Can physical stress be measured in urine using the parameter antioxidative potential?

Although regular exercise is known to promote health, it is also well known that competetive sports can lead to an increase of free radical production, and thus to a drop in antioxidative potential. Thus, the present study examined the effect of competetive sports on the antioxidative potential (AOP). Using chemoluminescence, the AOP was measured in the spontaneous urine of leisure and semi-professional athletes during a training camp. Further, the parameters creatinin and uric acid were measured. It was shown that physical stress led to a drop in the antioxidant potential of up to approximately 50%. To compensate for this decline, special antioxidant food is recommended.

Introduction

The antioxidant system is a mechanism which protects the body’s cells against endogenous and exogenous free radicals, and establishes an equilibrium that allows endogenous radicals to perform necessary cell functions, while preventing damaging effects by exogenous radicals [1]. This results in a decrease of toxic radical effects and decreased damage to the organism. Substances which react with radicals and neutralize their radical nature are termed antioxidants [2]. The antioxidative metabolic system is complex and connected to many other metabolic systems of the body. A developing imbalance can cause diseases or aggravate existing ones. It has been proven that the activity of the immune system is strongly connected to the antioxidant metabolic system [3], [4], [5]. Similarly, there is a broad consensus that physical stress leads to radical formation (among other things) [6], so that the formation of free radicals is much higher during physical stress than during resting (basal) phases [7]. Under training conditions, up to 5% of the oxygen molecules taken up are converted into superoxide radicals [8]. In addition, a local inflammatory reaction in the muscle tissue can lead to increased formation of free radicals. This is caused by the infiltration of leukocytes into the stress-inflamed muscle tissue [9], where the leukocytes produce free radicals, which function as second messengers [10]. The consequence of radical formation is decreased antioxidative potential.

In vivo detection of radicals is hardly possible, since they are extremely reactive and short-lived. Nevertheless, different biological markers exist which are the reaction products of radicals, e.g., oxidized guanosine (8-oxo-dG), is a marker for radical-damaged DNA, and is excreted in the urine; exhaled ethane is a marker for the lipoperoxidation of n-3 fatty acids by radicals [11], [12]; and the β-carotin content is a measure of the antioxidative potential of the skin [13], [14], [15].

Substances that can react with radicals by interrupting the radikal chain reaction have a reductive (an antioxidative) character. The sum of the antioxidative substances available in a system is called the AOP (antioxidative potential), a term which was introduced 1989 by Popov and Lewin [16]. The AOP is determined by a set of compounds which are present in different concentrations [16], [17]. It is expedient to differentiate between the antioxidative capacity of water-soluble (ACW) and fat-soluble components (antioxidative capacity of lipid soluble components, ACL). The ACW value is usually given in ascorbic acid equivalents, and the ACL usually in Trolox equivalents.

AOP is represented by different substances in different biological systems. In humans, blood, plasma, uric acid, ascorbic acid, bilirubin-bound albumin, and ceruloplasmin are regarded as the main components of the ACW [18]. The biological value of these compounds can differ widely. In plasma, uric acid is the chief component of the AOP. A further important component of the AOP is ascorbic acid. If the AOP is measured as a sum parameter, for instance, with chemoluminescence, a number of unknown compounds which likewise work as radical inhibitors will also be measured.

As opposed to normal, recreational sports or exercise, is competetive sports are a form of stress for the human body [19]. In psychology, stress is defined as a continuous adverse situation, the avoidance of which appears subjectively important, but the individual lacks the certainty of being able to terminate it [20]. One must differentiate between controllable positive stress and uncontrollable negative stress. Medically speaking, stress is a factor which endangers an individual’s health. If the sympathetic nervous system is activated by stress, the body increases its release of the stress hormones adrenalin and noradrenalin. Stress can also exert negative effects via the hormone cortisol. In addition, stress leads to the generation of free radicals [21], [22]. The clinical chemical measurement of hormones is complex and the correlation with total stress is frequently questionable. The parameter AOP could represent an an alternative to this, since it is easily detectable as a sum parameter and provides a measure of the radical load. Therefore, the present study examined whether the AOP is suitable as a screening parameter to measure physical stress.

