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Chronic cardiomyopathy and weakness or acute coma in children with a defect in carnitine uptake.

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Chronic Cardiomyopathy and Weakness or
Acute Coma in Chddren
with a Defect in
..
Larmtlne Uptake
A
T
T
1
Charles A. Stanley, MD,* Susan DeLeeuw,* Paul M. Coates, PhD,? Christine Vianey-Liaud, PhD,S
Priscille Divry, MD,$ Jean-Paul Bonnefont, MD,§ Jean-Marie Saudubray, MD,§ Morey Haymond, MD,I'
Friedrich K. Trefz, MD,' Galen N. Breningstall, MD,+* Rebecca S. Wappner, M D , t t Dennis J. Byrd, PhD,$S
Claude Sansaricq, MD,@ Ingrid Tein, MD,lI1'Warren Grover, MD," David Valle, MD,"*"
S. Lane Rutledge, M D , W and William R. Treem, MD$S$
A defect in intracellular uptake of carnitine has been identified in patients with severe carnitine deficiency. To define
the clinical manifestations of this disorder, the presenting features of 15 affected infants and children were examined.
Progressive cardiomyopathy, with or without chronic muscle weakness, was the most common presentation (median
age of onset, 3 years). Other patients presented with episodes of fasting hypoglycemia during the first 2 years of life
before cardiomyopathy had become apparent. A defect in carnitine uptake was demonstrable in fibroblasts and leukocytes from patients. The defect also appears to be expressed in muscle and kidney. Concentrations of plasma carnitine
and rates of carnitine uptake in parents were intermediate between affected patients and normal control subjects,
consistent with recessive inheritance. Early recognition and treatment with high doses of oral carnitine may be
life-saving in this disorder of fatty acid oxidation.
Stanley CA, DeLeeuw S, Coates PM, Vianey-Liaud C , Divry P, Bonnefont J-P, Saudubray J-M,
Haymond M, Trefz FK, Breningstall GN, Wappner RS, Byrd DJ, Sansaricq C, Tein I, Grover W,
Valle D, Rutledge SL, T r e e m WR. Chronic cardiomyopathy and weakness or acute coma in children
with a defect in carnitine uptake. Ann Neurol 1991;30:709-716
Carnitine is a small, water-soluble molecule required
for the oxidation of long-chain fatty acids by mitochondria. Its function is to shuttle long-chain fatty acids, in
the form of their acyl-carnitine esters, across the barrier
of the inner mitochondria1 membrane and into the mitochondrial matrix where they serve as fuels for oxidative phosphorylation { 11. Carnitine is supplied by both
diet and endogenous synthesis from trimethyllysine
{2]. Intracellular carnitine concentrations are maintained 20 to 50 times greater than those of the extracellular space by a sodium-dependent plasma membrane
transport system 11). We recently reported a defect in
carnitine uptake by cultured skin fibroblasts from an
infant with hypoketotic hypoglycemia, severe carnitine
deficiency, and evidence of impaired carnitine transport in muscle and kidney [3]. Eriksson and co-workers {4,51 described a similar carnitine uptake defect in
fibroblasts from a child with cardiomyopathy associated
with severe carnitine deficiency. In both of these patients, the signs of impaired fatty acid oxidation were
corrected by oral carnitine therapy.
This report describes the findings in 15 infants and
children with a defect in the uptake of carnitine demonstrated in cultured skin fibroblasts. The results, together with information on 5 other reported cases
14-61, indicate that this is a recessively inherited disor-
From the *Endocrine/Diabetes and ?Gastroenterology & Nutrition
Divisions, The Children's Hospital of Philadelphia. Philadelphia, PA;
Received Feb 6, 1991, and in revised form Apr 16. Accepted for
publication Mav 12. 1991.
klinik, Medizinische Hochschule-Hannover, Hannover, Germany;
§§New York University Medical Center, New York, N Y 'l1lHospital
for Sick Chddren, Toronto, Ontario, Canada; ##St Christopher's
Hospital, Philadelphia, PA; """Howard Hughes Laboratory of Genetics, Johns Hopkins University School of Medicine, Baltimore,
MD; ???University of Alabama at Birmingham, Birmingham, AL;
and $$$Hartford Hospital, Hartford, CT.
