Streptokinase
Streptokinase is an extracellular metallo-enzyme produced by beta-haemolytic streptococcus and is used as an effective and cheap clot-dissolving medication in some cases of myocardial infarction (heart attack) and pulmonary embolism.
It belongs to a group of medications known as fibrinolytics, and works by activating plasminogen through cleavage to produce plasmin.
The half life of streptokinase is approximately 20 minutes (quoted in SPC).
Mechanism of action
Plasmin is produced in the blood to break down the major constituent of blood clots fibrin, therefore dissolving clots once they have fulfilled their purpose in stopping bleeding. Extra production of plasmin caused by streptokinase breaks down unwanted blood clots, for example, in the lungs (pulmonary embolism). Streptokinase forms a complex in the plasma with plasminogen to form an activator complex. This complex then forms plasmin from unbound plasminogen.[1]
Administration
It is given intravenously as soon as possible after the onset of a heart attack (acute phase - myocardial infarction) to dissolve clots in the arteries of the heart wall. This reduces the amount of damage to the heart muscle. Streptokinase is a bacterial product so the body will build up an immunity to it. It is recommended that this medication should not be used again after four days from the first administration, as it may not be as effective and can also cause an allergic reaction. For this reason, it is usually given only for a person's first heart attack. Further thrombotic events could be treated with tPA. Overdose of Streptokinase or tPA can be treated with Aminocaproic acid.
Saturday, February 7, 2009
Hybridoma Techniques Antibodies
Introduction to Hybridoma Techniques
Antibodies
highly specific proteins - bind to "antigens" specificity is a very important property of antibodies some uses of antibodies
Antibodies and therapy New Developments in Monoclonal Antibodies Are Hopeful
differentiate between very similar molecular structures detect presence of various molecular structures diagnose and treat certain cancers neutralize biological toxins (e.g., tetanus toxin) hundreds of clinical lab tests purify mixtures of molecular substances (affinity chromotography) identify microorganisms detect infected cells selectively eliminate cells enhance phagocytosis inhibit cellular interactions interfere with virus infections
antibodies are produced by stimulated B-lymphocytes
one B-lymphocyte secretes antibodies of only one specificity
desire large amounts of a single specific antibody
poly-clonal antibodies (produced in vivo)
mixture of many different antibodies of many different specificities many different B-lymphocytes producing antibodies limited time for antibody production (animal will eventually die) low levels of antibodies (animal normally produces only what is needed by that animal)
expensive to maintain animals
dependent on existing genetics of animal
cannot easily select antibodies with different biological properties
mono-clonal antibodies
eliminate mixture of antibodies by isolating B-lymphocytes in tissue culture
grow one isolated B-lymphocyte ---->one " clone" of identical B-cells
problems encountered
isolated B-lymphocyte will often not remain viable in culture don't divide very rapidly very little antibody is produced
need to "add"
viability in tissue culture rapid cell division increased antibody production
tumor cells
many types remain very viable in tissue culture most divide very rapidly but, DON'T produce any antibodies
Combine properties of
B-lymphocyte
+
Tumor Cell
produces antibodies
tissue culture viability rapid cell division
"fuse" B-lymphocyte + tumor cell into one cell
---> “hybrid" cell
culture hybrid cell
continue to grow indefinitely in culture rapid cell division (produces one "clone") antibody production (by one[mono] clone) large amounts of antibodies secreted can manipulate genetics of isolated lymphocytes can select antibodies with certain biological properties much less expensive than maintaining animals
Antibodies
highly specific proteins - bind to "antigens" specificity is a very important property of antibodies some uses of antibodies
Antibodies and therapy New Developments in Monoclonal Antibodies Are Hopeful
differentiate between very similar molecular structures detect presence of various molecular structures diagnose and treat certain cancers neutralize biological toxins (e.g., tetanus toxin) hundreds of clinical lab tests purify mixtures of molecular substances (affinity chromotography) identify microorganisms detect infected cells selectively eliminate cells enhance phagocytosis inhibit cellular interactions interfere with virus infections
antibodies are produced by stimulated B-lymphocytes
one B-lymphocyte secretes antibodies of only one specificity
desire large amounts of a single specific antibody
poly-clonal antibodies (produced in vivo)
mixture of many different antibodies of many different specificities many different B-lymphocytes producing antibodies limited time for antibody production (animal will eventually die) low levels of antibodies (animal normally produces only what is needed by that animal)
expensive to maintain animals
dependent on existing genetics of animal
cannot easily select antibodies with different biological properties
mono-clonal antibodies
eliminate mixture of antibodies by isolating B-lymphocytes in tissue culture
grow one isolated B-lymphocyte ---->one " clone" of identical B-cells
problems encountered
isolated B-lymphocyte will often not remain viable in culture don't divide very rapidly very little antibody is produced
need to "add"
viability in tissue culture rapid cell division increased antibody production
tumor cells
many types remain very viable in tissue culture most divide very rapidly but, DON'T produce any antibodies
Combine properties of
B-lymphocyte
+
Tumor Cell
produces antibodies
tissue culture viability rapid cell division
"fuse" B-lymphocyte + tumor cell into one cell
---> “hybrid" cell
culture hybrid cell
continue to grow indefinitely in culture rapid cell division (produces one "clone") antibody production (by one[mono] clone) large amounts of antibodies secreted can manipulate genetics of isolated lymphocytes can select antibodies with certain biological properties much less expensive than maintaining animals
PRODUCTION OF VACCINES
PRODUCTION OF VACCINES
3.1 Process of Vaccine Production
Materials for vaccines are generally pathogens, bacteria and virus,
use infectious diseases. The quality control of vaccines is most important and regulated by the Minimum Requirements for Biological Products. Each vaccine is controlled by the standards for biological substance production and specific processes and techniques are requested. Vaccines being used in Japan should clear these regulations and pass the tests by the National Institute of health (N.I.H.). Vaccines are classified into live vaccines, inactivated vaccines, and toxoids, as shown in Table 3.1. Recently, recombinant vaccines have been developed using the DNA recombination technique. Processes of the vaccine production are shown in Figures 3.1, 3.2, and 3.3.
3.1.1 Materials for vaccines
a. Vaccine strain: A specified strain of pathogen is nominated as the vaccine strain by the Standard. Influenza virus, however, mutate easily, so the vaccine strain is decided every year by N.I.H. according to the prevalence strain isolated from patients, antigenic analysis, and antibodies found in the patients' blood. The vaccine strain of the year is distributed by N.I.H. Vaccine strains for the live vaccines are kept as the seed lots in N.I.H. The number of transfers of the culture from the seed lot is limited 5 at the maximum.
Media for Vaccine Production
Cultured cells have long been known to serve as excellent hosts for propagation of many types of viruses. The ability of cell culture systems to produce large quantities of attenuated viral particles has served as the basis for the production of both human and veterinary vaccines. Traditional methods have relied on the production of viral agents in cells cultured in medium supplemented with serum, most commonly fetal bovine serum. The animal serum in cell cultures used in production processes can cause a number of problems for manufacturers. The increased costs of raw materials and post-production processing associated with serum have prompted interest in the development of serum-free media for vaccine production. More recently, the potential for contamination by adventitious agents present in serum has heightened regulatory concerns regarding the use of animal-derived components in media used for pharmaceutical manufacturing. With these factors in mind, Sigma has developed new media for vaccine production with reduced levels or completely devoid of animal-derived components. At the same time, we will continue our commitment to traditional manufacturing methods by maintaining and expanding the range of basal media for use with serum supplementation and serve as the basis for customization into serum-free formulations.
The ability to genetically engineer viral particles for use as therapeutic agents to treat genetic diseases (gene therapy) represents one exciting new avenue that science and technology are bringing to modern medicine. Cultured cells have long been known to serve as excellent hosts for the propagation of many types of viruses. Traditional methods have relied on the growth of viral agents in cells cultured in serum-supplemented media. A great concern is the potential for contamination by adventitious agents introduced into the manufacturing process through the use of animal-derived materials. This has heightened regulatory concerns regarding the use of such components in media employed in pharmaceutical manufacturing, particularly in the case of therapeutic injectables. These concerns have led to recommendations that all animal-derived components be avoided when therapeutic agents are manufactured. It is likely that these recommendations will become rigid requirements in the near future. With these factors in mind, Sigma has developed new media tailored to the needs of two of the more popular cell lines used for the propagation of viral particles employed in gene therapy. These media are formulated without the use of animal-derived components.
Vaccine Production in Cells
For decades, vaccines have provided effective protection from influenza for Americans. While they have traditionally been produced in chicken eggs, a new technology-cell-based vaccine production-could save hundreds of thousands of lives in the event of an outbreak of pandemic influenza, or some other infectious disease.
