Medical science reveals to us some vitamins are fundamental to living healthy lives. They have been described as the building block of the body and thought to be nature's method of repairing cells, damaged tissues and bones that the human body comprises of. The National Institute of Health literally makes known to us that vitamins are substances needed for growth by the body as well as normal development. The body needs 13 vitamins and each vitamin performs a specific role.
The B vitamins are a group of eight water soluble vitamins that function significantly in cell metabolism. They are generally essential in that they aid cell growth and cell division, improve the function of the nervous system as well as the immune function, boost the rate of metabolism, sustain good muscle tone and healthy skin to mention a few. Providing energy to the body which takes place during the conversion of glucose to carbohydrate is another responsibility of these vitamins, not to mention that they are critically involved in proteins and fats. Before now, the B12 were thought to be a single vitamin but research has shown them to be chemically distinct. Of particular interest is the B12 vitamin.
1.2 Chemistry of Vitamin B12
Discovered by Dorothy Hodgkin, vitamin B12 with the IUPAC name α-(5, 6- dimethyl benzimidazole) cobomidcyanide and empirical formula C61-64H84-90N14O13-14PCo is an orgarnometallic compound and the only known biomolecule with a stable carbon metal bond. The molecule is a corrin ring that has various attached sidegroups. The ring has 4 pyrrole subunits which have a C-CH3 methylene link at opposite sides. The two pyrroles are joined directly by a C-CH3 methylene link. One bridging methylene group is removed, making it resemble a porphyrin. Each atom of nitrogen is aligned to the cobalt atom at the centre of the molecule. A nitrogen of a 5, 6- dimethylbenzimidazole is the 6th ligand beneath the ring. The other nitrogen is connected to a 5 carbon-sugar which is also linked to a phosphate group and then through one of the seven amide groups it goes back into the corrin ring.
Structure of Vitamin B12
vitamin b12
Fig 1.1 The vitamin B12 molecule.
1.4 Vitamin B12
Vitamin B12 is water-soluble and can be found in some foods naturally. To some foods it is added artificially, augmented for in the form of supplements and given as medications prescribed. Vitamin B12 is needed in the synthesis of DNA, proper formation of red blood cells and neurological functions (Zittoun and Zittoun, 1999). It also plays a role as a cofactor for methionine synthase and also L-methylmalonyl-CoA mutase. The latter acts as a catalyst in the conversion of homocysteine to methionine (Clarke, 2008), the reaction of which is essential for the synthesis of the universal methyl donor S-adenosylmethionine. Hormones, DNA, lipids, RNA, proteins are amongst 100 distinct substrates produced. L- methylmalonyl-CoA is converted to succinyl- CoA by L-methylmalonyl-CoA mutase in the breakdown of propionate (Combs, 1992). This is a vital biochemical reaction in the metabolism of proteins and fat. Succinyl- CoA is also needed for the manufacture of haemoglobin. A glycoprotein, an intrinsic factor (IF) produced by the parietal cells of the stomach binds to the free vitamin B12 and the emerging complex is absorbed by in the distal ileum by receptor- mediated endocytosis (Klee, 2000). Of a 1mcg oral dose of vitamin B12, about 56% is taken in but if the volume of intrinsic factor is surpassed, absorption diminishes greatly (Klee, 2000).
1.5 Forms of vitamin B12
The compound vitamin B12 refers to cobalt-containing cobalamins that possess vitamin activity with the exception of nonactive cobalamin analogs which could also be found in some foods and tissues. They include compounds like 5-deoxyadenosylcobalamin and Methylcobalamin (physiological forms), hydroxocobalamin (produced by bacteria), cyanocobalamins(synthetic).
1.6 Sources of vitamin B12
Sources of vitamin B12 include animal products such as liver, oysters, milk and milk products, fish, poultry, meat, clams, eggs, fortified breakfast cereals, some natural yeast, dietary supplements in the form of cyanocobalamins which is altered to active forms such as 5-deoxyadenosylcobalamin and methylcobalamin.
