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Merry Christmas and haPPy New yeAr

Harper's illustrated biochemistry , Volume 26

Extensively revised and updated, this authoritative biochemistry text is known worldwide for its comprehensive and up-to-date coverage. Extensively illustrated and user-friendly, the text offers examples pf how knowledge of biochemistry is essential for understanding the molecular basis of health and disease. The 26th edition also features expanded content on results of the Human Genome Project. Perfect as both text and USMLE review.

Contents:

SECTION I. STRUCTURES & FUNCTIONS OF PROTEINS & ENZYMES 

 

SECTION II. BIOENERGETICS & THE METABOLISM OF CARBOHYDRATES & LIPIDS

SECTION III. METABOLISM OF PROTEINS & AMINO ACIDS 

SECTION IV. STRUCTURE, FUNCTION, & REPLICATION OF INFORMATIONAL MACROMOLECULES 

SECTION V. BIOCHEMISTRY OF EXTRACELLULAR AND INTRACELLULAR 

COMMUNICATION

SECTION VI. SPECIAL TOPICS

What's an SSD?

Image via Wikipedia

An SSD provides data storage functionality just like an ordinary hard drive does in your computer, but it uses next-generation technology that makes it faster, smaller—and more expensive.

Left: SSD; Right: hard drive

As improvements in technology allow us to make our computing devices even lighter and faster than ever before, hard drives are beginning to have to compete with solid state drives (SSDs) that use cutting-edge “flash” technology to read and store information. The newer flash-based technology differs in that it’s completely electronic—meaning that SSDs don’t contain spinning disks and movable read/write heads that can slow down or delay operations. Instead, SSDs use microchips to contain data.

You’re probably already familiar with small flash-based storage devices that you can plug into a USB port on your PC to transfer a few files and tote around in your pocket. SSDs take this to the next level with much higher

capacity—enough to replace the hard drive in some instances.

Flash-based storage can already be found as an alternative to a hard drive in some newer lightweight laptops and tablet PCs: if you've recently purchased a new netbook or ultraportable PC, you may already have an SSD inside. Even if you don't currently own a device with an SSD, as the trend toward lighter and more portable computing devices continues, you'll definitely see SSDs becoming an increasingly commonplace and affordable data storage option. Tech enthusiasts and early adopters are increasingly purchasing SSDs separately to swap into existing PCs or laptops as an aftermarket performance-boosting upgrade. 

Continue reading here

M.Pharm.Ph.D Integrted Programme

 Central University of Punjab is one of the fastest growing universities of India with a commitment to have state-of-the-art infrastructure, world class teaching faculty, laboratories, enriched library and computing facilities of global standards. The University will be providing its students scholarships, benefits of credit transfer mechanism to other universities, campus wide internet connectivity, ejournals, well equipped scientific and research laboratories to cater for research needs.

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Principles and Methods of Bioassay

Bioassay is defined as the estimation of the potency of an active principle in a unit quantity of preparation or detection and  easurement of the concentration of the substance in a preparation using biological methods (i.e. observation of pharmacological effects on living tissues, microorganisms or  immune cells or animal). Hence micro bioassay, radioimmunoassay are also regarded as `bioassay'. Recently `biotechnology' has also been considered for bioassay. Bioassay of the products like erythropoietin, hepatitis-B vaccine etc. is being done through biotechnology. 

 Importance of Bioassay

Bioassays, as compared to other methods of assays (e.g. chemical or physical assay) are less accurate, less elaborate, more laborious, more troublesome and more expensive. However, bioassay is the only method of assay if

(1) Active principle of drug is unknown or cannot be isolated, e.g. insulin, posterior pituitary extract etc. 

(2) Chemical method is either not available or if available, it is  too complex and insensitive or requires higher dose e.g. insulin, acetylcholine.

(3) Chemical composition is not known, e.g. long acting thyroid stimulants. 

(4) Chemical composition of drug differs but have the same pharmacological action and vice-versa, e.g.  cardiac glycosides, catecholamines etc.

Moreover, even if chemical methods are available and the results of bioassay conflict with those of the chemical assay, the bioassay is relied upon and not the chemical assay, since it is the assessment on living organism.

The purpose of bioassay is to ascertain the potency of a drug and hence it serves as the quantitative part of any screening procedure (Research). Other purpose of bioassay is to standardize the preparation so that each contains the uniform specified pharmacological activity. In this way, it serves as a pointer in the Commercial Production of drugs when chemical assays are not available or do not suffice. From the  clinical  point of view, bioassay may help in the diagnosis of  various conditions, e.g. gonadotrophins for pregnancy. 

