lipids & dyslipoproteinemia

LIPIDS & DYSLIPOPROTEINEMIA

Merlyn A. Baraclan, RN, RMT

TOPIC OUTLINE Overview on Lipids Lipoproteins,

Apoliporoteins & Related Proteins

Lipid Transport & Lipoprotein Metabolism

Lipid & Lipoprotein Measurement

The NCEP Guidelines Lipids, Lipoproteins &

Disease References

LIPIDS are biological

compounds which are soluble in nonpolar organic solvents but relatively insoluble in polar solvents.

MAJOR BODY LIPIDS

Triglycerides / TAG / TG Cholesterol / C Phospholipids

Glycolipids

BIOLOGIC ROLES OF LIPIDS

Primary source of fuel Components of cell membranes &

many cell structures Provide stability to cell membranes Means of transmembrane transport

LIPOPROTEINS

The function of lipoprotein particle is to transport lipids around the body in the blood.

Contain cholesterol in 2 forms: _ free cholesterol_ cholesterol ester

Lipoproteins have a micellar structure.

LIPOPROTEINS The transport proteins of lipids

having a micellar structure composed of two parts as follows:

Central corecontains TAG and cholesterol ester

nonpolar moiety

Surface coatcontains PL, free cholesterol, and apolipoproteins

4 MAJOR LIPOPROTEIN CLASSES

- Chylomicrons (CM) - very low density lipoproteins

(VLDL)

- low density lipoproteins (LDL)

- high density lipoproteins (HDL)

LIPOPROTEINSCLASS DENSITY

(g/ml)MEAN

DIAMETER

(mm)

source Principal function

Electropho

retic mobility

CM < 0.95 500 intestine Transport ofExogenous

TAG

Remains at Origin

VLDL 0.96 -1.006

43 liver Transport of Endogenous

TAG

Pre β

IDL 1.007-1.019

27 Catabolism of VLDL

Precursor of LDL

“broad β”

LDL 1.020-1.063

22 Catabolism of VLDL via IDL

CholesterolTransport

β

HDL 1.064-1.210

8 Liver,intestine,catabolism of CM & VLDL

“reversecholesteroltransport”

α

CHEMICAL COMPOSITION OF MAJOR CLASSES OF PLASMA PROTEINS

CLASS PROTEIN%

TAG%

CHOLESTEROL%

PHOSPHO LIPID

%

Chylomicron/ CM

1-2 85 - 95 3 -5 5 -10

VLDL 10 60 -70 10 -15 10 – 15

LDL 15 -25 5 - 10 45 20 -30

HDL 50 Verylittle

20 30

LIPOPROTEINS , APOLIPOPROTEINS & RELATED PROTEINS

MAJOR LIPOPROTEINS

1. CHYLOMICRONS / CM- Produced by the intestine- Transport lipids of dietary origin- Poor in free cholesterol- Has a “very high lipid/protein ratio- “milky plasma”- “Floating creamy layer”- Removed by the liver

MAJOR LIPOPROTEINS

2. VERY LOW DENSITY LIPOPROTEINS / VLDL

- Produced by the liver- Supplies the body w/ TAG of

endogenous origin & also cholesterol- “ turbid plasma”

MAJOR LIPOPROTEINS

3. LOW DENSITY LIPOPROTEINS / LDL

- Produced by the metabolism of VLDL- The particles do not scatter light- Removed by the liver & macrophages

MAJOR LIPOPROTEINS 4. HIGH DENSITY LIPOPROTEIN /

HDL

- Consists mostly of proteins- Produced by the liver- “reverse cholesterol transport”- Cardioprotective

MINOR & ABNORMAL LIPOPROTEINS

1. Lipoprotein (a) / Lp (a)- Similar to LDL - Synthesized in the liver- “lipid staining pre β band”- Has atherogenic properties- 2. LPX Lipoprotein- Seen in patients with obstructive biliary

disease, & with LCAT deficiency- 3. β – VLDL - “Floating β lipoprotein”

EXOGENOUS (DIETARY) LIPID PATHWAYFood intestinal absorption

CM High TAG,Low Chol, Apo B48

TAG in adipose tissue

Muscle & FFA

chylomicron remnants taken by the liver

ENDOGENOUS LIPID PATHWAY FFA TAG synthesis in liver, intestine VLDL

High TAG

Low Chol

FFA Apo B100

LPL Apo E

IDL

Apo E Binds onto hepatocytes through Apo E

LDL LDL binds to the receptors in liver (70%)

& other tissues (30%)

HDL PATHWAYliver secretes Apo A1 + other Apos + PLs Nascent

HDL

Cholesterol from tissues

HDL 3

Esterification of Cholestrol by LCAT

LDL

uptake by liver

Cholesterol transfer to VLDL

excretion into bile

C.1 BLOOD SAMPLING & STORAGE

BIOLOGIC VARIATION FASTING POSTURE

VENOUS VS. CAPILLARY

PLASMA VS. SERUM STORAGE

BLOOD SAMPLING & STORAGE

BIOLOGIC VARIATION- Cholesterol level rises w/ age - Women have lower levels than

men (except in childhood & after the early 50’s)

- Age related variation is the basis of NCEP recommendation that cholesterol screening be repeated every 5 years.

