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A reappraisal of leukocyte dehydroascorbate reductase
Biochimica et Biophysica Acta 839 (1985) 119-121 Elsevier
119
BBA Report
BBA 20091
A reappraisal of leukocyte dehydroascorbate reductase R o b e r t L. Stahl, L e o n a r d F. L i e b e s a n d R o b e r t Silber * Department of Medicine, New York University School o/Medicine, 550 First Avenue, New York, N Y 10016 (U.S.A.) (Received August 7th, 1984) (Revised manuscript received January 17th, 1985)
Key words: Dehydroascorbate reductase; Chronic lymphocytic leukemia; (Human leukocyte)
Dehydroascorbate reductase (glutathione:dehydroascorbate oxidoreductase, EC 1.8.5.1) activity was determined in human leukocyte homogenates using a direct spectrophotometric assay. Despite previous studies, using a less sensitive coupled assay, which reported that this enzyme was present in leukocytes, we found that neither neutrophil nor chronic lymphocytic leukemia lymphocyte extracts had detectable activity. Furthermore, when the product was quantitated by HPLC, protein-dependent generation could not be demonstrated. Mixing experiments with a partially purified enzyme preparation from spinach leaves provided no evidence for the presence of an inhibitor in neutrophil homogenates. These findings suggest that in human leukocytes, dehydroascorbate reduction does not occur enzymatically.
Dehydroascorbate reductase (glutathione:dehydroascorbate oxidoreductase, EC 1.8.5.1) catalyzes the glutathione-dependent reduction of dehydroascorbate to ascorbic acid. The presence of the enzyme was originally established in plant tissues using dichlorophenol-indophenol titration [1,2]. Several methods have subsequently been used to measure its activity, among them a coupled assay which monitors the oxidation of NADPH by glutathione reductase in mammalian tissues [3]. The enzyme has been detected in various animal cells [4-6], and its activity is said to be linked to cellular dehydroascorbic acid transport in human leukocytes [6]. Recently, we developed a direct spectrophotometric assay based on the change in absorbance at 265 nm associated with the generation of ascorbic acid [7]. The dehydroascorbate reductase activity of a partially purified spinach standard as determined by this direct assay agreed well with that reported by others using a dichloro-
* To whom correspondence should be addressed.
phenol-indophenol titration method [8]. Since our previous studies had shown elevated ascorbic acid [9] and dehydroascorbic acid [10] contents in lymphocytes from patients with chronic lymphocytic leukemia, we decided to determine the activity of dehydroascorbate reductase in human leukocytes using the new assay. Heparinized blood was obtained from normal subjects and from patients with untreated chronic lymphocytic leukemia. Following Ficoll-Hypaque density centrifugation [11], normal polymorphonuclear neutrophils were obtained by sedimenting the cells in the Ficoll-Hypaque infranatant through 6% Dextran 252 in normal saline [11]. Contaminating erythrocytes were removed by lysis with icecold 0.87 g NH4C1/100 ml 0.9% NaC1. Chronic lymphocytic leukemia lymphocytes were isolated from the Ficoll-Hypaque mononuclear cell interface after removing monocytes by adherence to Falcon plastic culture dishes [12]. The degree of cellular purity was assessed by cell sizing using a Z B~Coulter Counter (Coulter Electronics, Hialeah, FL). Cell extracts for most experiments were pre-
0304-4165/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
120
