CHOLESTEROL METHODS.pptx

CHOLESTEROL METHODS, Triglycerides, OFONMBUK UMOH, LIPIDS, CHOLESTEROL, TRIGLYCERIDE, lipoprotein, fatty acids, Chylomicrons, TC, TG, HDL-C, LDL-C, Apo-A, Apo-B, Apo-C, LDL & VLDL, Liebermann-Burchardt Rxn, Modified Abell Kendall, Bloors, Van Handel & Zilversmith, Isotope Dilution Mass Spectrometry, GAS CHROMATOGRAPHY – MASS SPECTROMETRY, Fridewald Equation

1. LIPID ASSAY METHODOLOGY DR. OFONMBUK UMOH RESIDENT, DEPT OF CHEMICAL PATHOLOGY UNIVERSITY OF UYO TEACHING HOSPITAL

2. OUTLINE Introduction Pre-analytical procedures Analytical methods Important considerations Conclusion References

3. INTRODUCTION LIPIDS are a class of hydrophobic compounds of carbon and hydrogen, which are soluble in organic solvents, nearly insoluble in water; and on hydrolysis, yields fatty acids and complex alcohols CHOLESTEROL and TRIGLYCERIDE are the plasma lipids of most interest in the diagnosis and management of lipoprotein disorders

4. Cholesterol: a 27-carbon sterol, derived from diet or synthesized from acetate. Transported bound to lipoproteins: HDL, LDL, VLDL Triglycerides: Glycerol esters with 3 fatty acids. Forms a major component of human body fat. Intro…

5. Specimen Collection and Storage: Patient should fast for 12hours before sampling . The concentration of LDL-C/HDL-C declines after eating Chylomicrons are cleared within 6–9hrs and their presence after 12hrs fast is abnormal . Patient to be seated for 5min prior to sampling to prevent hemo -concentration . PRE-ANALYTICAL PROCESSES

6. Tourniquet should not be applied for more than minutes or two as prolonged venous occlusion leads to increase in cholesterol concentration by 10–15% Sample bottle: Plain bottle – blood sample allowed to clot and serum separated for use Anticoagulant sample – spun and plasma separated into plain bottle Pre-analytical…

7. Anticoagulants : EDTA is preferred anticoagulant even though TC and TG concentration in EDTA plasma are 3 % lower than in serum. EDTA inhibits oxidation of lipids and proteolysis of apolipoprotein TC values of EDTA plasma should be multiplied by 1.03 to make it comparable to serum values. Some anticoagulants such as citrate exert large osmotic effect resulting in falsely low plasma lipid and lipoprotein concentration. Heparin, because of its high M.W can alter electrophoretic mobility of lipoproteins . Pre-analytical…

8. Storage: TC, TG, HDL-C can be satisfactorily analyzed in frozen samples. Serum or plasma must be stored at – 70 o C if stored for long time. For short time storage (up to a month or two ) the sample can be kept at – 2 o . Pre-analytical…

9. Age : cholesterol levels increase with age. Sex : women have lower level than men except in childhood & after early 50’s. Exercise: Mild exercise produces a slight decrease in conc. of cholesterol and TG that may persists for several days. Those who walk for about 4 hours each week have an average cholesterol conc. 5% lower and HDL-C conc 3.4% higher than inactive persons Biological / Physical Variations:

10. Posture: It has been observed that after 20 minutes recumbence , there is a d ecrease of as much as 10% in conc. of TC, LDL-C, HDL-C, Apo-A and B. Season : cholesterol levels are slightly higher in cold periods. Food intake: daily intake of fat increases cholesterol levels. Patients should be on their usual diet for 2wks and are neither gaining nor losing weight. Variation factors…

11. Medical conditions: thyroid, liver, and kidney diseases Acute illness : It is recommended that lipoproteins measurement should be made no sooner than 8weeks after any form of trauma or acute bacterial/viral infection and 3 – 4 months after child birth Life-style : higher in sedentary and poor diet habits Alcohol ingestion: When moderate amount of alcohol is ingested for 1wk, the serum TG concentration is increased by more than 20mg/ dL . Variation factors…

