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Chemical structure and properties:

NH2׀

R – CH – COOH

•Amino acids are organic compounds containing both,

• Carboxylic (COOH) and amino (NH2) gps.

R= side chain with vaiable size.The chemical properity of this side chain responsible for unique charactristics of protein molecules present in human body.

The a.a. are classified according to the R gp into :

1. Aliphatic : Neutral : - hydrocarbon side chain : eg. glycineSulphur containing a.a. : eg. Cysteine Hydroxyl containing a.a : eg. serineAcidic : aspartic a. – glutamic a.Basic : arginine – lysine

2 Aromatic : phenyl alanine tyrosine2. Aromatic : phenyl alanine – tyrosine

3. Heterocyclic : proline – hydroxyproline – tryptophanAlthough more than 150 different a.a. are known biologically, yet only 22 are present in the body as significant constituents of peptides & proteins .

.What makes peptides and proteins different from each others in terms of structure and function are the characteristic acid base properties of individual a.a., as well as the variety of possible R gp. interaction.

AMINO ACID are classified into :Non essential a.a.Essential a.a.

GlycineValineAlanineLeucineSerineIsoleucineCysteineMethionineCystineThreonine CystineThreonineAspartic acidArginineGlutamic acidLysineHydroxylysinePhenylalanineTyrosineHistidineProlineTryptophaneHydroxyproline------

Essential a.a. :

Those which can not be adequately synthesized by the body and should be supplied in diet.

Metabolism of amino acids :

The 1ry supply of a.a. for endogenous protein synthesis.

Digestion & absorption :

Proteolytic enzymes (pepsin, trypsin, chymotrypsin, endo/expeptidases) in the GIT (stomach & intestine) act on ingested proteins,

releasing a.a. that are absorbed from the jejunum into the blood and subsequently become part of the body pool of a.a.

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Dietary Proteins

Body Proteins

Amino Acids

(Some) Specific pathways

Niccotinamidederivatives

Creatinine

SO4

Ammonia (NH3) α Keto acidsAmmonia (NH3)

Urea cycle

Urea

Kidneys

Urine

α Keto acids

-CHO intermediates

Most

Kreb’s cycle

CO2 H2O

(Some)

FA & intermediates

Body handling of a.a. :

The liver & other tissues draw from this pool for synthesis of plasma & IC proteins. The liver & kidneys are also involved in the interconvertion of a.a. by transamination as well as their degradation by deamination.

NH4+ are the deamination products, where they are rapidly consumed in urea synthesis, that is excreted by the kidneys.

Plasma a.a. concentration :

They vary during the day by about 30% ( highest in early afternoon and lowest in early morning = diurnal variation),

Reference range for total amino acids in adults (as amino acids nitrogen) :

Variable depending on the method used in their assay.

mg/dl0753Plasma : mg/dl0 .7–5 .3Plasma :

hrs urine24 mg/200 –50 :Urine

Clinical significance of plasma a.a.:Increase in plasma a.a. level may be :• physiological : following intake of a high protein diet.

• pathological :↑body protein breakdown (catabolism → negative N2 balance) eg : leukemia, sepsis…

1. severe hepatic insult, eg :acute hepatic necrosis (values may reach 100 – 200 mg/dl) dt ↓↓ of oxidative deamination of a.a..reach 100 200 mg/dl) dt ↓↓ of oxidative deamination of a.a..

2. severe renal disease eg : ESRD.

3. inherited metabolic block (most common in neonates & infants).

Decrease in plasma a.a. level is not important clinically, and was reported to occur following insulin injection.

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Clinical significance of urinary a.a.:AMINOACIDURIATypes of aminoaciduria (mechanisms):1. Overflow aminoaciduria:Occurs when the plasma level of 1or more a.a. exceeds the renal threshold.(Renal threshold: plasma a.a. level above which a.a. appears in urine. It is dt. Liver disease or inherited metaolic defect dt;enzyme dificiency

ex;PKU, MSUD.

