EFFECTS OF THIAMINE PYRIDOXINE AND BIOTIN ON BLOOD GLUCOSE CONCENTRATION AND RENAL FUNCTION PARAMETERS OF ALLOXAN-INDUCED DIABETIC RATS

ABSTRACT

The aim of this study was to investigate the effects of thiamine, pyridoxine and biotin on the concentrations of blood glucose, serum electrolytes and renal functions of alloxan-induced diabetic rats. A total of twenty seven (27) adult male albino rats of Wistar strain weighing between 160-200 g were used for the study. Twenty four (24) of the animals were rendered diabetic by a single and freshly prepared alloxan monohydrate dissolved in 0.9% ice cold normal saline solution and injected intraperitoneally at a dose of 100 mg/kg body weight. Forty eight (48) hours after confirmation of experimental diabetes, the rats were randomly divided into nine (9) experimental groups of three (3) rats each. Group 1 served as the normal control while Group 2 served as the diabetic control (diabetic untreated). In group 3 (standard control), metformin was used as a reference standard drug at a dose of 100 mg/kg body weight. Group 4 (diabetic rats treated with 25 mg/kg body weight of thiamine), Group 5 (diabetic rats treated with 25 mg/kg body weight of pyridoxine), Group 6 (diabetic rats treated with 0.5 mg/kg body weight of biotin). Group 7 (diabetic rats treated with 100 mg/kg of metformin and 25 mg/kg of thiamine), Group 8 (diabetic rats treated with 100 mg/kg of metformin and 25 mg/kg of pyridoxine) and Group 9 (diabetic rats treated with 100 mg/kg of metformin and 0.5 mg/kg of biotin). Blood glucose concentrations, serum electrolytes and renal function parameters were analysed. The results obtained showed that oral administration of thiamine, pyridoxine and biotin, after the seventh day of treatment significantly (p < 0.05) lowered blood glucose concentrations when compared to the values obtained for Group 2 (untreated) rats. Co-administration of thiamine and biotin with the metformin however, was observed to be more efficacious as they significantly lowered blood glucose concentration when  compared  to  the  values  obtained  for  groups  2,  4  and  6.  Sodium,  chloride  and bicarbonate concentrations in groups 4 and 9 were observed to be significantly (p < 0.05) lower than the value obtained for the untreated group, while potassium ion concentration in these groups were significantly (p < 0.05) higher than the value obtained for group 2. Groups

5 and 8 registered significantly (p < 0.05) lower concentrations of sodium and chloride ions and non-significantly (p > 0.05) lower concentrations of potassium and bicarbonate ions when compared to the values obtained for the untreated group 2 animals. Sodium, chloride and bicarbonate concentrations in groups 6 and 9 rats were observed to be significantly lower than the values obtained for the untreated group. However, the decrease in potassium concentration of group 6 was non-significantly (p > 0.05) lower than the values obtained for group 2. Urea and blood urea nitrogen (BUN) concentrations of all the groups treated with thiamine, pyridoxine and biotin, and those which received co-administration of the vitamins and metformin were observed to be significantly (p < 0.05) lower than the values obtained for the diabetic untreated group 2 rats. Uric acid concentrations of groups 5 and 6 were observed to be non-significantly (p > 0.05) lower than the value obtained for group 2. The result also indicated significantly (p < 0.05) lower concentrations of creatinine in all the treated groups when compared to the values obtained for the untreated group 2 animals. Conclusively, this study showed that thiamine, and biotin decreased blood glucose concentration, and to a large extent, improves electrolyte imbalance and renal functions of diabetic animals. The roles of pyridoxine, thiamine and biotin in this study prove their usefulness in blood glucose control; hence these vitamins can be used as adjuvant with standard anti-diabetic drugs for improving glycaemic control, electrolyte imbalance and renal functions of diabetics.

CHAPTER ONE

INTRODUCTION

Diabetes mellitus, commonly referred to as diabetes, is a metabolic disorder characterized by high blood sugar (glucose) levels over a prolonged period of time and results from defects in insulin secretion, or its action, or both (Kitabchi et al., 2009). Normally, blood glucose levels are tightly controlled by insulin, a hormone produced by the beta cells of the pancreas. Insulin is a water soluble hormone whose receptor is a tyrosine kinase (Rang et al., 2012). It functions primarily to lower blood glucose level by providing a mechanism for the uptake and utilization of glucose. When the blood glucose elevates (for example after a carbohydrate rich meal), insulin is secreted from the pancreas to normalize the blood glucose level. The inability of the pancreas  to  produce  sufficient  insulin  or  the  cells  of  the  body to  respond  to  the  insulin produced results in a state of hyperglycaemia and subsequently causes diabetes (David and Dolores, 2011). The prevalence of diabetes for all age groups worldwide was estimated to be 2.8% in 2000 and 4.4% by 2030. The total number of people with diabetes is projected to rise from 171 million in 2000 to 366 million in 2030 (Wild et al., 2003).

Vitamins are organic compounds occurring in small quantities in different natural foods necessary for growth and maintenance of good health in humans and animals. They cannot be synthesized by the body, hence are required in diets and food supplements in other to maintain adequate amounts. They are classified as fat soluble and water soluble vitamins. All water soluble B vitamins help the body to convert food (carbohydrates) into fuel (glucose), which is used  to  produce  energy  (Vasudevan  and  Sreekumari,  2007).  Thiamine  (Vitamin  B1), pyridoxine (vitamin B6) and biotin (vitamin B7) are part of the B complex group of vitamins. These B vitamins, often referred to as B complex vitamins, also help the body metabolize fats and protein. The body needs thiamine, pyridoxine and biotin to metabolize carbohydrates, fats, and amino acids. Since they are water-soluble, the body does not store them. However, some can be synthesized from bacteria in the intestine while others can be obtained in small amounts in a variety of foods (Badr et al., 2015). Thiamine functions as a coenzyme (thiamine pyrophosphate) for pyruvate dehydrogenase, α-ketoglutarate, transketolase and pyruvate decarboxylase (for alcohol fermentation) in carbohydrate metabolism. Pyridoxine serves as a coenzyme for many biological processes such as transamination and decarboxylation of amino acids, heme synthesis, glycogenolysis (where glycogen phosphorylase breaks down glycogen to release glucose into the blood during starvation), production of niacin, etc. Biotin is also important for normal embryonic growth, making it a critical nutrient during pregnancy (Mock et al., 2002; Báez-Saldaña et al., 2004). Biotin also plays important role in biotinylation (post translational modification of histone proteins in gene transcription), and as a co enzyme involved in the transfer of carboxylic groups between biomolecules (Zempleni et al., 2012).

Metformin belongs to the biguanide class of oral anti-diabetic drugs, and is generally recommended as a first line medication used for treatment of type 2 diabetes as there is good evidence  that  it  decreases  mortality (Roussel  et  al.,  2010;  Olokoba  et  al.,  2012).  It  was approved by the Food and Drug Administration (FDA) for use in the United States in 1995 (Modi, 2007). Metformin is a rather safe drug and its anti-hyperglycaemic property has been generally attributed to combination of a decreased rate of intestinal absorption of carbohydrate, decreased hepatic gluconeogenesis and improvement of peripheral glucose utilization (Pournaghi  et  al.,  2012).  Beyond  its  effect  on  glucose  metabolism,  metformin  has  been reported to reduce fatty liver, and to lower microvascular and macrovascular complications associated with type 2 diabetes (Okonkwo and Okoye, 2014). In spite of the introduction of hypoglycaemic drugs,  diabetes  and  related  complications  continue to  be a major medical problem (Nammi et al., 2003).

Renal function is an indication of the state of the kidney and its role in renal physiology (Stevens et al., 2006). Diabetic nephropathy remains the cause of end-stage renal  failure worldwide, with a prevalence rate expected to double over the next decade (Locatelli et al., 2003). Type 2 diabetes accounts for 90% of patients with diabetic nephropathy. This is as a result of chronic hyperglycaemia which is found to increase the production of free radicals that are associated with long-term damage, dysfunction, and failure of various organs, especially the eyes (retinopathy), kidneys (nephropathy), nerves (neuropathy), heart (cardiomyopathy), and blood vessels (Baynes, 1991; Mohamed et al., 1999; Gispen and Biessels, 2000). Despite conventional therapies of glycaemic and renal control, many patients still exhibit evidence of renal damage (Svensson et al., 2003). Since thiamine, pyridoxine and biotin are all representatives of water soluble vitamins involved in energy metabolism, it is important to investigate their role in blood glucose metabolism, electrolyte balance and renal functions of diabetic rats.

1.1 Thiamine (Vitamin B1)

Thiamine is a water-soluble B vitamin, also known as vitamin B1  or aneurine (Tanphaichitr,

1999).  Thiamine was  isolated and  characterized  in  the 1930s  as  one of the first  organic compounds to be recognized as a vitamin (Rindi, 1996). It occurs in the human body as free thiamine and as various  phosphorylated forms:  thiamine monophosphate (TMP), thiamine triphosphate (TTP), and thiamine pyrophosphate (TPP), which is also  known as thiamine diphosphate.

1.1.1 Coenzyme Functions

The synthesis of TPP from free thiamine requires magnesium, adenosine triphosphate (ATP), and the enzyme, thiamine pyrophosphokinase. TPP is required as a coenzyme for four multi- component enzyme complexes associated with the metabolism of carbohydrates and branched- chain each dehydrogenase complex  requires a  niacin-containing coenzyme (NAD), a  riboflavin- containing coenzyme (FAD), and lipoic acid (Hutson et al., 2005).

