10/21/2015 Dr Atef Masad Carbohydrates 1 Clinical Chemistry Chapter 3 Carbohydrates Dr Atef Masad PhD Biomedicine UK

04/20/23 Dr Atef Masad Carbohydrates 1

Clinical Chemistry

Chapter 3Carbohydrates

Dr Atef MasadPhD Biomedicine

UK

Carbohydrates

•Living systems depend on the oxidation of complex organic compounds to obtain energy.•Three types of organic compounds are used as a source of energy, Carbohydrates, Amino Acids, and Lipids•Carbohydrates are the primary source of energy for brain, erythrocytes, and retinal cells.•Carbohydrates are stored in liver and muscle as glycogen.

• Polysaccharides are formed by the linkage of many monosaccharide units.

• On hydrolysis, Polysaccharides yield more than ten monosaccharides.

• The most common Polysaccharides are starch and glycogen.


Glucose Metabolism•Glucose is the primary source of energy for humans.•The nervous system, including the brain, totally dependant on glucose from the surrounding extracellular fluid "ECF" for energy.•Nervous tissue cannot concentrate carbohydrates nor store it. •Therefore, it is critical to maintain a steady supply of glucose to the tissues. •When the concentration of glucose falls below a certain level, the nervous tissues lose their primary energy source and are incapable of maintaining normal function.


• Major energy pathways involved either directly or indirectly with glucose metabolism

1- Glycolysis– Breakdown of glucose for energy production

2- Glycogenesis

– Excess glucose is converted and stored as glycogen– High concentrations of glycogen in liver and skeletal

muscle

3- Glycogenolysis– Breakdown of glycogen into glucose– Glycogenolysis occurs when plasma glucose is decreased


4. Gluconeogenesis– Conversion of non-carbohydrate carbon substrates to

glucose, takes place, mainly in the liver

5. Lipogenesis– Conversion of carbohydrates into fatty acids– Fat is another energy storage form, but not as quickly

accessible as glycogen

6. Lipolysis– Decomposition of fat


Destiny of Glucose

•Salivary and Pancreatic amylase digests dietary starch and glycogen into dextrins and disaccharides.•Maltase, sucrase and lactase hydrolyze them to monosaccarides.

• Maltase is an enzyme released by intestinal mucosa that hydrolze maltose to two glucose units

• Sucrase hydrolyze sucrose to glucose & fructose • Lactase: hydrolyze lactose to glucose & galatose.

•Monosaccarides are absorbed by the gut and transported to the liver by the hepatic portal venous blood supply.•All sugars must be converted into glucose which enters the cell.


•The ultimate goal of the cell is to convert glucose into carbon dioxide and water.•Glucose is converted into G-6-P using ATP and hexokinase.•G-6-P can enter glycolysis or HMS (hexose monophosphate shunt) pathways or can be converted into glycogen. Glycolysis and HMS generate energy.


Scheme of glycogen synthesis and degradation


•Regulation of Carbohydrate Metabolism•The liver, Pancreas, and other endocrine glands are all involved in the controlling of blood glucose concentrations.•During a brief fast, glucose is supplied to the ECF from the liver through glycogenolysis.•When the fasting period is longer than 1 day, glucose is synthesized from other sources through gluconeogenesis.


Glycogenolysis in skeletal muscle and liver. Glycogen stores serve different functions in muscle cells and liver. In the muscle and most other cell types, glycogen stores serve as a fuel source for the generation of ATP. In the liver, glycogen stores serve as asource of blood glucose.


Sources of blood glucose in the fed, fasting, and starved states.


•Insulin and glucagon control blood glucose.•Insulin is released when glucose levels are high and is not released when glucose levels are decreased. •It decreases blood glucose by increasing the transport entry of glucose in muscle and adipose tissue by way of nonspecific receptors. •Insulin is referred to as a hypoglycemic agent.•Glucagon is the primary hormone responsible for increasing glucose levels, it is released during stress and fasting from panc α-cells. •It is a hyperglycemic agent

http://en.wikipedia.org/wiki/Alpha_cell


•Hyperglycemia•Hyperglycemia or increased plasma glucose, is caused by an imbalance of hormones.


•Epinephrine increases plasma glucose •Stimulates conversion of hepatic glycogen to gluocose(glycogenolysis ) and promoting lipolysis.•Glucocorticoids e.g cortisol, are released from adrenal cortex increases blood glucose•Glucocorticoids stimulates formation of glucose from non-carbohydrate sources (gluconeogenesis).•Growth hormone "GH" and ACTH promote increased plasma glucose.•GH increases plasma glucose by decreasing the entry of glucose into the cells and by decreasing glycolysis.


