03 血浆脂蛋白及其代谢紊乱

nullLIPOPROTEIN METABOLISM AND MEASUREMENT LIPOPROTEIN METABOLISM AND MEASUREMENT Fang Zheng The Department of Laboratory Medicine Wuhan University School of Medicine Vocabulary Vocabulary lipoprotein 脂蛋白 apolipoprotein载脂蛋白 cholesterol胆固醇 phospholipid 磷脂 triglyceride甘油三酯 cholesteryl ester 胆固醇酯 spherical球形的 hydrophobic疏水的 hydrophilic亲水的 aqueous液相的,液体的 chylomicron乳糜微粒 endogenous内源性的 synthesize合成 dietary饮食的 extrahepatic肝外的 macrophage巨嗜细胞 intestine小肠 liver肝脏 receptor受体 metabolism代谢 exogenous外源性的 scavenger清道夫 ultercentrifugation超速离心 electrophoresis电泳 fatty acid脂肪酸 glycerol甘油 lecithin卵磷脂 cholesterol acyl transferase胆固醇酰基转移酶 integrity完整性 syringe注射器 amylase淀粉酶 molecular weight分子量 density密度 atherogenic chronic慢性的;患慢性病者 abdominal a. 腹部的 emergency急诊;急救 osmolality克分子渗透压浓度 nullhypertriglyceridaemia高甘油三酯血症 alcohol乙醇;酒精 discrepancy差异, 差别 phenotype现[型] xanthelasma黄斑瘤 sequelae后遗症 pancreatitis胰腺炎 thrive繁荣, 成功 cataract白内障 xanthomas黄色瘤 manifestation临床表现 eruptive a. 暴发的, 喷火的 primary主要的 hypercholesterolaemia高胆固醇血症 hyperlipidaemia abetalipoproteinaemia无β脂蛋白血症 analphalipoproteinaemia neurological神经学上的 diabetes mellitus糖尿病 diuretic利尿剂,利尿的 hypothyroidism甲状腺功能减退 nephrotic 肾病的 cirrhosis硬化 thyroiditis甲状腺 arcus senilis老年环 pathogenic病原性的 essence要素;香精 advocate n. 提倡者, 拥护者; v. 主张, 提倡 pancreas 胰脏 myocardial infarction心肌梗死 adjunct附属物 ContentContentThe Lipoprotein involved Five Main Lipoprotein Main apolipoprotein Lipoprotein Metabolism Lipoprotein Measurement null Structure of LipoproteinsThe lipoprotein system evolvedThe lipoprotein system evolved A lipoprotein is a complex spherical structure which has a hydrophobic core wrapped in a hydrophilic coating. So fats can be transported around the body in the aqueous environment of the plasma. The five main lipoproteins and their function The five main lipoproteins and their functionIsolation of lipoprotein using ultercentrifugationIsolation of lipoprotein using ultercentrifugationThe electrophoresis pattern of lipoproteinsThe electrophoresis pattern of lipoproteinsTable Properties of some human apolipoproteinsTable Properties of some human apolipoproteinsApolipoprotein Molecular Site of synthesis Function weight A-I 28 000 Intestine, liver Activates LCAT A-II 17 000 Intestine, liver - B100 549 000 Liver Triglyceride and cholesterol transport. Binds to LDL receptor B48 264 000 Intestine Triglyceride transport C-I 6600 Liver Activates LCAT C-II 8850 Liver Activates LPL C-III 8800 Liver ?Inhibits LPL E 34 000 Liver, intestine Binds to LDL receptor and macrophage probably also to another specific liver receptor 187000~ 662000 Liver Inhibits the fibrinolysin Apolipoproteins are the protein components of the lipoproteins. They are important in: Apolipoproteins are the protein components of the lipoproteins. They are important in:  maintaining the structure integrity of the lipoproteins regulation certain enzymes which act in lipoproteins receptor recognition The gene structure of ApoAI, AII, AⅣ，B100, CⅡ，CⅢ and E The gene structure of ApoAI, AII, AⅣ，B100, CⅡ，CⅢ and E The VLDLThe VLDLThe LDLThe LDLnull The structure of apoE The structure of apoEApo(a)Apo(a)The key enzyme systems are involved in lipoprotein metabolism, e.g. : The key enzyme systems are involved in lipoprotein metabolism, e.g. : Lipoprotein lipase(LPL) releases free fatty acids and glycerol(n.甘油, 丙三醇) from chylomicrons and VLDL into the tissues. Lecithin(n.[生化] 蛋黄素, 卵磷脂): cholesterol acyl(n.[化]酰基) transferase(LCAT)(n.[生化]转移酶) forms cholesteryl esters from free cholesterol and fatty acids. 3-hydroxy-3-methylglutaryl-CoA (HMGCoA)　synthase ACAT(脂酰辅酶CoA胆固醇脂酰转移酶) Other Enzyme and ProteinOther Enzyme and ProteinHepatic lipase(HL) or hepatic triglyceride lipase(HTGL) releases free fatty acids and glycerol(n.