Medicines

body systems

2010-02-06T12:55:00.000-08:00

Basic Anatomy - Tissues & Organs There are many different types of cells in the human body. None of these cells function well on there own, they are part of the larger organism that is called - you.
Tissues Cells group together in the body to form tissues - a collection of similar cells that group together to perform a specialized function. There are 4 primary tissue types in the human body: epithelial tissue, connective tissue, muscle tissue and nerve tissue.
Epithelial Tissue - The cells of epithelial tissue pack tightly together and form continuous sheets that serve as linings in different parts of the body. Epithelial tissue serve as membranes lining organs and helping to keep the body's organs separate, in place and protected. Some examples of epithelial tissue are the outer layer of the skin, the inside of the mouth and stomach, and the tissue surrounding the body's organs.
Connective Tissue - There are many types of connective tissue in the body. Generally speaking, connective tissue adds support and structure to the body. Most types of connective tissue contain fibrous strands of the protein collagen that add strength to connective tissue. Some examples of connective tissue include the inner layers of skin, tendons, ligaments, cartilage, bone and fat tissue. In addition to these more recognizable forms of connective tissue, blood is also considered a form of connective tissue.
Muscle Tissue - Muscle tissue is a specialized tissue that can contract. Muscle tissue contains the specialized proteins actin and myosin that slide past one another and allow movement. Examples of muscle tissue are contained in the muscles throughout your body.
Nerve Tissue - Nerve tissue contains two types of cells: neurons and glial cells. Nerve tissue has the ability to generate and conduct electrical signals in the body. These electrical messages are managed by nerve tissue in the brain and transmitted down the spinal cord to the body. Organs Organs are the next level of organization in the body. An organ is a structure that contains at least two different types of tissue functioning together for a common purpose. There are many different organs in the body: the liver, kidneys, heart, even your skin is an organ. In fact, the skin is the largest organ in the human body and provides us with an excellent example for explanation purposes. The skin is composed of three layers: the epidermis, dermis and subcutaneous layer. The epidermis is the outermost layer of skin. It consists of epithelial tissue in which the cells are tightly packed together providing a barrier between the inside of the body and the outside world. Below the epidermis lies a layer of connective tissue called the dermis. In addition to providing support for the skin, the dermis has many other purposes. The dermis contains blood vessels that nourish skin cells. It contains nerve tissue that provides feeling in the skin. And it contains muscle tissue that is responsible for giving you 'goosebumps' when you get cold or frightened. The subcutaneous layer is beneath the dermis and consists mainly of a type of connective tissue called adipose tissue. Adipose tissue is more commonly known as fat and it helps cushion the skin and provide protection from cold temperatures.
A cross-section of skin
Epidermis
Dermis
Subcutaneous layer
Organ Systems Organ systems are composed of two or more different organs that work together to provide a common function. There are 10 major organ systems in the human body, they are the:
Skeletal System:

Image courtesy of A. McGann
Major Role: The main role of the skeletal system is to provide support for the body, to protect delicate internal organs and to provide attachment sites for the organs.
Major Organs: Bones, cartilage, tendons and ligaments.
Muscular System:
Image courtesy of G. Huang
Major Role: The main role of the muscular system is to provide movement. Muscles work in pairs to move limbs and provide the organism with mobility. Muscles also control the movement of materials through some organs, such as the stomach and intestine, and the heart and circulatory system.
Major Organs: Skeletal muscles and smooth muscles throughout the body.

Circulatory System:
Image courtesy of G. Huang
Major Role: The main role of the circulatory system is to transport nutrients, gases (such as oxygen and CO2), hormones and wastes through the body.
Major Organs: Heart, blood vessels and blood.
Nervous System:
Image courtesy of G. Huang
Major Role: The main role of the nervous system is to relay electrical signals through the body. The nervous system directs behaviour and movement and, along with the endocrine system, controls physiological processes such as digestion, circulation, etc.
Major Organs: Brain, spinal cord and peripheral nerves.
Respiratory System:

Image courtesy of A. McGann
Major Role: The main role of the respiratory system is to provide gas exchange between the blood and the environment. Primarily, oxygen is absorbed from the atmosphere into the body and carbon dioxide is expelled from the body.
Major Organs: Nose, trachea and lungs.
Digestive System:

Image courtesy of A. McGann
Major Role: The main role of the digestive system is to breakdown and absorb nutrients that are necessary for growth and maintenance.
Major Organs: Mouth, esophagus, stomach, small and large intestines.
Excretory System:
Image courtesy of G. Huang
Major Role: The main role of the excretory system is to filter out cellular wastes, toxins and excess water or nutrients from the circulatory system.
Major Organs: Kidneys, ureters, bladder and urethra.
Endocrine System:
Image courtesy of G. Huang
Major Role: The main role of the endocrine system is to relay chemical messages through the body. In conjunction with the nervous system, these chemical messages help control physiological processes such as nutrient absorption, growth, etc.
Major Organs: Many glands exist in the body that secrete endocrine hormones. Among these are the hypothalamus, pituitary, thyroid, pancreas and adrenal glands.
Reproductive System:
Female:
Male: Images courtesy of G. Huang
Major Role: The main role of the reproductive system is to manufacture cells that allow reproduction. In the male, sperm are created to inseminate egg cells produced in the female.
Major Organs: Female (top): ovaries, oviducts, uterus, vagina and mammary glands. Male (bottom): testes, seminal vesicles and penis.
Lymphatic/Immune System:

Image not available
Major Role: The main role of the immune system is to destroy and remove invading microbes and viruses from the body. The lymphatic system also removes fat and excess fluids from the blood.
Major Organs: Lymph, lymph nodes and vessels, white blood cells, T- and B- cells. For more information on human anatomy, try these other sites:
The National Library of Medicine has an excellent page that includes links to Medline, a searchable medical research database, and the Visible Human Project's animations, which include anatomical illustrations from human cadavers and an animated trip through the Visible Human male cryosections (770k movie linked here).
The Informative Graphics Corp. has put together a wonderful Human Anatomy On-line program.
The University of Washington's Digital Anatomist Interactive Atlas has some interesting computer generated illustrations of the brain, the heart and a knee cross-section.
Andrew McGann's Look Inside the Human Body has more information on some organ systems.
The Upper Freehold Regional School District's AP Biology class has put together a nice summary of the Human Organ Systems.
The Indianapolis-Marion County Public Library's Inside the Human Body site has organ system info.

2009-12-27T02:27:00.001-08:00

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General Principles: Chemotherapy

2009-08-14T06:04:00.000-07:00

General Principles of Chemotherapy
Many of the same basic principles apply to antimicrobial, antiparasitic and anticancer chemotherapy.
Selective Toxicity:
Selective toxicity refers to the ability of the drug to targets sites that are relative specific to the microorganism responsible for infection.
Sometimes these sites are unique to the microorganism or simply more essential to survival of the microorganism than to the host.
Examples of such specific or relatively specific sites include specific fungal or bacterial cell wall synthesizing enzymes, the bacterial ribosomal or the molecular machinery of viral replication.
Chambers, H.F., Hadley, W. K. and Jawetz, E. Introduction to Antimicrobial Agents in Basic and Clinical Pharmacology, (Katzung, B. G., ed) Appleton-Lange, 1998, p. 723.
Chemotherapeutic Drug Targets
Targets for Antimicrobial/Antiviral Drugs
Bacterial Cell Wall Synthesis Inhibitors
Agents that Increase Cell Membrane Permeability
Protein Synthesis Inhibitors: interfere with 30S or 50S bacterial ribosome function

Drugs that Bind to the 30S bacterial ribosomal subunit, leading to cell death
Agents that interfere with nucleic acid synthesis
Antimetabolites
Inhibitors of Viral Replication

Bacterial cell wall synthesis inhibitors:
penicillins
cephalosporins
cycloserine
vancomycin (Vancocin)
bacitracin
miconazole (Monistat)(imidazole antifungal)
ketoconazole (Nizoral)(imidazole antifungal)
clotrimazole (Mycelex)(imidazole antifungal)
Agents that increase cell membrane permeability
polymixins (detergent)
colistimethate (detergent)
nystatin (Mycostatin)(polyene antifungal)
amphotericin B (Fungizone, Amphotec)(polyene antifungal)

Protein synthesis inhibitors: interfere with 30S or 50S bacterial ribosome function.
Bacteriostatic
chloramphenicol (Chloromycetin)
tetracyclines
erythromycin estolate (Ilosone)
clindamycin (Cleocin)
Drugs that bind to the 30S bacterial ribosomal subunit, leading to cell death.
Bacteriocidal
Aminoglycosides (e.g.gentamicin (Garamycin), tobramycin (Nebcin))

Agents that interfere with nucleic acid synthesis
rifamycins (rifampin (Rimactane)): inhibits DNA-dependent RNA polymerase
quinolones: inhibit gyrase

Antimetabolites
sulfonamides
trimethoprim (generic)

Some Nucleic Acid Analogs (Antivirals): inhibitors of viral replication
zidovudine (Retrovir, AZT, azidothymidine)
ganciclovir (DHPG, Cytovene)
vidarabine (Vira-A)
acyclovir (Zovirax)
Chambers, H.F and Sande, M.A. Antimicrobial Agents in,In, Goodman and Gillman's The Pharmacologial Basis of Therapeutics,(Hardman, J.G, Limbird, L.E, Molinoff, P.B., Ruddon, R.W, and Gilman, A.G.,eds) TheMcGraw-Hill Companies, Inc.,1996, pp.1029-1030

Disease caused by bacteria and the the role of the host response in outcome of chemotherapeutic intervention.
Importance of Host Response
Host response as manifest in the inflammatory response is crucial for both the interruption resolution of the infection and for the basis of the infection's signs and symptoms.
The ability of effective antibiotic chemotherapy depends not only on appropriate selection of medication(s), dosage, and interval, but also on the host immune response.
Pier, G.B. Molecular Mechanism of Bacterial Pathogenesis., In Harrison's Principles of Internal Medicine (Isselbacher, K.J., Braunwald, E., Wilson, J.D., Martin, J.B., Fauci, A.S. and Kasper, D.L., eds) McGraw-Hill, Inc (Health Professions Division), 1994, p. 592.
Disease Manifestations of Bacterial Disease
Disease manifestation of bacterial infection involves:
Colonization
Invasion: Bacterial invasion refers to the presence of bacteria in tissue sites normally sterile.
Examples:
Gram-negative rods in the blood stream result in septisemia and bacteremia without requiring endotoxin involvement
Pneumococcal pneumonia is due the growth of Streptococcus pneumoniae in the lung while bacterial endotoxins do not appear to play a role.
Disease that occurs after bactermia and invasion of the meninges by meningitis-producing bacteria (N. meningitidis, H. influenzae, E. coli, K1 and group B streptococci) is due to tissue destruction secondary to bacterial growth and host inflammation.
Toxin production and release
Toxin Elaboration
Clinicial manifestation of some bacterial infections are primarily due to toxin elaboration.
For example: Botulinum toxin: C. botulinum, Tetanus toxins: C. tetani, Diptheria toxin causes the disease due to infection with C. diphtheriae
Some specific aspects of bacterial disease are caused by elaborated toxins
Enterotoxins cause the diarrhea associated with E. coli, Salmonella, Shigella, Staphylococcus and V. cholerae.
Toxins involved in Toxic shock syndrome caused by Staphylococci, steptococci, P. aeruginosa and Bordatella: include: Toxic shock syndrome toxin (TSST), erythrogenic toxin, exotoxin A and pertussis toxin.
Staphylococcal enterotoxins, TSST-1 and streptococcal exotoxins have been classified as superantigens which are capable of inducing certain T cell proliferation without processing of the protein toxin by antigen-presenting cells.
This process involves, in part, the elaboration of IL-1 and TNF-alpha which may cause many clinical features seen in staphylococcal toxic shock syndrome, scarlet fever, streptococcal toxic shock syndrome.
Pier, G.B. Molecular Mechanism of Bacterial Pathogenesis., In Harrison's Principles of Internal Medicine 14th edition, (Isselbacher, K.J., Braunwald, E., Wilson, J.D., Martin, J.B., Fauci, A.S. and Kasper, D.L., eds) McGraw-Hill, Inc (Health Professions Division), 1998, p. 855.
Endotoxins (lipid A portion of gram-negative LPS) may cause many clinical presentations seen in gram-negative bacterial sepsis: Toxins involve include: IL-1 and TNF-alpha--
Clinical presentations include:fever, intravascular coagulation, shock, and muscle proteolysis.
Pier, G.B. Molecular Mechanism of Bacterial Pathogenesis., In Harrison's Principles of Internal Medicine 14th edition, (Isselbacher, K.J., Braunwald, E., Wilson, J.D., Martin, J.B., Fauci, A.S. and Kasper, D.L., eds) McGraw-Hill, Inc (Health Professions Division), 1998, p. 855.
Importance of Host Response
Host response as manifest in the inflammatory response is crucial for both interruption and infection resolution and the infection's signs and symptoms.
The ability of effective antibiotic chemotherapy depends not only on appropriate selection of medication(s), dosage, and interval, but also on the host immune response.
Pier, G.B. Molecular Mechanism of Bacterial Pathogenesis., In Harrison's Principles of Internal Medicine (Isselbacher, K.J., Braunwald, E., Wilson, J.D., Martin, J.B., Fauci, A.S. and Kasper, D.L., eds) McGraw-Hill, Inc (Health Professions Division), 1994, p. 592.
Pharmacokinetic barriers that reduce the effectiveness of treatment
Introduction
Pharmacokinetic components include absorption, distribution, biotransformation (metabolism) and excretion.
Pharmacokinetic "profile" of antibacterial describes the drug concentration in tissues and serum as a function of time after administration.
The profile depends on the agent's absorption, distribution, biotransformation and excretion.
In therapeutics "trough" concentration (low) and "peak" concentrations (high) are important.
Pharmcokinetic information is used to establish dose and dosing intervals.
Absorption
Oral Therapy predominates for the following principal reasons: lower costs and fewer adverse effects
Another consideration, however, is that the oral route of administration is associated with a substantial range of bioavailabililty. For example, oral availability is only about 10% to 20% erythromycin estolate (Ilosone) and penicillin G, whereas bioavailability is about 100% for clindamycin (Cleocin), doxycycline (Vibramycin, Doryx) methonidazole, and trimethoprim (generic) sulfamethoxazole (Gantanol).
Bioavailability differences are NOT clinically important if the concentration of drug at the site of infection is sufficient to inhibit (bacteriostatic) or to kill (bacteriocidal).
Factors that can influence oral bioavailability include the presence of food in the digestive tract and drug interactions for example quinolones with metal cations.
Intramuscular
Administration by this route offers 100% bioavailable, but not widely used because of injection site pain can be usually ease of intravenous administration in the hospital inpatient setting.
Intravenous Administration:
Intravenous administration, however, would be appropriate if for example, oral agents proved ineffective, if there is a special concern about bioavailability, if larger doses are required relative to those typically obtained from oral dosing or because the bioavailability will be known to be 100%.
Distribution:
Antibacterial concentrations must exceed that required to inhibit bacterial growth (MIC).
Given that most infections are located outside the blood stream, the drug must distribute to those sites.
Drug concentration at most sites are similar to serum levels.
Some sites are "protected", however. these protected sites include the eye, prostate, and cardiac sites where there may be vegetative growth (e.g. around valves in a bacterial endocarditis setting)
High parentral doses may be required in these circumstances.
Poor efficacy may be related to adequate concentrations, but local unfavorable conditions.
For example, the activity of aminoglycosides is reduced in acidic pH, common at sites of infection. Biological constituants present in absesses may also inhibit the activity of antibacterials, thus requiring surgical dranage prior to efficacious therapy.
Archer,G.L. and Polk, R.E. Treatment and Prophylaxis of Bacterial Infections, In Harrison's Principles of Internal Medicine 14th edition, (Isselbacher, K.J., Braunwald, E., Wilson, J.D., Martin, J.B., Fauci, A.S. and Kasper, D.L., eds) McGraw-Hill, Inc (Health Professions Division), 1998, p. 860.

