Clinical Chemistry 44:4
810 – 816 (1998)
Automation and
Analytical Techniques
Determination of free and total carnitine with a
random-access chemistry analyzer
Lijun Wan and Richard W. Hubbard*
long-chain fatty acids as an energy source (1, 2, 10). Because long-chain fatty acids are involved in the synthesis
of phospholipids such as lung surfactants, it is possible
that carnitine deficiency contributes to the aggravation of
cystic fibrosis and to some 10% to 15% of sudden infant
death syndromes with a genetic deficiency of carnitine
acyl dehydrogenase (1, 13). Therefore, the major and acute
role of carnitine deficiency in a variety of abnormalities
makes the provision of a widely available and easily
performed carnitine assay highly desirable. This is particularly true for premature infants and infants under treatment for seizures with valproate and related anticonvulsants that suppress carnitine. The suppression of carnitine
is also related to some chronic diseases associated with
the aging process (6).
There are a number of methods to measure carnitine,
such as radioisotopic (1, 14, 15), radioenzymatic (3,
13, 16 –18), spectrophotometric (4, 18–25), radioisotopic
exchange with HPLC (13, 16, 26 –29), and tandem mass
spectrometry (11, 30). Sample preparation steps for these
methods include filtration and concentration (19, 22, 26, 31),
solid-phase extraction (3, 12–18, 26 –29), and dialysis
chamber extraction (32). Cederblad et al. (22) described an
automated spectrophotometric method for carnitine determination with high precision, low reagent cost, and a
short analysis time on a Cobas Bio centrifugal analyzer
(22); it correlates well with the standard carnitine radioenzymatic assay (REA). Our method is compared with both
the spectrophotometric method of Cederblad et al. and
the standard radioenzymatic method (14). Compared
with the method of Cederblad et al. (22), our method has
greater sensitivity and linearity, random accessibility, stat
capability, appreciably lower reagent costs, and no requirement for special heating equipment or expensive
filtration apparatus; moreover, it provides exactly the
same sample matrices for both free and total carnitine,
enabling both values to be determined with one calibration curve, one dilution factor, and one reagent cartridge.
With slight modifications in the procedure as used on the
Beckman Synchron CX4, this method is easily adaptable
on almost any of today’s random access chemistry ana-
Carnitine deficiency presents as a major problem in
fatty acid oxidation. The use of a plasma carnitine assay
can rapidly help to describe this deficiency. The method
we describe here requires two simple steps of sample
preparation, followed by automated analysis with the
Beckman Synchron CX4 random-access chemistry analyzer. The goal of this method development was to
reduce the cost of analysis and to allow a greater number
of laboratories to perform this assay on demand within
1 h for both free and total carnitine. The method has a
linearity of 0 –150 mmol/L and a detection limit of 5
mmol/L. The inter- and intraday CVs are <20%. The
method agreed closely with both the widely used RIA
and spectrophotometric methods.
l-Carnitine (3-hydroxy-4-N-trimethylammonium butyrate) is the carrier molecule for long-chain fatty acids to
cross the mitochondrial membrane (1–3). It is synthesized
in the liver, requiring lysine and methionine as precursors
and vitamin C as cofactor (4, 5). There are two isoforms of
the enzyme (E.C.2.3.1.21)—the carnitine palmitoyl transferase (CPT)1 I, which is located at the inner side of the
outer mitochondrial membrane, transesterifies the longchain fatty acid and transports the acyl CoA across the
inner mitochondrial membrane, where CPT II is located,
to break the acyl bond releasing the fatty acid, and
recycling CoASH (2, 6 – 8). The consequences of primary
carnitine deficiency and secondary deficiency caused by
carnitine acyl dehydrogenase deficiency range from mild
forms of muscle weakness (3) to severe forms of hypoglycemia and cardiomyopathy (9 –11), from Reye-like syndrome (10, 12) to lipidosis, myopathy, and abnormal
organic acid production due to the inability to utilize
Department of Pathology & Laboratory Medicine, School of Medicine,
Loma Linda University, Clinical Laboratory, Loma Linda University Medical
Center, Loma Linda, CA 92350.
