Determination of dehydroascorbic acid in mouse tissues and plasma by using tris(2-carboxyethyl)phosphine hydrochloride as reductant in metaphosphoric acid/ethylenediaminetetraacetic acid solution

Article abstract of DOI:10.1248/bpb.33.364

Ascorbic acid (AA) has a strong anti-oxidant function evident as its ability to scavenge superoxide radicals in vitro. Moreover, AA is an essential ingredient for post-translational proline hydroxylation of collagen molecules. Dehydroascorbic acid (DHA), the oxidized form of AA, is generated from these reactions. In this study, we describe an improved method for assessing DHA in biological samples. The use of 35mM tris(2-carboxyethyl)-phosphine hydrochloride (TCEP) as a reductant completely reduced DHA to AA after 2 h on ice in a 5% solution of metaphosphoric acid containing 1mM ethylenediaminetetraacetic acid (EDTA) at pH 1.5. This method enabled us to measure the DHA content in multiple tissues and plasma of 6-weeks-old mice. The percentages of DHA per total AA differed markedly among these tissues, i.e., from 0.8 to 19.5%. The lung, heart, spleen and plasma had the highest levels at more than 10% of DHA per total AA content, whereas the cerebrum, cerebellum, liver, kidney and small intestine had less than 5% of DHA per total AA content. This difference in DHA content may indicate an important disparity of oxidative stress levels among physiologic sites. Therefore, this improved method provides a useful standard for all DHA determinations.

Full text of DOI:10.1248/bpb.33.364

364
Biol. Pharm. Bull. 33(3) 364—369 (2010)
Vol. 33, No. 3
Determination of Dehydroascorbic Acid in Mouse Tissues and Plasma by
Using Tris(2-carboxyethyl)phosphine Hydrochloride as Reductant in
Metaphosphoric Acid/Ethylenediaminetetraacetic Acid Solution
Yasunori SATO,a,# Takayuki UCHIKI,a,# Mizuki IWAMA,b Yuki KISHIMOTO,a Ryoya TAKAHASHI,a and
,a
*
Akihito ISHIGAMI
a Department of Biochemistry, Faculty of Pharmaceutical Sciences, Toho University; Chiba 274–8510, Japan: and
b Department of Geriatrics and Vascular Medicine, Tokyo Medical and Dental University; Tokyo 113–8510, Japan.
Received November 20, 2009; accepted December 10, 2009; published online December 18, 2009
Ascorbic acid (AA) has a strong anti-oxidant function evident as its ability to scavenge superoxide radicals
in vitro. Moreover, AA is an essential ingredient for post-translational proline hydroxylation of collagen mole-
cules. Dehydroascorbic acid (DHA), the oxidized form of AA, is generated from these reactions. In this study, we
describe an improved method for assessing DHA in biological samples. The use of 35 mM tris(2-carboxyethyl)-
phosphine hydrochloride (TCEP) as a reductant completely reduced DHA to AA after 2 h on ice in a 5% solution
of metaphosphoric acid containing 1 mM ethylenediaminetetraacetic acid (EDTA) at pH 1.5. This method en-
abled us to measure the DHA content in multiple tissues and plasma of 6-weeks-old mice. The percentages of
DHA per total AA differed markedly among these tissues, i.e., from 0.8 to 19.5%. The lung, heart, spleen and
plasma had the highest levels at more than 10% of DHA per total AA content, whereas the cerebrum, cerebel-
lum, liver, kidney and small intestine had less than 5% of DHA per total AA content. This difference in DHA
content may indicate an important disparity of oxidative stress levels among physiologic sites. Therefore, this im-
proved method provides a useful standard for all DHA determinations.
