Determination of medium chain fatty acids in serum

Clinica Chimica Acta, 172 (1988) 233-238

233

Elsevier

CCA 04091

Determination

of medium chain fatty acids in serum

Carlo Dionisi Vici, Claude Bachmann, Marianne and Jean Pierre Colombo Department (Received

of Clinical Chemistry

Inselspital,

University

6 July 1987; revision received 29 September

Key words: Medium

ofBerne, Berne (Switzerland)

1987; accepted

chain fatty acid; acyl-CoA dehydrogenase Dodecanoic acid

Gradwoht

after revision 20 October

deficiency;

Octanoic

acid; Decanoic

1987)

acid;

Summary The determination of medium chain fatty acids in serum is a useful approach to the diagnosis of medium chain acyl-CoA dehydrogenase deficiency, ,an increasingly recognized cause of Reye-like syndrome in infants. A reliable and practical method requiring 0.5 ml of serum is presented by which results are obtained within 2.5 h. The preparation of the samples is done by solid phase extraction on reverse phase cartridges, the separation and quantitation by gas chromatography. Reference values for children (n = 24) and adults (n = 40) are given for octanoic, decanoic and dodecanoic acids.

Introduction Acyl-CoA dehydrogenase deficiencies cause of Reye like syndrome in infancy chain acyl-CoA dehydrogenase deficiency acids appears to be a simple and sensitive however not be sensitive enough or time We present a reliable, sensitive and termination of medium chain fatty acids by reverse phase and gas chromatographic

have been increasingly recognized as a since 1983. For the diagnosis of medium the determination of medium chain fatty approach. Methods reported so far might consuming [1,2]. practical method for the quantitative dein serum based on solid phase extraction separation and quantification.

Permanent address for CDV: Servizio di Patologia Metabolica, Ospedale Pediatrico Bambino Gesu Istituto di Ricerca Scientifica, Salita S. Onofrio 4, 00165, Rome, Italy. Correspondence and requests for reprints to: Claude Bachmann, Department of Clinical Chemistry, Inselspital, University of Beme, CH 3010, Beme, Switzerland.

0009-8981/88/$03.50

0 1988 Elsevier Science Publishers

B.V. (Biomedical

Division)

234

Materials and method The sodium salts of nonanoic acid, purchased from Sigma (St Louis, MO, obtained from Fluka (Basel, Switzerland). 97%. The standards were dissolved in NaCl (purified; Behringwerke, Marburg, FRG)

decanoic acid and dodecanoic USA). Octanoic acid (sodium The purity of these standards (9 g/l) containing 40 g/l and sonified (5 min).

human

acid were salt) was exceeded albumin

Sample preparation To 0.5 ml of serum or standard solution 100 ~1 of nonanoic acid (internal standard, 200 pmol/l) and 5 ml of sodium acetate buffer (0.5 mol/l; pH 4.6) were added. Sep-Pak Cl8 cartridges (Waters Assoc., Milford, MA, USA) were activated with 5 ml of methanol and then washed with 5 ml of sodium acetate (0.5 mol/l; pH 4.6). The samples were slowly injected into the cartridges. These were washed with 6 ml of distilled water. The elution of medium chain fatty acids was done with 3 ml of methanol. The eluate was alkalinized with 0.5 ml of aqueous ammonia 0.5 mol/l and dried under nitrogen at 50” C in conical glass tubes. The residue was derivatized with 50 ~1 BSTFA (containing 1% TMCS, Pierce Eurochemie BV, Oud-Beijerland, The Netherlands) at 90 o C for 60 min. All the glassware was silanized. Gas chromatography A Sigma 3B gas chromatograph (Perkin Elmer, Norwalk, CN, USA) was equipped with a glass column (2 m, 2 mm i.d.) filled with 3% Dexsil 400 on 100/120 mesh Supelcoport (Supelco Inc., Bellafonte, PA, USA) as stationary phase. Nitrogen was used as carrier gas at 10 ml/mm. After an initial time of 3 min at 105’C, the temperature was programmed at 4”C/min up to 200°C. The temperature of the injector and flame ionisation detector was 250 o C. The peaks were quantified by measuring the peak heights and relating them to the internal standard. Results The retention times of the medium chain fatty acids with the conditions used were: octanoic 7.6 min, nonanoic (internal standard) 10.6 min, decanoic 13.7 min and dodecanoic 19.7 min. Figure 1 illustrates the separation with a serum sample obtained from a one day old neonate with an enzymatically proven medium chain acyl-CoA dehydrogenase deficiency. Reliability The recoveries of octanoic, decanoic and dodecanoic acids spiked to serum at concentrations of 4 and 20 pmol/l are presented in Table I. This table shows in addition the intraseries precisions (CVW) of these determinations. The interseries

