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Annals of Clinical & Laboratory Science 32:181-187 (2002)
© 2002 Association of Clinical Scientists

A New Method for Measuring Oxidative Stress in Claudicants during Strenuous Exercise using Free Radical Derivatives of Antipyrine as Indicators: a Pilot Study.

Stefan A. J. Coolen1, Marc H. W. A. Wijnen2, Jetse C. Reijenga1, Huib L. Vader1,3, Rudi M. H. Roumen1 and Fred A. Huf1
1 University of Technology Eindhoven, Laboratory of Instrumental Analysis, Eindhoven,
2 Department of Paediatric Surgery, University Hospital, Nijmegen, and
3 Department of Surgery, Saint Joseph Hospital, Veldhoven, The Netherlands

Address correspondence to Marc H. W. A. Wijnen, M.D., Department of Paediatric Surgery, University Hospital Nijmegen, P.O. Box 9101, 6500 HB, Nijmegen, The Netherlands; tel 31 49 754 2361; fax 31 24 361 3547; e-mail: mhwijnen{at}worldonline.nl.


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients with intermittent claudication disease suffer from temporary lack of oxygen in the legs, caused by narrowing of arteries, resulting in ischemia and followed by reperfusion. The degree of oxidative stress present in 16 patients during strenuous exercise was determined using several indicators. Two derivatives of an exogenous marker, antipyrine (AP), (ie, p-hydroxyantipyrine, p-APOH, and o-hydroxyantipyrine, o-APOH), were assayed in plasma using HPLC-tandem-MS. Plasma malondialdehyde (assayed as thiobarb-ituric acid reactive species, TBARS) was also determined. The branchial/ankle blood pressure index (b-a index) was used to assess the severity of intermittent claudication disease, and plasma lactate concentration was also measured as an indicator of the ischemic situation. Plasma TBARS level did not change significantly after exercise. During the ischemic situation as well as during reperfusion, both free radical derivatives of antipyrine increased significantly in plasma (p <0.01). Because p-APOH is also formed enzymatically in humans, the plasma ratio of o-APOH to AP appeared to be the most specific marker for oxidative stress in patients with intermittent claudication.

(received 13 October 2001; accepted 22 December 2002)

Keywords: antipyrine, intermittent claudication, malondialdehyde, lactate, HPLC-MS/MS


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Intermittent claudication is caused by a temporary lack of oxygen during exercise, usually in the lower extremities. This results in repetitive low-grade ischemia and calf or buttock pain that subsides when the exercise is stopped and the reperfusion begins. It has been demonstrated that, during reperfusion, production of oxygen derived radicals and neutrophil activation causes additional damage [1–3]. This results in local inflammation and also has systemic effects [4]. Some authors suggested that the systemic inflammatory response is responsible for additional atherosclerosis and may be one of the reasons for an increase in ischemic heart disease in claudicants [5–7]. There is evidence that an increase in scavenging activity is beneficial in claudicants and that scavengers can reduce the systemic and local inflammatory response during ischemia-reperfusion (I-R) in humans [8–15]. Modulation of the oxygen derived free radical (ODFR) activity in claudicants would therefore be an attractive therapeutic option in these patients.

In vivo measurement of ODFR activity is problematical owing to the extremely short half-lifetime of the radicals. Therefore, most studies use indirect markers to assess free radical damage [16]. In many studies, the products of lipid peroxidation are measured by the thiobarbituric acid (TBA) assay. Despite its simplicity, the results of the TBA test can lead to misinterpretation of the free radical damage. One reason is malonaldehyde’s instability. Free malondialdehyde produced in vivo is quickly metabolised (eg, in mitochondria by aldehyde dehydrogenase to yield carbon dioxide and acetic acid).

Another disadvantage of the TBA test is cross-reactivity. Metabolites such as biliverdin, acetaldehyde-sucrose, deoxyribose, deoxyglucose, methionine, glutamic acid, and others react with TBA to form chromogens identical to that produced when malondialdehyde reacts with TBA [17–19]. Measuring the fluorescence of the malondialdehyde-TBA adduct is one way to improve the specificity of the TBA reaction. At present, the best technique is to separate the malondialdehyde-TBA product from the interfering chromogens by HPLC.