Transketolases  catalyse  critical  reactions  in  another  metabolic  pathway  occurring  in  the cytosol, known as the pentose phosphate pathway. One of the most important intermediates of this pathway is ribose-5-phosphate, a phosphorylated 5-carbon sugar required for the synthesis of the high-energy ribonucleotides, such as adenosine triphosphate (ATP) and guanosine triphosphate (GTP). Nucleotides are the building blocks of nucleic acids, DNA, and RNA. The pentose phosphate pathway also supplies various anabolic pathways, including fatty acid synthesis, with the niacin-containing coenzyme NADPH, which is essential for a number of biosynthetic reactions (Tanphaichitr, 1999). Because transketolase decreases early in thiamine deficiency and, unlike most thiamine-dependent enzymes, is present in red blood cells, measurement of its activity in red blood cells has been used to assess thiamine nutritional status (Rindi, 1996).

1.1.2 Thiamine Deficiency

Beriberi is a disease condition resulting from severe thiamine deficiency. Thiamine deficiency affects the cardiovascular, nervous, muscular, gastrointestinal, central and peripheral nervous systems  (Rindi,  1996).  Dry  beriberi  presents  symptoms  such  as  abnormal  (exaggerated) reflexes, as well as diminished sensation and weakness in the legs and arms. Muscle pain and tenderness and difficulty rising from a squatting position have also been observed (McDowell,

2000). Wet beriberi presents symptoms such as rapid heart rate, enlargement of the heart, severe swelling (oedema), difficulty in breathing, and ultimately congestive heart failure (Yamasaki et al., 2010). Cerebral beriberi affects the central nervous system with symptoms such as abnormal eye movements, stance and gait ataxia, and cognitive impairments (Doss et

al., 2003). Gastrointestinal beriberi is associated with nausea, vomiting, abdominal pain and lactic acidosis (Donnino, 2004).

Alcoholism is the primary cause of thiamine deficiency in industrialized countries (Saad et al., 2010). Cases of thiamine deficiency may result from inadequate thiamine intake (Rindi, 1996), increased requirement for thiamine (e.g strenuous physical exercise, fever, malaria, HIV/AIDS, pregnancy, breast-feeding, and adolescent growth) (Stanga et al., 2008), excessive loss of thiamine from the body (Sica, 2007), consumption of anti-thiamine factors in food (tea, coffee and betel nuts) or a combination of these factors (Ventura et al., 2013).

1.1.3 Disease Prevention and Treatment

Research has shown that thiamine plays vital role in prevention of disease conditions such as cataract (Jacques et al., 2005), diabetes mellitus and vascular complications (Alaei et al.,

2013). Thiamine has also been studied for its role in the treatment of disease conditions such as Alzheimer’s   disease   (Dumont   and   Bael,   2011),   congestive   heart   failure(Leslie   and Gheorghiade, 1996), metabolic diseases such as (thiamine-responsive pyruvate dehydrogenase complex deficiency(Lee et al., 2006), maple syrup urine disease (Chuang et al., 2006), thiamine-responsive megaloblastic anaemia (Shaw-Smith et al., 2012) and biotin-responsive basal ganglia disease (Alfadhel et al., 2013).

1.1.4 Sources and Supplements of Thiamine

Humans obtain thiamine from dietary sources and from the normal microflora of the colon (LeBlanc et al., 2013). However, food sources of thiamine include whole-grains, cereals and nuts. Lean pork and yeast are rich sources of thiamine. Others include lentils, green peas, long- grain brown rice, long-grain white rice, whole-wheat bread, enriched white bread, wheat germ, pecans, spinach, cantaloupe, milk and egg (Tanphaichitr, 1999). Since most of the thiamine is lost during the production of white flour and polished (milled) rice; white rice and foods made from white flour (for example bread and pasta), are fortified with thiamine in many western countries. Thiamine is available in nutritional supplements and for fortification as thiamine hydrochloride and thiamine nitrate (Rieck et al., 1999).

1.1.5 The Recommended Dietary Allowance

The recommended dietary allowance for thiamine, revised in 1998 by the Food and Nutrition

Board of the Institute of Medicine, was based on the prevention of deficiency in generally

healthy individuals (FNB, 1998). The Linus Pauling Institute supports the recommendation by the Food and Nutrition Board of 1.2 mg/day of thiamine for men, and 1.1 mg/day for women (Russell and Suter, 1993).

1.2 Pyridoxine (Vitamin B6)

Vitamin B6 is a water-soluble vitamin that was first isolated in the 1930s. The term vitamin B6 refers to the various forms, namely pyridoxal, pyridoxine (pyridoxol), pyridoxamine, and their phosphorylated forms. The phosphate ester derivative pyridoxal 5′-phosphate (PLP) is the bioactive   coenzyme   form,   and   is   involved   in   over   4%   of   all   enzymatic   reactions (Dakshinamurti and Dakshinamurti, 2007; Galluzzi et al., 2013). Pyridoxal 5`-phosphate (PLP) is the phosphate ester derivative of pyridoxal, and is a cofactor of most vitamin B6-dependent enzymes in the body (McCormick, 2006).

1.2.1 Biosynthesis of Pyridoxal 5’-phosphate (PLP)

Pyridoxal 5’-phosphate is the active form of vitamin B6, whereas pyridoxamine, pyridoxal and pyridoxine as well as their phosphate esters form the vitamin B6  complex. There are two different routes of denovo PLP synthesis present in different organisms. In the DOXP- dependent first route, the PLP precursor pyridoxine 5’-phosphate (PNP) is produced from 4- phosphohydroxyl-L-threonine (4PHT) and 1-deoxy-D-xylulose-5-phosphate (DOXP) by the actions  of  the  enzymes  PdxA  and  PdxJ.  These  precursors  are  synthesised  from  two independent pathways from metabolites of carbohydrate metabolism. PNP can then be converted to PLP by the enzyme pyridoxal 5’-phosphate synthase (pyridoxamine/pyridoxine

5’-phosphate oxidase). In the DOXP-independent second route, PLP synthesis is catalysed by the actions of Pdx1 and Pdx2 with glutamine, ribulose 5-phosphate (or ribose 5-phosphate) and glyceraldehyde 3-phosphate (or glycerone phosphate) as substrates (Dakshinamurti and Dakshinamurti, 2007). The pyridoxine, pyridoxamine and pyridoxal can be phosphorylated by the action of the enzyme, pyridoxal kinase (PdxK). Pyridoxine and pyridoxamine can be converted to pyridoxal by the action of pyridoxal 5’-phosphate synthase.

1.2.2 Coenzyme Functions of Vitamin B6

Vitamin B6 must be obtained from the diet because humans cannot synthesise it. PLP plays a vital role in the function of over 100 enzymes that catalyse essential chemical reactions in the human body (DaSilva et al., 2012). PLP-dependent enzymes have been classified into five structural classes namely: Fold type I (aspartate aminotransferase family), fold type II (tryptophan synthase family), fold type III (alanine racemase family), fold type IV (D-amino acid aminotransferase family), and fold type V (glycogen phosphorylase family) (Eliot and Kirsch, 2014).

PLP-dependent enzymes are involved in essential biological processes, such as haemoglobin and  amino  acid  biosynthesis,  as  well  as  fatty  acid metabolism.  PLP  also  functions  as  a coenzyme for glycogen phosphorylase, an enzyme which catalyses the release of glucose from stored glycogen. Much of the PLP in the human body is found in muscle bound to glycogen phosphorylase. PLP is also a coenzyme for reactions that generate glucose from amino acids, a process known as gluconeogenesis (Leklem, 1999).

1. Amino acid metabolism: Pyridoxal phosphate serves as a cofactor in the biosynthesis of five important neurotransmitters: serotonin, dopamine, epinephrine, norepinephrine, and gamma-amino butyric acid (GABA) and also in the synthesis of histamine. It is also involved in the breakdown of amino acids by enzymes which catalyses the transfer of amine groups from  one amino  acid  to  another.  Serine  racemase  which  synthesizes  the neuromodulator, serine, is a PLP-dependent enzyme. PLP is also a coenzyme needed for the proper function of the enzymes  cystathionine synthase  and  cystathionase.  These enzymes  work  to  transform methionine into cysteine. Seleno-methionine is the primary dietary form of selenium. PLP is needed as a cofactor for the enzymes that allow selenium to be used from the dietary form. PLP also plays a cofactor role in releasing selenium from seleno-homocysteine to produce hydrogen selenide, which can then be used to incorporate selenium into seleno-proteins. PLP is required for the conversion of tryptophan to niacin, so low vitamin B6  status impairs this conversion (Lichstein et al., 1945; Combs, 2008).

2. Glucose metabolism: PLP is a required coenzyme of glycogen phosphorylase, which breaks down glycogen to glucose and glucose-1-phosphate (glycogenolysis). PLP can catalyse transamination reactions that are essential for providing amino acids as a substrate for gluconeogenesis (Combs, 2008).

3. Nervous system function: In the brain, the PLP-dependent enzyme aromatic L-amino acid decarboxylase  catalyses  the  synthesis  of  two  major neurotransmitters:  serotonin  from  the amino acid tryptophan and dopamine from L-3,4-dihydroxyphenylalanine (L-Dopa). Other neurotransmitters, including glycine, D-serine, glutamate, histamine, and γ-aminobutyric acid (GABA), are also synthesized in reactions catalysed by PLP-dependent enzymes (Clayton, 2006).

4. Haemoglobin synthesis: PLP functions as a coenzyme of 5-aminolevulinic acid synthase, which is involved in the synthesis of heme, an iron-containing component of haemoglobin. Haemoglobin is found in red blood cells and is critical to their ability to transport oxygen

throughout the body. Both pyridoxal and PLP are able to bind to the haemoglobin molecule and affect its ability to pick up and release oxygen. However, the impact of this on normal oxygen delivery to tissues is not known (Leklem, 1999). Vitamin B6  deficiency may impair haemoglobin synthesis and lead to microcytic anaemia (Combs, 2008).

