KBP Chemistry of Lipids

Chemistry of Lipids

Definisi

•        Lipids / lemak adalah komponen organik yang dibentuk terutama dari kombinasi alkohol dan asam lemak terikat dalam bentuk ester

•       lemak tidak larut dalam air, tetapi larut dlm pelarut organik (ether, chloroform, benzene, acetone).
•         Lipid/lemak termasuk asam lemak, minyak dan komponen yg berhubungan
•         Terdistribusi luas di alam pada tanaman dan hewan

 Konstanta analitik

 

•Specific Gravity

      Rasio dari berat lemak terhadap air pada temperatur yg sama atau spesifik. Mengukur dg menggunakan piknometer. Metoda resmi pd suhu 25ºC  Karakteristik lemak .

        minyak kelapa spesifik gr. 0.926 pd 15ºC, 0.9188 pd 25ºC.

 

•Index of Refraction

        Bila chy melewati pd sudut dr satu media ke media lain, mk arah akan berubah pd permukaan kedua media  adalah rasio dari sinus sudut dtg/insiden dan sinus sudut refraksi/bias.

•Titik leleh (melting point)

        gliserida solid mempunyai titik leleh yg berbeda. 

ANALYTICAL METHODS TO MEASURE THE CONSTANTS OF FATS AND OILS

1.  Acid Value

      Number of mgs of KOH required to neutralize the Free Fatty Acids in 1 g of fat.  

2.  Saponification Value

 Saponification - hydrolysis of ester under alkaline condition

 Saponification Value of Fats and Oils

 Saponification Value Determination

 

Saponification # --mgs of KOH required to saponify 1 g of fat.

1.  5 g in 250 ml Erlenmeyer.

2.  50 ml KOH in Erlenmeyer.

3.  Boil for saponification.

4.  Titrate with HCl using phenolphthalein.

5.  Conduct blank determination.

 B - ml of HCl required by Blank.

S - ml of HCl required by Sample.

3.  Iodine Value 

        Number of iodine (g) absorbed by 100 g of oil.

        Molecular weight and iodine number can calculate the number of double bonds.  1 g of fat adsorbed 1.5 g of iodine value  = 150.

Iodine Value Determination

        Iodine Value = (ml of Na2S2O3 volume for blank -  ml of  Na2S2O3 volume for sample) N of Na2S2O3 ´ 0.127g/meq ´ 100

  Weight of Sample (g)

Excess unreacted ICl

 

Iodine Numbers of Triglycerides 

Fatty Acids	of Double-bonds	Iodine
Palmitoleic Acid	1	95
Oleic Acid	1	86
Linoleic Acid	2	173
Linolenic Acid	3	261
Arachidonic Acid	4	320

Compositions (%) of Fatty Acids of Fats 

Fat	C4	C6	C10	C16	C18	C18:1	C18:2	C18:3	C20:4
1	5	5	20	40			30
2			20	35	40	5
3			10	50		40
4			20	40	40
5			10	20	20	10	20	20
6									100

 

4.  GC Analysis for Fatty Acids 

• 1.  Extract fat.
• 2.  Saponify (hydrolysis under basic condition).
• 3.  Prepare methyl ester (CH3ONa).
• 4.  Chromatography methyl ester.
• 5.  Determine peak areas of fatty acids. Fatty acids are identified by retention time.
• 6.  Compare with response curve of standard. 

Fatty Acids Methyl Esters:

 GC condition:  10% DEGS Column (from supelco) Column temperature 200C.

5.  TRIGLYCERIDE ANALYSIS BY LIQUID CHROMATOGRAPHY  

•  Soybean Oil
•  Solvent CH3CN/HF
•  Column 84346 (Waters Associates) 

Oleate-containing triglycerides in olive oil

Fatty Acid Composition	Total Acyl Carbons:Unsaturation	Equivalent Carbon Number
OL2	54:5	44
02L	54:4	46
OPL	52:3	46
O3	54:3	48
OSL	54:3	48
O2P	52:2	48
O2S	54:2	50
OPS	52:1	50
OS2	54:1	52

6.CHOLESTEROL DETERMINATION

Enzymatic Determination: Cholesterol Oxidase

Spectromertic Absorption Standard Curve of Cholesterol 

Cholesterol by GLC

1.  Prepare cholesterol butyrate.

2.  Analyze by GLC.

     time in GC - 15 min.

     sensitivity - 10-7 g.

LIPID CONTENT ANALYSES

 

1.  Gravimetric Method

     (1) Wet extraction - Roese Gottliegb & Mojonnier.

For Milk: 

  1) 10 g milk + 1.25 ml NH4OH mix. Solubilizes protein and neutralizes.

  2) + 10 ml EtOH - shake.  Begins extraction, prevents gelation of proteins.

  3) + 25 ml Et2O - shake and mix.

  4) + 25 ml petroleum ether, mix and shake. 

     (2) Dry extraction - Soxhlet Method. 

          Sample in thimble is continuously extracted with ether using Soxhlet condenser.   

          After extraction, direct measurement of fat

         - evaporate ether and weigh the flask.

          Indirect measurement - dry thimble and weigh thimble and sample. 

 Soxhlet Method

 

 2.  Volumetric Methods (Babcock, Gerber Methods) 

Theory:

1.Treat sample with H2SO4 or detergent.

2.Centrifuge to separate fat layer.

3.Measure the fat content using specially calibrated bottles.

Methods:

1.  Known weight sample.

2.  H2SO4 - digest protein, liquefy fat.

3.  Add H2O so that fat will be in graduated part of bottle.

4.  centrifuge to separate fat from other materials completely.

 

Biological Importance of Lipids:

1.They are more palatable and storable to unlimited amount compared to carbohydrates.

2.They have a high-energy value (25% of body needs) and they provide more energy pergram than carbohydrates and proteins but carbohydrates are the preferable source of energy.

3.Supply the essential fatty acids that cannot be synthesized by the body.

4.Supply the body with fat-soluble vitamins (A, D, E and K).

5.They are important constituents of the nervous system.

6.Tissue fat is an essential constituent of cell membrane and nervous system. It is mainly phospholipids in nature that are not affected by starvation.
 

7-Stored lipids “depot fat” disimpan dalam semua sel manusia yg berperan sebagai:

• Simpanan energi.
• Lemak/bantalan utk organ internal utk melindungi dari outside shocks.
• Insulator panas dari  subcutaneous terhdp kehilangan panas tubuh

 

8-Lipoproteins, kompleks lemak dan protein, konstituen sellular penting yg ada pd membran

  sellular dan subsellular
 

9-Cholesterol masuk dalam struktur membran dan digunakan utk sintesis adrenal cortical 

hormones, vitamin D3 and bile acids.

10- Lipids provide bases for dealing with diseases such as obesity, atherosclerosis, lipid-

storage diseases, essential fatty acid deficiency, respiratory distress syndrome,

Classification of Lipids

1. Simple lipids (Fats & Waxes)
2. Compound or conjugated lipids
3. Derived Lipids 
4. Lipid-associating substances

Fatty alcohols

1-Glycerol:

• It is a trihydric alcohol (i.e., containing three OH groups) and has the popular name glycerin.
• It is synthesized in the body from glucose.
• It has the following properties:

1. Colorless viscous oily liquid with sweet taste.
2. On heating with sulfuric acid or KHSO4 (dehydration) it gives acrolein that has a bad odor. This reaction is used for detection of free glycerol or any compound containing glycerol. 

3-It combines with three molecules of nitric acid to form trinitroglycerin (TNT) that is used as  explosive and vasodilator.
4-On esterification with fatty acids it gives:

•Monoglyceride or monoacyl-glycerol: one fatty acid + glycerol.

•Diglyceride or diacyl-glycerol: two fatty acids + glycerol.

•Triglyceride or triacyl-glycerol: three fatty acids + glycerol.

5-It has a nutritive value by conversion into glucose and enters in structure of phospholipids.

Uses of Glycerol:

1.Glycerol enters in pharmaceutical and cosmetic preparations.

2.Reduces brain edema in cerebrovascular disease.

3.Nitroglycerin is used as vasodilator especially for the coronary arteries, thus it is used in treatment of angina pectoris. Also, enters in explosives manufacturing.

4.Glycerol is used in treatment of glaucoma (increased intraocular pressure)due to its ability to dehydrate the tissue from its water content.

2-Sphingosine:

• - It is the alcohol(monohydric) present in sphingolipids.
• - It is synthesized in the body from serine and palmitic acid.
• - It is not positive with acrolein test.

Fatty Acids 

Definition:

• Fatty acids are aliphatic mono-carboxylic acids that are mostly obtained from the hydrolysis of natural fats and oils.
• Have the general formula R-(CH2)n-COOH and mostly have straight chain (a few exceptions have branched and heterocyclic chains).  In this formula "n" is mostly an even number of carbon atoms (2-34) with a few exceptions that have an odd number.
• Fatty acids are classified according to several bases as follows:

 

I. According to presence or absence of double bonds they are classified into:

• A-Saturated Fatty Acids
• they contain no double bonds with 2-24 or more carbons.
• They are solid at room temperature except if they are short chained.
• They may be even or odd numbered.
• They have the following molecular formula, CnH2n+1COOH. 

Saturated fatty acids (no double )

A-Short chain Saturated F.A. (2-10 carbon).

a-Short chain Saturated volatile F.A.(2-6 carbon).

b- Short chain Saturated non volatile F.A.(7-10 carbon).

