Stability of Glutathione Peroxidase in Swine Plasma Samples ... - NCBI

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porcs, plasma, entreposage, activite enzymatique. INTRODUCTION. Since the discovery that selenium is a component of the enzyme glutathi- one peroxidase ...
Stability of Glutathione Peroxidase in Swine Plasma Samples under Various Storage Conditions W.R. Zhang, P.K. Ku, E.R. Miller and D.E. Ullrey*

ABSTRACT

posage a 40C ou a -15°C, de sorte qu'elle afficha une difference sensible The stability of plasma glutathione (P . 0,01) d'avec sa valeur initiale. peroxidase under different tempera- L'entreposage a -15°C, en presence tures (40C vs. -150C), various dura- d'azote, ralentit la vitesse de la tions of storage (0, 1, 2, 3, 7, 14, 28 and diminution de l'activite enzymatique, 56 d), and storage under inert gas mais il n'en maintint pas l'activite (nitrogen (N2)) vs air is described. The initiale. Pour l'obtention de valeurs glutathione peroxidase activity of absolues, on suggere de mesurer swine plasma decreased consistently l'activite de la glutathion-peroxydase with storage at either 40 C or -15° C 1- plasmatique porcine, immediatement 56 d after collection, and differed apres la centrifugation des echantil(P < 0.01) from the initial values. Ions de sang ou dans les 24 heures qui Storage under N2 at -15° C slowed the suivent l'entreposage a -150 C, en rate of enzyme activity decrease but presence d'azote. Si des differences did not maintain the initial activity. relatives de manipulation se revelent For absolute measurements, it is satisfaisantes en ce qui concerne suggested that swine plasma glutathi- l'activite de l'enzyme precite, on peut one peroxidase activity be measured alors la mesurer apries des periodes immediately after separation from the controlees d'entreposage. blood cells or be assayed within 24 h in plasma samples stored at -15°C with Mots cles: glutathion-peroxydase, air space displaced by N2. If relative porcs, plasma, entreposage, activite treatment differences in enzyme enzymatique. activity are satisfactory, then assays can be conducted after controlled periods of storage. INTRODUCTION

Key words: Glutathione peroxidase, swine, plasma, storage, enzyme activity. RESUME

Cet article decrit la stabilite de la glutathion-peroxydase plasmatique porcine, a 40 C et a -15° C, apres 0, un, deux, trois, sept, 14, 28 et 56 jours d'entreposage, en presence ou en l'absence d'azote. L'activite de l'enzyme precite diminua constamment, dans les 56 jours ulterieurs a l'entre-

Since the discovery that selenium is a component of the enzyme glutathione peroxidase (GSH-Px) in 1973 (1), much information has been published on the relationship between dietary selenium and the activity of this seleno-enzyme in blood and body organs of various species (2-4). There is a significant correlation between plasma GSH-Px and selenium (Se) status in swine (5). However, sometimes it is impossible to determine GSH-Px activity immediately when many samples are obtained at once, and the effects of storage conditions

on swine plasma GSH-Px activity are not well documented. Thus, the purposes of this study were: 1) to measure the effect of storage temperature and duration of storage on swine plasma GSHPx activity and 2) to determine the effect of N2 displacement of air prior to sample storage at -15° C on

plasma GSH-Px activity. In addition, the distribution of GSH-Px activity between swine plasma and erythrocytes (RBC) was

estimated. MATERIALS AND METHODS Blood samples (25 mL) were collected from the anterior vena cava of 32 cross-bred nursing pigs (23 to 25 days old, averaging 6.79 kg) from eight litters (two males and two females per litter) using preheparinized syringes and test tubes (heparin, 1000 USP units per mL, ICN, Nutritional Biochemical Co., Cleveland, Ohio). Their dams were fed a corn-soybean meal-based lactation diet supplemented with 0.1 ppm selenium from sodium selenite and all other known nutrients recommended by the National Research Council (6). The blood was kept at ambient temperature during transportation to the laboratory. Samples were centrifuged immediately after arrival at the laboratory for 20 minutes at 3000 x g at 40 C. Plasma was harvested and divided into two aliquots for GSH-Px activity analysis immediately after collection and after storage at either

*Visiting Scholar from Shanxi Agricultural University, People's Republic of China (Zhang) and Department of Animal Science, Michigan State University, East Lansing, Michigan 48824 (Ku, Miller and Ullrey). Reprint requests to Dr. P.K. Ku. Supported by the Michigan Agricultural Experiment Station, East Lansing, Michigan. Journal article no. 11692. Submitted August 15, 1985.

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Can J Vet Res 1986; 50: 390-392.

