Clinical laboratory Methods for ph.pptx your

Clinical lab note

1. Clinical Laboratory Methods (Clinical Chemistry and urinalysis By Jemal H 1

2. Outline Introduction to clinical chemistry Definition, significance, routine tests(single and panel tests) Liver function tests renal function tests Lipid and lipoprotein analysis for cardiac risk assessment Glucose tests for abnormalities in CHO metabolism(diabetes mellitus) 2

3. Introduction Clinical Chemistry It is an area in laboratory sciences that deals with chemical analysis of body fluids. It deals with the biochemistry of diseases 3

4. Introduction cont’d It links the knowledge of general, organic, Inorganic analytical & biochemistry with an understanding of human physiology . Measures change in biochemical compounds as an indicator of health status or disease processes. 4

5. Significance The main purpose of Clinical Chemistry tests To assess the physiological function of our body systems or organs To diagnose and monitor diseases To follow up response to treatment 5

6. Common clinical chemistry analytes 6

7. Panel tests Some tests are very specific for a disease and can be used for diagnosis. Many tests are not specific for a certain disease process. An individual chemistry test often lacks sufficient sensitivity and specificity to categorically identify a specific disease state. Many times a panel of tests is used in diagnosing a disease process. Multiple tests are frequently requested as a small group of tests in a panel 7

8. Common panel tests 8

9. Liver function tests 9

10. introduction Liver is largest internal organ in our body. Weight about 1.2-1.6kg in healthy adult Located at the right side of our body Two portal system of blood supply Hepatic arteries: supply oxygenated blood(25%) Portal vein : supply nutrient reach blood ( 75%) 10

11. Physiologic role of liver Metabolic function Excretory function Synthesis of all plasma proteins(except gamma-globulin) and coagulation factors Storage Function Protective function 11

12. Production of bile Production of certain proteins for blood plasma Production of cholesterol and special proteins to help carry fats through the body Conversion of excess glucose into glycogen for storage Regulation of blood levels of amino acids, which form the building blocks of proteins 12

13. Processing of hemoglobin for use of its iron Conversion of poisonous ammonia to urea Clearing the blood of drugs and other poisonous substances Regulating blood clotting Resisting infections by making immune factors and removing bacteria from the bloodstream Clearance of bilirubin 13

14. Tests for liver function Approaches used in diagnosis of liver disease: Clinical approach Anatomical approach Physiological approach: evaluation of biochemical and physiological alterations caused by disorder using chemical tests . 14

15. Chemical tests are grouped as: Tests of hepatic excretory function bilirubin , bile acids bromsulfophthalein(BSP), Hippuric acid test Tests measuring hepatic synthetic ability albumin , immunoglobulins, clotting factors Enzymes tests in liver disease AST, ALT, ALP, GGT, LDH 15

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17. Bilirubin Used to assess hepatic excretory function The principal orange-yellow pigment in the bile Source: 85% from the breakdown of hemoglobin 15% from the destruction of other heme -containing proteins 17

18. Metabolism of bilirubin Formation of bilirubin from Hgb and other source Hgb destroyed→ Globin and Heme(Fe and Porphyrin) Porphyrine-further break down as waste product and excreted After Fe and globine is removed→biliverdin(green) In RES, biliverdin is reduced to bilirubin(lipid soluble) 18

19. Bilirubin leaves RE cell and bound to albumin and transported to liver as bilirubin-albumin complex In liver, bilirubin separated from albumin and taken-up by hepatocytes Conjugation of bilirubin in ER of liver cells by uridyldiphosphate glucuronyl transferase(UDPG-T) Secretion of conjugated bilirubin into bile canaliculi→bile duct→ intestine 19

20. Metabolism of bilirubin cont’d In small intestine An enzyme in the higher pH environment of the small bowel convert conjugated bilirubin back into unconjugated bilirubin. Then bacteria convert it to three colorless compounds collectively known as urobilinogen(sterco-, meso-, and urobilinogen) . 20% of the urobilinogen reabsorbed and enter enterohepatic circulation 20

21. Majority of them taken by liver cells(for re-excretion into the bile) Small portion(2-5%) that escape excretion by hepatocyte, reach peripheral circulation(excreted by kidney in urine) About 80% of urobilinogen(not absorbed in the colon) is oxidized spontaneously to urobilin(orange brown) →excreted in stool. 21

22. From circulation & RES – hemolysis of senescent RBCs releases Hgb Unconjugated Bilirubin + Albumin Liver Intestines Globin to protein reserves & Iron to Iron stores Albumin removed in hepatic sinusoids Unconj. Bili conjugated to glucoronic acid by hepatocytes → Conjugated Bilirubin (soluble) enters biliary tree Conjugated bil →Unconjugated bilirubin Bil+ bacterial flora + alk. pH → Urobilinogen Blood Portal Vein Kidney 85% in feces 15% reabsorbed 2%-5% reabsorbed urobilinogen re-enters circulation and excreted in urine.