pared from (0.5-1.0).10 ~ cells/ml in 50 mM phosphate buffer with a glass-Teflon Dounce homogenizer at 4000 rpm for 4 min at 0°C. Neutrophils were also lysed in 0.2% Triton X-100 or by freeze-thawing three times. Lysates were centrifuged at 20000 x g for 30 min at 4°C and the supernatant fluid used for enzyme assays. Dehydroascorbic acid activity was determined by the direct assay from the change in absorbance at 265 nm associated with the formation of ascorbic acid [7]. The relatively high extinction coefficient of ascorbic acid at 265 nm (emM = 14.7) allowed measurement of as little as 2 nmol/ml. The reaction mixture contained 50 mM phosphate b u f f e r / 0.27 mM E D T A / 2 . 0 mM G S H / 1 . 0 mM dehydroascorbic acid/0.1 ml cell extract in a final volume of 1 ml (pH 6.8). Dehydroascorbic acid was prepared from ascorbic acid by the procedure of Staudinger and Weis [13] and quantitative conversion to dehydroascorbic acid was documented by HPLC [10]. The coupled assay, which monitors the decrease in absorbance at 340 nm, containing the following: 50 mM phosphate buffer/0.27 mM E D T A / 0 . 2 mM N A D P H / 2 . 0 mM G S H / 2 I.U. glutathione reductase/1.0 mM dehydroascorbic acid/0.1 ml cell extract in 1 ml (pH 6.8). Phosphate buffer replaced homogenate in the blank for both assays. The dehydroascorbate reductase activity of normal neutrophil and chronic lymphocytic leukem,~a lymphocyte homogenates was determined by the direct and coupled assays. An unexpected discrepancy was noted between the results obtained with the direct and coupled assays. While neutrophil dehydroascorbate reductase activity measured by the coupled assay was 56.7 _+ 8.9 n m o l / min per 10 s cells (mean _+ S.E., n = 11), the activity with the direct assay was 3.4 _+ 1.2 n m o l / m i n per 108 cells (n = 10), at the threshold of detection for the assay [7]. Furthermore, lymphocytes from patients with chronic lymphocytic leukemia had 17.2 and 13.7 n m o l / m i n per 108 cells (n = 2), while no activity was noted with the direct assay, 0 n m o l / m i n per 108 cells (n = 3). The results of a representative experiment using the direct assay with a neutrophil extract is shown in Fig. 1. Identification of the coupled assay reaction product was performed by HPLC as previously described [15]. The conversion of dehydroascorbic
1.5 c A I
c
3 0.5
B I
0 0
I 1 Time (rain)
I 2
Fig. 1. Spectrophotometric recording of change in absorbance at 265 nm in a direct assay mixture containing neutrophil homogenate (A) or a phosphate buffer blank (B). Data were collected at a scanning speed of 600 readings/min on a Beckman DU7 spectrophotometer.
acid to ascorbic acid by a neutrophil extract was followed by removing aliquots from the incubation mixture at 0 s and after 180 s. Ascorbic acid was identified by its characteristic retention time and was quantified by its absorbance at 254 nm. In the presence of neutrophil extract, ascorbic acid was generated from dehydroascorbic acid at a rate of 47.0 n m o l / m i n per 108 cells which was not different from that of the blank which lacked enzyme, 54.8 n m o l / m i n (Fig. 2). To determine whether an inhibitor was present in the leukocyte homogenate, a mixing experiment was carried out using a partially purified spinach extract [8] as a standard source of dehydroascorbate reductase. Equal volumes of spinach extract and either neutrophil homogenate or phosphate buffer were added to the direct assay reaction mixture. The activity of the spinach extract standard alone, 107.1 n m o l / m i n per mg protein, did not differ from that observed when the neutrophil homogenate was added, 112.3 n m o l / m i n per mg protein. The data presented above indicate that when either neutrophil or chronic lymphocytic leukemia lymphocyte extracts were assayed using a simpler direct spectrophotometric assay, dehydroascorbate reductase activity could not be detected. This failure, despite the greater sensitivity of the method used (emM = 14.7 vs. 6.22 for N A D P H in the coupled assay), raises the question as to whether dehydroascorbate reductase is present in human leukocytes. Furthermore, when the coupled assay