Mechanisms for chemotherapeutic drugs resistance
Bacterial resistance may occur because the drug does not reach its target site, drug is inactivated, or there is some sort of molecular alteration in the target itself, possibly due to mutation.
Resistance may occur because enzymes at or near the cell surface inactivate the antibiotic; the cell membrane is impermeable to the drug; there is an absence of aqueous channels (porins) through which the drug will reach the cell interior; there is a lack of a necessary transport system to support drug translocation; the transport mechanism is present but inoperative due to anaerobic metabolism; there are target site changes that results in reduced or absent antibacterial drug efficacy.
How Bacteria Acquire Resistance
Resistance may be acquired by vertical transfer, i.e. acquired by mutation and then passed to daughter cells
Mutations: Specific genetic mutations are the molecular basis for resistance to streptomycin (ribosomal mutation), to quinolones (DNA gyrase gene mutation) and to rifampin (Rimactane) (RNA polymerase gene mutation)
The mutation to rifampin (Rimactane) is an example of a single-step mutation: In this case E. coli or Staph. aureus exposure to rifampin results in highly resistant strain due to a point mutation in the RNA polymerase gene such that the polymerase protein no longer binds rifampin.
More usually, acquired by horizontal transfer of resistance factors from a donor cell, perhaps of a different species by transformation which involves the incorporation of DNA found free environment into the bacterial genome.
An example of this process is the basis of penicillin resistance in. pneumococci and Neisseria gonorrhoeae.
Penicillin-resistant pneumococci produce different PBPs (penicillin-binding proteins). These different PBPs exhibit relatively low affinity for penicillin compared to wild type pneumococci.
These different PBPs arise from integration of foreign DNA which were most likely from a closely related streptococcal strain into the PBP gene by a process of homologous recombination.
Transduction-based resistance occurs when a bacteriophage which includes bacterial DNA in its protein coat infects the bacteria. This bacterial DNA may contain a gene confiring resistance to antibacterial drugs.
Examples of this process:
Staphylococcus aureus strain resistance development to penicillin may occur by transduction (Some bacteriophages carry plasmids [extrachromosomal self-replicating DNA] that code for penicillinase
Other phages can transfer genes which confer resistance to tetracycline (Achromycin), erythromycin estolate (Ilosone), and chloramphenicol (Chloromycetin).
Conjugation is another important mechanism for single and multi-drug resistance development. In conjugation direct passage of resistance-confering DNA between bacteria proceeds by way of a bridge
The genetic material transfer in conjugation requires two elements: an R-determinant plasmid which codes for the resistance and a resistance-transfer factor (RTF) plasmid which contains the genes necessary for the bacterial conjugation process. Occasionally two plasmids join to form a complete R factor
Some genes that are responsible for resistance are located on transposons which can move from location to location within plasmid and bacterial genomes.
Conjugation mediated resistance is particularly important in gram-negative bacilli.
Enterococci may contain plasmids that spread resistance among gram-positive organisms
Vancomycin (Vancocin) resistance in enterococcal strains appears to occur as a result of the conjugation mechanism.
Conjugation is not a high efficiency mechanism for resistance development. Unfortunately, antibiotic use provides selection pressures which facilitate the elaboration of resistance bacteria. Furthermore, enteric bacteria carrying plasmids for multidrug resistance is now a worldwide, serious concern.
Resistance acquired by horizontal transfer can disseminate rapidly through the bacterial population by clonal spread as well as by continuing genetic material exchange between cells of the same or different susceptible strains.
Chambers, H.F and Sande, M.A. Antimicrobial Agents in,In, Goodman and Gillman's The Pharmacologial Basis of Therapeutics,(Hardman, J.G, Limbird, L.E, Molinoff, P.B., Ruddon, R.W, and Gilman, A.G.,eds) TheMcGraw-Hill Companies, Inc.,1996, pp.1031.
Microbial Resistance and Specific Drugs
Resistance: ß-Lactams
Most common among several mechanisms by which bacteria develop resistance to ß-Lactam antibiotics is by elaboration of the enzyme ß-lactamase, which hydrolyzes the ß-lactam ring.
Beta-lactamase genes may be found in both gram-positive and gram-negative bacteria. Resistance may be reduced by agents which bind to some beta-lactamases. Examples of these drugs include clavulanic acid and sulbactam.
Another mechanism by which bacteria may develop resistance to beta-lactam antibiotics is by changes in penicillin-binding proteins (PBPs). These changes may occur either by mutation of existing PBP genes or more often by the acquisition of new PBP genes. For the latter case, unimportant example is staphylococcal resistance to methicillin (Staphcillin).
In addition to mutation of existing PBP genes, bacterial also acquiring new "pieces or segments" of PBP genes. This process appears important in resistance development for certain pneumococcal, gonococcal, and meningococcal strains)
Yet another mechanism is observed in gram-negative bacteria and follows from alteration of genes that code for certain outer membrane proteins (porins). The expression of altered porins reduce membrane permeability to penicillins. This process appears important in cephalosporin-resistance of Enterbacteriaceae and that of Pseudomonas species to ureidopenicillins.
Multiple resistance mechanisms can exist within the same bacterial cell.
Archer,G.L. and Polk, R.E. Treatment and Prophylaxis of Bacterial Infections, In Harrison's Principles of Internal Medicine 14th edition, (Isselbacher, K.J., Braunwald, E., Wilson, J.D., Martin, J.B., Fauci, A.S. and Kasper, D.L., eds) McGraw-Hill, Inc (Health Professions Division), 1998, p. 859.
Chloramphenicol (Chloromycetin) resistance:
This resistance occurs because of the formation of an acetylated chloramphenicol (Chloromycetin) derivative which is not biologically active.
A plasmid-encoded enzyme, chloramphenicol acetyltransferase catalyzes the acetyl group transfer which would otherwise not occur
Vancomycin (Vancocin) resistance is an important problem in antibiotic chemotherapy. For example, vancomycin (Vancocin)-resistant enterococci strains are worldwide.
The resistance mechanism involves transfer between cells and is plasmid mediated.
The specific alteration is a change in the peptidoglycan stem peptide which causes a loss of the vancomycin (Vancocin) binding target.
Often significant vancomycin (Vancocin) resistance is observed with enterococci strains all the same time most staphylococci aureus and staphylococci epidermis remain vancomycin (Vancocin) sensitive.
Resistance to tetracyclines:
The most common mechanism for gram-negative bacterial resistance follows from insertion of a plasmid-encoded active-efflux pump which translocates tetracycline (Achromycin) out of the cell.
For gram-positive bacteria, resistance may follow from the above mechanism (active efflux) or is a result of changes in the tetracycline ribosomal target site.
Aminoglycoside resistance:
The most common resistance mechanism is antibiotic inactivation by enzyme-mediated covalent bond modification. Modifications occur as a result of acetyl, adenyl or phosphate group transfer.
Enzymes which catalyze these group transfers and confer antibody resistance are plasmid localized.
As a consequence of these molecular modifications, the modified antibiotic becomes less active secondary to reduced transport and attenuated ribosomal target site binding.
Aminoglycoside-modifying enzymes occur in both gram-negative and gram-positive bacteria.
Mupirocin resistance: Resistance to this topical drug is due to alteration in the target site which is an isoleucine tRNA synthetase enzyme. Following modification, the enzyme no longer binds mupirocin (Bactriban)
Macrolides and Licosamides resistance. Antibiotic from these categories target gram-positive bacteria.
Resistance to these antibiotics result from plasmid-mediated ribosomal RNA methylation that interferes with antibiotic binding.
Specific antibiotic susceptible to this type of resistance include erythromycin, newer macrolides and clindamycin (Cleocin).
Quinolone-resistance:
Quinone resistance including resistance to newer fluoroquinolones has developed quickly in Staphylococcus and Pseudomonas strains.
The resistance mechanism involves a mutation in the drug target site, which is DNA gyrases. The mediated DNA gyrases are not susceptible to inhibition by quinolones.
Other resistance mechanisms involve in gram-negative strains, mutations in porins that cause the bacterial membrane to lose its permeability to the drug.in gram-positive organisms, resistance follows the development of the capability that enables the cell to actively pump out the drug from the cell.
Rifampin (Rimactane) resistance can rapidly develop as a result of target site mutations. The target in this case is RNA polymerase. Following mutation of the polymerase, rifampin (Rimactane) no longer binds.
Rapid strain resistance development has become a major limiting factor in rifampin (Rimactane) use in management of susceptible staphylococci thus requiring rifampin (Rimactane) to be combined with another antistaphylococcal drug.
Multiple Antibiotic Resistance Summary
It is increasingly common for one bacterium to be resistant to several antibacterial drugs.
Mechanisms:
Acquisition of multiple, unrelated resistance genes (several steps required)
Mutation in a single gene which results in resistance to unrelated drugs. (single step)
Bacteria resistant due to acquisition of new genes:
hospital-associated gram-negative bacteria
enterococci
staphylococci
community-acquired salmonellae strains
gonococci
pneumococci.
Single gene mutations: usually affecting porins of gram-negative bacteria involve:
ß-lactams
quinolones
tetracycline
chloramphenicol (Chloromycetin)
trimethoprim (generic)
Strains resistant to all known antibacterial drugs have been identified.
Archer,G.L. and Polk, R.E. Treatment and Prophylaxis of Bacterial Infections, In Harrison's Principles of Internal Medicine 14th edition, (Isselbacher, K.J., Braunwald, E., Wilson, J.D., Martin, J.B., Fauci, A.S. and Kasper, D.L., eds) McGraw-Hill, Inc (Health Professions Division), 1998, p. 859.
Clinical indications and rationale for the use of multiple drugs at the same time (combination chemotherapy)
Single Agent Chemotherapy
Single most specific drug is preferable if the infecting bacteria has been identified.
Administration of a single drug with a narrow spectrum of action is desirable because (a) alteration of normal flora is minimized (which in turn reduces the likelihood of overgrowth of resistant nosocomial bacteria (e.g. Candida albicans, enterococci, Clostridium difficile), (b) reduces toxicity which may be associated with multiple drug regimens and (c) reduces cost.
Archer,G.L. and Polk, R.E. Treatment and Prophylaxis of Bacterial Infections, In Harrison's Principles of Internal Medicine 14th edition, (Isselbacher, K.J., Braunwald, E., Wilson, J.D., Martin, J.B., Fauci, A.S. and Kasper, D.L., eds) McGraw-Hill, Inc (Health Professions Division), 1998, p. 862.
Combination Chemotherapy
Combination chemotherapy may be warrented to:
Decrease the likelihood of emergence of resistant mutants. A single agent will be effective against sensitive organisms, but not against those that have developed a mutated "target" site, which is no longer susceptible or has diminished susceptibility to the drug. In this case the single drug will select out the mutant, resistant strain. This effect is more likely when the concentration of the antibacterial agent approximates the MIC (minimum inhibitory concentration).
Examples:
rifampin (Rimactane) : staphylococci
ciprofloxacin (Cipro): staphylococci and Pseudomonas.
imipenem: Pseudomonas
aminoglycosides: staphylococci
A second agent, which acts by a different mechanism, may prevent the emergence of the resistant strain (e.g. impenem + aminoglycoside for systemic Pseudomonas).
To take advantage of additive/synergistic action against some bacteria. Synergistic or additive activity occurs if the MIC or MBC of each agent is lowered in the presence of the other. Accordingly, each drug is more efficacious when combined with the other. (1) Certain ß-lactam-aminoglycoside combinations are effective against enterococci, viridans streptococci, and P. aeruginosa. (2) Combination of trimethoprim-sulfamethoxazole (Bactrim) is effective against many enteric gram negative bacteria. (3) Most other combinations are not clinically surperior compared to administration of the more efficacious single drug component.
Some combinations are less effective than a single agent: penicillin plus tetracycline (Achromycin) against pneumonococci.
To provide therapy when multiple pathogens may be or are known to be present . If a mixture of pathogens is thought to be present and/or the patient is critically ill, combination therapy may be warrented. [Multiple Pathogens may be present in intra-abdominal or brain abscesses and limb infection in diabetic patients; in critical illness fevers in neutropenic patients, acute aspiration pneumonia (oral flora) by hospitalized patients, septic shock or sepsis syndrome. However, monotherapy should be started if a single infecting bacterium that can be appropriately treated with a single drug has been identified
Archer,G.L. and Polk, R.E. Treatment and Prophylaxis of Bacterial Infections, In Harrison's Principles of Internal Medicine 14th edition, (Isselbacher, K.J., Braunwald, E., Wilson, J.D., Martin, J.B., Fauci, A.S. and Kasper, D.L., eds) McGraw-Hill, Inc (Health Professions Division), 1998, p. 862.

Rationale for chemoprophylaxis
Chemoprophylaxis
A manifestation of antibiotic misuse is that 30% to 50% of the time, the antibiotic is prescribed to prevent rather treat an infection.
Prophylaxis is more likely to be effective if a single, effective, nontoxic drug is used to prevent infection by a specific organism or to eliminate a recently established infection.
Examples of effective chemoprophylaxis:
Penicillin G prevents infection by group-A streptococci.
Intermittent use of trimethoprim (generic) sulfamethoxazole (Gantanol) prevents recurrent urinary tract infections
Prevention of endocarditis in patients with valvular heart lesions who are to undergo a surgical procedure.
The most extensive use of chemoprophylaxis is prevention of wound infections following surgery.
Chambers, H.F and Sande, M.A. Antimicrobial Agents in,In, Goodman and Gillman's The Pharmacologial Basis of Therapeutics,(Hardman, J.G, Limbird, L.E, Molinoff, P.B., Ruddon, R.W, and Gilman, A.G.,eds) TheMcGraw-Hill Companies, Inc.,1996, pp.1049-1050
Appropriate and Inappropriate Uses of Chemotherapy
Appropriate Use
Before the organism is identified: either combination therapy or a single broad spectrum agent may be used.
After the organism is identified, a low-toxicity regimen with a narrow-spectrum drug is indicated.
Selection of the drug should be goverened by its selectivity for the most likely involved bacteria and its toxicity.
First decide if an antibiotic is indicated since antibiotics may be toxic. Inappropriate use may hinder diagnosis, and can result in development of resistant bacterial strains
Some Clinical Issues
Optimal empirical treatment requires knowledge of the antibiotic sensitivity of the organisms which is most likely causing the infection.
Assessment with Gram's stain and other tests must be used to narrow the list of pathogens.
In life threatening situations, the selection of a single narrow-spectrum agent may not be possible and broad coverage would be indicated until a definitive identification is possible.
Chambers, H.F and Sande, M.A. Antimicrobial Agents in,In, Goodman and Gillman's The Pharmacologial Basis of Therapeutics,(Hardman, J.G, Limbird, L.E, Molinoff, P.B., Ruddon, R.W, and Gilman, A.G.,eds) TheMcGraw-Hill Companies, Inc.,1996, pp.1054-1055
Inappropriate Uses
Treatment of Untreatable Infections:
The infection is viral.
Antimicrobial treatment of measles, mumps and 90% of upper respiratory infections are inappropriate.
Treatment of fever of unknown origin:
Antimicrobials are not antipyretic agents.
Pyrexia of short duration, without localizaing signs, is most likely due to viral infection.
Three infections may be associated with prolonged fever:
tuberculosis
hidden intra-abdominal abscesses
infective endocarditis.
other causes: cancer metabolic disorders hepatitis atypical rheumatoid arthritis
Improper dosage:
Some drugs, such as the aminoglycosides, are frequently administered at subtherapeutic dosages because of concern about toxicity.
Clinical treatment failure and selection of resistant strains may result.
Inappropriate Dependence on Chemotherapy Alone:
Some disorders require both chemotherapy and a surgical procedure, especially if significant amount of necrotic tissue is present.
Example : Pneumonia in a patient with empyema (accumulation of pus) may be effectively manage following drainage
Lack of Adquate Bacteriological Information:
About one-half of antimicrobial therapy is given to hospitalized patients without support from microbiological data. [clinical judgment alone];
Antimicrobial therapy must be individualized, not administered based on prescribing habit alone.

Chambers, H.F and Sande, M.A. Antimicrobial Agents in,In, Goodman and Gillman's The Pharmacological Basis of Therapeutics,(Hardman, J.G, Limbird, L.E, Molinoff, P.B., Ruddon, R.W, and Gilman, A.G.,eds) TheMcGraw-Hill Companies, Inc.,1996, pp.1054-1055

Chemical aspects of drugs

2009-07-08T03:10:00.000-07:00

Drug shape
The shape of the drug is an important factor in defining the nature of the drug-receptor interaction. The three-dimensional shape of the drug is thought to interact with a complementary structural binding region of the receptor, typically a protein. The specific nature of the interaction defines whether the drug acts as an agonist promoting a change in cellular function or as an antagonist which blocks the receptor usually resulting in no direct biological effect.
For example, let's consider acetylcholine or a synthetic analogue bethanechol (Urecholine). Interaction of these molecules with receptor (nicotinic or muscarinic cholinergic receptor) causes a physiological response -- a decrease in heartbreak for instance. By contrast, a muscarinic antagonist such as atropine may bind even more tightly than acetylcholine to muscarinic receptor but causes no direct effect. However, following administration of antagonist a biological response may be observed as a result of receptor blockade.
A clinical example would be bradycardia following acute myocardial infarction. Bradycardia in this context might be due to excessive parasympathetic (cholinergic) tone and might cause unacceptably low cardiac output or predispose tomore serious arrhythmias. Administration of atropine, by blocking the muscarinic receptor blunts the action of acetylcholine and accordingly may reverse bradycardia.
Now let's consider the specific example,acetylcholine, as the 2D planar structure:
On the left side of the molecule note the quaternary (always positively charged) Nitrogen, which is part of the choline component of acetylcholine. The synthesis of acetylcholine proceeds by combination of choline and acetate (as Acetyl-CoA)-see below
Now let's examine the 3-D structure of choline (pressing and holding down your left mouse button allows you to rotate the choline 3-D interactive structure). By convention, nitrogen atoms are blue, oxygen red and carbon gray.
Similarly we can examine interactively the 3-D structure of acetylcholine:

Note above the presence of an "ester" linkage [O in red] between the choline moiety and the seal group.
This ester bond is susceptible to hydrolysis, i.e. breakage which may be catalyzed by esterases (acetylcholinesterase is an example).
Acetycholine:

Although acetylcholine is depicted as a "static" molecule in terms of internal rotation,, acetylcholine and many other drugs exhibit free rotation around internal bonds.
For acetylcholine,tau1, tau2, tau3, represent torsion angles and refer to the degree of twist around these bonds of free rotation
Specific additional analysis is required to determine which three-dimensional form of acetylcholine appears to be preferred for binding to the cholinergic receptor. The configuration of acetylcholine and solution is quite different than the configuration when bound to the nicotinic cholinergic receptor (using two-dimensional NMR to estimate bond angles)

Above figures adapted from Principles of Drug Action: The Basis of Pharmacology, Third Edition, edited by William . B. Pratt and Palmer Taylor, Churchill Livingston, New York, 1990. pp 20-23. Above Acetylcholine conformation figure -- original work: Behling, RW, Yamane T, Gavon G, Jelinski LW: Conformation of Acetylcholine Bound to the Nicotinic Acetylcholine Receptor. Proc Natl Acad Sci USA 85:6721, 1988.
Some short-acting pharmacological agents are in fact short-acting because they are rapidly hydrolyzed at an ester linkage.
Ester-type local anesthetics
Esmolol (Brevibloc)
Remifentanil (Ultiva).
return to Table of Contents

The biological action acetylcholine is terminated by hydrolysis, catalyzed by the enzyme acetylcholinesterase: The overall reaction is shown below --
Acetylcholinesterase itself is a large, complex protein which has its primary catalytic function the extremely rapid hydrolysis of the neurotransmitter acetylcholine.
Acetylcholinesterase
The image below illustrates the relationship between the very small molecule, acetylcholine, and its specific interaction within the very large molecule, acetylcholinesterase.
This image illustrates how the neurotransmitter acetylcholine represented above the in ball-and-stick form is recognized by specific amino acids within acetylcholinesterase's active site.
The positive charge of acetylcholine (due to the permanently positive quaternary nitrogen) interacts with tryptophan-84 (Trp-84) and phenylalanine-330 (Phe-330), through cationic (+ charged)- π-electron interactions}
This part of the acetylcholinesterase molecule is referred to as the "aromatic gorge"
The negatively charged amino acid, glutamatic acid-199 (Glu-199) is thought to interact with acetylcholine through ionic-type interactions

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2009-05-28T01:54:00.000-07:00

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The Importance of Microbiology in the Contamination Control Plan for Aseptic, Terminally Sterilized and Non-sterile Manufacturing

