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Conservation of vitamin C by uric acid in blood
Journal of Free Radicals in Biology & Medicine. Vol. I. pp. 117--124. 1985
0748-5514/85 $3.00 + .00 © 1985 Pergamon Press Ltd.
Printed in the USA. All rights reserved.
CONSERVATION
OF VITAMIN
C BY URIC
ACID
IN B L O O D
ALEX SEVANIAN,*t KELVIN J. A . DAVIES,*~ a n d PAUL HOCHSTEIN*:]: *Institute for Toxicology and Departments of ";'Pathology and :l:Biochemistry, The University of Southern California, 1985 Zonal Avenue, Los Angeles, California 90033, U.S.A. (Received l August 1984; Revised 5 February 1985; Accepted 18 March 1985)
Abstract--Urate at physiological concentrations increased the stability of ascorbate in human serum approximately fivefold. These measurements were made by depleting human serum of urate with uricase. In model experiments (using phosphate buffer instead of serum), urate protected against iron-induced ascorbate oxidation and, to a lesser extent, pH-induced ascorbate oxidation. In both human serum and in phosphate buffer, urate exerted its protective effect without itself undergoing measurable oxidation as determined by spectrophotometric and high pressure liquid chromatography (HPLC) techniques. These experiments suggest an important physiological role for urate in preserving ascorbate in blood and other biological fluids. Keywords~Uric acid, Ascorbic acid, Free radicals, Oxidation, Transition metals, Uricase, Primate evolution
equate tissue levels. It has been proposed 12 that the inability to synthesize ascorbic acid may be the consequence of a mutation in higher primates which occurred simultaneously with the loss of uricase. Ames et al. m3suggested that the high concentrations of urate in the higher primates afforded an antioxidant alternative to ascorbate. They demonstrated that urate served as a potent antioxidant against free radical damage to biological membranes. At physiological concentrations, urate effectively protected against organic hydroperoxide-dependent lipid peroxidation and hydroperoxide.dependent hemolysis of erythrocytes. Because urate levels in biological fluids, notably blood, remain reasonably constanP 4 despite dietary variations in other purines, it occurred to us that urate by virtue of its high concentrations may also protect ascorbate from oxidative reactions in these fluids.
INTRODUCTION
The role of vitamin C (ascorbic acid) in biochemical reactions has been widely studied for decades and, among the many functions described in mammals, its antioxidant properties have received considerable attention. This antioxidant capacity appears, in part, to result from its reducing potential and its participation in redox reactions with a variety of compounds. In this respect, ascorbate may serve to minimize oxidative or free radical reactions which can be damaging to the cell.~-4 This property is based upon a facile one-electron cycling between the dihydro-, semidehydro-, and dehydroascorbate forms (i.e., the quinone, semiquinone radical, and quinol)) The rates of these interconversions are influenced by the presence of transition metals, 6'7 hydrogen ion concentration, 8 and the state of other biological oxidation-reduction couples. L9-~ Some, or perhaps all, of these reactions may be responsible for the instability of ascorbate in biological fluids. In humans and higher primates, the tendency of ascorbate to oxidize, combined with a metabolic lesion (the inability to synthesize ascorbate acid) requires the continuous intake of the vitamin in order to maintain ad-
MATERIALS AND METHODS
Measurements of ascorbate oxidation were carried out in 100 mM potassium phosphate buffer, pH 7.4, using various concentrations of ascorbate (Sigma, St. Louis, MO) in the presence and absence of various concentrations of sodium urate (Sigma, St. Louis, MO). Uricase (type I from porcine liver) and catalase (C- 100) were also obtained from Sigma. Similar measurements were also made using human serum to which ascorbate was added to produce the final concentrations indicated.
Address for corresponding--Dr. Alex Sevanian, University of Southern California, Institute for Toxicology. 1985 Zonal Avenue, Los Angeles. CA 90033, U.S.A. 117
118
A. SEVANIAN.K. J. A. D~,VlES, and P. HOCHSTEI>~
Polarographic measurement of ascorbate oxidation The rates of ascorbate oxidation in potassium phosphate buffer were determined using a calibrated Gilson 5/6A oxygraph equipped with a Clark-type electrode. Comparisons were made for the rates of oxidation with FeCI3 added in concentrations ranging from 0 to 100 MM in the presence of 100/,tM ascorbate, and 0 to 400 tiM urate. The effects of pH on oxidation rates were studied by dissolving ascorbic acid in water and adjusting the pH by additions of dilute KOH or HCI. All incubations were conducted at 21°C. The rate of ascorbate oxidation in the presence of 10 #M FeC13 was also examined in the presence of urate and under conditions where samples were first incubated for 1 h with 100 to 400 gM urate plus 0.025 U/ml uricase. In a similar manner, the effects of the iron chelators, EDTA, diethylenetriaminepentaacetic acid (DTPA) and transferrin on ascorbate oxidation rates were determined by measurement of oxygen consumption.
Spectrophotometric measurement of ascorbate oxidation Ascorbate disappearance was also monitored at 265 nm in 100 mM potassium phosphate buffer, in the presence of various FeCI3 and urate concentrations; ascorbate concentrations ranged from 10 to 100 #M and all determinations were made at 21 °C using a Perkin-Elmer 552 spectrophotometer.
plished by monitoring the HPLC eluent at 265 and 212 nm. Details of the HPLC method for urate and ascorbate measurement are described elsewhere.~5~ In order to determine whether urate preserves ascorbate in serum, a method was selected which removed urate and enabled comparisons to be made of ascorbate oxidation in serum in the presence and absence of urate. Normal serum was divided into equal aliquots which were incubated with or without uricase at 0.025 U/ml serum for 60 min at 37°C. Similarly. samples containing added uricase plus 100 U/ml catalase were prepared. The oxidative loss of urate was followed by analyzing serum aliquots for urate and formation of allantoin by HPLC. Subsequent analysis of the serum samples established the remaining concentration of urate, as well as that of ascorbate after treatment with uricase. Following the addition of 25 to 100 #M ascorbate, the serum samples were incubated at 37°C for various time intervals. At specified time points aliquots were prepared for HPLC by a column extraction procedure described previously. ~6The approach described here circumvented the impediments associated with measurements of urate and ascorbate levels in serum due to a plethora of interfering substances. Alternately, serum ascorbate levels were estimated using the colorimetric dinitrophenylhydrazine method. ~7 Comparisons were made of time dependent changes in ascorbate levels for samples containing predetermined amounts of urate, as well as in samples in which urate was depleted by treatment with uricase.
HPLC Analysis of ascorbate/urate oxidation The extent of ascorbate oxidation in potassium phosphate buffer was measured by directly injecting 20 to 100/tl aliquots of the samples, incubated over various time intervals, into an HPLC. The analytical system consisted of a Perkin-Elmer Series 4 liquid chromatograph equipped with a 5-#m, 4.6 × 250-mm Spherisorb amine column (Chromanetics Corp., Jessup, MD). Samples were eluted with acetonitrile :0.04 M NaH2PO4 (75:25, v/v) at a flow rate of 1.5 ml/min. The eluent was monitored at 265 nm using a Perkin-Elmer model 85 detector equipped with a stop-flow scanning autocontroller, and determinations of the amount of ascorbate in the samples were made by comparison to a calibration curve obtained using ascorbate standards dissolved in HzO. This method permitted simultaneous determinations employing both the spectrophotometric and HPLC techniques. The content of urate was also measured by monitoring the HPLC eluent at 292 rim, which greatly increased the analytical sensitivity as compared to 265 nm. In some cases, concurrent measurement of ascorbate and urate as well as their oxidation products, dehydro-ascorbate and allantoin, was accom-
RESULTS
Experiments performed in buffered solutions The oxidation of ascorbate in potassium phosphate buffer (pH 7.4) was concentration dependent (Fig. 1). Oxidation rates were also dependent on ferric ion concentration, although Fe 3+ was found to produce a bimodal effect (data not shown). Increasing concentrations of FeCI3, prepared in 0.001 M HCI, were added in aliquots not exceeding 25 Mi. Under these conditions the pH varied less than 0.05 units. Using 100 MM solutions of ascorbate, maximal oxidation rates were observed in the presence of 10-20 MM Fe 3+ and minimum rates were measured when no iron was added. The rates of ascorbate oxidation in buffer varied between 11.3 and 11.7 nmol O2/min in the absence of added iron. When Fe 3+ concentrations exceeded 30 #M, oxidation rates gradually decreased, reaching a plateau of approximately 12.5 nmol 02 consumed/min. Addition of 100 #M EDTA or DTPA to samples containing 10 #M FeCI3 and 100/tM ascorbate reduced oxygen consumption rates by approximately 85%, demonstrating the
Uric acid protects vitamin C 25 .,..o E .%
3
20
c
15
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0 m E
~
I0
0
-EDTA
X 0
75
150
225
300
375
Ascorbic Acid Concentrotion
450
(uM)
Fig. 1. Ascorbateoxidation rates determined polarographicallywere measured as a function of ascorbate concentration. Measurements were made using a 2.0-ml cell fitted with a Yellow Springs, Clarktype electrode and maintained at 21°C. All measurements were performed in the presence of 10~uMFeCh in 100 mMpotassiumphosphate buffer, pH 7.4. Also shown are the rates of oxidation for 100 ltM solutions of ascorbate in the presence of 100 pM EDTA (-) and 100 ,uM DTPA (A).
