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A mass spectrometric approach to investigate cardiolipin metabolism in Barth syndrome

Valianpour, F.

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Citation for published version (APA):Valianpour, F. (2004). A mass spectrometric approach to investigate cardiolipin metabolism in Barth syndrome.

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Download date: 27 Jun 2020

CHAPTERR 5 Cardiolipi nn deficiency in X-linked cardioskeletal myopathy and neutropenia

(Barthh syndrome, MIM 302060): a study in cultured skin fibroblasts

Fredoenn Valianpour, Ronald JA Wanders, PhD, Henk Overmars,

Peterr Vreken, PhD, Albert H. van Gennip, PhD, Frank Baas, PhD, Barbara Plecko,

MD,, Rene Santer MD, Kolja Becker MD, Peter G. Barth MD

Journall of Pediatrics, 2002, 141(5):729-33

C h a p t err 5

A B S T R A C TT OBJECTIVE

Too determine cardiolipm concentrations in cultured skm fibroblasts of patients with

X-hnkedd cardioskeletal myopathy and neutropenia (Barth syndrome, MIM 302060) and in

twoo groups of controls and to evaluate whether specific differences exist between these

groups.. Method: High Performance Liquid Chro-matography-Electrospray Mass spectrometry

(HPLC-ES-MS)) was used to quantify total cardiolipin and subclasses of cardiolipin molecular

speciess in cultured skin fibroblasts. Groups: normal controls (n=10), patients with various

mitochondriall disorders (n=8) and patients with Barth syndrome (n=5). Results were com-

paredd in the three groups by using a t-student test for all individual analyses.

R E S U L T S S

Totall cardiolipin and cardiolipm subclasses were decreased in BTHS patients as com-

paredd to normal controls and disease controls. Conclusion: Barth syndrome patients have a

specificc decrease of various cardiolipin molecular species, foremost tetralineoyl-cardiolipin.

Thereforee the analysis of cardiolipin in fibroblasts offers a specific biochemical approach to

detectt this disorder.

I N T R O D U C T I O NN X-linked cardioskeletal myopathy and neutropenia (Barth syndro-

me,, MIM 302060) is an X-linked recessive dilated cardiomyopathy, neutropenia and skeletal

myopathyy (1). Biochemical findings include variable mitochondrial respiratory chain dys-

functionn in skeletal muscle and in cultured fibroblasts, increased urinary excretion of 3-

methylglutaconicc acid, 3-methy-lglutaric acid, and 2-ethylhydracrylic acid and moderately

decreasedd serum cholesterol. The gene involved in the disease is the Tafazzin (TAZ) gene,

whichh is localized on Xq28. Mutations have been identified in nearly all Barth syndrome

(BTHS)) patients.

Inn 1997 Neuwald (2) reported that the TAZ gene shares homology with acyltrans-

ferases,, involved in phospholipid biosynthesis and/or remodeling, suggesting that a specific

glycerophospholipidd might be lacking in BTHS. We studied the biosynthesis and remodeling

off the phospholipids phosphatidylglycerol (PG) and cardiolipin, which are mainly confined

too mitochondrial membranes, in cultured skin fibroblasts from BTHS patients and controls.

Thesee studies revealed reduced levels of CL and a disturbance in the remodeling of PG and

CL.. In particular the incorporation of linoleic acid into PG and CL was dramatically reduced,

whereass the incorporation of other fatty acids into these phospholipids was normal (3).

Thesee studies proved defective remodeling of cardiolipin as a characteristic and probably

basicc disturbance in BTHS patients. The method however is laborious and involves incor-

porationn of radioactive labeled linoleic acid into skin fibroblasts. We therefore set out to

70 0

Cardiolipinn deficiency in X-linked cardioskeletal myopathy and neutropenia

developp a method that is less laborious and avoids the use of radioactively labeled chemicals.

Thee quantitative and compositional analysis of CL in cultured skin fibroblasts by

normall phase High Performance Liquid Chromatography - Electrospray Mass Spectrometry

iss a more direct and rapid method and therefore can be useful to identify patients with BTHS.

M A T E R I A LL A N D M E T H O D S PATIENTS

Fivee patients with BTHS and known mutations in the TAZ gene were included in this

study.. The group included a patient with ventricular non-compaction, a previously identified

subgroupp of patients with a TAZ gene mutation. Individual patient data are presented in table 1.