The new approach would use mammalian cells (kidney cells are often used) to grow the influenza viruses. Cell-based vaccine production could more easily meet "surge capacity needs" because cells could be frozen and stored in advance of an epidemic or developed rapidly in response to an epidemic. Cell-based vaccine production dramatically reduces the possibility for contamination and promises to be more reliable, flexible, and expandable than egg-based methods.
In place of eggs, cell-based vaccine production utilizes laboratory-grown cell lines that are capable of hosting a growing virus. The virus is injected into the cells where it multiplies. The cells' outer walls are removed, harvested, purified, and inactivated. A vaccine can be produced in a matter of weeks. Polio vaccine is currently produced using the cell-based method.
While both methods could produce an equally effective vaccine against a virus such as H5N1, egg-based production is physically limited by the availability of specialized eggs and alone may not be able to meet the accelerated demands of a global influenza pandemic. Cell-based vaccines offer the potential to increase production surge capacity and save lives:
• In order to produce 300 million doses of vaccine, egg-based production would require some 900 million eggs. In the case of an avian flu pandemic, egg-producing flocks could decline, jeopardizing vaccine production capabilities.
• While eggs are perishable, cell lines can be safely kept frozen indefinitely, increasing the capability to rapidly produce vaccines if an influenza pandemic were to occur.
• Vaccine manufacturers are able to bypass the steps needed to adapt the virus strains to grow in eggs
• People allergic to eggs cannot receive vaccines produced from chicken eggs, but can be immunized with a cell-based vaccine.
In March 2005, the Department of Health and Human Services issued a five-year contract to Sanofi-Pasteur for $97.1 million to develop cell-based influenza vaccine technology and conduct clinical trials, with the goal of obtaining an FDA license for this vaccine. Under this advanced development contract, the company has committed to develop a plan to establish a U.S. cell-based influenza vaccine manufacturing facility, capable of producing at least 300 million doses of a pandemic influenza vaccine over a one year period.
In May 2006, HHS awarded five contracts totaling more than $1 billion to accelerate development and production of new technologies for influenza vaccines within the U.S. These five contracts support the advanced development of cell-based production technologies for influenza vaccines and will help to modernize and strengthen the nation's influenza vaccine production by creating an alternative to producing influenza vaccines in eggs. The funds are part of $3.3 billion proposed by the President and appropriated by Congress to HHS for fiscal year 2006 to help the nation prepare for a pandemic. (News release).
There is no H5N1 pandemic so it has not been possible to develop an H5N1 pandemic vaccine; however, "pre-pandemic vaccines" have been created, are being refined and tested, and do have some promise both in furthering research and preparedness for a possible pandemic.
3.1 Process of Vaccine Production
Materials for vaccines are generally pathogens, bacteria and virus,
use infectious diseases. The quality control of vaccines is most important and regulated by the Minimum Requirements for Biological Products. Each vaccine is controlled by the standards for biological substance production and specific processes and techniques are requested. Vaccines being used in Japan should clear these regulations and pass the tests by the National Institute of health (N.I.H.). Vaccines are classified into live vaccines, inactivated vaccines, and toxoids, as shown in Table 3.1. Recently, recombinant vaccines have been developed using the DNA recombination technique. Processes of the vaccine production are shown in Figures 3.1, 3.2, and 3.3.
3.1.1 Materials for vaccines
a. Vaccine strain: A specified strain of pathogen is nominated as the vaccine strain by the Standard. Influenza virus, however, mutate easily, so the vaccine strain is decided every year by N.I.H. according to the prevalence strain isolated from patients, antigenic analysis, and antibodies found in the patients' blood. The vaccine strain of the year is distributed by N.I.H. Vaccine strains for the live vaccines are kept as the seed lots in N.I.H. The number of transfers of the culture from the seed lot is limited 5 at the maximum.
Media for Vaccine Production
Cultured cells have long been known to serve as excellent hosts for propagation of many types of viruses. The ability of cell culture systems to produce large quantities of attenuated viral particles has served as the basis for the production of both human and veterinary vaccines. Traditional methods have relied on the production of viral agents in cells cultured in medium supplemented with serum, most commonly fetal bovine serum. The animal serum in cell cultures used in production processes can cause a number of problems for manufacturers. The increased costs of raw materials and post-production processing associated with serum have prompted interest in the development of serum-free media for vaccine production. More recently, the potential for contamination by adventitious agents present in serum has heightened regulatory concerns regarding the use of animal-derived components in media used for pharmaceutical manufacturing. With these factors in mind, Sigma has developed new media for vaccine production with reduced levels or completely devoid of animal-derived components. At the same time, we will continue our commitment to traditional manufacturing methods by maintaining and expanding the range of basal media for use with serum supplementation and serve as the basis for customization into serum-free formulations.
The ability to genetically engineer viral particles for use as therapeutic agents to treat genetic diseases (gene therapy) represents one exciting new avenue that science and technology are bringing to modern medicine. Cultured cells have long been known to serve as excellent hosts for the propagation of many types of viruses. Traditional methods have relied on the growth of viral agents in cells cultured in serum-supplemented media. A great concern is the potential for contamination by adventitious agents introduced into the manufacturing process through the use of animal-derived materials. This has heightened regulatory concerns regarding the use of such components in media employed in pharmaceutical manufacturing, particularly in the case of therapeutic injectables. These concerns have led to recommendations that all animal-derived components be avoided when therapeutic agents are manufactured. It is likely that these recommendations will become rigid requirements in the near future. With these factors in mind, Sigma has developed new media tailored to the needs of two of the more popular cell lines used for the propagation of viral particles employed in gene therapy. These media are formulated without the use of animal-derived components.
Vaccine Production in Cells
For decades, vaccines have provided effective protection from influenza for Americans. While they have traditionally been produced in chicken eggs, a new technology-cell-based vaccine production-could save hundreds of thousands of lives in the event of an outbreak of pandemic influenza, or some other infectious disease.
The new approach would use mammalian cells (kidney cells are often used) to grow the influenza viruses. Cell-based vaccine production could more easily meet "surge capacity needs" because cells could be frozen and stored in advance of an epidemic or developed rapidly in response to an epidemic. Cell-based vaccine production dramatically reduces the possibility for contamination and promises to be more reliable, flexible, and expandable than egg-based methods.
In place of eggs, cell-based vaccine production utilizes laboratory-grown cell lines that are capable of hosting a growing virus. The virus is injected into the cells where it multiplies. The cells' outer walls are removed, harvested, purified, and inactivated. A vaccine can be produced in a matter of weeks. Polio vaccine is currently produced using the cell-based method.
While both methods could produce an equally effective vaccine against a virus such as H5N1, egg-based production is physically limited by the availability of specialized eggs and alone may not be able to meet the accelerated demands of a global influenza pandemic. Cell-based vaccines offer the potential to increase production surge capacity and save lives:
• In order to produce 300 million doses of vaccine, egg-based production would require some 900 million eggs. In the case of an avian flu pandemic, egg-producing flocks could decline, jeopardizing vaccine production capabilities.
• While eggs are perishable, cell lines can be safely kept frozen indefinitely, increasing the capability to rapidly produce vaccines if an influenza pandemic were to occur.
• Vaccine manufacturers are able to bypass the steps needed to adapt the virus strains to grow in eggs
• People allergic to eggs cannot receive vaccines produced from chicken eggs, but can be immunized with a cell-based vaccine.
In March 2005, the Department of Health and Human Services issued a five-year contract to Sanofi-Pasteur for $97.1 million to develop cell-based influenza vaccine technology and conduct clinical trials, with the goal of obtaining an FDA license for this vaccine. Under this advanced development contract, the company has committed to develop a plan to establish a U.S. cell-based influenza vaccine manufacturing facility, capable of producing at least 300 million doses of a pandemic influenza vaccine over a one year period.
In May 2006, HHS awarded five contracts totaling more than $1 billion to accelerate development and production of new technologies for influenza vaccines within the U.S. These five contracts support the advanced development of cell-based production technologies for influenza vaccines and will help to modernize and strengthen the nation's influenza vaccine production by creating an alternative to producing influenza vaccines in eggs. The funds are part of $3.3 billion proposed by the President and appropriated by Congress to HHS for fiscal year 2006 to help the nation prepare for a pandemic. (News release).
There is no H5N1 pandemic so it has not been possible to develop an H5N1 pandemic vaccine; however, "pre-pandemic vaccines" have been created, are being refined and tested, and do have some promise both in furthering research and preparedness for a possible pandemic.
Protease
Protease
A protease is any enzyme that conducts proteolysis, that is, begins protein catabolism by hydrolysis of the peptide bonds that link amino acids together in the polypeptide chain.
Classification
Proteases are currently classified into six groups:
• Serine proteases
• Threonine proteases
• Cysteine proteases
• Aspartic acid proteases
• Metalloproteases
• Glutamic acid proteases
The threonine and glutamic acid proteases were not described until 1995 and 2004, respectively. The mechanism used to cleave a peptide bond involves making an amino acid residue that has the cysteine and threonine (peptidases) or a water molecule (aspartic acid, metallo- and glutamic acid peptidases) nucleophilic so that it can attack the peptide carbonyl group. One way to make a nucleophile is by a catalytic triad, where a histidine residue is used to activate serine, cysteine, or threonine as a nucleophile.
