Enclonar, Kimberly / MLS 3A

• Amphipathic
Lipids o Only hydroxyl group in A ring is hydrophilic
September 22, 2020
• Found in surface of lipid layers
Ace Ronald Sarabia, RMT, MHPEd
• Synthesized in liver
• Precursor of 5 major classes of steroids
Fats o Progestin
• Biochemical compounds medically referred to as lipids o Glucocorticoids
• Mostly C-H bonds o Mineralocorticoids
• Calorie - unit of energy o Androgen
o Present: Joles
• Can exist in an esterified form (cholesterol ester)
• Functions:
• Not readily catabolized by the cell
o Rich source of energy/storage of excess calories
• Converted in the bile → bile acids → cholesterol,
o Hormone or hormone precursor
cholic acid
▪ Adrenal steroids are from cholesterol
• Can be converted to Vitamin D3 (active form)
o Structural cell component o From 7-dehydrocholesterol
o Insulation
• Dyslipidemia = abnormal lipoprotein concentration Lipoproteins
• Plasma lipids = lipoproteins = atherosclerosis
• Composed of lipids and proteins (apolipoproteins)
o LDL = lechon de leche
• Class of proteins synthesized by the liver or intestines
o Leading cause of death
• There proteins form a "transport vesicle" with the
lipids to facilitate lipid circulation
Major Lipids • 10-1200 nm
• Fatty Acid • Size variation is attributed to TAG and cholesterol
• TAG content
• Phospholipids • The bigger the LP, the higher the lipid content
• Cholesterol o ↑ density = ↑ protein = ↑ lipid
o ↑ lipid = ↑ size
Fatty Acid o ↑density = ↑ size
• Short-chained, 4-6 carbon chain • Core
o Medium chain 6-12 o Triglyceride
o Long chain 12-26 o Cholesteryl ester (esterified form)
• Small amount is present in plasma in un-esterified • Surface
form o Free cholesterol
• Derived from the hydrolysis of TAG o Phospholipid
• Reference Value 9-15 mg/dL o Apolipoproteins
▪ Responsible for synthesis and
Triglycerides breakdown of lipoproteins
• 3 molecules of fatty acids, 1 molecule of glycerol ▪ Activator and inhibitors of enzyme
• Hydrophobic ▪ Also called, Lipid exchange mediator
• Do not contain charges • Fractions
• Main storage lipid of man (95%) o Chylomicrons
• Predominant form: glyceryl ester o VLDL
• Allows the body to store long-carbon chain o LDL
• Used in fasting state o HDL
• Synthesize 60-130 g daily
o Resynthesized in intestine and combine with Lipid Metabolism
cholesterol and Apolipo-b-protein 1. Emulsification of fats
• Fat found in food is is 1-2% a. Food is ingested
• 2 kinds: b. Proteins and sugars are degraded by salivary
Saturated Solid at RT enzymes
c. Fat cannot be digested in the mouth. Initial
Unsaturated w/ bends and kinks Oil at RT
digestion occurs in the stomach. Most digestion
occurs in intestines.
Phospholipid d. Gallbladder contracts releasing bile acids.
• Contain 2 fatty acids ▪ Large fat globule (bile acids) → micelles
• Most abundant lipids derived from phosphatidic acid (pancreatic lipase) → monoglycerol +
• Originated in liver and intestines fatty acids
• Formed by 2 conjugations ▪ Fatty acids
o Fatty acid • Short →
o Phosphorylated glycerol • Lipid carrier protein →
• Amphoteric • Liver
• Saturated fatty acid content of plasma Independent • Long →
risk factor of atherosclerosis • TAG →
• Sphingomyelin - reference for 3rd trimester • Chylomicrons →
o >2.0 L/S ratio, baby can survive • Lymph
▪ Monoglycerol
Cholesterol • TAG →
• Unsaturated steroid alcohol • Chylomicrons →
• Most complex • Lymph
• Contain 4 rings: A, B, C, D
 Enclonar, Kimberly / MLS 3A