121
Z
i®
c
AA
5
10 0 5 10 Elution Time (rain)
I
0
5
I
10
I
0
5
10
Elution Time (min)
reaction p r o d u c t was q u a n t i t a t e d by H P L C , the a m o u n t of ascorbic acid generated in the presence of h o m o g e n a t e was no greater t h a n that formed by the b l a n k which lacked p r o t e i n b u t c o n t a i n e d a n equal c o n c e n t r a t i o n of G S H . Mixing experiments provided n o evidence for an i n h i b i t o r in neutrophil h o m o g e n a t e s in keeping with the absence of a true d e h y d r o a s c o r b a t e reductase in these cells. O u r inability to d e m o n s t r a t e either a reaction rate using a simpler direct assay or p r o t e i n - d e p e n d e n t p r o d u c t g e n e r a t i o n when followed b y H P L C is consistent with the suggestion that only a n o n e n zymatic conversion of dehydroascorbic acid to ascorbic acid is taking place in leukocytes. I n previous experiments using spinach extract as a s t a n d a r d source of d e h y d r o a s c o r b a t e reductase [7], the a m o u n t of N A D P H oxidized, as measured by the coupled assay, was approx. 1.4-fold greater t h a n the a m o u n t of ascorbic acid generated. Although the discrepancy between the direct a n d coupled assays r e m a i n s unresolved, the possibility exists that with the coupled assay G S S G generation by a n o t h e r p a t h w a y in h o m o g e n a t e s accounts for a n a p p a r e n t reaction rate above that of the n o n - e n z y m a t i c blank. The i n t e r r e l a t i o n s h i p between ascorbic acid a n d dehydroascorbic acid continues to be worthy of further studies, since increased levels of these c o m p o u n d s have been described in leukemic cells [9,10], where they m a y play a role with G S H in the a n t i - o x i d a n t defense system of the cell.
Fig. 2. HPLC analysis of reaction product was performed with a 5 ~tm SAX column as described by Liebes [14]. Methanol extraction was omitted and chromatographic elution time was 20 min. Samples were removed at 0 s (A) and 180 s (B) during the reaction. Panel I shows the profiles obtained with a neutrophil homogenate and panel II shows the profiles when phosphate buffer was used instead of cell extract in the blank. Elution conditions: flow, 1.5 ml/min; isocratic elution with buffer (0.007 M KH2PO4/0.007 M KC1 (pH 4.0)). Dehydroascorbic acid (DHA) eluted at 2-3 min along with nucleosides and bases. Ascorbic acid (AA) eluted from the column at 7.2 min and was quantitated by integration of the area under the peak. AFS = 0.01 units.
This work was supported by National Institutes of Health Grant No. CA 28376. R.L.S. is the recipient of a National Research Service Award (T32HL07151) from the National Institutes of Health. L . F . L . is a Scholar of the Leukemia Society of America. References 1 Crook, B.M. (1941) Biochem. J. 35, 226-236 2 Yamaguchi, M. and Joslyn, M.A. (1952) Arch. Biochem. Biophys. 38, 451-465 3 Anderson, E.I. and Spector, A. 91971) Invest. Ophthalmol. 10, 41-53 4 Hughes, R.E. (1964) Nature (Lond.) 203, 1068-1069 5 Grimble, R.F. and Hughes, R.E. (1967) Experientia 23, 362 6 Bigley,R., Riddle, M., Layman, D. and Stankova, L. (1981) Biochim. Biophys. Acta 659, 15-22 7 Stahl, R.L., Liebes, L.F., Farber, C.M. and Silber, R. (1983) Anal. Biochem. 131, 341-344 8 Foyer, C.H. and Halliwell, B. (1977) Phytochemistry 16, 1347-1350 9 Liebes, L., Krigel, R., Kuo, S., Nevrla, D., Pelle, E. and Silber, R. (1981) Proc. Natl. Acad. Sci. USA 78, 6481-6484 10 Farber, C.M., Kanengiser, S., Stahl, R., Liebes, L. and Silber, R. (1983) Anal. Biochem. 134, 355-360 11 Boyum, A. (1968) Scand. J. Clin. Lab. Invest. (Suppl. 97) 21, 77-89 12 Rabinovitch, M. and DeStefano, M.J. (1975) In Vitro 11, 379-381 13 Staudinger, H. and Weis, W. (1964) Z. Physiol. Chem. 337, 284-285 14 Liebes, L.F., Kuo, S., Krigel, R., Pelle, E. and Silber, R. (1981) Anal. Biochem. 118, 53-57