Copyright 0 1991 by the American Neurological Association
709
der that may present with fasting hypoglycemia; more
frequently it is associated with progressive cardiomyopathy and skeletal myscle weakness.
Case Reports
The following case histories briefly illustrate the major types
of clinical presentations in this series of patients: hypoglycemic coma, symptomatic cardiomyopathy, and presymptomatic cardiomyopathy.
Hypog/ycemic C p m ~
PATIENT #lo. This 2-year-old Indian boy was admitted to
the hospital for gastroenteritis and dehydration. For 5 days
prior to admission he had fever, vomiting, watery diarrhea,
and diminished oral intake except for liquids. On admission,
he was semicomatose, afebrile, and appeared dehydrated.
Plasma glucose levels were not measured before treatment
was begun, but hypoglycemia was inferred from the improvement in his mental status following administration of intravenous glucose and fluids. He had mild acidosis (serum bicarbonate, 15 mmo//l), n o r 4 blood urea nitrogen (14 mg/
dl), slightly prolonged protbrombin time (14 sec), and elevated ammonia levels (173 pg/dl). A urine test for ketones
was negative. Because he was suspected to have Reye’s syndrome, he was transferred to a lager medical center. Over
the next 2 days, his liver enlarged to 4 cm below the costal
margin, his aspartate transaminase concentration increased tQ
55 1 U, his alanine transminase concentration increased to
7 16 U, and his creafinine phosphokinase concentration increased to 775 LJ. A liver biopsy revealed fatty infiltration
consistent with Reye’s syndrpme. He had a 3/6 systolic murmur but a normal cardiac silhouette, and an electrocardiogram showed minimal left ventricular hypertrophy. Serum
amino acids and urine organic acid profiles were normal. By
7 days, the serum enzyme levels returned toward normal
and he was discharged. One month later, a study of fasting
adaptation showed a normal increase in plasma concentrations of ketone8 (6-hydroxybutyrate, 3.07 mmolll; acetoacetate, 1.89 mmol/l) but a very low plasma total carnitine level
(1.8 pmol/l). The levels of urinary dicarboxylic acids during
the fast were elevated (adipic 1,023; suberic, 233; octenedioic, 121; sebacic, 58 mg/gm creatinine) but not above the
normal fasting range (adipic < 1,500; suberic, (300; sebacic,
< l o 0 mg/gm creatininej. His dietary sources of carnitine
included milk and cheese but not meat or eggs. N o weakness
was observed, but after treatment with oral r-carnitine was
begun, his parents commented that he seemed less irritable
and his activity level increased. Studies of both leukocytes
and cultured skin fibroblasts revealed a defect in carnitine
uptake (see Results section). In the 9 months since starting
oral 1.-carnitine treatment (50 mg/kg/day), the boy has been
well. The parents both come from southern India but are
unrelated. Their plasma total cwnitine levels were slightly
below pormal (Table 1).An older brother is healthy and has
normal plasma carnitine levels.
Cardiomyopathy
PATIENT #14. This 6.5-year-old boy was admitted to the
hospital for rapidly progressive heart failure. H e had a history
of asthma and had had recurrent episodes of pneumonia dur-
710 Annals of Neurology
Vol 30 N o 5
ing the previous year. Six weeks prior to admission, he developed increasing fatigue, dyspnea, and congestive heart failure. A chest radiograph showed a marked increase in heart
size compared to a radiograph taken 4 months earlier for
pcpsible pneumonia. Physical examination revealed a 2/6
short systolic murmur and no hepatomegaly. There was mild
proximal muscle weakness. Cardiac catheterization showed a
dilated cardiomyopathy, and he was begun on treatment with
digoxin and other cardiotonics. His plasma total carnitine
concentration was found to be very low (1.2 pmolil). A muscle biopsy revealed the presence of lipid storage and a low
total carnitine level (see Table 1j. Urinary organic acid levels
were normal. Within a week after beginning oral L-carnitine
(2 gmiday) treatment, his color and energy had improved;
his activity level returned to normal within 1 to 2 months,
although his heart remained enlarged for 6 months. After 1
year of treatment, he still has mild proximal muscle weakness, but his exercise tolerance and heart size appear normal.
