CLIN. CHEM.

38/11, 2215-2220 (1992)

Automated Analysis for Free and Short-Chain Acylcarnitine in Plasma with a

Centrifugal Analyzer
Diane S. Roe,’ Naoto Terada,2 and David S. Milllngton’

We describe a fully automated, spectrophotometnc assay ter, thus limiting convenience and increasing the poten-
of free and total camitine in plasma ultrafiltrates. The tial for error.
method, suitable for routine application in most hospital The most widely used alternative method is a radio-
laboratories, incorporates the hydrolysis of acylcarnitines isotopic exchange assay that uses [1-’4C]acetyl-CoA as
to free carnitine within the program of a Cobas Fara ii substrate for the enzyme and measures the [1-’4CJace-
centrifugal analyzer. The hydrolysis is monitored and tylcarnitine produced (10). This method is generally
calibrated with standard solutions containing octanoyicar- accepted as one of the most accurate, and several mod-
nitine. Results correlated well with those from a reference ifications have been described (4, 11 ). However, the
isotope-dilution mass spectrometric assay. The ability to procedure is labor intensive and unsuitable for automa-
analyze a batch of samples for both free and total tion and is therefore not used routinely in clinical
carnitine within 90 mm enables analysis of 100 samples laboratories.
per day. Used in conjunction with acylcarnitine species Here we describe in detail a modified version of the
identification by mass spectrometry, the Cobas assay spectrophotometric enzyme assay, adapted for use with
facilitates the diagnosis of carnitine-deficiency syndromes a centrifugal analyzer and validated by a recently
and specific metabolic disorders. developed isotope-dilution mass spectrometric Refer-
ence Method (12). The new assay uses plasma ultrafil-
AddItional Keyphrases: camitine . heritable disorders of metab- trates directly and incorporates the hydrolysis of acyl-
olism . pediatric chemistr,’ carnitines to free carnitine.

Increased awareness of the biochemical functions of Materials and Methods

L-carnitine and the clinical consequences of carnitine Apparatus
deficiency (1, 2) have generated considerable demand The Cobas Fara II centrifugal analyzer was from
for a convenient, routine assay ofL-carnitine in plasma. Roche Diagnostics (Montclair, NJ). The VG QUATIRO
Several methods have been published, each having tandem quadrupole mass spectrometer fitted with a
specific advantages and limitations (3, 4). The most cesium atom gun for secondary ion mass spectrometry
widely used methods are dependent on the enzyme was from Fisons-VG Instruments (Danvers, MA). Ultra-
carrntine acetyltransferase (CAT; EC 2.3.1.7), which ifitration was carried out by using Amicon Centrifreetm
catalyzes the reaction filters with YMT membranes (Amicon Corp., Beverly,
MA).
Acetyl-CoA + L-carnitine±acetylcamitine + CoASH
Matenals
where CoASH is the liberated free coenzyme.3 A popu- All chemicals were obtained from Sigma Chemical
lar spectrophotometric assay is based on the reaction of Co. (St. Louis, MO) except where noted. The reagents
the liberated CoASH with 5,5’-dithiobis(2-nitrobenzoic and solutions required for the assay are listed below,
acid) (DTNB), producing the thiophenylate ion, which some of which are similar to those described previously
absorbs at 412 nm (5). The assay is repeated after (13). All solutions were stored at 0-4 #{176}C. Distilled,
alkaline hydrolysis, enabling measurement of free and de-ionized water from an in-house still was used to
total carnitine, hence the acylcarnitine fraction by dif- prepare and dilute solutions.
ference. Most methods currently used are based on a Solution 1 comprised DTNB (2.7 mmolIL), 4-(2-hy-
partially automated version ofthis procedure (6-9), but droxyethyl)-1-piperazineethanesulfonic acid (HEPES,
these methods require several steps to be performed 0.5 mollL), and EDTA (10 mmollL), adjusted to pH 7.5
external to the automated analyzer or spectrophotome- with NaOH (1 molfL). Solution 2, prepared daily, was
acetyl-CoA (12 mmollL). Solution 3 comprised 3-EN-
1 Division of Genetics and Metabolism, Department of Pediat- morpholinoipropanesulfonic acid (MOPS, 20.93 g) in 80
rica, Box 3028, Duke University Medical Center, Durham, NC mL of 1 mollL HC1 made up to 100 mL with distilled
27710. water (MOPS-HC1, 1 molfL). Solution 4 was KOH (1
2 Department of Pediatrics, Kyoto Prefectural School of Medi-
cine, Kawaramachi Hirokoji Kamigyo-ku, Kyoto 602, Japan. moIJL). Primary reagent mixture was prepared daily:
3 Nonstandard abbreviations: CAT, carnitine acetyltransferase; Solution 1 (1.8 mL) was mixed with solution 2 (0.3 mL)
CoASH, free coenzyme A; DTNB, 5,5’-dithiobis(2-nitrobenzoic to provide final concentrations (mmol/L) as follows:
acid); HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfomc DTNB, 2.3; HEPES, 428; EDTA, 8.6; and acetyl-CoA,
acid; MOPS, 3-[N-morpholino}propanesulfothc acid; and MCAD,
medium-chain acyl-CoA dehydrogenase. 1.7. This amount was sufficient for one cuvette rotor (23
Received April 15, 1992; accepted July 22, 1992. samples plus 5 calibration standards). Enzymatic start

