University of Veterinary and Pharmaceutical Sciences Brno Faculty of Pharmacy Disorders of amino acid metabolism 1Patobiochemistry-AA Metabolic disorders Patobiochemistry-AA 2 Metabolic disorders • metabolic changes of proteins, carbohydrates, fats and water management (mostly mental or physical disability) • 7000 described metabolic disorders (7% of total population), 700-800 -inherited • Heredity - genetically determined enzyme defect causes metabolic block with pathological consequences • enzymopathy- most often is a metabolic disorder caused by a defect in the enzyme - defective enzyme has reduced enzymatic aktivity or activity is completely missing • primary - genetic background secundary – due to other disorders Principes of metabolic disorders substrate product mutation in genome mutation in mitochondrial genome defect protein multiorgan defects Patobiochemistry-AA 3 Types of metabolic disorders • Enzymopathy - totaly described more than 200 defect enzymes function -phenylketonurie - acummulation – spatial problem (glycogen storage disease, lipidosis) - increased toxicity (cysteinuria, gout) - conversion to other harmful metabolite - inhibits the metabolism of another enzyme, transporter, lack of product • Receptors and their disorders- receptor - familial hyperlipidemia (hypercholesterolaemia) • Disorders of molecular transport- cystic fibrosis • Defect of structure of cells - muscular dystrophy • Regulation of sex differentiation- gen SRY • Mitochondrial disease- Leber´s optic atrophy • Genes with so far unknown mechanism of action- Syndrome of fragile X (triplet disease) • Wrong endocrine regulation- diabetes mellitus Type of defect Disability examples Defect of enzymes PKU, galactosemia, adenosine deaminase deficiency Defect of receptors testicular feminization, hypercholesterolaemia Defect of molecular transport cystic fibrosis, hypertension Defect of cell structure Duchenn and Becker´s muscular dystrophy Defect of homeostasis antihemophilic globulin, immunoglobulins Defect of regulation of growth and differentitation sex determination, inactivation of X chromosome, tumor suppressors Defect of intercellular communication inzuline, growth hormone, sex differentiation Defect of mitochondria Leber´s optic atrophy TEST Patobiochemistry-AA 4 Metabolism of AA and proteins • proteins from AA linked by peptide bonds (CO-NH) structure: • – primary - 20 AA • – secundary – hydrogen and disulfide bonds - globulin, β-sheet • – tertiary – conformation in space • – quaternary – association of several protein subunits • under physiol. pH mostly negative charge • – buffers (capability of binding H+) • constant renewal and degradation of proteins associated with the synthesis and catabolism AA • proteins significantly differ its half-life • regulatory proteins, enzymes and transcription factors, usually several hours • - albumin 10 days • - muscle proteins ~180 days • - hemoglobin ~120 days • - collagen several years • – turnover in a 70 kg human of about 300 g proteins/day • – about 30g/day per day is required for the substrates for synthesis of nucleotides, glucose, ketone bodies and neurotransmitters • – about 35 – 55g/day is oxidized to water, CO2 and nitrogen (irreversibly eliminated as urea) there is no storage form of proteins • – AA “pool” is just such as it is an immediate need • – rest is oxidized and eliminated • losses (and essential AA) must be paid by dietary protein intake • – essential: His, Val, Leu, Ile, Lys, Met, Thr, Phe, Trp • – nonessential AA may be formed esp. of intermediates of citric acid cycle • nitrogen release from the AA in the form of ammonia and ammonium salts is toxic, therefore it is in the liver processed in the urea cycle to a non-toxic urea, which is excreted in the urine Patobiochemistry-AA 5 Protein requirements at diet Patobiochemistry-AA 6 Protein digestion and resorption AA in GIT proteins in GIT – ~50% from diet, very different "digestibility" proteins - little digested elastin, keratin, mucin enzyme digestion of proteins, resorption AA and di- and tripeptides by enterocytes of small intestine via transporters (SLC, solute carriers – many types), concentration AA in cell is generally much higher than extracellularly therefore is active maintaining • – Na+ -dependent transport – act. transport Na+ facilitated diffusionNa+/AK (=symport) • – Na+ -independent – facilitated diffusion(=uniport) resorption whole proteins in diet – limited potential - through endocytosis - and/or at the sites of epithelia – it's used for systemic enzyme therapy (capsule resistant to the effect of HCl and pancreatic enzymes) Patobiochemistry-AA 7 Digestion of Proteins proteins pepsin Aminopeptidase and dipeptidase Carboxypeptidase, trypsin, chymotripsin Carboxypeptidase pepsin pepsinogens Aminopeptidase dipeptidase epitel S S S S A A Basic AA neutral AA acidic AA Portalblood TEST Patobiochemistry-AA 8 Disfunctions of protein´s metabolism AMINOACIDS • basic structural components (structural proteins, enzymes,hormones, purines, •plasma proteins, amines, heme) • source of energy carbonaceous residues of the amino acids incorporated into Crebs cycle, by protein metabolism is produced ammonia→ urea → ornithin cycle • proteins are not stored in stock incidence affected enzyme hyperphenyla laninemia 1:6500 (ČR), 1:13 000 (world) phenylalaninhydroxylase(98 %) , tetrahydrobiopterin (2 %) tyrosinemia I 1:100 000 (world) fumarylacetoacetathydrolase tyrosinemia II rare tyrosinaminotransferase tyrosinemia III rare 4-hydroxyphenylpyruvate