Key words ascorbic acid; dehydroascorbic acid; dithiothreitol; tris(2-carboxyethyl)phosphine hydrochloride; metaphosphoric
acid
Ascorbic acid (AA) functions as an electron donor and HPLC coupled with detection by UV light are widely used
scavenges free radicals such as superoxide radicals1) and hy- for such analyses.10) Other means of AA determination sub-
droxyl radicals2) in vitro. Moreover, AA is essential for post- sequently developed are the ketone derivatization method
translational proline hydroxylation of collagen molecules,3) with 2,4-dinitrophenylhydrazine (DNPH) or o-phenylene-
after which hydroxyproline residues play a critical role in sta- diamine and HPLC coupled with electrochemical detection
bilizing the triple helical structure of collagen.4) Dehy- (ECD) system.11—13) HPLC-ECD is widely used at present,
droascorbic acid (DHA), the oxidized form of AA, is gener- because of this assay’s great sensitivity and specificity.14)
ated as a result of these reactions. The biological activity of However, for DHA determination, the UV detector is not
DHA has been considered equivalent to that of AA, because suitable, because DHA’s maximum absorbance at 220 nm
intracellular DHA is rapidly reduced to AA by dehydroascor- provides only insensitive and nonselective results. DHA is
bic acid reductase.5) However, Ogiri et al. reported that DHA also difficult to detect with HPLC-ECD because of its low
had less than 10% of AA’s biological efficiency on a molar electroactivity. Therefore, a DHA concentration is typically
basis, judging from experiments with the inherently scorbutic calculated by subtracting AA concentration from the total
ODS rat, which is a convenient animal model for investigat- AA (AAꢀDHA) concentration determined in a different
ing the metabolism of AA in humans.6) Moreover, May et al. chromatographic run. The total AA concentration is usually
noted that the human mature red blood cells they studied did calculated by reducing the DHA to AA with sulfhydryl com-
not have sodium-dependent vitamin C transporters (SVCT1 pounds including dithiothreitol (DTT) (Fig. 1), mercap-
and SVCT2) yet manifested a very low late uptake of radio- toethanol and homocysteine.15) However, the reducing ability
labeled AA.7) In contrast, human mature red blood cells effi- of these sulfhydryl compounds is limited to a narrow neutral
ciently took up DHA from blood via a glucose transporter 1 pH range. In general, metaphosphoric acid (MPA) and EDTA
(Glut1).8) Erythrocyte Glut1 and associated DHA uptake are are used to stabilize the AA and DHA in biological sam-
unique traits of humans and the few other mammals that have ples.16) Therefore, raising the samples’ pH to neutral is
lost the ability to synthesize AA in vivo. Furthermore, DHA essential to effectively reduce DHA to AA. Since AA and
is known to enter mitochondria via Glut1 and undergo reduc- DHA are unstable at neutral pH, using sulfhydryl compounds
tion to AA in the mitochondria.9) AA, which enters mito- as a reductant may not be a suitable.
chondria as DHA, also protects mitochondria from oxidative
Tris(2-carboxyethyl)phosphine hydrochloride (TCEP),
injury. Thus, because DHA possesses unique physiological which is easily soluble and stable in aqueous solution, has re-
features, distinct from those of AA, establishment of a sim- ducing activity at pH 1.5 to pH 9.0 and has been applied to
ple and accurate method of assessing the presence of DHA is DHA reduction at pH 4.3 (Fig. 1).17),18) Recently, Wechters-
very important.
bach and Cigic evaluated the reduction of DHA with TCEP
AA in biological samples is routinely evaluated by using at low pH and reported that DHA is fully reduced with
HPLC systems. AA’s maximum absorbance at 265 nm in 10 mM TCEP in a 2% MPA solution at 25 °C.19) Therefore,
© 2010 Pharmaceutical Society of Japan
∗ To whom correspondence should be addressed. e-mail: ishigami@phar.toho-u.ac.jp
#
These authors contributed equally to this work.