1.7 Vitamin B12 deficiency
Symptoms of a deficiency of vitamin B12 include weakness, loss of appetite and weight, exhaustion, constipation and megaloblastic anaemia (Herbert and Das, 1994). Other symptoms include depression, impaired memory, mental deterioration, soreness of the mouth or tongue, disorientation and struggle to maintain balance (Bottiglieri, 1996), neurological changes, for example tingling and lack of sensation in the upper and lower extremities (Healton et al, 1991) which can take place without anaemia and so, if diagnosed early, irreversible damages may not occur (Clarke, 2008). For infants, an indication of vitamin B12 deficiency includes developmental delays, failure to thrive, megaloblastic anaemia and movement disorders (Monsen and Ueland, 2003).
1.8 Vitamin B12 and Health
When a Medline search was done in 2007 dating back from 1999 on the connection between Vitamin B12 and Health, 200 results were found of which 129 was deemed to be important. These searches were carried out using the keyword Vitamin B12 and they greatly provided level II evidence. Mental health problems, cancer, adverse birth outcomes and cardiovascular disease were suggested by articles studied to be health effects of vitamin B12 deficiency.
1.8.1 Vitamin B12 and cerebrovascular disease
In a study by Mendrano et al. (2000) vitamin B6 absorption and folate were found reduce susceptibility to cerebrovascular diseases while on the other hand He et al (2004) demonstrated that the combination of B12 and folate had an inverse association with ischemic stroke. Quinlivan and colleagues (2002) showed that dependency of plasma homocysteine on folate could be reduced by supplementing folate with vitamin B12. This work implied that folate and vitamin B12 were beneficial in reducing vascular disease. The relationship between vitamin levels and vascular diseases depends on the type of stroke.
1.8.2 Vitamin B12 and cardiovascular disease
A very important risk factor for cardiovascular disease (CVD) has been shown to be elevated plasma homocysteine levels. Research has demonstrated that homocysteine levels can be reduced by combining folic acid with vitamin B12 ((Schwammenthal and Tanne, 2004; Stanger et al, 2003). Fasting plasma homocysteine levels were reduced by 32 percent in patients with CVD after ingestion of supplements of 250µg of B12 and 5mg of folic acid (Lee et al, 2004). What is not clear is the contribution of vitamin B12 alone in reducing homocysteine levels (Lewerin et al, 2003). This study showed an increase in risk of CVD especially for the elderly if they had low levels of vitamin B12.
1.8.3 Vitamin B12 and cancer
Folic acid, B6 and B12 deficiencies are to be associated with cancer. (Ames, 2001). Instead of the appropriate base, uracil is incorporated into human DNA leading to breaks in chromosomes (Ames, 2001). A protective role of vitamin B in cancer of the cervix was observed amongst Hawaiian women in a study conducted by Hernandez et al. in 2003 where there was a decrease in premalignant cervical lesions when absorption of vitamin B12 was high (Hernandez et al, 2003).
1.8.4 Vitamin B12 and birth outcome
An increase in the risk of Neural tube defects (NTD's) by up to 5 times has been associated with low levels of vitamin B12 and a mutation in the MTHTR gene together with polymorphism in methionine synthase (Ray and Blom, 2003). Many other studies (Gueant- Rodriguez et al, 2003) show that association between enzyme activity and genotypes have potential in increasing the susceptibility to spina bifida. Other researchers show that genetics might affect the transport of vitamin B12 to tissues by transcobalamin II (TCII). There is probably diminished affinity for vitamin B12 when there is a genetic variation in the TC II gene (Afman et al, 2001). The variations in abnormal birth outcomes throughout the world could be the association between nutrition and genotype which affects 1-carbon metabolism. Polymorphism in MTRR (methionine synthase reductase) and MTHFR have been linked to greater susceptibility to Down syndrome. Other polymorphisms may be pointers to NTD's (Gueant et al, 2003).