235

A

I

20

1

15

1

s

10

0

TIME

Fig. 1. Chromatogram of medium chain fatty acids in the serum of a one-day-old newborn (V.M.) with medium chain acyl-CoA dehydrogenase deficiency. The peaks are: A, octanoic acid (200 pmol/l); B, internal standard 40 amoI/l; X, decenoic acid (?); C, decanoic acid (23.7 nmoI/l); D, dodecanoic acid (8.4 pmol/l). For conditions see text.

TABLE Recovery

I and intraseties

precisions

Acid

of 4 and 20 pmol/l

chain

fatty acids added

to serum

Octanoic R a SD

Decanoic

Dodecanoic

X SD

X SD

1.7kO.2 4.8 f 0.1 78.1 f 1.9 2.5 2.5 f 0.3 23.2* 1.1 103.6 f 5.6 5.4

2.9 f 0.1 6.7rt0.2 93.7zt3.4 3.6 2.9 f 0.1 20.4 f 0.6 88.6*2.X 3.2

6.4f 0.3 10.6+ 0.7 101.2 f 18.6 17.9 6.2+ 0.5 2!3.2* 1.5 110.1 f 7.7 7.0

Serum alone: + 4 nmol of fatty acids found: Recovery (W) Precision (CV b, %) Serum alone: + 20 pmol/l of fatty acids found: Recovery (W) Precision (CV,W) a Mean and SD of 6 determinations b CV, coefficient of variation.

of medium

(pmoI/I)

236 TABLE

II

Reference

values in adults

and children

(p mol/l) Adults (n = 40)

Children (n=24)

2.5 1.1-7.0 2.7 1.2-6.0

2.0 0.9-3.0 2.0 1.2-3.5

5.0 1.0-15.0 4.6 1.6-13.4

3.3 1.2-6.9 3.3 1.3-8.2

8.5 2.7-14.5 7.3 3.7-15.7

8.2 2.9-27.8 8.3 2.5-28.0

Octanoic Median Range Mode Log normal 95% Confidence

range ’

Decanoic Median Range Mode Log normal 95% Confidence

range

Dodecanoic Median Range Mode Log normal 95% Confidence ’ Calculated

range

from K f 2

SD

of log values.

precisions of serum (with 20 pmol/l of each acid added) amounted to 4.6%, 6% and 8.2% (CVW, n = 6) for octanoic, decanoic and dodecanoic acids respectively. The method was linear for the three medium chain acids in a range tested between 4-200 pmol/l (r: octanoic 0.9998; decanoic 0.9999; dodecanoic 0.9996). The intersections with the y axis were not statistically different from zero. Reference

values

Reference values were determined from adult blood donors (n = 40) and selected pediatric in-patients (age 3 mth to 13 yr, n = 24) not receiving an MCT-containing diet (Table II). The observed distribution of the reference values was tested for Gaussian or log-Gaussian distributions by using the Kolmogoroff Smirnov and the x2 test. Significant deviations from normality were found for octanoic and decanoic acids in adults and for dodecanoic in children (x2: p < 0.005). In contrast the log normal distribution fitted well, showing throughout lower quantile differences between found and expected values in the Kolmogoroff Smirnov test than when using Gaussian distribution. The adult population differed from the pediatric reference population (Mann-Whitney-Wilcoxon test: octanoic p -C 0.01, decanoic p -C 0.02). No difference was found within the pediatric group between samples of children below 3 yr of age and the older ones.