dioxygenase alkaptonuria 1:100 000 - 1:1 000 000 (world), 1:19 000 (Slovakia) homogentisate-1,2- dioxygenase homocystinur ia 1-9:1 000 000 (world) cystationin ß-syntase cystinuria 1:7000 (USA) defect of renal transport of certain amino acids maple syrup disease 1:185 000 dehydrogenase of branchedchain alfaketoacids izovaleric acidemia 1:230 000 (world) isovaleryl-CoA dehydrogenase glutaric aciduria 1:40 000 (whites) glutaryl-CoA dehydrogenase methylmaloni c aciduria rare metylmalonyl-CoA mutase propionic acidemia rare propionyl-CoA carboxylase urea cycle disorders 1:30 000 (world) Disfunction of AA metabolisms or its transporters intestin blood tissue kidney urea liver proteins of tissue proteins AA AA Plasmitic proteins, urea, glycogen Patobiochemistry-AA 9 Disorders of AA metabolism • 1) Aminoacidopathy • 2) Organic aciduria • 3) Disorders of ammonia detoxification Disorders of AA transport Disorders of peptides metabolism TEST Patobiochemistry-AA 10 TEST Patobiochemistry-AA 11 incidence affected enzyme hyperphenylalani nemia 1:6500 (ČR), 1:13 000 (world) phenylalaninhydroxylase(98 %), tetrahydrobiopterin (2 %) tyrosinemia I 1:100 000 (world) fumarylacetoacetathydrolase tyrosinemia II rare tyrosinaminotransferase tyrosinemia III rare 4-hydroxyphenylpyruvate dioxygenase alkaptonuria 1:100 000 - 1:1 000 000 (world), 1:19 000 (Slovakia) homogentisate-1,2-dioxygenase homocystinuria 1-9:1 000 000 (world) cystationin ß-syntase cystinuria 1:7000 (USA) defect of renal transport of certain amino acids maple syrup disease 1:185 000 dehydrogenase of branched-chain alfaketoacids izovaleric acidemia 1:230 000 (world) isovaleryl-CoA dehydrogenase glutaric aciduria 1:40 000 (whites) glutaryl-CoA dehydrogenase methylmalonic aciduria rare metylmalonyl-CoA mutase propionic acidemia rare propionyl-CoA carboxylase urea cycle disorders 1:30 000 (world) TEST Patobiochemistry-AA 12 Disorders of AA metabolism • A). AMINOACIDOPATHY • Accummulation of AA, variations in degradation of AA in the cytosol • Ammonia accumulation • Carbon skeleton accumulations of organic acids • Product deficiency • Deficiencies of mitochondrial enzymes (dehydrogenase of ketoacids with branched chain) leucinesis • It doesn´t include CoA-activated metabolites • Accummulation of toxic metabolites, phenylalanin, phenylpyruvate, phenylacetate –PHENYLKETONURIA (specific organ damage) • Diagnosed by determination of metabolites level • diets Patobiochemistry-AA 13 B) ORGANIC ACIDURIAS • Deficiency of the enzyme in the mitochondrial metabolism, CoA-activated carboxylic acids Patobiochemistry-AA 14 Disorders of aromatic AA metabolism (Phenylalanin,Tyrosin) PHENYLALANIN → TYROSIN (phenylalanine hydroxylase) 1) hyperphenylalaninemia deficiency of phenylalaninehydroxylase – classical phenylketonuria (PKU) defect dihydrobiopterinreductase – hyperphenylalaninemia type II and III defect of dihydrobiopterin biosynthesis (cofactor) – hyperphenylalaninemia type IV and V AR hereditary disease accumulation of phenylalanine and metabolites (phenylpyruvic, phenyllactic, phenylacetate, o-hydroxyphenylacetate acid) disbalance of plasmatic AA: damage brain development, phenylalanine inhibits enteral resorption of tyrosine (compete together for a transporter)→ impaired catecholiamines and melanines synthesis (skin pigmentation and hair is reduced) → irreversible mental retardation (high level of Phe harms brain), seizures, psychosis, eczema, urine odor after mice, light pigmentation (blond hair and blue eyes even in the case that there is no genetic predisposition) 2) hypertyrosinemia Tyrosinemia type 1 – deficiency of fumaryl acetoacetate hydroxylase enzyme metabolite sukcynilaceton accumulates in the blood, which damages the liver, kidneys, CNS and PNS, particular manifestation of sick is self- harming Tyrosinemia type 2- deficiency of the enzyme tyrosine aminotransferase (disability eyes, skin and the CNS) Transient tyrosinemia or hyperphenylalanemia in newborns- Delaying the enzyme phenylalanine hydroxylase, A transient increase tyrosine in plasma in the first 2 weeks of life, given the delayed maturation enzymes of tyrosinaminetransferase or 4-hydroxyphenylpyruvatedioxygenase in liver 3) alkaptonuria defect of homogentisate oxygenase high concentration of homogentisic acid (oxidation homogentisate on benzochininacetate → generalized pigmentation binder, sclerae, ears, skin), arthritis (hips, ankles, spine), kidney damage (urolithiasis) and heart valves (aortic or mitral regurgitation valves), calcification of the aorta, urine darkens on light (brown pigment alkapton) TEST Patobiochemistry-AA 15 TEST Patobiochemistry-AA 16 Disorders of aromatic AA metabolism (Phenylalanin,Tyrosin) PHENYLALANIN → TYROSIN (phenylalanine hydroxylase) 1) hyperphenylalaninemia a) deficiency of phenylalaninehydroxylase – classical phenylketonuria (PKU) b) defect dihydrobiopterinreductase – hyperphenylalaninemia type II and III (HPA) c) defect of dihydrobiopterin biosynthesis (cofactor) – hyperphenylalaninemia type IV and V AR hereditary disease accumulation of phenylalanine and metabolites (phenylpyruvic, phenyllactic, phenylacetate, o-hydroxyphenylacetate acid) The frequency of 1 / 10,000 individuals - disbalance of plasmatic AA: damage brain development, - phenylalanine inhibits enteral resorption of tyrosine (compete together for a transporter)→ - impaired catecholiamines and melanines synthesis (skin pigmentation and hair is reduced) → - irreversible mental retardation (high level of Phe harms