B-Long chain Saturated F.A.(more the10 carbon)

 

a-Volatile short-chain fatty acids:

• They are liquid in nature and contain (1-6) carbon atoms.
• water-soluble and volatile at room temperature, e.g., acetic, butyric, and caproic acids.
• Acetic F.A. (2C )      CH3-COOH.
• Butyric F.A. (4C )    CH3-(CH2)2-COOH.
• Caproic F.A. (6C )    CH3-(CH2)4-COOH.

b-Non-volatile short-chain fatty acids:

• They are solids at room temperature and contain 7-10 carbon atoms.
• They are water-soluble and non-volatile at room temperature include caprylic and capric F.A.
• Caprylic (8 C )    CH3-(CH2)6-COOH.
• Capric (10 C )       CH3-(CH2)8-COOH.

B-Long-chain fatty acids:

• They contain more than 10 carbon atoms.
• They occur in hydrogenated oils, animal fats, butter and coconut and palm oils.
• They are non-volatile and water-insoluble
• Include palmitic, stearic, and lignoceric F.A.
• palmitic(16C)     CH3-(CH2)14-COOH
• stearic (18 C )       CH3-(CH2)16-COOH
• lignoceric (24C )    CH3-(CH2)22-COOH

B-Unsaturated Fatty Acids

They contain double bond

• monounsaturated

they contain one double bonds .

(CnH2n-1 COOH)

• polyunsaturated

they contain more the one double bond (CnH2n-more than 1 COOH).

 

1-Monounsaturated fatty acids:
1-Palmitoleic acid :

• It is found in all fats. 
• It is C16:1∆9, i.e., has 16 carbons and one double bond located at carbon number 9 and involving carbon 10.

     CH3-( CH2 )5CH  = CH-(CH2)7 –COOH

 

2-Oleic acid

• Is the most common fatty acid in natural fats. 
• It is C18:1∆9, i.e., has 18 carbons and one double bond located at carbon number 9 and involving carbon 10.

CH3-(CH2)7- CH=CH – (CH2)7-COOH

 

3-Nervonic acid

(Unsaturated lignoceric acid).

• It is found in cerebrosides.
• It is C24:1D15, i.e., has 24 carbons and one double bond located at carbon number 15 and involving carbon 16.

CH3 – (CH2)7 CH= CH – (CH2)13- COOH

2-Polyunsaturated fatty acids :

(Essential fatty acids):

• Definition: 
• They are essential fatty acids that can not be synthesized in the human body and must be taken in adequate amounts in the diet.
• They are required for normal growth and metabolism 
• Source: vegetable oils such as corn oil, linseed oil, peanut oil, olive oil, cottonseed oil, soybean oil and many other plant oils, cod liver oil and animal fats.
• Deficiency: Their deficiency in the diet leads to nutrition deficiency disease.
• Its symptoms include: poor growth and health with susceptibility to infections, dermatitis, decreased capacity to reproduce, impaired transport of lipids, fatty liver, and lowered resistance to stress.

 

•Function of Essential Fatty Acids:

1. They are useful in the treatment of atherosclerosis by help transporting blood cholesterol and lowering it and transporting triglycerides.
2. The hormones are synthesized from them.
3. They enter in structure of all cellular and subcellular membranes and the transporting plasma phospholipids.
4. They are essential for skin integrity, normal growth and reproduction.
5. They have an important role in blood clotting (intrinsic factor).
6. Important in preventing and treating fatty liver.
7. Important role in health of the retina and vision.
8. They can be oxidized for energy production.

 

1-Linoleic:

• C18:2D9, 12.
• It is the most important since other essential fatty acids can be synthesized from it in the body.

CH3-(CH2)4-CH = CH-CH2-CH=CH-(CH2)7-COOH

 

2-Linolenic acid:

• C18:3D9, 12, 15,
• in corn, linseed, peanut, olive, cottonseed and soybean oils.

CH3-CH2-CH=CH-CH2-CH=CH-CH2-CH=CH-(CH2)7-COOH

 

3-Arachidonic acid:

• C20:4D5, 8, 11, 14.
• It is an important component of phospholipids in animal and in peanut oil from which prostaglandins are synthesized.

CH3-(CH2)4-CH=CH-CH2-CH=CH-CH2-CH=CH-CH2-CH=CH-(CH2)3-COOH

 

1-Simple Lipids

A-Neutral Fats and oils (Triglycerides)

Definition:

• They are called neutral because they are uncharged due to absence of ionizable groups in it.
• The neutral fats are the most abundant lipids in nature. They constitute about 98% of the lipids of adipose tissue, 30% of plasma or liver lipids, less than 10% of erythrocyte lipids. 
• They are esters of glycerol with various fatty acids.  Since the 3 hydroxyl groups of glycerol are esterified, the neutral fats are also called “Triglycerides”. 
• Esterification of glycerol with one molecule of fatty acid gives monoglyceride, and that with 2 molecules gives diglyceride.

 Types of triglycerides      

1.         Simple triglycerides: If the three fatty acids connected to glycerol are of the same type the triglyceride is called simple triglyceride, e.g., tripalmitin.
2.        Mixed triglycerides: if they are of different types, it is called mixed triglycerides, e.g., stearo-diolein and palmito-oleo-stearin.

•          Natural fats are mixtures of mixed triglycerides with a small amount of simple triglycerides.

• The commonest fatty acids in animal fats are palmitic, stearic and oleic acids. 
• The main difference between fats and oils is for oils being liquid at room temperature, whereas, fats are solids. 
• This is mainly due to presence of larger percentage of unsaturated fatty acids in oils than fats that has mostly saturated fatty acids. 

Physical properties of fat and oils:

1. Freshly prepared fats and oils are colorless, odorless and tasteless.Any color, or taste is due to association with other foreign substances, e.g., the yellow color of body fat or milk fat is due to carotene pigments(cow milk).
2. Fats have specific gravity less than 1 and, therefore, they float on water.
3. Fats are insoluble in water, but soluble in organic solvents as ether and benzene. 
4. Melting points of fats are usually low, but higher than the solidification point, 

Chemical Properties of fats and oils:

1-Hydrolysis:

• They are hydrolyzed into their constituents (fatty acids and glycerol) by the action of super heated steam, acid, alkali or enzyme (e.g., lipase of pancreas).
• During their enzymatic and acid hydrolysis glycerol and free fatty acids are produced. 

 

2-Saponification. 

 Alkaline hydrolysis produces glycerol and salts of fatty acids (soaps).

•        Soaps cause emulsification of oily material this help easy washing of the fatty materials

 

Saponification number (or value):

•        Definition: It is the number of milligrams of KOH required to completely saponify one gram of fat.
•         Uses:
•       Since each carboxyl group of a fatty acid reacts with one mole of KOH during saponification, therefore, the amount of alkali needed to saponify certain weight of fat depends upon the number of fatty acids present per weight.
•        Thus, fats containing short-chain acids will have more carboxyl groups per gram than long chain fatty acids and consume more alkali, i.e., will have higher saponification number.

3-Halogenation

• Neutral fats containing unsaturated fatty acids have the ability of adding halogens (e.g., hydrogen or hydrogenation and iodine or iodination) at the double bonds.
• It is a very important property to determine the degree of unsaturation of the fat or oil that determines its biological value 

 

1-Iodine number (or value): 

•         Definition: It is the number of grams of iodine absorbed by 100 grams of fat or oil. 
•         Uses: It is a measure for the degree of unsaturation of the fat, as a natural property for it. 
•         Unsaturated fatty acids absorb iodine at their double bonds, therefore, as the degree of unsaturation increases iodine number and hence biological value of the fat increase. 
•         It is used for identification of the type of fat, detection of adulteration and determining the biological value of fat.

4-Hydrogenation or hardening of oils:

• It is a type of addition reactions accepting hydrogen at the double bonds of unsaturated fatty acids.
• The hydrogenation is done under high pressure of hydrogen and is catalyzed by finely divided nickel or copper and heat.
• It is the base of hardening of oils (margarine manufacturing), e.g., change of oleic acid of fats (liquid) into stearic acid (solid).
• It is advisable not to saturate all double bonds; otherwise margarine produced will be very hard, of very low biological value and difficult to digest.

 Advantages for hydrogenated oil or fat are as follows:

1.It is more pleasant as cooking fat.

2.It is digestible and utilizable as normal animal fats and oils.

3.It is less liable to cause gastric or intestinal irritation.

4.It is easily stored and transported and less liable to rancidity.

 Disadvantages of hydrogenated

           fats include lack of fat-soluble vitamins (A, D, E and K) and essential fatty acids

5-Oxidation(Rancidty)

• This toxic reaction of triglycerides leads to unpleasant odour or taste of oils and fats developing after oxidation by oxygen of air, bacteria, or moisture. 
• Also this is the base of the drying oils after exposure to atmospheric oxygen.

Example is linseed oil, which is used in paints and varnishes manufacturing

 

Rancidity

Definition:

•It is a physico-chemical change in the natural properties of the fat leading to the development of unpleasant odor or taste or abnormal color particularly on aging after exposure to atmospheric oxygen, light, moisture, bacterial or fungal contamination and/or heat.

• Saturated fats resist rancidity more than unsaturated fats that have unsaturated double bonds.

Types and causes of Rancidity:

1.Hydrolytic rancidity

2.Oxidative rancidity

3.Ketonic rancidity

1-Hydrolytic rancidity:

• It results from slight hydrolysis of the fat by lipase from bacterial contamination leading to the liberation of free fatty acids and glycerol at high temperature and moisture.