•Decreased levels of cortisol stimulate the anterior pituitary to release ACTH

• ACTH then stimulates the adrenal cortex to release cortisol and increases plasma glucose levels by converting liver glycogen to glucose, and by promoting gluconeogenesis.

•Thyroxine increases blood glucose by increasing glycogenolysis, gluconeogenesis and intestinal absorption of glucose.•Somatostatin which is produced by D cells of the islets of langerhans of the pancreas increases plasma glucose levels by the inhibition of insulin.


Islets of Langerhans and insulin secretion• The islets of Langerhans secrete hormones directly into

the blood flow by (at least) five different types of cells:- • α-cells, producing glucagon (15-20% of total islet cells)• β-cells, producing insulin and amylin (65-80% of total

islet cells) • δ-cells, producing somatostatin (3-10% of total islet

cells) • PP cells, producing pancreatic polypeptide (3-5% of total

islet cells) • ε-cells, producing *Ghrelin (<1% of total islet cells)

http://en.wikipedia.org/wiki/Glucagon


• Insulin acts through chemical responses to receptors on the cells of target tissues.

• In the muscle, insulin stimulates glucose

uptake into cells and enhances glycogenesis.

• In adipose tissue, insulin stimulates glucose uptake into cells and enhances lipogenesis.

• In the liver, insulin has a negative effect, inhibiting gluconeogenesis and glycogenolysis.


Types of diabetes mellitus.


Type 1 diabetes• Is characterized by lack of insulin

production and secretion by the beta cells of the pancreas.

• One cause of the hyperglycemia of type 1

diabetes mellitus is an autoimmune destruction of the beta cells of the pancreas.

• The cell mediated response causes

infiltration of the pancreas and reduction in the volume of beta cells.


• Autoantibodies are present in the circulation of many individuals with type 1 diabetes.

• There appears to be a genetic susceptibility to development of autoantibodies, with certain histocompatibility antigens predominant in the type 1 diabetes population.

• The development of disease is complex; triggering factors, such as rubella, mumps, and other viral infection, and chemical contact may be necessary for progression of disease.


• The most common form of diabetes is type 2 diabetes. About 90 to 95 % of people with diabetes have type 2.

• This form of diabetes is associated with older age, obesity, family history of diabetes, previous history of gestational diabetes, physical inactivity, and

ethnicity. • About 80 % of people with type 2 diabetes are

overweight. Type 2 diabetes is increasingly being diagnosed in children and adolescents.

Type 2 diabetes


• When type T2DM is diagnosed, the pancreas is usually producing enough insulin, but for unknown reasons, the body cannot use the insulin effectively, a condition called insulin resistance.

• After several years, insulin production decreases. The result is the same as for type 1 diabetes - glucose builds up in the blood and the body cannot make efficient use of its main source of fuel.

• T2DM is a major risk factor for cardiovascular disease, from which 60–80% of the patients die at a relatively young age


• Its prevalence and incidence continue to rise in most, if not all countries.

• The global figure of people with diabetes is set to rise from about 118 million in 1995 to 220 million in 2010 and 300 million in 2025.

• Between 1995 and 2010, the global prevalence of diabetes will rise by 55% from 2.1 to 3.2%


Type 2 diabetes• Is characterized by decline in insulin action due

to the resistance of tissue cells to the action of insulin.

• The problem is increased by the inability of the

beta cells of the pancreas to produce enough insulin to counteract the resistance.

• Type 2 D. M. constitutes the majority of the diabetes cases, most patients are obese, it is associated with genetic predisposition

• Type 2 diabetes is a disorder of both insulin resistance and relative deficiency of insulin.


• Insulin resistance syndrome, also known as metabolic syndrome and syndrome X, affects the metabolism of many nutrients, including glucose, triglycerides, and high-density lipoprotein (HDL) cholesterol.

• Individuals who are diagnosed with metabolic syndrome may show abdominal obesity and high blood pressure.

• Such individuals are at increased risk for

cardiovascular disease.


Gestational diabetes

• is similar in etiology to type 2 diabetes; • it is defined as diabetes that is diagnosed in

pregnancy. • Pregnancy is associated with increased tissue cell

resistance to insulin. • The hyperglycemia of gestational diabetes

diminishes after delivery; however, the individual who has developed gestational diabetes is at higher risk for the development of type 2 diabetes thereafter.


