Degradation studies of glutathione peroxidase in rat organs at different post-mortem intervals.

doi:10.21203/rs.3.rs-4869490/v1

Download PDF

Research Article

Degradation studies of glutathione peroxidase in rat organs at different post-mortem intervals.

https://doi.org/10.21203/rs.3.rs-4869490/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

Glutathione peroxidase (GPx) is a seleno-enzyme with an antioxidant function that counteracts reactive oxygen species generated during irrigation-reperfusion injury in transplant organs. GPx activity increased post mortem by seleno-methionine (SeMet) administration in vivo. GPx degradation was studied in the organs of rats supplemented with SeMet to obtain insights into organ thanatochemistry and improve its survival. Studies were performed in the liver, kidneys, and heart of rats. After death, organs were ablated at different post mortem intervals (PMI). GPx1 was extracted and determined by size exclusion chromatography (SEC) and affinity chromatography (AC) coupled to inductively coupled plasma mass spectrometry (ICP-MS). Degradation products were studied by analyzing low molecular weight selenium fractions (< 10 kDa). GPx1 degradation velocity (V_GPx1) in the different organs was evaluated by data analysis of GPx1 concentration variations according to PMI. GPx1 was determined in concentrations ranging from 0.19–0.76 mg kg^− 1 in the ablated organs in a PMI of 12 hs. SeMet administration increased GPx1 concentration, especially in the liver. GPx1 concentration decreased in all the studied tissues during a PMI of 12 hours. The degradation velocity of GPx1 (V_GPx1) was higher in the liver, followed by the kidneys and heart. Se degradation products were found in the range of 0.1 to 0.4 µg g^− 1, higher in the liver. In kidneys, no differences in GPx1 degradation were observed in control and SeMet-administered rats. GPx1 degradation was lower in SeMet-administered rats. SeMet administration increased GPx1 concentration and decreased its degradation post mortem in the organs of rats.

Selenium. Glutathione peroxidase. Rats. Transplant organs. Post mortem interval. Protein degradation

Organ transplantation is the preferred treatment option for patients diagnosed with end-stage organ failure (Black et al. 2018). However, primary graft dysfunction (PGD) is the main cause of early mortality after this procedure (Steffen et al. 2023). Several risk factors can contribute to PGD, including donor health, procurement technique, tissue ischemia-reperfusion injury (IRI), and recipient health (Cloer et al. 2023). IRI is traditionally defined as a mismatch between the supply of oxygen and energy substrates, and the cellular requirements for proper function and survival (Ughetto et al. 2023). IRI is characterized by producing reactive oxygen species (ROS), exceeding antioxidative defense mechanisms (Mauerhofer et al. 2021).

ROS are generated by inflammation (Teoh et al. 2002) and mitochondrial damage (Di Lisa et al. 2007). Anoxia uncouples mitochondrial oxygen consumption and ATP production producing superoxides. Superoxides react with lipids, proteins, and DNA to generate ROS (Zorov et al. 2014). Superoxide is spontaneously or enzymatically converted to hydrogen peroxide (Hauck and Bernlohr 2016). Glutathione peroxidase (GPx), superoxide dismutase, peroxiredoxins, and catalase are scavenging enzymes of hydrogen peroxide (Teoh et al. 1992).

GPx is a member of a family of antioxidant enzymes that reduce hydrogen peroxide and soluble lipid hydroperoxides using glutathione (GSH) as a source of reducing equivalents (Handy and Loscalzo 2022). Particularly, GPx1 is one member of a family of proteins that contain the amino acid selenocysteine (SeCys), an amino acid similar to cysteine but containing an atom of selenium at the position where sulfur resides in cysteines (Hatfield and Gladyshev 2002). GPx1 is ubiquitously expressed in all tissues and can be found in the cytosol and mitochondria. GPx1 is sensitive to the selenium nutrition status of individuals, being downregulated during hypoxia in ablated organs (Lubos et al. 2011).

To overcome the severe shortage of organs for transplantation, a strategy that can be adopted is improving the function and survival of transplanted organs (Akalay and Hosgood 2023). In this sense, it has been stated that agents increasing the expression of GPx1 can be potentially therapeutic in IRI (Chen et al. 2021). GPx1 expression is sensitive to the concentration of the micronutrient selenium, decreasing with low levels of selenium (Burk and Hill 2015; Sunde and Raines 2011). Therapeutically, the most likely successful strategies would target GPx1 overexpression to modulate excess oxidative stress associated with IRI. Selenium supplementation is clearly of consideration in this regard (Lubos et al. 2011). Recent studies have shown that selenomethionine (SeMet) administration decreases the oxidative stress induced by IRI in different organs of rats (Hasuoka et al. 2021). After SeMet administration for 21 days, a significant increase in GPx1 activity was observed in the liver, kidneys, and heart. However, GPx1 activity decreased after 3 hs post mortem. More studies are necessary to understand the molecular fate of GPx1 in ablated organs.