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Fig. 1. Reduction Scheme of Dehydroascorbic Acid (DHA) to Ascorbic Acid (AA) by Tris(2-carboxyethyl)phosphine Hydrochloride (TCEP) and Dithio-
threitol (DTT)
The reduction ability of TCEP has a wide pH range from 1.5 to 9.0, but the reduction ability of DTT is restricted to a pH range from only 6.5 to 9.0.
the reduction of DHA with TCEP at an acidic low pH can (MPA/EDTA) solution, after which 6.3 mM quantities of
now be considered more reliable. However, TCEP has not yet DHA were placed in brown-colored vials filled with argon
been used as a reductant for testing AA and DHA levels in gas for storage at ꢁ80 °C until use. DHA solution was used
biological samples such as murine tissues. Therefore, as pre- within one week.
sented here, we investigated the reduction of DHA with
Determination of AA AA was analyzed by using a
TCEP in MPA solution on ice. Using this improved method HPLC-ECD method, as described,21—23) consisting of a Wa-
to stabilize AA and DHA, we determined the DHA content ters 2695 separations module coupled with a Waters 2465
in mouse tissues and plasma and found significant variability electrochemical detector (Nihon Waters, Tokyo, Japan). A
in the percentages of DHA per total AA among the materials vacuum degasser, quaternary pump, and thermostated au-
examined.
tosampler were included in the Waters 2695 separations
module. Separation was achieved on an Atlantis dC18 5 mm
column (4.6ꢃ150 mm) combined with an Atlantis dC18
5 mm guard column (4.6ꢃ20 mm) both from Nihon Waters.
MATERIALS AND METHODS
Materials L-Ascorbic acid (AA), metaphosphoric acid The mobile phase consisted of 50 mM phosphate buffer (pH
(MPA), bromine, thiourea and dithiothreitol (DTT) were pur- 2.8), 540 mM EDTA and 2% methanol. The flow rate was
chased from Wako Pure Chemicals (Osaka, Japan). Ethylene- 1.3 ml/min. Temperatures for the column and Waters 2465
diaminetetraacetic acid (EDTA) was purchased from Dojindo electrochemical detector were set at 30 °C and for the ther-
Laboratories (Kumamoto, Japan). Tris(2-carboxyethyl)phos- mostated autosampler at 4 °C. Ten microliters of the sample
phine hydrochloride (TCEP) was purchased from Tokyo was injected into the HPLC-ECD system for AA analysis.
Chemical Industry (Tokyo, Japan).
Electrical signals were recorded by using an electrochemical
Animals Male C57BL/6N Cr Slc mice (6 weeks old) detector with a glassy carbon electrode at ꢀ0.6 V. All electri-
were purchased from Japan SLC (Shizuoka, Japan). cal signal data from the electrochemical detector were col-
Throughout the experiments, mice were maintained on a 12- lected by the Waters Empower2 software (Nihon Waters).
h light/dark cycle in a controlled environment. All experi-
Reduction of DHA in MPA/EDTA Solution by TCEP
mental procedures using laboratory animals were approved and DTT For reduction of DHA, 100 mM, 175 mM, and
by the Animal Care and Use Committee of the Toho Univer- 350 mM portions of TCEP and 350 mM of DTT in 5%
sity.
MPA/EDTA were freshly prepared before use. DHA solution
Preparation of Plasma and Tissues Mice were sacri- was gently thawed and diluted to 6.3 mM DHA with 5%
ficed, and their blood was collected from the inferior vena MPA/EDTA. Eight hundred and ten microliters of 6.3 mM
cava. Then, 500 ml of blood was gently mixed with 5 ml of DHA solution (final conc. 5.7 mM DHA) was incubated with
0.5 M EDTA and centrifuged at 880 g for 15 min at 4 °C. The 90 ml of 100 mM, 175 mM and 350 mM TCEP (final conc.
resulting supernatants were used as plasma for further analy- 10 mM, 17.5 mM and 35 mM TCEP, respectively) or 350 mM
sis. Afterward, mice were systemically perfused with ice-cold DTT (final conc. 35 mM DTT) on ice for 4 h. The reaction
phosphate buffered saline through the left ventricle to wash mixture was checked and confirmed as having a pH value of
out remaining blood cells, and tissues of interest were col- 1.5. After each incubation time, 90 ml of reaction mixture
lected and stored at ꢁ80 °C until use.
