June 2019

Medical Mass Spectrometry Vol. 3 No. 1

Short Communication

2-Methylacetoacetylcarnitine in blood of beta-ketothiolase deﬁciency
and HSD10 disease
111

2

3Yosuke Shigematsu *, Miori Yuasa , Ikue Hata , Hideki Nakajima , Go Tajima ,4

5

6Nobuyuki Ishige , Toshiyuki Fukao , Yasuhiro Maeda
1

Department of Pediatrics, University of Fukui,23‒3 Matsuoka-shimoaizuki, Eiheiji-cho, Yoshida-gun, Fukui 910‒1193, Japan
2
Mass Screening Laboratory, National Research Institute for Child Health and Development,2‒10‒1 Okura, Setagaya-ku, Tokyo 157‒8535, Japan3
Department of Pediatrics, Hiroshima University Graduate School of Biomedical Sciences,1‒2‒3 Kasumi, Minami-ku, Hiroshima-shi, Hiroshima 734‒8553, Japan
4Tokyo Health Service Association, 1‒2 Ichigaya Sadohara-cho, Shinjuku-ku, Tokyo 162‒8402, Japan
5
Department of Pediatrics, Graduate School of Medicine, Gifu University,
1‒1 Yanagito, Gifu 501‒1194, Japan
6
Department of Hospital Pharmacy, Graduate School of Pharmaceutical Sciences, Nagoya City University,
1 Aza-Kawasumi, Mizuhocho, Mizuho-ku, Nagoya-shi, Aichi 467‒8601, Japan
Abstract

Together with 2-methyl-3-hydroxybutyrylcarnitine, 2-methylacetoacetylcarnitine has been newly identiﬁed insigniﬁcantly increased amounts in serum and dried blood spot of patients with β -ketothiolase deﬁciency. In patients withHSD10 disease, however, the amounts of 2-methylacetoacetylcarnitine were considerably low as compared with those inpatients with β -ketothiolase deﬁciency, and the decreased ratio of 2-methylacetoacetylcarnitine to 2-methyl-3-hydroxybutyrylcarnitine in dried blood spot could be an additional index to discriminate HSD10 disease from β -ketothiolase deﬁciency.
Key words: beta-ketothiolase deﬁciency, HSD10 disease, hydroxy-pentanoylcarnitine, organic aciduria

Introduction

organic acid analysis typically shows elevated excretion of
2-methyl-3-hydroxybutyrate (MHBA) and tiglylglycine in

Mitochondrial 2-methylacetoacetyl-CoA thiolase deﬁciency, or β -ketothiolase deﬁciency (KTD), is a rare autoso-

both disorders, although increased 2-methylacetoacetate2)

mal recessive disorder of isoleucine and ketone bodymetabolism. HSD10 disease is a rare X-chromosomal dis-(MAAA) has been detected only in ΚΤ D . However, dueease caused by a moonlighting protein encoded by theHSD17B10 gene, and this protein catalyzes the 2-methyl-abnormalities of these disorders, the chemical diagnosis
seems problematic in not a few patients with these disor-

3-hydroxybutyryl-CoA dehydrogenation (MHBD) reaction

ders, and characteristic metabolites in urine were reportedly

to broad spectrum in clinical pictures and biochemical1)

in isoleucine metabolism . Chemical diagnosis for thesedisorders is based on the elevated concentrations of metab-missing or obscure in the patients with a milder form of ΚΤ2‒7)

D or those with atypical forms of HSD10 disease . On
the other hand, elevated hydroxy-pentanoylcarnitines (C5-olites originating from isoleucine breakdown, and urinaryOH) and tiglylcarnitine (C5:1) in dried blood spots were

* Corresponding author

observed in tandem mass spectrometric screening for8)
KTD , and 2-methyl-3-hydroxybutyrylcarnitine (MHBC) inYosuke Shigematsu
Tel.: ＋81‒776‒61‒3111, Fax: ＋81‒776‒61‒8129

serum of KTD patients has been reportd in liquid-chromatography tandem mass spectrometric (LC-MS/MS) analy-

E-mail address: yosuke@u-fukui.ac.jp
Received August 23, 2018.

Accepted October 17, 2018.

Epub December 21, 2018.

9)
sis . In the present study, we have investigated 2-methylac-

DOI: 10.24508/mms.2019.06.002

etoacetylcarnitine (MAAC) and the other metabolites related43June 2019

Medical Mass Spectrometry Vol. 3 No. 1

to these disorders in blood by LC-MS/MS in order to clar-

The mixture of 5μL of serum specimen and 167μL ofify how useful these metabolites are as diagnostic markers.

