Triglycerides

Properties of Triglycerides
Triglycerides
or

Triacylglycerols (TAGs)
are nonComplexes of non-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 reacting with fatty acids.

Properties of Triglycerides..cont.
Triglycerides
TAG are the major form of

Neutral Fat
found in nature.

Properties of Triglycerides .cont.

TAG is the

Principal Fat
found in foods, body tissues and blood.
Mammalian tissues also contain some diglycerides and monoglycerides, these occur in trace levels when compared withTriglycerides.

Properties of Triglycerides ..cont.

Fat in food becomes fat (triglycerides) in our blood.
It is the main constituent of vegetable oils and animal fats.

A diet high in carbohydrates (sugar and starches) may also raise blood triglyceride levels.

Properties of Triglycerides ..cont.

TAG is the main fuel store in the body.

Structure ofTriglycerides
Triglycerides (Triacylglycerol,TAG) are Esters of
=

Glycerol
Reacting with

Three Fatty Acids

Ester Linkage

Glycerol, Free Fatty Acids and TAG Structures

Structure of Triglyceride cont.

Fatty Acids of TAG
The three fatty acids can be all different, all the same, or only two the same. They can be saturated or unsaturated fatty acids. Chain lengths of the fatty acids in naturally occurring triglycerides can be of varying lengths but 16, 18 and 20 carbons are the most common.

Structure of TAG

= Ester Bond

Structure of Triglyceride cont.

Saturated and Non-Saturated FFAs

Formation of TAG from Glycerol and FFAs

+
3H2O

Sources of Plasma Triglycerides

Plasma TAG are derived from 2 sources: (I) Intestine
Intestinal TAG are derived from dietary fat.

(II)
Liver

Exogenous Sources

Endogenous Sources

Sources of Triglycerides
1) Exogenous Sources
An adult human ingest about 60- 150 g of lipid per day. Triglycerides consistitute more than 90% of this intake. Excess dietary fat can only be stored as TAG in adipose tissue.

Digestion of Exogenous TAG by Stomach and Small Intestine

TAG in the diet are digested in:
1 Stomach Gastric Lipase
(Limitted Effect)

2 Small Intestine Pancreatic lipase

2-Monoacylglycerols + 2 Free fatty acids.

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 reaches the duodenum largely unaltered. In the intestine, TAG is solubilized by bile acids, which are secreted from liver / gall bladder. High CMC (critical micellar concentration) of bile acids ensures rapid action.

2. Pancreatic Lipase. After emulsification by bile acids, solubilized TAG is degraded by

pancreatic lipase (that is secreted into duodenum).

How Bile Acids Emulsify Lipids ?

Intestinal Lipolysis and Absorption of Triglycerides

After TAG emulsification (micelles formation) by the bile :

Pancreatic lipases breakdown TAG

Lipolysis
TAG molecules are split into

Glycerol + Free fatty acids Which are then moved into the cells lining the intestine (absorptive enterocytes).

How Non-polar TAG is Transported from Intestine to the Liver and other Tissues by the Blood ?
The TAG are rebuilt in the enterocytes from their fragments:

FFA + glycerol

How Non-polar TAG is Transported from Intestine to the Liver and other Tissues by the Blood? ..cont.
Since TAG are insoluble in water, the problem of how to transport TAG in the aqueous blood is solved by the ability of enterocytes to associate: 1) Nonpolar lipids (TAG and cholestryl esters) 2) Amphipathic lipids ( phospholipids and cholesterol) 3) Proteins in large particals; chylomicrons.
.

After digestion and uptake by intestinal cells, triacylglycerol is resynthesized and packaged into chylomicrons
Protein

Fatty acids, 2-MAG

Gut lumen

Fatty acids, 2-MAG ATP ADP Triacylglycerol Apolipoproteins Chylomicrons

intestinal epithelium

Triacylglycerol

Chylomicrons

lymphatics

The Role of Chylomicrons in Transporting TAG

Transport of TAG from Intestine to Liver and other Tissues

Chylomicrons; water-miscible lipoproteins ,are excreted from the intestinal cells and collected by the lymph system. Chylomicrons are responsible for the transport of all dietary lipids from intestine to the circulation, however, TAG is the predominant lipid in chylomicrons.

Transport of Triglycerides from Intestine to Liver and other tissues Various tissues can capture
chylomicrons, which will release TAG into the cells to be used as a source of energy.