• Volatile short-chain fatty acids have unpleasant odor.

 

2-Oxidative Rancidity:

•It is oxidation of fat or oil catalyzed by exposure to oxygen, light and/or heat producing peroxide derivatives which on decomposition give substances, e.g., peroxides, aldehydes, ketones and dicarboxylic acids that are toxic and have bad odor.

•This occurs due to oxidative addition of oxygen at the unsaturated double bond of unsaturated fatty acid of oils.

 

3-Ketonic Rancidity:

•It is due to the contamination with certain fungi such as Asperigillus Niger on fats such as coconut oil.

• Ketones, fatty aldehydes, short chain fatty acids and fatty alcohols are formed.

•Moisture accelerates ketonic rancidity.

 

•Prevention of rancidity is achieved by:

1.Avoidance of the causes (exposure to light, oxygen, moisture, high temperature and bacteria or fungal contamination).  By keeping fats or oils in well-closed containers in cold, dark and dry place (i.e., good storage conditions).

2.Removal of catalysts such as lead and copper that catalyze rancidity.

3.Addition of anti-oxidants to prevent peroxidation in fat (i.e., rancidity).  They include phenols, naphthols, tannins and hydroquinones. The most common natural antioxidant is vitamin E that is important in vitro and in vivo.

Absorptive rancidity 

•Ketengikan krn absorpsi.

•Banyak substansi yg sangat lrt dlm lemak

•Bila lemak terpapar dg bau/rs dr substansi tsb maka bau akan diabsorbsi

•Mentega atau lemak bila terpapar dgn bawang, cat, gas atau buah akan mengabsorbsi bau tsb sehingga tdk layak utk dikonsumsi

   

Hazards of Rancid Fats:

1.The products of rancidity are toxic, i.e., causes food poisoning and cancer.

2.Rancidity destroys the fat-soluble vitamins (vitamins A, D, K and E).

3.Rancidity destroys the polyunsaturated essential fatty acids.

4.Rancidity causes economical loss because rancid fat is inedible.   

Analysis and Identification of fats and oils

(Fat Constants)

•Fat constants or numbers are tests used for:

1.Checking the purity of fat for detection of adulteration.

2.To quantitatively estimate certain properties of fat.

3.To identify the biological value and natural characteristics of fat.

4.Detection of fat rancidity and presence of toxic hydroxy fatty acids. 

  

3-Acids Number (or value): 

•Definition: 

•It is the number of milligrams of KOH required to neutralize the free 

fatty acids present in one gram of fat. 

•Uses: 

•It is used for detection of hydrolytic rancidity because it measures the amount of free fatty acids present.  

4-Reichert- Meissl Number (or value): 

•Definition: It is the number of milliliters of 0.1 N  KOH required to neutralize the water-soluble fatty acids distilled from 5 grams of fat. Short-chain fatty acid (less than 10 carbons) is distillated by steam. 

•Uses:  This studies the natural composition of the fat and is used for detection of fat adulteration. 

•  Butter that has high percentage of short-chain fatty acids has highest Reichert-Meissl number compared to margarine.

5-Acetyl Number (or value): 

•Definition: It is number of milligrams of KOH needed to neutralize the acetic acid liberated from hydrolysis of 1 gram of acetylated fat (hydroxy fat reacted with acetic anhydride). 

•Uses: The natural or rancid fat that contains fatty acids with free hydroxyl groups are converted into acetylated fat by reaction with acetic anhydride. 

• Thus, acetyl number is a measure of number of hydroxyl groups present.  It is used for studying the natural properties of the fat and to detect adulteration and rancidity. 

B-Waxes

• Definition: Waxes are solid simple lipids containing a monohydric alcohol (with a higher molecular weight than glycerol) esterified to long-chain fatty acids. Examples of these alcohols are palmitoyl alcohol, cholesterol, vitamin A or D.
• Properties of waxes: Waxes are insoluble in water, but soluble in fat solvents and are negative for acrolein test. 
• Waxes are not easily hydrolyzed as the fats and are indigestible by lipases and are very resistant to rancidity. 
• Thus they are of no nutritional value.

Type of Waxes:

•         Waxes are widely distributed in nature such as the secretion of certain insects as bees-wax, protective coatings of the skins and furs of animals and leaves and fruits of plants. They are classified into true-waxes and wax-like compounds as follows:

A-True waxes: include:

•          Bees-wax is secreted by the honeybees that use it to form the combs.  It is a mixture of waxes with the chief constituent is mericyl palmitate.

B-Wax-like compounds:

•Cholesterol esters: Lanolin (or wool fat) is prepared from the wool-associated skin glands and is secreted by sebaceous glands of the skin.

•It is very complex mixture, contains both free and esterified cholesterol, e.g., cholesterol-palmitate and other sterols. 

	Waxes	Neutral lipids
1.Digestibility:	Indigestible (not hydrolyzed by lipase).	Digestible (hydrolyzed by lipase).
2-Type of alcohol:	Long-chain monohydric alcohol + one fatty acid.	Glycerol (trihydric) + 3 fatty acids
3-Type of fatty acids:	Fatty acid mainly palmitic or stearic acid.	Long and short chain fatty acids.
4-Acrolein test:	Negative.	Positive.
5-Rancidability:	Never get rancid.	Rancidible.
6-Nature at room temperature.	Hard solid.	Soft solid or liquid.
7-Saponification	Nonsaponifiable.	Saponifiable.
8-Nutritive value:	No nutritive value.	Nutritive.
9-Example:	Bee & carnuba waxes.	Butter and vegetable oils.

 

2-Compound Lipids

Definition:

• They are lipids that contain additional substances, e.g., sulfur, phosphorus, amino group, carbohydrate, or proteins beside fatty acid and alcohol.
• Compound or conjugated lipids are classified into the following types according to the nature of the additional group:

1.Phospholipids 

2.Glycolipids.

3.Lipoproteins 

4.Sulfolipids and amino lipids.

 

A-Phospholipids 

Definition: Phospholipids or phosphatides are compound lipids, which contain phosphoric acid group in their structure.

Importance:

1. They are present in large amounts in the liver and brain as well as blood. Every animal and plant cell contains phospholipids.
2. The membranes bounding cells and subcellular organelles are composed mainly of phospholipids. Thus, the transfer of substances through these membranes is controlled by properties of phospholipids.
3. They are important components of the lipoprotein coat essential for secretion and transport of plasma lipoprotein complexes. Thus, they are lipotropic agents that prevent fatty liver.  
4. Myelin sheath of nerves is rich with phospholipids. 
5. 5-Important in digestion and absorption of neutral lipids and excretion of cholesterol in the bile.
6. Important function in blood clotting and platelet aggregation.  
7. They provide lung alveoli with surfactants that prevent its irreversible collapse.  
8. Important role in signal transduction across the cell membrane.  
9. Phospholipase A2 in snake venom hydrolyses membrane phospholipids into hemolytic lysolecithin or lysocephalin.
10. They are source of polyunsaturated fatty acids for synthesis of eicosanoids.

Sources: They are found in all cells (plant and animal), milk and egg-yolk in the form of lecithins.

Structure: phospholipids are composed of:

1.Fatty acids (a saturated and an unsaturated fatty acid).

2.Nitrogenous base (choline, serine, threonine, or ethanolamine).

3.Phosphoric acid.

4.Fatty alcohols (glycerol, inositol or sphingosine).

•         Classification of Phospholipids are classified into 2 groups according to the type of the alcohol present into two types:

         A-Glycerophospholipids: They are regarded as derivatives of phosphatidic acids that are the simplest type of phospholipids and include:

1.Phosphatidic acids.

2.Lecithins

3.Cephalins.

4.Plasmalogens.

5.Inositides.

6.Cardiolipin.

B-Sphingophospholipids: They contain sphingosine as an alcohol and are named Sphingomyelins.

   



A-Glycerophospholipids                                               

1-Phosphatidic acids:They are metabolic intermediates in synthesis of triglycerides and  

glycerophospholipids in the body and may have function as a second messenger.

They exist in two forms according to the position of the phosphate

2-Lecithins: 

•Definition: 

Lecithins are glycerophospholipids that contain choline as a base beside phosphatidic 

acid.  They exist in 2 forms a- and b-lecithins.  Lecithins are a common cell constituent 

obtained from brain (a-type), egg yolk (b-type), or liver (both types).  Lecithins are important 

in the metabolism of fat by the liver. 

•Structure:

Glycerol is connected at C2 or C3 with a polyunsaturated fatty acid, at C1 with a saturated 

fatty acid, at C3 or C2 by phosphate to which the choline base is connected. The common 

fatty acids in lecithins are stearic, palmitic, oleic, linoleic, linolenic, clupandonic or 

 arachidonic acids.  

Lysolecithin causes hemolysis of RBCs. This partially explains toxic the effect of snake venom,. The venom contains lecithinase, which hydrolyzes the polyunsaturated fatty converting lecithin into lysolecithin. Lysolecithins are intermediates in metabolism of phospholipids.

•Lung surfactant

• Is a complex of dipalmitoyl-lecithin, sphingomyelin and a group of apoproteins called apoprotein A, B, C, and D.  
• It is produced by type II alveolar cells and is anchored to the alveolar surface of type II and I cells.
• It lowers alveolar surface tension and improves gas exchange besides activating macrophages to kill pathogens.
• In premature babies, this surfactant is deficient and they suffer from respiratory distress syndrome.
• Glucocorticoids increase the synthesis of the surfactant complex and promote differentiation of lung cells.

