Download Serum carnitine concentrations in patients with

Clinical Science (1999) 97, 493–501 (Printed in Great Britain)
Serum carnitine concentrations in patients with
idiopathic hypertrophic cardiomyopathy:
relationship with impaired myocardial
fatty acid metabolism
Tomoki NAKAMURA*, Hiroki SUGIHARA†, Noriyuki KINOSHITA*, Kazuki ITO*,
Yoshihiko ADACHI*, Satoshi HIRASAKI*, Akiko MATSUO*, Akihiro AZUMA*,
Naoki KODO‡ and Masao NAKAGAWA*
*Second Department of Medicine, Kyoto Prefectural University of Medicine, Kyoto 602-0841, Japan, †Department of Radiology,
Kyoto Prefectural University of Medicine, Kyoto 602-0841, Japan, and ‡Department of Pediatrics, Kyoto Prefectural University
of Medicine, Kyoto 602-0841, Japan
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We evaluated the clinical significance of serum carnitine concentrations in determining the
severity of impaired myocardial fatty acid metabolism in idiopathic hypertrophic cardiomyopathy (HCM). We studied 56 asymptomatic or mildly symptomatic patients with HCM.
Serum levels of free carnitine and acylcarnitine were measured by the enzymic cycling method.
Myocardial scintigraphy with 123I-labelled 15-(p-iodophenyl)-3-R,S-methylpentadecanoic acid
(BMIPP) was performed, and the images were analysed quantitatively and semi-quantitatively.
Serum free carnitine levels were significantly higher in HCM patients than in normal subjects
(52.5p9.5 and 42.3p5.5 nmol/ml respectively ; P 0.0001). On the other hand, serum
acylcarnitine levels and acyl/free carnitine ratios were lower in HCM patients than in normal
subjects (10.2p4.0 nmol/ml and 0.19p0.08, compared with 13.2p3.9 nmol/ml and 0.32p0.11
respectively ; P 0.0001). Clinical characteristics were not significantly different between the
patients showing high and normal free carnitine levels, although female patients with high free
carnitine levels were few (P l 0.02). Both quantitative and semi-quantitative analyses revealed
that the severity of decreased myocardial BMIPP uptake was significantly correlated with serum
free carnitine levels (quantitative analysis : r l k0.422, P 0.0012 ; semi-quantitative analysis :
r l 0.633, P 0.0001). In the presence of reduced carnitine uptake into the myocardium in HCM,
there may also be reduced transport of acylcarnitines out of the myocardium into the plasma.
Although inborn errors of fatty acid metabolism and carnitine deficiencies are reported to
provoke secondary HCM and are associated with low serum carnitine concentrations, this study
has revealed that the levels of carnitine are, in contrast, increased in idiopathic HCM. Moreover,
serum carnitine concentrations are a sensitive indicator of the severity of impaired myocardial
fatty acid metabolism even in asymptomatic patients with HCM.
Key words : carnitine, fatty acid metabolism, hypertrophic cardiomyopathy, "#$I-15-(p-iodophenyl)-3-R,S-methylpentadecanoic
acid (BMIPP).
Abbreviations : APH, apical hypertrophic cardiomyopathy ; BMIPP, "#$I-labelled 15-(p-iodophenyl)-3-R,S-methylpentadecanoic
acid ; HCM, hypertrophic cardiomyopathy ; HNCM, hypertrophic non-obstructive cardiomyopathy ; HOCM, hypertrophic
obstructive cardiomyopathy.
Correspondence : Dr Tomoki Nakamura (e-mail tomoki-nakamura!mail.goo.ne.jp).
# 1999 The Biochemical Society and the Medical Research Society
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INTRODUCTION
MATERIALS AND METHODS
Approximately 70 % of myocardial energy requirements
are derived from the β-oxidation of non-esterified fatty
acids at rest under normal aerobic conditions, while
carbohydrates provide the majority of the remaining
30 % of energy production [1,2]. Long-chain fatty acids
are the major substrate for β-oxidation in the myocardium. Non-esterified fatty acids bind to albumin in
the bloodstream and are transported to the myocyte via
long-chain-fatty-acid-binding protein located in the
plasma membrane. Once fatty acids are taken up into the
cytoplasm, they bind to heart-type fatty-acid-binding
proteins and are activated by long-chain acyl-CoA
synthetase [3]. Acyl-CoA is then transferred into the mitochondria via carnitine, carnitine palmitoyltransferase I,
carnitine :acylcarnitine translocase, and carnitine palmitoyltransferase II. In the mitochondria, long-chain acylCoA is metabolized by the β-oxidation enzyme system.
