Cystic Fibrosis CFTR- the Gene

• 1989 gene described: Chromosome 7, codes for protein CFTR
• autosomal recessive: carrier rates-1/30 caucasian decent, 1/64 african-american decent, 1/70 asian decent
• no disease manifests in carriers

Cystic Fibrosis CFTR- the Gene

• CF Transmembrane Conductance Regulator 
• Gene coding for protein on chromosome 7
• ATP binding cassette transporter
• located at apical surface of epithelial cells
• involved in salt and fluid transport9regulates water content of luminal secretions

CFTR Structure and function

• main mechanism to transport chloride out of cell
• also regulates transport of sodium and water
• down-regulates amiloride-sensitive epithelial sodium channel
• up-regulates an alternate chloride channel(outwardly rectifying channel)

Physiologic effects of CFTR Dysfunction

• increased Na and water absorption
• thin airway surface liquid layer
• thick, tenacious mucus
• cilia dysfunction
• can lead to pulmonary disease which involves bronchial obstruction, infection, inflamation and bronchiectasis (major cause of morbididy, measured with FEV1)
• pancreas, liver, reproductive and endocrine also effected

Pulmonary Manifistations

Cystic Fibrosis

• chronic cough and sputum production(pneumonitis, bronchitis)
• radiographic abnormalities (atelectasis, bronchiectasis, pneumothorax)
• chronic airway obstruction(wheezing, air trapping, dyspnea)
• chronic pansinusitis and nasal polyps
• chronic persistent, recurrent airway infections(s aureus, h influenza, p aeruginosa, b cepacia)

Pancreatic Insufficiency

Gastrointestinal disease

cystic fibrosis

• cystic dilatation and fibrosis of acinar ducts
• unable tos ecrete pancreatic enzymes to digest food(malabsorption, failure to thrive, hypoproteinemia, edema)
• fat malabsorption
• bowel obstruction(meconium ileus, distal intestinal obstructive syndrome

Other organ involvment

• reproductive (male infertility: CBAVD, female sub-fertility)
• endocrine (CF related diabetes, delayed growth and puberty)
• liver disease
• sinus disease

Treatment of Airway obstruction

• bronchodilators(beta2agonists, albuterol)
• airway clearance: get mucus out(chest percussion and drainage, hypertonic saline and high frequency chest wall oscillation
• mucolytics: break up mucus(DNAase)
• anti-inflammatoris: high dose ibuprofen, azithromycin

Classes of CFTR mutations

• Class I mutation: nonsense G542X
• Class II: missense: AA deletion ΔF508 (block in processing
• Class III missense: G551D protein gets to cell surface but doesnt function
• some strategies to stop the premature stop codons in Class I

Aminoglycoside antibiotics

• target bacterial small ribosome subunit
• bind to ribosome decoding site and change conformation
• can also happen to some degree in human small ribosome subunit
• conformational change can allow insertion of appropriate tRNA at subunit to allow translation to continue
• targets nonsense class I mutations in cystic fibrosis
• cause renal damage and hearing loss

VX-770 Potentiator

• large, multi-center trial in patients with CF(patients had atleast 1 copy of G551D
• few serious adverse events(rash and elevated blood glucose)
• efficacy: within subject improvement in CFTR funtion, some small improvements in lung function
• some treatments use potentiators and correctors

ATPase Activity and CFTR

• 2ATP or ATP and drug needed to open the CFTR channel(Cl-)
• ATPase hydrolyzes a phosphate to close the channel

Functions of plasma proteins

• maintain osmotic pressure
• substance transport/binding
• immune-related 9immunoglobulins, complement, protease inhibitors)
• coagulation/fibrinolysis(serum=plasma-clotting factors)

Platelet Structure

• megakaryocyte-derived anucleate cytoplasmic fragment
• open canicular system(SCCS) increases membrane SA
• Microfilament(SMF) and microtubular systems shape change to increase SA
• dense granules(dense bodies) for Ca, ADP, ATP and serotonin
• alpha granules(G): vWF, fibrinogen, factors V and VIII, PDGF, TGFbeta, βthromboglobulin, platelet factor 4

Platelet Function

• adhesion: platelets adhere to site of vessel injury
• GPIa binding: platelet shape change from disk to spiny sphere, which allows...
• GPIb binding: via von wilebrand factor(vWF), which exposes...
• GPIIb/IIIa: for binding vWF and fibrinogen
• secretion: release granule contents, especially ADP
• ADP binds to platelet membrane receptor: unmasks additional GPIIb/IIIa
• ADP induces platelet swelling: facilitates platelet-platelet contact/adherence
• aggregation: platelet-platelet adherence mediated by fibrinogen binding to GPIIb/IIIa

von WIlebrand Factor

• protein multimer of various molecular weights
• synthesized in endothelial cells and megakaryocytes as high molecular weight form
• cleaved into lower molecular weight forms by plasma metalloprotease(ADAMTS13)
• secreted into subendothelial matrix, stored in platelet alpha granules, circulates in plasma
• mediates platelet adhesion to subendothelial collagen
• carrier molecular for factor VIII: prevents degradation

Platelet interaction with soluble coagulation 

• thrombin binds to platelet surface receptor: stimulates platelet secretion
• platelet membrane phospholipids provide srface for activation of soluble coagulation cascade
• fibrinogen, factor V, and factor VIII released from platelet alpha granules