2. Exogenous Pathway
o Use of chylomicrons

Chylomicron
• Density: <0.93 g/mL
• High lipid content; larger
• Cholesterol poor
• Contains apo B-48
• Largest LP and least dense
• Presence of chyle (chylous sample) VLDL / pre-beta lipoprotein
• Rich in exogenous TAG (85-95%) • Density: 0.93-1.006 g/mL
• Poor in cholesterol • Smaller than chylo
• Packed with dietary lipids in the intestines • Rich in endogenous TAG
• Of dietary origin • Produced in the liver and has apo B-100, apo E, and
• Contribute to turbidity of specimen apo Cs
• Seen in postprandial plasma • Cause turbidity in fasting hyperlipidemic plasma
• Fasting samples should not be chylous, but may be • Not of dietary origin
lipemic • Lower L:P ratio compared to chylo
• Principal role: • Pathway
o Delivery of dietary lipids to the liver and o VLDL to IDL to LDL
peripheral cells o LDL is one of the major cholesterol transporter
▪ Nascent chylomicrons - newly
synthesized chylos
▪ Apo E allows chylos to enter liver
▪ Apo CII activates LPL
▪ Hydrolysed by lipoprotein lipase to form
chylomicron remnants

LDL / beta lipoprotein

• Density: 1.019-1.063 g/mL
• 50% of total LP's in plasma
• Contains apo B-100
• Contains 50% cholesterol
• Cholesterol rich
• Smaller than VLDL = traverses the vessel walls more
easily
• Forward cholesterol transport
 Enclonar, Kimberly / MLS 3A
• Major lipoproteins responsible for the delivery of Apolipoprotein B-48
exogenous cholesterol to peripheral cells due to the • Main distribution: Chylomicron
efficient uptake of LDL by LDL receptors • Not recognized by LDL receptor
• Good marker for coronary heart disease • Synthesized in intestine, encode by same gene and
• Bad cholesterol same amino terminus as apo B-100

HDL / alpha lipoprotein Apolipoprotein C-1

• Density: 1.063-1.21 g/mL • Main distribution: CM and VLDL
• Smallest LP, most dense • May inhibit hepatic uptake of VLDL and CETP
• Synthesized by the liver and intestines (cholesterol ester transfer lipoprotein)
• Least TAG, highest protein
• 50% proteins, 20% cholesterol, 30% phospholipid, Apolipoprotein C-II
traces of TAG • Main distribution: CM and VLDL
• Has antiatherogenic property • Activates lipoprotein lipase
o Collects excess cholesterol • Deficiency causes reduced clearance of triglyceride-
• Good cholesterol rich lipoproteins
• Reverse cholesterol transport
• 2 major types: HDL2 and HDL3 Apolipoprotein C-III
o HDL3 (discoidal) via Apo A1 • Main distribution: VLDL, HDL
o HDL2 (spherical) • Inhibits lipolysis of TAG-rich lipoproteins, decreases
• Negative marker for coronary heart disease clearance rate of remnant particles
• Deficiency causes reduced clearance of TAG-rich
lipoproteins

Apolipoprotein D
• Main distribution: HDL
• Activates LCAT

Apolipoprotein E
• Main distribution: CM, VLDL, IDL, remnants and HDL
• Recognition factor that targets CM and VLDL
remnants to hepatic receptor; also binds to cell
Apolipoproteins surface LDL receptors and proteoglycans
• It helps the lipids in solution during circulation • Isoforms:
through the blood stream o Apo E-2
• Interact with specific cell-surface receptors and ▪ Type 3 hyperlipoproteinemia
directs the lipid to the correct target organ and tissues o Apo E-3
in the body o Apo E-4
• Contain structural motif called "amphipathic helix" ▪ Associated with high LDL-C
• Aids in the solubilization of the lipids and transfer ▪ Higher risk for CHD and Alzheimer's
from GT to the liver disease

