LIPID

= Commonly known as fats

= functions:
 Source of fuel
 stability of cell membrane
 Steroid hormone production

= With special transport mechanism

= Phospholipid, Cholesterol, Triglycerides, Fatty acids and Fat-soluble vitamins (ADEK)
= Vitamin A – Retinol
= Vitamin D - Cholecalciferol
= Vitamin E – Alpha Tocopherol
= Vitamin K – Phytonadione

PHOSPHOLIPIDS
= conjugated lipids
= most abundant type of lipid
= not an energy source
= synthesis – liver and intestine
= combination of 2 fatty acids and 1 glycerol
= amphipathic
= lungs (type II pneumocytes)
= reference range – 150-380 mg/dl
= functions
 Surfactant – prevent the collapse of alveoli
 Cellular metabolism – permeability of cell membrane
 Blood coagulation
= Forms
 Lecithin/Phosphatidyl choline – 70%
 Sphingomyelin – 20%
 Cephalin – 10%
= phosphatidyl ethanolamine
= phosphatidyl serine
= Lysolecithin + inositol phosphatide

SPHINGOMYELIN
= Not derived from glycerol
= Sphingosine + fatty acids
= essential component of cell membranes
= Niemann Pick Disease
 accumulation of sphingomyelin in the liver and spleen
 deficiency in the enzyme sphingomyelinase

PHOSPHOLIPIDS

= for the assessment of fetal lung maturity

= specimen – amniotic fluid
= mature lung = L/S ratio ≥ 2
= not part of the lipid panel

CHOLESTEROL
= not a source of fuel
= synthesized in the liver
= transport and excretion – promoted by estrogen
= should be measured in adults 20 years of age and older – at least once every 5 years
= reference range
 < 200 mg/dl = desirable
 200-239 mg/dl = borderline high
 ≥ 240 mg/dl = high
= Functions
 precursor of steroid hormones
 fluidity of the cell membrane
  Bile acid formation
 Vitamin D formation

= steroid hormone
 Progesterone
 Cortisol
 Aldosterone
 Androgen
 Estrogen
= Diagnostic significance
 evaluate risk for atherosclerosis, myocardial and coronary arterial occlusions
 thyroid function test
 Liver function test
 renal function test
 Monitor effectiveness of lifestyle chage
= Sources
 Endogenous – liver
 Exogenous - Diet
= Forms
a. Cholesterol Ester – 70%
 Plasma and serum
 bound to Fatty acid
 LCAT – esterification
Lecithin cholesterol acyl transferase
Removal of fatty acid from lecithin
Liver- synthesis
Apo A-1 – activator of LCAT
b. Free cholesterol – 30%
 serum, plasma and RBC
 non-esterified
 hydrolysis

Patient preparation
= usual diet for 2 weeks prior to testing
= fasting is not a requirement

Measurement
= Total cholesterol is measured rather than its forms

Methods for Cholesterol

Chemical Method
A. Liebermann-Burchardt Reaction
= end color – green
= end product – cholestadienyl monosulfonic acid
= reagent – LB reagent

B. Salkowski Reagent
= end color – red
= end product – cholestadienyl disulfonic acid
= reagents – Salkowski reagent

Precautions:
= avoid hemolyzed sample – False Inc
= avoid icteric samples – false inc
= avoid water contamination
= precise and accurate timing for color development

Enzymatic Method
= routine
= principle – H2O2 production
= hemolysis – false inc
= bilirubin – false inc and false dec
CDC Reference Method
= Abell, Levy and Brodie method
= KOH
= Hexane
= LB reagent

Increased Cholesterol
= biliary cirrhosis
= DM
= Alcoholism
= Hypothyroidism
= Nephrotic syndrome
= Hyperlipoproteinemia

Decreased Cholesterol
= Severe liver disease
= Malnutrition
= Hyperthyroidism
= Malabsorption syndrome

TRIGLYCERIDES
= Triacylglycerol
= main storage lipid in man
= adipose tissue
= source of energy
= thermal insulation
= low caloric intake – low TGL
= fasting requirement – 12-14 hrs
= reference range
- <150 mg/dl – normal
- 150-199 mg/dl – borderline high
- 200-499 – high TAG
- >500 mg/dl – very high TAG
= Diagnostic significance
- evaluate suspected atherosclerosis
- measure the ability to metabolize fat
- ≥200 mg/d l = increase risk for CVD