Metallomic approaches have been proposed to determine and quantify seleno-proteins in blood, serum, and plasma samples (Lamarche et al. 2021). Different chromatographic modes were coupled to inductively coupled plasma mass spectrometry (ICP-MS), for selenium monitoring, to determine GPx1, selenoprotein P (SePP), and selenoalbumin (SeAlb) (García-Sevillano et al. 2013). In ablated organs, protein degradation begins 4 min after death with autolysis (C. Zapico et al. 2014). Autolysis corresponds to the self-digestion or self-degradation of cells or tissues by their enzymes, involving lysosomes and hydrolytic enzymes (Madea et al. 2023). Post mortem seleno-proteins degradation by hydrolytic enzymes can be studied by metallomic approaches.

The present research reveals the fate of GPx1 in rats' kidneys, liver, and heart during post mortem interval (PMI). To this end, GPx1 was isolated by size exclusion chromatography (SEC) and determined by affinity chromatography (AC). ICP-MS was coupled to liquid chromatography for selenium monitoring. GPx1 analysis was performed at different PMIs. Selenoproteins degradation to lower molecular weight fractions was studied by Se analysis in < 10 kDa fractions. GPx1 degradation velocity was analyzed according to GPx1 concentration variations during PMIs. According to GPx1's role in decreasing oxidative stress in transplant organs, studies of its degradation during PMI are most important.

2.1. Reagents.

Bovine glutathione peroxidase (GPx1) was obtained from Sigma (St. Louis, MO). Recombinant Selenoprotein P1, Plasma (SEPP1), was obtained from Wuhan USCN Business, China. Ultrapure water with a resistivity of 18.2 mΩ cm^− 1 was obtained from a Milli-Q system (Millipore, Billerica, MA, USA). 1X PBS saline phosphate buffer was prepared by placing 8.06 g of NaCl, 0.22 g of KCl, 1.15 g of Na₂HPO_4, and 0.20 g of KH₂PO₄, in a beaker with 800 mL of Milli-Q water. Finally, pH was adjusted to 7.4 with H₃PO₄, and the volume was completed to 1000 mL. Methanol and ultrapure water were acquired from Fisher Scientific (Fair Lawn, NJ, United States. Digestion reagents like HNO₃ 65% (vv-1), H₂O₂ 30% (v v^− 1), perchloric acid 70% (v v^− 1), and NaOH were obtained from Sigma-Aldrich (St. Louis, United States).

2.2. Experimental design in rodents.

Sixty female Wistar rats between 180 and 200 g weight and 70–75 days of age were obtained in the Central Bioteria of the National University of San Luis. CICUA, Institutional Animal Care and Use Committee evaluated the research activity protocols by Protocol F-293/18. The study lasted 21 days. Rats were randomly placed in metal cages with sterilized wood chips. Chip was replaced daily. Rooms were bright, clean, and ventilated, with a uniform temperature (24 ± 2°C) day and night. The animals received balanced food based on plant and animal elements with minerals and vitamins appropriate for normal growth. The selenium content of the food was 532 ± 65 µg Se kg^− 1 (ICP-MS analysis) in agreement with other studies reported in the literature (Bermingham et al. 2014; Suzuki et al. 2010). The approximate food consumption of each rat was between 15 to 25 grams per day (4 to 6 pellets). The basal intake of selenium is between 8 to 13 ug Se per day. The water supply was completely renewed daily, eliminating the residual contents in the beverage bottle. These bottles were disinfected once a week, thus ensuring that the water quality was acceptable. Rats were administered orally by a probe with a dose of 150 ug of SeMet kg^− 1 day^− 1 in 1 mL of distilled water. SeMet doses and treatment periods were established based on previous studies (Kim et al. 2000). SeMet was selected for supplementation since its incorporation into GPx1 remains for larger periods (Evenson and Sunde 2021). Considering the basal Se diet and SeMet treatment, a total Se diet of 100 µg^− 1 day^− 1 was reached in rats. This Se diet is not toxic according to the maximum level of 1000 µg Se kg^− 1 day^− 1 established in other studies (Sunde et al. 2016). A control group was established (n = 15) and administered orally with 1 mL of water. The control group provides information regarding the basal selenium status of rats. The remaining rats (n = 45) were divided into three groups and administered with SeMet for 7, 14, and 21 days respectively. At the end of the administration period, the animals were sacrificed by inhalation of carbon dioxide (chemical method). Death was confirmed by cervical dislocation. The liver, kidneys, and heart were removed from different rats at different PMIs (0, 1, 3, 6, and 12 h). Organs were freeze-dried to stop all oxidative processes and stored at -20°C before analysis.

2.3. Seleno-proteins extraction from transplant organs.

Extraction was performed according to previous studies (Hasuoka et al. 2021) with modifications. To perform the extraction, 0.2 g of tissue (liver, heart, and kidney) was homogenized in 2 mL of 1X PBS, pH 7.4, for 5 minutes. Then the suspension was centrifuged at 10000 rpm for 30 minutes at 4°C. Finally, the supernatants were collected.

