‫بسم هللا الرحمن الرحیم‬

‫اختالالت متابولیکی و بیماریهای‬ ‫مربوط به اسیدهای آمینه و‬ ‫پروتئین ها‬

‫اهداف درس‪:‬‬ ‫پس از مطالعه این بخش شما باید مطالب زیر را بطور کامل بدانید‪:‬‬ ‫• آشنای ی با آختالالت مهم جذب‬

‫• تعریف آمینوآسیدوری و آنوآع آن‬ ‫• آشنای ی با آختالالت مهم متابولیسم آسیدهای آمینه‬ ‫• آشنای ی با بیماریهای سیکل آوره‬ ‫• آشنای ی با عوآرض‪ ،‬نقص آنزیمی‪ ،‬مولکولی و وآکنشهای بیوشیمیای ی مرتبط با آختالالت فوق‬ ‫• آشنای ی با آصول درمان بیماریهای فوق‬ ‫• آشنای ی با روش های آزمایشگاهی برآی تشخیص موآرد باال‬

Some Amino Acid Transport Systems 1.

Netural AA with short & polar side chain (Ala, Ser, Thr)

2.

Neutral AA with non-polar & aromatic side chain (Phe, Tyr, Met, Val, Leu, Ile) (Defective in HARTNUP DISEASE)

3.

Imino AAs (Pro & Hyp) & Gly

4.

Cystine and basic AAs [COAL](Cystine, Orn, Arg, Lys) (Defective in CYSTINURIA)

5.

Acidic AA (Asp, Glu)

6.

Basic amino acids (Orn, Arg, Lys) (Defective in LYSINURIC PROTEIN INTOLERANCE)

Amino acidurias • Primary amino acidurias

– Inherited enzyme defects of amino acids transport or metabolism – More than 70 inherited disease are known • Disorders of amino acid transport • Disorders of ammonia removal (Urea cycle defects) • Disorders of amino acid Metabolism – Aminoacidopathies – Organic acidaemias

– Individually rare but collectively important since many are treatable if diagnosed early

• Secondary amino acidurias – Disorders of the liver – Renal tubular dysfunction – Wasting, Malnutrition

Some disorders of amino acid transport

• Cystinuria • Hartnup Disease • Lysinuric protein intolerance

Genetic defect seen in cystinuria Cystine crystallises above >1250μM at pH7.5

Disorders of Amino Acid transport (i) Disorder resulting from the defect

Biochemistry

Diagnosis

Clinical Features

Cystinuria

Kidney tubular reabsorption of Cys & basic amino acids (Cys, Lys, Arg, Orn) (COAL)

Urine: Cys, Lys, Arg & Orn

• kidney stone • Treatment:

Plasma: Normal amino acids

- High fluid intake (>3L/24h) - Alkalinisation of the urine - Thiol compounds that form water soluble disulfides with cys (eg Pencillamine, mercaptopropionylglycine)

Disorders of Amino Acid transport (ii) Disorder resulting from the defect

Biochemistry

Hartnup disease Reabsorption defect of Neutral amino acids (Ala, Ser, Val, Leu, Ile, Phe, Tyr, Thr, Trp, His, Gln, Asn)

Diagnosis

Urine: Ala, Ser, Val, Leu, Ile, Phe, Tyr, Thr, Trp, His, Gln, Asn

Clinical Features

-Most patients asymptomatic -Photodermatitis - Neurological involvements (resemble Niacin deficiency) -Trp deficiency → Niacin and - Degradation of serotonin deficiency unabsorbred Trp by • Treatment: intesinal bacteria produces indoles -Oral Nicotinamide urinary Trp-induced -Trp ethyl ester indoles Plasma: N- Neutral amino acids

Disorders of Amino Acid transport (iii) Disorder resulting from the defect

Biochemistry

Diagnosis

Clinical Features

Lysinuric protein intolerance

(Re)absorpti on defect of Dibasic amino acids (Lys, Arg, Orn)

Urine: • Lys, Arg, Orn Plasma: • N- Lys, Arg, Orn • in blood NH3 after protein-rich meal

-Arg & Orn→ Interruption of urea cycle →hyperammonaemia - Arg → NO →Disturbed immune function Lys → -Failure to thrive -Osteoporosis (easy fracture in children) -Anaemia -Skeletal abnormalities -Mental retardation Treatment: -Prevent hyperammonaemia -Citrulline supplement

Disorders of ammonia detoxification (Urea cycle defects)

+ Glutamate + Acetyl-CoA

Arginine

NAGS

2ATP

NH4+ + HCO3-

The Urea Cycle

N-Acetylglutamate

+

2ADP

CPS I

Carbamoyl phosphate

OTC

Citrulline

Ornithine

Mitochondrion

T

Orotic Acid

Cytosol

Citrulline Aspartate

Ornithine

ATP ASS

Arginase ADP

Arginosuccinate

O ll H2N-C-NH2

Urea

Arginine ASL

Urine Fumarate T: ornithine transporter

11

Differential diagnosis of hyperammonaemia Acquired – – – – –

Transient hyperammonaemia of the newborn Liver failure or impairment Infection with urease-positive bacteria Renal Failure Certain medications (eg Valproate, Asparginase)

Inherited disorders – Urea cycle enzyme defects – Organic acidurias – Other inborn errors of metabolism

12

Ammonia toxicity Ammonia is one of the most toxic compound produced in the body!