12. Diet: A high fat diet increases serum T Gs . Ingestion of mono & polyunsaturated fat reduces cholesterol. Plasma TG conc. is reduced when sucrose intake is reduced. Individuals who eat many small meals throughout the day tend to have conc. of total LDL and HDL-C that are lower than when same type and amount of food is eaten as square meals. Large protein meals at lunch or in the evening also increase the serum cholesterol for at least 1 hour after a meal. In vegetarian individuals, conc. of LDL & VLDL-C are reduced by 37 % and 12% respectively. Variation factors…

13. Menstrual cycle: The plasma cholesterols and TG concentration tend to be highest at mid-cycle , the time of maximum estrogen secretion The cyclical variation in cholesterol is not observed with annovulatory cycles Smoking : The plasma cholesterol, triglyceride and LDL cholesterol conc. are higher by about 3%, 9.1% and 1.7% respectively in smokers than in non smokers. HDL cholesterol is lower in smokers than in non smokers Variation factors…

14. ANALYTICAL METHODOLOGY CHOLESTEROL : Chemical Method : Liebermann- Burchardt R xn Modified Abell Kendall Method (Reference method) Bloors Method Enzymatic Method: Cholesterol oxidase (Routine Lab) GC–MS Method (Reference Method) Isotope Dilution Mass Spectrometry (Definitive Method) TRIGLYCERIDE : Chemical Method Van Handel and Zilversmith Enzymatic Method Glycerol Kinase GC–MS Method (Reference Method)

15. Analytical methods could be: Reference methods: G old standard or fully validated methods that have been developed by standardized bodies, and used as accuracy targets for the more common analytes . Routine methods: S imple and more practical methods that are used in place of Reference methods which are complex, time consuming, at least partially manual and requires a high level of expertise for reliable operation.

16. Desktop portable analyzers and POCT devices C apable of accurately measuring TC, HDL-C & TGs in a few microliter of whole blood, serum or plasma, within few minutes Better suited for public Cholesterol screening programs. The National Institute of Standards and Technology (NIST, USA) has approved the modified Abell -Kendall protocol and Isotope-dilution mass spectrometry (ID-MS) method as reference measurement procedures (RMPs) for blood cholesterol quantification .

17. Cholesterol Estimation CHEMICAL METHODS: Abell Kendall Method Reference Method: Principle: 3 step Cholesterol is hydrolyzed with alcoholic KOH Unesterified cholesterol is extracted with petroleum jelly Measured using the L-B Reaction Liebermann-Burchardt Reaction (L-B Reaction): Cholesterol + Sulfuric acid + Acetic anhydride => bluish green solution Read at 500nm wavelength in a spectrophotometer.

18. Bloors Method: Principle: 2 step Cholesterol is extracted using an alcohol ether mixture Then measured using the L-B Reaction

19. ENZYMATIC METHOD: Cholesterol Oxidase Method (Routine Lab – Assay of Choice): Principle: Cholester y l ester + H 2 Free C holesterol + Fatty Acids cholesterol hydrol ase - > cholesterol oxid ase - > Free Cholesterol + O 2 C holest -4 en-3-one + H 2 2 Trinders Reaction: H 2 2 + Phenol + 4-amino antipyrine peroxidase -> Quinoneimine dye (red) + 2 H 2 O Commercial reagents with the above chemical complexes exists that requires measured sample and reagent to be applied and incubated for the above reactions to take place, under controlled conditions for generation of a colour reaction. Read at 500nm wavelength – Linear up to 600 – 700mg/dL (15.54 – 18.13mmol/L ) …Cholesterol Estimation

20. Advantages: Precise and accurate Less interferences – bilirubin, ascorbic acid, Hb Smaller sample quantity Rapid; does not require preliminary extraction step Can be used to measure unesterified cholesterol by omitting de- esterification step Mild reagents; better suited for automated analyzers …Cholesterol Estimation