*Author for correspondence. Fax 909-824-4832; e-mail [email protected].
1
Nonstandard abbreviations: CPT, carnitine palmitoyl transferase; REA,
radioenzymatic assay; CAT, carnitine acetyltransferase; DTNB, 5,59-dithiobis2-nitrobenzoate; TNB, 5-thio-2-nitrobenzoate; and UDR, user-defined reagent.
Received August 27, 1997; revision accepted November 4, 1997.
810
Clinical Chemistry 44, No. 4, 1998
lyzers, and can be used to determine carnitine in most
tissues and biological fluids (3, 18, 31).
Materials and Methods
principle
l-Carnitine reacts with acetyl CoA catalyzed by carnitine
acetyltransferase (CAT) to form acetyl l-carnitine and
CoASH. CoASH reacts nonenzymatically with 5,59-dithiobis-2-nitrobenzoate (DTNB) to form 5-thio-2-nitrobenzoate (TNB). The concentration of TNB is measured spectrophotometrically at 410 nm.
811
paring a zero blank (type I water) and a 150 mmol/L
calibrator, which was performed biweekly or monthly,
since the calibration curve was stable for at least 1 month.
Every 6 months, a full linearity curve with all five
calibrators was performed as required by CAP and CLIA
regulations. Serum-based controls were made by addition
of stock calibrator and 1 mmol/L palmitoyl carnitine
aqueous calibrator (Sigma P4509) to Biocell serum to
produce a low control and a high control. Both controls
were stored at 217 °C, and the ranges were established by
running triplicates everyday for at least 7 days. Controls
were run with each batch of patient samples.
instrument
The instrument is a Beckman Synchron CX4 Chemistry
Analyzer. An Eppendorf pipet is used for acid and base
dispensing.
reagents
Three reagents were stored on board the instrument in a
single user-defined reagent (UDR) cartridge. We dissolved 20 mg of DTNB (Sigma D8130) in 100 mL of 50
mmol/L HEPES buffer and placed it in compartment A of
the UDR cartridge. It was sufficient for 500 tests. To make
the HEPES buffer, 5 mL of 1 mol/L HEPES (Sigma
H-7523) was diluted to 100 mL with phosphate buffer,
which was made from dissolving 1.19 g of potassium
phosphate monobasic (KH2PO4, JT Baker 1–3246) and
2.83 g of potassium phosphate dibasic (K2HPO4, JT Baker
3252–1) in 100 mL of type I water. The pH was adjusted to
7.5 with 5 mol/L NaOH before adding HEPES. Twentyfive milligrams of acetyl CoA (Sigma A2181) were dissolved in 10 mL of type I water and placed it in compartment B. It was sufficient for 450 tests. Fifty microliters of
carnitine acetyltransferase (EC 2.3.1.7, Sigma C 8757) was
diluted 1:100 by volume with type I water and stored in
compartment C. It was only sufficient for 125 tests;
therefore, three more refills of CAT could be made before
the entire cartridge was discarded. All three reagents
were stable for 3 months in the instrument reagent
compartment, which was maintained at 2– 6 °C. We made
the protein-precipitating reagent by diluting 18 mL of 70%
perchloric acid (HClO4, Malinkrodt 2766) to 100 mL with
type I water. It was stable at room temperature for 12
months. The 2 mol/L KOH at 11.2 g/L solution (JT Baker
3140 – 01, does not compensate for K2CO3 impurities) was
used for both hydrolyzing the sample for total carnitine
and neutralizing the HClO4. It was stable for 6 months at
room temperature. Biocell serum (Biocell Laboratory) was
used to produce low and high controls.
calibrators and controls
Stock l-carnitine standard (5.0 mmol/L; Sigma C7518)
was made by dissolving 98.7 mg of l-carnitine in 100 mL
of type I water. Five working calibrators of 0.0, 10.0, 35.0,
75.0, and 150.0 mmol/L were made from stock l-carnitine
calibrator with type I water. Once established, the calibration curve was verified by a two-point calibration com-
sample collection and patient preparation
Adult patients fasted for at least 4 h before venipuncture;
children and pediatric patients fasted for at least 2 h. A
lipemic specimen does not itself interfere with the assay,
yet it can cause redistribution of the carnitine fractions in
vivo (10, 34). Many foods, such as meat, dairy products,
asparagus, and avocados (6, 34) contain carnitine, thus
mandating the 4-h fasting for adults and 2 h for children.