Platelet interaction with blood vessels

• release of serotonin and synthesis of thromboxane A2: vasoconstriction
• release of PDGF: vascular endothelial proliferation

Platelet-Related Disorders

• deficiency of platelet number or function is typically associated with ucocutaneous bleeding: easy bruising/ecchymosis, petechial hemorrhages. epistaxis, GI bleeding, menorrhagia
• platelet glycoprotein receptor deficiences: GPIb/IIIa(glanzmann thrombasthenia, deficient platelet aggregation) and GPIb/IXa(bernard-soulier syndrome, deficient platelet adhesion)
• platelet granule deficiences: dense body deficiency(delta granules) and grey platelet syndrome(Alpha granule deficiency)
• von Willebrand disease: deficient platelet adhesion

Thrombotic Thrombocytopenia Purpura(TPP)

• deficiency of ADAMTS13(congenital or aquired/autoimmune)
• usually large circulating von Willebrand multimers (no broken up into normal smaller multimers)
• formation of microthrombi: mechanical damage to red blood cells and consumption of platelets
• classic pentad: hemolytic anemia, thrombocytopenia, neurologic symptoms, renal failure, fever

Soluble Coagulation: Coagulation Cascade

• accelerated localized and regulated generation of clot
• conversion of fibrinogen to fibrin (thrombin)
• proenzymes II, VII, IX, X, XI: serine proteases activate site serine cleaves peptide bond; seauential activation cleave next proenzyme in cascade
• cofactor proteins VI, VIII, tissue factor(TF) provide binding sites for other factors, V, VIII circulate and stored as procofactors, TF integral membrane protein and no activation is required

Coagulation Cascade

• Extrinsic Pathway: trauma activates VII, which activates X using Tissue factor(TF)
• Intrinsic pathway: Factor XI activates factor IX
• Intrinsic path: Factor IX along with VIII activates X
• Factor X along with cofactor V converts prothrombin to thrombin
• thrombin converts fibrinogen to fibrin(soft clot)

Factor VII 

• in coagulation cascade and has autocatalytic properties
• activates factor X directly with the help of Tissue Factor(TF)
• also activates factor IX, which then goes on to activate factor X with the help of VIII

Intrinsic Pathway

• pathway in the coagulation cascade
• internal activation mechanisms, contact with negativly charged surface(active platelet membrane, monocyte membrane, glass)
• sustains coagulation
• factors XI, IX, VIII used to activate factor X 

Coagulation Cascade Laboratory Testing

• Extrinsic pathway: prothrombin time (PT) and international normalized ratio(INR)
• intrinsic pathway: activated partial thromboplastin time(aPTT)
• specific factor assay for common congenital deficiences: Factor VIII(hemophilia A), Factor IX(hemophilia B, Christmas disease): both x linked recessive and result in joint and deep muscular bleeding

Thrombin

• converted from prothrombin Via factor X and cofactor V
• thrombin then converts fibrinogen to fibrin(soft clot)
• also converts XIII to its active form, which converts fibrin soft clot to cross-linked clot(hard clot)

Fibrinogen/Fibrin-related Laboratory Testing

• fibrinogen assay(clauss method)
• fibrin degradation products(FDP) and D-dimer: produced by degradation of fibrin by fibrinolytic system(vide infra)
• urea solubility test: factor XIII deficiency screen

Clotting Regulation: Factor Complexes

• localize complex assembly and thrombin generation to platelet surface
• markedly increase rate of thrombin generation 
• coordination complexes of factor+Ca+membrane phospholipid: negatively charged glutamate residues on factors II, VII, IX, X(y-carboxylation), negatively charged platelet membrane phospholipids, positively charged calcium ions
• extrinsic tenase complex: TF, VIIa
• intrinsic tenase complex: IXa, VIIIa
• prothormbinase complex: Va, Xa

Y-Carboxylation

• occurs in synthesis of factors II, VII, IX, X
• occurs in hepatocyte prior to secretion of protein
• Vitamin K dependent process (reduced form is oxidized)
• reduced vitamin k is then reformed via epoxide reductase and vitamin k reductase
• can be manipulated pharmacologically 

Warfarin Anticoagulation

• AKA 4-hydroxycoumarin, Coumadin
• Vitamin K antagonist
• prevents y-carboxylation 
• non-carboxylated proteins are non-functional (dont gain negative charge to form coagulation complexes)
• level of anticoagulation monitored by prothrombin time and international normalized ratio(PT/INR) 

Feed back amplification by Thrombin

• activation of factors V, VII, VIII, XI: increased rate of clot formation
• release of factor VIII from vWF
• platelet actiavtion promotes platelet aggregation

Feedback inhibition by thrombin

• binds to thrombomodulin receptor on endothelial cell surface
• allows thrombin to activate protein C
• protein C and S: Vitamin K dependent factors
• protein C(serine protease) active form is APC
• protein S forms complex with APC, Ca and platelet phospholipid
• APC cleaves factors Va and VIIIa: decreases thrombin generation
• increase endothelial PGI2 secretion(inhibits platelet aggregation), increasestPA relase and inactivates PAI-1(fibrinolysis)