23. Excretion >99% bilirubin: in feces(in the form of urobilin) <1% bilirubin: in urine(in the form of urobilinogen) Low concentration of bilirubin(0.2-1 mg/dl) found in serum The majority is unconjugated form Small portion(0-0.2mg/dl): conjugated form 23

24. Disorder of bilirubin metabolism Jaundice Yellowish pigmentation of skins and sclera due to accumulation of bilirubin or retention of bilirubin in general circulation Manifested when serum bilirubin >2 mg/dl(though the upper limit is 1 mg/dl) Cause: Increased bilirubin load to liver cell Disturbance in uptake and transport within the liver Defect in conjugation or excretion Obstruction of large bile duct before bilirubin reach small intestine 24

25. Jaundice cont’d Jaundice classification Prehepatic jaundice Hepatic jaundice Post hepatic 25

26. Pre-Hepatic Jaundice(hemolytic jaundice) Characterized by unconjugated hyperbilirubinemia Laboratory Results: ↑ serum bilirubin (mostly unconjugated), ↑ urine urobilinogen, ↓ Hgb ↓ Hct MCV ↓ MCHC Bilirubin not detected in urine Urobilinogen is greatlly increased in urine and faeces(urobilin) Bilirubin rarely exceeds 5 mg/dl

27. Hepatic Jaundice Associated with liver disease The defect associated with liver disease include: Conjugation failure Preconjugation transport failure: ↑unconjugated bilirubin, ↓urobilinogen Post conjugation transport failure: ↑conjugated bilirubin, slight ↓urobilinogen Diffuse hepatocellular damage or necrosis: by viral hepatitis, toxic drugs, cirrhosis 27

28. Post-Hepatic Jaundice Result from impaired excretion of bilirubin Caused by mechanical obstruction of the flow of bile into intestine During complete obstruction No bilirubin glucuronides reach the intestine→no urobilinogen is produced Urobilin is lacking, faeces appear light brown to chalky white, no urobilinogen in urine, but urine contain bilirubin In sever cases Conjugated bilirubin : 12 mg/dl Total bilirubin : 18 mg/dl

29. Measuring Bilirubin in Serum or Plasma By photometric methods based upon the diazo reaction Bilirubin + diazotized sulfanilic acid → azobilirubin (green to blue-green) Conjugated bilirubin (direct bilirubin) Unconjugate bilirubin (indirect bilirubin) Total Bilirubin Methods: Malloy- evelyn method Jendrassic grof method

30. Reference Ranges for Bilirubin Compare patient result with reference range Newborn range for full term,1-2 days old Patient conjugated (direct) bilirubin total bilirubin unconj . + conjugated Urine bilirubin, conjugated Newborn (1-2 d old) Not applicable < 12.0 mg/ dL Adult 0-0.2 mg/ dL 0.3-1.2 mg/ dL negative

31. Clinical Correlations ** Urobilinogen = UBG; *Total Bili = (conjugated + unconjugated) Jaundice Type Clinical Condition Serum Conjugated Bilirubin Serum Total Bilirubin * Urine Bilirubin Urine (UBG)** None Normal Normal level Normal level Neg Normal <1 mg/ dL Pre-hepatic Hemolytic anemia Normal or Sl Increase Increase Neg Increase Hepatic Hepatitis Increase Increase Positive Normal or increase Post-hepatic Obstruct-ion of bile duct Increase Increase Positive Normal or none.

32. Liver Enzymes Used to aid in diagnosis and treatment of liver disease. Enzymes that are synthesized within cellular organelles carry out their functions within cells and are released into body fluids when those cells become diseased. Enzyme activity levels in body fluids can reflect: Leakage from cells due to cellular injury, or changes in enzyme production rate or actual enzyme induction due to metabolic or genetic states or proliferation of neoplasms. 32

33. Liver Enzymes cont’d Damage to liver tissue LDH, ALP, ALT, and AST, Cellular death (necrosis) will release mitochrondrial sources of ALT and AST. ALP and GGT are more concentrated in the biliary ducts or tissues of the small ducts (canaliculi) AST, ALT, and LD are found mainly in structural (parenchymal) hepatic cells 33

34. Serum enzyme tests can be grouped into two categories: 34

35. Correlation of hyperbilirubinemia pattern and enzymes Bilirubin concentration Enzymes Prehepatic jaundice Normal serum conjugated bilirubin , Increased unconjugated bilirubin , Increased urinary urobilinogen Normal serum levels of hepatocellular and hepatobiliary enzymes, with the exception of LDH and possibly AST Hepatic jaundice Increased serum conjugated and unconjugated bilirubin Increased urinary urobilinogen increased serum levels of hepatocellular enzymes. Post-hepatic jaundice Normal serum unconjugated bilirubin Increased conjugated bilirubin Decreased urinary urobilinogen pronounced elevations of hepatobiliary enzymes but normal to slightly elevated serum levels of hepatocellular enzymes. 35