Apolipoprotein A-I Apolipoprotein F

• Main distribution: HDL • Main distribution: HDL, LDL, VLDL
• Activates lecithin-cholesterol acyltransferase (LCAT) • Regulates CETP function
that esterifies cholesterol in plasma
• Ligand for ABCA 1 Apolipoprotein H
o ATP binding cassette protein 1 • Main distribution: CM, HDL, VLDL
o Mediates secretion of extracellular lipids • Relative to the activation of LPL; TAG metabolism
o Key factor in cholesterol homeostasis • Associated with hyperthrombosis and stroke
• Synthesized in the liver and intestine
• For HDL biosynthesis Apolipoprotein J
• Cell aggregating factor in Sertoli cells
Apolipoprotein A-II • Inhibitor of the C5b*7 complement complex
• Main distribution: HDL • Cholesterol trafficking in brain
• May inhibit lipoprotein and hepatic lipase and • Involved in apoptosis
increases plasma TAG • Also known as clusterin

Apolipoprotein A-IV Apolipoprotein L

• Main distribution: HDL. CM, and free in plasma • Main distribution: HDL
• Cofactor for LCAT; increased during fat reabsorption; • May be linked to reverse cholesterol transport
HDL biosynthesis
Apolipoprotein (a)
Apolipoprotein B-100 • Main distribution: Lp(a)
• Main distribution: VLDL and LDL • Homologous to plasminogen, may be prothrombic
• Carboxy-terminal recognition signal targets LDL to the • Bound to Apo B-100 by disulfide linkage
LDL (apoB, E) receptor
 Enclonar, Kimberly / MLS 3A
Minor Lipoproteins Analyte Reference Values IU (mmol/L) CF
Total 140-200 mg/dL 3.6-5.2 0.026
Intermediate Density Lipoprotein
Cholesterol
• A product of VLDL catabolism
• Converted to LDL - "subclass of LDL" HDL-C 40-75 mg/dL 1.0-2.0 0.025
• Migrates either in the pre B or B region LDL-C 50-130 mg/dL 1.3-3.4 0.026
• Defective clearance of IDL in type 3
hyperlipoproteinemia is probably due to deficiency of TAG 60-150 mg/dL 0.7-1.7 0.0113
APO E-II
• Density: 1.006-1.019 kg/L causing it to float on 1.63 Arteriosclerosis/atherosclerosis
density potassium bromide solution • Leading cause of mortality
• Not normally measured in healthy serum • Esterified cholesterol deposits
o Plaque formation
Lipoprotein (a)/Lp (a) • Different sites of formation:
• Similar to LDL (density and composition) o Peripheral vascular disease
• Has LDL-like particles linked to Apo B-100 o Coronary artery disease
• Variable migration: Pre B, or sometimes between LDL • Patho-physiology
and albumin
• "Sinking pre-B lipoprotein" Laboratory Determination
o Same mobility with VLDL, but same density
with LDL Importance of Determination
• Isolated in the LDL-HDL density range by • Important indicators of coronary heart disease
ultracentrifugation • Risk monitoring for atherosclerosis
o Ultracentrifugation - reference method for
• Assessment of healthy diet/lifestyle
lipoproteins
• Complex structure is same with plasminogen
Fatty Acids
• Increased level may indicate premature coronary
• Gas liquid chromatography
disease and stroke
• Independent risk factor for atherosclerosis
• Contains Apo B-100 Phospholipids
• Density: 1.045-1.080 kg/L • Most abundant phospholipid in the BODY
• Values can range from <20-1500 mg/L o 1st = Phosphatidylcholine (70-75%)
o 2nd = sphingomyelin
Abnormal Lipoproteins • Most abundant in the LIVER
o Phosphatidylcholine
o Phosphatidyl ethanolamine
Lipoprotein X
• Does not indicate CHD
• Abnormal lipoprotein found in obstructive jaundice
Method: enzymatic assays
and LCAT deficiency
• Specific and sensitive indicator of cholestasis
• Lipid content: Phospholipid and free cholesterol (90%) Cholesterol
• Contains Apo C and albumin • Does not require a fasting specimen if tested alone
• For lipid profile, a 12-hour fasting specimen is a must
B-VLDL • Specimen: Serum/Plasma
• "floating B lipoprotein" o EDTA, heparin
• Known as "abnormally migrating B-VLDL" • If analysis is delayed, refrigerate at 4 degrees Celsius.
o Density of VLDL during ultracentrifugation, but • Methods
migrates to LDL in the B region during o Chemical methods
electrophoresis o Enzymatic methods
• Found in type 3-hyperlipoproteinemia or
dysbetalipoproteinemia Chemical Methods
• VLDL is rich in cholesterol instead of TAG 1. Liebermann Burchardt
o Principle: Dehydration and oxidation of
cholesterol to form a colored compound
Lipid Disorders o End product: Cholestadienyl monosulfonic acid
(green end color)
Dyslipidemia o Reagent:
• Lipid concentration abnormalities ▪ Glacial acetic acid
• Associated with ▪ Acetic anhydride
o Arteriosclerosis ▪ Concentrated H2SO4
o CHD 2. Salkowski
• Important factors: o Principle: Dehydration and oxidation of
o Lifestyle cholesterol to form a colored compound
o Inheritance/Family genetic background o End product: Cholestadienyl disulfonic acid (red
• Hyperlipoproteinemia end color)
o Hypercholesterolemia o Reagent:
o Hypertriglyceridemia ▪ Glacial acetic acid
o Combined hyperlipidemia ▪ Ferric chloride
• Hypolipoproteinemia ▪ Concentrated H2SO4
3. One-step method (colorimetry)
 Enclonar, Kimberly / MLS 3A
4. Two-step method (extraction + colorimetry) • Total glycerol
o Reagent for extraction = petroleum ether o Single-cuvette blank
5. Three-step method (Saponification + extraction + • Measures the amount of hydrogen peroxide produced
colorimetry) in the reaction
o Saponification • Amount of H2O2 = glycerol
▪ Hydrolysis of lipids • Measured at 500 nm
▪ Reagent = alcoholic KOH
6. Four-step method (Saponification + extraction +
colorimetry + precipitation)
o Precipitate all other elements except
cholesterol (for interference)