role of iron in the oxidation of ascorbate under these conditions. Analysis of these buffers by atomic absorbtion spectroscopy showed that the iron contamination was =< 1.5 //M in potassium phosphate and =< 0.6 pM in tris-HCl buffer. This amount of iron is sufficient to account for the background levels of ascorbate oxidation noted here. The rate of ascorbate oxidation is also known to increase with pH, and we observed a continuous increase in oxygen consumption rates from 11.7 +- 0.20 to 36.1 --- 0.72 nmol/min when 100/.tM ascorbate was dissolved in buffers (100 mM phosphate and 50 mM tris-HC1) ranging in pH from 6.5 to 9.5. The addition of I00 pM urate to phosphate buffer decreased the rates of ascorbate oxidation from 16.1 --- 0.16 to 11.3 --0.31 at pH 6.5, but had a diminishing effect with increasing pH such that at pH 9.5 the rates of ascorbate oxidation were not significantly affected. A concentration-dependent inhibition of ascorbate oxidation was evident with increasing amounts of urate at pH 7.4 (Fig. 2). For example, the oxidation rate of a 100 tiM solution of ascorbate decreased by 20% and 51% in the presence of 25 and 100 pM urate, respectively. At 300/JM urate the oxidation of ascorbate was reduced by 73%. Spectrophotometric analysis of ascorbate (100/tM) in the presence of FeCI3 (10 pM) confirmed that urate markedly inhibits the rate of ascorbate disappearance (Fig. 3, a, b, c). This was evidenced by examining the time-dependent decrease in ascorbate absorbance at 265 nm in the absence and presence of equimolar urate. As
119
shown in Fig. 3, a and b, ~rate appears only to decrease the rate of ascorbate oxidation without producing any change in the ascorbate spectrum. The effect of transferrin on ascorbate oxidation in buffer was also examined. Purified apotransferrin was loaded with iron by a previously described method ~ and then added to phosphate buffer solutions containing 100 pM ascorbate. We found that 25.0 pg of apotransferrin loaded with 2.5 pg Fe 2+, as Fe 2. (NH4)2 (SO4)2, did not stimulate ascorbate oxidation. The final concentration of iron in the assay was calculated to be 6.25 /~M. In fact, both spectrophotometric and polarographic analyses demonstrated a slight suppression in the rates of ascorbate oxidation (data not shown). The rate of ascorbate oxidation was stimulated 25% only when the iron loading was increased to l0/~g per 25 /~g apotransferrin. This would be equivalent to 25 pM iron bound to transferrin in the ferric state. Because an examination of urate's effect on ascorbate stability in human serum would require depletion of urate, we considered as an expedient approach the removal of urate using uricase. In order to validate this procedure, experiments were first performed in potassium phosphate buffer. A 300-pM solution of urate was divided into equal aliquots, one of which was treated with uricase as described in the Methods section. Treatment with uricase reduced the concentration of urate to the limit of detection by HPLC (_-< 20 pM). At this point, ascorbate was added to the samples to yield an initial concentration of 100/tM.
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i,
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75
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I
I
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150
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Fig. 2. Theeffectofurateontherateofascorbateoxidationisshown. Initial rates of oxidation were measured polarographically as described in Fig. I, using 100 ,UMascorbate and 10 I~MFeCI~. A 2-raM urate solution in 100 mM potassium phosphate buffer (pH 7.4) was prepared by sonication in a Bransonic sonicatorbath for 20 rain. The solution was maintained at 50°C during sonication which facilitated dissolution of urate. Increasing aliquots of this solution were used to produce the final concentrations shown.
120
A.
K. J. A.
SEVANIAN,
and P. HOCHSTEIN
DAVIES.
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Fig. 3. A spectrophotomctric analysis of ascorbate oxidation performed in 100 mM potassium phosphate buffer, pH 7.4, containing 10 #M FeCI> Analyses of the ultraviolet spectrum of a 100 /~M solution of ascorbate were made against potassium phosphate buffer. Repetitive scans were taken every 10 rain at a scanning rate of 120 nm/min. The absorbance maximum for the instrument was set at 2.0 and measurements were made at room temperature. The original spectrum for freshly added ascorbate is indicated as tracing (i) in panels A and B. Panel A shows the progressive oxidation of ascorbate in the presence of 10/~u Fe'+ over a 50-rain interval. Panel B represents a similar analysis in the presence of 100 gM urate added to both the sample and reference cuvettes. Panel C is a plot of the data derived from panels A and B showing the nanomoles of ascorbate oxidized as a function of time. The data from panel A are represented by the solid circles and data from panel B are represented by the open circles.
The results of the HPLC determination of ascorbate content in potassium phosphate buffer over 2.5 h of incubation are shown in Fig. 4. The figure shows that less than 20% of the ascorbate was oxidized in the presence of 300 /IM urate. When these preparations were treated with uricase, approximately 90% of the ascorbate was oxidized by 2.5 h. Uricase in the absence of urate did not affect ascorbate oxidation rates. Considerable amounts of H202 are produced by uricase oxidation of urate. The quantity of H202 formed in potassium phosphate buffer was sufficient to significantly increase ascorbate oxidation rates. Thus, ascorbate oxidation was not only accelerated by the depletion of urate, but also by the formation of H202 during the urate-uricase reaction. The amount of ascorbate oxidized under these circumstances was doubled, from 20% to 40% over 1 h, when ascorbate was added to urate solutions previously oxidized with uricase. However, the addition of catalase to uricase-treated samples, either during or after oxidation of urate, eliminated the pro-oxidant effect and yielded ascorhate oxidation rates similar to those of solutions containing only ascorbate. Ascorbate oxidation rates were mark-
edly increased by addition of 300/JM H202; however, these rates were typically 40% to 50% less than the rates observed when urate-containing solutions were treated with uricase (Fig. 4). In the presence of catalase we found that following urate depletion by uricase, ascorbate oxidation rates were similar to those observed for ascorbate alone in buffer. We attribute this effect to the degradation by catalase of the H202 formed during uricase-induced oxidation of urate. If urate were behaving as a typical scavenging antioxidant, its content should decrease by an amount approximately equivalent to the moles of ascorbate spared from oxidation, moles of iron reduced, or the difference in moles of oxygen consumed. This is estimated to be approximately 80 gM urate oxidized over 2.5 h, which is well within the sensitivity of the assay as previously demonstrated. 16 As seen in Fig. 4, there was no indication of urate oxidation under conditions where ascorbate oxidation was clearly inhibited. Table 1 shows the results of a spectrophotometric determination of the rates of ascorbate oxidation. We found that ascorbate oxidation rates increased over sixfold in buffer preparations of ur'ate treated with uricase.