Carnitine is thus an essential substance that transports
long-chain fatty acids into mitochondria, where βoxidation takes place. Carnitine also modulates the intramitochondrial CoA\acyl-CoA ratio. In the absence of
carnitine, β-oxidation ceases, glycogen is depleted, triacylglycerols accumulate, and organ dysfunction results [4].
On the other hand, medium- and short-chain fatty acids
are transported directly into mitochondria without any
association with carnitine. Medium- and short-chain
acyl-CoA synthetases are localized in the mitochondrial
matrix. This explains why the oxidation of medium- and
short-chain fatty acids is carnitine-independent [5]. The
serum level of free carnitine is fairly constant under a
variety of conditions [6]. Fatty acid metabolism is thus a
significant factor in the pathophysiology of heart diseases.
Single-photon emission computed tomography with
"#$I-labelled 15-(p-iodophenyl)-3-R,S-methylpentadecanoic acid (BMIPP) is an important tool for exploring
myocardial fatty acid metabolism. Application of this
method in patients with hypertrophic cardiomyopathy
(HCM) has revealed reduced BMIPP uptake, chiefly in
the hypertrophied myocardium, which is independent of
myocardial perfusion [7]. Moreover, a decrease in myocardial BMIPP uptake is considered to be the most
sensitive indicator of metabolic abnormalities in HCM,
and the degree of decreased BMIPP uptake relates to
mortality [8,9].
The plasma free carnitine concentration is reported to
be elevated in patients with congestive heart failure and
some kinds of cardiomyopathies, and is associated with
poor prognosis [10,11], but this has not been fully
investigated in idiopathic HCM. Therefore the aim of our
study is to assess the clinical significance of serum
carnitine levels and to elucidate the relationship between
serum carnitine concentration and impairment of myocardial fatty acid metabolism in asymptomatic or mildly
symptomatic patients with HCM.
Subjects
# 1999 The Biochemical Society and the Medical Research Society
We examined 56 patients with idiopathic HCM (42 men,
14 women ; mean age 56p12 years, range 17–73 years).
HCM subgroups were classified as follows : 36 patients
with hypertrophic non-obstructive cardiomyopathy
(HNCM), seven with hypertrophic obstructive cardiomyopathy (HOCM), and 13 with apical hypertrophic
cardiomyopathy (APH). Twelve patients had a family
history of HCM. The diagnosis of HCM was based on
clinical findings, i.e. echocardiographic demonstration of
a hypertrophied, non-dilated left ventricle in the absence
of other cardiac or systemic diseases known to cause left
ventricular hypertrophy [12,13]. For our study, only
asymptomatic or mildly symptomatic patients (40
patients in New York Heart Association functional class
I and 16 in class II) were selected. Patients who had
coronary artery disease, congestive heart failure, hypertension, diabetes mellitus or hepatorenal dysfunctions
were excluded from the study, because such conditions
affect myocardial metabolism and the kinetics of BMIPP,
and carnitine is mainly synthesized in the liver and
excreted in the urine [10,14–17].
The age-matched control group consisted of 80 normal
healthy volunteers (40 men and 40 women ; mean age
54p13 years) who were receiving no medication. All
patients and volunteers signed informed consent forms
before the study, and the study protocol was approved
by the Research Council of our institution.
Determination of serum carnitine
concentrations
A 10-ml blood sample was drawn from the antecubital
vein of each patient at rest under fasting conditions.
Blood samples were centrifuged at 3000 rev.\min at 3 mC
for 10 min, and serum samples (4 ml) were stored at
k70 mC until assayed. The serum concentration of free
carnitine and acylcarnitine was measured by the new
enzymic cycling method using NADH, thio-NAD+ and
carnitine dehydrogenase, as this is a highly sensitive and
specific procedure [18,19]. Enzymes and assay kits for
the measurement of total and free carnitine levels were
purchased from Kainos Laboratories Co. (Tokyo, Japan).