Methods of TGL Measurement

Chemical method
= Van Handel and Zilversmith (Blue end color)

Enzymatic Method
= Specific, rapid and easy
= interference – glycerol
= glycerol kinase method

CDC Reference Method

= Modified Van Handel and Zilversmith
= Hydrolysis
= Extraction
= Adsorption
= Colorimetry

Increased TAG
= Hyperlipoproteinemia
= Alcoholism
= Nephrotic syndrome
= Hypothyroidism
= Pancreatitis

Decreased TAG
= Malabsorption syndrome
= Hyperthyroidism
= malnutrition
Lipoproteins
= Lipids + proteins
= Transporter
= Cholesterol and TAG – bound to lipoprotein

Apolipoproteins
= react with receptor
= amphipatic helix
= maintain structural integrity of the LPP complex
Major Lipoproteins
A. Chylomicrons
= Largest
= least dense
= cleared within 6-9 hours post prandial
= produced by the intestine
= transport exogenous TAG
= Apo B-48, Apo A-1, Apo-C and Apo-E

B. VLDL
= secreted by the liver
= transport endogenous TAG
= Atherogenic
= Apo B-100, Apo C and Apo E

C. HDL
= smallest
= most dense
= liver and intestine
= reverse cholesterol transport
= reference method – ultracentrifugation
= Apo A-1, Apo A-II and Apo C
= Phospholipid content – high
= <35 mg/dl – high risk
= > 60 mg/dl – high (protective)

D. LDL
= liver
= end product of VLDL catabolism
= most abundant lipoprotein
= transport cholesterol to peripheral tissues
= most cholesterol-rich
= most atherogenic
= better marker of CHD risk
= primary target of cholesterol lowering therapy
= Apo B-100 and Apo E
= Reference range
 <100 mg/dl – optimal
 100-129 – above optimal
 130-159 mg/dl – borderline high
 160-189 mg/dl – high
 ≥190 – very high

Minor Lipoproteins
1. Intermediate Density Lipoprotein (IDL)
= Product of VLDL catabolism
= VLDL remnant
= Migrates either in the pre-beta or beta region
= major lipoprotein – Apo B-100

2. Lipoprotein a / Lp (a)
= Similar to LDL (density and composition)
= Migration – pre-beta region
= Known as sinking pre-beta lipoprotein
= isolated in the LDL-HDL density range by ultracentrifugation
= increased – premature coronary heart disease and stroke
= major apolipoprotein: Apo B-100 and Apo a

Abnormal Lipoproteins
1. Lipoprotein X
= found in obstructive jaundice and LCAT deficiency
= sensitive indicator of cholestasis
= lipid content is mostly phospholipid and free cholesterol (90%)
= Contains Apo C and Albumin

2. Beta-VLDL
= Floating beta lipoprotein
= abnormally migrating beta-VLDL
= Density – VLDL
= Migration – LDL
= VLDL rich in cholesterol

Lipoprotein methodologies
= Generally, TC, Tag and HDL-C can be satisfactorily analyzed in frozen samples, and LDL-C concentration can be
estimated with the Friedwald Equation
= Lipemic samples – non-fasting, patient with hyperlipidemia, TPN therapy
= Lipemic samples -pretreated by ultracentrifugation to remove lipids
= TC – HDL-C = Non-HDL-C

Sample Requirements:
= Using serum separator tubes is the preferred sample
= Plasma or serum is acceptable
= EDTA is preferred for ultracentrifugation or electrophoretic methods
= Cholesterol is 3% lower in serum
= heparin – can affect electrophoretic mobility

Patient Preparation:
1. Fasting
= 12-14 hours
= Required when TAG and LDL-C are being measured
= Fasting state – TAG is present in VLDL
= Non-fasting state – TAG is present in chylomicrons
= non-fasting sample – TC and HDL-can be measured

2. Posture
= standing patient reclines, extravascular fluid transfers to the vascular system
= seated for 5 minutes

Recommendation:
= Initial screening – 20 years old
= Testing should be repeated at least once every 5 years

Physical properties
= Density - Ultracentrifugation
= Charge and Size - Electrophoresis
= Apolipoprotein content – Immunochemical methods

