Carbohydrates: Complete Guide (PDF)

Chapter 5 CARBOHYDRATES By Alazar Tamirat (BSc, MSc in Clinical Chemistry) 1  Outline Chemistry of carbohydrates Role of Laboratory in DM Digestion and absorption of Glycated protein: CHO...

Chapter 5 CARBOHYDRATES By Alazar Tamirat (BSc, MSc in Clinical Chemistry) 1  Outline Chemistry of carbohydrates Role of Laboratory in DM Digestion and absorption of Glycated protein: CHO Glycosylated Hemoglobin, Metabolism of CHO Fructosamine, AGE Regulation of blood glucose Ketone bodies concentration Microalbuminuria Carbohydrate metabolism Islet Autoantibody Testing disorders Insulin Testing Diabetes mellitus (DM) Complication of DM 2 Chemistry of carbohydrates 3  Carbohydrates Carbohydrates are compounds containing C, H, and O. The general formula for a carbohydrate is Cx(H2O)y. All carbohydrates contain C=O and -OH functional groups. Some carbohydrate can be formed by the addition of other chemical groups, such as phosphates, sulfates, and amines. Are polyhydroxy aldehydes or ketones, or substances that yield such compounds upon hydrolysis. 4  Function To provide energy through their oxidation – primary source of energy for brain, erythrocytes, and retinal cells in humans To serve as a stored form of chemical energy Precursor of organic compounds, ribose (RNA), deoxyribose (DNA) Participate in structure of cell membrane: glycoprotein, glycolipid Serve as structural component; cellulose in plants, chitin in insects Play role in cellular intercommunication, cell signaling and immunity Involved in detoxification; glucuronic acid And lubricate skeletal joints Non digestible carbohydrates cellulose, agar, gum and pectin serves as dietary fibers 5  Classification of Carbohydrates based on different properties: – the size of the base carbon chain – the location of the CO function group, – the number of sugar units, – the stereochemistry of the compound 6  Carbohydrates with an aldehyde as their most oxidized functional group are called aldoses Carbohydrates with a ketone as their most oxidized functional group are called ketoses 7  Classification number of sugar units Monosaccharides (simple sugars) Disaccharides Oligosaccharides Polysaccharides 8  Monosaccharides Simple sugars & cannot be hydrolysed further. Frequently named using the suffix –ose Possess the molecular formula (CH2O)n , where n = the number of carbon atoms ≥3. Linked by glycosidic bonds to create larger structures Termed aldose or ketose, according to the position of the carbonyl group 9 Cont….. vary in the stereochemical configuration at one or more carbon centers Important fuel molecules and building blocks for NAs Colorless, crystalline solids, freely soluble in water but insoluble in nonpolar solvents Most have a sweet taste Glycosidic bonds formed b/n the OH group on the anomeric carbon of a monosaccharide with an –OH or an –NH group of another compound may be  or  N-glycosidic bonds are found in NAs and nucleotides O-glycosidic bonds, join sugars to each other or attach sugars to the hydroxyl group of an amino acid on a protein  Disaccharides Two monosaccharides connected by a glycosidic bond. The bond may be α or β as in cyclic monosaccharides. The structures include glycosidic bonds that create α 1,4 link between C1 of one monosaccharide and C4 of the second monosaccharide o Maltose =glucose + glucose o Lactose= galactose+ glucose o Sucrose= glucose + fructose) 10 Disacc… 11 Sucrose – common table sugar – obtained from cane or beet – formed by plants – α glucose with β fructose (α,1,2) – cleaved by sucrase 14 Lactose – disaccharide of milk – consists of galactose and glucose (β-1,4) – hydrolyzed by lactase in humans and by β- galactosidase in bacteria 15 Maltose – contains two D-glucose residues (α -1,4) – comes from the hydrolysis of starch – hydrolyzed by maltase 16 Figure 11.11. Common Disaccharides. Sucrose, lactose, and maltose are commo Isomaltose – a disaccharide of glucose (α-1,6) – formed during the hydrolysis of starch Trehalose – a disaccharide of glucose (α-1,1) – a major constituent of hemolymph of insects, serving as an energy-storage compound – found in algae, mushrooms, and other fungi 17  Oligosaccharides Contain from three to about ten monosaccharide units, E.g. Maltotriose. (Glucose + Glucose + Glucose). 