CLINICAL CHEMISTRY, Vol. 38, No. 11, 1992 2215

reagent was prepared daily: CAT from pigeon-breast
Table 1. Cobas Analyzer Settings for Assay of Free
muscle (100 p.L, protein 2.0 gIL; 86 kU/g protein) was
Camftlne and Total Carnltlne In Plasma Ultraflftrates
diluted with water (900 pL) to give a final concentration
Fros
ofl7.2 kUIL. camftlns Total carnftlns
The stock solution contained 1 mmol/L each of L-car- General
nitine and octanoyl-L-carnitine, prepared by dissolving Measurement mode: ABS ABS
L-carnitine hydrochloride (4.93 mg) and octanoyl-DL- Rxn mode: P-I-SRi -A S-l-W-SR1 -I-Ri-
carnitine hydrochloride (16.16 mg) in distilled water (25 I-SR2-A
mL). Solutions containing 100, 40, 30, 20, and 10 moL1L Calibration mode: Linreg Unreg
of each standard were prepared by diluting the stock Reagent blank: Reag/dil Reag/dil
solution. Wavelength: 412 412
Temperature: 37#{176}C 37#{176}C
Sample Preparation Unit: unoVL /LITIOVL

Cobas. For the analysis for both free and total car- Malysis
mtine on the Cobas Fara II, serum or plasma collected P (5k)
Sample volume: 25 L 401LL
with EDTA or heparin (350 L minimum per assay) was Diluent name: H20 KOH
eluted through a Centrifree filter by centrifugation in a Vol: 5 L 5;A.L
450 fixed-angle rotor at 1000-2000 X g for 30 mm. To Cleaner cycle: Off Off
allow for a sample-cup dead volume of 50 p.L, at least Reagent volume: 30 L
150 L ofthe ultrafiltrate was placed in a sample cup for Incubation: 150s 480 s
assay. The aqueous standards for calibration were pro- Ml: 20s No
pared similarly to compensate for losses of acylcar- M2: 140s No
mtines on the filter. wa Wait time: 480 a
Mass spectrornetry. For analysis by tandem mass SRi Start reag I: l0&L 5141
spectrometry, samples were prepared by a modification Diluent name: H20 H2O
of the method described previously (12). To plasma or Vol: 51LL. 0LI
serum (100 L) were added the following internal stan- Wet Cycle: On Off
ia Incubation: 240 a
dards: [2H3-methyfl-L-carnitine, 4 nmol; acetyl-[2H3-
methyl]-L-carnitine, 0.5 nmol; [2H5lpropionyl-L-car-
M: No
M: No
mtine, 0.1 nmol; isovaleryl-[2H3-methyll3-DL-carnitine
Rla Reag 1: 30 p1.
(0.1 nmol), and octanoyl-[2H3-methyl]-L-carnitine (0.1
Incubation: 150s
nmol). Ethanol (800 L) was added and the mixture was
Ml: 20s
vortex-mixed and centrifuged (13 000 x g, 5 mm). The
M2: l4Os
supernate was washed with hexane (1 mL) and evapo-
SR2 Start reag 2: 104
rated under nitrogen, and the residue was incubated Diluent name: H20
with boron trifluoride, 140 gIL, in n-butanol (from All- Vol: 5;hL