Prolonged fasting (.24 h), on the other hand, will cause
an increase of acyl carnitine (10, 17, 22). Whole blood was
collected in a 4.5-mL K3EDTA Vacutainer Tube (Beckton
Dickinson, 366536). For pediatric samples, we collected
blood in a microtainer (Becton Dickenson, 5974) containing K3EDTA. Blood was centrifuged at 3000g for 10 min,
and plasma was separated and stored at 210 °C to 220 °C
until assayed.
assay procedure
Two sets of 12 3 100 mm test tubes were added for each
control or patient sample. Two hundred microliters of
control or patient sample were added to both sets of the
test tubes. To the first set, 10 mL of 2 mol/L KOH was
added with an Eppendorf pipette, mixed gently, covered,
and incubated at room temperature for 45 min to hydrolyze the ester bond. At the end of the incubation, 40 mL of
180 mL/L diluted HClO4 was added to precipitate the
protein; it was vortexed immediately for 10 s; another 30
mL of 2 mol/L KOH was added to neutralize the acidity;
it was vortexed immediately for 10 s and centrifuged at
3000g for 5 min. The supernatant was transferred into a
sample cup without causing foam and run on the analyzer
for total carnitine. While the first set of tubes was incubating, the second set was processed by adding 40 mL of
the 180 mL/L diluted HClO4 to precipitate the protein;
vortexed immediately for 10 s. After 40 mL of 2 mol/L
KOH was added to neutralize the acidity, it was vortexed
immediately again for 10 s, and centrifuged at 3000g for 5
min. The supernatant was carefully transferred into a
sample cup of the CX4 and free carnitine was measured.
Free and total carnitine shared the same user-defined
chemistry, reagent cartridge, calibration curve, and dilution factor. The parameters for the CX4 user-defined
chemistries are shown in Table 1.
Because the controls and patient samples have a dilu-
812
Wan and Hubbard: Free and total carnitine determination
Table 1. User-defined chemistry for free and total carnitine
on Beckman CX-4.
Chemistry name
Test name
Reaction type
Reaction direction
Units
Decimal precision
Calculation factor
Math model
Cal time limit
Number of calibrators
Primary wavelength
Secondary wavelength
Sample volume
Primary injection reagent
A
B
Secondary injection reagent
C
Add time
Calibrators
#1
#2
#3
#4
#5
#6
Multipoint span
1–2
2–3
3–4
4–5
5–6
6–1
Reagent blank
Start read
End read
Low absorbance limit
High absorbance limit
Reaction
Start Read
End Read
Low absorbance limit
High absorbance limit
Usable range
Lower limit
Upper limit
Substrate depletion
Initial rate
Dabsorbance
Recovery/sensitivity
SD (conc)
CV, %
SD (mA)
Threshold
CAT
Free and total carnitine
Rate 1
Increasing
mmol/L
3.3
0
1
336 h
5a
410 nm
600 nm
25 mL
180 uL
20 uL
35 uL
368 sec
0.0
10.0
35.0
75.0
150.0
0.00
0.000
0.000
0.000
0.000
0.000
0.000
tion factor of 1.4, whereas the calibrators are not diluted,
the concentrations of the calibrators can be entered in the
user-defined chemistry with this 1.4 factor, i.e., 35 mmol/L
will be 49 mmol/L, and 75 mmol/L will be 105 mmol/L;
thus the final instrument printout will give values corrected for the dilution.
statistics
Statistical analysis was performed by simple linear regression with SYSTAT software version 6.0.1 published by
SPSS, 1996.
