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1
University of Texas Southwestern Medical Center, Department of Pathology, and Children’s Medical Center of Dallas, TX 75235.
2
University of Colorado Health Science Center, Department
of Pediatrics, Denver, CO 80262.
3
University of Padua, Department of Pediatrics, I-35128
Padua, Italy.
4
Mayo Clinic, Department of Laboratory Medicine and
Pathology, Rochester, MN 55905.
a Address correspondence to this author at: Children’s Medical Center, Department of Pathology, 1935 Motor St., Dallas, TX 75235. Fax 214-456-6199; e-mail PJONES{at}CHILDMED.DALLAS.TX.US
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: We used stable isotope dilution gas chromatography-mass spectrometry (GC-MS) with electron impact ionization and selected ion monitoring. Natural and isotope-labeled compounds were synthesized for the assay.
Results: The assay was linear from 0.2 to 50 µmol/L for all six 3-OH-FAs. CVs were 5–15% at concentrations near the upper limits seen in healthy subjects. In 43 subjects, the medians (and ranges) in µmol/L were as follows: 3-OH-C6, 0.8 (0.3–2.2); 3-OH-C8, 0.4 (0.2–1.0); 3-OH-C10, 0.3 (0.2–0.6); 3-OH-C12, 0.3 (0.2–0.6); 3-OH-C14, 0.2 (0.0–0.4); and 3-OH-C16, 0.2 (0.0–0.5). 3-OH-FAs were increased in infants receiving formula containing medium chain triglycerides. Two patients diagnosed with LCHAD deficiency showed marked increases in 3-OH-C14 and 3-OH-C16 concentrations. Two patients diagnosed with SCHAD deficiency showed increased shorter chain 3-OH-FAs but no increases in 3-OH-C14 to 3-OH-C16.
Conclusion: Measuring blood concentrations of the 3-OH-FAs with this assay may be a valuable tool for helping to rapidly identify deficiencies in LCHAD and SCHAD and may also provide useful information about the status of the FAO pathway.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Disorders of fatty acid metabolism form a group of inborn errors that often represent a serious diagnostic challenge (4)(5)(6)(7). Clinical symptoms and abnormal concentrations of characteristic biochemical metabolites may become detectable only under conditions of acute metabolic decompensation. Symptoms of FAO disorders may be mild to life threatening, depending on the specific defect and the amount of flux through the pathway (8).
Currently, a diagnostic work-up frequently starts with abnormal metabolites detected in urine by organic acid and acylglycine analyses and in plasma by acylcarnitine profiling. Follow-up testing includes FAO studies on fibroblast culture to determine flux through the overall pathway, and specific catalytic assays in leukocytes or fibroblast culture (9)(10). In recent years, molecular testing of many disorders has become a reality, following the identification of the genes responsible for many of the mitochondrial FAO disorders (11)(12)(13)(14)(15)(16)(17)(18). Unfortunately, the initial finding of abnormal metabolites in urine and plasma often is critically dependent on the timing of sample collection, the most informative being when the FAO pathway is activated during fasting or metabolic stress. If an acutely ill patient is treated with conventional emergency measures, the intravenous infusion of glucose is likely to rapidly shut down FAO in favor of glycolysis; subsequently, abnormal metabolites will disappear, and the diagnosis may be missed.
Costa et al. (19) recently reported plasma free fatty acid and 3-hydroxy-fatty acid (3-OH-FA) concentrations in various FAO disorders. The method described here improves on their assay by using stable isotope-labeled calibrators to quantify the free 3-OH-fatty acids, and confirms the clinical utility of their measurement in plasma for the biochemical diagnosis of selected FAO disorders.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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synthesis of natural and isotope-labeled
3-OH-FAs
3-OH-FAs, both natural and isotope-labeled compounds, were
synthesized using the Reformatsky reaction (20). All
glassware used in the synthesis was oven dried at 110 °C overnight.
All chemicals were dried before use as follows:
1,2-13C2-ethyl bromoacetate over
K2CO3, each aldehyde over
Na2SO4, and diethyl ether and benzene were
distilled from CaH2 and stored over MgSO4. The
mossy zinc was washed with ether twice, and then dried overnight in an
oven at 110 °C. Immediately before use, the zinc was chopped into
very small pieces, washed with ether, and dried in an oven at 110 °C
for 15 min.