Uric acid protects vitamin C
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Table I. Spectrophotometric Measurement of Ascorbic Acid Oxidation in Phosphate Buffer Uric Acid (,UM)
Uricase* Treatment
Initial Rate of Ascorbic Acid Oxidation';" (nmol/min)
-300 < 20
--+
28.0 - 0.90 8.0 -+ 0.32 49.6 -+ 1.63
+
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180
TIME OF INCUBATION (min)
*Uricase was added to samples as indicated. Samples were incubated with 0.025 U/ml uricase for I h at 37°C, and uric acid levels were subsequently measured by HPLC as described in Materials and Methods. tMean and standard deviation of three measurements of the oxidation rate for a 100/IM solution of ascorbate acid in 100 mM potassium phosphate buffer. The rate of absorbanc¢ decrease at 265 nm was used to calculate the rate of ascorbate acid oxidation using Emax 265 nm = 7 x 10~.
Fig. 4. The results of an HPLC analysis of ascorbate oxidation. A 300-/ZM solution of urate was prepared in 100-raM potassium phosphate buffer, pH 7.4, containing 10 ~uMFeCI3. Samples were incubated at 37°C in a shaking water bath for 5 rain and ascorbate was then added to yield an initial concentration of 100/ZM. Aiiquots of 100 ,ul were removed and injected onto the HPLC column at the times indicated, O-----Q. Also shown is the effect of urate depletion by incubating the urate solution with uricase, as described in the text, prior to the addition of ascorbate, C)..... O. The rates of ascorbate oxidation alone in phosphate buffer, • A ; ascorbute plus 300-/zM H20:, A ..... A; and ascorhate added to a sample pretreated with 300-/ZM urate plus uricase plus 100-U/ml catalase, • l ; are shown. The eluent in the above experiments was monitored at 265 nm. Included in the figure are the levels of urate (1"7..... []) in samples not treated with ur•case. The levels of urate were determined by simultaneous monitoring of the eluent at 292 and 265 nm using a Perk•nElmer LC 85 detector equipped with a stop-flow autocontroller.
1108
1
The initial rate of oxidation for a 100/ZM solution of a s c o r b a t e a l o n e w a s n o t s i g n i f i c a n t l y d i f f e r e n t in t h e presence or absence of uricase. ~0"-.
-O . . . . .
60
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Experiments using human serum T h e H P L C p r o c e d u r e w a s a l s o u s e d to m o n i t o r t h e r a t e o f a s c o r b a t e o x i d a t i o n in s e r u m s a m p l e s . W e f o u n d c o n s i d e r a b l e v a r i a t i o n in s e r u m u r a t e c o n c e n t r a t i o n s from volunteer donors simultaneously with a large vari a b i l i t y in a s c o r b a t e o x i d a t i o n r a t e s a m o n g t h e s e s a m p l e s . In e v e r y i n s t a n c e , h o w e v e r , t h e r e w a s a n o t a b l e i n c r e a s e in a s c o r b a t e o x i d a t i o n r a t e s w h e n s e r u m s a m p l e s w e r e d e p l e t e d o f u r a t e . T h i s is s h o w n in Fig. 5, which represents a typical experiment using human s e r u m . In t h i s i n s t a n c e , t h e s e r u m u r a t e w a s 2 8 0 / Z M , and following uricase treatment urate levels decreased to b e l o w t h e l i m i t o f d e t e c t i o n . U p o n a d d i t i o n o f 100 /~M a s c o r b a t e to w h o l e s e r u m , w e f o u n d t h a t 7 0 % o f the ascorbate remained after 2 h, whereas only 10% of t h e o r i g i n a l a s c o r b a t e r e m a i n e d in t h e s e r u m s a m p l e
0
10
30
120
150
TIME OF INCUBATION (rain) Fig. 5. An HPLC analysis of ascorbate oxidation in human serum. The serum was divided into equal aliquots and incubated with or without 0.25 U/ml uricase for 60 rain at 37°C. Ascorbate was then added to the serum samples to yield an initial concentration of 100/ZM. The rates of ascorbate oxidation are shown for both the uricase treated O ..... O, and untreated H serum. Also shown are the rates of ascorbate oxidation in the presence of 300-~UMH20,, A A; serum pretreated with uricase plus 100 U/ml catalase, [ ] ..... I-q; and the levels of urate in serum samples not treated with uricase or H.,O2, • • . The effect of the chelating agents, EDTA, • • ; and DTPA, ~7..... ~7; on the rate of ascorbate oxidation in whole serum are included in the figure. The conditions for analyses are otherwise as described in Fig. 4, and serum samples were prepared for HPLC as described in the Methods section.
122
A. SEVANIAN,K. I. A. DAVIES.and P. HOCHSTEIN
treated with uricase. This pattern was evident in all serum samples analyzed. The half-life of added ascorbate (100/IM) in serum samples containing the normal range of urate for humans (100-380/tM) was approximately five times longer than in samples depleted of urate. Similar to the situation noted for ascorbate oxidation in buffer, uricase treatment of serum caused ascorbate to undergo rapid oxidation. In contrast to buffer, however, catalase addition did not markedly reduce this effect (Fig. 5). Expression of catalatic activity in serum was indicated by the ability of added catalase to inhibit ascorbate oxidation following addition of H202. It appears that H20,. oxidizes ascorbate in serum, but in the absence of urate the rates of ascorbate oxidation are markedly increased. It can be seen in Fig. 5 that less than 50% of the ascorbate underwent oxidation following addition of H202 to a final concentration of 300 ~M in serum, which represents the total amount of H202 that could be formed by the complete oxidation of ~ 300,ttM urate. Thus, factors other than the H202 formed in the serum are likely to account for the rapid oxidation of ascorbate in the absence of urate. Also shown in Fig. 5 is the effect of added EDTA and DTPA on the oxidation of ascorbate added to otherwise untreated serum. Both chelating agents at 100/~M markedly reduced the rate of ascorbate oxidation, DTPA being more effective than EDTA. EDTA stabilized ascorbate for approximately 45 min and thereafter a significant oxidation of ascorbate was noted. It should be noted that conventional methods for measuring ascorbate levels in serum are inappropriate under conditions where ascorbate oxidation takes place. One common method used clinically for ascorbate analysis is a colorimetric assay using dinitrophenylhydrazine. 17 Table 2 shows that this method cannot differentiate between dihydroascorbate and its oxidized products. No significant change in ascorbate content was found when a 200-/.tM solution in serum was analyzed following incubation for intervals up to 15 min. The levels were also unaffected by the absence of urate. Table 2. Oxidation of Ascorbic Acid in Serum Measured by the DinitmphenylhydrazineMethod mg
Incubation Time (rain) 0 5 I0 15
Ascorbic Acid/100 ml Serum*
Complete Serum 3.5 3.1 3.3 3.2
-+ 0.4 - 0.2 -4- 0.4 +- 0.5
Serum + Uricase 3.3 3.6 3.5 3.4
-+ 0.4 - 0.4 +--0.5 _ 0.3
*Results are presented as mean + S.D. of four experiments, performed as described in Materials and Methods.
