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Addition of quantitative 3-hydroxy-octadecanoic acid to the stable isotope gas chromatography-mass spectrometry method for measuring 3-hydroxy fatty acids.

Mitochondrial fatty acid oxidation (FAO) is a catabolic pathway that supplies energy for the normal physiologic functioning of many tissues when glucose is unavailable, and it also supplies energy for some tissues even when glucose is available (1, 2). The FAO pathway is complex and not fully understood. Quantitative measurement of the concentrations of 3-hydroxy-fatty acids (3-OHFAs) in plasma or serum samples from individuals who are suspected of having a deficiency in FAO, especially in the enzyme step involving the L-3-hydroxyacyl-CoA-dehydrogenases, is a useful tool to aid in diagnosis (3, 4). This study adds the quantitative measurement of 3-hydroxyoctadecanoic acid (3-OH-C18) to the previously reported assay (4) that measures the six shorter chain-length FAO intermediates, 3-hydroxy-hexanoic acid (3-OH-C6), 3-hydroxyoctanoic acid (3-OH-C8), 3-hydroxy-decanoic acid (3-OH-C10), 3-hydroxy-dodecanoic (3-OH-C12), 3-hydroxytetradecanoic acid (3-OH-C14), and 3-hydroxyhexadecanoic acid (3-OH-C16).

3-OH-C18 was synthesized by the method of Jones et al. (4), with the following changes. The precursor for 3-OH-C18 was not commercially available; thus the 3-OH-C18 precursor, hexadecanal, was synthesized first by the method of Landini et al. (5). A saturated solution of potassium chromate (0.55 mol/L) in 300 mL/L aqueous sulfuric acid was reacted with 0.01 mol of 1-hexadecanal dissolved in 60 mL of methylene chloride in the presence of 0.001 mol of tetrabutylammonium hydrogen sulfate as a catalyst (ratio of 1-hexadecanol to catalyst, 10:1). The unlabeled and [1,2]-[.sup.13][C.sub.2]-labeled 3-OH-C18 were then synthesized from the hexadecanal as described previously (4). The methylene chloride, 1-hexadecanol, and tetrabutylammonium hydrogen sulfate were obtained from Aldrich Chemical Co. Analysis of the naturally occurring and stable-isotope 3-OH-C18s after synthesis was also performed as described previously (4). This analysis demonstrated that the naturally occurring 3-OH-C18 was 89% pure with the expected composition, and the stable-isotope 3-OH-C18 was 95% pure with the expected composition. Mass spectra revealed patterns that reflected the structure of the authentic compounds and demonstrated that the impurities would not affect the ions used for quantification of 3-OH-C18. Because of the synthetic process, very little of the naturally occurring compound was expected in the isotope-labeled 3-OH-C18, and the mass spectra confirmed this by showing 0.06%.

The 3-OH-C18 stable-isotope-labeled calibrator used in the assay and the natural compound used for linearity and precision studies were made by weighing the 3-OH-C18 species and dissolving each to a concentration of 500 [micro]mol/L in dichloromethane.

Control samples were serum, heparinized plasma, or EDTA-plasma, left over from previous studies, from apparently healthy individuals. The control samples included those from nonfasting individuals (n = 35) who were not on any special diet. The control individuals ranged in age from 2 days to 53 years (15 females and 20 males).

In addition to the control group, we assayed samples from 10 ketotic patients who had increased concentrations of the short-chain 3-OHFAs. These patients showed no increases in their 3-OH-C18 concentrations above the control group. Samples were also obtained from two patients diagnosed with deficiency in long-chain L-3-hydroxyacyl-CoA-dehydrogenase (LCHAD). Both of these patients were being managed with medium-chain triglyceride supplementation and carnitine. Nonfasting samples were collected at routine clinical follow-up visits.

Samples were extracted, derivatized, and assayed as described previously (4), except that 10 [micro]L of 500 [micro]mol/L 3-OH-C18 calibrator was added to the sample separately, and six other 3-OHFA stable-isotope calibrators were added. Gas chromatographic-mass spectrometric analysis was carried out on an Agilent Technologies 6890 gas chromatograph with a 5973Network Series quadrupole mass spectrometer as described previously (4).

Calibration curves were constructed for 3-OH-C18 to determine the linearity, precision, and accuracy of the assay. These curves were constructed by adding 0.01-25 [micro]mol/L of the naturally occurring 3-OH-C18 into an essentially fatty-acid-free albumin at physiologic protein concentrations and then adding 5 nmol of the isotope-labeled calibrator to each sample, as described above. The signal-response ratio of the naturally occurring compound to isotope-labeled calibrator was then plotted against the known analyte concentration. We subjected these calibration curves to linear regression analysis using the signal-response ratio as the dependent variable. To evaluate the method, we repeated calibration curves (n = 6) and used the signal-response ratios to back-calculate concentrations from the derived regression equations.