4°C or -15°C at the following times after collection: 1, 2, 3, 7, 14, 28 and 56 d. Plasma samples from seven of the above 32 pigs were also divided into aliquots to test the protective effect of N2 on plasma GSH-Px activity. These samples were stored at -15°C. Onehalf of them had the air space above the plasma displaced with N2 introduced in a gentle stream by a Pasteur disposable pipet before capping. The other one-half served as negative controls until GSH-Px activity was assayed at 7, 14 and 28 d after collection. Glutathione peroxidase activity was determined in triplicate by the coupled assay procedure (7, 8) using a Beckman DU/ Gilford spectrophotometer with an automatic cuvette positioner that was connected to a linear chart recorder (Model 250A, Linear Instrument Co., Irvine, California). Therefore, enzyme activity of each plasma sample could be monitored continuously and recorded. The assay mixture consisted of 0.1 mM NADPH, 2 mM reduced glutathione (GSH) and 1.0 unit glutathione reductase per mL in 100 mM phosphate buffer (pH 7.0) containing 3 mM EDTA and 1 mM sodium azide. The assay was performed by adding 25 ,uL of plasma and 115 ,uL of distilled, deionized water to 0.85 mL of the assay buffer mixture; then the mixture was incubated in the cuvette cell compartment to reach the reaction temperature (25° C). The reaction was initiated by adding 10 ,L of 0.12 mM hydrogen peroxide. Glutathione peroxidase activity was determined by measuring the rate of oxidation of NADPH (change of absorbance (AA) at 340 nm). To determine the nonenzymatic oxidation, a blank was prepared by adding 140 ,uL of distilled, deionized water instead of a plasma sample to the reagent-buffer mixture. The AA of the blank was subtracted from AA of the sample. The corrected A A was divided by the molar absorptivity (6.22 x 103) for NADPH at 340 nm, multiplied by two (two moles GSH oxidized per mole NADPH oxidized) and divided by 0.025 mL to express the GSH-Px activity as enzyme units (,umoles GSH-Px oxidized/min) per mL plasma (EU/mL). The distribution of GSH-Px activ-

TABLE I. Effect of Storage Time and Temperature on Glutathione Peroxidase (GSH-Px) Activity in Swine Plasma

GSH-Px Activity 40C -150C Percentage Percentage Time of 40C -150C Decrease Decrease Storage EU/mL EU/mLa Oh 0 0 0.56 ± 0.03b 0.56 ± 0.02 I d 0.46 ± 0.02 0.51 ± 0.03 17.8 8.9 2d 31.2 22.6c 0.39 ± 0.01 0.43 ± O.Olc 3d 0.37 ± 0.01 34.6 27.6c 0.41 ± 0.0IC 28.2c 7d 0.34±0.01 0.40±0.0Ic 40.1 14 d 42.6 33.3c 0.32 ± 0.02 0.37 ± O.Olc 28 d 58.5 48.5d 0.23 ± 0.01 0.29 ± 0.01d 56 d 0.19 ± 0.01 0.24 ± 0.01d 66.8 56.9d = aEU Enzyme units (I ,umol GSH oxidized/min) bValues are means ± standard error of plasma samples from 32 pigs cSignificantly (P < 0.05) differs from corresponding mean value of plasma stored at 40C dHighly significant (P < 0.01) difference from corresponding mean value of plasma stored at 40C

ity in the blood of swine was estimated on samples from 12 pigs. Whole blood GSH-Px activity was determined by the same procedure used for plasma, except 25 MAL whole blood lysate (0.1 mL whole blood in 2.0 mL distilled, deionized water) was used instead. Hematocrits were determined by the microprocedure of McGovern et al (9). Erythrocyte GSH-Px activity was calculated using the formula of Thompson et al (10). Details are presented in Table III, footnote a. Data obtained in this study were analyzed by one way analysis of variance, and statistical significance of differences between treatment means was determined by Student's t test.

plasma stored at the two temperatures for different durations are presented in Table I. Activity of GSH-Px was considerably decreased with increasing storage time. As compared to initial values, this difference was significant (P < 0.01) after 2 d of storage. The reduction of selenoenzyme activity at 40 C was much greater than in samples stored at -15°C. Glutathione peroxidase activity was reduced to only one third of the initial value after 56 d of storage at either 4° C or -15° C. Results of placing seven plasma samples under N2 on GSH-Px activity are shown in Table II. Nitrogentreated plasma samples had consistently higher GSH-Px activities than negative controls, and the differences RESULTS were statistically significant (P < 0.01). However, with plasma Glutathione peroxidase activities of samples stored under N2 at -15°C, the

TABLE II. Effect of Storage Time at -15° and Treatment with Nitogren on Plasma Glutathione Peroxidase (GSH-Px) Activity GSH-Px Activity

-150C

-150C + N,

-150C + N, Percentage Percentage Decrease Decrease EU/mL 0 0 0.43 ± 0.02 34.4 15.2c 0.28 + 0.01 0.36 + 0.02C 37.2 23.9c 0.27 ± 0.01 0.35 ± 0.0lc 25.5c 38.4 4 wk 0.26 + 0.01 0.32 ± O.Olc 'EU = Enzyme units (1I mol GSH oxidized/min) bValues are means ± standard error of plasma samples from seven pigs cHighly significant (P < 0.01) difference from corresponding mean value of plasma not stored under