3-Cephalins (or Kephalins):

• Definition: They are phosphatidyl-ethanolamine or serine. Cephalins occur in association with lecithins in tissues and are isolated from the brain (Kephale = head).
• Structure: Cephalins resemble lecithins in structure except that choline is replaced by ethanolamine, serine or threonine amino acids. 
• Certain cephalins are constituents of the complex mixture of phospholipids, cholesterol and fat that constitute the lipid component of the lipoprotein “thromboplastin” which accelerates the clotting of blood by activation of prothrombin to thrombin in presence of calcium ions.

4-Plasmalogens:

• Definition: Plasmalogens are found in the cell membrane phospholipids fraction of brain and muscle (10% of it is plasmalogens), liver, semen and eggs.
• Structure: Plasmalogens resemble lecithins and cephalins in structure but differ in the presence of a,b-unsaturated fatty alcohol rather than a fatty acid at C1 of the glycerol connected by ether bond.
• At C2 there is an unsaturated long-chain fatty acid, however, it may be a very short-chain fatty acid 
• Properties: Similar to lecithins.

5-Inositides:

• Definition: 
• -They are phosphatidyl inositol.
• Structure: They are similar to lecithins or cephalins but they have the cyclic sugar alcohol, inositol as the base. They are formed of glycerol, one saturated fatty acid, one unsaturated fatty acid, phosphoric acid and inositol

• Source: Brain tissues.
• Function:
• Phosphatidyl inositol is a major component of cell membrane phospholipids particularly at the inner leaflet of it.
• They play a major role as second messengers during signal transduction for certain hormone..
• On hydrolysis by phospholipase C, phosphatidyl-inositol-4,5-diphosphate produces diacyl-glycerol and inositol-triphosphate both act to liberate calcium from its intracellular stores to mediate the hormone effects.

6-Cardiolipins:

• Definition: They are diphosphatidyl-glycerol. They are found in the inner membrane of mitochondria initially isolated from heart muscle (cardio). It is formed of 3 molecules of glycerol, 4 fatty acids and 2 phosphate groups.
• Function: Used in serological diagnosis of autoimmunity diseases.

B-Sphingophospholipids

1-Sphingomyelins

• Definition: Sphingomyelins are found in large amounts in brain and nerves and in smaller amounts in lung, spleen, kidney, liver and blood.
• Structure: Sphingomyelins differ from lecithins and cephalins in that they contain sphingosine as the alcohol instead of glycerol, they contain two nitrogenous bases: sphingosine itself and choline.
• Thus, sphingomyelins contain sphingosine base, one long-chain fatty acid, choline and phosphoric acid. 
• To the amino group of sphingosine the fatty acid is attached by an amide linkage. 
• Ceramide This part of sphingomyelin in which the amino group of sphingosine is attached to the fatty acid by an amide linkage. 
• Ceramides have been found in the free state in the spleen, liver and red cells.

B-Glycolipids

• Definition: They are lipids that contain carbohydrate residues with sphingosine as the alcohol and a very long-chain fatty acid (24 carbon series).
• They are present in cerebral tissue, therefore are called cerebrosides
• Classification: According to the number and nature of the carbohydrate residue(s) present in the glycolipids the following are

1. Cerebrosides. They have one galactose molecule (galactosides).
2. Sulfatides. They are cerebrosides with sulfate on the sugar (sulfated cerebrosides).
3. Gangliosides. They have several sugar and sugaramine residues.

  1-Cerebrosides: 

• Occurrence:  They occur in myelin sheath of nerves and white matter of the brain tissues and cellular membranes.  They are important for nerve conductance. 
• Structure:  They contain sugar, usually b-galactose and may be glucose or lactose, sphingosine and fatty acid, but no phosphoric acid.

•Types: According to the type of fatty acid and carbohydrate present, there are 4 different types of cerebrosides isolated from the white matter of cerebrum and in myelin sheaths of nerves. Rabbit cerebrosides contain stearic acid.

1.              Kerasin contains lignoceric acid (24 carbons) and galactose. 
2.           Cerebron (Phrenosin) contains cerebronic acid (2-hydroxylignoceric acid) and galactose. 
3.            Nervon contains nervonic acid (unsaturated lignoceric acid at C15) and galactose. 
4.          Oxynervon contains oxynervonic acid (2-hydroxynervonic acid) and galactose. 

2-Sulfatides:

•They are sulfate esters of kerasin or phrenosin in which the sulfate group is usually attached to the –OH group of C3 or C6 of galactose. Sulfatides are usually present in the brain, liver, muscles and testes.

Top 300 Pharmacy Drug Cards

Top 300 Pharmacy Drug Cards

Preface A: Anatomy of a Flash Card

Medication Name
Both generic an common bran names are listed .
Class
Medications are grouped into classes (“families”) base on their chemical, pharmacological, or clinical properties. It is often useful to study medications on a class-by-class basis, identifying similarities and differences among members of each class.
Controlled Substance Schedule
Title 21 of the Unite States Co e (USC) is the Controlled Substances Act of 1970. It regulates medications with potential for abuse. These Federal regulations are overseen by the Drug Enforcement A ministration, but many States have enacted more strict regulations base on them. Medications are place into schedules base on their clinical use an their risk of abuse and dependence. It is important to note that some States change the Federal scheduling of certain medications. Under Federal law, a State cannot place a medication in a lower schedule than where it is place by the Federal government (eg, States cannot change a rug place in Federal Schedule II to Schedule III, IV, or V), but States can and do place certain medications in higher schedules (eg, changing a drug place in Federal Schedule V into Schedule II, III, or IV, or changing a drug which is not a controlled substance under Federal law into a controlled substance within that State).
• Schedule I: No medical use, high abuse, and dependence potential.
• Schedule II: Legitimate medical use, high abuse, and dependence potential.
• Schedule III: Legitimate medical use, abuse, and dependence potential somewhat less than Schedule II.
• Schedule IV: Legitimate medical use, abuse, and dependence potential less than Schedule III.
• Schedule V: Legitimate medical use, limited abuse, and dependence potential.
Dosage Forms
The most common dosage forms an strengths are listed. Other dosage forms may exist, an may be reference in the Clinical Pearls section.

Common FDA Label Indication, Dosing, and Titration
The US Foo an Drug A ministration (FDA) approves medications for market, an also approves specific indications for use an the doses for those uses. Some medications are approve for only one indication, while others are approve for many indications. In most cases, all FDA-approve (“labeled”) indications are listed with their approve doses.
O -Label Uses
While every medication must be approve by the FDA for at least one indication before it is marketed, FDA approval is not always sought for subsequent indications. Prescribers are legally entitle to prescribe medications for any indication they feel is appropriate an clinically justified. In most cases, prescribers limit their use of medications to indications for which evidence supports safety an efficacy, as demonstrate in published clinical trials. While these may not be FDA-approve indications, “off-label” use is common an often completely appropriate. Common off-label uses are inclu e , along with dosing recommendations.
MOA (Mechanism o Action)
The MOA is a succinct summary of the pharmacological properties of each me ication.
Drug Characteristics
Each car includes a table summarizing key rug parameters, as outline below.
Dose Adjustments Hepatic
A Child-Pugh Score can be use to assess hepatic dysfunction. The score employs five clinical measures of liver disease. Each is score 1-3, with 3 indicating the most severe derangement of that measure. Base on the number of points for each measure, liver disease can be classified into Chil -Pugh class A, B, or C.

measure	1 point	2 point	3 point
total bilirubin, mg/dL	<2	2,0-3,0	>3
Serum albumin, g/L	>35	28-35	<28
INR	<1.7	1.71-2.20	>2.20
Ascites	None	Mil	Severe
Hepatic encephalopathy	None	Gra e I-II	Gra e III-IV

Dose Adjustments Renal

Dose adjustments for some (but not all) of medications that are renally eliminate are necessary in patients with renal dysfunction and hepatically eliminate medications in patients with hepatic dysfunction. Dose adjustments are ma e by either lowering the dose or dosing less frequently (eg, reducing from ti to daily dosing). The degree of renal dysfunction usually determines the degree of the dose adjustment. Definitions of renal an hepatic dysfunction are often inconsistent, but the recommended dose adjustments included in these flash car s are drawn from product package inserts an other sources. Clinicians should always exercise caution when treating patients with liver an /or kidney disease, an monitor carefully for signs of toxicity, even if dose adjustments are made.

KBP Chemistry of Cooking

Kimia Bahan Pangan

Chemistry of Cooking

• Why is my toast brown ?
• What happens to meat when it is cooked ?
• What causes the odour of roasted meats ?
• what causes the flavour of roasted coffee ? 
• Does cooking introduce harmful by products ? 

