Paxil

• specific uptake inhibitor that decreases depression and anxiety (the CYP2D6 catalytic cycle)
• in presynaptic neuron serotnin can be packaged in vesicles or deaminated by monamine oxidase A to form 5 hydroindole3acetaldehyde
• elevated blood serotinin causes contraction of blood vessels(hypertension), overstimulation of 5HT receptor, leading to: nausea, anxiety, insomnia and headaches

Dextromethorphan

(nyquil)

• non-competitive antagonist channel blocker of NMDA receptor(glutamate receptor) suppresses medullary cough center
• liver congerts dextromethorphan to active dextrorphan (uses CYP2D6)
• p450 are monooxygenase(inserts on oxygen to water and other to sub) and can be an oxidase producting hydrogen peroxide if metab two diff substrates, substrate overwhelm catalytic site, metabolize substrates have high km for p450 disrupting it

Liver Detoxification overview

• solute ligand carriers (SLC)
• cytochrome P450 oxidation
• transferase-conjugation
• transporter -ATP binding cassette(ABC)
• function of drug detoxification system is to take a hydrophobic chemical and make it more hydrophilic so it can be eliminated from the body

Cytochrome P450 functions

• bile acid syn, cholesterol catabolism, fatty acid metabolism, vitamin d metabolism, syn of cholesterol CYP51, synthesis of steroid hormones
• CYP17A1 implicated in sex steroid deficiency
• CYP21 deficiency results in increase adrenal gland androgen formation (life threatening in neonates from salt loss due to aldosterone and cortisol def)
• CYP19(aromatase) is target for breast cancer to lower estrone and estradiol levels in hormone responsive tumors

Drug Detoxification System

• Phase I: enzymes insert oxygen into drug, chemical, carcinogen(monooxygenase, possible mixed func oxidase)
• Phase II: enzymes are transferases that attach a polar group to hydroxylated drug: UDP glucuronyl transferase or glutathione S-transferase
• Phase III: enzymes use ATP to transport conjugated drug from cell: Multidrug resistance protein(MDR), multi resistance protein(MRP): increase in these ABC transporters in cancer cell is major reason for tumor resistance to chemo drugs

Cytochrome P450 characteristics

• heme protein found in SER and mitochondria
• insert one oxygen into substrate and one to water to make a lipophilic substrate hydrophilic
• have multi-substrate specificity
• recieve reducing equivalents from NADPH cytochrome P450 reductase
• coupling reaction inefficent and decoupling leads to production of ROS

CYP Gene Family Nomenclature

• family if >40% AA identity
• A sub family if >55% identity
• 1 member if 55%-95% identity
• *1 variant if >95%<100% identity 

CYP3A4

• metabolizes more than 40% of all drugs currently prescribed
• accounts for 50% of the total liver content of P450 and induced by steroids, st johns wort, and antiHIV drugs
• metabolizes caclium hannel blocker-nifedipine, statins, antibiotics and antirejection drugs
• inhibited by grapefruit, and antifungal agents

CYP2 family members

• metabolize 15% of drugs and account for 20% of liver content
• induced by barbituites (phenobarbital) 

CYP2D6 

• metabolize over 15% of prescribed drugs and accounts for 10% of the liver content 
• major P450 in which genetic polymorphisms account for adverse drug reaction in the human population with 10% of caucasians expressing a varient with low activity 
• metabolizes codeine and dextromethorphan
• inhibited by quindin and serotonin reuptake inhibitors 
• paxil is metabolized by CYP2D6 but its metabolite inhibts enzyme and catalytic cycle(suicide inhibitor)

CYP2E1 Toxicity 

• ethanol induces CYP2E1 in the liver and is metabolizes P4502E1
• also metabolizes the NSAID, acetaminophen, to NAPQ1 toxic metabolites
• NAPQI causes oxidative stress, but can be conjugated and excreted by glutathione-s-transferase
• Nacetyl cysteine induces GSH syntehsis and helps preventNAPQI toxicity
• given to patients in liver failure

Determinants of Drug Response

• pharmacodynamics: biological effects of a drug; drug targets(receptors, enzymes, ion channels)
• phamacokinetics: absorption distribution, metabolism, excretion, transport, sequestration, metabolizing enzymes, drug interactions