Protein S and C related disorders

• heterozygous deficiences of protein C or S: risk factors form thrombosis; risk for coumarin-induced skin necrosis
• homozygous(or compound heterozygous) deficiences of protein C or S: neonatal purpura fulminans
• activated protein C resistance: risk factor for thrombosis: most often due to factor V leiden
• V leiden: single point mutation in factor V gene; arginine replaced by glutamine: mutant factor V resistant to cleavage by protein C

Antithrombin III(ATIII)

• serine protease inhibitor(SERPIN)
• forms irreversible complex with thrombin at thrombin active site(arginine reacts with serine)
• also binds and inactivates factors Xa, IXa, XIa
• heparin markedly increases activity of ATIII 

Heparin 

• markedly increases activity of ATIII
• consists of GAG of various molecular weights
• has specific pentasaccharide sequence which binds to ATIII
• binding causes allosteric alteration in ATIII: better exposes arginine residue (this attaches to serine in thrombin)
• relased when ATIII binds thrombin: can be reused

Pharmacologic use of heparin 

• unfractionated heparin(monitored w/ aPTT) and Low molecular weight heparin: monitored by anti-Xa activity assay
• heparin resistance: congenital or aquired deficiency of antithrombin III
• heparin therapy complications: bleeding, osterporosis, heparin-induced thrombocytopenia(HIT)(autoantibody to heparin-platelet factor 4 complex, thrombocytopenia and thrombosis)

Vascular Endothelium Nonthrombogenic Surface

• negatively charged: repels platelets
• synthesis of PGI2 and nitric oxide(NO): vasodilators, inhibit platelet aggregation 
• thrombomodulin expression: protein C activation
• heparan sulfate: glycosaminoglycan similar to heparin; weaker activator of antithrombin III

Fibrinolysis

• degradation of fibrin clot by plasmin(serine protease)
• plasminogen is an inactive precursor with high affinity for fibrin clot (act most affective of clot surface)
• endogenous activators are tPA, and urokinase(serine protease)
• exogenous activators are Streptokinase
• plasmin in circulation is scavanged by α2-antipalasmin(not efficient at clot bound plasmin
• plasminogen activator inhibitor 1 blocks tPA action
• plasmin has low specificity(cleave fibrin, fibrinogen, plasminogen, factor V, VIII, GPIb, GPIIb/IIIa

difference btw intra and extra vascular hemolysis

• extravascular hemolysis just increases bilirubin
• intravascular hemolys increases bilirubin and free hemeoglobin as well as decreases haptoglobin 

Hereditary spherocytosis

• most common hereditary hemolytic anemia caused by a red cell membrane structural defence(autosomal dominant)
• incidence of 1 in 2000 in Northern Europeans
• variable severity, depends on the protein involved and the nature of mutation
• gallstones are a major problem. Hemolytic crisis can be caused by infection. another major problem is aplastic cirsis associated especially with parvovirus infection
• usual management is removal of gallbladder if symptomatic and removal of spleen once vaccinated for encapsulated organisms 

Pyruvate Kinase Deficiency 

• second most common red cell enzymopathy, very heterologous disorder as many mutations and most cases result from unique combination of mutant parental alleles
• mutations have been noted in PEP binding site, ADP binding site adn failure of F1,6DP to activate
• inheritance is autosomal recessive, parents have PK levels of 50-70% normal and no symptoms
• PK is a tetramer, different tissues express different forms which result for locus heterozygosity(M chr 15, L/R chr 1) and alternate splicing
• elevated 2,3 DPG helps unload oxygen from hemoglobin and so patients often tolerate a lower level of hemoglobin

G-6-PD deficiency

• x linked recessive
• several types exist:B is normal form, A+ normal function but weird migration, A- mutant with decreased stabability and found commonly in blacks
• high frequency of the gene is assumed to result from the protection it provides against malaria(G6PD red cells are removed from circ before the parasite can complete its cycle
• the A- mutant usually has no symptoms unless exposed to a stressing drug. other G6PD mutants(rare) are more severe and present early in life with a chronic hemolytic anemia

Heme 

• porphyrin ring coordinated with an iron atom
• 4 pyrrole rings joined by methenyl bonds
• 8 side chains(2 on each pyrrole): Methyl, vinyl, propionyl: order =MVMVMPPM
• heme syn in erythroid precursors: constituent of hemoglobin
• heme syn in hepatocytes: constituent of cytochrome P450
• also required for other important hemoproteions(myoglobin, caalase, peroxidase)

Heme Synthesis

• succinyl CoA plus glycine yeales Aminolevulinic  acid(ALA) (done in mitochondria)
• 2 ALA form a pyrrole(uses Dhase) (done in cytosol)
• 4 pyrryole come together to form hydroxymethylbilane 
• this is cyclilized into a preporforin
• porforin then has iron added to it to form heme(iron 2+ or ferous) 

Heme Synthesis Regulation

• heme regulation of ALA synthase(especially in liver): repress synthesis of enzyme, directly inhibits enzyme activity by allosteric modification
• iron regulation of erythroid heme synthesis: iron availability regulates translation of erythroid-specific ALA synthase(ALAS2) mRNA
• heme promotion of hemoglobin synthesis: maintains ribosomal initial complex for globin synthesis in active state by inhibiting phosphorylation of initiation factor eIF2

