Clinical Chemistry - 08 Proteins And Aminoacidopathies

CLINICAL CHEMISTRY CLASSIFICATION OF PROTEINS PROTEINS AND AMINOACIDOPATHIES • OUTLINE • Proteins o Functions of Proteins o Classification of Proteins • Plasma Protein o Two Major Groups o Albumin Group o Globulin Group • Cardiac Proteins o Myoglobin o Troponins o B-Type Natriuretic Peptide • Renal Protein o Cystatin C • Proteins in Body Fluids o Urinary Proteins o CSF Protein • Aminoacidopathies o Alkaptonuria o Homocystinuria o Maple Syrup Urine Disease o Phenylketonuria o Tyrosinemia • PROTEINS (CHON) • • • • • • • • Proteis – first rank of importance o Proteins have numerous physiologic functions Complex macromolecules composed of amino acids linked by peptide bonds Synthesized by the liver (except immunoglobulins) o Immunoglobulins - produced by B cells Amphoteric in nature o Amphoteric - positively or negatively charge o Zwitterions - Isoelectric point Could act as weak acid or weak base o Good blood buffer (maintains blood pH) 12-20% - total daily body energy requirement 50-70% of cell’s dry weight May be classified as: o Acute phase reactant - substances in the blood that react with infammation o Positive Acute Phase Reactant - blood analyte that increase during infection o Negative Acute Phase Reactant - decrease in concentration during inflammation PLASMA PROTEIN • • • • • • • • • Structural component – Collagen, elastin, keratin Transport – proteins bind and transport substances o Prealbumin, albumin, ceruloplasmin, α-2 macroglobulin, transferrin, hemopexin, haptoglobin, lipoprotein Blood Coagulation – Clotting factors o All clotting factors are proteins except Ca (electrolyte) Immune defense – immunoglobulins (antibodies) used for elimination of foreign antigens o IgG, IgM, IgA, IgE, IgD Osmotic pressure – maintains water distribution between cells and tissue, interstitial compartments & blood vessels. Blood buffers – proteins are weak acid or weak base due to the presence of positive and negative charge on their molecular structure Biocatalysts – enzymes act as catalyst of biochemical reactions (enzymes are made up of proteins) Hormones – chemical messengers that controls the activity of some cells or organs Source of energy – serves as precursor of glucose o Glucogenic amino acid – can undergo gluconeogenesis to form glucose and used as energy source. Proteins present in plasma Most commonly analyzes proteins in clinical laboratory TWO MAJOR GROUPS • • • FUNCTIONS OF PROTEINS • • Simple Proteins – contains peptide chain of amino acids. o Globular protein – water soluble symmetrical proteins ▪ Functions as transporter, enzymes, & messengers ▪ Examples: albumin, hemoglobin, immunoglobulins (IgA, IgM, IgG) o Fibrous protein – long protein filaments, asymmetrical and usually inert. ▪ Generally water insoluble because of their hydrophobic R groups ▪ Provide structures to cells ▪ Examples: fibrinogen, troponin, collagen Conjugated Proteins – consist of proteins & a non-protein. o non-protein part – referred to as prosthetic group i. Metalloprotein – with ions; ferritin, ceruloplasmin, hemoglobin, flavoproteins ii. Glycoprotein – haptoglobin, α1-antitrypsin (10-40% CHO) iii. Lipoprotein – lipids w/ specialized proteins (apolipoprotein); VLDL, LDL, HDL, Chylomicrons iv. Mucoproteins/Proteoglycans – mucin (higher CHO than CHON) v. Nucleoprotein – chromatin (combined w/ nucleic acid) Depending on their electrophoretic mobility (migration) Albumin group – function as transporter, maintains osmotic pressure, and amino acid reserves. o Example: Albumin, pre-albumin Globulins – subdivided into four groups each with specific properties and functions. o α1 globulin o α2 globulin o β globulin o γ globulin (immunoglobulins only) ALBUMIN GROUP PRE-ALBUMIN • • • • • • • • • Transthyretin Indicator of nutrition o Protein-poor diet result to decrease source of amino acid for protein synthesis by the liver thus decreasing the level of protein (pre-albumin, albumin, β-globulin) Binds thyroid hormones and retinol (vit. A) Named because