Determination of xanthine oxidoreductase forms: influence of reaction conditions

Clinica Chimica Acta 303 (2001) 117–125 www.elsevier.com / locate / clinchim

Determination of xanthine oxidoreductase forms: influence of reaction conditions ´ Encarna Varela, Carles Pascual, Rosa M. Segura* Ramon Martı, ´ , Hospital General Universitari Vall d’ Hebron, 119 -129, 08035 Barcelona, Spain Servei de Bioquımica Received 17 May 2000; received in revised form 18 September 2000; accepted 25 September 2000

Abstract Xanthine oxidoreductase (XOR) has been implicated in ischaemia-reperfusion injury, and increases in this enzyme have been found in plasma of patients with different illnesses. The catalytic concentrations of the XOR forms found in plasma, using various reaction conditions, greatly differ in the related literature. We studied the effect of the assay conditions on the xanthine oxidation rate catalysed by the XOR forms. Our results demonstrate inhibition of XOR by the reaction products and a time-dependent decrease in the reaction rates of XOR forms. Substrate consumption and inhibition by the products did not account for this decrease. Determination at 60 min incubation leads to catalytic concentrations up to 80% lower for the XOR forms than those obtained at 10 min. We conclude that elimination of the reaction products (NADH, H 2 O 2 and O 2 ) from the reaction mixture, and short incubation times, are necessary for accurate measurement of the XOR activities.  2001 Elsevier Science B.V. All rights reserved. Keywords: Xanthine oxidase determination; Xanthine dehydrogenase; Ischaemia reperfusion injury; Reactive oxygen species

1. Introduction Xanthine oxidoreductase catalyses the two last steps in the degradation of purines leading to the production of uric acid as the end product. In mammals, this enzyme has two forms that vary in their selectivity to the oxidative substrate: xanthine dehydrogenase (XD; EC 1.1.1.204), which is NAD 1 dependent, and xanthine oxidase (XO, EC 1.2.3.2), which has oxygen as the electronic acceptor and generates the reactive oxygen species (ROS), H 2 O 2 and O 22 , as products of its catalytic action. XD can be transformed into the XO form by two mecha-

*Corresponding author. Fax: 134-93-274-6831. E-mail address: [email protected] (R.M. Segura).

nisms: reversible oxidation of thiol groups (XO rev ) or irreversible partial proteolysis (XO irr ) [1]. There is general agreement as to the causative role of ROS in ischaemia-reperfusion injury, but the contribution of XOR to ROS production in these processes is a matter of controversy [2–4]. It has been postulated that, in conditions of ischaemia, XD (the main XOR form found in tissues in physiological conditions) is converted into XO. According to this hypothesis, during ischaemic episodes, decreases in the cellular energetic charge due to ATP degradation lead to increases in the cytosolic Ca 21 concentration, thus activating Ca 21 or Ca 21 -calmodulin dependent proteases, which transform XD into XO irr by limited proteolysis. At the same time hypoxanthine accumulates in tissues as a consequence of ATP degradation. During the reperfusion phase, both

0009-8981 / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0009-8981( 00 )00390-9