12  Cont……. Most do not occur as free entities, often found attached through N- or O-glycosidic bonds to non-sugar molecules (lipids or proteins) in glycoconjugates 19  Polysaccharides Contain greater than ten monosaccharide units. Can be hundreds of sugar units in length. Important polysaccharides include: – branched glycogen (from animal sources) and starch (plant sources) – Unbranched cellulose (plant sources) Can function as compact energy storage units in starch and glycogen or As strong, protective fibers in cellulose and chitin. 13  Polysaccharides Form linear chains or branched structures Most carbohydrates found in nature occur as polysaccharides which differ from each other in  component monosaccharide units  length of their chains  types of bonds linking the units  degree of branching 21 Glycogen  a storage polysaccharide in animal cells Abundant in the liver (7% of the wet weight) and in skeletal muscle Dextrans Bacterial and yeast polysaccharides made up of glucose Dental plaque is rich in dextrans Synthetic dextrans are used in several commercial products e.g Sephadex Cellulose linear, unbranched homopolysaccharide found in plant cell walls, particularly in stalks, stems, trunks and all the woody portions cannot be used as a fuel paper, cardboard, rayon, insulating tiles, etc are derived from cellulose 22 Chitin – Serves a structural function – the second most abundant polysaccharide – principal component of the hard exoskeletons of several species of arthropods; insects, lobsters, and crabs – also found in mushrooms – cannot be digested by vertebrates Inulin a polysaccharide of fructose moiety found in tubers and roots of certain plants it’s inert, but readily soluble in water used to determine GFR 23  Agar – a mixture of sulfated heteropolysaccharides made up of D- galactose and L-galactose. – a component of cell walls of certain marine red algae, including some of the sea-weeds – composed of two major components; the unbranched polymer agarose and a branched, agaropectin Peptidoglycan component of bacterial cell walls, preventing bacterial cell swelling and lysis lysozyme targets the -1,4 glycosidic bond Penicillin and related antibiotics prevents synthesis of the cross-links 24 Peptidoglycan network structure 25 Glycoconjugates – biologically active molecules of carbohydrates covalently joined to a protein or a lipid – include Proteoglycans Glycoproteins glycolipids 26  Others……. Glycosaminoglycans (GAGs) Hyaluronic acid Dermatan sulfate Chondroitin sulfate Keratan sulfates Heparin 27  Isomerism Isomers are compounds having the same chemical formula but different structures. 14 Structural Isomers Vs Stereoisomers 15  Structural Isomers Structural Isomers: compounds that have identical molecular formulas but differing in the order in which the individual atoms are connected 16  Stereoisomerism The isomers have the same molecular and structural formula and connectivity but differ in the spatial arrangement of the atoms in the molecule Fructose, glucose, mannose, and galactose are all isomers of each other, having the same chemical formula, C6H12O6 Enantiomers: non super impossible mirror images of one another Diastereomers: All compounds that are not mirror images of one another; eg Cis/trans isomers, conformational isomers 17  Enantiomers A special type of isomerism is found in the pairs of structures that are mirror images of each other. D- and L-sugars are referred to as enantiomers. Their structures are mirror images of each other. Only D-glucose or D- sugars are utilized by humans 18  Epimerism Carbohydrate isomers that differ in configuration around only one specific carbon atom are defined as epimers of each other. Glucose and galactose are C-4 epimers—their structures differ only in the position of the –OH group at carbon 4. Glucose and mannose are C-2 epimers. 19  Cyclization of monosaccharide 6 CH 2OH 6 CH 2OH 5 O 5 O H H H OH H H 4 H 1 4 H 1 OH OH OH OH OH H 3 2 3 2 H OH H OH a-D-glucose b-D-glucose Cyclization of glucose produces a new asymmetric center at C1. The 2 stereoisomers are called anomers, a & b. Haworth projections represent the cyclic sugars as having essentially planar rings, with the OH at the anomeric C1: a (OH below the ring) b (OH above the ring).  