diagnosis of fatty acid oxidation disorders by mass
TRANSCRIPT
DIAGNOSIS OF FATTY ACID OXIDATION DIAGNOSIS OF FATTY ACID OXIDATION DISORDERS BY MASS SPECTROMETRYDISORDERS BY MASS SPECTROMETRY
Eric LawEric LawChemical Pathology, PWHChemical Pathology, PWH
15 January 2010
Outline of talkOutline of talk
•• Basic of mitochondrial fatty acid oxidation (FAO)Basic of mitochondrial fatty acid oxidation (FAO)•• Laboratory investigations of FAODLaboratory investigations of FAOD•• New combined FAO rate and probe assay New combined FAO rate and probe assay
Fatty acidsFatty acids
•• ShortShort--chainchain: 2 to 4 carbon atoms: 2 to 4 carbon atoms•• MediumMedium--chainchain: 6 to 12 carbon atoms: 6 to 12 carbon atoms•• LongLong--chainchain: 14 to 18 carbon atoms: 14 to 18 carbon atoms•• Very longVery long--chainchain: 20 to 26 carbon atoms: 20 to 26 carbon atoms
COOH
Plasma
Membrane OCTN2
Outer mito. Membrane
Inner mito. Membrane
CPT I
LCFA TRANSPORTER
SC/MCFA
CPT IICACT
L-Carnitine
Acyl-CoA
LCFA
Acylcarnitine
Carnitine
Acyl-CoA
Enoyl-CoA
3-hydroxylacyl-CoA
3-ketoacyl-CoAAcetyl-CoA
Ketogenesis
TCA cycle
steroidogenesis
CoASH
CoASH
FAD+
FADH2
NAD+
NADH+H+
S/MCFAs
LC/VLCFA
Acyl-CoA
L-Carnitine
Carnitine
Acylcarnitine
VLCAD,SCAD, MCAD
LCHAD,S/MCHAD
ECH
KAT
Incidence of FAODIncidence of FAOD
•• CUD 1 in ~ 40,000 (Japanese, ?similar CUD 1 in ~ 40,000 (Japanese, ?similar in Chinese) to 1 in ~100,000 (other in Chinese) to 1 in ~100,000 (other countries)countries)
•• MCAD 1 in ~10,000 to 20,000MCAD 1 in ~10,000 to 20,000•• VLCAD 1 in ~75,000VLCAD 1 in ~75,000•• LCHAD 1 in ~75,000LCHAD 1 in ~75,000•• TFP 1 in ~100,000TFP 1 in ~100,000•• Others less than 1 in ~100,000Others less than 1 in ~100,000
American College of Medical Genetics. Newborn Screening: Toward a Uniform Screening Panel and System. Final Report, March 8, 2005
All FAOD 1 in ~ 14,000 to as high as 1 in ~4000
Congenital Hypothyroidism 1 in ~3300
PKU 1 in ~16,000
Approach to investigations of FAODApproach to investigations of FAOD
•• PrePre--symptomatic screening through symptomatic screening through expanded newborn screening programsexpanded newborn screening programs
•• Laboratory investigations of symptomatic Laboratory investigations of symptomatic patientspatients
•• Prenatal diagnosisPrenatal diagnosis•• Postmortem diagnosisPostmortem diagnosis
Combined oxidation rate and acylcarnitine profiling
into one culture experiment
MethodologiesMethodologies
•• Skin fibroblast cultures fed with Skin fibroblast cultures fed with 22HH3131--palmitate palmitate •• Measurement of deuterium labeled acylcarnitines Measurement of deuterium labeled acylcarnitines
in culture medium using an ESIin culture medium using an ESI--MSMS--MS methodMS method•• Measurement of deuterated water (DHO) Measurement of deuterated water (DHO)
formation resulted from overall mitochondrial formation resulted from overall mitochondrial fatty acid fatty acid ββ--oxidation using an isotope ratio oxidation using an isotope ratio mass spectrometer mass spectrometer
FADD2
NADD
D2O
D2O H2O
H2O
H2O
H2O
D D
D
D
D
D
D
D
D
D
D
2D
D2O
Skin fibroblasts are cultured until confluence is reached
Subculture into culture plate
Leave to adhere overnight
Replace with medium contained Deuterium labelled fatty acids and L-carnitine
72 to 96 hours
Inhibitors
glucose
Activators
Analyze deuterium labelled acylcarnitine species by MS/MS
1811111433412N =
COMPLEX IV
OTHER IMD
SCHAD
LCHAD
TFP
SCAD
MCAD
VLCAD
MAD
CPT-II
CACT
CPT-I
CUD
Per
cent
age
of c
ontro
l med
ian
of d
eute
rate
d w
ater
enr
ichm
ent
160
140
120
100
80
60
40
20
0
-20
Fig.1. Box & whisker plot showing the percentages of control median of deuterated water enrichment (2H2O) in control, FAOD and other IMD cell lines incubated at 37ºC in humidified 5% CO2 and 95% air for 96 hours with 0.2 mM 2H31- palmitate and 0.4 mM L-carnitine in DMEM no glucose, without serum supplemented, in the presence of 0.4% defatted BSA and 2 mM L-glutamine. Red line shows 60% of the control median (60.9 ppm/mg protein/96 h).
