DEVELOPMENT OF NOVEL DETECTION TECHNOLOGIES
The kidney is a very important organ which performs many functions that are essential for regulation of constant environment and maintenance of metabolic homeostasis. Renal disease is a worldwide public health problem (its end stage is renal failure), which then requires renal replacement therapy such as haemodialysis or kidney transplant to maintain life. The estimates of the number of people receiving renal replacement therapy is on the increase and may not reach a steady state for about another 25 years, which costs over 2% of the total NHS budget. The condition has been found to affect between 1- 4 persons in every 1000. Due to this high figure, the cost of healthcare is soaring high and runs into billions of pounds annually. Treatment options are better if there is an early diagnosis of the disease. The aim of the proposed project is to develop a device which can monitor renal function ideally by patient or clinician at the point of care.
In the management of critically ill persons, renal dysfunction is a major problem. And regardless of its causes, its main outcomes are renal failure, reduced renal function, and cardiovascular disease (Liangos et al., 2006).
According to Remuzzi G. et al., 2002, increasing evidences have shown that these adverse outcomes can be prevented by early identification and monitoring of relevant changes in renal function and then treated immediately. However, renal disease is most under diagnosed and under treated, which then results in the inability of the early detection to prevent the adverse outcome of the disease (Mc Clellan WM et al., 1997). This can be due to lack of the application of simple tests for early detection and evaluation.
The incidence of chronic renal disease has been found to complicate about 1-7% of all hospital admissions (Liangos et al., 2006). It also complicates 1-25% of all intensive care unit admissions (de Mendonca et al., 2000). The risk factors for chronic renal disease include diabetes mellitus, hypertension, oxidative stress, family history, age and race ethnic status (Johnson CA et al., 2004).
Diabetes mellitus, mostly called diabetes is the major risk factor for chronic renal development and progression in most developed counties (Stengel B et al., 2006). It is not alarming for people with chronic renal disease to also have an abnormal lipid level (Keith DS et al., 2004). The abnormal increase in lipid level - hyperlipidemia increases the risk of atherogenesis, which is a contributing factor to cardiovascular death (Chertow GM et al., 2004). In comparison to the normal population, cardiovascular death remains 10-20 times higher in people with End- stage Renal Disease (Rigalleau V et al., 2005).
Hypertension, which is the second risk factor of renal disease, has statistically been found to have a significant link with serum creatinnine increase (Rosansky SJ et at., 1990).National Heart, Lung, and Blood Institute (NHLBI) 2004, suggests that ineffective treatment of hypertension as been found to worsen the condition of chronic renal disease and also make it difficult to control high blood pressure. And the uncontrolled high blood pressure can result in a direct damage to small blood vessels within the nephron unit (Glassock RJ, 1998) and as times continues the kidney loses its function of auto-regulating glomerula filtration flow and pressure. Thus, the increase in the arterial pressure in the kidney brings about hyperfiltration, which manifests as proteinuria and albuminuria (glassock RJ, 1998).
According to the free radical theory of ageing, endogenous oxygen radicals have been found to be produced in cells after the metabolism of oxygen resulting in highly toxic by-product called reactive oxygen species (ROS) which results in a pattern of cumulative damage (Harman D, 1957; Leichert and Jakob, 2004).
Thiols are derived from organic sulphur and their function is to stabilise protein structures through the formation of disulfide bonds that are covalent in nature (Himmelfab et al., 2004). Thiol groups are very vulnerable to non-specific reactions during oxidative stress, which is due to their increased reactivity rate (Leichert and Jakob, 2004). These sulphurhydryl thiols have abilities to operate as biomarkers which are of great importance in the regulation of several biochemical processes and are essential to maintain life (White, P. C.; Lawrence, N. S.; Davis, J.; Compton, R. G., 2001). This is possible due to the possession of antioxidant properties which aids in the regulations of the biochemiscal processes.
Oxidative stress has been found to be linked to the pathogenesis of chronic renal failure, hypertension and so many complications of these conditions (Vaziri and Nosratola, 2004). The treatment of renal disease requires renal replacement therapy such as hemodialysis or kidney transplant to maintain life. Early detection and management of people with established or high risk of renal disease can significantly reduce complications and healthcare cost by reducing the need of steady hospital visits (Chertow GM, 2004). However, failure to start or make reasonable changes in drug therapy or ineffective treatment can elevate a swift disease progression and result in death.