Reducing sugars If the hydroxyl group on the anomeric carbon of a cyclized sugar is not linked to another compound by a glycosidic bond, the ring can open. The sugar can act as a reducing agent, and is termed a reducing sugar. Such sugars can react with chromogenic agents (e.g. Benedict’s reagent or Fehling’s solution) causing the reagent to be reduced and colored, with the aldehyde group of the acyclic sugar becoming oxidized. 21  Complex carbohydrates Carbohydrates can be attached by glycosidic bonds to non-carbohydrate structures, including: purine and pyrimidine base (found in nucleic acids), aromatic rings (such as those found in steroids and bilirubin) proteins (found in glycoproteins and proteoglycans Lipid (found in glycolipid) 22  Digestion of carbohydrates The dietary carbohydrates can be divided into three groups: – A. Ready-to-absorb carbohydrates Do not require digestion and are absorbed as such, e.g., monosaccharides: glucose, mannose, galactose, fructose and pentoses. – Digestible carbohydrates: completely digested into their respective monosaccharides. These include starch, glycogen, maltose, sucrose, and lactose (oligosaccharides and polysaccharides). – Non-digestible carbohydrates: The indigestibility of these dietary fibers is primarily due to the absence of specific digestive enzymes. Most of these indigestible carbohydrates are plant polysaccharides like cellulose, pentosans, hemicellulose, lignin, gums and pectins. Carbohydrates Digestion 24  Digestion in mouth Saliva contains a carbohydrate splitting enzyme called salivary amylase (ptyalin). – requires Cl- ion for Salivary amylase : a 1-4 endoglycosidase, activation and optimum pH 6-7 G G G G G G G G a Limit dextrins G G G G G G G G α-amylase G G G G G G G a 1-6 link G G G maltotriose a 1-4 link G G G G G G maltose G G isomaltose  Stomach Ptyalin action stops in stomach when pH falls to 3.0 No carbohydrate splitting enzymes are available in gastric juice Not much carbohydrate digestion Acid and pepsin to unfold proteins HCl may hydrolyze some dietary sucrose to equal amounts of glucose and fructose. Digestion of dietary disaccharides in small intestine Dietary disaccharides include trehalose, lactose, and sucrose Dietary disaccharides do not require α-amylase digestion Intestinal brush border enzymes initiate digestion of disaccharides to monosaccharides trehalase catalyzes trehalose → glucose + glucose lactase catalyzes lactose → glucose + galactose sucrase catalyzes sucrose → glucose + fructose 41  Digestion in small intestine Food reaches the duodenum from stomach where it meets the pancreatic juice. Pancreatic juice contains a carbohydrate-splitting enzyme – Pancreatic amylase: hydrolyzes terminal α-(1,4), glycosidic linkage in polysaccharides and Oligosaccharide molecules liberating free glucose – Lactase – Maltase – Sucrase – a dextrinase, peptidase, 27  Absorption of Carbohydrates Products of digestion of dietary carbohydrates are practically completely absorbed almost entirely from the small intestine. Some disaccharides, which escape digestion, may enter the cells of the intestinal lumen by “pinocytosis” and are hydrolyzed within these cells. No carbohydrates higher than the monosaccharides can be absorbed directly in to the blood stream. Mechanism of Absorption – Simple Diffusion: depend on sugar concentration gradients between the intestinal lumen – “Active “Transport Mechanisms 28  Carbohydrate absorption Monosaccharides are too large for passive diffusion across brush border membrane. It use facilitated diffusion to absorb these molecules. Glucose and galactose use a sodium-glucose symport (SGLUT1) Fructose uses the Glut5 apical basolateral Active and Passive Transport 45  Glucose transporter The transport of glucose into cells is modulated by two families of proteins: – Intestinal Sodium-dependent glucose co-transporter 1 (SGLT-1) Energy-requiring process that requires a concurrent uptake of sodium ions Promotes the uptake of glucose and galactose from the lumen of the small bowel and their reabsorption from the urine in the kidney – Facilitative glucose transporters (GLUTS ) Located on the surface of all cells and designated GLUT1- GLUT12. 