5. Tryptophan metabolism: Deficiency in another B vitamin (niacin) is easily prevented by adequate dietary intakes. The dietary requirement for niacin and the niacin coenzyme, nicotinamide adenine dinucleotide (NAD),  can  also  be met  to  a fairly limited extent,  by the catabolism  of  the  essential  amino  acid  tryptophan  through  the  tryptophan-kynurenine pathway. Key reactions in this pathway are PLP-dependent. In particular, PLP is the cofactor for the enzyme kynureninase, which catalyses the conversion of 3-hydroxykynurenine to 3- hydroxyanthranilic acid. A reduction in PLP availability appears to primarily affect kynureninase activity, limiting NAD production and leading to higher concentrations of kynurenine, 3-hydroxykynurenine, and xanthurenic acid in blood and urine (Rios-Avila et al.,

2013). Thus, while dietary vitamin B6  restriction impairs synthesis of NAD from tryptophan, Adequate PLP levels helps to maintain NAD formation from tryptophan (Oxenkrug, 2013).

6. Nucleic acid synthesis: PLP serves as a coenzyme for serine hydroxymethyl transferase, which catalyses the simultaneous conversions of serine to glycine and tetrahydrofolate (THF) to 5, 10-methylene THF. The latter molecule is the one-carbon donor for the generation of dTMP from dUMP by thymidylate synthase.

7.  Lipid  metabolism:  PLP  is  an  essential  component  of  enzymes  that  facilitate  the biosynthesis of sphingolipids. Particularly, the synthesis of ceramide requires PLP. In this reaction, serine is decarboxylated and combined with palmitoyl-CoA to form sphinganine, which is combined with a fatty acyl-CoA to form dihydroceramide. Dihydroceramide is then further desaturated to form ceramide. In addition, the breakdown of sphingolipids is also dependent on vitamin B6 because S1P lyase, the enzyme responsible for breaking down sphingosine-1-phosphate, is also PLP-dependent (Combs, 2008).

1.2.3 Deficiency of Vitamin B6

Severe deficiency of vitamin B6  is not very common. However, alcoholics are thought to be most at risk of vitamin B6  deficiency due to low dietary intakes and impaired metabolism of the vitamin (Bowman and Russel, 2006). Decreased levels of vitamin B6 can be seen in women with type 1 diabetes and in patients due to systemic inflammation, liver disease, rheumatoid arthritis, and those infected with HIV (Massé et al., 2012; Ulvik et al., 2014). The use of oral contraceptives and treatment with certain anticonvulsants, theophylline, isoniazid, cycloserine, penicillamine,  and  hydrocortisone  negatively  impact  vitamin  B6   status  (Bhagavan,  1985; Wilson et al., 2014). Haemodialysis as well, reduces vitamin B6  plasma levels (Corken and Peter, 2011).

Neurologic symptoms noted in severe vitamin B6 deficiency include seizures, peripheral neuritis, irritability, depression, and confusion. Additional symptoms include inflammation of the tongue, sores or ulcers of the mouth, and ulcers of the skin at the corners of the mouth (Leklem, 1991; Clayton, 2006). Dermatological manifestations (pellegra), hematological manifestations such as xanthurenic aciduria and homo cystinuria are associated with vitamin B6 deficiency (Vasudevan and Sreekumari, 2007). Treatment of vitamin B6 deficiency involves replacement with pyridoxine hydrochloride, orally, as a nasal spray, or for injection when in its solution form.

1.2.4 Medical Applications of Vitamin B6

Vitamin B6 supplements at pharmacologic doses have been used in an attempt to treat a wide variety of disease conditions such as metabolic diseases (Pearl and Gospe, 2014), morning sickness (Magee et al., 2002), premenstrual syndrome (Wyatt et al., 1999) and carpal tunnel syndrome(Ellis et al., 1976; Ellis et al., 1979).  Vitamin B6 has been used to treat nausea and vomiting in early pregnancy for decades, commonly in conjunction with other medications such as metoclopramide or doxylamine. Alone, it has been found safe and effective, though any woman’s prenatal care-giver must help guide treatment for these symptoms (Angley et al.,

2007). Vitamin B6  has  been shown in at least two small-scale clinical studies to have a beneficial effect on the carpal tunnel syndrome, particularly in cases where no trauma or overuse aetiology is known (Ellis et al., 1979; Kasdan and Janes, 1987). Pyridoxine may help balance hormonal changes in women and aid the immune system (Kashanian et al., 2007).

1.2.5 Sources and Supplement of Vitamin B6

Many plant food contains a unique form of vitamin B6 called pyridoxine glucoside; this form of vitamin B6  appears to be only about half as bioavailable as vitamin B6  from other food sources or supplements (Clayton, 2006). Vitamin B6  in a mixed diet has been found to be approximately 75% bioavailable (FNB, 1998). Some foods that are relatively rich in vitamin B6 include cereal, salmon, turkey, potato, spinach, chicken, avocado, banana etc. Vitamin B6 is available  as  pyridoxine  hydrochloride  in  multivitamin,  vitamin  B-complex,  and  vitamin B6 supplements (Morris et al., 2008).

1.2.6 Recommended Dietary Allowance (RDA)

The Recommended Daily Allowance of vitamin B6  according to the Institute of Medicine is

1.3 mg/day for a 19 to 50 year old adult. For older adults (60 years and above) require 1.7 mg/day for men and 1.5 mg/day for women (Paul et al., 2013). However, the Linus Pauling Institute recommends that older adults (>50 years) take a multivitamin/mineral supplement, which generally provides at least 2.0 mg of vitamin B6 daily. During pregnancy and lactation, the requirement is increased to 2.5mg/day (Vasudevan and Sreekumari, 2007).

1.3 Biotin (Vitamin B7)

Biotin is a water-soluble vitamin that is generally classified as a B-complex vitamin. After its initial  discovery  in  1927,  40  years  of  additional  research  was  required  to  unequivocally establish biotin as a vitamin (FNB, 1998). Biotin is required by all organisms but can be synthesized by some strains of bacteria, yeast, mould, algae, and some plant species. It has an unusual structure with two rings fused together via one of their sides. The two rings are ureido and thiophene moieties (Mock, 2007).

1.3.1 Biosynthesis of Biotin

Biotin is a heterocyclic sulphur-containing monocarboxylic acid. It is made from two precursors, alanine and pimeloyl-CoA by the action of three enzymes: 8-Amino-7- oxopelargonic acid synthase (which is PLP dependent), Dethiobiotin synthethase which catalyzes the formation of the ureido ring via a DAPA carbamate activated with ATP, and biotin synthase which reductively cleaves S-adenosyl methionine into a deoxyadenosyl radical which was found to be the iron-sulfur (Fe-S) centre contained in the enzyme (Marquet et al.,

2001).A majority of  bacteria that normally colonize the small and large intestine (colon) synthesize biotin (Magnusdottir et al., 2015). The uptake of free biotin into intestinal cells via the  human  sodium-dependent  multivitamin  transporter  (hSMVT)  has  been  identified  in cultured cells derived from the lining of the small intestine and colon (Said, 2009; Zempleni et al., 2009), implying that humans may be able to absorb biotin produced by enteric bacteria: a phenomenon documented in swine.

1.3.2 Roles of Biotin

Biotin plays important roles in the following processes:

1. Biotinylation

2. Cofactor for many metabolic enzymes.

1.   Biotinylation:   This   is   the   covalent   addition   of   biotin   to   molecules,   including apocarboxylases (catalytically inactive carboxylases) and histones. The covalent attachment of biotin to the apocarboxylase is catalysed by the enzyme, holocarboxylase synthetase (HCS). The enzyme catalyses post translational biotinylation of the epsilon amino group of a lysine residue at the active site of each apocarboxylase converting the inactive apocarboxylase into a fully active holocarboxylase (Zempleni et al., 2011).

The holocarboxylase synthase catalyse the transfer of biotin to a specific lysine residue to the active site of apocarboxylase, converting the enzyme to a fully active holocarboxylase.

The biotinylation of apocarboxylases is catalysed by holocarboxylase synthase whereas the enzyme (biotinidase) catalyses the release of biotin from biotinylated histones and from the peptide products of holocarboxylase breakdown.

Figure 9: Biotin recycling. Source: (Zempleni et al., 2012).

2.  Enzyme  cofactor:  Biotin  functions  as  a  covalently  bound  cofactor  required  for  the biological activity involving transfer of carboxylic groups (COO-) between five known mammalian  biotin-dependent  carboxylases.  Five  mammalian  carboxylases  are  known  to require biotin for their activity. They include: acetyl-CoA carboxylase 1, acetyl-CoA carboxylase 2,  pyruvate  carboxylase,  methylcrotonyl-CoA carboxylase  and  propionyl-CoA carboxylase.

(a) Acetyl-CoA Carboxylase 1 and Acetyl-CoA Carboxylase 2: These are biotin-containing enzymes which catalyses the conversion of acetyl-CoA to malonyl-CoA using bicarbonate and ATP. Malonyl CoA generated via acetyl-CoA carboxylase1 is a rate-limiting substrate for the synthesis of fatty acids in the cytosol, and malonyl CoA generated via acetyl-CoA carboxylase 2 inhibits carnitine-palmitoyl tranferase 1 (CPT1), an outer mitochondrial membrane enzyme, important in fatty acid oxidation as indicated in Fig. 10. Acetyl-CoA carboxylase 1 is found in all tissues and is particularly active in lipogenic tissues (liver, white adipose tissue, and mammary gland), heart, and pancreatic islets whereas acetyl-CoA carboxylase 2is abundant in skeletal muscle and heart (Saggerson, 2008).

In the cytosol of liver cells, fatty acids are converted to acyl-CoA, and glucose undergoes glycolysis to produce pyruvate. Acyl-CoA is then shuttled into the mitochondria via CPT1- mediated transport and undergoes β-oxidation that generates acetyl-CoA. In addition, pyruvate is converted to acetyl-CoA in the mitochondria which then condense with oxaloacetate to form citrate. Citrate can be exported into the cytosol and cleaved to oxaloacetate and acetyl-CoA. The acetyl CoA is then used to generate malonyl-CoA in a reaction catalysed by biotin0- containing acetyl CoA carboxylase 1 (ACC 1) in the presence of ATP and Bicarbonate (Saggerson, 2008).

