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Miftah lipoprotein Presentation Transcript

  • 1. 1PEMERIKSAANKOLESTEROL HDL DANKOLESTEROL LDLMiftahul ilmiah,dr/ Dra. Soehartini, MS, Apt
  • 2. Pendahuluan2  Kolesterol HDL dan kolesterol LDL merupakan prediktor kuat untuk penyakit jantung koroner  Kolesterol LDL sebagai faktor kunci dalam patogenesis atherosclerosis dan penyakit jantung koroner  Kolesterol HDL merupakan antiatherogenesis dan mencegah penyakit jantung koroner
  • 3. Kolesterol HDL3  Densitas 1,063 – 1,21  Kandungan protein tinggi (Apo A,C,E, tu A1)  Fungsi: mengambil kolesterol dari jaringan perifer ke hati, lalu didegradasi atau diekskresi empedu (removal / reverse transport dari kolesterol)  Dari degradasi VLDL & kilomikron. Inti HDL = kolesterol ester yang diambil di jaringan perifer dengan bantuan enzim LCAT.
  • 4. 4
  • 5. Kolesterol LDL5  d = 1,006 – 1.063  Kaya akan kolesterol Dibentuk di plasma, berasal dari degradasi VLDL.  Fungsi: mengangkut kolesterol ke jaringan perifer.  Apoprotein: B100, B48
  • 6. 6
  • 7. Pemeriksaan7 Persiapan pasien :  Tidak ada perubahan pola makan selama 2 minggu sebelum pemeriksaan  Tidak ada pertambahan atau penurunan berat badan  Tidak melakuan aktivitas berat 24 jam sebelum pemeriksaan  Sebaiknya puasa 12 jam sebelum pengambilan sampel
  • 8. 8 Teknik pengambilan sampel:  Posisi standar duduk 5 menit sebelum pengambilan sampel.  Tourniket tidak digunakan > 2 menit.  Plasma atau serum dapat digunakan.  Antikoagulan EDTA merupakan pilihan utama jika menggunakan plasma  Sampel dapat disimpan pada suhu 4⁰C selama 3-4 hari sebelum dianalisis.  Suhu -20⁰C bertahan beberapa bulan.  Suhu -70 ⁰C bertahan sampai beberapa tahun.
  • 9. Metode pemeriksaan HDL9 Ultrasentrifugasi  Plasma atau serum disesuaikan pada densitas 1,063 g/ml dengan KBr (415 mg/5 mL) dan disentrifus pada 105 000 g selama 24 jam  Semua lipoprotein dipisahkan menurut densitasnya, HDL pada 1,063-1,21 g/mL
  • 10. Metode pemeriksaan HDL Ultrasentrifugasi  Plasma/serum + larutan KBr (415mg/5 mL) Sesuaikan densitas pada 1,063 g/mL Sentrifus sampel pada 105.000 g selama 24 jam pada suhu 16⁰ C Buang supernatant ( VLDL dan LDL) Analisis kadar kolesterol dalam infranatan10
  • 11. 11 Kromatografi kolom  Kolesterol HDL diisolasi dan dipisahkan dengan cara kromatografi penukar ion (ion- exchange) atau ukuran molekul (gel permeation)  Sulit untuk menentukan kontrol kondisi kromatografi yang tepat
  • 12. metode elektroforesis12  HDL dipisahkan dari lipoprotein lain berdasarkan muatan dan ukurannya  Beberapa metode elektroforesis untuk penentuan HDL-C : - starch block electroforesis digunakan untuk isolasi HDL yang berjumlah besar, tapi tidak yang bkuantitatif - agarose gel electroforesis presisi tidak adekuat untuk penggunaan klinik - polyacrylamide gel electroforesis untuk level HDL yang dibawah perkiraan jarang digunakan
  • 13. Prosedur: isolasi HDL dengan heparin- manganese chloride (metode presipitasi)13 Reagen  Larutan Mangaan klorida 1 mol/L. Larutkan 19,791 g MnCl2.4H₂O ke dalam distilled water, dan bawa sampai volume 100 mL. Larutan ini stabil sampai 3 bulan pada 4⁰C.  Larutan Heparin 4000 U/mL. Encerkan 1 mL heparin dengan 1,5 mL larutan salin 0,9%. Larutan ini stabil selama 1 minggu pada suhu 4⁰C.