30  Transport of glucose involves: –Absorption through intestine SGLT at apical membrane and GLUT 2 at basolateral membrane –Uptake into pancreatic beta cells: GLUT-2 –Uptake into liver: GLUT-2 –Uptake into other cells (muscle, adipose tissues) GLUT 4 is commonly used 31  Transport of glucose involves: GLUT1 erythrocyte; GLUT2 hepatocyte; GLUT3 brain; GLUT4 muscle and fat; GLUT5 small intestine 48 32 Carbohydrate Metabolism 33 Glycolysis 51 Carbohydrate metabolism 34 35  Alternative Catabolic Pathways Triglycerides Glycogen Proteins Fatty Acids Glycerol Glucose Amino Acids Pyruvic cytoplasm acid Acetyl-CoA mitochondrion Citric Acid Electron Transport Cycle ATP Production 36  Regulation of blood glucose concentration Is regulated by a complex interplay of multiple pathways, modulated by several hormones The liver, pancreas, and other endocrine glands Control of blood glucose is under two major hormones: insulin and glucagon Insulin is the primary hormone responsible for the entry of glucose into the cell. Glucagon is the primary hormone responsible for increasing glucose levels Other hormones increases plasma glucose: Epinephrine, Glucocorticoids (cortisol), growth hormone, ACTH 37 Cont… Insulin and glucagons are both produced in the pancreas, insulin in the beta cells and glucagons in alpha cells of the islets of Langerhans. 56 38  Carbohydrate metabolism disorders Derangement in glucose metabolism –Diabetes Mellitus. Inborn deficiency of carbohydrate metabolism – due to deficiency or absence of an enzyme that participates in carbohydrate metabolism Glycogen storage disorders – the result of defects in the processing of glycogen synthesis or breakdown within muscles, liver, and other cell types Galactosemia – Is a rare genetic metabolic disorder that affects an individual's ability to metabolize the sugar galactose properly. 39  Hyperglycemia Is an increase in plasma glucose levels. It is caused by an imbalance of hormones. In healthy individuals, during a hyperglycemia state, insulin is secreted by the β cells of the pancreatic islets of Langerhans. – it enhances membrane permeability to cells in the liver, muscle, and adipose tissue. – It also alters the glucose metabolic pathways. 40  Hypoglycemia Is a blood glucose concentration below the fasting value, the most widely used cutoff is 50 mg/dl It occurs frequently in both type 1 and type 2 diabetes, more frequent in type 1. Its symptoms vary among individuals, and none is specific – increased hunger, sweating, nausea and vomiting, dizziness, nervousness and shaking, blurring of speech and sight, and mental confusion. Laboratory findings – decreased plasma glucose levels and extremely elevated insulin levels in patients with pancreatic B-cell tumors (insulinoma). 41 42  Diabetes mellitus (DM) It refers to a group of common metabolic disorders that share the phenotype of hyperglycemia. factors contributing to hyperglycemia include reduced insulin secretion , decreased glucose utilization, and increased glucose production. As the disease progresses, individuals are at increased risk for the development of specific complications, including: – Retinopathy (which may lead to blindness), renal failure, neuropathy (nerve damage), and atherosclerosis. The last condition may result in stroke, gangrene, or coronary artery disease. The metabolic dysregulation associated with DM causes secondary pathophysiologic changes in multiple organ systems 43 Classification 44  Symptoms of diabetes, such as: - polyuria - excessive secretion and discharge of urine polyphagia - excessive hunger polydipsia - excessive thirst 45  Type 1 Diabetes Mellitus Known as IDDM, type I, or juvenile-onset diabetes Characterized by lack of insulin production and secretion beta cells of the pancreas (absolute deficit of insulin) Account 5% to 10% of all individuals with diabetes mellitus Individuals depend on insulin treatment Peak incidence in childhood and adolescence. 