was diluted with 810 ml of 5% MPA/EDTA. Then, the mix-
Preparation of DHA DHA was prepared by bromine ture was transferred to an autosampler vial, and AA content
oxidation of AA.20) Briefly, 5 ml of bromine (purity ꢂ99.9%) was analyzed by HPLC-ECD for the percentage of DHA re-
was added to 10 ml of 10 mM AA solution for oxidation of duction.
AA to DHA. Immediately thereafter, 20 ml of 5% thiourea
Reduction Efficiency of DHA Added to the Mouse
was added to the solution to remove the small amount of ex- Liver Mouse livers were homogenized with 14 volumes of
cess bromine and obtain a clear-colored DHA solution. DHA ice-cold 5.4% MPA/EDTA and centrifuged at 21000 g for
solution was diluted with 5% MPA containing 1 mM EDTA 10 min at 4 °C. AA or DHA (0.57 mmol/g tissue) was added

366
Vol. 33, No. 3
to the centrifuged supernatant and reduced with 35 mM TCEP RESULTS
on ice for 2 h. After incubation, the reaction mixture was di-
luted with 5% MPA/EDTA and analyzed for AA by HPLC-
ECD.
Chromatograms of AA Since MPA and EDTA are usu-
ally used for stabilizing AA in biological samples,17,24,25) our
Determination of DHA in Tissues and Plasma by Using analysis began with those materials. To confirm AA’s chro-
TCEP and DTT as Reductant To compare the reduction matographic separation profiles, AA was dissolved in 5%
abilities of TCEP and DTT, we applied both agents sepa- MPA/EDTA then analyzed with our HPLC-ECD system. Fig-
rately to tissues and plasma obtained from five mice and ures 2A and B show a typical chromatogram of AA dissolved
measured total AA (AAꢀDHA) and AA content. Tissues in 5% MPA/EDTA and 5% MPA/EDTA alone, respectively.
were homogenized with 14 volumes of ice-cold 5.4% Although the retention times changed depending on column
MPA/EDTA and centrifuged at 21000 g for 10 min at 4 °C. batch, flow speed and consistency of the mobile phase, the
Plasma was mixed with 1 volume of 10% MPA/EDTA and AA peak was detected at 2.5 min of retention time and was
centrifuged at 21000 g for 10 min at 4 °C. Total AA (AAꢀ completely separate from MPA/EDTA peaks in this HPLC
DHA) and AA contents in centrifugal supernatants were de- condition (Figs. 2A, B).
termined individually, and DHA content was calculated by
Characterization of DHA Prepared by Bromine Oxida-
subtraction of AA from total AA. For determination of total tion of AA For this study, we prepared DHA solution from
AA by using TCEP as reductant, 90 ml of the centrifugal su- AA by bromine oxidation, because commercial DHA powder
pernatants were reduced with 10 ml of 350 mM TCEP (final formed DHA dimers, a structure that is insoluble in aqueous
conc. 35 mM TCEP) for 2 h on ice. After reduction, the reac- solution.26) To ensure the AA was completely oxidized to
tion mixture was diluted with 5% MPA/EDTA and analyzed DHA, DHA solution prepared with bromine was tested by
for total AA by HPLC-ECD. For determination of AA, HPLC-ECD. No AA peak was present (Fig. 2C). Next, the
100 ml of the centrifugal supernatant was diluted with 5% DHA preparation was reduced again to AA with TCEP to ex-
MPA/EDTA and analyzed by HPLC-ECD. To determine total clude the possibility of DHA and AA breakdown. That is, the
AA with DTT applied as reductant, we used the method de- soluble DHA preparation was reduced with 35 mM TCEP for
scribed by Lykkesfeldt et al.11) with a slight modification. 2 h on ice as described in the next section and then analyzed
Briefly, 100 ml of centrifugal supernatants were reduced with for AA with the HPLC-ECD system. As Fig. 2D depicts, the
100 ml of 5 mM DTT (final conc. 2.5 mM DTT) in 0.5 M Tris– AA peak was detected at 2.5 min of retention time, and re-
HCl buffer (pH 9.0) for 5 min at 25 °C. The pH value in the covery of AA from the theoretical DHA content was 99.9%.