The Porphyrias

• classified according to site of overproduction and accumulation of porphyrin precursors or porphyrins
• heapatic porphyrias(liver) and erythropoietic prophyrias(bone marrow erythroid cells)
• acute hepatic porphyrias: acute intermittent, hereditary coproporphyria(HCP), variegate porphyria(VP), ALA dehydratase deficient porphyria(ADP)
• chronic/cutaneous porphyrias: hepatic cutaneous(porphyria cutanea tarda(PCT) and erythropoietic cutaneous porphyrias

Acute hepatic porphyrias

• life threatening neurologic attacks, usually after puberty (around 30s)
• env or hormonal factors induce ALAS1, causing overproduction of ALA and PBG(only ALA in ADP)
• ALA and PBG may interact with y-aminobutyric acid or glutamate receptors
• autosomal dominant inheritance 
• clinically normal in btw attacks
• pregnancy can precipitate acute attacks

Acute Hepatic prophyrias treatment

• narcotics and phenothiazines for symptomatic treatment
• carbohydrate loading(IV glucose) for mild attacks
• hemin for moderate to severe attacks (breakdown product of heme derived from RBC)(iron in Fe3+ form)
• allogeneic liver transplantation 

Porphyria Cutanea Tarda

(Werewolf) 

• usually presents in adults, sporadic and AD inheritance(shows penetrance)
• skin blistering, friability, atrophy, scarring, milia, hyperpigmentation, hypertrichosis(excessive hair)
• orange-red urine which fluoresces red under UV
• diagnosis: increased urinary porphyrins; increased fecal isocoproporphyrin; UROD mutation analysis
• pathogenesis: decreased hepatic URO-decarboxylase activity(inhibited through iron overload)
• therapy: sunlight avoidance and phlebotomy 

Chronic Erythropoietic Porphyria

• deficient URO-synthase, autosomal recessive 
• severe cutaneous photosensitivity beginning in infancy, secondary infection of skin lesions
• porphyrin accumulation in teeth and bones: bone resorp., red brown teeth(erythrodonitia, fluoresce under long wave UV)
• hemolytic anemia due to accumulation of porphyrins in RBC, splenomegaly
• diagnosis: increased uroporhyrin and coproporphyrin in RBC, plasma, urine, feces; decreased URO-synthase enzyme; UROS gene mutation analysis
• treatment: sunlight avoidance; transfusions for anemia; bone marrow/cord blood transplantation

Erythropoietic Protoporphyria and XLP

• EPP(FECH mutation, autosomal recessive); XLP (ALAS2 gain of function, X linked dominant)
• clinically indistiguishable: photosensitivity, absent or mild hemolysis and may have mild to severe liver disease
• diagnosis: markedly increased erythrocyte protoporphyrin levels, erythrocytes fluoresce red or mutation analysis
• treatment: sunlight avoidance; absorbent substances(cholestyramine) to promote fecal protoporphyrin excretion; plasmapheresis; hemin; liver transplant; bone marrow transplant

Iron Absorption-Small intestine

• heme iron from meat readily absorbed: Heme carrier protein 1(HCP1), iron liberated from heme by heme oxygenase
• none-heme irion from plants not readily absorbed: plant substances form insoluble precipitates with iron, vitamin C increases non-heme iron abs
• inside cell iron in Fe2+ form or stored in cell as ferritin(Fe3+)
• iron is oxidized prior to blood transport (Fe3+)
• transported in blood in transferrin complex

Iron uptake

• transferrin(iron transporter) receptors on cell surface(hepatocytes)
• receptor uses clathrin coated pit and endosome
• acidification of endosome causes release of iron which is transported into cytoplasm via DMT1 transport protein
• either goes to mitochondria or stored as ferritin(Fe3+) or hemosiderin 
• main sites of storage are liver, bone marrow and cells of reticular endothelial system(spleen and lymp)

Iron Storage-Ferritin 

• iron stored intracellulary as ferritin: apoferritin(24 subunits which form a shell around 2,000 to 4500 iron atoms; ferritin has ferroxidase activity(oxidizes iron to the 3+ state in which it is stored
• major storage sites-liver, spleen, bone marrow
• hemosiderin: iron deposits exceeding ferritin storage capacity, comprised of ferritin, denatured ferritin, other substances(iron not readily available)
• hemosiderin usually in macrophages and often associated with hemorrhage

Serum Iron Test

• test of iron metabolismmeasures iron bound to transferrin
• indirect measure of rate of delivery to tissues
• iron deficiency: iron rapidly removed from transferrin, iron level low
• iron overload: iron level high

Total Iron-binding capacity(TIBC)

• test of iron metabolism
• estimate of transferrin concetration(most serum iron bound to fransferrin)
• rate of transferrin production inversely proportional to iron stores
• decreased iron stores: increased transferrin
• increased iron stores: decreased transferrin

Transferrin Saturation

• test for iron metabolism
• ratio of serum iron to iron-binding capacity
• iron deficiency: low saturation
• iron overload: high saturation

Ferritin and Zinc protoporphyrin tests

• iron metabolism tests
• ferritin: serum level directly related to total body iron stores, good marker of iron deficiency
• zinc protoporphyrin: zinc incorporated into heme if iron not available, ZPP increased in iron deficiency, lead posioning