Principle of Bioassay  

The basic principle of bioassay is to compare the test substance with the International Standard preparation of the same and to find out how much test substance is required to produce the same biological effect, as produced by the standard. The standards are internationally accepted samples of drugs maintained and recommended by the Expert Committee of the Biological Standardization of W.H.O. They represent the fixed units of activity (definite weight of preparation) for drugs. In India, standard drugs are maintained in Government institutions like Central Drug Research Institute, Lucknow, Central Drug Laboratory, Calcutta, etc.

The problem of biological variation must be minimized as far as possible. For that one should keep uniform experimental conditions and assure the reproducibility of the responses. 

Methods of Bioassay for Agonists 

An agonist may produce graded response or quantal response. Graded response means that the response is proportional to the dose and response may lie between no response and the maximum response. By quantal, it is meant that the response is in the form of "all or none", i.e. either no  response or maximum response. The drugs producing quantal effect can be bioassayed by end point method. The drugs producing graded responses can be bioassayed by 

(1) Matching or bracketing method or (2) Graphical method.

1. End Point Method: Here the threshold dose producing a positive effect is measured on each animal and the comparison between the average results of two groups of animals (one receiving standard and other the test) is done. e.g. bioassay of digitalis in cats. Here the cat is anaesthetized with  chloralose and its blood pressure is recorded. The drug is slowly infused into the animal and the moment the heart stops beating and blood pressure falls to zero, the volume of fluid infused is noted down. Two series of such experiments-one using standard digitalis and the other using test preparation of digitalis is done and

then potency is calculated as follows: 
                                      
 Conc. of Unknown = Threshold dose of the Standard X Conc. of Std.                                                   Threshold dose of the Test 

       In case, if it is not possible to measure individual effective dose or if animals are not available, fixed doses are injected into groups of animals and the percentage of mortality at each dose level is determined. The percentage of mortality is taken as the response and then the comparison is done in the same way as done for graded response. 

2. Matching Method: In this method a constant dose of the test is brack doses of standard till the exact match is obtained between test dose and the standard dose.
Initially, two responses of the standard are taken. The doses are adjusted such that one is giving response of approximately 20% and other 70% of the maximum. The response of unknown which lies between two responses of standard dose is taken. The panel is repeated by increasing or decreasing the dose s of standard till all three equal responses are obtained. The dose of test sample is kept constant. At the end, a response of the double dose of the standard and test which match each other are taken. These should give equal responses. Concentration of the test sample can be determined as follows:

                                       Dose of the Standard
Conc. of Unknown =   -------------------------- X Conc. of Std.
                                          Dose of the Test 

This method has following limitations:

1.  It occupies a larger area of the drum as far as tracings are concerned.

2.  The match is purely subjective, so chances of error are there and one cannot determine them.

3.  It does not give any idea of dose-response relationship.
 

However, this method is particularly useful  if the sensitivity of the preparation is not stable. Bioassay of histamine, on guinea pig  ileum is preferably  carried out by this method.

3. Graphical method:  This method is based on the assumption of the dose-response relationship. Log-dose-response curve is plotted and the dose of standard producing the same response as produced by the test sample is directly read from the graph. In simpler design, 5-6 responses of the graded doses  of the standard are  taken and then two equiactive responses of the test sample are taken. The height of contraction is measured and plotted against the log-dose. The dose of standard producing the same response as produced by the test is read directly from the graph and the concentration of test sample is determined by the same formula as mentioned before. 

 

The characteristic of log-dose response curve is that it is linear in the middle (20-80%). Thus, the comparison should be done within this range only. In other words, the response of test sample must lie within this range. 

Advantage of this method is that, it is a simple method and chances of errors are less if the sensitivity of the preparation is not changed. Other methods which are based on the dose-response relationship include 3 point, 4 point, 5 point and 6 point methods. In these  methods, the responses are repeated several  times and the mean of each is taken. Thus, chances of error are minimized in these methods. In 3 point assay method 2 doses of the standard and one dose of the test are used. In 4 point method 2 doses of standard and 2 doses of the test are used. In 6 point method 3 doses of standard and 3 doses of the test are used. Similarly one can design 8 point method also. The sequence of responses is followed as per the Latin square method of randomization in order to avoid any bias. 

 The mean responses are calculated and plotted against log-dose and amount of standard producing the same response as produced by the test is determined graphically as well as mathematically:

 

n1 = Lower Standard dose                               
n2 = Higher Standard dose
t    = Test dose
S1 = Response of n1
S2 = Response of n2
T   = Response of test (t)
Cs = Concentration of standard 

Similarly, in 4 point method, amount of standard producing the same response as produced by the test can be determined by graphical method. It is determined mathematically as follows: 

 

t1 = lower dose of test; t2 = higher dose of test; T1 = response of t1; T2 = response of t2. 