•Signs and symptoms include•Polydipsia "excessive thirst".•Polyphagia "increased food intake".•Polyuria "excessive urine production".•Rapid weight loss. •Weight loss despite polyphagia•Blurred vision•Growth impairment•Susceptibility to infections.


•Mental confusion and possible loss of consceiousness due to increased glucose to brain.•Complications include microvascular problems such as nephropathy, neuropathy, retinopaty, increased heart disease.


DIABETES MELLITUS

Complications • K etosis, coma• A theroma swelling in artery walls - CVD, Strokes• N europathy : Peripheral & Autonomic • G angrene: neuropathy, atheroma, infection• I nfection: increased susceptibility• R enal: nephropathy, UTI• O phthalmic: retinopathy, cataracts• O bstetric: recurrent poor obstetric history



•Pathophysiology of D. M•Glucosuria happens when the glucose concentration of plasma exceeds roughly 180 mg/dl in an individual with normal renal function and urine output.•If the hepatic glucose overproduction continues, the plasma glucose concentration reaches a plateau around 300 mg/dl to 500 mg/dl.

•This leads to polyuria (an osmotic diuresis), which, in turn, leads to volume depletion and hemoconcentration that causes a further increase in blood glucose level.


•In type 1 there is an absence of insulin with an excess of glucagon, this permits gluconeogenesis and lipolysis to occur.

•If type 2 insulin is present therefore glucagon is attenuated, fatty acid oxidation is inhibited.•Individuals with type 1 have tendency to produce ketones while type 2 patients have greater tendency to develop hyperosmolar nonketotic states (diabetic coma).•Diabetic patient with ketoacidosis tend to reflect dehydration, electrolyte disturbances and acidosis.


•Acetoacetate, -hydroxybutyrate, acetone are produced from the oxidation of fatty acids.•Serum osmolality is high due to hyperglycemia.

•Sodium concentration tends to be low because of the losses "polyuria" and the shift of water from cells due to hyperglycemia.

Hyperkalemia is present due to the displacement of potassium from cells in acidosis.•The Na-K ATPase ion pump moves 3 Na ions out of the cell in exchange for 2 K ions moving into the cell as ATP is Converted into ADP. buy



Islet amyloid and T2DM

• Amyloid fibrils are insoluble protein deposits which are a common link between many apparently unrelated diseases.

• Associated with considerable damage to tissues.

• Could be due to generation of free radicals and reactive oxygen species (ROS) by aggregating proteins.

• Amyloid deposits derived from a peptide called ‘amylin’ are found in the pancreas in the vast majority of cases of T2DM.

• CRITICAL THINKING..WAT is amyloidoses?


• Loss of β-cell mass contributes to progressive β-cell failure in T2DM.

• Islet amyloid could play an important role in loss of islet cells and decline in insulin secretion.

• Increased production of amylin, associated with increased demand for insulin, results in accumulation and aggregation of amylin.

• hA (but not rA) aggregates to form fibrils that are toxic to islet β-cells in culture.

Islet amyloid and β-cell toxicity


•Criteria for the diagnosis of D. M.•All adults older than age 45 years should have a measurement of fasting blood glucose every 3 years. Testing should be carried out at an earlier age or more frequently in individuals who display:•Obesity•Family history of D. M•History of gestational D. M•Hypertension•Elevated triglycerides•Low HDL


•Diagnostic criteria for DM•Random plasma glucose 200 mg/dL "11.1 mmol/L".

•Plus symptoms of diabetes•Fasting plasma glucose 126 mg/dL "7.0 mmol/L".•2 hours plasma glucose 200 mg/dL "11.1 mmol/L".

•during an oral glucose tolerance test.•N:B any of the three criteria must be confirmed on a subsequent day by any of the three methods.


•Categories of Fasting Plasma Glucose•Normal fasting glucose < 110 mg/dL•Impaired fasting glucose 110 mg/dL and 126 mg/dL•Provisional diabetes diagnosis 126 mg/dL

•Categories of oral glucose Tolerance•Normal glucose tolerance 2 h< 140 mg/dL•Impaired glucose tolerance 2 h 140 mg/dL and 200 mg/dL•Provisional diabetes diagnosis 200 mg/dL


Hypoglycemia

• Hypoglycemia involves decreased plasma glucose levels.•Signs of hypoglycemia are related to the CNS.•Epinephrine act with glucagon to increase plasma glucose. •In addition cortisol and GH are released which increased glucose metabolism.•Hypoglycemia can be classified as:•1- Postabsorptive "fasting", an individual has a loss of glycemic control during fasting state. -Healthy individuals rely on gluconeogenesis to maintain the extracellular glucose concentration.