  • 14. 14
  • 15. Kolesterol HDL15 Pemeriksaan kuantitatif menggunakan vitros chemistry product
  • 16. HDL + non-HDL PTA/MgCl2 HDL + non HDL HDL Emulgen B-66 Cholesterol + HDL cholesterol esters + proteins HDL kolesterol Cholesterol ester hydrolase Cholesterol + fatty esters + H2O acids Cholesterol + O2 Cholesterol oksidase Cholest-4-en-3 + H2O2 H2O2 + leuco dye Peroxidase Dye + H2O216
  • 17. Reagen : Bahan Kadar • Emulgen B-66 0,7 mg • Phosphotungstic acid 0,3 mg • Magnesium chloride 0,2 mg • Cholesterol oxidase (Cellulomonas) 0,8 U • Cholesterol ester hydrolase (Candida rugosa) 1,2 U • Peroxidase (horseradish root) 2,2 U • 2-(3,5-dimethoxy-4-hydroxyphenyl)-4,5-bis-(4- dimethylaminophenyl) imidazole leuco dye) 0,02 mg • Bahan lain: Pigment, Binders, Buffer, Surfactants, stabilizers, scavenger, cross-linking agent17
  • 18. Kolesterol HDL18 Metode Homogeneous enzymatic colorimetric assay (cobas)
  • 19. Bahan Reagen HEPES buffer: 10.07 mmol/L; CHES 96.95 mmol/L, 1 pH 7.4; Dextran sulfate: 1.5 g/L; magnesium nitrate hexahydrate: >11.7 mmol/L; HSDA: 0.96 mmol/L; ascorbate oxidase (Eupenicillium sp., recombinant): >50 μkat/L; peroxidase (horseradish): >16.7 μkat/L; preservative Reagen HEPES buffer: 10.07 mmol/L, pH 7.0; 2 PEG-cholesterol esterase (Pseudonomas spec.): >3.33 μkat/L; PEG-cholesterol oxidase (Streptomyces sp., recombinant): >127 μkat/L; peroxidase (horseradish): >333 μkat/L;19 4-amino-antipyrine: 2.46 mmol/L; Preservative
  • 20. Prinsip metode20  Magnesium sulfat, dekstran sulfat membentuk kompleks water-soluble dengan LDL, VLDL, dan kilomikron yang tahan terhadap enzim PEG-modified  Kadar kolesterol pada HDL kolesterol ditentukan secara enzimatis oleh kolesterol esterase dan kolesterol oksidase yang bergabung dengan PEG menjadi kelompok amino (sekitar 40%)
  • 21. HDL-cholesterol PEG-cholesterol esterase HDL-cholesterol + esters + H2O RCOOH HDL-cholesterol PEG-cholesterol oxidase ∆4-cholestenone + + O2 H2O2 2H2O2 + 4-amino-antipyrine + Peroxidase Purple-blue HSDA + H+ + H2O pigment21
  • 22. 22 Kalkulasi  Faktor konversi : mmol/L x 38,66 = mg/dL mmol/L x 0,3866= g/L mg/dL x 0,01 = g/L mg/L x 0,0259 = mmol/L Interpretasi hasil HDL: Mg/dL Mmol/L g/L Rendah <40 < 1,03 < 0,40 Tinggi > 60 > 1,55 > 0,60
  • 23. interference23 Pemeriksaan HDL kolesterol dengan alat cobas tidak terpengaruh:  kadar bilirubin konjugasi sampai kira-kira 30 md/dL, bilirubin tidak terkonjugasi 60 mg/dL  Kadar hemoglobin sampai 1200 mg/dL  Kadar trigliserida sampai 1200 mg/dL  Kadar asam askorbat sampai kadar 50 mg/dL  Peningkatan kadar asam lemak bebas dan denaturasi protein menyebabkan false elevated HDL kolesterol
  • 24. Pemeriksaan LDL24  Ultrasentrifugasi  Elektroforesis  Friedwald calculation :  Kol-LDL = Kol Total – (Kol-HDL) – TG/5  Syarat: TG < 400 mg/dl (Kol-VLDL≠TG/5)
  • 25. .25 Metode pemeriksaan LDL pemeriksaan kuantitatif menggunakan vitros chemistry product