This method does not distinguish between the dihydroand dehydro- forms of ascorbate, whereas large decreases in ascorbate levels are found by 15 rain using the HPLC method (Fig. 5). DISCUSSION In this report, we demonstrate that urate, at concentrations approximating physiological levels, stabilizes ascorbate in human serum and phosphate buffer. A striking feature of this effect is the absence of measurable urate disappearance despite a marked inhibition of ascorbate oxidation. Ascorbate oxidation in the presence and absence of urate was measured using three different methods. In order to study the effect of urate depletion on ascorbate stability in potassium phosphate buffer and human serum, the oxidation of urate by uricase was employed (uric acid ~ allantoin + H_,O2). This procedure, however. introduced a potentially complicating factor to the experiments. The rapid oxidation of ascorbate under these conditions can be partially attributed to the H202 formed during the oxidation of urate by uricase; however, H202 was not solely responsible for the enhanced ascorbate oxidation rates. This was demonstrated by the ability of catalase to reduce significantly the extent of ascorbate oxidation, producing rates equivalent to those found for solutions containing ascorbate alone. In phosphate buffer, ascorbate alone underwent oxidation at rates significantly greater than in solutions also containing urate. HPLC analysis of serum showed that ascorbate at concentrations in the range found in normal humans underwent oxidation at rates considerably slower than those measured in serum samples depleted of urate by pretreatment with uricase. The enhanced oxidation of ascorbate is not attributable to HaO~ because comparable oxidation rates were observed in the presence or absence of catalase. These findings suggest that the stabilization of ascorbate in serum may be an important biological function of urate. Our findings also suggest that catalysts such as iron may be responsible for the oxidation of ascorbate in serum, and that urate may inhibit this catalytic process. There is an ongoing controversy over the role of iron as a catalyst for in vivo free-radical reactions. Gutteridge et a1.19 maintain that " c a t a l y t i c " iron occurs in vivo and may be in forms such as highly loaded ferritin, 2° iron complexes of phosphate esters (e.g., ADP or ATP), 21 iron chelates such as EDTA-Fe 3+ , and even low levels of iron salts.22 Winterbourne23 and others z4'25 disagree with the notion that free iron salts exist, but maintain that oxidation-reduction reactions between ascorbate and a variety of in vivo forms of iron are
Uric acid protects vitamin C possible. Reactions such as these may account for the catalysis of ascorbate oxidation in serum; however, transferrin, the most abundant iron depot in serum, did not stimulate ascorbate oxidation. Based on our observations, we tentatively conclude that serum component(s) other than transferrin are likely to he responsible for ascorbate oxidation. The pH of serum (7.4-7.6 in the case of these experiments) may account for some of this oxidation, but other catalysts of ascorbate oxidation may also exist. The protective effect of urate might occur by one of the following hypothetical mechanisms: (l) a competition between urate and ascorbate for the reduction of iron; (2) rereduction of ascorbate by urate; (3) sequestration of iron via a uric acid " c h e l a t e " which cannot act as an oxidant of ascorbate; (4) inhibition by a chainbreaking antioxidant mechanism; and (5) stabilization of ascorbate via hydrogen bonding to urate. Possibilities (1) and (2) are unlikely because there was no evidence of stoichiometric urate oxidation. It should be noted that approximately 20 /ZM urate represents the limit of reliable detection by the methods employed. Because only -- 5% ( ~ 15 ,t/M) of the urate was lost after 2.5 h of incubation (Fig. 5), it would be difficult to conclude whether this loss was significant. The third possible mechanism remains open to investigation, because Albert 26 has reported that urate and other purines have the capacity to bind iron. Unfortunately, the binding of iron by urate remains poorly understood. A chain-breaking antioxidant effect of urate is a possible mechanism of action. If the oxidation of ascorbate is proceeded by means of a long propagative radical chain reaction, very little urate should be required to provide an effective scavenging action. Large amounts of vitamin C might be spared from metal catalyzed oxidation and concomitantly small, and perhaps undetectable quantities of urate would be consumed over considerable time intervals. The fifth possible mechanism for the protective action of urate is deduced from the studies of Jamaluddin et al. 27 Because the oxidation of ascorbate occurs through the abstraction of its enolic hydrogen by oxygen, 2s and becomes more facile at basic pH, it has been proposed that hydrogen bonding between a purine (adenine or guanine) and ascorbate would make the enolic hydrogen less available for abstraction. A similar hydrogen bonding could take place with urate such that equimolar or greater concentrations should effectively retard the rate of ascorbate oxidation. This hydrogen bonding effect should reduce the degree of both iron-dependent and pH dependent ascorbate oxidation, as observed in the present studies. Kittridge and Willson :9 have recently shown that urate reacts with .OH at rates exceeding those measured for mannitol, a commonly used .OH scavenger. In ad-
123
dition to the observations of Ames et al.,J3 Smith and Lawing a° have found that urate is an effective erythrocyte membrane lipid antioxidant. In each of these instances, the antioxidant effect of urate was associated with its stoichiometric loss. The effective levels for antioxidant action were in the range found in normal human serum. Urate was recently shown to protect against oxidative damage to DNA, 31 and a similar antioxidant protection was found against hydroxyl radical-induced oxidation of certain proteins (K. J. A. Davies, unpublished observations). The ability to scavenge oxygen free radicals as well as free radical intermediates indicates potentially important antioxidant roles for urate in which this purine undergoes extensive oxidation. The stabilizing effect we describe in this paper appears distinct from previously described stoichiometric scavenging functions. Urate prevents the oxidation of ascorbate by an as yet undefined mechanism that does not involve extensive oxidation of urate to allantoin and other products. This stabilizing effect, readily observed in human serum and phosphate buffer, may have important consequences for the conservation of ascorbate in other biological fluids. The evolutionary loss of the ability to synthesize ascorbate may have been accompanied by a complementary loss of uricase. The latter mutation would insure levels of urate sufficiently high to stabilize extracellular ascorbate, derived from dietary sources, for ultimate utilization at intracellular sites. Acknowledgements--This work was supported in part by grants from the National Institutesof Health (HLI 5162) and the Arco Foundation. The authors acknowledge the technical assistance of Ms. Laurie
McLeod. REFERENCES
1. D. C. Borg, K. M. Schaich, J. J. Elmore, and J. A. Bell. Cytotoxic reactions of free radical species of oxygen. Photochem. Photobiol. 28:887-907 (1978). 2. H. Ogura, M. Murata, and M. Kondo. Radiolysisof ascorbic acid in aqueous solution. Radioisotopes 19:89-91 (1970). 3. B. Liebovitz and B. V. Siegel. Aspects of free radical reactions in biological systems: Aging. J. Geromol. 35:45-56 (1980). 4. V. G. Zannoni, E. J. Holsztynska, and S. S. Lau. Biochemical functions of ascorbic acid in drug metabolism. In: Ascorbic Acid: Chemistry, Metabolism and Uses. (P. A. Sieb and B. M. Tolbert. eds. ) pp. 349-368, Advances in ChemistrySeries 200, American Chemical Society, Washington, DC (1982). 5. B. H. J. Bieiski. Chemistryof ascorbic acid radicals. In: Ascorbic Acid Chemistry, Metabolism and Uses. (P. A. Seib and B. M. Tolbert, eds.), pp. 81-100, Advances in Chemistry Series 200, American Chemical Society, Washington, DC (1982). 6. A. E. Martell. Chelatesofascorbic acid. In:Ascorbicacid:Chemistry, Metabolism and Uses. (P. A. Seib and B. M. Tolbert, eds.) pp. 153-178, Advances in Chemistry Series 200, American Chemical Society, Washington, DC (1982). 7. A. R. Morgan, R. L. Cone, and T. M. EIgert. The mechanism of DNA strand breakage by vitamin C and superoxide and the protective roles of catalase and superoxide dismutase. Nucleic Acids Res. 3:1139-1149 (1976).
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A. SEVANIAN,K. J. A. DAVIES. and P. HOCHSTEIN
8. B. H. J. Bielski, A. O. Allen, and H. A. Schwarz. Mechanism of disprotionation of ascorbic radicals. J. Am. Chem. Soc. 103: 3516-3518 (1981). 9. W. Weiss. Ascorbic acid and electron transport. Ann. N. Y. Acad. Sci. 258:190-200 (1975). 10. C. C. Kratzing and R. J. Willis. Decreased levels of ascorbic acid in lung following ozone exposure. Chem. Biol. Interact. 30:53-56 (1980). 11. H. Staudinger, K. Krisch, and S. Leonhauser. Role of ascorbic acid in microsomal electron transport and the possible relationship to hydroxylation reaction. Ann. N.Y. Acad. Sci. 92: 195207 (1961). 12. P. Proctor. Similar functions of uric acid and ascorbate in man. Nature 228:868 (1970). 13. B. N. Ames, R. Cathcart, E. Schwiers, and P. Hochstein. Uric acid provides an antioxidant defense in humans against oxidantand radical-causnd aging and cancer: A hypothesis. Proc. Natl. Acad. Sci. USA 78:6858-6862 (1981). 14. S. Akizuki. S¢rum urate acid levels among thirty-four thousand people in Japan. Ann. Rheum. Dis. 41:272-274 (1982). 15. P. Hochstein, L. L. Hatch, and A. Sevanian. Methods Enzymol. 105:162-166 (1984). 16. L. L. Hatch and A. Sevanian. Measurement of uric acid, ascorbic acid and related metabolites in biological fluids. Anal. Biochem. 138:324-328 (1984). 17. P. J. Garry, G. M. Owen, D. W. Lashley, and P. C. Ford. Automated analysis of plasma and whole blood ascorbic acid. Clin. Biochem. 7:13 ! - 145 (1974). 18. J. Martincz-Medcllin and H. M. Schulman. The kinetics of iron and transferrin incorporation into rabbit erythroid cells and the nature of stromal bound iron. Biochim. Biophys. Acta 264: 272289 (1972). 19. J. M. C. Gutteridge, D. A. Rowley, and B. Halliwell. Superoxide dependent formation of hydroxyl radicals and lipid peroxidation in the presence of iron salts. Biochem. J. 206: 605609 (1982).