tech Associates, Deerfield, IL; 100 L; 65 #{176}C for 15 mm). Wet cycle: On
The solution was again evaporated and reconstituted in A Readings Off
50 L of an equivolume solution of methanol:glycerol First: 1 .0 S 1 .0 S
containing sodium octyl sulfate, 10 gIL. Number 27 27
Interval 20 s 205
Assay Procedures No No
Cobas. The Cobas Fara II test variables were pro- No No
grammed as indicated in Table 1. For free carnitine,
Calculation
reagent programming was set up for a two-reagent
Reaction direction: Increase Increase
chemistry, with the primary reagent mixture in position
Check: On On
1 and the enzymatic start reagent (CAT) in position 2.
Calculation step A: Kinetic Kinetic
For total carnitine, programming was for a three-re-
First:
agent chemistry, with the primary reagent mixture in Last: 27 27
position 1, solution 3 in position 2, and CAT in position
3. Solution 4 was programmed as an alternative “di- Calibration
luent solution” to be pipetted in parallel with sample Cal. interval: Each run Each run
and placed in an assigned position on the same reagent Number of stds: 5 5
rack. The standards and quality control were loaded Position of std 1:
into the calibration rack and patients’ samples were Std 1: 10.0 20.0
loaded into an assigned sample rack. All solutions and Std 2: 20.0 40.0
reagents were placed in their assigned rack positions, a Std 3: 30.0 60.0

cuvette rotor was installed, and analysis was started. Std 4: 40.0 80.0
Std 5: 100.0 200.0
When programmed as a “profile,” free carnitine was
a Fc total camitine analysis only.
assayed first, followed by total carnitine. The procedure

2216 CLINICAL CHEMISTRY, Vol. 38, No. 11, 1992

 1000
took -90 mAn for a full rotor of 29 cuvettes, including
set-up time. For free carnitine determination only, a
0 FREE
minimum sample volume of 70 pL was required and a
full rotor was analyzed in only 15 mm. Results were * TOTAL
identical when either end-point or kinetic calculations 0
were used. The kinetic method was used here. E
Mass spectrorn.etry. For analysis by tandem mass .
spectrometry, --2 /AL of sample was placed on the C
a)
sample probe. Ions were produced by bombardment with E
cesium ions at 10 keV. Selective detection of carnitine a,
and its internal standard was accomplished by means of 0.

the “precursors of m/z 103” scan function previously

reported (12). The acylcarrntines were analyzed in the
same sample by switching to a “precursors of m/z 85”
scan
signals
mtine
function.
at
and
m/z
To quantify
218 and m/z
[2H3-methylJ-carnitine,
free
221,
carnitine,
corresponding
was
the