1.8.7 Vitamin B12 and mental health
Biochemical factors such as homocysteine as researchers have shown are also involved in brain function. Vitamin B12 deficiency brings about an accumulation of homocysteine in blood (Clarke, 2008). Positive associations have been revealed between raised homocysteine levels and the occurrence of both dementia and Alzheimer's disease (Seshadri et al, 2002). In patients with Alzheimer disease, those who had lower levels of vitamin B12 presented more common psychological and behavioural symptoms of dementia than those with normal levels (Miens et al, 2000). Low levels of the vitamin also cause a decline in cognition (Clarke et al, 2007). However, another study proves that evidence is still insufficient to say that the cognitive function of people with dementia can be improved with B12. There has been known to be an interrelationship between vitamin B12 found in serum and frontotemporal dementia (Engelborghs et al, 2004)
1.9 Methods for measuring quantity of vitamin B12
Measurement is a concept that is easily understood but difficult to measure in laboratories. Even after ten decades, no reliable and specific method has been developed for the detection of vitamin B12. It is therefore important to review methods that exist. These methods include microbiological assay (Kelleher et al, 1990), radioisotopic assay (Muhhamad et al, 1993), Chromatographic assays, spectroscopic assays, chemiluminescence assay -CL (Kazuyoshi et al, 2001; Zhenghua and Sheung, 2003) and recently, biosensors (Yaling et al, 2008)
1.9.1 Microbiological assays
Lactobacillus lactis is known to grow with difficulty in culture media in the absence of vitamin B12 (Shorb 1947). Based on this, test for vitamin B12 were designed to involve the use of vitamin B12 and vitamin B12 dependent bacteria and measuring growth. Other organisms that have been used include: Ochromonas malhamensis, Arthrobacter lockhead, Escherichia coli mutant, lactobacillus delbrueckii (formally called Lactobacillus leichmanni) (Watanabe et al, 2002) and Euglena gracilis (Schneider and Stroinski, 1987).
This method is not reliable because some of these bacteria were found to use substances similar in structure to vitamin 12 (Kumar et al 2010)
1.9.2 Radioisotopic dilution assays
This method involves vitamin B12 and a 57Co-labeled vitamin B12 competing for a binding site on an R-protein or IF. Compared to the microbiological method this method is more sensitive but the R-protein involved will behave differently in plasma, than it does in the reaction kit (Kumar et al 2010). Also, due to the cost of Co-labelled B12, Radioisotopic is an expensive method.
1.9.3 Chromatographic methods
In nature vitamin B12 is found bound to proteins as coenzymes like cobalamin coenzyme. Chromatographic separation of cobalamin coenzyme using Thin layer Chromatography (TLC) has been used to determine the concentration cobalamin coenzyme. Other methods that have been used here include Gas liquid chromatography which may be coupled with mass spectrometry and high performance liquid chromatography (HPLC) with fluorescence or electrochemical detection methods. The accuracy of this test depends on the amount of the vitamin B12 bound to the coenzyme and it is less sensitive when compared to the microbiological methods (Iwase and Ono, 1997). A lot of skills are required to carry out separation techniques and in HPLC, specificity is a problem when only small amount of vitamin B12 are assayed.
1.9.4 Spectroscopic methods
Various methods have been used to determine the absorption spectra of cyanocobalamins. Atomic Absorption Spectrometry -AAS (Akatsuka and Atsuya, 1989), UV/ Vis spectrophotometry, capillary electrophoresis - CE (Baker and Miller-Ihli, 2000), matrix- assisted laser desorption / ionization -MALDI, time-of-flight mass spectrometry -TOFMS (Fei et al, 1996) This method is used only when large quantities of vitamin B12 are to be assayed. The success of this method is usually based on the efficiency of the extraction procedures adopted and detection errors are common with these tests because other corrinoids, analogues of B12, and other compounds that had similar absorption peaks (Quesada- Chanto et al, 1998).
1.9.5 Enzyme-linked immunosorbent assay (ELISA)
This involves the reaction of antibodies with antigen or the use of naturally occurring vitamin binding proteins with radiolabels or enzyme labels. The most commonly used ELISA has a combination of vitamin and protein fixed on the well surface. This method fell short of good end point detection systems and also extraction / clean up procedures.
1.9.6 Chemiluminescence (CL)
In CL, chemical reactions bring about the emission of light. In this method, a labelled vitamin B12 derivative interacts with a specific binding site protein, IF (Watanabe et al, 1998; Miyamoto et al, 2006). IF is then replaced with a vitamin B12 targeting site on a suitable microorganism (Sato et al, 2002). Using a luminol-hydrogen peroxide system as chemical, vitamin b12 acting as a catalyst is reduced producing light. This is the only method that has allowed analysis to b done at the pictogram level. CL techniques are sensitive but specificity is still questionable because metal ions interfere in biological matrix.