36. Clinical Significance of AST Hepatocellular inflammation releases cytoplasmic AST Hepatocellular necrosis releases mitochondrial AST. Associated with liver inflammation (hepatitis) Drugs overdose or toxicity Infections from Viruses Alcohol Other organ diseases Myocardia infarction

37. Analytical Methodology of ALT and AST Continuous Monitoring Method is used Reference range for serum/plasma ALT Adult male: <45 U/L Adult female: <34 U/L Reference range for serum/plasma AST Adult male <35 U/L Adult female <31 U/L

38. Clinical Significance of ALP High concentration of ALP in hepatobiliary cells Biliary inflammation or ductal obstruction Cholestasis may cause ALP increased 3-10 X the normal levels. Cellular inflammation and necrosis Increased with bone diseases of osteoblastic activity Reference ranges for ALP 53 -128 U/L

39. Albumin It is the most abundant(about half the plasma protein mass) Increased concentrations of albumin are present only in acute dehydration and have no clinical significance. Decreased concentrations are seen in: Hepatic Disease : Acute and chronic inflammation: Urinary loss and Gastrointestinal Loss Edema Protein Energy Malnutrition Adult serum albumin: 3.5 - 5.0 g/dl

40. Renal function tests 40 Anatomy and physiology Renal pathophysiology Laboratory tests Factors affecting renal function tests Urinalysis Serum BUN and creatinine Clearance tests

41. Major Components of Kidney Nephron: functional unit of the kidney Arterioles: Afferent Efferent Glomerulus Bowman’s capsule Tubules: PCT, Loop of henle, DCT, Collecting tubules 41

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43. Physiology of kidney Removal of unwanted substances Homeostasis function Water balance Ionic (electrolytes) Equilibrium Acid – base balance 3. Endocrine regulation Secretion of erythropetin ,rennin and prostagladin . Kidney is the target organ for catabolism of insulin, glucagons , aldosterone and vitamin D. 43

44. Urine formation requiers : Urine Formation Glomerular Filtration Due to differences in pressure water, small molecules move from the glomerulus capillaries into the glomerular capsule Tubular reabsorption Many molecules are reabsorbed from the nephron into the capillary ( diffusion, facilitated diffusion, osmosis, and active transport) Tubular secretion Substances are actively removed from blood and added to tubular fluid (active transport) ie.  H+, creatinine, and some drugs are moved by active transport from the blood into the distal convoluted tubule a) b) c) 44

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46. Renal Pathophysiology Glomerular disease Acute glomerulonephritis Chronic glomerulonephritis Nephrotic syndrome Tubular diseases Decreased excretion/reabsorption of certain substances or reduced urinary concentrating capability. Renal tubular acidosis(RTA) Urinary tract infection Renal obstruction Renal calculi Renal failure: acute and chronic 46

47. Renal function tests Glomerular function tests : All the clearance tests (innulin, creatinine, urea) Tubular function test : Urine concentration or dilution test or urine acidification test Osmolality measurements, Specific proteinurea, Glycouria, Aminoaciduria Analysis of blood/serum blood urea, serum creatinine, protein and electrolytes Urinalysis Appearance, Specific gravity and osmolality, pH, Glucose, Protein, ketone bodies, Urinary sediments 47

48. Factors affecting Renal function tests Pre-renal factors Affect blood volume, blood flow or blood pressure Include: Dehydration due to pyloric & intestinal obstruction and diarrhea, shock Excessive loss of blood due to severe intestinal bleeding Cardiac failure: the defect lies in the blood supply before if reaches the kidney due to cardiovascular system failure and consequent hypovolemia 48

49. Factors affecting RFT cont’d Renal Factors The defect on the kidney Include: Disease affecting the GFR and tubular function mainly due to damage of the nephrones from glomerulus nephritis, nephrotic syndrome Changes in the renal vascular system that decrease the blood flow 49

50. Factors affecting RFT cont’d Post Renal factors The defect lies in the urinary tract after it exits kidney Due to any obstruction of the urine flow such as: Enlargement of the prostate gland Stones in the urinary tract Carcinomas or tumors that may compress the ureters, urethra or bladder opening. 50

51. Types of analysis Macroscopic Examination Chemical Analysis (Urine Dipstick) Microscopic Examination Urinalysis 51 Urinalysis Appearance Specific gravity and osmolality pH Glucose Protein Urinary sediments

52. Macroscopic Examination Color: Colorless Diluted urine Deep Yellow Concentrated Urine, Riboflavin Yellow-Green Bilirubin / Biliverdin Red Blood / Hemoglobin Brownish-red Acidified Blood Turbidity Typically cells or crystals 52