brain), seizures, psychosis, eczema, - urine odor after mice, - light pigmentation (blond hair and blue eyes even in the case that there is no genetic predisposition) Diet therapy (until the end of the development of the CNS - i.e. up to about the 20th year of life), serving saptoterin (Kuvan) → synthetic versions of the natural THBP (increases the activity of phenylalanine hydroxylase, whether the problem is faulty enzyme or THBP), serving LDOPA (substitution for the formation of catecholamines), LNAA trasporter (large neutral amino acids trasporter – blocking transfer of Phe at high levels through the blood brain barrier Patobiochemistry-AA 17 Phenylalanine hydroxylase → monooxygenase = engages only one O, arises from the second water H2 donor for the formation of water is tetrahydrobiopteridin (THBP), after releasing H2 arises dihydrobiopteridin (DHBP), it is reduced DHBP reductase back to THBP Phe is accumulated (hyperphenylalaninemia - up to 150-630mg /l plasma) and is converted to phenylpyruvate and phenyllactate and excreted in the urine, often is excreted as phenylacetylglutamine Patobiochemistry-AA 18 defect phenylalanine hydroxylase – classical phenylketonuria (PKU) • The first enzyme in the catabolic pathway for phenylalanine (Fig. 17-26), phenylalanine hydroxylase, catalyzes the hydroxylation of phenylalanine to tyrosine. A genetic defect in phenylalanine hydroxylase is responsible for the disease phenylketonuria (PKU). Phenylketonuria is the most common cause of elevated levels of phenylalanine (hyperphenylalaninemia). Phenylalanine hydroxylase inserts one of the two oxygen atoms of O2 into phenylalanine to form the hydroxyl group of tyrosine; the other oxygen atom is reduced to H2O by the NADH also required in the reaction. This is one of a general class of reactions catalyzed by enzymes called mixed-function oxidases (see Box 20-1), all of which catalyze simultaneous hydroxylation of a substrate by O2 and reduction of the other oxygen atom of O2 to H2O. Phenylalanine hydroxylase requires a cofactor, tetrahydrobiopterin, which carries electrons from NADH to O2 in the hydroxylation of phenylalanine. During the hydroxylation reaction the coenzyme is oxidized to dihydrobiopterin (Fig. 17-27). It is subsequently reduced again by the enzyme dihydrobiopterin reductase in a reaction that requires NADH. Patobiochemistry-AA 19 defect dihydrobiopterinreductase – hyperfenylalaninemia type II and III defect of dihydrobiopterin biosynthesis (cofactor) – hyperphenylalaninamia type IV and V Patobiochemistry-AA 20 21 Metabolites of phenylalanine COOH CHH2N CH2 transaminace COOH C CH2 O fenylalanin fenylpyruvát oxid. dekarboxylace hydrogenace COOH CH2 COOH CH CH2 OH fenylacetát fenyllaktát When phenylalanine hydroxylase is genetically defective, a secondary pathway of phenylalanine metabolism, normally little used, comes into play. In this minor pathway phenylalanine undergoes transamination with pyruvate to yield phenylpyruvate (Fig. 17-28). Phenylalanine and phenylpyruvate accumulate in the blood and tissues and are excreted in the urine: hence the name of the condition, phenylketonuria. Much of the phenylpyruvate is either decarboxylated to produce phenylacetate or reduced to form phenyllactate. Phenylacetate imparts a characteristic odor to the urine that has been used by nurses to detect PKU in infants. The accumulation of phenylalanine or its metabolites in early life impairs the normal development of the brain, causing severe mental retardation. Excess phenylalanine may compete with other amino acids for transport across the blood-brain barrier, resulting in a depletion of some required metabolites. Alternative pathways for catabolism of phenylalanine in phenylketonurics. Phenylpyruvate accumulates in the tissues, blood, and urine. Phenylacetate and phenyllactate can also be found in the urine. Patobiochemistry-AA disease is included in the newborn screening (method of tandem mass spectrometry (from 1. 10. 2009); earlier Guthrieho test – collected blood of a child /4.–5.day after childbirth/ is added to colony of Bacillus subtilis, bacillus survives just in the blood rich in Phe) Patobiochemistry-AA 22 23 Hyperphenylalaninemia + Phenylketonuria • consequence of untreated disorders - mental retardation • treatment - strict diet with low intake of Phe to about 15 years of age • later less strict diet • many products contains sweetener aspartame, unsuitable for phenylketonurics, hydrolysis releases phenylalanine H2N N HOOC O H O O CH3 L-aspartyl-L-fenylalanine methyl ester (180× more sweeter than sucrose)Patobiochemistry-AA Function of Phe in org. • Phenylalanine occurs in 3 forms: L-phenylalanine, What is the natural form of which is found in proteins, D-phenylalanine, which is a mirror image of L-phenylalanine produced in the laboratory, and DLphenylalanine, a combination of D- and L-forms . L-Phe form is part of the protein, while the D-form acts as a painkiller. • The amino acid – Phe is the immediate precursor of tyrosine (Tyr), is converted primarily to that amino acid which is used in the biosynthesis of the dopamine and norepinephrine neurotransmitters Patobiochemistry-AA 24 Treatment - dietary measures • For a whole life • The high content of Phe have these foods : • eggs, milk, cheese, meat, poultry, fish, dried beans and legumes, high content of protein - excluded from food • Treatment with BH4 -tetrahydrobiopterin, some patients don´t respond • responsive to BH4 therapy depending on their PAH gene mutation. Sapropterin