Application of the TBA assay to body fluids also measures the malondialdehyde that is a breakdown product of the endoperoxides formed by cyclo-oxygenase during prostaglandin biosynthesis. The dietary intake of fatty acids influences the quantitation of malondialdehyde. Many researchers use the so-called thiobarbituric acid reactive species (TBARS) assay to assess free radical damage; TBARS includes all substances that react with TBA to form adducts with spectral absorption at 532 nm. MDA can be formed during the TBA reaction, owing to the degradation of fatty acids and other biomolecules that occurs at 95°C. Finally, the intensity of the color formed during the TBA reaction depends on the type and strength of the acid used. Since researchers use many different TBA assays, it is hard to compare analytical results from different laboratories.

Some prior studies in patients with intermittent claudication have focused on neutrophil activation during reperfusion and on the diminution of total antioxidant capacity that occurs after ischemia-reperfusion [3,20,21]. As an alternative, organ function tests have been used to assess the remote damage induced by lipid peroxidation. With respect to the kidney as a target organ, increased micro-albumin/creatinine ratio in the urine may be an indicator of endothelial damage caused by oxidative stress [2,21,22]. Another approach is to administer an exogenous marker that will react in vivo with free radicals, leading to metabolites that are a direct result of free radical attack.

The ideal way to determine radical damage with an exogenous marker is to measure a product that cannot be formed by other metabolic pathways. The determination of the plasma ratio of free radical product(s) of the exogenous marker versus the marker itself can be an indication of the free radical generation in different individuals. The exogenous marker must fulfill several conditions. The free radicals reactions should be of the competitive type; ie, the exogenous marker and endogenous molecules present at the site of radical formation should compete for reaction with the free radicals. Since the endogenous molecular targets are present in high concentrations, the exogenous marker must have a high reaction rate constant with free radicals, in the order of 109 – 1010 L· mol-1·sec-1. These are so-called diffusion-controlled reactions. Since the reaction rates depend not only on the reaction rate constants, but also on the concentration of the exogenous marker, this concentration must be relatively high (in the order of 0.1 to 1 mM).

Another requisite is non-toxicity of the xenobiotic to be administered as well as the free radical products of the xenobiotic. Also, side effects such as induction of enzymes by the administered xenobiotic should be avoided. The radical products that are formed should be stable so that they can be determined in biological fluids. The radical products should be hydrophilic enough to be excreted, in order to prevent their accumulation in the tissues. The xenobiotic and its radical products should not occur naturally in vivo and the metabolic pathways of the xenobiotic should be known.

Since the hydroxyl radical is one of the most aggressive radicals formed in vivo, most exogenous methods are based on the hydroxylation of phenyl-containing compounds. These compounds are highly susceptible to free radical attack. Reaction of phenilic compounds with hydroxyl radicals will lead to phenolic products. Xenobiotics such as benzoic acid, tryptophan, phenylalanine, salicylic acid, and nitrophenol have previously been used.

In the present study, antipyrine and its free radical derivatives were employed, since they fulfil the requirements for an exogenous marker. Previous experiments showed that three phenolic derivatives of antipyrine are formed when an aqueous solution of antipyrine is exposed to {gamma}-radiation, which induces the formation of the highly reactive hydroxyl radicals [23]. An increase in oxidative stress with free radical damage can lead to formation of phenolic derivatives of antipyrine.

Antipyrine (2,3-dimethyl-phenyl-3-pyrazolin-5-one) is an antipyretic drug that was developed at the end of the 19th century and was formerly used for its antipyretic and analgesic properties. The pathways of antipyrine metabolism are well known. During the past several decades, antipyrine has not been used clinically as an antipyretic, but it has been used to investigate hepatic cytochrome P450 activity [24,25]. Antipyrine has a high reaction rate constant with hydroxyl radicals (>1010 L·mol-1·s-1) [26]. Since a high dose of antipyrine can be administered (eg, 1 g, 4 times/day, po), it is possible to attain tissue concentrations of antipyrine that are sufficient to compete with endogenous biomolecules for reaction with hydroxyl radicals.

Antipyrine is a typical example of a low excretion, low clearance drug; the amount of antipyrine that can be presented to the liver is high in relation to the enzymatic activity of the hepatic P450 system. Moreover, the hepatic metabolism of antipyrine is relatively insensitive to fluctuations of hepatic blood flow. This last property of antipyrine makes it suitable for use in studies of free radical damage during exercise, since hepatic blood flow changes during strenuous exercise.