Results
linearity
The linearity of this method was between 0 and 150
mmol/L (Fig. 1). Five aqueous calibrators of 0, 10, 35, 75,
and 150 mmol/L gave values of 0.1, 9.8, 35, 75, and 150
mmol/L, respectively (r 5 1.000). The linear range encompassed the reference range of carnitine values of 20 to 100
mmol/L. A serial dilution of serum-based low control is
presented in Fig. 2.
precision
The intraday and interday (1 month) CVs for three
concentrations were ,20% (Table 2).
accuracy
Recovery of the method was 99.8% 6 0.7% for aqueous
solution (n 5 10) and 97.6% 6 3.9% for serum-based
materials (n 5 20). The method agreed well with the
standard REA (x) for both free (n 5 29) and total carnitine
(n 5 28): for free carnitine, y 5 1.059x 2 15.97, r 5 0.942,
SE 5 0.072; for total carnitine, y 5 1.09x 2 12.357, r 5
0.997, SE 5 0.017 (Figs. 3 and 4). Consequently, the
correlation for esterified carnitine was y 5 1.077x 1 5.71,
336 s
352 s
20.5
0.5
64 s
144 s
21.5
1.5
0.00
99999.00
99.999
1.5
0.000
0.000
0.000
9999.00
a
To calibrate the instrument with two calibrators, change the number of
calibrators to 2, and change the concentrations of the first and second
calibrators to 0 and 150, respectively.
Fig. 1. Linearity of five aqueous calibrators.
813
Clinical Chemistry 44, No. 4, 1998
Fig. 2. Serial dilution of serum-based control (n 5 4 for each concentration; graph represents mean 6 SD).
Fig. 3. Comparison of free carnitine values measured by the proposed
method and the radioenzymatic method.
r 5 0.996, n 5 27. The method agreed also with spectrophotometric method of Cederblad et al (x): for free
carnitine, y 5 0.881x 1 0.998, r 5 0.989, SE 5 0.032, n 5 29;
for total carnitine, y 5 0.861x 2 0.029, r 5 0.995, SE 5
0.026, n 5 23 (Figs. 5 and 6). Consequently, the regression
for esterified carnitine was y 5 0.824x 1 0.233, r 5 0.983,
n 5 23. The filtrates were stable at room temperature for
at least 1 h.
Our preliminary ranges of reference values are free carnitine 25– 80 mmol/L and total carnitine 31–100 mmol/L,
with females being slightly lower and newborns ,3
months giving significantly lower values.
detection limit
Method validation against the standard radioenzymatic
method (14) run at Los Angeles Children’s Hospital gave
estimates of mean reference values
Discussion
We established the detection limit of the method by
assaying 15 aqueous calibrators of 3.0 mmol/L, which
yielded a mean of 2.48 and SD of 1.69. The 3.0 mmol/L
(rather than 0) was used because the CX4 does not accept
readings less than zero. Taking the mean plus 3 SD gives
a value of 7.55 mmol/L. We then subtracted the mean of
2.48 from 7.55 to give the estimated detection limit of 5.07
mmol/L.
stability of control materials
The serum-based controls were assessed for their stability
when stored at 217 °C over a period of 6 months. The
CVs of free and total carnitine in both the low and high
controls are presented in Table 3.
Table 2. Interday and intraday assay variations.
Intraday concentration, mmol/L
35
Interday concentration, mmol/L
10
35
75
Mean
SD
CV, %
n
36.94
5.04
13.6
14
10.09
32.17
71.85
2.42
3.89
4.76
24
12.1
6.6
20
20
20
Fig. 4. Comparison of total carnitine values measured by the proposed
method and the radioenzymatic method.
814
Wan and Hubbard: Free and total carnitine determination
Table 3. CVs for low and high controls over a
6-month period.
Fig. 5. Comparison of free carnitine values measured by the proposed
method and the spectrophotometric method of Cederblad et al. (20).
excellent correlation as shown in the results. In addition,
it correlated well with the spectrophotometric method of
Cederblad et al. (22), which is used at Corning Nichols
Institute. Because that method has a positive bias compared with that of the radioenzymatic method with a 1.12
average slope (22), the current method apparently has
offset this bias and improved the agreement with the
radioenzymatic method, although there is still a positive
bias with an average slope of 1.06. There were only a few
specimens used in both method comparisons that belong
Fig. 6. Comparison of total carnitine values measured by the proposed
method and the spectrophotometric method of Cederblad et al. (22).