reaction mixture was 6.1. After reduction, 50 ml of 0.2 M These results indicate that AA was completely oxidized to
H2SO4 was added to the reaction mixture followed by deter- DHA by bromine. In addition, a peak of TCEP was also de-
mination of AA. In the final step, 100 ml of the centrifugal tected at 3.1 min of retention time, and this peak was com-
supernatant diluted with 5% MPA/EDTA was subjected to pletely separate from the AA and MPA/EDTA peaks in this
HPLC-ECD for analysis of AA.
HPLC condition. Therefore, this HPLC-ECD system was
Statistical Analysis Results are expressed as meansꢄ used throughout the following study.
S.E.M. The probability of statistical differences between
DHA Reduction by TCEP in MPA/EDTA Solution
experimental groups was determined by paired t-test using The pH value of 5% MPA/EDTA solution was 1.5. To deter-
KaleidaGraph software (Synergy Software, Reading, PA, mine if DHA was reduced with TCEP and DTT in 5%
U.S.A.). Statistical differences were considered significant at MPA/EDTA solution, 5.7 mM DHA in 5% MPA/EDTA was
pꢅ0.05.
incubated with 10 mM, 17.5 mM, 35 mM TCEP and 35 mM
Fig. 2. Representative Chromatograms of AA, DHA, MPA/EDTA, and TCEP
Samples were analyzed by HPLC-ECD as described in Materials and Methods. (A) AA dissolved in 5% MPA/EDTA. (B) 5% MPA/EDTA. (C) DHA dissolved in 5%
MPA/EDTA. (D) Reduced DHA dissolved in 5% MPA/EDTA by 35 mM TCEP for 2 h on ice.

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DTT for 4 h on ice (Fig. 3). DHA reduction by 10 mM TCEP tion in a biological sample, we combined 0.57 mmol/g tissue
was approximately 80% at 4 h. Furthermore, DHA reduction of AA or DHA with the centrifugal supernatant of mouse
by 17.5 mM and 35 mM TCEP approximated 100% at 4 h and liver then added 35 mM TCEP for a 2 h incubation on ice.
2 h, respectively. However, no DHA reduction was evident The total AA (AAꢀDHA) contents in a centrifugal super-
when 35 mM DTT was added to the 5% MPA/EDTA solution. natant of mouse liver with added AA, added DHA or no
From these results, we chose to achieve DHA reduction with additive were 1.88ꢄ0.07 mmol/g tissue, 1.89ꢄ0.07 mmol/g
35 mM TCEP incubated for 2 h in 5% MPA/EDTA solution tissue and 1.26ꢄ0.07 mmol/g tissue, respectively (Fig. 4).
on ice.
These results indicate that TCEP completely reduced the
Reduction of DHA Added to the Mouse Liver To de- added DHA in a biological sample, and its reduction effi-
termine whether TCEP effectively induced this DHA reduc- ciency was almost 100%.