Occurrence
Proteases occur naturally in all organisms. These enzymes are involved in a multitude of physiological reactions from simple digestion of food proteins to highly-regulated cascades (e.g., the blood-clotting cascade, the complement system, apoptosis pathways, and the invertebrate prophenoloxidase-activating cascade). Peptidases can either break specific peptide bonds (limited proteolysis), depending on the amino acid sequence of a protein, or break down a complete peptide to amino acids (unlimited proteolysis). The activity can be a destructive change, abolishing a protein's function or digesting it to its principal components; it can be an activation of a function, or it can be a signal in a signaling pathway.
Bacteria also secrete proteases to hydrolyse the peptide bonds in proteins and therefore break the proteins down into their constituent monomers.
Proteases are also a type of exotoxin, which is a virulence factor in bacteria pathogenesis. Bacteria exotoxic proteases destroy extracellular structures. Protease enzymes are also found used extensively in the bread industry in Bread improver.
Proteases, also known as proteinases or proteolytic enzymes, are a large group of enzymes. Proteases belong to the class of enzymes known as hydrolases, which catalyse the reaction of hydrolysis of various bonds with the participation of a water molecule.
Proteases are involved in digesting long protein chains into short fragments, splitting the peptide bonds that link amino acid residues. Some of them can detach the terminal amino acids from the protein chain (exopeptidases, such as aminopeptidases, carboxypeptidase www A); the others attack internal peptide bonds of a protein (endopeptidases, such as trypsin, chymotrypsin, pepsin, papain, elastase).
Proteases are divided into four major groups according to the character of their catalytic active site and conditions of action: serine proteinases, cysteine (thiol) proteinases, aspartic proteinases, and metalloproteinases. Attachment of a protease to a certain group depends on the structure of catalytic site and the amino acid (as one of the constituents) essential for its activity.
Proteases are used throughout an organism for various metabolic processes. Acid proteases secreted into the stomach (such as pepsin) and serine proteases present in duodenum (trypsin and chymotrypsin) enable us to digest the protein in food; proteases present in blood serum (thrombin, plasmin, Hageman factor, etc.) play important role in blood-clotting, as well as lysis of the clots, and the correct action of the immune system. Other proteases are present in leukocytes (elastase, cathepsin G) and play several different roles in metabolic control. Proteases determine the lifetime of other proteins playing important physiological role like hormones, antibodies, or other enzymes -- this is one of the fastest "switching on" and "switching off" regulatory mechanisms in the physiology of an organism. By complex cooperative action the proteases may proceed as cascade reactions, which result in rapid and efficient amplification of an organism's response to a physiological signal.
Inhibitors
The function of peptidases is inhibited by protease inhibitor enzymes. Examples of protease inhibitors are the class of serpins (serine protease or peptidase inhibitors), incorporating alpha 1-antitrypsin. Other serpins are complement 1-inhibitor, antithrombin, alpha 1-antichymotrypsin, plasminogen activator inhibitor 1 (coagulation, fibrinolysis) and the recently discovered neuroserpin.
Natural protease inhibitors include the family of lipocalin proteins, which play a role in cell regulation and differentiation. Lipophilic ligands, attached to lipocalin proteins, have been found to possess tumor protease inhibiting properties. The natural protease inhibitors are not to be confused with the protease inhibitors used in antiretroviral therapy. Some viruses, with HIV among them, depend on proteases in their reproductive cycle. Thus, protease inhibitors are developed as antiviral means.
Degradation
Proteases, being themselves proteins, are known to be cleaved by other protease molecules, sometimes of the same variety. This may be an important method of regulation of peptidase activity.
Serine protease
Crystal structure of Trypsin, a typical serine protease.
Serine proteases or serine endopeptidases (newer name) are proteases (enzymes that cut peptide bonds in proteins) in which one of the amino acids at the active site is serine.
They are found in both single-cell and complex organisms, in both cells with nuclei (eukaryotes) and without nuclei (prokaryotes).
Serine proteases are grouped into clans that share structural similarities (homology) and are then further subgrouped into families with simiar sequences.
The major clans found in humans include the chymotrypsin-like, the subtilisin-like, the alpha/beta hydrolase, and signal peptidase clans.
In evolutionary history, serine proteases were originally digestive enzymes. In mammals, they evolved by gene duplication to serve functions in blood clotting, the immune system, and inflammation.
Serine proteases are paired with serine protease inhibitors, which turn off their activity when they are no longer needed.[1]
Digestive serine proteases
Members
Chymotrypsin-clan
The three serine proteases of the chymotrypsin-like clan that have been studied in greatest detail are chymotrypsin, trypsin, and elastase. All three enzymes are synthesized by the pancreatic acinar cells, secreted in the small intestine, and are responsible for catalyzing the hydrolysis of peptide bonds. All three of these enzymes are similar in structure, as shown through their X-ray structures. The differing aspect lies in the peptide bond that is being cleaved; this is called the scissile bond. The different enzymes, like most enzymes, are highly specific in the reactions they catalyze. Each of these digestive serine proteases targets different regions of a polypeptide chain, based upon the side chains of the amino acid residues surrounding the site of cleavage:
• Chymotrypsin is responsible for cleaving peptide bonds following a bulky hydrophobic amino acid residue. Preferred residues include phenylalanine, tryptophan, and tyrosine, which fit into a snug hydrophobic pocket.
• Trypsin is responsible for cleaving peptide bonds following a positively-charged amino acid residue. Instead of having the hydrophobic pocket of the chymotrypsin, there exists an aspartic acid residue at the base of the pocket. This can then interact with positively-charged residues such as arginine and lysine on the substrate peptide to be cleaved.
• Elastase is responsible for cleaving peptide bonds following a small neutral amino acid residue, such as Alanine, glycine, and valine. (These amino acid residues form much of the connective tissues in meat). The pocket that is in "trypsin" and "chymotrypsin" is now partially filled with valine and threonine, rendering it a mere depression, which can accommodate these smaller amino acid residues.
The combination of these three enzymes make an incredibly effective digestive team and are primarily responsible for the digestion of proteins.
Subtilisin
Subtilisin is a serine protease in prokaryotes. Subtilisin is evolutionary unrelated to the chymotrypsin-clan, but shares the same catalytic mechanism utilising a catalytic triad, to create a nucleophilic serine. This is the classic example used to illustrate convergent evolution, since the same mechanism evolved twice independently during evolution.
Catalytic mechanism
The main player in the catalytic mechanism in the chymotrypsin and subtillisin clan enzymes mentioned above is the catalytic triad. The triad is located in the active site of the enzyme, where catalysis occurs, and is preserved in all serine protease enzymes. The triad is a coordinated structure consisting of three essential amino acids: histidine (His 57), serine (Ser 195) (hence the name "serine protease") and aspartic acid (Asp 102). Located very near one another near the heart of the enzyme, these three key amino acids each play an essential role in the cleaving ability of the proteases.
In the event of catalysis, an ordered mechanism occurs in which several intermediates are generated. The catalysis of the peptide cleavage can be seen as a ping-pong catalysis, in which a substrate binds (in this case, the polypeptide being cleaved), a product is released (the N-terminus "half" of the peptide), another substrate binds (in this case, water), and another product is released (the C-terminus "half" of the peptide).
Each amino acid in the triad performs a specific task in this process:
*The serine has an -OH group that is able to act as a nucleophile, attacking the carbonyl carbon of the scissile peptide bond of the substrate.
• A pair of electrons on the histidine nitrogen has the ability to accept the hydrogen from the serine -OH group, thus coordinating the attack of the peptide bond.
• The carboxyl group on the aspartic acid in turn hydrogen bonds with the histidine, making the pair of electrons mentioned above much more electronegative.
The whole reaction can be summarized as follows:
• The polypeptide substrate binds to the surface of the serine protease enzyme such that scissile bond is inserted into the active site of the enzyme, with the carbonyl carbon of this bond positioned near the nucleophilic serine.
• The serine -OH attacks the carbonyl carbon, and the nitrogen of the histidine accepts the hydrogen from the -OH of the [serine] and a pair of electrons from the double bond of the carbonyl oxygen moves to the oxygen. As a result, a tetrahedral intermediate is generated.
• The bond joining the nitrogen and the carbon in the peptide bond is now broken. The covalent electrons creating this bond move to attack the hydrogen of the histidine, breaking the connection. The electrons that previously moved from the carbonyl oxygen double bond move back from the negative oxygen to recreate the bond, generating an acyl-enzyme intermediate.