it migrates before albumin (SPE) o SPE - Serum Protein Electrophoresis Used to confirm the specimen is CSF o It crosses more easily into the CSF that other proteins Half-life: 2 days o Time required to become half in concentration Increased: alcoholism, chronic renal failure, steroid treatment Decreased: poor nutrition Reference value: 18-45 mg/dL (CF to mg/L: 10) o o ALBUMIN • • • • • The most abundant protein in plasma o Liver synthesis: 9-12 g/day Negative acute phase reactant (decrease in inflammation) Decreased: inflammation, liver disease, renal disease o Acute nephrotic syndrome: increased albumin excretion; lowest plasma albumin level Reference value: 3.5-5.0 g/dL (CF to g/L: 10) Functions: o A general transport protein ▪ Electrolytes: Ca2+, Mg2+, Iron ▪ Bile pigment: Unconjugated bilirubin ▪ Hormones: thyroid hormones ▪ Insoluble drugs: aspirin ▪ Fatty acids o Maintains osmotic pressure o Indicator of nutritional status (several amino acids for protein synthesis in liver are required from diet) o Included in assessing the synthetic function of liver o Sensitive and prognostic marker of cystic fibrosis o Indicator of short-term hyperglycemic control • GLOBULIN GROUP GLOBULIN • • • • Group of proteins consists of α1, α2, β, and γ fractions Measurement: Globulin = Total Protein - Albumin Increased: early cirrhosis o Compensates to the loss of albumin resulting to normal level of total protein Reference value: 2.3-3.5 g/dL (CF to g/L: 10) • α1 GLOBULIN GROUP • • α1 – Antitrypsin (AAT) o Positive acute phase reactant (↑ in inflammation) o Most abundant protein in the α1-globulin band (90%) o Major inhibitor of protease activity ▪ Prevents self-destruction of tissues o Neutralizes trypsin-like enzymes: ▪ Neutrophil elastase: primary neutrophilic granules that combats infection but may also destroy pulmonary alveoli resulting to emphysema o Increased: inflammation, pregnancy and contraceptive use o Decreased: emphysema, juvenile hepatic cirrhosis, SERPINA1 gene mutation ▪ SERPINA1 gene - encode for α1-antitrypsin ▪ Decreased level result to almost lack of α1 band on SPE (majority of α1 band is AAT) o Reference value: 145-270 mg/dL (CF to g/L: 0.01) α1–Fetoprotein (AFP) o Migrates between albumin and α1globulin band o It is synthesized by the fetal yolk sac and then by the fetal parenchymal cells of the liver. o It has no known function in adults. o Normally detectable in maternal serum (7-8th month of pregnancy) o Specimen: maternal serum, amniotic fluid, (assessing fetal health); serum (cancer screening) o Method: immunochemical test (uses specific antibodies against AFP), RIA, EIA ▪ RIA - radioimmunoassay Reference value: 5 ng/mL (adult and children sera) Diagnostic significance (AFP): ▪ Maternal serum – sample for screening any fetal condition ▪ Amniotic fluid – confirmatory sample ▪ Increased AFP: • Maternal serum (twin fetus) • Peaks in fetus at 13th week of gestation o Gestational markers: Neural tube defects, anacephaly, spina bifida, GIT atresia, fetal distress, ataxiatelangiectasia, tyrosinosis, HDN • Serum: hepatoma (>1000 ng/mL), gonadal CA ▪ Decreased AFP: Down Syndrome (Trisomy 21), Edward’s syndrome (Trisomy 18) (Maternal serum/Amniotic fluid) α1–Acid Glycoprotein (AAG) o Orosomucoid o Major plasma glycoprotein o Positive acute phase reactant o Contains high percentage of carbohydrate (45%) and sialic acid (11-12%) o Similar amino acid sequence with immunoglobulins o Diagnostic tool for neonatal bacterial infections o Binds progesterone and quinidines (cardioactive drug) o Increased: pregnancy, cancer, pneumonia, AMI, RA (rheumatoid arthritis), and cell proliferation ▪ AMI - acute myocardial infarction o Methods: Radial immunodiffusion, immunoturbidity, nephelometry, immunofixation. o Reference value: 50-120 mg/dL (CF to g/L: 0.01) α1–Antichymotrypsin (α1-x) o Member of the serine protease inhibitor (serpin) family ▪ Inhibits by cleaving (conformation of shape) • cathepsin G, pancreatic elastase, mast cell chymase, chymotrypsin o Positive acute phase reactant o