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
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.
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
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.
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%)
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
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
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
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
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
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.
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
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)