Malonyl-CoA  is  an  essential  substrate  for the  biosynthesis  of  fatty acids  and  subsequent triglycerides, phospholipids and lipoproteins. It is also a regulator of fatty acid β-oxidation. It is generated from acetyl-CoA by another biotin-containing enzyme known as  acetyl-CoA carboxylase 2 (ACC 2). This enzyme is localised at the mitochondrial membrane and has been shown to downregulate the β-oxidation of fatty acids in the mitochondria by inhibiting CPT1 (Saggerson, 2008).

(b) Pyruvate carboxylase: This is a critical enzyme in gluconeogenesis which catalyses the ATP-dependent incorporation of bicarbonate into pyruvate, producing oxaloacetate. This is a typical anaplerotic reaction of the TCA cycle. Oxaloacetate can then be converted to phosphoenolpyruvate and eventually to glucose.

Figure 11: Pyruvate carboxylase and acetyl-CoA carboxylase 1 Source: (Saggerson, 2008).

Biotin-containing pyruvate carboxylase supplies the tricarboxylic acid (TCA) cycle with oxaloacetate by catalysing the conversion of pyruvate to oxaloacetate using bicarbonate and ATP.   In the liver, oxaloacetate can be used as a precursor for gluconeogenesis. It is first converted phosphoenolpyruvate (PEP) by PEP carboxykinase and then to glucose via gluconeogenesis. In the tricarboxylic acid cycle, oxaloacetate can also be condensed with acetyl-CoA to produce citrate, which can be exported from the mitochondria. In the liver, adipose tissue and skeletal muscle, citrate is cleaved to oxaloacetate and acetyl-CoA in the cytosol.  Acetyl  CoA  is  converted  to  malonyl-CoA  by  another  biotin-containing  enzyme known as acetyl-CoA carboxylase 1. The malonyl-CoA is then used by fatty acid synthase 1 to generate long chain fatty acids (Saggerson, 2008).

(c) Methylcrotonyl-CoA carboxylase: These enzyme catalyses an essential step in the catabolism of leucine, an essential branched-chain amino acid. The enzyme contains biotin and catalyses the production of 3-methylglutaconyl-CoA from methylcrotonyl-CoA (Saggerson,2008).

Figure 12: Biotin containing carboxylases in the metabolism of branched chain amino acids, odd chain fatty acids and cholesterol

Source: (Saggerson, 2008).

Two biotin-containing enzymes (propionyl-CoA carboxylase and methylcrotonyl-CoA carbocylase) are reqiuired in the metabolism of branched chain amino acids (BCAAs) namely: leucine, valine and isoleucine, the oxidation of odd chain fatty acids, and the degratation of cholesterol side chain. The metabolic pathways generate acetyl-CoA and succinyl CoA, which then enter the tricarboxylic acid cycle (Saggerson, 2008).

(d) Propionyl-CoA carboxylase: This is a biotin containing enzyme produces D-malonyl- malonyl-CoA from propionyl-CoA, a by-product in the β-oxidation of fatty acids with an odd number of carbon atoms. The conversion of propionyl-CoA to D-malonylmalonyl-CoA is also required in the catabolic pathways of two branched-chain amino acids (isoleucine and valine), methionine, threonine as shown in Fig. 13, and the side chain of cholesterol as seen in Fig. 12.

1.3.3 Biotin Deficiency

Although overt biotin deficiency is very rare, the human requirement for dietary biotin has been demonstrated in three different situations: prolonged intravenous feeding (parenteral) without biotin supplementation, infants fed an elemental formula devoid of biotin, and consumption of raw egg white for a prolonged period (Mock, 2014a). Raw egg white contains an antimicrobial protein known as avidin that can bind biotin and prevent its absorption. Cooking egg white denatures avidin, rendering it susceptible to digestion and therefore unable to prevent the absorption of dietary biotin  (Zampleni et al., 2012). Signs of overt biotin deficiency include hair loss (alopecia) and a scaly red rash around the eyes, nose, mouth, and genital area. Neurologic symptoms in adults have included depression, lethargy, hallucinations, numbness and tingling of the extremities, ataxia, and seizures (Elrefai and Wolf, 2015). Aside from prolonged consumption of raw egg white or total intravenous nutritional support for those lacking biotin, other conditions may increase the risk of biotin depletion. Smoking has been associated with increased biotin catabolism (Sealy et al., 2004). The rapidly dividing cells of the developing foetus require biotin for synthesis of essential carboxylases and histone biotinylation;  hence,  the  biotin  requirement  is  likely  increased  during  pregnancy  (Mock,

2014a; Perry et al., 2014). Additionally, certain types of liver disease may decrease biotinidase activity and theoretically increase the requirement for biotin (Pabuccough et al., 2002).

1.3.4 Disease Prevention and Treatment

Biotin  plays  vital  role  in  prevention  of  disease  conditions  such  as  congenital  anomalies (Takechi et al., 2008). Biotin has also been studied for its role in the treatment of disease conditions such as biotin-responsive basal ganglia disease(Alfadhel et al., 2013), multiple sclerosis (Sedel et al., 2015), diabetes mellitus(Revilla-Monsalve et al., 2006; Badr et al.,

2015; Valdés-Ramos et al., 2015 ), brittle fingernails (onychorrhexis) and hair loss (alopecia) (Famenini and Goh, 2014).

1.3.4.1 Role of Biotin in the Treatment of Diabetes mellitus

Overt biotin deficiency has been shown to impair glucose utilization in mice (Larrieta et al.,

2012) and cause fatal hypoglycaemia in chickens. Overt biotin deficiency likely also causes abnormalities in glucose regulation in humans. One early human study reported lower serum biotin concentrations in 43 patients with type 2 diabetes mellitus compared to 64 non-diabetic control subjects, as well as an inverse relationship between fasting blood glucose and biotin

concentrations (Maebashi et al., 1993). In a small, randomized, placebo-controlled intervention study in 28 patients with type 2 diabetes, daily supplementation with 9 mg of biotin for one month resulted in a mean 45% decrease in fasting blood glucose concentration (Maebashi et al., 1993). Yet, another study in 10 patients with type 2 diabetes and 7 non-diabetic controls found no effect of biotin supplementation (15 mg/day) for 28 days on fasting blood glucose concentrations  in  either  group  (Baez-Saldana  et  al.,  2004).  A  more  recent  double-blind, placebo-controlled study by the same research group showed that the same biotin regimen lowered plasma triglyceride concentrations in both diabetic and non-diabetic patients with hypertriglyceridemia (Revilla-Monsalve et al., 2006). In this study, biotin administration did not affect blood glucose concentrations in either patient group. Additionally, a few studies have shown that co-supplementation with biotin and chromium picolinate may be a beneficial adjunct therapy in patients with type 2 diabetes (Singer and Geohas, 2006; Geohas et al., 2007; Albarracin et al., 2007;Albarracin et al., 2008). However, administration of chromium picolinate alone has been shown to improve glycaemic control in diabetic subjects (Suksomboon et al., 2014).

As a cofactor of carboxylases required for fatty acid synthesis, biotin may increase the utilisation of glucose for fat synthesis. Biotin has been found to stimulate glucokinase, a liver enzyme that increases synthesis of glycogen, the storage form of glucose. Biotin also appeared to trigger the secretion of insulin in the pancreas of rats and improve glucose homeostasis (Lazo de la Vega-Monroy et al., 2013). Yet, reduced activity of ACC1 and ACC2 would be expected to reduce fatty acid synthesis and increase fatty acid oxidation, respectively. It is currently unclear whether pharmacologic doses of biotin could benefit the management of hyperglycaemia in patients with impaired glucose tolerance. Moreover, whether supplemental biotin lowers the risk of cardiovascular complications in diabetic patients by reducing serum triglycerides and LDL-cholesterol remains to be proven (Revilla-Monsalve et al., 2006; Singer and Geohas, 2006; Geohas et al., 2007; Albarracin et al., 2007).

1.3.5 Sources and Supplement of Biotin

Biotin is found in many foods, either as the free form that is directly taken up by enterocytes or as biotin bound to dietary proteins. Egg yolk, liver, whole wheat, pork, cheese, salmon, rasp berries, cauliflower, avocado and yeast are rich sources of biotin (Staggs et al., 2004). Biotin is available as a single-nutrient supplement in various doses and is often included in B-complex and multivitamin-mineral supplements (Mock, 2015).

1.3.6 The Adequate Intake

According to the recommendations of the Food and Nutrition Board (FNB) of the Institute of Medicine (IOM), the Adequate Intake for adults is (30 micrograms per day). Dietary intakes of generally healthy adults have been estimated to be 40-60 µg/day of biotin (FNB, 1998). The requirement for biotin in pregnancy may be increased (Mock, 2014b).

1.4 Diabetes Mellitus

Diabetes mellitus (DM), commonly referred to as diabetes is a group of metabolic disorder characterized by high blood sugar (glucose) levels over a prolonged period of time and results from defects in insulin secretion, or its action, or both (Kitabchi et al., 2009). It was first identified as a disease associated with “sweet urine” and excessive muscle loss in the ancient world. Elevated levels of blood glucose (hyperglycaemia) lead to spillage of glucose into the urine, hence the term ‘sweet urine’. Diabetes mellitus is classified into four broad categories: type 1 diabetes, type II diabetes, gestational diabetes, and other specific types (David and Dolores, 2011).