Let's compare

• Grilling (BBQ) gas or with hard wood coals : char broiled

• Oven broiled
• Oven roasting (baked)
• Boiled
• Steamed
• Microwave

What is known for sure

• BBQ (Grilling) of high fat content meats at high temp produces Polynuclear aromatic hydrocarbons (PNAH's)
• Planar molecules : intercalate into the major groove of the DNA double helix

• Can be cancer inducing if DNA directed synthesis of protein gets out of control

First cancerous lesions

• Observed in chimney sweeps in UK in 1700's and workers in coal tar industry
• Exposure to soot on skin
• PNAH's common in soot from burning of coal

That BBQ steak flavour

• 15 different PNAH's have been isolated from the outer layer of charcoal broiled steak
• 8 microgram of 1,2-benzopyrene per kilo of steak
• Arise from decomposition of fat that drips on to the glowing charcoal and the subsequent vaporization of the hydrocarbons and deposition on the surface of the meat

Other possible carcinogens

• Heterocyclic amines (HCA's) added to list of known carcinogens in 2005
• Arise from reaction of creatine (an amino acid found in muscle) and carbohydrate
• Higher temps from grill, frying or oven broiling increase the concetrations

How to minimize these risks

• Use lean meats or remove fiat
• Cook at lower temperatures (ie allow coals gas grill move food to an upper rack
• Use merinades (olive oil or citrus based)
• Avoid overcooking

Marination : also denaturation

• Long time (days) exposure to acid (in vinegar) will denature some protein and tederize some meat
• Also kills salmonella, but not E coli

Cooking fish on the grill

• Leave skin on and do most of the grilling skin side down-easly separated when fish is cooked
• Use cedar grilling planks to impart a rich smoky flavour-keeps fish moist and no charring

Happy BBQ's (a summer tradition)

• Should be a treat. not every night ! Enjoy !

In general

• Long slow cooking (baking, roasting) are best due to lower temperature used
• Minimizes formation of potential carcinogens
• BUT, real dangers from undercooked food containing harmful bacteria (E coli : can be fatal almost immediately)
• Hamburger (large surface area) and poultry

Let's think positively about cooking

• Where do those wounderful aromas come from (ie, baking bread). coffee brewing, cookies from the oven and the traditional Sunday roast beef dinner ( with oven browned potatoes, carrots etc)

Traditional (for some)

The Maillard browning reaction

• Louis-Camille Maillard investigated this ~1910
• Reaction between an amino acid (in protein) and a sugar (from starch)
• Accelerated in a basic environment : amino group becomes non protonated

What is the mechanism ?

• The N atom of the amino acid is nucleophilic (ie it is seeking a partially positive target)
• The C = O in the open form of sugars (aldohexoses and aldoketoses) ie glucose and fructose has a partially positive C, due to the fact that O is more electronegative than C

Nucleophilic addition

• Lone pair of electrons on N "attacks" partially positive C of C=O group

 Can you name this compound ?

• Probably not !
• 2-acetyl-3,4,5,6-tetrahydropyridine
• Oops-it is a heterocyclic amine (HCA)

Acrylamide structure

• Planar, thus a potential DNA intercalator

 Note the similarity to acrylamide structure

• So..........could acrylamide be generated by the Maillard reaction ?
• Yes ! (J. Agr. Food Chem. 53, 4628-4632 (2005)

Glucose reacts with Asparagine

• N attacks C=O !

 Many Steps later .. a bit of ..

• Exact mechanism unknown for acrylamide formation
• Many other products !

What about acrylamide ?

•  No evidence yet of human cancer induction
• Considered a probable carcinogen based on animal studies
• First noted in Swedish study in 2002
• Particularly in deep fried foods
• Low levels suggest not a critical issue

Maillard reactions of Tryptophan

• In turkey : reacts with glucose to produce a glycoside

 Tryptophan : glycoside

Maillard reaction in roasted nuts

• Almost all nuts are roasted before
• consumption; kills bacteria and increases flavor
• Peanut roasting has been studied in detail due to widespread allergies
• Allergy is protein induced
• Some of the Maillard products may increase the allergenicity of peanuts

Summary

• Maillard reaction produces hundreds of compounds which are responsible for pleasant odour and taste of protein containing foods.
• It also produces trace amounts of acrylamide (the price we pay for flavor !)

Other Browning reactions

• Maillard "browning" often accompanied by carmelization if more carbohydrate (sugar) is present
• Carmelized onions : both protein and sugar naturally present in the onion (Demo).
• Heating of sucrose alone can cause carmelization (browning) (Demo).

ACE inhibitors + Diuretics; Loop, Thiazide and related

ACE inhibitors + Diuretics; Loop, Thiazide and related

The combination of captopril or other ACE inhibitors with loop or thiazide diuretics is normally safe and effective, but ‘first dose hypotension’ (dizziness, lightheadedness, fainting) can occur, particularly if the dose of diuretic is high, and often in association with various predisposing conditions. Renal impairment, and even acute renal failure, have been reported. Diuretic-induced hypokalaemia may still occur when ACE inhibitors are used with these potassium-depleting diuretics.

Clinical evidence

(a) First dose hypotensive reactionThe concurrent use of captopril or other ACE inhibitors and loop or thiazide diuretics is normally safe and effective, but some patients experience ‘first dose hypotension’ (i.e. dizziness, lightheadedness, fainting) after taking the first one or two doses of the ACE inhibitor. This appears to be associated with, and exaggerated by, certain conditions (such as heart failure, renovascular hypertension, haemodialysis, high levels of renin and angiotensin, low-sodium diet, dehydration, diarrhoea or vomiting) and/or hypovolaemia and sodium depletion caused by diuretics, particularly in
high doses. A study describes one woman whose blood pressure of 290/150 mmHg failed to respond to a 10-mg intravenous dose of furosemide.
After 30 minutes she was given captopril 50 mg orally and within 45 minutes her blood pressure fell to 135/60 mmHg, and she required an infusion of saline to maintain her blood pressure.1 In another study, a man developed severe postural hypotension shortly after furosemide was added to captopril treatment.2
Starting with a low dose of the ACE inhibitor reduces the risk of firstdose hypotension. In a study in 8 patients with hypertension, treated with a diuretic (mainly furosemide or hydrochlorothiazide) for at least 4 weeks, captopril was started in small increasing doses from 6.25 mg.
Symptomatic postural hypotension was seen in 2 of the 8 patients, but was only mild and transient.3
Hypotension is more common in patients with heart failure who are receiving large doses of diuretics. In a study in 124 patients with severe heart failure, all receiving furosemide (mean dose 170 mg daily; range 80 to 500 mg daily) and 90 also receiving the potassium-sparing diuretic spironolactone, the addition of captopril caused transient symptomatic hypotension in 44% of subjects. The captopril dose had to be reduced, and in 8 patients it was later discontinued. In addition, four patients developed symptomatic hypotension after 1 to 2 months of treatment, and captopril was also discontinued in these patients.4
There is some evidence that in patients with heart failure the incidence of marked orthostatic hypotension requiring treatment discontinuation in the first 36 hours was lower with perindopril 2 mg once daily than captopril 6.25 mg three times daily (6 of 357 cases versus 16 of 368 cases, respectively).5

(b) HypokalaemiaIn one study, the reduction in plasma potassium was greater with hydrochlorothiazide 25 mg daily than with hydrochlorothiazide combined with cilazapril 2.5 mg daily, showing that cilazapril reduced the potassium-depleting effect of hydrochlorothiazide.6 In one analysis, 7 of 21 patients taking potassium-depleting diuretics given ACE inhibitors for heart failure developed hypokalaemia. This was corrected by potassium supplementation in 2 cases, an increase in the ACE inhibitor dose in 3 cases, and the use of a potassium-sparing diuretic in the remaining 2 cases.7 In another report, a woman taking furosemide 80 to 120 mg daily remained hypokalaemic despite also taking ramipril 10 mg daily and spironolactone 50 to 200 mg daily.8 However, note that the addition of ‘spironolactone’, (p.23) to ACE inhibitors and loop or thiazide diuretics has generally resulted in an increased incidence of hyperkalaemia.

(c) HyponatraemiaAn isolated report describes a patient who developed severe hyponatraemia 3 days after bendroflumethiazide 10 mg daily was added to treatment with enalapril 20 mg daily and atenolol 100 mg daily. However, on 2 other occasions she only developed mild hyponatraemia when given bendroflumethiazide alone.9 An earlier study reported changes in sodium balance due to captopril in all 6 patients with renovascular hypertension and in 11 of 12 patients with essential hypertension; sodium loss occurred in 12 of the 18 patients.1

(d) Impairment of renal functionThe risk of ACE inhibitor-induced renal impairment in patients with or without renovascular disease can be potentiated by diuretics.10-13 In an analysis of 74 patients who had been treated with captopril or lisinopril, reversible acute renal failure was more common in those who were also treated with a diuretic (furosemide and/or hydrochlorothiazide) than those who were not (11 of 33 patients compared with 1 of 41 patients).12
Similarly, in a prescription-event monitoring study, enalapril was associated with raised creatinine or urea in 75 patients and it was thought to have contributed to the deterioration in renal function and subsequent deaths in 10 of these patients. However, 9 of these 10 were also receiving loop or thiazide diuretics, sometimes in high doses.14 Retrospective analysis of a controlled study in patients with hypertensive nephrosclerosis identified 8 of 34 patients who developed reversible renal impairment when treated with enalapril and various other antihypertensives including a diuretic (furosemide or hydrochlorothiazide). In contrast, 23 patients treated with placebo and various other antihypertensives did not develop renal impairment.
Subsequently, enalapril was tolerated by 7 of the 8 patients without deterioration in renal function and 6 of these patients later received diuretics.15 One patient was again treated with enalapril with recurrence of renal impairment, but discontinuation of the diuretics (furosemide, hydrochlorothiazide, and triamterene) led to an improvement in renal function despite the continuation of enalapril.16
Renal impairment in patients taking ACE inhibitors and diuretics has also been described in patients with heart failure. A patient with congestive heart failure and pre-existing moderate renal impairment developed acute non-oliguric renal failure while taking enalapril 20 mg daily and furosemide 60 to 80 mg daily, which resolved when the sodium balance was restored.17 In a study involving 90 patients with severe congestive heart failure who were receiving furosemide and spironolactone, a decline in renal function occurred in 18 patients during the first month after initiation of captopril treatment; mean serum creatinine levels rose from 220 to 300 micromol/L. All the patients were receiving high daily doses of furosemide and all had renal impairment before receiving the first dose of captopril.4
Acute, fatal, renal failure developed in 2 patients with cardiac failure within 4 weeks of being treated with enalapril and furosemide, and in 2 similar patients renal impairment developed over a longer period.18 Reversible renal failure developed in a patient with congestive heart failure when captopril and metolazone were given.19

