Estimation of serum cholesterol

Estimation of serum cholesterol

1. Estimation of Serum Cholesterol Dr. Azad Alam Siddiqui Assistant Professor Department of B.Voc (MMDT) Km. Mayawati Govt.GirlsPG College Badalpur, G.B. Nagar (U.P.)

2. Wh at I s C h o le s t e r ol? W e may a ss o c iat e c h o le s t e r ol w i t h fat t y f o o d s, b u t m os t of the w a xy s u bs t a n ce i s m a d e b y o u r o w n b o di e s . T h e l iv e r p r o du c e s 75% of th e c h ol e st e r o l t hat c i r c ulat e s i n o u r b l oo d . T h e o t h e r 25% c o me s fro m fo o d . A t n o r m al l e v el s, c h o le s t e r ol ac t u al ly p la ys an i m p o r t a n t r o l e i n h el p i n g c e lls d o t h e i r j o bs . B u t c h o l e s t er o l l e v el s a r e p r eca r i o usl y h i g h i n m o re t h an 100 m i l lio n In d i a n s .

3. S y m p t o m s o f H i g h C h o le s t e r o l H i g h c h o le s t e r ol d o e s n o t c au s e any s y m p t o m s . B u t it d o e s c au s e d a ma g e d e e p w i thi n t h e b o d y . O v er t i me, t oo m u c h c h o le s t er o l m a y l e a d t o a build-up of p l a q ue i n sid e t h e a r t e r i e s . K n o w n as a t h e r o s c le r o sis, t hi s c o nd i t i on n arr o w s th e spa c e a v a i labl e f o r b l o o d f l o w a n d c an t r i g g er h e a r t d i s e a s e . T h e g o o d n e w s i s h i g h c h ol e st e r ol i s s i m p le t o d e t ec t , and th ere are m a n y w a ys t o b r i n g it d o w n .

4. ESTIMATION OF TOTAL CHOLESTEROL

5. At the end o f this pr a c t ica l , you sh o uld be a b le t o : Expl a in role o f cho l esterol and li p o p rotei n s i n he a lth and dise a se S t ate t h e d esi r a b l e le v els o f total ch o l esterol and dif f erent li p o p rotei n s S t ate t h e pr i n c ip l e o f c holes t er o l esti m ation & H DL esti m at i on, i nt erpret t h e results ob t a i n ed and correla t e t h em wi t h t h e clin i c al findings

6. Steroid and Sterol

7. Cy c lopen t t a a n n o o pe rh y d d r r o o ph e nan t h r e n e r ing Steroid

8. Cy c lopen t t a a n n o o pe rh y d d r r o o ph e nan t h r e n e r ing Sterol Cholesterol

9. P h ytosterols Zoost e rols Ergosterol Sitost e r o l Sigmas t er o l Fungi P r otozoa Chol e sterol Sterol

10. Fu n ctions of Cholesterol

11. In plasma membr ane M e m b rane f luidity Lipid r afts P r ec ursor mol ecule of: Ster o id horm o n e s Vitamin D Bile Acids Nerve co nduction Signal tr a ns duction Vitamin D

12. Cholesterol Synthesis Fro m Ace t yl CoA; HMG CoA Reducta s e Mai n ly in Live r ER a n d C y to s ol Transpor t ed to p e rip hera l tiss ues Bef o re tra n sport, chol e ster o l is e ster i f i ed t o f o rm c h oles t erol e s t e rs

13. Lipoproteins

14. Lipop ro tein Apo- lipop r ote i n + Lipid  Lipopro t e in

15. C l a ssi f i ca t ion o f L i p o p r o teins L i p o p r o t ei n s c an b e cl a s si f ied- B as e d on d e nsit y - T h ey a r e se p arated b y u l t r ace n tr if u g a t i o n . d e p e n d i n g u p o n t h e flo a t a t i o n c o n s t a n t (SF) , f i v e ma j o r g r o u p s o f l i p o p r o t e i n s h a v e b een i d e n t i f ied th at are i m p o rta n t p h y si ol o g i c a ll y a n d i n cl i n i c a l di a g n o s i s . ( i) C hy l o m ic r o n - De ri v e d f r o m i n te s t i n al ab s o r p ti o n o f tr i acy l g l yc e r ol a n d o t h er l i p i d s ; transport lipids absorbed from the intestine to adipose, cardiac, and skeletal muscle tissue, where their triglyceride components are hydrolyzed by the activity of the  lipoprotein lipase , allowing the released  free fatty acids  to be absorbed by the tissues. when a large portion of the triglyceride core has been hydrolyzed , chylomicron remnants are formed and are taken up by the liver, thereby also transferring dietary fat to the liver. d e n si t y i s g e n er all y l e s s t h a n 0. 9 5 wh i l e t h e me a n d i am e t er l i e s b e t w een 1 00 - 5 n m