measured
ratio
to car-
di-
of
-
#{149}-1 F ‘ U

1000
redly and compared with a standard curve. Similarly, Actual (pmol/ L)
the amounts of acetyl-, propionyl-, isovaleryl (C5)-, and Fig. 1 . Calibration curve for camftlne standard solutions (average of
octanoyl (C8)-carnitines were quantified by using their 10 conSecutive assays)
respective internal standards and ion ratios. To quan-
tify C4 and C6 acylcarnitines, we used the internal free carnitine after hydrolysis, was 101% ± 3%. The
standard for isovalerylcarnitine. The sum of individual recoveries for these standards added to plasma before
short-chain acylcarnitine determinations was compared ifitration were 97 ± 3% and 77 ± 3%, respectively,
with the short-chain acylcarnitine value from the Cobas indicating the partial binding of octanoylcarnitine to
assay. plasma proteins, which are retained by the filter. The
calculated recovery of decamoylcarmitine from plasma
Statistical Methods was -53%, and species with a longer chain length were
Linear-regression analysis was used for data from not recovered from the filter because of their binding to
both assays and for comparing the two methods. plasma proteins (4). For free and acylcarnitines up to
C8, the analytical recovery was essentially 100%.
Results
Specificity. A comparison offree carnitine determima-
Hydrolysis tions in untreated plasma and plasma UltrafIltrates
The effectiveness of the programmed hydrolysis for both with and without preliminary thiol oxidation con-
total carnitine determination was assessed by using a firmed that ultraffitration effectively removes the bio-
mixture of free L-carnitine and octanoyl-L-carnitine in logical interferences that make thiol oxidation neces-
equimolar amounts in the standard stock solution. Stan- sary for untreated plasma (13). Icteric, uremic, and
dard curves were obtained from the five standard solu- slightly hemolyzed samples apparently do not affect the
tions for both free carnitine and total carnitine (Figure assay, but grossly hemolyzed samples gave inaccurate
1). The values obtained from the Cobas were consis- results as judged by comparison with the Reference
tently within ± 5% of the expected values of 10, 20, 30, Method. Other investigators reported similar results (6,
40, and 100 mol/L for free carnitine and 20, 40, 60, 80, 8).
and 200 pmol/L for total carnitine. The errors were Comparison of >100 individual measurements of free
randomly distributed about the mean. Linear-regres- and total carnitine with duplicate values from the mass
sion analysis of the data from 10 consecutive calibra- spectrometric Reference Method indicated that the Co-
tions for free and total carnitine indicated a slope of 1.00 bas assay consistently gave values --11% higher than
(r2 0.999). the Reference Method. The reason for this discrepancy is
Samples of plasma ultrafiltrates hydrolyzed outside not clear.
the Cobas, then analyzed for free carnitine, gave the Linearity. The assay was linear from 2 to 500 mol/L.
same results as duplicate samples analyzed for total To improve precision, we used calibration ranges of
carnitine under the programmed conditions. A solution 10-100 pinol/L for free and 20-200 p.mol/L for total
ofthe pure octanoylcarnitine standard used in the assay carnitine, with one set of standards.
(100 pmoIIL) contained no detectable free carnitime by Sensitivity. The detection limit for free carnitine by
the Cobas method. the Cobas method was 2 miol/L.
Precision. Intra-assay precision was determined by
Analytical Vanables repeated analysis ofa pooled plasma ultrafiltrate by the
Analytical recovery. The recovery of added L-carnitine Cobas method. The values (mean ± SD) for free and
(50 and 200 mol/L) in plasma ultrafiltrates determined total carnitine were 48.6 ± 0.85 and 57.6 ± 0.70 pznol/L,
by the Cobas method was 96% ± 3% (mean ± SD), and respectively (n = 16). Interassay variation was deter-
the recovery of added octanoylcarnitine, determined as mined by analysis of the same pooled plasma over --3

CLINICAL CHEMISTRY, Vol. 38, No. 1 1 , 1992 2217

weeks. Mean values were 50 ± 2.0 and 57.6 ± 1.3 100
Acetyl (C-2) A
l.Lmol/L for free and total carnitine, respectively (n =
12). The interassay values obtained for the same speci-
mens by the tandem mass spectrometric method were
46.6 ± 1.89 and 53.5 ± 2.18 p.mollL. i.s.
The quality-control sample used in each batch of 50
assays was also a portion of a pooled plasma sample.
Over a 3-month period, analysis of this control plasma
by the Cobas method under routine clinical assay con-
ditions gave 55.9 ± 3.7 mol/L (range 51-64) for free U)

carnitine
carnitine
within
variation
and 62.8 ± 3.9 pmol/L
(n = 29). The
the previously
for L-carnitine
interassay
reported
(range