118

R. Martı´ et al. / Clinica Chimica Acta 303 (2001) 117 – 125

hypoxanthine and oxygen are present in the tissue, together with a high proportion of the XO form, therefore large amounts of ROS can be produced [5]. This hypothesis has been studied in several experimental works over the last years. In the majority, the authors attempted to prove the conversion of XD into XO through episodes of experimental ischaemia in animal models, measuring XOR and XO activities in tissues that had undergone different degrees of ischaemia [6,7]. In other studies, blood XO, or XO and XOR, were assessed after spontaneous or provoked ischaemic episodes [8]. Increases in circulating XO levels have also been reported in some diseases not directly related to ischaemia-reperfusion injury including jaundice [10], acute respiratory distress syndrome [11], rheumatoid and autoimmune diseases [12], scleroderma [13], hypertension [14] and alcoholism [15], and its detrimental role as a producer of ROS species is stressed. Several methods have been used to measure the forms of XOR. The most frequent are spectrophotometric methods following uric acid formation at 295 nm during incubation of the sample with xanthine or hypoxanthine [1]. More sensitive methods have been developed for measuring low levels of the enzyme, or for use in samples (i.e., plasma) which show high absorbance at the wavelength of maximum uric acid absorption. These techniques include endpoint determination or monitoring of uric acid formation by fluorimetry [16], chemiluminescence [17] and chromatographic endpoint methods separating the substrate (radiolabelled or not) and product by HPLC [18–20]. The chromatographic measurement of the uric acid formed after a fixed time of reaction with the substrate has often been applied to determine circulating levels of XOR and XO [20–23]. The reaction conditions employed vary greatly with respect to the addition of auxiliary reagents (to avoid product inhibition of the enzyme), and, particularly, regarding the incubation time (from 10 min to 7 h). Some authors even recommend different incubation periods depending on the expected sample activity [19]. Data on the linearity of uric acid appearance along the incubation period are rarely given. Moreover, the catalytic concentration of the different XOR forms and proportions found in normal plasma and in several clinical conditions, considerably differ among the studies [2,6,9–12,20,24–28]. This fact

suggests that the reaction conditions and incubation periods could have a great influence on the values obtained. An immunologic assay that measures XOR as protein has recently been described [29]. This method cannot distinguish among the different XOR forms, it measures both inactive and active protein and it does not differentiate XD, XO rev and XO irr . Therefore it does not allow the evaluation of the contribution of XOR forms to the ROS production. Despite its high sensitivity and possibility of routine application, its usefulness is limited by these restrictions. The aims of this work were: (1) to determine whether variations in assay conditions can explain discrepancies in the XOR concentration values found by different authors; and (2) to establish the conditions that facilitate more accurate measurement of the XOR forms.

2. Materials and methods

2.1. Reagents Uric acid and xanthine were purchased from Fluka (Buchs, Switzerland). Hydrogen peroxide and EDTA (disodium salt) were obtained from Sigma Chemical Co. (St. Louis, MO) and mercaptoethanol from BioRad (Hercules, CA). Purified XO (from cow’s milk), superoxide dismutase (SOD, from bovine erythrocytes) and catalase (from beef liver) were from Boehringer–Mannheim (Mannheim, Germany). Sephadex G-25 columns were obtained from Pharmacia (Uppsala, Sweden). All the other reagents were of analytical grade and were purchased from Merck (Darmstadt, Germany).

2.2. Sample processing Samples of pig liver (|0.5 g) were homogenised with phosphate buffer 50 mmol / l pH 7.8 (10 ml per g of tissue) containing EDTA 1 mmol / l, phenylmethanesulfonyl fluoride (PMSF) 1 mmol / l. Homogenisation was carried out on an ice bath with a Potter homegeniser at 500 rpm. The homogenates were centrifuged for 1 h at 40 0003g and 48C. The serum samples were pools of sera from patients with

R. Martı´ et al. / Clinica Chimica Acta 303 (2001) 117 – 125

119

various liver diseases (ALT.1000 U / l). To eliminate endogenous uric acid, and low Mr inhibitors, both the serum samples (0.5 ml) and supernatants of pig liver homogenates (raw extract, 2.5 ml) were desalted before analysis through Sephadex G-25 columns eluted with the reaction buffer (potassium phosphate 200 mmol / l, pH 8.0; EDTA 1 mmol / l, PMSF 1 mmol / l).

ditions were as follows: sample injection volume 80 ml; mobile phase potassium phosphate 20 mmol / l, 1% methanol, pH 5.0; flow 1 ml / min and detection at 295 nm, 0.02 absorbance units full scale. Uric acid concentration in the samples was calculated by interpolation of its area peak on an aqueous uric acid standard curve.