CONTROLL GROUPS

1.. Fibroblast lines from healthy controls (n=10) and 2. Fibroblast lines from patients

withh disorders of energy metabolism (n=10) including mitochondrial chain disorders: isolated

complexx I deficiency (n=l), isolated complex IV deficiency (n=3), MERRF (tRNALys

G8344AA mutation) (n=l), Leigh syndrome with NARP (T8993C mutation) (n=l), autosomal

dominantt intergenomic signalling defect (n=l), and a female patient with 3-methylglutaconic

aciduria,, leucoencephalopathy and lactic acidosis (n=l).

MATERIAL L

Alll solutions were of analytical quality and were purchased from Merck (Darmstadt,

Germany).. (C14:0)4-CL (internal standard) was purchased from Avanti lipids (Alabaster,

USA).. Commercially available (C18:2)^.-CL was purchased from Fluka BioChemika (Fluka

artt No. 71238, Switzerland). Analytical HPLC LiChrospher Si 60 column (2 x 250 mm, 5 um

particlee size) was purchased from Merck (Darmstadt, Germany).

CELLL CULTURE

Fibroblastss were grown in HAM-F10 medium supplemented with 10% FCS and

incubatedd for 10 days at 37 °C. After trypsinization, cells were collected and centrifuged at

5000 g for 5 min and washed twice with 2 ml of phosphate buffered saline (PBS). The pellets

weree suspended in 1 ml of water and sonificated 2 times during 10 seconds. Ten uL of this

solutionn was used for protein determination.

LIPIDD E X T R A C T I O N

Lipidss were extracted by using the method of Bligh & Dyer (5) and were dissolved in

2000 uL chloroform/methanol/water (50:45:5, v/v/v, lipid extract). A portion of this lipid

extractt was used to measure the total organic phosphorous which is direct reflection of the

totall amount of phospholipids.

HPLC C

Thee HPLC system consisted of an HP 1100 series binary gradient pump, a vacuum

71 1

C h a p t err 5

degasser,, and a column temperature controller (all from Hewlett Packerd) and a Gilson 231

XLL autosampler (Gilson). The column temperature was maintained at 22°C. The lipid extract

wass injected onto a 2.1 x 250 mm silica column, 5 um particles diameter. The phospholi-

pidss were separated from interfering compounds by a linear gradient between solution B

(chloroform)) and solution A (methanol/water, 9:1 v/v). Both solutions contained 0.01 % of

aa 25% aqueous ammonia solution. The gradient (0.3 ml/min) was as follows: 0-3 min: 20 %

AA to 100 % A, 3-8 min: 100% A, 8-8.1 mm to 0% A and 8.1-14 mm equilibration with 0% A.

Alll gradient steps were linear, and the total analysis time, including the equilibration, was

144 min A splitter between the HPLC column and the mass spectrometer was used and

300 ul/min of eluant was introduced into the mass spectrometer. An electrically operated valve

wass used so that only the eluant from 4 to 8 min was introduced into the mass spectrometer.

MASSS SPECTROMETRY

AA Quattro II triple-quadrupole mass spectrometer (Micromass), was used in the negative

ESII mode. Nitrogen was used as nebulizing gas. Argon was used as collision gas at a pressure

off 2.5 x 10-3 mBar. The capillary voltage used was 3kV The source temperature was set at

800 °C. Optimal cone voltage energy was 30 V MS and MS/MS (daughter fragments, optimal

collisionn energy of 40 eV) analysis was performed to identify the fatty acid composition of

PGG and CL molecular species (as indicated in tables 3 and 4).

Alll CL molecular species including IS were measured by Single Ion Monitoring of

theirr double charged ion in negative mode.

VALIDATION N

Thee method was validated using control fibroblasts, commercially available (C18:2)A-

CLL as standard analyte and (C14:0)^-CL as internal standard. A standard procedure for

validationn of analytical methods was followed.

STATISTICS S

PP values are calculated for each CL molecular species in both control groups versus

thee same CL molecular species in the BTHS group and were obtained by using the t-student

testt for all individual analysis.