2009-05-02T08:08:00.000-07:00

Introduction
The development of a contamination control program is critical to the effort to get a new facility qualified, and to maintain the facility in a state of control once qualified. The design and successful execution of a contamination control program requires a plan. The creation of a specific document allows the company philosophy, goals, and expectations to be formalized and agreed to by all parties. It also provides the goals and metrics by which the state of control for the facility can be measured in the annual review. The business reasons for this are obvious in terms of reduced regulatory risk and reduction of rejected/recalled batches (Lowry 2001).
This plan is important no matter what type of facility is being developed. Although it is most frequently used in the Quality plan for commissioning an aseptic facility, this is also important and should be used for commissioning and controlling facilities using terminal sterilization, and for non-sterile manufacturing facilities.
Why be concerned with contamination control in a nonsterile manufacturing facility? In many ways contamination control is more of a concern in a non-sterile facility than in sterile product production facilities. The sterile production facility knows there is a problem with contamination and cross-contamination of batches, the non-sterile facility has a great temptation to belief they are not touched by these issues. This can lead to an extremely cavalier attitude about contamination control by the operators and management. The non-sterile manufacturer is responsible for all aspects of his product, including any objectionable organisms present (Sutton, 2006) as described in a recent newsletter (PMF Newsletter v12 n7).
The API manufacturer is also concerned with contamination control. The FDA has explicit instruction on this score (FDA 1998) out of CBER. The EMEA guidance on API manufacture also includes guidance on control of bioburden and cross-contamination of batches (EMEA 2000).
This essay will not be able to provide more than an overview of issues in the space available this month. However, it is hoped that the need for an adequate contamination control plan for a facility will be made clear, and the beginnings of the content of such a plan explained. The interested reader is referred to the articles listed in the “References” and the “Further Readings” sections.
Scope
The Contamination Control Plan should be developed as part of the facility commissioning effort. As such, there will be four distinct phases of the facility operations that will need to be addressed:
Commissioning and initial start-up
Ongoing Operations
Shut-down for regular maintenance
Start-up after scheduled shut-down.
These phases will not have the same level of contamination control. In fact, the third and fourth phases may well have different levels of control to be addressed. A good plan will discuss the concerns specific to each of these phases.
This program, and the protocol governing the program, are essential documents useful in documenting the rationale and methods used to accomplish three tasks:
Minimizing the bioburden throughout the manufacturing processes
Minimizing the level of batch residual cross-over contamination
Minimizing the level of cleaning material residual contamination
As the SME (Subject Matter Expert) in microbiology, we will be most heavily involved in the first of these three tasks, minimizing bioburden. However, all three will be discussed (at least briefly) in this essay for context.
Minimizing Bioburden

Validated methods
All measures of bioburden in a facility will be indirect. We cannot count bacterial cells on a surface or in the air. We must transfer the microorganisms to an agar plate (or some other mechanism) and count colony forming units. If we make the assumption that the transfer of microorganisms from the air or from a surface to agar is consistent, then we can use these numbers to estimate trends over time. This assumes that the nutrient agar is capable of growing the microorganisms to visible colonies. As residual disinfectant on a surface may impede the growth of microorganisms, neutralizers are frequently incorporated into the growth media (Dey-Engley agar, MCTA, etc.). All sampling methods must be validated for the conditions of use.
The facility should be disinfected regularly using validated sanitizers and sporicides. The contamination control plan should describe the methods for testing and rationale for acceptance of materials to be used in the ongoing program of disinfection. The plan should ideally describe the in vitro or laboratory tests to evaluate the sanitizers, including the identification of the most resistant microorganisms found in the facility as well as the most difficult-to-disinfect materials in the facility. This is also where the method for on-going evaluation of the sanitizers based on environmental monitoring data will be recorded. The choice of disinfection regimens should be reevaluated annually, and the contamination control plan should describe how this evaluation will occur.
Know the enemy
A successful contamination control program is geared to providing the most useful information on the microorganisms present while at the same time showing some fiscal responsibility. The FDA aseptic processing guidance document recommends genetic identification of all organisms isolated from the manufacturing environment on a regular basis. (FDA, 2004) This is a laudable goal, but few of us have anything near the required budget to accomplish this task, and in all honesty it is reasonable to wonder if it is really necessary. The numbers of CFU from validated sites (viable air and surface, non-viable) is sufficient to provide a measure of the state of control of the facility. However, periodic cataloging of the resident microflora will provide you with a good check on the continued effectiveness of the disinfectants in use. Shifts of bioburden to spore forming microorganisms will be strong evidence of the need for use of a sporicidal agent. Occasionally, this effort will also pick up shifts among non-spore-forming organisms – this is not due to “resistance” but rather ecological shifts towards species more naturally resistant to the disinfectant in use.
Control incoming bioburden
The first step in any control program is to control contamination at the very beginning of the process. This includes raw materials (excipients, API, water, etc) and the primary containers. All materials should be tested for incoming bioburden against documented acceptance criteria. Part of the incoming bioburden will also be any water used as an excipient to the process. A good guide for the water bioburden is the EMEA guidance on the subject (EMEA 2002).
Appropriate gowning
The gowning methods and materials are of critical importance to minimization of contamination. Although most attention is placed on aseptic gowning procedures, the appropriate use of gowning precautions will be a great boon to most non-sterile manufacturing facilities as well. All personnel should be well-trained in appropriate gowning practice and behavior. The contamination control plan should describe the rationale for the level of gowning chosen, the frequency of gown cleaning, behavior and the acceptable gown materials for the type of manufacturing process.
Training
Operator training is critical to contamination control. No supervisor can be present at all locations at all times. Each operator must be aware of his or her role in contamination control and how to minimize the risk to batch integrity. The PDA has published a technical report that speaks to some of these training requirements from the microbiological perspective (PDA 2001).
Controlled Environments
Control and monitoring of the environment is another critical element of the contamination control plan. Large portions of this can be addressed by the corporate Environmental Monitoring Master Plan (which provides rationale and consistency for a single EM philosophy across the different facilities of the corporation) or the site Environmental Master Plan (which provides consistency and detailed instruction for the various manufacturing buildings at a given site). However, the Contamination Control Plan should cite the relevant documents and their role in contamination control. Those interested in more on environmental monitoring should refer to the PDA’s treatment of the subject for a good overview (PDA 2001).
The appropriate Environmental Monitoring (EM) plan for non-sterile manufactures and for API manufacturers is not well-defined from a regulatory sense. There are no strong recommendations such as those seen for the environmental monitoring of aseptic facilities; however the absence of regulatory guidance is not the same thing as the absence of need for the activity. EM is useful for determining the state of control of the facility (not so much, perhaps an indicator of the finished product quality) and so is an important part of the monitoring program for all manufacturers.
Well-defined and Understood Manufacturing Processes
The manufacturing process should be evaluated for its potential to limit or eliminate bioburden. The two common methods for performing this is either a HACCP-type (Jahnke and Kuhn 2003) or a FMEA approach. The use of organic solvents, heat, or other inhospitable activities can greatly reduce bioburden of a process. The contribution of compression (and associated shear), for example, should be evaluated for a potential reduction in risk of excessive microbial contamination (Blair 1991). The contribution of the finished product water activity should also contribute to this analysis (USP 2007).
Of particular importance in this evaluation for the potential for microbial contamination of the process are cleaning steps, equipment hold times, HVAC, control level of environments for critical tasks, open-system vs closed-system operations, and bioburden monitoring (among others specific to your process). As an example of the importance of the bioburden control point issue, there is a strong regulatory expectation in Europe that products sterilized by filtration should have a pre-filtration bioburden of not more than 10 CFU/100 mL immediately before the sterilizing filter.
Finally the Contamination Control Plan should cite the need clear SOPs on all aspects of manufacturing, monitoring and control. These SOPs are critical for training, documentation and batch release.
Minimization of Batch Residual Cross-over Contamination
The contamination control plan should also address the potential for a batch to be contaminated by material from the previous batch manufactured using that equipment. Obviously, the contamination control plan should describe the methods by which this likelihood is minimized.
The concern over batch residual cross-over is most relevant when there is more than one product manufactured at a site. This concern has little to do with the sterility of the finished product, and is relevant to sterile and non-sterile manufacture alike.
Minimization of Cleaning Material Residual Contamination
Validation of cleaning procedures is essential to demonstrate not only that the cleaning procedure effectively cleans and sanitizes the manufacturing equipment, but also that residual cleaning material is removed to prevent contamination of the next batch manufactured.
Conclusions
The Contamination Control Plan is an important document designed to formalize the rationale, methods and validation of contamination control procedures in a manufacturing facility. This plan is a valuable tool for pharmaceutical, medical device and personal product manufactures and should be written to address all phases of the facilities life cycle. The Contamination Control Plan should specifically address:
Minimizing the bioburden throughout the manufacturing processes
Minimizing the level of batch residual cross-over contamination
Minimizing the level of cleaning material residual contamination
The microbiologist, as SME, has a critical role to play in the first of these three primary goals, and this essay has therefore been directed at that first topic. Minimization of bioburden in the manufacturing process occurs through (but is not limited to):
Minimizing bioburden in the process
Control incoming bioburden
Appropriate Gowning
Controlled Environments
Well-defined Standard Operating Procedures; and
Well-defined and understood manufacturing processes.

Microbial Limits Tests: The Difference Between “Absence of Objectionable Microorganisms” and “Absence of Specified Microorganisms”

2009-05-02T08:04:00.000-07:00

We have to note from the outset that USP and FDA frequently are interested in the same thing. From the vantage point of USP, there is a need to have a test for sterility, for antimicrobial efficacy, for Antibiotic/ Vitamin Potency, for Bacterial Endotoxin, for Microbial Limits etc. The need for these tests is not driven by any concern over “Good Manufacturing Process” (GMP). It is governed by the USP monographs found in the National Formulary (NF). If there is a monograph that requires a test for antimicrobial efficacy, then chapter <51> Antimicrobial Effectiveness Test” is the referee test used to demonstrate that characteristic.
FDA has similar, but separate concerns. Where the requirements are identical, the referee chapters in USP (those numbered under <1000>) are enforced. However, there are situations where the FDA’s concerns are not covered by a USP referee test method. One such situation is with the CFR requirement that medicines be “free of objectionable microorganisms.” 21CFR 211.113 under the section “Control of microbiological contamination. (a)” states “Appropriate written procedures, designed to prevent objectionable microorganisms on drug products not required to be sterile, shall be established and followed.” This is reinforced by 21 CFR 211.165 which states “Testing and release for distribution... (b) There shall be appropriate laboratory testing, as necessary, of each batch of drug product required to be free of objectionable microorganisms.”
So, here we have a problem. The USP monograph for a product (as provided in the current National Formulary) may require “Absence of Pseudomonas aeruginosa.” There is a test in the Microbial Limits chapter to demonstrate the absence of Pseudomonas aeruginosa. However, although this test may be required to demonstrate compliance with the monograph requires as laid out in NF it does not meet the FDA concern that any organism in the final product be acceptable to the product and the target population (i.e. are not “objectionable”).
The FDA Concern
FDA will enforce the GMP requirement that if your product approval to market submission contained a statement that you would test the finished product by the Microbial Limits Tests that in fact you must do that. This is purely a GMP concern. However, the Agency has been absolutely clear on the concern over objectionable microorganisms in the product, and that fact that testing to the USP chapter might be necessary, but it is not sufficient to demonstrate microbial quality. In fact, in the 1993 instructional guide for inspections of QC Microbiology Labs (1) the FDA states:
“For a variety of reasons, we have seen a number of problems associated with the microbiological contamination of topical drug products, nasal solutions and inhalation products. The USP Microbiological Attributes Chapter <1111> provides little specific guidance other than "The significance of microorganisms in nonsterile pharmaceutical products should be evaluated in terms of the use of the product, the nature of the product, and the potential hazard to the user." The USP recommends that certain categories be routinely tested for total counts and specified indicator microbial contaminants. For example natural plant, animal and some mineral products for Salmonella, oral liquids for E. Coli [sic], topicals for P. aeruginosa and S. Aureus [sic], and articles intended for rectal, urethral, or vaginal administration for yeasts and molds. A number of specific monographs also include definitive microbial limits.
As a general guide for acceptable levels and types of microbiological contamination in products, Dr. Dunnigan of the Bureau of Medicine of the FDA commented on the health hazard. In 1970, he said that topical preparations contaminated with gram negative organisms are a probable moderate to serious health hazard. Through the literature and through our investigations, it has been shown that a variety of infections have been traced to the gram negative contamination of topical products. The classical example being the Pseudomonas cepacia contamination of Povidone Iodine products reported by a hospital in Massachusetts several years ago.
Therefore, each company is expected to develop microbial specifications for their nonsterile products. Likewise, the USP Microbial Limits Chapter <61> provides methodology for selected indicator organisms, but not all objectionable organisms. For example, it is widely recognized that Pseudomonas cepacia is objectionable if found in a topical product or nasal solution in high numbers; yet, there are no test methods provided in the USP that will enable the identification of the presence of this microorganism.
A relevant example of this problem is the recall of Metaproterenol Sulfate Inhalation Solution. The USP XXII monograph requires no microbial testing for this product. The agency classified this as a Class I recall because the product was contaminated with Pseudomonas gladioli/cepacia. The health hazard evaluation commented that the risk of pulmonary infection is especially serious and potentially life-threatening to patients with chronic obstructive airway disease, cystic fibrosis, and immuno- compromised patients. Additionally, these organisms would not have been identified by testing procedures delineated in the general Microbial Limits section of the Compendia. . . .
Microbiological testing may include an identification of colonies found during the Total Aerobic Plate Count test. Again, the identification should not merely be limited to the USP indicator organisms.
The importance of identifying all isolates from either or both Total Plate Count testing and enrichment testing will depend upon the product and its intended use. Obviously, if an oral solid dosage form such as a tablet is tested, it may be acceptable to identify isolates when testing shows high levels. However, for other products such as topicals, inhalants or nasal solutions where there is a major concern for microbiological contamination, isolates from plate counts, as well as enrichment testing, should be identified.”
Why is this a concern? To understand this we have to go back to the 1970’s. USP had a test for the “Bacteriological Examination of Gelatin” as early as 1942 (2). However, most non-sterile medications in the US were not required to assay for microbiological quality attributes until the introduction of the Microbial Limits Tests in 1970 (3). In the late 1960’s several outbreaks of disease were traced back to pathogencontaminated medications, and this prompted increased attention to the microbial content of non-sterile drugs (4). Later in the 1980’s there was a series of articles appearing in the literature describing contamination by P. cepacia (currently Burkholderia cepacia) (5, 6) and its survival in disinfectants(7 – 11). This lead to the addition of requirements in the 21 CFR to ensure that there are not objectionable organisms in product released to market (see above). Add to this the knowledge that the USP “Absence of Pseudomonas aeruginosa” assay will not identify presence of B. cepacia (as discussed).
The USP Concern
The USP is on record as early as 1982 verifying that the demonstration of “absence of objectionable microorganisms” is not the intent of the chapter. In a one page Stimuli to the Revision Process the microbiology committee of the time states:
“The tests described in the Microbial Limits Tests <61> were not designed to be all-inclusive, i.e., to detect all potential pathogens. To accomplish this, an extensive text on laboratory detection of microorganisms would be required. The procedures in USP were designed to detect the presence of specific “index” or “indicator” organisms. Nevertheless, the present chapter does not preclude the detection of Ps. Cepacia – the organism requires subsequent differentiation. The chapter does not provide specific methods for this, nor does it provide procedures for detecting thousands of other potentially pathogenic organisms. Individual monographs include requirements for limits on total aerobic counts and/or absence of one or more of the four selected “indicator” organisms. The chapter on Microbial Limits Tests provides methods to assure that one may test for those microbial requirements in the individual monographs...
In conclusion, the Microbial Attributes and Microbial Limit Tests chapters accomplish their intent. If a manufacturer needs particular tests for any specific organisms that are potential problems in a process or a final product, the quality control microbiologist can provide specific detection procedures. Many such procedures are published in several laboratory texts on microbiology.”
Conclusions
On the question of the microbial quality of non-sterile pharmaceuticals, the USP and the FDA are in agreement – the product must be safe for use. The NF monograph requirements for absence of specific organisms is a minimal requirement, and should not be taken as proof that the product is suitable for sale from a microbiological perspective.
The manufacturer is responsible for the quality and safety of the product marketed, and it is the clear expectation of FDA (as described in CFR) that this will include a determination of the microbial safety – i.e. the “absence of objectionable microorganisms” from the product. These positions have been publicly stated for decades and should not come as a surprise to anyone. The harmonized microbial limits tests only address the “absence of specified microorganisms” and leave the determination of the “absence of objectionable microorganisms” in the capable hands of each company’s appropriately educated and well-trained microbiology group.

The Harmonization of the Microbial Limits Test - Absence of Specified Organisms

2009-05-02T08:00:00.000-07:00

The last issue of the PMF Newsletter (vol 12, no. 3) contained a review of the harmonization status of the Microbial Limits Tests – Enumeration. In addition, the article provided a brief overview of the compendial harmonization process as agreed to by the Pharmacopeial Discussion Group (PDG). In this article, I would like to focus on the other side of the Microbial Limits Tests – the “Absence of Specified Microorganisms” component.
USP
EP
<61> Microbiological Examination Of Nonsterile Products: Microbial Enumeration Tests
2.6.12 Microbiological Examination Of Nonsterile Products: Microbial Enumeration Tests
<62> Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms
2.6.13 Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms
<1111> Microbiological Quality of Nonsterile Pharmaceutical Products
5.1.4 Microbiological Quality of Nonsterile Pharmaceutical Products
Table 1: Harmonized Chapter Numbering Scheme
There is a significant controversy in the United States over the intent of this evaluation. The FDA is bound by the concern expressed in the Code of Federal Regulations (21CFR 211.113 and 21CFR 211.165) relating to the importance of “objectionable microorganisms.” This is not the concern of the compendial chapters. The controversy is worthy of discussion, but not the topic of this review – it will be discussed in length in the final of three articles on the harmonization of the microbial limits tests to be published in next month’s newsletter.
What follows is a tabular presentation of the existing “Microbial Limits – Absence of Specified Microorganisms” tests from the current USP and Pharm Eur, as well as the draft harmonized document (the finalized document is extremely close to this version, but not release to the industry). It is provided as an aid to evaluation, and may assist in determining whether revalidation of method suitability studies is needed. It should be noted that this harmonization draft represents a true compromise by all parties, with (at least in the author’s opinion) significant changes from the current USP, Pharm Eur and JP chapters.

