lipid metabolism & measurement Flashcards
lipoprotein metabolism pathways
- Absorption pathway ( intracellular - transport pathway
- Exogenous pathway
- Endogenous pathway
- reverse cholesterol transport pathway
absorption, exogenous & endogenous pathways
absorption, exogenous & endogenous pathways
all depend on Apo-B containing lipoprotein
processes to transport dietary & liver synthesized lipids to peripheral cells
net result of these 3 processes can lead to atherosclerosis
peripheral cells are prone to cholesterol accumulation because :
- they synthesize their own cholesterol
- no enzymatic pathway to break down cholesterol like the liver does
- cholesterol is water insoluble - cant diffuse away from the site of deposition or synthesis
reverse cholesterol pathway helps maintain cholesterol equilibrium
- mediated by HDL
Absorption pathway
during digestion pancreatic lipase converts lipid into more polar compunds
triglycerides –> monoglycerides + diglycerides
cholesterol esters–> free cholesterol
these lipids from large aggregates with bile acids which are called micelles ***
Absorption of lipids occurs when micelles come in contact with the microvilli of intestinal mucosal cells
short & long chain fatty acids in absorption pathway
short chain fatty acids ( _<10 ) pass directly into the portal circulation & are transported to the liver buy albumin ***
Long chain fatty acids, monoglycerides & diglycerides are reesterified in the intestine to form triglycerides & cholesterol esters
- these are then packaged unto chylomicrons->gives milky appearance
90% of dietary triglycerides, half of cholesterol & a small fraction of plant sterols are absorbed by the intestines
Exogenous pathway
Function: transport dietary lipids from intestine to liver & peripheral cells
the chylomicrons synthesized in the intestine enter circulation though thoracic duct
they interact with proteoglycans (ex. heparin sulphate) & a specific protein on capillary surfaces to promote binding to lipoprotein lipase ( LPL)
LPL hydrolyses triglycerides on the chylomicrons ti glycerol & free fatty acids which can then be taken up by cells for energy use
excess fatty acids are reesterified into the triglycerides for long- term storage
Exogenous pathway hormones
epinephrine & cortisol
- mobilize & hydrolyze triglycerides from adipocytes
insulin
- prevents lipolysis by adipocytes
- promotes fat storage & glucose utilization
Exogenous pathway after a meal
a few hours after a meal, chylomicrons are converted into chylomicron remnants which are quickly taken up by the liver
enzymes breakdown the remnants, releasing fatty, free cholesterol & amino acids
some cholesterol is converted into bile acids
- bile acids & cholesterol are excreted directly into bile
- almost all bile acids are reabsorbed & reused by the liver for bile production
- any cholesterol that is not reabsorbed will appear in the stool
Endogenous pathway
function : transfer lipids synthesized in the liver to peripheral cells for energy
most triglycerides in the liver are packaged into VLDL
lipolysis of triglycerides from the core of VLDL transform it to IDL & then to LDL
LDL is a major lipoprotein involved in transporting exogenous cholesterol to peripheral cells for energy due to efficient uptake by LDL receptors
VLDL –> IDL –> LDL –> Cells
endogenous pathway - abnormal LDL receptor function
abnormalities in LDL receptor function leads to :
- elevated LDL levels in the circulation
- hypercholesterolemia
- premature atherosclerosis
familial Hypercholesterolemia ( FH)
- Half the normal LDL receptors = decreased uptake of LDL by the liver
- Patients have increased plasma LDL
- Leads to development of CHD by mid adulthood
( hetero–> midadulthood, homo–> less than that )
Reverse Cholesterol Transport Pathway
Function : remove excess cholesterol from peripheral cells & transport it to the liver for excretion
this process is mediated by HDL
lecithin cholesterol acyltransferase ( LCAT) converts cholesterol to cholesteryl esters which remain trapped in the core of HDL until they are removed by the liver
~1/2 cholesterol on HDL is returned to liver by LDL receptors
- cholesterol is transferred form HDL to LDL via cholesteryl ester transfer protein ( CETP)
Cholesterol Metabolism - Summary
Most cholesterol is synthesized endogenously from acetyl CoA in the liver & intestine
70% of cholesterol in plasma/ serum is esterified with fatty acids (30%= free cholesterol)
cholesterol is transported to the cells via LDL
Carried back to the liver via HDL
- cells do not have an enzyme to catabolize cholesterol & it is not H2O soluble, so it cannot diffuse away from cell
- HDL is needed to transport cholesterol to the liver to be excreted via bile
Triglyceride Metabolism -Summary
obtained from the diet & packaged into VLDL in the liver
Excess triglycerides are stored in adipose tissue
During fasting or a deficiency of carbohydrates, triglycerides are hydrolyzed to fatty acids for energy
Fatty acids –>carried to tissues(bound to albumin)–> ß-oxidation–>ATP
Lipid Population Distribution
Women tend to have higher HDL & lower total cholesterol & triglycerides than men
Levels of total cholesterol, LDL, & triglycerides increase in both men & woman as they age
Societies that eat less animal fat & more grain, fruits & vegetables have lower LDL concentrations & lower rates of heart disease when compared to societies that ingest more fat
Genetics & lifestyle factors both play a role in cholesterol concentrations
Clinical Significance of Cholesterol
Heart disease is strongly associated with cholesterol concentrations ( especially LDL)