Content of DHA Reduced by TCEP or DTT in Multi-
Fig. 4. Reduction Efficiency of DHA Added to Mouse Liver Samples
Livers were homogenized with 14 volumes of ice-cold 5.4% MPA/EDTA and cen-
trifuged at 21000 g for 10 min at 4 °C. 0.57 mmol/g tissue of AA or DHA were added to
the centrifugal supernatant of livers and then incubated with 35 mM TCEP for 2 h on
Fig. 3. Reduction of DHA to AA by Using TCEP and DTT in 5% MPA/
EDTA
5.7 mM DHA was incubated with 10 mM, 17.5 mM, 35 mM TCEP and 35 mM DTT in
5% MPA/EDTA on ice for 4 h. The amount of AA produced by reduction was analyzed
by HPLC-ECD system. Values are expressed as meansꢄS.E.M. of three independent
experiments.
ice. Total AA (AAꢀDHA) contents of AA-added, DHA-added or additive-free cen-
trifugal supernatants of livers were analyzed by HPLC-ECD. Slash area represents the
estimated AA and DHA content added to the centrifugal supernatant of liver. Values are
expressed as meansꢄS.E.M. of three animals.
Fig. 5. Comparison of Reductant Ability between TCEP and DTT, and DHA Content in Mouse Tissues and Plasma
DHA was reduced with either TCEP or DTT as described in Materials and Methods and total AA (AAꢀDHA) content measured. AA content (closed column) was determined
as described in Materials and Methods and DHA content (slashed column) calculated by subtraction. Values are expresseed as meansꢄS.E.M. of five animals.

368
Vol. 33, No. 3
ple Mouse Tissues and Plasma To compare the reduction
abilities of TCEP and DTT, we applied both agents sepa-
rately to matching tissue preparations and measured total AA
(AAꢀDHA) and AA content. In addition to plasma, the tis-
sues tested were cerebrum, cerebellum, lung, heart, liver,
spleen, kidney, adrenal gland, stomach, small intestine and
large intestine of 6 weeks old mice. In this experiment, we
used conventional DTT reduction methods as describe by
Lykkesfeldt et al.,11) and DHA content was calculated by
subtraction. Amounts of DHA in the cerebrum, cerebellum,
liver, spleen, kidney, adrenal gland, stomach, small intestine
and large intestine were not significantly different when ei-
ther TCEP or DTT was used for reduction (Fig. 5). However,
DHA contents in the lung, heart and plasma were signifi-
cantly higher when TCEP was the reduction agent compared
to that by DTT. With TCEP reduction, the percentages of
DHA per total AA in the cerebrum, cerebellum, lung, heart,
liver, spleen, kidney, adrenal gland, stomach, small intestine,
large intestine and plasma were 3.4%, 0.8%, 14.3%, 14.7%,
1.3%, 19.5%, 4.1%, 8.6%, 7.2%, 3.5%, 7.4% and 19.2%,
respectively. Moreover, TCEP reduction provided total AA
(AAꢀDHA) yields in the cerebrum, cerebellum, lung, heart,
Fig. 6. DHA Determination Protocol in Tissues and Plasma
DHA in the centrifugal supernatant was reduced by using 35 mM TCEP in 5%
MPA/EDTA for 2 h on ice and analyzed by HPLC-ECD. DHA content was calculated
by the subtraction method.
liver, spleen, kidney, adrenal gland, stomach, small intestine, ity. Lykkesfeldt et al. reported that DHA reduction in
large intestine and plasma of 3.00ꢄ0.03 mmol/g tissue, MPA/EDTA solution by TCEP was performed at pH 4.3 and
2.98ꢄ0.09 mmol/g tissue, 2.55ꢄ0.08 mmol/g tissue, 0.47ꢄ 6.2.17) However, their method required the addition of Trizma
0.02 mmol/g tissue, 1.41ꢄ0.06 mmol/g tissue, 2.56ꢄ0.03 buffer or McIlvaine buffer containing TCEP to raise the pH
mmol/g tissue, 0.87ꢄ0.07 mmol/g tissue, 7.14ꢄ0.22 mmol/g value of MPA/EDTA solution. For our improved method,
tissue, 0.90ꢄ0.02 mmol/g tissue, 2.86ꢄ0.07 mmol/g tissue, though, the pH value of the MPA/EDTA solution did not
1.48ꢄ0.02 mmol/g tissue and 62.06ꢄ1.58 mM, respectively.