75% acquire the disease before 30 years of age More prone to ketoacidosis 46  Type 1 Diabetes Mellitus Cause of the hyperglycemia of type 1 diabetes mellitus Autoimmune destruction of the beta cells of the pancreas Autoantibodies are present in the circulation of many individuals with type 1 diabetes(about 90 %) Idiopathic (type 1B ): Less than 10% have no evidence of pancreatic B cell autoimmunity Genetic: Susceptibility to type 1 diabetes is inherited, but the mode of inheritance is complex and has not been completely defined Environmental factors(triggering factors) viruses: rubella, mumps, and coxsackie virus B 47  Type 2 Diabetes Mellitus Known as NIDDM, Adult onset diabetes Characterized by decline in insulin action due to the resistance of tissue cells to the action of insulin. The problem is intensified by the inability of the beta cells of the pancreas to produce enough insulin to counteract the resistance – Type 2 diabetes is a disorder of both insulin resistance and relative deficiency of insulin. Constitutes about 90% of all cases of diabetes. Patients have minimal symptoms, rarely Ketoacidosis and may not dependent on insulin. Occurs predominantly in adults, but it is now encountered in children and adolescents 48  Etiology of type 2 diabetes Insulin resistance – It is a decreased biological response to normal concentrations of circulating insulin (defect in insulin action) Loss of β-Cell Function – Inability of the pancreas to produce sufficient insulin to compensate for the insulin resistance. Genetic factors: 30 mmol/L) without significant hyperketonaemia (7.3, bicarbonate >15 mmol/L plus Osmolality >320 mosmol/kg HHS and DKA can have marked effects on cerebral function and be associated with transient changes in mental performance and also with longer term effects 57 DKA Vs HHS 58  Chronic complications of DM It affects many organ systems and are responsible for the majority of morbidity and mortality associated with the disease The risk of complications increases with the duration of hyperglycemia Usually become apparent in the second decade of hyperglycemia. In most T2DM patients, chronic complication is identified at the time of diagnosis due to a long asymptomatic period of hyperglycemia 59  Chronic complications of DM cont’d The vascular complications Non-vascular complications – Micro-vascular: – Gastro paresis, Retinopathy, – Infections Neuropathy, – Skin change Nephropathy – Macro-vascular: coronary artery disease (CAD), peripheral arterial disease (PAD), Cerebro-vascular disease. 60 61  Advanced glycation end products (AGE) None enzymatic attachment of glucose to long lived molecules, such as tissue collagen, produces stable Amadori early-glycated products. These undergo a series of additional rearrangements, dehydration, and fragmentation reactions, resulting in stable advanced glycation end products (AGE). The amounts of these products do not return to normal when hyperglycemia is corrected, and they accumulate continuously over the lifespan of the protein. Hyperglycemia accelerates the formation of protein-bound AGE, and patients with DM have increased AGE in their body tissues. AGE may contribute to the microvascular and macrovascular complications of diabetes mellitus. 62 Advanced glycation end products (AGE) cont’d 63  Role of Laboratory in differential diagnosis and management of patients with glucose metabolic alterations The demonstration of hyperglycemia or hypoglycemia under specific conditions is used to diagnose diabetes mellitus and hypoglycemic conditions. Other laboratory tests have been developed: – to identify insulinomas and – to monitor glycemic control – To monitor development of renal complications. 64 65  Criteria for the Diagnosis of DM 1. Symptoms of diabetes plus RBG concentration ≥ 200mg/dl (11.1 mmol/L) OR 2. FBS≥ 126mg/dl(7.0 mmol/L) OR 3. Two-hour plasma glucose ≥200mg/dl (11.1 mmol/L) during an oral glucose tolerance test. – The test should be performed using a glucose load containing th equivalent of 75 g anhydrous glucose dissolved in water; not recommended for routine clinical use. Random is defined as without regard to time since the last meal. Fasting is defined as no caloric intake for at least 8 h. The classic symptoms of diabetes include polyuria, polydipsia, polyphagia, and unexplained weight loss The 2-hour OGTT is not recommended for routine clinical use 66 67 68  Specimen for glucose measurements Specimen: whole blood, plasma, serum, CSF, pleural fluid, and urine for a variety of diagnostic and management purposes – The standard clinical specimen is venous plasma glucose. Glucose is metabolized at room temperature at a rate of 7 mg/dL/hour – The rate of metabolism is higher with bacterial contamination or leukocytosis. A serum specimen is appropriate for glucose analysis if serum is separated from the cells within 30 minutes, if