Adipose tissues are ( after the liver ) the main

tissue that clear chylomicrons from the circulation and taking TAG, however, this is mainly for storage and not for energy production.

Sources of Triglyecrides
2) Endogenous Sources Biosynthesis of TAG
Most mammalian tissues convert fatty acids to triacylglycerols by a common sequence of reactions : (1) Liver (2) Adipose tissue These 2 tissues carry out this process to the greatest extent than others.

Endogenous Sources of TAG ..cont.

(I) Biosynthesis of TAG by the Liver (from Exogenous FFAs) Chylomicron remenants remaining after digestion by lipoprotein lipase are cleared from the blood by the liver. TAG present in the remenants are hydrolzed by lysosomal lipase.

The role of Lipoprotein Lipase in Utilizing TAG in Chylomicrons and VLDL by Different Tissues
Lipoprotein lipase is expressed in
1. Adipose tissues 2. Cardiac muscle 3. Skeletal muscle Allowing these tissues to utilize TAG from lipoproteins.

Utilization of Chylomicrons in Peripheral Tissues

Fat cell Triacylglycerol

capillary Fatty acids, glycerol Chylomicron remnants

Chylomicron Lipoprotein lipase

Muscle cell

Acetyl-CoA

TAG Synthesis

Sources of Triglycerides cont.

2) Endogenous Sources
I) Biosynthesis of TAG by the Liver
From Endogenous Sources of FFAs Exogenous Sources of FFAs

Transport of TAG from Liver to Extrahepatic Tissues

1. FFA
comes from the chylomicrons re-esterified with

2. Glycerol -3- phosphate

(derived from free glycerol and glucose)

TAG
which is then packaged into

VLDL
which is secreted into the bloodstream to go to extrahepatic tissues.

Transport of Triglycerides from Liver to Extrahepatic Tissues.cont.

VLDL
are vehicles of transport of

TAG
from the liver to the extrahepatic tissues.

Transport of lipids between organs

1. Lipoproteins. These basically are lipid droplets with a hydrophilic protein coat. Important examples are:
Chylomicrons. Distribute triacylglycerol (TAG) from intestine to peripheral organs (bypassing the liver) VLDL = very low density lipoprotein. Moves TAG and some other 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 peripheral organs to liver Free fatty acids. These are bound to albumin. This is the major transport mechanism for release of fat from fat tissue .

Where are Triglycerides Stored in the Body?

TAG is confined largely to storge sites in adipose tissue. TAG synthesis in liver is primarly for the production of plasma lipoproteins, rather than for energy storage. Some storage of TAG also occurs in skeletal and cardiac muscle, but only for local consumption.

Where are Triglycerides Stored in the Body?

Synthesis Storage Site Storage for Local Site Consumption to Export to to Produce Different Tissues Energy Storage Site for Lipoproteins Production

Adipose Tissues Liver

TAG (mainly in this tissue) TAG (mainly in this tissue)

TAG

TAG TAG (mainly in this tissue)

TAG

Skeletal Muscle and Cardiac Muscle

TAG

TAG (mainly in this tissue)

TAG Transport in the Fasted State

When the body needs FFAs, TAG stored in adipose tissue are mobilized for use as fuel in the fasted state. This process is initiated by the hormone sensitive lipase, which is located within adipocytes. Insulin inhibits the activity of this enzyme. During fasting, glucagon, epinephrene and norepinephrene signals the breakdown of TAG by increasing the activity of this lipase to release free fatty acids.

Effect of Hormones on Hormone-Sensitive Lipase Activity

Function of Triglycerides
1) The primary function of TAG is to provide energy to the cell. TAG are directly utilized by many tissues
as an energy source. They contain more than twice as (9 much energy (9kcal/g) as carbohydrates and proteins.

2) TAG, as major components of very low density chylomicrons, lipoproteins (VLDL) and chylomicrons, play an important role in the body metabolism as energy sources. 3) FFAs produced from TAG hydrolysis, can be converted by many tissues to phospholipids which are important consistituents of membranes.

Levels of TAG in Plasma

The Concentration of TAG
in the plasma at any given time is a balance between:

(I) The Rate of Entry into the Plasma

and

(II) The Rate of Removal.

A change in the concentration may therefore be a result of a change in either or both of these factors.

Metabolism of Triacylglycerol Overview

Triacylglycerol
Sugars Amino acids

ADP

ATP CO 2 +H O 2

Acetyl- Co.A

Triacylglycerol

Cholesterol

Ketone bodies

TAG

Catabolism

-Oxidation

Why Hypertriglyceridemia is Dangerous?