Incubations were carried out at 37 mC for 3 min in a
10 mm pathlength cuvette, and contained 1 ml of
100 mmol\l Tris\HCl buffer (pH 9.5), 5 mmol\l thioNAD+, 0.2 mmol\l NADH and 100 k-units\l carnitine
dehydrogenase (where 1 unit is defined as the amount of
enzyme producing 1 µmol of NADH\min at 37 mC). The
rate of thio-NADH production, which is proportional to
the amount of L-carnitine, was measured by determining
absorbance at 415 nm between 1 and 6 min after the
addition of 50 µl of serum specimen or L-carnitine
standard solution.
Carnitine in hypertrophic cardiomyopathy
For the determination of total carnitine, 1 k-unit\l
acylcarnitine hydrolase was added to the above reagent,
as acylcarnitine is hydrolysed to L-carnitine by this
enzyme. The acylcarnitine level was calculated as the
difference between total carnitine and free L-carnitine
concentrations. The concentration of L-carnitine was
calculated by comparison with the rate obtained
using a 50 µmol\l L-carnitine standard solution. We
used a Model UV-250 spectrometer (Shimadzu Seisakusho Co., Kyoto, Japan) and a Cobas-Fara analyser
(Roche Co., Nutley, NJ, U.S.A.).
Determination of serum non-esterified fatty
acid concentrations
Serum non-esterified fatty acids were analysed by a highperformance chemiluminescent detection method [20].
Serum samples (25 µl) were incubated at 37 mC for 10 min
with 25 µl of KH PO buffer (50 mmol\l, pH 7.2)
# %
containing 5i10% units of bilirubin oxidase and 20 g\l
sodium cholate. Then we added 1 ml of KH PO buffer
# %
(50 mmol\l, pH 7.0) containing 400 units of acyl-CoA
synthetase, 0.3 mmol of CoA, 1.5 mmol of ATP-Na ,
#
1 mmol of MgCl and 5 g of Triton X-100, and the
#
samples were incubated at 37 mC for 5 min. After the
second incubation, we added 2 ml of KH PO buffer
# %
(10 mmol\l, pH 6.8) containing 7i10$ units of acylCoA oxidase and 1.2 mmol\l N-ethylmaleimide, followed by incubation at 37 mC for 1 min. This reaction
mixture (100 µl) was used for the chemiluminescent
measurement, mixed with 1.5 ml of NaHCO buffer
$
(0.4 mol\l, pH 9.5) containing 0.12 mmol of isoluminol
and 0.5 µmol of microperoxidase. Then we measured the
intensity of light emitted for 1 min using a Luminometer
UPD-8000 (Meidensha Electric Mfg. Co., Tokyo, Japan).
BMIPP scintigraphy
A dose of 111 MBq of BMIPP (Nihon Medi-Physics Co.,
Nishinomiya, Japan) was administered intravenously at
rest under fasting conditions. Data acquisition for singlephoton emission computed tomography was performed
15 min after injection using a rotating digital scintillation
camera (Gamma-camera 901A ; Toshiba Co., Tokyo,
Japan) equipped with a collimator. A total of 30 projection images (40 s per frame) were acquired over 180 m
from the left posterior oblique 45 m to the right anterior
oblique 45 m. The images were reconstructed using a
Shepp–Logan filter without correction for attenuation.
BMIPP scintigraphy was also performed in 20 people (11
men and 9 women) out of 80 normal healthy volunteers.
BMIPP imaging of normal subjects and HCM patients is
shown in Figure 1.
Figure 1 BMIPP imaging of normal subjects and HCM
patients
SA, short axis ; VLA, vertical long axis. Decreased myocardial uptake is not seen in
any segment of the myocardium in normal subjects. However, myocardial BMIPP
uptake is prominent in the anterior and posterior junctions of the hypertrophied
ventricular septum in HNCM and HOCM patients, and is marked in the left
ventricular apex in APH patients.
titative analysis, we calculated the heart\mediastinum
uptake ratio from the average scintillation counts.
Regions of interest were assigned in the heart and
mediastinum on the anterior planar image, and the
average scintillation counts in each segment were calculated. For semi-quantitative analysis, the left-ventricular
tomograms were divided into 17 segments (Figure 2). The
short-axis slices were separated into eight segments at the
basal and midventricular levels. Vertical long-axis slices
were used to evaluate the apical portion of one segment.
