Neda Rezaei BSc; Leila Shafeghat BSc; Mohammad TaghiShams PhDSedighehMD, AshtianiHaghi

Pediatrics Center of Excellence Children’s Medical Center TehranPediatrics Center of Excellence, Children s Medical Center, Tehran University Of Medical sciences , Iran

Introduction

Amino acids are precious components of the human organism

* building blocks of proteins

*Integral components of metabolic cycles

*Neurotransmitters*NeurotransmittersInherited defects of amino acid catabolism, biosynthesis, or transport have been known for many yearsp y y

.

A i id thi ll   lt f  i h it d d f t  Aminoacidopathies usually result from inherited defects 

in enzymes of the metabolism of amino acids(inborn errors 

of metabolism)

Clinical symptom s caused by the accumulation of amino Clinical symptom s caused by the accumulation of amino 

acids or their intermediate metabolites. Most of the amino 

acid disorders are treatable with dietary restriction of 

protein if it is diagnosed rapidly before onset of irreversible protein if it is diagnosed rapidly before onset of irreversible 

organ damage. 

Application of Measurement A.A

*inborn errors of metabolism*sensitive markers of the nutritional state

*function of various organs such as the liver the kidneys the intestine and theliver, the kidneys, the intestine, and the muscles..

Th   h  i   i   id  t ti  The changes in amino acid concentrations in (acquired) malfunctioning of these 

   b   btlorgans may be subtleThe low levels of amino acids in the 

b l fl d ( ) d hcerebrospinal fluid (CSF) and the neurotransmitter  action of several amino 

dacidsthere is a necessity to correctly identify increased and decreased amino acid levels

Laboratory Diagnosis Diagnosis is through analysis of plasma ,CSF and urinary concentration of amino acids

Chemical  MethodsP  Ch   d TLCPaper Chr. and TLCIon Exchange Chr. HPLCHPLCTandem Mass 

FeClFeCl33 in urinein urineDisease/AnalyteDisease/Analyte ColorColor

PKU/PKU/phenylpyruvatephenylpyruvate GreenGreenPKU/PKU/phenylpyruvatephenylpyruvate GreenGreen

Tyrosinemia/Tyrosinemia/pp--hydroxyphenylpyruvic hydroxyphenylpyruvic acidacid

Rapidly fading greenRapidly fading green

MSUD/ MSUD/ branched chain ketoacidsbranched chain ketoacids Greenish grayGreenish gray

Alkaptonuria/Alkaptonuria/homogentisic acidhomogentisic acid Effervescent blueEffervescent blue--greengreen

AcetaminophenAcetaminophen GreenGreen

SalicylatesSalicylates purplepurple

example fast

li bl

example fast

but

Dr. Z. Lukacs - University Hospital Hamburg

unreliable

Bacterial Inhibition Assay (BIA)Bacterial Inhibition Assay (BIA)

•Phenylalanine-free agar•B. subtilis spores• β 2 thienylalanine (inhibitor)

Dr. R. Guthrie

• β-2-thienylalanine (inhibitor)

Dried blood spots are t thput on the agar.

Areas of intense growth indicate high phenylalanine concentrations

Dr. Z. Lukacs - University Hospital Hamburg

concentrationsGuthrie R, Susi A. Pediatrics 1963; 32: 338-43

Phenylalanine + H O Phenylpyruvate + NH +

UV/VIS Spectroscopy IIUV/VIS Spectroscopy IIPDHPDHPhenylalanine + H2O Phenylpyruvate + NH4

NAD+ NADH/H+

Electron acceptor (reduced) Electron acceptor (oxidized)

Tetrazolium Formazan

Paper chro.             TLC    

lid

plate

start

eluent

sample

eluent

Amino Acid Analyzer and HPLCAmino Acid Analyzer  and HPLC 

TandemMass SpectrometryTandem Mass Spectrometry

Material and MethodsIn a descriptive –cross sectional study, all data of amino acid 

analysis by HPLC during 2012 collected.y y g

Data from patients with Phenylketonuria and those with 

mildly increase or decrease levels of a range of amino acids mildly increase or decrease levels of a range of amino acids 

( indicating not follow the diet ) excluded .