Synthesized by the liver o Binds and inactivates PSA ▪ Major form of PSA found in plasma bound w/ α1-x o Pathognomonic of Alzheimer’s disease ▪ α1-x is an integral component of the amyloid deposits in Alzheimer’s disease o Mutations have been identifies among patients with Parkinson’s disease and COPD (chronic obstructive pulmonary disease) o Increased: infection, malignancy, burn, AMI, Alzheimer’s disease o Decrease: liver disease (↓ synthetic function) o Reference value: 30-60 mg/dL (CF to g/L: 0.01) OTHER PROTEINS (α1 GLOBULIN BAND) • • Inter-α-trypsin inhibitor o Positive acute phase reactant o Member of serine protease inhibitor family (serpin) o Plays an important role in inflammation & carcinogenesis. o Increased: inflammatory disorders α1-lipoprotein (High density lipoprotein) o Transports excess cholesterol from the peripheral tissues to the liver. o Indirect marker for atherosclerosis o Increased (≥65 mg/dL): cardioprotective o Decreased (<35 mg/dL): risk for CHD, AMI ▪ CHD - coronary/chronic heart disease • Group-specific component globulin (Gc globulin) o Vitamin D-binding protein o Major carrier protein for vitamin D and its metabolites o Also transports fatty acids and endotoxins o Migrates towards the α1 and α2 bands (SPE) o Increased: pregnancy (3rd trimester), oral contraceptives (estrogen) o Decreased: liver disease (decreased synthesis) o Reference value: 20-55 mg/dL (CF to g/L: 0.01) o Method: radial immunodiffusion β-GLOBULIN GROUP • α2 GLOBULIN GROUP • • • Haptoglobin o Positive acute phase reactant o Synthesized by the liver o A tetramer(4 globin chain): 2 α chain, 2 β chain o Binds free hemoglobin by its α chain ▪ Preventing loss of hemoglobin and its constituent iron into the urine o Marker for intravascular hemolysis (HTN, HTR) o Marker to evaluate rheumatic disease o Increased: stress, myoglobinuria, inflammation o Decreased: Intravascular hemolysis, hemoglobinuria ▪ Intravascular hemolysis - hemolysis w/in blood vessel where Hgb are extravagated which will be bound with haptoglobin o Method: radial immunodiffusion, immunonephelometry o Reference value: 26-185 mg/dL (CF to g/L: 0.01) Ceruloplasmin o Copper-binding glycoprotein (enzyme) ▪ 90% copper – bound to ceruloplasmin ▪ 10% copper – bound to albumin o Positive acute phase reactant o Imparts blue color to protein o Marker for Wilson’s Disease (0.1 g/L ceruloplasmin) ▪ Characterized with copper deposition in skin, liver, brain and cornea (Kayser-Fleisher rings) o Increased: inflammation, cancer, pregnancy o Decreased: Wilson’s disease, malnutrition, malabsorption, nephrotic disease, Menkes’ Syndrome (kinky hair) o Reference value: 18-45 mg/dL (CF to mg/L: 10) α2-Macroglobulin (AMG) o Largest major non-immunoglobulin protein in plasma ▪ IgM - immunoglobulin o Forms complex with PSA (Prostate-Specific Antigen) o Inhibits proteases: trypsin, thrombin, kallikrein, plasmin o Removed by reticuloendothelial system after inhibiting proteases. o Increased: ▪ Nephrosis (ten folds or more) – reciprocal markers of nephrosis (Albumin & AMG) • Albumin & AMG - ↓ in nephrosis ▪ Pregnancy (↑20%) ▪ Diabetes ▪ Liver disease ▪ Contraceptive medication o Method: radial immunodiffusion, immunonephelometry, ELISA and latex agglutination o Reference value: 150-420 mg/dL (CF to g/L: 0.01) • • • Hemopexin o Positive acute phase reactant o Binds heme released by degradation of hemoglobin (1:1) ▪ 1 hemopexin only bind 1 heme molecule ▪ Strongest affinity for heme o Increased: inflammation, diabetes, Duchenne-type muscular dystrophy, melanomas o Decreased: urinary loss, decreased liver synthesis, hemolytic anemia, intravascular hemolysis ▪ Heme in extravagated Hgb are bound to Hemopexin o Reference value: 50-120 mg/dL (CF to g/L: 0.01) β2-Microglobulin o Light chain component of the Major Histocompatibility Complex or Human Leukocyte Antigen ▪ Found in most nucleated cells (highest on lymphocytes) o Needed in production of CD8 cells (T cytotoxic cells) o Freely filtered (glomerulus) and reabsorbed (proximal tubules) by the