-Glucose must be given to the patient to relieve symptoms. -In -cell tumors "insulinoma" there is elevated insulin levels.•2- Postprandial "reactive" hypoglycemia•Not serious-Excess insulin results in decrease glucose levels below normal fasting level.-Spontaneous recovery of glucose level as insulin levels return to normal.



•Genetic Defects in Carbohydrate Metabolism•Glycogen storage disease

•result from enzyme defects that affect the processing of glycogen synthesis or breakdown within muscles, liver, and other cell types•Nine distinct diseases considered to be glycogen storage diseases•Glucose – 6 – phasphatase deficiency type 1. "Glycogenolysis is inhibited".

•An autosomal recessive disease.•Characterized by severe hypoglycemia match with metabolic acidosis, ketonemia, and elevated lactate and alanine.


Galactosemia•Congenital deficiency in one of three enzymes involved in galactose metabolism resulting in increased plasma galactose.•1-Galactose-1-phosphate uridyl transferase •2-Galactokinase•3-UDP galactose epimerase •Galactose must be removed from diet.•If untreated, patient may develop mental retardation and cataract.


•leading to toxic levels of galactose to build up in the blood, resulting in hepatomegaly (an enlarged liver), cirrhosis, renal failure, cataracts, brain damage, and ovarian failure. •Without treatment, mortality in infants with galactosemia is about 75%.•Symptoms may include hypoglycemia.


•Methods of glucose Measurement•Glucose oxidase or hexokinase are the most used methods of glucose analysis•Glucose oxidase is specificly reacts with -D-glucose and converts it to gluconic acid. •O2 is consumed and H2O2 is produced.•The reaction can be monitored either by measuring the rate of disappearance of oxygen using an oxygen electrode or by consuming H2O2 in a side reaction.•Horseradish peroxidase is used to catalyze the second reaction and H2O2 is used to oxidize a dye compound.


Glucose + O2 + H2O glucose

Gluconicacid + H2O2

oxidase

H2O2 + reduced chromogen peroxidase

oxidized chromogen + H2O

1- Glucose oxidase

May be coupled with a peroxidase indicator reaction or may be assessed by measuring oxygen consumption, using an oxygen electrode.

specific for beta-D-glucose


The hexokinase method •the reference method for glucose determination•considered more accurate than glucose oxidase methods because the coupling reaction using G-6-PD is highly specific. •The increase in absorbance of NADPH at 340 nm is measured as directly proportional to glucose. •The hexokinase reaction may also be coupled to an indicator reaction and measured through the development of a colored product.


Glucose + ATP G-6-P + ADPHexokinase

G-6-P + NADP G6PD

NADPH + H+

++ 6-Phosphogluconate

2- Hexokinase

3- Glucose dehydrogenaseinvolves the measurement of NADH production:

Glucose + NAD+

glucose dehydrogenase

D-gluconolactone+ NADH + H+


The Specimen•Glucose in serum, plasma, and cerebrospinal fluid (CSF) may be measured by these methods. •Glucose levels in serum or plasma are 10% to 15% higher than those in whole blood. •Serum or plasma must be separated within 1 hour to prevent degradation by glycolysis. •Glucose is stable for 24 hours in whole blood when preserved with sodium fluoride.Reference RangesSerum or plasma (fasting) 74–110 mg/dLCSF 60% of serum or plasma level


Glucose Tolerance and 2 h Post prandial TestsOral glucose tolerance test "OGTT".•The patient should be on a normal to high carbohydrate intake for 3 days before the test.•The patient should be fasting at least 10 h and not more than 16 h.•The test should be performed in the morning because of the hormonal diurnal effect on glucose.•Medications such as large doses of salicylates, diuretics, anticonvalsants, oral contraceptives and corticosteroids may affect the tolerance results.


•The test is performed as the following:•Collect blood sample while fasting.•The patient is given 50-75 gm of glucose orally.•Collect blood samples at 30, 60, 90, 120, 150 and 180 minutes.•Analyze the samples and draw a chart.•In normal persons, a return to the fasting level occurs in 2 or at most 2½ h.•In diabetics, the peak is higher and there is a delay in the return of the blood glucose to a fasting level.•Urine remains free from glucose throughout the test in normal individuals and becomes positive in about 60 minutes in diabetics.


•OGTT has been replaced by 2 h postprandial test in which 75 g glucose in solution is administered and a specimen for plasma glucose measurement is drawn 2 h later.