53. Chemical Analysis Urine Dipstick Glucose Bilirubin Ketones Specific Gravity Blood pH Protein Urobilinogen Nitrite Leukocyte Esterase 53

54. Negative Trace (100 mg/dL) + (250 mg/dL) ++ (500 mg/dL) +++ (1000 mg/dL) ++++ (2000+ mg/dL) The Urine Dipstick Glucose Glucose + 2 H 2 O + O 2 ---> Gluconic Acid + 2 H 2 O 2 Glucose Oxidase 3 H 2 O 2 + KI ---> KIO 3 + 3 H 2 O Horseradish Peroxidase Chemical Principle Read at 30 seconds RR: Negative

55. Specific gravity This is a semi-quantitative measure of concentration. A higher specific gravity indicates a more concentrated urine. PH Urine is usually acidic Osmolality Serves as general marker of tubular function. The ability to concentrate the urine is highly affected by renal diseases. If the urine osmolality is 600mosm/kg or more, tubular function is usually regarded as intact When the urine osmolality does not differ greatly from plasma (urine: plasma osmolality ratio=1), the renal tubules are not reabsorbing water Urine dipstick 55

56. Urine sediments Microscopic examination of sediment from freshly passed urine involves looking for cells, casts, fat droplets Blood: haematuria is consistent with various possibilities ranging from malignancy through urinary tract infection to contamination from menstruation. Red Cell casts could indicate glomerular disease Crystals Leucocytes in the urine suggests acute inflammation and the presence of a urinary tract infection. Microscopic examination 56

57. Urinary casts Are cylindrical structures produced by the kidney and present in the urine in certain disease states. They form in the distal convoluted tubule and collecting ducts of nephrons , then dislodge and pass into the urine, where they can be detected by microscope. They form via precipitation of mucoprotein which is secreted by renal tubule cells, and sometimes also by albumin. 57

58. Red blood cell cast in urine White blood cell cast in urine 58

59. Urinary crystals. (A) Calcium oxalate crystals; (B) uric acid crystals (C) triple phosphate crystals with amorphous phosphates ; (D) cystine crystals. Crystals 59

60. Non-protein Nitrogenous (NPN) substances These are compounds that contain nitrogen, but are not proteins Formed in the body as a result of the derivative metabolism of nucleic acids, amino acids and proteins The kidneys play an essential role in the excretion of these metabolic waste products. Measurement of these compounds can be used to assess kidney function 60

61. Include >15 compounds Amino acids: from protein catabolism (breakdown) Ammonia: from amino acid catabolism Urea: from ammonia catabolism Creatinine: from creatine breakdown in the muscle Uric acid: from nucleic acid catabolism 61

62. Urea Urea constitutes half of the NPN substances in blood It is synthesized in the liver Urea is transported by the plasma to the kidney where it is readily filtered from the plasma by the glomerulus. More than 90% of urea excreted through the kidney and < 10% urea excreted through GIT and skin. 62

63. It is neither actively reabsorbed nor secreted by the tubules (but is filtered freely by the glomeruli) About 40% is reabsorbed by passive diffusion during passage of the filtrate through the renal tubules. Plasma level is affected by renal function the protein content of the diet level of protein catabolism , and hydration of the patient 63

64. Urea Vs blood urea nitrogen(BUN) Urea is different from BUN The structure of urea is NH 2 -CO-NH 2 with a molecular weight of 60g, the two Nitrogen atoms represent 28g, Mol. Wt of urea = 60/28 = 2.14 At wt of Nx2 (BUN in mg /dl) (2.14)= Urea in mg/dl 64

65. Clinical significance of urea Azotemia: an elevated level of urea in the blood Uremia or uremic syndrome: Very high levels of plasma urea accompanies by renal failure Cause of decreased urea levels Decreased protein intake Severe liver disease Conditions that leads to increased protein synthesis such as normal pregnancy and infancy Severe vomiting & diarrhea 65

66. Cont…… Increased urea level Pre renal Decreased renal blood flow due to congestive heart failure shock, hemorrhage dehydration. Increased protein catabolism and intake of high protein diet Renal Acute & chronic renal failure Glomerular nephritis Tubular necrosis Post renal Obstructions to the flow of urine any where in urinary tract 66

67. Cont….. In conjunction with serum creatinine, BUN/creatinine ratio used to differentiate between pre renal & post renal azotemia The normal ratio is 10:1 to 15:1 and High ratio with normal creatinine -Prerenal azotemia High ratio with increased creatinine-Post renal obstruction or renal azotemia Urea clearance is a poor indicator of GFR. urea clearance generally underestimates GFR 67

68. Reference range for Blood Urea N (BUN) For adults (Serum/plasma)……………….. 6-20 mg/dl New born up to one week( Serum/plasma)… 3-25mg/dl Adult over 60 (Serum/plasma) …….8-23mg/dl Urine: 12-20 g/24hrs 68