119
BBA Report
BBA 20091
A reappraisal of leukocyte dehydroascorbate reductase R o b e r t L. Stahl, L e o n a r d F. L i e b e s a n d R o b e r t Silber * Department of Medicine, New York University School o/Medicine, 550 First Avenue, New York, N Y 10016 (U.S.A.) (Received August 7th, 1984) (Revised manuscript received January 17th, 1985)
Key words: Dehydroascorbate reductase; Chronic lymphocytic leukemia; (Human leukocyte)
Dehydroascorbate reductase (glutathione:dehydroascorbate oxidoreductase, EC 1.8.5.1) activity was determined in human leukocyte homogenates using a direct spectrophotometric assay. Despite previous studies, using a less sensitive coupled assay, which reported that this enzyme was present in leukocytes, we found that neither neutrophil nor chronic lymphocytic leukemia lymphocyte extracts had detectable activity. Furthermore, when the product was quantitated by HPLC, protein-dependent generation could not be demonstrated. Mixing experiments with a partially purified enzyme preparation from spinach leaves provided no evidence for the presence of an inhibitor in neutrophil homogenates. These findings suggest that in human leukocytes, dehydroascorbate reduction does not occur enzymatically.
Dehydroascorbate reductase (glutathione:dehydroascorbate oxidoreductase, EC 1.8.5.1) catalyzes the glutathione-dependent reduction of dehydroascorbate to ascorbic acid. The presence of the enzyme was originally established in plant tissues using dichlorophenol-indophenol titration [1,2]. Several methods have subsequently been used to measure its activity, among them a coupled assay which monitors the oxidation of NADPH by glutathione reductase in mammalian tissues [3]. The enzyme has been detected in various animal cells [4-6], and its activity is said to be linked to cellular dehydroascorbic acid transport in human leukocytes [6]. Recently, we developed a direct spectrophotometric assay based on the change in absorbance at 265 nm associated with the generation of ascorbic acid [7]. The dehydroascorbate reductase activity of a partially purified spinach standard as determined by this direct assay agreed well with that reported by others using a dichloro-
* To whom correspondence should be addressed.
phenol-indophenol titration method [8]. Since our previous studies had shown elevated ascorbic acid [9] and dehydroascorbic acid [10] contents in lymphocytes from patients with chronic lymphocytic leukemia, we decided to determine the activity of dehydroascorbate reductase in human leukocytes using the new assay. Heparinized blood was obtained from normal subjects and from patients with untreated chronic lymphocytic leukemia. Following Ficoll-Hypaque density centrifugation [11], normal polymorphonuclear neutrophils were obtained by sedimenting the cells in the Ficoll-Hypaque infranatant through 6% Dextran 252 in normal saline [11]. Contaminating erythrocytes were removed by lysis with icecold 0.87 g NH4C1/100 ml 0.9% NaC1. Chronic lymphocytic leukemia lymphocytes were isolated from the Ficoll-Hypaque mononuclear cell interface after removing monocytes by adherence to Falcon plastic culture dishes [12]. The degree of cellular purity was assessed by cell sizing using a Z B~Coulter Counter (Coulter Electronics, Hialeah, FL). Cell extracts for most experiments were pre-
0304-4165/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
120