1.9.7 Biosensor- based assay
Biosensors recently designed for assaying vitamin B12 utilizes SPR technology immobilizing a vitamin B12 binding protein on a chip and studying the biomolecular interactions with various cobalamins (Cannon et al, 2002). Affinity biosensors are also made to assay cobalamins couple antibodies with binding protein and interpret results through electrochemical, optical or piezoelectrical transducers (Indyk et al, 2002).
Despite the fact that there are a lot of assays to quantify vitamin b12 available, there still is a need to develop an assay to make better the shortcomings of the previous techniques. Recognised setbacks were insensitivity, problem of standardization, inability to use where high volumes were involved. They also involved centralized laboratory devices, skilled labour and the use of expensive equipments. Attempts have been to develop such assays but they have not been successful due to the small size and complexity of the molecule. There remains an urgent need to develop a reliable, accurate and precise reference method for assaying cyanocobalamins.
1.9.8 Chemical Sensors
Chemical sensors provide advantages over conventional methods in that they are able to overcome issues previously stated and simultaneously analyse vitamins. Specific recognition of an analyte by a chemical system exploited by chemical sensors has the potential to decrease extensive and time wasting preparation of samples. Furthermore, the specific recognition of an analyte brought about by the intimate contact of the recognition element with a physicochemical transducer allows for real time measurement. The sensors utilize chemical components such as polymers, organic molecules, ceramics, inorganic crystals, functionalization reagents located near the surface of the transducer and also enjoy the advantage of selectivity, rapid response time and simplicity in operation and hence, measure up to the requirements of regulatory bodies.
1.9.8.1 Types and principles of chemical sensors
Many chemical sensors have been introduced for different purposes. A chemical sensor typically consists of a recognition element and a transducer. The basic components for the construction of a chemical sensor are shown in Table 1.1. Depending on the proposed application, researchers adopt various approaches for the selection of the recognition element and signal transducer in order to achieve the analytical requirement.
Common assays utilized in diagnostic medicine
Analyte
Method of assay
Hepatitis B
Chemiluminescent immunoassay
Candida albicans
Piezo-electric immunoassay
Sodium
Glass ion-selective electrode
Potassium
Ion-exchange-selective electrode
Calcium
Ionophore ion-selective electrode
pH
Glass ion-selective electrode
Table 1.1 shows different kinds of electrodes utilized by chemical sensors and their recognition element.
Chemical sensors integrate recognition element with a transducer in analytical devices to generate a signal which is commensurate to the concentration of the sought analyte. The signal could be in the form of light or heat emission, release or uptake of gases or change in the concentration of proton. This signal is then converted by the transducer into a measurable response such as temperature change, current, absorption of light or potential by thermal, optical or electrochemical mechanisms. Chemical sensors are categorized into major three groups depending on the type of transducer used. They include optical sensors, thermal sensors and electrochemical sensors.
1.9.8.1.1 Thermal sensors
The principle of thermal sensors is on the basis that the amount of substrate generated can be determined calorimetrically by the heat given off in an enzymatic reaction (Mandenius et al., 1984). In this type of sensor, the chemical substance is either put in the temperature -controlled column or adhered precisely to the temperature transducer and as the sample flows through the column, the heat of reaction is quantified by noting the rise in temperature between the inlet and outlet.
1.9.8.1.2 Optical sensors
Optical sensors are based on the principle of monitoring changes in properties as fluorescence and reflectance and chemiluminescence, UV/Vis absorption resulting from the association of the target analyte with the catalyst. By quantifying NAD(P)H fluorescence, the oxidation and reduction of NAD(P)H can be monitored and the substrate concentration related to changes in fluorescence intensity.
1.9.8.1.3 Electrochemical sensors
For designing chemical electrodes, Potentiometric, amperometric and conductometric are the three types of electrochemical transducers employed.
A lot of chemical-catalysed reactions as a result of a change in solution electrical conductivity lead to a change in ionic species. The conductometric transducer measures the change in the electrical conductivity of the analyte. Because this measurement is non-specific, conductometric sensors are limited because specificity is plays a key function.