69. Creatinine Waste product formed in muscle from phosphocreatine Excreted into the circulation at a relatively constant rate, proportional to the muscle mass of the individual Removed from the circulation by glomerular filtration. 69

70. Normally, creatinine clearance exceeds the glomerular filtration rate by 10% to 40%. A small but significant amount of creatinine is secreted by the PCT, better indicator of renal function than urea serum creatinine is not affected by diet, exercise, or hormones, factors that influence the levels of urea 70

71. Clinical significance Elevated level of creatinine is mainly associated with abnormal renal function. However, it is relatively insensitive monitor and may not be measurably increased until renal function has deteriorated more than 50%. Its serum level increases in disease associated with extensive muscle destruction 71

72. Excellent analyte for the assessment of renal function Strong relationship between plasma creatinine and the GFR Plasma creatinine level are relatively constant and unaffected by diet The serum creatinine level in combination with BUN is used to differentiate between pre renal & post renal cause of azotemia. 72

73. Creatinine cont’d Reference Range Serum Adult male: 0.6-1.1 mg/dl Adult female: 0.5-0.8 mg/dl Child: 0-0.6 mg/dl Urine Male: 800-2000 mg/24hr Female: 600-1800 mg/24hr Amniotic fluid: 1-2 mg/dl 73

74. BUN/Creatinine Ratio Used to differentiate causes of azotemia: Pre-renal Post-renal Calculated: serum BUN (mg/dl) serum creatinine (mg/dl) Normal ratio: 10-20 with majority around 12-16 74

75. BUN/Creatinine Ratio Differentiation of azotemia Increased ratio with increased BUN, normal creatinine Tend to be caused by pre-renal conditions: Congestive Heart Failure Shock, hemorrhage Dehydration Increased protein metabolism Increased protein catabolism 75

76. BUN/Creatinine Ratio Differentiation of azotemia Increased ratio with dysproportionate increased BUN, slightly increased creatinine Tend to be caused by post-renal conditions that obstruct urine flow: Stone Tumor Sever infection 76

77. BUN/Creatinine Ratio Differentiation of azotemia Increased ratio with increased BUN, increased creatinine Tend to be caused by - renal conditions that decrease kidney function: Acute renal failure Chronic renal failure Glomerulonephritis Tubular necrosis 77

78. BUN/Creatinine Ratio Decreased ratio with decreased BUN Tend to be caused by conditions of decreased urea production: Low protein diet Liver disease 78

79. Renal clearance tests / Estimate of GFR Extremely useful, effective and sensitive way of measuring the actual excretory capacity of the kidney. Detect much earlier stages of the disease Clearance tests are more sensitive and clinically more useful than the other function tests. The concentration of a substance excreted in the urine is measured and compared to the concentration of the same substance in the plasma The renal clearance of a substance is defined as the volume of plasma from which the substance is completely cleared by the kidney per unit time 79

80. Clearance test cont’d The amount of substance cleared by the kidney is generally expressed as a volume of plasma which contains the quantity of the substance excreted in the urine. ml plasma cleared = Uc x V , Uc=conc of subs in urine in mg/dl Pc Pc= conc of subs in plasma in mg/dl V=urine volume excreted per minute in ml (urine flow rate) The clearance rate is proportional to the size of the kidney and the patients body surface area. ml plasma clearance/ min= Uc x V x 1.73 Pc A 1.73= A factor generally accepted average body surface area in m 2 A=the body surface area of the patient 80

81. Markers used for renal clearance test Substance used to monitor GFR must meet the following criteria Freely filterable at the glomerular barriare Not reabsorbed by the tubules Not secreted by the tubules Present at a stable plasma concentration Include: Exogenous markers: inulin, manitol& iohexol Endogenous markers: creatinine, urea & low molecular proteins For the benefit of patients and ease of procedure creatinine is the best choice for renal clearance tests 81

82. Creatinine clearance Used to approximate GFR. Creatinine is ideal substance for the measurement of clearance It is an endogenous metabolic product synthesized at a constant rate for a given individual It is cleared essentially only by glomerular filtration (it is not reabsorbed and only sightly secreted by the PCT) It can be analyzed inexpensively by readily available colorimetric methods. Creatinine clearance has become the standard laboratory assay for determination of Early renal failure It over estimates actual GFR by 10 – 20% 82

83. Procedure Hydrate the patient by administering a minimum of 600ml of water Caffeine containing compounds (coffee, tea), heavy exercise and drugs should be avoided The water intake promote good urine flow Have the patient void and discard the urine specimen Collect a 4, 12 or 24 hrs specimen and record the exact times of starting and completing each collection. Although a full 24 hr period is ideal, the excretion rates of an endogenous marker (creatinine) is sufficiently constant that a 4-h period may be used Blood sample can be collected at any time during the urine collection (ideally taken at mid point of 24 hour urine collection) Determine urine & blood creatinine and Calculate the clearance 83