PATIENT #13.
The 5.5-year-old sister of Patient #14 was
investigated when her brother was found to have carnitine
deficiency. She had previously been considered to be healthy,
but on examination was found to have a systolic heart murmur. Chest radiography and echocardiography revealed an
enlarged heart. She had no weakness. Plasma and muscle
total carnitine cqncentrations were found to be low, similar
to her brother (see Table 1).O n oral treatment with 1.5 gmi
day L-carnitine, her heart size returned to normal over a period of 3 to 4 months. The parents of Patients #13 and #14
are unrelated, of German and Scandinavian extraction, and
have plasma total carnitine levels slightly below the normal
range (see Table 1).
Materials and Methods
Patients
Cultured skin fibroblasts were obtained from the 15 patients
and shipped to The Children’s Hospital of Philadelphia for
studies of carnitine uptake. Clinical details of Patients #1,
#6, #8, and #12 have been reported previously 13, 7-31.
Where possible, fibroblasts were also obtained from the parents of patients. The response to withdrawal of carnitine for
5 days was examined in Patient # 11 using the protocol previously reported for Patient #1 [3}. These studies were approved by the Institutional Review Bowd of The Children’s
Hospital of Philadelphia A11 references to carnitine indicate
the L-isomer unless otherwise stated.
Fibroblast and Leukocyte Carnitine Uptake
The uptake of labeled carnitine was measured in fibroblast
monolayers grown in 7.5-cm2 wells on RPMI-1640 medium
(Sigma, St Louis, MO) supplemented with 5% fetal calf serum. The medium was replaced with fresh RPMI, without
serum, containing 2.5 pCi L-[methyl-iH)carnitine and 0 to
50 wmol/l unlabeled L-carnitine. A carnitine concentration of
10 mmolil was used to correct for nonspecific uptake. All
experiments were carried out in duplicate. After incubation
for 4 hours at 37°C in humidified 57% CO,, the monolayer
was washed 4 times with phosphate-buffered saline (pH, 7.4;
4°C) and hydrolyzed with 0.5 normal sodium hydroxide.
Cell-bound radioactivity was measured In an aliquot using
November 1791
Table 1 Clinical Featares of Infants and Children with a Defect in Carnitine Uptake
Total Carnitine"
Clinical
Presentation
Patient [reQ
Plasma (pmolll)
Plasma
Total Carnitine
(pmolll)
Muscle (nmollgm)
After
Fasting
Age
Before
After
Before
Sex (yrlmo) Type Treatment Treatment Treatment Treatment Ketosis
Present Series
1 [31
F
2b
M
3
M
4
M
5
M
6 c71
F
7
F
F
8 C81
9
M
10
M
11
F
M
12 E91
F
13'
-
013
018
0111
110
l/l
116
116
118
118
210
310
314
516
14'
M
616
lSb
M
710
Other reported cases
16 ~ 4 , 5 1
F
410
17 E61
F
112
18 E63
M
115
19 E63
F
216
20 E6l
F
514
H
0.7
H
H
H,C
C
...
35
No
No
No
Yes
...
...
44
14
80
...
...
...
...
(4.1)
160
132
...
270
0
No
Yes
Yes
Yes
22
...
...
...
...
...
...
NolYesd . . .
40
70
30
35
38
...
36
35
30-40
46-50
...
(0.01)
c
1.2
4
...
(0.01)
...
...
1.2
W,C 19
w 1.2
H
O
c
9
...
<40
170
(1.1)
c,w
Father
-
~
4
1.4
2.1
1.5
H,C 4.2
H
O
H,C 9
W
1.4
H
1.8
C
0.3
C
4.2
c
1.2
c
Sibling
Death Mother
25-60
9-28
...
...
...
...
.
6
...
...
(0.35)
...
...
...
(0.97)
I
...