X-Linked sideroblastic Anemia 

• deficiency of δ-aminolevulinin acid synthase-2(ALAS2)(products of erythroid-specific ALA gene)
• decreased heme (and hemoglobin) synthesis stimulates erythropoiesis, which is ineffective
• ineffective erythropoises stimulates increased iron absoprtion(erythropoietic hemochromatosis)
• progressive iron accumulation in tissues, which may be fatal if untreated
• presents with mild to severe microcytic/hypochromic anemia, evidence of iron overload(increased ferritin level, icnreased transferrin saturation) and toxicity
• pathognomic lesion in bone marrorw(non ferritin iron accumulation)
• treat with pyridoxine and phlebotomy or chelation

Heme Degradation

• heme is degraded to bilirubin in macrophages
• converted to an open chain that can be excreted
• heme is oxidized and cleaved to form biliverdin(linear tetrapyrrole)
• biliverdin is reduced to bilirubin
• associated protein is cleaved to its constituent amino acids
• iron atom is recycled and stored
• also generates carbon monoxide

Bilirubin Transport

• bilirubin is virtually insoluble in aqueous solution at physiologic pH(unconjugated bilirubin)
• circulates in plasma tightly bound to albumin(small franction of unbound bilirubin in plasma)
• cannot be excreted in urine
• removed from circulation by liver(dissociates from albumin prior to entering hepatocyte)
• light on bilirubin causes changes in interpyrrolic bridges, subsequent cyclization, and more polar structual isomers that can be excreted in bile

Bilirubin Conjugation in hepatocyte

• bilirubin conjugated to UDP-glucuronate to form bilirubin monoglucuronide and bilirubin diglucuronide(predominant 70-90%)
• UDP-glucuronate attached to P side chains
• conjugated bilirubin more water soluble 

Bilirubin Excretion

• conjugated bilirubin excreted into bilary tree as constituent of bile
• bile released into intestine
• bacterial β-glucuronidases deconjugate bilirubin and convert it to urobilinogens(colorless)
• most urobilinogens oxidized to urobilins(stercobilin) and excreted into feces
• 20% of urobillinogens reabsorbed in ileum and colon, transported to liver via enterohepatic circulation and re-excreted into bile(small amount excreted in urine)

Jaundice and hyperbilirubinemia

(unconjugated)

• excessive bilirubin production: hemolytic anemia, resorption of blood from hemorrhage, ineffective erythropoiesis
• decreased hepatic uptake: drug interference with membrane carrier systems
• impaired conjugation: physiologic jaundice of newborn, breast milk jaundice, genetic deficiency of UDP-glucuronate transferase(Crigler-najjar and gilbert syndromes), hepatocellular disease(hepatitis, cirrhosis)

Jaundice and hyperbilirubinemia

(conjugated)

• decreased hepatocellular excretion: dubin-johnson syndrome, rotor syndrome, hepatocellular disease
• impaired intrahepatic and extrahepatic bile flow: paucity of intrahepatic bile ducts, extrahepatic biliary atresia, inflammatory bile duct destruction(biliary cirrhosis and sclerosing cholangitis), extrahepatic obstruction by cysts, stones, tumors

 Jaundice and hyperbilirubinemia

• jaundice is deposition of unconjugated or conjugated bilirubin in tissues, causing yellow discoloration
• unconjugated bilirubin cannot be excreted in urine: unbound fraction may diffuse into tissues, may cause kernicterus in infant brain
• conjugated bilirubin is nontoxic, only loosely albumin bound: excretable in urine and can become covalently bound to albumin(delta fraction or biliprotein, long plasma half life)

Cigler-Najjar syndrome

• type I(autosomal recessive): complete absence of UDP-glucuronate transferase activity; unconjugated hyperbilirubinema, invariably fatal, death due to kernicterus
• Type II: marked reduction in UGT activity(autosomal recessive), unconugated hyperbilirubinemia, some risk of kernicterus, most develop normally
• Gilber syndrome is also reduced UGT activity due to mutations in UGT1A1 promoter 

Dubin-Johnson Syndrome

• impaired canalicular excretion of conjugated bilirubin due to defect in transport protein(cMOAT)
• autosomal recessive
• predominantly conjugated hyperbilirubinemia;delta-bilirubin
• benign; chronic fluctuating or recurrent jaundice
• liver biopsy: hepatocellular cytoplasmic melanin-like granules
• urinary coprophyrins: >80% is coproporphyrin 1(nomrally >75% is coproporphyrin III); useful diagnostic finding

Rotor Syndrome

• unknown organic anion transport defect
• autosomal recessive
• benign conjugated hyperbilirubinemia
• reduced bilary excretion of coproporphyrins; increased urinary coproporphyrin excretion

Physiologic Jaundice of Newborns

• transient, mild unconjugated hyperbilirubinemia which affects newborns and which occurs due to increased bilirubin production and immature conjugation and excretion mechanisms
• β-glucuronidases in milk deconjugate bilirubin glucuronides in gut, leading to increased reabsorption of unconjugated bilirubin
• newborns lack intestinal bacter that can convert unconjugated bilirubin to urobilinogen

Treatment of Jaundice

• phototherapy
• exchange transfusion
• metalloporphyrins: inhibitors of heme oxygenase