Th  f   f  i   id di d  i   dditi  t    The frequency of amino acid disorders in addition to mean ±

SD concentration of each amino acid according to sex and 

gender were reported.

M th dMethod:

OPA‐HPLC Specimen: Heparinized PlasmaDeproteinization :  with MethanolDerivatization :OPA‐2MEIS=HomoserineColumn =C18M bil   h     h lMobile phase : methanol, Sodium acetate, waterD t t  FlDetector: Flueroscence

Asp,Glu,Asn,Ser,GlnAsp,Glu,Asn,Ser,Gln,,His,Gly,Thr,Cit,Arg,TauHis,Gly,Thr,Cit,Arg,Tau,,Ala,TyrAla,Tyr, α, α‐‐ aminobytricaminobytric ,,Trp,Met,Val,Phe,IleuTrp,Met,Val,Phe,Ileu,,Leu,Orn,LysLeu,Orn,Lys

Results:Total HPLC :1778

Ab l  86   E t  Ph CAbnormal =86   Except  Phe,Cys

Female :36  Male :50 

Youngest 2days  Oldest 42Y 

F f A A di dFrequency of A.A disordersN % % in Total HPLC(n=1778)

Tyr 40 46.5 2.2Gly 14 16.3 0.8L Il V l 6 6Leu,Ileu,Val 10 11.6 0.6Gln 7 8.1 0.4Orn 6 7 0 3Orn 6 7 0.3Gln+Cit/Arg/Ala/Lys 4 4.7 0.2

Gl t i idGlutamic acid 3 3.5 0.2Met 1 31.2 0.1Ala 1 1 2 0 1Ala 1 1.2 0.1Total  86 100 4.8

Distribution of patients acc to Gender and AgeDistribution of patients acc. to Gender and AgeA.A Number of 

Patients Age (months )

Female Male Mean ±SD Youngest Oldest

Tyr 20 20 37±39 13d 10Y

l dGly 3 11 28±25 9d 6Y

Leu,Ileu,Val 5 5 45±59 17d 13Y

Gln ‐ 7 27±48 6d 11YGln 7 27±48 6d 11Y

Orn 4 2 362±100 (30±8.4y)

264 (22y) 504 (42y)

l dGln+Cit,Arg/Ala/Lys 1 3 1±1.6 2d 4m

Met 1 240(20y) ‐ ‐

Ala 1 13d ‐ ‐

Mean,SD,Minimum.Maximum of A.A Concentration A.A Concentration (µmol/L)

Mean SD Min Max Lab Reference Interval 

Tyr 652 292 229 1362 30‐120

Gly 1239 912 721 4284 140‐490(80‐320 for 1‐3m)

Leu 1269 613 292 2269 60‐230(35‐180 for 1‐3m)60 30(35 3 )Ileu 408 126 175 603 30‐130

Val 497 85 389 609 140‐350

Gln 1581 786 795 2989 396‐746(100‐1200 for 1‐3 m)Gln 1581 786 795 2989 396 746(100‐1200 for 1‐3 m)

Orn 737 82 655 839 20‐135

GLu 305 90 209 388 10‐120

Cit 675 662 0 2 1342 8 47Cit 675 662 0.2 1342 8‐47

Arg 13.7 ‐ ‐ ‐ 40‐160(10‐130 for 1‐3m)Lys 610 ‐ ‐ ‐ 80‐250(45‐200 for 1‐3m)Met 833 ‐‐ ‐ ‐ 6‐49

Ala 1492 ‐ 935 2050 240‐600(100‐400 for 1‐3m)

d ( l/ )Amino Acids concentration (µmol/L)patients VS. Reference Interval (RI)

1400

1600

1000

1200

Min‐RI

600

800Min RI

Max_RI

Mean ‐P

0

200

400

0Tyr  Gly  Leu  Ileu  Val  Gln  Orn  GLu  Cit  Arg  Lys  Met  Ala 

Tyrosine A.A Concentration 

(µmol/L)

Mean SD Min Max

Lab Reference 

Type I : Fumaryl acetoacetateHydrolase(FAH)‐Hepatorenalx Reference 

Interval 

Tyr 652 292 229 1362 30‐120

Hepatorenal

Type II: Tyrosine yp yAminotransferase (TAT)Eye and Skin Lesions

A.A Number of Patient

Age (months )