1.4.1 Type 1 Diabetes Mellitus

Type 1 diabetes mellitus is also referred to as insulin-dependent diabetes mellitus (IDDM) or juvenile diabetes. It is characterized by loss of the insulin-producing beta cells of the islets of Langerhans in the pancreas, leading to insulin deficiency and can be further classified as immune-mediated or idiopathic. The majority of type 1 diabetes is of the immune-mediated nature, in which a T-cell-mediated autoimmune attack leads to the loss of beta cell function and thus insulin (Rother, 2007).  Sensitivity and responsiveness to insulin are usually normal, in the early stages of type 1 diabetes. It can be accompanied by irregular and unpredictable high blood sugar levels, frequently with ketosis, and sometimes with serious low blood sugar levels. Other complications include an impaired counter regulatory response to low blood sugar, infection, gastroparesis which leads to erratic absorption of dietary carbohydrates and endocrinopathies  for  example  Addison’s  disease.  Type 1  diabetes  is  partly  inherited  with multiple genes, including certain HLA genotypes, known to influence the risk of diabetes (Dorner et al., 1977).

1.4.2 Type II Diabetes Mellitus

Type II diabetes is also referred to as non-insulin-dependent diabetes mellitus (NIDDM) or adult-onset diabetes. It is characterized by insulin resistance, which may be combined with relatively reduced insulin secretion (David and Dolores, 2011). The defective responsiveness of body tissues to insulin is believed to involve the insulin receptor. However, the specific defects are not known. In the early stage of type II diabetes, the predominant abnormality is reduced insulin sensitivity. At this stage, hyperglycaemia can be reversed by a variety of measures and medications that improve insulin sensitivity or reduce glucose production by the liver. Type II diabetes is primarily due to lifestyle factors (obesity, lack of physical activity, poor diet, stress, and urbanization) and genetics (Risérus et al., 2009).

1.4.3 Gestational Diabetes

Gestational diabetes mellitus (GDM) is the third main type of diabetes, and it occurs when pregnant women without a previous history of diabetes develop a high blood sugar level. It resembles type II diabetes in several respects, involving a combination of relatively inadequate insulin secretion and responsiveness. It occurs in about 2-10% of all pregnancies and may improve  or  disappear  after  delivery.  However,  after  pregnancy  approximately  5-10%  of women with gestational diabetes are found to have diabetes mellitus, most commonly type II (NDIC, 2011).

Gestational diabetes is fully treatable, but requires careful medical supervision throughout the pregnancy. Management may include dietary changes, blood glucose monitoring, and in some cases insulin may be required. If untreated, gestational diabetes can damage the health of the foetus or mother. Risks to the baby include macrosomia (high birth weight), congenital cardiac and central nervous system anomalies, and skeletal muscle malformations. Increased foetal insulin may inhibit foetal surfactant production and cause respiratory distress syndrome. A high blood bilirubin level may result from red blood cell destruction. In severe cases, perinatal death may occur as a result of poor placental perfusion due to vascular impairment (NDIC,2011).

1.5 Clinical Features and Diagnosis of Diabetes Mellitus

The classical symptoms of diabetes mellitus includes: polyuria, polydipsia, polyphagia, fatigue and weight loss (Cooke and Plotnick, 2008). Other important clinical features include: collapse of sexual function (Nabipour, 2003), blurring of vision and skin rashes collectively known as

diabetes dermadromes which usually develops in 30-70% of diabetic patients (Izaki, 2000). Diabetes mellitus is characterized by recurrent or persistent high blood sugar, and is diagnosed by demonstrating any one of the following:

  Fasting plasma glucose level ≥ 7.0 mmol/l (126 mg/dl) Plasma glucose ≥ 11.1 mmol/l (200 mg/dl) two hours after a 75 g oral glucose load as in a glucose tolerance test Symptoms of high blood sugar and casual plasma glucose ≥ 11.1 mmol/l (200 mg/dl) Glycated haemoglobin (HbA1C) ≥ 48 mmol/mol (WHO, 1999).

1.6 Prevention and Management of Diabetes Mellitus

Diabetes mellitus is a chronic disease, for which there is no known cure. Management concentrates on keeping blood sugar levels as close to normal, without causing hypoglycaemia. Prevention and management can be accomplished with a healthy diet, physical exercise, not using tobacco and weight loss. Blood pressure control and proper foot care are also important for people with the disease. Type 1 diabetes is managed with  insulin injections. Type II diabetes may be treated with medications with  or without insulin.  Insulin and some oral medications such as metformin, glibenclamide, acabose, sitagliptin, repaglinide, rosiglitazone etc., can lower blood sugar (Rippe and Irwin, 2010).Weight loss surgery in those with obesity is sometimes an effective measure in those with type 2 diabetes mellitus (Picot et al., 2009). Gestational diabetes usually resolves after the birth of the baby (Cash, 2014).

1.7 Thiamine, Pyridoxine and Biotin in Diabetes Mellitus

The B vitamins are important to glucose metabolism. They usually serve as cofactors in cellular reactions utilizing glucose. Hence, they have been extensively studied to determine their benefits for controlling blood sugar levels. Since they are water soluble, they are easily excreted from the body along with urine. This is particularly important for diabetics as they easily develop deficiencies of the B vitamins (Badr et al., 2015).

Thiamine  is  a  coenzyme  in  the  metabolism  of  keto  sugars.  It  is  also  important  for  the breakdown of pyruvic acid, a product released during glucose metabolism. Hence it can help improve how cells utilize glucose, leading to better control of blood sugar levels (Manzetti et al., 2014). Available studies do not always agree on the importance of Thiamine supplementation for diabetics. Clinical data show that patients with Type 1 diabetes usually have low vitamin B1 levels and can, therefore, benefit from thiamine supplements. On the other hand, Type 2 diabetes patients usually have normal blood levels of thiamine. However, one study   demonstrated   that   although   diabetics   have   normal   levels   of   this   vitamin,   its transportation across tissues is impaired. Therefore, even normal levels of the vitamin may not be sufficient to effectively control blood glucose levels in diabetics. Vitamin B1 supplementation has been proven to prevent and treat neuropathy (nerve damage) caused by diabetes (González-Ortiz et al., 2010).

Pyridoxine is another important coenzyme. It is involved in amino acid and carbohydrate metabolism (Hellman and Mooney, 2010). Its deficiency is common among diabetics and those with poor blood sugar control almost always have low plasma levels of vitamin B6. In addition, clinical data show that diabetics who take insulin have lower vitamin B6 levels than those still placed on oral anti-diabetics. This shows that vitamin B6  levels get even lower as diabetes progresses (Ahn et al., 2011). Studies also show that vitamin B6 deficiency is strongly associated with  glucose  intolerance and  reduced  secretion  of insulin  and  glucagon.  Since pyridoxine is required for tryptophan metabolism, diabetics with low levels of the vitamin cannot  properly  metabolize  tryptophan.  This  leads  to  the  accumulation  of  intermediate products  such  as  xanthurenic  acid  and  hydroxkynurenine.  Xanthurenic  acid  binds  to  and inhibits insulin. This can directly translate to poor blood sugar control (Rios-Avila et al., 2013; Oxenkrug, 2013). There is clinical evidence to show that vitamin B6  supplementation can improve glucose tolerance but only in those who are deficient. However, most studies do not conclusively prove that  vitamin  B6   supplementation  can  affect  blood  glucose levels  even though it may improve some other measures of glycaemic control. Experts agree that vitamin B6  supplementation can help prevent/relieve neuropathy (nerve damage) caused by diabetes (Paul and Nalia, 2010).

Biotin is another B-complex vitamin that is necessary for both metabolism and growth. Biotin is also involved in the manufacture and utilization of protein, fats and carbohydrates. Biotin works in synergy with insulin in the body, and independently increases the activity of the enzyme glucokinase (Valdés-Ramos et al., 2015). Glucokinase is responsible for the first step of glucose utilisation, and is therefore an essential component of normal bodily functioning. Glucokinase occurs only in the liver, and in sufferers from diabetes its concentration may be extremely low. Supplements of biotin may have a significant effect on glucose levels for both type 1 and type 2 diabetics (Badr et al., 2015).

1.8 Experimental Animal Models of Diabetes

Diabetes mellitus has been considered as one of the major health concerns all around the world today (Stolar et al., 2008; Kruger et al., 2012). Experimental animal models are one of the best strategies for the understanding of the pathophysiology of any disease in order to design and develop the drugs for its treatment (Rees and Alcolado, 2005; Chatzigeorgiou et al., 2009). Numerous animal models have been developed for the past few decades for studying diabetes mellitus and testing anti-diabetic agents that include chemical, surgical and genetic manipulations (Srinivasan and Ramarao, 2007; Etuk, 2010). One of the most potent methods to induce experimental  diabetes  mellitus  is  the  chemical  induction  by alloxan  (Etuk,  2010). Alloxan is a well-known diabetogenic agent that is used to induce Type I diabetes in experimental animals (Viana et al., 2004).

Alloxan is a urea derivative which causes selective necrosis of the β-cells of pancreatic islets. In addition, it has been widely used to produce experimental diabetes in animals such as rabbits, rats, mice and dogs with different grades of disease severity by varying the dose of alloxan used (Etuk, 2010; Iranloye et al., 2011). As it has been widely accepted that alloxan selectively destroys the insulin-producing beta-cells found in the pancreas, hence it is used to induce diabetes in laboratory animals. The toxic action of alloxan on pancreatic beta cells involve oxidation of essential sulphydryl (-SH groups), inhibition of glucokinase enzyme, generation of free radicals and disturbances in intracellular calcium homeostasis (Szkudelski,

2001). The underlying mechanism involves the selective uptake of the compound due to its structural similarity to glucose as well as highly efficient uptake mechanism of the pancreatic beta-cells (Lenzen, 2008; Viswanathaswamy et al., 2011).

1.8.1 Alloxan

Figure 14: Structure of alloxan. Source: (NCBI, 2016).