The Harmonization of the Microbial Limits Test - Enumeration

2009-05-02T07:55:00.000-07:00

The USP and the European Pharmacopoeia (EP, Pharm Eur) Microbial Limits Tests are in the final stages of harmonization. They were signed off to Stage 6A at the November, 2005 meeting of the Pharmacopeial Discussion Group (PDG) held in Chicago, IL USA (USP 2006a). However, the signed-off versions have yet to be published. This makes the description of the test a bit difficult, as the current tests will be disappearing, and the final, harmonized test is not yet public knowledge. However, we do know that the harmonized tests do not differ greatly from the drafts published in 2003 (USP 2003a, USP 2003b, USP 2003c), and so we will use those drafts as the description of the finalized test.
The Microbial Limits Tests are actually two chapters in the current USP: Current USP <61> Microbial Limits Tests (USP 2006b) and <1111> Microbiological Attributes of Nonsterile Pharmaceutical Products (USP 2006c). This will be modified in the harmonized version to mirror the European format:
USP
EP
<61> Microbiological Examination Of Nonsterile Products: Microbial Enumeration Tests
2.6.12 Microbiological Examination Of Nonsterile Products: Microbial Enumeration Tests
<62> Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms
2.6.13 Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms
<1111> Microbiological Quality of Nonsterile Pharmaceutical Products
5.1.4 Microbiological Quality of Nonsterile Pharmaceutical Products
Table 1: Harmonized Chapter Numbering Scheme
This review will only address the microbial enumeration portions of the harmonization effort – that which will become USP chapter <61> and Pharm. Eur. chapter 2.6.12.
The microbial enumeration test is a basic, simple design to count the number of CFU in a nonsterile product or raw material. The preferred method is to put the material into solution and then plate aliquots to determine the CFU/gram (or mL) of initial material. If the product cannot be put into solution, there are provisions to use the Most Probable Number method (MPN – see FDA BAM website). The method of plating can be either pour plate, spread plate or the filtration of material and then placing the membrane filter on the surface of an agar plate. The membrane filtration method should only be used when there are few expected colony forming units in the material to be tested as it is a good method to test a large volume of liquid, but can only count up to approximately 100 CFU/membrane.
The harmonized method provides a great deal more detail than any of the current pharmacopeial methods in terms of demonstration of method suitability (validation of the method) and in terms of media growth promotion.
The demonstration of method suitability should be performed using the challenge organisms listed (see Table 2 below) in accordance with the recommendations found in USP chapter <1227> (USP 2006d). Growth promotion is an area of some ambiguity in the compendial text. Although media growth promotion is not described in the tests, demonstration of media suitability is required, and the draft USP Chapter <1117> (USP 2004) provides assistance in designing the studies using 10-100 CFU of the challenge organisms.
A major concern of many QC workers is if the changes in the harmonized chapter will necessitate revalidation of existing assays to meet the requirements of the harmonized test. There are several considerations that might lead to revalidation – a required change in media, in volume of material required for testing, in general testing conditions. It is difficult to determine whether all product types would require revalidation, and so a summary table is provided (Table 2) describing the critical aspects of the current Microbial Limits Tests (Enumeration) and the draft harmonization text. The summaries provided in Table 2 are only meant as an aid, the decision as to whether or not revalidation is necessary rests with each individual facility for their particular products.

Streaking for Single Colonies: An Essential First Step in Microbial Identification

2009-05-02T07:45:00.000-07:00

From the outset, let’s admit that the current state of microbial identification is a little confusing. We can ID by traditional biochemical tests (API Strips) or by elegant refinements of the traditional methods (for example, the Vitek 2 Compact). We can identify microorganisms by carbohydrate utilization (Biolog systems) or by the GC pattern of the cell’s fatty acids (Sherlock System). If you want to go genotypic, then you currently have a choice between the Dupont RiboPrinter or Applied Biosystems MicroSeq systems. Your identification (genus and species) may well depend on which system you use as there is no objective standard, and each system is reliant on its proprietary database to assign an identification to the data.
Virtually all of these systems require a preliminary Gram stain to accurately identify the sample. The Gram stain requires a relatively fresh culture for best results (PMF Newsletter, Feb. 2006). This is the first reason to restreak for single colonies after isolation of a contaminant. However, each system also is dependent on the presentation of a monoclonal sample for accurate results. In fact, only one of these systems provides you with enough information to recognize if you have a contaminated (polyclonal) sample (no, I am not going to tell which one it is).
The basic fact is that acceptable microbiological practice (I am not even going to say “good lab practice” or “best lab practice” but “acceptable” or, if you prefer, “adequate”) requires streaking for well isolated, single colonies of good health for identification purposes. This is not difficult.
The best starting material is a relatively “clean looking” colony on your primary plate. This colony should not show obvious signs of being multiple pinprick colonies that merged into a single CFU. Using a sterile loop, sample from the center of the colony and begin a heavy streak onto a new plate of appropriate agar media. This is quadrant #1 (see accompanying figure).
Streaking in quadrant #1 (and all subsequent streaking events) should be in the same direction, with the same part of the loop in contact with the agar. After the completion of the streaking in this quadrant, the loop should be resterilized (thoroughly flamed or discarded for a new, sterile disposable loop) and the plate streaked into quadrant #2 by drawing the fresh loop across two or three lines in quadrant #1. This should be done once or twice, then subsequent streaks performed without touching any of the previous line in the agar surface. The loop is resterilized, and the process is repeated in quadrants #3 and #4, each time the loop becoming contaminated by drawing it across a few lines in the previous quadrant. The idea is a successive dilution of the level of CFU in each quadrant, on each successive line after the initial inoculation in the prior quadrant. The plate is then incubated overnight for colony growth. Single, well-isolated colonies should be evident in quadrant #4 or quadrant #3. Care should be taken not to accidentally contaminate the colony when harvesting.
One note of caution. This streaking for single colony isolates should be conducted a second time if the original plate was heavily contaminated, or if there are multiple colony morphologies evident on this initial streaked plate. The integrity of the microbial identification process requires a monoclonal colony (a colony that is from a single bacterial strain).
Remember – the only assurance you have of a correct identification is proper preparation of the monoclonal sample. To this concern, the final isolation plate should never be used as a storage device – the single well-isolated colony chosen should be restreaked on a separate plate or agar-slant tube for storage.
This seems like a lot of work, and requires an additional day (at least) to the turn-around time for identification of a contaminant when compared to the time required if single-colony isolation is omitted from the process. However, if microbial identification is attempted directly from the colony on the primary test plate (environmental monitoring or bioburden plate) any resultant microbial identification must be suspect as there is no assurance that you are working with a pure culture. When auditing your microbiology lab (or your contract lab), check to see if an SOP is in place requiring this step, and also check the refrigerators and incubators to see if you can find evidence that this is, in fact, occurring. It is an unfortunately common practice to omit this essential step in microbial identification in an ill-advised attempt to save time and money. However, as there are few quality controls possible on the microbial identification process, you have to build the quality into the process to avoid the GIGO phenomenon.
We are in a period of high regulatory interest in environmental monitoring identifications (as part of aseptic production controls), and in the demonstration of absence of objectionable microorganisms in nonsterile finished drug products. This is not the time (if there ever was one) to save a few pennies by omitting a step necessary to the accurate and confident identification of a microbial colony.

Counting Colonies

2009-05-02T07:41:00.000-07:00

Who Cares?
What is the fuss about in determining the number of colony forming units? After all, the CFU is only an estimate of the number of cells present. It is a skewed estimate at best as the only cells able to form colonies are those that can grow under the conditions of the test (incubation media, temperature, time, oxygen conditions, etc). Even among that group of microorganisms a colony does not represent a single cell, but rather cells that happened to be well separated on the plate and so can be distinguished after growth. A colony could arise from one cell, or several thousand. So why the fuss?
One reason for concern is that microbiology has a well-deserved reputation for being highly variable. Our lax attention to precision and accuracy in our measurements helps further this perception. We have allowed specifications for environmental monitoring, raw material bioburden, in-process bioburden and finished product bioburden to be imposed by regulation without regard for the ability of the method to support those specifications.
A second reason for concern is that now we are trying to introduce alternate microbiological methods into the lab. Being obsessive by training, we are trying to exceed measures of accuracy and precision in this exercise that the traditional methods cannot come close to matching. A good example of this is the Pharm Eur “Precision” requirement for an alternate method (quantification) to have a Relative Standard Deviation (RSD) in the range of 10-15% (1). While you might get lucky and hit this with dilutions whose plate counts are in the 150-250 CFU/plate range, - at lower plate counts this target value imposed by regulation will virtually guarantee a long, difficult and quite possibly unsuccessful, validation exercise.
Countable Range on a Plate
Literature
The general ranges in common acceptance for countable numbers of colonies on a plate are 30 – 300 and 25 – 250. The origin of those ranges is worth examination. Breed and Dotterrer published a seminal paper on this topic in 1916 (2). They set out to determine the “limit in the number of colonies that may be allowed to grow on a plate without introducing serious errors…in connection with the proposed revisions of standard methods of milk analysis.” They note that “the kind of bacteria in the material under examination will have an influence on the size of the colonies, and consequently, on the number that can develop on a plate.” They also note that food supply can be an issue, that colonies close to each other on the plate may merge, and that neighbor colonies may inhibit growth or conversely stimulate growth. “Because of these and other difficulties certain plates in any series made from a given sample are more satisfactory for use in computing a total than are others. The matter of selecting plates to be used in computing a count becomes therefore a matter requiring considerable judgment.”
Breed and Dotterrer chose their countable plates from triplicate platings of each dilution, requiring acceptable plates to be within 20% of the average. On this analysis, plates with more than 400 CFU were unsatisfactory, as were those of less than 30 CFU, with best results in the range of 50-200 CFU/plate.
The major paper from Tomasiewicz et al (3) provides an excellent review of the continued evolution of the appropriate number of CFU per plate from milk. They took data from colony counts of raw milk from three different experiments (each dilution plated in triplicate) and used to determine a mean-squared-error of the estimate for all plates. Their recommendation at the end of the study was for a countable range of 25-250 CFU/plate in triplicate. It is interesting to note that although the authors note that CFU follow a Poisson distribution, no mention is made of any data transformation used to approximate a normal distribution prior to the use of normal statistical analytical tools. Tomasiewicz et al provide excellent cautionary advice:
“The data presented herein are not necessarily applicable to other systems. For automated equipment, the optimum range may well vary with the instrument…Furthermore, even if automation is not used appropriate numbers of colonies that should be on a countable plate can very widely, depending on many other variables. With soil fungi for example…”
The compendia have recently harmonized a microbial enumeration test (4), and in this test recommend that the technician “Select the plates corresponding to a given dilution and showing the highest number of colonies less than 250 for TAMC and 50 for TYMC.” In determination of the resistance of biological indicators, USP recommends a range of “20 to 300 colonies, but not less than 6” (5). However, the most complete description of the countable range is found in the informational chapter <1227> (6):
“The accepted range for countable colonies on a standard agar plate is between 25 and 250 for most bacteria and Candida albicans. This range was established in the food industry for counting coliform bacteria in milk. The range is acceptable for compendial organisms, except for fungi. It is not optimal for counting all environmental monitoring isolates. The recommended range for Aspergillus niger is between 8 to 80 cfu per plate. The use of membrane filtration to recover challenge organisms, or the use of environmental isolates as challenge organisms in the antimicrobial effectiveness testing, requires validation of the countable range.”
ASTM provides countable ranges of 20-80 CFU/membrane, 20-200 for spread plates and 30-300 for pour plates (7). The FDA Bacterial Analytical Manual (BAM) recommends 25-250 CFU/plate as a countable range (8).
Upper Limit
The upper limit of plate counts is dependent on a number of factors, as described previously. The major issues include the colony size and behavior (swarming?), and the surface area of the plate. The size particularly comes into play with plating a membrane for determination of CFU as the surface area of that membrane is so much smaller than that of a standard plate.
TNTC can be reported out several ways. ASTM (7) recommends reporting this out as >”upper limit”. For example, a 1:10 dilution with more than 200 CFU on a spread plate would be reported as “>2,000 CFU/mL (or gram). FDA’s BAM recommends counting the colonies from the dilution with plates giving counts closest to 250, counting a portion of the plate, estimating the total number and then using that number as the Estimated Aerobic Count. It is not clear to the author how this is greatly superior to guessing. In my opinion this is an invalid plating and needs to be done correctly at a later date (note I am strenuously avoiding the use of the word retest. This result invalidates the plating and therefore the test was not performed correctly.) I know this is a hardship to the lab, who were trying to reduce the plating load initially by not plating out sufficient dilutions. However, making a mistake initially is not a reasonable excuse to avoid doing it correctly after the mistake is recognized. If the lab wishes to use this “estimated count” it should, at a minimum, have it clearly described in their “counting CFU” SOP with a rationale as to when the plate counts are not critical and can be estimated in this fashion.
There are methods available if you should want to accurately determine the upper limit for a unique plating surface or a unique colony type. One is presented in the USP informational chapter <1227> (5) which is based on a pair-wise comparison of counts from a dilution series. This is based on the assumption that at the upper limit the observed numbers of CFU will fall off the expected numbers at some point (see Figure 1). This divergence will become significant at some point – that defines the upper limit of CFU/plate.
Figure 1. Difference between Expected and Observed CFU with Increasing Numbers

Lower Limit
A central concern in this determination is the reporting of the Limit of Quantification (which is what we are really interested in reporting) against the Limit of Detection (1 CFU). This is an important distinction if we are being held to specifications in the lower range.
ASTM recommendations focus on the LOD, and urge the user to report that answer out if no colonies are recovered (ie <10 CFU/mL for a 1:10 dilution) (7). If countable colonies are present, but below the countable range, count them anyway and report an estimated count.
USP (6) does not have a specific recommendation on how to report out these low numbers, but does note “Lower counting thresholds for the greatest dilution plating in series must be justified.”
FDA BAM (8) recommends a different reporting format. In the FDA BAM method, all counts are recorded in the raw data, but the information is reported out as ). This leads to graphs such as in Figure 2 which shows us that as the CFU/plate drops below the countable range, the error as a percent of the mean increases rapidly. This confusion between the Limit of Detection and the Limit of Quantification for plate counts has led to some very difficult situations (as discussed below).
Figure 2. Increase in Error with Decreasing Numbers
Unusual Situations
What About Two Dilutions with Countable Colonies?
Ideally you would never see two separate dilutions with counts in the countable range, as the countable ranges cover a ten-fold range of CFU. However, this is microbiology. ASTM recommendations (7) urge you to take both dilutions into account, determining the CFU/mL (or gram) separately for each, then averaging the results for the final result. Breed and Dotterrer (2) also used several dilutions if the numbers fit the QC requirements (see below). FDA BAM has no recommendations in this situation.
While the argument can be made to use all counts, this is a stronger argument if triplicate plates are used and QC limits are in place to discard erroneous plates.
A strong argument can also be made to take the dilution providing the larger number of CFU in the countable range. This approach minimizes two concerns, that the errors in the estimates increase with increasing serial dilutions, and that the error in the estimate increases with decreasing plate counts. Use of the smaller dilution (eg 1:10 vs 1:100) could be justified from this perspective.
Whichever method used should be documented and justified in the “Counting CFU” SOP.
What about QC Limits on Replicate Plate Counts?
Periodically there are recommendations to establish Quality Control limits on replicate plate counts. Breed and Dotterrer in their 1916 paper (2) required valid plate counts from triplicate plates to provide estimates of CFU/mL within 20% of the mean. In other words, all plates were counted, each plate’s CFU count was used to estimate the original CFU/mL, then each estimate was evaluated. If the individual plate’s estimate was within 20% of the mean, it was deemed acceptable. This method is not practical in the QC lab.
Establishment of QC limits for plate counts works best if you have at least three replicate plates for each dilution. The average of the dilution replicates can be determined, variant counts (hopefully no more than one plate per triplicate plating) discarded and the final average determined. If you try this with duplicate plates you frequently end up with trying to average the results of one plate. While this method looks good on paper, the prudent lab manager will evaluate some historical data before instituting it as a practice.
The method used to QC individual plate counts, if used, should be documented and justified in SOP, along with the response to finding variant counts.
Can I plate 10 1 mL samples to plate a Total of One 10 mL Sample?
There have been suggestions that a larger volume of material may be plated across several plates, and the results reported out for the larger volume. For example, plating 10 1 mL samples on 10 different plates, and then reporting it as if a 10 mL sample was plated. This approach is flawed in that it ignores several sources of variability in plating including sampling error, plating errors, growth/incubation error and counting errors (9, 10). The correct interpretation for this situation that you have just plated 1 mL ten times, not 10 mL once. The numbers might be averaged, they cannot be added.
Rounding and Averaging
To discuss this we need to determine what the significant figures might be in the measure. For raw colony counts, common practice determines that the CFU observed determine the significant figure, and that the average is one decimal to the right of that number (sticklers for accuracy will report the geometric mean rather than the arithmetic mean given the Poisson distribution followed by CFU). In reporting, it is common practice to report out as scientific notation using two significant figures. This requires rounding.
USP (11) and ASTM (7) both round up at five if 5 is the number to the right of the last significant figure. FDA BAM has a more elaborate scheme, rounding up if the number is 6 or higher, down if 4 or lower. If the number is 5, BAM looks to the next number to the right and rounds up if it is odd, down if it is even.
This is one of those situations where you want every-one to do the calculations the same way (I am hard pressed to come up with a situation in a lab where you want everyone to do it differently). Be sure to include direction and its justification in the “Counting CFU” SOP if it does not already exist in a separate SOP.
Impact on Specifications and Environmental Monitoring Control Levels
We are back to the question of WHO CARES?
If you are faced with a finished product bioburden of NMT (Not More Than) 100 CFU/gram, and your method suitability study requires a 1:100 dilution of the product to overcome any antimicrobial effects, then how are you to test it? Common practice is to perform the 1:100 dilution, perform a pour plate of 1 mL in duplicate and if 2 colonies grow on each plate, the product fails specification. This common practice is scientifically unsupportable – it confused the Limit of Detection with the Limit of Quantification for the plate count method.
Let’s take a look at environmental monitoring alert and action levels for aseptically produced products. Hussong and Madsen (12) recently published a thoughtful review of this topic where they argue that the levels of acceptable CFU for many room classifications are below the noise level of plate count technology (eg in the range of 1-2 CFU/m3). In addition, environmental data can be extremely variable, much more so than controlled lab studies as the numbers of microorganisms, the physiological state of the isolates, even the species are completely out of the control of the investigator. In addition the numbers do not conform to a normal distribution as there are sporadic counts with a count of “zero” CFU predominating. They conclude that since the numbers are unreliable, the trend in the data is the only important consideration, and that EM counts cannot be used for product release criteria. A separate treatment of this subject was presented by Farrington (13) who argues that the relationship between EM data and finished product quality is a widely held, but unproven belief, compounded by the problems in accuracy with the low counts generated by plate count methodology.
Conclusions
In conclusion, all methods have limitations. One of the major limitations to the plate count method is the relatively narrow countable range (generally considered to be 25-250 CFU bacteria on a standard petri dish). The currently prevailing confusion between the Limit of Detection (1 CFU) and Limit of Quantification (25 CFU) for the plate count method creates a larger degree of variability in microbiology data than is necessary. An unfortunate regulatory trend in recent years is to establish expectations (specifications, limits, levels) for data generated by the plate count method that the accuracy of the method cannot support. This is a real opportunity for modification of current practice to approach the goal of “science-based regulations”.