Type III: 4 Hydroxy Phenyl Pyruvate Dioxigenade (HPPD)

Patients Fema

Male

Mean ±SD

Younge

Oldest y g ( )

legst

Tyr 20 20 37±39 13d 10Y

HT1

http://www.newbornscreening.info/Parents/aminoaciddisorders/Images/tyrosinemia_type1.gif

TreatmentsDietary restriction of tyrosine and phenylalanine. (aim is below 500 μmol/L)Treatement with NTBC (2‐(2‐nitro‐4‐trifluoro‐Treatement with NTBC (2‐(2‐nitro‐4‐trifluoro‐methylbenzoyl)‐1,3‐cyclohexanedione) to block metabolism.

h b f ll lPro‐ Inhibitor of HPPD.  NTBC structurally similar to HPPDPro‐ Protects patients from fatal, end stages of diseasep , gCon‐ However, increases plasma tyrosine levels and requires several follow‐ups.

Li er transplant (popular in  980s)Liver transplant (popular in 1980s)

Nonketotic hyperglycinemiaNonketotic hyperglycinemia*Defect in glycine Glycine NH3 + CO2catabolism

autosomal recessiveautosomal recessive

symptoms in first 24 hours

hypotonia/encephalopathy seizures burst suppression EEGhypotonia/encephalopathy, seizures, burst suppression EEG

increased CSF/plasma glycine

Tx: benzoate, dextramethorphan, p

poor prognosis, diet ineffective

*Diagnosis based on elevated CSF/Plasma glycine ratio

MSUD

A.A Concentration (µmol/L)

M SD Mi M L b R f  

MSUD

Enz deficien: branched-chain alpha-keto acidMea

nSD Min Ma

xLab Reference 

Interval 

Leu 1269 613 292 2269

60‐230(35‐180 for 1‐3m)

chain alpha-keto acid dehydrogenase (BCKD)

presents with odor to Ileu 408 126 17 5 603 30‐130

Val 497 85 389 609 140‐350

urine and CNS problems

A.A Number of Patients 

Age (months )

Female

Male Mean ±SD

Youngest

Oldest

Leu,Ileu,Val 5 5 45±59 17d 13Y

http://www.newbornscreening.info/tools/GraphicsLib/MSUD.jpg

Types of MSUDClassical: little or no enzyme activity 

TreatmentRestrict their diet to foods without leucine, 

isoleucine, and valine

Must continue throughout life or symptoms will 

reoccur

During times of metabolic decompensation, 

patients can be treated with intra‐venous 

hyperalimentationhyperalimentation.

Supplements can be taken so that patients receive 

those essential amino acidsthose essential amino acids.

Urea Cycle DisordersUrea Cycle DisordersDIET

C b lProtein ↑NH4+ + HCO3 Carbamoyl PhosphateHyperammonemia

b lii hOrnithine

Citrulline

UREA

OTC

U l di d

metabolic withoutacidosis (usually have

s)alkalosirespiratory

Arginine

urea(2N)

UREACYCLE Asp

(N)

•Urea cycle disorders:•Ornithinetranscarbamylasedeficiency (X-linked)

Argininosuccinic Acid

y ( )•Carbamoyl phosphate synthase deficiency (AR)•Citrullinemia (AR)•Argininosuccinic•Argininosuccinicacidemia (AR)

•Argininemia (AR)

OrnitineGyrate atrophy (GA) is a rare hereditary disease aracterized by markedly high serum ornithinel l   l i  f   h  d fi i   f  i hi dlevels resulting from the deficiency of ornithine‐d‐amino transferase (OAT), a mitochondrial matrix enzyme degenerativeenzyme.degenerative,Hyperornithinaemia is accompanied by lysinuriaand reduced lysine plasma levels in GAand reduced lysine plasma levels in GA

Conclusion High performance Liquid Chromatography (HPLC) is a Chromatography (HPLC) is a method with high sensitivity, high 

l d l l hresolution and relatively short analysis time. It is suitable for early a a ys s t e. t s su tab e o ea ydiagnosis of patients that is crucial t   id i ibl    dto avoid irreversible organ damage.