serum is in contact with cells for > 30 min, a preservative such as sodium fluoride that inhibits glycolysis should be added. With long-term specimen storage, even at −20° C, glucose values decrease significantly and progressively 69 70  Hexokinase Method Principle Hexokinase catalyzes the phosphorylation of glucose from ATP which yields G6P – (glucose-6-phosphate) The second enzyme G6P-dehydrogenase oxidizes G6P and reduces NAD+ to NADH NADH causes an increase in absorbance at 340 nm, proportionate to the glucose conc 71  Hexokinase Method Advantages of hexokinase method: – Hexokinase considered reference method because – More accurate, less susceptible to interference, as enzyme G6PD is very specific to G6P Limitations of hexokinase method: – Interference: hemolyzed serum or plasma (>0.5g/dL), lipemia (>500 mg/dL trig), bilirubin, some drugs – Requires near UV wavelength (340 nm) 72  Glucose oxidase method Principle Glucose oxidase catalyzes oxidation of glucose to gluconic acid and H2O2 Peroxidase catalyzes the reduction of H2O2 to water and a reduced chromogenic dye (colorless) is oxidized – Dyes used in various systems: o – dianisidine, 4- aminoantipyrine, or tetramethylbenzidine Production of oxidized colored dye is proportionate to H2O2 produced Increased colored dye → increased absorbance Intensity of the color is proportional to the amount of glucose – Absorbance is read at 546 nm 73  Limitations of glucose oxidase method: Glucose oxidase very specific to ß-D-glucose – Mutarotase must be included or incubation increased to detect α-D-glucose subject to positive and negative interference – Increased levels of uric acid, bilirubin, and ascorbic acid can cause falsely decreased values B/C these substances being oxidized by peroxidase, which then prevents the oxidation and detection of the chromogen. – Strong oxidizing substances, such as bleach, can cause falsely increased values. 74  Glucose Dehydrogenase method Principle: GDH Glucose + NAD+ D-glucono-δ-lactone +NADH + H+ The increase in absorbance at 340 nm is measured. The enzyme glucose dehydrogenase [GDH] catalyzes the oxidation of glucose to gluconolactone. The amount of NADH generated is proportional to the glucose concentration. 75  Reference Ranges Serum or plasma (fasting) =74–100 mg/dL CSF= 60% of serum or plasma level(44.4-60 mg/dl) Fasting Plasma Glucose(FPG) Concentrations – FPG concentrations exceeding 126 mg/dL (7 mmol/L) on more than one occasion are diagnostic of diabetes mellitus – The diagnosis of most cases of diabetes mellitus is established with this criterion 76  Oral Glucose Tolerance Test(OGTT) Is a serial measurement of plasma glucose before and after a specific amount of glucose given orally But it is affected by a large number of factors that result in poor reproducibility The sensitivity of FPG is lower than the sensitivity of the OGTT for diagnosing diabetes Plasma glucose should be measured fasting, then every 30 minutes for 2 hours after an oral glucose load. Although more sensitive than FPG determinations, GTT is affected by a large number of factors that result in poor reproducibility. 20% of OGTTs fall into the non-diagnostic category 77 Self-monitoring (Glucometers) Point-of-Care Testing(POCT) At- or near-patient monitoring by POCT with home meters provides information so that therapeutic glucose intervention may be initiated immediately The Reaction Principle: Glucometers use the same chemical reactions that are used in glucose analysis in the laboratory: glucose oxidase, hexokinase, and dehydrogenase. Most systems use dehydrated reagents embedded in pads on plastic strips. The strip is inserted in the meter, where the reaction is measured. The reaction may be a color change 78  Self-monitoring cont’d The Specimen: Capillary or anti-coagulated whole blood is measured. The sample is placed directly on the test pad. Reportable range varies widely from instrument to instrument. Readings beyond the reportable range of the instrument should be measured on different equipment Reference range: Whole blood glucose is approximately 10% to 15% lower than serum or plasma glucose levels. 