How to Determine if an Organism is “Objectionable

2009-05-02T07:34:00.000-07:00

since the microbial limits tests do not address themselves to “objectionable” microorganisms, how is the manufacturer to determine if there are “objectionables” in a lot of product awaiting release? One approach is suggested by FDA - once all organisms grown in the total count studies (total aerobic as well as total yeast and mold) are identified, a qualified microbiologist would conduct a risk analysis on the presence of that organism in that medication (4). This risk analysis should incorporate a minimum of four separate analyses: Absolute numbers of organisms seen Microorganism’s Characteristics Product Characteristics Potential Impact on Patients
Absolute Number of Organisms Seen
Although high numbers of non-pathogenic organisms may not pose a health hazard, they may affect product efficacy and/or physical /chemical stability. An unusually high number of organisms seen in the product may also indicate a problem during the manufacturing process, or an issue with a raw material. The high bacterial counts may indicate that the microorganisms are thriving in the product. If a preserved product this could indicate that the product’s preservative system is not functioning or worse, the preservative was missing or incorrectly formulated.
The Characteristics of the Microorganism
The characteristics of the microorganism can be determined by a search of textbooks, or library work, by internet searches, or a combination of all of these. It is always a good idea to remember that you are interested in the microbiology of the situation – do not restrict the search to pharmaceutical sources as most of the best information will come from food, environmental, clinical and perhaps cosmetic microbiology sources in addition to the pharmaceutical field.
During this search look for synonyms of the organisms current name. With the widespread use of genetic techniques in taxonomy the names of some organisms have undergone multiple changes. The national culture collections are a good source of synonyms and all name variants should be researched.
First of all, determine if the organism is a known pathogen. A good place to start on this search is the FDA web site the “Bad Bug Book.” This is only a guide, but a good one (5). One approach is to do a preliminary evaluation for any organism that appears on the FDA/CFSAN list and immediately classify that organism as “objectionable.” However, it is also important to consider the route of administration and the susceptible population in this evaluation.
A second characteristic of the microorganism that must be taken into account is the potential for the organism to cause spoilage of the product. Make a list of substances used by the microorganism for growth. This can be from the literature, or from the identification equipment. For example, the Vitek 2 Compact will provide an extensive list of compounds the microorganism can metabolize, the Biolog a list of carbohydrates utilized, etc. Use the information gained during the identification of the organism. Compare these to the product formulation for potential issues. A microorganism is also objectionable if it has the potential to degrade the product on stability. Evaluate the microorganism’s tolerance to unusual conditions: low or high pH high salt concentration high sugar concentration (osmotic conditions) Low water activity Growth temperature, etc. It can also be useful to determine if the microorganism has a recognized proclivity for harboring plasmid-mediated antibiotic resistance. This is a special concern in regards to horizontal transmission of the trait within an vulnerable patient population.
Product Characteristics
The dosage form is important to consider. Is the product anhydrous or water based? This can have an effect on the ability of microorganisms to proliferate. Does it have sufficient free water to support microbial growth (6, 7). Is the container designed to minimize contamination and subsequent spoilage? Closure design can have a major effect on in-use stability of a product. Is the container adequately designed to retard access to the environment, and to prevent contamination from the environment. Give special consideration to the likelihood of an anhydrous medication’s exposure to water, providing the potential for microbial proliferation. The route of administration is also important. A medication orally administered can tolerate some microorganisms that would be disastrous in a medication meant to be applied topically to abraded skin or to rashes. Similarly, some microorganisms that could be tolerated in a topical would cause severe distress to a patient if taken orally. Inhalants, although not required to be sterile, are a particularly sensitive area and great care should be taken in classifying any contaminate as “non-objectionable.” Other product-related considerations should include a review of the production records and the environmental monitoring trends, A review of field complaints is also useful (is this contaminant one that causes eventual returns?).
Patient Population
Finally, a consideration of the targeted patient population is in order. The manufacturer cannot control, and should be held accountable, for patient abuse of a product or off-label use of the product by physicians. However, reasonable use of the product should be considered and part of the risk analysis. Are patient populations that are likely to use this product at increased risk if exposed to the particular microorganism?
Summary
The FDA’s concern with non-sterile dosage format is that the product not contain “objectionable” organisms. This FDA concern has been made clear since the 1970’s. However, many companies continue to mistakenly believe that if their non-sterile product meets the requirements in USP, it will be safe from FDA dispute. This not the case. The manufacturer is responsible for all contents of his drug product. Should question arise over the appropriateness of a particular organism, the manufacturer is expected to have a justification for the presence of that organism, preferably as part of the batch release document. Presented here is a brief description of some factors to consider in determining if an organism is objectionable. These considerations include: Absolute number of organisms present Microorganism characteristics Product characteristics Patient Population These are not meant to be a comprehensive listing of all issues, but rather a starting point for the non-sterile manufacturer to use in establishing their program to qualify finished product bioburden

Measurement of Cell Concentration in Suspension by Optical Density

2009-05-02T07:29:00.000-07:00

A common issue for the microbiology lab is the determination of starting inoculum concentration. If the inoculum concentration is determined by plating, the inoculum is several days old before use. This essay describes the use of turbidity to estimate microbial concentration in a suspension, using the Antimicrobial Efficacy Test as the example.
Determination of Inoculum for the AET
The compendial antimicrobial efficacy test (AET) requires inoculation of the product with microorganisms to a final concentration of approximately 106 CFU/mL. Although this seems to be a minor point, it does serve to illustrate some of the inherent difficulties in microbiological testing and the need for experienced and academically trained microbiologists to head the laboratory.
Let’s look at the compendial guidance. The Pharm Eur (1) instruction on preparing the inoculum for the AET states:
“To harvest the … cultures, use a sterile suspending fluid … Add sufficient suspending fluid to reduce the microbial count to about 108 micro-organisms per milliliter…Remove immediately a suitable sample from each suspension and determine the number of colony-forming units per milliliter in each suspension by plate count or membrane filtration (2.6.12). This value serves to determine the inoculum and the baseline to use in the test. The suspensions shall be used immediately.”
There are, of course, two problems with these instructions. The first is that the technician is instructed to use an inoculum of about 108 microorganisms per milliliter and then instructed to determine this by plate count. Colony forming units (CFU) and cells are two different measures and this will inevitably lead to difficulties as the unfortunate lab worker cannot guarantee the number of cells in the suspension, only the number of CFU found. However, we can accept the scientific inaccuracy as the numbers will generally work out. The more serious problem is the instruction to use the plate count CFU for determination of the inoculum for the test, and that the suspension shall be used immediately. This quite frankly cannot be done. If you use the suspension immediately, the plate counts are unavailable, if you use the plate counts to set the inoculum, then the suspension is at least a day old.
Contrast these instructions with those in the USP (2) for the same exercise:
“To harvest the … cultures, use sterile saline … Add sufficient … to obtain a microbial count of about 1 x 108 cfu per mL…[Note: The estimate of inoculum concentration may be performed by turbidimetric measurements for the challenge organisms. Refrigerate the suspension if it is not used within 2 hours].
Determine the number of cfu per mL in each suspension …to confirm the initial cfu per mL estimate. This value serves to calibrate the size of the inoculum used in the test.”
These USP instructions have the advantage of being physically possible to perform, an obvious advantage to the lab worker. However, the turbidometric measure of the cells is also only an approximation of CFU. Thus the instruction to confirm the numbers (after the test is underway) with the plate count is an important control on the test.
This article will explore the turbidometric approximation for cell numbers, and important controls on the process as well as potential pitfalls to the method.
Theory
Light scattering techniques to monitor the concentration of pure cultures have the enormous advantages of being rapid and nondestructive. However, they do not measure cell numbers nor do they measure CFU. Light scattering is most closely related to the dry weight of the cells (3).
Light is passed through the suspension of microorganisms, and all light that is not absorbed is re-radiated. There is a significant amount of physics involved in this, and those interested are referred to optical treatises, particularly those discussing Huygens’ Principle (a good choice is Light Scattering by Small Particles by H C Van De Hulst). For our purposes it is enough to say that light passing through a suspension of microorganisms is scattered, and the amount of scatter is an indication of the biomass present in the suspension. In visible light, this appears “milky” or “cloudy” to the eye (3). It follows from this that if the concentration of scattering particles becomes high, then multiple scattering events become possible.
Methods
McFarland Turbidity Standards
McFarland standards can be used to visually approximate the concentration of cells in a suspension. The McFarland Scale represents specific concentrations of CFU/mL and is designed to be used for estimating concentrations of gram negative bacteria such as E. coli. Note that this estimate becomes uncertain with organisms outside the normal usage as different species of bacteria differ in size and mass, as do yeast and mold. Use of this method would require calibration and validation.
McFarland Standards are generally labeled 0.5 through 10 and filled with suspensions of Barium salts. (Note - latex bead suspensions are now also available which extend the shelf life of the material). The standards may be made in the lab by preparing a 1% solution of anhydrous BaCl2 and a 1% solution of H2SO4 – mix them in the proportions listed in the table. They should be stored in the dark, in a tightly sealed container at 20-25oC and should be stable for approximately 6 months (4).
The advantage of the use of these standards is that no incubation time or equipment is needed to estimate bacterial numbers. The disadvantage is that there is some subjectivity involved in interpreting the turbidity, and that the numbers are valid only for those microorganisms similar to E. coli. In addition, the values are not in the appropriate range for the AET inoculum and so further dilutions may be required.
Approximate E. coli concentrations on McFarland Scale
McFarland Scale
CFU (x106/mL)
1% BaCl2/ 1% H2SO4 (mL)
0.5
<300
0.05/9.95
1
300
0.1/9.9
2
600
0.2/9.8
3
900
0.3/9.7
4
1200
0.4/9.6
5
1500
0.5/9.5
6
1800
0.6/9.4
7
2100
0.7/9.3
8
2400
0.8/9.2
9
2700
0.9/9.1
10
3000
1.0/9.0

Spectrophotometer
The spectrophotometer method measures turbidity directly. The best case (i.e. most sensitive) would be to have a narrow slit and a small detector so that only the light scattered in the forward direction would be seen by the detector. This instrument would give larger apparent absorption readings than other instruments.
As should be obvious, each spectrophotometer used must be independently calibrated for use in estimating microbial concentrations. Not only is the apparent absorption affected by the width of the instrument’s slit, the condition of the filter, and the size and condition of the detector, but also each time the lamp is changed the calibration needs to be repeated as different bulbs may vary in total output.
The correlation of absorption to dry weight is very good for dilute suspensions of bacteria (5), and this relationship seems to hold regardless of cell size (although the relationship of absorption to CFU does not). However, in more concentrated suspensions this correlation (absorption to dry weight) no longer holds. The linear range of absorption to estimated CFU is of limited scope and for this reason the calibration study must demonstrate the linear range of the absorbance vs CFU values and the relevant values.
Procedure
As there are a variety of different instruments, there cannot be one single procedure. In general, the spectrophotometer can be set at a wavelength of 420 – 660 nm. This wavelength must be standardized and may need to be adjusted specifically to the material being tested. Different vegetative cells, bacterial spores and spores of Aspergillus niger may not have the same maximal absorbance wavelength.
It is important to have the cells in known physiological state of growth. That is to say, as the cell size varies with phase of growth (lag, log, stationery) the approximate relationship between absorbance and CFU will also vary. A recommended practice might be to pass a single well-isolated colony twice on overnight cultures surface streaks from the refrigerated stock, harvesting the rapidly growing culture from the second passage for preparation of vegetative cells. This also will serve to minimize a source of variability for the AET (6).
A second source of concern might be the cuvette used for the measurement – care must be taken to maintain the correct orientation of the cuvette, and to protect it from damage that could affect the passage of light. Finally, it is necessary to blank the spectrophotometer (adjust the absorbance reading to zero) using a standard, either water or the suspending fluid, and maintain this practice.
Calibration
It must be stressed that this calibration should be done for all organisms. The size of the organism, any associated pigments, the preparation of the suspension, and other factors all influence the readings. This calibration study should also be rechecked after changing the bulb on the light source, and should be reevaluated throughout the life of the light bulb.
The calibration itself is simple to perform. Prepare a concentrated solution of the organism, grown under the conditions that will be used for the test. Make a series of dilutions to cover the range of absorption measurements of interest; 5 to 8 dilutions are recommended. Immediately take the spectrophotometer readings in sequence, and then take a confirmatory reading of the first in series to confirm that no growth has occurred. The dilutions are then immediately plated for viable count (serial dilution of the suspensions will be necessary). Graph the relationship between the absorbance and the CFU/mL after the plate counts are available and use values in the linear range of this graph.
As there are several factors that can affect this curve (quality of lamp output, size of slit, condition of filter, condition of detector, microorganism characteristic, etc) this calibration should be confirmed when the conditions of the assay change.
Conclusions
The use of optical density to estimate CFU in a suspension is possible, if basic precautions are taken. It is important to control:
The physiological state of the organism
The species of the organisms (i.e. don’t calibrate the instrument using E. coli and expect the numbers to work for Candida albicans)
The nature and condition of the equipment
Despite the inherent inaccuracy of the method, if the procedure is adequately controlled and calibrated the estimation of microbial numbers by optical density (either by McFarland Standards or spectrophotometrically) is sufficiently accurate for use in preparing inocula for QC testing and offers the overwhelming advantages of being rapid, low cost and non-destructive

The Gram Stain

2009-05-02T07:24:00.000-07:00

Gram staining is an empirical method of differentiating bacterial species into two large groups (Gram-positive and Gram-negative) based on the chemical and physical properties of their cell walls. The method is named after its inventor, the Danish scientist Hans Christian Gram (1853-1938), who developed the technique in 1884 (Gram 1884). The importance of this determination to correct identification of bacteria cannot be overstated as all phenotypic methods begin with this assay.
The Basic Method
1. First, a loopful of a pure culture is smeared on a slide and allowed to air dry. The culture can come from a thick suspension of a liquid culture or a pure colony from a plate suspended in water on the microscope slide. Important considerations:
· Take a small inoculum – don’t make a thick smear that cannot be completely decolorized. This could make gram-negative organisms appear to be gram-positive or gram-variable.
· Take a fresh culture – old cultures stain erratically.
2. Fix the cells to the slide by heat or by exposure to methanol. Heat fix the slide by passing it (cell side up) through a flame to warm the glass. Do not let the glass become hot to the touch.
3. Crystal violet (a basic dye) is then added by covering the heat-fixed cells with a prepared solution. Allow to stain for approximately 1 minute.
4. Briefly rinse the slide with water. The heat-fixed cells should look purple at this stage.
5. Add iodine (Gram's iodine) solution (1% iodine, 2% potassium iodide in water) for 1 minute. This acts as a mordant and fixes the dye, making it more difficult to decolorize and reducing some of the variability of the test.
6. Briefly rinse with water.
7. Decolorize the sample by applying 95% ethanol or a mixture of acetone and alcohol. This can be done in a steady stream, or a series of washes. The important aspect is to ensure that all the color has come out that will do so easily. This step washes away unbound crystal violet, leaving Gram-positive organisms stained purple with Gram-negative organisms colorless. The decolorization of the cells is the most “operator-dependent” step of the process and the one that is most likely to be performed incorrectly.
8. Rinse with water to stop decolorization.
9. Rinse the slide with a counterstain (safranin or carbol fuchsin) which stains all cells red. The counterstain stains both gram-negative and gram-positive cells. However, the purple gram-positive color is not altered by the presence of the counter-stain, it’s effect is only seen in the previously colorless gram-negative cells which now appear pink/red.
10. Blot gently and allow the slide to dry. Do not smear.

What’s Going On?
Bacteria have a cell wall made up of peptidoglycan. This cell wall provides rigidity to the cell, and protection from osmotic lysis in dilute solutions. Gram-positive bacteria have a thick mesh-like cell wall, gram-negative bacteria have a thin cell wall and an outer phospholipid bilayer membrane. The crystal violet stain is small enough to penetrate through the matrix of the cell wall of both types of cells, but the iodine-dye complex exits only with difficulty (Davies et al. 1983)
The decolorizing mixture dehydrates cell wall, and serves as a solvent to rinse out the dye-iodine complex. In Gram-negative bacteria it also dissolves the outer membrane of the gram-negative cell wall aiding in the release of the dye. It is the thickness of the cell wall that characterizes the response of the cells to the staining procedure. In addition to the clearly gram-positive and gram-negative, there are many species that are “gram-variable” with intermediate cell wall structure (Beveridge and Graham 1991). As noted above, the decolorization step is critical to the success of the procedure.
Gram’s method involves staining the sample cells dark blue, decolorizing those cells with a thin cell wall by rinsing the sample, then counterstaining with a red dye. The cells with a thick cell wall appear blue (gram positive) as crystal violet is retained within the cells, and so the red dye cannot be seen. Those cells with a thin cell wall, and therefore decolorized, appear red (gram negative).
It is a prudent practice to always include a positive and negative control on the staining procedure to confirm the accuracy of the results (Murray et al 1994) and to perform proficiency testing on the ability of the technicians to correctly interpret the stains (Andserson, et al. 2005).

Excessive Decolorization
It is clear that the decolorization step is the one most likely to cause problems in the gram stain. The particular concerns in this step are listed below (reviewed in McClelland 2001)
Excessive heat during fixationHeat fixing the cells, when done to excess, alters the cell morphology and makes the cells more easily decolorized.
Low concentration of crystal violetConcentrations of crystal violet up to 2% can be used successfully, however low concentrations result in stained cells that are easily decolorized. The standard 0.3% solution is good, if decolorization does not generally exceed 10 seconds.
Excessive washing between stepsThe crystal violet stain is susceptible to wash-out with water (but not the crystal violet-iodine complex). Do not use more than a 5 second water rinse at any stage of the procedure.
Insufficient iodine exposureThe amount of the mordant available is important to the formation of the crystal violet - iodine complex. The lower the concentration, the easier to decolorize (0.33% - 1% commonly used). Also, QC of the reagent is important as exposure to air and elevated temperatures hasten the loss of Gram’s iodine from solution. A closed bottle (0.33% starting concentration) at room temperature will lose >50% of available iodine in 30 days, an open bottle >90%. Loss of 60% iodine results in erratic results.
Prolonged decolorization95% ethanol decolorizes more slowly, and may be recommended for inexperienced technicians while experienced workers can use the acetone-alcohol mix. Skill is needed to gauge when decolorization is complete.
Excessive counterstainingAs the counterstain is also a basic dye, it is possible to replace the crystal violet—iodine complex in gram- positive cells with an over-exposure to the counterstain. The counterstain should not be left on the slide for more than 30 seconds.

