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Department of Laboratory Medicine, Box 359743, Harborview Medical Center and University of Washington, Seattle, WA 98104-2499.
a Author for correspondence. Fax 206-731-3930; e-mail labbe{at}mail.labmed washington.edu.
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Abstract |
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Key Words: indexing terms: antioxidants • centrifugal analyzer • dehydroascorbic acid
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Given the increasing interest in nutrition testing, and especially in nutrients that serve as antioxidants such as AA, an automated procedure designed for the clinical laboratory is desirable. To meet this need, we have developed an easily adaptable procedure that has significant advantages over chromatographic and spectrophotometric methods. This enzymatic end point procedure was developed by using the Cobas Fara centrifugal analyzer. In principle, AA is selectively and completely oxidized to DHAA by AA oxidase. The product is then coupled with o-phenylenediamine (OPDA) to yield a chromophore, the absorbance of which is measured at 340 nm.
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Materials and Methods |
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A protein precipitating agent (MPA/DTT) consisted of a 6.13 mol/L (40%) MPA solution containing 53.5 mmol/L DTT. An MPA/DTT diluent solution was then prepared by diluting this precipitating agent 1:10 in deionized water.
A stock calibrator solution of AA was prepared by dissolving 2.83 mmol/L in MPA/DTT diluent. This stock solution was stable for 1 year when stored at -20 °C. For working calibrator solutions, dilutions of the stock calibrator were made in the MPA/DTT diluent to concentrations of 283, 170, 113, 56.6, and 28.3 µmol/L. These diluted calibrator solutions were stable for 1 week when stored at 4 °C.
A 0.10 mol/L phosphate buffer was prepared by dissolving 11.547 g of NaH2PO4 and 3.885 g of Na2HPO4·7H2O in 800 mL of deionized water, adjusting to pH 6.5, then diluting to 1.0 L. A stock solution of AA oxidase was prepared by diluting with this buffer the assayed activity of the enzyme indicated on the vial from the supplier to obtain an activity of 200 U/mg per mL. A 100-µL aliquot of this stock enzyme solution was further diluted with 2.4 mL of the phosphate buffer to obtain an enzyme activity of 8 kU/L or 40 mg of protein/L to be used as a working reagent.
OPDA was dissolved to a concentration of 4.6 mmol/L in 0.1 mol/L phosphate buffer, pH 6.5. DTT was then added to a concentration of 0.6 mmol/L. This solution was stable for 2 weeks when stored at 4 °C in a brown bottle.
Sample preparation.
AA is very labile and subject to
oxidation to DHAA, which in itself is also labile (3). For
this reason, specimens are preferably collected in EDTA to minimize
metal-catalyzed oxidation. The anticoagulated whole-blood specimens
were collected and handled to ensure minimal exposure to air until
deproteinized and stabilized. With plasma, only fasting specimens
should be used for assessing AA status. After centrifugation of the
specimen, plasma (500 µL) was removed and quickly treated with 50
µL of MPA/DTT as a stabilizer. The treated sample was vortex-mixed
for 30 s, then centrifuged for 10 min at 1500g in a
refrigerated centrifuge at 4 °C. The supernatant was stored at
-20 °C until assayed. This filtrate can be stored at -20 °C for
1 month. Longer storage may be acceptable, but has not been clearly
established. Perchloric acid, sometimes used as a protein precipitant
and stabilizer in AA determination (4), gave results that
were comparable with those obtained with MPA/DTT (unpublished
observation).
Instrumentation.
This procedure was developed with a
Roche Diagnostic Systems (Montclair, NJ) Cobas Fara centrifugal
analyzer. For each control and unknown, 200 µL of the deproteinized
plasma supernatant was loaded into the Cobas cups and placed into a
Fara sample rack. AA concentrations of 28.4, 56.8, and 170 µmol/L
were loaded into Cobas cups and placed into positions 8, 9, and 10 of
the Fara calibrator rack. An amount of OPDA/DTT reagent sufficient for
the number of samples was placed in a 15-mL reagent cup. The working AA
oxidase solution was placed in a 4-mL reagent cup. Both reagents were
placed in the programmed position on a reagent 5 rack, the AA oxidase
going into the left or 1 position. The program parameters for the
Cobas Fara were set as outlined in Table 1
. An enzymatic end point was measured with a
direct printout in concentration being obtained after 300 s of
incubation, i.e., 30 readings at 10-s intervals. The blank was
automatically subtracted and the calculation was performed internally
according Beer's Law. All results were multiplied internally by a
factor of 1.1 to correct for dilution of the specimen by MPA/DTT.