determinations
medically
57-70)
variation
for total
is well
acceptable
of 9.3 mol/L
I Octanoyl (C-B) B
(7).
i.s.
Accuracy: correlation with tarukm mass spectrometry.
-J
Free and total carnitine were determined by both the 50
Cobas and mass spectrometry methods for 92 random
plasma samples from mostly pediatric patients with C-1O:1
C-2
various clinical problems. The differences between mdi- C-6
vidual measurements ranged from 1% to 30%, the mean
difference being 10.5%. Linear-regression analysis of
I t
250 300 350 400 450
the results for free carnitine, which ranged from 10 to
168 pinolfL, indicated a slope of 1.17 (r = 0.90; y = m/z
1.17x - 3.5). For the total carnitine data, the slope was Fig. 2. Acylcamitine profiles generated
by tandem mass spectromet-
nc analysiS of plasma (A) a normal patient and (B) a patient
from
1.08 (r = 0.93; y = 1.08x - 1.7). Thus the Cobas values
with MCAD deficiency, a defect of fatty acid catabolism
for carnitine are typically -11% higher than those The molecular species of the normal metaboltte, acetylcarnitlne, and a major
given by the Reference Method. In previous comparative diagnostic metabolfte of MCAD deficiency, octanoylcamltine, are observed at
studies, spectrophotometric assays have given values m/z 218 and 302, respectively. The ISOtOpICaIPy labeled internal standards
added for quantification account for the signals at m 221 and 305. Both
higher than those determined by radioenzymatic assay patients had normal carnitine concentrations
by about the same amount (8, 14). The mass spectro-
metric assay compared well with the radioenzymatic
assay in our hands (12). diagnosed metabolic diseases) exhibited normal acylcar-
From the 92 patients who provided the samples re- nitine profiles by tandem mass spectrometry (15). A
ferred to above, 47 were selected as a control group typical acylcarnitine proffle from a metabolically
according to the following criteria: age between 1 month healthy child is shown in Figure 2A. The profile clearly
and 12 years, no definitive diagnosis or biochemical shows acetylcarnitine as the dominant species, with
evidence for known metabolic diseases, and not receiv- small amounts of C3 and C4 species. By contrast, a
ing carnitine or valproic acid therapy. The values deter- patient with medium-chain acyl-CoA dehydrogenase
mined by the Cobas assay (mean ± SD) were as follows: (MCAD) deficiency, the most common disorder of fatty
free carnitine, 37.9 ± 10.8 moL1L (range 16.7-64.4); acid catabolism (16), gave the proffle shown in Figure
total carnitine, 46.5 ± 10.5 p,mol/L (range 22.6-72); and 2B. The increase in C6, C8, and C10:1 acylcarnitines
short-chain acylcarnitine (by difference), 8.6 ± 5.2 confirmed the diagnosis. The Cobas assay gave values of
lhmol/L (range 1-24.9). Because this was not a normal 43 (free) and 53 mol/L (total) for the normal patient
pediatric population, these values can be considered and 43 (free) and 57 molfL (total) for the MCAD
provisional reference values for this age group for the patient. This example shows that plasma carnitine
Cobas method. concentrations alone are not sufficient for identifying
patients with metabolic diseases.
Summary of the First 500 Assays
During the first5 months of assay use, 535 assays Discussion
were performed, mostly on samples from pediatric pa- Determining free carnitine and total short-chain acyl-
tients with suspected or known metabolic diseases. carnitine concentrations in plasma by using the auto-
Some of these patients were also receiving carnitine mated spectrophotometric assay is an efficient and con-
therapy. In this skewed population, 37 (7%) had free venient alternative to radioenzymatic methods. The
carnitine values <20 mol/L with a normal or low incorporation of programmed hydrolysis into the
acylcarnitine, 55 (10%) had free carnitine <20 mol/L method further simplifies the assay and enhances its
with high acylcarnitine (>15 mol/L), and 443 (83%) suitability for routine use in hospital laboratories. The
were in the normal range. Most patients with abnormal use ofstandard solutions containing a chemically stable
values or high ratios of acylcarnitine to free carnitine acylcarnitine (octanoylcarnitine) enables the hydrolysis
from the Cobas assay (except those with previously step to be monitored and provides accurate calibration