R E S U L T SS The recovery of the method was 92.8% +/- 3.8. Both (C18:2)4-CL and

(C14:0)4-CLL (IS) ions were suppressed by 23.1 +/- 2.9% and 25.9 +/- 2.1. The intra-assay

variationn and inter-assay variation were 8.6% and 12.1% respectively. The response factor

wass calculated as the ratio of (CI 8:2)^.-0- / (C14:0)^-CL from the analysis of calibration

mixturee before each set of analyses. The method was not validated for a quantitative measu-

rementt of PG.

72 2

Card io l i p inn def ic iency in X- l inked card ioske le tal myopathy and neu t ropen ia

Patients s

1 1

2 2

3 3

4 4

5 5

Agee at onset andd presenting

symptom m

1.33 y muscle weakness s

0.55 y failur e to thriv e e

1.11 y cardiac decompensation n

66 wk cardiac decompensation n

1stt month non-specific c

illness,, abnor-maall cardiac echochram m

Age e

11.2 2

Died d 1.1 1

14.5 5

ioa a

1.7 7

Dilated d cardiomyopaty y

+ +

+ +

+

+ +

+ + ventricular r

noncompaction n

Mil d d peroximal l

muscle e weakness s

+ +

+ +

+ +

+ +

+ +

Neutro--penia a

+ +

+ +

+ +

+ +

+ +

Statural l growth h

-SD D

-3 3

-2 2

-3 3

-2 2

-2 2

Organic c asiduria a

3-mcga, , 3-mgra, , 2-eha a

3-mcga, , 3-mgra, , 2-eha a

3-mcga, , 3-mgra, , 2-eha a

3-mcga a

3-mcga a 3-mgra a

TAZZ gene mutation n andd effect

C441GG premature stopcodonn exon 2

G527-11 G>C

428dell3 3 rrameshift t

exon2 2

G877T T glycinee >valine

exon8 8

exon2,, AG > AC splicee acceptor

site e

Li < >M « f L U ^ . . . ; » ! . ^ 1 ^ « U f ^ ««u iu^ » Reference sequence Genebank Nr NMJD00116, Tafazzin,

TableTable 1: Clinical, biochemical and genetic findings. TA2 AbbrJations: 3.mgca 3-methyi-giutaconic add; 3 mgramgra 3-methylgiutaric acid; 2-eha 2 ethyl-hydracrylic acid

CLL and PG were successfully separated from the non-polar lipids and other phos-

pholipidss by HPLC resulting in a more pure fraction. The purity of this fraction was not

establishedd by other methods. However the MS spectra showed some co-eluting components

inn the mass range lower than m/z 500. After separation by HPLC, individual CL and PG

molecularr species were measured by scanning of respectively negatively double and single

chargedd ions. The levels of the most abundant CL species (C18:2)4-CL, (0.8:2)3/(0.8:1)-

CLL and (08:2)2/(08:1)2-CL) in cultured fibroblasts from the individual samples belonging

too BTHS patients and control groups were then quantified (table 2).

Tablee 2 shows the median and the range of the concentrations (nmol/mg protein) of

differentt CL molecular species for each group. The levels of CL species were decreased in

BTHSS patients while there were no significant differences between the normal control group

andd the group of mitochondrial disorders. The fatty acid composition of vanous CL molecular

speciess was determined by MS/MS in fibroblasts of all groups. No differences were observed

inn the fatty acid compositions of molecular species with the same mass in the different

groupss used in this study. Linoleic acid forms the predominant fatty acid in the CL fraction

(tablee 2). The fatty acid composition of various PG molecular species was established by

MS/MSS analysis of each PG compound. In comparison to CL, the fatty acids of the PG

molecularr species contain more C18:l, 0 6 : 0, C20:4 and C20:2. Although the PG molecular

73 3

Chapterr 5

speciess were not quantified in this study, we detected qualitative differences between the

controll groups and BTHS group. The relative levels of PG molecular species containing lino-

leicc acid compared to PG molecular species containing other fatty acids were very low. One

PGG compound (at m/z 797.6) had the fatty acid composition C18:l/C20:3 in controls while

thee same mass in BTHS patients had both C18:l/C20:3 (major) and C18:2/C20:2 (minor).

Thee median, lowest and the highest concentrations (nmol/mg protein) of different CL mole-

cularr species in controls (n=10), disease controls (n=8) and BTHS patients (n=5) are shown.