84. Creatinine clearance cont’d Normal values Males 85-125ml/min Female 75-115ml/min Decreased creatinine clearance It is sensitive indicator of decreased GFR It can be caused by acute or chronic damage to the glomerulus, reduced blood flow to the glomeruli, and acute tubular damage. 84

85. Lipid analysis 85

86. Lipid and lipoprotein analysis for cardiac risk assessment Lipids Function, classification triglycerides Cholesterol Lipoproteins 86

87. Lipids Lipids are organic compounds that are poorly soluble in solutions such as water and soluble in organic solutions such as ether. Classification Simple lipids Neutral fats, waxes, triglycerides Conjugated lipids Phospholipids, glycolipids, lipoproteins Derived lipids Fatty acids, Glycerol, Fat soluble vitamins(A,D,E,K) Sterols: bile acids, cholesterol , ergot ester, and steroid hormones. 87

88. Function of lipids Structural and functional elements of cell membranes Source of biochemical fuels Provide energy storage Act as insulator Allow nerve conduction Prevent(retard) loss of body heat Serve as hormones or precursors to other hormones and to other essential substances 88

89. Triglycerides (Triacylglycerols) Esterification products of fatty acids with glycerol Composed of one molecules of glycerol with 3 fatty acid molecules. glycerol first FA second FA third FA 89

90. Triglycerides cont’d Function Provide energy storage Provide insulation to vital organs Transported in plasma mostly in the form of large triglyceride rich molecules(chylomicrons and VLDL) Triglycerides are hydrolyzed to form free fatty acid and glycerol The free fatty acids are oxidised to form many acetyl CoA( β -oxidation) Acetyl CoA enters krebs cycle to form ATP The glycerol is recycled into additional triglycerides 90

91. Cholesterol Principal body sterol Complex solid unsaturated alcohol formed of four rings (perhydrocyclopentanthroline ring)/sterane skeleton and a side chain of 8-carbon atoms Pure cholesterol is solid at body temperature It has 27 C, 46 hydrogen and 1 oxygen(C27H46O) Has one double bond between C5 and C6, one –OH at C3(responsible for most of reaction) 91

92. Function of cholesterol Minimum levels of cholesterol are essential for: Used for the manufacture and repair of cell membrane It regulates membrane fluidity over a wide range of temperature Used for the synthesis of bile acids(help to digest fats) and vitamin D Major precursor of various steroid hormones(progestin, estrogen, adrogens, glucocorticoids, mineralocorticoids) 92

93. Lipoproteins Organic compounds composed of both lipids and protein (apolipoproteins) Outer coat: - Apoproteins - Phospholipids - Cholesterol (Unesterified) Inner core : - TG - Cholesterol ester 93

94. Classification of lipoproteins Classes of lipoproteins Have distinctive physical properties, structure and function Each differ in size, density and electrophoretic mobility Have specific sets of apolipoproteins(in membrane) and different mixtures of lipids(in the core) Lipoproteins have been categorized based on differences in their hydrated densities, as determined by ultracentrifugation. Chylomicrons Very-low density lipoprotein (VLDL) Intermediate-density lipoprotein (IDL) Low­ density lipoprotein (LDL) High-density lipoprotein (HDL) lipoprotein(a) [Lp(a)]. 94

95. Chylomicrons The largest and the least dense lipoproteins Produced by the intestine Transport dietary lipid (mostly exogenous triglycerides) from intestine to fat/muscle cells Mostly lipid, almost 90% triglyceride (TG), 1% protein 95

96. Very low density lipoprotein(VLDL) Produced by the liver Transport liver synthesized fat (mostly TG) to fat/muscle cells Mainly lipid, 55% TG, 10% protein 96

97. Low density lipoprotein(LDL) More cholesterol rich Transport cholesterol from liver to peripheral tissues LDL delivers cholesterol either to the liver for bile acid formation or to other tissues for use as a structural components of new cell membranes, as a precursor of steroid hormones or for storage as cholesteryl esters. Proatherogenic Better marker for CHD risk 97

98. High density lipoprotein(HDL) The smallest and most dense lipoprotein Synthesized by liver and intestine Mainly protein (Apo A-I, A-II) Lipid: cholesterol, phospholipid Involved in "reverse transport" of cholesterol from tissue to liver Carry 20-35% total plasma cholesterol from tissue and return it to liver where it is excreted, lowering the risk of arteriosclerosis 98

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100. Clinical significance of lipids Diseases associated with abnormal lipid concentrations can be caused: Directly by genetic abnormalities Through environmental/lifestyle imbalances or Secondarily, as a consequence of other diseases. Many, but not all, lipid disorders, regardless of etiology, are associated with CHD, or arteriosclerosis. 100