kidneys o Has tendency to fold into a β sheet configuration = amyloid formation ▪ Common cause of dialysis-associated amyloidosis o Increased: renal failure, multiple myeloma, rheumatoid arthritis, systemic lupus, HIV ▪ Renal failure - impaired renal filtration & absorption ▪ Myeloma - increased lymphocytes o Method: Immunoassay o Reference value: 0.2-2.8 μg/dL Transferrin o Siderophilin o Negative acute phase reactant o Major β-globulin protein (90%) o Transporter of ferric ions (from iron stores to bone marrow); iron is essential for RBC synthesis o Routinely measured to determine cause of anemia o Prevents loss of iron through the kidney o Increased plasma level appears as paraprotein (psuedoparaproteinemia) ▪ Paraprotein - increase conc. of total protein o Decreased level: ▪ Impairs hemoglobin production leading to anemia • Ferric ions are still bound in transferrin ▪ Accumulation of iron in apoferritin or histiocytes ▪ Precipitate in tissues as Hemosiderin o Increased: hemochromatosis (bronze diabetes), IDA o Decreased: Liver disease, malnutrition, nephrotic syndrome o Method: immunodiffusion, immunonephelometry o Reference Value: (CF to g/L: 0.01) ▪ Male: 215-365 mg/dL ▪ Female: 250-380 mg/dL Fibrinogen o Clotting factor I ▪ Converted into fibrin by the action of thrombin o Positive acute phase reactant o One of the largest proteins in the blood o Most abundant coagulation factor o Marker for long-term prognosis of cardiovascular disease o ↑ Fibrinogen = shorter ESR = coating red cells allowing them to sediment faster o Increased: inflammatory disorders, pregnancy, use of oral contraceptives o Decreased: extensive coagulation o Reference value: 200-400 mg/dL (CF to g/L: 0.01) • • Complement o Positive acute phase reactant o Natural defense mechanism of body against infection ▪ Important proteins for complement system ▪ End point is activation of membrane attack complex (MAC) o Circulates in inactivated form (nonfunctional precursor) o C3 is the most abundant form o C4 – 2nd most abundant o C3 & C4 – marker for RA and SLE ▪ Most commonly measured complement protein o Increased: inflammation o Decreased: ▪ C3: autoimmune disease, neonatal respiratory distress syndrome, bacteremia, tissue injury, chronic hepatitis ▪ C4: DIC, acute glomerulonephritis, chronic hepatitis, hemolytic anemia and malnutrition. • DIC - Disseminated Intravascular Coagulation o Method: immunonephelometry, turbidimetry. C-Reactive Protein (CRP) o Member of pentaxin protein family o Most popular acute phase reactant (positive) o First acute phase reactant to rise in response to inflammation o Termed as it precipitates with the C-substance of pneumococci o Undetectable in blood of healthy individuals. o Early Cardiac Marker ▪ Reflects severity of CHD o Used as rapid test for presumptive diagnosis of bacterial vs viral infection OTHER β-GLOBULIN PROTEINS • • Pre-β lipoprotein o Very Low Density Lipoprotein (VLDL) o Transport lipids (mainly TAG) from liver to the peripheral tissue β-lipoprotein o Low Density Lipoprotein (LDL) o Transport lipids (mainly Cholesterol) from liver to the peripheral tissue o The most atherogenic lipoprotein (transports most atherogenic lipids) o Reference value (Optimal): <100 mg/dL ▪ Above Optimal: 100-129 mg/dL ▪ Borderline: 130-159 mg/dL ▪ High: 160-189 mg/dL ▪ Very high: ≥190 mg/dL CARDIAC PROTEINS • MYOGLOBIN • • • • • • • • • • • Only proteins found in γ-region in SPE Synthesized by plasma cells/B-cells (lymphocyte) o Only globulins not synthesized in liver Examples: IgG, IgA, IgM, IgD, IgE Increased: hepatic disease, infections, toxoplasmosis, cytomegalovirus, rubella, herpes, syphilis, allergic reaction, multiple myeloma Decreased: HIV and other immunodeficiencies o HIV - attack lymphocytes which impair synthesis Method: nephelometry, turbidimetry, radial immunodiffusion, immunoassay The primary oxygen-carrying protein found in striated skeletal and cardiac muscle. o Has higher affinity for oxygen than hemoglobin o Transports and stores oxygen from hemoglobin to intracellular