Microsoft PowerPoint - Triglycerides Estimation

8/2/2019 Microsoft PowerPoint - Triglycerides Estimation

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Triglycerides


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Properties of Triglycerides

Triglyceridesor

Triacylglycerols (TAGs)

are

Complexes of nonComplexes of nonComplexes of nonComplexes of non----polar lipids.polar lipids.polar lipids.polar lipids. Lipid is a general term that describes substances that are relatively

hydrophobic: water-insoluble and extractable by non-polar solvents.

TAGs are esters of the alcohol glycerol reactingwith fatty acids.


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Properties of Triglycerides .cont.

TAG is the

found infoods, body tissues and blood.

Mammalian tissues also contain some diglyceridesand monoglycerides, these occur in trace levels when

compared withTriglycerides.


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Properties of Triglycerides ..cont.

FatFatFatFat in food becomes fat (triglycerides) inin food becomes fat (triglycerides) inin food becomes fat (triglycerides) inin food becomes fat (triglycerides) in

our blood.our blood.our blood.our blood.

It is the main constituent ofvegetable oils andanimal fats.

A diet high in carbohydrates (sugar andstarches) may also raise blood triglyceride

levels.


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Properties of Triglycerides ..cont.

the body.


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Triglycerides (Triacylglycerol,TAG) are

Esters of=

Structure of Triglycerides

Glycerol

Three Fatty Acids

Reacting with


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Glycerol,

Free FattyAcids and

Ester Linkage

Structures


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Structure of Triglyceride cont.

Fatty Acids of TAG

The three fatt acids can be all different all the

same, or only two the same. They can besaturated or unsaturated fatty acids.

Chain lengths of the fatty acids in naturallyoccurring triglycerides can be of varying lengths

but 16, 18 and 20 carbons are the most common.


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Structure

ofTAG

= Ester Bond


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Saturated and Non-Saturated FFAs

Structure of Triglyceride cont.


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Formation of TAG from Glycerol andFFAs

3H2O

+


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Sources of Plasma Triglycerides

Plasma TAG are derived from 2 sources:

(I)

(II)Liver

IntestineIntestinal TAG

are derived

from

dietary fat.

Endogenous

Sources

Exogenous

Sources

+


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Sources of Triglycerides

1) Exogenous Sources

An adult human ingest about 60- 150 g of

lipid per day. Triglycerides consistitute morethan 90% of this intake.

Excess dietary fat can only be stored as TAG

in adipose tissue.


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Digestion of Exogenous TAG byStomach and Small Intestine

TAG in the diet are digested in:TAG in the diet are digested in:TAG in the diet are digested in:TAG in the diet are digested in:1

2

2-Monoacylglycerols + 2 Free fatty acids.

omacGastric Lipase(Limitted Effect)

Pancreatic

lipase


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Digestion of Exogenous TAG in

Stomach and Small Intestine .cont.1. Gastric lipase. The effect of gastric lipase on

TAG to break it down is a limited effect because fat

is not yet emulsified in the stomach. TAG reachesthe duodenum largely unaltered.

2. Pancreatic Lipase. After emulsification bybile acids, solubilized TAG is degraded bypancreatic lipase (that is secreted into duodenum).

In the intestine, TAG is solubilized by bile acids, which aresecreted from liver / gall bladder. High CMC (critical

micellar concentration) of bile acids ensures rapid action.


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How Bile Acids Emulsify Lipids ?


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Intestinal Lipolysis and Absorption of

Triglycerides After TAG emulsification (micelles formation) by the

bile :

Pancreatic lipases breakdown TAG

Glycerol + Free fatty acids

Which are then moved into the cells lining the

intestine (absorptive enterocytes).

TAG molecules are split into


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How Non-polar TAG isTransported from Intestine to the Liver

and other Tissues by the Blood ?

The TAGTAGTAGTAG are rebuilt in the enterocytes from

FFA + glycerol


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How Non-polar TAG isTransported from Intestine to the Liverand other Tissues by the Blood? ..cont.

Since TAG are insoluble in water, the problem ofhow to transport TAG in the aqueous blood is

1) Nonpolar lipids (TAG and cholestryl esters)

2) Amphipathic lipids ( phospholipids and

cholesterol)

3) Proteins

in large particals; chylomicrons.