• Symptoms of ammonia intoxication: – Tremors )‫ رعشه‬،‫(لرزه‬ – Slurred speech – Blurred vision – Irreversible brain damage

– Coma – Death

Urea cycle disorders – Complex group of disorders with very variable clinical phenotype: • Hyperammonaemia • Failuer to thrive • Ataxia • Mental retardation • Learning difficulty • Anorexia • Vomiting • Lethargy • Coma • Death

Urea cycle disorders 1. 2. 3. 4. 5. 6. 7.

N-Acetylglutamate synthase deficiency Carbomyl Phosphate synthase I deficiency The ornithine transporter deficiency OTC deficiency (X-linked) Arginosuccinate synthase deficiency

Arginosuccinate Lyase deficiency Arginase deficiency

15

Urea cycle disorders • Genetic deficiencies of all the enzymes of the urea cycle have been described

• Overall incidence 1 in 10,000 • OTC deficiency (x-linked) is the most common of these disorders

• All of the other defects autosomal recessive • Complex group of disorders with very variable clinical phenotype

• Most present with hyperammonaemia (ie blood

[ammonia]>100µM) during the first weeks of life 16

Diagnosis of Urea Cycle Disorders • Ammonia Measurement • Plasma Amino Acid Measurement

Normal plasma Amino Acid profile

Plasma AA profile in arginosuccinic aciduria

Treatment of Urea Cycle Disorders 1. Maintain anabolic state 2. Limit protein intake (Replace natural proteins with essential amino acids)

3. Give Arginine (in all disorders except arginase deficiency) 4. Citrulline may be given in severe OTC and CPS deficiencies

5. Citrate to provide a supply of Krebs cycle intermediates 6. Remove ammonia (Benzoate, phenylbutyrate, dialysis) 7. Give lactulose The aim is to keep plasma ammonia levels below 80 umol/l and plasma glutamine levels below 800 umol/l with concentrations of essential AAs within the normal range

Some disorders of amino acid Metabolism

• Phenylketonuria • Tyrosinaemias • Alkaptanuria • Homocystinuria • Maple Syrup Urine Disease (MSUD)

Disorders of Phenylalanine and Tyrosine Metabolism

Degradation of Phe & Tyr & associated metabolic disorders

Phenylalanine metabolism in normal & PKU

Biosynthetic reactions involving tetrahydrobiopterin & amino acids

Phenylketonuria (PKU) Enzyme: Phenylalanine Hydroxylase (or BH4 deficiency) Symptoms: slow growth, poor feeding, vomiting, Smell of mouse urine, hyperactivity, hypopigmentation Clinical: If untreated causes severe and irreversible brain damage with mental retardation Classification:

1. Phenylalanine Hydroxylase deficiency (non-BH4 responsive) 2. BH4 sensitive PKU Diagnosis: plasma [Phe], N- plasma [Tyr]

Treatment and management of PKU

• Restriction of dietary Phe • Phe is an essential AA so too much dietary restriction can also cause poor growth and neurological symptoms • Tyrosine becomes essential • Regular monitoring of blood Phe level • Dietary restriction should be for life but particularly important during first decade of life and pregnancy: – Hyperphenylalaninaemia in in the mother may damage the fetus causing microcephaly, heart defect and mental retardation

Goal: 1st – 10th yr: [Phe]: 40 - 240μM During pregnancy: [Phe]: 120 - 360μM

Contrast-untreated and treated PKUs. The 11y old boy is severely retarded,

whereas his 2y old sister, diagnosed in treated in early infancy, is normal Centerwall, S. A. et al. Pediatrics 2000;105:89-103

Tyrosinaemia Type I (Hepatorenal tyrosinaemia) Enzyme: Fumarylacetoacetae Hydrolase Clinical: severe liver failure, vomiting, Renal tubulopathy, Fanconi syndrome, Hepatomegaly, cirrhosis, growth retardation, rickets, tubulopathy, neurological crises

Diagnosis: Organic Acids (urine) Therapy: Nitisinone (NTBC)(Inhibitor of 4-OHPhenylpyruvate dioxygenase; Phe + Tyr restricted diet

Other disorders of Tyrosine metabolism Disorder

Enzyme defect

Tyrosinaemia Type II (Oculocutaneous tyrosinaemia)

Alkaptanuria (Black urine disease)

