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Technical Briefs |
1
Neo Gen Screening, PO Box 219, Bridgeville, PA 15017
2
Centers for Disease Control and Prevention, 4770 Buford Hwy. NE, Atlanta, GA
a author for correspondence: fax 412-220-0784, e-mail dhchace{at}neogenscreening.com
The use of tandem mass spectrometry (MS-MS) in the analysis of filter paper blood spots from newborns for acylcarnitines and amino acids has expanded significantly in recent years (1)(2). With estimates of 1 million specimens analyzed by MS-MS per year throughout the world, the demand is acute for assay standardization and harmonization. Programs exist at the CDC for amino acid standardization and quality assurance pertaining to newborn screening (3)(4). This program is being extended to include acylcarnitines, and the data in this report stem from that extension.
Five metabolites are key in the diagnosis of several disorders of fat and organic acid metabolism. Preliminary results demonstrated excellent linearity for each of the five acylcarnitines added to blood. However, an extremely unusual result was observed for octanoylcarnitine: the recovery of octanoylcarnitine was significantly lower than that of other acylcarnitines. This observation is supported by Turner and Dalton (5), who report a 40% loss of octanoylcarnitine after addition to whole blood and plasma. We investigated the cause of this loss of octanoylcarnitine so that this serious error could be prevented or accounted for. The results of this study demonstrate the importance of assay standardization and the validation required in clinical screening that goes well beyond this particular quality-assurance/quality-control program for acylcarnitines.
We obtained isotope-labeled internal standards (L-2H3-propionylcarnitine, L-2H3-butyrylcarnitine, L-2H3-octanoylcarnitine, L-2H9-myristoylcarnitine, and L-2H3-palmitoylcarnitine) from Cambridge Isotope Laboratories. Unlabeled standards (D,L-octanoylcarnitine, D,L-myristoylcarnitine, and L-palmitoylcarnitine) were obtained from Sigma, and L-propionylcarnitine, L-butyrylcarnitine, and L-octanoylcarnitine were obtained from Life Sciences Resources. The unlabeled standards were used to prepare a series of blood specimens at the CDC using procedures described previously (3)(4) with the following modifications: L-propionylcarnitine, L-butyrylcarnitine, D,L-octanoylcarnitine, D,L-myristoylcarnitine, and L-palmitoylcarnitine were added to whole blood containing EDTA, whole blood containing heparin, and lysed cells at final concentrations of 0–14 µmol/L. Blood (25 µL) was applied to S&S Type 903 filter paper (Schleicher & Schuell), dried, and sent to Neo Gen Screening for analysis by MS-MS. A smaller subset of blood specimens containing heparin were prepared at Neo Gen Screening by the addition of an equimolar solution (31 µmol/L) of D,L-octanoylcarnitine or L-octanoylcarnitine to the internal standard, L-2H3-octanoylcarnitine.
For specimens obtained from the CDC, a 4.8 mm diameter spot was punched from each dried blood specimen, extracted with methanol containing deuterated L-acylcarnitine internal standards, and derivatized using a procedure described previously (6) with the following modification: dry, derivatized sample extracts were reconstituted immediately before analysis in acetonitrile-water (50:50 by volume) containing 0.2 mL/L formic acid and analyzed using electrospray MS-MS as described below. Specimens that contained both the internal standard and its unlabeled analog were extracted using methanol without the internal standard.
An Applied Biosystems/MDS Sciex Model API 3000 tandem mass spectrometer equipped with an electrospray ionization source was used for all analyses. A 10-µL aliquot of each specimen was injected using a Gilson 215 sample handler fitted with a Rheodyne Model 7010 injector and a Perkin-Elmer LC Pump operating at a flow rate of 18 µL/min with acetonitrile-water (50:50 by volume) containing 0.2 mL/L formic acid as the mobile phase. Precursors of 85 Da scans and 103 Da scans were used, representing analyses for acyl and free carnitines (6). Concentration calculations were obtained by the following method: raw data (ion intensity data) were processed using an Apple script followed by its exportation to an Excel spreadsheet for further data reduction and calculations.
Excellent linearity (R2
>0.98) was obtained for acylcarnitine calibration curves from blood
containing either EDTA or heparin or in which the red cells were lysed.
The slopes and intercepts for these addition assays are provided in
Table 1
. The results for the addition containing
D,L-octanoylcarnitine had a slope of 0.59, which
suggests a significantly reduced recovery. Similar results were
reported by Turner and Dalton (5), who reported a
significant loss of octanoylcarnitine of 40%. The stereoisomeric
forms used in their study for octanoylcarnitine, however, were not
noted. In an experiment in which
L-octanoylcarnitine was used in an addition
analysis, no significantly reduced recovery of
L-octanoylcarnitine was observed (slope =
0.93). Repeat preparation of the
L-octanoylcarnitine calibration curve using
EDTA-treated blood did not show reduced recovery of
L-octanoylcarnitine (data not shown).