Polymorphism vs mutation

• polymorphism is a genetic variation that occurs at a frequency > 1% in a population
• mutation is a frequency less than 1%

SIngle Nucleotide Polymorphism (SNP)

• most common source of variation
• 100-300 bp in genome
• coding, nonsynonymous (cause changes in protein function) 
• coding, synonymous (although same amino acid, changes in regulatory sites)
• noncoding SNPs

Cytochrome P450 enzymes w/ polymorphisms

• CYP2C9
• CYP2D6
• CYP2C19
• CYP2A6
• CYP1A2
• CYP3A4/5

CYP2C9

• 30 variant alleles
• metabolizes 16% of drugs
• 20% of hepatic CYPs
• 40% of cacasians are poor metabolizers
• 3-5% of asians and africans are poor metabolizers
• substrates: NSAIDs, Angiotensin receptive bolckers, anti diabetic drugs and warfarin 
• *2 variant leads to 50% reduction in cyp activity and *3 leads to 90% reduction in activity (heterzygotes have reduction too)

Warfarin

• inhibits the synthesis/maturation of clotting factors (VKORC1 used to remake cosubstrate for carboxylase reaction that makes prothrombin)
• commonly prescribed anticoagulant
• narrow therapeutic window (high rate of adverse affects)(INR 2-3)
• CYP2C9 metabolizes it 
• dose to high leads to hemorage and dose to low are clots 
• *2 variant leads to 50% reduction in cyp activity and *3 leads to 90% reduction in activity (require lower warfarin dose)

Potential benefits of genotyping

• lower initial dosing= reaach target INR faster
• less time at non-therapeutic INR
• cost benefit 

CYP2D6

• 70 variant alleles, most phenotypic variation of CYPs
• metabolizes up to 25% of drugs and makes up 2-5% of liver CYPs
• substrates: beta blockers, antidepressants, opiods, tamoxifen
• shows all 4 phenotypes, with extensive metabolizers being the norm and many regional groups that are ultrametabolizers 
• metabolizes codeine to morphine(active)

Tamoxifen

• selective estrogen receptor modulator for ER+ breast cancer
• antagonist at receptor in cancer tissue(estrogen dependent)
• aromatase important enzyme that can also be inhibited in cancer treatment 
• halves recurrence of breast cancer and reduces mortality by 30%
• metabolized by CYP2D6 to endoxifen and 4hydroxyTAM (higher affinity then TAM) so metabolizm activates 

CYP2C19

• 25 variant alleles
• metabolizes up to 10% of drugs
• 25% of asians are poor metabolizers and 15% of all persons are poor metabolizers
• substrates: proton pump inhibit (this is good thing in this case), clopidogrel, diazepam, antidepressants

Clopidogrel

• antiplatelet agent, clot prevention
• often used in place or in combination w/ aspirin
• it is activated by CYP2C19
• poor metabolizers of CYP2C19 have higher risk of MI, stroke when on clopidrogel

CYP2A6

• 35 variant alleles
• metabolizes up to 3% of drugs
• 10% of all CYPs
• 20% of asians are poor metabolizers
• substrates: nicotine, coumarins, mostly minor

CYP1A2

• 15 variant alleles
• metabolizes up to 5-10% of drugs
• substrates are caffeine and antipsychotics

Ethanol Metabolism

• ethanol to acetaldehyde(ADH1(low km) and ADH2(high km)) uses NAD+ cofactor
• acetaldehyde to acetate using either ALDH1(cytosolic 20%) or ALDH2 (mitochondrial, 80%)
• Acetate to acetyl CoA using ACS1(cytosol in fatty acid synthesis) and ACS II (heart, muscle)
• disulfiram inhibits ALDH causing nausea and vomiting
• asian population has variant of ALDH that has high km
• CYP2E1 also used as an alternate pathway(requires NADPH) to send ethanol ot acetaldehyde