20, J. M. C. Gutteridge, B. Halliwelt, A. Treffry, P. M. Harrison. and D. Blake. Effect of ferritin-containing fractions with different iron loading on lipid peroxiclation. Biochem. J. 2.09: 557560 (1983). 21, E. H. Morgan. Studies on the mechanism of iron release from transferrin. Biochim. Biophys Acta 580:312-326 11979). 22. B. Halliwell. The biological effects of the superoxide radical and its products, Bull. Eur. Physiopathol. Respir. 17:21-28 (1981). 23 C. C. Winterboume. Hydroxyl production in body fluids. Biochem. J, 198:125-131 (1981), 24. H. Rosen and S. J. Klebanoff. Role of iron and ethylenediamine tetracetic acid in the bactericidal activity of superoxide aniongenerating systems. Arch. Biochem. Biophys. 208:512-519 (1981). 25. J. A. Fee. On the question of superoxide toxicity and the biologic function of superoxide dismutases. In: Proceedings of the Third International Congress on oxidases and related redox systems, (T. E. King, H. S. Mason, and M. Morrison. eds.) pp. 76-87, Pergamon Press, Oxford (1982). 26. A. Albert. Quantitative studies of the acidity of naturally occurring substances for trace metals. Biochem. J. 54:646-654 (1953). 27. M. Jamaluddin, S. RajaLakshmi, and D. S. R. Sarma. Evidence for a direct interaction of L-ascorbate with rat liver DNA. Biochim. Biophys. Acta 653:423-427 (1981). 28. E. J. Nanni, M. D. Stallings, and D. T. Sawyer. Does superoxide ion oxidize catechol, ~-topocherol and ascorbic acid by direct electron transfer? J. Am. Chem. Soc. 102:4481-4485 (1980). 29. K, 3. Kittridge and R L. Willson. Superoxide free radicals, uric acid, and enzyme inactivation, Int. J. Radiat, Biol. (in press), 30. R. C. Smith and L. Lawing. Antioxidant activity of uric acid and 3-N-ribosylurate acid with unsaturated fatty acids and erythrocyte membranes. Arch. Biochem. Biophys. 223:166-172 (1983). 31. A. M. Cohen, R. E. Aberdoth, and P. Hochstein. Inhibition of free radical-induced DNA damage by uric acid. FEBS Lett. 174: 147-150 (1984).
0748-5514/85 $3.00 + .00 © 1985 Pergamon Press Ltd.
Printed in the USA. All rights reserved.
CONSERVATION
OF VITAMIN
C BY URIC
ACID
IN B L O O D
ALEX SEVANIAN,*t KELVIN J. A . DAVIES,*~ a n d PAUL HOCHSTEIN*:]: *Institute for Toxicology and Departments of ";'Pathology and :l:Biochemistry, The University of Southern California, 1985 Zonal Avenue, Los Angeles, California 90033, U.S.A. (Received l August 1984; Revised 5 February 1985; Accepted 18 March 1985)
Abstract--Urate at physiological concentrations increased the stability of ascorbate in human serum approximately fivefold. These measurements were made by depleting human serum of urate with uricase. In model experiments (using phosphate buffer instead of serum), urate protected against iron-induced ascorbate oxidation and, to a lesser extent, pH-induced ascorbate oxidation. In both human serum and in phosphate buffer, urate exerted its protective effect without itself undergoing measurable oxidation as determined by spectrophotometric and high pressure liquid chromatography (HPLC) techniques. These experiments suggest an important physiological role for urate in preserving ascorbate in blood and other biological fluids. Keywords~Uric acid, Ascorbic acid, Free radicals, Oxidation, Transition metals, Uricase, Primate evolution
equate tissue levels. It has been proposed 12 that the inability to synthesize ascorbic acid may be the consequence of a mutation in higher primates which occurred simultaneously with the loss of uricase. Ames et al. m3suggested that the high concentrations of urate in the higher primates afforded an antioxidant alternative to ascorbate. They demonstrated that urate served as a potent antioxidant against free radical damage to biological membranes. At physiological concentrations, urate effectively protected against organic hydroperoxide-dependent lipid peroxidation and hydroperoxide.dependent hemolysis of erythrocytes. Because urate levels in biological fluids, notably blood, remain reasonably constanP 4 despite dietary variations in other purines, it occurred to us that urate by virtue of its high concentrations may also protect ascorbate from oxidative reactions in these fluids.
INTRODUCTION
The role of vitamin C (ascorbic acid) in biochemical reactions has been widely studied for decades and, among the many functions described in mammals, its antioxidant properties have received considerable attention. This antioxidant capacity appears, in part, to result from its reducing potential and its participation in redox reactions with a variety of compounds. In this respect, ascorbate may serve to minimize oxidative or free radical reactions which can be damaging to the cell.~-4 This property is based upon a facile one-electron cycling between the dihydro-, semidehydro-, and dehydroascorbate forms (i.e., the quinone, semiquinone radical, and quinol)) The rates of these interconversions are influenced by the presence of transition metals, 6'7 hydrogen ion concentration, 8 and the state of other biological oxidation-reduction couples. L9-~ Some, or perhaps all, of these reactions may be responsible for the instability of ascorbate in biological fluids. In humans and higher primates, the tendency of ascorbate to oxidize, combined with a metabolic lesion (the inability to synthesize ascorbate acid) requires the continuous intake of the vitamin in order to maintain ad-
MATERIALS AND METHODS
Measurements of ascorbate oxidation were carried out in 100 mM potassium phosphate buffer, pH 7.4, using various concentrations of ascorbate (Sigma, St. Louis, MO) in the presence and absence of various concentrations of sodium urate (Sigma, St. Louis, MO). Uricase (type I from porcine liver) and catalase (C- 100) were also obtained from Sigma. Similar measurements were also made using human serum to which ascorbate was added to produce the final concentrations indicated.
Address for corresponding--Dr. Alex Sevanian, University of Southern California, Institute for Toxicology. 1985 Zonal Avenue, Los Angeles. CA 90033, U.S.A. 117
118
A. SEVANIAN.K. J. A. D~,VlES, and P. HOCHSTEI>~
Polarographic measurement of ascorbate oxidation The rates of ascorbate oxidation in potassium phosphate buffer were determined using a calibrated Gilson 5/6A oxygraph equipped with a Clark-type electrode. Comparisons were made for the rates of oxidation with FeCI3 added in concentrations ranging from 0 to 100 MM in the presence of 100/,tM ascorbate, and 0 to 400 tiM urate. The effects of pH on oxidation rates were studied by dissolving ascorbic acid in water and adjusting the pH by additions of dilute KOH or HCI. All incubations were conducted at 21°C. The rate of ascorbate oxidation in the presence of 10 #M FeC13 was also examined in the presence of urate and under conditions where samples were first incubated for 1 h with 100 to 400 gM urate plus 0.025 U/ml uricase. In a similar manner, the effects of the iron chelators, EDTA, diethylenetriaminepentaacetic acid (DTPA) and transferrin on ascorbate oxidation rates were determined by measurement of oxygen consumption.