...
NO
No
...
.
...
...
Yes
Yes
...
...
...
...
...
...
...
...
"low"
...
...
...
...
No
Yes
Yes
(2.7)
.
.
I
...
...
...
...
34
33
...
26
34
...
...
...
35
32
25
38
34
(see Patient
#13)
(see Patient # 2 )
34
28
25
...
...
...
36
21
...
Yes
...
61
35
...
...
...
...
20
20
22
...
22
30
~~
"Normal ranges: plasma, 40-60 pmolil; muscle 2,500-3,500 nmoligm or (17.7
bCases 2 and 15 are sibling pairs.
'Cases 13 and 14 are sibling pairs.
dSee caSe report.
H
=
hypoglycemia; C = cardiomyoparhy; W
=
f
3.7 nmollmg protein, mean
I
SD).
weakness.
ScintiVerse E (Fisher, Pittsburgh, PA) at an efficiency of
40%. Cell protein was measured by the method of Lowry
and colleagues [ l o } . The effect of D-carnitine and acylcarnitines on carnitine uptake by fibroblasts was determined
using unlabeled acyl-carnitines at final concentrations ranging
from 0.1 to 200 pmolll. Sodium dependence of carnitine
uptake in fibroblasts was examined by measuring uptake of
labeled carnitine in Krebs-Henseleit bicarbonate buffer with
lithium chloride substituted for sodium chloride. For studies
of carnitine uptake in white blood cells, mononuclear leukocytes were isolated from 20 ml of freshly drawn blood using
Ficoll-Hypaque. No specific uptake of carnitine was found
in red cells. The leukocytes were resuspended in 3 ml RPMI
1640 and incubated at 37°C in 9596 O2:5%CO, for 2 hours
with labeled and unlabeled carnitine, at the same concentrations used with fibroblasts. At the end of the incubation period, 0.5 ml of the suspension was added to 0.5 ml of ice-cold
RPMI-1640, it was centrifuged, and the cell pellet was
washed 3 times with ice-cold phosphate-buffered saline. The
pellet was hydrolyzed with 0.5 ml of 0.5 normal sodium
hydroxide and counted using ScintiVerse E.
FibroblaJt Steadystate Carnitine Levels
The cquilibrium concentration of carnitine was determined
in fibroblasts after incubation with labeled and unlabeled carnitine until the uptake of labeled carnitine reached plateau
(48 hr). Fibroblast monolayers in 9.5-cm2 wells were incubated in 2 ml of RPMI-1640 with 0.25 pCi of r-{methyl3H1-carnitine, unlabeled carnitine at concentrations from 0
to 200 pmolil, and 0.067 pCi {'4C}-polyethylene glycol as
an extracellular water marker. The incubation was terminated
after 4 8 hours and the cell-associated radioactivity was measured in the same manner as described.
Fibroblast Futty Acid Oxidation
Fibroblasts were incubated with 0 to 50 pmolll L-carnitine
in RPMI-1640 medium without serum on the bottom of
25-cm2 flasks for 4 8 hours. The medium was replaced with
Krebs-Henseleit bicarbonate buffer with 0.25 pCi [U-'*C]oleate and 50 pmolll unlabeled oleate complexed to fatty
acid-free albumin at a 1:1 molar ratio and L-carnitine at concentrations from 0 to 50 pmolll. Flasks were gassed with
95% 0 2 : 5 % , CO,, stoppered with hanging center wells, and
Stanley et al: Carnitine Uptake Defect
711
incubated at 37°C for 6 hours. Oxidation was stopped by
injecting 0.15 rnl 3 normal perchloric acid; the '*C02 was
trapped in hyamine hydroxide for 1 hour and then counted
with ScintiVerse E.