101. Arteriosclerosis Pathological conditions in which there is thickening or hardening of the wall of the arteries due to accumulation of lipid material in the veins and arteries. Stem from the deposition of lipids mainly in the form of esterified cholesterol in the wall of the arteries The lipid deposition starts with thin layers called fatty streaks then develop over time into plagues which partially block(occlude) the flow of blood. 101

102. Arteriosclerosis cont’d Deposition of lipids, mainly, esterified cholesterol in artery walls = fatty streaks infiltration of LDL particles into the space below the endothelial lining of blood vessel initiates an immune response that triggers the local accumulation of inflammatory cells that degrade LDL particles causing the cholesterol to be released LDL can also be taken up by macrophages = foam cells Accumulation of foam cells deposit in artery walls = fatty streaks Fatty streaks can develop over time into plaques which can partially block or occlude blood flow. 102

103. Arteriosclerosis cont’d Peripheral vascular disease(PVD) when the plague formation develops in arteries of the arms or legs Coronary artery disease(CAD) when it develops in the heart. It is associated with angina and myocardial infraction Cerebrovascular disease(CVD) when it develops in the vessels of the brain. It is associated with stroke. 103

104. Lipid and Lipoprotein Analysis Lipids and lipoproteins are important indicators of CHD risk This is the major reason for their measurement in clinical practice. Diagnosis of dyslipidemia is dependent upon the measurement of: 1- Total cholesterol 2- Triglycerides 3- HDL cholesterol 4- LDL cholesterol 104 Lipid Panel or profile

105. Determination of Triglycerides cont’d Methods Based on the assay of glycerol content of the triglyceride Chemical colorimetric method Enzymatic colorimetric Enzymatic UV Based on the assay of fatty acid content of triglyceride- not used for routine lab 105

106. REFERENCE LIMITS Reference range for triglyceride Broad and varies with age and gender Male (25–29 years old): 45–204 mg/dL Females (25–29 years old): 42–159 mg/dL DESIRABLE: < 200 mg/dL (< 2.3 mmol/L) BORDERLINE HIGH: 200-399 mg/dL (2.3-4.5 mmol/L) HIGH: > 400-1000 mg/dL (4.5-11.3 mmol/L) VERY HIGH: > 1000 mg/dL (> 11.3 mmol/L 106

107. Reference values for total cholesterol Low at birth, but increase about 40% by third day(reach 125 mg/dl) Increase slightly with age Cholesterol concentration of women less than men until menopause, after menopause it exceeds the concentration of men Reference values: Male (25–29 year old): 130–234 mg/dL Female (25–29 years old): 130–231 mg/dL 107

108. Total cholesterol cont’d Result of cholesterol grouped into 3 categories: DESIRABLE : < 200 mg/dL (< 5.2 mmol/L) Low risk of heart disease BORDERLINE HIGH : 200-240 mg/dL (5.2-6.2 mmol/L) Moderate risk of heart disease Differentiation is required whether the increased cholesterol is due to ↑LDL or ↑HDL or any other risk factor HIGH: > 240 mg/dL (> 6.2 mmol/L) A serious risk factor for development of heart disease 108

109. Reference limits for HDL Men (25–29 years old) 31–63 mg/dL Women (25–29 years old) 37–83 mg/dL DESIRABLE : > 35 mg/dL (> 0.9 mmol/L) NEGATIVE RISK : > 60 mg/dL (> 1.6 mmol/L) HIGH RISK : < 35 mg/dL (> 0.9 mmol/L) 109

110. Reference limits for LDL Male (25–29 years old) 70–165 mg/dL Females (25–29 years old) 71–164 mg/dL DESIRABLE : < 130 mg/dL (< 3.4 mmol/L) BORDERLINE HIGH : 130-160 mg/dL (3.4-4.2 mmol/L) HIGH: > 160 mg/dL (> 4.2 mmol/L) 110

111. Glucose analysis for diagnosis of diabetes mellitus Carbohydrate Digestion, Metabolism Regulation of CHO metabolism Disorder(diabetes mellitus) Types Diagnosis 111

112. Digestion of carbohydrates 112

113. Carbohydrate Metabolism 113

114. Regulation of carbohydrate metabolism Carbohydrate metabolism is regulated by: Hormone control Hormone that decrease plasma glucose: Insulin Hormones that increase blood glucose (counter regulatory hormones): Glucagon, Epinephrine/adrenalin, Growth hormone & ACTH(adrenocorticotropic hormone), Glucocortico steroids, Thyroid hormone, Somatostatin 114