2.3. Determination of XOR forms

3. Results

The spectrophotometric or HPLC measurements of uric acid formation rate were based on previously reported methods [22]. The samples (800 ml of desalted homogenate; 300 ml of desalted serum) were incubated at 378C in reaction buffer and aerobic conditions. NAD 1 600 mmol / l was added to the reaction medium when XOR (XD1XO) was measured. The XO forms were measured in the same conditions but without NAD 1 , with mercaptoethanol (10 mmol / l) for assessment of XO irr and without mercaptoethanol for assessment of XO rev 1XO irr . Mercaptoethanol was also added, when indicated, to check its effect on total XOR determination. The concentrations of other auxiliary reagents, used occasionally, are indicated in the Results section. The reaction was always started by the addition of xanthine 100 mmol / l. All the concentrations are given as final concentrations in the reaction mixture. Sample (without xanthine) and reagent blanks were processed in each measurement. Urate production was continuously monitored at 295 nm in a UVIKON 860 (Kontron Instruments, Milan, Italy) at 378C when XOR forms were analysed in tissue extracts or purified XO preparations. The activities were calculated using the differential coefficient of absorptivity, ´295 (urate)2´295 (xanthine)59.97 l / mmol?cm, experimentally obtained by us in our reaction medium. In the serum samples, the catalytic reaction was stopped by adding HClO 4 (final concentration 1 mol / l) to aliquots of the incubation mixture taken at different times. The pellet was separated by centrifugation and urate was quantified in the supernatant by HPLC using Kontron equipment. HPLC separation of uric acid was performed in a mBondapak C 18 column with a Nova-pak C 18 precolumn (both from Waters, Milford, MA). The chromatographic con-

The production of uric acid over time by the catalytic action of XO on xanthine decreased with time. From 0 to 20 min the catalytic concentration (U / l) was 0.50 for pig liver extract XO, 0.36 for cow’s milk XO, and 0.016 for XO from human serum. The corresponding rates from 20 to 60 min were 14, 19 and 19% of the above respectively, and from 60 to 120 min, reaction rates decreased to 0, 8 and 19% (Fig. 1a). We also checked the reaction profile for the different pig liver XOR forms. Mercaptoethanol (10 mmol / l) was added to the reaction mixture for XO irr measurement. The effect of thiol addition on XD1 XO assessment was also tested (Fig. 1b). Addition of mercaptoethanol resulted in a constant slope during the monitored period when XO irr was determined, and increased the linearity in the second hour of the XD1XO catalytic reaction. To prove whether decreases in the reaction rate with time could be due to substrate consumption, we studied the effect of xanthine concentration on the catalytic reaction of purified XO and human serum XO. Results are shown in Fig. 2. Maximal activities were obtained at xanthine 75 mmol / l and 25 mmol / l respectively, higher concentrations leading to slight XO inhibition. Nevertheless, it is apparent from this figure that xanthine consumption does not explain either the plateau observed during the second hour of reaction with pig liver XO, or the decrease of reaction rate with cow’s milk purified XO and XO from human serum (Fig. 1a). In fact, after 2 h of reaction, the concentration of xanthine remaining was at least 85 mmol / l in pig liver extract and cow’s milk purified XO, and 99 mmol / l in serum sample. Since it has been reported that XO can be inhibited by its products (H 2 O 2 , O 2 2 and uric acid) [22], we tested the influence of these species on the

120

R. Martı´ et al. / Clinica Chimica Acta 303 (2001) 117 – 125

Fig. 2. Influence of xanthine concentration on uric acid formation rate. (j) Human serum XO; (1) Cow’s milk purified XO. Reaction was followed for 4 min.

Fig. 1. Monitoring of uric acid formation by XOR. (A) (top): Reaction catalysed by XO from different sources: ( j ) Pig liver extract; (1) Cow’s milk purified XO; (h) Human Serum (*). (B) (bottom): Reaction catalysed by different forms of XOR in pig liver extract: ( j ) XO; (1) XO irr ; (h) XD1XO without mercaptoethanol addition; (m) XD1XO with 10 mmol / l mercaptoethanol.