dihydrochloride (Kuvan, BioMarin Pharma) is an orally active synthetic form of BH4. • Note: the same symptoms showed similar disease (hyperphenylalaninemia), which, however, has a different basis. The cause of this disease is a deficiency of one of four enzymes that are involved in the formation and metabolism of tetrahydrobiopterin BH4 - see herein above • Another possibility is therapy with enzyme replacement Patobiochemistry-AA 25 Content of Phe in food Food Average content of PHE in 1g of protein (mg) Fresh fruits 27 Fresh vegetables 35 Fresh mushrooms 29 Potatoes and products made from it 49 Milk and dairy products 51 Bakery products 58 Pork meat 44 Beef meat 48 Smoked meat 46 Fish 43 Nuts 51 Corn 55 Yolk 49 Egg white 69 Candies, chocolate, cookies 50 Average content of phenylalanine (PHE) in various types of foods Patobiochemistry-AA 26 Large Neutral Amino Acid Supplementation Other novel therapeutic approaches can be categorized by the site of action or target organ (Figure 2) [17]. These categories include enteral, systemic, liver-directed approaches. Dietary restriction of Phe intake is an example of enteral approach. Alternatively Large Neutral Amino Acid (LNAA) can be used. LNAA can compete with the same transporter of Phe across the gastrointestinal and blood brain barrier to reduce Phe absorption and entry into the brain [18]. A double blind, placebo-controlled study indicated a significant decline in blood Phe concentration in patients with PKU treated with LNAA for 2 weeks suggesting that LNAA compete with the transport of Phe in the gastrointestinal trac [19]. These studies suggest that adding LNAA to the diet of patients with PKU could reduce blood Phe concentrations. Patobiochemistry-AA 27 Disorders in metabolism of aromatic AA (Phenylalanine, Tyrosine) PHENYLALANINE → TYROSINE (phenylalanine hydroxylase) 2.hypertyrosinemia Tyrosinemia type 1 – deficiency of enzyme fumarylacetoacetate hydroxylase Metabolite succinylacetone accumulated in blood, damaging liver, kidneys, PNS a CNS. Particular manifestation is is self-harming of the pacient Tyrosinemia type 2- deficiency of enzyme tyrosine-aminotransferase (impairment of eyes, skind and CNS) Transient tyrosinemia or hyperphenylalaninemia of the newborn – delayed activation of enzyme phenylalanine hydroxylase, Transient increase of tyrosin levels in plasma in first 2 weeks of life, caused by delayed maturation maturation of enzyme tyrosine aminotransferase or 4-hydroxyfenylpyruvate dioxygenase in liver 3. alkaptonuria Defect of homogentisate oxygenase High concentration of homogentisic acid (oxidation of homogentisate to benzoquinone acetate → generalised pigmentation of connective tissue, sclera, auricles, skin), arthritis (hip, ankle, spine), kidney damage (urolithiasis) and heart valves (regurgitation of aortal or mitral heart valve), calcification of aorta, urine darkens on light (brown pigment alkapton) Patobiochemistry-AA 28 2. hypertyrosinemia Tyrosinemia type 1 - deficit of enzyme fumaryl acetoacetate hydroxylase - sukcynilaceton metabolite is accumulated in the blood and damages the liver, kidneys, CNS and PNS particular manifestation is a self-harm of sick person Tyrosinemia type 2- deficiency of the enzyme tyrosine aminotransferase (disability eyes, skin and the CNS) Transient tyrosinemia or hyperphenylalanemia in newborns - Delayed activation of enzyme phenylalanine hydroxylase, A transient increase of tyrosine in plasma in the first 2 weeks of life, given the delayed maturation of enzymes tyrozinaminotransferázy or 4-hydroxyfenylpyruvátdioxygenázy in liver Patobiochemistry-AA 29 Patobiochemistry-AA 30 Tyrosinemia type 1 - deficit of enzyme fumaryl acetoacetate hydroxylase (liver) - sukcynilaceton metabolite is accumulated in the blood and damages the liver, kidneys, CNS and PNS particular manifestation is a self-harm of sick person Tyrosinemia type I defect in fumarylacetoacetate hydrolase (expressed mainly in the liver and kidneys) and probably also in maleinylacetacetatehydrolase, AR hereditary high level Tyr (60-120mg / l plasma) and Met, the high level of metabolites affect activity of other enzymes and transport systems - severe pathology hepatorenal failure (cirrhosis of the liver, hepatomegaly, coagulopathy, Fanconi syndrome - a disease of the proximal tubules of the kidneys, excretion of phosphate → hypophosphatemic infliction of CNS (cramps, hyperextension, self-injury, respiratory arrest), ascites, accumulated metabolites (maleylacetoacetate, fumarylacetoacetate) and their derivatives (succinylacetone and succinylacetoacetate) make glutathione derivatives (removal of function of one antioxidant) - tissue damage caused by radicals Patobiochemistry-AA 31 •acute tyrosinosis without treatment → diarrhea, vomiting •smell of cabbages, die within 6-8 months (liver failure) • chronic tyrosinemia → symptoms are the same, but weaker individuals die within 10 years • sooner treatment diet without Phe, Tyr; Today NTBC → p-blocker of hydroxyfenylpyruvathydroxylase ●the drug NTCB inhibits phydroxyphenylpyruvate dioxygenase, intercepting the degradative pathway upstream of the toxic metabolites ●dietary restriction of tyrosine required to prevent neurological deficit Treatment: Metabolic defect is treated by diet with lowincome of tyrosine (Tyr) and (Phe) into adulthood, in serious cases → failure of liver functions, liver transplantation Patobiochemistry-AA 32 Treatment Nitisone - known as NTBC, substance originally developed as a herbicide. Now - substance