For the synthesis of each compound, a three-necked flask was fitted with a stirrer, a water-chilled condenser fitted with drying tube, and a dropping funnel, and then was placed in a water bath. Zinc (2 g) and a crystal of iodine were placed in the flask. The following mixture was placed in the dropping funnel: 0.025 mol of ethyl bromoacetate, 0.030 mol of the specific aldehyde needed (i.e., butyraldehyde to make 3-OH-hexanoic acid), 4 mL of benzene, and 1 mL of ether. Ten to 20 drops of the mixture in the funnel were added to the zinc in the flask to start the reaction (formation of fine bubbles). Once the reaction had started, the remaining solution was added over a 30-min period. The mixture was heated to reflux for an additional 30 min. The contents of the three-necked flask were cooled to 5 °C, and 10 mL of 100 mL/L H2SO4 was added with vigorous stirring. The mixture was transferred to a separatory funnel, and the aqueous layer was separated and discarded. The organic layer was washed twice with 5 mL each time of 100 mL/L H2SO4, once with 2 mL of 100 g/L Na2CO3, and then twice with 2 mL each time of water. The aqueous layer was discarded after each wash. The organic layer was then transferred to a 25-mL round-bottomed flask, and the solvents were removed on a rotary evaporator. The remaining 3-OH-ester was hydrolyzed by adding 2 mL of 100 g/L NaOH and refluxing for 15 min. The mixture was cooled and made acidic with 6 mol/L HCl. The aqueous mixture was extracted with ether (three times with 5 mL each time), and the ether layers were separated and combined. The combined ether layers were dried over Na2SO4, filtered, and dried under nitrogen gas. The products were either a slightly yellow oil or crystalline. For the synthesis of the isotope-labeled species, 1,2-13C2-ethyl bromoacetate was used, which substituted 13C in place of the 12C in positions one and two of the corresponding compound and produced isotope-labeled compounds with weights that were 2 atomic mass units higher than the corresponding natural compound. Each of the natural and isotope-labeled compounds was subjected to the following chemical evaluations after synthesis to test for purity: (a) melting point after recrystallization for those compounds that were not liquid at room temperature (3-OH-C10 to 3-OH-C16); (b) gas chromatography (GC) to determine retention time index and ensure a single peak; and (c) mass spectrometry (MS) of the trimethylsilyl derivatives to ensure that the molecular weight was correct for the natural compounds and for the addition of two 13C atoms to the isotope-labeled compounds. All analyses demonstrated pure compounds of expected composition. Mass spectra revealed a pattern that reflected the structure of the authentic compounds. Because of the synthetic process, none of the natural compound was expected in the corresponding isotope-labeled compound, and the mass spectra confirmed this by showing no natural compound mass ions.
stable isotope-labeled calibrators
The stable isotope-labeled calibrators used in the assay, and the
natural compounds used for linearity and precision studies were made by
weighing the individual 3-OH-FAs and dissolving each to a concentration
of 500 µmol/L. The stable isotope-labeled and natural compounds for
3-OH-hexanoic acid (3-OH-C6) were dissolved in
anhydrous ethanol. The isotope-labeled and natural compounds for
3-OH-octanoic acid (3-OH-C8), 3-OH-decanoic acid
(3-OH-C10), 3-OH-dodecanoic acid
(3-OH-C12) and 3-OH-tetradecanoic acid
(3-OH-C14) were dissolved in chloroform. The
natural 3-OH-hexadecanoic acid (3-OH-C16) was
dissolved in chloroform, and the isotope-labeled form was dissolved in
dichloromethane.
control samples
Control samples were serum, heparinized plasma, or EDTA plasma
from healthy subjects. Citrated plasma was not acceptable because of
citrate interference in the mass spectrometric analysis. The control
group included 43 total samples from individuals who were nonfasted and
not on any special diet. Twenty of these were adults ranging in age
from 20 to 48 years. The other 23 were pediatric patients ranging in
age from 1 week to 18 years. The distribution statistics of the
3-OH-FAs analyzed in the two groups showed no significant difference;
therefore, the groups were combined into a single control group. Serum
samples were also obtained from 20 pediatric patients who were
receiving medium-chain triglycerides (MCTs) in their formulas.