甘油, 丙三醇) from β－VLDL, VLDL and VLDL remnant into the tissues. HMGCoA reductase is the key enzyme in the synthesis of cholesterol(to synthesise mevalonic acid) nullCETP(cholesterol ester transfer protein)ATP-binding cassette transporter A1(ABC1)nullThe exogenous lipid cycle The endogenous lipid cycle Lipoprotein Metabolism Lipoprotein MetabolismThe metabolism of CMThe metabolism of CMThe metabolism of VLDL and LDLThe metabolism of VLDL and LDLThe metabolism of HDLThe metabolism of HDL The LDL receptor pathway The LDL receptor pathway The structure of LDLR and VLDLRThe structure of LDLR and VLDLRThe scavenger receptorThe scavenger receptorThe factors which are relevant to atherosclerosisThe factors which are relevant to atherosclerosisLipoprotein remnant Modified LDL LDL-C Lp(a) HDL ApoAIMeasurementsMeasurementsTotal Cholesterol Total Triglyceride HDL-Chol LDL-Chol ApoAI ApoB Lp(a) ApoE genotype   Preanalytical Factors Affecting Lipid Test Results     Preanalytical Factors Affecting Lipid Test Results   Biological factors: individual variations, gender, age, and race, etc. Studies showed that TC, TG, HDL-C, LDL-C, apoA1, apoB and Lp(a) have average biological variations of 6.1%~11%, 23%~40%, 7%~12%, 9.5%, 7%~8%, 6.5%~10%, and 8.6% respectively. Life-style factors: diet, obesity, smoking, stress, alcohol, coffee, and exercise, etc.nullClinical factors: (1) Concurrent illnesses (e.g. endocrine or metabolism disorders, kidney and liver diseases, or others); (2) drug-induced malfunctions (antihypertensive agents, immunosuppressants, and estrogen) etc. Collecting and handling of specimens: fasting conditions, hemoconcentration, use of anticoagulants and preservatives, capillary blood against venesection, and storage conditions of the specimen etc. The reference method CholesterolThe reference method CholesterolA 0.5-mL aliquot of serum is treated with 5.0 mL alcoholic KOH to hydrolyze the cholesteryl esters. TC is then extracted from the mixture with 10mL hexane, and then is treated with mixture of acetic acid, acetic anhydride乙酸酐and sulfuric acid硫酸for color development. After 30 min, absorbance is read at 620nm. mg/dL=mmol/L38.7TriglyceridesTriglyceridesThe TG are first extracted quantitatively with chloroform to remove water-soluble interfering substances. TG+KOHfatty acids+glycerol Glycerol+periodate高碘酸盐formic Acid蚁酸+formaldehyde甲醛 Formaldehyde+chromotropic acid铬变酸chromogen色原体 The absorbance is measured at 570nm. mg/dL=mmol/dL×88.5HDL-cholesterolHDL-cholesterolThe CDC reference method uses a combination of ultra-centrifugation and poly-anion precipitation to prepare the HDL-containing fraction.Routine method CholesterolRoutine method CholesterolEnzymatic method The reagent usually is mixed with 3-10µL aliquot of serum or plasma, incubated under controlled conditions for color development. These methods can give inaccurate results in the presence of high concentrations of bilirubin, ascorbic acid维生素 C, or other substances that containing H2O2. Thus cholesterol methods are best thought of as “measurement systems” composed of reagent, calibrator and instrument.Reference valueReference valueAdult: 2.8~5.2mmol/L Children:＜4.4mmol/L TriglycerideTriglycerideTG are measured enzymatically directly in plasm or serum. As for cholesterol, reagents combining all the required enzymes, cofactors, and buffers are available from various manufacturers. Triglyceride Blanks(Correction for Endogenous Glycerol)Reference valueReference valueAdult: 0.56~1.70mmol/L Children:0.36~1.50mmol/L PhospholipidPhospholipidChemical Method Enzymatic method(PL-D) Adult: 1.3~3.2mmol/L FFAFFAEnzymatic method LPL Adult: 0.4~0.9mmol/L High-Density Lipoprotein CholesterolHigh-Density Lipoprotein CholesterolHDL-cholesterol is most commonly measured in the supernatant after the precipitation of the apoB100-containing lipoprotein. Dextran Sulfate右旋糖酐硫酸-MgCl2 Method Heparin-MnCl2 Method Ultracentrifugation to Remove Trig-Rich LipoproteinReference valueReference value＞0.9mmol/LLow-density lipoprotein cholesterolLow-density lipoprotein cholesterolIndirect methods The Friedewald Equation [LDL