Alternatives to the Gram Stain
Gram’s staining method is plainly not without its problems. It is messy, complicated, and prone to operator error. The method also requires a large number of cells (although a membrane-filtration technique has been reported; Romero, et al 1988). However, it is also central to phenotypic microbial identification techniques.
This method, and it’s liabilities, are of immediate interest to those involved in environmental monitoring programs as one of the most common isolates in an EM program, Bacillus spp., will frequently stain gram variable or gram negative despite being a gram-positive rod (this is especially true with older cultures). The problems with Gram’s method have lead to a search for other tests that correlate with the cell wall structure of the gram-positive and the gram-negative cells. Several improvements/alternatives to the classical gram stain have appeared in the literature.

KOH String Test
The KOH String Test is done using a drop of 3% potassium hydroxide on a glass slide. A visible loopful of cells from a single, well-isolated colony is mixed into the drop. If the mixture becomes viscous within 60 seconds of mixing (KOH-positive) then the colony is considered gram-negative. The reaction depends on the lysis of the gram-negative cell in the dilute alkali solution releasing cellular DNA to turn the suspension viscous. This method has been shown effective for food microorganisms (Powers 1995), and for Bacillus spp (Carlone et al 1983, Gregersen 1978), although it may be problematic for some anaerobes (Carlone et al 1983, but also see Halebian et al 1981).
This test has the advantage of simplicity, and it can be performed on older cultures. False negative results can occur in the test by using too little inoculum or too much KOH (DNA-induced viscosity not noticeable). False positive results can occur from too heavy an inoculum (the solution will appear to gel, but not string), or inoculation with mucoid colonies. This can serve as a valuable adjunct to the tradition gram stain method (von Graevenitz and Bucher 1983).

Aminopeptidase Test
L-alanine aminopeptidase is an enzyme localized in the bacterial cell wall which cleaves the amino acid L-alanine from various peptides. Significant activity is found almost only in Gram-negative microorganisms, all Gram-positive or Gram-variable microorganisms so far studied display no or very weak activity (Cerny 1976, Carlone et al. 1983). To perform the test, the reagent is used to make a suspension (with the bacteria). Aminopeptidase activity of the bacteria causes the release of 4-nitroaniline from the reagent, turning the suspension yellow. The test is especially useful for non-fermenters and gram-variable organisms, and is a one step test with several suppliers of kits. Results of the test are available in 5 minutes.

Fluorescent Stains
A popular combination of fluorescent stains for use in gram staining (particularly for flow-cytometry) involves the use of the fluorescent nucleic acid binding dyes hexidium iodide (HI) and SYTO 13. HI penetrates gram-positive but not gram-negative organisms, but SYTO 13 penetrates both. When the dyes were used together in a single step, gram-negative organisms are green fluorescent by SYTO 13 while gram-positive organisms are red-orange fluorescent by HI which overpowers the green of SYTO 13 (Mason et al 1998). There are commercial kits available for this procedure, which requires a fluorescent microscope or a flow cytometer.
Sizemore et al (1990) developed a different approach to fluorescent labeling of cells. Fluorescence-labeled wheat germ agglutinin binds specifically to N-acetylglucosamine in the outer peptidoglycan layer of gram-positive bacteria. The peptidoglycan layer of gram-negative bacteria is covered by a membrane and is not labeled by the lectin. A variant of this method has also been used to “gram stain” microorganisms in milk for direct measurement by flow cytometry.

LAL-based Assay
Charles River Laboratories has just released a product to be used with their PTS instrument – the PTS Gram ID (Farmer 2005). This methodology makes use of the same reaction used for the chromogenic LAL test. Gram-negative organisms, with bacterial endotoxin, initiate the LAL coagulase cascade which results in activation of the proclotting enzyme, a protease. In the LAL test, this enzyme cleaves a peptide from the horseshoe crab coagulen, resulting in a clot. It can also cleave a peptide from a synthetic substrate, yielding a chromophore (p-nitroaniline) which is yellow and can be measured photometrically at 385 nm (Iwanaga 1987). Gram-positive organisms, lacking endotoxin, do not trigger the color change in this method, while gram-negative organisms do trigger it. Results are available within 10 minutes.

Summary
The differentiation of bacteria into either the gram-positive or the gram-negative group is fundamental to most bacterial identification systems. This task is usually accomplished through the use of Gram’s Staining Method. Unfortunately, the gram stain methodology is complex and prone to error. This operator-dependence can be addressed by attention to detail, and by the use of controls on the test. Additional steps might include confirmatory tests, of which several examples were given. As with all microbiology assays, full technician training and competent review of the data are critical quality control steps for good laboratory results.

Microbial Recovery

2009-05-02T07:15:00.000-07:00

Introduction
The PMFList is a source of great ideas for review and for further thought. One that keeps coming up on the list is the question of 70% recovery (as described in USP chapter <1227> Validation of Microbial Recovery from Pharmacopeial Articles) and 50% recovery as described in the harmonized chapter <61> Microbiological Examination Of Nonsterile Products: Microbial Enumeration Tests.
The questions and discussion seem to fall into two distinct groups – the first a discussion about when to apply 70% and when to apply 50% as your recovery criteria (with frequent complaints about the inferred lack of consistency in USP) and the second a discussion of what types of tests we are talking about. We will look at these issues separately.
What are we talking about?
The first thing to do is to establish the scope of the discussion. For starters, let’s begin by stating that the compendial chapters are, by definition, validated. This refers to those chapters in the USP that number under 1000. We therefore cannot really “validate” the test method, instead we are trying to demonstrate the suitability of the recovery method. This has been referred to as “verification” (Porter 2007) and in the harmonized Microbial Limits chapters as “method suitability.”
The point of a method suitability study in microbiology is not to validate the assay, but rather to demonstrate that our specific test method is suitable; that the recovery scheme allows recovery of viable microorganisms. In other words, microorganisms are not prevented from growing in the experimental system by residual antimicrobial activity of the product
This demonstration is critical in accurate determination of disinfecting efficacy, bioburden, sterility or any test that requires determination of surviving microorganisms in a product containing antimicrobial properties. Failure to confirm adequate neutralization and recovery could result in under-reporting of surviving microorganisms. This expectation of 70% recovery can also be applied to media growth promotion studies, where a new batch of media is compared to a previously qualified batch for its ability to support at least 70% of a standard inoculum.
A convenient method for this neutralization is through the use of recovery diluents designed to neutralize commonly used antimicrobials. A number of reagents are used in this regard (reviewed by Russell 1981; Furr & Rogers 1987). However, some of these compounds may be toxic to the test organisms (Reybrouck 1978) and so it is also important to determine the potential toxicity of the neutralizing medium (recovery diluent). These two activities, neutralizer efficacy and growth promotion (or neutralizer toxicity), are equally important in this consideration. A schematic of a design for this type of study is presented below, where a consistent inoculum is added to the product in the recovery diluent, peptone in the recovery diluent (use the same volume of peptone as that of the product), and into peptone. These are then plated 5-6 times to provide a good estimate of the number of organisms present (Wilson and Kullman 1931). The Neutralizer Efficacy is determined by comparing the recovery in the peptone suspension to that in the Product + Recovery Diluent suspension, Neutralizer Toxicity by comparing the Peptone suspension to the Peptone + Recovery Diluent (USP 2007a).

What is not part of this discussion?
It should be obvious from the previous discussion that the “method suitability” study is highly controlled. A standard inoculum is added to three tubes, and then replicate aliquots are removed and immediately plated. In a perfect world the numbers would be in agreement 100% of the time, but we work in microbiology. Even in such a simple design the opportunity for variability is enormous, and there are workers in the field who are vehement that no better than 50% should be expected between replicates of this type. One wonders if this is a limitation of the test system or of their laboratory training program. In any event, the discussion of 50% to 70% between the populations applies only to this design (and those closely related to it).
The recommendation in USP of 70% recovery was never meant to apply to studies of microbial recovery from solid surfaces. These studies are extremely complicated, and are confounded by issues of recovery efficacy of swabs, contact plates, and other methods (Buggy, et al 1983, Rose et al 2004, Whyte 1989). In addition, if vegetative cells are used for the study, there is the additional problem of die-off due to dessication (Potts 1994).
Recovery studies looking at bioburden of solid surfaces (facility, equipment, medical device or personnel) are not part of the 50% to 70% debate. They have their own set of issues and will be discussed in a later newsletter.

Is there any support for these numbers?
There are two studies which directly support the 70% recovery acceptance criterion.
Proud and Sutton (1992) describe the development of a “universal” diluting fluid for membrane filtration sterility testing using a modification of the design described above. The product was placed in a filtration apparatus containing 100 mL of the diluting fluid, and then passed through the membrane, followed by two additional 100 mL rinses. The membrane was then removed and placed on the surface of a nutrient agar plate for incubation and enumeration. Each treatment was performed at least three times. CFU were converted to their log10 values, and ANOVA analysis performed on the replicates. When all was said and done, a recovery of 75% of the inoculum count (raw CFU – untransformed) passed the ANOVA analysis.
Sutton, et al. (2002) conducted a large study on methods to recover microorganisms in the presence of surface disinfectants. “Neutralizer efficacy (NE) ratios were determined [in this study] by comparing the recovery of identical inocula from the neutralizing solution in the presence, or the absence, of a 1:10 dilution of the biocide. Neutralizer toxicity (NT) ratios were determined between recovery of viable microorganisms incubated for a short period in peptone, and in the neutralizing medium without the biocide. An effective and non-toxic neutralizer was initially identified by NE and NT ratios of ≥ 0.75. Statistical evaluation of the data was performed by ANOVA, with Dunnett’s test for multiple comparisons used to confirm failures. By this analysis, 239/244 identified failures were confirmed by ANOVA of 588 NT and NE comparisons (5 presumptive failures were not confirmed by statistical analysis). We therefore conclude that recovery of 75% is a suitable criterion (2% false negative rate) for neutralizer evaluations.”
A side issue to this discussion is the occasional use of 70-130% recovery as the acceptance criteria. I have trouble with this one – would you really disqualify a method because it improves your recovery over expectations? In my opinion the acceptance criteria should be that the test treatment should recover at least 70%, with no consideration of recovery by the test in excess of the comparator treatment.
Which should you use?
I am of the opinion that 70% is easily attainable if the technicians are proficient and the recovery method works. This may require 5-6 replicates, rather than the usual duplicate plates per sample. However, this is a “verification” study or a “method suitability” study (or whatever we wish to call it) and so may be worth a bit more work.
So, how did they get different criteria in the USP? Chapter <1227> was developed to address a specific concern – that of providing information on microbial recovery studies (not limited to neutralizer efficacy) for use in the pharmaceutical industry. This work was well in progress by 1996 (USP 1996). The harmonization program discussed this point much later, and after negotiation agreed to the 50% so that agreement could occur. No data was presented to support the assertion that 50% was appropriate (by my records), it was, however, the number that could be accepted.
The harmonized USP chapter <61> (USP 2006b) cites a 50% recovery frequency and so this is the official acceptance criteria for this test. If you wish to use 50% for the acceptance criteria for all method suitability studies (non-compendial bioburden tests, method suitability studies for disinfectancy tests, Antimicrobial Efficacy tests, media growth promotion, etc) I would strongly urge a solid rationale for failing to observe the recommendation of chapter <1227>. In addition, I would be prepared to answer questions of technician proficiency as the suspicion may be that your lab is not confident of reproducibility to 70% even between identical samples.

Manipulating Carbohydrates

2009-05-01T06:20:00.000-07:00

The application of manipulating carbohydrates while preparing for a contest is done with intent on the muscle glycogen and water content. The thought is for you to have as much glycogen in your muscles as you can on the show day of the competition. From every gram of glycogen in your body, you have another three grams of water added into your muscle cells also. That means that a weight gain of two to four pounds could be predicted if you do it correctly.
Using Creatine before a contest
Loading yourself with creatine is done in an attempt to saturate and control a larger amount of creatine in your muscle cells. The creatine you take in helps you out with the creatine phosphate section of the kreb cycle to create energy for your working cells.
Side note: Kreb cycle â€“ The kreb cycle is what is found in all plants and animals, a variety of enzymatic reactions in the mitochondria of cells, used to create a lot of energy phosphate compounds that are a main source of cellular energy.
Protocols for Creatine:
- First off, 20-25 grams per day for the period of 5-7 days
- Afterwards, 2-5 grams per day for 21-37 days
It has also been shown that creatine raises your total body water and glycogen while not using fluid distribution. A two week study was done to show the results that combining both the carbohydrate loading after you use creatine supplementation has lead to prove that it gives a super concentration of glycogen and water in your muscle cells.
Competitive Bodybuilders: Usage Suggestion
10 Days out: Only have an intake of a high carbohydrate based diet.
7 Days out: Put five grams of creatine into your body daily.
5 Days out: Start the period of carbohydrate depletion for the process of three days which is followed with a cessation of creatine.
2 Days of carbohydrate loading and gradual water intake decreases.
Another suggestion is for you to takea potassium supplement which could help out with any cramping you may be having.
While more research is needed on this topic to come to a conclusion, we all know that for certain people, certain choices will be made. While professional bodybuilders use a certain type of training and preperation to make sure their muscles are as good as possible for performing at the show they are featured in, other less competitive bodybuilders may not worry as much about this issue.
Either way, it is best for you to stick around and find out any conclusion that is discovered.

Pharmacokinetics

2009-04-30T03:17:00.000-07:00

Pharmacokinetics is the study of what the body does to a drug.
Pharmacodynamics is the study of what a drug does to the body.
Routes of Drug Administration:
Intravenous
Oral
Buccal
Sublingual
Rectal
Intramuscular
Transdermal
Subcutaneous
Inhalational
Topical
Of all of these routes you are most likely to be asked about the transdermal, as it is fashionable.
Otherwise, most other basic pharmacology questions tend to concern the pharmacology of intravenous agents; that is what is discussed below.
First Order Kinetics:
A constant fraction of the drug in the body is eliminated per unit time. The rate of elimination is proportional to the amount of drug in the body. The majority of drugs are eliminated in this way.

What follows concerns drugs which follow first order kinetics.

The Volume of Distribution (Vd) is the amount of drug in the body divided by the concentration in the blood. Drugs that are highly lipid soluble, such as digoxin, have a very high volume of distribution (500 litres). Drugs which are lipid insoluble, such as neuromuscular blockers, remain in the blood, and have a low Vd.
The Clearance (Cl) of a drug is the volume of plasma from which the drug is completely removed per unit time. The amount eliminated is proportional to the concentration of the drug in the blood.
The fraction of the drug in the body eliminated per unit time is determined by the elimination constant (kel). This is represented by the slope of the line of the log plasma concentration versus time.
Cl = kel x Vd
Rate of elimination = clearance x concentration in the blood.
Elimination half life (t1/2): the time taken for plasma concentration to reduce by 50%. After 4 half lives, elimination is 94% complete.
It can be shown that the kel = the log of 2 divided by the t1/2 = 0.693/t1/2.
Likewise, Cl = kel x Vd, so, Cl = 0.693Vd/t1/2.
And t1/2 = 0.693 x Vd / cl
The rate of elimination is the clearance times the concentration in the plasma
Roe = Cl x Cp
Fraction of the total drug removed per unit time = Cl/Vd.
If the volume of distribution is increased, then the kel will decrease, the t1/2 will increase, but the clearance won't change.
Confused?
Example: You have a 10ml container of orange squash. You put this into a litre (ok 990ml!) of water. The Vd of the orange squash is 1000ml. If, each minute, you empty 10ml of the orange liquid into the 10ml container, discard this, and replace it with 10ml of water. The clearance is 10 ml per minute. The elimination half life is: 70 minutes . The kel is Cl/Vd = 10/1000 = 0.01. Shown the other way, 0.693/50 = 0.01.
If the volume of the container is increased to 2000ml, then the clearance remains the same, but the Vd, and consequently the t1/2, increases (to 140 minutes).
Simple, isn't it?
What is described above is a single compartment model, what would occur if the bloodstream was the only compartment in the body (or if the Vd = the blood volume). But the human body is more complex than this: there are many compartments: muscle, fat, brain tissue etc. In order to describe this, we use multicompartment models.
Multicompartment Models:
Why does a patient wake up after 5 minutes after an injection of thiopentone when we know that it takes several hours to eliminate this drug from the body? What happens is that, initially the drug is all in the blood and this blood goes to "vessel rich" organs; principally the brain. After a few minutes the drug starts to venture off into other tissues (fat, muscle etc) it redistributes, the concentration in the brain decreases and the patient wakes up! The drug thus redistributes into other compartments.
If you were to represent this phenomenon graphically, you would follow a picture of rapid fall in blood concentration, a plateau, and then a slower gradual fall. The first part is the rapid redistribution phase, the alpha phase, the plateau is the equilibrium phase (where blood concentration = tissue concentration), and the slower phase, the beta phase, is the elimination phase where blood and tissue concentrations fall in tandem. This is a simple two compartment model and is as much as you need to know.