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After digestion and

uptake by intestinal

cells, triacylglycerol

is resynthesized

and packaged into

chylomicrons

Fatty acids, 2-MAG

Fatty acids, 2-MAG

ATP

Gut lumen

Triacylglycerol

Apolipoproteins

Chylomicrons

Chylomicrons

intestinalepithelium

lymphaticsTriacylglycerol


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The Role of Chylomicrons in

Transporting TAG


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Transport of TAG fromIntestine to Liver and other Tissues

Chylomicrons; water-miscible lipoproteins ,areexcreted from the intestinal cells and collected

by the lymph system.

Chylomicrons are responsible for the transport

of all dietary lipids from intestine to thecirculation, however, TAG is the predominant lipid

in chylomicrons.


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Transport of Triglycerides from

Intestine to Liver and other tissues

Various tissuesVarious tissuesVarious tissuesVarious tissues can capture chylomicrons, whichwill release TAG into the cells to be used as a

Adipose tissues are ( after the liver ) the maintissue that clear chylomicrons from the

circulation and taking TAG, however, this is

mainly for storage and not for energy

production.


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Sources of Triglyecrides

2) Endogenous Sources

Biosynthesis of TAG

triacylglycerols by a common sequence of

reactions :

(1) Liver (2) Adipose tissueThese 2 tissues carry out this process to the

greatest extent than others.


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Endogenous Sources of TAG

..cont.

(I) Biosynthesis of TAG by the Liver

(from Exogenous FFAs)

Ch l mi r n r m n nt r m inin

after digestion by lipoprotein lipaseare cleared from the blood by the liver.

TAG present in the remenants arehydrolzed by lysosomal lipase.


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The role of Lipoprotein Lipase in

Utilizing TAG in Chylomicrons andVLDL by Different Tissues

Lipoprotein lipase is expressed in

1. Adipose tissues2. Cardiac muscle

3. Skeletal muscle

Allowing these tissues to utilize TAG

from lipoproteins.


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Utilization of Chylomicrons in Peripheral Tissues

Fat cell

Fatty acids,

Triacylglycerol

capillary

g ycero

Lipoprotein lipase

Chylomicron

Muscle cell Acetyl-CoA

Chylomicronremnants


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TAG Synthesis


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Sources of Triglycerides cont.

2) Endogenous Sources

I) Biosynthesis of TAG by the LiverBiosynthesis of TAG by the LiverBiosynthesis of TAG by the LiverBiosynthesis of TAG by the Liver

Exogenous

Sources of

FFAs

Endogenous

Sources ofFFAs

From

T t f TAG f Li t


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Transport of TAG from Liver to

Extrahepatic Tissues1. FFA

comes from the chylomicrons

re-esterified with

2. Glycerol -3- phosphate

(derived from free glycerol and glucose)

TAGwhich is then packaged into

VLDLwhich is secreted into the bloodstream to go to extrahepatic tissues.


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Transport of Triglycerides fromLiver to Extrahepatic Tissues.cont.

VLDL

from

the liver to the extrahepatic tissues.

TAG


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Transport of lipids between organs

Chylomicrons. Distribute triacylglycerol (TAG) from intestine toperipheral organs (bypassingthe liver)

VLDL = ver low densit li o rotein. Moves TAG and some other

1. Lipoproteins. These basically are lipid droplets witha hydrophilic protein coat. Important examples are:

lipids from liver to periphery

LDL = low density lipoprotein. Moves lipids (particularly cholesterol)from liver to periphery

HDL = high density lipoprotein. Moves excess cholesterol from peripheralorgans to liver

.. This is the majoralbumin. These are bound toFree fatty acidstransport mechanism for release of fat from fat tissue


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Where are Triglycerides Stored in the

Body?

TAG is confined largely to storge sites inadipose tissue.

TAG synt esis in ver is primar y or t eproduction of plasma lipoproteins, rather thanfor energy storage.

Some storage of TAG also occurs in skeletaland cardiac muscle, but only for local

consumption.

Where are Triglycerides Stored in the Body?


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Where are Triglycerides Stored in the Body?

Storage Sitefor

Lipoproteins

Production

Storage

Siteto Export to

Different

Tissues

Storage Site

for LocalConsumption

to Produce

Energy

Synthesis

TAG

Adipose

this tissue)

TAG

(mainly in

this tissue)

TAG

TAG

(mainly in

this tissue)

Liver

TAG

(mainly in

this tissue)

TAG

Skeletal

Muscle and

Cardiac

Muscle


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TAG Transport in the Fasted State When the body needs FFAs, TAG stored in

adipose tissueare mobilized for use as fuel inthe fasted state. This process is initiated by thehormonesensitive lipase, which is located

within adipocytes. Insulin inhibits the activity of this enzyme.

