This document discusses HDL cholesterol and LDL cholesterol testing. It provides information on: 1. HDL cholesterol functions to remove cholesterol from tissues and transport it to the liver for degradation or excretion, playing an anti-atherogenic role. LDL cholesterol transports cholesterol to peripheral tissues and is a key factor in atherosclerosis pathogenesis. 2. Methods for measuring HDL and LDL cholesterol include ultracentrifugation, electrophoresis, homogeneous enzymatic assays, and calculation methods like the Friedwald equation. 3. The cobas method for HDL and LDL cholesterol measurement is a homogeneous enzymatic colorimetric assay that uses cholesterol esterase, cholesterol oxidase, and peroxidase enzymes to produce a colored end product that is measured

1. 1
PEMERIKSAAN
KOLESTEROL HDL DAN
KOLESTEROL LDL
Miftahul ilmiah,dr/ Dra. Soehartini, MS, Apt

2. Pendahuluan
2
 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 HDL
3
 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 LDL
5
 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. Pemeriksaan
7
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 HDL
9
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 infranatan
10

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 elektroforesis
12
 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 HDL
15
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 + H2O2
16

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 agent
17

18. Kolesterol HDL
18
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 metode
20
 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 pigment
21

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. interference
23
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 LDL
24
 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. prinsip
26
 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 + O2
28

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 4H2O
29

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 preservative
33

34. Interpretasi hasil
34
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. Interference
35
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 presipitasi
39
 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
HDL
40

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 androgens
42 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

45. 45

46. 46

47. 47

48. 48

49. 49

50. 50

51. 51

52. ‹#›

53. ‹#›

54. ‹#›

55. 55

56. HDL-C Cobas
56
 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 cobas
62
 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 cobas
63
 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