Results
Table 1 summarizes the features of the 15 patients in
this series and of the 5 cases reported elsewhere C4-61
in whom a defect in carnitine uptake has been demonstrated in cultured skin fibroblasts. The clinical feature
most commonly noted at initial presentation was progressive cardiomyopathy, which occurred in 12 of the
20 patients between 1 and 7 years of age (median, 3
yr). Nine patients presented with an episode of hypoglycemia between 3 months and 2.5 years of age (median, 1.5 years). Evidence of cardiomyopathy was present at the time of the initial hypoglycemic episode in
3 of these 9 infants. In one family, one boy (Patient
#2) presented with hypoglycemia in early infancy,
whereas h s brother (Patient # 15) presented with cardiomyopathy at 7 years of age. Skeletal muscle weakness was described in 4 of the patients at the time of
first presentation and was the sole manifestation in 2
(Patients #9, #18). The diagnosis of carnitine deficiency was delayed for 1 to 6 years in 9 patients; during
this time all developed cardiomyopathy and 8 were
reported to have muscle weakness. In some of the
other 12 patients, muscle weakness may have been
present but not specifically noted because it was mild
(e.g., Patients #lo, #14) or overshadowed by the severity of the cardiomyopathy (Patient #11).
In the patients with hypoglycemia, ketogenesis was
defective, at least during acute episodes. Hepatic fatty
acid oxidation was not impaired in 1 after recovery
(Patient #lo) and was also normal in 1 older infant
who presented with cardiomyopathy (Patient # 12) 19).
All of the patients with cardiomyopathy and weakness responded to L-carnitine treatment and experienced dramatic improvement in symptoms within a
few weeks and reduction of heart size toward normal
within a few months. In one patient (Patient #1), impaired fasting ketogenesis was corrected with carnitine
therapy 131; she later died from complications of intestinal adhesions following placement of a feeding gastrostomy. Eighteen of the other 19 patients continue
to be healthy on carnitine therapy after periods of 1
to 10 years. Patient #15 is moderately impaired with
weakness and cardiomyopathy at age 20, possibly due
to poor compliance.
Plasma total carnitine concentrations were reduced
in all 20 patients; 18 had values less than 5 pmol/l
(10% of normal mean). Muscle total carnitine concentrations in 13 patients ranged from 0.05 to 17% of the
normal mean. Liver total carnitine was 5% of normal
in the 1 patient tested (Patient #1) [3}. In all patients
tested, urinary organic acid profiles were described as
normal or showed only modest elevations of mediumchain dicarboxylic acids within the range found in
fasting children. Plasma carnitine concentrations approached normal during carnitine therapy but were
rarely elevated because of impaired renal conservation
of carnitine. In Patient #11, acute withdrawal of oral
carnitine resulted in a rapid decrease in plasma total
carnitine levels from 18 to 3.3 pmol/l over 3 days,
whereas the fractional excretion of free carnitine
ranged between 74 and 230% of the filtered load (normal, <5%). Similar data showing poor renal conservation of carnitine have been previously reported in Patients #1, #6, and #16 r3-5, 7). During treatment
with oral carnitine, muscle carnitine concentrations increased only slightly. In contrast, liver carnitine levels
increased nearly into the normal range during treatment in Patient #1 (750 vs 900-1,800 nmol/gm) [S).
The ethnic background of the 18 families shown in
Table 1 include European, Afro-American, North African Arab, Asian Indian, Mexican, and Chinese. In
5 of the families, the parents were consanguineous.
Plasma total carnitine concentrations were low in all
11 mothers and in 10 of 11 fathers tested. In 8 families,
other children had died with illnesses compatible with
the same carnitine transport defect.
Studies in Cultured Fibroblasts
As shown in Tables 2 and 3 , carnitine uptake by cultured skin fibroblasts was sodium-dependent. Uptake
was stereoselective, with greater affinity for L-carnitine
than the D-isomer. Straight-chain acyl-carnitines, especially medium-chain and long-chain acyl-carnitines,
were potent inhibitors of carnitine uptake by fibroblasts.