NeoSMMAT kit solution was centrifuged at 10,000 rpm,
and the supernatant was collected. The samples (10μL)

Materials and Methods

were introduced into the LC mobile phase ﬂow of 0.4 mL/min using 150 mm×3.0 mm Scherzo SS-C18 column

Biological samples
Three patients with KTD and two with HSD 10 disease(Imtakt, Portland, USA). Gradient elution of the analyte
was achieved using a program with mobile phase A (aque-were included in the present study. Patient A with KTD had
increased levels of MHBA, TG and MAAA in urine, con-ous 0.5% formic acid) and mobile phase B ((0.5 M ammonium formate/0.5 M NH4OH＝9:1)/methanol＝1 : 9) as fol-stantly: serum specimen and dried blood spot (DBS) werecollected in stable condition at the age of 19 years. Patient
B with KTD had also increased levels of the three metabo-

lows: 10% B for 1 min, 10% B to 40% B in 4 min, 40% B

lites mentioned above in urine during a ketoacidotic epi-

additional 7 min at 100% B, then back to 10% B in 0.1 min
and re-equilibration for 5 min.

to 70% B in 8 min, 70% B to 100% B in 0.1 min plus ansode at the age of 12 months: DBS was collected during

For the measurement of short-chain acylcarnitines inDBS and serum specimen by electrospray-ionization

this episode. Patient C with KTD had very mildly increased
excretion of these metabolites during his ﬁrst ketoacidotic
episode at the age of 18 months: serum specimen was col-LC-MS/MS, a model API 4000 triple-stage mass spectrometer (AB Sciex, Tokyo, Japan) equipped with a model
LC10Avp HPLC system and a model SIL-20AC auto-injec-lected during this episode. Patient D with HSD10 disease
had typical clinical phenotype and increased levels of10)

was collected for newborn screening at the age of 5 days

tor (Shimadzu, Kyoto, Japan) was used . The positive ion
MS/MS analysis was performed in multiple-reaction moni-and was stored in freezer before the present analysis.
3)
Patient E with HSD10 disease had atypical clinical pheno-

toring (MRM) mode with the following transitions; m/z
2260＞85 for MAAC, m/z 271＞85 for [ H9]3-hydroxyisova-type and had increased excretion of MHBA and TG during

lerylcarnitine, m/z 251＞85 for [ H9]isovalerylcarnitine,

a ketoacidotic event: the serum specimen was collectedduring this event. The diagnosis for these patients was con-and m/z 244＞85 for tiglylcarnitine. Suitable measurementﬁrmed by mutation analysis.

with the automatic tune function of the Analyst software. Inaddition, the positive ion MS/MS was performed in prod-Chemicals
NeoSMAAT kit for MS/MS newborn screening, which

uct-ion scan mode, in order to get the product ion spectra of
any peak using variable collision-energy settings. The con-contains labeled acylcarnitines, including [ H9]3-hydroxy2
isovalerylcarnitine and [ H9]isovalerylcarnitine, was pur-dition for the transition m/z 262＞103 for MHBC wasdetermined based on the data of product-ion scan measure-

MHBA and TG in urine during ketoacidotic events: DBS

2

conditions for the designated transitions were identiﬁed

2chased from Sekisui Medical CO. (Tokyo, Japan). 2-Methy-

ment of MHBC peak in DBS of patient A.

lacetoacetylcarnitine was synthesized by mixing carnitinehydrochloride and 2-methylacetoacetyl chloride in triﬂuoro-injection. For quantiﬁcation, the recorded peak areas of theThe data were recorded for 13 min after every sample

acetic acid, which was formed from ethyl 2-methylacetoacetate (Sigma-Aldrich CO., Tokyo, Japan). Tiglylcarnitine

designated MRM ion set were used.

was synthesized from carnitine hydrochloride and tiglyl

Results
In Fig. 1, MRM chromatogram for serum acylcarnitines

chloride (Tokyo Chemical Industry Co., Tokyo, Japan).

of a patient with KTD was shown. Three peaks of MHBC,
2a single peak of MAAC, and a large peak of [ H9]isova-Methods
One punch (1/8 inch of diameter) of DBS was extractedlerylcarnitine, were observed from 7.8 to 8.8 min of reten-with 100μL of methanol solution of NeoSMAAT kit (0.07522nmol/ml [ H9]3-hydroxyisovalerylcarnitine and [ H9]isova-tion time. As shown in Fig. 2, product ion mass spectra forlerylcarnitine) of by the routine manner for newborn

Since MHBC could not be quantitated separately from
3-hydroxyisovalerylcarnitine (HIVC), using transition of m/8)

3 peaks of MHBC agreed with the reported mass spectra .

screening, and the extract was dried under the nitrogenstream and was re-dissolved in 2% formic acid solution.

z 262＞85, as shown in the lowest column of Fig. 3, the44June 2019

Fig. 1.