  • 26. prinsip26  Terdapat 2 langkah pemeriksaan: penambahan reagen 1 Non LDL kolesterol dieliminasi pada oleh reaksi kolesterol esterase dan kolesterol oksidase menjadi kolestenon dan hidrogen peroksida. Hidrogen peroksida bereaksi dengan enzim katalase menjadi scavenger
  • 27. 27  Penambahan reagen 2 Katalase dinonaktifkan oleh sodium azide Surfaktan memisahkan kolesterol dan kolesterol ester dari partikel LDL Kolesterol ester bereaksi dengan kolesterol esterase menjadi kolesterol dan asam lemak Kolesterol bereaksi dengan kolesterol oksidase menjadi kolestenon dan hidrogen peroksida Hidrigen peroksida bereaksi dengan TOOS dan 4- aminoantipirin bereaksi dengan peroksidase membentuk pewarna berwarna quinon yang diukur dengan spektrofotometer pada 600 nm
  • 28. Langkah I: Penambahan Reagen1 – Eliminasi kolesterol dari kilomikron, VLDL, dan HDL. Non-LDL cholesterol Cholesterol esterase Cholesterol + esters fatty acid Cholesterol + O2 Cholesterol oxidase Cholestenone + H2O2 2H2O2 Catalase+ 2H2O + O228
  • 29. Langkah 2: penambahan Reagen2 – Pengukuran spesifik LDLC LDL particles Surfactans Cholesterol + cholesterol esters + protein Cholesterol esters Cholesterol esterase Cholesterol + fatty acid Cholesterol + O2 Cholesterol oxidase Cholestenone + H2O2 2H2O2 + 4-aminoantipyrine Peroxidase Quinine pigment + + TOOS 4H2O29
  • 30. Reagen Bahan Kadar Reagen Cholesterol esterase (Pseudomonas sp.) 600 U/L 1 Cholesterol oxidase (Microorganism) 500 U/L Catalase (corynebacterium glutamicum) 1 200 000 U/L Surfactant (polyoxyethlene compound) 0,3% Dye precursor (N-Ethyl-N-[2-hydroxy-3- 2,0mM sulfopropyl]-3-methylaniline [TOOS]) Bahan lain (buffer, garam anorganik, scavenger, preservative, processed water) Reagen Peroxidase (Horseradish) 5 000U/L 2 4-aminoantrpyrine 4,0 mM Catalase inhibitor (sodium azide) 0,05% Polyoxyethilene alkylphenyl ether 1%30 Bahan lain (buffer, preservative, processed
  • 31. Pemeriksaan LDL dengan Metode Homogeneous enzymatic colorimetric assay (cobas)31 Prinsip Kolesterol ester oleh enzim kolesterol esterase menjadi kolesterol bebas dan asam lemak bebas Dengan adanya oksigen, kolesterol pada LDL kolesterol dioksidasi oleh enzim kolesterol oksidase menjadi kolestenon dan hidrogen peroksida Dengan adanya enzim peroksidase, H2O2 bereaksi dengan 4-aminoantipirin dan HSDA membentuk pewarna purple-blue Intensitas warna dari pewarna ini diukur dengan fotometer pada 585 nm untuk mengetahui kadar kolesterol
  • 32. Metode Homogeneous enzymatic colorimetric assay (cobas) LDL-cholesterol Detergent Cholesterol+ free fatty acids esters + H2O (selective micellary Cholesterol esterase solubilization) LDL-cholesterol Cholesterol oxidase ∆4- cholestenone + + O2 H2O2 Peroxidase Purple – blue 2H2O2+ 4-aminoantrpyrine + pigment+5H2O (Abs. HSDA+H2O +H+ max.= 585 nm)32