16. C l a ssi f i ca t ion o f L i p o p r o teins B a s e d on d e n s i t y ( c o nt d . ) ( ii) V e r y l o w d e n s i t y l i p o pr o t e ins ( V L D L ) , der i v ed f r o m th e l i v er f o r th e e x p o rt o f t ri acy l g l yc er o l ; The VLDL particles mainly carry triglycerides to the tissues. VLDL is similar to LDL cholesterol, but LDL mainly carries cholesterol to the tissues instead of triglycerides. d e n si t y l i e s b e t w een . 9 5 - 1. 00 6 a n d t h e m ean d i a meter l i e s b e t w een 3 - 8 n m . ( i i i ) Int e r m e d i a te d e nsit y l i p o pr o t e ins (I D L ) are d er i v ed fr o m t h e c ata b ol is m of VL DL,w i t h a de n s i ty r a n g i n g i n ter m ed i ate b e t w een V LDL a n d L DL l i p o p r o te i n s i. e . r a n g i n g b e t w e e n 1.0 6 - 1 . 1 9 a n d t h e m e an d i a m eter r a n g e s b e t w e e n 2 5 - 5 n m .

17. C l a ssi f i ca t ion o f L i p o p r o teins B as e d on d e nsit y ( c o nt d. ) i v) Lo w - d e n s i t y l ip o pr o t e i n s (LDL) , re p r e se n t i n g a f i n al s t a g e i n t h e c a t a b ol is m of VL DL; transport cholesterol from liver to other tissues, de n s i t y l i e s b e t w een 1 .0 1 9 - 1 . 63 a n d mean d i a meter l ie s b e t w een 1 8 - 2 8 n m ( i v) H i g h- d e nsit y l i p o p r o t e ins ( H DL) , i n volv ed i n c h o l e s ter o l t r a n s p o r t from peripheral tissues to liver a n d a l s o i n VL DL a n d c h yl o m i c r o n meta b ol is m . D e n s i t y ra n g e s b e t w een 1. 6 3 - 1. 1 2 1 a n d t h e me a n d i ameter v ar ie s b e t w een 5 - 15 n m .

18. A p o - B 4 8 (Str u ct u r a l) A po - E ( B i nd s to A po - E r ec ep t o r) A p o - C 2 ( A ct i v ate s LpL ) A po - A 1 (Str u ct u r a l) A po - B 100 (Str u ct u r a l, B i nd s to LD L - R ece p t o r) A po - E A po - C2 A po - B 100

19. L i pop r o tein L i p ase A p o C 2 A p o E Dietary fats and Cholesterol

20. Endogenous Fats and Cholesterol Lipopro t e in Lipase Hep a tic Lipase A po C2 A po E A po B- 100

21. After c o n v ers i on to bile ac i ds Di r ectly i n to t he bile E x f o li a tion of cells Excretion of Cholesterol

22. Ultra-centrifugation Electrophoresis Separation of Lipoproteins

23. Hy perlipide m ias and Hypercholesterolemia

24. Why is hyperlipidemia dangerous?

25. Causes of Hyperlipidemia Ty p e 2 Dia b etes Mel l i t us Hyp o thyroidism Nep h rot i c syndrome Alcoh o lism High carb o hydra t e i nt a k e Glyc o gen s t orage dis o rders Cushing syndrome Diet Obesity Pregnancy Acute hepatitis

26. Hy po cholesterolemia

27. Hypocholesterolaemia can be caused by several things: Anaemia (a low amount of red blood cells) Hyperthyroidism Malnutrition, or a lack of food Liver disease The body being unable to absorb food (malabsorption) Rare genetic conditions, such as hypobetalipoproteinaemia and abetalipoproteinemia Tangier disease

28. D e sirable L e ve l s

29. NC E P - A T P III G u i d e li n es Total Cholesterol < 2 00 mg/dL Desirable 20 -239 mg/dL Bor d erline high > 240 mg/dL Hi g h HDL Cholester o l < 4 mg/dL L o w ≥ 60 mg/dL High Tr i -Ac y l Gly c er o l < 1 50 mg/dL N o rmal 150-199 mg/dL Hi g h 200-499 mg/dL H y p e r t rigly c e r id e mic >500 mg/dL Very high LDL Cholester o l < 70 mg/dL In pa t ients with risk fact o rs < 1 00 mg/dL Optim a l 160-189 mg/ d L Hi g h >190 mg/dL Very high