Spectrophotometric measurement of ascorbate oxidation Ascorbate disappearance was also monitored at 265 nm in 100 mM potassium phosphate buffer, in the presence of various FeCI3 and urate concentrations; ascorbate concentrations ranged from 10 to 100 #M and all determinations were made at 21 °C using a Perkin-Elmer 552 spectrophotometer.
plished by monitoring the HPLC eluent at 265 and 212 nm. Details of the HPLC method for urate and ascorbate measurement are described elsewhere.~5~ In order to determine whether urate preserves ascorbate in serum, a method was selected which removed urate and enabled comparisons to be made of ascorbate oxidation in serum in the presence and absence of urate. Normal serum was divided into equal aliquots which were incubated with or without uricase at 0.025 U/ml serum for 60 min at 37°C. Similarly. samples containing added uricase plus 100 U/ml catalase were prepared. The oxidative loss of urate was followed by analyzing serum aliquots for urate and formation of allantoin by HPLC. Subsequent analysis of the serum samples established the remaining concentration of urate, as well as that of ascorbate after treatment with uricase. Following the addition of 25 to 100 #M ascorbate, the serum samples were incubated at 37°C for various time intervals. At specified time points aliquots were prepared for HPLC by a column extraction procedure described previously. ~6The approach described here circumvented the impediments associated with measurements of urate and ascorbate levels in serum due to a plethora of interfering substances. Alternately, serum ascorbate levels were estimated using the colorimetric dinitrophenylhydrazine method. ~7 Comparisons were made of time dependent changes in ascorbate levels for samples containing predetermined amounts of urate, as well as in samples in which urate was depleted by treatment with uricase.
HPLC Analysis of ascorbate/urate oxidation The extent of ascorbate oxidation in potassium phosphate buffer was measured by directly injecting 20 to 100/tl aliquots of the samples, incubated over various time intervals, into an HPLC. The analytical system consisted of a Perkin-Elmer Series 4 liquid chromatograph equipped with a 5-#m, 4.6 × 250-mm Spherisorb amine column (Chromanetics Corp., Jessup, MD). Samples were eluted with acetonitrile :0.04 M NaH2PO4 (75:25, v/v) at a flow rate of 1.5 ml/min. The eluent was monitored at 265 nm using a Perkin-Elmer model 85 detector equipped with a stop-flow scanning autocontroller, and determinations of the amount of ascorbate in the samples were made by comparison to a calibration curve obtained using ascorbate standards dissolved in HzO. This method permitted simultaneous determinations employing both the spectrophotometric and HPLC techniques. The content of urate was also measured by monitoring the HPLC eluent at 292 rim, which greatly increased the analytical sensitivity as compared to 265 nm. In some cases, concurrent measurement of ascorbate and urate as well as their oxidation products, dehydro-ascorbate and allantoin, was accom-
RESULTS
Experiments performed in buffered solutions The oxidation of ascorbate in potassium phosphate buffer (pH 7.4) was concentration dependent (Fig. 1). Oxidation rates were also dependent on ferric ion concentration, although Fe 3+ was found to produce a bimodal effect (data not shown). Increasing concentrations of FeCI3, prepared in 0.001 M HCI, were added in aliquots not exceeding 25 Mi. Under these conditions the pH varied less than 0.05 units. Using 100 MM solutions of ascorbate, maximal oxidation rates were observed in the presence of 10-20 MM Fe 3+ and minimum rates were measured when no iron was added. The rates of ascorbate oxidation in buffer varied between 11.3 and 11.7 nmol O2/min in the absence of added iron. When Fe 3+ concentrations exceeded 30 #M, oxidation rates gradually decreased, reaching a plateau of approximately 12.5 nmol 02 consumed/min. Addition of 100 #M EDTA or DTPA to samples containing 10 #M FeCI3 and 100/tM ascorbate reduced oxygen consumption rates by approximately 85%, demonstrating the
Uric acid protects vitamin C 25 .,..o E .%
3
20
c
15
E
0 m E
~
I0
0
-EDTA
X 0
75
150
225
300
375
Ascorbic Acid Concentrotion
450
(uM)
Fig. 1. Ascorbateoxidation rates determined polarographicallywere measured as a function of ascorbate concentration. Measurements were made using a 2.0-ml cell fitted with a Yellow Springs, Clarktype electrode and maintained at 21°C. All measurements were performed in the presence of 10~uMFeCh in 100 mMpotassiumphosphate buffer, pH 7.4. Also shown are the rates of oxidation for 100 ltM solutions of ascorbate in the presence of 100 pM EDTA (-) and 100 ,uM DTPA (A).
role of iron in the oxidation of ascorbate under these conditions. Analysis of these buffers by atomic absorbtion spectroscopy showed that the iron contamination was =< 1.5 //M in potassium phosphate and =< 0.6 pM in tris-HCl buffer. This amount of iron is sufficient to account for the background levels of ascorbate oxidation noted here. The rate of ascorbate oxidation is also known to increase with pH, and we observed a continuous increase in oxygen consumption rates from 11.7 +- 0.20 to 36.1 --- 0.72 nmol/min when 100/.tM ascorbate was dissolved in buffers (100 mM phosphate and 50 mM tris-HC1) ranging in pH from 6.5 to 9.5. The addition of I00 pM urate to phosphate buffer decreased the rates of ascorbate oxidation from 16.1 --- 0.16 to 11.3 --0.31 at pH 6.5, but had a diminishing effect with increasing pH such that at pH 9.5 the rates of ascorbate oxidation were not significantly affected. A concentration-dependent inhibition of ascorbate oxidation was evident with increasing amounts of urate at pH 7.4 (Fig. 2). For example, the oxidation rate of a 100 tiM solution of ascorbate decreased by 20% and 51% in the presence of 25 and 100 pM urate, respectively. At 300/JM urate the oxidation of ascorbate was reduced by 73%. Spectrophotometric analysis of ascorbate (100/tM) in the presence of FeCI3 (10 pM) confirmed that urate markedly inhibits the rate of ascorbate disappearance (Fig. 3, a, b, c). This was evidenced by examining the time-dependent decrease in ascorbate absorbance at 265 nm in the absence and presence of equimolar urate. As
119
shown in Fig. 3, a and b, ~rate appears only to decrease the rate of ascorbate oxidation without producing any change in the ascorbate spectrum. The effect of transferrin on ascorbate oxidation in buffer was also examined. Purified apotransferrin was loaded with iron by a previously described method ~ and then added to phosphate buffer solutions containing 100 pM ascorbate. We found that 25.0 pg of apotransferrin loaded with 2.5 pg Fe 2+, as Fe 2. (NH4)2 (SO4)2, did not stimulate ascorbate oxidation. The final concentration of iron in the assay was calculated to be 6.25 /~M. In fact, both spectrophotometric and polarographic analyses demonstrated a slight suppression in the rates of ascorbate oxidation (data not shown). The rate of ascorbate oxidation was stimulated 25% only when the iron loading was increased to l0/~g per 25 /~g apotransferrin. This would be equivalent to 25 pM iron bound to transferrin in the ferric state. Because an examination of urate's effect on ascorbate stability in human serum would require depletion of urate, we considered as an expedient approach the removal of urate using uricase. In order to validate this procedure, experiments were first performed in potassium phosphate buffer. A 300-pM solution of urate was divided into equal aliquots, one of which was treated with uricase as described in the Methods section. Treatment with uricase reduced the concentration of urate to the limit of detection by HPLC (_-< 20 pM). At this point, ascorbate was added to the samples to yield an initial concentration of 100/tM.
....) E
= o
12
ca
i,
O t.J
X 0
i
0
75
#
I
I
I
150
225
300
375
450
Uric Acid Concentrotion (uM)
Fig. 2. Theeffectofurateontherateofascorbateoxidationisshown. Initial rates of oxidation were measured polarographically as described in Fig. I, using 100 ,UMascorbate and 10 I~MFeCI~. A 2-raM urate solution in 100 mM potassium phosphate buffer (pH 7.4) was prepared by sonication in a Bransonic sonicatorbath for 20 rain. The solution was maintained at 50°C during sonication which facilitated dissolution of urate. Increasing aliquots of this solution were used to produce the final concentrations shown.
120
A.
K. J. A.
SEVANIAN,
and P. HOCHSTEIN
DAVIES.