115. glycogenesis Glycogenolysis triglyceride synthesis Ketogenesis Gluconeogenesis glucose uptake protein synthesis Protein degradation glycogenesis Glycogenolysis glucose uptake triglyceride storage Lipolysis Endocrine Effects of Insulin Stimulates Inhibits Liver Skeletal Muscle Adipose tissue Promotes anabolic processes Inhibits catabolic processes DECREASES HIGH BLOOD GLUCOSE LEVELS 115

116. Disorders of Carbohydrate metabolism Hyperglycemia An increased plasma glucose concentration Hypoglycemia: A decreased plasma glucose concentration Inborn errors of CHO metabolism Deficiency or absence of an enzyme that participates in carbohydrate metabolism may result in accumulation of monosaccharides 116

117. Diabetes Mellitus It is a group of metabolic disorders of CHO metabolism in which glucose in under utilized It is characterized by hyperglycemia resulting from defects in insulin production, secretion &/or action It is a chronic complex syndrome induced by absolute or relative deficit of insulin which is characterized by metabolic disorders of carbohydrates, lipids and proteins. 117

118. Classification cont’d 118

119. Type 1 Diabetes Mellitus Known as IDDM, type I, or juvenile-onset diabetes Characterized by lack of insulin production and secretion by the 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 119

120. 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 120

121. 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 121

122. 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: <5% Environmental factors Obesity, lack of physical activity family history of type 2 diabetes (genetic predisposition) Previous diagnosis of gestational diabetes 122

123. Diagnosis of diabetes mellitus The diagnosis of diabetes mellitus depends solely on the demonstration of hyperglycemia Diagnostic criteria Classic symptoms of diabetes Demonstration of significant hyperglycemia Random plasma glucose Fasting plasma glucose Oral glucose tolerance test 123

124. Diagnosis cont’d 124

125. Fasting Plasma Glucose 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. 125

126. Glucose tolerance test (GTT) GTT is a test used to diagnose mild or hidden cases of diabetes individuals Who are not clearly abnormal at the time of testing Who do have normal or border line fasting glucose levels Demonstrate impaired glucose tolerance post prandially Non-preferred method of diabetes diagnosis Methods: Oral glucose tolerance Intravenous glucose tolerance test 126

127. Test procedure and interpretation of OGTT The patient should eat an adequate CHO diet containing at least 150mg for three days prior to test The test should be performed in the morning after the patient has fasted for 10 to 16 hours only water is permitted Obtain fasting blood and urine specimen, and measure glucose concentration A glucose dose of 75g of dissolved in 300 ml flavored (added Lemmon to avoid nausea…) water is administered orally Plasma and urine glucose is measured every 30 minutes for 2 hours 127

128. OGTT cont’d The shape of glucose tolerance curve is useful in evaluating the OGTT Healthy subjects peak at ½ hr & return to fasting level at hrs Diabetics peak late (approx. 1 hr) or even show plateau at 2 -3 hrs & return to the base line value after 3 hrs Glucose value > 200mg/dl is suggestive of diabetic mellitus 128

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130. Methods for glucose analysis The most popular method for determination of plasma glucose because of their specificity, rapidity, use of small sample quantities and ease of automation Common enzymatic methods include: Glucose oxidase Glucose hexokinase Glucose dehydrogenase Enzymatic methods are specific for glucose. They will not measure other mono- or disaccharides, i.e., fructose, galactose , sucrose or lactose 130

131. Tests of Diabetes Control and Disease Progression (Long-Term Monitoring) Laboratory testing for diabetes after diagnosis of the disease is directed toward the assessment of the progression of disease. Help to determine the extent of glycemic control and the risk for the severe consequences of hyperglycemia Can be done by analysis of glycated hemoglobin, ketones, creatinine, microalbuminuria, acid-base status, lipids, and other measurements 131

132. Long-Term Monitoring cont’d 132

133. Urine Glucose The monitoring of urine glucose lacks sensitivity and specificity and provides no information about blood glucose concentrations below the renal threshold (usually 180 mg/dL). Diabetes cannot be diagnosed by testing urine for glucose Urine glucose may be measured either qualitatively or quantitatively 133

134. Glucose in CSF Glucose oxidase method used to measure glucose in CSF Normally CSF glucose is about 60% to 75% of plasma level Bacteria and other cells may contaminate CSF, so it should be analyzed immediately or centrifuged and stored at 4 ° C It is useful in the diagnosis of menigitis. Many bacteria and leukocytes in the infected CSF rapidly metabolize glucose and lower its conc. The concentration of glucose in normal individuals varies from 45 to 70 mg/dl but is usually less than 30 mg/dl in various types of bacterial meningitis 134

135. Thank you! 135

136. quiz 1. What are the hormones involved in regulation of carbohydrate metabolism and describe their action? 2. Describe the differences between type 1 and type 2 diabetes mellitus 3. Explain the criteria used for diagnosis of diabetes? 4. What are the sample requirements for glucose analysis? 5. What re the four lipid profile done for cardiac assessment?