Materials and methods

Two separate studies were conducted. Study I took place during a recreational Tae kwondo training camp. 17 subjects (14 men, 3 women) participated. For 8 days, the participants completed two 1.5-hour training units per day. Two urine samples per participant and day were collected, one in the morning before training and one in the evening after the second training unit. The samples were coded and frozen immediately at –20°C. In addition, the body weight, blood pressure and pulse of each subject were recorded. In the urine samples, the parameters AOPtotal, uric-acid-independent AOP (AOPU) were determined, as well as the uric acid and creatinine content. Before analysis, the samples were allowed to thaw overnight in the refrigerator and were centrifuged after mixing briefly, in order to separate suspended matter and sediment.

The participants in study II were five male Taekwondo athletes (Taekwondo) preparing for a championship. The training level of these athletes was higher than that of the participants in study I and the load much more intensive. In contrast to the participants in study I, all Taekwondo were uniformly trained and received exactly the same diet. The study lasted 5 days. All parameters were measured and substances sampled using the same procedures as in study I.

AOPtotal and AOPU were determined by means of chemoluminescence using a Photochem® device (Analytik Jena, Jena, Germany). The software version 5.1.12 was used. Photometric measurements were taken with the spectrometer Ultrospec 4000 (Pharmacia Biotech, Sweden). The chemicals employed had p.a quality? unless noted otherwise. For all investigations, high-purity water was used (Reinstwasser-System, SG Wasseraufbereitung und Regenerierstation GmbH, Barsbüttel, Germany). The uricase used (Serva, Heidelberg, Germany) had an activity of 4.43 U/mg. The reaction solution made of it was good for 7 h at 4°C.

For the determination of the AOPtotal and the AOPU, the ACW kit (Analytik Jena, Jena) was used. The uric acid was determined with the “5+1 fluid uric acid kit” (MTI DIAGNOSTICs), and creatinine with the DRI® Creatinine Detect® test (microgenetics GmbH).

To determine the AOPtotal, the reagents contained in the kit – R 1 (diluent), R 2 (buffer pH 14), R 3 (photosensitizer) and R4 (ascorbic acid calibration solution) – were used in the quantities indicated in Table 1 (Tab. 1).

Table 1

Determination of AOPtotal and AOPU with the ACW kit (Analytik Jena)

The centrifuged urine diluted for the ACW x and 1:10 for the AOPU. The measurement temperature was 4°C. For AOPU determination, 10 µl uricase solution (8 U/10µl) was added to 10 µl of the centrifuged and diluted urine sample (1:10) (Table 1 (Tab. 1)). The resulting solution was then incubated for 5 min at 37°C. Subsequently, the reagents were added according as presented in Table 1 (Tab. 1). For measurement, it is crucial that R1 and R2 be at ambient temperature. On the other hand, the photosensitizer (R3) and the ascorbic acid solution (R4) must be kept in a separate box at 4°C until the moment it is added to the reaction mixture [23].

The ascorbic acid equivalents are calculated using the formula:

The statistical analysis was performed with the program PASW Statistics 18® (IBM). The parameters were intended for the descriptive statistics, and Pearson’s correlation was calculated and tested for significance.

Results

Study I

In the urine samples, the parameters uric acid, AOPtotal and AOPU were determined and standardized to the creatinine excretion. The results are summarized in Table 2 (Tab. 2).

Table 2

Descriptive statistics of the results of study I (mg/g creatinine)

In order draw conclusions about the relation between the parameters AOPtotal and AOPU, the proportion of uric acid (as the difference between AOPtotal and AOPU) in the parameter AOPtotal was determined (Table 3 (Tab. 3)).

Table 3

Percent of uric acid in the parameter AOPtotal

For each participant, graphs were plotted of the uric acid level vs. AOPtotal and AOPU. Odd numbers designate the morning values and even numbers the evening values.

For all participants, the uric acid level and the AOPtotal correlated highly significantly (r=0.42, p=0.001, Figure 1 (Fig. 1)). One example of is shown in Figure 2 (Fig. 2).