used for slowing the effects of tyrosinemia type I. For the first time for this indication was used in 1991 averted the need of using transplantation of liver damaged by this disease such as the treatment of first choice. It is studied in connection with alkaptonuria. Commercial name of the drug - Orfadin. The mechanism of action of nitisinon involves reversible inhibition of 4-hydroxyphenylpyruvate oxidase and performs preventive forming of maleylacetoacetic acid and fumarylacetoacetic acid. Patobiochemistry-AA 33 Tyrosinemia type II. (Richter-Hanhart syndrome) • defect in liver tyrosintransaminase • Conversion of tyrosine to acetate - manifested by disease called keratosis • very rare disease, AR hereditary • elevated levels of tyrosine (40-50mg / l plasma) • mild mental retardation, hyperkeratosis (on the palms and soles feet), inflammation of the conjunctiva, corneal ulceration, nystagmus, glaucoma (turbidity by tyrosine crystals), tyrosine and its metabolites are present in urine • treatment by diet • Patobiochemistry-AA 34 Patobiochemistry-AA 35 Metabolic disorders of aromatic AAs (phenylalanine, tyrosine) FENYLALANINE → TYROSINE 3). alkaptonuria defect of homogentisateoxygenase high homogentisic acid concentration (oxidation of homogentisate to benzochininacetate → generalized binder pigmentation, sclera, ears, skin), arthritis (hips, ankles, spine), kidney damage (urolithiasis) and heart valves (regurgitation of the aortic or mitral valve), calcification of the aorta, urine darkens on light (brown pigment alkapton) • Treatment – diet, ascorbic acid (vit. C) prevents the binding of homogentisicacid. to binder, administration of NTBC mutation in the HGD gene Patobiochemistry-AA 36 37 • BCAA (branched chain amino acids) • all three are essential • the first reactions of catabolism are similar (transamination oxide. decarboxylation dehydrogenation) final products are different • leucine - ketogenic AA • after eating, their representation in blood is high (about 70% of all AA), because the liver do not use them (lack of aminotransferases) • most utilized by muscle and CNS • favorably affect the catabolic states (infusion) Metabolic disorders of branched AMK (Val, Leu, Isoleucine) Patobiochemistry-AA Metabolism of branched AMK overview • the first three reactions common to all these AMK, AMK are going through liver • Common reactions transamination (first reaction) by common transaminase (highest activity in myocardium and skeletal muscle, low in the liver) – corresponding 2-oxo acids are formed (2-val → oxoisovalerate, leuoxokapronate → 2, 2 → ile-oxometylvalerate); hypervalinemic block • decarboxylation (the second reaction) and dehydrogenation (the third reaction) - takes place in the mitochondria → specific multienzyme dehydrogenase → acyl-CoA which is one carbon shorter than the original oxoacids is formed; block (2 – dekarboxylation) at the maple syrup disease; block (3 – dehydrogenace) at isovaleric acidemia • result: • Val → methylakryoloyl-CoA • Leu → β-metylkrotonoyl-CoA • Ile → tigloyl-CoA 1. transamination (transaminase)→ 2-oxoacids (val → 2-oxoisovalerate, leu → 2-oxokapronate, ile → 2-oxometylvalerate 2. dekarboxylation 3. dehydrogenation (specific multienzyme dehydrogenase) Patobiochemistry-AA 38 39 Compare the final products Leucine acetyl-CoA + acetoacetate ketogenic AA Isoleucine acetyl-CoA + sukcinyl- CoA mixed AA Valine sukcinyl-CoA glukogenic AA B12 B12 Patobiochemistry-AA Metabolic disorders of branched AMK (Val, Leu, Isoleucine) 1. transamination (transaminase)→ 2-oxoacids (val → 2-oxoisovalerate, leu → 2-oxokapronate, ile → 2-oxometylvalerate 2. dekarboxylation 3. dehydrogenation (specific multienzyme dehydrogenase) Hypervalinemia low activity of transaminase common for valine, a very rare disease, Maple syrup disease (leucinosis) deficit or insufficient activity decarboxylase/dehydrogenase complex increased levels of Val, Leu and Ile, and 2-oxoacids brain damage, failure to thrive, drowsiness, coma and later vegetative nerve problems (abnormal heart activity - bradycardia, hypothermia), severe dehydration Intermittent forms of leucinosis less severe modification of decarboxylase, metabolism of Val, Ile, and Leu is reduced but maintained, symptoms of leucinosis appear later and occasionally, (after ingestion of large amounts of AMK) Isovaleric acidemia deficit of isovaleryl-CoA-dehydrogenase metabolic acidemia (pH 7,3), ketonuria, hyperammonemia, hypocalcemia, hyperlactémie, odor of breath, body fluids, coma after ingestion of large amounts of protein, Generalized pancytopenia methylmalonic aciduria Caused by avitaminosis B12. B12 is a cofactor of the enzyme which converts methylmalonyl-CoA to succinylCoA (radical isomerization) metabolic acidosis transamination Dekarbocylation, dehydrogenation TEST Patobiochemistry-AA 40 maple syrup disease 1:185 000 dehydrogenase of branched alfaketoacids #24860 #24861 #24861 #24690 izovaleric acidemia 1:230 000 (worldwide) isovaleryl-CoA dehydrogenase #24350 glutaric aciduria 1:40 000 (white people) glutaryl-CoA dehydrogenase #23167 metylmalonic aciduria rare metylmalonyl-CoA mutase #25100 propionic acidemia rare propionyl-CoA karboxylase #60605 Metabolic disorders of branched AMK (Val, Leu, Isoleucine) Patobiochemistry-AA 41 Maple syrup disease • Disorder in Leu, Ile, Val mtb - branched amino acids – deficiency of dehydrogenase/decarboxylase complex of branched AA, • organic acids are accumulated (alpha -ketoacids derivatives) – severe toxicity, increased levels