Major classes of Extracellular matrix proteins

• collagens
• elastin
• fibronectin
• laminins
• proteoglycans 

Collagen Structure and function

• 27 different types of collagen(some common structural features)
• triple helix domain with repeating amino acid sequence Gly-X-Y with proline and hydroxyproline appearing frequently in the X and y positions respectivly
• glycines (33% of molecule) on inside and prolines on the outside and replacement of glycine with larger AAs have serious consequences for collagen stability and function
• hard to denature collagen because the prolines make it rigid, they also provide means for lateral association with other triple helices or proteins
• collagens are either fibril forming or non-fibring forming 

Collagen synthesis and fibrillar assembly 

• syn in RER, then modified in RER and Golgi and ultimately in extracellular space
• forms are preprocollagen, procollagen, tropocollagen and collagen(insoluble)
• hydroxylation of proline and lysine residues, adddition of oligosaccharides some disulfide bond stuff at N and C ends, exocytosis and removal of N and C terminal peptides and lateral association of triple helices
• one fibril of collage consist of multiple chians of collagen and fibrils come together to form fiber

Elastin

• non collagen ECM protein 
• principal constituent of elastic fibers of teh vasculature, lungs, ligament and skin
• only one gene responsible for elastin 
• rich in proline and glycine like collagen but more hydrophobic(lots of valine) and little hydroxy AAs
• lysines in elastine contribute to intermolecular crosslinking as in collagen and valine-valine interactions in hydrophobic regions of the molecule impact elasticity 
• lysyl oxidase important for crosslinking of elastin and desmosine most important

Fibronectin 

• non collagenous ECM glycoprotein that is soluble
• functions in cell adhesion, migration, differentiation and morphology as well as cytoskeleton organization
• two subunits joined by disulfide bonds and each su composed of distinct domains with seveal homologous repeating units
• su identified for affinities for binding to specific ECM components including collagen, heparin, fibrin and cell surface(RGD domain)
• PXSRN sequence motif binds to the integrin proteins on cell surfaces
• integrins are transmembrane proteins that bind both EC proteins and cytoskeleting providing a bridge for extracellular communication

Laminins

• non-collagenous glycoproteins found in basement membranes (large heterotrimers composed of alpha beta and gama chains arranged to generate an asymmetric molecule linked by disulfide bonds 
• in presence of calcium, laminins may reversibly self-assemble to form polymers
• have domains like fibronectin that are functionally the sites of binding to other molecules or to cells
• laminin alpha chains bind to integrins, heparin and heparan sulfate
• laminin polymers bind to type IV collagen and nidogen/entactin, which itself has an RGD sequence for integrins
• laminin through nidogen/entactin bridges cells ECM molecules (Type IV collagen) in the basement membrane

Proteoglycans

• found in joint, ocular fluid, cartilage, bone, tendon, skin, heart valves, blood vessels, mast cells, liver, cornea and cell surface
• cotain protein and sugar
• oligosccharide chains comprised of repeating disaccharide units usually one amino sugar and one uronic acid
• chains are extremely high charged as result of negative carboxyl groups of uronic acid and neg sulfate groups of hydroxyl or amino groups of sugars
• electrical repulsion causes chains to be highly extended and rigid in conformation
• carbohydrate protion known as GAG
• six types: hyaluronic acid, chondroitin sulfats, dermaton sulfate, heparan sulfate, heparin and keratan sulfate(only one w/o normal disacch combo)
• except for hyaluronic acid and keratan sulfates, all GAGs linked by Gal-Gal-Xyl to a serine or threonine on the protein component 

Synthesis and degradation of Proteoglycans

• syn is thru a series of glycosyl transferases, epimerases adn sulfotransferases
• syn begins in the RER with oligosacch being added to the protein core
• repeatin oligosacch and post trans mod. happen in golgi
• degradation of proteoglycans in lysosomes (first step endoglycosidic cleavage by heparanase)
• lysosomal proteases do the protein portion and lysosomal acid hydrolases do the sequentialy degradation of GAG chains begining from external end
• undergraded molecules accum in lysosome
• mucopolysaccharidoses are lysosomal storage deiseases caused by accum of GAGs

Epidermolysia bullosa

• rare heritable disorder results in severe blistering of skin and epithelial tissue
• can be identified as simplex, junctional and dystrophic
• simplex blistering in epidermis caused by defects in keratin filaments
• junctional blistering dermal-epidermal junc(laminin defect)
• dystrophic is blistering in dermis caused by mutations in gene encoding type VIII collage
• all three have similar clinical features but distinct causative factors