reaction profile. Uric acid formation, after xanthine oxidation catalysed by purified cow’s milk XO, was monitored in two aliquots from the same sample. In one of the aliquots 50 mmol / l H 2 O 2 was added to the reaction mixture before starting the reaction. Fig. 3a shows that 50 mmol / l H 2 O 2 inhibited XO; however, the initial slope, for the first 10 min, of the added sample was more pronounced (uric acid formed per minute50.68 mmol / l) than the slope of the second hour of incubation of the non-added sample (uric acid formed per minute50.035 mmol / l). Moreover, the H 2 O 2 formed in the non-added sample was less than 12 mmol / l, much less than that furnished to the added sample. This fact allowed us

to rule out that the peroxide formed during the catalytic reaction caused the decrease in the reaction rate observed in the non-added sample. The effect of the accumulation of reaction products on the reaction profile was also tested by adding catalase (to eliminate the H 2 O 2 formed), SOD to eliminate the O 22 formed, and both catalase and SOD. As shown in Fig. 3b, the results confirm XO inhibition by peroxide and evidence that the superoxide also inhibits the enzyme. However, addition of the O 2 2 and H 2 O 2 scavengers did not change the reaction profile. The effect produced with the addition of other reported inhibitor product, uric acid [20,30] and NADH [1] on XOR activities is summarized in Table 1. Data from this table demonstrate that uric acid up to 24 mmol / l did not result in XOR inhibition; in contrast, addition of 100 mmol / l NADH to the reaction mixture of XOR clearly inhibited the reaction catalysed by XD1XO. Furthermore the decrease of catalytic concentrations calculated at longer incubation times, evidences that the profile of the reaction does not change, the reaction rate also decreases with time. Apart from the effect of substrate concentration and product inhibition on the catalytic activity of the XOR forms, the figures presented in this work demonstrate that, due to the reaction profile, the time of incubation has a great influence on the XOR values obtained with endpoint methods. This is

R. Martı´ et al. / Clinica Chimica Acta 303 (2001) 117 – 125

121

times. Long incubation times resulted in lower XOR activities in all its forms as a consequence of the time-dependent decrease of the slopes shown in Fig. 1b. The estimated ratio of XO decreases with incubation time, whereas that of XO irr increases, particularly when mercaptoethanol is omitted to assess total XOR.

4. Discussion

Fig. 3. Influence of peroxide and superoxide on xanthine oxidation catalysed by cow’s milk purified XO. (A) (top): Influence of H 2 O 2 addition: ( j ) No addition; (1) 50 mmol / l H 2 O 2 . (B) (bottom): Influence of catalase and / or SOD addition: ( j ) No addition; (1) Catalase 1200 kU / l; (h) SOD 50 kU / l; (m) Catalase 1200 kU / l plus SOD 50 kU / l.

shown in Table 1 and more clearly illustrated in Table 2 where the catalytic concentrations of all the XOR forms were calculated for different incubation

Due to its ability to produce ROS, xanthine oxidoreductase, particularly in the oxidase form, has been implicated in ischaemia-reperfusion injury [5]. Moreover, it has recently been proposed that the dehydrogenase form can also give rise to ROS production, although to a lesser extent [31,32]. Inhibitors of the enzyme have been empirically included in the preservation solutions used for organ transplantation [33]. The liver and intestine are recognised as the tissues containing highest XOR specific activities, with little differences among works [34]. More striking differences are observed in plasma levels of the forms of this enzyme. XOR catalytic concentrations range from 0 to 4200 mU / l in plasma from human healthy subjects [9–12,24– 27]. The extraction methods and ‘‘in vitro’’ conversion of the XD to the XO forms can account for the differences found in tissues, but the conditions used to assess the activity of the XOR forms, both in tissue extracts and plasma, must also be considered when comparing the results. Due to the inhibition of the enzyme by the reaction products, activities can be underestimated. XOR inhibition by the NADH formed in the catalytic

Table 1 Effect of uric acid on XO and of NADH on XOR activities a XO (mU / l) (Cow’s milk purified XO) Addition of uric acid

10 min 30 min 60 min 120 min a

XOR:XD1XO (mU / l) (Pig liver extract) Addition of NADH

No

24 mmol / l

No

100 mmol / l

1260 728 445 261

1218 721 455 263

2538 1852 1385 824

1050 802 649 474

Catalytic concentrations are calculated at different reaction times.