of Val, Leu and Ile, and 2-oxoacids • deficit or insufficient activity decarboxylase • Excess of toxic metabolites - always after increased amount of branched AA, eg. an infant postpartum weight loss, protein degradation -fever, starvation, diet, disease • clinical manifestation: (sweat, urine – breath odour after maple syrup, caramel, dried fruit) • Newborn - soon after birth, lethargy, or does not accept breastfeeding - weak suck, irritability, a milder form - mental retardace.Hyperacidosa, hyperammonemia → convulsions, coma - death without treatment !!! brain damage, failure to thrive, drowsiness, coma and later vegetative nerve problems (abnormal heart activity - bradycardia, hypothermia, ažapnoe), severe dehydration Treatment: Diet with limited leucine and valine and izoleucine intake, additions spec. Nutrition with AA important for growth (contribution of health insurance company) Precautions: avoid starvation, limiting protein intake - substituting for. Glucose / in sickness etc.). Start treatment as soon as possible!!! after 14.days of starting the treatment – impairment of intellect, rarely - normal intellect Patobiochemistry-AA 42 Leucinosis Intermittent forms of minor modifications of decarboxylase metabolism of Val, Ile, and Leu is reduced but maintained. Leucinosis symptoms appear later and occasionally (after ingestion of large amounts of AMK) Patobiochemistry-AA 43 Metylmalonic aciduria Caused by avitaminosis B12. B12 is a cofactor of the enzyme which converts methylmalonyl-CoA to succinyl-CoA (radical isomerization) Metabolic acidosis belongs to the group of organic acidurias. • methylmalonyl-CoA mutse disorder (converts isoleucine, valine, methionine and threonine, involved in the synthesis of CHOL and MK). • AR hereditary. • Clinical picture • Short symptomless period after birth; then vomiting; lethargy; progressive impairment of consciousness; brain edema; liver and kidney failure; children die of sepsis, bleeding or shock state • in the acute stage - ketoacidosis and laboratory evidence of liver and renal failure • There is higher level of glycine, valine, methionine and some organic acids (especially methylmalonic acid) in urine and blood. Patobiochemistry-AA 44 • Diagnosis • examination of organic acids and AAs in urine and blood; • exact type of defect is determined by enzymatic examination of cultured fibroblasts • Therapy: with suspicion, the supply of protein should be stopped and must avoid catabolism - glc concentrated infusion; prognosis is good if treated early • critically ill patients must undergo elimination methods to detox • with the recessionary effect: hemodialysis, hemodiafiltration, peritoneal dialysis and exchange transfusion; a lifelong diet is needed with low intake of natural protein with the addition of a mixture of essential AAs without isoleucine, valine, methionine and threonine. Patobiochemistry-AA 45 Isovaleric acidemia isovaleryl-CoA-dehydrogenase deficiency metabolic acidemia (pH 7,3), ketonuria, hyperamonemia, hypocalcemia, hyperlactemia, odour of breath, body fluids, coma after ingestion oflarge amounts of proteins, generalized pancytopenia Patobiochemistry-AA 46 Hypervalinemia low activity of common transaminase for valine, very rare disease, Intermittent forms of leucinosis minor modifications of decarboxylase metabolism of Val, Ile, and Leu is reduced but maintained. Leucinosis symptoms appear later and occasionally (after ingestion of large amounts of AMK) Isovaleric acidemia isovaleryl-CoA-dehydrogenase deficiency metabolic acidemia (pH 7,3), ketonuria, hyperamonemia, hypocalcemia, hyperlactemia, odour of breath, body fluids, coma after ingestion oflarge amounts of proteins, generalized pancytopenia Patobiochemistry-AA 47 Disorders of metabolism of sulfur AAs Homocystinuria disorder in β-cystathionine synthetase activity (transsulfuration of methionine to cystine) manifestations are quite diverse and affect various tissues and organs impairment of mental development, marfanoid phenotype (tall slender figure, arachnodactyly, kyfosa, scoliosis, osteoporosis) ectopia of lenses, glaucoma and central and peripheral thromboembolic events Cystinosis is a consequence of deficiency of lysosomes to release cystine, which is then accumulated therein, accumulation affects RES (spleen, liver, lymph nodes and bone marrow renal impairment - glycosuria, phosphaturia, albuminuria, hyperaminoacidurie, chronic acidosis and uremia Cystinuria AR disorder of AAs transport – cystine, lysine, ornithine and arginine in the kidney and the gut, renal and intestinal problems, cystine crystallizes in the urine TEST Patobiochemistry-AA 48 Homocystinuria disorder in β-cystathionine synthetase activity (causes transsulfuration of methionine to cystine) AR hereditary frequency 1 : 200 000[3] clinical picture: symptoms are not apparent at birth, but in the further development leads to symptoms affecting various tissues and organs • appear in the toddler or preschool age - impaired mental development (psychomotor retardation in 60% of cases [4]), marfanoid phenotype (tall slender figure, arachnodactyly, kyfosa, scoliosis, osteoporosis) and ectopic lenses, glaucoma and central and peripheral thromboembolic events • dislocation of the lens causing strong myopia, thromboses occur most frequently at the base of the skull and life-threatening, gangrena of organs occurs, which usually ends the patient's life in 20 to 30 year • optic nerve atrophy, cor pulmonale, hypertension • laboratory: increase of homocysteine