(e) Pharmacokinetic and diuresis studies

1. Furosemide. A study in healthy subjects given single doses of enalapril and furosemide found no evidence of any pharmacokinetic interaction between these drugs.20 Another study in hypertensive patients found that captopril did not affect the urinary excretion of furosemide, nor its subsequent diuretic effects.21 However, a further study in healthy subjects showed that, although captopril did not alter urinary excretion of furosemide, it did reduce diuresis.22 Yet another study in healthy subjects found that captopril reduced the urinary excretion of furosemide, and reduced the diuretic response during the first 20 minutes to approximately 50%, and the natriuretic response to almost 30%, whereas enalapril and ramipril did not significantly alter the diuretic effects of furosemide.23 In
one single-dose study in healthy subjects the concurrent use of benazepril and furosemide reduced the urinary excretion of furosemide by 10 to 20%, whereas benazepril pharmacokinetics were unaffected.24 Lisinopril did not alter plasma levels or urinary excretion of furosemide in one study, nor did it alter urinary electrolyte excretion.25 Similarly, furosemide did not affect the pharmacokinetics of lisinopril either in single-dose or multipledose regimens.26

2. Hydrochlorothiazide. In a single-dose, randomised, crossover study in 19 elderly patients the pharmacokinetics of enalapril 10 mg were unaffected by hydrochlorothiazide 25 mg. However, there was a significant reduction in renal clearance and a significant increase in the AUC of its metabolite, enalaprilat, resulting in higher serum levels of the active drug. This acute interaction was not thought to be clinically significant for long-term use.27
No pharmacokinetic interaction occurred between cilazapril and hydrochlorothiazide in healthy subjects or patients with hypertension.6 Similarly, no significant pharmacokinetic interaction occurred between imidapril and hydrochlorothiazide in healthy subjects28 and neither captopril nor ramipril altered the diuresis induced by hydrochlorothiazide.23
The manufacturer of spirapril briefly noted in a review that there was no clinically relevant pharmacokinetic interaction between spirapril and hydrochlorothiazide.
29 Furthermore no pharmacokinetic interaction was found when spirapril and hydrochlorothiazide were given together as a bi-layer tablet.30 There was no clinically important pharmacokinetic interaction when moexipril was given with hydrochlorothiazide in a singledose study in healthy subjects.31

Mechanism
The first dose hypotension interaction is not fully understood. One suggestion is that if considerable amounts of salt and water have already been lost as a result of using a diuretic, the resultant depletion in the fluid volume (hypovolaemia) transiently exaggerates the hypotensive effects of the ACE inhibitor.
The cases of hypokalaemia are simply a result of the potassium-depleting effects of the diuretics outweighing the potassium-conserving effects of the ACE inhibitor. The converse can also occur.
Thiazides can cause hyponatraemia, but this enhanced effect may have been due to an alteration in renal haemodynamics caused by the ACE inhibitor; sustained angiotensin-converting enzyme blockade can produce natriuresis.1
Marked decreases in blood pressure may affect renal function, and in addition, the renin-angiotensin system plays an important role in the maintenance of the glomerular filtration rate when renal artery pressure is diminished.11 However, diuretic-induced sodium depletion may also be an important factor in the renal impairment sometimes observed with ACE inhibitors.

Importance and management
The ‘first dose hypotension’ interaction between ACE inhibitors and diuretics is well established. The BNF in the UK notes that the risk is higher when the dose of diuretic is greater than furosemide 80 mg daily or equivalent, 32 and suggest that, in patients taking these doses of diuretics, consideration should be given to temporarily stopping the diuretic or reducing its dosage a few days before the ACE inhibitor is added. If this is not considered clinically appropriate, the first dose of the ACE inhibitor should be given under close supervision. In all patients taking diuretics, therapy with ACE inhibitors should be started with a very low dose, even in patients at low risk (e.g. those with uncomplicated essential hypertension on low-dose thiazides). To be on the safe side, all patients should be given a simple warning about what can happen and what to do when they first start concurrent use. The immediate problem (dizziness, lightheadedness, faintness), if it occurs, can usually be solved by the patient lying down. Taking the first dose of the ACE inhibitor just before bedtime is also preferable.

Any marked hypotension is normally transient, but if problems persist it may be necessary temporarily to reduce the diuretic dosage. There is usually no need to avoid the combination just because an initially large hypotensive response has occurred.
A number of products combining an ACE inhibitor with a thiazide diuretic are available for the treatment of hypertension. These products should be used only in those patients who have been stabilised on the individual components in the same proportions.
The use of ACE inhibitors in patients taking potassium-depleting diuretics does not always prevent hypokalaemia developing. Serum potassium should be monitored.
There is only an isolated report of hyponatraemia, but be aware that ACE inhibitors may affect the natriuresis caused by diuretics.

The cases of renal impairment cited emphasise the need to monitor renal function in patients on ACE inhibitors and diuretics. If increases in blood urea and creatinine occur, a dosage reduction and/or discontinuation of the diuretic and/or ACE inhibitor may be required. In a statement, the American Heart Association comments that acute renal failure complicating ACE inhibitor therapy is almost always reversible and repletion of extracellular fluid volume and discontinuation of diuretic therapy is the most effective approach. In addition, withdrawal of interacting drugs, supportive management of fluid and electrolytes, and temporary dialysis, where indicated, are the mainstays of therapy.13 Combined use of ACE inhibitors, diuretics and NSAIDs may be particularly associated with an increased risk of renal failure, see ‘ACE inhibitors + NSAIDs’, p.28. 

The possibility of undiagnosed renal artery stenosis should also be considered. 

None of the pharmacokinetic changes observed appear to be clinically significant.
1. Case DB, Atlas SA, Laragh JH, Sealey JE, Sullivan PA, McKinstry DN. Clinical experience with blockade of the renin-angiotensin-aldosterone system by an oral converting-enzyme inhibitor (SQ 14,225, captopril) in hypertensive patients. Prog Cardiovasc Dis (1978) 21, 195–206.
2. Ferguson RK, Vlasses PH, Koplin JR, Shirinian A, Burke JF, Alexander JC. Captopril in severe treatment-resistant hypertension. Am Heart J (1980) 99, 579–85.
3. Koffer H, Vlasses PH, Ferguson RK, Weis M, Adler AG. Captopril in diuretic-treated hypertensive
patients. JAMA (1980) 244, 2532–5.
4. Dahlström U, Karlsson E. Captopril and spironolactone therapy for refractory congestive heart failure. Am J Cardiol (1993) 71, 29A–33A.
5. Haïat R, Piot O, Gallois H, Hanania G. Blood pressure response to the first 36 hours of heart failure therapy with perindopril versus captopril. French General Hospitals National College of Cardiologists. J Cardiovasc Pharmacol (1999) 33, 953–9.
6. Nilsen OG, Sellevold OFM, Romfo OS, Smedsrud A, Grynne B, Williams PEO, Kleinbloesem CH. Pharmacokinetics and effects on renal function following cilazapril and hydrochlorothiazide alone and in combination in healthy subjects and hypertensive patients. Br J Clin Pharmacol (1989) 27 (Suppl 2), 323S–328S.
7. D’Costa DF, Basu SK, Gunasekera NPR. ACE inhibitors and diuretics causing hypokalaemia. Br J Clin Pract (1990) 44, 26–7.
8. Jolobe OM. Severe hyperkalaemia induced by trimethoprim in combination with an angiotensin  converting enzyme inhibitor in a patient with transplanted lungs. J Intern Med (1997) 242, 88–9.
9. Collier JG, Webb DJ. Severe thiazide-induced hyponatraemia during treatment with enalapril. Postgrad Med J (1987) 63, 1105–6.
10. Watson ML, Bell GM, Muir AL, Buist TAS, Kellett RJ, Padfield PL. Captopril/diuretic  combinations in severe renovascular disease: a cautionary note. Lancet (1983) ii, 404–5.
11. Hoefnagels WHL, Strijk SP, Thien T. Reversible renal failure following treatment with captopril and diuretics in patients with renovascular hypertension. Neth J Med (1984) 27, 269–74.
12. Mandal AK, Markert RJ, Saklayen MG, Mankus RA, Yokokawa K. Diuretics potentiate angiotensin converting enzyme inhibitor-induced acute renal failure. Clin Nephrol (1994) 42, 170–4.