30. Labo r ato ry Est i mati o n

31. Lipid p r o file ◉ Total Chol e ster o l, T r i -Acyl Gl y ce r ol, LDL and HDL Fasting sample f o r T r i - Acyl Gl y ce r ol esti m ation T e sts should be r e pe a ted on a di f f e r e nt o c c a si o n Fri e dwald equa t io n : T o tal Chol e ster o l = HDL + LDL + VLDL VLDL = TAG/5 Not valid i f TAG > 400 mg/dL

32. Protocol for Total Chole s te r ol Es t ima t ion Zak’s Method Enzymatic method

33. Zak ’ s Method • T h e pr o tei n s present in t he serum sa mple ar e first pr e cipitated by ad d ing Ferric chl o rid e - Acetic acid re a gen t . T h e pr o tein free filtrate is treated with conc. H 2 SO4. C h o l ester o l ↓ de h ydr ation C hol e st a - 3 - 5 -di e ne (2 m o l e cul e s) ↓ oxidation Bis cho l esta- 3 - 5 -di e ne ( 1 m o l e cul e ) ↓ sulpho n ation Lib e r m a n -Bur c hard r e acti o n Mon o s u l p honic Acid d e rivatives (Gr e en c o l o u r ) Sal k ows k i r e acti o n Disulphonic Acid d e rivatives ( Red c o l o u r ) F e 3+

34. 1 . Take .1 mL se r um, add 9 .9 mL o f FeC l 3 - CH 3 C O O H mixt ure and mix thoroughly with gla s s ro d . 2. Centrifuge at 2000 rp m f o r 10 min 3. Take 3 test tu b es and m a rk them as B, S and T Blan k Stan d ard Superna tan t ----- - ------ 4. Mi x well and k e ep in water b a th at 5 0-60 ° C f o r 10 mi n. C o ol to room te mp e r a ture and me a sure the O D at 540 n m. Test 5 mL FeCl 3 -CH 3 C OO H 5 m L 4. 9 mL ----- - 0. 1 mL 3 m L 3 mL ------ Stan d ard (200 mg /d L ) ------ Con c.H 2 S O 4 3 mL

35. Enzymatic Method (CHOD/POD method) C hole sterol ester Ch o leste r ol + Fatty a c id Chole sterol + O 2 Chol e s t -4-en-3- o n e + H 2 O 2 H 2 O 2 + 4- AP + Phen o l 2H 2 O + Quin o n e - imi n e ◉ Absorbance of Quin o n e imi n e at 5 1 nm is d i r ectly pr o p o rti o n a l to the c o n c e n trati o n of chol e ster o l in serum C E h y d r o l ase Cho l este r o l Oxi d ase Per o x i d ase

36. Assay Procedure: Blood drawn from patients fasting for 12 to 16 hours. Mix well and incubate for 10 min at 37 C and measure the absorbance of standard and test sample against the reagent blank at 510nm. Pip ette into tubes marked Blank Standard Test Working Reagent 1.O ml 1.O ml 1.O ml Double distilled water 20µl --------------------- -------------------------- Serum sample ----------------------- --------------------- 20µl Standard (200mg/dl) ----------------------- 20µl ---------------------------

37. Calculation: Total serum cholesterol in mg/dl = --------------------- Χ concentration of standard Result: ----------mg/dl Absorbance of Test Absorbance of standard

38. Protocol for HD L -Cholesterol Estimati o n

39. T o 1 mL serum, add .1 mL Phospho t ungst a te re a gent an d 5 0 µl MgCl 2 solutio n . Ce n tri f uge a t 250 rpm fo r 1 0 minutes. LDL, VLDL an d chylomi c rons are precipitat e d by polyani o ns i n the pr e sence o f metal io n s to le a ve HDL i n solutio n . C oll e ct the super n ata nt and estimate cholester o l b y the total chol e ster o l method.

40. Pr e ca u tions

41. Gl a ci a l ac i d i s ex t r e mely volatil e , i r r itant an d co r r o si v e to muc o us membr a n e . M o uth pipetting o f a c etic a c id should be str i ctly avoided . N o mouth pip e tti n g o f H 2 SO 4 H 2 SO 4 co n tai n ing so l ution shou l d be hand l ed care f ully and c o nt a ct with skin should b e a v o i ded Alwa y s add a c id to water Sta n da r d pr e c a utions should be f o llowed fo r handl i ng serum.