Table 2. Sodiam Dependence of L-carnitine Uptake by
Fibroblasts from Normal Control Subjects
Sodium Concentration V,
(mrnolil)
(pmolirninimgprotein)
Km
(Wrnolil)
124
24
3.42
2.49
3.42
1.18
Table 3 . Substrate Concentrations Giving Half-mximal
Inhibition of L-{methyl-3H}-Carnitine Uptake by Fibroblasts"
Substrate
(brnolil)
L-carnitine (n = 9)
D-carnitine (n = 3)
3.05
7.5
Acetyl-carnitine(n = 3 )
Octanoyl-carnitine (n = 3 )
Myristoyl-carnitine(n = 3)
Palmityl-carnitine (n = 3)
4.6 2 0.5
2.9 2 0.4
0.16 k 0.017
0.37 0.06
an experiments in 2-4 control cultures, mean
712 Annals of Neurology Vol 30 No 5 November 1991
k
*
&
SD.
0.31
* 1.0
I .2-
.
1.0-
8
120,
eo
60
W
11. (llpmolll)
40
-.-
* T
E
a
L
a
Patient
0
2
4
6
8
10
v
-
CARNlTlNE (pwlll)
Fig 1. Uptake of {jH)-L-carnitine by cultured skin fibroblasts
from a patient with a defect in camitine uptake, a parent, and
a control subject. Fibroblasts were incubated with labeled and
various concentrations of unlabeled camitine for 4 hours. lnset
shows a double reciprocal plot of carnitine uptake velocity versus
medium carnitine concentration in parent and control fibroblasts.
Figure 1 illustrates the effect of unlabeled carnitine
on uptake of E3H]-~-carnitineby cultured skin fibroblasts from an affected patient, a parent, and a control
subject. Uptake of labeled carnitine was markedly
lower in cells from the patient compared with the control subject and was intermediate in cells from the parent. The rates of high-affinity carnitine uptake by fibroblasts at a carnitine concentration of 5 bmol/l
showed essentially no overlap between patients, parents, and control subjects (Fig 2). In the father of 1
patient, both the V,
and the uptake velocity at 5
p,mol/i carnitine were within normal range. It is not
known whether this finding might reflect nonpaternity.
Excluding this one outlier, the mean uptake velocity at
5 pmol/l carnitine in parent cells was approximately
40% of control (0.34 k 0.16 vs 0.93 & 0.13 pmol/
min/mg protein [mean .t SD]; p < 0.001). In parents'
cells, the V,, was also 40% of the control subjects'
(0.64 f 0.32 vs 1.69 f 0.32 pmol/min/mg protein; p
< 0.001), but the Km was similar to control levels
(3.26 t 1.32 vs 2.67 t 0.55 pmol/l). Studies using
peripheral blood mononuclear leukocytes from Patient
#10 demonstrated the same defect in carnitine uptake
that was found in fibroblasts from this patient, although
the rate of uptake in leukocytes was only 10% of that
found in fibroblasts (uptake velocity at 5 pmol/l carnitine: 0.008 pmol/min/mg protein in Patient #10 vs
0.082 f 0.009 in 6 control subjects).
Figure 3 shows the effect of the carnitine uptake
defect on steady-state levels of intracellular carnitine in
fibroblasts equilibrated with different concentrations of
carnitine for 48 hours. In control subjects, intracellular
carnitine increased steeply with extracellular carnitine
concentrations in the range of the Km for carnitine
uptake. Beyond 10 p.mol/l, intracellular carnitine in-
g6
0.8-
E
.
.f
I
E
X
..: I
.
Q
Y
0.6
x
W
Y
2n
3
I=
0.4-
z
a
4
0
0.2-
"
Patients
Parents
Controls
Fig 2. Carnitine uptake rates at an extraceflularcarnitine concentration of 5 pnollf by jbmbfasts fmm patients with a d - c t
in camitine uptake, parents, and control subjects (mean 5 SO).
Experiments were cawied out as described in F i g 1 and in Materials and Methods section.
creased linearly with extracellular carnitine, probably
reflecting uptake by passive diffusion. In contrast, in
patient fibroblasts, intracellular carnitine only increased
linearly with extracellular carnitine. Intracellular carnitine concentrations in fibroblasts from parents were
intermediate between patients and control subjects and
followed a curvilinear relationship to extracellular carnitine similar to control subjects. Assuming a fibroblast
protein content of 70 gm/l [ 5 ] , intracellular carnitine
concentrations in patient fibroblasts were essentially
the same as in the medium (e.g., 70 pmol/l intracellular
vs 50 pmol/l extracellular), which is consistent with
passive diffusion of carnitine into the cells.