Quantification of the naturally occurring 3-OH-C18 in patient samples was accomplished as described previously (4) for the other six 3-OH species.

The [[M-CH3].sup.+] fragment ions used to identify and quantify 3-OH-C18 were m/z 429 for the naturally occurring compound and m/z 431 for the isotope-labeled calibrator. 3-OH-C18 also had ions resulting from the 3-OH fragments, which were m/z 233 for the naturally occurring compound and m/z 235 for the isotope-labeled calibrators. These fragments were used to help identify the correct compounds, but could also be used to quantify the naturally occurring compound.

The linear regression analysis of the calibration curves led to a linear regression equation of y = 0.0849x + 0.0163 ([r.sup.2] = 0.9992) for the m/z 429/431 pair. For the m/z 233/235 pair, the equation was y = 0.0792x + 0.0168 ([r.sup.2] = 0.9988). For both ion pairs, the 3-OH-C18 was linear in the concentration range of 0.2-10 [micro]mol/L. The lower limit of detection, defined as 3 SD above the mean value for the blank, was 0.12 [micro]mol/L. Imprecision (CV) at 8, 1.5, and 0.45 [micro]mol/L was 5.3%, 6.8%, and 13%, respectively, for the m/z 429/431 ion pair, and 5.0%, 6.5%, and 6.8%, respectively, for the m/z 233/235 ion pair. The differences from the target concentrations at the concentrations given above were 2.5%, -4.7%, and 10% for the m/z 429/431 ion pair and -6.2%, -3.3%, and -4.0% for the m/z 233/235 ion pair.

The upper limit of the reference interval for 3-OH-C18 for both free and total species in controls (n = 35) was <0.5 [micro]mol/L. For free 3-OH-C18, this follows the pattern seen previously (4), with concentrations decreasing as chain length increases. For total 3-OH-C18, there appears to be little to no conjugated 3-OH-C18 in plasma or serum, even under rigorous hydrolysis conditions (10 mol/L NaOH, 70[degrees]C, 45 min). Only 4 of 35 control samples showed any increase in concentrations between the free and total 3-OH-C18 values, and these increases ranged from 20% to 25%. This is in contrast to 3-OH-C14 and 3-OH-C16, which are ~25-50% conjugated even in apparently healthy individuals (6). Representative concentrations and a representative pattern in a control sample are demonstrated in Fig. 1, which also shows the abnormal pattern and concentrations displayed by an LCHAD-deficient patient. In LCHAD deficiency, there was a marked increase of 3-OHFAs of chain lengths 3-OH-C14 and -C16 and an increase in 3-OH-C18. Although the 3-OH-C18 concentration was above the upper limit of the reference interval, it did not show the pattern of increase seen with the 3-OH-C14 and -C16 species, nor did it show any difference in free and total 3-OH-C18. For example, for the LCHAD-deficient patient depicted in Fig. 1, the 3-OH-C14 concentration was 5-fold higher (2.0 [micro]mol/L) than the upper limit of the 3-OH-C14 reference interval (0.4 [micro]mol/L), and the 3-OH-C16 concentration was 20-fold higher (10 [micro]mol/L) than the upper limit of the 3-OH-C16 reference interval (0.5 [micro]mol/L). The 3-OH-C18 concentration was only threefold higher (1.5 [micro]mol/L) than the upper limit of the 3-OH-C18 reference interval (0.5 [micro]mol/L). Another LCHAD-deficient patient, who was in better metabolic control, had a concentration that was 1.5-fold above the upper limit of the reference interval for 3-OH-C14, 3.4-fold above the upper limit of the reference interval for 3-OH-C16, and 1.2-fold above the upper limit of the reference interval for 3-OH-C18. Again, the 3-OH-C18 concentration did not follow the pattern of increasing metabolite accumulation established by 3-OH-C12 to -C16.

Mitochondrial fatty acid [beta]-oxidation defects are often difficult to diagnose (7-9). This is attributable, in part, to the fact that routinely detectable metabolites are often absent when a patient is clinically well and, in part, to the fact that this pathway is still not fully understood. Assays that contribute to the knowledge base for this pathway are useful not only for diagnostic purposes, but also to increase understanding of the mechanisms involved. The addition of quantitative 3-OH-C18 to our original assay has the potential to be useful in these ways.