2.4. Instrumentation.

The chromatographic separation involved a Series 200 Perkin-Elmer chromatograph (Thornhill, Canada). SEC was performed with a Phenomenex BioSep-SEC-s2000 (300 x 7.8 mm x 250 mm) column. AF was performed with a heparin-sepharose (HEP-HP) column, 1 mL x 0.7 x 2.5 cm (GE Healthcare, Bio-Sciences AB, Uppsala, Sweden). The separation conditions were adapted from the studies of Steinbrenner et al. (Steinbrenner et al. 2006). Separation conditions can be observed in Table 1SI of Supplementary Information. Bovine serum albumin (66 kDa), alcohol dehydrogenase (150 kDa), β-amylase (200 kDa), thyroglobulin (669 kDa), apoferritin (443 kDa), and equine myoglobin (17 kDa) were employed for calibration. SEC analysis was applied to determine Se distribution within these molecular weight fractions. S was determined simultaneously to investigate the presence of peptides and proteins.

Selenium fractions identified by SEC-ICP-MS were collected employing a Fraction Collector (Gilson, FC203B with valve, 220 V). Once collected, fractions were concentrated with 10 kDa molecular weight cut-off filters (Amicon (R) Ultra-4 Millipore; Billerica, MA, USA).

Selenium was monitored with an ICP-MS (Perkin-Elmer SCIEX, ELAN DRC-e Thornhill, Canada). The carrier gas, Ar with a purity of 99.996%, was supplied by Air Liquide (Río Cuarto, Córdoba, Argentina). Table 1SM shows the operation conditions of the ICP-MS. For sample introduction the following devices were used: an automatic injector (S10 AS-90 Perkin-Elmer autosampler), an HF-resistant high-performance fluoroacetate (PFA) nebulizer model PFA-ST and a quartz cyclonic fogging chamber with a drain line and internal cover, cooled with ESI PC3 system (Omaha - NE, USA).

2.5. Data analysis.

ANOVA analysis was performed to define significant differences between experimental groups. Previous analysis data groups were verified by Grubbs test, Ryan-Joiner test and the equality test of variances. The results were analysed as the mean ± standard error and the statistical significance of the differences between groups was calculated using ANOVA with an error rate of α = 0.05 (equivalent to a 95% confidence level) subsequent comparison by Tukey- Fisher. The Minitab 17® program was used for statistical and graphical analysis.

GPx1 degradation velocity was obtained by employing a differential method of kinetic analysis. Velocity was calculated using a numeric method of finite differences to obtain GPx1 concentration derivate against the time of post mortem interval. Calculus was performed with GNU Octave software (octave.org).

3.1. Size exclusión chromatography.

Figure 1 shows SEC-ICP-MS analysis of tissue extracts from rat organs. High molecular weight fractions of 669 and 443 kDa were disregarded since the molecular weight is too elevated to be considered proteins. Selenoalbumin and GPx1 were not detected (Limit of detection, 0.36 mg L^− 1) in SEC chromatograms of liver, kidney, and heart extracts after SeMet administration to rats compared to standards analysis. These observations are not coincident with Sandre et al. studies where SeAlb and GPx1 were determined in plasma, red blood cells (RBC), and tissues (liver, kidney, muscle, and brain) of rats (Akalay and Hosgood 2023). To achieve GPx1 detection in tissue extracts the fraction corresponding to the enzyme (83–95 kDa) from SEC analysis was collected and preconcentrated in MWCO filters to be determined by AC in a second-dimension chromatography.

Figure 1. SEC chromatogram of liver extract, 21 days SeMet administration, PMI 0; GPx standard (1 mg L^− 1), Albumin standard (1 mg L^− 1). Molecular weight markers are shown at the top. Se and S signals were determined by ICP-MS.

3.2. Affinity chromatography.

Previous studies have shown that SeMet administration promotes the activity of endogenous seleno-dependent antioxidants, particularly those of the GPx and thioredoxin reductase systems (Reyes et al. 2019). To elucidate if GPx1 increased activity is related to increased GPx1 synthesis, the protein was quantified by AF-ICP-MS and compared to controls. Figure 1SI presents a chromatogram obtained after optimizing GPx1 separation by AF-ICP-MS. The first Se peak in the chromatogram represents GPx1, which is not retained, and the second peak corresponds to SePP which elutes after 8 min. GPx1 and SePP were not separated by SEC since their molecular weight are similar (95 and 60 kDa respectively). SePP contains multiple seleno-cysteine (SeCys) residues, whereas all other selenoproteins contain only one SeCys (Lamarche et al. 2021). SePP is synthesized in the liver of rats and secreted to plasma (McConnell et al. 1979; Motsenbocker and Tappel 1982). SePP is a marker of nutritional selenium status (Persson-Moschos et al. 1995).

Two more chromatograms are also depicted in Fig. 1SM, Liver Control and Liver-SeMet. The comparison of GPx1 peak height shows a higher concentration of this enzyme in the liver of rats supplemented with SeMet. Nevertheless, quantification of GPx1 in liver, kidney, and heart extract at different PMI must be performed to obtain information about GPx1 thanatochemistry.

3.3. GPx1 quantification at different post mortem intervals.

GPx1 concentration in liver, heart, and kidney extracts at different post mortem intervals can be observed in Table 1. Different GPx1 concentrations were found in the liver, kidneys, and heart according to selenium availability and GPx1 expression in cell types (Handy and Loscalzo 2022).

Table 1. GPx1 concentration in tissue extracts at different post mortem intervals.