respiratory enzymes of contractile cells Screening test for myocardial infarction (>100 μg/L). o The earliest marker for MI o Rise: 1-3 hours o Peak: 5-12 hours (highest concentration) o Normalize: 18-30 hours (ave. 24 hrs) It is approximately 2% of the total muscle protein A potential nephrotoxin (Must be excreted when in excess) Myoglobinuria may cause false-positive reaction in urine strip for hemoglobin due to its pseudoperoxidase activity. Increased: AMI, angina, rhabdomyolysis, muscle trauma, strenuous exercise, intramuscular injection, acute renal failure. Method: immunoassay TROPONINS (Tn) • • • Gold standard in diagnosis of AMI A complex protein that resides in the filaments of cardiac (94-97%) and skeletal muscle (3-6%) o Almost exclusive to cardiac muscle Regulators of actin & myosin (contracts & relaxes muscles) 3 SUBUNITS • • • IMMUNOGLOBULINS (γ-GLOBULINS) • • Assess acute myocardial infarction or congestive heart disease • • Troponin T o Tropomyosin-binding subunit o Has been found in fetal skeletal muscle and deceased skeletal muscles o Sensitive marker for diagnosis of unstable angina o ≥1.5 ng/mL – suggestive of AMI o Absent from normal serum Troponin I o Inhibitory subunit o Cardiac-specific & sensitive (released w/ slight damage) o Only found in the myocardium o Absent from normal serum Troponin C o Calcium-binding subunit o Regulates muscle contraction o Not cardiac specific Rise Peak Normalize Troponin I 3-6 hours 12-18 hours 5-10 days Troponin T 3-4 hours 10-24 hours 7 days Method: Immunoassay Reference values depends on the antibodies and calibrators used in the immunoassay B-TYPE NATRIURETIC PEPTIDE (BNP) • • • • • A popular marker for congestive heart failure Increases in response to peptide ventricular systolic and diastolic dysfunction & is diagnostic of congestive heart failure Half-life: 60-120 minutes Methods: RIA, microparticle enzyme immunoassay, ECLIA Reference value: <100 pg/mL CSF PROTEIN RENAL PROTEIN CYSTATIN C • • • • • • A cysteine proteinase inhibitor Freely filtered by the glomerulus and almost completely reabsorbed and catabolized by the proximal tubular cells. Newly proposed as a new sensitive endogenous serum marker for glomerular filtration rate. Alternative for creatinine and creatinine clearance test to screen and monitor kidney dysfunction. o Not affected by age, diet, gender and muscle mass (unlike creatinine) o Useful for cases wherein creatinine clearance test is not appropriate (liver disease, obesity, muscular disease) Increased: renal disease Method: particle-enhanced immunoturbidimetry, immunonephelometry PROTEINS IN BODY FLUIDS • • • • • • • CSF OLIGOCLONAL BANDING • URINARY PROTEINS • • • • • Most urinary proteins are from blood Albumin in urine is abnormal (even in trace amount) o Albumin is filtered by glomerulus o Damage in glomerulus allow passage of albumin Normal albumin excretion rate = <20 μg/min Albuminuria after strenuous exercise is observed in some healthy individuals Urine protein ≥6.0 mg/dL = positive albumin urine dipstick TYPES OF PROTEINURIA • • • • Glomerular proteinuria (albuminuria) – most common and serious type of proteinuria Tubular proteinuria – appearance of low molecular weight proteins in the urine due to defective reabsorption with slightly increased urine albumin excretion rate Overload proteinuria – hemoglobinuria, myoglobinuria, and Bence-Jones Proteins Postrenal proteinuria – refers to protein coming from the urinary tract caused by infection, bleeding or malignancy CSF – ultrafiltrate of plasma formed in the choroid plexus of the brain ventricles. CSF Glucose and Protein analysis – blood sample should be analyzed first prior to collection of CSF sample. o Blood glucose and protein conc. is used for comparison Method: TCA, SSA, Coomasie Brilliant Blue (dye), Lowry and Kinetic Biuret Reaction Increased: bacterial, viral and fungal meningitis, traumatic tap, multiple sclerosis, intracerebral hemorrhage, myxedema, drug toxicity Decreased: intracranial hypertension, hyperthyroidism, CSF leakage