• Now, water comes in to the reaction. Water replaces the N-terminus of the cleaved peptide, and attacks the carbonyl carbon. Once again, the electrons from the double bond move to the oxygen making it negative, as the bond between the oxygen of the water and the carbon is formed. This is coordinated by the nitrogen of the histidine. which accepts a proton from the water. Overall, this generates another tetrahedral intermediate.
• In a final reaction, the bond formed in the first step between the serine and the carbonyl carbon moves to attack the hydrogen that the histidine just acquired. The now electron-deficient carbonyl carbon re-forms the double bond with the oxygen. As a result, the C-terminus of the peptide is now ejected.
Additional stabilizing effects
It was discovered that additional amino acids of the protease, Gly 193 and Ser 195, are involved in creating what is called an oxyanion hole. Both Gly 193 and Ser 195 can donate backbone hydrogens for hydrogen bonding. When the tetrahedral intermediate of step 1 and step 3 are generated, the negative oxygen ion, having accepted the electrons from the carbonyl double bond fits perfectly into the oxyanion hole. In effect, serine proteases preferentially bind the transition state and the overall structure is favored, lowering the activation energy of the reaction. This "preferential binding" is responsible for much of the catalytic efficiency of the enzyme.
Zymogens
There are certain inhibitors which resemble the tetrahedral intermediate, and thus fill up the active site, preventing the enzyme from working properly. Trypsin, a powerful digestive enzyme, is generated in the pancreas. Inhibitors prevent self-digestion of the pancreas itself.
Zymogens are the usually inactive precursors of an enzyme. If the digestive enzymes were active when synthesized, they would immediately start chewing up the synthesizing organs and tissues. Acute pancreatitis is such a condition, in which there is premature activation of the digestive enzymes in the pancreas, resulting in self-digestion (autolysis). It also complicates postmortem investigations, as the pancreas often digests itself before it can be assessed visually.
Zymogens are large, inactive structures, which have the ability to break apart or change into the smaller activated enzymes. The difference between zymogens and the activated enzymes lies in the fact that the active site for catalysis of the zymogens is distorted. As a result, the substrate polypeptide cannot bind effectively, and proteolysis does not occur. Only after activation, during which the conformation and structure of the zymogen change and the active site is opened, can proteolysis occur.
Zymogen Enzyme Notes
Trypsinogen trypsin
When trypsinogen enters the small intestine from the pancreas, secretions from the duodenal mucosa cleaves the lysine 15 - isoleucine 16 peptide bond of the zymogen. As a result, the zymogen trypsinogen breaks down into trypsin. Recall that trypsin is also responsible for cleaving lysine peptide bonds, and thus, once a small amount of trypsin is generated, it participates in cleavage of its own zymogen, generating even more trypsin. The process of trypsin activation can thus be called autocatalytic.
Chymotrypsinogen chymotrypsin
After the Arg 15 - Ile 16 bond in the chymotrypsinogen zymogen is cleaved by trypsin, the newly generated structure called a pi-chymotrypsin undergoes autolysis (self digestion), yielding active chymotrypsin.
Proelastase elastase
It is activated by cleavage through trypsin.
As can be seen, trypsinogen activation to trypsin is essential, because it activates its own reaction, as well as the reaction of both chymotrypsin and elastase. It is therefore essential that this activation doesn't occur prematurely. There are several protective measures taken by the organism to prevent self-digestion:
• The activation of trypsinogen by trypsin is relatively slow
• The zymogens are stored in zymogen granules, capsules that have walls that are thought to be resistant to proteolysis.
Inhibition
Serine proteases are inhibited by a diverse group of inhibitors, including synthetic chemical inhibitors for research or therapeutic purposes, and also natural proteinaceous inhibitors. One family of natural inhibitors called "serpins" (abbreviated from serine protease inhibitors) can form a covalent bond with the serine protease, inhibiting its function. The best-studied serpins are antithrombin and alpha 1-antitrypsin, studied for their role in coagulation/thrombosis and emphysema/A1AT respectively. Artificial irreversible small molecule inhibitors include AEBSF and PMSF.
Role in disease
Mutations may lead to decreased or increased activity of enzymes. This may have different consequences, depending on the normal function of the serine protease. For example, mutations in protein C, when leading to insufficient protein levels or activity, predispose to thrombosis.
Diagnostic use
Determination of serine protease levels may be useful in the context of particular diseases.
• Coagulation factor levels may be required in the diagnosis of hemorrhagic or thrombotic conditions.
• Fecal elastase is employed to determine the exocrine activity of the pancreas, e.g. in cystic fibrosis or chronic pancreatitis.
• Prostate specific antigen is used to determine prostate cancer risk
Aspartate protease
Aspartic proteases are a family of eukaryotic protease enzymes that utilize an aspartate residue for catalysis of their peptide substrates. In general, they have two highly-conserved aspartates in the active site and are optimally active at acidic pH. Nearly all known aspartyl proteases are inhibited by pepstatin.
Eukaryotic aspartic proteases include pepsins, cathepsins, and renins. They have a two-domain structure, probably arising from ancestral duplication. Retroviral and retrotransposon proteases (Pfam PF00077) are much smaller and appear to be homologous to a single domain of the eukaryotic aspartyl proteases.
Examples
• HIV-1 protease - a major drug-target for treatment of HIV
• Chymosin (or "rennin", with two "n"s)
• Renin (with one "n")
• Cathepsin D
• Pepsin
• Plasmepsin
Mechanism
Proposed mechanism of peptide cleavage by aspartyl proteases[1]
While a number of different mechanisms for aspartyl proteases have been proposed, the most widely accepted is a general acid-base mechanism involving coordination of a water molecule between the two highly-conserved aspartate residues.[1][2] One aspartate activates the water by abstracting a proton, enabling the water to attack the carbonyl carbon of the substrate scissile bond, generating a tetrahedral oxyanion intermediate. Rearrangement of this intermediate leads to protonation of the scissile amide.
A protease is any enzyme that conducts proteolysis, that is, begins protein catabolism by hydrolysis of the peptide bonds that link amino acids together in the polypeptide chain.
Classification
Proteases are currently classified into six groups:
• Serine proteases
• Threonine proteases
• Cysteine proteases
• Aspartic acid proteases
• Metalloproteases
• Glutamic acid proteases
The threonine and glutamic acid proteases were not described until 1995 and 2004, respectively. The mechanism used to cleave a peptide bond involves making an amino acid residue that has the cysteine and threonine (peptidases) or a water molecule (aspartic acid, metallo- and glutamic acid peptidases) nucleophilic so that it can attack the peptide carbonyl group. One way to make a nucleophile is by a catalytic triad, where a histidine residue is used to activate serine, cysteine, or threonine as a nucleophile.
Occurrence
Proteases occur naturally in all organisms. These enzymes are involved in a multitude of physiological reactions from simple digestion of food proteins to highly-regulated cascades (e.g., the blood-clotting cascade, the complement system, apoptosis pathways, and the invertebrate prophenoloxidase-activating cascade). Peptidases can either break specific peptide bonds (limited proteolysis), depending on the amino acid sequence of a protein, or break down a complete peptide to amino acids (unlimited proteolysis). The activity can be a destructive change, abolishing a protein's function or digesting it to its principal components; it can be an activation of a function, or it can be a signal in a signaling pathway.
Bacteria also secrete proteases to hydrolyse the peptide bonds in proteins and therefore break the proteins down into their constituent monomers.
Proteases are also a type of exotoxin, which is a virulence factor in bacteria pathogenesis. Bacteria exotoxic proteases destroy extracellular structures. Protease enzymes are also found used extensively in the bread industry in Bread improver.
Proteases, also known as proteinases or proteolytic enzymes, are a large group of enzymes. Proteases belong to the class of enzymes known as hydrolases, which catalyse the reaction of hydrolysis of various bonds with the participation of a water molecule.
Proteases are involved in digesting long protein chains into short fragments, splitting the peptide bonds that link amino acid residues. Some of them can detach the terminal amino acids from the protein chain (exopeptidases, such as aminopeptidases, carboxypeptidase www A); the others attack internal peptide bonds of a protein (endopeptidases, such as trypsin, chymotrypsin, pepsin, papain, elastase).
Proteases are divided into four major groups according to the character of their catalytic active site and conditions of action: serine proteinases, cysteine (thiol) proteinases, aspartic proteinases, and metalloproteinases. Attachment of a protease to a certain group depends on the structure of catalytic site and the amino acid (as one of the constituents) essential for its activity.
Proteases are used throughout an organism for various metabolic processes. Acid proteases secreted into the stomach (such as pepsin) and serine proteases present in duodenum (trypsin and chymotrypsin) enable us to digest the protein in food; proteases present in blood serum (thrombin, plasmin, Hageman factor, etc.) play important role in blood-clotting, as well as lysis of the clots, and the correct action of the immune system. Other proteases are present in leukocytes (elastase, cathepsin G) and play several different roles in metabolic control. Proteases determine the lifetime of other proteins playing important physiological role like hormones, antibodies, or other enzymes -- this is one of the fastest "switching on" and "switching off" regulatory mechanisms in the physiology of an organism. By complex cooperative action the proteases may proceed as cascade reactions, which result in rapid and efficient amplification of an organism's response to a physiological signal.
