CLS-333 Clinical Biochemistry (III)

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CLS-333Clinical Biochemistry (III)

• Course-
• Carbohydrates
• Amino acids
• Proteins
• Lipids
• Liver function
• Blood gases

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• 7. Elecrolytes
• 8. Calcium and inorganic phosphate
• 9. Renal function
• 10. Gastrointestinal function
• 11. Endocrine function test
• 12. Bioassay/ Modern tests, thyroid hormones
• 13. Ovarian / testicular function tests

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Assessments

• First mid term - 15
• Second mid term exam 15
• Laboratory work and reports 15
• Final practical exam - 15
• Final theoretical exam - 40

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Reference books

• 1. Clinical chemistry, principal, procedure
  and correlation
• Written by Michael C Bishop, Edward P fody, Larry
  E Schoeff
• Fundamentals of clinical biochemistry
• written by Teitz

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• First mid term exam- 4th march,2013
• Second mid term exam 8th april,2013
• practical exam -

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Carbohydrates

• Carbohydrates are polyhydroxy aldehyde or ketones
  or compounds which produce them on hydrolysis.
• They present in food in various forms
• simple sugars - monosaccharides
• ex. Glucose,
  fructose,
  galactose
• 2. complex chemical units -
• a. Disaccharides - lactose, maltose,
  sucrose
• B. polysaccharides - starch, cellulose,
  glycogen

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Carbohydrates

• Monosaccharide's simple sugars.
• Also known as reducing sugars
• Monosaccharides, give positive test with
  benedicts solution and Fehling solution.
• hydroxyl group near an aldehyde or ketone group
  can react with Cu2, converting it to Cu

D-glucose
D-galactose
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• Disaccharides
• Two monosaccharide units attached together with
  glycosidic linkage.
• Disaccharide reducing sugars - are lactose and
  maltose as one reducing end is available for the
  reaction.

reducing end
Lactose
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reducing end
Maltose
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• Sucrose is non reducing disaccharide- as no any
  reducing end is available for further reaction.
• Both end involved in bond formation between two
  monomer units.

Sucrose
not a reducing substance
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• Polysaccharides polymers of monosaccharides
• A. Homopolysaccharides - starch, cellulose,
  glycogen
• B. Heteropolysaccharides hyaluronic acid,
  mucopolysaccharides, agar, heparin

reducing end
Cellulose
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Regulation of blood glucose

• An adult body contain about 18 gm free glucose in
  blood.
• This amount is just sufficient to meet basal
  energy requirement of the body for one hour.
• The liver has about 100 gm stored glycogen .
• Beside this it is capable of producing about
  125-150 mg glucose /minute.

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Normal plasma glucose level

• Fasting plasma glucose level-70-110 mg
• Post parandial glucose level -100 140 mg
• When values fall below- 50 mg , the condition
  is known as hypoglycemia
• Urine glucose .. lt detectable limit (Nil)
• CSF glucose 60 - 90 mg/dl

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• An increase in blood glucose level above to the
  normal- hyperglycemia
• Excretion of glucose in urine- glycosuria.
• Normally glucose is not filtered in urine because
  it is reabsorbed by the kidney tubules.
• But if sugar level is more than 180 mg/dl,
  (kidney threshold) urine contains sugar.

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• Regulation of blood glucose
• Regulation of blood glucose is essential to have
  continuous supply of glucose to the brain.
• Factors maintaining blood sugar-
• a. Major process by which entry of glucose into
  blood are-
• a. absorption of glucose from
  intestine
• b. glycogenolysis- degradation of
  glycogen in liver
• produce
  free glucose.
• c. gluconeogenesis degradation of
  glycogen in
• muscles, result
  in the formation of lactate.
• - breakdown of fat in adipose tissues
  produce free glycerol and propionate. Lactate,
  glycerol, propionate and some amino acide are
  good precursor for glucose synthesis.

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• Factors leading to depletion of glucose in blood
  are-
• a. Utilization of glucose by tissues for
  
• energy.
• b. Glycogen synthesis
• c. conversion of glucose into fat
• (lipogenesis)

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Blood glucose regulation
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• c. Hormones
• make the balance possible between glucose
  entering and leaving the extra cellular fluid.
• List of various hormones which regulates
  blood sugar level are-
• Insulin produced by beta cells of islet
  of langerhans in response to hyperglycemia means
  lowers blood glucose level.
• Glucagon synthesized by alpha cells of islet
  of langerhans of pancreas .
• Hypoglycemia stimulates its production ,
  hence elevate blood glucose level.
• it enhances gluconeogenesis and
  glycogenolysis.
• C. Epinephrine - secreted by adrenal medulla ,
  increases blood glucose level