BLOOD SAMPLING & STORAGE FASTING- 12 hours before venipuncture- Chylomicrons are completely cleared

w/in 6 – 9 hours - NCEP Adult Treatment Panel III( ATP III),

has recommended that patients fast for at least 9 hours before blood specimens are taken for lipid & lipoprotein analysis

POSTURE- Current NCEP guidelines recommend

that patients be seated for 5 minutes prior to sampling.

BLOOD SAMPLING & STORAGE VENOUS VS. CAPILLARY SAMPLES- Measurements in capillary blood samples

are lower than venous samples & tend to be more variable.

PLASMA VS. SERUM- Either plasma or serum can be used when

only TAG, cholesterol & HDL are measured, & LDL – C is calculated from these three measurements.

- Plasma is preferred when lipoproteins are measured by ultracentrifugal or electrophoretic methods

BLOOD SAMPLING & STORAGE PLASMA VS. SERUM Cont.- EDTA is the preferred anticoagulant - ♥- Heparin – X- Citrate – X STORAGE- When serum or plasma must be stored

for long periods it should be maintained at a temperature of -70⁰C.

- For short term storage, the samples can be kept at – 20⁰C.

LIPID & LIPOPROTEIN MEASUREMENT A. TAG measurementChemical Nonenzymatic MethodsGeneral Steps1. Extraction- To remove TAG from LP’s- Accomplished by using MeOH, EtOH,

isopropyl alcohol, Folch’s rgt & diethyl ether

- - removal of interferences by zeolite

A. TAG MEASUREMENT

2. Hydrolysis of TAG into FFA & Glycerol- By saponification w/ alcoholic KOH

3. Measurement of Glycerol- The glycerol liberated is oxidized by

periodate to HCHO & quantified by using any of the ff:

- Eegrine reaction- Schryver’s reaction- Pay’s reaction- Hantzsch reaction (method of choice)

A. TAG MEASUREMENT Enzymatic Methods- Based on the hydrolysis of TAG & the

measurement of glycerol that is released in the reaction:

LPS- TAG + 3H2O glycerol + fatty acid- Methods:- Boculo David - Megraw - Winartasaputra- Nagele - Trinder

B. CHOLESTEROL MEASUREMENT Chemical Nonenzymatic Methods4 General Steps1. Extraction- Using Bloor’s rgt (3:1 EtOH – ether) or

zeolite extraction2. Saponification- Using KOH3. Purification- Using digitonin4. Color development- May proceed w/ the Leibermann-

Burchardt reaction or Salkowski reaction

B. CHOLESTEROL MEASUREMENT

Leibermann - Burchardt reaction- Uses sulfuric acid & acetic anhydride to

produce an unstable green cholestadienyl monosulfonic acid; color stabilized by sodium sulfate

Zak or Salkowski reaction- Uses sulfuric acid & ferric ions to

produce a stable red to red – violet cholestadienyl disulfonic acid

CHOLESTEROL MEASUREMENT NONENZYMATIC 1 Step Methods- Zlatkis – Zak Boyle method- Ferro – Ham method- Pearson – Stern – MacGavack- Wybenga et al 2 Step Methods- Carr – Drekter 3 Step Methods- Abell – Kendall method (Standard reference

method) 4 Step Methods- Schoenheimer – Sperry - Parek – Jung- Sperry - webb

CHOLESTEROL MEASUREMENT: ENZYMATIC (CHOD – PAP) cholesterol ester

Cholesterol ester + H2O cholesterol + FFA

hydrolase

cholesterol

Cholesterol + O2 cholest – 4 – en – 3 – one + H2O2

oxidase

peroxidase

H2O2 + phenol + 4 aminoantipyrine quinoneimine dye

+ 2 H2O

LIPOPROTEIN MEASUREMENT 1. HDL Measurement- Polyanion precipitation- Electrophoresis – spectrophotometric

det’n- Ultracentrifugation ( reference method) 2. Chylomicrons / CM- Standing Plasma Test 3. Lipid Profile- Use of the Friedewald equation

THE NCEP GUIDELINES Testing & TreatmentCholesterol Goals:ATP III recommends a complete

lipoprotein profile as the initial test for evaluating blood cholesterol.