Clinical Bilirubin measurement

• most laboratories do not report concentration of each bilirubin fraction; instead report total and direct bilirubin
• diazo reaction: conversion of bilirubin to azopyrroles by reaction with diazo reagent , formation of colored product
• direct-reaction bilirubin reaction occurs within minutes(5% unconj., 70-90% of conj, 100% of delta fraction
• indirect reaction bilirubin reaction occurs in presence of accelerator substrate such as methanol or caffeine(measures total bilirubin and then you subtract direct, roughly corresponds to unconjugated bilirubin)

Erythropoietin

• primarily made in the kidney
• pushes cells downt he RBC pathway
• activates steps that make CFU-E, Pronormoblast and reticulocyte
• kidney senses blood oxygen levels and secretes erythropoietin when it is low 
• uses JAK/STAT receptor, 2 stats promotes SOCS
• JAK mutation that keeps JAK and creates lots of red blood cells

hemoglobin Switch

• BCL IIA is a newly discovered gene that encodes for a protein found to be a transcriptional repressor for fetal Hb Y gene expression
• the protein has been found to interact with nucleosome remodeling and histone deacetylase complexes 

Reduced Cytochrome b5

• this is oxidized the convet Fe3+ hemoglobin to Fe2+(iron that coordinates with oxygen) hemoglobin
• the oxidized cytochrome is converted back to reduced cytochrome using NADH

Heinz Bodies

• Glucose6phosphate dhase deficiency laeads to a lack in production of NADPH
• with out NADPH one cannot convert GS-SG to glutathione(GSH)
• an accumulation of GS-SG leads to heinz bodies in erythrocyte 

Reduction of oxygen

(3 ways)

• input of energy to excite one of the unpaired electrons to higher orbit inverting spin and forming a singlet oxygen species
• interaction of oxygen with a metal atom containing its own unpaired electrons(iron, copper) will enable the direct divalent reduction of oxygen
• spin restriction can be bypassed by adding electrons one at a time(univalent reduction)
• each electron transfered increases oxidizing power

Oxidizing agent and Reducing Agent

• oxidizing agent accepts e- to become reduced (reactive oxygen species)
• reducing agent donates e- to become oxidized(antioxidant) 

Singlet Oxygen

• produced during inflammation when neutrophiles produce hypochlorous acid(HOCl) and hydrogen peroxide react to generate singlet oxygen
• singlet oxygen is used medically in photodynamic therapy of cancer where porphyrin sensitizers are taken up by tumors, which are then irradiated leading to singlet oxygen production and cell death
• in disease of porphyria deposition of porphyrins in tissues leads to avoidance of sun-ligh by porphyria stricken people

Characteristics of ROS

• four electron will reduce molec oxygen to water
• one electron reduction of molecular oxygen produces ROS(superoxide, hydrogen peroxide, hydroxyl radical)
• the hydroxyl radical has the greatest oxidizing potential and therefore most damaging
• hydrogen peroxide can go through membranes
• harbor-weiss reaction leads to formation of hydroxyl radical (superoxide condenses with hydrog perox) 
• fenton reaction with reduce Fe2+ or Cu+ produces hydroxyl radical (hydrogen perox. substrate)
• both reduced copper and iron can serve as one e- donor in the fenton reaction

Oxidases

• enzyme that prodecuses ROS
• insert one oxygen into substrate and other to produce hydrogen peroxide 

Xanthine dehydrogenase

• normally functions in blood vessels as a dehydrogenase reducing NAD to NADH, however during oxidative stress the enzymes cystein amino aid are oxidized resulting in the enzyme functioning as an oxidase by transfering electrons to oxygen to produce hydrogen peroxide
• conversion of this enzyme is believed to be the major source of ROS in ischemia-repurfusion injury 

Dioxygenase and Oxygenase

• dioxygenase insert two molecules of oxygen into substrate thereby forming hydroperoxides(LOOH)
• oxygenase(cytochrome P450) insters one molecule oxygen into a substrate and other into water
• however cytochrome p450 can function as an oxidase to produce hydrogen peroxide, superoxide and hydroxyl radicals when drug that is not normally metabolized by that particular cytochrome p450 resulting in drug toxicity (also happens when to many drugs to be metabolized)

Lipid peroxidation

• hydroxyl radical is able to abstract a hydrogen from PUFA leading to formation of a lipid radical
• this radical forms a lipid peroxide in the presence of oxygen
• he lipid peroxide abstracts H from another lipid forming lipid hydroperoxide and a lipid radical (propagating lipid peroxidation)
• lipid hydroperoxide degrades to malondialdehyde and HNE
• malondialdehyde functions to attract immune cells to injury
• HNE is highly reactive and capable of oxidizing protein, lipid, DNA
• lipid peroxidation stoped by VitE donating electron to lipid radical forming LH(can do this twice)

ROS and proteins

• hydroxyl radicals attack amino acids causing carbonyl stress by hydroxylation
• leads to protein not able to fold properly (disulfide bonds causing crosslinking btw proteins)
• altered proteins targeted for cellular proteolysis
• extensive damage can lead to aggregates that re resistant to degradation
• proteins aggregates observed in parkinsons, alzheimers and huntingtons
• can increase oxidative damage in cell by reducing the cellular level of GSH(oxidation of cysteine residues)