Elevated Levels of

Triglycerides in Plasma
have been identified as risk factor related to

Atherosclerotic Disease.

Hypertriglyceridemia
FACTORS THAT CONTRIBUTE TO ELEVATED SERUM TRIGLYCERIDES Excess weight or obesity Physical inactivity Stress Excessively high carbohydrate diets (<60% of the caloric intake) Type II diabetes Chronic renal failure Drugs (such as corticosteroids, estrogens, retinoids, high does of beta adrenergic blocking agents) Certain genetic metabolic disorders (including familial combined hyperlipidemia, and familial hypertriglyceridemia.

HIGH TRIGLYCERIDES LEVELS

can be controlled in most cases, but not cured. To do so you must make permanent, beneficial changes in your lifestyle.

How can Blood Triglycerides be Lowered?

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. How can Blood Triglycerides be Lowered?
1. Achieve and maintain your ideal body weight.
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).

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?

2. Increase your Activity If 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 cases, but not cured. To do so you must make permanent, beneficial changes in your life style..cont.
How can Blood Triglycerides be Lowered?

3. Decrease your Calorie Intake - Take smaller portion sizes at each meal. Use low calorie foods
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
Hypotriglyceridemia is a state of

Low Triglyceride Levels

Differential diagnosis of underlying causes Hyperthyroidism Malabsorption syndrome Hereditary abetalipoproteinemia Hypobetalipoproteinemia

(I) OBJECTIVE OF THE EXP.

To determine the level of TAG in a serum or plasma sample.

(II) METHOD PRINCIPLE The standard method used for the measurements of triglycerides concentration ,which is used in this experiment , is an enzymatic one. This formulation makes use of the enzymatic hydrolysis of TAG and quantification since it is specific and not subject to interference by phospholipids.

(I) METHOD PRINCIPLE..cont.

The present procedure involves hydrolysis of triglyerides by lipase. The glycerol concentration is then determined by enzymatic assay coupled with Trinder reaction, which measure the H2O2 activity,that terminates in the formation of a Quinoneimine dye. The amount of the dye formed, determined by its absorption as 505 + nm is directly proportional to the concentration of triglycerides present in the sample.

Principle of the Test

The enzymatic reaction sequence employed in the assay of triglycerides is as follows:
Lipase

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

Glycerol Kinase

Glycerol + ATP --- -

Glycerol 3 Phosphate + ADP

Glycerol phosphate oxidase (GPO)

Glycerol -3 Phosphate + O2 --- Dihydroxyacetone phosphate + H2O2

Peroxidase

H2O2 + 4- Aminoantipyrine + 4- Cholorophenol ---- Quinoneimine Dye ( red dye) + 2H2O

RANGE OF EXPECTED TAG VALUES IN SERUM Normal distributions vary with age , the following concentrations if exceeded, clearly indicate hyperlipidemia. 0-29 30-30 40-49 50-59 years years years years 10-140 mg/dl 10-150 mg/dl 10-160 mg/dl 10-190 mg/dl

(III) MATERIALS REQUIRED

REAGENTS 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, Glycero-30phosphate Oxidase 7000 U/L Sodium Azide 0.01% Lipase 200, 000 U/L Glycerol 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 azide 0.01% Added as a preservative.

(III) MATERIALS REQUIRED

MATERIALS

Tg- Buffer Reagent ( R1), Tg Enzyme Reagent ( R1a) Tg Standard ( 200 mg/dl ) Spectrophotometer, Cuvettes Pipettes Constant temperature incubator set at 37 oC Timer and Distilled water.

(III) MATERIALS REQUIRED

MATERIALS
SPECIMEN
SERUM Fresh , non- hemolyzed serum from fasting pastients is recommended. Triglycerides in serum appear stable for 3 days when stored at 2-8 o C Prolonged storage of the samples at room temperature is not recommended since other glycerol containing compounds may hydrolyze, releasing free glycerol with an apparent increase in total triglycerides content. Blood collection devices lubricated with glycerin ( glycerol) should not be used.

(IV) CALCULATIONS
CALCULATIONS
CONCENTRATION IN TEST ( mg/dl) = A (TEST) x CONC. OF STD ( mg/dl) = A ( STANDARD ) A= Absorbance

EXAMPLE :

0.17 x 200 mg / dl = 154. 5mg/dl 0.22