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Results |
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Precision.
Commercial lyophilized control material was
supplemented with AA calibrators at concentrations of 93.4 and 37.9
µmol/L. The within-run CVs were 0.51% and 2.7%, respectively
(n = 22). The between-run CVs were 4.2% and 8.4%, where n =
19 and 20, respectively.
Recovery.
Recovery was determined by using five
different concentrations of AA, ranging from 55.5 to 160 µmol/L added
to plasma. Recovery ranged from 93.8% to 119%, with a mean of 104%.
Interferences.
The addition of bilirubin at a
concentration of 1860 µmol/L to both a control and a serum pool
produced no effect on assay results. Similarly,
Intralipid® added to a concentration of 18 g/L to create a
severe lipemia showed no effect on results. A hemoglobin concentration
of 1.3 g/L had no effect, but at 2.5 g/L (equivalent to extensive
hemolysis) the resulting AA concentration was falsely low by a
significant 17%.
Correlation.
For comparison with HPLC as a reference
method, 62 specimens were assayed by the two procedures. The range of
AA concentrations was 1.1 to 183 µmol/L. The correlation between the
methods was defined by AAEnz = 0.953AAHPLC +
0.0047 and r2 = 0.957.
Reference range.
The reference range for plasma AA
concentration in healthy adults, including both men and women, was
26.1–84.6 µmol/L, based upon fasting specimens (n = 20).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The described automated procedure was chosen and developed to keep specimen processing as simple as possible so that the test would be most convenient and practical for the clinical laboratory, yet give results that were comparable with or superior to a reference HPLC procedure (4). The automated procedure has been utilized for patient care as well as research in our institution for ~2 years. In daily practice, experience has shown that running the test in duplicate is not necessary to obtain clinically reliable results.
Results obtained through participation in a proficiency program conducted by NIST indicate that this enzymatic procedure has a routine negative bias averaging ~4% (6). Data are not available for a more detailed analysis of this feature of the procedure. However, nearly all participants in the NIST program use HPLC as a method of determination, and we found the same bias with our laboratory's HPLC results as noted above in the correlation evaluation. Any negative bias may be explained in part by the initial blanking before starting the enzymatic reaction, a procedural step that removes endogenous DHAA. A negative bias might result also from a greater specificity introduced by the enzymatic reaction, although this seems less likely. A possible role of DTT, added as a preservative, in preventing complete enzymatic oxidation of AA seems unlikely since the coupling of DHAA with OPDA should pull the reaction to completion. Since the bias has not invalidated the results and appears to have no clinical significance, we have not pursued further an interpretation or a correction.
In considering either the assessment or the monitoring of AA status of patients, it may be noteworthy that both AA and DHAA are biologically available, but normally only a trace (much less than 5%) of total AA equivalents circulate in plasma as DHAA (3)(7). Since this analytical procedure blanks out any DHAA before initiating the enzymatic reaction, only the AA form is detected. For nutritional status determinations in clinical practice, this is of no significance. However, the failure to detect DHAA due to blanking may be noteworthy for some research applications.
The assessment of AA status is commonly believed to be most accurate when measured in leukocytes as an indication of body tissue stores. While leukocyte concentrations may be appropriate for healthy individuals, the same conclusion cannot be arbitrarily extended to hospitalized patients (3)(7). In addition to the effects of trauma, many commonly used drugs as well as states of hyperglycemia and stress can dramatically alter the flux of AA across cell membranes (3). For this reason, we believe that a fasting plasma specimen is preferred, at least for hospitalized patients. Of further importance for the clinical laboratory, plasma preparation is far more practical than is leukocyte preparation, even if the isolation of leukocytes is limited to the buffy coat. In either case, fasting specimens are preferred for the most dependable determination of AA status.
Although the roles of AA in metabolism remain to be fully elucidated, the requirements of AA for optimal wound healing and immune function are of prime importance to hospitalized patients. Thus, assessment of AA status on hospital admission and monitoring of AA therapy would seem to be prudent until outcome studies demonstrate the true value of maintaining AA repletion. The features of this method will permit a more frequent and cost-effective test for this important, commonly deficient nutrient (6) to be offered on a routine basis for patient care.
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Acknowledgments |
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Footnotes |
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