Inhibitors
The function of peptidases is inhibited by protease inhibitor enzymes. Examples of protease inhibitors are the class of serpins (serine protease or peptidase inhibitors), incorporating alpha 1-antitrypsin. Other serpins are complement 1-inhibitor, antithrombin, alpha 1-antichymotrypsin, plasminogen activator inhibitor 1 (coagulation, fibrinolysis) and the recently discovered neuroserpin.
Natural protease inhibitors include the family of lipocalin proteins, which play a role in cell regulation and differentiation. Lipophilic ligands, attached to lipocalin proteins, have been found to possess tumor protease inhibiting properties. The natural protease inhibitors are not to be confused with the protease inhibitors used in antiretroviral therapy. Some viruses, with HIV among them, depend on proteases in their reproductive cycle. Thus, protease inhibitors are developed as antiviral means.
Degradation
Proteases, being themselves proteins, are known to be cleaved by other protease molecules, sometimes of the same variety. This may be an important method of regulation of peptidase activity.
Serine protease
Crystal structure of Trypsin, a typical serine protease.
Serine proteases or serine endopeptidases (newer name) are proteases (enzymes that cut peptide bonds in proteins) in which one of the amino acids at the active site is serine.
They are found in both single-cell and complex organisms, in both cells with nuclei (eukaryotes) and without nuclei (prokaryotes).
Serine proteases are grouped into clans that share structural similarities (homology) and are then further subgrouped into families with simiar sequences.
The major clans found in humans include the chymotrypsin-like, the subtilisin-like, the alpha/beta hydrolase, and signal peptidase clans.
In evolutionary history, serine proteases were originally digestive enzymes. In mammals, they evolved by gene duplication to serve functions in blood clotting, the immune system, and inflammation.
Serine proteases are paired with serine protease inhibitors, which turn off their activity when they are no longer needed.[1]
Digestive serine proteases
Members
Chymotrypsin-clan
The three serine proteases of the chymotrypsin-like clan that have been studied in greatest detail are chymotrypsin, trypsin, and elastase. All three enzymes are synthesized by the pancreatic acinar cells, secreted in the small intestine, and are responsible for catalyzing the hydrolysis of peptide bonds. All three of these enzymes are similar in structure, as shown through their X-ray structures. The differing aspect lies in the peptide bond that is being cleaved; this is called the scissile bond. The different enzymes, like most enzymes, are highly specific in the reactions they catalyze. Each of these digestive serine proteases targets different regions of a polypeptide chain, based upon the side chains of the amino acid residues surrounding the site of cleavage:
• Chymotrypsin is responsible for cleaving peptide bonds following a bulky hydrophobic amino acid residue. Preferred residues include phenylalanine, tryptophan, and tyrosine, which fit into a snug hydrophobic pocket.
• Trypsin is responsible for cleaving peptide bonds following a positively-charged amino acid residue. Instead of having the hydrophobic pocket of the chymotrypsin, there exists an aspartic acid residue at the base of the pocket. This can then interact with positively-charged residues such as arginine and lysine on the substrate peptide to be cleaved.
• Elastase is responsible for cleaving peptide bonds following a small neutral amino acid residue, such as Alanine, glycine, and valine. (These amino acid residues form much of the connective tissues in meat). The pocket that is in "trypsin" and "chymotrypsin" is now partially filled with valine and threonine, rendering it a mere depression, which can accommodate these smaller amino acid residues.
The combination of these three enzymes make an incredibly effective digestive team and are primarily responsible for the digestion of proteins.
Subtilisin
Subtilisin is a serine protease in prokaryotes. Subtilisin is evolutionary unrelated to the chymotrypsin-clan, but shares the same catalytic mechanism utilising a catalytic triad, to create a nucleophilic serine. This is the classic example used to illustrate convergent evolution, since the same mechanism evolved twice independently during evolution.
Catalytic mechanism
The main player in the catalytic mechanism in the chymotrypsin and subtillisin clan enzymes mentioned above is the catalytic triad. The triad is located in the active site of the enzyme, where catalysis occurs, and is preserved in all serine protease enzymes. The triad is a coordinated structure consisting of three essential amino acids: histidine (His 57), serine (Ser 195) (hence the name "serine protease") and aspartic acid (Asp 102). Located very near one another near the heart of the enzyme, these three key amino acids each play an essential role in the cleaving ability of the proteases.
In the event of catalysis, an ordered mechanism occurs in which several intermediates are generated. The catalysis of the peptide cleavage can be seen as a ping-pong catalysis, in which a substrate binds (in this case, the polypeptide being cleaved), a product is released (the N-terminus "half" of the peptide), another substrate binds (in this case, water), and another product is released (the C-terminus "half" of the peptide).
Each amino acid in the triad performs a specific task in this process:
*The serine has an -OH group that is able to act as a nucleophile, attacking the carbonyl carbon of the scissile peptide bond of the substrate.
• A pair of electrons on the histidine nitrogen has the ability to accept the hydrogen from the serine -OH group, thus coordinating the attack of the peptide bond.
• The carboxyl group on the aspartic acid in turn hydrogen bonds with the histidine, making the pair of electrons mentioned above much more electronegative.
The whole reaction can be summarized as follows:
• The polypeptide substrate binds to the surface of the serine protease enzyme such that scissile bond is inserted into the active site of the enzyme, with the carbonyl carbon of this bond positioned near the nucleophilic serine.
• The serine -OH attacks the carbonyl carbon, and the nitrogen of the histidine accepts the hydrogen from the -OH of the [serine] and a pair of electrons from the double bond of the carbonyl oxygen moves to the oxygen. As a result, a tetrahedral intermediate is generated.
• The bond joining the nitrogen and the carbon in the peptide bond is now broken. The covalent electrons creating this bond move to attack the hydrogen of the histidine, breaking the connection. The electrons that previously moved from the carbonyl oxygen double bond move back from the negative oxygen to recreate the bond, generating an acyl-enzyme intermediate.
• Now, water comes in to the reaction. Water replaces the N-terminus of the cleaved peptide, and attacks the carbonyl carbon. Once again, the electrons from the double bond move to the oxygen making it negative, as the bond between the oxygen of the water and the carbon is formed. This is coordinated by the nitrogen of the histidine. which accepts a proton from the water. Overall, this generates another tetrahedral intermediate.
• In a final reaction, the bond formed in the first step between the serine and the carbonyl carbon moves to attack the hydrogen that the histidine just acquired. The now electron-deficient carbonyl carbon re-forms the double bond with the oxygen. As a result, the C-terminus of the peptide is now ejected.
Additional stabilizing effects
It was discovered that additional amino acids of the protease, Gly 193 and Ser 195, are involved in creating what is called an oxyanion hole. Both Gly 193 and Ser 195 can donate backbone hydrogens for hydrogen bonding. When the tetrahedral intermediate of step 1 and step 3 are generated, the negative oxygen ion, having accepted the electrons from the carbonyl double bond fits perfectly into the oxyanion hole. In effect, serine proteases preferentially bind the transition state and the overall structure is favored, lowering the activation energy of the reaction. This "preferential binding" is responsible for much of the catalytic efficiency of the enzyme.
Zymogens
There are certain inhibitors which resemble the tetrahedral intermediate, and thus fill up the active site, preventing the enzyme from working properly. Trypsin, a powerful digestive enzyme, is generated in the pancreas. Inhibitors prevent self-digestion of the pancreas itself.
Zymogens are the usually inactive precursors of an enzyme. If the digestive enzymes were active when synthesized, they would immediately start chewing up the synthesizing organs and tissues. Acute pancreatitis is such a condition, in which there is premature activation of the digestive enzymes in the pancreas, resulting in self-digestion (autolysis). It also complicates postmortem investigations, as the pancreas often digests itself before it can be assessed visually.
Zymogens are large, inactive structures, which have the ability to break apart or change into the smaller activated enzymes. The difference between zymogens and the activated enzymes lies in the fact that the active site for catalysis of the zymogens is distorted. As a result, the substrate polypeptide cannot bind effectively, and proteolysis does not occur. Only after activation, during which the conformation and structure of the zymogen change and the active site is opened, can proteolysis occur.
Zymogen Enzyme Notes
Trypsinogen trypsin
When trypsinogen enters the small intestine from the pancreas, secretions from the duodenal mucosa cleaves the lysine 15 - isoleucine 16 peptide bond of the zymogen. As a result, the zymogen trypsinogen breaks down into trypsin. Recall that trypsin is also responsible for cleaving lysine peptide bonds, and thus, once a small amount of trypsin is generated, it participates in cleavage of its own zymogen, generating even more trypsin. The process of trypsin activation can thus be called autocatalytic.