The management of people with chronic renal disease are targeted at cardiovascular risk reduction, delayed disease progression, improved quality life outcomes, and reduced cost. People with high risk of chronic renal disease should be identified and assessed for presence of mico- or macro- albuminuria with their GFR estimated annually (Johnson CA et al., 2004).
Glomerulus filtration rate (GFR), which the measurement of glomerular filtrate formed in the kidneys (a substance that is similar to plasma but has no proteins), can be used to evaluate the ability of the kidney to excrete waste products from the body. It can also be used for the early renal damage and deterioration of the kidney.
Serious investigative efforts have resulted in the detection and evaluation of many serum and urinary proteins as biomarkers of chronic renal disease (Emeigh Hart, 2005; Trof et al., 2006; Vaidya and Bonventre, 2006).
Several endogenous plasma markers of GFR such as Urea, Creatinine, and some small molecular weight proteins have been used as a point-in-time measurement in order to assess GFR (Price and Fenneey, 2000).
Urea, which is synthesized in the liver from ammonia and carbon IV oxide through the urea cycle and its enzymes, is the primary end product of protein catabolism. When released into the plasma, its subsequent excretion is predominantly above 90% through the kidney and a lesser value is excreted through sweat and gastrointestinal tract (Finco, 1997; Newman and Price, 1999).
The changes in glomerular function and structure were previously thought to be initiated by microalbuminuria. However, recent evidence in rats shows that the normal glomerular filter may leak albumin at levels that are higher than the previously expected. This condition can indicate changes in the proximal tubule cell retrieval pathway (Russo et al., 2007). Thus, protein albumin is a useful biomarker of renal disease and proximal tubular cell damage (Koch Nogneiva et al., 1998; Kern et al., 2000).
The metabolism of Creatine results in creatinine which is a muscle derived by-product of creatine. Presently, creatinine is the most common biomarker for renal function; however, its vulnerability is hampered due to the fact that about 50% kidney function must take place before creatinine level rises ( Naud and Leblanc,2008). This process leads to a delay in the diagnosis of acute renal failure and that of other biomarkers that may be available. For instance, most of the following biomarkers- cystin C, actin, urinary low-molecular weight proteins, tubular cell proteins, are assayed using western blot and enzyme linked immunosorbent assay and not by lab-on- a chip device (Naud and LeBlanc, 2008). Due to the limitations of present biomarkers used in the diagnosis of renal disease, their inefficient methods, and time wasting, this proposed project will investigate the detection of a novel biomarker- Glutathione (GSH) antioxidant thiol using a portable screening device which will be developed in the course of the project.
An electrochemical screening device (sensor) will be developed which will be capable of measuring glutathione.
Naphthoquinone derivatives that are reactive towards physiological thiols (GSH) will be selected. Quinone thiol conjugates will be formed in which during the absence of GSH, the NQ indicator which is mainly in the oxidised form (I) will in the presence of GSH be converted to the reduced form (II) as shown in Figure 1.
The changes in the redox balance will then be detected at a screen-printed electrode assembly. This difference in electric potential within the system will then be related to the concentration of glutathione. Monitoring difference in redox potential is a consequence of NQ reactions with GSH. In the past few years the mechanism of the potentiometric response of simple benzoquinone-thiol system has been determined, and a similar approach is used for NQ systems (Diga A et al., 2003). It involved;
- All reagents used being in the highest grade available and used without further purification.
- All solutions and subsequent dilutions being prepared daily by using deionised water and refrigerating when not in use.
- All solutions generally being used within one hour of preparation to minimise losses due to aerial oxidation.
- Electrochemical measurement being recorded using a µ-auto lab computer controlled potentiostart with standard III-electrode configuration and a typical cell volume of 20cm3.
- The glassy carbon acting as the carbon electrode, platinum wire wound into a spiral acting as the counter electrode with a colomel reference electrode completing the cell assembly.
- All measurements obtained at a temperature of 22+/-2oC under argon flow.
- The electrodes being polished between each set of experiments with diamond plates of decreasing particular size.
The primary advantage of this propose methodology over convention lab systems is that the method can be used for near patient testing or point of care. It is very easy to use unlike other methods and it is not expensive. The detection pattern and the instrumentation needed to bring about the measurements are readily accessible resulting in a wider uptake and extensive development of the method.
Early detection and management off people with established or high risk renal disease can significantly reduce complications and healthcare cost. However, present biomarkers used for this early detection have got limitations, making them not to function effectively. And this brings the need of a novel biomarker. The success of this proposed project will result in the development of a novel biomarker which can detect early onset of renal disease.
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