Clinical Chemistry 43: 273-278, 1997;
(Clinical Chemistry. 1999;43:273-278.)
© 1999 American Association for Clinical Chemistry, Inc.
Stability of long-chain and short-chain 3-hydroxyacyl-CoA dehydrogenase activity in postmortem liver
Daniel E. Hale1,a,
John E. Cornell2 and
Michael J. Bennett3
1
Department of Pediatrics, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dr., San Antonio, TX 78284.
2
Geriatric Research, Education and Clinical Center
and the South Texas Veterans Health Care System, Audie L. Murphy
Division; and Department of Medicine, University of Texas Health
Science Center at San Antonio, San Antonio, TX 78284.
3
Departments of Pathology and Pediatrics, University
of Texas Southwestern Medical Center, Children's Medical Center of
Dallas, 1935 Motor St., Dallas, TX 75235.
a Author for correspondence. Fax 210-567-6921; e-mail hale{at}uthscsa.edu
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Abstract
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Inherited enzyme defects in mitochondrial fatty acid oxidation (FAO) are
associated with acute metabolic crisis and sudden death. Necropsy
findings may be subtle, yielding no diagnosis and precluding genetic
counseling. Preliminary identification of an FAO disorder requires the
use of sophisticated tools (e.g., GC/MS) and specific body fluids, and
the diagnosis rests on molecular analysis or enzyme assay. At present,
confirmation of long-chain or short-chain 3-hydroxyacyl-CoA
dehydrogenase deficiency relies on measurement of enzyme activity.
Here, we report our examination of the effect of storage temperature
(25, 4, -20, and -70 °C) and the postmortem interval on enzyme
activities in rat and human liver. Enzyme activity decreases 50% in
30 h in samples stored at 25 °C, whereas 55 h at 4 °C
is required to reach this value; freezing minimizes this loss.
Regardless of rate of degradation, however, the short-chain to
long-chain activity ratio remains constant—which should make it
possible to differentiate postmortem degradation from enzyme
deficiency.
Key Words: indexing terms: sudden infant death syndrome • fatty acid oxidation • inherited metabolic disease • pediatric chemistry • sample handling • rats
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Introduction
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Inherited defects of mitochondrial fatty acid oxidation
(FAO) are a recognized cause of mortality in infants and
children who die
unexpectedly.1
Affected infants are often given the diagnosis of sudden
infant death syndrome (SIDS) (1)(2)(3)(4). Clues to
the correct diagnosis can be derived from observations of subtle
pathological findings such as hepatic steatosis or from measurements of
metabolic intermediates that accumulate in body fluids (blood, urine,
bile, vitreous humor) or tissues (5)(6)(7)(8)(9). The pattern of
abnormal intermediates can suggest the probable site of defect;
however, the confirmation of a defect relies on either direct assay for
a particular enzyme in the appropriate tissue or on the demonstration
of homozygosity for known disease-causing genetic mutations.
Frequently, one cannot confirm a diagnosis enzymatically because of
lack of information about the stability of specific enzymes in
postmortem tissues. Knowledge of enzyme stability is important because
most autopsies are performed several hours, or even days, after the
death of a child.
Long-chain and short-chain 3-hydroxyacyl-CoA dehydrogenases
(LCHAD and SCHAD, respectively) are
chain-length-specific forms of the third enzyme in the mitochondrial
ß-oxidation sequence. LCHAD activity resides in a membrane-bound
multienzyme complex that also contains long-chain 3-enoyl-CoA hydratase
and long-chain 3-ketoacyl-CoA thiolase activities (trifunctional
protein) (10)(11). SCHAD is a single activity
enzyme found on the mitochondrial matrix. Defects involving these
enzymes are increasingly recognized as a significant cause of morbidity
and mortality in infancy and childhood (12)(13)(14)(15)(16)(17)(18)(19). Correct
identification of the defect in an affected child has clear
implications for siblings, present or potential (12). It
also has ramifications for the mother during subsequent pregnancies
because LCHAD deficiency in the fetus has been associated with a severe
obstetric complication, acute fatty liver of pregnancy
(15). The present study was undertaken to establish the
postmortem lability of LCHAD and SCHAD activity in liver as a
prerequisite for confirming enzyme-based diagnoses in infants dying
suddenly. We present data from stability studies of the enzymes in both
rat and human liver tissues.
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Materials and Methods
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Rat liver tissue.