	GPx1 concentration (mg kg^-1) tissue extract
Postmortem Interval (hours) Supplementation SeMet (days)	0	1	3	6	12
	Liver
Control	0,42±0,05	0,40±0,05	0,36±0,03	0,27±0,03	0,19±0,04
7	0,64±0,07	0,57±0,06	0,51±0,05	0,42±0,07	0,43±0,07
14	0,70±0,05	0,64±0,06	0,42±0,04	0,45±0,05	0,39±0,04
21	0,76±0,002	0,74±0,07	0,62±0,07	0,59±0,06	0,41±0,04
	Kidney
Control	0,41±0,03	0,35±0,04	0,38±0,04	0,27±0,03	0,26±0,02
7	0,59±0,06	0,55±0,05	0,57±0,06	0,46±0,06	0,39±0,07
14	0,64±0,05	0,53±0,06	0,52±0,06	0,45±0,04	0,34±0,08
21	0,70±0,07	0,56±0,07	0,62±0,05	0,51±0,06	0,46±0,04
	Heart
Control	0,35±0,04	0,31±0,04	0,31±0,03	0,29±0,05	0,20±0,02
7	0,51±0,06	0,53±0,06	0,45±0,06	0,41±0,04	0,39±0,04
14	0,56±0,07	0,55±0,05	0,46±0,07	0,43±0,04	0,37±0,08
21	0,53±0,06	0,51±0,05	0,47±0,06	0,40±0,05	0,36±0,05

After 7 days of SeMet administration, an increase in GPx1 concentration was demonstrated in all tissue extracts compared to the control (p > 0.01 Tukey and Fisher test). These observations correlate with those of Evenson et al. where the incorporation of Se into GPx is detected after 24 hours of administration (Evenson and Sunde 2021). As observed in Table 1, a higher GPx1 concentration was found in the liver (0.76 mg kg^− 1) after 21 days of SeMet administrations. It has been reported that in the liver when Se is administered in the form of selenite, it is incorporated into GPx1, with a maximum of 24 hours, and then decreased. However, when selenium was administered in the form of SeMet, incorporation into GPx1 (cytosolic) was maintained for a longer time (Evenson and Sunde 2021). The explanation for this difference is that SeMet is efficiently metabolized and mixes with the common Se metabolite pool, where Se is preferentially incorporated as SeCys into selenoproteins (Bierla et al. 2023). Other studies showed that GPx1 activity in the liver of rodents reaches a plateau after a 28-day diet supplemented with multiple graded levels of dietary selenium, provided as inorganic sodium selenite (Sunde et al. 2016). These results contrast with the results obtained in this study, where GPx1 concentration increases according to SeMet administration for 5 days. In this sense, the importance of determining GPx1 concentration and activity is remarked.

GPx1 concentrations after SeMet administration in kidneys and heart were lower compared to the liver but with no significant difference between the administration periods (p = 0.05). These differences in GPx1 concentrations between the liver, kidneys, and heart coincide with Se accumulation observed in previous studies (Hasuoka et al. 2021), where Se was accumulated mostly in the liver.

GPx1 concentration decreased in all the studied tissues in a PMI of 0–12 hours. Figure 2 shows the GPx1 decrease in percentage at different PMIs in the studied organs between different SeMet supplementation periods. In organs post mortem, proteins undergo degradation and denaturation processes (Madea et al. 2023). This research reveals that GPx1 follows a degradation process, rather than denaturalization, according to a concentration decrease. During post mortem ischemia, irreversible damage occurs in the stability of the cell membrane, protein synthesis, and the cellular respiratory chain. Furthermore, hydrolytic enzymes are released into the cytoplasm after digestion of different cellular constituents.

After 7 days of SeMet administration, the concentration of GPx1 in the liver at a PMI of 12 hours decreased to 68.8% compared to GPx1 concentration immediately after death (PMI 0, Fig. 2a). In the control group (not administered with SeMet) GPx1 concentration decreased to 44.8%, 12 hrs PMI. These results suggest that the administration of SeMet had a protective effect on GPx1 degradation in the liver. The administration of SeMet reduces lipid lipoperoxidation of cell membranes by acting as an antioxidant and increasing the activity of antioxidant enzymes such as GPx1 (Hasuoka et al. 2021). At 14 and 21 days of SeMet administration GPx1 degradation decreased to 55.27 and 54.25% respectively. In the liver, higher GPx1 degradation with higher SeMet administration periods can be related to a toxic effect of Se in this organ (Hasuoka et al. 2021).

In kidneys (Fig. 2b), a different phenomenon was observed since no significant differences were observed between controls and SeMet-administered groups of rats in GPx1 degradation at a PMI of 12 h (p < 0.05) in the studied organs. However at early PMI, 3 hs, degradation was lower in rats administered with SeMet during 7 days, 96.94%, compared to controls, 81.85%. This observation agrees with those reported by Alkalai and Hosgood (Akalay and Hosgood 2023) where the protective effects of agents increasing GPx concentration in kidneys are not completely understood and need more studies.