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• Glucocoticoids
• produced by adrenal cortex
• help in increasing blood glucose
  level.
• E. Growth hormones and ACTH -
• anterior pituitary gland secrets GH
  and ACTH.
• decreases glucose utilization .
• F. Thyroxine
• hormone of thyroid
  gland
• Elevates blood glucose
• Insulin lowers the blood glucose level while rest
  oppose the action of insulin

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Regulation of blood glucose by hormones
stimulates
Somatostatin
inhibits
d
Pancreatic Islet
a
b
Cortisol Growth hormone
glucose
Insulin
glucagon
epinephrine
Glucose uptake Glycolysis
Glycogenolysis Gluconeogenesis
Glucose uptake Lipogenesis
Muscle
Liver
Adipose tissue
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• Postprandial blood sugar regulation-
• After a meal, glucose is absorbed from intestine
  and enters into the blood.
• Increased level of glucose in blood stimulates
  the secretion of insulin by beta cells of islets
  of langerhans of pancreas.
• Insulin helps in the storage of glucose as
  glycogen or its conversion to fat.

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• Regulation in fasting state
• Normally ,2 2.5 hrs after a meal blood glucose
  level falls to near fasting state.
• it may go down further but it is prevented by
  glycogenolysis.
• Liver is the main organ that supplies glucose
  during fasting state and maintain glucose level
  in blood.

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Hypoglycemia

• Reasons are-
• insulin overdose
• drugs
• sulfonylureas
• antihistamines
• alcoholism (long term)
• insulinoma
• galactosemia
• glycogen storage diseases

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Hypoglycemia

• glucose concentration falls to less than 45
  mg/dl .
• Symptoms are- headache, anxiety, confusion,
  sweating, slurred speech, seizures and coma.

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Inborn errors leading to hypoglycemaia

• Galactosemia
• Hereditary fructose intolerance
• Glycogen storage disease
• Disorders of gluconeogenesis
• Organic acedemia
• Maple syrup disease
• Disorders of fatty acid oxidation

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• Galactosemia-
• Serious inborn error , because of the deficiency
  of enzyme galactose 1 phosphate uridyl
  transferase.
• due to the block in this enzyme galactose 1
  phosphate will accumulate in liver.
• This inhibits galactokinase and glycogen
  phosphorylase enzyme activity .
• Resulting hypoglycemia .

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Hereditary fructose intolerance

• It is an autosomal recessive inborn error of
  metabolism.
• Defect is in enzyme aldolase B, hence fructose 1
  phosphate can not be metabolized.
• Accumulation of this product inhibits glycogen
  phosphorylase because allosterically inhibits
  liver phosphorylase and blocks glycogenolysis.
• It leads to the accumulation of glycogen in liver
  and associated with hypoglycemia.

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Fructose Intolerance
Glycogen
Fructose

• Lack of Pi
• Allosteric Inhibition by F-1-P

ATP
Glucose-6-P
Glucose
ADP
Fructose-1-P
1o Deficiency F-1-P Aldolase
F-1,6-BP

• Effects
• F-1-P
• Pi
• Glycogenolysis
• Gluconeogenesis
• Blood Glucose
• Reducing sugar ( ) in urine

DHAP Glyceraldehyde
DHAP G-3-P
Pyruvate

• Treatment
• Limit Fructose/Sucrose

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Disorders of gluconeogenesis

• Pyruvate Carboxylase Deficiency
• It is a defect in the first step of
  gluconeogenesis which is the production of
  oxaloacetate from pyruvate.
• Fasting results in hypoglycaemia .
• Fructose-1,6-Bisphosphatase Deficiency
• Impaired gluconeogenesis and
• accumulation of precursors of
  gluconeogenesis
• lactate, pyruvate, alanine, ketones.
• The only glucose source is - dietary or via
  glycogenolysis.

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Diabetes mellitus

• condition is known as hyperglycemia.
• Two types-
• Insulin dependent diabetes mellitus
• ( IDDM) - type- 1
• Non insulin dependent diabetes mellitus
• (NIDDM) - type - 2

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Type 1 diabetes Mellitus

• almost total deficiency of insulin due to
  destruction of beta cells of pancreas.
• It may be due to -
• -Drugs,
• - viruses,
• - or autoimmunity.
• patients must take insulin to survive.

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Type 2 Diabetes Mellitus

• More commonly occurs in obese persons.
• Obesity act as a diabetogenic factor in persons
  by increasing the resistance to the action of
  insulin.
• It is due to the decrease in insulin receptors
  on insulin responsive cells.
• Circulating insulin level is normal or mildly
  elevated or slightly decreased.

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Metabolic changes in diabetes

• hyperglycemia- elevation in blood glucose level.
• It is due to
• a. reduced glucose uptake by tissues and
• b. increased production via
  gluconeogenesis and glycogenolysis.
• 2. Ketoacid
• osis increased mobilization of fatty acids
  results in overproduction of ketone bodies, leads
  to ketoacidosis

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Metabolic dearrangement of insulin
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• 3. hyper triglyceridemia
• Conversion of fatty acids to triacylglycerols
  and the secretion of VLDL and chylomicrone is
  high in dabeties.
• But activity of enzyme lipoprotein lipase is low.
• so hypercholesterolemia is also seen in
  diabetics.