R. Martı´ et al. / Clinica Chimica Acta 303 (2001) 117 – 125

122

Table 2 XOR catalytic concentrations calculated at different endpoint times* (mU / l) Form

Sample

Mercapto-ethanol

10 min

30 min

60 min

120 min

XO

Serum Cow’s milk Pig liver Pig liver Pig liver Pig liver

– – – 10 mmol / l – 10 mmol / l

23.6 507 677 135 788 728

13.9 288 408 103 515 505

8.2 176 234 88.7 314 343

5.8 106 112 75.3 153 228

Pig Pig Pig Pig

a

85.9 93.0 17.1 18.5

79.2 80.8 20.0 20.4

74.5 68.2 28.2 25.9

73.2 49.1 49.2 33.0

XO irr XD1XO

%XO %XO irr

liver liver liver liver

b c d

a

Calculated from (XO) /(XD1XO; without mercaptoethanol. Calculated from (XO) /(XD1XO; with 10 mmol / l mercaptoethanol. c Calculated from (XO irr ) /(XD1XO; without mercaptoethanol. d Calculated from (XO irr ) /(XD1XO; with 10 mmol / l mercaptoethanol. * Results from data of Fig. 1a and b. b

reaction of XD was described by Della-Corte and Stirpe [1] and confirmed in the present work; however elimination of the reduced coenzyme is not a general rule, and the products O 2 2 and H 2 O 2 , which also inhibit the enzyme, are not always eliminated [2,6]. Our results confirm that the addition of SOD and catalase to the reaction mixture leads to higher estimates of XOR activity; thus it is evident that these enzymes should be added to the reaction mixture for accurate XOR measurement. The addition of a NADH-dependent dehydrogenase and the corresponding substrate is also necessary to avoid inhibition by NADH when XD1XO is assessed [22,23]. We have determined the enzyme in presence of lactate dehydrogenase 5.2 kU / l and pyruvate 1.75 mmol / l, concentrations that effectively eliminate the NADH formed in the reaction mixture for liver and plasma samples in our conditions. Particular attention must be paid to the influence of the catalytic reaction profile of the XOR forms on the values obtained at different incubation times. When XOR, in any of its forms, is measured in raw tissue extracts or cells, short incubation times (10 min or less) are usual [17,35]. However when circulating XO (or XO and XOR) is assessed, the endpoint approaches, with long incubation times, are more frequently used [20–23]. Our results show that

the formation of uric acid, catalysed by XO from different samples (human serum, raw extract from pig liver, purified XO from cow’s milk) does not have a linear kinetics over time. Moreover, uric acid production among the different forms of pig liver XOR presents profiles that do not coincide. The rate of catalysis by XD1XO or by XO decreases gradually during the first hour of reaction, whereas the catalysis by XO irr presents a more linear behaviour (Fig. 1b). It is clear from this figure that measuring these enzyme forms at incubation times greater than 10 min will lead to underestimation of XD1XO and, consequently, to overestimation of the XO irr / XD1 XO ratio, especially when XD1XO are determined in the absence of thiol reducing agents, which is the most common approach [2,6]. Our results demonstrate that neither substrate consumption nor reversible inhibition of the enzyme by the products that accumulate during the reaction course are responsible for the non-linear XOR kinetics. Inhibition of XOR by high xanthine concentrations has been reported for milk XO [36,37]. Hausladen and Fridovich [38] have interpreted this as a spectrophotometric artefact due to stray light, rather than true inhibition. In disagreement with these authors, we observed slight inhibition of XOR at xanthine concentrations higher than 100 mmol / l

R. Martı´ et al. / Clinica Chimica Acta 303 (2001) 117 – 125

123

Table 3 Proposed conditions for the measurement of XOR forms a Total XOR (XD1XO)

Total XO (XO rev 1XO irr )

Desalting of the sample: Sephadex G-25 Elution with:

Potassium buffer pH 8.0 (mmol / l) EDTA (mmol / l) PMSF (mmol / l) Mercaptoethanol (mmol / l)

200 1 1 –

200 1 1 –

Addition to the desalted sample:

Catalase (kU / l) SOD (kU / l) NAD 1 (mmol / l) LD (kU / l) Pyruvate (mmol / l)

1200 50 600 5.2 1.75

1200 50 – – –

Xanthine (mmol / l)

100 100 Incubate the mixtures 10 min at 378C

Starting the reaction with:

XO irr

200 1 1 10 b 1200 50 – – –

100

a

Final concentrations in the reaction mixture. Preincubation of this eluate 20 min at 378C to allow XO rev to XD reduction. Calculation of XD from: Total XOR–Total XO. Calculation of XO rev from: Total XO–XO irr . b

when uric acid formation was monitored by both spectrophotometry and chromatography. On the other hand, the same experiment allowed us to rule out that xanthine consumption was the cause of the decrease of XOR activity over time. Although inhibition of XO by its products has been reported [20,22,30], our results show that the absence of linearity cannot be attributed to this cause. We found that peroxide 50 mmol / l inhibited XO (Fig. 3a), but the initial slope of the sample containing 50 mmol / l peroxide was higher than the final slope of the non-added sample, where less than 12 mmol / l of this product was formed. Therefore, peroxide could not be the agent producing the decrease of the reaction rate. Moreover, the elimination of peroxide (with catalase), superoxide (with SOD) and both products from the reaction mixture increased the rates of urate production at all reaction times (thus confirming XOR inhibition by these products). However, this inhibitory effect does not explain the decreases in the rate because such decreases were also observed when SOD and catalase were present. Inhibition by uric cannot either be the cause of the

decreasing rate; the levels of uric acid formed in the reactions of the present work did not seem to be inhibitory. In fact, previously reported inhibition of XOR by uric acid was observed at higher concentrations [20,30]. Some authors assume a linear behaviour of the reaction during 20 min or more time, depending on the specific work, but none of them show results that demonstrate reaction linearity [2,36]. Independently of the causes that give rise to the non-linear oxidation of xanthine catalysed by the XOR forms, Table 2 shows the aftermath of XOR being measured by endpoint methods. Horizontal examination of the upper part of the table demonstrates that measurements made at 120 min of incubation resulted in activities more than 80% lower than those measured at 10 min. In fact, the reported levels of XO in normal peripheral human plasma are very low (range 0–17 mU / l or 1.7 mU / g prot) when measured by endpoint methods with one h or longer incubation times [10,11,24]. However, those obtained with follow-up methods, probably with short incubation times, range from 500 to 4200 mU / l [9,12,25–27]. Similarly, Tan et al. [20], using an endpoint method

124

R. Martı´ et al. / Clinica Chimica Acta 303 (2001) 117 – 125

(60 min incubation at 378C) and HPLC measurement of uric acid in cord venous plasma, obtained XOR values of 2.5 mU / l; the corresponding values found by Supnet et al. [28], with a spectrophotometric method and short incubation time, were 1000–2000 mU / l, even though incubations were carried out at 258C. The lower part of this table also evidences that the ratio XO /(XD1XO) is underestimated and the ratio XO irr /(XD1XO) is overestimated at long incubation times. Moreover, mercaptoethanol or other thiol group reducing agents are frequently used to measure XO irr but not XD1XO [2,6]. We found that this reagent also influences the reaction kinetics of XD1 XO, thereby changing the ratios of XOR forms when they are assessed at long incubation times. The results of the present work and the differences in values reported in plasma suggest that part of the discrepancies regarding the importance of XOR in ischaemia-reperfusion processes and the relevance of XOR in several syndromes, are due to methodological differences. From our results, we believe that prolonged incubation times should be avoided if results are to be interpreted as activities, and that the same conditions and times of incubation should be used to compare results. We conclude that for accurate XOR measurement, NADH, peroxide and superoxide must be eliminated from the reaction mixture. Therefore we recommend the assay conditions indicated in Table 3. A short incubation time (10 min) is advisable to obtain higher reaction rates and to avoid thiol reagent addition when XO1XD is measured to estimate the XO irr /(XO1XD) ratio. Short incubation will make it necessary to quantify uric acid by methods with higher sensitivity than spectrophotometry, particularly in samples with low XOR activities, such as plasma. In this case the reaction can be stopped by adding perchloric acid 1 mol / l (final concentration) followed by centrifugation and quantification of uric acid in the supernatant.

Acknowledgements ´ Garcıa, ´ Dolors Palau and M. We thank Sofıa ´ Angeles Vega for their expert technical contribution

and Celine Cavallo for her excellent linguistic advice.