231

Discussion The presented method is reliable and practical. As shown in Table I the recoveries and precisions were satisfactory especially considering the low concentrations tested. The detection ,was linear in a critical range (4-200 pmol/l) for patients suffering from heritable defects such as medium chain acyl-CoA dehydrogenase (MCAD) deficiency. In contrast with the reported undetectable levels of octanoic and decanoic acid in healthy subjects [3,4], Table II shows that small amounts of these acids were always present in our controls. This is probably due to the higher sensitivity of our method. Haidukewych et al [3] found octanoic and decanoic acid only in children receiving a medium chain triglyceride supplemented diet while Duran et al detected appreciable levels of these acids only in MCAD deficient patients [4]. The values of octanoic in our adult control subjects agree with those reported by Staeffen et al (51 who use a time consuming extraction and need larger volumes of serum. In contrast our method allows to obtain results within 2.5 h and requires only 0.5 ml of serum. It thus seems more practical, and especially useful for diagnostic purposes in critically ill pediatric patients [6]. The determination of medium chain fatty acids is mainly needed in newborns and infants presenting as Reye’s syndrome in order to exclude acyl CoA dehydrogenase deficiencies. These defects have been increasingly recognized since 1983 [7]. In our experience the medium chain fatty acids in serum are a better indicator of the MCAD deficiency than dicarboxylic acids in urine [8] which in several instances were not increased while the serum medium chain fatty acid pattern was abnormal. This latter includes not only an elevation of the saturated C8-Cl0 acids but also the presence of a peak (X, Fig. 1) tentatively identified as decenoic acid by mass-spectrometry. No further attempts for identification were made so far since we lack a standard. This compound could not be found in any of four patients receiving a medium chain triglyceride supplemented diet who had increased medium chain fatty acids in plasma and dicarboxylic aciduria. Acknowledgement This work was partly no. 3.910.0.85.

supported

by the Swiss National

Science Foundation

grant

References 1 Rabinowitz JL, Staeffen J, Aumonier P, et al. A method for serum octanoate in hepatic cirrhosis and hepatic encephalopathy. Clin Chem 1977;23:2202-2206. 2 Duran M, Ketting D, Van Vossen R, et al. Octanoylglucuronide excretion in patients with a defective oxidation of medium chain fatty acids. Clin Chim Acta 1985;152:253-260, 3 Haidukewych D, Forsythe WI, Sills M. Monitoring octanoic and decanoic acids from children with intractable epilepsy treated with medium-chain triglyceride diet. Clin Chem 1982;28:642-645.

238 4 Duran M, Mitchell G, De Klerk JBC, et al. Octanoic acidemia and octanoylcarnitine excretion with dicarboxylic aciduria due to defective oxidation of medium-chain fatty acids. J Pediatr 1985;107:397-404. 5 Staeffen J, Rabinowitz JL, Aumonier P, et al. Hyperoctanoatemie de l’encephalopathie htpatique des cirrhoses. Nouv Press Med 1979;8:1663-1666. 6 Editorial. Sudden infant death and inherited disorders of fat oxidation. Lancet 1986;ii:1073-1075. 7 Treem WR, Witzleben CA, Piccoli DA, et al. Medium-chain and long-chain acylCoA dehydrogenase deficiency: clinical, pathologic and ultrastructural differentiation from Reye’s syndrome. Hepatology 1986;6:1270-1278. 8 Mortensen PB, Gregersen N. Medium-chain triglyceride as a pitfall in the diagnosis of non-ketotic C6-ClO-dicarboxylic acidurias. Clin Chim Acta 1980;103:33-37.