in blood, frequent metabolic osteopathy – necessary to confirm at the enzymatic and molecular level • dif.dg: homocysteinemia occurs also at methylmalonic acid metabolism impairment, cobalamin or B12 deficiency • therapy: proportion of patients (approximately 50% [4]) responds favorably to high doses of pyridoxine (vitamin B6) (in an amount of 300-900 mg / d [4]), which regulates the activity of cystathionine β-synthetase – it is necessary to initiate dietary treatment with a limited supply of methionine – prenatal diagnosis is available Patobiochemistry-AA 49 Homocysteine• Homocysteine (systematic name 2-amino-4-sulfanylbutanic acid) is an amino acid that is produced during normal metabolism in humans and other mammals from the amino acid methionine. Normally is degraded to form amino acid called cysteine under the influence of B vitamins (especially B6, B12, folic acid). • The lack of these vitamins, e.g. in vegetarian diets, or a rare hereditary disease "homozygous homocystinuria" [1] may lead to elevated levels of homocysteine in the blood. TEST Patobiochemistry-AA 50 Patobiochemistry-AA 51 2) Neuropathic cystinosis • AR hereditary • frequency 1 : 50 000 - 1 : 1 000 000[4] • This is a defect of lysosomal cystine transport, which leads to its deposition • accumulation affects RES (spleen, liver, lymph nodes and bone marrow), deposits can be proved even in the cells of the kidney tubules and conjunctiva • clinical manifestations are evident only in the kidney, where there is a serious breach of their function • laboratory: signs of kidney damage - glycosuria, phosphaturia, albuminuria, hyperaminoacidurie, chronic acidosis and uremia – generalized aminoaciduria is due to a decrease in GF, which will soon result in kidney failure • therapy: symptomatic treatment tubular dysfunction, usually high doses of vitamin D are required [4] – Supplementation with cysteamine which acts in two ways • binding to cystine results in cysteine formation that can be secreted from the lysosome via cysteintransporter[4] • binding to cystine results in cysteine-cysteamine formation, which can be secreted from the lysosome via lysinetransporterudisulfide[4] Patobiochemistry-AA 52 cystinosis • Cystinosis - inborn disorder of metabolism of cystine with autosomal-recessive inheritance; free cystine accumulates in the lysosomes of cells of the whole organism, esp. in the reticuloendothelial system, bone, kidney and retina. • In the foreground is renal tubular atrophy and variously extensive bone disease, if there is also an accumulation of ferritin in lysosomes, retinopathia retinitis pigmentosa is developped. Infantile c. - severe form of renal rickets resistant to vitamin D with a dwarf in stature, renal imairment is manifested by tubular acidosis with hypokalemia and aminoaciduria, further retinopathy. Juvenile c. - In the foreground is especially renal impairment. Glomerular proteinuria and progressive renal failure, retinopathy. C. adults benign form, the disorder can be proved in the laboratory and histological crystals of cystine, kidney function is not significantly impaired. Syn. Abderhald-Kaufmann-Lignac syndrome, Fanconi-Lignac syndrome, Fanconi-Abderhalden sydrome specially type IPatobiochemistry-AA 53 Patobiochemistry-AA 54 3) Cystinuria disorder of AAs transport – cystine, lysine, ornithine and arginine in the kidney and the intestine, renal and intestinal problems, cystine crystallizes in the urine AR hereditary • frequency 1 : 2000 - 1 : 7000[4] • congenital disorder transport dibasic AMK - cystine, lysine, ornithine and arginine in renal tubules and in the gut • clinical picture: cystine nephrolithiasis, which is caused by poor solubility of cystine in water, and its crystallization in an acidic environment • Diagnosis: elevated levels of cystine, ornithine, arginine, lysine in the urine; sono kidney and urinary system[4] • therapy: the goal is to prevent the formation of nefrolitiasis; fluid intake coupled with a night drinking is recommended – In severe cases it is possible to consider medical therapy by Dpenicillamine or mercaptopropionylglycine which cause the formation of more soluble bisulfites with cystine[4] Patobiochemistry-AA 55 Patobiochemistry-AA 56 Hartnup disease -AR disorder of transporters of neutral AAs in kidney tubules and small intestine tryptophan deficiency - skin changes • AR hereditary. • substrate is abnormal resorption of neutral AAs in the intestine and kidneys. • usually does not cause any clinical symptoms. • eventually. photosensitivity of skin is at the forefront Disorders of metabolism of Tryptophan and Tyrosine essential AAs, among others. formation of nicotinic acid and serotonin. Patobiochemistry-AA 57 Organic acidurias Organic acidurias are a group of several tens of diseases with common characteristics: carboxylic acid excretion in urine. Organic acids accumulate in the body during metabolism disorder in particular amino acids, as well as fatty acids and carbohydrates, rarely other substances. Heredity: •AR Pathogenesis: •impairment of cytosolic, mitochondrial or peroxisomal pathway (deficiency of the enzyme, cofactor deficiency) •accumulation of the substrate before failure Symptoms: •are different depending on the type aciduria, often nonspecific •highly suspicious is strange odor •often metabolic acidosis •often hyperammonemia TEST Patobiochemistry-AA 58 •Forms: 1.Acute neonatal •serious impairment of the intermediary metabolism •manifests in the first days or weeks of life 2. Running intermittently •partial enzyme deficiencies that suffices for the intermediate metabolism under normal conditions •stimulus is increased catabolism (eg. operations), increased protein intake, long starvation •manifest themselves by attacks of acute encephalopathy, acidosis, hypoglycemia 3.Chronically ongoing •less common, progressive, difficult to influence •CNS disorders Organic aciduria investigations within the nationwide newborn screening in the Czech Republic include: •glutaric aciduria type I (GA I) •isovaleric acidemia (IVA) •leucinosis (MSUD) Patobiochemistry-AA 59 Glutaric aciduria typ I (GA I) • belongs among organic acidurias, is caused by the inability of the body to process the amino acids lysine and tryptophan due to deficiency of glutaryl-CoA dehydrogenase. The enzyme glutaryl-coenzymeA dehydrogenase is stored in mitochondria. In the liver, kidney, fibroblasts and leukocytes catalyzes the oxidative decarboxylation of glutaryl-CoA to crotonyl-CoA. Deficiency of this enzyme leads to increased levels of glutaric acid and toxic metabolites.[1] • Flooding the body with toxic metabolites occurs after increased amount of lysine and tryptophan (eg. In the normal weight loss in the neonatal period, when the breakdown of body protein, child with fever and starvation, when common infections after surgery and in similar stressful situations) .[1] • GA 1 is AR hereditary disease (gene GCDH- at 19p13.2, OMIM #231670). Since 1. 10. 2009 is part of a nationwide newborn screening in the Czech Republic. Increased C5-DC acylcarnitine testifies for GA I incidence. When GA I is suspected, analysis of organic acids in urine is immediately carried out. Elevated levels of glutaric acid and 3hydroxyglutaric to confirm the diagnosis. If the analysis does not confirm a diagnosis, specialist DMP considers the analysis of glutarylkarnitine in urine and 3-hydroxyglutaric acid in the blood and cerebrospinal fluid, analysis of enzymes in fibroblasts and molecular analysis of GCDH gene.[1] • Incidence of GA I: 1:40 000 in white populations and 1:30 000 in Sweden.[1] http://www.wikiskripta.eu/index.php/Glutarov%C3%A1_acidurie Patobiochemistry-AA 60 Urea cycle disorders group of enzymatic disorders that result in the accumulation of nitrogen in the form of ammonia, which is very toxic for the body and causes irreversible brain damage Hyperamonemia - cramps, vomiting, coma, psychomotor retardation, behavioral disorders, repetitive cerebellar ataxia, headache, metabolic acidosis Gout (arthritis urika) defect in the breakdown of purines (uricase) - excessive production of uric acid. High levels of uric acid in the blood causes crystallisation of this compound in joints and other tissues (inflammation) may cause gout attacks of the joints, kidney stones blockade of urinary tract. The crystals of uric acid may also block tubules of the kidney and cause the kidney insufficiency. Hyperuricemia - industrialized countries (high intake of purines in the diet, alcohol, obesity, lead in food) Hyperammonemia I Hyperammonemia II Citrulinemia Argininsukcinateuria Argininemia TEST Patobiochemistry-AA 61 Urea cycle disorders group of enzymatic disorders that result in the accumulation of nitrogen in the form of ammonia, which is very toxic for the body and causes irreversible brain damage Hyperamonemia - cramps, vomiting, coma, psychomotor retardation, behavioral disorders, repetitive cerebellar ataxia, headache, metabolic acidosis damaged enzyme location heredity type Hyperammonemia I karbamoylphosphatesynt hetase (CPS1) mitochondria AR hereditary Hyperammonemia II ornitinkarbamoyltransfer ase (OTC) mitochondria X conjugated representation in heterozygous girls Citrulinemia argininsukcinatesyntetas e (ASS) cytosol AR hereditary Argininsukcinateuria argininsukcinase (ASL) cytosol AR hereditary Argininemia arginase (ARG1) cytosol AR hereditary TEST Patobiochemistry-AA 62 • Diagnostics • hyperamonemia; hyperamonemia; ABR – first respiratory alkalosis, metabolic acidosis later • chromatography of amino acids in plasma: increased concentration of glutamine and glutamic acid, decreased level of arginine; increased concentration of amino acids before enzymatic defect and decreased concentrations of the amino acids after defect • orotic acid in the urine, increased when disorders of all enzymes except CPS1 occurs • Liver biopsy: determination of the enzymatic activity of liver tissue • mutations analysis[2] • Therapy • First aid: catabolism to anabolism conversion (even high doses of glucose with insulin, high caloric parenteral nutrition) and detoxification (sodium benzoate, sodium phenylbutyrate, ev. hemodialysis, hemofiltration) • substitution of the missing amino acids (usually arginine and citrulline) • lifelong reduction of protein intake and their substitution by a mixture of essential amino acids • severe impairment leads to liver transplantation[2] Patobiochemistry-AA 63 Hyperlysinemia - (hyperammonemia) -high conc. Lys in blood, serum -block of enzym arginase (in ornithine, urea cycle) - high concentration of NH3 and arginine - illness, mental disability. Related to protein intake, difficulties decrease when restricting the proteins intake. Hyperprolinaemia I -high values of Pro (low activity of Pro-dehydrogenase) - causes renal malformation, hematuria, renál insuficience = renal failure, decreased hearing, deafness - these disorders = so called Alport syndrome Hyperprolinaemia II - does not affect the kidneys, slowing growth and mental developmentPatobiochemistry-AA 64