diagnosis of fatty acid oxidation disorders by mass

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

http://mchb.hrsa.gov/screening



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


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-)


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


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



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



S192 Pal car A2 96h

m/z200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500

%

0


4.75e5418

361

305

249

221 229 260

414

446

CPT-IID/CACTD at 96 hour, in d31-palmitate

d31-C16

d27-C14C5



S195 Pal car A2 96h

m/z200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500

%

0


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





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



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


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-)


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

diagnosis of fatty acid oxidation disorders by mass

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