due to trauma Reference value: 15-45 mg/dL CSF Albumin: 10-30 mg/dL (2/3 of CSF total protein) • • • • • Seen in multiple sclerosis o Presence or 2 or more IgG bands in the γ region o 90% cases show oligoclonal bands in the γ region of the protein electrophoresis. o Indication of antibody production within the CNS Primary purpose of CSF protein electrophoresis: o To observe this type of banding representing inflammation within the CNS (+) result for CSF: ≥ 2 bands in CSF not present in serum Other disorders with 2 or more bands in the CSF: o Encephalitis, neurosyphilis, Guillain-Barre syndrome, neoplastic disorders Disorders with serum banding appearing in CSF: o Leukemia, lymphoma, viral infection Supporting medium and stain: o Agarose gel & Coomasie Brilliant Blue MICROALBUMINURIA • • • • • • • • • Early indicator of glomerular dysfunction and precedes nephropathy associated with type 1 DM Urine albumin-creatinine ratio of 30-300 μg/mg creatinine. Not detected by urine reagent strips Positive microalbuminuria: 2 out 3 specimens collected within 3-6 months Physiologic increase: physical exercise during the previous 24 hours (in normal healthy individuals) Increased: diabetic nephropathy, fever, infection, & hypertension Specimen: Random-urine (reject bloody urine) Method: random-spot albumin-creatinine ratio Reference value: 0-29 μg/mg creatinine. o Microalbuminuria: 30-300 μg/mg creatinine. o Clinical albuminuria: >300 μg/mg creatinine. ▪ With positive urine strip for protein AMINOACIDOPATHIES • • • Inherited disorders associated with amino acid metabolism Enzyme activity defect or membrane transport defect Detected in newborn screening ALKAPTONURIA • • • Inborn error of metabolism characterized by the absence of homogentisate-1,2-dioxidase in the tyrosine pathway o Inability to convert homogentisic acid to maleylacetoacetic acid Clinical feature: ochronosis (tissue pigmentation) Diagnostic indicator: darkening of urine upon standing at room temperature PHENYLKETONURIA • • • • • • HOMOCYSTINURIA • • • • Characterized by impaired activity if cystathionine β-synthetase Results to elevated levels of homocysteine and methionine in blood and urine Clinical features: physical defects, thrombosis, osteoporosis, eye lens abnormality, mental retardation Screening test: Modified Guthrie Test (L-methionine sulfoximine – antagonist) TYROSINEMIA • • • • MAPLE SYRUP URINE DISEASE (MSUD) • • • • • • Characterized by markedly reduced or absence of αketoacid decarboxylase It results to accumulation of branched-chain amino acids (leucine, isoleucine and valine) in blood, urine and CSF Clinical feature: failure to thrive, muscular ridgidity, mental retardation, hypoglycemia Screening test: Modified Guthrie Test (4-azaleucine – antagonist) Diagnostic test: amino acid analysis (HPLC - High Performance Liquid Chromatography) Indicator: 4 mg/dL leucine – indicative of MSUD Autosomal recessive trait characterized by the deficiency of phenylalanine hydrolase (PAH)/phenylalanine-4monooxygenase Characterized by the presence of elevated phenylpyruvic acid (prime metabolite) in both blood and urine Deficiency of tetrahydrobiopterin (BH4) = elevated phenylalanine Reference value (phenylalanine): 1.2-3.4 mg/dL (70-200 umol/L) Clinical and Diagnostic features: o Mental retardation (infants and children) o >1000 mg/dL serum phenylalanine o Musty odor urine Screening test: Guthrie Bacterial Inhibition Assay o Bacillus subtilis spores & β2-thienylalanine antagonist ▪ B. subtillis is inoculated in agar plate ▪ β2-thienylalanine - inhibit bacterial growth o Positive: Bacterial growth (phenylalanine >4 mg/dL) ▪ Phenylalanine diminishes antagonist activity Deficiency of either of these enzymes in tyrosine pathway: o Tyrosine aminotransferase (Tyrosinemia II) o 4-hydroxyphenylpyruvic acid oxidase (Tyrosinemia III) o Fumarylacetoacetate hydrolase/FAA hydrolase (Tyrosinemia I) Deficiency of these enzymes would lead to liver damage or cirrhosis Accompanied by elevated methionine and phydroxyphenylpyruvic acid in blood