Lathyrism

• caused by a dietary deficiency 
• deformities of spine and relatively easy dislocation of joints, dimineralization of bone, aortic aneurysms and joint hemorrhage
• do not have functional lysyl oxidase(required for collagen cross linking(
• inhibition can be caused by chronic ingesting of sweet pea(lathyrus odoratus, beta aminopropionitrile) a irreversible inhibitor
• penicillamine(chelation therapy) can also cause it by possible chelation of copper required as a cofactor in enzyme or by rx with hydoxyallysine needed for collagen crosslinking

Marfan Syndrome

• mutation of the gene for fibrillin, the glycoprotein that is principle constituent of microfibrils surrounding elastic fibers
• individuals with marfan have long arms and legs, tall stature and long, spider like fingers(arachnodactylyl)
• mild form char by loose joints, deform of the spine and flexible mitral vavles and cardiac regurgitation, and lens dislocation(eye problems)
• severe form leads to weak aortic wall that frequently rupture because of defects in elastic fiber formation

Osteogenesis imperfecta

• heterogenous group of diseases char by brittle bones and in some cases dentinogenesis imperfecta(soft mottled teath prone to fracture and carries)
• severe forms result in perinatal death while mild forms result only in increase of fractures
• caused by mutation in type I collagen gene (swapping glycine for large AA)
• mutations may shift the assembly of collagen arrays so that hole and overlap regions dont allign 
• could lead to failure of the mineral to fuse properly to form large mineral aggregates(result weak brittle bones)

Ehlers-Danlos syndrome

• heterogenous group char by joint hypermobility and skin fragility and maybe aortic aneurysms
• defect in either type I or type III collagen or defects in post trans mod enzymes(failure to cross link or remove propeptides)

SLC26A1 abnormalities

• abnormalities of resting cartilage
• failure of sulfate transport for activation and crosslinking of matrix proteoglycans aggrecan, biglycan, glypican and chondroitin result in lack of matrix, inheritedin autosomal recessive manner 
• some SLC26A mutations have more residual enzyme(diastrophic dysplasia and multiple epiphyseal dysplasia) and are milder forms 

COL2A1 abnormalities

• syn of abnormal type II collagen chain results in some decrease in matrix and alterations in types of collagen presnt
• chondrocytes have inclusions in cytoplasm, inherited in an autosomal dominant manner
• some milder forms like spondyloepiphysia dysplasia that do not make abnormal collagen that interfere with triple helix formation and linking

Thanatophoric dysplasia

• death loving: infants usually die in hours due to pulmonary hypoplasia
• pregnancy is sometimes complicated by polyhydramnios (
• considered to be a new dominant mutation(used to think it was autosomal recessive)
• targeted mutation analysis for the three mutations in FGFR3 gene detects a mutation in aprox 90% of cases, sequence analysis of FGRF3 detects a mutation in 99% of cases
• resting cartilage looks normal but abnormal growth plate
• most common type of short limb dwarfism is a dominant FGFR3 mutation(achondroplasia)

Achondroplasia 

• 80% of cases of achondroplasia arise as a result of a new mutation risk for older fathers
• targeted mutation analysis allows molecular diagnosis-98% of cases have a G to A point mutation at nucleotide 1138, and 1% have a G to C mutation at nucleotide 1138
• the mutations result in a constitutive activation of the FGF receptor which negatively regulates bone growth
• homozygous achondroplasia is lethal 
• little people is a strong organization that advocates shit about it 

Prenatal diagnosis of Dwarfism

• suspect on the basis of ultrasound-there are good data about normal longbone lengths related to gestational age
• ultrasound for associated features such as facial features can often suggest a particular group of disorders(FGFR3 mutations)
• amniocentesis and molecular analysis can often arrrive at an etiology
• size of the thorax is a major determinant of survival

