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.