13. Schoolwerth AC, Sica DA, Ballermann BJ, Wilcox CS. Renal considerations in angiotensin converting enzyme inhibitor therapy. A statement for healthcare professionals from the Council on the Kidney in Cardiovascular Disease and the Council for High Blood Pressure Research of the American Heart Association. Circulation (2001) 104, 1985–91.
14. Speirs CJ, Dollery CT, Inman WHW, Rawson NSB, Wilton LV. Postmarketing surveillance of enalapril. II: Investigation of the potential role of enalapril in deaths with renal failure. BMJ (1988) 297, 830–2.
15. Toto RD, Mitchell HC, Lee H-C, Milam C, Pettinger WA. Reversible renal insufficiency due to angiotensin converting enzyme inhibitors in hypertensive nephrosclerosis. Ann Intern Med (1991) 115, 513–19.
16. Lee H-C, Pettinger WA. Diuretics potentiate the angiotensin converting-enzyme inhibitor associated acute renal dysfunction. Clin Nephrol (1992) 38, 236–7.
17. Funck-Brentano C, Chatellier G, Alexandre J-M. Reversible renal failure after combined treatment with enalapril and frusemide in a patient with congestive heart failure. Br Heart J (1986) 55, 596–8.
18. Stewart JT, Lovett D, Joy M. Reversible renal failure after combined treatment with enalapril and frusemide in a patient with congestive heart failure. Br Heart J (1986) 56, 489–90.
19. Hogg KJ, Hillis WS. Captopril/metolazone induced renal failure. Lancet (1986) 1, 501–2.
20. Van Hecken AM, Verbesselt R, Buntinx A, Cirillo VJ, De Schepper PJ. Absence of a pharmacokinetic interaction between enalapril and frusemide. Br J Clin Pharmacol (1987) 23, 84–7.
21. Fujimura A, Shimokawa Y, Ebihara A. Influence of captopril on urinary excretion of furosemide in hypertensive subjects. J Clin Pharmacol (1990) 30, 538–42.
22. Sommers De K, Meyer EC, Moncrieff J. Acute interaction of furosemide and captopril in healthy salt-replete man. S Afr Tydskr Wet (1991) 87, 375–7.
23. Toussaint C, Masselink A, Gentges A, Wambach G, Bönner G. Interference of different ACE-inhibitors with the diuretic action of furosemide and hydrochlorothiazide. Klin Wochenschr (1989) 67, 1138–46.
24. De Lepeleire I, Van Hecken A, Verbesselt R, Kaiser G, Barner A, Holmes I, De Schepper PJ. Interaction between furosemide and the converting enzyme inhibitor benazepril in healthy volunteers. Eur J Clin Pharmacol (1988) 34, 465–8.
25. Sudoh T, Fujimura A, Shiga T, Tateishi T, Sunaga K, Ohashi K, Ebihara A. Influence of lisinopril
on urinary electrolytes excretion after furosemide in healthy subjects. J Clin Pharmacol (1993) 33, 640–3.
26. Beermann B. Pharmacokinetics of lisinopril. Am J Med (1988) 85 (Suppl 3B) 25–30.

27. Weisser K, Schloos J, Jakob S, Mühlberg W, Platt D, Mutschler E. The influence of hydrochlorothiazide on the pharmacokinetics of enalapril in elderly patients. Eur J Clin Pharmacol (1992) 43, 173–7.
28. Breithaupt-Grögler K, Ungethüm W, Meurer-Witt B, Belz GG. Pharmacokinetic and dynamic interactions of the angiotensin-converting enzyme inhibitor imidapril with hydrochlorothiazide, bisoprolol and nilvadipine. Eur J Clin Pharmacol (2001) 57, 275–84.
29. Grass P, Gerbeau C, Kutz K. Spirapril: pharmacokinetic properties and drug interactions. Blood Pressure (1994) 3 (Suppl 2), 7–13.
30. Schürer M, Erb K, Junge K, Schäfer HF, Schulz H-U, Amschler S, Krupp S, Hermann R. Drug interaction of spirapril hydrochloride monohydrate and hydrochlorothiazide. Arzneimittelforschung (2003) 53, 414–19.
31. Hutt V, Michaelis K, Verbesselt R, De Schepper PJ, Salomon P, Bonn R, Cawello W, Angehrn JC. Lack of a pharmacokinetic interaction between moexipril and hydrochlorothiazide. Eur J Clin Pharmacol (1996) 51, 339–44.
32. British National Formulary. 53rd ed. London: The British Medical Association and The Pharmaceutical Press; 2007. p. 98–100.

Anticoagulants

Anticoagulants

The blood clotting process
When blood is lost or clotting is initiated in some other way, a complex cascade of biochemical reactions is set in motion, which ends in the formation of a network or clot of insoluble protein threads enmeshing the blood cells. These threads are produced by the polymerisation of the molecules of fibrinogen (a soluble protein present in the plasma) into threads of insoluble fibrin. The penultimate step in the chain of reactions requires the presence of an enzyme, thrombin, which is produced from its precursor prothrombin, already present in the plasma. This is initiated by factor III
(tissue thromboplastin), and subsequently involves various factors including activated factor VII, IX, X, XI and XII, and is inhibited by antithrombin III. Platelets are also involved in the coagulation process.
Fibrinolysis is the mechanism of dissolution of fibrin clots, which can be promoted with thrombolytics. For further information on platelet aggregation and clot dissolution, see ‘Antiplatelet drugs and thrombolytics’, (p.697).

Mode of action of the anticoagulants
Anticoagulants may be divided into direct anticoagulants, which have an immediate effect, and the indirect anticoagulants, which inhibit the formation of coagulation factors, so have a delayed effect as they do not inactivate coagulation factors already formed. See ‘Table 12.1’, (p.359), for a list.
 

(a) Direct anticoagulants
The direct anticoagulants include heparin, which principally enhances the effect of antithrombin III, thereby inhibiting the effect of thrombin (factor IIa) and activated factor X (factor Xa). Low-molecular-weight heparins are salts of fragments of heparin and act similarly, except that they have a
greater effect on factor Xa than factor IIa. They have a longer duration of action than heparin and usually require less monitoring. The heparinoids (such as danaparoid) are similar. A more recent introduction is the synthetic polysaccharide fondaparinux, which is an inhibitor of factor Xa.
The other group of direct anticoagulants are the thrombin inhibitors, which bind to the active thrombin site. These include recombinant forms or synthetic analogues of hirudin such as bivalirudin and lepirudin. Megalatran and its oral prodrug ximelagatran act similarly, but have been withdrawn because of liver toxicity.

(b) Indirect anticoagulants
The indirect anticoagulants inhibit the vitamin K-dependent synthesis of factors VII, IX, X and II (prothrombin) in the liver, and may also be referred to as vitamin K antagonists. The most commonly used are the coumarins, principally warfarin, but also acenocoumarol and phenprocoumon. The indanediones such as phenindione are now less frequently used. The indirect anticoagulants have the advantage over currently available direct anticoagulants in that they are orally active. They are often therefore referred to as oral anticoagulants, but this term may become misleading with the development of direct-acting oral anticoagulants, such as ximelegatran, which have different monitoring requirements and interactions.

Coagulation tests
During anticoagulant therapy the aim is to give protection against intravascular clotting, without running the risk of bleeding. To achieve this, doses of heparin and oral anticoagulants should be individually titrated until the desired response is attained. With the coumarin and indanedione oral anticoagulants, this procedure normally takes several days because they do not act directly on the blood clotting factors already in circulation, but on the rate of synthesis of new factors by the liver. The successful titration is determined by one of a number of different but closely related laboratory tests, see ‘Table 12.2’, (p.360) and below. Note that routine monitoring of anticoagulant effect is not required for low-molecular weight heparins or heparinoids, except in patients at increased risk of bleeding, such as those with renal impairment or who are overweight. Also, note that these tests cannot be used to monitor the anticoagulant effect of fondaparinux or the direct thrombin inhibitors, and these require no routine monitoring.

(a) Prothrombin timeThe prothrombin time test (PT, Pro-Time, tissue factor induced coagulation time) is the most common method employed in clinical situations. It measures the time taken for a fibrin clot to form in a citrated plasma sample containing calcium ions and tissue thromboplastin. The PT is usually reported as the International Normalised Ratio (INR).
1. International normalised ratio (INR). The INR was adopted by the WHO in 1982 to standardise (using the International Sensitivity Index) oral anticoagulant therapy to take into account the sensitivities of the different thromboplastins used in laboratories across the world. The formula for calculating the INR is as follows:
INR = (patient’s prothrombin time in seconds/mean normal prothrombin time in seconds)ISI
The PT values obtained from the patient’s sample are compared to a control, and this gives the INR. The higher the INR, the higher the PT value so if the patient’s ratio is 2, this means the PT (and therefore clotting) is twice as long as the normal plasma. The British Corrected Ratio is essentially the same, but was calculated to a standard British thromboplastin.
2. Quick Value. The Quick Value is expressed as a percentage; the lower the value, the longer the blood takes to coagulate. Therefore as the Quick Value increases, the corresponding INR value gets smaller and vice versa.

(b) Activated partial thromboplastin time
The activated partial thromboplastin time (aPTT) is the second most common method for monitoring anticoagulant therapy, measuring all the clotting factors in the intrinsic pathway as opposed to the PT test, which measures the extrinsic pathway.

(c) Other methods of assessing clottingOther tests used, which in some instances offer more sensitivity to specific aspects of therapy, include the prothrombin-proconvertin ratio (PP), the thrombotest, the thrombin clotting time test (TCT, activated clotting time, activated coagulation time), the platelet count and the bleeding time test.
The use of the most appropriate test will depend on the situation and the desired result.
 