chol]=[Total chol]-[HDL chol]-[Triglyceride]/5 In several of direct methods, LDL was selectively precipitated with polyvinyl sulfate聚乙烯硫酸or heparin at low pHReference valueReference valueAdult: 2.1~3.1mmol/L Children:＜2.8mmol/L Apolipoprotein AI and B-100Apolipoprotein AI and B-100Immuno-turbidimetry and immuno-nephelometry are widely used to measure apoAI and apo B 100.Lipoprotein(a)Lipoprotein(a)At present, Lp(a) measurements are not standardized, and most of the Lp(a) assays have not been evaluated for their apo(a) size sensitivity Reference valueReference value0~1000mg/L ＜30mg/LApoE genotypingApoE genotypingnullnull PCR-RFLP法检测ApoE第四外显子区的原理nullApoE PAGE/RFLP带型Mε4/4ε3/3ε2/2ε3/2ε4/3ε4/2nullThe Principle of Multi-PCR/ARMS MethodnullMulti-PCR/ARMS Method 5 primers was designed: P1、P2→Cys/Arg(158) P3、P4→Cys/Arg(112) P5 was common primer. P1+P5 PCR product size: 451bp P2+P5 PCR product size: 451bp P3+P5 PCR product size: 588bp P4+P5 PCR product size: 588bp分别组成两个体系nullThe sizes of products in A/B PCR system null null The electrophoresis pattern of ApoE genotypes determined by PCR/ARMS methodApoCIIApoCIIVariable number of tandem repeats, VNTRApo(a)Apo(a)Pentanucleotide repeats, PNRnullThe electrophoresis patternnullThe scheme of TaqMan techniquenullDNA chipNCEP recommendations for lipid and lipoprotein measurementsNCEP recommendations for lipid and lipoprotein measurementsDatabase linkage Reference method Routine method Cholesterol measurements Blood samples Specimen storage Serial samples Glycerol blanking Goals for analytical performance Database linkageDatabase linkageLaboratories that provide lipid and lipoprotein measurements should maintain linkage with the existing epidemiological databases relating lipid and lipoprotein concentration to risk for CHD. CDC reference methods for cholesterol, triglyceride, HDL cholesterol serve as the basis for judging the accuracy of other methods.Reference methodReference methodReference methods should provide serum equivalent values.Routine method Routine method In most cases, lipid and lipoprotein measurements can be made using specimens of either serum or plasma. EDTA plasma should be used when lipoproteins are to be analyzed using ultracentrifugation.Cholesterol measurements Cholesterol measurements Cholesterol measurements can be made either in fasting or non-fasting samples. Triglyceride, HDL cholesterol and LDL cholesterol measurements should be made in samples collected after a 12-h fast.Blood samples Blood samples Blood samples should be standardized to be taken in the sitting position whenever possible.Specimen storage Specimen storage Serum or plasma should be removed from cells with 3h of venipuncture静脉穿刺. Specimens can be stored for up to 3d at 4℃, up to several wk at -20 ℃ in a non-self-defrosting freezer; and at -70 ℃ or lower for longer period.Serial samples Serial samples The measurements should be made at least two serial samples collected at least 1 wk apart.Glycerol blankingGlycerol blankingAll labs should offer a glycerol-blanked triglyceride analysis. Reports from the lab should clearly state whether the triglycerid analysis was glycerol-blanked. Glycerol-blanking of triglycerid analysis should be mandatory. Glycerol-blanking of triglycerid analysis need not be routinely conducted on outpatient samples, unless economically feasible. But all in-patient samples should be glycerol-blanked.Goals for analytical performanceGoals for analytical performanceNECP goals are stated in terms of total error. SummarySummaryLipoproteins are complexes of lipid and proteins which facilitate lipid transport. Their metabolism can be though of as two interconnected cycles centred on the liver. Lipoproteins are defined by their density and differ in composition, structure and function. Apolipoproteins have a functional as well as structural importance. Cholesterol can only be excreted from the body by way of the liver.CLINICAL DISORDERS