The present study was performed to assess whether or not the free radical derivatives of antipyrine can serve as markers of oxidative stress in claudicants during strenuous exercise. Using a group of patients with stable intermittent claudication, we induced oxidative stress during a standard treadmill walking test and assayed serially the plasma concentrations of lactate, thiobarbituric acid reactive species (TBARS), and free radical derivatives of antipyrine.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Antipyrine (99%) was from Genpharma. Methanol (HPLC grade) was from Biosolve (Valkenswaard, The Netherlands). Ammonium acetate (>99%) and acetic acid were from Merck (Darmstadt, Germany). Deionized water was from a MilliQ apparatus (Millipore, Bedford, MA, USA). All solid-phase experiments used C-18 columns (200 mg, 3 ml vol, Alltech Associates Inc., Deerfield, IL, USA).

Analytical procedures.  Isocratic separation and solid-phase extraction of the plasma phenolic derivatives of antipyrine were performed by HPLC-MS, as previously described [27,28].]

To assay thiobarbituric acid rd`Stive species (TBARS) in plasma, 100 µl of plasma and 900 µl of thiobarbituric acid solution (6 g/L in deionized water) were mixed in an Eppendorf cup and vortexed. The cup was placed in a waterbath at 95°C for 1 hr. The samples were cooled to room temperature and the absorption was measured at 532 nm, using a Spectronic 1001 UV-VIS spectrophotometer (Meyvis, Bergen op Zoom, The Netherlands).

Plasma lactate concentrations were measured with a Vitros 950 analyser using the Ektachem slide technique (Eastman Corp, Rochester, NY, USA).

Clinical procedures.  Sixteen patients with intermittent claudication were included in this pilot study. Their age, gender, and clinical characteristics are summarized in Table 1Go. All had been stable for >1 yr in respect to brachial/ankle blood pressure index (b-a index) and walking distance. In all of the patients, the b-a index was <0.8 and the decrease of b-a index after the standard walking test was >=0.3 in one or both legs. Patients with renal dysfunction and those who were unable to perform the walking test were excluded from the study.


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  		Table 1. Characteristics of the 16 patients with intermittent claudication who were selected as test subjects
 
The experimental protocol is outlined in Table 2Go. Upon each patient’s arrival in the vascular laboratory, a blood sample (T0) was withdrawn through an iv catheter in the medial cubital vein and placed on ice in EDTA containing tubes. The patient was given 15 mg/kg of antipyrine po. The patient was then seated for 1 hr to avoid ischemia prior to the treadmill test. A second blood sample (T1) was drawn immediately before the treadmill walking test. Then a standard walking test was performed (5 min, 3 km/hr, 8° incline). At the end of the walking test and at 5 and 50 min there-after, blood samples T3, T4, and T5 were collected. The patient’s b-a index was measured before the walking test and at 1 and 5 min after the test. The blood samples were centrifuged and were promptly analysed for plasma lactate and TBARS. Plasma samples for assay of antipyrine and its free radical derivatives were stored at -20°C until analysis.


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  		Table 2. Schedule of the experimental protocol
 
Statistics.  Data are presented as medians ± SE. The data were analyzed by the Wilcoxon signed-rank test using the exact calculation method. Since only a rise in oxidative stress parameters was expected, a one-sided test was performed. Statistical significance was set at p <0.05.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
As shown in Fig. 1Go, plasma lactate concentration increased significantly during the exercise period (T1 to T2, p = 0.002) consistent with the occurrence of hypoxemia. During the reperfusion period (T2 to T3), plasma lactate concentration did not change significantly. During the post-reperfusion period (T3 to T4), plasma lactate concentration decreased to the level observed prior to exercise (T1).



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  		Fig. 1. Plasma lactate concentrations during the treadmill walking test (median ± SE).

 
As shown in Fig. 2Go, the TBARS concentration in plasma decreased progressively during the period of observation. A slight rise in the concentration of TBARS was noted during the reperfusion period (T2 to T3), but this fluctuation was insignificant.



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  		Fig. 2. Plasma TBARS concentrations (expressed as malondialdehyde) during the walking test (median ± SE).

 
As shown in Fig. 3Go, the plasma antipyrine level increased until approximately the start of exercise. The slight increases of plasma antipyrine levels at 5 and 10 min after the exercise began were statistically insignificant.



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  		Fig. 3. Relative levels of plasma antipyrine during the treadmill walking test (median ± SE).