21. Disadvantages: They are not absolutely specific for cholesterol. Cholesterol oxidase reacts with other sterols e.g plant sterol Ascorbic acid and Bilirubin interfere by consuming H 2 2 Bilirubin interference can produce falsely high or low values Significant only at conc >5mg/dL decreasing Chol values by 5 – 15 % …Cholesterol Estimation

22. GC–MS METHOD (Reference Method): GAS CHROMATOGRAPHY – MASS SPECTROMETRY Specifically measures cholesterol and does not detect related sterols A gaseous mobile phase is used to pass the sample through a stationary phase. The mobile phase is typically an inert gas such as Nitrogen, Argon, Helium or Hydrogen. They are also referred to as the carrier gas The gas chromatograph is coupled to a mass spectrometer to measure the cholesterol Shows good agreement with the Definitive Method - Isotope Dilution – Mass Spectrometry …Cholesterol Estimation

23. Isotope Dilution – Mass Spectrometry (ID-MS) method A mass spectrometer consists of an ion-source – which forms ions from analytes ; a vacuum pump, mass analyzer and ion detectors It is considered to be a universal detector because it can be used to measure anything that has mass, and can be coupled to almost all analytical techniques. In ID-MS, an analogous compound with a stable isotope and fixed conc. i s mixed with one with a naturally occurring one in sample with unknown conc. i n a process known as ISOTOPE DILUTION ANALYSIS. The compound labelled with stable isotope serves as the internal standard because it behaves nearly identical to the native compound during chromatographic analysis.

24. ID-MS contd … This chromatograph is coupled to a specific mass spectrometric technique. This mass spectrometer simultaneously differentiates and quantifies the compound with normal abundance, or naturally occurring one from the analog ones (with stable isotope) This is the principle of ISOTOPE DILUTION – MASS SPECTROMETRY. This technique has been used to develop definitive methods for a number of clinically relevant analytes .

25. HDL–C Estimation: PRECIPITATION METHOD: Precipitating reagents such as divalent cations and polyanions are used to remove all lipoproteins except HDL Enzymatic method for total cholesterol ( Cholesterol Oxidase ) is used to quantitate HDL–C Demerit: Interference from elevated TG levels causing incomplete sedimentation after centrifuging which results in over estimation of HDL-C

26. MAGNETIC METHOD Similar to the HDL-C precipitation method but uses a precipitant that is complexed to magnetic particle This sediments and does not require centrifugation Has been adapted for use in automated clinical chemistry analysers because, It allows the supernatant to be analysed without the need to remove it from the sedimented complex. …Cholesterol Estimation

27. HOMOGENOUS ASSAY (Direct HDL-C Assay): Enzymatic method: First reagent – “blocks” non-HDLs Use of Antibodies or Polymers or complexing agent e.g Cyclodextrin Modification of cholesterol esterase and oxidase enzymes which makes them selective for HDL-C Use of blanking step that selectively consumes cholesterol from non-HDL species Second reagent – quantifies accessible HDL-C Highly precise and reasonably accurate but lacks specificity for HDL in unusual specimens e.g liver or kidney disease Does not require pretreatment …Cholesterol Estimation

28. The “Three-step Procedure”(Reference method for HDL-C estimation): Ultracentrifugation to remove VLDL Heparin manganese precipitation to remove LDL Analysis of supernatant: cholesterol by the Abell Kendall assay It is more specific Although more tedious and expensive …Cholesterol Estimation

29. LDL–C Estimation INDIRECT METHODS: Fridewald Equation(Calculation Method) – Routine. LDL–Chol(mmol/L) = [TC – HDL-Chol] – Plasma TG/2.175; or, LDL-Chol(mg/dL) = [TC – HDL-Chol] – Plasma TG/5 VLDL (mg/dL) = [TAG]/5 or VLDL (mmol/L) = [TAG]/2.175 The factor [TAG]/5 is an estimate of the VLDL cholesterol and is based on the average ratio of triglyceride to cholesterol in VLDL Equation assumes patient fasted and plasma [TAG] does not exceed 5.0mmol/L …Cholesterol Estimation