fao disorder samples
Samples were obtained from two patients diagnosed with
long-chain 3-hydroxy-acyl-CoA dehydrogenase (LCHAD) deficiency and two
patients diagnosed with short-chain 3-hydroxy-acyl-CoA dehydrogenase
(SCHAD) deficiency. None of the patients was in metabolic crisis at the
time the samples were collected. The LCHAD-deficient patients both were
being managed with MCT supplementation and carnitine and were not to be
fasted. The samples were collected at routine follow-up clinic
visits. The first LCHAD-deficient patient presented at 9 months of age
with lethargy, severe hypoglycemia, cardiomegaly, hepatomegaly,
hypotonia, and pigmentary retinal degeneration. The second
patient presented at 15 months with acute respiratory failure, severe
cardiomyopathy, and peripheral myopathy. Urine organic acid analysis of
both patients by GC-MS during acute metabolic decompensation showed
dicarboxylic aciduria and 3-hydroxy-dicarboxylic aciduria.
Acylcarnitine analysis demonstrated increased concentrations of
long-chain acylcarnitines and long-chain 3-hydroxy acylcarnitines
(C14 to C18). Diagnosis was
confirmed in both patients by analysis of LCHAD activity in cultured
fibroblasts (B. Garavaglia, Besta Institute, Milan). Diagnoses were
also supported by immunological analysis for mitochondrial
trifunctional protein (TFP), of which LCHAD is encoded on the
-subunit. The first patient showed absent and ß subunits, and
the second patient showed absent subunit and severely decreased ß
subunit by Western blot with purified antibody to human TFP (R.J.A.
Wanders, University of Amsterdam).
The first patient with SCHAD deficiency presented in the first year of life with fulminant liver failure, eventually requiring liver transplantation at 9 months of age. This patient demonstrated developmental delay, hepatosplenomegaly, and intermittent hypoglycemia. At the time the sample was collected, the patient was not in metabolic crisis but was experiencing increasingly severe liver failure. SCHAD deficiency is a relatively newly described FAO disorder; most cases described to date have been postmortem (21). This patient was shown to have deficient C4 hydroxy-acyl-CoA dehydrogenase (CHAD) activity in biopsied liver tissue (172.2 nmol · min-1 · mg protein-1; controls, 668.3–1146.1 nmol · min-1 · mg protein-1) with normal C16 CHAD activity (213.5 nmol · min-1 · mg protein-1; controls, 184.1–426.7 nmol · min-1 · mg protein-1). Both enzymes in skeletal muscle demonstrated normal activity [C4 CHAD activity, 380.9 nmol · min-1 · mg protein-1 (controls, 337.4–560.8 nmol · min-1 · mg protein-1); C16 CHAD activity, 98.8 nmol · min-1 · mg protein-1 (controls, 78.9–178.7 nmol · min-1 · mg protein-1)]. All tissue enzyme analyses were carried out as described previously (21).
The second SCHAD-deficient patient was an infant with failure to thrive, whose sibling had been diagnosed postmortem as having deficient liver SCHAD activity. The sample for this assay was drawn at 1 month of age when the infant was not metabolically decompensated. The infant died suddenly at 2 months of age, and enzyme activity studies performed on liver tissue demonstrated SCHAD deficiency. Liver C4 CHAD activity was 194.1 nmol · min-1 · mg protein-1 (controls, 472.6–785.2 nmol · min-1 · mg protein-1). Liver C16 CHAD activity was 248.0 nmol · min-1 · mg protein-1 (controls, 213.6–305.4 nmol · min-1 · mg protein-1).
sample preparation
To 500 µL of sample was added 5 nmol of each of the six 3-OH-FA
isotope-labeled calibrators, 3-OH-C6 to
3-OH-C16. These calibrators were added by
pipetting 10 µL of each 500 µmol/L calibrator into the sample
separately and then vortex-mixing the mixture hard for 10 s. In
this way, adequate mixing of the 60 µL of isotope-labeled calibrators
and the 500 µL of sample was achieved. The samples were acidified
with 125 µL of 6 mol/L HCl and then extracted twice with 3 mL of
ethyl acetate, each time by vortex-mixing vigorously for 30 s and
then centrifuging to separate the layers. The two extracted ethyl
acetate layers were combined and dried over anhydrous
Na2SO4. The samples were
centrifuged again, decanted into clean tubes, and then dried down at
37 °C under nitrogen.
derivatization
After drying, the samples were derivatized by the addition of 100
µL of BSTFA + TMCS (99:1). The samples were allowed to react for 45
min at 75 °C, and 1 µL was used for analysis.
gc-ms analysis
GC-MS analysis was carried out on a Hewlett-Packard 5890 Series II
gas chromatograph with a 5972 Series quadrupole mass spectrometer.