2. Renal aminoaciduria:Occurs when there is a defect in the renal tubular reabsorption system (theOccurs when there is a defect in the renal tubular reabsorption system (the plasma a.a. level is normal) either;

A-selactive( dt. Transport system defect ex; cystinuria , Hart nup )B- generalized ; congenital ; as fanconi, HFI , wilsson disease.

acquired; heavy metal poisoning (pb , Hg ,cd )

3. No threshold aminoaciduria:Occurs when excessive amounts of an a.a. (arising from an inherentmetabolic block) pass in urine, due to saturation of the PCRT transportsystem responsible for this a.a. reabsorption ( but with normal pl. a.a.level since all the a.a. are excreted eg; homocystinuria.

Phenylketonuria=PKU(Hyperphenylalaninemi)

CH2 CH COOH

NH2

CH2 CH COOH

NH2

HO

phenylalaninehydroxylase

O2

TyrosineTetrahydrobiopterinL- Phenylalanine

Phenyl Pyruvic acid

Phenyl Lactic acidPhenyl Acetic acid

O- Hydroxy Phenyl Acelic acid

Transamination

dehydrogenase

Phenylketonuria=PKU (Hyperphenylalaninemia)

Definition:They are a gp. of disorders resulting from impaired conversion of phenylalanine to tyrosine.

Remark:Normally, the major metabolic pathway for conversion of phenylalanine totyrosine involves the enzyme phenylalanine hydroxylase whichtyrosine involves the enzyme phenylalanine hydroxylase which is foundonly in liver & kidneys. Tetrahydrobiopterin (THBP) is an obligatorycofactor, and is kept in the active reduced form via a THBP reductaseenzyme.

Causes (Pathophysiological mech):-97% of cases of PKU : are dt phenylalanine hydroxylase deficiency.-1-3 % of cases : are dt THBP reductase defect or one of the steps involved in biopterin (BP) formation.

Types of PKU:

Classical PKU:

contributes to 1/2 cases of PKU, and is dt total absence ofphenylalanine hydroxylase activity.

phenylalanine accumulation inblood, urine and CSF (levels ecceed 20 mg/dl)( g )(N adult URL: 2mg/dl).

activation of alternative minor pathways phenylketones +other metabolites production which are rapidly cleared by kidneys into urine.

PKU variants: (Less severe forms of PKU)

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:Transient neonatal hyperphenylalaninemiadt delayed hepatic maturation of phenylalanine hydroxylase system.it is not inherited andblood Phe.ala. level may exceed 12mg/dl initially, but level returns tonormal later.

: Maternal hyperphenylalaninemia• likely to occur in adult pregnant females who were treated from PKU when y p gthey were infants.

High maternal levels of plasma phenylalanine cross placenta causingdeleterious foetal defects.

To reduce or prevent this, dietary restriction of phenylalanine before conception and throughout pregnancy .(including synthetic sweetener aspertam that is a dipeptide ofphenylalanine and aspartic acid).

Clinical picture:

• Severe neurological defects and mental retardation in untreated cases

• bec. phenylalanine reduces synth. of myelin, norepinephrine and serotonin.

• Hypopigmentation may occur dt hyperphenylalaninemia.

• Bec.phenylalanine is a competitive inhibitor of tyrosinase enzyme responsible for initiating melanogenesis.,

Diagnosis of PKU:

:In the neonate• Early screening for enzyme and cofactor defects is needed.This includes

serum and urinary measurements of phe. Alanine, phe. pyruvate, phe.lactate and phe. acetate.

Evaluation should include measurement of THBPand BP in urine and serum.

:Prenatal diagnosis• Very promising for DNA analysis,

Treatment:

Dietary restriction of phenylalanine before onset of brain damage to keep

plasma phenylalanine concentration not exceeding 8 mg/dl.

(Even if diagnosis is made later (4 – 6 months) diet restriction decreases the rate of mental retardation.

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Methods for phenylalanine determination in biological samples:

They include:1. Chromatographic methods

2. Spectrophotometric methods

3. Fluorometric methods

•

Fluorometric methods:Phenylalanine is reacted with ninhydrin, in the presence of dipeptide LleucylL- alanine, to form a fluorescent product that is proportional to thephe. Ala. concentration. The reaction is carried at a pH =5.88, and with theaddition of copper tartarate to stabilize the fluorescent product.

Normal values for phenylalanine in serum of :

• Adults : 0.8 – 1.8 mg/dl

• Neonates :Full term N W newborn : 1.2 – 3.4 mg/dl

Preterm LBW newborn: 2.0 – 7.5 mg/dl

.