Alloxan (2,4,5,6-Pyrimidinetetrone also known as 1,3-Diazinane-2,4,5,6-tetrone by IUPAC, and mesoxalylurea and 5-oxobarbituric acid) is an oxygenated pyrimidine derivative which is present as alloxan hydrate in aqueous solution (Ankur and Shahjad, 2012). Brugnatelli originally isolated alloxan in 1818 and the name was given by Wohler and Liebig in 1838. Moreover, the compound was discovered by von Liebig and Wohler in 1828 and has been regarded as one of the oldest named organic compounds that exist. The name Alloxan emerged from the merging of two words, i.e. Allantoin and Oxaluric acid. Allantoin is a product of uric acid excreted by the foetus in the allantois while oxaluric acid has been derived from oxalic acid and urea that is found in urine. Additionally, the alloxan model of diabetes induction was first described in rabbits by Dunn, Sheehan and McLetchie in 1943 (Dunn et al., 1943).

Alloxan was originally prepared by the oxidation of uric acid by nitric acid. The monohydrate is simultaneously prepared by oxidation of barbituric acid by chromium trioxide (Holmgren and Wilhelm, 1963). Moreover, alloxan has been regarded as a strong oxidizing agent that forms a hemiacetal with its reduced reaction product; dialuric acid, in which a carbonyl group is reduced to a hydroxyl group, that is called alloxantin (Ankur and Shahjad, 2012). The drug has been noted to exert its diabetogenic action when administered intravenously, intra- peritoneally or subcutaneously. Furthermore, the dose of alloxan required for inducing diabetes depends on the animal species, route of administration and nutritional status (Federiuk et al.,

2004). Moreover, alloxan has been demonstrated to be non-toxic to the human beta-cells, even in very high doses, the reason of which may be attributed to the differing glucose uptake mechanisms in humans and rodents (Eizirik et al., 1994; Tyrberg et al., 2001).

1.8.2 Phases of Diabetes Induction

Alloxan has been used to induce experimental diabetes due to the selective destruction of the insulin-producing pancreatic beta-islets. Alloxan induces a multiphasic blood glucose response when injected into to an experimental animal, which is accompanied by corresponding inverse changes in the plasma insulin concentration followed by sequential ultra-structural beta cell changes ultimately leading to necrotic cell death.

The first phase that comes into view within the first minutes after alloxan injection is transient hypoglycaemic phase that lasts maximally for 30 minutes (Wrenshall et al., 1950; Lenzen, 2008). This little hypoglycaemic response has been noted to be the result of a transient stimulation  of insulin  secretion  that  was  confirmed by an  increase  of  the plasma  insulin concentration  (Kliber  et  al.,  1996).  The  underlying  mechanism  of  this  transient  hyper- insulinaemia may be attributed to a temporary increase in ATP availability due to inhibition of glucose phosphorylation through glucokinase inhibition (Ankur and Shahjad, 2012).

The second phase appearing one hour after administration of alloxan leads to rise in blood glucose concentration. Moreover, the plasma insulin concentration has been noted to decrease at the same time. This is the first hyperglycaemic phase after the first contact of the pancreatic beta cells with the toxin (West et al., 1996; Lenzen, 2008). This hyperglycaemic phase lasts for 2-4 hours which is accompanied by decreased plasma insulin concentrations. These changes are a result of inhibition of insulin secretion from the pancreatic beta cells that is attributed to the induction due to their beta cell toxicity (Ankur and Shahjad, 2012).

The third phase is again a hypoglycaemic phase that is noted 4-8 hours after the alloxan injection, which lasts for several hours (West et al., 1996; Lenzen, 2008). The flooding of circulation with insulin occurs as a result of the alloxan-induced secretory granule and cell membrane rupture resulting in severe transitional hypoglycaemia (Banerjee and Bhattacharya, 1948). In addition, other subcellular organelles are also ruptured that include cisternae of rough endoplasmic reticulum and the Golgi complex. Moreover, the outer and inner membranes of the mitochondria loose structural integrity in this particular phase. These changes are irreversible and highly characteristic for a necrotic cell death of pancreatic islets (Jorns et al.,1997; Mythili et al., 2004).

The last phase(the fourth phase) of the blood glucose response is the final permanent diabetic hyperglycaemic phase during which complete degranulation and loss of the integrity of the beta cells within 24-48 hours after administration of the alloxan takes place (Mythili et al.,

2004; Lenzen, 2008). Surprisingly, the non-beta cells and other endocrine and non-endocrine islet cell types along with extra-pancreatic parenchyma remain intact, providing the evidence of selective toxic action of alloxan (Lenzen et al., 1996; Jorns et al., 1997). Thus, alloxan injection has been noted to induce an insulin-dependent type I like diabetes syndrome and all the morphological features of beta cell destruction are characteristic for a necrotic cell death (Peschke et al., 2000; Lenzen, 2008).

1.8.3 Mechanism of Action of Alloxan

Alloxan-induced diabetes has been commonly employed as an experimental model of insulin dependent diabetes mellitus. The mechanism of alloxan action has been thoroughly studied which   currently   can   be   characterized   quite   well.   Several   experimental   studies   have

demonstrated that alloxan evokes a sudden rise in insulin secretion in the presence or absence of glucose which appeared just after alloxan treatment (Szkudelski et al., 1998; Lachin and Reza, 2012). This particular alloxan-induced insulin release occurs for short duration followed by the complete suppression of the islet response to glucose even when high concentrations of glucose were used (Kliber et al., 1996).

Furthermore, the alloxan action in the pancreas is preceded by its rapid uptake by pancreatic beta cells that have been proposed to be one of the important features determining alloxan diabetogenicity.  Moreover,  in  pancreatic  beta  cells,  the  reduction  process  occurs  in  the presence of different reducing agents like glutathione (GSH), cysteine, ascorbate and protein- bound sulfhydryl (-SH) groups (Lenzen and Munday, 1991; Zhang, 1992). Alloxan reacts with two sulfhydryl groups in the sugar binding site of glucokinase resulting in the formation of the disulphide bond and inactivation of the enzyme. As a result of alloxan reduction, dialuric acid is  formed  which  is  then  re-oxidized  back  to  alloxan  establishing  a  redox  cycle  for  the generation of reactive oxygen species (ROS) and superoxide radicals (Das et al., 2012). The superoxide radicals liberate ferric ions from ferritin and undergo dismutation to yield hydrogen peroxide (H2O2) in the presence of superoxide dismutase. As a result, highly reactive hydroxyl radicals are formed according to the Fenton reaction in the presence of ferrous iron and H2O2 (Sakurai and Ogiso, 1995).

Another mechanism that has been reported is the effect of ROS on the DNA of pancreatic islets. The fragmentation of DNA takes place in the beta cells exposed to alloxan that causes DNA damage, which stimulates poly ADP-ribosylation, a process participating in DNA repair. Antioxidants  like  superoxide  dismutase,  catalase  and  the  non-enzymatic  scavengers  of hydroxyl radicals have been found to protect against alloxan toxicity (Ebelt et al., 2000). In addition, the disturbance in intracellular calcium homeostasis has also been reported to constitute an important step in the diabetogenic action of alloxan. It has been noted that alloxan elevates cytosolic free Ca2+  concentration in the beta cells of pancreatic islets (Park et al., 1995). The calcium influx follows the ability of alloxan to depolarize pancreatic beta cells that further opens voltage-dependent calcium channels and enhances calcium entry into pancreatic cells. The increased concentration of Ca2+ ion further contributes to supra-physiological insulin release  that  alongwith  ROS  has  been  noted  to  ultimately cause  damage  of  beta  cells  of pancreatic islets (Szkudelski, 2001; Lenzen, 2008; Etuk, 2010).

1.8.4 Biological Effects

Alloxan is a hydrophilic and unstable chemical compound which has similar shape as that of glucose, which is responsible for its selective uptake and accumulation by the pancreatic beta cell (Gorus, 1982).  Similarity in the shape allows it to be transported into the cytosol by the glucose transporter (GLUT2) located in the plasma membrane of beta cell (Gorus, 1982; Elsner et al., 2002).

Another biological effect of alloxan has been attributed to the thiol group reactivity that allows selective inhibition of glucose-induced insulin secretion through inhibition of glucokinase. This inhibition of glucose-induced insulin secretion has been regarded as the major pathophysiological effect of alloxan, which results from the thiol group reactivity of alloxan. The thiol groups of glucokinase, the glucose phosphorylating enzyme, are particularly sensitive to oxidation by alloxan (Lenzen and Panten, 1988).

Glucokinase inhibition reduces glucose oxidation and ATP generation that further suppresses glucose-induced insulin secretion (Lenzen and Mirzaie-Petri, 1992; Tiedge et al., 2000). Moreover, the insulin biosynthesis is also inhibited by alloxan through the same mechanism. Alloxan inhibits many cellular functions at higher concentrations such as the ability to oxidize thiol groups of many functionally important enzymes like hexokinase, phosphofructokinase, calmodulin-dependent protein kinase, aconitase and other proteins (Lenzen and Mirzaie-Petri, 1992;  Lenzen,  2008).  Hence,  it  is  evident  that  the  inhibition  of  glucose-induced  insulin secretion by alloxan is the result of the thiol reactivity of the glucokinase. Another biological effect of alloxan is pancreatic beta cell toxicity and diabetogenicity that may be attributed to alloxan-induced redox cycling and ROS generation.

The mechanism underlying the cytotoxic action of alloxan to insulin-producing cells may be ascribed as the reduction by interaction with intracellular thiols such as glutathione (Elsner et al., 2006). The resultant formation of cytotoxic ROS is the result of a cyclic reaction between alloxan and its reduction product, dialuric acid, which by autoxidation generates superoxide radicals, hydroxyl radicals and H2O2  (Winterbourn and Munday, 1989). Induction of diabetes in the laboratory animals by alloxan injection is the result of selective uptake of alloxan via GLUT2 into a pancreatic beta cell (Elsner et al., 2002). The effective prevention of redox cycling and generation of ROS can prevent pancreatic beta cell death and counteract the development of alloxan diabetes in vivo (Jorns et al., 1999; Elsneret al., 2006; Lenzen, 2008; Zhang et al., 2009). Hence, it can be summarized that the alloxan-induced pancreatic beta cell toxicity and  the resultant  diabetogenicity is  due to  the redox  cycling and  the toxic ROS generation in combination with the hydrophilicity and the glucose similarity of the molecular shape of alloxan.