Medical Mass Spectrometry Vol. 3 No. 1

MRM chromatogram for serum acylcarnitines of apatient with β -ketothiolase deﬁciency.

Fig. 3.

MRM chromatograms using transition of m/z262＞103 (1), m/z 262＞145 (2), and m/z 262/85 (3).

Fig. 4.

Two peaks in MRM chromatogram using transition of m/z 260＞85 and product ion mass spectrain the measurment of 2-methylacetoacetylcarnitine.

Every MRM transition is shown in parenthesis: see text.

Fig. 2.

Three peaks in MRM chromatogram using transition of m/z 262＞103 and product ion mass spectrain the measurement of 2-methyl-3-hydroxybutyrylcarnitine.

(1) MRM chromatogram using transition of m/z 260＞
85, (2) Product ion mass spectra of precursor ion (m/z260) for two peaks, (3) supposed fragment ions from＋
[M＋H] of 2-methylacetoacetylcarnitine.

(1) MRM chromatogram using transition of m/z 262＞
103, (2) Product ion mass spectra of precursor ion (m/z 262) for three peaks, (3) supposed fragment ions from＋
[M＋H] of 2-methyl-3-hydroxybutyrylcarnitine.

45June 2019

Medical Mass Spectrometry Vol. 3 No. 1

Table 1.

Characteristic acylcarnitines in HSD10 disease and β-ketothiolase deﬁciency
Tiglylcarnitine

MHBC

MAAC

MAAC/MHBC

Pt-A: KTD (serum)
Pt-A: KTD (DBS)

0.874

0.634

0.1471

0.232

0.615

3.610

0.05900.016

Pt-B: KTD (DBS)
Pt-C: KTD (serum)

0.485

4.844

0.1360

0.028

0.036

0.048

0.0014

0.029

Pt-D: HSD10 (DBS)
Pt-E: HSD10 (serum)

0.247

0.550

0.0005

0.001

0.440

0.135

0.0016

0.012

Controls DBS (n＝15)

0.003±0.001

0.014±0.004

mean±SD (range)(0.002‒0.004)(0.008‒0.018)(＜0.0004)

Unit of acylcarnitines is nmol/mL.
22The internal standards were [ H9] isovalerylcarnitine for tiglylcarnitine and [ H9]3-hydroxyisovalerylcarnitine for 2-methyl-3-hydroxybutyrylcarnitine and 2-methylacetoacetylcarnitine, respectively; see text for details.
MHBC; 2-methyl-3-hydroxybutyrylcarnitine, MAAC; 2-methylacetoacetylcarnitine,KTD; β-ketothiolase deﬁciency, DBS; dried blood spot

lacetoacetyl-CoA, which is a substrate of mitochondrial
β -ketothiolase. Therefore, it is speculated that MAAC cantransition of m/z 262＞103 for MHBC was used, and thesum of the areas of three peaks was used for calculation.
measured by the transition m/z 262＞103 were compared tobe produced from accumulated 2-methylacetoacetyl-CoA11)
by carnitine acetyltransferase . In our experience, how-

those by m/z 262＞85 in DBS of patient B, who had aever, MAAA seems unstable in body ﬂuids, and sometime

markedly large amount of MHBC, but a very low amount

could not be detected in the urine of the patients with KTD,
especially after long-time storage. Since MAAA was notBecause authentic MHBC was not available, the peak areasof HIVC; the mean ratio of the former areas to the latter

commercially available unfortunately, the stability of
MAAA could not be tested and LC-MS/MS measurement

were 0.32 (n＝7).
As shown in Fig. 4, the product ion mass spectrum forof MAAA has not been performed, yet. Although MAAC
may not be stable enough, it could be detected in consider-Peak D was identical to that of synthesized MAAC. Theproduct ion mass spectrum for Peak E, which was observed

able amounts in blood samples of the patients with KTD,
which were stored in freezer for up to 3 years. Blood sam-constantly in both of control subjects and the patients, wassimilar to that of MAAC.
The concentrations of MHBC and MAAC, which were2
calculated using [ H9]3-hydroxyisovalerylcarnitine as anple, instead of urine, was suggested to be a useful substituteinternal standard, and tiglylcarnitine, which were calculated

Increased MHBC, which was assumed to be produced
from accumulated 2-methyl-3-hydroxybutyryl-CoA, hasfor chemical diagnosis of KTD.