need to rise, because TCEP was easily dissolved in MPA/
EDTA and sufficiently effective at pH 1.5 on ice. Thus, the
advantage of our method is that reduction of DHA can be
performed in MPA/EDTA solution without any change in
DISCUSSION
The present study documents an improved method for cal- composition or requirement to alter the pH.
culating DHA content in biological samples by using TCEP
One can accelerate the reduction rate of DHA by TCEP in
as a reduction agent. Here, 35 mM TCEP completely reduced MPA/EDTA solution by increasing the reaction temperature
DHA to AA after tissue samples from numerous organs from on ice to 25 °C (data not shown), but we performed all
and plasma of mice were incubated for 2 h on ice in 5% these experimental procedures on ice to avoid possible DHA
MPA/EDTA solution. MPA and EDTA at low pH solution and AA breakdown at the higher temperature and to develop
have been used widely for stabilizing AA and DHA in bio- the simplest method possible for DHA determination.
logical samples.17,24,25) The AA was easily separated and
DHA is known to be transported intracellularly via the
usable in an HPLC-ECD system for identification of DHA. glucose transporters, Glut1, Glut3 and Glut4.29) Once DHA
Actual DHA content in biological samples was calculated enters a cell’s interior, intracellular DHA is readily reduced
by subtracting amounts of the non-treated preparation (AA) by dehydroascorbic acid reductase and changed to AA.30)
from TCEP treated total AA (AAꢀDHA) as illustrated in Thereby, levels of DHA in tissues have been kept at low con-
Fig. 6.
centrations. Using our improved method, we have determined
DTT is widely used as reductant in biological samples; the DHA content in various tissues and plasma of 6 weeks
however, the reduction ability of DTT is restricted to a nar- old mice (Fig. 5). This method indicated that amounts of
row pH range from 6.5 to 9.0, and the resulting solution is DHA per total AA differed markedly at separate sites from
unstable.5,27) Therefore, it is essential to neutralize the pH of 0.8 to 19.5%. Especially high DHA levels were found in the
biological samples in MPA solution before applying DTT for lungs, heart, spleen and plasma, i.e., over 10% of DHA per
the reduction of DHA, and re-acidification is required for sta- total AA content. On the other hand, cerebrum, cerebellum,
bilization of the AA until its injection into HPLC. These pro- liver, kidney and small intestine had less than 5% DHA per
cedures are very complicated, and the breakdown of DHA to total AA content. This variability of DHA content may indi-
2,3-diketogulonic acid, which occurs readily, yields an irre- cate the different oxidative stress levels among the organs
versibly hydrolyzed compound of DHA in the neutralized so- and may represent the related amounts of dehydroascorbic
lution.28) In contrast, TCEP retains its reduction ability in acid reductase activity. Sasaki et al. reported that dehy-
wide pH range from 1.5 to 9.0 and is stable in the solution. In droascorbic acid reductase activity was highest in the liver
fact, 35 mM TCEP completely reduced 5.7 mM DHA to AA and second highest in the brains of chickens.31) Moreover,
during only 2 h in the MPA/EDTA solution at pH 1.5 on ice dehydroascorbic acid reductase activity was lower in the
(Fig. 3). Thus, DHA in MPA/EDTA solution does not need spleens and lungs of these chickens. Paolicchi et al. also re-
neutralization for reduction and re-acidification or AA stabil- ported that the highest levels of dehydroascorbic acid reduc-

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Acknowledgments This study is supported by a Grant-
in-Aid for Scientific Research from the Ministry of Educa-
tion, Culture, Sports, Science and Technology of Japan (to
A.I. 21590974), and a Grant-in-Aid from the Smoking Re-
search Foundation, Japan (to A.I.). We thank Prof. Yuusaku
Yokoyama for help with DHA preparation and Ms. P. Minick
for the excellent English editorial assistance.
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