Five adult male Sprague–Dawley rats
were fasted overnight and decapitated at 0700. The abdomen was incised
and the liver surgically removed. Each liver was divided into six
pieces of approximately equal size and each piece was placed on a large
glass slide. These pieces were then stored as follows: (a)
at room temperature, (b) at room temperature in a humidified
chamber, (c) at 4 °C, (d) at 4 °C in a
humidified chamber, (e) at -20 °C, and (f) at
-70 °C. After 12, 24, 36, 48, 72, and 96 h, a small piece
(30–50 mg) was removed from each specimen, weighed, labeled, and
frozen at -70 °C. Livers from three additional rats were frozen at
-70 °C and sampled repeatedly over 5 years. We also took samples
from various lobes of the rat liver (anterior right, posterior right,
left, middle) and stored them frozen at -70 °C. All procedures
performed on animals were in accordance with ethical standards and were
approved by the Animal Use Committee of the Children's Hospital of
Philadelphia.
Human liver tissue.
Although studies similar to those
with rat liver cannot be performed with fresh, normal human liver, we
examined enzyme activity loss in livers from three SIDS victims in whom
the time of death could be determined from the history with a high
degree of certainty (±2 h). The time between discovery and sample
collection and freezing was short (<4 h after discovery; therefore,
4–6 h after demise). Small portions (0.1–0.2 g) of the livers were
sliced off and either prepared for immediate assay or stored at 4 °C
under humidified conditions for 6–72 h before assay.
We also examined the relation between the postmortem interval and the
change in LCHAD and SCHAD in liver tissue from 42 infants—samples that
were part of a series of liver samples collected for a large study of
the relation between FAO disorders and sudden death (FAO Study,
(1)). Of these children, 22 had a final diagnosis of SIDS,
8 died traumatically, and 12 had a specific nonmetabolic pathological
diagnosis; none had organic acids in urine suggestive of an FAO
disorder. Determination of the postmortem interval (time from death
until sample collection) was based on standard forensic markers and on
a direct interview with the family to ascertain the approximate time of
demise. All samples were stored at -70 °C from the time of sample
collection until the day of assay.
In both the SIDS and FAO studies, liver specimens were routinely taken
from the anterior portion of the right lobe of the liver. All studies
with human tissue were performed with the approval of the Ethical
Committee (Children's Hospital, Sheffield, UK) or the Institutional
Review Board (Children's Hospital of Philadelphia).
Sample preparation.
A portion (~20 mg) of tissue was
added to 1 mL of chilled (4 °C) 100 mmol/L potassium phosphate
buffer (pH 6.5, containing 0.1 mmol/L EDTA) and then was sonicated for
3 x 5 s at 40 W with a tissue sonicator. After each 5-s
sonication, the sample tube was returned to the ice bucket for at least
1 min. After the third (final) sonication, the solution was
homogeneous. We then centrifuged this homogeneous solution at
30 000g for 20 min at 4 °C and transferred the
supernatant liquid to a clean, labeled vial, which we placed on ice
until the enzyme assays were performed, usually within 2 h and
always within 4 h of tissue preparation. Preliminary experiments
demonstrated that enzyme activity toward C16
(3-ketopalmityl-CoA; Sigma Chemical Co., St. Louis, MO) and
C4 (acetoacetyl-CoA; Laroden Fine Chemicals,
Malmo, Sweden) substrate in liver supernate did not decline for at
least 4 h under these conditions.
Determination of enzyme activity.
LCHAD and SCHAD
activities were determined spectrophotometrically at 340 nm with the
C4 and C16 substrates and with NADH as the
electron donor. The final assay mixture contained, in a final volume of
1 mL, 100 mmol/L potassium phosphate, pH 6.5, 0.1 mmol/L EDTA, and 0.1
mmol/L NADH. Buffer was warmed to 30 °C and all assays were carried
out in a water-jacketed chamber maintained at 30 °C. Samples (1–20
µL, containing 2–10 µg of soluble protein) were added to this
mixture and the basal rate (for 1–3 min) was determined. The baseline
was generally stable and showed minimal change over 10 min. The
reaction was initiated by the addition of substrate (final
concentrations: C4, 50 µmol/L; C16, 20
µmol/L). Preliminary experiments did not reveal any difference in the
basal rate according to whether substrate or enzyme source was added
last. The subsequent reaction was linear for at least 5 min.
Each sample was assayed with each substrate 3–6 times. Intraassay
variability ranged from 0.3% to 5.7% and interassay variability
ranged from 0.5% to 8%. An aliquot of each sample was assayed for
protein according to the modified Lowry method, and enzyme activity is
expressed as nanomoles of NADH reduced per minute per milligram of
soluble protein. The results reported for each liver are the mean of
all assays run with a specific substrate.