References [1] Della Corte E, Stirpe F. The regulation of rat liver xanthine oxidase. Involvement of thiol groups in the conversion of the enzyme activity from dehydrogenase (type D) into oxidase (type O) and purification of the enzyme. Biochem J 1972;126:739–45. [2] Kooij A, Schiller HJ, Schijns M, Van Noorden CJF, Frederiks WM. Conversion of xanthine dehydrogenase into xanthine oxidase in rat liver and plasma at the onset of reperfusion after ischemia. Hepatology 1994;19:1488–95. [3] Brass CA. Xanthine oxidase and reperfussion injury: a major player or minor irritant? (letter). Hepatology 1995;21:1757– 8. [4] Kooij A, Frederiks WM, Van Noorden CJK. Xanthine oxidase and reperfusion injury: a major player or minor irritant? (reply). Hepatology 1995;21:1758–17560. [5] McCord JM. Oxygen derived free radicals in postischemic tissue injury. N Engl J Med 1985;312:159–63. [6] Brass CA, Narciso J, Gollan JL. Enhanced activity of the free radical producing enzyme xanthine oxidase in hypoxic rat liver. Regulation and pathophysiologic significance. J Clin Invest 1991;87:424–31. ¨ [7] Angermuller S, Schunk M, Kusterer K. Alteration of xanthine oxidase activity in sinusoidal endothelial cells and morphological changes of kupffer cells in hypoxic and reoxygenated rat liver. Hepatology 1995;21:1594–601. ¨ [8] Muller MJ, Vollmar B, Friedl HP, Menger MD. Xanthine oxidase and superoxide radicals in portal triad crossclamping-induced microvascular reperfusion injury of the liver. Free Radic Biol Med 1996;21:189–97. [9] Friedl HP, Smith DJ, Till GO, Thomson PD, Louis DS, Ward PA. Ischemia-reperfusion in humans. Appearance of xanthine oxidase activity. Am J Pathol 1990;136:491–5. [10] Giler SH, Sperling O, Brosh S, Urca de Vries A. Serum xanthine oxidase in jaundice. Clin Chim Acta 1975;63:37– 40. [11] Grum CM, Ragsdale RA, Ketai LH, Simon RH. Plasma xanthine oxidase in patients with adult respiratory distress syndrome. J Crit Care 1987;2:22–6. [12] Miesel R, Zuber M. Elevated levels of xanthine oxidase in serum of patients with inflammatory and autoimmune rheumatic diseases. Inflammation 1993;17:551–61. [13] Murell D. A radical proposal for the pathogenesis of scleroderma. J Am Acad Dermatol 1993;28:78–85. [14] Newaz MA, Adeeb NNN, Muslim N, Razak TA, Htut NN. Uric acid, xanthine oxidase and other risk factors of hypertension in normotensive subjects. Clin Exp Hypertens 1996;18:1035–50. ´ L, Stıpek ´ [15] Zima T, Novak S. Plasma xanthine oxidase level and alcohol administration. Alcohol Alcohol 1993;28:693–4.

