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Improved stable isotope dilution-gas chromatography-mass spectrometry method for serum or plasma free 3-hydroxy-fatty acids and its utility for the study of disorders of mitochondrial fatty acid [beta]-oxidation.

In the fasting state, mitochondrial (beta]-oxidation of fatty acids is activated to maintain energy homeostasis. The utilization of this pathway varies between tissues. Tissues with a high energy demand (skeletal and cardiac muscle) are heavily dependent on fatty acid oxidation (FAO) [5] other tissues (brain) have limited ability to metabolize fatty acids but can readily utilize the ketone body end-products synthesized by the liver (1, 2). When energy demands increase or glycogen reserves are exhausted, e.g., at the time of fasting, increased muscular activity, or febrile illness, [beta]-oxidation becomes the principle supply of oxidizable substrates to sustain energy production. In infants, neonates, and young children who have limited glycogen reserves, FAO plays an even greater role under fasting conditions (3).

Disorders of fatty acid metabolism form a group of inborn errors that often represent a serious diagnostic challenge (4-7). 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 (11-18). 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.

Materials and Methods

CHEMICALS

Chemicals used in the synthesis of the natural and stable isotope-labeled 3-OH-FA compounds were of analytical grade or better. The following chemicals were obtained from Aldrich: butyraldehyde, hexyl aldehyde, octyl aldehyde, decyl aldehyde, dodecyl aldehyde, tetradecyl aldehyde, ethyl bromoacetate, and mossy zinc. The ethyl bromoacetate-(1,2-[sup.13][C.sub.Z]) used to make the isotope-labeled species was obtained from Cambridge Isotope Laboratories. Chloroform (HPLC grade), bovine albumin (essentially fatty acid free), iodine, calcium hydride, and anhydrous potassium carbonate, sodium carbonate, magnesium sulfate, and sodium sulfate were obtained from Sigma. Dichloromethane (reagent grade), ethyl acetate (HPLC grade), concentrated [H.sub.2]S[O.sub.4], and NaOH pellets were obtained from Mallinkrodt through Baxter Scientific. HCI (6 mol/L) was obtained from Ricca Chemical. Anhydrous ethanol was obtained from Electron Microscopy Sciences. The derivatizing reagent N,O-bis(trimethylsilyl)trifluoroacetamide and trimethylchlorosilane (BSTFA + TMCS, 99:1) was obtained from Supelco.

