FASTING BLOOD SUGAR ANALYSIS AND BLOOD SUGAR REGULATION

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FASTING BLOOD SUGAR ANALYSIS AND BLOOD SUGAR REGULATION

CHAPTER ONE

INTRODUCTION

1.1Introduction:

The blood sugar concentration or blood glucose level is the amount of glucose present in the blood of human. Normally in mammals, the body maintains the blood glucose level at reference range between 64.8        and 104.4 mg/dl. The human body naturally tightly regulates blood         glucose level as a part of metabolic hemostasis (Baker L, et al, 1969).         Glucose can measure in whole blood, serum or plasma (Richard, 2001). Collection of blood specimen for measurement of blood glucose level should be doing on the day and time requested. This is because collection times related to food intake (Ochei and Kolhatkar, 2005). There are two different method of determining glucose level. They include the chemical method and the enzymatic method. The chemical method exploits the non-specific reducing property of glucose in reactions with an indicator substance, which concomitantly changes color on its reduction. While the enzymes such as Glucose Oxidase and Hexokinase (Louie, et al, 2002). The enzymatic method has reached and advanced stage where                      the enzymes could immobilized in electronic machines or devices               for easier and faster analysis (Chernow, et al, 1996). Glucose estimation using plasma or whole blood requires the use of an anticoagulant, which are compounds that help prevent the clotting of blood. When blood is shed or collected, the cell does not die immediately. They continue to metabolize and use up glucose as a source of energy, via the glycolytic process. Glucose thus disappears from whole blood on standing over a period. Glycolysis can prevent with an enzyme inhibitor (Lawrence et al, 2008).

The commonest inhibitor for this purpose is sodium fluoride, which usually used in conjunction with an anticoagulant potassium oxalate. Fluoride actually inhibits the enzyme Enolase that found in the metabolic pathway of glucose and has a little effect on glucose Oxidase and Peroxidase enzymes. It also inhibits bacterial growth (Lawrence, et al, 2008). Other widely used anticoagulant is Ethylene Di amine Tetra – acetate (EDTA); when EDTA added to blood sample; it chelates the calcium needed for blood clotting thereby preventing the formation of fibrin. It forms an insoluble calcium salt by chelation.

1.2 Literature Review:

1.2.1Carbohydrates:

Carbohydrates are organic compounds that composed of atoms of   carbon, hydrogen and oxygen in a ratio of one carbon atom,                      two hydrogen atoms, and one oxygen atom. Some carbohydrates                are relatively small molecules; the most important to us is glucose, which has six carbon atoms. These simple sugars called monosaccharide. The primary function of carbohydrates is for short-term energy storage (sugars are for Energy).  A secondary function is intermediate-term energy storage (as in starch for plants and glycogen for animals). Other carbohydrates are involved as structural

components in             cells, such as cellulose, which found in the cell of plants.

http://www.biweb.wku.edu/courses/bio/115/wyatt/biochem es/carbos.html

1.2.2 Definition of Glucose:   

Glucose is by far the most common carbohydrate and classified as a monosaccharide, an aldose, a hexose, and is a reducing sugar.               It also known as dextrose, because it is dextrorotatory; it means that as an optical isomer is rotates plane polarized light to the right and an origin for the D designation. It also called blood sugar as it circulates                           in the blood at a concentration of 65-110 mg/dl (or 65-110 mg/100 ml)             of blood. Glucose initially synthesized by chlorophyll in plants using carbon dioxide from the air and sunlight as an energy source; then it further converted to starch for storage. (http://www.chemiwiki.ucdavis.edu/biological.chemistry/ca rbohyderates/monosacchrides/glucose).

 

Glucose is primary source of energy for human; the nervous system including the brain totally depending on glucose from the surrounding extracellular fluid (ECF) for energy. Nervous tissue cannot concentrate or store carbohydrate, it is critical to maintain steady supply of glucose to tissue for this reason the concentration of ECF must be maintained in narrow range. The normal blood glucose concentration in person who has not eaten meal with in the past 3 or 4 hours is about 90mg/dl, after meal containing large amount of carbohydrate this level seldom rise above 140mg/dl unless the person has diabetes mellitus (Bishop, 2005).

1.2.2.1 Ring Structure for Glucose:

An aqueous sugar solution contains only 0.02% of the

glucose in                      the chain form, the majority of the structure is in the cyclic chair form. Since carbohydrates contain alcohol and aldehyde or ketone functional group, the straight-chain form easily converted into the chair form – hemiacetal    ring structure.   Due to the tetrahedral geometry of carbons that ultimately make a six membered stable ring, the -OH on carbon, number (5) converted into ether linkage to close the ring with carbon number (1). This makes a 6 member ring-five carbons and oxygen. (http://www.chemiwiki.ucdavis.edu/biological.chemistry/ca rbohyderates/mosacchride/glucose).