Tyrosinemia & Tyrosinuria

Tyrosinase

L- TyrosineTyrosine -3-hydroxylase

CH2 CH COOH

NH2

HO Tyrosineaminotransferase

P – OH Phe Lactic (PHPLA)

P- hydroxyphenyl pyruvic acid (P-HPPA)

PHPPA Oxidase CO2

Homogentisic acid (HGA)

(TAT)

NADNADH+H

Melanins DOPA

Dopamine

Epinephrin L- Norepinephrin

(HGA)

Malylacetoacetic acid (MAA)

Homogentisic acid Oxidase

HGA Oxidase

MAA isomeraseFumaryl acetoacetic acid

(FAA)FAA Hydrolase

Acetoacetic acid Fumaric acid

Tyrosinemia & Tyrosinuria

Tyrosinemia has several forms, each of which is accompanied by tyrosinuria and phenolic aciduria.

:RemarkThe body obtainsTYROSINE from dietary proteins, as well as from hydroxylation of phenylalanine in vivo.hydroxylation of phenylalanine in vivo.

TYROSINE essential for protein synthesis and serves as a precursor for

Thyroxine, Melanins and Catecholamines.

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Types of tyrosinemias:

Tyrosinemia I (tyrosinosis or hepatorenal tyrosinemia) :

Tyrosinemia II:Transient neonatal tyrosinemia:

Tyrosinemia I (tyrosinosis or hepatorenal y ( y ptyrosinemia) :

: Incidence1:100,000 births – common among the French, Canadianpopulation

Causes:-primary defect is reduced FAA hydrolase activity.

-less commonly reduced PHPPA oxidase activity

The resulting biochem. defects are:

1. plasma & urinary tyrosine level

22- Urinary excretion of DOPA & other tyrosine metabolites(alternative pathway is enhanced).

Clinical picture:

effects on:

Liver:varies from acute hepatic failure and death ( in infants) to chronicliver dis (cirrhosis) later in life.

Kidneys: generalized tubular transport failure develops resulting in Fanconi syndrome (phosphaturia & glycosuria & generalized a.a.uria & y (p p g y gvitamin D resistant rickets).

Treatment:

Dietary tyrosine restriction corrects the biochem changes, but notthe progressive liver damage.

Tyrosinemia II:

Cause:deficiency of the hepatic enzyme tyrosine aminotransferase (catalyzes the formation of PHPPA)

Cl. features:

1-Eye lesions: corneal erosions

2 Skin lesions: mainly in palms and soles2. Skin lesions: mainly in palms and soles

3. Mental retardation: might occur(both 1&2 are dt IC tyrosine crystal formation that induce inflammation)

Biochem findings:

Tyrosine in plasma & urine

conc of urinary phenolic acids

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Serum tyrosine level in:

1. Normal adults : 0.8 – 1.3 mg/dl.

2. Full term newborn : 1.6 – 3.7 mg/dl.

3. Preterm infant : higher dt immature liver enzyme.

4. Tyrosinosis : 4 – 10 mg/dl

Remark:

In type I tyrosinemia, 24 hrs urinary PHPPA conc. may reach up to

1.6 mg/24h which is diagnostic (i.e. 25 times its normal urinary conc)

Maple syrup urine disease (MSUD)Maple syrup urine disease (MSUD)The branched chain a.a. – leucine, isoleucine, valine – are normally converted by transamination to their corresponding ketoacids, that by oxidative decarboxylation are then converted to Acyl Co-A derivatives.

An inherited defect of the decarboxylation stepaccumulation of the branched chain a.a. and their corresponding ketoacids in blood, urine & CSF.

Incidence: 1:250,000Clinical findings:

several types have been identified clinically & biochemically

Classical type:Affected neonate appears normal at birth, but later develops frequentvomiting and failure to thrive.Acute ketoacidotic episodes: often triggered by recurrent infection, dt organic acids production. Severe neurological deficits can develop (seizures – coma – respiratory failure) death or mental retardation in survivors.

Milder forms:Identified by enzyme analysis of leucocytes & fibroblasts.