An increase in cholesterol & triglycerides causes plaque to build up on arterial walls which leads to arteriosclerosis ( hardening)
causes eventual decreased blood flow ( and oxygen ) to the heart which can cause angina or a myocardial infarct
increasing HDL concentrations helps reduce the risk of CHD
Cholesterol reference ranges
total cholesterol by itself is not a good indicator of high risk patients (LDL could be increased while HDL is decreased )
the ratio of total cholesterol / HDL should be 5/1 ; optimal range is < 3.5/1
lipid acceptable reference ranges
total cholesterol < 5.2
LDL < 3.4
triglycerides < 1.7
HDL >0.9
elevated cholesterol
elevated cholesterol can be seen in the following conditions :
- diabetes mellitus ( inc. fatty acids from acetyl CoA )
- Nephrotic syndrom
- Hypothyroidism
- Biliary cirrhosis
Gallstones are composed of precipitated cholesterol
elevated triglycerides
elevated triglycerides can be seen in :
- arthrosclerosis & CHD
- diabetes
- pancreatitis ( affects CHO metabolism )
- acoholism
- Nephrotic syndrome
- gout
- obesity
AKA
dyslipidemia –> diseases associated with elevated lipids
Coronary heart disease risk factors
age _> 45 for men, _>55 for women family history hypertension elevated LDL borderline to high LDL with other risk factors low HDL diabetes mellitus
increased: triglycerides, LDL, total cholesterol
decreased: HDL
Methods of triglyceride Analysis
methods:
Gas Chromatography –> reference method
methods based on enzymatic determination of glycerol ( these 3 all HAVE SAME 1ST STEP )
- -> NADH consumption
- -> colorimetric ( used most often )
- -> fluorescent
Trig enzymatic method- NADH consumption
lipase
1. Triglycerides —————>glycerol + 3 fatty acids *****
glycerokinase 2. Glycerol + ATP --------------->glycerol- 3 -phosphate + ADP
pyruvate kinase 3.ADP + phosphoenolpyruvate ------------------>ATP + pyruvate
lactate dehydrogenase 4.Pyruvate + NADH + H^+---------------------------> lactate + NAD^+ *****
the decrease in absorbance of NAD^+ @ 340 nm is measured ***
this is an earlier method that was susceptible to interferences & side reactions and has largely been replaced
Trig enzymatic methods - colorimetric method
lipase
1. Triglycerides —————>glycerol + 3 fatty acids *****
glycerokinase 2. Glycerol + ATP --------------->glycerol- 3 -phosphate + ADP
glycerophosphateoxidase 3. Glycerol-3-phosphate + O2-------------------> Dihydroxyacetone + H2O2
peroxidase 4. H2O2 + 4-cholorophenol ---------------------> Quinoneimine dye + H2O
an increase absorbance from the dye is directly proportional to the amount of triglycerides in the sample**
Trig enzymatic methods - Fluorometric method
lipase
1. Triglycerides —————>glycerol + 3 fatty acids *****
glycerol-3-phosphate dehydrogenase 2. Glycerol + NAD^+ -------> dihydroxyacetone phosphate + NADH+ H^+
diaphorase 3.NADH + H^+ + Resazurin ---------------> Resorufin + NAD^+
fluorescence produced by resorcin ( fluorescent dye ) is measured *****
it absorbs short wavelengths/ high energy & emits long wavelengths / low energy in the visible region
Methods of cholesterol analysis
gas chromatography —-> reference method
Enzymatic methods
- —> H2O2 measurement
- —> O2 consumption
- —> electrode
- —> 4- aminoantipyrine
- —> A NAD
CHolesterol enzymatic methods - H2O2 measurement
cholesteryl esterase
1. Cholesteryl esters ——–> cholesterol ***** + fatty acids
cholesterol oxidase 2.cholesterol + O2 ---------------> cholesterol-4-ene-3-one****** + H2O2
peroxidase 3. H202+ 4- aminophenanone + phenol-------> Quinonemine dye + H2O
the intensity of color of dye ( measured around 500 nm ) is proportional to the amount of cholesterol in the sample
cholesterol enzymatic mehtods - oxygen electrode
GOOD Q: measures consumption of oxygen ( decrease in oxygen ) as the reaction proceeds
cholesterol oxidase cholesterol + O2--------------> Cholesterol-4-ene-3-one + H2O2
HDL measurement - precipitation method
precipitation method:
a precipitation is used to precipitate all lipoprotein fractions except HDL
after centrifugation, HDL is measured from the supernatant
interference from high triglyceride levels is a problem
no longer routinely used in clinical lab
HDL measurement - direct
2 reagents used :
1st regent blocks non- HDL lipoproteins
2nd reagent contains enzymes that will allow quantification of HDL
better suited to the clinical lab as there is no pre-treatment & direct methods can be automated
LDL measurement
reference method for LDL is ß- Quantitation :
- combines ultracentrifugation with chemical precipitation
- tedious - not performed in clinical lab
LDL can be calculated by the Friedewald Formula***
HDL + LDL + VLDL = TOTAL cholesterol
LDL= TOTAL cholesterol -( HDL+ VLDL) ——-> VLDL = trig/2.2
LDL= TOTAL cholesterol- ( HDL + triglycerides/2.2)
LDL measurement notes
use trig/5 if units are in mg/dL
LDL cant be calculated if triglycerides are abnormally high (>4.52mmol/L )
patient protocol for lipids collection
optimum conditions include:
no change in dietary habits for 3 weeks prior to test
fast for 12-16 hrs
- triglycerides rise 2 hrs post- prandial & peak at 4-6 hrs
no alcohol consumption for 72 hrs prior to test
- causes temporary increases in triglycerides
specimen collection for lipids
fresh serum or plasma can be collected
- collect in SST or heparin
- DO NOT USE sodium fluoride - lower cholesterol by ~52 %
- don’t used EDTA - can form micro clots; clogging analyzer
prolonged tourniquet use causes hemoconcentration
- increased filtration pressure across capillary wall
- falsely increases lipids
