamine and HbA1c in various studies have also been reported (Islam et al. 1993; Braatvedt et al. 1997; Cohen et al. 2003). This finding can be explained by the fact that both markers reflect a glycaemic status over greatly different time frames as a consequence of very diverse half-lives (Nayak et al. 2011).
As the protein content in a keratinised structures (including nails) is approximately 80% of the total mass (Goldsmith 1983), glycation of nail proteins has been observed (Bakan & Bakan 1985; Masuta et al. 1989; Seven et al. 1993; Márová et al. 1995; Kobayashi & Igimi 1996; Yajima et al. 2005). Nail clippings can be easily obtained and finger nail fragments rather than blood could be considered as specimens for the analysis. In this pilot study, we explored the possibilities of measuring glycation of nail proteins as a non-invasive method to diagnose and monitor diabetes.
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Nail proteins are subject to non-enzymatic glycation (Bakan & Bakan 1985; Masuta et al. 1989; Seven et al. 1993; Márová et al. 1995; Kobayashi & Igimi 1996; Yajima et al. 2005). The present study shows, for the first time, the successful application of a nitroblue tetrazolium-based test for measuring nail protein glycation in human fingernails. The presented glycated nail protein assay turned out to be reproducible. In contrast to glucose determinations (Chan et al. 1989), assaying nail protein glycation does not imply important pre-analytical problems, as even prolonged storage (up to 2 weeks) at 37 °C did not affect test results.
The nail is a specialised keratinous skin appendage that grows approximately 2–3 mm per month, with complete replacement achieved in 6–9 months (Cashman & Sloan 2010). As variation in human nail length is smaller than hair length, this test can be better standardised. The choice of the finger is not critical, as nail growth velocity is comparable for all fingers (Yaemsiri et al. 2010). Use of toe nails is not recommended as diabetic foot injury is associated with trophic disturbances and nail problems (Tantisiriwat & Janchai 2008). Although glycation of nail proteins could also be determined in hair, hair is less suited as a sample because of variation in its length, which creates standardisation problems. Also previous treatment of hair with shampoos might cause analytical interferences.
As the protein content of human nails is about 80% (Goldsmith 1983), the physiological fructosamine/protein ratio is on the order of 2–2.5 μmol/g nail protein, which is a bit lower than the values found by Bakan and Bakan (1985) who used a thiobarbituric acid method, a different standardisation (based on fructose and 5-hydroxymethylfurfural) and reference values for plasma protein glycation. Typical fructosamine/protein ratios of 3.5 μmol/g protein were found. It is clear that diffusion of molecules in nails (Walters & Fly 1983; Elkeeb et al. 2010) is slower than in serum, which explains the lower degree of glycation in nails, despite the longer life span of a finger nail segment.
The test provides information about the period that the nail was exposed to extracellular fluid glucose. The presented method may be regarded as an alternative for assessing long-term regulation of glycaemia. Our data are in agreement with literature data on nail protein glycation obtained in stratum corneum, nails (Bakan & Bakan 1985) and hair (Masuta et al. 1989; Seven et al. 1993; Kobayashi & Igimi 1996). The increase in reference values upon ageing may be explained by the slower growth rate of nails (Cashman & Sloan 2010), by altered chemical composition of nail proteins (Dittmar et al. 2008) and by the increasing reference range for serum glucose upon ageing (Ghasemi et al. 2011).
The sensitivity obtained in the present study for glycated nail proteins was better than that of the trivial assessment of urinary glucose (van der Sande et al. 1999). Also, the pre-analytical care (often a problem in rural communities in developing countries) is far less critical for glycated nail proteins. Glucosuria is strongly affected by diet and diuresis. In this respect, analyses of glycation in nails definitely offer advantages.
Despite the marked differences observed in glycated nail protein values between diabetics and non-diabetics, glycated nail protein values correlated poorly with HbA1c values. Discordances between HbA1c and other measures of glycaemic control are common in clinical practice as these markers reflect different exposure times, but these differences remain incompletely understood (Cohen et al. 2003). In contrast to HbA1c formation, which is a slow intracellular process occurring during the entire life span of an erythrocyte (about 120 days), exposure of nail proteins to extracellular glucose is a longer process (>6 months), as the transfer of the basal zone to the distal part of the nail takes several months. Interestingly, HbA1c is not a substrate for deglycating enzymes as HbA1c values depend on glycation of valine linked glycation. Lysine-linked glycation is a substrate for fructosamine 3 kinase (Delpierrre et al. 2004; Delplanque et al. 2004). This enzyme was not discovered at the time HbA1c was introduced as a long-term diabetes marker. Currently, we know that fructosamine 3 kinase is polymorphic. Besides biochemical reasons, also glucose diffusion rate from the blood vessel to the nail bed may show variation. These differences may contribute to the observed lack of correlation between glycated nail protein and HbA1c.
The presented method may have an important clinical value in sub-Saharan Africa. In rural Africa, where many people have an aversion to blood sampling, collecting fingernail clippings is an interesting alternative because of its much lower psychological threshold. Moreover, collecting nail clippings does not require medically trained personnel. In comparison with conventional glycaemia determinations, the pre-analytical phase is much less critical as the nail clippings can be stored for a long period, even at 37 °C. As the reagent cost for our glycated nail protein assay is very small (reagent cost: ±0.1 dollar/test), the reagents can be easily prepared and the required equipment is simple. Thus, the test is excellently suited for developing countries. Our results suggest that the determination of glycated nail proteins may have the potential to serve as a diagnostic marker of diabetes mellitus and may provide useful information for management of diabetic complications in situations where blood sample analysis is not possible. As this is a pilot study, further studies are needed to investigate the in vivo kinetics of nail protein glycation and to assess the clinical value of the assay. It could be interesting to further investigate the correlation between glycated nail protein and other markers of glycaemic control, such as estimated average glucose, fructosamine and glycated albumin.