An couple of interesting pieces of information can be derived from the log concentration versus time graph. If you extrapolate back the elimination line to the y axis, then you get to a point called the CP0 - a theoretical point representing the concentration that would have existed at the start if the dose had been instantly distributed (dose/Vd). From this new straight line you can figure out how long it takes for the concentration to drop by 50%: the elimination half life. Likewise, a similar procedure can be performed on the α phase: the redistribution half life.
While it is very important that you understand these concepts, the reality is that most drugs are infinitely more complicated that this, and computer calculations are required to derive this data.
Bioavailability
This is the fraction of the administered dose that reaches the systemic circulation. Bioavailability is 100% for intravenous injection. It varies for other routes depending on incomplete absorption, first pass hepatic metabolism etc. Thus one plots plasma concentration against time, and the bioavailability is the area under the curve.
Zero Order Elimination
Why if I have 10 pints of beer before midnight will I fail a breathalyser test at 8 am the following morning? Either this is due to alcohol having a very long half life (which it does not) or that alcohol is cleared in a different way.
What happens is that the metabolic pathways responsible for alcohol metabolism are rapidly saturated and that clearance is determined by how fast these pathways can work. The metabolic pathways work to their limit. This is known as zero order kinetics: a constant amount of drug is eliminated per unit time. This form of kinetics occours with several important drugs at high dosage concentrations: phenytoin, salicylates, theophylline, and thiopentone (at very large doses). Because high dose thio is very slow to clear, we no longer use it in infusion for status epilepticus (as it takes ages for the patient to wake up!).
Dosage regimens
The strategy for treating patients with drugs is to give sufficient amounts that the required theraputic effect arises, but not a toxic dose.
The maintenance dose is equal to the rate of elimination at steady state (i.e.at steady state, rate of elimination = rate of administration):
Dosing rate = clearance x desired plasma concentration.
Drugs will accumulate within the body if the drug has not been fully eliminated before the next dose. Steady state concentration is thus arrived at after four half lives. This is all very well if you are willing to wait 4 half lives for the drug to be fully effective, but what if you are not? What you may need to do is to "load" the volume of distribution with the drug to achieve target plasma concentrations rapidly: the loading dose.
The loading dose = the volume of distribution x the desired concentration (i.e. the concentration at steady state).
You can figure this out by: Loading dose = usual maintenance dose / usual dosage interval x kel (t1/2/0.693).
Hepatic Drug Clearance
Many drugs are extensively metabolised by the liver. The rate of elimination depends on 1) The liver's inherent ability to metabolise the drug, 2) the amount of drug presented to the liver for metabolism. This is important because drugs administered orally are delivered from the gut to the portal vein to the liver: the liver gobbles up a varying chunk of the administered drug (pre-systemic elimination) and less is available to the body for theraputic effect. This is why you have to give a higher dose of morphine, for examole, orally, than intravenously.
Hepatic drug clearance (i.e. the amount of each drug gobbled up by the liver) depends on:
1) The Intrinsic clearance (Cl int).
2) Hepatic blood flow.
These two factors are independent of one another, and their combined effect is the proportion of drug gobbled up: the extraction ratio.
For drugs that have a low intrinsic clearance, this effect can be increased by giving a second agent that boosts the effect of the liver's enzyme system; these are enzyme inducers. Examples of such drugs are cigarrettes, antiepileptics (carbamazepine & phenytoin), rifampicin, griseofulvin, alcohol and spironolactone (CAR GAS) [also barbiturates]. Consequnetly if a drug addict is given rifampicin or tuberculosis, a higher dose of heroin is required for the same effect. Enzyme inhibitors have the opposite effect: examples are flagyl, allopurinol, cimetidine, erythromycin, dextropropoxyphene, imipramine, (the) pill (FACE DIP).
Likewise, if the blood flow increases, the liver has less chance to gobble up the drug, and the extraction ratio falls. This is particularly the case, as you would expect, of the intrinsic clearance is low.
Illustration: Think of factory workers picking bad apples out of a pile on a conveyor belt, if only one person (low intrinsic clearance) is doing the picking and the speed of the conveyor belt is increased, more bad apples get through. If there are several pickers (high intrinsic clearance) then they are much more able to cope with an increase in the speed of the conveyor belt, but there will come a rate at which they will become overwhelmed, and bad apples will get through.
From this example you can take home this message: for drugs with a low extraction ratio, the kinetics (the body's ability to deal with the drug) depends on enzymatic activity (giving an enzyme inducer effectively gives the single picker 4 arms!). For high extraction ratio drugs, kinetics depends on liver blood flow - the slower the flow the higher the extraction, the higher the flow the lower the extraction ratio.
Drug distribution
When a drug is introduced into the body, where it ends up depends on a number of factors:
1) blood flow, tissues with the highest blood flow receive the drug first, 2) protein binding, drugs stuck to plasma proteins are crippled, they can only go where the proteins go (and that's not very far!), 3) lipid solubility and the degree of ionisation, this describes the ability of drugs to enter tissues (highly lipid soluble / unionised drugs can basically go anywhere).
Protein Binding
Most drugs bind to proteins, either albumin or alpha-1 acid glycoprotein (AAG), to a greater or lesser extent. Drugs prefer to be free, it is in this state that they can travel throughout the body, in and out of tissues and have their biological effect. The downside of this is that they are easy prey for metabolising enzymes.
As you would expect, more highly bound drugs have a longer duration of action and a lower volume of distribution. Generally high extraction ratio drugs' clearance is high because of low protein binding and, conversely, low extraction ratio drugs' clearance is strongly dependent on the amount of protein binding.
Why is this important? If a drug is highly protein bound, you need to give loads of it to get a theraputic effect; as so much is stuck to protein. But what happens if another agent comes along and starts to compete with the drug for the binding site on the protein? Yes, you guessed it, the amount of free drug is increased. This is really important for drugs that are highly protein bound: if a drug is 97% bound to albumin and there is a 3% reduction in binding (displaced by another drug), then the free drug concentration doubles; if a drug is 70% bound and there is a 3% reduction in binding, this will make little difference.
The drugs that you really need to keep an eye on are: warfarin, diazepam, propranolol and phenytoin. For example, a patient on warfarin is admitted with seizures, you treat the patient with phenytoin, next thing you know - his INR is 10.
The amount of albumin does not appear to be hugely relavent. In disease states such as sepsis, the serum albumin drops drastically, but the free drug concentration does not appear to increase
Degree of ionisation
This is really important with regard to local anaesthetics. The essential fact to know is that highly ionized drugs cannnot cross lipid membranes (basically they can't go anywhere) and unionised drugs can cross freely. Morphine is highly ionised, fentanyl is the opposite. Consequently the latter has a faster onset of action. The degree of ionisation depends on the pKa of the drug and the pH of the local environment. The pKa is the the pH at which the drug is 50% ionised. Most drugs are either weak acids or weak bases. Acids are most highly ionised at a high pH (i.e. in an alkaline environment). Bases are most highly ionised in an acidic environment (low pH). For a weak acid, the more acidic the environment, the less ionised the drug, and the more easily it crosses lipid membranes. If you take this acid, at pKa it is 50% ionised, if you add 2 pH points to this (more alkaline), it becomes 90% ionised, if you reduce the pH (more acidic) by two units, it becomes 10% ionised. Weak bases have the opposite effect.
Local anaesthetics are weak bases: the closer the pKa of the local anaesthetic to the local tissue pH, the more unionised the drug is. That is why lignocaine(pKa 7.7) has a faster onset of action than bupivicaine (pKa 8.3). If the local tissues are alkalinised (e.g. by adding bicarbonate to the local anaesthetic), then the tisssue pH is brought closer to the pKa, and the onset of action is hastened.

Xenobiotic metabolism

2009-04-30T03:05:00.000-07:00

Xenobiotic metabolism is the set of metabolic pathways that modify the chemical structure of xenobiotics, which are compounds foreign to an organism's normal biochemistry, such as drugs and poisons. These pathways are a form of biotransformation present in all major groups of organisms, and are considered to be of ancient origin. These reactions often act to detoxify poisonous compounds; however, in some cases, the intermediates in xenobiotic metabolism can themselves be the cause of toxic effects.
Xenobiotic metabolism is divided into three phases. In phase I, enzymes such as cytochrome P450 oxidases introduce reactive or polar groups into xenobiotics. These modified compounds are then conjugated to polar compounds in phase II reactions. These reactions are catalysed by transferase enzymes such as glutathione S-transferases. Finally, in phase III, the conjugated xenobiotics may be further processed, before being recognised by efflux transporters and pumped out of cells.
The reactions in these pathways are of particular interest in medicine as part of drug metabolism and as a factor contributing to multidrug resistance in infectious diseases and cancer chemotherapy. The actions of some drugs as substrates or inhibitors of enzymes involved in xenobiotic metabolism are a common reason for hazardous drug interactions. These pathways are also important in environmental science, with the xenobiotic metabolism of microorganisms determining whether a pollutant will be broken down during bioremediation, or persist in the environment.

MTT assay

2009-04-30T03:01:00.000-07:00

The MTT assay and the MTS assay are laboratory tests and standard colorimetric assays (an assay which measures changes in color) for measuring the activity of enzymes that reduce MTT or MTS + PMS to formazan, giving a purple color. It can also be used to determine cytotoxicity of potential medicinal agents and other toxic materials, since those agents would result in cell toxicity and therefore metabolic dysfunction and therefore decreased performance in the assay.
Yellow MTT (3-(4,5-Di methyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole) is reduced to purple formazan in living cells.[1] A solubilization solution (usually either dimethyl sulfoxide, an acidified ethanol solution, or a solution of the detergent sodium dodecyl sulfate in diluted hydrochloric acid) is added to dissolve the insoluble purple formazan product into a colored solution. The absorbance of this colored solution can be quantified by measuring at a certain wavelength (usually between 500 and 600 nm) by a spectrophotometer. The absorption maximum is dependent on the solvent employed.
MTS is a more recent alternative to MTT. MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium), in the presence of phenazine methosulfate (PMS), produces a water-soluble formazan product that had an absorbance maximum at 490-500 nm in phosphate-buffered saline.[2] It is advantageous over MTT in that (1) the reagents MTS + PMS are reduced more efficiently than MTT, and (2) the product is water soluble, decreasing toxicity to cells seen with an insoluble product.
These reductions take place only when reductase enzymes are active, and therefore conversion is often used as a measure of viable (living) cells. However, it is important to keep in mind that other viability tests (such as the CASY cell counting technology) sometimes give completely different results, as many different conditions can increase or decrease metabolic activity. Changes in metabolic activity can give large changes in MTT or MTS results while the number of viable cells is constant. When the amount of purple formazan produced by cells treated with an agent is compared with the amount of formazan produced by untreated control cells, the effectiveness of the agent in causing death, or changing metabolism of cells, can be deduced through the production of a dose-response curve

DNase footprinting assay

2009-04-30T02:42:00.000-07:00

A DNase footprinting assay[1] is a DNA footprinting technique from molecular biology/biochemistry that detects DNA-protein interaction using the fact that a protein bound to DNA will often protect that DNA from enzymatic cleavage. This makes it possible to locate a protein binding site on a particular DNA molecule. The method uses an enzyme, deoxyribonuclease (DNase, for short) to cut the radioactively end-labelled DNA, followed by gel electrophoresis to detect the resulting cleavage pattern.
For example, the DNA fragment of interest may be PCR amplified using a 32P 5' labeled primer, with the end result being many DNA molecules with a radioactive label on one end of one strand of each double stranded molecule. Cleavage by DNase will produce fragments, the smaller of which will move further on the electrophoretic gel. The fragments which are smaller with respect to the 32P-labelled end will appear further on the gel than the longer fragments. The gel is then used to expose a special photographic film.
The cleavage pattern of the DNA in the absence of a DNA binding protein, typically referred to as free DNA, is compared to the cleavage pattern of DNA in the presence of a DNA binding protein. If the protein binds DNA, the binding site is protected from enzymatic cleavage. This protection will result in a clear area on the gel which is referred to as the "footprint".
By varying the concentration of the DNA-binding protein, the binding affinity of the protein can be estimated according to the minimum concentration of protein at which a footprint is observed.
This technique was developed by David Galas and Albert Schmitz at Geneva in 1977

Reversible inhibitors

2009-04-30T02:13:00.000-07:00

[edit] Enzyme activity
Enzyme activity = moles of substrate converted per unit time = rate × reaction volume. Enzyme activity is a measure of the quantity of active enzyme present and is thus dependent on conditions, which should be specified. The SI unit is the katal, 1 katal = 1 mol s-1, but this is an excessively large unit. A more practical and commonly-used value is 1 enzyme unit (EU) = 1 μmol min-1 (μ = micro, x 10-6). 1 U corresponds to 16.67 nanokatals.[1]

[edit] Specific activity
The specific activity of an enzyme is another common unit. This is the activity of an enzyme per milligram of total protein (expressed in μmol min-1mg-1). Specific activity gives a measurement of the purity of the enzyme. It is the amount of product formed by an enzyme in a given amount of time under given conditions per milligram of enzyme. Specific activity is equal to the rate of reaction multiplied by the volume of reaction divided by the mass of enzyme. The SI unit is katal kg-1, but a more practical unit is μmol mg-1 min-1. Specific activity is a measure of enzyme processivity, usually constant for a pure enzyme.

[edit] Related terminology
The rate of a reaction is the concentration of substrate disappearing (or product produced) per unit time (mol L − 1s − 1)
The % purity is 100% × (specific activity of enzyme sample / specific activity of pure enzyme). The impure sample has lower specific activity because some of the mass is not actually enzyme. If the specific activity of 100% pure enzyme is known, then an impure sample will have a lower specific activity, allowing purity to be calculated.

[edit] Types of assay
All enzyme assays measure either the consumption of substrate or production of product over time. A large number of different methods of measuring the concentrations of substrates and products exist and many enzymes can be assayed in several different ways. Biochemists usually study enzyme-catalysed reactions using four types of experiments:[2]
(1) Initial rate experiments. When an enzyme is mixed with a large excess of the substrate, the enzyme-substrate intermediate builds up in a fast initial transient. Then the reaction achieves a steady-state kinetics in which enzyme substrate intermediates remains approximately constant over time and the reaction rate changes relatively slowly. Rates are measured for a short period after the attainment of the quasi-steady state, typically by monitoring the accumulation of product with time. Because the measurements are carried out for a very short period and because of the large excess of substrate, the approximation free substrate is approximately equal to the initial substrate can be made. The initial rate experiment is the simplest to perform and analyze, being relatively free from complications such as back-reaction and enzyme degradation. It is therefore by far the most commonly used type of experiment in enzyme kinetics.
(2) Progress curve experiments. In these experiments, the kinetic parameters are determined from expressions for the species concentrations as a function of time. The concentration of the substrate or product is recorded in time after the initial fast transient and for a sufficiently long period to allow the reaction to approach equilibrium. We note in passing that, while they are less common now, progress curve experiments were widely used in the early period of enzyme kinetics.
(3) Transient kinetics experiments. In these experiments, reaction behaviour is tracked during the initial fast transient as the intermediate reaches the steady-state kinetics period. These experiments are more difficult to perform than either of the above two classes because they require rapid mixing and observation techniques.
(4) Relaxation experiments. In these experiments, an equilibrium mixture of enzyme, substrate and product is perturbed, for instance by a temperature, pressure or pH jump, and the return to equilibrium is monitored. The analysis of these experiments requires consideration of the fully reversible reaction. Moreover, relaxation experiments are relatively insensitive to mechanistic details and are thus not typically used for mechanism identification, although they can be under appropriate conditions.
Enzyme assays can be split into two groups according to their sampling method: continuous assays, where the assay gives a continuous reading of activity, and discontinuous assays, where samples are taken, the reaction stopped and then the concentration of substrates/products determined.

Temperature-controlled cuvette holder in a spectrophotometer.

[edit] Continuous assays
Continuous assays are most convenient, with one assay giving the rate of reaction with no further work necessary. There are many different types of continuous assays.

[edit] Spectrophotometric
In spectrophotometric assays, you follow the course of the reaction by measuring a change in how much light the assay solution absorbs. If this light is in the visible region you can actually see a change in the color of the assay, these are called colorimetric assays. The MTT assay, a redox assay using a tetrazolium dye as substrate is an example of a colorimetric assay.
UV light is often used, since the common coenzymes NADH and NADPH absorb UV light in their reduced forms, but do not in their oxidised forms. An oxidoreductase using NADH as a substrate could therefore be assayed by following the decrease in UV absorbance at a wavelength of 340 nm as it consumes the coenzyme.[3]
Direct versus coupled assays

Coupled assay for hexokinase using glucose-6-phosphate dehydrogenase.
Even when the enzyme reaction does not result in a change in the absorbance of light, it can still be possible to use a spectrophotometric assay for the enzyme by using a coupled assay. Here, the product of one reaction is used as the substrate of another, easily-detectable reaction. For example, figure 1 shows the coupled assay for the enzyme hexokinase, which can be assayed by coupling its production of glucose-6-phosphate to NADPH production, using glucose-6-phosphate dehydrogenase.

[edit] Fluorometric
Fluorescence is when a molecule emits light of one wavelength after absorbing light of a different wavelength. Fluorometric assays use a difference in the fluorescence of substrate from product to measure the enzyme reaction. These assays are in general much more sensitive than spectrophotometric assays, but can suffer from interference caused by impurities and the instability of many fluorescent compounds when exposed to light.
An example of these assays is again the use of the nucleotide coenzymes NADH and NADPH. Here, the reduced forms are fluorescent and the oxidised forms non-fluorescent. Oxidation reactions can therefore be followed by a decrease in fluorescence and reduction reactions by an increase.[4] Synthetic substrates that release a fluorescent dye in an enzyme-catalyzed reaction are also available, such as 4-methylumbelliferyl-β-D-galactoside for assaying β-galactosidase.

[edit] Calorimetric

Chemiluminescence of Luminol
Calorimetry is the measurement of the heat released or absorbed by chemical reactions. These assays are very general, since many reactions involve some change in heat and with use of a microcalorimeter, not much enzyme or substrate is required. These assays can be used to measure reactions that are impossible to assay in any other way.[5]

[edit] Chemiluminescent
Chemiluminescence is the emission of light by a chemical reaction. Some enzyme reactions produce light and this can be measured to detect product formation. These types of assay can be extremely sensitive, since the light produced can be captured by photographic film over days or weeks, but can be hard to quantify, because not all the light released by a reaction will be detected.
The detection of horseradish peroxidase by enzymatic chemiluminescence (ECL) is a common method of detecting antibodies in western blotting. Another example is the enzyme luciferase, this is found in fireflies and naturally produces light from its substrate luciferin.

[edit] Light Scattering
Static Light Scattering measures the product of weight-averaged molar mass and concentration of macromolecules in solution. Given a fixed total concentration of one or more species over the measurement time, the scattering signal is a direct measure of the weight-averaged molar mass of the solution, which will vary as complexes form or dissociate. Hence the measurement quantifies the stoichiometry of the complexes as well as kinetics. Light scattering assays of protein kinetics is a very general technique that does not require an enzyme.

[edit] Discontinuous assays
Discontinuous assays are when samples are taken from an enzyme reaction at intervals and the amount of product production or substrate consumption is measured in these samples.

[edit] Radiometric
Radiometric assays measure the incorporation of radioactivity into substrates or its release from substrates. The radioactive isotopes most frequently used in these assays are 14C, 32P, 35S and 125I. Since radioactive isotopes can allow the specific labelling of a single atom of a substrate, these assays are both extremely sensitive and specific. They are frequently used in biochemistry and are often the only way of measuring a specific reaction in crude extracts (the complex mixtures of enzymes produced when you lyse cells).
Radioactivity is usually measured in these procedures using a scintillation counter.