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Experiments were designed to further clarify and confirm the loss of
D,L-octanoylcarnitine in blood. An equimolar mixture of
D,L-octanoylcarnitine with
L-2H3-octanoylcarnitine
or of L-octanoylcarnitine with
L-2H3-octanoylcarnitine
was first analyzed as pure compounds. The MS-MS analyses of
these mixtures demonstrated molar equivalents of D,L- or
L-octanoylcarnitine (Fig. 1,
A and B
). An aliquot of this equimolar mixture was then added to
a blood specimen and analyzed. In this case, the relative concentration
of D,L-octanoylcarnitine was substantially different from
that of L-octanoylcarnitine (Fig. 1, C and D
). The peak
representing the racemic mixture of D,L-octanoylcarnitine
was clearly lower than that of L-octanoylcarnitine alone.
However, addition of this equimolar mixture to whole blood containing
EDTA produced a vastly different result. We observed a substantial loss
of D,L-octanoylcarnitine but not
L-octanoylcarnitine (Fig. 1
).
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In one final observation, the concentration of free carnitine was
measured in the CDC blood standard preparations at the highest
concentration (8 µmol/L) and the controls for both
D,L-octanoylcarnitine addition and
L-octanoylcarnitine. Because each blood pool contained
different concentrations of endogenous acylcarnitine and free
carnitine, the ratio of free carnitine in the supplemented pool to that
in the endogenous pool was calculated. The results showed that the
relative increase of free carnitine was 12.4% for blood containing
L-octanoylcarnitine compared with 47% for
D,L-octanoylcarnitine. The 
10–15% increase of free
carnitine resulted from the hydrolysis of added standards during sample
preparation.
These experiments show that loss of octanoylcarnitine observed in MS-MS analyses is presumably attributable to hydrolysis of octanoylcarnitine, as observed by the increase of free carnitine in the enriched specimens. Furthermore, because the difference between standards is D-octanoylcarnitine, which comprises 50% of the octanoylcarnitine in a racemic mixture of D,L-octanoylcarnitine compared with L-octanoylcarnitine alone, the hydrolysis must be specific to D-octanoylcarnitine. The mass spectrometer does not distinguish stereoisomers of octanoylcarnitine. This presumed hydrolysis is supported by evidence provided by Turner and Dalton (5), who showed in a series of experiments a 40% decrease in octanoylcarnitine added to blood. It is presumed that they used D,L-octanoylcarnitine rather than L-octanoylcarnitine. They also observed that this loss of octanoylcarnitine did not occur in longer-chain acylcarnitines (i.e., myristoyl- or palmitoylcarnitine). We do not have evidence that this hydrolysis does not occur in shorter-chain racemic mixtures of acylcarnitines, in part because of the lack of information in the abstract by Turner and Dalton (5), and D,L racemic mixtures of other acylcarnitines were not available for purchase. It is noteworthy that D-octanoylcarnitine is not commercially available. Experimental use of D-octanoylcarnitine would have provided important further evidence to support our hypotheses.
This report provides important information about the development of quality-assurance/quality-control programs for acylcarnitines and other stereoisomeric standards. The use of a racemic mixture of octanoylcarnitine for standardization would produce an overestimation of the concentration of octanoylcarnitine in routine blood specimen analyses. Noting the stereoisomeric form of biologically active molecules in future publications, a point lost in many recent reports observed in the literature, will be important. Furthermore, several organizations that provide newborn screening programs may be unaware of this problem and may miscalculate the true concentrations of certain acylcarnitines in blood. In addition, questions concerning the reasons that octanoylcarnitine is hydrolyzed in blood preparations may be more properly answered. On the basis of our study, the conclusion of Turner and Dalton (5), that blood or serum should be calibrated against aqueous calibrators rather than blood, appears false. Furthermore, their conclusion that an enzyme in plasma of both healthy subjects and patients with medium chain acylCoA dehydrogenase deficiency hydrolyzes octanoylcarnitine is also false because the physiologically active form of octanoylcarnitine is L-octanoylcarnitine. Furthermore, because L-octanoylcarnitine is not hydrolyzed in the blood, we must conclude that the conversion of L-octanoylcarnitine to free carnitine is unlikely. However, nonspecific hydrolytic enzymes in the blood appear to remove D-octanoylcarnitine from the blood. This fact is certainly an interesting observation and may stimulate further interest in its mechanism and its role in blood.
References
The following articles in journals at HighWire Press have cited this article:
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  D. H. Chace, J. C. DiPerna, T. A. Kalas, R. W. Johnson, and E. W. Naylor Rapid Diagnosis of Methylmalonic and Propionic Acidemias: Quantitative Tandem Mass Spectrometric Analysis of Propionylcarnitine in Filter-Paper Blood Specimens Obtained from Newborns Clin. Chem., November 1, 2001; 47(11): 2040 - 2044. [Full Text] [PDF]
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