In the heart extracts GPx1 decomposed at a lower degree compared to liver and kidney (Fig. 2c). At a PMI of 12 hs GPx1 degradation corresponds to 74.6% compared to a 0 h PMI in rats administered during 7 days with SeMet. In the control group, GPx1 degradation reached 57.8%. Selenium is an important micronutrient that maintains cardiovascular health. Selenium deficiency leads to Keshan's disease cardiomyopathy (Handy and Loscalzo 2022). Our study adds insights to the protective role of SeMet in the heart by avoiding GPx1 degradation after heart stop.

3.4. Velocity degradation studies of GPx post mortem.

Proteins are in a dynamic state of synthesis and degradation. The stability of individual proteins can vary under different physiological conditions. Protein degradation was thought to follow an exponential decay process (McShane et al. 2016). However, there is substantial evidence that protein degradation does not always follow first-order kinetics. Variations of GPx1 concentrations were observed in the studied rat organs at different PMIs. Data analysis of these variations during different PMI (time) was performed to calculate degradation velocity in transplant organs according to Eq. 1:

$$\:{\text{V}}_{\text{GPx1}}=\frac{{d\text{C}}_{\text{GPx1}}}{{d\text{t}}_{\text{PMI}}}$$

where V_GPx1 corresponds to degradation velocity of GPx1, dC_GPx1 /dt_PMI corresponds to derivative of GPx1 concentration against time (PMI).

The velocity of protein degradation does not occur homogeneously in the corpse, differing between organs (Kocsmár et al. 2023). As observed in Fig. 3, degradation velocity is higher in the liver, followed by the kidneys and heart.

V_GPx1 is similar in the liver of SeMet-administered rats and controls (Fig. 3a). In kidneys (Fig. 3b), V_GPx1 is constant in 7-day SeMet-administered rats. However, at higher SeMet supplementation periods V_GPx1 increases through PMI. The kidney is an organ with fast degradation kinetics (Kocsmár et al. 2023). SeMet administration for 7 days is sufficient to reduce oxidative stress (Hasuoka et al. 2021), maintaining low GPx1 degradation during PMI in this organ. In the heart, V_GPx1 is constant through PMI, being lower in 21-day SeMet-administered rats (Fig. 3c). This observation agrees with studies showing that in the heart increased GPx1 expression minimizes oxidative stress and mitochondrial dysfunction generated by stressors like IRI (Handy and Loscalzo 2022).

3.5. Variations of low molecular weight selenium fractions at different post mortem intervals.

Selenoproteins can undergo denaturation or degradation into low molecular weight peptides during the early stages of the PMI. Accordingly, an analysis of < 10kDa molecular weight selenium fractions, may indicate which of these two phenomena are involved, contributing to the understanding of GPx1 thanatochemistry. During IRI, the excessive and uncontrolled formation of lipid hydroperoxides, the product of lipid peroxidation by ROS excess formation, leads to plasma membrane rupture (Akalay and Hosgood 2023). The cell membrane rupture initiates an autolysis process, during which hydrolytic enzymes are released from lysosomes to self-digest cellular components. (Madea et al. 2023). In this sense, it was decided to study the effects of autolysis on selenoproteins by studying seleno-protein degradation products, as low molecular weight selenium fractions (< 10 kDa). The study involved the analysis of selenium by ICP-MS in the residue after filtration with 10 kDa MWCO filters of liver, kidney, and heart tissue extracts at different PMIs. The results are depicted in Table 2. Se concentrations in the < 10kDa fractions were found in the range of 0.1 to 0.4 µg g^− 1. These low concentrations highlight the sensitivity of ICP-MS for selenium and confirm its compatibility with this type of study.

Table 2

Se concentration in < 10 kDa fractions of tissue extracts at different supplementation periods and *post mortem* intervals.
SeMet Supplementation (days)	Postmortem Interval (hours)	Se ⁸² concentration (µg g^− 1) tissue extract
		Liver		Kidney		Heart
		Control	Sample	Control	Sample	Control	Sample
7	0	0,15 ± 0,01	0,24 ± 0,03	0,13 ± 0,01	0,22 ± 0,02	0,11 ± 0,01	0,24 ± 0,02
	1	0,15 ± 0,01	0,26 ± 0,03	0,14 ± 0,01	0,24 ± 0,02	0,12 ± 0,01	0,25 ± 0,03
	3	0,19 ± 0,01	0,27 ± 0,03	0,2 ± 0,02	0,26 ± 0,03	0,15 ± 0,02	0,26 ± 0,03
	6	0,20 ± 0,02	0,28 ± 0,03	0,2 ± 0,01	0,27 ± 0,03	0,18 ± 0,02	0,26 ± 0,03
	12	0,22 ± 0,02	0,28 ± 0,03	0,21 ± 0,01	0,28 ± 0,02	0,19 ± 0,02	0,26 ± 0,02
14	0	0,15 ± 0,01	0,25 ± 0,03	0,13 ± 0,01	0,28 ± 0,03	0,11 ± 0,01	0,24 ± 0,001
	1	0,15 ± 0,01	0,27 ± 0,03	0,14 ± 0,01	0,29 ± 0,03	0,12 ± 0,01	0,23 ± 0,02
	3	0,19 ± 0,02	0,28 ± 0,03	0,2 ± 0,02	0,28 ± 0,02	0,15 ± 0,02	0,27 ± 0,03
	6	0,20 ± 0,03	0,28 ± 0,03	0,20 ± 0,02	0,30 ± 0,03	0,18 ± 0,02	0,27 ± 0,03
	12	0,22 ± 0,02	0,30 ± 0,03	0,21 ± 0,02	0,34 ± 0,04	0,19 ± 0,02	0,27 ± 0,03
21	0	0,15 ± 0,01	0,33 ± 0,04	0,13 ± 0,01	0,3 ± 0,03	0,11 ± 0,01	0,27 ± 0,03
	1	0,15 ± 0,02	0,32 ± 0,04	0,14 ± 0,01	0,3 ± 0,03	0,12 ± 0,01	0,25 ± 0,02
	3	0,19 ± 0,02	0,35 ± 0,05	0,2 ± 0,02	0,31 ± 0,03	0,15 ± 0,01	0,28 ± 0,03
	6	0,20 ± 0,03	0,35 ± 0,04	0,2 ± 0,03	0,31 ± 0,03	0,18 ± 0,02	0,30 ± 0,03
	12	0,22 ± 0,002	0,4 ± 0,001	0,21 ± 0,02	0,35 ± 0,04	0,19 ± 0,02	0,36 ± 0,04