Diagnosis

Clinical Features

Therapy

Tyrosine Plasma: aminotransferase Tyr, Phe Urine: Tyrosine metabolites

- Corneal lesion - Hyperkeratosis - Mild mental retardation

Phe & Tyr restricted diet

Homogentisate dioxygenase

- Joint damage Low protein diet - Arthritis - Onset in the 3rd4th decade of life

Urine: Homogentisic acid

Tyrosinaemia Type II

Deposition of Homogentisic acid in Alkaptonuria

Disorders of Homocysteine Metabolism

Degradation & resynthesis of methionine

Causes of homocystinuria Genetic enzyme deficiencies, eg: • Cystathionine -synthase deficiency (classical homocystinuria)

• Methionine synthase deficiency Nutritional deficiencies: • Vit B12 deficiency • Folic Acid Deficiency • Anti folate drugs (eg Methotrexate)

Classical Homocystinuria Enzyme: Cystathionine β-synthase deficiency Clinical: Mental retardation, dislocated lens, thromboembolism, osteoprosis, scoliosis, psychiatric symptoms Biochem: Accumulation of Hcy →linkage with collagen → Collagen disorder Diagnosis:

[Hcy] in plasma & Urine

NBS in some countries (eg incident 1:1800 in Qatar)

Therapy: Aim: plasma [Hcy]<30uM; Normal [Cys] ~50% of patients respond to pharm doses of vit B6 + folic acid Diet: low Met/High Cys; Hydroxycobalamine

Maple Syrup Urine Disease Enzyme: Branched-Chain 2-keto Acid dehydrogenase (BCKDH) complex Diagnosis:

Characteristic smell of Maple syrup AA (P): Val, Ile, Leu OA (U): Branched-Chain 2-keto- and hydroxy acids

Some Inherited disorders of BCAA metabolism

Branched-Chain Keto Acid Dehydrogenase Complex

MSUD Isovalery-CoA Dehydrogenase

IVA

Maple Syrup Urine Disease Therapy: Acute: Glucose + Insulin iv Long term: Diet (limit and monitor plasma Val, Leu & Ile) Aim is to keep the level as close to normal as possible

Incidence:

USA: 1:200,000 Mennonites: 1:150 Turkey: 1:50,000

Other amino acid disorders Organic acidaemias • Inborn errors of amino acid metabolism in which •

the parent amino may not be elevated About 50 disorders known (includes inborn errors of fat oxidation)

• Characterised by increased excretion of

carboxylic acids derived from carbon skeleton of amino acids

• Complex group of disorders with a variety of clinical and biochemical problems

Classical Organic acidaemias Neonatal presentation • • • • • • •

Hypoglycaemia / Hyperglycaemia  Lactate  Ammonia Ketosis / Ketoacidosis Unusual body odour Jaundice Fits

Isovaleric Acidaemia Enzyme: Isovaleryl-CoA Dehydrogenase Clinical: Vomiting, lethargy, coma, odor of “sweaty feet”, acidosis, ketosis

Diagnosis: OA Urine: isovalerylglycine, isovalerylcarnitine, Therapy: L-carnitine, Glycine. Leucine restricted or low protein diet

Isovaleric Acidaemia low protein diet

Laboratory Investigation of a suspected Amino Acid Disorder

• DNA Analysis • Specific Enzyme Assay • Metabolite Assay

Laboratory Investigation of a suspected Amino Acid disorder Metabolite Assay: • Analysis of Plasma Ammonia • Analysis of Plasma and urine amino acids • Analysis of Urinary organic acids – Gas Chromatography Mass Spectrometry (GCMS)

• Analysis of Plasma and urine carnitine & Acylcarnitines

Newborn screening blood specimen collection

Photo curtsey of Whatman Inc.

Urine spot tests: Ferric chloride test (Folling reaction) This test is used for detection of oxo-acids: Color

• • • •

Blue-green Transient blue Green-gray Transient Green

Compound

Disorder

Phenylpyruvic acid Homogentisic acid Branched chain oxoacids p-OH-phenylpyruvic acid

Classical PKU Alkaptonuria MSUD Tyrosinemia types 1 &2

Reacts also with ketones & some drug metabolites (eg Salicylates)

A positive ferric chloride (Folling) urine test in a patient with untreated PKU

Urine spot tests: Dintrophenyl Hydrazine (DNPH) DNPH reacts with 2-oxo-acids to form Hydrazones which form yellow precipitates: Compound

• Phenylpyruvic acid • Branched chain oxoacids • p-OH-phenylpyruvic acid

Disorder

Classical PKU MSUD Tyrosinemia types 1 & 2

Urine spot tests: Nitroprusside test (Brand reaction) This test reacts with sulfur containing Amino acids to form a red-purple color complex Positive compound • Cystine

Disorder – Source

• Homocystine

Homocystinuria

Cystinuria Generalized aminoaciduria Fanconi syndrome

Reacts with some drugs: eg. ampicilin, penicillamine and others

Detection of phenylalanine by paper chromatography and ninhydrin staining

PKU

Paper chromatographic amino acid screening

Plasma AA profile in PKU

Urinary AAs profile in Homocystinuria

Urinary organic acids analysed by GC/MS

Isovaleric Acidaemia

IV-Gly

*

IV-Glu

*

Normal