Figure 4 compares the effect of extracellular carnitine concentration on oxidation of oleate by fibroblasts
from a control subject and a patient. Similar results
were obtained in experiments examining the oxidation
of a tracer dose of oleate by fibroblasts from 3 patients
and 4 control subjects (data not shown). In the absence
of added carnitine, oleate oxidation rates were similar
Stanley et al: Carnitine Uptake Defect
713
I
t
1
/
Controla (4)
a
E
3.0
E
I
Y
I
t
CARHITINE (pmolll)
Fig 3. Steady-state intracellular carnitine concentration in
fibvoblasts from patients with a defect in camitine uptake,
parents, and control subjects (mean SDi. Fibroblasts were
incubated for 48 hours with {3H)-L-rarnitineand various concentrations of unlabeled camitine using {'4C)-polyethyleneglycol
as an extracellular water marker. Results from 0 t o 5 pmoltl
camitine are from a single experiment.
*
,-0
I
0
10
20
30
40
50
carniiine (~mol:l)
4. EHect of extracellular carnitine concentration on oxidation of 50 pmolll {U-'4C)-oleate by fibroblastsfrom a patient
with a defect in carnitine ujitah and a control subject. Fibroblasts were preincubated for 48 hours in medium containing
various concentrations o f carnitine, and 14C02production wer
6 hours was measured (means of duplicates).
Fig
714 Annals of Neurology Vol 30 No 5
in patient and control cells. The addition of carnitine
did not affect oxidation rates in control cells, but it
stimulated oleate oxidation two-fold in patient cells.
Maximal oxidation rates in patient cells required a carnitine concentration of 10 Fmolil.
Discussion
The results of this study indicate that the carnitine deficiency disorder associated with a defect in intracellular uptake of carnitine most commonly presents with
signs of progressive cardiomyopathy in late infancy and
early childhood. Patients are normal at birth and may
appear healthy for several years before they develop
signs of heart failure that may be rapidly fatal unless
treatment with carnitine is begun. Patients with the
same carnitine uptake defect may also present with
episodes of hypoglycemic coma. Patients with this presentation tended to be younger infants and had developed little or no evidence of cardiomyopathy. Because
both types of presentations occurred in one family (Patients #2, #15), it is clear that the differences in clinical manifestations do not reflect different underlying
genetic defects. Rather, it appears that other circumstances related to fasting stress, as in Patient #lo, or
a carnitine-deficient diet, as in Patient #1, may provoke an acute episode of illness before the cardiomyopathy is fully developed.
Several features of carnitine uptake defect distinguish it from other genetic disorders of fatty acid oxidation 111). Plasma and tissue carnitine deficiency is
more severe than that seen with the secondary carnitine deficiency associated with P-oxidation enzyme defects, such as medium-chain or long-chain acyl coenzyme A (acyl-CoA) dehydrogenase deficiency, in which
carnitine levels are 25 to 50% of normal. Abnormal
increases in urinary dicarboxylic acids, seen during fasting with P-oxidation enzyme defects, are not a feature
in patients with carnitine uptake defect. This finding
may reflect the high level of block in the pathway of
fatty acid oxidation, because an abnormal dicarboxylic
aciduria is also not found with the hepatic form of
carnitine palmityl-transferase 1 deficiency [ 12). Finally,
in contrast to all other known fatty acid oxidation defects, the most common clinical manifestation of carnitine uptake defect is cardiomyopathy, rather than hypoglycemic coma.