  • 33. Reagen 1 MOPS (3-morpholinopropane sulfonic acid) buffer: 20.1 mmol/L, pH 6.5; HSDA: 0.96 mmol/L; ascorbate oxidase (Eupenicillium spec., recombinant): ≥50 μkat/L; peroxidase (horseradish): ≥167 μkat/L; preservative Reagen 2 MOPS (3-morpholinopropane sulfonic acid) buffer: 20.1 mmol/L, pH 6.8; MgSO4·7H2O: 8.11 mmol/L; 4-aminoantipyrine: 2.46 mmol/L; cholesterol esterase (Pseudomonas spec.): ≥50 μkat/L; cholesterol oxidase (Brevibacterium spec., recombinant): ≥33.3 μkat/L peroxidase (horseradish): ≥334 μkat/L; detergent; dan preservative33
  • 34. Interpretasi hasil34 Klasifikasi mg/dL mmol/L g/L Optimal < 100 < 2,59 <1 Mendekati 100-129 2,59-3,34 1-1,29 optimal Borderline 130-159 3,36-4,11 1,30-1,59 Tinggi 160-189 4,14-4,89 1,60-1,89 Sangat tinggi >190 >4,91 > 1,90
  • 35. Interference35 Pemeriksaan kolesterol LDL dengan alat cobas tidak terpengaruh:  kadar bilirubin konjugasi sampai kira-kira 30 mg/dL, bilirubin tidak terkonjugasi 60 mg/dL  Kadar hemoglobin sampai 1000 mg/dL  Kadar asam askorbat sampai kadar 50 mg/dL
  • 36. 36
  • 37. 37  TERIMA KASIH
  • 38. 38  TERIMA KASIH
  • 39. Metode presipitasi39  Polyanions (heparin, dekstran sulfat), phosphotungstate, poly ethylene glycol, in presence of divalentcations, digunakan untuk presipitasi ukuran lebih besar, lipoprotein densitas rendah  Pengukuran HDL secara kuantitatif dengan menggunakan supernatan
  • 40. Lipoproteins: HDL Nascent HDL Liver C HDL PERIPHER recept AL or Lecithin: TISSUES cholesterol Cholesterol acyltransferase ester + Apo HDL40
  • 41. 41
  • 42. Fig. 25-5 Metabolism of high-density lipoprotein (HDL) in reverse cholesterol transport. (LCAT, lecithin:cholesterol acyltransferase; C, cholesterol; CE, cholesteryl ester; PL, phospholipid; A-I, apolipoprotein A-I; SR-B1, scavenger receptor B1; ABCA 1, ATP binding cassette transporter A1.) Preβ-HDL, HDL2, HDL3 - see Table 25–1. Surplus surface constituents from the action of lipoprotein lipase on chylomicrons and VLDL are another source of pre -HDL. Hepatic lipase activity is increased by androgens42 and decreased by estrogens, which may account for higher concentrations of plasma HDL 2 in women.
  • 43. LDL – Does Size Matter? “LDL size correlates positively with plasma HDL levels and negatively with plasma triglyceride concentrations, and the combination of small, dense LDL, decreased HDL cholesterol and increased triglycerides has been called the „atherogenic lipoprotein phenotype‟. This partly heritable trait is a feature of the metabolic syndrome, and is associated with increased cardiovascular risk. LDL size seems to be an important predictor of cardiovascular events and progression of coronary artery disease, and a predominance of small, dense LDL has been accepted as an emerging cardiovascular risk factor by the National Cholesterol Education Program Adult Treatment Panel III. However, other authors have suggested that LDL subclass measurement does not add independent information to that conferred by the simple LDL concentration, along with the other standard risk factors.7 Thus it remains debatable whether to measure LDL particle size for cardiovascular risk assessment, and if so, in which categories of patients.”43 Rizzo & Berneis, Q. J. Med. 2006; 99:1-14.