Each segment was graded visually using a score between
0 and 3 (0, normal ; 1, mildly reduced uptake ; 2,
moderately reduced uptake ; 3, markedly reduced
uptake\absent activity) in a blinded manner by two
experienced nuclear cardiologists. Differences of opinion
were resolved by consensus. The sum of each score was
defined as the total defect score, reflecting the severity of
impaired myocardial fatty acid metabolism.
Echocardiographic study
Analysis of BMIPP imaging
BMIPP imaging was analysed quantitatively and semiquantitatively as reported previously [21]. For quan-
The echocardiographic study consisted of M-mode, twodimensional and Doppler blood flow measurements with
a Hewlett-Packard ultrasound system (Sonos 2500). The
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T. Nakamura and others
Figure 2
Semi-quantitative analysis of BMIPP imaging
BMIPP tomograms were divided into 17 segments : 1 and 9, anteroseptal ; 2 and 10, superoseptal ; 3 and 11, inferoseptal ; 4 and 12, septoinferior ; 5 and 13, lateroinferior ;
6 and 14, inferolateral ; 7 and 15, superolateral ; 8 and 16, anterolateral ; 17, apical.
distributions of hypertrophy, wall thickness, cavity size
and wall motion were evaluated from multiple windows
[22]. The morphology of the left-ventricular apex was
assessed from the apical view for the diagnosis of APH.
We also determined the presence or absence of complete
end-systolic cavity obliteration at the papillary muscle
level in the short-axis view for the diagnosis of HOCM.
Cardiac catheterization
Cardiac catheterization was performed in all the HCM
patients by the percutaneous Seldinger technique. Baseline ventricular pressure was evaluated using an end-hole
water-filled catheter before cine-angiography. A diagnosis of HOCM was established when a patient had a
pressure gradient above 20 mmHg in the left-ventricular
outflow tract without provocation. Left ventriculography was then performed in the right anterior oblique
projection. Selective coronary angiograms were obtained
in multiple projections.
Statistical analysis
Data are expressed as meanspS.D. An unpaired t test or
Chi-square test for independence was used to analyse
differences between variables. One-way analysis of variance with subsequent Scheffe! ’s multiple-range tests was
used to compare the data among the three subtypes of
HCM. Correlations between continuous variable data
were determined by linear-regression analysis. Statistical
significance was defined as P 0.05.
# 1999 The Biochemical Society and the Medical Research Society
RESULTS
Clinical data for HCM patients are summarized in
Table 1.
Serum levels of free carnitine and
acylcarnitine, and of non-esterified fatty
acids, in patients with HCM
Table 2 shows the serum concentrations of free carnitine
and acylcarnitine and of non-esterified fatty acids in
normal subjects and in patients with HCM. Free carnitine
levels in HCM patients significantly exceeded those in
normal subjects (52.5p9.5 and 42.3p5.5 nmol\ml respectively ; P 0.0001), but demonstrated no statistically
significant differences among the HCM subtypes.
Acylcarnitine concentrations in HCM patients were
significantly lower than those of normal subjects
(10.1p4.0 and 13.2p3.9 nmol\ml respectively ; P
0.0001), although again there were no significant
differences among the HCM subtypes. Acylcarnitine\
free carnitine ratios in HCM patients were significantly
lower than those of normal subjects (0.19p0.08 and
0.32p0.11 respectively ; P 0.0001). Levels of nonesterified fatty acids, however, showed no statistically
significant differences among normal subjects or any
HCM subtype groups.
Relationship between carnitine levels and
analysis of BMIPP imaging
We investigated the relationship between serum carnitine
levels and quantitative or semi-quantitative analysis of
BMIPP imaging for identification of impaired myocardial
Carnitine in hypertrophic cardiomyopathy
Table 1
Clinical data for 56 patients with HCM
Abbreviations : LV, left ventricular ; LVEDP, left-ventricular end-diastolic pressure : NYHA class, New York Heart Association
functional class. Significance of differences : *P 0.05 compared with APH patients.
Clinical characteristics
Age (years)
Male/female
Family history of HCM
NYHA class I/II
Echocardiographic findings
Ventricular septum (mm)
LV posterior wall (mm)
Maximal wall thickness (mm)
Fractional shortening (%)
Haemodynamic features
Ejection fraction (%)
LVEDP (mmHg)
Table 2
HNCM (n l 36)
HOCM (n l 7)
APH (n l 13)
55p13
26/10
8
23/13
53p13
5/2
3
5/2*
59p10
11/2
1
12/1
19p5*
11p3
23p6
41.4p7.6
21p5*
13p2
26p5
43.6p8.4
15p3
13p2
21p5
47.3p6.7
76p8
13p6
80p6
14p4
78p8
11p8
Serum concentrations of carnitine and non-esterified fatty acids in normal subjects and patients with HCM
Significance of differences : *P
0.05, **P
0.005, ***P
0.0005, ****P
0.0001 compared with normal subjects.