Higher Se concentrations were found in < 10kDa fractions in the liver, followed by those in the kidney and heart. Although this fraction is expected to be composed mainly of seleno-peptides and seleno-amino acids, other seleno-species like selenosugars (Evenson and Sunde 2021), were determined.

Selenium concentrations in fractions < 10kDa were higher in tissue extracts from rats supplemented with SeMet rather than control, increasing proportionally to the administration period. This increase is especially marked in the liver and kidney. After 21 days of SeMet administration, a marked increase in < 10 kDa selenium fractions was observed in the liver, possibly by SeMet not being incorporated into proteins.

Elevated concentrations of proteins such as proteases and proteasomes have been detected in the kidney, increasing autolysis in this organ. Heart and liver show slower degradation and practically no degradation after 12 hours of PMI. Even in the heart, selenium concentration slightly decreases in fractions < 10kDa. These observations agree with other studies where it is established that protein degradation is not observable between 12 and 24 hours of PMI and becomes apparent only at 48 hours of PMI (Kocsmár et al. 2023).

Temperature is an important factor affecting the evolution of protein degradation, such as GPx1, after death. When cadaveric cooling occurs rapidly, to 4°C, the proteome profile remains unchanged for up to 24 h. However, since our studies were performed at room temperature, 25°C, a decrease in the proteome was observed at a PMI of 12 h, coincident with a higher Se concentration in < 10 kDa fractions during this PMI in the different organs studied (Tavichakorntrakool et al. 2008).

Oxidative stress and ROS levels are factors influencing autolytic processes. Previous studies showed how selenium administration as SeMet increases the activity of GPx1 at different PMI in rat liver, heart, and kidney (Hasuoka et al. 2021), decreasing oxidative stress. Se concentration in < 10kDa fractions does not undergo modifications, or increases slightly at different PMI in the heart, liver, and kidney when SeMet is administered to rats. Considering reduced oxidative stress according to the high antioxidant activity of GPx1, indirectly, elevated GPx1 concentration prevents its degradation during post mortem autolysis.

This research showed GPx1variations in transplant organs of SeMet-administered rats at different PMIs. SeMet administration increased GPx1 concentration in the liver, kidney, and heart, being higher in the liver. In the studied PMIs, GPx1 concentration decreased following a degradation pathway rather than denaturalization. In the heart and liver, GPx1 degradation was lower in SeMet-administered rats according to the inherent antioxidant activity of SeMet and increased expression of antioxidant proteins like GPx1 itself. Elevated SeMet administration periods should be avoided since they increase GPx1 degradation in the liver. SeMet did not avoid GPx1 degradation in kidneys. A study of the degradation velocity of GPx1 during the proposed PMI showed a faster decay in the kidney followed by the liver. In the heart, the degradation velocity of GPx1 was constant through the PMI in SeMet-administred and control groups of rats. Elevated concentrations of low molecular weight seleno-species in kidney and liver confirmed the higher and fastest degradation of GPx1 in these organs. The information obtained from this study is important to improve graft survival after transplant surgeries by SeMet administration to living donors according to the protective antioxidant role of GPx1 during IRI.

Conflicts of interests:

The authors declare no conflict of interest.

Ethics approval:

All animal experimentation was reviewed and approved by the Animal Care and Use Institutional Committee of the Universidad Nacional de San Luis (Ord. 006 − 02, Protocol N ° F-293/18 UNSL).

CRediT authorship contribution statement.

Pablo Pacheco conceived the study. Paul Hasuoka extracted and analyzed data. All authors interpreted the data. Paul Hasuoka prepared the first draft of the manuscript with the contributions of Pablo Pacheco and Franco Tonelli. All authors read and approved the final manuscript.

Funding:

This work was supported by the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) [1 grant, Hasuoka], the Agencia Nacional de Promoción Científica y Técnica (ANPCyT) and the Universidad Nacional de San Luis (UNSL).