Law LK et al Clinica Chimica Acta 382;25-30, 2007
Biomedical Mass Spectrometry Biomedical Mass Spectrometry Laboratory, CUHKLaboratory, CUHK
D14 Pal car A1 0h
m/z200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500
%
0
100ACYLH1514 1 (4.003) Sm (Mn, 2x0.75); Sb (1,40.00 ) 1: Parents of 99ES+
5.21e5418
361
305
249
221
Control at 0 hour, in d31-palmitateInternal standards: 221 (d3-acetyl-); 249 (d3-butyryl-); 305 (d3-octanoyl-);361 (d3-dodecanoyl-); 418 (d3-palmitoyl-)
(methyl acylcarnitine esters)
D14 Pal car A1 96h
m/z200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500
%
0
100ACYLH1520 1 (4.003) Sm (Mn, 2x0.75); Sb (1,40.00 ) 1: Parents of 99ES+
5.40e5418
361
305
249
221
232 246
260
253285284
302
350
317
330
382
446
Control at 96 hour, in d31-palmitate
d31-C16
d23-C12
d19-C10
d15-C8
d11-C6C5C3
d7-C4
Internal standards: 221 (d3-acetyl-); 249 (d3-butyryl-); 305 (d3-octanoyl-);361 (d3-dodecanoyl-); 418 (d3-palmitoyl-)
(methyl acylcarnitine esters)
Mass spectra of typical acylcarnitine profiles using 2H31-palmitate as substrate for feeding cultured fibroblasts incubated at 37ºC in humidified 5% CO2 and 95% air for 96 hours with 0.5 mM L-carnitine in DMEM no glucose, without serum supplemented, in the presence of 0.4% defatted BSA and 2 mM L-glutamine. Diagnostic intermediate acylcarnitines found in each FAOD are bold. Control skin fibroblast cell line; C3-, propionyl-; C4:0-, butyryl-; C6:0-, hexanoyl-; C8:0-, octanoyl-; C10:0-, decanoyl-; C12:0-, dodecanoyl-; C16:0-, palmitoyl-.
S196 Pal car A3 96h
m/z200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500
%
0
100ACYLH1548 1 (4.003) Sm (Mn, 2x0.75); Sb (1,40.00 ) 1: Parents of 99ES+
5.04e5418
361
305
249
221
260
350
317
311
382
446
CUD at 96 hour, in d31-palmitate
d31-C16d23-C12
d19-C10
d15-C8
Internal standards: 221 (d3-acetyl-); 249 (d3-butyryl-); 305 (d3-octanoyl-);361 (d3-dodecanoyl-); 418 (d3-palmitoyl-)
(methyl acylcarnitine esters)
S191 Pal car A1 96h
m/z200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500
%
0
100ACYLI2221 1 (4.003) Sm (Mn, 2x0.75); Sb (1,40.00 ) 1: Parents of 99ES+
7.44e5361
305
249
221
246260
418
CPT-ID at 96 hour, in d31-palmitate
C5
Internal standards: 221 (d3-acetyl-); 249 (d3-butyryl-); 305 (d3-octanoyl-);361 (d3-dodecanoyl-); 418 (d3-palmitoyl-)
(methyl acylcarnitine esters)
S192 Pal car A2 96h
m/z200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500
%
0
100ACYLH1946 1 (4.003) Sm (Mn, 2x0.75); Sb (1,40.00 ) 1: Parents of 99ES+
4.75e5418
361
305
249
221 229 260
414
446
CPT-IID/CACTD at 96 hour, in d31-palmitate
d31-C16
d27-C14C5
Internal standards: 221 (d3-acetyl-); 249 (d3-butyryl-); 305 (d3-octanoyl-);361 (d3-dodecanoyl-); 418 (d3-palmitoyl-)
(methyl acylcarnitine esters)
S195 Pal car A2 96h
m/z200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500
%
0
100ACYLH1534 1 (4.003) Sm (Mn, 2x0.75); Sb (1,40.00 ) 1: Parents of 99ES+
3.29e5418
361
305
252249
221
246
285284
267260302
350
317
382
414 445
SCHADD at 96 hour, in d31-palmitate
d27-C14 d31-C16d23-C12
d19-C10
d15-C8
d11-C6
d5-C4OH
d7-C4
Internal standards: 221 (d3-acetyl-); 249 (d3-butyryl-); 305 (d3-octanoyl-);361 (d3-dodecanoyl-); 418 (d3-palmitoyl-)
(methyl acylcarnitine esters)
Internal standards: 221 (d3-acetyl-); 249 (d3-butyryl-); 305 (d3-octanoyl-);361 (d3-dodecanoyl-); 418 (d3-palmitoyl-)
(methyl acylcarnitine esters)