Bioassay of Antagonists

Commonly used method for the bioassay of antagonist is simple graphical method. The responses are determined in the form of the percentage inhibition of the fixed dose of  agonist. These are then plotted against  the log dose of the antagonist and the concentration of unknown is determined by finding out the amount of standard producing the same effect as produced by the test.

In this method, two responses of the same dose of agonist  (sub maximal giving approximately 80% of the maximum response) are taken. The minimum dose of standard antagonist is added in the bath and then the response of the same dose of agonist is taken in presence of antagonist. The responses of agonist are repeated every ten min till recovery is obtained. The higher dose of standard antagonist is added and responses are taken as before. Three to four doses of the standard antagonist are used and then one to two doses of test sample of the antagonist is used similarly. The percentage inhibition is calculated, plotted against log dose of antagonist and the concentration of unknown is determined as usual. 

Bioassay of Some Important Drugs 

 Depending upon pharmacological action of various drugs, different preparations may be used. Following chart gives different preparations and the pharmacological activity for which a particular drug is assayed: 

 

Shampoos

Introduction : 

Definition: A shampoo is a preparation of a surfactant (i.e. surface active material) in a suitable form – liquid, solid or powder – which when used under the specified conditions will remove surface grease, dirt, and skin debris from the hair shaft and scalp without adversely affecting the user.

Requirements of a Shampoo:

•  It should effectively and completely remove dust or soil, excessive sebum or other fatty substances and loose corneal cells from the hair.
•  It should produce a good amount of foam to satisfy the psychological requirements of the user
• It should be easily removed on rinsing with water.
•  It should leave the hair non-dry, soft, lustrous with good manageability and minimum fly away.
•   It should impart a pleasant fragnance to the hair.
•  It should not cause any side-effects / irritation to skin or eye. It should not make the hand rough and chapped.

Types of Shampoo : 

• ·         Powder Shampoo
• ·         Liquid Shampoo
• ·         Lotion Shampoo
• ·         Cream Shampoo
• ·         Jelly Shampoo
• ·         Aerosol Shampoo
• ·         Specialized Shampoo
• ·         Conditioning Shampoo
• ·         Anti- dandruff Shampoo
• ·         Baby Shampoo
• ·         Two Layer Shampoo

PRODUCT INGREDIENTS : 

Surfactants are the main component of shampoo. Mainly anionic surfactants are used.

The raw materials used in the manufacture of shampoos are:

Principal surfactants: Provide detergency and foam.

Secondary surfactants: Improve detergency, foam and hair condition.

Other additives. CLEANSING ACTION OF SHAMPOO A surfactant consists of two part- one hydrophilic (water loving) while the other is hydrophobic in nature.

Surfactants : 

Surfactants Anionic surfactants are mostly used (good foaming properties). The hydrophilic portion carries a negative charge which results in superior foaming, cleaning and end result attributes. Non-ionic surfactants have good cleansing properties but do not have sufficient foaming power. Cationic surfactants are toxic and are hence not used. However, they may be used in low concentration in hair conditioners. Ampholytics, being expensive, are generally not used. However, they are mainly used as secondary surfactants and good hair conditioners.

ADDITIVES : 

Conditioning agents: Lanolin, mineral oil, herbal extracts, egg derivatives.

Foam builders: Lauroyl monoethanolamide, sarcosinates

Viscosity modifiers : Electrolytes – NH4Cl, NaCl Natural gums – Gum Karaya, tragacanth, alginates Cellulose derivatives – Hydroxy ethyl cellulose, methyl cellulose Carboxy vinyl polymers – Carbopol 934 Others – PVP, phosphate esters.

Sequestering agents: EDTA

Opacifying agents: Alkanolamides of higher fatty acids, propylene glycol, Mg, Ca and Zn salts of stearic acid, spermaceti, etc.

Clarifying agents: Solubilizing alcohols – ethanol, isopropanol Phosphates – Non-ionic solubilizers – polyethoxyated alcohols and esters.

Perfumes : Herbal, fruity or floral fragnances.

Preservatives : Methyl and propyl paraben, formaldehyde (most effective).

Anti-dandruff agents: The shampoos contain small amount of these actives, which are in contact with the scalp for only a short time. In order to be effective the active ingredient must work in the oil-water environment of the scalp and must be readily substantive to the scalp for continuing activity.

Eg: Selenium sulfide, zinc pyrithone, salicylic acid.