1.2.2.1.1 Steps in the ring closure (hemiacetal synthesis):

Figure 1.2: chemical structure of glucose

  1. The electrons on the alcohol oxygen used to bond the carbon number (1) to make ether.
  2. The hydrogen transferred to the carbonyl oxygen to make a new alcohol group. The chair structures always written with the orientation depicted on the left to avoid confusion.

(http://www.chemiwiki.ucdavis.edu/biological.chemistry/ca rbohyderates/monosacchrides/glucose).

1.2.2.1.2 Hemiacetal Functional Group:

Carbon number 1 now called the anomeric carbon and is the center of a hemiacetal functional group. A carbon that has both ether oxygen and an alcohol group is a hemiacetal.

1.2.2.1.3 Comparison between Alpha and Beta Glucose in the Haworth Structures:

The Beta position defined as the -OH being on the same side of the ring as the C number (6). In the Haworth structure, this results in an upward projection. The Alpha position defined as the –OH    being on the opposite side of the ring as the C number (6).

In the Haworth structure, this also results in downward projection.

1.2.2.1.4 Comparison between Alpha and Beta Glucose in the Chair Structures:

The position of the -OH group on the numeric carbon (1) is an important distinction for carbohydrate chemistry. The Beta position defined as the -OH being on the same side of the ring as the C number 6. In the chair structure, this results in a horizontal projection. The alpha position defined as the -OH being on the opposite side of the ring as the C number 6.  In the chair structure, this results in a downward projection. The alpha and beta label not applied to any other carbon – only the numeric carbon.

(http://www.chemiwiki.ucdavis.edu/biological.chemistry/ca rbohyderates/monosacchrides/glucose).

1.2.2.3 Physical Properties of Glucose:

1.2.2.3.1 Solution:

All forms of glucose are colorless and easily soluble in water, acetic acid, and several other solvents. They are only sparingly soluble in methanol and ethanol. The open-chain form is thermodynamically unstable, and it spontaneously isomerizes to the cyclic forms. (Although the ring closure reaction could in theory create four- or three-atom rings, these would be highly strained and are not observed) In solutions at room temperature, the four cyclic isomers interconvert over a time scale of hours, in a process called mutarotation (McCurry and John E, 1988). Starting from any proportions, the mixture converges stable ratio of α: β 36:64. The ratio would be α: β 11:89 if it were not for the influence of the numeric effect (juarrais, et al, 1988).

Mutarotation is considerably slower at temperatures close to 0 °C; it consists of a temporary reversal of the ring-forming reaction, resulting in the open-chain form, followed by a re-forming of the ring. The ring closure step may use a different -OH group than the one recreated by the opening step (thus switching between pyranose and furanose forms), and/or the new ahemiacetal group created on C-1 may have the same or opposite handedness as the original one (thus switching between α and β forms). Thus, even though the open-chain form is barely detectable in solution, it is an essential component of the equilibrium (Fraser-Reid, et al 2009).

1.2.2.3.2 Solid state:

Depending on conditions, three major solid forms of glucose can crystallized from water solutions: α-glucopyranose, β-glucopyranose, and β-glucopyranose hydrate (Schenck, 2006).

1.2.2.3.3 Optical activity:                                                                                             

Whether in water or in the solid form, D-glucose is dextrorotatory, meaning that it will rotate the direction of polarized light clockwise. The effect is due to the chirality of the molecules, and indeed the mirror-image isomer, L-glucose, is levorotatory (rotates polarized light counterclockwise) by the same amount. The strength of the effect is different for each of the five tautomers. Note that the D- prefix does not refer directly to the optical properties of the compound. It indicates that the C-2 chiral center has the same handedness as that of D-glyceraldehydes (which was labeled because it is dextrorotatory). The fact that D-glucose is dextrorotatory is a combined effect of its four chiral centers, not just of C-2; and indeed some of the other D-aldohexoses are levorotatory (Fraser-Reid, et al, 2009).