Author Contribution

Data availability statement:

Additional/raw data can be made available upon request

Akalay S, Hosgood SA (2023) How to Best Protect Kidneys for Transplantation—Mechanistic Target. J Clin Med. 12 doi:10.3390/jcm12051787
Bermingham EN, Hesketh JE, Sinclair BR, Koolaard JP, Roy NC (2014) Selenium-enriched foods are more effective at increasing glutathione peroxidase (GPx) activity compared with selenomethionine: A meta-analysis. Nutrients. 6:4002–4031 doi:10.3390/nu6104002
Bierla K, Szpunar J, Lobinski R, Sunde RA (2023) Selenomethionine supplementation and expression of selenosugars, selenocysteine, and other selenometabolites in rat liver. Metallomics 15:mfad067 doi:10.1093/mtomcs/mfad067
Black CK, Termanini KM, Aguirre O, Hawksworth JS, Sosin M (2018) Solid organ transplantation in the 21st century. Ann Transl Med 6
Burk RF, Hill KE (2015) Regulation of Selenium Metabolism and Transport. vol 35. doi:10.1146/annurev-nutr-071714-034250
C. Zapico S, Menéndez ST, Núñez P (2014) Cell death proteins as markers of early postmortem interval. Cell Mol Life Sci. 71:2957–2962 doi:10.1007/s00018-013-1531-x
Cloer CM et al. (2023) Mitochondrial transplant after ischemia reperfusion promotes cellular salvage and improves lung function during ex-vivo lung perfusion. J Heart Lung Transplant. 42:575–584 doi:10.1016/j.healun.2023.01.002
Chen Y, Fan H, Wang S, Tang G, Zhai C, Shen L (2021) Ferroptosis: A Novel Therapeutic Target for Ischemia-Reperfusion Injury. Front Cell Dev Biol. 9 doi:10.3389/fcell.2021.688605
Di Lisa F, Canton M, Menabò R, Kaludercic N, Bernardi P (2007) Mitochondria and cardioprotection. Heart Fail Rev. 12:249–260 doi:10.1007/s10741-007-9028-z
Evenson JK, Sunde RA (2021) Metabolism of Tracer 75Se Selenium From Inorganic and Organic Selenocompounds Into Selenoproteins in Rats, and the Missing 75Se Metabolites. Front Nutr. 8 doi:10.3389/fnut.2021.699652
García-Sevillano MA, García-Barrera T, Gómez-Ariza JL (2013) Development of a new column switching method for simultaneous speciation of selenometabolites and selenoproteins in human serum. J Chromatogr A. 1318:171–179 doi:10.1016/j.chroma.2013.10.012
Handy DE, Loscalzo J (2022) The role of glutathione peroxidase-1 in health and disease. Free Radic Biol Med. 188:146–161 doi:10.1016/j.freeradbiomed.2022.06.004
Hasuoka PE, Iglesias JP, Teves M, Kaplan MM, Ferrúa NH, Pacheco PH (2021) Selenomethionine administration decreases the oxidative stress induced by post mortem ischemia in the heart, liver and kidneys of rats. BioMetals 34:831–840 doi:10.1007/s10534-021-00310-3
Hatfield DL, Gladyshev VN (2002) How selenium has altered our understanding of the genetic code. Mol Cell Biol. 22:3565–3576 doi:10.1128/mcb.22.11.3565-3576.2002
Hauck AK, Bernlohr DA (2016) Oxidative stress and lipotoxicity. J Lipid Res. 57:1976–1986 doi:https://doi.org/10.1194/jlr.R066597
Kim HC, Jhoo WK, Shin EJ, Bing G (2000) Selenium deficiency potentiates methamphetamine-induced nigral neuronal loss; comparison with MPTP model. Brain Res. 862:247–252 doi:10.1016/s0006-8993(00)02085-0
Kocsmár É et al. (2023) Proteome alterations in human autopsy tissues in relation to time after death. Cell Mol Life Sci. 80 doi:10.1007/s00018-023-04754-3
Lamarche J, Ronga L, Szpunar J, Lobinski R (2021) Characterization and Quantification of Selenoprotein P: Challenges to Mass Spectrometry. Int J Mol Sci. 22:6283
Lubos E, Loscalzo J, Handy DE (2011) Glutathione peroxidase-1 in health and disease: From molecular mechanisms to therapeutic opportunities. Antioxid Redox Signal. 15:1957–1997 doi:10.1089/ars.2010.3586
Madea B et al. (2023) Autolysis, Putrefactive Changes and Post-mortem Chemistry. In: Estimation of the Time Since Death: Fourth Edition. pp 187–281. doi:10.1201/9781003244974-7
Mauerhofer C, Grumet L, Schemmer P, Leber B, Stiegler P (2021) Combating ischemia-reperfusion injury with micronutrients and natural compounds during solid organ transplantation: Data of clinical trials and lessons of preclinical findings. Int J Mol Sci. 22 doi:10.3390/ijms221910675
McConnell KP, Burton RM, Kute T, Higgins PJ (1979) Selenoproteins from rat testis cytosol. Biochim Biophys Acta Gen Subj. 588:113–119 doi:10.1016/0304-4165(79)90376-3
McShane E et al. (2016) Kinetic Analysis of Protein Stability Reveals Age-Dependent. Degradation. Cell. 167:803–815.e821 doi:https://doi.org/10.1016/j.cell.2016.09.015
Motsenbocker MA, Tappel AL (1982) A selenocysteine-containing selenium-transport protein in rat plasma. Biochim Biophys Acta Gen Subj. 719:147–153 doi:10.1016/0304-4165(82)90318-x
Persson-Moschos M, Huang W, Srikuniar TS, Åkesson B, Lindeberg S (1995) Selenoprotein P in serum as a biochemical marker of selenium status. The Analyst. 120:833–836 doi:10.1039/an9952000833
Reyes L, Bishop DP, Hawkins CL, Rayner BS (2019) Assessing the efficacy of dietary selenomethionine supplementation in the setting of cardiac ischemia/reperfusion injury. Antioxid. 8 doi:10.3390/antiox8110546
Steffen SP, Tenório DF, Cirillo GCG, Gaspar SF, de Oliveira KAS, Gaiotto FA, Jatene FB (2023) Severe and Moderate Primary Graft Dysfunction in Adult Heart Recipients. Braz J Cardiovasc Surg. 38:214–218 doi:10.21470/1678-9741-2022-0107
Steinbrenner H, Bilgic E, Alili L, Sies H, Brenneisen P (2006) Selenoprotein P protects endothelial cells from oxidative damage by stimulation of glutathione peroxidase expression and activity. Free Radic Res. 40:936–943 doi:10.1080/10715760600806248
Sunde RA, Li J-L, Taylor RM (2016) Insights for Setting of Nutrient Requirements, Gleaned by Comparison of Selenium Status Biomarkers in Turkeys and Chickens versus Rats, Mice, and Lambs. Adv Nutr. 7:1129–1138 doi:https://doi.org/10.3945/an.116.012872
Sunde RA, Raines AM (2011) Selenium regulation of the selenoprotein and nonselenoprotein transcriptomes in rodents. Adv Nutr. 2:138–150 doi:10.3945/an.110.000240
Suzuki Y, Hashiura Y, Matsumura K, Matsukawa T, Shinohara A, Furuta N Dynamic pathways of selenium metabolism and excretion in mice under different selenium nutritional statuses. In: Metallomics, 2010. pp 126–132. doi:10.1039/b915816b
Tavichakorntrakool R et al. (2008) Serial analyses of postmortem changes in human skeletal muscle: A case study of alterations in proteome profile, histology, electrolyte contents, water composition, and enzyme activity. Proteomics Clin Appl. 2:1255–1264 doi:10.1002/prca.200800051
Teoh KH, Mickle DAG, Weisel RD, Li RK, Tumiati LC, Coles JG, Williams WG (1992) Effect of oxygen tension and cardiovascular operations on the myocardial antioxidant enzyme activities in patients with tetralogy of Fallot and aorta- coronary bypass. J Thorac Cardiovasc Surg. 104:159–164 doi:10.1016/s0022-5223(19)34848-2
Teoh N, Pena AD, Farrell G (2002) Hepatic ischemic preconditioning in mice is associated with activation of NF-κB, p38 kinase, and cell cycle entry. Hepatol. 36:94–102 doi:10.1053/jhep.2002.33134
Ughetto A et al. (2023) Heart graft preservation technics and limits: an update and perspectives Frontiers in Cardiovascular Medicine 10 doi:10.3389/fcvm.2023.1248606
Zorov DB, Juhaszova M, Sollott SJ (2014) Mitochondrial Reactive Oxygen Species (ROS) and ROS-Induced ROS Release. Physiol Rev. 94:909–950 doi:10.1152/physrev.00026.2013