•If the level of glucose is 200 mg/dL and is confirmed on a subsequent day, the patient is diagnosed with diabetes.


•Glycosylated Hemoglobin•Hb A1c, a glycated hemoglobin, is an indicator of long-term glycemic control.

•The term glycated hemoglobin describes a chemically stable conjugate of any of the forms of hemoglobin with glucose.

•In adults, hemoglobin is a mixture of three forms: Hb A1, Hg A2, and Hb F, with HbA1 predominating. Hemoglobin A1 consists of three subforms: Hb A1a, Hb A1b, and Hb A1c, with Hb A1c predominating.


•Glycated forms of hemoglobin are formed slowly, nonenzymatically, and irreversibly at a rate that is proportional to the concentration of glucose in the blood.

•Form a labile aldimine "Schiff base", the product can then undergo an Amadori rearrangement to form glycosylated Hb "ketoamine".

•The ketoamine is stable and cannot revert back to Hb and glucose.

•This fraction is formed by the reaction of the -chain of HbA with glucose.


•level provides a glycemic history of hemoglobinglycation over the life span of the erythrocyte

•Clinically, glycated hemoglobin is used to reflect glycemic control over the previous 90 to 120 days.• •Measurements are unaffected by daily variation of glucose from diet and exercise.

•CRITICAL THINKING•WAT is the effect of Sickle cell disease and hemolytic disease on glycated hemoglobin results?


C

C

C

OHH

C

C

HOH

OH

OH

H

H

OH

CH2OH

Val NH2

C

C

C

OHH

C

C

HOH

OH

OH

H

H

H

CH2OH

C

C

C

C

C

HOH

OH

OH

H

H

H

CH2OH

Val N

O

H

Val N

HbA +

Fast

HbA HbA

Slow amadori

rearrangment

Hb A Glucose Unstable Schiff Stable Ketoamine base HbA1C

Aldimine"pre- HbA1C


•Preferred method of HbA1C measurement is using high affinity chromatography. •In this method, the glycosylated Hb attaches to the boronate group of the resin and is then selectively eluted from the resin bed by using a buffer.•Other methods include electrophoresis, and isoelectric fucosing.

Glycated Albumin can be used as a sensitive indicator of short term hyperglycemic control than HBA1c since its life spane 15-21 days


C-peptide

•Insulin is stored in the pancreas as the biologically inactive protein proinsulin.•Proinsulin is cleaved into the active hormone, insulin, and an inactive peptide, C-peptide.•Concentration of C-peptide represents the concentration of the endogenous insulin that is produced in the pancreas and is not affected by insulin antibody interference.


Proinsulin, C-peptide, and insulin


•The presence of autoantibodies to pancreatic islet markers confirms a diagnosis of type 1 diabetes.•Islet cell cytoplasmic autoantibodies are seen at the onset of 70% to 80% of type 1 diabetes cases.•Glutamic acid decarboxylase autoantibodies are also found at the onset of 70% to 80% of type 1 diabetes cases.


Hypertension and Microalbuminuria•Hypertension, defined as blood pressure greater than or equal to 140/90 mm Hg, is a common complication of diabetes. •Hypertension is associated with other microvascular complications of the disease and is risk factor for the development of cardiovascular disease.•Cardiovascular disease is the major cause of death in diabetics.


•Renal disease is a serious complication of diabetes, occurring in 20% to 40% of patients with diabetes.•Microalbuminuria is an early marker of diabetic nephropathy.•Microalbuminuria is a term that is used to describe albumin in the urine in amounts that are slightly above normal. •Quantitative tests for urine albumin use nephelometry or immunoassay methodology.•Nephelometry measures the complexes that are formed with antibodies to albumin.•Immunoassay measures the radioactive or enzyme labels that change when albumin binds an antibody


Ketones•In the absence of cellular glucose, fatty acids are oxidized for production of energy. •Ketones are by-products of excessive betaoxidation and degradation of fatty acids to A (acetyl-CoA) . •The ketones composed of beta-hydroxybutyric acid 78% , acetoacetate 20%,and acetone 2% are products of this process. •The extent of ketosis correlates well with the degree of acidosis. •Ketones are produced in cases of carbohydrate deficiency or decreased carbohydrate use such as diabetes mellitus, starvation, prolonged vomiting, and glycogen storage disease.

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10/21/2015 Dr Atef Masad Carbohydrates 1 Clinical Chemistry Chapter 3 Carbohydrates Dr Atef Masad PhD Biomedicine UK