79  Glycated protein Glucose and protein initially form a labile glycosylamine or Schiff Base, which undergoes an irreversible Amadori rearrangement to produce a more stable ketoamine 80  Glycosylated Hemoglobin Measurement of glycated proteins, primarily GHb, is effective in monitoring long-term glucose control in people with DM. It provides a retrospective index of the integrated plasma glucose values over an extended period of time Is not subject to the wide fluctuations observed when assaying blood glucose concentrations. GHb concentrations are a valuable and widely used adjunct to blood glucose determinations for monitoring of long-term glycemic control. GHb is a measure of the risk for the development of complications of diabetes. 81  Glycosylated Hemoglobin cont ’d Formation of GHb is essentially irreversible. The concentration of GHb in the blood depends on : – the lifespan of the red blood cell (average 120 days) – the blood glucose concentration. The rate of formation is directly proportional to the concentration of glucose in the blood Helps to assess glucose control because GHb values are: – free of day to-day glucose fluctuations – unaffected by recent exercise or food ingestion 82  Glycosylated Hemoglobin cont’d Substantial reduction in GHb is due to: – Hemolytic disease or – Other conditions that shorten red blood cell – Significant blood loss owing to a higher fraction of young erythrocytes; GHb will then increase as the cells age, allowing time for the hemoglobin to have glucose attached to form GHb. The absolute risks of retinopathy and nephropathy were directly proportional to the mean Hb Alc. 83  Glycosylated Hemoglobin cont’d Human adult hemoglobin (HbA) usually consists of: – HbA1 (97% of the total) Chromatographic analysis of HbA1 identifies several minor Hbs , namely HbAla, HbAlb, and HbA1c HbA1 is fast hemoglobins HbA1c is the major fraction, constituting approximately 80% of HbAl – HbA2 (2.5%), – HbF (0.5%). 84  Glycosylated Hemoglobin cont’d Factors that influence HBA1c and its measurement – Chronic liver disease – Genetic or chemical alterations in haemoglobin E.g. haemoglobinopathies – Alcoholism, chronic renal failure – Erythrocyte destruction 85  The Specimen The preferred specimen is whole blood collected in EDTA, heparin ,or fluoride anticoagulant. Capillary blood may be used for some procedures such as immunoassay. A hemolysate of washed RBCs is tested. Interference – Falsely increased glycated Hb (HbA1c) may be reported for blood with increased HbA1a levels. – Falsely decreased HbA1c may be reported in individuals with disorders in which RBC survival is decreased 86  Test methodology for Hb A1c Glycated hemoglobin may be separated and identified on the basis of charge and structure differences. Methods of analysis include: – Ion-exchange or affinity chromatography, – Electrophoresis – Iso-electric focusing, and – Immunoassay Reference range for HbA1c – HbA1c is reported as a percentage of the total hemoglobin: – HbA1c = 4.0%–6.0% 87 88  Fructo-samine It is the generic name for plasma protein Ketoamines. Structure of the ketoamine rearrangement product formed by the interaction of glucose with lysine residues of albumin. Used as an index of the average concentration of blood glucose over an extended (but shorter) period of time Non-ezymatic attachment of glucose to amino groups of proteins other than hemoglobin (e.g., serum proteins, membrane proteins, and lens crystallins) to form ketoamines also occurs In selected patients with diabetes mellitus (e.g., GDM or change in therapy), there may be a need for assays that are more sensitive than GHb to shorter-term alterations in average blood glucose levels. 89  Fructosamine cont’d Serum proteins turn over more rapidly than erythrocytes (the circulating half-life for albumin is about 20 days), – the concentration of glycated serum albumin reflects glucose control over a period of 2 to 3 weeks. All glycated serum proteins are fructosamine Albumin is the most abundant serum protein, Measurement of fiuctosamine is thought to be largely a measure of glycated albumin 90  Fructosamine cont’d Purpose: fructosamine test is ordered in patient with: – Diabetes – Hemoglobinopathy – Sickle cell disease – Anemia – Recent loss of blood – Conditions affecting RBC count/lifespan 91  Ketone Ketone bodies are produced by the liver through metabolism of fatty acids to provide a ready energy source from stored lipids at times of low carbohydrate availability. The