The  chemical  induction  of  diabetes  appears  to  be  the  most  popularly  used  procedure  in inducing diabetes mellitus in experimental animals. The foremost drug-induced diabetic model is the alloxan diabetes that is capable of inducing type I diabetes mellitus in experimental animals. The surgical and genetic methods of diabetes induction are associated with a high percentage of animal morbidity and mortality. Hence, alloxan-induced diabetes model appears to be the most reliable and easily reproducible method of inducing diabetes mellitus in experimental animals (Ankur and Shahjad, 2012).

1.9 Metformin

Metformin is an oral anti-diabetic medication of the biguanide class. It is the first-line drug of choice for the treatment of type 2 diabetes, in particular, in overweight and obese people and those with normal kidney function (ADA, 2009). Its use in  gestational diabetes has been limited by safety concerns. It is also used in the treatment of polycystic ovary syndrome, and has been investigated for other diseases where insulin resistance may be an important factor such as non-alcoholic fatty liver disease. Metformin works by suppressing glucose production by the  liver  (Rang  et  al.,  2012).  Limited  evidence  indicates  metformin  may  prevent  the cardiovascular and possibly the cancer complications of diabetes. It helps reduce LDL cholesterol and triglyceride levels and is not associated with weight gain; in some people, it promotes weight loss (El Messaoudi, 2011). Metformin is one of only two oral anti diabetic drug in the World Health Organization model list of essential medicines, the other being glibenclamide (WHO, 2010).

Metformin causes few adverse effects when prescribed appropriately (the most common is gastrointestinal upset) and has been associated with a low risk of having a low blood sugar. Lactic acidosis (a build-up of lactate in the blood) can be a serious concern in overdose and when it is prescribed to people with contraindications, but otherwise, no significant risk exists (Lipska et al., 2011). Metformin is contraindicated in people with any condition that could increase the risk of lactic acidosis, including kidney disorders, lung disease and liver disease (Jones et al., 2003). Metformin has an oral bioavailability of 50-60% under fasting conditions, and is absorbed slowly. Peak plasma concentrations (Cmax) are reached within one to three hours of taking immediate-release metformin and four to eight hours with extended-release

formulations (Heller, 2007). Metformin is not metabolized, it is usually cleared from the body by tubular secretion. It has a half-life of about 3 hours and is excreted unchanged in the urine (Rang et al., 2012).

1.9.1 Mechanism of Action

Metformin  originally  found  in  French  lilac  (Galega  officinalis)  is  the  only  drug  of  the biguanide class which is used clinically (Rang et al., 2012). Biguanides have several mechanisms of action. They act by reducing hepatic glucose production (gluconeogenesis) which is markedly increased in type II diabetes, increase uptake and utilization of glucose in skeletal muscle hence reducing insulin resistance. They reduce carbohydrate absorption, increase fatty acid oxidation and reduce circulating level of low-density lipoprotein and very low-density lipoprotein (Rang et al., 2012). The mechanism by which metformin reduces hepatic gluconeogenesis  involves activation of AMP protein kinase (AMPK) which is an important enzyme responsible for metabolic control in the hepatocytes (Trowler and Hardie, 2007). Activation of AMP protein kinase increases expression of a nuclear receptor which inhibits expression of genes that are important for gluconeogenesis in the liver (Kim et al., 2008).

1.10 Blood Glucose Concentration

The blood sugar concentration or blood glucose level is the amount of glucose present in the blood of a human or animal. Naturally, the body tightly regulates blood glucose levels as a part of metabolic homeostasis (Van Soest, 1994).With some exceptions, glucose is the primary source of energy for the body’s cells, and blood lipids (in the form of fats and oils) are primarily a compact energy store (Young, 1977). Glucose is transported from the intestines or liver to body cells via the bloodstream, and is made available for cell absorption via the hormone insulin, produced by the body primarily in the pancreas. Glucose levels are usually lowest in the morning, before the first meal of the day (fasting level), and rise after meals for an hour or two by a few milli molar. Increase in blood sugar levels above the normal range may be an indicator of a medical condition. A persistently high fasting blood glucose level (above 200 mg/dl) is referred to as hyperglycaemia whereas low levels (below 40 mg/dl) is referred to as hypoglycaemia, which is a potentially fatal condition. Symptoms may include lethargy, impaired mental functioning, irritability, shaking, twitching, weakness in the arms and leg muscles, pale complexion, sweating, paranoid or aggressive mentality and loss of consciousness.  Mechanisms  that  restore  satisfactory  blood  glucose  levels  after  extreme hypoglycaemia must be quick and effective to prevent extremely serious consequences of insufficient glucose (Warade, 2014).

Diabetes mellitus is characterized by persistent hyperglycaemia from any of several causes, and is the most prominent disease related to failure of blood sugar regulation. Many factors affect  a  person’s  blood  sugar  level.  A  body’s  homeostatic  mechanism,  when  operating normally, restores the fasting blood sugar level to a narrow range of about 4.4-6.1 mmol/l (79.2 to 110 mg/dl). The normal blood glucose level (tested while fasting) for non-diabetics, should be between 3.9 and 5.5 mmol/L (70 to 100 mg/dl). However, this level fluctuates throughout the day. Blood sugar levels for those without diabetes and who are not fasting should be below

6.9  mmol/l  (125 mg/dl).  The  blood  glucose  target  range  for  diabetics,  according  to  the American Diabetes Association, should be 5.0-7.2 mmol/l (90-130 mg/dl) before meals and less than 10 mmol/l (180 mg/dl) after meals (ADA, 2006).

1.11 Electrolytes

An electrolyte is a substance that produces an electrically conducting solution when dissolved in a polar solvent such as water. The dissolved electrolyte separates into cations and anions which disperse uniformly through the solvent. They can also be seen as minerals formed in the blood and other body fluids that carry an electric charge. These are positively and negatively charged ions that are found when mineral or other salts dissociate in water. They include sodium (Na+), potassium (k+), chloride (Cl-) bicarbonate (HCO3-) ions. Due to their charges, electrolytes possess the ability to conduct electrical current in water (Estevez et al., 2008). This characteristic of electrolytes is important because the current enables electrolytes to regulate how and where fluids are distributed throughout the body and keeping water from floating freely across the cell membranes (Kamil et al., 2011).

In other to control fluid passage across cell membranes, cells regulate the inward and outward movement of electrolytes thus causing water to follow the charged particles around (Estevez et al., 2008). These actions help to maintain a state of fluid balance. The difference in electrical balance inside and outside of the cells also allows for transmission of nerve impulses, contraction or relaxation of muscles, blood pressure control and proper gland functioning. The presence of electrolytes determine the acidity or alkalinity of some fluids especially the blood (Kamil  et  al.,  2011).  The concentrations  of these ions  in  the blood  stream  remain  fairly constant and an imbalance can lead to hypernatremia or hyponatremia and hyperkalaemia or hypokalaemia (Kamil et al., 2011).

1.11.1 Sodium

Sodium is the major cation of extracellular fluid (Reynolds et al., 2006). It plays a central role in the maintenance of the normal distribution of water and the osmotic pressure in the various fluid compartments. The main source of body sodium is sodium chloride contained in ingested foods. Only about one-third of the total body`s sodium is contained in the skeleton since most of it is contained in the extracellular body fluids. Excess sodium obtained from dietary sources is excreted in the urine. Sodium ions regulate the total amount of water in the body and the transmission of sodium into and out of the individual cells also play a role in critical body functioning (Rrosner and Kirven, 2006). Many processes in the body especially in the brain, nervous system and muscles require electrical signals for communication (Reynolds et al.,

2006; Schrier, 2010). The movement of sodium ions is critical in the development of these electrical signals (Adrogue and Madias, 2000). Hyponatremia (low serum sodium level) is found in a variety of conditions including the following: severe polyuria, metabolic acidosis, Addison`s  disease,  diarrhoea,  and  renal  tubular  disease.  Hypernatremia  (increased  serum sodium  level)  is  found  in  the  following  conditions:  hyperadrenalism,  severe  dehydration, excess treatment with sodium salts, and diabetic coma after therapy with insulin (Adrogue and Madias, 2000).

1.11.2 Potassium

Potassium is an essential dietary mineral and electrolyte. Normal body function depends on tight regulation of potassium concentrations both inside and outside of cells (Peterson, 1997). It functions in the maintenance of membrane potential which is critical for nerve impulse transmission, muscle contraction, and heart function (Sheng, 2000).It also play important role as  cofactor  for enzymes  which  require the  presence of potassium  for  their activity.  This involves pyruvate kinase, an important enzyme in carbohydrate metabolism (Sheng, 2000). Potassium is the principal cation of the intracellular fluid. It is also an important constituent of the extracellular fluid due to its influence on muscle activity. Its intracellular function is parallel to that of its extracellular function, namely influencing acid-base balance and osmotic pressure, including water retention (Reynolds et al., 2006).

Elevated potassium levels (hyperkalaemia) are often associated with renal failure, dehydration shock or adrenal insufficiency. Decreased potassium levels (hypokalaemia) are associated with malnutrition, negative nitrogen balance, gastro intestinal fluid losses and hyperactivity of the adrenal cortex. An abnormally low plasma potassium concentration is referred to as hypokalaemia. Hypokalaemia is most commonly a result of excessive loss of potassium, e.g., from  prolonged  vomiting,  the  use  of  some  diuretics,  some  forms  of  kidney  disease,  or metabolic disturbances. The symptoms of hypokalaemia are related to alterations in membrane potential and cellular metabolism. They include fatigue, muscle weakness and cramps, and intestinal paralysis, which may lead to bloating, constipation, and abdominal pain. Severe hypokalaemia   may  result   in   muscular   paralysis   or   abnormal   heart   rhythms   (cardiac arrhythmias) that can be fatal (Sheng, 2000; FNB, 2005).