During fasting, glucagon, epinephrene andnorepinephrene signals the breakdown of TAGby increasing the activity of this lipase to release

free fatty acids.


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Effect of Hormones

on Hormone-Sensitive Lipase Activity


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Function of Triglycerides1111)))) The primary function of TAG is to provide energyThe primary function of TAG is to provide energyThe primary function of TAG is to provide energyThe primary function of TAG is to provide energy

to the cell.to the cell.to the cell.to the cell. TAG are directly utilized by many tissuesTAG are directly utilized by many tissuesTAG are directly utilized by many tissuesTAG are directly utilized by many tissues

as an energy source. They contain more than twice asas an energy source. They contain more than twice asas an energy source. They contain more than twice asas an energy source. They contain more than twice asmuch energy (much energy (much energy (much energy (9999kcal/g) as carbohydrates and proteins.kcal/g) as carbohydrates and proteins.kcal/g) as carbohydrates and proteins.kcal/g) as carbohydrates and proteins.

,,,,

lipoproteins (VLDL) andlipoproteins (VLDL) andlipoproteins (VLDL) andlipoproteins (VLDL) and chylomicronschylomicronschylomicronschylomicrons, play an, play an, play an, play animportant role in the body metabolism as energyimportant role in the body metabolism as energyimportant role in the body metabolism as energyimportant role in the body metabolism as energysources.sources.sources.sources.

3333) FFAs produced from TAG hydrolysis, can be) FFAs produced from TAG hydrolysis, can be) FFAs produced from TAG hydrolysis, can be) FFAs produced from TAG hydrolysis, can beconverted by many tissues to phospholipids whichconverted by many tissues to phospholipids whichconverted by many tissues to phospholipids whichconverted by many tissues to phospholipids whichare importantare importantare importantare important consistituentsconsistituentsconsistituentsconsistituents of membranes.of membranes.of membranes.of membranes.


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Levels of TAG in Plasma The Concentration of TAG

in the plasma at any given time is a balance between:

I The Rate of Entr into the Plasma

and

(II) The Rate of Removal.

A change in the concentration mayA change in the concentration mayA change in the concentration mayA change in the concentration maytherefore be a result of a change in eithertherefore be a result of a change in eithertherefore be a result of a change in eithertherefore be a result of a change in either

or both of these factors.or both of these factors.or both of these factors.or both of these factors.

Metabolism of Triacylglycerol


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Sugars Amino acids

ADP ATP

Triacylglycerol

Metabolism of Triacylglycerol

Overview

Cholesterol

CO2 + H2O

Ketone bodies

Triacylglycerol

Acetyl- Co.A


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TAGTAGTAGTAGCatabolismCatabolismCatabolismCatabolism

-Oxidation


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Why Hypertriglyceridemia isDangerous?

Elevated Levels ofElevated Levels ofElevated Levels ofElevated Levels of

Triglycerides in PlasmaTriglycerides in PlasmaTriglycerides in PlasmaTriglycerides in Plasma

have been identified as

risk factor related to

Atherosclerotic DiseaseAtherosclerotic DiseaseAtherosclerotic DiseaseAtherosclerotic Disease.

H t i l id i


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Hypertriglyceridemia

FACTORS THAT CONTRIBUTE TO ELEVATED SERUMTRIGLYCERIDES

Excess weight or obesity Physical inactivity

Stress

Excessively high carbohydrate diets (


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HIGH TRIGLYCERIDES

LEVELScan be controlled in most cases,but not cured.

To do so you must makepermanent, beneficial changes in

your lifestyle.


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Howcan Blood

r g ycer es eLowered?

High triglyceride levels can be controlled in


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High triglyceride levels can be controlled in

most cases, but not cured. To do so you mustmake permanent, beneficial changes in your

life style.

How can Blood Triglycerides be Lowered?

. .

To do that, examine your eating habits. Are you overeating,

eating only one large, late meal a day, having a bedtime

snack?

There are many reasons for overeating, not just hunger

(stress, boredom).


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High triglyceride levels can be controlled in most

cases, but not cured. To do so you must make

permanent, beneficial changes in your life

style.cont.

How Can Blood Triglycerides be Lowered?. ncrease your c v yIf you are overweight, you have taken in more

calories than you have used up. "Burn up"

calories by exercising - moderate brisk walking(1/2 hour 3 - 4 times per week, or as directed

by your physician).

High triglyceride levels can be controlled in most


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3. Decrease your Calorie Intake

- Take smaller portion sizes at each meal. Use low calorie foods

cases, but not cured. To do so you must make

permanent, beneficial changes in your lifestyle..cont.