MCADD at 96 hour, in d31-palmitate/d27-myrisate
d15-C8
d19-C10
d11-C6
C181 no adr 1 Pal car A1 96h
m/z200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500
%
0
100ACYLC0734 1 (4.003) Sm (Mn, 2x0.75); Sb (1,40.00 ) 1: Parents of 99ES+
3.93e5417
361
305
249
221215 246
302
414
382
446
d27-C14
d23-C12
d31-C16
Internal standards: 221 (d3-acetyl-); 249 (d3-butyryl-); 305 (d3-octanoyl-);361 (d3-dodecanoyl-); 418 (d3-palmitoyl-)
(methyl acylcarnitine esters)
VLCADD at 96 hour, in d31-palmitate
N2 Pal car A1 96h
m/z200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500
%
0
100ACYLH0340 1 (4.017) Sm (Mn, 2x0.75); Sb (1,40.00 ) 1: Parents of 99ES+
9.67e5361
305
249
221
232 267260 284
349317
418
382
414
446
460C3
C5 d11-C6 d15-C8 d19-C10
d23-C12
d27-C14d29-C16OH
d31-C16
LCHADD/TFPD at 96 hour, in d31-palmitateInternal standards: 221 (d3-acetyl-); 249 (d3-butyryl-); 305 (d3-octanoyl-);361 (d3-dodecanoyl-); 418 (d3-palmitoyl-)
(methyl acylcarnitine esters)
Strengths and Strengths and weaknessesweaknesses•• All FAO disorders studied could be differentiated from All FAO disorders studied could be differentiated from
the control and nonthe control and non--FAOD IMD groups using the FAOD IMD groups using the combined functional assaycombined functional assay
•• The present method requires only one culture The present method requires only one culture experiment, is relatively simple and nonexperiment, is relatively simple and non--radioactiveradioactive
•• Acylcarnitine profile is similar between CUD and control, Acylcarnitine profile is similar between CUD and control, however total amount of acylcarnitine intermediates however total amount of acylcarnitine intermediates produced can be used as a specific marker for CUDproduced can be used as a specific marker for CUD
•• Identical profiles observed between CACT and CPTIdentical profiles observed between CACT and CPT--II or II or LCHAD and MTP (alternative substrates needed, Roe DS LCHAD and MTP (alternative substrates needed, Roe DS et al Mol Genet Metab 2006)et al Mol Genet Metab 2006)
•• Not many laboratories equipped with cell culture facilities, Not many laboratories equipped with cell culture facilities, isotope ratio mass spectrometer and ESIisotope ratio mass spectrometer and ESI--MSMS MSMS
Carnitine deficiency, systemic primary CDSP Carnitine deficiency, systemic primary CDSP or carnitine uptake defect CUDor carnitine uptake defect CUD
CUD is the most common FAOD in Japanese, and CUD is the most common FAOD in Japanese, and probably also in Chinese probably also in Chinese
recurrent hypoketotic hypoglycaemia encephalopathy, recurrent hypoketotic hypoglycaemia encephalopathy, failure to thrive, sudden deathfailure to thrive, sudden death
carnitinecarnitine--responsive cardiomyopathy with or without responsive cardiomyopathy with or without weakness weakness
very low serum and tissue carnitine concentrations, very low serum and tissue carnitine concentrations, decrease renal reabsorption of carnitinedecrease renal reabsorption of carnitine
laboratory investigations : serum and urine carnitine, laboratory investigations : serum and urine carnitine, radioactive carnitine uptake assay in fibroblast culture and radioactive carnitine uptake assay in fibroblast culture and OCTN2 mutation analysisOCTN2 mutation analysis