The carnitine deficiency associated with the uptake
defect is dual in nature. First, renal conservation of
carnitine is impaired, resulting in extremely low plasma
concentrations of carnitine. Second, tissues that express the defect, such as muscle and fibroblasts, cannot
concentrate carnitine, and, therefore, intracellular carnitine levels increase very little when carnitine is given
to increase plasma levels to normal (see Table 1, Fig
3 ) . As we and others have noted [ 3 , 4, 61, the clinical
and in vitro findings in patients with a carnitine uptake
November 1991
defect are compatible with a disorder of a plasma membrane transport system for carnitine [13- 171. The specific transport system that may be affected appears to
be one that is expressed by the kidney, as well as by
heart and skeletal muscle, but not the liver. This inference is based on the observations in many patients
that renal conservation of carnitine is impaired and that
muscle carnitine levels respond poorly to treatment,
whereas, in the 1 patient who was tested, liver carnitine
levels increased to nearly normal on treatment. In addition, the low Km and the preference for the L-isomer
of carnitine that characterize fibroblast carnitine uptake
are similar to those found in studies of carnitine transport in heart and muscle {13, 141 but very different
from the findings reported in liver [15}.
The carnitine uptake defect appears to have more
severe consequences for muscle than for other tissues,
such as liver or fibroblasts. If it is true that the disorder
does not affect the liver system for carnitine uptake,
the liver may be able to maintain adequate concentrations of carnitine for fatty acid oxidation unless plasma
concentrations decrease too close to zero. For example,
ketogenesis was impaired in Patient #10 following several days of illness and 40 carnitine intake, but was
normal a month later when he was healthy and on his
usual carnitine-containing diet. In addition, heart and
skeletal muscle may have a higher requirement for carnitine than other tissues. The Km for capitine of carnitine palmityl-transferase 1 in cardiac and skeletal muscle has been reported to be 5- to 10-fold higher than
for the isoform of the enzyme in liver [IS}. Fibroblasts
also express the liver isoform of carnitine pdmityltransferase 1 [19}, which may explain why very little
carnitine was required to maximize fatty acid oxidation
in fibroblasts with the uptake defect (see Fig 4).
The carnitine uptake system expressed in fibroblasts
is potently inhibited by acyl-carnitines, particularly the
esters of medium-chain and long-chain fatty acids (see
Table 3). These data are also consistent with those
reported by Bohmer and associates 120) on carnitine
transport by myocytes. This observation may have relevance to the development of secondary carnitine deficiency in fatty acid P-oxidation enzyme disorders, such
as medium-chain acyl-CoA dehydrogenase deficiency
[1l}. Patients with medium-chain acyl-CoA dehydrogenase deficiency have been reported to have an impairment in the renal threshold for free carnitine 121231. It is possible that the medium-chain or long-chain
acyl-carnitines that accumulate in these patients cause
carnitine deficiency by inducing a defect in cellular uptake of free carnitine similar to but less severe than
that found in patients with a carnitine uptake defect.
The data on parents of patients affected with the
carnitjne uptake defect indicate an autosomal recessive
mode of inheritance. Although parents are asymptomatic, the carnitine uptake defect is p d y expressed.
They have modestly reduced plasma levels of carnitine,
which presumably reflects some impairment in renal
conservation of carnitine. Their fibroblasts are unable
to maintain normal levels of carnitine. Tissue levels
of carnitine were not measured in parents, but it is
reasonable to expect that muscle carnitine levels would
be lower in heterozygotes both because of their lower
plasma levels and because of their partial defect in muscle uptake.
In patients with the carnitine uptake defect, most
of the clinical signs of myopathy were corrected with
carnitine treatment, even though skeletal muscle carnitine concentrations remained far below normal. The
persistently low levels in muscle are consistent with
the data in fibroblasts showing that, ip the absence Qf
high-affinity carnitine uptake, intracellular concentrations of carnitine passively follow the extracellular concentrations. Thus, it is possible that carnitine does not
become a limiting factor for fatty acid oxidation in muscle as long as the intracellular concentration is 30 to
50 p-mol/l or more (i.e., approximately 2-496 of normal). If these observations can be applied to other settings, it would appear unlikely that clinical disease of
skeletal muscle can be ascribed to deficiency of carnitine unless the tissue concentration is below this range.
This work was supported in part by grants from the National Institutes of Health (NS 17752 and RR 00240) and Sigma Tau, Inc.
The authors thank Mrs Norma Govens for assistance in preparing
the manuscript.
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