Figure 1

Correlation between uric acid and AOPtotal

Figure 2

Uric acid levels, total antioxidative potential (AOPtotal) and the uric acid-independent antioxidative potential (values standardized to creatinine excretion values) in participant 12 over time

The AOPU values were clearly below the AOPtotal values. Two examples are represented in Figure 3 (Fig. 3) and Figure 4 (Fig. 4). Participant 12 (21-year-old male) was selected, because he corresponded to the typical course in study II. The other participants in study I showed very different courses. This was apparently due to various causes, such as obesity, diet, different training load and different age (5 participants under 17 years old).

Figure 3

Uric-acid-independent antioxidative potential in participant 12 over time (standardized to creatinine excretion values)

Figure 4

Course of the uric-acid-independent antioxidative potential (AOPU) of the study manager (standardized to creatinine excretion values)

Figure 3 (Fig. 3) (participant 12) clearly shows that the AOPU decay to the end of the second training. After adaptation to the training load, the AOPU values rise somewhat at first, then drop again, and finally remain low with small fluctuations. Frequently, the values are higher in the morning than in the evening (Figure 3 (Fig. 3)).

In the manager (male, 42 years, only organizing, no sportive activities), the AOPU exhibited different course. Up to the 5th day the values rise; afterwards, they return to approximately the initial value. In the evening, the values are frequently higher than in the morning (Figure 4 (Fig. 4)).

Study II

In this study, a similar reaction pattern was present in four of five participants, so that the values were averaged (Figure 5 (Fig. 5)). A curve similar to that of Figure 3 (Fig. 3) results. With increasing training duration, the AOPU decreases, although starting from measuring point 4, higher values of AOPU were found in the morning than in the evening. The standard deviation shows a clear tendency to decrease from the beginning of training to the end (Figure 5 (Fig. 5)).

Figure 5

Average AOPU values of 4 competetive athletes in study II (standardized to creatinine excretion values)

The athlete in whom the AOPU deviated from that of the other 4 study-II participants (students orapprentices) was employed. His initial AOPU value was at about the same level of the others’ initial values, but then dropped drastically to almost zero. Despite a short recovery phase, a general decline was observed (Figure 6 (Fig. 6)).

Figure 6

Deviating course of the participant’s (study II) AOPU

Discussion

This study aimed to determine whether a connection exists between physical stress and change of the antioxidative potential (AOP). According to the study design, only spontaneous urine was available for analysis. Analyzing 24-h urine collection would have been preferable, but was logistically impossible. An evaluation of parameters in spontaneous urine is complicated, since this can be more or less diluted. Particularly after physical stress, as in the present study, more concentrated urine is to be expected due to sweating. In order to obviate this problem, the creatinine level in urine is also measured. Provided that the creatinine excretion is relatively constant over 24 h, the mean creatinine excretion can serve as a reference for the measured valuesexcretion. Generally, an average creatinine excretionexcretion of 1 g/24 h is assumed. Similarly, reference to creatinine is common in screening for iodine deficiency, and supplies comparable results to those of 24-h urine collection [24]. Thus, all measured values were standardized to the creatinin excretion in this study. It must be borne in mind that age, sex, muscle mass, and diet all have an influence on creatinine excretion [25], and that the reference to creatinine excretion is only an approximation [26]. With children under 6–8 years of age, this method is not applicable, since less creatinine is excreted due to the small muscle mass [27]. The present study, however, did not include participants of this age group.

High scatter was found in particular for the parameters uric acid and AOPtotal in study I (Table 2 (Tab. 2)), i.e., there are large individual differences. The uric acid contents vary between 2.24 and 1179.5 mg/g creatinine with an average value of 203.5 mg/g creatinine. The AOPtotal values range between 19.1 and 4993.6, with an average value of 497.2 mg/g creatinine. The average AOPU value is with 85.4 mg/g creatinine about 17.2% of the AOPtotal and ranges from 4.2 to 588.2 mg/g creatinine (Table 2 (Tab. 2)).

A series of studies [28], [29], [30], [31] show that uric acid constitutes a substantial proportion of the AOPtotal. However, data are lacking to date on the relationship AOPU/AOPtotal and/or on the proportion of the AOPtotal in urine samples that is due to uric acid. Comparisons between the two parameters are well-known only from serum and/or blood plasma. Here, the proportions range from 33 to 60% (Table 4 (Tab. 4)) [28], [29], [30], [31].