Chymotrypsinogen chymotrypsin
After the Arg 15 - Ile 16 bond in the chymotrypsinogen zymogen is cleaved by trypsin, the newly generated structure called a pi-chymotrypsin undergoes autolysis (self digestion), yielding active chymotrypsin.
Proelastase elastase
It is activated by cleavage through trypsin.
As can be seen, trypsinogen activation to trypsin is essential, because it activates its own reaction, as well as the reaction of both chymotrypsin and elastase. It is therefore essential that this activation doesn't occur prematurely. There are several protective measures taken by the organism to prevent self-digestion:
• The activation of trypsinogen by trypsin is relatively slow
• The zymogens are stored in zymogen granules, capsules that have walls that are thought to be resistant to proteolysis.
Inhibition
Serine proteases are inhibited by a diverse group of inhibitors, including synthetic chemical inhibitors for research or therapeutic purposes, and also natural proteinaceous inhibitors. One family of natural inhibitors called "serpins" (abbreviated from serine protease inhibitors) can form a covalent bond with the serine protease, inhibiting its function. The best-studied serpins are antithrombin and alpha 1-antitrypsin, studied for their role in coagulation/thrombosis and emphysema/A1AT respectively. Artificial irreversible small molecule inhibitors include AEBSF and PMSF.
Role in disease
Mutations may lead to decreased or increased activity of enzymes. This may have different consequences, depending on the normal function of the serine protease. For example, mutations in protein C, when leading to insufficient protein levels or activity, predispose to thrombosis.
Diagnostic use
Determination of serine protease levels may be useful in the context of particular diseases.
• Coagulation factor levels may be required in the diagnosis of hemorrhagic or thrombotic conditions.
• Fecal elastase is employed to determine the exocrine activity of the pancreas, e.g. in cystic fibrosis or chronic pancreatitis.
• Prostate specific antigen is used to determine prostate cancer risk
Aspartate protease
Aspartic proteases are a family of eukaryotic protease enzymes that utilize an aspartate residue for catalysis of their peptide substrates. In general, they have two highly-conserved aspartates in the active site and are optimally active at acidic pH. Nearly all known aspartyl proteases are inhibited by pepstatin.
Eukaryotic aspartic proteases include pepsins, cathepsins, and renins. They have a two-domain structure, probably arising from ancestral duplication. Retroviral and retrotransposon proteases (Pfam PF00077) are much smaller and appear to be homologous to a single domain of the eukaryotic aspartyl proteases.
Examples
• HIV-1 protease - a major drug-target for treatment of HIV
• Chymosin (or "rennin", with two "n"s)
• Renin (with one "n")
• Cathepsin D
• Pepsin
• Plasmepsin
Mechanism
Proposed mechanism of peptide cleavage by aspartyl proteases[1]
While a number of different mechanisms for aspartyl proteases have been proposed, the most widely accepted is a general acid-base mechanism involving coordination of a water molecule between the two highly-conserved aspartate residues.[1][2] One aspartate activates the water by abstracting a proton, enabling the water to attack the carbonyl carbon of the substrate scissile bond, generating a tetrahedral oxyanion intermediate. Rearrangement of this intermediate leads to protonation of the scissile amide.
General Principles of Toxicology
General Principles of Toxicology
Know the following definitions:
Toxicology: relation of hazardous effects of chemicals, including drugs, to biological systems
Acute toxicity: adverse effect resulting from a single, usually large, exposure to a toxin
Subacute toxicity: Somewhat acute, between acute and chronic (online med. dictionary)
Chronic toxicity: harmful effect from repeated exposures to a toxin for 3+ months
Therapeutic index: LD50/ED50
Risk: probability that injury will result from exposure to a substance for given conditions, dose, and route
Threshold limit values (TLV): maximum safe ambient air concentrations of chemicals during a workweek,a 15 minute interval, and instantaneously
Know the following general mechanisms by which drugs or chemicals can cause toxicity:
Alterations in receptor-ligand interactions
Nicotine, most drugs
Alterations in membrane function
Local anesthetics, hydrocarbons
Interference with cellular energy mechanisms
Cyanide, Pentachlorophenol
Covalent binding to biomolecules
Organophosphates, alkylating agents
Interference with calcium homeostasis
Oxalates
By causing non-lethal alterations in somatic cells
Carcinogens, e.g. aflatoxin
Alterations in ligand-activated transcription factors
Dioxins
By inducing programmed cell death (apoptosis)
Acetaminophen
· Understand the primary determinants of toxicity
Dose and dose rate
Duration of exposure: long duration: bad.
(Principle) routes of exposure: inhalation, transdermal, and oral
· Understand how the following factors modify toxicity
Biotransformation
Parathion à Paraxon
Methanol à Formaldehyde à Formic Acid (MeOH shares pathway with EtOH)
Immune function
Review the hypersensitivities, e.g. Mosby 1-9 or perhaps even your long term memory
Phototoxicity: drug intermediates accumulate in skin, exposed to UV à toxic compounds
Examples: tetracycline, sulfonamides
Age: pharmodynamics, pharmokinetics vary over a lifetime
Gender: no specifics were discussed
· Understand how to manage a poisoned patient - know the ABCD's
Airway: should be cleared of vomitus or other obstruction and an airway or ET tube inserted
Breathing:Assesed by observation and measurements of arterial gases
Intubate and mechanically ventilate if necessary
Circulation
Monitor pulse rate, blood pressure, and urinary output
Start IV and draw blood for glucose and other labs
Dextrose: to every patient with altered mental status (thinking about hypoglycemic problems)
100mg thiamine to alcoholic and malnourished to prevent Wernicke-Korsakoff’s
· Understand the importance of the history and physical examination in treating a poisoned patient
Oral history may be unreliable for a number of reasons
Be on the lookout for classical OD effects of common meds and poisons
Treat the patient, not the poison
· Understand the importance of the following laboratory analysis and procedures
Arterial blood gases
CO2: increased with hypoventilation
PO2: low with aspiration pneumonia or drug-induced pulmonary edema
Reduced with poor tissue oxygenation due to hypoxia, hypotension, or Cn poisoning
May appear normal in CO because dissolved O2 is measured, not oxyhemoglobin
Electrolyte analysis (an ion gap)
(Na+ + K+) + (HCO3
- + Cl-) (this is just major cations minus anions)
normal = 12 plus/minus 4
elevated by:
renal failure
diabetic ketoacidosis
shock-induced lactic adicosis
Drug-induced metabolic acidosis (salicylates, methanol, ethylene glycol, isoniazid, Fe)
Renal and liver function tests
Renal tests: In the UA look for:
BUN, creatinine (nitrogen load and glomerular filtration checks, respectively)
CK and myoglobin (muscle insults)
oxlate crystals: suggest ethylene glycol poisoning
Liver tests: transaminases, look at the PT
Osmolar gap: useful in alcohols poisonings; alcohols will increase the osmolar gap (K. table 59-4)
Calculated serum osmolality: [ 2Na+ + glucose/18 + BUN/3]
Osmolar gap: measured osmolality – calculated osmolality
EKG examples:
Wide QRS: tricyclics, quinidine
Long QT: quinidine, phenothiazine, tricyclics
Variable AV block, screwy rhythms: dig. overdose
Ischemic changes: hypoxemia due to CO
· Understand how toxins can be removed or elimination can be enhanced
Gastric lavage
> 30 minutes have passed since the ingestion of a corrosive material
ingestion of hydrocarbons
coma, stupor, delerium, unconsciousness, convulsions
Induced emesis (know contraindications)
Syrup of Ipecac is often used; same contraindications as above
Increased rate of excretion
Catharsis: increased clearance of intestinal contents
Sorbitol is the preferred agent (hellooooo, apple juice), MgSO4 can be used if kidneys are ok
osmotic diuretics: e.g. mannitol, urea, etc.