Helium was used as the carrier gas. A split/splitless injector at
270 °C introduced the sample onto a Hewlett-Packard HP-5MS capillary
column [30 m x 0.25 mm (i.d.)] coated with a 0.25-µm film of
cross-linked 5% PH ME Siloxane. The initial oven temperature
was 80 °C for 5 min. The oven temperature was then programmed to
rise 3.8 °C/min to a temperature of 140 °C, rise 2.3 °C/min to
a temperature of 200 °C, and then rise 15.0 °C/min to 290 °C,
where it remained for 6 min. The column was inserted directly into the
ion source at an interface temperature of 290 °C. This system
utilizes electron impact ionization. 3-OH-FAs were detected by
selected-ion monitoring for the [M -
CH3]+ fragments for the
natural compounds and isotope-labeled calibrators. The common
3-OH-signature ion at m/z 233 for the natural 3-OH-FAs and
at m/z 235 for the isotope-labeled 3-OH-FA calibrators was
also detected by selected-ion monitoring. A dwell time of 50 ms was
used.
assay validation and 3-oh-fa quantification
Calibration curves were constructed for each of the six natural
3-OH-FAs and for a combination of all six to determine the linearity,
precision, and accuracy of the assay. These curves were constructed by
adding 0.01–200 µmol/L of the six natural 3-OH-FAs into essentially
fatty-acid-free albumin at physiological protein concentrations and
then adding 5 nmol of each of the isotope-labeled calibrators to each
sample, as described above. The signal-response ratio of natural
compound to isotope-labeled calibrator was then plotted against the
known analyte concentration that was added. These calibration curves
were subjected to linear regression analysis, using the signal-response
ratio as the dependent variable. To evaluate the method, we repeated
calibration curves (n = 4) and used the signal-response ratios to
back-calculate concentrations from the derived regression equations.
Quantification of the six natural 3-OH-FAs in patient samples was accomplished by use of the regression equation for each compound’s calibration curve with the mean slope and intercept. The measured signal responses of the [M - CH3]+ fragment ions from the natural compounds and the isotope-labeled calibrators were first obtained, and the ratio of natural compound to isotope-labeled calibrator was calculated. This signal-response ratio was then plugged back into the specific regression equation to calculate the amount of natural compound in the patient sample.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. These [M -
CH3]+ ions varied with the
molecular weight of the compound. All of the 3-OH-FAs also had ions
resulting from the 3-OH fragments, which were m/z 233 for
the natural compound and m/z 235 for the isotope-labeled
calibrators. These fragments were used to help identify the correct
compounds. The m/z 233 ions were common to any compound
having a 3-OH group. The [M -
CH3]+ ion for
3-OH-C12 (m/z 347) is also a fragment
of citrate, which unfortunately has a very similar retention time. This
was found to interfere if the citrate concentration in the sample was
excessively increased, i.e., citrated plasma. This impediment was
resolved by using the m/z 233/235 ion pair to quantify
3-OH-C12 and the m/z 345/347 pair to
confirm identity, instead of the reverse.
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The data from the linear regression analysis of the calibration curves
are shown in Table 2
. All six 3-OH-FAs were linear in the concentration range
0.2–50 µmol/L. The lower limit of detection, defined as 3 SD above
the measured blank average, was 0.2 µmol/L. The precision and
accuracy data for the assay are shown in Table 3
.