 
During and after exercise, significant changes were observed in the plasma concentrations of the free radical products of antipyrine (Fig. 4Go). The level of p-APOH increased during the exercise period (T1 to T2, p = 0.005) and the reperfusion period (T2 to T3) (p = 0.006). The plasma o-APOH level paralleled that of p-APOH, but the progressive increase of o-APOH level was significant (p = 0.03) only during the reperfusion period (T2 to T3).



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  		Fig. 4. Relative plasma levels of free radical products of antipyrine (p-hydroxyantipyrine, p-APOH, o-hydroxyantipyrine, o-APOH) during the treadmill walking test (median & SE).

 
As shown in Fig. 5Go, the plasma ratio of the level of hydroxylated product p-APOH to that of antipyrine increased during the exercise period (T1 to T2) and the reperfusion period (T2 to T3) and subsequently declined slightly (T3 to T4). For the o-APOH/AP ratio, there is a significant increase (p = 0.03) during the reperfusion period (T2 to T3) and a significant decrease (p = 0.04) during the post-exercise period (T3 to T4).



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  		Fig. 5. Ratios of plasma levels of free radical products (FRP) to antipyrine during the treadmill walking test. The FRPs are p-hydroxyantipyrine (p-APOH) and o-hydroxyantipyrine (o-APOH) (median & SE).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Claudicants suffer from chronic injury from ischemia-reperfusion. Every time they exercise ischemia is induced, followed by reperfusion. This not only damages the muscles and other tissues that are exposed to ischemia-reperfusion, but also causes a systemic response and remote organ impairment. If free radicals are responsible for the oxidative damage during reperfusion, administering anti-oxidants to patients could reduce this damage. This approach has been successfully applied in clinical trials. [29–32].

Increased plasma concentration of TBARS is generally regarded as an indicator of lipid peroxidation. As discussed in the introduction, there are several reasons why the plasma concentrations of TBARS can lead to erroneous interpretations of oxidative stress. Therefore, we evaluated a new method of detecting and quantifying oxidative stress, based on administering antipyrine to patients and measuring its free radical reaction products in plasma. The plasma levels of both of the free radical reaction products (o-APOH and p-APOH) changed significantly during a period of ischemia-reperfusion. However, they showed different response patterns.

The increase of plasma antipyrine before exercise and the fairly stable antipyrine concentration after exercise started are important, since the amounts of free radical products depend on the antipyrine concentration at the site of free radical formation, which is assumed to be reflected by the plasma concentration. The fact that p-APOH increased not only during the recovery period but also during exercise indicates that p-APOH is formed during both the reperfusion and ischemic periods. Other mechanisms than oxidative stress may also be involved in the production of p-APOH. Since p-APOH is also formed enzymatically (eg, in the liver by action of cytochrome P450), p-APOH is not by itself an ideal marker for oxidative stress. In contrast o-APOH concentrations only increased significantly during the reperfusion period. At this time the highest degree of oxidative stress is expected. Since o-APOH is evidently not formed by pathways other than free radical reactions, the plasma o-APOH level may be a specific marker for oxidative stress.

Slopes of the time-response curves for plasma levels of p-APOH and o-APOH (Fig. 4Go) were steeper during reperfusion than during the other periods of observation. Since the amounts of free radical products that are formed depends on the local concentration of antipyrine, it seems advisable to use the ratio of free radical products/antipyrine levels as a marker for oxidative stress. In the authors’ opinion, when antipyrine is used as the test agent, the o-APOH/AP ratio is probably the best indicator for oxidative stress during ischemia-reperfusion.

In conclusion, the results of this pilot study on the oxidative damage in patients suffering from intermittent claudication, as measured with several alternative markers, shows some promising features. The increase of plasma lactate indicates that oxidative stress occured in the claudicants during exercise. However, the plasma TBARS level did not increase significantly during the experiment. In respect to the free radical products of antipyrine, the plasma p-APOH level increased during exercise and reperfusion, while the plasma o-APOH level increased significantly only during the reperfusion.

The use of antipyrine free radical products, especially o-APOH, appears to be a good approach to measure oxidative stress and to assess the effects of antioxidant therapy in patients suffering from ischemia-reperfusion damage. Further research is needed to seek a quantitative relationship between the measured increase in antipyrine reaction products in plasma and the amounts of local and remote organ damage from oxidative stress.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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