90
A
C
B 1
80
800"
70
'~ 700'
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0 0 to
I
240
i
I
280
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i
I
320 200 Wavelength (nm)
I
240
I
280
i'
320 Time
(min)
Fig. 3. A spectrophotomctric analysis of ascorbate oxidation performed in 100 mM potassium phosphate buffer, pH 7.4, containing 10 #M FeCI> Analyses of the ultraviolet spectrum of a 100 /~M solution of ascorbate were made against potassium phosphate buffer. Repetitive scans were taken every 10 rain at a scanning rate of 120 nm/min. The absorbance maximum for the instrument was set at 2.0 and measurements were made at room temperature. The original spectrum for freshly added ascorbate is indicated as tracing (i) in panels A and B. Panel A shows the progressive oxidation of ascorbate in the presence of 10/~u Fe'+ over a 50-rain interval. Panel B represents a similar analysis in the presence of 100 gM urate added to both the sample and reference cuvettes. Panel C is a plot of the data derived from panels A and B showing the nanomoles of ascorbate oxidized as a function of time. The data from panel A are represented by the solid circles and data from panel B are represented by the open circles.
The results of the HPLC determination of ascorbate content in potassium phosphate buffer over 2.5 h of incubation are shown in Fig. 4. The figure shows that less than 20% of the ascorbate was oxidized in the presence of 300 /IM urate. When these preparations were treated with uricase, approximately 90% of the ascorbate was oxidized by 2.5 h. Uricase in the absence of urate did not affect ascorbate oxidation rates. Considerable amounts of H202 are produced by uricase oxidation of urate. The quantity of H202 formed in potassium phosphate buffer was sufficient to significantly increase ascorbate oxidation rates. Thus, ascorbate oxidation was not only accelerated by the depletion of urate, but also by the formation of H202 during the urate-uricase reaction. The amount of ascorbate oxidized under these circumstances was doubled, from 20% to 40% over 1 h, when ascorbate was added to urate solutions previously oxidized with uricase. However, the addition of catalase to uricase-treated samples, either during or after oxidation of urate, eliminated the pro-oxidant effect and yielded ascorhate oxidation rates similar to those of solutions containing only ascorbate. Ascorbate oxidation rates were mark-
edly increased by addition of 300/JM H202; however, these rates were typically 40% to 50% less than the rates observed when urate-containing solutions were treated with uricase (Fig. 4). In the presence of catalase we found that following urate depletion by uricase, ascorbate oxidation rates were similar to those observed for ascorbate alone in buffer. We attribute this effect to the degradation by catalase of the H202 formed during uricase-induced oxidation of urate. If urate were behaving as a typical scavenging antioxidant, its content should decrease by an amount approximately equivalent to the moles of ascorbate spared from oxidation, moles of iron reduced, or the difference in moles of oxygen consumed. This is estimated to be approximately 80 gM urate oxidized over 2.5 h, which is well within the sensitivity of the assay as previously demonstrated. 16 As seen in Fig. 4, there was no indication of urate oxidation under conditions where ascorbate oxidation was clearly inhibited. Table 1 shows the results of a spectrophotometric determination of the rates of ascorbate oxidation. We found that ascorbate oxidation rates increased over sixfold in buffer preparations of ur'ate treated with uricase.
Uric acid protects vitamin C
~
100
° o
,~
.
1,,o 1 ot,oo 1
o .......................
..o .........
BO
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121
Table I. Spectrophotometric Measurement of Ascorbic Acid Oxidation in Phosphate Buffer Uric Acid (,UM)
Uricase* Treatment
Initial Rate of Ascorbic Acid Oxidation';" (nmol/min)
-300 < 20
--+
28.0 - 0.90 8.0 -+ 0.32 49.6 -+ 1.63
+
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180
TIME OF INCUBATION (min)
*Uricase was added to samples as indicated. Samples were incubated with 0.025 U/ml uricase for I h at 37°C, and uric acid levels were subsequently measured by HPLC as described in Materials and Methods. tMean and standard deviation of three measurements of the oxidation rate for a 100/IM solution of ascorbate acid in 100 mM potassium phosphate buffer. The rate of absorbanc¢ decrease at 265 nm was used to calculate the rate of ascorbate acid oxidation using Emax 265 nm = 7 x 10~.
Fig. 4. The results of an HPLC analysis of ascorbate oxidation. A 300-/ZM solution of urate was prepared in 100-raM potassium phosphate buffer, pH 7.4, containing 10 ~uMFeCI3. Samples were incubated at 37°C in a shaking water bath for 5 rain and ascorbate was then added to yield an initial concentration of 100/ZM. Aiiquots of 100 ,ul were removed and injected onto the HPLC column at the times indicated, O-----Q. Also shown is the effect of urate depletion by incubating the urate solution with uricase, as described in the text, prior to the addition of ascorbate, C)..... O. The rates of ascorbate oxidation alone in phosphate buffer, • A ; ascorbute plus 300-/zM H20:, A ..... A; and ascorhate added to a sample pretreated with 300-/ZM urate plus uricase plus 100-U/ml catalase, • l ; are shown. The eluent in the above experiments was monitored at 265 nm. Included in the figure are the levels of urate (1"7..... []) in samples not treated with ur•case. The levels of urate were determined by simultaneous monitoring of the eluent at 292 and 265 nm using a Perk•nElmer LC 85 detector equipped with a stop-flow autocontroller.
1108
1
The initial rate of oxidation for a 100/ZM solution of a s c o r b a t e a l o n e w a s n o t s i g n i f i c a n t l y d i f f e r e n t in t h e presence or absence of uricase. ~0"-.
-O . . . . .
60
9-
O--
Experiments using human serum T h e H P L C p r o c e d u r e w a s a l s o u s e d to m o n i t o r t h e r a t e o f a s c o r b a t e o x i d a t i o n in s e r u m s a m p l e s . W e f o u n d c o n s i d e r a b l e v a r i a t i o n in s e r u m u r a t e c o n c e n t r a t i o n s from volunteer donors simultaneously with a large vari a b i l i t y in a s c o r b a t e o x i d a t i o n r a t e s a m o n g t h e s e s a m p l e s . In e v e r y i n s t a n c e , h o w e v e r , t h e r e w a s a n o t a b l e i n c r e a s e in a s c o r b a t e o x i d a t i o n r a t e s w h e n s e r u m s a m p l e s w e r e d e p l e t e d o f u r a t e . T h i s is s h o w n in Fig. 5, which represents a typical experiment using human s e r u m . In t h i s i n s t a n c e , t h e s e r u m u r a t e w a s 2 8 0 / Z M , and following uricase treatment urate levels decreased to b e l o w t h e l i m i t o f d e t e c t i o n . U p o n a d d i t i o n o f 100 /~M a s c o r b a t e to w h o l e s e r u m , w e f o u n d t h a t 7 0 % o f the ascorbate remained after 2 h, whereas only 10% of t h e o r i g i n a l a s c o r b a t e r e m a i n e d in t h e s e r u m s a m p l e
0
10
30
120
150
TIME OF INCUBATION (rain) Fig. 5. An HPLC analysis of ascorbate oxidation in human serum. The serum was divided into equal aliquots and incubated with or without 0.25 U/ml uricase for 60 rain at 37°C. Ascorbate was then added to the serum samples to yield an initial concentration of 100/ZM. The rates of ascorbate oxidation are shown for both the uricase treated O ..... O, and untreated H serum. Also shown are the rates of ascorbate oxidation in the presence of 300-~UMH20,, A A; serum pretreated with uricase plus 100 U/ml catalase, [ ] ..... I-q; and the levels of urate in serum samples not treated with uricase or H.,O2, • • . The effect of the chelating agents, EDTA, • • ; and DTPA, ~7..... ~7; on the rate of ascorbate oxidation in whole serum are included in the figure. The conditions for analyses are otherwise as described in Fig. 4, and serum samples were prepared for HPLC as described in the Methods section.