Enzymatic Methods
• Most common method for measuring cholesterol
• Measures the amount of hydrogen peroxide produced
in the reaction Modified Van Handel Zilversmith
o Amount of H2O2 is also the amount of o CDC reference method
cholesterol present o Uses chloroform for extraction
• Measured at 500 nm o Alcohol KOH for saponification
• Intensity of color produced is directly proportional to o Uses silicic acid - to remove phospholipids
cholesterol concentration o End color: Pink
• Advantage: No extraction step needed
• Interferences: Vitamin C, bilirubin, hemoglobin GCMS
o 2 weeks preparation • Most recent reference method
• Cholesteryl ester (70% of cholesterol)
• Free cholesterol (30%) Lipoprotein
• Fasting specimen is required (12-14 hours)
• Specimen: Serum/Plasma
• Methods:
o Ultracentrifugation
o Electrophoresis
o Chemical Precipitation

Ultracentrifugation
Abell Levy, and Brodie Method • Reference method for quantitation of lipoproteins
• CDC reference method • Based on the protein and TAG contents
• Uses hexane for extraction • Expressed in Svedberg (S) units
• Alcohol KOH for saponification • Reagent: Potassium bromide solution with 1.063
• Reaction with Liebermann Burchardt Reagent density
• End color: green • Chylos form "creamy" layer on top of tube
• Lp(a) sinks in the LDL region.
GCMS • B-VLDL floats in the VLDL region.
• Most recent reference method
Analyte Densities (kg/L)
Triglyceride
• Fasting specimen is required (12-14hours) CM <0.95
• Specimen: serum/plasma VLDL 0.95-1.006
• Measurement is dependent on glycerol
LDL 1.019-1.063
• Methods
o Chemical methods HDL 1.063-1.21
o Enzymatic Methods
HDL 2 1.063-1.25
Chemical methods HDL 3 1.125-1.21
1. Van Handel & Zilversmith
o Principle: Colorimetry
o End color: Blue
o Reagent:
▪ Alcohol KOH
▪ Periodic acid
▪ Chromotropic acid