ROS and DNA

• hydroxyl radicals attack guanosine as well as other DNA and RNA bases resulting in formation of 8-hydroxyguanosine
• altered bases can be misread by DNA polym resulting in point mutations
• 8-OH G in damaged DNA strand is recognized as T and thus during DNA rep an A is inserted instead of C
• hydroxyl radicals can also induce single strand DNA breaks by attacking phosphodiester bonds of DNA
• as we age amount of 8-OH G increases 

Nitric Oxide Synthase 

• NO is a radical which also functions as a NT and vasodilator of smooth muscles
• activates guanylyl cyclase to produce cGMP
• nitroglycerin can also stimulate cGMP(vasodilator)
• phosphodiesterase metabolizes cGMP to GMP and is inhibited by VIagra 
• neuronal and endothelial NOS constitutively expressed and activated by Ca2+
• immune cell activated NOS(iNOS) produces NO that reacts with superoxide in neutrophils to generate strong oxidizing agent peroxynitrite

Peroxynitrite

• immune cell activated NOS(iNOS) produces NO that reacts with superoxide in neutrophils to generate strong oxidizing agent peroxynitrite
• most damaging NO agent
• compound increases cellular oxidative stress b increasing amount of nitrosylated of intracellular proteins
• primary amino acid attacked by ONOO is tyrosine and the primary DNA base attacked is guanosine

RNOS

• phagocytic cells of the immune system respond to infections agents and tissue damage by a rapid consumption of oxygen, which is known as the respiratory burst
• this is the major mechanism by which neutrophil and macrophages produce superoxide, hdyrogen, peroxide, hydroxyl radical and hypochlorous acid as part of the bodys anti-microbial defense
• three enzymes activated during respiratory bursts: NADPH oxidase, myeloperoxidase and iNOS
• activation of these systems lead to inflamation
• continual act of immune cells with release of ROS in tissue during chronic infl is seen as pathological changes in tissues in numerous diseases

Antioxidants

• non-enzymatic are Vit E, C, A, ubiquinone(CoQ), lipoic acid, uric acid, melatonin
• non-enzym are unique in that they will donate two electrons to free radicals becoming fully oxidized
• enzymatic antioxidant defense enzyme also functions to donate two H or electrons to the free radical(influde superoxide dismutase, glutathione peroxidase and catalase
• Glutathione peroxidase controls level of reduced glutathione(GSH) and also maintain the reduced state of cysteine residues and therefore inhibits cross linking of protein thru disulfide bonds

Superoxide dismutase(SOD)

• reduces superoxide to hydrogen peroxide
• cytosol uses both Cu2+ and Zu2+ at its catalytic site
• lou gehrigs disease, a familial form of amyotrophic lateral sclerosis, there is a genetic deficiency of SOD-1 gene
• extracellular form of SOD is believed to be important in prevention of superoxide induced damage to endothelial cells
• mitochondrial form of SOD using Mn2+ in the catalytic site funct to remove superoxide generations during mit respiration
• catalase found primarily in liver and kidney peroxisome functions to remove hydrogen peroxide produced during beta oxidation by acyl-CoA oxidase

Vitamin C (ascorbic Acid)

• in the reduced form functions to regenerate the reduced form of vitamin E by donation of two electrons
• reduced ascorbic acid plays a major role in reducing oxidized LDL lipids that are the major cause of plaque formation and coronary heart disease
• the oxidized formation of ascorbic acid can be either eliminated from the body since it is water soluble or regenerated by the thioredoxin/thioredoxin reductase system

Vitamin A (β-carotene)

• not only functions as precursor of retinoic acid but participates in the termination of free radical lipid oxidation by donating single electron to lipid peroxyl radicals(LOO)
• the reason Vit A is not as good as Vit E is either tis one electron donation and form of Vit A radical or because its hydrophobic nature makes it un accesible to recieve eletron from Vit C
• Vit A may have a benefit in photodynamic therapy to prevent singlet oxygen damage to normal tissues

Flavonoid 

• strong reducing agents donating electrons and becoming fully oxidzed have ability to stabilize radical forms by changing resonance
• flavonoids found in wine, green tea and chocolate
• have ability to donate two electrons and become fully oxidized
• can bind both copper and iron used in the fenton reaction
• inhibit enzyme responsible for superoxide formation

Induction of antioxidant defense

• keap1-Nrf2-ARE signaling pathway is responsible for turning on the adaptive response against oxidant stress
• during enhanced ROS the cysteine residues in keap-1 are modified or oxidized causing the dissociation of Nrf2 and translocation of it to the nucleus and binding to the ARE(antioxidant response element)

cardinal signs of inflammation

• rubor(redness)
• calor(heat)
• tumor(swelling, edema)
• dolor(pain)
• functio laesa (loss of function)

LTB4 and PGE2

and acute inflamation 

• eicosanoids that act as chemoattractants to recruit neutrophils that phagocytose invading microorganisms and cellular debris
• activated neutro[phils with newly formed phagosomes form phagolysosomes by fusing with lysosomal granules containing degradative enzymes
• resulting resp burst produces reactive oxygen, ntriogen species(ROS-RNS) that kill microorganisms and degrade cellular debris in acute inflamation
• in resolution of acute infl. lipoxins recruit macrophages to site of injury to clean and repair damaged tissue (PGE2 and PGD2 used here for growth and resolution)
• chronic inflamation never goes to stage of resolution, goes to fibrosis