Subtypes of HCM
Free carnitine (nmol/ml)
Acylcarnitine (nmol/ml)
Acylcarnitine/free carnitine ratio
Non-esterified fatty acids (µEq/l)
Table 3
Normal subjects (n l 80)
HCM (n l 56)
HNCM (n l 36)
HOCM (n l 7)
APH (n l 13)
42.3p5.5
13.2p3.9
0.32p0.11
335.0p139.0
52.5p9.5****
10.1p4.0****
0.19p0.08****
318.2p124.3
53.0p9.0****
10.7p4.2**
0.20p0.09****
326.9p117.7
53.7p7.7****
9.8p3.0*
0.18p0.05****
312.6p140.0
50.4p11.8***
8.8p3.7***
0.17p0.05****
297.3p141.2
Scintigraphic data with BMIPP in normal subjects and patients with HCM
H/M ratio, heart/mediastinum uptake ratio of BMIPP. Significance of differences : *P
0.005, **P
0.001, ***P
0.0001 compared with normal subjects.
Subtypes of HCM
H/M ratio
Total defect score
Normal subjects (n l 20)
HCM (n l 56)
HNCM (n l 36)
HOCM (n l 7)
APH (n l 13)
2.33p0.16
–
2.11p0.12***
14p6
2.10p0.12***
14p7
2.10p0.17*
14p4
2.14p0.12**
12p6
fatty acid metabolism. Quantitative analysis disclosed
that the heart\mediastinum uptake ratio in HCM patients
was significantly lower than that of normal subjects
(2.11p0.12 and 2.33p0.16 respectively ; P 0.0001 ;
Table 3). However, no statistically significant differences
were seen among the HCM subtypes. In semi-quantitative analysis, normal subjects demonstrated no visually reduced BMIPP uptake in any segment of the
myocardium, as shown in Figure 1. The total defect score
showed no statistically significant difference among
HCM subtypes (Table 3). Both quantitative and semiquantitative analyses demonstrated significant corre-
lations with free carnitine levels (r l k0.422, P l 0.0012
and r l 0.633, P 0.0001 respectively) (Figure 3). Both
of the analyses tended to correlate with acylcarnitine
levels, although these correlations were not statistically
significant (quantitative analysis : r l k0.251, P l 0.062 ;
semi-quantitative analysis : r l 0.247, P l 0.066).
Characteristics of HCM patients showing
high and normal free carnitine levels
To investigate the relationship between free carnitine
levels and patient characteristics, patients with HCM
were divided into two groups : 28 patients with high free
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with a low free carnitine level (below 2 S.D. of the mean)
was excluded from this category. There were no significant differences in patient characteristics, such as clinical
characteristics, echocardiographic findings or haemodynamic features, between the two groups, although
there were significantly fewer female patients in the
group with high free carnitine levels (P l 0.02 ; Table 4).
DISCUSSION
Kinetics of carnitine and fatty acid
metabolism in patients with HCM
Figure 3 Scatterplots showing relationships of serum free
carnitine level with heart/mediastinum (H/M) uptake ratios
of BMIPP (upper panel) and total defect score (TDS) of BMIPP
imaging (lower panel)
carnitine levels (above meanj2 S.D. of the mean obtained
from normal subjects) and 27 patients with normal free
carnitine levels (within p2 S.D. of the mean). One patient
Carnitine is synthesized endogenously from lysine and
methionine. In humans, the final reaction in carnitine
synthesis, i.e. the hydroxylation of γ-butyrobetaine,
occurs mainly in the liver, not in the heart [5]. The
myocardial concentration of carnitine is about 80–140fold higher than the plasma concentration [15]. Therefore
carnitine uptake in the myocardium occurs against a large
concentration gradient. A binding protein (carriermediated) [6,23], active transport [24], diffusion [6] and
an exchange system [25] have been proposed as the
transport mechanism for carnitine. Approx. 90 % of
the cellular carnitine is located in the cytoplasm, with the
remaining 10 % in the mitochondria [26].