SCHAD and LCHAD exhibit some overlap in substrate chain-length
specificity; in particular, SCHAD contributes ~10% of the total
activity measured with C16 (20).
Extramitochondrial enzymes with affinity for C4 may
contribute to measured SCHAD activity, but this cannot be evaluated in
postmortem frozen liver samples; LCHAD, however, has no activity with
C4. For purposes of simplicity and clarity, in the
remainder of this paper we refer to activity measured with
C16 substrate as LCHAD activity and to activity measured
with C4 substrate as SCHAD activity.
Statistics.
Repeated measures ANOVAs were used to test
for mean differences between storage conditions and duration of storage
for each enzyme (21). Storage conditions and enzyme type
were tested as the independent groups, and time was the repeated
measure. Dunnett's test for contrasts was used to evaluate specific
mean differences for significance of interactions between enzymes, type
of storage conditions, and duration of storage conditions. Measurements
taken at various times were contrasted with the baseline value at time
0. For some analyses, we examined the rate of change of enzyme activity
with time. An ANOVA of the rate of change was used to evaluate
differences in rate of loss of enzyme activity with time and storage
conditions. We evaluated mean differences in assay values across
pathology groups from the FAO study by ANCOVAs and used the Tukey
multiple comparison test to examine specific mean differences. Time was
the covariate in these analyses. All statistical tests were considered
significant at P <0.05.
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Results
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Enzyme activities in rat liver stored at -20 and -70 °C were
determined repeatedly over 5 years. Although SCHAD activity in tissue
stored at -70 °C varied somewhat, there was no difference between
the initial rates and the rates after 5 years. In contrast, there was a
slow but steady loss of SCHAD activity at -20 °C over the course of
the studies, which was significant at and beyond 24 months
(Fig. 1
, top). There was essentially no change in LCHAD activity over
the first 52 months at either temperature; after 60 months at
-20 °C, however, LCHAD activity was significantly different from
the initial values in the samples. There was no change in the
SCHAD/LCHAD ratio in any specimen over the 5 years of the study
(Fig. 1
, bottom). Moreover, LCHAD and SCHAD activity in the
four sampled regions of the rat liver were not significantly different
from each other, and the SCHAD/LCHAD ratio was the same in all regions
examined (data not shown).
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Figure 1. Effects of long-term storage on (top)
3-hydroxyacyl-CoA dehydrogenase activity and (bottom) the
SCHAD/LCHAD ratio in rat liver.
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The temperature of the liver, the storage conditions, and the time from
death until sample collection and freezing distinctly affect LCHAD and
SCHAD activity. In rat liver, 30–40% of LCHAD and SCHAD activity
is lost in the first 24 h when the tissue is stored at 25 °C
(Fig. 2
, top and middle panels). Storage at 4 °C retards the loss of
enzyme activity, and freezing the tissue at -20 or -70 °C prevents
any reduction in activity for 96 h. Samples stored in a
nonhumidified environment at either 25 or 4 °C showed no change in
activity from the basal rate in the first 12 h. Subsequently, the
rate at which activity was lost in the nonhumidified sample was similar
to that of the humidified specimen. After 12 h, the rate of loss
of activity was significantly more rapid at 25 than at 4 °C.
However, there was no statistical difference in the SCHAD/LCHAD ratio
under any condition (Fig. 2
, bottom).
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Figure 2. Effects of storage conditions and time on
(top) LCHAD activity, (middle) SCHAD
activity, and (bottom) the SCHAD/LCHAD ratio in rat liver.
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In the three SIDS samples stored in a humidified environment at
4 °C, increased storage time resulted in a loss of both LCHAD and
SCHAD activity; freezing the samples prevented any loss of activity. As
with rat liver, the SCHAD/LCHAD ratio remained constant throughout the
duration of the experiment (Fig. 3
). In addition, repeated sampling over the 5 years from four of
the frozen (-70 °C) human livers from the FAO study (two with high
activity and two with low activity at the time of the original assays;
data not shown) showed that neither the individual enzyme activities
nor the SCHAD/LCHAD activity ratio changed significantly during that
time. We did not examine the regionality of enzyme activity in human
liver.