2using [ H9]isovalerylcarnitine, were shown in Table 1. HSD8)

been reported in patients with KTD . In the reported
LC-MS/MS measurement, there were three isomer peaks of

10 patients had large amounts of tiglylcarnitine and MHBC,
but very low amounts of MAAC, while KTD patients hadMHBC, which possesses two chiral carbons and exists infour isomers. It is thought that the second peak of 2-meth-large amounts of tiglylcarnitine, MHBC together withincreased amounts of MAAC, which were more clearlypatient 3 had higher concentrations of MHBC in DBS thanyl-3-hydroxybutyrylcarnitine in Fig. 2 may contain two of
the four isomers and three peaks of 2-methyl-3-hydroxybu-in serum, while those of MAAC in DBS were lower than intyrylcarnitine appeared.

shown by the ratio of MAAC/MHBC. In addition, KTD

On the other hand, MAAC has no chiral carbon andserum.

showed a single peak in our LC-MS/MS measurements.

Discussion

Interestingly, one peak with a product ion mass spectrum

In the present study, accumulated MAAC has been ﬁrst

similar to that of MAAC was observed close to the peak ofMAAC. While it appeared in small amounts even in con-identiﬁed in blood of KTD patients. Increased MAAA in
urine of KTD patients suggested the accumulated 2-methy-trols and the ingredient of the peak has not been clariﬁed, it46

June 2019

Medical Mass Spectrometry Vol. 3 No. 1

should not be counted as MAAC, accidentally. In ouratypical presentation. J Hum Genet 59: 609‒614, 2014.

MRM measurements, in addition, very small amounts of4) Fukao T, Maruyama S, Ohura T, Hasegawa Y, Toyoshima

MAAC were quantitated even in patients with HSD10, andM, et al: Three Japanese patients with beta-ketothiolase

the peak of MAAC may contain some unknown substancedeﬁciency who share a mutation, c.431A＞C (H144P) in

which could inﬂuence on the quantitation of MAAC in very

ACAT1: subtle abnormality in urinary organic acid analy-low concentration range, using the MRM transition m/z 260

sis and blood acylcarnitine analysis using tandem mass＞85.

spectrometry. JIMD Reports 3: 107‒115, 2012.
9)

In the report mentioned above , MAAC was not5) Fukao T, Scriver CR, Kondo N, the T2 Collaborativeobserved. It may be partly because the concentrations ofWorking Group: The clinical phenotype and outcome of

MAAC might not be high enough, or simply because theMRM measurement to detect MAAC was not be per-mitochondrial acetoacetyl-CoA thiolase deﬁciency ( β -ke-formed.

and mutation-deﬁned patients. Mol Genet Metab 72: 109‒

tothiolase or T2 deﬁciency) in 26 enzymatically proved

In the present study, the accumulation of MHBC was

114, 2001.

more evident in DBS, as compared with that of tiglylcarni-6) Sarafoglou K, Matern D, Redlinger-Grosse K, Bentler K,tine, than in serum. The dominant accumulation of
3-hydroxyisovalerylcarnitine in DBS of the infants withGaviglio A, et al: Siblings with mitochondrial acetoacetyl-CoA thiolase deﬁciency not identiﬁed by newborn
screening. Pediatrics 128: e246‒e250, 2011.

12)

biotin deﬁciency has been reported , and MHBC in DBS
could be a better marker for KTD and HSD10 than that in7) Akagawa S, Fukao T, Akagawa Y, Sasai H, Kohdera U, etserum. As shown in KTD patient C, MAAC was rather low

al: Japanese male siblings with 2-methyl-3-hydroxybutyr-in KTD patient with very mildly increased amounts ofyl-CoA dehydrogenase deﬁciency (HSD10 Disease) with-

tiglylcarnitine and MHBC, while the ratio of MAAC toMHBC seems to be a better marker than MAAC concentra-

out neurological regression. JIMD Rep 32: 81‒85, 2017.
8) Han LS, Ye JW, Qiu WJ, Gao XL, Wang Y, et al: Selec-tion itself to discriminate KTD from HSD10.

tive screening for inborn errors of metabolism on clinicalpatients using tandem mass spectrometry in China: A

Acknowledgement

four-year report. J Inherit Metab Dis 30: 507‒514, 2007.

This study was supported in part by a Grant-in-Aid for9) Penga M, Fangb X, Huanga Y, Caia Y, Lianga C, et al:Young Scientist (B), Japan Society for the Promotion ofSeparation and identiﬁcation of underivatized plasma

Science.

acylcarnitine isomers using liquid chromatography‒tandem mass spectrometry for the differential diagnosis ofConﬂict of Interest

organic acidemias and fatty acid oxidation defects. JChromatogr A, 1319: 97‒106, 2013.

The authors declare that they have no competing interests.

10) Shigematsu Y, Hata I, Tajima G: Useful second-tier tests

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