30. Limitations: not appropriate in Samples with TG > 400mg/ dL Patients with suspected Dysbetalipoproteinaemia Other limitations: Does not account for cholesterol assosciated with IDL and Lp (a) Underestimate LDL-C in chronic alcoholics Unsuitable for monitoring Mis -classifies 15 – 40 % of patients when TG levels are between 200 to 400 mg/ dL …Cholesterol Estimation

31. Beta-Quantification (Reference method ) for LDL-C : Tedious – reserved for samples where Fridewald equation is inappropriate 2 steps Ultracentrifugation to remove VLDL leaving behind LDL and HDL as well as IDL and Lp(a) Chemical Precipitation of HDL-C from either the whole serum or the infranate obtained from the ultracentrifugation LDL-C is calculated as difference btw Cholesterol measured in infranate and in the HDL fraction VLDL-C is usually calculated as the difference btw that in whole serum and the amount in the infranate fraction

32. VLDL-C/Plasma TG ratio : may be useful in evaluation of Type III hyperlipoproteinemia Expressed in mol / mol or mass/mass Ranges 0.230-0.575 Type III subjects have ratio > 0.689; usually in range of 0.689 – 0.0919

33. Homogenous method: Selectively measures LDL after masking non-LDL cholesterol, OR By selectively solubilizing LDL Advantages: Does not involve measurement of TGs thus non-fasting samples can be used Comparable to calculated LDL results from beta- quantitation on normolipemic specimens

34. T riagl y ceride s : CHEMICAL METHOD: First, Lipids are extracted using chloroform and phospholipids and removed by zeolite absorption Van Handel and Zilversmith Method (former Reference Method): Principle: TAG alcoholic KOH - > Glycerol + Fatty acids Glycerol + periodic acid ---------------> Formaldehyde Formaldehyde + Chromotropic acid-------------> Blue solution Demerit: – Tedious, poorly charaterized

35. ENZYMATIC METHOD: Glycerol Kinase Method: Principle : TAG + 3H 2 lipase -> Glycerol + 3fatty acids Glycerol + ATP glycerol kinase -> Glycerophosphate + ADP Glycerophosphate Glycerophosphate Oxidase -> Dihydroxyacetone + H 2 O 2 Trinder’s reaction: peroxidase -> H 2 O 2 + Chromogen Pink compound + H 2 O Read absorbance at 500nm wavelength, and linear up to 700mg/dL Merits: Fairly specific

36. Triglycerides Desirable Level: <176 mg/dl (<2.0mmol/L); Conversion factor: 0.011 Demerits : Glycerol kinase reacts with endogenous free glycerol causing interference which is clinically insignificant except in DM, emotional stress, IV admin of glycerol containing drugs or nutrients, contamination of blood collecting devices, and before measuring TG

37. QUALITY CONTROL Use of commercial control materials or Produced in-house from freshly collected patient serum, aliquoted into vials quick ly, frozen and stored at -70 o C – Less subject to matrix inter a ction compared to commercial ones Whichever control option is used, a t least two pools should be analysed with levels near decision points for each analyte.

38. Some important considerations Classification of Total, HDL and LDL – Cholesterol Clinical identification of Metabolic syndrome Major risk factors of Coronary Heart Disease (CHD) Categories of risk for LDL – Cholesterol goals

39. Conversion factor for Cholesterol, from mg/dl to mmol /L is 39

40. CONCLUSION Disorders of lipids is of immense importance to medical practice owing to its strong relations to Arteriosclerosis and thus obesity, HTN, DM and other debilitations. E arly detection of deranged blood lipid profile in the management of these conditions usually results in good prognosis.

41. REFERENCES Tietz textbook of Clinical Chemistry and Molecular Diagnostics; fifth ed., by Burtis et al. Clinical Chemistry; Principles, Techniques and Correlations, 7th ed., by Bishop et al. HENRY’S clinical diagnosis & management by lab. methods 21/e

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