Carnitine controls
Mean
CV, %
n
Low control–free
Low control–total
High control–free
High control–total
23.3
35.3
96.9
125.6
12.7
15.8
8.2
8.1
37
31
33
42
to the “abnormal” range, which bear the potential to
leverage the linear regression line generally used to
interpret the acceptance of the comparison. However,
when those few abnormal specimens were not used in
constructing the linear regression curve and a new linear
regression line was constructed solely on the basis of the
group of normal values, the slopes changed slightly
within the standard error, and extrapolation indicates that
these few abnormal specimens did not alter the linear
regression line or possible clinical significance with our
assay values. We verified the ability of the assay to detect
carnitine ,20 mmol/L for infants older than 3 months,
and ,10 mmol/L for infants younger than 3 months (Fig.
2). The results indicate that we can distinguish carnitine
deficiencies of ,10 mmol/L and between 10 to 20
mmol/L.
The CX4 takes ,8 min to measure the first sample and
30 s each sample thereafter; therefore, from the beginning
of the operation, through incubation, until the reporting
of both free and total carnitine is ;1 h, with labor
involvement of approximately ;1 min per sample. The
reagents are very stable compared with some published
methods, which call for freshly made reagents (22, 23).
Because we use rate measurement instead of end point,
there are ,2 min from the addition of CAT until the end
of the measurement of absorbance, and the reported
inactivation of CAT by DTNB is negligible (23). The CX4
chemistry analyzer and a common laboratory centrifuge
are required. To perform the analysis with a manual
spectrophotometer method requires considerable skill
and expertise, which greatly increase the test cost and
running time.
Our method requires a reasonably small amount of
sample, with 400 mL of plasma for the determination of
both free and total carnitine. Plasma separated from red
cells is stable for 24 h at 2– 8 °C; however, 217 °C or below
in a glass vial is recommended for prolonged storage. The
unique design of using acid and then base (40 mL each) in
free carnitine, and using base, acid, base (10, 40, and 30
mL, respectively) in total carnitine makes the final reaction
matrices the same for both free and total carnitine, which
allows the use of the same UDR reagent cartridge, same
calibration curve, and same dilution factor (31.4 vs calibrators). Plasma specimens containing a high amount of
protein consistently agglutinated with 20 mL of 2 mol/L
KOH. We reduced the initial amount of KOH from 20 mL
to 10 mL for the hydrolysis of the ester bonds and
lengthened the incubation time from 30 min to 45 min to
Clinical Chemistry 44, No. 4, 1998
equate the hydrolysis of ester bond. Because the protein
precipitate begins to dissociate at pH 4 and completely
dissolves at pH 6 (35), repeated experiments were carried
out to determine the types and the minimum amount of
acids to use. Sulfosalicylic acid, metaphosphoric acid, and
perchloric acid were compared. Perchloric acid was chosen because metaphosphoric acid was too weak and very
unstable, and residual sulfosalicylate remaining after neutralization by alkali tended to hinder the following enzymatic process. Perchloric acid, however, is a strong acid
with a small molecular structure, with its protein precipitation property derived solely from its acidity and ionic
strength. Hence the residual acidity in the filtrate to
prevent the dissolution of the precipitate is buffered by
the first reagent in compartment A and, therefore, does
not denature the enzyme when CAT is added.
We conclude that this carnitine assay is suitable for a
large tertiary-care hospital, and also is usable in small
clinical and research laboratories possessing automated
chemistry analyzers. It achieves our goals for a rapid,
low-cost method to measure free and total plasma carnitine.
We graciously thank Lawrence Sweetman and the Biochemistry Laboratory of Children’s Hospital of Los Angeles for their continuous support in providing samples
for method validation, and the Clinical Trial Department
of Corning Nichols Institute for providing method validation samples. We also appreciate James Westerngard
for his statistical expertise and advice. Financial support
from the Department of Pathology, School of Medicine is
greatfully acknowledged.
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