A literature survey on animal peroxisomes reveals that most studies are confined to laboratory mammals and humans. The scarcity of knowledge on other vertebrate and invertebrate species, and the need for more comparative studies has been recently emphasised [1–3]. Nevertheless, it is known that fish peroxisomes have enzymatic systems similar to those reported in mammals. Catalase, D-amino acid oxidase, urate oxidase, allantoinase, allantoicase, L-α-hydroxyacid oxidase, alanine:glyoxylate aminotransferase, fatty acyl-CoA oxidase, enoyl-CoA hydratase and carnitine acetyltransferase are among the enzymes that were detected in fish peroxisomes [3–10]. It is also known that peroxisomal metabolism is lower in fish than in mammals. For example, in the liver of rainbow trout (Onchorynchus mykiss) and Japanese medaka (Oryzias latipes) peroxisomal enzyme activities are at least four folds lower than in rodent liver. These results were either assigned to a reduced number of peroxisomes per hepatocyte, or a lower amount of peroxisomal enzymes, or both [9].

The research in fish revealed that environmental factors like water temperature, salinity, season and feeding habits, exerted changes in peroxisomal enzyme activities that, additionally, vary greatly among species [1]. It was also discovered that season, age and gender affect the morphology of fish liver peroxisomes. In the gray mullet (Mugil cephalus) hepatic peroxisomes tend to be larger in summer and in aged animals [11]. Moreover, in the liver of brown trout (Salmo trutta) both the individual dimensions of peroxisomes and their total volume per hepatocyte, but not their number, change during the annual breeding cycle in both genders [12]. Yet, in the latter species hepatic peroxisomes are significantly smaller in females than in males during the breeding season [12]. These observations raised the question about the kind of biochemical changes that may be associated with those morphological variations detected in fish liver peroxisomes. To measure some peroxisomal enzyme activities in brown trout liver, a method originally applied for mammalian peroxisomal hydrogen peroxide producing oxidases [13] was tested for the first time in fish. Catalase, a well-known peroxisomal marker enzyme [14], was also included in this work. Since the temperature is an important factor that affects enzyme activities, its influence on several trout peroxisomal enzymes was investigated. This work also aimed at studying the distribution of several peroxisomal enzymes in crude cell fractions. To control the amount of cross contamination in the crude peroxisomal fraction by other organelles, standard spectrophotometric procedures were used in order to measure the activity of marker enzymes of mitochondria (succinate dehydrogenase), lysosomes (aryl sulphatase) and microsomes (NADPH cytochrome c reductase). Finally, this paper reports the first data about the activities of peroxisomal enzymes in the liver of brown trout.

One of the purposes of this study was to test in brown trout liver the spectrophotometric method originally used by Cablé et al. [13] for measurement of peroxisomal oxidase activities in mammalian tissues. However, some modifications were introduced and the method was extended to urate and glycolate oxidases. To calculate the amounts of H₂O₂ produced by these enzymes, instead of using the extinction coefficient previously published [13], a calibration line was made with known amounts of H₂O₂. Others also made similar calibration lines, but when using luminometric methods [15] and fluorometric techniques [16].

Since the physiological temperature is much lower in brown trout than in mammals, temperature influence in the activity of five peroxisomal enzymes was investigated. The results showed that the activities of these enzymes rose in a linear mode from 10 to 37°C, but while the activity of hydrogen peroxide producing oxidases were strongly influenced by temperature, catalase activity was only slight affected. This last observation is in total accordance with previous data regarding catalase activity in mammals [14]. Therefore, these enzymatic activities can be measured at whatever temperature within the above-referred range. However, at 37°C activities are higher and therefore easier to measure than at the physiological temperatures of brown trout (10 – 15°C). In fact, other authors have used a wide range of temperatures in studies involving fish peroxisomal enzymes. For example, in carp (Cyprinus carpio) catalase activity was determined at 25°C whereas urate oxidase, fatty acyl-CoA oxidase and glycolate oxidase were measured at 37°C [4, 5, 17] and in rainbow trout (O. mykiss) catalase activity was determined on ice whereas fatty acyl-CoA oxidase was measured at 15°C [18].

Some similarities are evident when crude cell fractions of rat liver [19] are compared with those obtained from brown trout liver. In both species relative specific activities of catalase and hydrogen peroxide producing oxidases are higher in the D fraction obtained using very similar centrifugation procedures. Moreover, catalase and glycolate oxidase (or L-α-hydroxyacid oxidase) present a high percentage distribution in the cytosolic fraction of rat and brown trout, has expected for soluble matrix enzymes released from peroxisomes due to membrane damage during homogenisation. On the other hand, urate oxidase is not detectable in the supernatant of rat liver [19] while in brown trout about 50% of urate oxidase activity can be found in the supernatant. This discrepancy is due to a fundamental difference between brown trout and mammalian urate oxidase, which behaves exactly like a soluble matrix enzyme in the former but is a crystallised core enzyme in the latter. Indeed cores were neither found in brown trout liver peroxisomes [12] or in other fish species [3]. Although urate oxidase was described as a membrane-linked enzyme in carp liver peroxisomes [17] in other fish species was considered a soluble matrix enzyme [6] as it seems to be in brown trout liver peroxisomes.

Differences between rat and brown trout crude liver fractions are also evident in the microsomal fraction, in which the percentage distributions of peroxisomal enzyme activities are higher in rat than in brown trout [19].

The absence of correlations between D fractions and total liver homogenates showed that enzyme activities in peroxisomal enriched fractions are not proportional to the activities in homogenates. The contamination of crude peroxisomal fractions with other organelles and the substantial loss of soluble matrix enzymes can explain this result. Because of these facts, and knowing that in rainbow trout the gradient centrifugation of the crude peroxisomal fraction produced a further decrease in the activities of some oxidases [18], in future seasonal and toxicological studies about brown trout peroxisomes total homogenates will be used instead of cell fractions. Moreover, variation coefficients suggest that enzyme activities have relevant individual variability in brown trout. Thus, very subtle differences due to experiments or natural events may pass unnoticed when working with a small number of animals.

In conclusion: 1) the spectrophotometric protocols used for measurement of mammalian peroxisomal enzymes can be successfully applied to brown trout; 2) the enzymatic activities of the peroxisomal enzymes studied by us can be correctly measured between 10° and 37°C, this because their activity rose in a linear mode with temperature, with catalase activity increasing only slightly; 3) for peroxisomal enzymes in brown trout liver homogenates there is a clear correlation between specific activity and activity per g of liver or per liver; 4) during preparation of cell fractions enriched with peroxisomes a significant amount of matrix enzymes are loosed, and the remaining enzyme activities are not correlated with the activities in liver homogenates.