R. Martı´ et al. / Clinica Chimica Acta 303 (2001) 117 – 125 [16] Beckman JS, Parks DA, Pearson JD, Marshall PA, Freeman BA. A sensitive fluorometric assay for measuring xanthine dehydrogenase and oxidase in tissues. Free Rad Biol Med 1989;6:607–15. [17] Bailey IA, Blackwell CP, Woodward B. Chemiluminescence measurement of xanthine oxidase and xanthine dehydrogenase activity in four types of cardiovascular cell. Basic Res Cardiol 1988;83:392–400. [18] Phan SH, Gannon DE, Varani J, Ryan US, Ward PA. Xanthine oxidase activity in rat pulmonary artery endothelial cells and its alteration by activated neutrophils. Am J Pathol 1989;134:1201–11. [19] Terada LS, Leff JA, Repine JE. Measurement of xanthine oxidase in biological tissues. In: Methods in Enzymology, Vol. 186, New York: Academic Press, 1990, pp. 651–6. [20] Tan S, Radi R, Gaudier F, Evans RA, Rivera A, Kirk KA, Parks DA. Physiologic levels of uric acid inhibit xanthine oxidase in human plasma. Pediatr Res 1993;34:303–7. [21] Jaansen M, Tavenier M, Koster JF, de Jong JW. In vitro and ex vivo xanthine oxidoreductase activity in rat and guinea pig hearts using hypoxanthine or xanthine as substrate. Biochim Biophys Acta 1993;1156:307–12. [22] Terada LS, Beehler CJ, Banderjee A, Brown JM, Grosso MA, Harken AH, McCord JM, Repine JE. Hyperoxia and self- or neutrophil-generated O 2 metabolites inactivate xanthine oxidase. J Appl Physiol 1988;65:2349–53. [23] Terada LS, Dormish JJ, Shanley PF, Leff JA, Anderson BO, Repine JE. Circulating xanthine oxidase mediates lung neutrophil sequestration after intestinal ischemia-reperfusion. Am J Physiol 1992;263:L394–401. [24] Tan S, Gelman S, Wheat JK, Parks DA. Circulating xanthine oxidase in human ischemia reperfusion. South Med J 1995;88:479–82. [25] Castelli P, Condemi AM, Brambillasca C, Fundaro` P, Botta M, Lemma M, Vanelli P, Santolli C, Gatti S, Riva E. Improvement of cardiac function by allopurinol in patients undergoing cardiac surgery. J Cardiovasc Pharmacol 1995;25:119–25. [26] Grattagliano I, Vendemiale G, Sabba` C, Buonamico P, Altomare E. Oxidation of circulating proteins in alcoholics: role of acetaldehyde and xanthine oxidase. J Hepatol 1996;25:28–36.

125

[27] Mathru M, Dries DJ, Barnes L, Tonino P, Sukhani R, Rooney MW. Tourniquet-induced exsanguination in patients requiring lower limb surgery. Anesthesiology 1996;84:14– 22. [28] Supnet MC, David-Cu R, Walther FJ. Plasma xanthine oxidase activity and lipid hydroperoxide levels in preterm infants. Pediatr Res 1994;36:283–7. [29] Battelli MG, Abbondanza A, Musiani S, Buonamici L, Strocchi P, Tazzari PL, Gramantieri L, Stirpe F. Determination of xanthine oxidase in human serum by a competitive enzyme-linked immunosorbent assay (ELISA). Clin Chim Acta 1999;281:147–58. [30] Radi R, Tan S, Prodanov E, Evans RA, Parks DA. Inhibition of xanthine oxidase by uric acid and its influence on superoxide radical. Biochim Biophys Acta 1992;1122:178– 82. [31] Nishino T, Nakanishi S, Okamoto K, Mizushima J, Hori H, Iwasaki T, Nishino T, Ichimori K, Nakazawa H. Conversion of xanthine dehydrogenase into oxidase and its role in reperfusion injury. Biochem Soc Trans 1997;25:783–6. [32] Harrison R. Human xanthine oxidoreductase: in search of a function. Biochem Soc Trans 1997;25:786–91. ` [33] Chazouilleres O, Calmus Y, Vaubourdolle M, Ballet F. Preservation-induced liver injury. Clinical aspects, mechanisms and therapeutic approaches (review). J Hepatol 1993;18:123–34. [34] Kooij A. A re-evaluation of the tissue distribution and physiology of xanthine oxidoreductase (review). Histochem J 1994;26:889–915. [35] Rinaldo JE, Clark M, Parinello J, Shepherd V. Nitric oxide inactives xanthine dehydrogenase and xanthine oxidase in interferon-gamma-stimulated macrophages. Am J Respir Cell Mol Biol 1994;11:625–30. [36] Hofstee BHJ. On the mechanism of inhibition of xanthine oxidase by the substrate xanthine. J Biol Chem 1955;216:235–44. [37] Muraoka S. Studies on xanthine oxidase. II. Mechanism of substrate inhibition and its reversal by histamine. Biochim Biophys Acta 1963;73:27–38. [38] Hausladen A, Fridovich I. Excess substrate inhibition of xanthine oxidase: a re-examination. Arch Biochem Biophys 1993;306:415–9.