  • 44. 44
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  • 52. ‹#›
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  • 54. ‹#›
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  • 56. HDL-C Cobas56  Calibration  Traceability:19 This method has been standardized against the designated CDC  reference method (designated comparison method).20 The standardization  meets the requirements of the “HDL Cholesterol Method Evaluation Protocol  for Manufacturers” of the US National Reference System for Cholesterol,  CRMLN (Cholesterol Reference Method Laboratory Network), November 1994.  S1: 0.9% NaCl  S2: C.f.a.s. Lipids  Calibration frequency  Two-point calibration is recommended:  • after reagent lot change  • as required following quality control procedures
  • 57. 57  Measuring range  0.08–3.10 mmol/L (3–120 mg/dL).  Determine samples having higher concentrations via the rerun function.  Roche/Hitachi 904, 911, 912, 917, MODULAR P analyzers:  Dilution of samples via the rerun function is a 1:2 dilution. Results from samples  diluted by the rerun function are automatically multiplied by a factor of 2.  On instruments without rerun function, manually dilute samples with  higher concentrations using 0.9% NaCl (e.g. 1 + 1). Multiply the  result by the appropriate dilution factor (e.g. 2).
  • 58. Presisi HDL (cobas)58 Within run Between run sample Mean Mean CV Mean Mean CV Mg/dl Mmol/L % Mg/dL Mmol/L % Human serum low 34,4 0,891 0,95 31,6 0,818 1,3 Human serum high 80,4 2,08 0,60 63,4 1,64 1,2 Precinorm L 47,2 1,22 0,90 41,8 1,08 1,1 Precipath HDL/LDL-C 35,9 0,930 0,81 33,0 0,855 1,8
  • 59. Precise LDL (cobas)59
  • 60. 60  Analytical sensitivity (lower detection limit)  0.08 mmol/L (3 mg/dL)  The detection limit represents the lowest measurable analyte level  that can be distinguished from zero. It is calculated as the value  lying three standard deviations above that of the lowest standard  (standard 1 + 3 SD, within-run precision, n = 21).
  • 61. The simple principle of the homogeneous assay for sd-LDL-C. Surfactant A (polyoxyethylene benzylphenyl ether derivative) reacts with TRLs and HDL, and cholesterol in these lipoproteins is eliminated by the action of cholesterol oxidase/esterase and catalase in step 1. Sphingomyelinase specifically reacts with lb-LDL, while surfactant B (polyoxyethylene styrenephenyl ether derivative) protects sd-LDL from the actions of sphingomyelinase and cholesterol oxidase/esterase (R1). The sd-LDL-C that escapes via the action of these enzymes is measured by the standard cholesterol assay in step 2.61
  • 62. Hdl cobas62  Passing/Bablok29 Linear regression  y = 0.984 x - 0.047 y = 0.986 x - 0.046  τ = 0.971 r = 0.998  Number of samples measured: 55  The sample concentrations were between 0.20 and 2.49 mmol/L  (7.7–96.3 mg/dL).
  • 63. Ldl cobas63  Measuring range  0.078–14.2 mmol/L (3–550 mg/dL or 0.03–5.5 g/L)  Determine samples with LDL-cholesterol concentrations > 14.2 mmol/L  (> 550 mg/dL) via the rerun function.  Dilution of samples via the rerun function is a 1:2 dilution. Results from samples  diluted by the rerun function are automatically multiplied by a factor of 2.  On instruments without rerun function, manually dilute samples with 0.9%  NaCl (e.g. 1 + 1). Multiply the result by the appropriate dilution factor (e.g. 2).
  • 64. 64  Expected values14  Levels in terms of risk for coronary heart disease:  Adult levels:  Optimal < 2.59 mmol/L (< 100 mg/dL)  Near optimal/above optimal 2.59–3.34 mmol/L (100–129 mg/dL)  Borderline high 3.37–4.12 mmol/L (130–159 mg/dL)  High 4.14–4.89 mmol/L (160–189 mg/dL)  Very high ≥ 4.92 mmol/L (≥ 190 mg/dL)  Each laboratory should investigate the transferability of the expected values to  its own patient population and if necessary determine its own reference range.
  • 65. 65  Analytical sensitivity (lower detection limit)  Detection limit: 0.078 mmol/L (3 mg/dL or 0.03 g/L)  The detection limit represents the lowest measurable analyte level  that can be distinguished from zero. It is calculated as the value  lying three standard deviations above that of the lowest standard  (standard 1 + 3 SD, within-run precision, n = 21).
  • 66. 66  EDTA plasma was the traditional choice in lipid research laboratories, especially for lipoprotein separations, because the anticoagulant enhances stability by chelating metal ions. (bishop page 305)
  • 67. 67