Enzymatic Method
• Commonly used in the laboratory
• Glycerol present in the serum/plasma can become an
interference Electrophoresis
o Double-cuvette blank • Allow separation of major lipoproteins
▪ One is without reagent • Preferred supporting medium: agarose gel
• Free glycerol o Provides faster, a more sensitive process.
▪ The 2nd has reagent o Provides clear background.
 Enclonar, Kimberly / MLS 3A
o Alkaline pH. ▪ TAG/5 is only applicable if TAG
• Basis is protein:lipid ratio; not just protein content. <400mg/dL
▪ TAG*0.16; if TAG is >400 mg/dL
HDL Alpha Most anodic. o LDL (mmol/L) = Total cholesterol - HDL -
lipoprotein Travels the farthest. (TAG/2.175)

VLDL Pre-beta Lp(a) also migrates in the pre-beta Lipid profile

lipoprotein region. • Total cholesterol
LDL Beta B-VLDL also migrates in the beta • HDL
lipoprotein region. • TAG
• LDL (calculated)
CM - Chylos usually do not move from
the point of application. When
(Henry's)
they do, they only move until the
gamma region. Total Cholesterol
<200 mg/dL Desirable
200-239 mg/dL Borderline high
≥240 mg/dL High cholesterol

TAG
<150 mg/dL Normal
Chemical precipitation 150-199 mg/dL Borderline high
• Uses polyanions (heparin, dextran sulfate) and 200-499 mg/dL High TAG
divalent cations (manganese, magnesium)
• Most commonly used for HDL ≥500 mg/dL Very high TAG
o Precipitates all the other proteins except HDL.
• Aggregation of lipoproteins relies on charges LDL-C

HDL-C Method <100 mg/dL Optimal

• Homogenous assay 100-129 mg/dL Near/above optimal
o Allows automation: highly precise and accurate
o Most commonly used in the laboratory 130-159 mg/dL Borderline high
o 1st reagent - "block" non-HDLs 160-189 mg/dL High
o 2nd reagent - quantifies HDL
≥190 mg/dL Very High
• CDC reference method:
o 3-step procedure
i. Removal of VLDL and CM via HDL-C
ultracentrifugation. <40 mg/dL High risk
ii. Heparin manganese precipitation to
remove LDL. ≥60 mg/dL Low risk
iii. Analysis via Abell-Kendall assay
(cholesterol measurement) Additional notes
• Initial screening must be done at the age of 20 or
LDL-C Method older
• Beta quantification (reference method) • Total cholesterol, HDL-C, LDL-C, TAG
o Combination of ultracentrifugation and • Testing should be repeated at least one every 5 years
chemical precipitation
i. Ultracentrifugation - remove VLDL and Cut-off points for cholesterol
CM (product 1: LDL & HDL) Age Moderate Risk High Risk
• Divide equally into 2 parts. Save
2-19 >170 mg/dL >180 mg/dL
the other one for later. Use the
other one for the next step. 20-29 >200 mg/dL >220 mg/dL
ii. Chemical precipitation - separate HDL 30-39 >220 mg/dL >240 mg/dL
from LDL (product 2)
iii. Cholesterol is measured in product 1 and 40 and over >220 mg/dL >260 mg/dL
2.
iv. LDL is calculated by subtracting
cholesterol measurements in product 2
from product 1.
• Friedewald equation
o Derived from total cholesterol = HDL + LDL +
VLDL
o LDL = Total cholesterol - HDL - VLDL
▪ VLDL is not measured in the lab, it is
calculated
▪ VLDL = Tag/5
o LDL (mg/dL) = Total cholesterol - HDL - (TAG/5)