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[degrees]C overnight. All chemicals were dried before use as follows: 1,2-[sup.13][C.sub.2] ethyl bromoacetate over [K.sub.2]C[O.sub.3], each aldehyde over [Na.sub.2]S[O.sub.4], and diethyl ether and benzene were distilled from Ca[H.sub.2] and stored over MgS[O.sub.4]. The mossy zinc was washed with ether twice, and then dried overnight in an oven at 110[degrees]C. Immediately before use, the zinc was chopped into very small pieces, washed with ether, and dried in an oven at 110[degrees]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[degrees]C, and 10 mL of 100 mL/L [H.sub.2]S[O.sub.4] 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 [H.sub.2]S[O.sub.4], once with 2 mL of 100 g/L [Na.sub.2]C[O.sub.3], 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 HCI. 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 [Na.sub.2]S[O.sub.4], 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-[sup.13][C.sub.2] ethyl bromoacetate was used, which substituted [sup.13]C in place of the [sup.12]C 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-[C.sub.10] to 3-OH-[C.sub.16]); (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 [sup.13]C 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 [micro]mol/L. The stable isotope-labeled and natural compounds for 3-OH-hexanoic acid (3-OH.-[C.sub.6]) were dissolved in anhydrous ethanol. The isotope-labeled and natural compounds for 3-OH-octanoic acid (3-OH-[C.sub.8]), 3-OH-decanoic acid (3-OH-[C.sub.10]), 3-OH-dodecanoic acid (3-OH-[C.sub.12]) and 3-OH-tetradecanoic acid ([3-OH-C.sub.14]) were dissolved in chloroform. The natural 3-OH-hexadecanoic acid (3-OH-[C.sub.16]) 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 ([C.sub.14] to [C.sub.18]). 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 [alpha]-subunit. The first patient showed absent [alpha] and [beta] subunits, and the second patient showed absent [alpha] subunit and severely decreased [beta] 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 [C.sub.4] hydroxy-acyl-CoA dehydrogenase (CHAD) activity in biopsied liver tissue (172.2 nmol * [min.sup.-1] * mg [protein.sup.-1]; controls, 668.3-1146.1 nmol * [min.sup.-1] * mg [protein.sup.-1]) with normal [C.sub.16] CHAD activity (213.5 nmol * [min.sup.-1] * mg [protein.sup.-1]; controls, 184.1-426.7 nmol * [min.sup.-1] * mg [protein.sup.-1]). Both enzymes in skeletal muscle demonstrated normal activity [[C.sub.4] CHAD activity, 380.9 nmol * [min.sup.-1] * mg [protein.sup.-1] (controls, 337.4-560.8 nmol *[min.sup.-1] * mg [protein.sup.-1]); [C.sub.16] CHAD activity, 98.8 nmol *[min.sup.-1] * mg [protein.sup.-1] (controls, 78.9-178.7 nmol * [min.sup.-1] * mg [protein.sup.-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 [C.sub.4] CHAD activity was 194.1 nmol * [min.sup.-1] * mg [protein.sup.-1] (controls, 472.6-785.2 nmol * [min.sup.-1] * mg [protein.sup.-1]). Liver [C.sub.16] CHAD activity was 248.0 nmol * [min.sup.-1] * mg [protein.sup.-1] (controls, 213.6-305.4 nmol * [min.sup.-1] * mg [protein.sup.-1]).

SAMPLE PREPARATION

To 500 [micro]L of sample was added 5 nmol of each of the six 3-OH-FA isotope-labeled calibrators, [3-OH-C.sub.6] to [3-OH-C.sub.16], These calibrators were added by pipetting 10 [micro]L of each 500 [micro]mol/L calibrator into the sample separately and then vortex-mixing the mixture hard for 10 s. In this way, adequate mixing of the 60 [micro]L of isotope-labeled calibrators and the 500 [micro]L of sample was achieved. The samples were acidified with 125 /,L 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 [Na.sub.2]S[O.sub.4]. The samples were centrifuged again, decanted into clean tubes, and then dried down at 37[degrees]C under nitrogen.

DERIVATIZATION

After drying, the samples were derivatized by the addition of 100 [micro]L of BSTFA + TMCS (99:1). The samples were allowed to react for 45 min at 75[degrees]C, and 1 [micro]L was used for analysis.

GC-MS ANALYSIS

GC-MS analysis was carried out on a Hewlett-Packard 5890 Series 11 gas chromatograph with a 5972 Series quadrupole mass spectrometer. Helium was used as the carrier gas. A split/splitless injector at 270[degrees]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-[micro]m film of cross-linked 5% PH ME Siloxane. The initial oven temperature was 80[degrees]C for 5 min. The oven temperature was then programmed to rise 3.8[degrees]C/min to a temperature of 140[degrees]C, rise 2.3[degrees]C/min to a temperature of 200[degrees]C, and then rise 15.0[degrees]C /min to 290[degrees]C, where it remained for 6 min. The column was inserted directly into the ion source at an interface temperature of 290[degrees]C. This system utilizes electron impact ionization. 3-OH-FAs were detected by selected-ion monitoring for the [[M - C[H.sub.3].sup.+] 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 [micro]mol/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 - C[H.sub.3]].sup.+] 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.

Results

The [[M - C[H.sub.3]].sup.+] fragment ions used to identify and quantify the 3-OH-FAs are shown in Table 1. These [[M - C[H.sub.3]].sup.+] 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 - C[H.sub.3]].sup.+] ion for [3-OH-C.sub.12] (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-C.sub.12] and the m/z 345/ 347 pair to confirm identity, instead of the reverse.