122
A. SEVANIAN,K. I. A. DAVIES.and P. HOCHSTEIN
treated with uricase. This pattern was evident in all serum samples analyzed. The half-life of added ascorbate (100/IM) in serum samples containing the normal range of urate for humans (100-380/tM) was approximately five times longer than in samples depleted of urate. Similar to the situation noted for ascorbate oxidation in buffer, uricase treatment of serum caused ascorbate to undergo rapid oxidation. In contrast to buffer, however, catalase addition did not markedly reduce this effect (Fig. 5). Expression of catalatic activity in serum was indicated by the ability of added catalase to inhibit ascorbate oxidation following addition of H202. It appears that H20,. oxidizes ascorbate in serum, but in the absence of urate the rates of ascorbate oxidation are markedly increased. It can be seen in Fig. 5 that less than 50% of the ascorbate underwent oxidation following addition of H202 to a final concentration of 300 ~M in serum, which represents the total amount of H202 that could be formed by the complete oxidation of ~ 300,ttM urate. Thus, factors other than the H202 formed in the serum are likely to account for the rapid oxidation of ascorbate in the absence of urate. Also shown in Fig. 5 is the effect of added EDTA and DTPA on the oxidation of ascorbate added to otherwise untreated serum. Both chelating agents at 100/~M markedly reduced the rate of ascorbate oxidation, DTPA being more effective than EDTA. EDTA stabilized ascorbate for approximately 45 min and thereafter a significant oxidation of ascorbate was noted. It should be noted that conventional methods for measuring ascorbate levels in serum are inappropriate under conditions where ascorbate oxidation takes place. One common method used clinically for ascorbate analysis is a colorimetric assay using dinitrophenylhydrazine. 17 Table 2 shows that this method cannot differentiate between dihydroascorbate and its oxidized products. No significant change in ascorbate content was found when a 200-/.tM solution in serum was analyzed following incubation for intervals up to 15 min. The levels were also unaffected by the absence of urate. Table 2. Oxidation of Ascorbic Acid in Serum Measured by the DinitmphenylhydrazineMethod mg
Incubation Time (rain) 0 5 I0 15
Ascorbic Acid/100 ml Serum*
Complete Serum 3.5 3.1 3.3 3.2
-+ 0.4 - 0.2 -4- 0.4 +- 0.5
Serum + Uricase 3.3 3.6 3.5 3.4
-+ 0.4 - 0.4 +--0.5 _ 0.3
*Results are presented as mean + S.D. of four experiments, performed as described in Materials and Methods.
This method does not distinguish between the dihydroand dehydro- forms of ascorbate, whereas large decreases in ascorbate levels are found by 15 rain using the HPLC method (Fig. 5). DISCUSSION In this report, we demonstrate that urate, at concentrations approximating physiological levels, stabilizes ascorbate in human serum and phosphate buffer. A striking feature of this effect is the absence of measurable urate disappearance despite a marked inhibition of ascorbate oxidation. Ascorbate oxidation in the presence and absence of urate was measured using three different methods. In order to study the effect of urate depletion on ascorbate stability in potassium phosphate buffer and human serum, the oxidation of urate by uricase was employed (uric acid ~ allantoin + H_,O2). This procedure, however. introduced a potentially complicating factor to the experiments. The rapid oxidation of ascorbate under these conditions can be partially attributed to the H202 formed during the oxidation of urate by uricase; however, H202 was not solely responsible for the enhanced ascorbate oxidation rates. This was demonstrated by the ability of catalase to reduce significantly the extent of ascorbate oxidation, producing rates equivalent to those found for solutions containing ascorbate alone. In phosphate buffer, ascorbate alone underwent oxidation at rates significantly greater than in solutions also containing urate. HPLC analysis of serum showed that ascorbate at concentrations in the range found in normal humans underwent oxidation at rates considerably slower than those measured in serum samples depleted of urate by pretreatment with uricase. The enhanced oxidation of ascorbate is not attributable to HaO~ because comparable oxidation rates were observed in the presence or absence of catalase. These findings suggest that the stabilization of ascorbate in serum may be an important biological function of urate. Our findings also suggest that catalysts such as iron may be responsible for the oxidation of ascorbate in serum, and that urate may inhibit this catalytic process. There is an ongoing controversy over the role of iron as a catalyst for in vivo free-radical reactions. Gutteridge et a1.19 maintain that " c a t a l y t i c " iron occurs in vivo and may be in forms such as highly loaded ferritin, 2° iron complexes of phosphate esters (e.g., ADP or ATP), 21 iron chelates such as EDTA-Fe 3+ , and even low levels of iron salts.22 Winterbourne23 and others z4'25 disagree with the notion that free iron salts exist, but maintain that oxidation-reduction reactions between ascorbate and a variety of in vivo forms of iron are
Uric acid protects vitamin C possible. Reactions such as these may account for the catalysis of ascorbate oxidation in serum; however, transferrin, the most abundant iron depot in serum, did not stimulate ascorbate oxidation. Based on our observations, we tentatively conclude that serum component(s) other than transferrin are likely to he responsible for ascorbate oxidation. The pH of serum (7.4-7.6 in the case of these experiments) may account for some of this oxidation, but other catalysts of ascorbate oxidation may also exist. The protective effect of urate might occur by one of the following hypothetical mechanisms: (l) a competition between urate and ascorbate for the reduction of iron; (2) rereduction of ascorbate by urate; (3) sequestration of iron via a uric acid " c h e l a t e " which cannot act as an oxidant of ascorbate; (4) inhibition by a chainbreaking antioxidant mechanism; and (5) stabilization of ascorbate via hydrogen bonding to urate. Possibilities (1) and (2) are unlikely because there was no evidence of stoichiometric urate oxidation. It should be noted that approximately 20 /ZM urate represents the limit of reliable detection by the methods employed. Because only -- 5% ( ~ 15 ,t/M) of the urate was lost after 2.5 h of incubation (Fig. 5), it would be difficult to conclude whether this loss was significant. The third possible mechanism remains open to investigation, because Albert 26 has reported that urate and other purines have the capacity to bind iron. Unfortunately, the binding of iron by urate remains poorly understood. A chain-breaking antioxidant effect of urate is a possible mechanism of action. If the oxidation of ascorbate is proceeded by means of a long propagative radical chain reaction, very little urate should be required to provide an effective scavenging action. Large amounts of vitamin C might be spared from metal catalyzed oxidation and concomitantly small, and perhaps undetectable quantities of urate would be consumed over considerable time intervals. The fifth possible mechanism for the protective action of urate is deduced from the studies of Jamaluddin et al. 27 Because the oxidation of ascorbate occurs through the abstraction of its enolic hydrogen by oxygen, 2s and becomes more facile at basic pH, it has been proposed that hydrogen bonding between a purine (adenine or guanine) and ascorbate would make the enolic hydrogen less available for abstraction. A similar hydrogen bonding could take place with urate such that equimolar or greater concentrations should effectively retard the rate of ascorbate oxidation. This hydrogen bonding effect should reduce the degree of both iron-dependent and pH dependent ascorbate oxidation, as observed in the present studies. Kittridge and Willson :9 have recently shown that urate reacts with .OH at rates exceeding those measured for mannitol, a commonly used .OH scavenger. In ad-
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dition to the observations of Ames et al.,J3 Smith and Lawing a° have found that urate is an effective erythrocyte membrane lipid antioxidant. In each of these instances, the antioxidant effect of urate was associated with its stoichiometric loss. The effective levels for antioxidant action were in the range found in normal human serum. Urate was recently shown to protect against oxidative damage to DNA, 31 and a similar antioxidant protection was found against hydroxyl radical-induced oxidation of certain proteins (K. J. A. Davies, unpublished observations). The ability to scavenge oxygen free radicals as well as free radical intermediates indicates potentially important antioxidant roles for urate in which this purine undergoes extensive oxidation. The stabilizing effect we describe in this paper appears distinct from previously described stoichiometric scavenging functions. Urate prevents the oxidation of ascorbate by an as yet undefined mechanism that does not involve extensive oxidation of urate to allantoin and other products. This stabilizing effect, readily observed in human serum and phosphate buffer, may have important consequences for the conservation of ascorbate in other biological fluids. The evolutionary loss of the ability to synthesize ascorbate may have been accompanied by a complementary loss of uricase. The latter mutation would insure levels of urate sufficiently high to stabilize extracellular ascorbate, derived from dietary sources, for ultimate utilization at intracellular sites. Acknowledgements--This work was supported in part by grants from the National Institutesof Health (HLI 5162) and the Arco Foundation. The authors acknowledge the technical assistance of Ms. Laurie
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