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• Long term effect of diabetes-
• A. atherosclerosis
• B. retinopathy
• C. nephropathy
• d. neuropathy

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Determination of glucose

• The blood for the estimation of glucose is
  collected using an anticoagulant and an inhibitor
  of glycolysis.
• Specimens used -
• whole blood
• plasma, serum
• CSF
• urine

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Glucose Methods

• Hexokinase
• glucose ATP
  gluc-6-PO4 ADP
• gluc-6-PO4 NAD
  6-phosphogluconate NADH H
• INT NADH H
  formazan NAD
• most widely used
• reference method against which others are
  compared
• serum, plasma and urine
• avoid hemolysis

HK
G6PD
PMS
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1. Enzymatic methods

• Glucose oxidase method

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2. Chemical methods

• Oxidation-reduction reaction (alkaline
• copper reduction method)
• This method depends on the reducing properties of
  glucose which reacts with cupric ions in alkaline
  medium producing the red colored cuprous oxide
  that can be measured colorimetrically

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Condensation reaction (o-toluidinemethod)
When heated with o-toluidine and glacial acetic
acid, glucose reacts with o-toluidine to form
N-glycosylamine. This compound has a blue-green
colour and its absorbance can be measured at 625
nm.
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• Take all absorbencies at 630nm against blank
• 630nm is the wavelength of red light, the
  complimentary color of green, which is the color
  of the imine complex product

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Calculations

• Calculations
• The concentration of glucose in the standard
  solution is 100mg/100ml.
• The concentration of glucose in urine is given
  by
• O.D. TEST
• ? 100 mg Glucose
  /100ml blood
• O.D. STANDARD

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Oral glucose tolerance test

• Patient is instructed to have good carbohydrate
  diet for 3 days prior to the test.
• but should in in fasting state on the testing
  day.
• In the morning time , a blood sample of patient
  in fasting state is collected. And urine sample
  is also collected at the same time.

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• This is denoted as the zero hr sample.
• A dose of 75 gm anhydrous glucose in 250-300 ml
  of water is than given to the patient.
• Sample collection-
• The blood and urine samples are collected at ½
  an hrs interval for the next 2 ½ hrs.
• So total 6 samples including zero hr sample will
  be collected.
• Glucose is estimated in all the blood samples and
  urine sample is te4sted for glucose
  qualitatively.

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OGTT curve
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Causes for abnormal GTT curve

• Impaired glucose tolerance-
• -glucose values are above than normal level
  but below the diabetic level.
• 2. Alimentary glycosuria -
• - fasting and 2 hours values of sugar are
  normal but rise in sugar is seen in other
  duration of samples.
• - this is due to increased rate of
  absorption of glucose from the intestine.
• - seen in hyperthyroidism and after
  gestrectomy.

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• 3. Gestational diabetes mellitus-
• - carbohydrate imbalance during pregnancy.
• 4. Renal glycosuria -
• - normal renal threshold for glucose is 175-
  180 mg?dl
• If level of glucose rises above this in
  blood , glucose start to appear in urine.
• - Diabetes Mellitus is the most commom cause
  of this.

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Ketone bodies

• Acetone ,acetoacetate and beta hydroxybutyrate
  are known as ketone bodies.
• Acetyl Co A, pyruvate and some amino acids are
  the precursor for ketone bodies.
• They are easily transported from liver to
  tissues, and provide energy to peripheral
  tissuses, skeletal muscles, cardiac muscles and
  renal cortex.
• - Normally, ketones do not appear in the urine,
  because all of the metabolized fat is completely
  broken down into carbon dioxide and water.

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• Clinical significance-
• The production of KB and its utilization become
  more significant when glucose is less in blood as
  seen in starvation or diabetes mellitus.
• During starvation KB are the major fuel source
  for the brain and CNS.
• In normal person , constant production of KB done
  by liver and normal conc. in blood is maintained
  as 1 mg/dl.

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• Ketonemia increased production of Kb but
  deficiency in their utilization
• Ketonuria- excretion of KB in urine.
• Both is commonly known as ketosis.
• Ketosis is most commonly associated with
  starvation and severe uncontrolled diabetes
  mellitus.

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Rotheras test

• Principle-
• acetone acetoacetic acid react with sodium
  nitroprusside in the presence of alkali to
  produce purple colour.
• Method-
• take 5ml of urine in a test tube saturate it
  with ammonium sulphate. Then add one crystal of
  sodium nitroprusside. Then gently add 0.5ml of
  liquor ammonia along the sides of the test tube.
• Change in colour indicates test

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