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 [micro]mol/L. The lower limit of detection, defined as 3 SD above the measured blank average, was 0.2 [micro]mol/L. The precision and accuracy data for the assay are shown in Table 3.

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 - C[H.sub.3]].sup.+] 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 - C[H.sub.3]].sup.+] 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 [C.sub.14] and [C.sub.16], whereas in SCHAD deficiency, the medium chain 3-OH-FAs, [C.sub.6] to [C.sub.10], predominate, with a [3-OH-C.sub.8] concentration higher than the [3-OH-C.sub.6] concentration and normal excretion of [3-OH-C.sub.14] and [3-OH-C.sub.16].

Discussion

Mitochondrial fatty acid [beta]-oxidation defects are difficult to diagnose, mainly because of the absence of routinely detectable metabolites in most clinically asymptomatic patients, especially in the fed state. Our results in healthy subjects indicate that measurable amounts of the 3-OH-FA intermediates of mitochondrial FAO can be determined in serum and plasma samples. This assay may be useful for helping to identify disorders of the 3-hydroxyacyl-CoA dehydrogenases (Fig. 2) 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 [beta]-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 [beta]-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-C.sub.8] concentration that is greater than the [3-OH-C.sub.6] 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-C.sub.8] concentration higher than the [3-OH-C.sub.6] conccentration, the ratio of [3-OH-C.sub.8] to [3-OH-C.sub.6] 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 [beta]-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.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

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 [beta]-oxidation pathway in other disorders and conditions that may lead to an accumulation of FAO intermediates.

Received July 29, 1999; accepted November 9, 1999.

References

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(6.) Kamijo T, Indo Y, Souri M, Aoyama T, Hara T, Yamamoto S, et al. Medium chain 3-ketoacyl-coenzyme A thiolase deficiency: a new disorder of mitochondrial fatty acid R-oxidation. Pediatr Res 1997; 42:569-76.

(7.) Nyhan WL. Diagnosing disorders of fatty acid oxidation. Clin Chem 1995;41:10-1.

(8.) Bennett MJ. Fatty acid oxidation: normal metabolism and genetic diseases. Diagn Endocrinol Metab 1997;15:13-20.

(9.) Roe CR, Coates PM. Mitochondrial fatty acid oxidation disorders. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The metabolic and molecular bases of inherited disease, 7th ed. New York: McGraw-Hill, 1995:1501-33.

(10.) Bennett MJ. The laboratory diagnosis of inborn errors of fatty acid oxidation. Ann Clin Biochem 1990;27:519-31.

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(12.) Kelly DP, Whelan AJ, Ogden ML, Alpers R, Zhang Z, Bellus G, et al. Molecular characterization of inherited medium-chain acyl-CoA dehydrogenase deficiency. Proc Natl Acad Sci U S A 1990;87: 9236-40.

(13.) Brackett JC, Sims HF, Steiner R, Nunge M, Zimmerman EM, deMartinville B, et al. A novel mutation in medium-chain acyl-CoA dehydrogenase causes sudden neonatal death. J Clin Investig 1994;94:1477-83.

(14.) Brackett JC, Sims HF, Rinaldo P, Shapiro S, Powell CK, Bennett MJ, Strauss AW. Two a-subunit donor splice mutations cause trifunctional protein deficiency. J Clin Investig 1995;95:2076-82.

(15.) IJIst L, Wanders RJA, Ushikubo S, Kamijo T, Hashimoto T. Molecular basis of long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency. Biochim Biophys Acta 1994;1215:347-50.

(16.) Sims HF, Brackett JC, Powell CK, Treem WR, Hale DE, Bennett MJ, et al. The molecular basis of pediatric long chain 3-hydroxyacyl-CoA dehydrogenase deficiency associated with acute fatty liver of pregnancy. Proc Natl Acad Sci U S A 1995;92:841-5.