1.2.2.3.4 Production of glucose:

1.2.2.3.4.1Biosynthesis:

In plants and some prokaryotes, glucose is a product of photosynthesis. In animals and fungi, glucose results from the breakdown of glycogen, a process known as glycogenolysis. In plants the breakdown substrate is starch. In animals, glucose is synthesized in the liver and kidneys from non-carbohydrate intermediates, such as pyruvate,lactate and glycerol, by a process known as gluconeogenesis. In some deep-sea bacteria, produce glucose by chemosynthesis  (Schenck, 2006).

1.2.2.3.4.2Commercial:

Glucose produced commercially via the enzymatic hydrolysis of starch. Many crops can use as the source of starch. Maize,rice,wheat,cassava,cornhusk and sago are used in various parts of the world. In the United States, cornstarch (from maize) used almost exclusively. Most commercial glucose occurs as a component of invert sugar, an approximately 1:1 mixture of glucose and fructose. In principle, cellulose could be hydrolysed to glucose, but this process is not yet commercially practical (Schenck, 2006).

1.2.2.4 Glucose metabolism:

Fructose and glactose are readily converted to glucose by the liver by the appropriate enzymes. Glucose is either converted to liver glycogen, stored there until it is required to maintain the normal glucose level, glucose then passes in to the circulating system, and transported to cells where it is undergoes remarkable metabolic changes. This includes energy releasing degradation of glucose to form simple product such as carbon dioxide and water (Godkar, 2008).  

The energy released particularly in the form of adenine tri phosphate (ATP) molecules can be used for energy requiring synthesis of new complex organic compounds and also for other cellular activities from glucose , formation of glycogen occurs in partially in every tissue of the body, mainly in the liver and muscles . The liver may contain about 57% glycogen following meal with high carbohydrate diet. After 12-18 hours of fasting, the liver is completely depleted from glycogen; muscle glycogen is rarely elevated above 1% of the wet weight of tissue. Since the amount of glycogen that can be stored is limited, the excess of glucose is converted to fatty acids which combined with glycerol molecules and deposited into adipose tissue as triglycerides (TG) (Godkar, 2008).  

Glycolysis: Is the oxidation of glucose or glycogen to pyruvate and lactate by the Embden Meyerhof pathway. The relative proportion of pyruvate and lactate depends on the degree of oxygen supply incomplete anaerobic environment only, thus only lactate formation takes place. The pyruvate formed is oxidized to carbon dioxide and water if adequate oxygen is available in citric acid cycle (Krebs cycle) if glucose is converted to lactate; 3 molecules of ATP are gained, when ATP molecules undergo hydrolysis by the action of adenosine triphosphate , energy is released and it  available for cellular activities (Godkar, 2008).

1.2.2.4.1Tri Citric acid cycle (TCA):

Oxidation of pyruvate to carbon dioxide and water takes place through the citric acid cycle in matrix of the mitochondria, which operates TCA cycle. However, TCA cycle does not take place in red blood cell due to lack of mitochondria. (Godkar, 2008).  

The Hexose Monophosphate Shunt (HMS) operates in the cytosol since the enzymes, which carry out the various reactions, are present in cytosol, it is an alternative pathway to the Emden Meyerhof pathway and citric acid cycle for the oxidation of glucose. NADPH molecules, which needed for lipogenesis, obtained by Hexose

Monophosphate Shunt pathways, liver, adipose tissue and mammary long require NADPH for lipogenesis, it provides pentose required for nucleotide and nucleic acids synthesis.  This pathway in red blood cells provides reduced NADPH for                 the reduction of oxidized glutathione to the reduced glutathione.                The reduced glutathione then removes H2O from erythrocytes accumulation of H2O2   may decrease the life span of erythrocyte by increasing the rate of oxidation of hemoglobin to methemoglobin (Godkar, 2008).

Glycogenesis: Is the synthesis of glycogen from glucose can occur in most tissue of the body. Liver and muscles are the most important sites of the glycogenesis, in the absence urgent demands of oxidative energy or conversion to any other compound. Excess of glucose is converted to glycogen and stored in the tissue; glycogen is available reserve of blood glucose in fasting conditions.

Glycogenolysis:  Is the breakdown of glycogen to glucose, both muscle and liver glycogen undergo glycogenolysis. Liver glycogenolysis gives glucose while in muscle glycogenolysis formation of lactic acid takes place due to the absence of glucose-6-phosphatase. This lactic acid reaches the liver by blood circulation where is converted to glucose. (Godkar, 2008).

Gluconeogenesis: Is the glucose synthesis from non-carbohydrate substances, these substances known as glycogenic substances such as lactic acid, glycerol, and pyruvate. These glycogenic substances converted into glucose or glycogen by reversal of glucolytic and citric acid cycle reaction, gluconeogenesis maintains continuous supply of glucose as a necessary source of energy especially for erythrocytes and nervous system (Godkar, 2008).