Time of Storage 0h I wk 2 wk

-150C EU/mmLa 0.43 ± 0.02b

N2

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TABLE III. Glutatione Peroxidase (GSH-Px) Activity in Swine Plasma and Erythrocytes (RBC) GSH-Px (EU/mL) No. of Whole Animals Blood Plasma RBCa 12 ± 3.22 ± 0.19 0.30 0.01 7.66 ± 0.44 = aRBC (EU/mL-) (W)-(l -Hct)(P) Hct W = GSH-Px activity in whole blood Hct = Hematocrit expressed in decimal form P = GSH-Px activity in plasma

Ratio RBC: Plasma 26: 1

conducted immediately after separation from the blood cells; or alternatively, assays should be conducted within 24 h on samples stored at -15° C with the air space displaced by nitrogen. If only relative treatment differences are to be measured, assays still must be conducted under identical conditions and periods of storage.

REFERENCES GSH-Px activity was still appreciably decreased after 7 d of storage as compared to values determined immediately after plasma collection. No differences were noted for plasma GSH-Px activity between male and female pigs. Glutathione peroxidase activities in whole swine blood, plasma and estimated values in RBC are presented in Table III. The estimated GSH-Px activity in RBC was about 26 times higher than in plasma.

DISCUSSION

Information on plasma GSH-Px activity is a potentially useful index for the diagnosis of selenium status in swine. This study attempted to define the normal physiological value of swine plasma GSH-Px activity and its stability. Results from the present study show that there is significant change in enzyme activity of plasma at either 4°C or -15°C after 2 d of storage. This is different from the results of Sheppard and Miller (11) who reported that ovine plasma separated from blood on day 0 and stored at -15°C retained its GSH-Px activity for at least 2 wk. However, they observed that samples of the same plasma stored at 40C retained only 16% of their initial activity after 24 h. This is in general agreement with our study in which pig plasma stored at 40 C for 24 h had GSH-Px activity 82% that of initial values. The reason for the variation (82% vs 16%) between these studies is not clear, but may be a reflection of the species difference in GSH-Px distribution or type. It is known that the activity of the enzyme in ovine plasma is very low. The distribution of GSH-Px between RBC and plasma has been proposed to be 392

99:1 in sheep (11), and 49:1 in cattle (12,13). In swine, assuming all nonplasma GSH-Px is in RBC, the ratio between RBC and plasma GSHPx activity is about 26:1 (Table III), although leukocytes and platelets undoubtedly account for significant activity in the cellular fraction (2). Nevertheless, due to the high GSH-Px activity in RBC, blood samples showing evidence of hemolysis should not be used for plasma GSH-Px assays. Koller et al (12) reported that GSH-Px activity in whole cattle blood stored at 40 C for 7 d remained similar to original levels. The present study demonstrated the advantage of storing plasma samples at -15°C rather than at 40C. More than 90% of the initial enzyme activity was retained when samples were stored at -15°C for 24 h versus 82% of initial enzyme activity in samples stored at 40 C. Levels of plasma GSH-Px measured in samples stored at -15°C and treated with nitrogen (Table II) indicated some benefit from nitrogen application. In comparison with values obtained on day 0, samples stored for 7 d at -15°C and treated with nitrogen had 85% of initial enzyme activity, while only 66% of initial GSH-Px activity was present in plasma samples stored at -15°C without nitrogen. Despite the nitrogen treatment, plasma GSH-Px activity continued to fall after 2 wk or 4 wk of storage at -15°C. It is apparent that nitrogen may delay the decrease in enzyme activity of plasma stored at -15°C. Whether this is true for samples treated with nitrogen and stored at 40C remains to be studied. Due to the unstable nature of swine plasma GSH-Px, if in vivo activities are to be estimated by in vitro assays, it is suggested that determinations be

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169. 8. LAWRENCE RA, SUNDE RA, SCHWARTZ GL, HOEKSTRA WG. Glutathione peroxidase activity in rat lens and other tissues in relation to dietary selenium intake. Exp Eye Res 1974; 18: 563569. 9. McGOVERN JJ, JONES AR, STEINBERG AG. The hematocrit of capillary blood. N Engl J Med 1955; 253: 308-312. 10. THOMPSON KG, FRASER AJ, HARROP BM, KIRK JA. Glutathione peroxidase activity in bovine serum and erythrocytes in relation to selenium concentrations of blood, serum and liver. Res Vet Sci 1980; 29: 321-324. 11. SHEPPARD AD, MILLER KR. Stability of glutathione peroxidase in ovine blood samples under various storage conditions and the response of this enzyme to different methods of selenium supplementation. NZ Vet J 1981; 29: 77-80. 12. KOLLER LD, SOUTH PJ, EXON JH, WHITBECK GA, MAAS J. Comparison of selenium levels and glutathione peroxidase activity in bovine whole blood. Can J Comp Med 1984; 48: 431-433. 13. SCHOLZ RW, HUTCHISON LJ. Distribution of glutathione peroxidase activity and selenium in the blood of dairy cows. Am J Vet Res 1979; 40: 245-249.