(17.) Ulst L, Ruiter JPN, Hoovers JMN, Jakobs ME, Wanders RJA. Common missense mutation G1528 in long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency. J Clin Investig 1996;98:1028-34.

(18.) Tyni T, Palotie A, Viinika L, Valanne L, Salo MK, von Dobeln U, et al. Long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency with the G1528C mutation. J Pediatr 1997;130:67-76.

(19.) Costa CG, Dorland L, Holwerda U, Tavares de Almeida I, Poll-The B-T, Jakobs C, Duran M. Simultaneous analysis of plasma free fatty acids and their 3-hydroxy analogs in fatty acid R-oxidation disorders. Clin Chem 1998;44:463-71.

(20.) Morrison RT, Boyd RN. Reformatsky reaction. Preparation of R-hydroxy esters. In: Morrison RT, Boyd RN. Organic chemistry, 3rd ed. Boston, MA: Allyn and Bacon, 1973:720-2.

(21.) Bennett MJ, Spotswood SD, Ross KF, Comfort S, Koonce R, Boriack RL, et al. Fatal hepatic short-chain L-3-hydroxyacyl-coenzyme A dehydrogenase deficiency: clinical, biochemical and patho logical studies on three subjects with this recently identified disorder of mitochondrial R-oxidation. Pediatr Develop Pathol 1999;2:337-45.

(22.) Thompson GN, Hsu BYL, Pitt JJ, Treacy E, Stanley CA. Fasting hypoketotic coma in a child with deficiency of mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase. N Engl J Med 1997;337: 1203-7.

[5] Nonstandard abbreviations: FAO, fatty acid oxidation; 3-OH-FA, 3-hydroxy-fatty acid; BSTFA, N,O-bis(trimethylsilyl)trifluoroacetamide; TMCS, trimethylchlorosilane; GC, gas chromatography; MS, mass spectrometry; MCT, medium-chain triglyceride; LCHAD and SCHAD, long- and short-chain 3-hydroxy-acyl-CoA dehydrogenase; FIT, trifunctional protein; and CHAD, hydroxy-acyl-CoA dehydrogenase.

PATRICIA M. JONES, [1] * REBECCA QUINN, [1] PAUL V. FENNESSEY, [2] SUSAN TJOA, [2] STEPHEN I. GOODMAN, [2] STEPHANY FIORE, [1] ALBERTO B. BURLINA, [3] PIERO RINALDO, [4] RICHARD L. BORIACK, [1] and MICHAEL J. BENNETT [1]

[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, 1-35128 Padua, Italy.

[4] Mayo Clinic, Department of Laboratory Medicine and Pathology, Rochester, MN 55905.

* 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@CHILDMED.DALLAS.TX.US.
Table 1. The 3-OH-FA natural and isotope-labeled ions.

 [M - C[H.sub.3]] + ion
3-OH-FA Natural Isotope-labeled

3-OH-Hexanoic acid 261 263
3-OH-Octanoic acid 289 291
3-OH-Decanoic acid 317 319
3-OH-Dodecanoic acid 345 347
3-OH-Tetradecanoic acid 373 375
3-OH-Hexadecanoic acid 401 403

Table 2. Linear regression analysis of calibration curves.

3-OH-FA Equation of the line *

3-OH-Hexanoic acid y = (0.098 [+ or -] 0.006)x -
 (0.005 [+ or -] 0.02)

3-OH-Octanoic acid y = (0.13 [+ or -] 0.003)x -
 (0.001 [+ or -] 0.01)

3-OH-Decanoic acid y = (0.105 [+ or -] 0.005)x -
 (0.006 [+ or -] 0.02)

3-OH-Dodecanoic acid (345/347) y = (0.103 [+ or -] 0.005)x -
 (0.005 [+ or -] 0.03)

3-OH-Dodecanoic acid (233/235) y = (0.102 [+ or -] 0.005)x +
 (0.003 [+ or -] 0.03)