How can Blood Triglycerides be Lowered?

and snacks. Have three meals a day rather than one large, late

meal.

- Choose whole grain, higher in fiber breads, cereals, crackers,

whole grain pastas, and rice.

- Try homemade, high fiber, low sugar baked goods.- Fruits contain natural sugars. Limit fruit juice and use only 100 %

fruit juice, no sugar added brands.

- Choose whole fruits more often.

- Use artificial sweeteners.

Hypotriglyceridemia


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Hypotriglyceridemia

Hypotriglyceridemia

is a state of

Low Triglyceride LevelsLow Triglyceride LevelsLow Triglyceride LevelsLow Triglyceride Levels

Di erentia iagnosis o un er ying causes

Hyperthyroidism

Malabsorption syndrome

Hereditary abetalipoproteinemia

Hypobetalipoproteinemia


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(I) OBJECTIVE OF THE EXP.

To determine the level of TAG in a

serum or plasma sample.


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(II) METHOD PRINCIPLE

The standard method used for the

measurements of triglyceridesconcentration ,which is used in this

, .formulation makes use of theenzymatic hydrolysis of TAG and

quantification since it is specific andnot subject to interference byphospholipids.

(I) METHOD PRINCIPLE cont


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The present procedure involves hydrolysisof triglyerides by lipase. The glycerol

concentration is then determined byenzymatic assay coupled with Trinderreaction which measure the H O

(I) METHOD PRINCIPLE..cont.

activity,that terminates in the formation of aQuinoneimine dye. The amount of the dyeformed, determined by its absorption

as 505 + nm is directly proportional to theconcentration of triglycerides present in thesample.

Principle of the Test


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The enzymatic reaction sequence employed in the assayof triglycerides is as follows:

Triglycerides +H2O ------> Glycerol + 3 Free Fatty Acids

Lipase

Glycerol

Kinase

ycero + --- - ycero osp a e +

Glycerol -3 Phosphate + O2 --- -

Dihydroxyacetone phosphate + H2O2

H2O2 + 4- Aminoantipyrine + 4- Cholorophenol ----

Quinoneimine Dye ( red dye) + 2H2O

Glycerol phosphate oxidase

)(GPO

Peroxidase

RANGE OF EXPECTED TAG VALUES IN


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RANGE OF EXPECTED TAG VALUES IN

SERUM

Normal distributions vary with age , the

following concentrations if exceeded, clearlyindicate hyperlipidemia.

0-29 years 10-140 mg/dl30-30 years 10-150 mg/dl

40-49 years 10-160 mg/dl50-59 years 10-190 mg/dl

(III) MATERIALS REQUIRED


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(III)MATERIALS REQUIREDREAGENTS COMPOSTION

R1. TRIGLYCERIDES BUFFER REAGENT : Pipes Buffer 40 mmol/L. pH 7.5

4-Chlorophenol 5.0 mmol /L. Magnesium ions 5.0 mmol /L.

R1a, TRIGLYCERIDES ENZYME REAGENT:

ATP 3.3 mM, 4- Aminoantipyrine 0.7 mM, G ycero-30p osp ate Oxi ase

7000 U/L Sodium Azide 0.01% Lipase 200, 000 U/LGlycerol Kinase 100 U/L and peroxidase 3,000 U/L

TRIGLYCERIDES STANDARAD ( 200 MG/ DL AS Triolein) :

2.2584 mmol/L of Glycerol with Surfactant. Sodium azide0.01% Added as a preservative.

( )


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MATERIALS

Tg- Buffer Reagent ( R1), Tg Enzyme Reagent ( R1a)

Tg tan ar mg Spectrophotometer, Cuvettes

Pipettes Constant temperature incubator set at 37 oC Timer and Distilled water.



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MATERIALS SPECIMEN SERUM

Fresh , non- hemolyzed serum from fasting pastientsis recommended.

Tri l cerides in serum a ear stable for 3 da s when

stored at 2-8 o C Prolonged storage of the samples at roomtemperature is not recommended since otherglycerol containing compounds may hydrolyze,

releasing free glycerol with an apparent increase intotal triglycerides content.

Blood collection devices lubricated with glycerin

( glycerol) should not be used.

(IV) CALCULATIONS


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(IV) CALCULATIONS

CALCULATIONS

CONCENTRATION IN TEST ( mg/dl) =

A= Absorbance

.

A ( STANDARD )

EXAMPLE : 0.17 x 200 mg / dl = 154. 5mg/dl0.22


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Documents

Microsoft PowerPoint - Triglycerides Estimation