Table 4

Percent of different compounds in the AOP (composition by [28])

When comparing the uric acid percentages, it must be mentioned that the values were obtained with different methods. The reason for the high uric acid content found with the Total Radical Trapping Antioxidant Parameter and Ferric Reducing Ability of Plasma (FRAP) method is that neither method differentiates between ACW and ACL, but rather determine a total value [28], [29], [30], [31]. In contrast, the Trolox Equivalent Antioxidant Capacity (TEAC) only measured the ACL. This investigation also refers to serum. The lower uric acid content found by Miller et al. resulted from the low fat-solubility of uric acid [32], but the proportion of proteins was higher due to their higher liposolubility.

In our group of participants, the AOPU accounts for approx. 25% of the AOPtotal. That means that the uric acid in urine samples constitutes about 75% of the AOP (Table 3 (Tab. 3)). The method used here is comparable to the TEAC method. However, differences exist in the determined parameter (ACW in our study, ACL in the TEAC method). One can assume, however, that the ACW is the crucial parameter for urine samples.

Since the AOPtotal reflects the uric acid level to an extent of only 77% with highly significant correlation (Figure 1 (Fig. 1)), the informative value of this parameter (AOPtotal) for medical studies is limited. The parallel courses of the curves for uric acid and AOPtotal demonstrate that the parameter AOPtotal in urine samples essentially reflects the fluctuations of the uric acid concentration (Figure 1 (Fig. 1) and Figure 2 (Fig. 2)). Due to the dominating influence of uric acid, the parameter AOPtotal loses considerable sensitivity, since uric acid obscures the influence of those compounds with antioxidative potential that are present at lower concentrations. Therefore, the determination of the AOPU appears more meaningful. Figure 2 (Fig. 2) clearly shows that this parameter is some orders of magnitude smaller than the AOPtotal. However, the individual differences were also considerable with this parameter (Table 2 (Tab. 2)). The statistical evaluation of all 17 participants – an epidemiological approach as in screening for iodine deficiency – proved to provide little useful information, since individual reaction patterns arose. The elimination of the data of persons under 17 years (consideration of the deviations in the creatinine metabolism of adolescents) likewise provided little distinguishing data, due to the heterogeneity of the participants, the different training conditions, differences in diet, and lower physical load/stress than in study II (data not shown). Rather, an evaluation of the AOP must be conducted on the individual level. This is described in the following 2 examples.

In athlete 12 (Figure 3 (Fig. 3)), the body made use of AOP reserves during the adaptation phase, which tended to decrease the AOPU. Afterwards, the AOP decreased by training. At night, regeneration occurs during the resting phase, so that the values are higher in the morning than in the evening. Furthermore, it was evident that a tendency existed for the AOPU to decrease during the training camp. Due to the physical load, the body relies on its antioxidative reserves, but regeneration is not 100%.

For the study manager, who did not participate actively in training but organized the training camp, the curve progressed differently (Figure 4 (Fig. 4)). The AOPU reached a maximum approximately halfway through the duration of the training camp. Contrary to athlete 12, the values were lower in the morning than in the evening. An explanation for this process might be the psychological stress of organizing each morning.

In study II, four of the athletes showed the same tendency as the participant 12 (study I). After an initial adaptation phase, in which the AOPU values dropped, the values became relatively stable, and were clearly lower at the end of the study than at the beginning. In three of the four participants, the morning values after the adjustment phase always exceeded the evening values. In the fourth participant, this was the case only on the last two days; prior to that, the morning values were lower than the evening ones. The higher morning values can be explained by the overnight recovery phase. The fourth participant organized the training camp and thus experienced high physical stress particularly at the beginning. The low morning values are thus possibly an expression of physical stress. As it happened, the one employed participant was also less physically fit. For the relatively high physical stress, the AOP reserves are apparently not sufficient.

In summary, it can be stated that the parameter AOPU is more sensitive than the parameter AOPtotal. For the parameter AOPU, individual curves are shown which indicate a correlation between psychological and physical stress, which were confirmed by the subjective stress level (data not shown). Under continuous physical stress, the AOPU decreases continuously. This is where preventive measures can be started, e.g., antioxidative diet (consumption of compounds with high AOP) and measures to counteract stress (e.g., sleep, recovery). Further studies are planned, in order to examine this connection, and simultaneously analyze individual interdependencies and introduce AOPU monitoring.