Evaluation of Shampoos : 

• ·         Evaluation of Shampoos Performance characteristics
• ·         Foam and foam stability
• ·         Detergency and cleaning action
• ·         Effect of water hardness
• ·         Surface Tension and wetting
• ·         Surfactant content and analysis
• ·         Rinsing Conditioning action Softness Luster Lubricity Body, texture and set retention
• ·         Irritation and toxicity
• ·         Dandruff control
• ·         Microbiological assay
• ·         Eye irritancy test
• ·         Product characteristics
• ·         Fragnance
• ·         Colour
• ·         Consistency
• ·         Package

1.Foam and foam stability: The Ross-Miles foam column test is accepted. 200 ml of surfactant solution is dropped into a glass column containing 50ml of the same solution. The height of the foam generated is measured immediately and again after a specified time interval, and is considered proportional to the volume. Barnett and Powers developed a latherometer to measure the effect of variables such as water hardness, type of soil and quantity of soil on foam speed, volume and stability. Fredell and Read titrated actual standard oiled heads of hair with additive increments of shampoo until a persistent lather end point appeared.

2.Detergency and cleaning action: Cleansing power is evaluated by the method of Barnet and Powers 5gm sample of soiled human hair is placed at 35°c in 200 cc of water containing of 1 gm of shampoo. The flask is shaken 50 times a minute for 4 minutes. Then washed once again with sufficient amount of water, then after filter the hair dried and weighed. The amount of soil is removed under these condition is calculated.

3. Wetting Action: Canvas disk sinking test: A mount veron cotton duck # 6 canvas disk 1 inch in diameter, is floated on the surface of a solution, and the time required for it to sink is measured accurately.

 4. Rinsing: Skilled beauticians are employed to make comparisons on the performance of several shampoos.

5. Conditioning Action: Conditioning action is a difficult property to assess. This is because it is basically dependent on subjective appraisal. No method has been published for measuring conditioning action. The degree of conditioning given to hair is ultimately judged by shampoo user who is making the evaluation on the basis of past experience and present expectations.

6. Microbiological assay: PREPARATION OF PRE-INOCULUM Take the loopful culture of staphylococcus aureus (ATCC6532) aseptically and transfer to sterilized and cooled 100 ml SCDM (broth). Mix well. Incubate the broth at 37oC for 24 hrs. PREPARATION OF MEDIA Soya bean casein digests medium, soya bean casein digest agar and nutrient agar. PREPARATION OF POUR PLATES Sterilized SCD agar (100 ml) is cooled to 40°C and mixed with 5 ml of 24 hrs old pre inoculated culture. This is immediately poured in plates (340 ml each) and allows to set. MAKING THE WELLS ON AGAR PLATES The wells are dig on agar plates with sterilised well digger aseptically. Take 100µml of each sample, add to well aseptically. Incubate the plates at 37oC for 24 hrs to 48 hrs. Observe the effectiveness of sample on culture growing on the agar plate and we can see the effectiveness of sample in the form of zone of inhibition around each well containing different sample.

7. Evaluation of eye irritancy: The test calls for dropping 0.1 ml of liquid shampoo in the conjunctiva sac of one eye of the rabbit , the other eye serving as control. In the case of the first three animals, the treated eye remains unwashed. Since washing the eye may or may not alleviate symptoms of injury. The six remaining animals are divided into two equal groups. In the first of these groups eyes instilled with the substances are washed with 20 ml of lukewarm water two seconds after treatment and in the second group after instillation. Readings are then made at 24, 48 and 72 hr and again four and seven days after treatment. If the lesions have not cleared up in seven days the test material is considered as severe irritant.

8. Viscosity: Viscosity of the liquid shampoo is determined using a Brookefield viscometer 100 mL of the shampoo is taken in a beaker and the spindle is dipped in it for about 5 min and then the reading is taken.

9.Oral toxicity  

1.        Oral toxicity can be given in terms of its lethal dose 50 ( LD/50 ) i.e., number if gms of the material per kg of body weight required to kill half of the test animals used.

2.       Fasting cage animals are taken & dosing is accomplished with the help of stomach tube.

3.       Lower the LD/50 the greater the toxicity

10.Test for pH

1.      Soap based shampoos are more effective in pH of 9.0-10.0.

2.       Synthetic detergent based shampoos are effective pH range of 6.0-9.0.

3.       pH can be measured with the help of pH meter

11.Skin irritation tests

Draize Test in rabbits

·         A set of 6 rabbits is used for testing each material.

·         Shampoos should be tested only for a short duration, that is, not more than 4 hrs as these products come in contact with the skin only for a short duration.