Conditions that increase the risk of hypokalaemia include; the use of potassium-wasting diuretics (e.g., thiazide diuretics or furosemide), alcoholism, severe vomiting or diarrhoea, overuse or abuse of laxatives, anorexia nervosa or bulimia, magnesium depletion, as well as congestive heart failure (Gennari, 1998). Low dietary intakes of potassium do not generally result in hypokalaemia (Gennari, 1998). However, research indicates that insufficient dietary potassium increases the risk of a number of chronic diseases such as Stroke (Bazzano et al., 2001; Green et al., 2002), osteoporosis, (Tucker et al., 1999; Zhu et al., 2009), kidney stones (Trinchieri et al., 2001; Curhan et al., 1997).

1.11.3 Chloride

Chloride is a major anion and is important in the maintenance of the cation/anion balance between intracellular and extracellular fluids (Zumdahl, 2009). This electrolyte is therefore essential to the control of power hydration, osmotic pressure and acid/base equilibrium. Low serum chloride values are found with extensive burns, excessive vomiting, intestinal obstruction, nephritis, metabolic acidosis, and in Addisonian crisis (Cambier  et al., 1998; Zumdahl, 2009). Elevated serum chloride values maybe seen in dehydration, hyperventilation, congestive heart valve and prostatic or other types of urinary obstruction (White, 1970; Tietz, 1976).

1.11.4 Bicarbonate

Bicarbonate is one of the principal blood gases. It plays major role in the transport of carbon dioxide. It is determined primarily as CO2. Carbon dioxide in serum or plasma exists primarily

as dissolved carbon dioxide (CO2) and bicarbonate anion (HCO3-) (Kaplan and Pesce, 1984). The plasma carbon dioxide content is decreased in metabolic acidosis and respiratory alkalosis, whereas the level is increased in metabolic alkalosis and respiratory acidosis (Latner, 1975). In pathologic condition, diabetes mellitus, glomerulonephritis, pyloric obstruction, diarrhoea etc, acidosis or alkalosis could be anticipated (Tietz et al., 1986). Therefore, determination of plasma carbon dioxide content as part of electrolyte profile can help establish, to a degree, the anticipated change in the patient.

1.12 Renal Function

Renal function is an indication of the state of the kidney and its role in renal physiology (Stevens et al., 2006). Glomerular filtration rate (GFR) which is the flow of filtered fluid through the kidney and creatinine clearance (CrCl) which is the volume of blood plasma that is cleared of creatinine per unit time may be calculated by comparative measurements of substances in the blood and urine (Guyton and Hall, 2006). The results of these tests are important in assessing the excretory function of the kidneys (National Kidney Foundation,

2002). Plasma concentrations of the waste substances of creatinine and urea as well as electrolytes can be used to determine renal function (Levey et al., 2006). However, blood urea nitrogen (BUN) and creatinine will not be raised above the normal range until 60% of total kidney function is lost (Guyton and Hall, 2006).

1.12.1 Urea/Blood Urea Nitrogen (BUN)

Urea is a major nitrogenous end product of protein and amino acid catabolism produced by liver. It is the first organic chemical compound to be synthesized. The liver produces urea in the urea cycle as a waste product of the degradation of protein (Landry and Basari, 2011). In the kidneys, urea is filtered out of blood by glomeruli and is partially reabsorbed with water (Corbett, 2008). The most frequently determined clinical indices for estimating renal function depends on the concentration of urea in the serum. It is useful in differential diagnosis of acute renal failure and pre-renal condition where blood urea nitrogen-creatinine ratio is increased (Mitchell and Kline, 2006). Urea clearance is a poor indicator of glomerular filtration rate as its overproduction  rate  depends  on  several  non-renal  factors,  including  diet  and  urea  cycle enzymes (Mitchell and Kline, 2006).

Blood urea nitrogen (BUN) is an indicator of renal health. 1 mg of urea corresponds to 0.467 mg of blood urea nitrogen (Tietz, 1995). Increased blood urea nitrogen (BUN) is seen to be associated with kidney disease or failure, blockage of the urinary tract by a kidney stone, congestive heart failure, dehydration, fever, shock, and bleeding in the digestive tract. The high BUN levels can sometimes occur during late pregnancy or result from eating large amounts of protein-rich foods. If the BUN level is higher than 100 mg/dl it points to severe kidney damage whereas decreased BUN is observed in fluid excess. Low levels are also seen in trauma, surgery, opioids, malnutrition, and anabolic steroid use (Pagana, 1998).

1.12.2 Uric Acid

Uric acid is a chemical formed when the body breaks down substances called purines. Purines are found in some foods and drinks such as liver, anchovies, mackerel, dried beans and peas, and beer. Most uric acid dissolves in blood and transported to the kidneys and excreted in urine. Excess production of uric acid or inability of the kidneys to remove excess uric acid from the blood may result in disease condition. A high level of uric acid in the blood is called hyperuricemia. High levels of uric acid can cause gout or kidney disease. Rapid weight loss, which may occur with such treatments, can increase the amount of uric acid in the blood (Goldman et al., 2011).

Normal values range between 3.5  and 7.2  mg/dl. Greater-than-normal  levels of uric acid (hyperuricemia)  may  be  due  to  acidosis,  alcoholism,  chemotherapy-related  side  effects , diabetes, excessive exercise , gout, hypoparathyroidism, lead poisoning, leukaemia, medullary cystic kidney disease, nephrolithiasis, polycythaemia vera, purine-rich diet, renal failure, toxaemia  of  pregnancy.  Lower-than-normal  levels  of  uric  acid  may  be  due  to  Fanconi syndrome,  low  purine  diet,  syndrome  of  inappropriate  antidiuretic  hormone  (SIADH) secretion, Wilson’s disease etc. Uric acid test can be performed in other disease conditions such as chronic gouty arthritis, chronic kidney disease, Injury of the kidney and ureter (Goldman et al., 2011).

1.12.3 Creatinine

Creatinine is an anhydride creatine, and a breakdown product of creatine phosphate in muscle. It is usually produced at a fairly constant rate by the body depending on muscle mass (Gross et al., 2005). It is a waste product formed by the spontaneous dehydration of the kidneys (Henry,

1974). Measuring serum creatinine is a simple test, and it is the most commonly used indicator of renal function. Most of the creatinine is formed in the muscle tissue where it is present as creatine phosphate and serves as a high energy storage reservoir for further conversion to ATP.

Independent of diet, serum creatinine concentrations depends almost entirely upon its excretion rate by the kidneys. For this reason, its elevation is highly specific for kidney diseases. Serum creatinine is an important indicator of renal health because it is an easily measured by-product of muscle metabolism that is excreted unchanged by the kidneys (Rule et al., 2004).

Creatine is synthesized primarily in the liver from the methylation of glycocyamine (guanidino acetate, synthesized in the kidney from the amino acids arginine and glycine) by S-adenosyl methionine. It is then transported through blood to the other organs, muscle, and brain, where, through phosphorylation, it becomes the high-energy compound phosphocreatine (Waikar and Bonventre, 2009). Creatinine is removed from the blood chiefly by the kidneys, primarily by glomerular filtration, but also by proximal tubular secretion (Metha et al., 2007). Little or no tubular reabsorption of creatinine occurs. If the filtration in the kidney is deficient, creatinine blood levels rise. Therefore, creatinine levels in blood and urine may be used to calculate the creatinine clearance (CrCl), which correlates with the glomerular filtration rate (GFR) (Myers et al., 2006). Blood creatinine levels may also be used alone to calculate the estimated GFR (Katie et al., 2014).

1.13 Rationale of the Study

The B vitamins are important to glucose metabolism in that they serve as coenzymes in cellular reactions involving glucose utilization (Badr et al., 2015). Therefore, they are being studied to determine their benefits for controlling blood sugar levels. Since they are water soluble, they are easily excreted from the body along with urine. This is particularly important for diabetics as they easily develop deficiencies of the B vitamins (Badr et al., 2015).

Metformin is an oral hypoglycaemic drug which is widely used in clinical practise for the treatment and management of diabetes mellitus (Rand et al., 2012). It lowers blood sugar level with moderately poor restoration of renal function. Available evidence supports the use of metformin in patients with mild to moderate kidney disease (Inzucchi  et al., 2014). This research was aimed investigating the efficacy of the drug action as well as its performance when administered as an adjuvant with the B vitamins involved in energy/carbohydrate metabolism. Also, since B vitamins such as thiamine, pyridoxine and biotin play vital co-factor roles in the metabolism of carbohydrates, it is important to investigate their effect on blood sugar levels and renal functions of diabetic rats when administered singly or as an adjuvant.

1.14 Aim of the Study

The aim of this research was to determine the effects of thiamine, pyridoxine and biotin on blood glucose levels and renal function parameters of alloxan-induced diabetic rats

1.14.1 Specific Objectives of the Study

The specific objectives included:

       To determine the effect of thiamine, pyridoxine and biotin, with metformin on blood glucose concentration of alloxan-induced diabetic rats.

       To determine the effects of thiamine, pyridoxine and biotin with metformin on serum electrolyte (sodium, potassium, chloride and bicarbonate) concentrations in alloxan- induced diabetic rats.

       To determine the effect of thiamine, pyridoxine and biotin, with metforminon serum urea concentration of alloxan-induced diabetic rats.

       To determine the effect of thiamine, pyridoxine and biotin, with metformin on blood urea nitrogen of alloxan-induced diabetic rats.

       To determine the effect of thiamine, pyridoxine and biotin, with metforminon serum uric acid concentration of alloxan-induced diabetic rats.

       To determine the effectof thiamine, pyridoxine and biotin, with metformin on creatinine concentration of alloxan-induced diabetic rats.

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