three ketone bodies are acetone (2%), acetoacetic acid (20%), and 3-B-hydroxybutyric acid (78%). A low level of ketone bodies are present in the body at all times. To meet energy needs, Ketone blood levels increase in cases of: – Carbohydrate deprivation or – Decreased carbohydrate use such as DM, – starvation/fasting, – high-fat diets, – prolonged vomiting, 92 – glycogen storage disease,  Ketone cont’d Measurement of ketones is recommended for patients with type 1 diabetes during: – acute illness, stress, pregnancy, or – elevated blood glucose levels above 300 mg/dL or – when the patient has signs of ketoacidosis. The specimen: fresh serum or urine; tightly stoppered and analyzed immediately. No method reacts with all three ketone bodies. 93  Reference ranges Sodium nitroprusside method for acetoacetic acid and acetone: Serum and =Negative or 10 mg/dl urine Beta-hydroxybutyrate dehydrogenase method for beta- hydroxybutrate: Serum = 0.02–0.27 mmol/L 94  Microalbuminuria DM causes progressive changes to the kidneys and results in diabetic renal nephropathy over years An early sign that nephropathy is occurring is an increase in urinary albumin. Microalbumin measurements are useful to assist in diagnosis at an early stage and before the development of proteinuria. An annual assessment of kidney function by the determination of urinary albumin excretion is recommended for diabetic patients. Microalbuminuria: persistent albuminuria in the range of 30 to 299 mg/24 h or an albumin-creatinine ratio of 30 to 300 g/mg. Clinical proteinuria or macroalbuminuria: albumin-creatinine ratio of >=300 mg/24 h or an albumin-creatinine ratio of >=300 g/mg. 95  Microalbuminuria cont’d Methods for microalbuminuria screening – Spot method : the use of a random spot collection for the measurement of the albumin-creatinine ratio is the preferred method. – 24-hour collection or a timed 4-hour overnight collection more burdensome to the patient and add little to prediction or accuracy, A patient is determined to have microalbuminuria when two of three specimens collected within a 3- to 6-month period are abnormal. Factors that may elevate the urinary excretion of albumin: – exercise within 24 hours, infection, fever, congestive heart failure, marked hyperglycemia, and marked hypertension. 96  Micro-albuminuria The specimen A timed urine collection is recommended : – 24-hour, – 8 to 12-hour, or – 1- to 2-hour timed specimens are acceptable. Test methodology Quantitative tests for urine albumin use: – Nephelometry: Nephelometry measures the complexes that are formed with antibodies to albumin – Immunoassay methodology: Immunoassay measures the radioactive or enzyme labels that change when albumin binds an antibody 97  Islet Autoantibody Testing The presence of autoantibodies to the B islet cells of the pancreas is characteristic of type 1 diabetes. – IAA(insulin autoantibody) – GAD antibody (glutamic acid decarboxylase antibodies) – IA2: Protein tyrosine phosphatase antibodies However, islet autoantibody testing is not currently recommended for routine screening for diabetes diagnosis. In the future, this testing might identify at-risk, prediabetic patients. 98  Insulin Testing The results of blood insulin tests would be the means for distinguishing between T1DM and T2 DM. However, the difficulty in measuring insulin accurately diminishes its usefulness for DM diagnosis. – Large differences in results have been noted between laboratories using the same analytical methods to measure insulin. – In addition, the specimen that is tested may contain exogenous insulin that the patient is receiving as insulin treatment Insulin measurements are not required for the diagnosis of diabetes mellitus But in certain hypoglycemic states, it is important to know the conc. of insulin in relation to the plasma glucose concentration. 99  C-peptide concentration Insulin is stored in the pancreas as the biologically inactive protein pro- insulin Pro-insulin is cleaved into the active hormone, insulin, and an inactive peptide , C-peptide C-peptide concentration reflects the production of endogenous insulin by the pancreas. As lack of analysis standardization, the ADA does not recommend the use of either insulin or C-peptide measurement for diagnosis of type 1 diabetes. 100 Any question?? 101