As demonstrated in Fig. 1, measurable amounts of the 3-OH-C18 can be found in serum and plasma samples; however, almost none of it is found in conjugated form. This finding is in contrast to 3-OH-C14 and 3-OH-C16, where a significant percentage of the circulating form is conjugated, much of which is probably conjugated to carnitine. In serum samples, linoleic acid (C18:2) and oleic acid (C18:1) contribute a large proportion of the total C18 fatty acid content. We have used the 3-OH-C18:0 stable isotope internal standard to attempt to approximate the amounts of 3-OH-C18:2 and -C18:1 present. We used the [[M-CH3].sup.+] ion pairs m/z 425/431 for 3-OH-C18:2 and m/z 427/431 for 3-OH-C18:1 and then m/z 233/235 for each to estimate these concentrations. We analyzed two serum samples from an LCHAD-deficient individual and four apparently healthy controls. For the control samples, the 3-OH-C18:1 and -C18:2 values varied from undetectable to 0.38 [micro]mol/L and undetectable to 0.24 [micro]mol/L, respectively. For the LCHAD-deficient individual, the sample drawn when the patient was not in metabolic control showed approximated concentrations of 3-OH-C18:1 and -C18:2 of 1.96 and 0.88 [micro]mol/L, respectively, compared with 0.78 [micro]mol/L for 3-OH-C18. For the sample drawn when the patient was in better metabolic control, the results of these three forms were 0.42, 0.16, and 0.45 [micro]mol/L, respectively. The two samples differed because the unsaturated 3-OH-C18 concentration was lower than the saturated when the patient was in relative metabolic control. The opposite was true when the patient was not in control. In addition, the unsaturated forms of the 3-OH-C18 species were more conjugated, with the concentrations after hydrolysis being 1.5- to 4-fold higher than the unhydrolyzed concentrations.


Interestingly, the 3-OH-C18:1 and -C18:2 values obtained from controls were comparable whether they were calculated with the [[M-CH3].sup.+] ion pair or the m/z 233/235 ion pair. In contrast, with the LCHAD-deficient patient, the values were quite different when calculated with the [[M-CH3].sup.+] vs the m/z 233/235 pair. For example, 3-OH-C18: 1 was 1.96 vs 1.0 [micro]mol/L, and 3-OH-C18:2 was 0.88 vs 0.42 [micro]mol/L. This finding held true for the other LCHAD-deficient sample also, with a 3-OH-C18:1 concentration of 0.42 vs 0.23 [micro]mol/L, and a 3-OH-C18:2 concentration of 0.16 vs 0.10 [micro]mol/L. These results illustrate that without a native compound calibrator to establish a credible calibration, the use of a native compound/internal standard ratio to calculate a concentration is, at best, an approximation. However, the results for the unsaturated species could still be used to monitor the status of a previously diagnosed LCHAD-deficient patient.

The results we obtained with the saturated and unsaturated species, as well as the finding that the 3-OH-C18 concentration in LCHAD-deficient individuals is not increased as much above the upper limit of the reference interval as 3-OH-C14 and -C16, suggest that stearic acid may be handled by a different mechanism than other long-chain fatty acids. The addition of 3-OH-C18 to this assay has increased its utility as a research and a diagnostic tool.

The synthesis of compounds for this work, and also for the original assay reported by Jones et al. (4), were partially funded by the Mental Retardation Research Center Core Grant No. PO1 HD 08315.


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(6.) Jones PM, Burlina AB, Bennett MJ. Quantitative measurement of total and free 3-hydroxy fatty acids in serum or plasma samples: short-chain 3-hydroxy fatty acids are not esterified. J Inherit Metab Dis 2000;23:745-50.

(7.) Bennett MJ, Rinaldo P, Strauss AW. Inborn errors of mitochondrial fatty acid oxidation. Crit Rev Clin Lab Sci 2000;37:1-44.

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

(9.) Roe CR, Ding J. Mitochondrial fatty acid oxidation disorders. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The metabolic and molecular bases of inherited disease, 8th ed. New York: McGraw-Hill, 2001:2297-326.

Patricia M. Jones, [1] * Susan Tjoa, [2] Paul V. Fennessey, [2] Stephen I. Goodman, [2] and Michael J. Bennett [1]

[1] University of Texas Southwestern Medical Center, Department of Pathology, and Children's Medical Center of Dallas, Dallas, TX 75235;

[2] University of Colorado Health Sciences Center, Department of Pediatrics, Denver, CO 80262;

* 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 or
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Title Annotation:Technical Briefs
Author:Jones, Patricia M.; Tjoa, Susan; Fennessey, Paul V.; Goodman, Stephen I.; Bennett, Michael J.
Publication:Clinical Chemistry
Date:Jan 1, 2002
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