Anticoagulant interactions
Stable oral anticoagulant therapy is difficult to achieve even during close monitoring. For example, in one controlled study in patients with atrial fibrillation, only 61% of INR values were within the target range of 2 to 3, despite monitoring the INR monthly and adjusting the warfarin dose appropriately.
1 A large number of factors can influence levels of coagulation, including diet, disease (fever, diarrhoea, heart failure, thyroid dysfunction), and the use of other drugs. It must therefore be remembered that it is particularly difficult to ascribe a change in INR specifically to a drug interaction in a single case report, and single case reports or a few isolated reports for widely used drugs do not prove that an interaction occurs.
Nevertheless, either the addition or the withdrawal of drugs may upset the balance in a patient already well stabilised on an anticoagulant. Some drugs are well known to increase the activity of the anticoagulants and can cause bleeding if the dosage of the anticoagulant is not reduced appropriately.
Others reduce the activity and return the prothrombin time to normal.
Both situations are serious and may be fatal, although excessive hypoprothrombinaemia manifests itself more obviously and immediately as bleeding and is usually regarded as the more serious. The interaction mechanism may be pharmacodynamic or pharmacokinetic: pharmacokinetic mechanisms are particularly well established and important for coumarin anticoagulants.

 

 

(a) Metabolism of the coumarins
The coumarins, warfarin, phenprocoumon and acenocoumarol, are racemic mixtures of S- and R-enantiomers. The S-enantiomers of these coumarins have several times more anticoagulant activity than the R-enantiomers.
Reports suggest for example, that S-warfarin is three to five times more potent a vitamin K antagonist than R-warfarin. The S-enantiomer of warfarin is metabolised primarily by the cytochrome P450 isoenzyme CYP2C9, and to a much lesser extent, by CYP3A4. The metabolism of Rwarfarin is more complex, but this enantiomer is primarily metabolised by CYP1A2, CYP3A4, and CYP2C19. S-warfarin is eliminated in the bile and R-warfarin is excreted in the urine as inactive metabolites. There is much more known about the metabolism of warfarin compared with other anticoagulants, but it is established that S-phenprocoumon and S-acenocoumarol are also substrates for CYP2C9 and that they differ from warfarin in their hepatic metabolism, and stereospecific potency.2
It makes sense to assume therefore, that an inhibitor of CYP2C9 (e.g. ‘fluconazole’, (p.387)) is likely to increase the concentration of the coumarin and enhance the anticoagulant effect. Drugs that induce CYP2C9 (e.g. ‘rifampicin’, (p.375)) reduce plasma levels of the coumarins by increasing the clearance.
‘Genetic differences’, (p.4), in the genes for these cytochrome P450 isoenzymes may have an important influence on drug metabolism of the coumarins. For example, different versions of the gene encoding CYP2C9 exist and the enzymatic activity of the most clinically important CYP2C9 variants, CYP2C9*2 and CYP2C9*3, is significantly reduced. Studies have suggested an association between patients possessing one or more of these variants and a low-dose requirement of warfarin. Similar observations have been seen with the CYP2C9*3 variant and acenocoumarol.
While the metabolism of the coumarins, especially warfarin, are well known, the numerous interaction pathways and the variability in patient responses, makes the clinical consequences difficult to predict.

(b) Other mechanisms for anticoagulant interactions
Some drugs, such as ‘colestyramine’, (p.393), may also prevent the absorption of the coumarins and reduce their bioavailability. See also ‘Drug absorption interactions’, (p.3). Additive anticoagulant effects can occur if anticoagulants are given with other drugs that also impair coagulation by other mechanisms such as ‘antiplatelets’, (p.700). Coumarins and indanediones act as vitamin K antagonists, and so dietary intake of ‘vitamin K’, (p.409) can also ‘reduce or abolish’, (p.9) their effects. ‘Protein-binding displacement’, (p.3) is another possible drug interaction mechanism but this usually plays a minor role compared with other mechanisms.3

 

Bleeding and its treatment

 
When prothrombin times become excessive, bleeding can occur. In order of decreasing frequency the bleeding shows itself as ecchymoses, blood in the urine, uterine bleeding, black faeces, bruising, nose-bleeding, haematoma, gum bleeding, coughing and vomiting blood.
Vitamin K is an antagonist of the coumarin and indanedione oral anticoagulants.
The British Society for Haematology has given advice on the appropriate course of action if bleeding occurs in patients taking warfarin, and this is readily available in summarised form in the British National Formulary.
If the effects of heparin are excessive it is usually sufficient just to stop the heparin, but protamine sulfate is a specific antidote if a rapid effect is required. Protamine sulfate only partially reverses the effect of low molecular weight heparins.
There is currently no known specific antidote for fondaparinux, or for the direct thrombin inhibitors.
1. Stroke Prevention in Atrial Fibrillation Investigators. Adjusted-dose warfarin versus low-intensity, fixed dose warfarin plus aspirin for high-risk patients with atrial fibrillation: Stroke Prevention in Atrial Fibrillation III randomised clinical trial. Lancet (1996) 348, 633–8.
2. Ufer M. Comparative pharmacokinetics of vitamin-K antagonists. Warfarin, phenprocoumon and acenocoumarol. Clin Pharmacokinet (2005) 44, 1227–46.
3. Sands CD, Chan ES, Welty TE. Revisiting the significance of warfarin protein-binding displacement interactions. Ann Pharmacother (2002) 36, 1642–4.

Tacrolimus + Phenytoin

Tacrolimus + Phenytoin

An isolated report describes an increase in serum phenytoin levels attributed to the use of tacrolimus. Phenytoin decreased tacrolimus levels in one case, and has been used to reduce tacrolimus levels after an overdose.

Clinical evidence

(a) Phenytoin levels
A kidney transplant patient taking phenytoin 500 and 600 mg on alternate days (and also taking azathioprine, bumetanide, digoxin, diltiazem, heparin, insulin and prednisone) had his immunosuppressant treatment changed from ciclosporin, to tacrolimus 14 to 16 mg daily.
About 7 weeks later he presented to hospital because of a fainting episode and his phenytoin levels were found to have risen from 18.4 to 36.2 micrograms/mL. The phenytoin was temporarily stopped until his serum levels had fallen, and he was then discharged on a reduced phenytoin dosage of 400 and 500 mg on alternate days with no further problems.1
The presumption is that the fainting episode was due to the raised serum phenytoin levels.
 

(b) Tacrolimus levels
In one kidney transplant patient taking phenytoin, tacrolimus 250 micrograms/kg daily was needed to give a blood level of 9 nanograms/mL. Three months later phenytoin was gradually stopped, with gradual tapering of the tacrolimus dose. The patient was eventually stabilised with a tacrolimus dose of 160 micrograms/kg daily giving a blood level of 11 nanograms/mL.2
Another report describes the use of an intravenous phenytoin infusion to treat acute tacrolimus overdoses in 2 patients, with the aim of enhancing tacrolimus metabolism.3

Mechanism
Tacrolimus is extensively metabolised by the cytochrome P450 isoenzyme CYP3A4, and phenytoin is a known inducer of this system. Phenytoin is therefore predicted to decrease tacrolimus levels. In the first case, it was suggested that tacrolimus might have inhibited the metabolism of phenytoin, although other factors may have had some part to play in the raised phenytoin levels.1
 

Importance and management
No interaction is established, but based on the known metabolism of these drugs it would be prudent to monitor tacrolimus levels in a patient given phenytoin. Similarly, based on the single case of phenytoin toxicity, it may also be advisable to monitor phenytoin levels.
1. Thompson PA, Mosley CA. Tacrolimus-phenytoin interaction. Ann Pharmacother (1996) 30, 544.

2. Moreno M, Latorre C, Manzanares C, Morales E, Herrero JC, Dominguez-Gil B, Carreño A, Cubas A, Delgado M, Andres A, Morales JM. Clinical management of tacrolimus drug interactions in renal transplant patients. Transplant Proc (1999) 31, 2252–3.
3. Karasu Z, Gurakar A, Carlson J, Pennington S, Kerwin B, Wright H, Nour B, Sebastian A. Acute tacrolimus overdose and treatment with phenytoin in liver transplant recipients. J Okla State Med Assoc (2001) 94, 121–3.

ACE inhibitors and Angiotensin II receptor antagonists + Heparins

ACE inhibitors and Angiotensin II receptor antagonists + Heparins

Heparin may increase the risk of hyperkalaemia with ACE inhibitors or angiotensin II receptor antagonists.
Clinical evidence, mechanism, importance and management An extensive review of the literature found that heparin (both unfractionated and low-molecular-weight heparins) and heparinoids inhibit the secretion of aldosterone, which can cause hyperkalaemia.1 The CSM in the UK suggests that plasma-potassium levels should be measured in all patients with risk factors (including those taking potassium-sparing drugs) before starting heparin, and monitored regularly thereafter, particularly if heparin is to be continued for more than 7 days.2 Note that ACE inhibitors and angiotensin II receptor antagonists are potassium sparing, via their effects on aldosterone. Some workers1 have suggested that the monitoring interval should probably be no greater than 4 days in patients at a relatively high risk of hyperkalaemia. Other risk factors include renal impairment, diabetes mellitus, pre-existing acidosis or raised plasma potassium.2 If hyperkalaemia occurs, the offending drugs should be stopped (although this may not be practical in the case of heparin). When the hyperkalaemia has been corrected (by whatever medical intervention is deemed appropriate) the drugs can cautiously be reintroduced.
1. Oster JR, Singer I, Fishman LM. Heparin-induced aldosterone suppression and hyperkalemia.
Am J Med (1995) 98, 575–86.
2. Committee on Safety of Medicines/Medicines Control Agency. Suppression of aldosterone secretion by heparin. Current Problems (1999) 25, 6.