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The data for apparently healthy individuals (n = 43) and for
infants receiving formula containing MCTs (n = 20) are shown in
Table 4
. The median values and ranges for these six 3-OH-FAs show a
pattern of decreasing concentration with increasing chain
length. Fig. 1
illustrates the scans obtained with the [M -
CH3]+ fragment ions used
to quantify the natural 3-OH-FAs from an abnormal patient (Fig. 1A
) and
a control patient (Fig. 1B
), and the [M -
CH3]+ isotope-labeled
calibrator (Fig. 1C
). Fig. 2
demonstrates the range pattern and concentrations in the
healthy controls and also shows the abnormal patterns and
concentrations displayed by patients who have been diagnosed with LCHAD
and SCHAD deficiencies. In LCHAD deficiency, there is a marked increase
in 3-OH-FAs of chain lengths C14 and
C16, whereas in SCHAD deficiency, the medium
chain 3-OH-FAs, C6 to C10,
predominate, with a 3-OH-C8 concentration higher
than the 3-OH-C6 concentration and normal
excretion of 3-OH-C14 and
3-OH-C16.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) in a timely manner. The assay can be performed
in <4 h, which supplies information about possible defects in these
dehydrogenases long before enzyme assays based on cultured fibroblasts
can be run. The assay may also be useful for providing information
about other conditions during which there is increased flux through the
FAO pathway. Infants receiving formulas containing MCTs demonstrated
increased concentrations of the medium-chain 3-OH intermediates of this
ß-oxidation pathway, as would be expected for the metabolism of these
compounds. Thompson et al. (22) reported a child
with 3-hydroxy-3-methylglutaryl-coenzyme A synthase deficiency
who showed increased medium-chain 3-OH-FAs after MCT feeding. With this
deficiency, the products of mitochondrial FAO could not be utilized for
ketogenesis, and so might accumulate. The assay may provide information
in disorders in enzymes outside the fatty acid ß-oxidation pathway
that could lead to accumulation of FAO metabolites, i.e., disorders in
the ketogenic pathways, or possibly in the respiratory chain. The use of a combination of the pattern of increases and the actual concentrations may be the most informative interpretation of the assay results. The "normal" pattern appears to be decreasing concentration with increasing chain length. Defects in the LCHAD and SCHAD enzymes appear to alter the pattern as well as increasing the concentrations. Apparently healthy patients show a normal pattern. Most of those patients who are receiving MCTs show the same normal pattern, but at increased concentrations. A flux through the FAO pathway appears to increase concentrations, but it generally does not alter the pattern of decreasing concentration with increasing chain length. To date, of 146 total patient samples assayed, an "abnormal" pattern with an increased 3-OH-C8 concentration that is greater than the 3-OH-C6 concentration was seen in six patients other than the two SCHAD patients. All six of these patients were infants <6 months of age, and all six were on MCT-containing formulas. Although these six patients did show a pattern similar to the SCHAD patients, with a 3-OH-C8 concentration higher than the 3-OH-C6 concentration, the ratio of 3-OH-C8 to 3-OH-C6 did not appear as high as in the patient having a defect in 3-hydroxyacyl-CoA dehydrogenase. More study will be necessary to determine whether this finding will continue to hold true.
This assay is useful for accurately measuring concentrations of the
3-OH-FAs in serum and plasma samples of individuals and in helping to
identify LCHAD and SCHAD deficiencies before the confirmatory enzyme
and molecular studies. Fig. 1
demonstrates the marked difference found
between a patient with abnormal accumulation of FAO intermediates and a
patient with normal FAO. As can be seen in Fig. 2
, patients with
deficiencies in the LCHAD and SCHAD enzymes do exhibit increased
concentrations of the 3-OH intermediates of the appropriate chain
lengths. This assay should be incorporated into metabolic testing
protocols, especially when mitochondrial FAO disorders are suspected,
because it could supply timely information in helping to identify these
disorders. Because the assay only measures increased concentrations of
the intermediates of the ß-oxidation pathway, however, it does not
distinguish between true LCHAD deficiency and TFP deficiency, in which
all three of the enzymes that make up the TFP are affected. Both
deficiencies would produce increased concentrations of long-chain
3-OH-FAs. Enzyme analysis of cultured cells and metabolic studies will
still be necessary for diagnostic confirmation.
The assay reported here is robust and, because of the sample extraction and the stable isotope dilution technique used, shows little interference by other components found in blood samples. Additional studies remain to be done, especially with more individuals having known disorders of FAO, to follow the effectiveness of this assay for diagnosis of the mitochondrial FAO defects. Because of the specific metabolites being measured, this assay will be most effective in helping to identify LCHAD and SCHAD deficiencies. Additional studies, however, will help to determine its possible utility for providing information about the mitochondrial fatty acid ß-oxidation pathway in other disorders and conditions that may lead to an accumulation of FAO intermediates.
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunit donor splice mutations cause trifunctional protein deficiency. J Clin Investig 1995;95:2076-2082.
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and •) and two LCHAD-deficient patients
(
and 
).