Testing should be performed on all adults aged 20 & older & should be repeated once every 5 years.

The need for a therapeutic lifestyle change & drug therapy

ATP III CLASIFICATION FOR LIPID VALUES

For Cholesterol: conversion factor to convert mg/dL to mM is 0.02586For triglyceride : conversion factor to convert mg / dL to mM is 0.011 LDL

CholesterolHDL

Cholesterol

Total Cholesterol

Triglyceride

<100 optimal < 40

low < 200

desirable < 150

normal

100-129

Near optimal / above optimal

≥ 90 high 200-239

Borderline

high

150 -199

Borderline high

130-159

Borderline

high

≥ 240 high 200 -499

high

160 -189

High ≥ 500 Very high

≥190 Very high

HYPERLIPIDEMIA DRUGS

Drug Class Mechanism of Action Example

Statins Lowers LDL cholesterol Lovastatin , simvastatin,Pravastatin, fluvastatin

Fibric acid derivatives

Lowers TAG Gemfibrosil, Fenofibrate

Bile acid resins

Lowers LDL cholesterol Colestipol, cholestyramine

Niacin (nicotinic acid)

Lowers TAG niacin

E. LIPIDS, LIPOPROTEINS & DISEASE

CHD (coronary heart

disease) Atherosclerosis

RISK FACTORS OF CHDPositive

Age: Male 45 yrs and above, Females 55 yrs and above or premature menopause without estrogen therapyFamily history of premature CHDCurrent cigarette smokingHypertension (equal or more than 140/90 mmHg or on antihypertensive therapy)Low HDL-Chol (<35 mg/dl)Diabetes mellitus

NegativeHigh HDL-Cholesterol (equal to or above 60 mg/dl)

HIGH CHOLESTEROL WITH HIGH LDL - CHOLESTEROL

hyperbetalipoproteine-

mia Elevated LDL – C Normal TAG High cardiac risk Commonly

encountered

SPECIFIC DISEASES- Polygenic (Nonfamilial)

Hypercholesterolemia- Familial

hypercholesterolemia/FH

- Familial Defective Apo B

- Sitosterolemia

HIGH TAG WITH NORMAL CHOLESTEROL

Due to the elevation of TAG rich particles

Hyperprebetalipo – proteinemia

secondary to excess alcohol & high carbohydrate intake

SPECIFIC DISEASES- Diabetic dyslipidemia- Familial

hypertriglyceridemia- Lipoprotein lipase

deficiency- Apo C II deficiency- Apo C III excess

LIPIDOSES

A group of inherited disorders characterized by the accumulation of lipids in tissues especially the “brain” due to a deficiency in a particular sphingolipid catabolic enzyme

LIPIDOSES: Niemann – Pick disease- Deficiency in sphingomyelinase & accumulation of

sphingomyelin Gaucher’s disease- Deficiency in β – D glucosidase & the

accumulation of glucocerebroside Krabbe’s disease- Deficiency in β – D galactosidase & the

accumulation of galactocerebrosides Fabry’s disease- Deficiency in α – D galactosidase & the

accumulation of ceramide trihexoside Tay – Sach’s disease- Deficiency in β – D hexaminidase & the

accumulation of β - sulfogalactocerebroside

KEY POINT SUMMARY Ultracentrifugation & electrophoretic techniques

are of historical significance, MOST useful lipid & lipoprotein testing are now enzymatic.

LDL – cholesterol can be measured directly , but is usually calculated using the Friedewald formula.

LDL – C is now considered the MOST important value in assessing cardiac risk & directing therapy.

The profile for initial adult screening aged 20 & above, includes TC, TAG, HDL – C & LDL – C & should be repeated every 5 years.

F. REFERENCES INTERNET SOURCES Bhagavan NV (2002).

Medical Biochemistry. San Diego: Harcourt/Academic Press. ISBN 0-12-095440-0. http://books.google.com/?id=vT9YttFTPi0C&printsec=frontcover. 

http://biology.clc.uc.edu/courses/bio104/lipids.htm

BOOK SOURCES Clinical Diagnosis and

Management by Laboratory Methods / John Bernard Henry.

20th ed. 224 – 248

Henry’s Clinical Diagnosis and Management by Laboratory

Methods / Richard McPherson & Mathhew Pincus. 21st ed. 200 –

219 Clinical Chemistry: Principles,

Procedures & Correlations / Michael Bishop, Janet Engelkirk

& Edward Fody. 4th ed. 232 – 259 Southwestern University

College of Medical Technology Clinical Internship Manual / 2005

ed. Southwestern University College of

Medical Technology: Lecture Handbook in Routine Clinical

Chemistry/ Julius Mario. 2008 ed. 44 -54

Danny Donor ♥

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