OF LIPID METABOLISMCLINICAL DISORDERS OF LIPID METABOLISMCLASSIFICATION ATHEROGENIC PROFILES ClassificationClassificationprimary hyperlipidaemia secondary hyperlipidaemia Xanthelasma黄色斑in younger individuals(age<40 years) usually indicate hypercholesterolaemia. In the elderly they do not carry the same significanceXanthelasma黄色斑in younger individuals(age<40 years) usually indicate hypercholesterolaemia. In the elderly they do not carry the same significanceTendon xanthomas . There are pathognomonic for familial hypercholesterolaemia and are often first seen on the achilles tendon as in this patientTendon xanthomas . There are pathognomonic for familial hypercholesterolaemia and are often first seen on the achilles tendon as in this patientEruptive xanthomas are the characteristic of hypertriglyceridaemiaEruptive xanthomas are the characteristic of hypertriglyceridaemia Arcus senilis Arcus senilis Clinical Note Clinical NoteThe physical signs of the hyperlipidaemias are not specific for any particular disease and may sometimes be present in normollipidaemic patients, e.g. arcus senilis. Tendon xanthomas are particularly associated with familial hypercholesterolaemia. Fredrickson(WHO ) classification of dyslipidaemia. This is based on the appearance of a fasting plasma sample after standing for 12 hours at 4C and analysis of its cholesterol and triglyceride content Fredrickson(WHO ) classification of dyslipidaemia. This is based on the appearance of a fasting plasma sample after standing for 12 hours at 4C and analysis of its cholesterol and triglyceride content Some genetic causes of lipidaemia Some genetic causes of lipidaemia Common Causes of secondary hyperlipidaemia Common Causes of secondary hyperlipidaemia Disease Usual dominant lipid abnormality Diabetes mellitus Increase triglyceride Alcohol excess Increase triglyceride Chronic renal failure Increase triglyceride Drugs, e.g. thiazide diuretics Increase triglyceride Non-selective -blocker Hypothyroidism Increase triglyceride Nephrotic syndrome Increase triglyceride Case history 53Case history 53A 53-year-old male was found to have the following results on a fasting blood sample: Total cholesterol Triglyceride Glucose GT mmol/l U/l 8.4 6.8 9.8 138 A non-smoker, his blood pressure was 145/95 mmHg and he was obese with central fat distribution. What other information and investigations would be helpful in this man’s management? Risk assessment table for men based on the National Health Committee New Zealand Guidelines 1997Risk assessment table for men based on the National Health Committee New Zealand Guidelines 1997nullMANAGEMENT OF HYPERLIPIDAEMIA MANAGEMENT OF HYPERLIPIDAEMIA CLASSIFICATION MANAGEMENT GUIDELINES DIETARY MANAGEMENT DRUG THERAPY Coronary heart disease risk factors Coronary heart disease risk factors Lipid-lowering dietary guideline Lipid-lowering dietary guideline Palmer xanthomas before(a) after (b) treatment Palmer xanthomas before(a) after (b) treatmentnull The abnormal lipid cutoff values that come with the China’s “Recommendations on Prevention and Treatment of Lipid Abnormalities” [血脂异常防治建议] were recommended to intervene and manage patients. nullnullCase history 52Case history 52Case history 54Case history 54As asymptomatic(无症状的) 38-year-old woman was screened at a well-woman clinic and found to have a non-fasting plasma cholesterol level of 8.7 mmol/l. What other information and investigation you require to help in the management of this woman? Management of hyperlipidaemiaManagement of hyperlipidaemiaAt least two fasting lipid profiles should be performed before starting any form of lipid lowering therapy First-line therapy always consists of dietary modification. Drug therapy, if required, should be viewed as an adjunct to dietary therapy Other risk factors for coronary heart disease must be managed along with hyperlipidaemiaThank you for your attention!Thank you for your attention!