AA significant difference is found between both controls groups and BTHS patients' fibroblasts.

Tabell 2: Concentrations (nmol/mg protein) of the most abundant CL molecular species inn cultured fibroblasts from all groups.

Normall control patients (n=I 0)

(C18:2)4-CL L (C18:2)3/(C18:1)1-CL L <C18:2)2/(C18:l)2-OL L

Mitochondria ll disease control patients (n=8)

(C18:2)4-CL L (C18:2)3/(C18:1)1-CL L (C18:2)2/(C18:1)2-CL L

BTHSS (n=5)

(C18:2)4-CL L (C18:2)3/(C18:1)1-CL L (C18:2)2/(C18:1)2-CL L

Median n

0.70 0 0.80 0 1.92 2

Median n

0.61 1 0.97 7 1.63 3

Median n

0.09 9 0.21 1 1.24 4

Range e

0.63-0.94 4 0.66-1.45 5 0.81-2.13 3

Range e

0.47-1.12 2 0.75-1.78 8 1.30-3.48 8

Range e

0.05-0.14 4 O.Ofrfl.42 2 0.09-0.34 4

Pvalne e

Vss BTHS group

.0003 3

.0006 6

.0003 3

Vss normal control patients

.000001 1 .0008 8 .00009 9

TheThe median, lowest and the highest concentrations (nmol/mg protein) of different CL molecular species in controls (n=10). diseasedisease controls (n=8) and BTHS patients (n=5) are shown. A significant difference is found between both controls groups and BTHSBTHS patients' fibroblasts.

D I S C U S S I O NN The method applied in this paper is a fast and reproducible method

forr quantitative measurement of CL and compositional analysis of PG in fibroblasts. In the

presentt study we show that vanous cardiolipin molecular species are deficient in fibroblasts

off BTHS patients. Because cardiolipin is normally synthesized in BTHS cells, as previously

shownn (3), this deficiency is most probably the result of its deficient fatty acid remodeling.

Inn mammalian tissues the biosynthesis of cardiolipin occurs via the cytidinediphosphate-

diacylglyceroll (CDP-DG) pathway (6). Newly formed CL undergoes extensive remodeling by

deacylationn and reacylation in order to produce the specific C18:2-C18:2 and C18:l-C18:2

diacyll combinations which are observed in mammalian CL (6-10).

Thesee studies indicate that tetralineoyl-cardiolipin is the most important cardiolipin

species.. Most of the cardiolipin is found in the inner mitochondrial membrane. Cardiolipin

74 4

Cardiolipinn deficiency in X-linked cardioskeletal myopathy and neutropenia

iss needed for proper functioning of respiratory chain components, especially cytochrome C.

Losss of cardiolipin from the inner mitochondrial membrane causes loss of cytochrome C into

thee cytosol and represents a crucial step in cellular apoptosis (11). Variable involvement of

severall respiratory chain complexes has been reported in some publications dealing with

mitochondriall function in BTHS (1).

Thee significance of CL for respiratory chain functioning has been shown previously

inn yeast cells deficient in cardiolipin (12-13) and Chinese hamster ovary (CHO) cells gene-

ticallyy deficient in cardiolipin (14). The precise role of cardiolipin in the functioning of the

innerr mitochondrial membrane is yet to be determined. Other, less well known roles for

cardiolipinn are suggested by papers reporting the presence of free cardiolipin in plasma

(15),, surface expression on apoptotic cells (16) and a bound form occurring together with

chromatinn (17).

Wee observed differences within the patients' group with respect to CL levels. Patient

44 has the lowest CL level (total CL level: 0.20 nmol/mg protein) while patient 1 has the

highestt total CL level of 0.53 nmol/mg protein. Patient 1 has a mutation in exon 2 resulting

inn a premature stopcodon. This mutation causes loss of the first of the two ATG-sites from

whichh 5 '-translation starts which causes loss of the membrane anchoring (N-terminal) end

off tafazzin protein, but still allows gene translation starting from the second ATG- site

onn exon 3. Patient 4 has a missense mutation in exon 8, leading to structural changes in

alll 10 tafazzin proteins, that can be predicted on the basis of the gene structure.