Chemotaxis of Neutrophils

• happens by diapedesis
• primary event in the initiation of inflammation
• largely mediated by Leukotriene B4(LTB4) with chemokines and cytokines

Rubor-redness

• cardinal sign of inflammation
• caused by vasodilation of vessels
• prostglandins(PGI2) vasodilation
• nitric oxidase
• hintamine-histidine arteriole dilation venous constriction 

Calor-Heat

• cardinal sign of inflammation
• caused by increased blood flow and fever
• prostaglandins PGE2 sets core body temperature
• IL-1 chemokinase
• TNF cytokine 

Dolor-pain

• due to increased pressure by accumulation of interstitial fluid and:
• prostaglandins PGE2 activating free nerve endings
• bradykinin arteriole dilation, venous constriction(edema)

Tumor-swelling

• due to extravascular fluid accumulation exudate by increase vascular permeability
• histamine and serotonin(platelets), bradykinin, substance P
• leukotrienes C4,D4,E4
• platelet activating factor and complement C3a and C5a

chemotaxis of leukocytes

• cardinal sign of inflammation
• leukotriene B4
• TNF, IL-1, chemokines, C3a, C5a, bacteria products
• C3 and C5a apear during the tumor-swelling sign 

Functio laesa-loss of tissue function

• cardinal sign of inflammation
• lysosomal enzyme release leukocytes
• reactive oxygen species and nitric oxide

Prostaglandin PGE2 

• sets core temperature
• posterior hypothalmus does below set point and anterior hypothalmus does above set point
• IL-1 (pyrogen) increases PGE2 synthesis and increases core temperature, causing fever 
• aspirin inhibits production of PGE2 leading to sweating and vasodilation

Eicosanoid metabolism 

• Produced from PUFA containing 20carbons(eicosa) by three different pathways (arachidonic acid made)
• activated by bacterial products, chemokinase-g, lipid mediators, or cytokines to release arachidonic acid from Sn2 position of phospholipids 
• most cells express COX1 and COX2(prostaglandins)
• different cytochrome P450 are expressed in different tissues
• lipoxygenase are primarily in in leukocytes

Release of eicosanoid from membrane

• attached to glycerol backbone of phospholipids at the Sn-2 position
• normally, oleic acid is atached but 5% of time its eicasanoids
• activation of phosholipase A2 by Ca2+ results in direct relase of arachidonic acid
• phospholipase C activation release DAG and arachidonic acid is released from DAG by the action of either diacyglycerol lipase or monoglycerol lipase

Linoleic acid and linolenic acid

• liver converts linoleic acid to omega 6 eicasanoid, arachidonic acid, which is pro-inflamatory (PGE2 and LTB4)
• linolenic acid is converted to omega 3 eicasanoid, eicosapentaenoic acid, an anti-inflammatory (less of inflam response)
• they are incorporated to Sn-2 position of phospholipids and transported to other tissues by VLDL particles
• these eicosanoids are released by capilary endothelial LPL inco cells
• omega6 eicosatrienoic acid produces less potent prostaglandins and leukotrienes
• balance btw omega 6 and 44 in phospholipids of cell mem determines the degre of a cells inflam response to tissue damage

Prostaglandins and thromboxanes 

• synthesis of these begins with cyclooxygenase
• COX adds four oxygen atoms to arachidonic acid to form an unstable thing that goes to hydroperoxide
• prostaglandin endoperoxide has cyclooxygenase and peroxidase activity (GSH used)
• COX1 is constitutively expressed in most tissues and COX2 induced by cytokines and growth factors
• COX2 expression is increased in macrophages and monocytes during inflam and rapidly proliferating cancers (activation of blood vessels or growth)

COX1 inhibitors 

• NSAIDS and aspirin both preferentially inhibit COX1 to a greater extent then COX2
• aspirin irreversibly inhibits and NSAID reversibly inhibits 
• steroidal drugs like hydrocortisone, prednisone and dexamethasone inhibit COX activity by inhibiting phospholipase A2 activity

COX2 inhibitors

• Vioxx and celebrex selectivly inhibit COX2 but have been associated with incresed adverse cardivascular events
• COX2 inhib decrease the antithrombotic production of prostacyclin(PGI2) in vascular endothelium
• while platelets COX1 syn of vaso-constrictive thrombaxane(TXA2) in platelets is not inhibited leading to increased vasculature vasoconstriction

Lipoxygenase pathway

• produces linear eicosanoids
• LOX have two activities, dioxygenase and dehydratase that transforme 5HPETE to LTA4
• LTA4 is a branch point in neutrophils in syn of LTB4 or conversion to LTC4 by adding GSH
• adding glutamate and glycine to LTC4 produces LTD and LTE4 which are slow reacting substances of anaphylaxis
• LTB4 provokes bonchoconstriction and infl by mediating immune cell chemotaxis, degranulation and release of lysosomal hydrolytic enzymes
• SRSAs are humoral agens that promote smooth muscle contraction and constriction of airway, increase capillary permeability and therefore tissue edema

HETE and diHETE

• made in cytochrome p450 pathway and epoxide is converted to these by epoxide hydratase
• one function of HETE is to inhibit the Na/K ATPase enzyme 

Inactivation ofEicosanoids

• involves cytochrome p450(a different one)
• alcohol follows the same pathway