pared from (0.5-1.0).10 ~ cells/ml in 50 mM phosphate buffer with a glass-Teflon Dounce homogenizer at 4000 rpm for 4 min at 0°C. Neutrophils were also lysed in 0.2% Triton X-100 or by freeze-thawing three times. Lysates were centrifuged at 20000 x g for 30 min at 4°C and the supernatant fluid used for enzyme assays. Dehydroascorbic acid activity was determined by the direct assay from the change in absorbance at 265 nm associated with the formation of ascorbic acid [7]. The relatively high extinction coefficient of ascorbic acid at 265 nm (emM = 14.7) allowed measurement of as little as 2 nmol/ml. The reaction mixture contained 50 mM phosphate b u f f e r / 0.27 mM E D T A / 2 . 0 mM G S H / 1 . 0 mM dehydroascorbic acid/0.1 ml cell extract in a final volume of 1 ml (pH 6.8). Dehydroascorbic acid was prepared from ascorbic acid by the procedure of Staudinger and Weis [13] and quantitative conversion to dehydroascorbic acid was documented by HPLC [10]. The coupled assay, which monitors the decrease in absorbance at 340 nm, containing the following: 50 mM phosphate buffer/0.27 mM E D T A / 0 . 2 mM N A D P H / 2 . 0 mM G S H / 2 I.U. glutathione reductase/1.0 mM dehydroascorbic acid/0.1 ml cell extract in 1 ml (pH 6.8). Phosphate buffer replaced homogenate in the blank for both assays. The dehydroascorbate reductase activity of normal neutrophil and chronic lymphocytic leukem,~a lymphocyte homogenates was determined by the direct and coupled assays. An unexpected discrepancy was noted between the results obtained with the direct and coupled assays. While neutrophil dehydroascorbate reductase activity measured by the coupled assay was 56.7 _+ 8.9 n m o l / min per 10 s cells (mean _+ S.E., n = 11), the activity with the direct assay was 3.4 _+ 1.2 n m o l / m i n per 108 cells (n = 10), at the threshold of detection for the assay [7]. Furthermore, lymphocytes from patients with chronic lymphocytic leukemia had 17.2 and 13.7 n m o l / m i n per 108 cells (n = 2), while no activity was noted with the direct assay, 0 n m o l / m i n per 108 cells (n = 3). The results of a representative experiment using the direct assay with a neutrophil extract is shown in Fig. 1. Identification of the coupled assay reaction product was performed by HPLC as previously described [15]. The conversion of dehydroascorbic
1.5 c A I
c
3 0.5
B I
0 0
I 1 Time (rain)
I 2
Fig. 1. Spectrophotometric recording of change in absorbance at 265 nm in a direct assay mixture containing neutrophil homogenate (A) or a phosphate buffer blank (B). Data were collected at a scanning speed of 600 readings/min on a Beckman DU7 spectrophotometer.
acid to ascorbic acid by a neutrophil extract was followed by removing aliquots from the incubation mixture at 0 s and after 180 s. Ascorbic acid was identified by its characteristic retention time and was quantified by its absorbance at 254 nm. In the presence of neutrophil extract, ascorbic acid was generated from dehydroascorbic acid at a rate of 47.0 n m o l / m i n per 108 cells which was not different from that of the blank which lacked enzyme, 54.8 n m o l / m i n (Fig. 2). To determine whether an inhibitor was present in the leukocyte homogenate, a mixing experiment was carried out using a partially purified spinach extract [8] as a standard source of dehydroascorbate reductase. Equal volumes of spinach extract and either neutrophil homogenate or phosphate buffer were added to the direct assay reaction mixture. The activity of the spinach extract standard alone, 107.1 n m o l / m i n per mg protein, did not differ from that observed when the neutrophil homogenate was added, 112.3 n m o l / m i n per mg protein. The data presented above indicate that when either neutrophil or chronic lymphocytic leukemia lymphocyte extracts were assayed using a simpler direct spectrophotometric assay, dehydroascorbate reductase activity could not be detected. This failure, despite the greater sensitivity of the method used (emM = 14.7 vs. 6.22 for N A D P H in the coupled assay), raises the question as to whether dehydroascorbate reductase is present in human leukocytes. Furthermore, when the coupled assay
121
Z
i®
c
AA
5
10 0 5 10 Elution Time (rain)
I
0
5
I
10
I
0
5