Hypophosphatasia

• deposition of calcium phosphate in bone matrix is regulated by levels of pyrophosphate. tissue nonspecific alkaline phosphatase breaks down pyrophosphate and allows mineralization to occur
• patients have a decreased serum alkaline phosphate levels and increased phosphethanolamine in urive. severe forms are autosomal recessive and milder forms are dominant
• classic mild presentation is rickets with decreased alkaline phosphatase levels
• mutation in ALPL gene is cause. many families have a unique mutation so genotype-phenotype is not well defined. best prediction of phenotype is correlation with level of alkaline phosphatease activity
• DDx in perinatal lethal form is OI

Chronic Inflamation 

• injury caused by viral infections, chronic infections, persistant injury, autoimmune diseases
• leads to angiogenesis, mononuclear cell infiltrate and fibrosis(scar)(loss of function)

types of tissue

• labile cells(continuously cycling cells): epidermis and endoderm(skin) and hematopoetic system
• quiescent(stable cells): make up bulk of solid organs, liver and endocrine organs- cell number remains stable, replenishes what it needs to if a drastic loss 
• Permanent cells: neurons. cariac myocytes: once they are dead they are dead (replaced by scar)

Pathology of Cirrhosis

• when there is injury to cells + disruption of matrix, orderly regeneration of the liver cannot occur 
• proliferation of matrix isolates clusters of regenerating cells-regenerating clusters develop circulatory compromise and injury and avortive regeneration occur again 

Ethanol Abuse and the liver

• ethanol met in the liver yields acetaldehyde, NADH and ROS 
• this impairs hepatocyte function and can cause hepatocyte breakdown
• ethanol causes the release of LPS from the gut which timulates kupffer cells to release mediatiors including PDGF
• inflam mediators activate and transform HSC to myofibroblastic cells that produce ECM(Collagen 1, 3, 5 , fibronectin, laminin, hyaluronan and proteoglycans)

Recurrent Injury Cause a Vicous cycle

• damaged hepatocytes release ROS and fibrogenic mediators and induce recruitment of blood white cells
• apoptosis of damaged hepatocytes stimulates fibrogenic actions of liver myofibroblasts
• inflammatory cells from the blood active more HSC to secrete collagen
• activated HSC(hepatic satalite cells) secrete inflam chemokines and modulate the activities of blood inflam cells causing cycle to repeat

Consequences of Fibrosis

(liver)

• surviving hepatocytes are stimulated to regenerate and form nodules w/i the confines of fibrous septa
• delivery of blood to the hepatocytes is severely compromised
• ability of hepatoctyes to secrete subs into the plasma is markedly impaired
• disruption of the interface btw the hepatic parenchyma and portal tracts also obliterates biliary channels leading to an inability to secrete into the bile

Therapeutic approaches for cirrhosis

• cessation of drinking
• angiotensin inhibitors-inhibit HSC activation-anti-fibronic effects in cell culture, experimental animals and some supportive retrospective data in humans
• interferon-α- inhibit HSC activation- anti-fibrotic effects in cell culture, experiment animasl and effective in patients with chronic hepatitis 

Skeletal Muscle of the heart

• under aerobic metabolism it uses fatty acids and Acetyl-CoA carboxylase 2 (ACC2) is the major control point (inh by palimitoyl CoA)
• acetyl CoA and NADH levels become high, leading to activation of PC and then citrate syn 
• Fatty Acyl CoA is stop point and does not go to FA syn in heart 
• Malonyl CoA then inhibts CPT-1 allowing acetylCoA levels to drop and PDH to activate 
• glucose not used as energy until NADH and acetyl CoA levels droped
• lactate increases due to PDH inhibiton while OAA increases

AMP and ischemia

• in heart AMP levels become high becaue no fatty acids to oxidize or O2
• AMP activates AMP kinase
• AMPK activates PFK2 with increased production of F2,6BP that increases PFK1 activity
• AMPK also inhibits ACC-2 and activates malonyl-CoA decarboxylase(gets rid of the malonyl CoA that normally inhibits CTP-1 (doesnt matter because no FA getting here)
• allows pyruvate to be converted to lactic acid to generate ATP (anaerobic glycolysis)
• asmotic swelling then happens because no energy for pumps and cell eventually explodes(necrosis)