No competing interests reported.

SupplementaryInformation.docx

Download PDF

Reviews received at journal
13 Sep, 2024
Reviews received at journal
01 Sep, 2024
Reviewers agreed at journal
12 Aug, 2024
Reviewers agreed at journal
12 Aug, 2024
Reviewers invited by journal
10 Aug, 2024
Editor assigned by journal
09 Aug, 2024
Submission checks completed at journal
09 Aug, 2024
First submitted to journal
06 Aug, 2024

You are reading this latest preprint version

Degradation studies of glutathione peroxidase in rat organs at different post-mortem intervals.

Status:

Version 1

Abstract

Figures

1. Introduction

2. Experimental

2.1. Reagents.

2.2. Experimental design in rodents.

2.3. Seleno-proteins extraction from transplant organs.

2.4. Instrumentation.

2.5. Data analysis.

3. Results and discussion

3.1. Size exclusión chromatography.

3.2. Affinity chromatography.

3.3. GPx1 quantification at different post mortem intervals.

3.4. Velocity degradation studies of GPx post mortem.

3.5. Variations of low molecular weight selenium fractions at different post mortem intervals.

4. Conclusion

Declarations

Conflicts of interests:

Ethics approval:

CRediT authorship contribution statement.

Funding:

Author Contribution

Data availability statement:

References

Additional Declarations

Supplementary Files

Status:

Version 1