Investigations:• Urine odour: maple syrup or burnt sugar odour is dt the high conc. of aliphatic ketoacids.• Amino acid analysis of blood & urine : high levels of valine, leucine and isoleucine• Alloisoleucine (normally not present in normal persons) is ( y p p )typically present in MSUD• Abnormal amounts of ketoacids in urine: detected by 2,4 –DNPH test.• 10% FeCl3 test: gives gray blue colour with MSUD.• Neonatal screening: possible with Guthrie test.• Antenatal diagnosis: possible with assay of decarboxylase activity in culturedcells from amniotic fluid.

Treatment:Diet restriction of branched chain a.a. (valine, leucine and isoleucine)

NB:•Daily analysis of urine (DNPH) and monthly analysis of•Daily analysis of urine (DNPH) and monthly analysis of plasma a.a.level.• (HPLC, GC/MS) are important in monitoring efficacy of dietary therapy

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Methods for determination of tyrosine in serum:

1-Ion exchange liquid chromatography:(reference method)Detects a substantial increase in seum tyrosine

2-Fluorometric method:

TCA protein free filtrate is allowed to react with a mixture of -nitroso -naphthol (ANBN) and nitrite to give a pinkish complexnaphthol (ANBN) and nitrite to give a pinkish complex.

Treatment of thecomplex with nitric acid converts the complex to a yellow fluorescence.

and the fluorescence is read at 570nm

3-Enzymatic methods:

catalyzes the oxidation of tyrosine to dopaquinone. Tyrosinase

TheConsumed O2 was measured amperometrically.Adv :

d f l d t i i ti t ti i b ti• no need for sample deproteinization, extraction or incubation• small sample vol (10 – 50 ul) is needed• sensitive

Alkaptonuria

L- Tyrosine

Tyrosine aminotransferase

P- hydroxyphenyl pyruvic acid

(TAT)

NH2

CH2

CH

COOH

OH

CH2

CH

COOH

OH

O

L Tyrosine(P-HPPA)

PHPPA Oxidase

CO2

Homogentisic acid (HGA) Malylacetoacetic acid

(MAA)

HGA Oxidase

COOHOH CH2

OH

CH2

CH

CH2

CHCOOH

COOH

C O

C O

AlkaptonuriaAetiology :

It is a rare disease resulting from a deficiency in HGA oxidase activity thatcatalyzes the conversion of HGA to MAA.

Pathogenesis:

• The resultant accumulation of HGA & its metabolites in cells & body

fluids causes their excretion in large amounts in urine.

• The monomeric and polymeric forms of HGA bind to collagen in

Cartilage and other C.T.

pigmentation of cartilage +degenerative arthritis.

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Cl. picture:

1. Disease is usually not diagnosed until middle age where arthritis develops.

2. It can de diagnosed in neonates when a dark stain is b d i th i h d diobserved in their unwashed diapers.

3. Patient develops dark blue to black pigmentation in cartilage & C.T.

Lab investigations:

1. Darkening of urine:

On prolonged standing – on exposure to air and sunlight – on addition ofalkali – occurs due to oxidation of HGA.

N.B: normally no HGA is present in urine – urine at an acidic pH won’tdarken in colour

N.B:Darkening of urine may occur dt other causes:Melanins – phenols – gentisic acid (a salicylate metabolite) – and indoxylsulphate (indican, a tryptophan metabolite formed by stagnant S.I. contents).

2. Reversed benedict test:

HGA reduces alkaline Cu reagent in the presence of an alkaline medium. The name reversed comes from the fact that the ratio of urine to benedict reagentis reversed (i.e. 4.5 ml of urine + 0.5 ml of reagent).

3. Ammoniacal silver nitrate test :

This test helps in differentiation between alkaptonuria & melanuria.

HGA in urine (0.5 ml) reacts with AgNO3 soln (5.0 ml) rapidly causing darkening of urine even before addition of ammonia, resulting in formation of colloidalsilver (brown to black ppt). Melanogens react slowly with silver nitratefollowed by dil NH3 addition.

4 10% F Cl3 t t i t i t d k bl l ith HGA4. 10% FeCl3 test: gives a transient dark blue colour with HGA.

5. Paper or thin layer chromatography: to identify HGA.

Treatment:

1. Dietary restriction of tyrosine or its precursor phe. Ala. may be beneficialin early diagnosed cases.

2. Vitamin C (Ascorbic acid) administration: known to be required formaximum activity of HGA oxidase