Altered urinary pH
Alkalination: useful for salicylate or phenobarbitol overdose
Acidification: not recommended b/c worsens renal effects of rhabdomyolysis
Peritoneal dialysis: simple and available, but inefficient for most drugs
Hemodialysis: especially useful in cases where electrolyte and fluid imbalances are present
Hemoperfusion: blood pumped from pt’s vein through a cartridge filled with adsorbent material
Especially effective for high molecular weight compounds
· Understand the importance of the following specific drugs in managing the poisoned patient
Activated charcoal: large surface area, suggested dose is 10:1 charcoal to est. weight of toxin
Good for: adsorption of many drugs and poisons
No good for: Fe, Li, K, Cn, alcohols, corrosive acids and alkali, methylcarbmate, tolbutamide
Ipecac syrup: emetic agent; use 30 ml for adults, 10-15 ml for kids, repeated q 15 minutes if necessary
Emetic contraindications still apply: not for corrosives, hydrocarbons, rapidly acting convulsants
Ammonium chloride: used to acidify the urine
Not used much clinically because of side effects, e.g. indirect kidney damage
urinary excretion of weak organic bases
Sodium bicarbonate: urine alkalinization
urinary excretion of weak organic acids
Magnesium sulfate: cathartic, contraindicated in renal compromise
Mannitol: used to renal clearance of toxins (also to intraocular and intracranial pressure)
Deferoxamine: use IV or IM; chelator of choice for Fe poisoning
Dimercaprol: single-agent therapy for arsenic and mercury, use for lead with EDTA
Edetate, calcium disodium (EDTA): efficient chelator of many di/trivalent metal ions, esp. lead
Penicillamine: chelator of Cu and Pb
used in Wilson’s cystinuria, resistant cases of rheumatoid arthritis
Succimer (Dimercaptosuccinic Acid, DMSA), analog of dimercaprol,
prevents and reverses metal-induced inhibition of sulfhydryl-containing enzymes
urinary Pb excretion, protects against lethal effects of As, kidney [Hg]
Acetylcysteine: acetaminophen OD antidote, give within 8-10 hours of insult
Digoxin-specific FAb antibody: binds Dig
Atropine: used for cholinesterase poisonings to block ACh binding
Pralidoxime (2-PAM): cholinesterase reactivator, given only for organophosphates
Physostigmine: suggested for antimuscarinic, anticholinergic agents, but not for TCA’s
Flumazenil: used for benzodiazepine overdoses
Cyanide antidote package: sodium nitrate, sodium thoisulfate, amyl nitrate
Glucagon: antidote for Beta-adrenoreceptor blockers, may reverse low BP, bradycardia
Ethanol: used in ethylene glycol poisoning to decrease kidney damage, in MeOH poisoning as well
Fomepizole: an alcohol dehydrogenase inhibitor used to treat MeOH and ethylene glycol accidents
Diazepam: used for chemical-induced convulsions
Pyridoxine (Vitamin B6): used for isoniazid OD
Methylene blue: used to convert methemoglobin to hemoglobin (nitrate poisoning)
Know the following definitions:
Toxicology: relation of hazardous effects of chemicals, including drugs, to biological systems
Acute toxicity: adverse effect resulting from a single, usually large, exposure to a toxin
Subacute toxicity: Somewhat acute, between acute and chronic (online med. dictionary)
Chronic toxicity: harmful effect from repeated exposures to a toxin for 3+ months
Therapeutic index: LD50/ED50
Risk: probability that injury will result from exposure to a substance for given conditions, dose, and route
Threshold limit values (TLV): maximum safe ambient air concentrations of chemicals during a workweek,a 15 minute interval, and instantaneously
Know the following general mechanisms by which drugs or chemicals can cause toxicity:
Alterations in receptor-ligand interactions
Nicotine, most drugs
Alterations in membrane function
Local anesthetics, hydrocarbons
Interference with cellular energy mechanisms
Cyanide, Pentachlorophenol
Covalent binding to biomolecules
Organophosphates, alkylating agents
Interference with calcium homeostasis
Oxalates
By causing non-lethal alterations in somatic cells
Carcinogens, e.g. aflatoxin
Alterations in ligand-activated transcription factors
Dioxins
By inducing programmed cell death (apoptosis)
Acetaminophen
· Understand the primary determinants of toxicity
Dose and dose rate
Duration of exposure: long duration: bad.
(Principle) routes of exposure: inhalation, transdermal, and oral
· Understand how the following factors modify toxicity
Biotransformation
Parathion à Paraxon
Methanol à Formaldehyde à Formic Acid (MeOH shares pathway with EtOH)
Immune function
Review the hypersensitivities, e.g. Mosby 1-9 or perhaps even your long term memory
Phototoxicity: drug intermediates accumulate in skin, exposed to UV à toxic compounds
Examples: tetracycline, sulfonamides
Age: pharmodynamics, pharmokinetics vary over a lifetime
Gender: no specifics were discussed
· Understand how to manage a poisoned patient - know the ABCD's
Airway: should be cleared of vomitus or other obstruction and an airway or ET tube inserted
Breathing:Assesed by observation and measurements of arterial gases
Intubate and mechanically ventilate if necessary
Circulation
Monitor pulse rate, blood pressure, and urinary output
Start IV and draw blood for glucose and other labs
Dextrose: to every patient with altered mental status (thinking about hypoglycemic problems)
100mg thiamine to alcoholic and malnourished to prevent Wernicke-Korsakoff’s
· Understand the importance of the history and physical examination in treating a poisoned patient
Oral history may be unreliable for a number of reasons
Be on the lookout for classical OD effects of common meds and poisons
Treat the patient, not the poison
· Understand the importance of the following laboratory analysis and procedures
Arterial blood gases
CO2: increased with hypoventilation
PO2: low with aspiration pneumonia or drug-induced pulmonary edema
Reduced with poor tissue oxygenation due to hypoxia, hypotension, or Cn poisoning
May appear normal in CO because dissolved O2 is measured, not oxyhemoglobin
Electrolyte analysis (an ion gap)
(Na+ + K+) + (HCO3
- + Cl-) (this is just major cations minus anions)
normal = 12 plus/minus 4
elevated by:
renal failure
diabetic ketoacidosis
shock-induced lactic adicosis
Drug-induced metabolic acidosis (salicylates, methanol, ethylene glycol, isoniazid, Fe)
Renal and liver function tests
Renal tests: In the UA look for:
BUN, creatinine (nitrogen load and glomerular filtration checks, respectively)
CK and myoglobin (muscle insults)
oxlate crystals: suggest ethylene glycol poisoning
Liver tests: transaminases, look at the PT
Osmolar gap: useful in alcohols poisonings; alcohols will increase the osmolar gap (K. table 59-4)
Calculated serum osmolality: [ 2Na+ + glucose/18 + BUN/3]
Osmolar gap: measured osmolality – calculated osmolality
EKG examples:
Wide QRS: tricyclics, quinidine
Long QT: quinidine, phenothiazine, tricyclics
Variable AV block, screwy rhythms: dig. overdose
Ischemic changes: hypoxemia due to CO
· Understand how toxins can be removed or elimination can be enhanced
Gastric lavage
> 30 minutes have passed since the ingestion of a corrosive material
ingestion of hydrocarbons
coma, stupor, delerium, unconsciousness, convulsions
Induced emesis (know contraindications)
Syrup of Ipecac is often used; same contraindications as above
Increased rate of excretion
Catharsis: increased clearance of intestinal contents
Sorbitol is the preferred agent (hellooooo, apple juice), MgSO4 can be used if kidneys are ok
osmotic diuretics: e.g. mannitol, urea, etc.
Altered urinary pH
Alkalination: useful for salicylate or phenobarbitol overdose
Acidification: not recommended b/c worsens renal effects of rhabdomyolysis
Peritoneal dialysis: simple and available, but inefficient for most drugs
Hemodialysis: especially useful in cases where electrolyte and fluid imbalances are present
Hemoperfusion: blood pumped from pt’s vein through a cartridge filled with adsorbent material
Especially effective for high molecular weight compounds
· Understand the importance of the following specific drugs in managing the poisoned patient
Activated charcoal: large surface area, suggested dose is 10:1 charcoal to est. weight of toxin
Good for: adsorption of many drugs and poisons
No good for: Fe, Li, K, Cn, alcohols, corrosive acids and alkali, methylcarbmate, tolbutamide
Ipecac syrup: emetic agent; use 30 ml for adults, 10-15 ml for kids, repeated q 15 minutes if necessary
Emetic contraindications still apply: not for corrosives, hydrocarbons, rapidly acting convulsants
Ammonium chloride: used to acidify the urine
Not used much clinically because of side effects, e.g. indirect kidney damage
urinary excretion of weak organic bases
Sodium bicarbonate: urine alkalinization
urinary excretion of weak organic acids
Magnesium sulfate: cathartic, contraindicated in renal compromise
Mannitol: used to renal clearance of toxins (also to intraocular and intracranial pressure)
Deferoxamine: use IV or IM; chelator of choice for Fe poisoning
Dimercaprol: single-agent therapy for arsenic and mercury, use for lead with EDTA
Edetate, calcium disodium (EDTA): efficient chelator of many di/trivalent metal ions, esp. lead
Penicillamine: chelator of Cu and Pb
used in Wilson’s cystinuria, resistant cases of rheumatoid arthritis
Succimer (Dimercaptosuccinic Acid, DMSA), analog of dimercaprol,
prevents and reverses metal-induced inhibition of sulfhydryl-containing enzymes
urinary Pb excretion, protects against lethal effects of As, kidney [Hg]
Acetylcysteine: acetaminophen OD antidote, give within 8-10 hours of insult
Digoxin-specific FAb antibody: binds Dig
Atropine: used for cholinesterase poisonings to block ACh binding
Pralidoxime (2-PAM): cholinesterase reactivator, given only for organophosphates
Physostigmine: suggested for antimuscarinic, anticholinergic agents, but not for TCA’s
Flumazenil: used for benzodiazepine overdoses
Cyanide antidote package: sodium nitrate, sodium thoisulfate, amyl nitrate
Glucagon: antidote for Beta-adrenoreceptor blockers, may reverse low BP, bradycardia
Ethanol: used in ethylene glycol poisoning to decrease kidney damage, in MeOH poisoning as well
Fomepizole: an alcohol dehydrogenase inhibitor used to treat MeOH and ethylene glycol accidents
Diazepam: used for chemical-induced convulsions
Pyridoxine (Vitamin B6): used for isoniazid OD
Methylene blue: used to convert methemoglobin to hemoglobin (nitrate poisoning)
CHOLECYSTOKININ & Substance P
CHOLECYSTOKININ
-Cholecystokinin is produced in duodenum causing contraction of gall bladder.