Differentiation of carnitine uptake defect from Differentiation of carnitine uptake defect from normal control and other FAOD based on total normal control and other FAOD based on total acylcarnitines production in cultured cellsacylcarnitines production in cultured cells
•• 2 (HK)2 (HK)•• 1 (Germany)1 (Germany)•• 1 (Netherlands)1 (Netherlands)•• 3 (Taiwan)3 (Taiwan)•• 2 (Genetic Repository, Coriell)* 2 (Genetic Repository, Coriell)* •• 22 (control human fibroblasts)22 (control human fibroblasts)•• 10 (other FAOD fibroblasts)10 (other FAOD fibroblasts)
822N =
Oxidation of d31-palmitate in Human Skin Fibroblast Cultures
CUDControl
95%
CI m
ean
of d
-enr
ichm
ent (
ppm
/mg
prot
ein/
96h)
140
120
100
80
60
40
20
Mann-Whitney U test p<0.05
Acylcarnitine Profiles in Human Skin Fibroblast Culture Medium
CUDControl
Mea
n ac
ylca
rnitine
inte
rmed
iate
s (n
mol
/mg
prot
ein/
96h)
4
3
2
1
0
C4:0-carnitine
C6:0-carnitine
C8:0-carnitine
C10:0-carnitine
C12:0-carnitine
C14:0-carnitine
C16:0-carnitine
Total Acylcarnitines in Human Skin Fibroblast Culture Medium
CUDControl
95%
CI
Mea
n of
tot
al a
cylc
arni
tine
inte
rmed
iate
s16
14
12
10
8
6
4
Mann-Whitney U test p<0.001Law LK et al J Inherit Metab Disease 30:816, 2007
ConclusionsConclusions
•• Total amount of acylcarnitine Total amount of acylcarnitine intermediates produced (C4:0 to C16:0 intermediates produced (C4:0 to C16:0 eveneven--chain acylcarnitines) under the chain acylcarnitines) under the standardized culture conditions can be standardized culture conditions can be used as a specific marker for CUD used as a specific marker for CUD
•• This method is relatively simple and may This method is relatively simple and may replace the radioreplace the radio--active uptake assay for active uptake assay for the diagnosis of carnitine uptake defectthe diagnosis of carnitine uptake defect
Clinical symptoms suggestive of FAOD
Routine biochemistries:↑NH3,LFT,CK,lactate,↓glucose
Organic acids, acylglycines
Free carnitine, acylcarnitines
Abnormal and characteristic
Confirmed by tissue enzyme assay or mutation study
Urine Serum/plasma/DBS
Prenatal Diagnosis
Amniotic fluid
Inconclusive
In vitro fibroblast functional assay – combined FAO rate and AC profiling
SIDS
Normal AC profile, TC>9.3 & 2H2O enrichment>55
FAOD
unlikely
Normal AC profile, TC>9.3 & 2H2O enrichment<55
Respiratory chain or non-FAO disorders
Normal AC profile, TC<9.3 & 2H2O enrichment<109
CUD, confirmed by mutation study of OCTN2
Abnormal AC profile, 2H2O enrichment <55
Defects: CPT-1, CPT-II/CACT, MAD, SCAD, MCAD, VLCAD, LCHAD/TFP, SCHAD
Confirmed by tissue enzyme assays or mutation study
No
Confirmed by tissue enzyme assays or mutation study
No
No No
Unidentified or new disorders
AcknowledgmentsAcknowledgments
•• Dept of Chemical Pathology, PWH/CUHKDept of Chemical Pathology, PWH/CUHK•• Prof CWK LamProf CWK Lam•• Prof N TangProf N Tang•• Dr CS HoDr CS Ho•• Mr Simon FungMr Simon Fung•• Mr Eric PangMr Eric Pang•• Ms Caroline LaiMs Caroline Lai
•• Dept of Paediatrics, PWH/CUHKDept of Paediatrics, PWH/CUHK•• Dr Joannie Hui Dr Joannie Hui •• Dr KL CheungDr KL Cheung•• Prof TF FokProf TF Fok
•• Laboratory for Genetic Metabolic Diseases, Laboratory for Genetic Metabolic Diseases, Academic Medical Centre, The NetherlandsAcademic Medical Centre, The Netherlands
•• Prof RJA Wanders Prof RJA Wanders •• Dr J RuiterDr J Ruiter