3-OH-Tetradecanoic acid y = (0.112 [+ or -] 0.01)x +
 (0.003 [+ or -] 0.04)

3-OH-Hexadecanoic acid y = (0.100 [+ or -] 0.006)x +
 (0.008 [+ or -] 0.04)

3-OH-FA [r.sup.2]

3-OH-Hexanoic acid 0.9989
3-OH-Octanoic acid 1.9998
3-OH-Decanoic acid 1.9999
3-OH-Dodecanoic acid (345/347) 1.9998
3-OH-Dodecanoic acid (233/235) 0.9992
3-OH-Tetradecanoic acid 0.9838
3-OH-Hexadecanoic acid 0.9990

(a) Each equation is expressed as mean [+ or -] SD (n = 4).

Table 3. Results of replicated calibration curves for the six 3-OH-FAs.

Target values

 30 [micro]mol/L
3-OH-FA Mean (a) CV, % Diff, % (b)

3-OH-[C.sub.6] 28.4 6.9 -5.3
3-OH-[C.sub.8] 29.3 7.3 -2.3
3-OH-[C.sub.10] 29.1 5.0 -3.0
3-OH-[C.sub.12] (c) 28.9 1.0 -3.7
3-OH-[C.sub.12] (d) 30.1 3.8 0.3
3-OH-[C.sub.14] 29.2 4.0 -2.7
3-OH-[C.sub.16] 28.2 10.5 -6.0

 8 [micro]mol/L
3-OH-FA Mean CV, % Diff, %

3-OH-[C.sub.6] 8.0 11.3 0.0
3-OH-[C.sub.8] 8.6 5.8 7.5
3-OH-[C.sub.10] 7.7 3.4 -3.8
3-OH-[C.sub.12] (c) 8.1 2.8 1.3
3-OH-[C.sub.12] (d) 7.9 3.1 -1.3
3-OH-[C.sub.14] 7.1 11.7 -11.2
3-OH-[C.sub.16] 8.1 8.6 1.3

 0.3 [micro]mol/L
3-OH-FA Mean CV, % Diff, %

3-OH-[C.sub.6] 0.31 12.1 3.3
3-OH-[C.sub.8] 0.295 12.5 -1.7
3-OH-[C.sub.10] 0.27 12.0 -10.0
3-OH-[C.sub.12] (c) 0.31 12.4 3.3
3-OH-[C.sub.12] (d) 0.28 5.2 -6.7
3-OH-[C.sub.14] 0.26 10.9 -13.3
3-OH-[C.sub.16] 0.28 15.4 -6.7

(a) Mean concentrations in [micro]mol/L. n = 4 for each compound
at each concentration.

(b) Diff, relative difference between target concentration
and calculated mean.

(c) 345/347 ions.

Table 4. 3-OH-FA concentrations (a) in controls and in
patients receiving MCT formula.

 Controls, MCT formula,
 [micro]mol/L [micro]mol/L
 3-OH-FA (n = 43) (n = 20)

3-OH-Hexanoic acid 0.8 (0.3-2.2) 4.2 (1.6-18.5)
3-OH-Octanoic acid 0.4 (0.2-1.0) 2.5 (0.4-8.3)
3-OH-Decanoic acid 0.3 (0.2-0.6) 0.5 (0.2-2.3)
3-OH-Dodecanoic acid 0.3 (0.2-0.6) 0.4 (0.2-1.4)
3-OH-Tetradecanoic acid 0.2 (0-0.4) 0.2 (0-0.9)
3-OH-Hexadecanoic acid 0.2 (0-0.5) 0.2 (0-0.8)

(a) Values are medians (ranges).
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Title Annotation:Molecular Diagnostics and Genetics
Author:Jones, Patricia M.; Quinn, Rebecca; Fennessey, Paul V.; Tjoa, Susan; Goodman, Stephen I.; Fiore, Ste
Publication:Clinical Chemistry
Date:Feb 1, 2000
Words:5178
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