ADP and hypoxic State

• this lead to the increased formation of dADP by the action of Ribonucleotide reductase
• dADP can be phosphorlated to dATP and induce apoptosis
• as  ADP increases, so does AMP, which activates phosphorylase b and PFK-1
• 2ADP can be converted to ATP and AMP (myokinase)
• also a breakdown of AMP to adenosine(vasodilator)

AMP Activated Glycogenolysis

• AMP directly activates glycogen phosphorylase b conversion to active form
• high intracellular Ca(low ATP cannont keep ion balance) increases calmodulin activation of phosphorylase kinase, which activates phosphorylase b
• phosphorylase kinase also activated by PKA
• beta adrenergic receptor activation by epinephrine
• AMP kinase can also activate GLUT4 receptors(usually insulin) and these as well as GLUT1 bring glucose to heart
• AMP kinase inhibits ACC-2 and activates malonyl CoA decarboxylase (no function during ischemia but adapting for when thrombis is gone)
• 
  

Ca and ischemia

• because of the increase in lactate formation, there is also an increase in H+ formation
• this causes an influx of sodium through the H+/Na exchanger
• Increase in Na then leads to Ca accumulation via the Na/Ca exchanger

Hypoxia induction of glycolysis

• hypoxia induces the transcription factor hypoxia inducing factor(HIF-1α)
• HIF-1α undergoes rapid prolyl hydroxylation under normoxic conditions by specific cellular prolyl hydroxylases, interacts with ubiquitin ligase and is targeted for destruction
• under hypoxia, hydroxylation does not occur because prolyl hydroxylase lack oxygen
• HIF heterodimerize with aryl hydrocarbon nuclear translocator(ARNT) and binds the hypoxia respnsive element of genes involved in glycolysis, erythropoiesis and formation of new blood vessels (angiogensis)

Repurfusion Injury

• the repurfusion of the heart with oxygen and FA result in the dramatic increase in the formation of ROS that cause lipid peroxidation and release of cytochrome c from the inter mitochonrdrial member
• low pH and high ATP conc can keep mitochondrial pore closed
• excessive Ca, Pi and ROS opens poor 
• BAX provides a tunnel for cytochrome C to exit the mit with dATP and the release of apoptosis activating factor one(Apaf-1)
• dATP activates complex that converts procaspase 9 to active caspase 9 that initiates apoptosis

Causes of Sudden Cardiac Death in Young

Individuals

• undetected congenital heart disease(anomalous coronary artery)
• unrecognized myocarditis(usually viral): coxsacki virus
• cardiomyopathy
• structurally normal heart with a mutation in a gated ion channel(channelopathy)
• commotio cordis(sharp blow to chest): usually problem in smaller younger children

Types of Cardiomyopathy

• dilated cardimyopathy when muscle is somewhat thin and the chambers are enlarged (cant generate force well)
• hypertrophic cardiomyopathy where walls are thickened and chambers are somewhat smaller(muscle gets in the way of normal heart function) (often asymetric and involves septum)
• restrictive cardiomyopathy is an individual with a rigid myocardium(usually an abnormal protein in wall muscle) 
• dilated and hyper(classic) are often unsuspected and lead to sudden death

Protein Mutations in cardiac muscle

• most common is myosin heavy chain(35%)
• then it is myosin binding protein C and Troponin T proteins 

Arrhythmogenic right ventricular dysplasia

• right heart becomes dilated
• autosomal dominant with considerable pentrance (some 50% some as low as 20%)
• seven genes known to be associated: TGFB3, RYR2, DSP, PKP2, DSG2, DSC2, THEMA43
• yellow fat becomes present in wall
• microscopic has area where muscle is lost, causing a very thin wall 
• expect to see early stages in young people that cause an arrhythmia 
• obese individuals have incresed amount of myogenic fat 
• alot of disagreement on diagnosing this

Channelopathy

• ion channels are diverse group of pore fomring proteins that cross lipid membranes and selectively conduct ions across a barrier
• associated with familial epilepsy, myotonia syndroms and familial cardiac arrhythmia sydnromes
• most notably long and short Q-T syndrome
• impaired k channesl decrease k efflux, increased Q-T interval (repolarization time)
• impaired Na Channels widen QRS, slow contraction sequence
• autosmal domin usually pure cardiac and recessive usually associated with sensorineural hearing loss