The present study has found that serum free carnitine
concentrations were significantly higher, and acylcarnitine concentrations and acyl\free carnitine ratios
were significantly lower, in patients with HCM than
in normal subjects. These results suggest that, in HCM,
carnitine is not adequately transported into the myocardium from the bloodstream, and β-oxidation subse-
Table 4 Comparison of characteristics of patients with high and normal free carnitine levels
in HCM
LV, left ventricular ; LVEDP, left-ventricular end-diastolic pressure ; NYHA class, New York Heart Association functional
class. Significance of differences : *P 0.05 compared with the group showing normal free carnitine levels.
High free carnitine levels (n l 28)
Normal free carnitine levels (n l 27)
Clinical characteristics
Age (years)
Male/female
Family history of HCM
NYHA class I/II
56p11
25/3*
6
19/9
55p13
17/10
6
17/10
Echocardiographic findings
Ventricular septum (mm)
LV posterior wall (mm)
Maximal wall thickness (mm)
Fractional shortening (%)
19p5
12p3
24p5
42.6p7.9
18p5
11p2
22p6
43.3p7.8
77p8
13p6
77p8
12p6
Haemodynamic features
Ejection fraction (%)
LVEDP (mmHg)
# 1999 The Biochemical Society and the Medical Research Society
Carnitine in hypertrophic cardiomyopathy
quently declines. York et al. [27] and Whitmer [28]
investigated carnitine transport and myocardial metabolism using Bio 14.6 Syrian hamsters, representing a
model for HCM. They reported that carnitine concentrations in the heart in Bio 14.6 Syrian hamsters were
significantly lower throughout life, and plasma carnitine
concentrations were higher from an early stage of life,
compared with normal controls. These findings were
observed before the hamsters began to show clinical
symptoms of HCM and before severe histological pathology was evident. Furthermore, carnitine-binding protein, which is located on the surface of the plasma
membrane, had a reduced maximal capacity for carnitine
binding and an increased dissociation constant. Hoppel
et al. [29] reported that the myocardial acylcarnitine\free
carnitine ratio was not markedly affected in Bio 14.6
Syrian hamsters, although the levels of both free carnitine
and acylcarnitine were markedly depressed. Mitochondrial enzymes, such as carnitine palmitoyltransferase and palmitoyl- and butyryl-CoA dehydrogenases, were unaffected, and myocardial total CoA
levels were not depressed. These authors speculated that
a ‘ steady-state ’ ratio of carnitine to its metabolites is
maintained, although they did not refer to the serum
acylcarnitine\free carnitine ratio. If these findings are
applicable to humans, the depressed serum acylcarnitine\
free carnitine ratio suggests that a disorder of the
myocardial plasma membrane, affecting uptake of free
carnitine into myocytes, is more closely related to the
pathophysiology of HCM than mitochondrial damage. It
is still unclear whether medium- and short-chain fatty
acids substitute for long-chain fatty acids as substrates
for β-oxidation in the affected myocardium of HCM
patients. A positron-emission tomographic study using
")F-labelled 2-deoxyglucose showed enhanced myocardial glucose metabolism [30], although a previous study
reported depressed glucose metabolism [31]. Therefore it
remains unclear whether glucose metabolism compensates for impaired myocardial fatty acid metabolism in
HCM.
Patient characteristics demonstrated a statistically
significant difference only with regard to the gender of
patients with high and normal free carnitine concentrations. The amount of serum free carnitine in normal
healthy volunteers has been reported to be greater in men
than in women [19], and this difference may have affected
our results. The increase in plasma carnitine is reported to
be more distinct in patients with a severely reduced leftventricular ejection fraction than in patients with a mildly
reduced ejection fraction [10]. However, the ejection
fraction was normal in all the HCM patients, and no
statistically significant differences between the patients
with high and normal free carnitine levels was observed.
Inborn errors of fatty acid metabolism and carnitine
deficiencies are reported to provoke secondary HCM,
with low serum carnitine concentrations [32,33] ; in
contrast, our study revealed that the levels of free
carnitine are increased in idiopathic HCM.