Ultracentrifugation
= Common in research laboratory
= uncommon in clinical laboratory
= reference method for lipoprotein quantitation
= Based on the protein and TAG content of lipoprotein (Density)
= Frozen samples are not appropriate for ultracentrifugal analysis because the TAG-rich LPP do not withstand freezing

Electrophoresis
= Visual display useful in detecting unusual or variant pattern
= Agarose gel
= Oil red O, Fat red &b or Sudan Balck
= Chylomicrons – remain at the origin
= More of qualitative
= Less desirable for lipoprotein quantitation because of poor precision

Chemical Precipitation
= Use of polyanions (Heparin and divalent cations such as manganese

Immunochemical Methods
= has the potential for both research and routine use
= use of antibodies specific for a certain lipoprotein
= Immunoturbidimetric assay - measurement of lipoprotein A
= Turbidity (apolipoprotein -antibody complex)

HDL measurement
= Measurement of HDL cholesterol has assumed greater importance in the latest NCEP treatment guidelines
= include HDL cholesterol measurement with total cholesterol in the first medical work-up
= Routine test – chemical precipitation

Chemical precipitation for HDL

= Heparin + manganese – produced interference with enzymatic assays
= Sodium phosphotungstate + magnesium – sensitive to reaction conditions and greater variability
= Dextran sulfate + Magnesium – common

CDC reference method for HDL measurement

= 3-step process
= Ultracentrifugation
= Heparin manganese precipitation
= Abell-Kendall Assay

LDL Measurement
= proven to be atherogenic lipoprotein
= primary basis for treatment decision
= most important value in assessing cardiac risk and redirecting therapy
= Reference method – beta quantification

Friedwald’s formula
= Routine method
= Common to both research and clinical laboratory
= invalid once the TAG concentration exceeds 4.5 mmol/l
= not suitable for non-fasting sample

VLDL (mmol/L) = Plasma TAG / 2.175

VLDL (mg/dl) = Plasma TAG / 5

LDL-C = Total Cholesterol – HDL – VLDL

LIPID DISORDERS
Tangier’s Disease
= Hypoalpha-lipoproteinemia
= High density lipoprotein deficiency
= Inherited disorder
= Significantly reduced levels of HDL
= Mutation of ABCA 1 gene
= Cholesterol accumulates within cells

Tay-Sachs Disease
• Def – hexosaminidase A
• Accumulation of sphingolipids in the brain

Abetalipoproteinemia
= Undetectable LDL concentration
= LDL is absent, not just deficient
= failure to synthesize Apo B
= Definitive diagnosis – immunochemical demonstration of the absence of LDL Apo B in the plasma

Hypobetalipoproteinemia
= Decreased LDL
= decreased cholesterol

Mixed Hyperlipoproteinemia
= Increased VLDL
= Increased Chylomicrons *fasted
= Can cause pancreatitis
= Common in adults
= LDL and HDL are normal
= Plasma is opaque
= Floating cream layer above the turbid plasma
= > 10000 mg/dl
= Creamy supernate

Dysbetalipoproteinemia
= Increased Chole and TAG
= Abnormal LDL (IDL)
= Abnromal LDL merges with the pre-beta band on electrophoresis to produce a broad-beta band
= Floating beta lipoprotein (beta VLDL)
= associated to hypothyroidism and alcoholism
= TAG and Chole – High
= VLDL – High
= LDL and CM - normal

Hyperprebetalipoproteinemia
= endogenous hyperlipemia
= carbohydrate-induced hyperlipemia
= LDL – normal
= Increased VLDL
= Increased TAG
= Standing plasma (4 C for 10-12 hrs) – no chylomicrons
= Electrophoresis – distinct pre-beta band

Combined hyperlipoproteinemia
= defined as the presence of elevated level of both serum total cholesterol and TAG
= Increase risk for CHD

Hyperchylomicronemia
= not associated with CHD
= can cause pancreatitis
= decreased LPL – no clearance of chylomicrons
= TAG – high
= Chole – N
= LDL – N
= VLDL – N
= CM - High

Features:
= Low cardiac risk
= eruptive xanthomas
= Recurrent pancreatitis
Creamy layer over clear plasma

Hypercholesterolemia
• Lipid abnormality most likely linked to heart disease
• Familial hypercholesterolemia
• Genetic
• LDL receptor - problem
• Homozygote – 800 -1000 mg/dl (LDL pheresis)
• Heterozygote – 300-600 mg/dl (statin)