•• Dept of Medical Genetics and Pediatrics, Dept of Medical Genetics and Pediatrics, National Taiwan University Hospital, Taiwan, National Taiwan University Hospital, Taiwan, ROCROC
•• Dr YH ChienDr YH Chien•• Dr WL HwuDr WL Hwu
•• Newborn Screening Service, Dept of Chem Path, Newborn Screening Service, Dept of Chem Path, WomenWomen’’s and Childrens and Children’’s Hospital, South Australias Hospital, South Australia
•• Mr Mr EnzoEnzo Ranieri Ranieri
Thank you for your attention
CACT (CACT (Carnitine:AcylcarnitineCarnitine:Acylcarnitine Translocase) Translocase) deficiencydeficiency
a mitochondrial carrier protein as one of the components of the a mitochondrial carrier protein as one of the components of the carnitine cyclecarnitine cycle
catalyzes a reversible exchange of carnitine and acylcarnitine catalyzes a reversible exchange of carnitine and acylcarnitine across across the mitochondrial membranesthe mitochondrial membranes
typical presentations include seizures, progressive liver and/otypical presentations include seizures, progressive liver and/or heart r heart failure, comafailure, coma
hypoketotic hypoglycaemia, hyperammonemia, lactic acidosis, hypoketotic hypoglycaemia, hyperammonemia, lactic acidosis, elecvatedelecvated CK and CK and transaminasestransaminases
dicarboxylic aciduriadicarboxylic acidurialow free and total plasma carnitine, increase in acylcarnitineslow free and total plasma carnitine, increase in acylcarnitines to free to free
carnitine ratiocarnitine ratiohigh plasma long chain acylcarnitines concentrationhigh plasma long chain acylcarnitines concentration
Plasma
Membrane OCTN2
Outer mito. Membrane
Inner mito. Membrane
CPT I
LCFA TRANSPORTER
SC/MCFA
CPT IICACT
L-Carnitine
Acyl-CoA
LCFA
Acylcarnitine
Carnitine
Acyl-CoA
Enoyl-CoA
3-hydroxylacyl-CoA
3-ketoacyl-CoAAcetyl-CoA
Ketogenesis
TCA cycle
steroidogenesis
CoASH
CoASH
FAD+
FADH2
NAD+
NADH+H+
S/MCFAs
LC/VLCFA
Acyl-CoA
L-Carnitine
Carnitine
Acylcarnitine
VLCAD
LCHAD
ECH
KAT
Prenatal diagnosis for a family with a case of Prenatal diagnosis for a family with a case of CACT deficiencyCACT deficiency
•• The proband was a male baby who died The proband was a male baby who died suddenly on day one after birthsuddenly on day one after birth
•• Postmortem examination was suggestive Postmortem examination was suggestive of FAOD, mutation analysis confirmed a of FAOD, mutation analysis confirmed a diagnosis of CACT deficiencydiagnosis of CACT deficiency
•• AminoticAminotic fluid was received during a fluid was received during a second pregnancysecond pregnancy
Oxidation of D31-palmitate in Human Aminocyte Cultures
FetusControl
Mea
n +
- 2 S
E D-e
nrichm
ent (
ppm
/mg
prot
ein/
96h)
240
220
200
180
160
140
120
100
80
Acylcarnitine profiles in human aminocyte culture medium
Fetus (prenatal)Control
Mea
n of
acy
lcar
nitin
e inte
rmed
iate
s (n
mol
/mg
prot
erin/9
6 h)
7
6
5
4
3
2
1
0
PALC4
PALC6
PalC8
PalC10
PalC12
Pal_C14
PalC16
ConclusionsConclusions
•• Combined functional assay for FAO Combined functional assay for FAO revealed normal FAO rate and revealed normal FAO rate and unremarkable acylcarnitine profileunremarkable acylcarnitine profile
•• The fetus was unaffectedThe fetus was unaffected•• Mutation analysis confirmed a carrier Mutation analysis confirmed a carrier
status of CACT deficiencystatus of CACT deficiency