Access to a large cohort of livers from children dying unexpectedly and
in whom the time of death was reasonably well-established (FAO study
livers) (1) allowed us to examine changes in LCHAD and
SCHAD activity from the perspective of the postmortem interval. Both
LCHAD and SCHAD activity decreased linearly with increasing postmortem
interval (Fig. 4
). Although the temperature of the liver between the time of the
child's death and sample collection and storage was unknowable, the
rate of activity loss was slightly more rapid than that of rat liver
stored at 4 °C and slower than that stored at 25 °C. The
specimens collected within 10 h after death had LCHAD and SCHAD
activities comparable with those found in the three closely studied
SIDS livers. The SCHAD/LCHAD ratio was constant, regardless of the
postmortem interval (Fig. 4
), and there were no significant
differences in the rate of loss of activity or in the SCHAD/LCHAD ratio
between those children dying from traumatic injuries, those with a
specific pathological diagnosis, and those having no pathologically
identifiable cause of death (SIDS).
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Figure 4. Effects of the postmortem interval on (A) LCHAD
activity, (B) SCHAD activity, and (C) the
SCHAD/LCHAD ratio in livers of children dying from various causes.
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Discussion
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Data from several studies suggest that the prevalence of the FAO
disorders in children is between 1:3000 and 1:10 000
(1)(9)(20)(22)(23). More accurate estimates of the frequency are not possible
because no single tool is currently sufficient to identify all children
with FAO defects. The association between inherited FAO disorders and
sudden unexpected death is well-documented
(1)(2)(3)(4)(8)(9)(10); however, the contribution of
the FAO disorders to the overall incidence of SIDS is still an area of
active investigation (20)(22)(23).
Analysis of metabolic intermediates such as organic acids,
acylglycines, and acylcarnitines is not sufficient to confirm a
diagnosis; instead, enzyme assays or molecular tests are needed for
confirmation.
LCHAD deficiency was first described in 1987 (13).
Presentation of this deficiency is varied and ranges from sudden
unexpected death through acute hepatic failure and chronic liver
disease (11)(12)(13)(14)(15)(16)(17)(18)(25)(26)(27). Muscle dysfunction
is also present, with both cardiomyopathy and skeletal muscle
involvement being recognized features. And ~50% of known cases
develop changes in retinal pigment and a peripheral neuropathy.
LCHAD is part of a multienzyme complex comprising four 
- and four
ß-subunits (10). LCHAD activity is associated with the
-subunit, as is the activity of long-chain enoyl-CoA hydratase. The
ß-subunit contains long-chain 3-ketoacyl-CoA thiolase activity. A
relatively common mutation (G1528
C) has been identified in children
with LCHAD (17)(19) and is thought to affect
the binding of NAD+ to the 3-hydroxyacyl-CoA dehydrogenase
region. This base substitution results in a new Pst 1
cleavage site and consequently permits a simple molecular approach to
diagnosis. However, the frequency of this mutation is considerably less
than the A985G mutation in medium-chain acyl-CoA dehydrogenase
deficiency; thus, screening for the G1528C mutation alone will
result in considerable underdiagnosis of LCHAD deficiency.
SCHAD deficiency was first described in 1991 (14); more
recently, an apparently liver-specific SCHAD deficiency has been
identified (28). Few patients have been identified to
date; therefore, little information on the clinical aspects of this
defect is available and no information on the molecular abnormalities
causing SCHAD deficiency. Accordingly, there is a need for direct
enzyme analyses for the diagnostic confirmation of both LCHAD and SCHAD
deficiencies. Our data, the first to be presented on the stability of
both enzymes in postmortem and stored tissues, demonstrate that
activity of both enzymes is lost at about the same rate. Consequently,
the ratio of SCHAD to LCHAD remains constant over many hours and in a
wide range of conditions—which should permit differentiation of
autolytic degradation from specific enzyme deficiency. As regards
enzyme degradation, the SCHAD/LCHAD ratio remains constant because both
enzyme activities are lost at the same rate. In contrast, the ratio
should shift upward in liver in LCHAD deficiency, as it does in
fibroblasts (13), and downward in SCHAD deficiency. Using
the direct enzyme assay described here gives valid results for at least
40 h in postmortem tissue, a time during which most necropsies are
performed.
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Acknowledgments
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We acknowledge the assistance of the Philadelphia County Medical
Examiner, Haresh Mirchandani, and his staff in obtaining small liver
specimens from children dying suddenly. We also thank Sadick Variend
and Rodney Pollitt for permitting us access to the liver tissues from
the Sheffield (UK) SIDS study. These studies were supported in part by
a fund for SIDS Research at The Children's Hospital of Philadelphia
and by the Foundation for the Study of Infant Deaths (London, UK).
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Footnotes
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1 Nonstandard abbreviations: FAO, fatty acid oxidation; SIDS, sudden infant death syndrome; LCHAD, long-chain 3-hydroxyacyl-CoA dehydrogenase; and SCHAD, short-chain 3-hydroxyacyl-CoA dehydrogenase. 
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Top
Abstract
Introduction