2218 CLINICAL CHEMISTRY, Vol. 38, No. 1 1 , 1992

of assays for total carnitine. All previously reported ciency (free carnitine <20 p.mollL, total s30 .unol/L)
methods lack this important feature. Ultrafiltration is a and to monitor treatment. Patients with free carnitine
simple and effective method for sample preparation that values between 20 and 30 molJL (total carnitine 30-40
obviates the need to dilute the samples or to treat them pinol/L) might be considered borderline and may re-
with thiol oxidizing reagent (13, 14) before analysis. A quire further evaluation, including a repeat assay. The
typical batch of analyses, including a five-point stan- values given here are subjective and based on experi-
dard curve, a quality control, a blank, and 22 samples, ences in our institute. The clinical diagnosis or symp-
takes <90 mm to complete. Free carnitine results can be torus must also be considered when deciding whether or
available within 15 min when the instrument is pro- not a patient has carnitine deficiency or insufficiency.
grammed to perform free carnitine analysis first. Be- Clinicians and laboratory analysts should be aware of
cause calibration is required only once per day and the variation of carnitine concentrations with age and
assuming sufficient quantities ofthe reagents requiring the differences in values between analytical methods.
daily preparation are available, the analysis of 27 sam- By radioenzymatic assay, it is normal to observe free
ples per batch is practicable after the first batch is carnitine values <20 mol/L during the first week
complete. Up to 100 samples per day can be analyzed postpartum and <30 pmol/L until age 1 month (21 ). The
with one instrument and one technician familiar with spectrophotometric assay gives results consistently
routine assays on the Cobas Fara II. -10% higher than those determined by radioenzymatic
In our hands, the few problems encountered with this assay, and this should be considered when results from
assay were ascribed mostly to instrument faults. En- these two methods are compared.
zyme concentration is critically important for reproduc- Carnitine deficiency can develop from various causes,
ibility of the assay. Because concentrations vary be- including disorders of fatty acid and branched-chain
tween lots and suppliers, care must be taken to amino acid catabolism, long-term carnitine-free nutri-
accurately dilute the enzyme solution to the specified tion, and valproic acid therapy. Regardless of the assay
final concentration. Because all features of the instru- used, the clinical significance of abnormal results is
ment are used for this assay, preventive maintenance often unclear without identification ofthe acylcarnitine
must not be overlooked. species.
We carefully evaluated the new assay in our labora-
tory for 5 months, during which time >500 assays were We acknowledge the valuable technical assistance of R. Wynne
performed. When sufficient material was available, as- and the generous support for the development of this assay from
says were performed in duplicate. Having seen no sig- Sigma Tau Pharmaceuticals, Inc., Gaithersburg, MD, and the
Division of Research Resources, National Institutes of Health
nificant discrepancy between duplicates, we now con- (General Clinical Research Center grant no. M01-RR-30).
sider routine duplication unnecessary. The authenticity
of unusually low values (<20 jnnoLfL) is checked by References
viewing the kinetic plots and by duplication if there is 1. Bieber LL, Emaus R, Valkner K, Farrell S. Possible functions of
sufficient sample. The accuracy ofthis spectrophotomet- short-chain and medium-chain carrntine transferases. Fed Proc
ric assay has been validated by isotope-dilution mass 1982;41:2858-62.
2. Stanley CA. New genetic defects in mitochondrial fatty acid
spectrometry, considered a Definitive Method (17). oxidation and carnitine deficiency. Adv Pediatr 1987;34:59-88.
As automated methods for measuring carnitine con- 3. Marzo A, Cardace C, Monti N, Muck 5, Arrigoni-Martelli E.
centrations are introduced to hospital laboratories, the Chromategraphic and non-chromategraphic assay of L-carnitine
family components [Review]. J Chromatogr 1990;527:247-58.
need for accurate interpretation of the results must be
4. Hoppel CL. Determination of carnitine. In: Hommes FA, ed.
stressed. Low plasma free carnitine concentration, often Techniques in diagnostic human biochemical genetics. New York:
associated with an increased ratio ofplasma short-chain Wiley-Lisa, 1991:309-26.
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carnitine concentrations in rat tissue. J Lipid Rae 1964;5:184-7.
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6. Seccombe DW, Dodek P, FrolichJ, Hahn P, SkalaJP, Campbell
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ugal analyzer. Clin Chem 1986;32:342-6.
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9. Maeda J, Dudrick SJ. Rapid spectrophotometric determination
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2220 CLINICAL CHEMISTRY, Vol. 38, No. 1 1 , 1992