[edit] Chromatographic
Chromatographic assays measure product formation by separating the reaction mixture into its components by chromatography. This is usually done by high-performance liquid chromatography (HPLC), but can also use the simpler technique of thin layer chromatography. Although this approach can need a lot of material, its sensitivity can be increased by labelling the substrates/products with a radioactive or fluorescent tag. Assay sensitivity has also been increased by switching protocols to improved chromatographic instruments (e.g. ultra-high pressure liquid chromatography) that operate at pump pressure a few-fold higher than HPLC instruments (see HPLC#Pump_pressure).[6]

[edit] Factors to control in assays
Salt Concentration: Most enzymes cannot tolerate extremely high salt concentrations. The ions interfere with the weak ionic bonds of proteins. Typical enzymes are active in salt concentrations of 1-500 mM. As usual there are exceptions such as the halophilic (salt loving) algae and bacteria.
Effects of Temperature: All enzymes work within a range of temperature specific to the organism. Increases in temperature generally lead to increases in reaction rates. There is a limit to the increase because higher temperatures lead to a sharp decrease in reaction rates. This is due to the denaturating (alteration) of protein structure resulting from the breakdown of the weak ionic and hydrogen bonding that stabilize the three dimensional structure of the enzyme. The "optimum" temperature for human enzymes is usually between 35 and 40 °C. The average temperature for humans is 37 °C. Human enzymes start to denature quickly at temperatures above 40 °C. Enzymes from thermophilic archaea found in the hot springs are stable up to 100 °C.[7] However, the idea of an "optimum" rate of an enzyme reaction is misleading, as the rate observed at any temperature is the product of two rates, the reaction rate and the denaturation rate. If you were to use an assay measuring activity for one second, it would give high activity at high temperatures, however if you were to use an assay measuring product formation over an hour, it would give you low activity at these temperatures.
Effects of pH: Most enzymes are sensitive to pH and have specific ranges of activity. All have an optimum pH. The pH can stop enzyme activity by denaturating (altering) the three dimensional shape of the enzyme by breaking ionic, and hydrogen bonds. Most enzymes function between a pH of 6 and 8; however pepsin in the stomach works best at a pH of 2 and trypsin at a pH of 8.
Substrate Saturation: Increasing the substrate concentration increases the rate of reaction (enzyme activity). However, enzyme saturation limits reaction rates. An enzyme is saturated when the active sites of all the molecules are occupied most of the time. At the saturation point, the reaction will not speed up, no matter how much additional substrate is added. The graph of the reaction rate will plateau.
Level of crowding, large amounts of macromolecules in a solution will alter the rates and equilibrium constants of enzyme reactions, through an effect called macromolecular crowding.[8]

[edit] List of enzyme assays
MTT assay
Overlay assay
Fluorescein diacetate hydrolysis

[edit] See also
Restriction enzyme
DNase footprinting assay
Enzyme kinetics

[edit] References
^ Nomenclature Committee of the International Union of Biochemistry (NC-IUB) (1979). "Units of Enzyme Activity". Eur. J. Biochem. 97: 319–20. doi:10.1111/j.1432-1033.1979.tb13116.x. http://www.blackwell-synergy.com/doi/pdf/10.1111/j.1432-1033.1979.tb13116.x.
^ Schnell, S., Chappell, M. J., Evans, N. D. and Roussel, M. R. The mechanism distinguishability problem in biochemical kinetics: The single-enzyme, single-substrate reaction as a case study. Comptes Rendus Biologies 2006; 329, 51-61. DOI: 10.1016/j.crvi.2005.09.005
^ Bergmeyer, H. U. "Methods of Enzymatic Analysis", Vol. 4, Academic Press (New York, NY:1974), pp.2066-2072.
^ Passonneau, J. V., and Lowry, O. H. "Enzymatic Analysis. A Practical Guide", Humana Press (Totowa, NJ:1993), pp.85-110.
^ Todd MJ, Gomez J. Enzyme kinetics determined using calorimetry: a general assay for enzyme activity? Anal Biochem. 2001 Sep 15;296(2):179-87.
^ Churchwella, M; Twaddlea, N; Meekerb, L; and Doergea, D. Improving Sensitivity In Liquid Chromatography-Mass Spectrometry. Journal of Chromatography B Volume 825, Issue 2, 25 October 2005, Pages 134-143
^ Cowan DA (1997). "Thermophilic proteins: stability and function in aqueous and organic solvents". Comp. Biochem. Physiol. A Physiol. 118 (3): 429–38. doi:10.1016/S0300-9629(97)00004-2. PMID 9406427.
^ Minton AP (2001). "The influence of macromolecular crowding and macromolecular confinement on biochemical reactions in physiological media". J. Biol. Chem. 276 (14): 10577–80. doi:10.1074/jbc.R100005200. PMID 11279227. http://www.jbc.org/cgi/content/full/276/14/10577.
v • d • e Medicine: Pathology
Principles of pathology
Disease/Medical condition (Infection, Neoplasia) - Hemodynamics (Ischemia) - Inflammation - Wound healing
Cell death: Necrosis (Liquefactive necrosis, Coagulative necrosis, Caseous necrosis) - Apoptosis - Pyknosis - Karyorrhexis - Karyolysis
Cellular adaptation: Atrophy - Hypertrophy - Hyperplasia - Dysplasia - Metaplasia (Squamous, Glandular)accumulations: pigment (Hemosiderin, Lipochrome/Lipofuscin, Melanin) - Steatosis
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Surgical pathology - Cytopathology - Autopsy - Molecular pathology - Forensic pathology - Dental pathology Gross examination - Histopathology - Immunohistochemistry - Electron microscopy - Immunofluorescence - Fluorescent in situ hybridization
Clinical pathology
Clinical chemistry - Hematopathology - Transfusion medicine - Medical microbiology - Diagnostic immunology - ImmunopathologyEnzyme assay - Mass spectrometry - Chromatography - Flow cytometry - Blood bank - Microbiological culture - Serology
Specific conditions
Myocardial infarction
v • d • e Proteins: key methods of study
Experimental
Protein purification - Green fluorescent protein - Western blot - Protein immunostaining - Protein sequencing - Gel electrophoresis/Protein electrophoresis - Protein immunoprecipitation - Peptide mass fingerprinting
Bioinformatics
Protein structure prediction - Protein-protein docking - Protein structural alignment - Protein ontology - Protein-protein interaction prediction
Assay
Enzyme assay - Protein assay - Secretion assay
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2009-04-26T04:10:00.000-07:00

Enzyme inhibitor
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HIV protease in a complex with the protease inhibitor ritonavir. The structure of the protease is shown by the red, blue and yellow ribbons. The inhibitor is shown as the smaller ball-and-stick structure near the centre. Created from PDB 1HXW.
Enzyme inhibitors are molecules that bind to enzymes and decrease their activity. Since blocking an enzyme's activity can kill a pathogen or correct a metabolic imbalance, many drugs are enzyme inhibitors. They are also used as herbicides and pesticides. Not all molecules that bind to enzymes are inhibitors; enzyme activators bind to enzymes and increase their enzymatic activity.
The binding of an inhibitor can stop a substrate from entering the enzyme's active site and/or hinder the enzyme from catalysing its reaction. Inhibitor binding is either reversible or irreversible. Irreversible inhibitors usually react with the enzyme and change it chemically. These inhibitors modify key amino acid residues needed for enzymatic activity. In contrast, reversible inhibitors bind non-covalently and different types of inhibition are produced depending on whether these inhibitors bind the enzyme, the enzyme-substrate complex, or both.
Many drug molecules are enzyme inhibitors, so their discovery and improvement is an active area of research in biochemistry and pharmacology. A medicinal enzyme inhibitor is often judged by its specificity (its lack of binding to other proteins) and its potency (its dissociation constant, which indicates the concentration needed to inhibit the enzyme). A high specificity and potency ensure that a drug will have few side effects and thus low toxicity.
Enzyme inhibitors also occur naturally and are involved in the regulation of metabolism. For example, enzymes in a metabolic pathway can be inhibited by downstream products. This type of negative feedback slows flux through a pathway when the products begin to build up and is an important way to maintain homeostasis in a cell. Other cellular enzyme inhibitors are proteins that specifically bind to and inhibit an enzyme target. This can help control enzymes that may be damaging to a cell, such as proteases or nucleases; a well-characterised example is the ribonuclease inhibitor, which binds to ribonucleases in one of the tightest known protein–protein interactions.[1] Natural enzyme inhibitors can also be poisons and are used as defenses against predators or as ways of killing prey.

List of biomolecules

2009-04-23T02:33:00.000-07:00

[edit] A
For substances with an A- or α- prefix such as α-amylase, please see the parent page (in this case Amylase).
A23187 (Calcimycin, Calcium Ionophore)
Abamectin
Abietic acid
Acetic acid
Acetylcholine
Actin
Actinomycin D
Adenosine
Adenosine diphosphate (ADP)
Adenosine monophosphate (AMP)
Adenosine triphosphate (ATP)
Adenylate cyclase
Adonitol
Adrenaline, epinephrine
Adrenocorticotropic hormone (ACTH)
Aequorin
Aflatoxin
Agar
Alamethicin
Alanine
Albumins
Aldosterone
Aleurone
Alpha-amanitin
Allantoin
α-Amanatin, see Alpha-amanitin
Amino acid
Anabolic steroid
Anethole
Angiotensinogen
Amylase (also see α-amylase)
Angiotensinogen
Anisomycin
Antidiuretic hormone (ADH)
Arabinose
Arginine
Ascomycin
Ascorbic acid (vitamin C)
Asparagine
Aspartic acid
Asymmetric dimethylarginine
Atrial-natriuretic peptide (ANP)
Auxin
Azadirachtin A – C35H44O16

2009-04-18T10:40:00.000-07:00

[edit] Use of carbohydrates as an energy source
See also carbohydrate metabolism
Glucose is the major energy source in most life forms. For instance, polysaccharides are broken down into their monomers (glycogen phosphorylase removes glucose residues from glycogen). Disaccharides like lactose or sucrose are cleaved into their two component monosaccharides.

[edit] Glycolysis (anaerobic)
Glucose is mainly metabolized by a very important and ancient ten-step pathway called glycolysis, the net result of which is to break down one molecule of glucose into two molecules of pyruvate; this also produces a net two molecules of ATP, the energy currency of cells, along with two reducing equivalents in the form of converting NAD+ to NADH. This does not require oxygen; if no oxygen is available (or the cell cannot use oxygen), the NAD is restored by converting the pyruvate to lactate (lactic acid) (e. g. in humans) or to ethanol plus carbon dioxide (e. g. in yeast). Other monosaccharides like galactose and fructose can be converted into intermediates of the glycolytic pathway.

[edit] Aerobic
In aerobic cells with sufficient oxygen, like most human cells, the pyruvate is further metabolized. It is irreversibly converted to acetyl-CoA, giving off one carbon atom as the waste product carbon dioxide, generating another reducing equivalent as NADH. The two molecules acetyl-CoA (from one molecule of glucose) then enter the citric acid cycle, producing two more molecules of ATP, six more NADH molecules and two reduced (ubi)quinones (via FADH2 as enzyme-bound cofactor), and releasing the remaining carbon atoms as carbon dioxide. The produced NADH and quinol molecules then feed into the enzyme complexes of the respiratory chain, an electron transport system transferring the electrons ultimately to oxygen and conserving the released energy in the form of a proton gradient over a membrane (inner mitochondrial membrane in eukaryotes). Thereby, oxygen is reduced to water and the original electron acceptors NAD+ and quinone are regenerated. This is why humans breathe in oxygen and breathe out carbon dioxide. The energy released from transferring the electrons from high-energy states in NADH and quinol is conserved first as proton gradient and converted to ATP via ATP synthase. This generates an additional 28 molecules of ATP (24 from the 8 NADH + 4 from the 2 quinols), totaling to 32 molecules of ATP conserved per degraded glucose (two from glycolysis + two from the citrate cycle). It is clear that using oxygen to completely oxidize glucose provides an organism with far more energy than any oxygen-independent metabolic feature, and this is thought to be the reason why complex life appeared only after Earth's atmosphere accumulated large amounts of oxygen.

[edit] Gluconeogenesis
Main article: Gluconeogenesis
In vertebrates, vigorously contracting skeletal muscles (during weightlifting or sprinting, for example) do not receive enough oxygen to meet the energy demand, and so they shift to anaerobic metabolism, converting glucose to lactate. The liver regenerates the glucose, using a process called gluconeogenesis. This process is not quite the opposite of glycolysis, and actually requires three times the amount of energy gained from glycolysis (six molecules of ATP are used, compared to the two gained in glycolysis). Analogous to the above reactions, the glucose produced can then undergo glycolysis in tissues that need energy, be stored as glycogen (or starch in plants), or be converted to other monosaccharides or joined into di- or oligosaccharides. The combined pathways of glycolysis during exercise, lactate's crossing via the bloodstream to the liver, subsequent gluconeogenesis and release of glucose into the bloodstream is called the Cori cycle

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[edit] Carbohydrates
Main articles: Carbohydrates, Monosaccharides, Disaccharides, and Polysaccharides

A molecule of sucrose (glucose + fructose), a disaccharide.
Carbohydrates have monomers called monosaccharides. Some of these monosaccharides include glucose (C6H12O6), fructose (C6H12O6), and deoxyribose (C5H10O4). When two monosaccharides undergo dehydration synthesis, water is produced, as two hydrogen atoms and one oxygen atom are lost from the two monosaccharides' carboxyl group.

[edit] Lipids
Main articles: Lipids, Glycerol, and Fatty acids

A triglyceride with a glycerol molecule on the left and three fatty acids coming off it.
Lipids are usually made up of a molecule of glycerol and other molecules. In triglycerides, or the main lipid, there is one molecule of glycerol, and three fatty acids. Fatty acids are considered the monomer in that case, and could be saturated or unsaturated. Lipids, especially phospholipids, are also used in different pharmaceutical products, either as co-solubilisers e.g. in Parenteral infusions or else as drug carrier components (e.g. in a Liposome or Transfersome).

[edit] Proteins
Main articles: Proteins and Amino Acids

The general structure of an α-amino acid, with the amino group on the left and the carboxyl group on the right.
Proteins are large molecules, and have monomers of amino acids. There are 20 different known kinds of amino acids, and they contain a carboxyl group, an amino group, and an "R" group. The "R" group is what makes each amino acid different. When Amino acids combine, they form a special bond called a peptide bond, and become a polypeptide, or a protein.

[edit] Nucleic Acids
Main articles: Nucleic acid, DNA, RNA, and Nucleotides

The structure of deoxyribonucleic acid (DNA), the picture shows the monomers being put together.
Nucleic acids are very important in biochemistry, as they are what make up DNA, something all cellular organism use to store their genetic information. The most common nucleic acids are deoxyribonucleic acid and ribonucleic acid. Their monomers are called nucleotides. The most common nucleotides are called adenine, cytosine, guanine, thymine, and uracil. Adenine binds with thymine and uracil, thymine only binds with adenine, and cytosine and guanine can only bind with each other.

[edit] Carbohydrates
Main article: Carbohydrate
The function of carbohydrates includes energy storage and providing structure. Sugars are carbohydrates, but not all carbohydrates are sugars. There are more carbohydrates on Earth than any other known type of biomolecule; they are used to store energy and genetic information, as well as play important roles in cell to cell interactions and communications.

[edit] Monosaccharides

Glucose
The simplest type of carbohydrate is a monosaccharide, which among other properties contains carbon, hydrogen, and oxygen, mostly in a ratio of 1:2:1 (generalized formula CnH2nOn, where n is at least 3). Glucose, one of the most important carbohydrates, is an example of a monosaccharide. So is fructose, the sugar that gives fruits their sweet taste. Some carbohydrates (especially after condensation to oligo- and polysaccharides) contain less carbon relative to H and O, which still are present in 2:1 (H:O) ratio. Monosaccharides can be grouped into aldoses (having an aldehyde group at the end of the chain, e. g. glucose) and ketoses (having a keto group in their chain; e. g. fructose). Both aldoses and ketoses occur in an equilibrium between the open-chain forms and (starting with chain lengths of C4) cyclic forms. These are generated by bond formation between one of the hydroxyl groups of the sugar chain with the carbon of the aldehyde or keto group to form a hemiacetal bond. This leads to saturated five-membered (in furanoses) or six-membered (in pyranoses) heterocyclic rings containing one O as heteroatom.

[edit] Disaccharides

Sucrose: ordinary table sugar and probably the most familiar carbohydrate.
Two monosaccharides can be joined together using dehydration synthesis, in which a hydrogen atom is removed from the end of one molecule and a hydroxyl group (—OH) is removed from the other; the remaining residues are then attached at the sites from which the atoms were removed. The H—OH or H2O is then released as a molecule of water, hence the term dehydration. The new molecule, consisting of two monosaccharides, is called a disaccharide and is conjoined together by a glycosidic or ether bond. The reverse reaction can also occur, using a molecule of water to split up a disaccharide and break the glycosidic bond; this is termed hydrolysis. The most well-known disaccharide is sucrose, ordinary sugar (in scientific contexts, called table sugar or cane sugar to differentiate it from other sugars). Sucrose consists of a glucose molecule and a fructose molecule joined together. Another important disaccharide is lactose, consisting of a glucose molecule and a galactose molecule. As most humans age, the production of lactase, the enzyme that hydrolyzes lactose back into glucose and galactose, typically decreases. This results in lactase deficiency, also called lactose intolerance.
Sugar polymers are characterised by having reducing or non-reducing ends. A reducing end of a carbohydrate is a carbon atom which can be in equilibrium with the open-chain aldehyde or keto form. If the joining of monomers takes place at such a carbon atom, the free hydroxy group of the pyranose or furanose form is exchanged with an OH-side chain of another sugar, yielding a full acetal. This prevents opening of the chain to the aldehyde or keto form and renders the modified residue non-reducing. Lactose contains a reducing end at its glucose moiety, whereas the galactose moiety form a full acetal with the C4-OH group of glucose. Saccharose does not have a reducing end because of full acetal formation between the aldehyde carbon of glucose (C1) and the keto carbon of fructose (C2).

[edit] Oligosaccharides and polysaccharides

Cellulose as polymer of β-D-glucose
When a few (around three to six) monosaccharides are joined together, it is called an oligosaccharide (oligo- meaning "few"). These molecules tend to be used as markers and signals, as well as having some other uses. Many monosaccharides joined together make a polysaccharide. They can be joined together in one long linear chain, or they may be branched. Two of the most common polysaccharides are cellulose and glycogen, both consisting of repeating glucose monomers.
Cellulose is made by plants and is an important structural component of their cell walls. Humans can neither manufacture nor digest it.
Glycogen, on the other hand, is an animal carbohydrate; humans and other animals use it as a form of energy storage.