Thee clinical involvement of patients 1 and 4 is approximately of the same degree. It is

thereforee not possible to relate absolute levels of CL in fibroblasts to specific TAZ mutations

orr to severity of clinical involvement. We did not quantify the PG molecular species. We ob-

servedd only some minor changes in fatty acid composition of PG molecular species in one of

BTHSS patients. The relative levels of PG molecularr species containing linoleic acid compared

too PG molecular species containing other fatty acids were very low while CL pool has a high

contentt of linoleic acid. These data indicate that the newly formed CL from PG has to be

remodeledd to form CL molecular species with high content of linoleic acid.

Wee conclude that analysis of cardiolipin in cultured skin fibroblasts by HPLC-ESI-MS

iss a fast and simple method to identify patients with BTHS which is less laborious and avoids

thee use of radioactively labeled chemicals. However, there is no evidence yet that the de-

creasedd level of CL is a primary effect of deficiency of one or more tafazzin proteins. Thus at

thiss time a gene mutation analysis has to be performed to complete the diagnosis. Compared

too our earlier published method (3), the method presented here gives more information

aboutt the compounds involved.

75 5

C h a p t err 5

A C K N O W L E D G M E N T SS The Prinses Beatrix Fonds financed this study, grant

95-1004.. P Mooyer and P Valtman for cell culturing.

R E F E R E N C E S S

1.. Banh PG, Wanders RJ, Vreken P X-hnked cardioskeletal myopathy and neutropenia (Barth syndrome)-

MIMM 302060 J Pediatr 1999, 135: 273-276.

2.. Neuwald AF Barth syndrome may be due to an acyltransferase deficiency. Curr Biol 1997, 7: R465-R466.

3.. Vreken P, Valianpour F, Nijtmans LG, Grivell LA, Plecko B, Wanders RJ et al. Defective remodeling of car-

diolipinn and phosphatidylglycerol in Barth syndrome. Biochem Biophys Res Commun 2000; 279(2):378-382.

4.. Bleyl SB, Mumford BR, Thompson V, Carey JC, Pysher TJ, Chin TK, Ward K. Neonatal, lethal noncom-

pactionn of the left ventricular myocardium is allelic with Banh Syndrome. Am J Hum Genet 1997, 61: 868-72.

5.. Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can Journal Biochem Physiol 200;

37:: 911-917

6.. Schlame M, Rua D, Greenberg ML. The biosynthesis and functional role of cardiolipm. Prog Lipid Res 2000;

39:: 257-288.

7.. Ma BJ, Taylor WA, Dohnsky VW, Hatch GM. Acylation of monolysocardiolipin in rat heart. J Lipid Res

1999;40:1837-1845. .

8.. Hatch GM. Cardiolipin: biosynthesis, remodeling and trafficking in the heart and mammalian cells (Review).

Intt J Mol Med 1998; 1:33-41.

9.. Schlame M, Brody S, Hosietler KY. Mitochondrial cardiolipin in diverse eukaryotes. Comparison of bio-

syntheticc reactions and molecular acyl species. Eur J Biochem 1993; 212:727-735.

10.. Schlame M, Rustow B. Lysocardiolipin formation and reacylation in isolated rat liver mitochondria.

Biochemm J 1990; 272:589-595.

11.. Ott M, Robertson JD, Gogvadze V, Zhivotovsky B, Orrenius S. Cytochrome c release form mitochondria

proceedss by a two-step process. Proc Nat Acad Science USA 2002; 99: 1259-1263.

12.. Koshkin V, Greenberg ML. Oxidative phosphorylation in cardiolipm-lacking yeast mitochondria.

Biochemm J 2000; 347 Pt 3:687-691.

13.. Ostrander DB, Zhang M, Mileykovskaya E, Rho M, Dowhan W Lack of mitochondrial anionic phospho-

lipidss causes an inhibition of translation of protein components of the electron transport chain. A yeast genetic

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14.. Rusnak A, Mangat R, Xu F, McClarty G, Hatch GM Cardiolipin remodeling in a Chinese hamster lung

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15.. Deguchi H, Fernandez JA, Hackeng TM, Banka CL, Griffin JH. Cardiolipin is a normal component of

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16.. Sorice M, Qrcella A, Misasi R, Pittoni V, Garofalo T, Cirelli A et al. Cardiolipin on the surface of apoptotic

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