Kinetics of BMIPP and fatty acid metabolism
in HCM patients
BMIPP is a radioiodinated long-chain-fatty-acid analogue used for the evaluation of cardiac fatty acid
metabolism. A binding protein is considered to be
associated with the transport of BMIPP from the
bloodstream to the myocardium [34]. After injection,
BMIPP is extracted extensively from the plasma into the
myocardium (74 % of the injected dose), and is retained
mainly in the triacylglycerol fraction (65.3 %). After
washout from the myocardium, most of the retained
radioactivity (8.7 %) is derived from metabolites of αand β-oxidation [35]. Using etomoxir, an inhibitor of
carnitine palmitoyltransferase I, Hosokawa et al. [36]
investigated the metabolic fate of BMIPP. BMIPP was
retained to a considerable extent in the myocardium,
despite etomoxir treatment, but the amount of full
metabolites of β-oxidation decreased significantly, and
back-diffusion of BMIPP increased. These authors concluded that BMIPP is very sensitive to etomoxir and is
suitable for assessing mitochondrial dysfunction [36]. In
addition, a close positive correlation was shown between
myocardial ATP content and BMIPP accumulation [37].
High serum levels of non-esterified fatty acids are
reported to be associated with slow clearance of BMIPP
from the myocardium [38]. However, none of our
patients showed high levels of serum non-esterified fatty
acids, so such an influence can be ruled out. The kinetics
of BMIPP suggest that an impairment of the mitochondria and\or the plasma membrane may be related to
the reduced or absent myocardial uptake of BMIPP in
patients with HCM.
Reduced BMIPP uptake is seen in the hypertrophied
myocardium, especially in the anterior and posterior
junctions of the ventricular septum in HCM patients (see
Figure 1). In addition, these segments correspond pathologically with myocardial disarray [39]. Maron et al. [40]
investigated changes in the myocardium in HCM patients
by electron microscopy. Operative biopsies in humans
revealed mitochondrial damage, such as swelling of the
mitochondria and disruption of the cristae, in 14 out of 15
patients. These abnormalities were more marked in the
patients with a hypertrophied ventricular septum than in
those with the left-ventricular free walls. In our
institution, moreover, BMIPP myocardial scintigraphy
and electron microscopic investigations were performed
on WKY\NCrj rats, which are useful models for HCM
[41]. BMIPP levels were reduced in the anterior and
posterior junctions of the ventricular septum, and disruption of the mitochondrial cristae was prominent in the
same region [42]. These results support our conclusions.
As shown in Figure 3, serum free carnitine levels
correlated significantly with depressed myocardial
# 1999 The Biochemical Society and the Medical Research Society
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T. Nakamura and others
BMIPP uptake. Because myocardial BMIPP is indicative
of fatty acid metabolism for the reasons described above,
increased serum free carnitine levels indicate an impairment of myocardial fatty acid metabolism. In conclusion, high free carnitine concentrations and low
acylcarnitine concentrations in serum indicate a
significant impairment of myocardial fatty acid metabolism, which implies a disturbance in the plasma membrane and\or mitochondria in the hypertrophied myocardium in HCM patients. Also, serum free carnitine
concentrations are increased even though the left-ventricular ejection fraction is normal, and they proved to be
sensitive indicators for detecting impaired myocardial
fatty acid metabolism even in the asymptomatic phase of
HCM.
Study limitations
We did not investigate myocardial carnitine levels in this
study. Myocardial carnitine levels have been shown to be
significantly lower in HCM patients than in normal
controls [27–29]. Transcatheter biopsy does not always
obtain the affected myocardium, and approx. 70 % of the
cellular carnitine is lost, even if the specimens are freshly
prepared [43]. Thus the myocardial carnitine concentration is difficult to measure and seems to be somewhat
unreliable ; instead, the serum carnitine concentration
may adequately indicate myocardial fatty acid metabolism.
ACKNOWLEDGMENTS
This work was supported by a Research Grant for
Cardiovascular Diseases (8A-5) from the Ministry of
Health and Welfare of Japan, Tokyo, Japan. We thank
Naoto Terada, Hiroshi Nakajima, Zenro Kizaki (Department of Pediatrics, Kyoto Prefectural University of
Medicine, Kyoto, Japan), and Mamoru Takahashi (Asahi
Chemical Industry, Shizuoka, Japan) for their support in
serum carnitine analysis and for helpful discussions.
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Received 16 February 1999/19 May 1999; accepted 23 July 1999
# 1999 The Biochemical Society and the Medical Research Society
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