-Popularly known as pancreozymin.
-When administered by IV injection causes increase in secretion of
Pancreatic enzymes and also stimulates gall bladder contraction.
-It is used as diagnostic aid for the purpose of testing pancreatic
function along with SECRETIN.
-It is also used as adjunct in cholecystography.
-Vasomotor reactions, abdominal discomfort and hypersensitivity has
been reported.
-Cholecystokinin is a Neuropeptide (octapeptide)
-cholecystokinin receptors are a group of G-protein coupled receptors.
-Antagonists to neuropeptide CCK proved to possess anxiolytic activity.
-*Loxiglumide is CCK antagonist.
SUBSTANCE.P
Substance .P is a neuropeptide .
It is a short chain with 11 amino acid polypeptide.
It functions as Neurotransmitter and neuromodulator.
It belongs to Tachykinin Family, Neuropeptide family.
In body it is majorily synthesized in CNS, bloodvessels, GIT, always and skin.
In CNS substance.P has been associated in regulation of mood
disorders, anxiety, stress, nausea, emesis, pain, respiratory rhythm,
neurogenesis and neurotoxicity.
Tachykinin Family includes NKA,NKB(NK-neurokinin) and Substance. P
*Pharmacological actions-
Action on Blood vessels-
It is a potent vasodilator.
This cause release of Nitric oxide from endothelium causing hypotension.
Action on Neurons-
Substance.P involved in transmission of pain impulses from peripheral
receptors to CNS.
Action on Vomiting center-
Vomiting center in brainstem (medulla oblongata).
Action on smooth muscle-
Causes contraction of smooth muscle and constriction of blood vessel.
*Substance P as a Pain Mediator-
It is a protein found in brain and spinal cord associated with
inflammatory process in joints.
-low back pain
- fibromyalgia
-Arthritis
Physiological Activities-
Vomiting reflex
Defensive behavior
Change in cardiovascular tone
Stimulation of salivary secret
Smooth contraction
Vasodilatation.
Receptors for Substance.P
The endogenous receptor for substance.p is NK-1, NK-1R.
These are G-protein receptors.
Treatment-
Capsaicin obtained from capsicum has been shown to reduce the levels
of substance p in nerve endings and decrease in pain by depletion of
c-fibre nerves.
Antagonist- Aprepitant
Structure- Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2.
-Cholecystokinin is produced in duodenum causing contraction of gall bladder.
-Popularly known as pancreozymin.
-When administered by IV injection causes increase in secretion of
Pancreatic enzymes and also stimulates gall bladder contraction.
-It is used as diagnostic aid for the purpose of testing pancreatic
function along with SECRETIN.
-It is also used as adjunct in cholecystography.
-Vasomotor reactions, abdominal discomfort and hypersensitivity has
been reported.
-Cholecystokinin is a Neuropeptide (octapeptide)
-cholecystokinin receptors are a group of G-protein coupled receptors.
-Antagonists to neuropeptide CCK proved to possess anxiolytic activity.
-*Loxiglumide is CCK antagonist.
SUBSTANCE.P
Substance .P is a neuropeptide .
It is a short chain with 11 amino acid polypeptide.
It functions as Neurotransmitter and neuromodulator.
It belongs to Tachykinin Family, Neuropeptide family.
In body it is majorily synthesized in CNS, bloodvessels, GIT, always and skin.
In CNS substance.P has been associated in regulation of mood
disorders, anxiety, stress, nausea, emesis, pain, respiratory rhythm,
neurogenesis and neurotoxicity.
Tachykinin Family includes NKA,NKB(NK-neurokinin) and Substance. P
*Pharmacological actions-
Action on Blood vessels-
It is a potent vasodilator.
This cause release of Nitric oxide from endothelium causing hypotension.
Action on Neurons-
Substance.P involved in transmission of pain impulses from peripheral
receptors to CNS.
Action on Vomiting center-
Vomiting center in brainstem (medulla oblongata).
Action on smooth muscle-
Causes contraction of smooth muscle and constriction of blood vessel.
*Substance P as a Pain Mediator-
It is a protein found in brain and spinal cord associated with
inflammatory process in joints.
-low back pain
- fibromyalgia
-Arthritis
Physiological Activities-
Vomiting reflex
Defensive behavior
Change in cardiovascular tone
Stimulation of salivary secret
Smooth contraction
Vasodilatation.
Receptors for Substance.P
The endogenous receptor for substance.p is NK-1, NK-1R.
These are G-protein receptors.
Treatment-
Capsaicin obtained from capsicum has been shown to reduce the levels
of substance p in nerve endings and decrease in pain by depletion of
c-fibre nerves.
Antagonist- Aprepitant
Structure- Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2.
PENTAGASTRIN
PENTAGASTRIN
Drug Category :
• Diagnostic aid
• gastric function
Indication: Used as a diagnostic aid for evaluation of gastric acid secretory function
Pharmacology: Pentagastrin is indicated as a diagnostic aid for evaluation of gastric acid secretory function. It is effective in testing for anacidity (achlorhydria) in patients with suspected pernicious anemia, atrophic gastritis, or gastric carcinoma. It is also effective in determining the reduction in acid output after operations for peptic ulcer, such as vagotomy or gastric resection.
Mechanism of Action: The exact mechanism by which pentagastrin stimulates gastric acid, pepsin, and intrinsic factor secretion is unknown; however, since pentagastrin is an analogue of natural gastrin, it is believed that it excites the oxyntic cells of the stomach to secrete to their maximum capacity. Pentagastrin stimulates pancreatic secretion, especially when administered in large intramuscular doses. Pentagastrin also increases gastrointestinal motility by a direct effect on the intestinal smooth muscle. However, it delays gastric emptying time probably by stimulation of terminal antral contractions, which enhance retropulsion.
Precautions-
It should be given with care to patients with
Acute peptic ulceration
Hepatic or biliary tract disease.
Adverse effects-
Pentgastrin may cause a no GIT effects. It causes Nausea and abdominal cramps.
Tachycardia
Arrhythmia
Dizziness, allergic reactions
Incidence less frequent or rare:- fast heartbeat ,usually transient chills, dizziness, faintness, or lightheadedness, drowsiness, feeling of heaviness of arms and legs, headache, increased sweating, numbness, tingling, pain, or weakness in hands or feet, shortness of breath, unusual tiredness
DOSAGE-
6 micrograms/kg by SC injection, IM or Nasal Inhalation.
Drug Category :
• Diagnostic aid
• gastric function
Indication: Used as a diagnostic aid for evaluation of gastric acid secretory function
Pharmacology: Pentagastrin is indicated as a diagnostic aid for evaluation of gastric acid secretory function. It is effective in testing for anacidity (achlorhydria) in patients with suspected pernicious anemia, atrophic gastritis, or gastric carcinoma. It is also effective in determining the reduction in acid output after operations for peptic ulcer, such as vagotomy or gastric resection.
Mechanism of Action: The exact mechanism by which pentagastrin stimulates gastric acid, pepsin, and intrinsic factor secretion is unknown; however, since pentagastrin is an analogue of natural gastrin, it is believed that it excites the oxyntic cells of the stomach to secrete to their maximum capacity. Pentagastrin stimulates pancreatic secretion, especially when administered in large intramuscular doses. Pentagastrin also increases gastrointestinal motility by a direct effect on the intestinal smooth muscle. However, it delays gastric emptying time probably by stimulation of terminal antral contractions, which enhance retropulsion.
Precautions-
It should be given with care to patients with
Acute peptic ulceration
Hepatic or biliary tract disease.
Adverse effects-
Pentgastrin may cause a no GIT effects. It causes Nausea and abdominal cramps.
Tachycardia
Arrhythmia
Dizziness, allergic reactions
Incidence less frequent or rare:- fast heartbeat ,usually transient chills, dizziness, faintness, or lightheadedness, drowsiness, feeling of heaviness of arms and legs, headache, increased sweating, numbness, tingling, pain, or weakness in hands or feet, shortness of breath, unusual tiredness
DOSAGE-
6 micrograms/kg by SC injection, IM or Nasal Inhalation.
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