10
Elution Time (min)
reaction p r o d u c t was q u a n t i t a t e d by H P L C , the a m o u n t of ascorbic acid generated in the presence of h o m o g e n a t e was no greater t h a n that formed by the b l a n k which lacked p r o t e i n b u t c o n t a i n e d a n equal c o n c e n t r a t i o n of G S H . Mixing experiments provided n o evidence for an i n h i b i t o r in neutrophil h o m o g e n a t e s in keeping with the absence of a true d e h y d r o a s c o r b a t e reductase in these cells. O u r inability to d e m o n s t r a t e either a reaction rate using a simpler direct assay or p r o t e i n - d e p e n d e n t p r o d u c t g e n e r a t i o n when followed b y H P L C is consistent with the suggestion that only a n o n e n zymatic conversion of dehydroascorbic acid to ascorbic acid is taking place in leukocytes. I n previous experiments using spinach extract as a s t a n d a r d source of d e h y d r o a s c o r b a t e reductase [7], the a m o u n t of N A D P H oxidized, as measured by the coupled assay, was approx. 1.4-fold greater t h a n the a m o u n t of ascorbic acid generated. Although the discrepancy between the direct a n d coupled assays r e m a i n s unresolved, the possibility exists that with the coupled assay G S S G generation by a n o t h e r p a t h w a y in h o m o g e n a t e s accounts for a n a p p a r e n t reaction rate above that of the n o n - e n z y m a t i c blank. The i n t e r r e l a t i o n s h i p between ascorbic acid a n d dehydroascorbic acid continues to be worthy of further studies, since increased levels of these c o m p o u n d s have been described in leukemic cells [9,10], where they m a y play a role with G S H in the a n t i - o x i d a n t defense system of the cell.
Fig. 2. HPLC analysis of reaction product was performed with a 5 ~tm SAX column as described by Liebes [14]. Methanol extraction was omitted and chromatographic elution time was 20 min. Samples were removed at 0 s (A) and 180 s (B) during the reaction. Panel I shows the profiles obtained with a neutrophil homogenate and panel II shows the profiles when phosphate buffer was used instead of cell extract in the blank. Elution conditions: flow, 1.5 ml/min; isocratic elution with buffer (0.007 M KH2PO4/0.007 M KC1 (pH 4.0)). Dehydroascorbic acid (DHA) eluted at 2-3 min along with nucleosides and bases. Ascorbic acid (AA) eluted from the column at 7.2 min and was quantitated by integration of the area under the peak. AFS = 0.01 units.
This work was supported by National Institutes of Health Grant No. CA 28376. R.L.S. is the recipient of a National Research Service Award (T32HL07151) from the National Institutes of Health. L . F . L . is a Scholar of the Leukemia Society of America. References 1 Crook, B.M. (1941) Biochem. J. 35, 226-236 2 Yamaguchi, M. and Joslyn, M.A. (1952) Arch. Biochem. Biophys. 38, 451-465 3 Anderson, E.I. and Spector, A. 91971) Invest. Ophthalmol. 10, 41-53 4 Hughes, R.E. (1964) Nature (Lond.) 203, 1068-1069 5 Grimble, R.F. and Hughes, R.E. (1967) Experientia 23, 362 6 Bigley,R., Riddle, M., Layman, D. and Stankova, L. (1981) Biochim. Biophys. Acta 659, 15-22 7 Stahl, R.L., Liebes, L.F., Farber, C.M. and Silber, R. (1983) Anal. Biochem. 131, 341-344 8 Foyer, C.H. and Halliwell, B. (1977) Phytochemistry 16, 1347-1350 9 Liebes, L., Krigel, R., Kuo, S., Nevrla, D., Pelle, E. and Silber, R. (1981) Proc. Natl. Acad. Sci. USA 78, 6481-6484 10 Farber, C.M., Kanengiser, S., Stahl, R., Liebes, L. and Silber, R. (1983) Anal. Biochem. 134, 355-360 11 Boyum, A. (1968) Scand. J. Clin. Lab. Invest. (Suppl. 97) 21, 77-89 12 Rabinovitch, M. and DeStefano, M.J. (1975) In Vitro 11, 379-381 13 Staudinger, H. and Weis, W. (1964) Z. Physiol. Chem. 337, 284-285 14 Liebes, L.F., Kuo, S., Krigel, R., Pelle, E. and Silber, R. (1981) Anal. Biochem. 118, 53-57