1.2.2.5 Glucose Regulation: 

The liver, pancreas and other endocrine glands are all involved in controlling the blood glucose concentration, during short fast glucose supplied to Extra Cellular Fluid (ECF) from the liver through glycogenolysis when the fasting period is longer than one-day glucose is synthesized from other source through gluconeogenesis (Bishop, 2005).   Muscle glycogen does not contribute directly to blood sugar, glycogenolysis which occur in muscle produce lactate that is converted to glucose in the liver, also kidney exerts regulatory effect by the completely re absorption of glucose by the tubule when blood glucose level is below 180 mg/dl (renal threshold), in normal conditions blood glucose level does not rise above its threshold level due to present of a certain hormones (Godkar, 2008).

Controlling of blood glucose is under the action of two hormones insulin and glucagon, both hormone are produced by pancreas; the action of them are appose each other. Other hormones and neuroendocrine substances also exert some control over of blood glucose concentration permitting the body to respond to increase demand the conservation of energy as lipid when excess substrates are ingested (Bishop, 2005).

Insulin is the primary hormone responsible for entry of glucose into the cells, it synthesized by β-cells of Islet of Langerhans in the pancreas, when these cells detect increase in body glucose they release insulin, which causes increase movement of glucose into the cell, and increase glucose metabolism.  Normally it secreted when glucose level is high so it decreases plasma glucose level by transport glucose into muscles, adipose tissue, enhances glycogenesis, lipogensis, glycolsis, and inhibits glycogenlysis. Insulin is the only hormone that decreases glucose level so it can refer to as a hypoglycemic agent (Bishop, 2005).

Glucagon is the primary hormone responsible for increasing glucose level, it synthesized by α-cells of pancreas and releases during fasting, stress; when the cells detect decrease glucose level. Glucagon acts to increase plasma glucose level by enhancing glycogenlysis in the liver, enhances gluconeogenesis so it can refer to as a hyperglycemic agent. Other hormones produced by adrenal gland affect carbohydrate metabolism are Epinephrine and Cortisol hormones. Epinephrine produced in stress condition to increase plasma glucose level by enhancing glycogenlysis and promoting lipolysis. Cortisol also acts to increase glucose level by decreasing intestinal entry into the cells and increasing gluconeogensis and lipolysis (Bishop, 2005).

1.2.2.6 Blood glucose concentration:

It is the amount of glucose present in the blood of a human or animal. The body naturally tightly regulates blood glucose levels as a part of metabolic homeostasis. Glucose is a primary source of energy for the body’s cells, and blood lipids, which are primarily a compact energy store. (There are exceptions. For example, because their dietary metabolizable carbohydrates tend to be used by rumen organisms (Van Soest,1994), ruminants tend to be continuously gluconeogenic (Young,1977), consequently their hepatocytes must rely on such primary energy sources as volatile fatty acids, absorbed from the rumen, rather than glucose). Glucose transported from the intestines or liver to body cells via the bloodstream, and made available for cell absorption via the hormone insulin, produced by the body primarily in the pancreas. The mean normal blood glucose level in humans is about 5.5 mill molar (mmol/L) or 100 milligrams per deciliter (mg/dl); however, this level fluctuates throughout the day. Glucose level is usually lowest in the morning, before the first meal of the day, and rises after meals for an hour or two by a few mmol/L. Normal blood glucose level (tested while fasting) for non-diabetics, should be between 70 and 100 mg/dl.        Blood glucose levels for those individuals without diabetes and who are non-fasting should be below

125 mg/dl (Glucose test – blood. NIH – National Institutes of Health). The blood glucose target range for diabetics, according to the American Diabetes Association, should be 90–130 (mg/dl) before meals and less than 180 mg/dl after meals (as measured by a blood glucose monitor) (Davidson and Moreland, 2011). Blood glucose levels outside the normal range may be an indicator of a medical condition. A persistently high level referred as hyperglycemia; low levels referred as hypoglycemia (Walker and Rodgers, 2006). Diabetes mellitus characterized by persistent hyperglycemia from any of several causes, and is the most prominent disease related to failure of blood glucose regulation. Intake of alcohol causes an initial surge in blood glucose, and later tends to cause levels to fall. In addition, certain drugs can increase or decrease glucose levels (Walker and Rodgers, 2006).

FASTING BLOOD SUGAR ANALYSIS AND BLOOD SUGAR REGULATION

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