Typical potentiometric chemical sensors comprise of a gas-sensing electrode coated with an ion-selective electrode. A change in potential which results from ions accumulating and depleting is generated by the enzymatic reaction with the analyte. Under zero current flow, potentiometric sensors quantify the variance between the transducing electrode and the reference electrode. ions such as K⺠, Naâº, Clâ», NHâ‚„âº, Ca²âº, Mg²âº. Because potentiometric sensors exhibit a logarithmic association between the analyte concentration and the electrode potential, tiny errors in the measured potential may lead to greater errors in the concentration of analyte published. For this reason, potentiometric sensors fail to meet the requirement of a very stable reference electrode (luong et al, 1991)
Contrary to potentiometric sensors, with respect to the reference electrode, amperometric sensors function at a fixed potential and quantify the current produced by the reduction or oxidation of species at the working electrode. Redox chemical catalysts form the basis for amperometric sensors. Molecular oxygen is utilized by these catalysts and they generate hydrogen peroxide when they react with their substrate. By quantifying the concentration of the substrate, the oxygen consumed or hydrogen peroxide produced can be determined. Because amperometric electrodes monitor all electron transfer reactions at applied potential, they appear to be less selective but since they are not dependent upon mass transfer interfaces, they usually respond faster than ion-selective electrodes.
Electrochemical sensors have been used before now to assay vitamin B12 and also to study the redox nature of cobalamins, finding applications in cyclic voltammetry as well as polarography.
Previous work done on the electrochemical detection of vitamin B12 was adsorptive stripping voltammetry immobilizing α-benzildioxime as a chelating agent on a mecury electrode. This method required pretreatment to liberate cobalt from its corrin ring system (Giroussi et al, 1997). Similarly, Smith and his colleagues also needed to remove cobalt by pretreating it with UV irradiation before chelation with 2-(5ʹ-bromo-2ʹ-pyridylazo)-5-diethylaminophenol. (Offspring et al, 1996). The problem with these methods was that metal ions interfered which was cause for concern.
Selecting a² "good" active material is key to achieving the desired end result. An organic material Trans-1, 2-dibromocyclohexane (trans-DBCH) used at the electrode has been known to act selectively with vitamin b12 (Tomcik et al, 2004). This reaction is on the basis that the Co(III) centre of vitamin B12 is electrochemically reduced to Co(I) which then reacts specifically with DBCH producing electrocatalytic currents, generating Co (II).
RBr² + Co(I)L(aq) → + Brâ» (aq) + Co(III)L Br(aq) + R′(oil) (i)
Co(III)LBr(aq) + Co(I)L(aq) → Br⻠(aq) + 2Co(II)L (ii)
The overall reaction is,
2Co(I)L(aq) + RBr² (oil) → 2Co(II)L(aq) + 2Brâ» (aq) + R′(oil) (iii)
Where, R is cyclohexene, RBr² represents trans-1,2-dibromocyclohexane, Co(III)L is vitamin B12a , Co(I)L is vitamin B12s and Co(II)L is vitamin B12r.
The conversion of the Co(III) into the Co(II) complex is not a simple one as it not only involves an electron transfer but the overall reaction goes from six-coordinated Co(III) complex to a five-coordinated Co(II) complex (Tomcik et al, 2004). One axial ligand is given off.
This research work attempts to use this organic material, trans-1,2-dibromocyclohexane immobilized at the electrode to quantify vitamin B12 since it has been established to be specific and sensitive to vitamin B12.
Chemical based sensors have some unique features:
They have a high degree of selectivity, specificity and reproducibility.
The systems are low cost and the chemical sensors are cost effective and available in large quantities.
Prolonged storage capacity and easy to operate.
They can detect very low concentration of analyte and are very stable.
They have good response and recovery time as well as excellent detection limits.
1.10 Aim and main objectives
This study recognises the advantages of amperometric chemical sensors and in particular, the potential application of these sensors in monitoring vitamin B12 levels. The aim of this study is to identify and characterize a dependable monitoring system of amperometric based chemical sensor for the determination (monitoring and quantifying) of vitamin B12 levels using trans 1, 2 dibromocyclohexane. The objectives are:
Selecting a chemical specific and sensitive to vitamin B12.
To characterize the sensor to monitor vitamin B12.
