Selenium Inhibits Renal Oxidation and Inflammation But Not Acute Kidney Injury in an Animal Model of Rhabdomyolysis

Tissue Se distribution

X-ray fluorescence microspectroscopy (XFM) was used to map the distribution and to determine Se concentration in tissues from sham, RM (glycerol-treated) and Se++ (dietary Se 5 ppm) groups that were sectioned (thickness 30 μm), and dehydrated onto silicon nitride (see Methods). The Se and K⁺elemental distribution and a corresponding hematoxylin and eosin-stained section from the same (Se++) sample are shown (Fig. 1A). The distribution of K⁺ was uniform across all kidney regions, as expected for this ubiquitous cation. The majority of Se was concentrated within the renal cortex and to a lesser extent the renal medulla (c.f., XFM and parallel H&E-stained images, Fig. 1A). Dietary Se (at 5 ppm) increased both renal Se levels ∼2-fold (Fig. 1B) and circulating GPx activity ∼1.5±0.1-fold (mean±SD; n=4) compared to the corresponding sham (Supplementary Fig. S2).

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FIG. 1.

X-ray fluorescence imaging reveals renal K⁺ and Se distribution. Animals were supplemented with 5 ppm dietary Se (Se++ group) as described in the Methods section. Kidneys were harvested 24 h after experimental RM (except the sham), frozen sections were obtained and applied to silicon nitride windows and incubated under vacuum (<0.02 Torr; 12 h) before transport to the Australian Synchrotron for assessment by XFM imaging. (A) The elemental distribution of Se and potassium ions were then determined by elemental mapping, and representative data is reported for tissue from a rat in the Se++ group together with a parallel renal section stained with hematoxylin and eosin. (B) Quantification of Se reported as area normalized density units. Images are representative of at least three different microscopic fields from n=2 kidneys.

Oxidative damage to the kidney

Levels of unesterified cholesterol (UC), vitamin E (as α-tocopherol; α-TOH), cholesteryl arachidonate (C20:4), and cholesteryl linoleate (C18:2), together referred to as cholesteryl esters (CE), were unchanged in all groups (Table 1). Lipid peroxidation increased markedly in both plasma and renal tissues after RM, as judged by comparing concentrations of cholesteryl ester-derived lipid hydro(per)oxides (CE-O(O)H; a marker of cell membrane oxidation) and F₂-isoprostanes (marker of polyunsaturated fatty acid oxidation) determined in the sham and RM groups. By contrast, Se supplementation decreased CE-O(O)H and F₂-isoprostane accumulation in both the plasma and renal tissues, although this did not reach the corresponding baseline concentrations determined in the sham (Table 1).

Urinary and plasma markers of AKI

Urinary clusterin, a marker for AKI that correlates with hypoxic tubular damage (28), and kidney injury molecule-1 (KIM-1), a glycoprotein released in the event of early proximal tubule injury (6, 59, 61), both increased significantly after RM (Fig. 2A and 2B). While urinary clusterin was unaffected by Se supplementation, KIM-1 decreased significantly in the Se++ group (vs. the RM group), although it remained elevated by comparison to the sham. Both urinary protein and the ratio of protein:creatinine increased significantly in all glycerol-treated animals, except in the Se++ group, where lower levels of proteinuria (and the corresponding protein:creatinine ratio) were indicative of reno-protection (Table 2). A marked decrease in the value of body weight normalized creatinine clearance (CCr) was determined after experimental RM (Table 2) and this was unaffected by Se supplementation. Almost all plasma biochemical markers were unchanged after experimental RM except blood urea nitrogen (BUN) and plasma creatinine, which increased in all animals after RM irrespective of Se supplementation (Table 2).

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FIG. 2.

Urinary markers of AKI after experimental RM. Animals were treated as described in the legend to Figure 1 before inducing experimental RM (except the sham) for a 24 h period during which urine was collected prior to harvest of blood and kidneys. Urinary concentrations of (A) clusterin (ng/ml) and (B) KIM-1 (ng/ml) were measured with a commercial Luminex assay. Data are expressed as mean±SD; n=4 or n=6 for clusterin and KIM-1, respectively. *Different to the sham; p<0.05. ^#Different to the RM group; p<0.05; **Different to the RM and Se+ group; p<0.01.

Vascular function

Binding of NO to its molecular target, soluble guanylyl cyclase, activates cGMP production (56). Induction of experimental RM yielded significantly lower cGMP levels than detected in the aorta (Fig. 3A) and kidney (Fig. 3B) from the sham group. Dietary Se supplementation restored cGMP content in the thoracic aortic to similar levels detected in the sham, indicating an improved vascular tone in large bore aortic vessels after experimental RM (Fig. 3A). By contrast, Se did not restore renal cGMP, with levels remaining similar to the RM group irrespective of the level of Se supplementation (Fig. 3B). These data indicate that, while Se enhanced vascular function in large arterial vessels, renal microvascular dysfunction remained unaffected.

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FIG. 3.

Aortic and renal levels of cGMP after experimental RM. Animals were treated as described in the legend to Figure 1 before inducing experimental RM (except the sham). After 24 h, (A) aortae and (B) kidneys were harvested, homogenized, and cGMP assessed with a commercial kit as described in the Methods section. Data are expressed as mean±SD; n=6 samples from different animals. *Different to the sham; p<0.01. ^#Different to the RM group; p<0.05.

Changes to gene expression and proteins after experimental RM

Gene expression of the antioxidant response elements (Cu-Zn)SOD-1, (Mn)SOD-2, and GPx-1 increased significantly after RM (Table 3). In case of SOD-1, RM stimulated an accumulation of monomeric and dimeric SOD-1 in renal tissues. This was marginally inhibited by dietary Se supplementation, with decreases in the active dimeric SOD evident in both the Se+ and Se++ groups (Fig. 4A). Consistent with this evaluation of protein accumulation, immunoactive SOD increased in tubule epithelial cells in all glycerol-treated groups (Supplementary Fig. S3A(i–iv)). Accumulation of SOD-1 was primarily in the renal tubule epithelia of the cortex and outer medullar regions—some weak staining was evident within glomeruli (compare insets in Supplementary Fig. S3A(i–iv)). Despite increases in renal SOD-1 after RM, total SOD activity showed only a weak trend to increase after RM (Fig. 4B) and this was ameliorated with Se supplementation, albeit this did not reach statistical significance.

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FIG. 4.

Accumulation of SOD-1, GPx-1, and phosphorylated P65 and corresponding protein activities in the kidney after experimental RM. Animals were treated as described in the legend to Figure 1 before inducing experimental RM (except the sham). Animals were sacrificed, and blood and kidneys were harvested to prepare plasma and renal homogenates as described in the Materials and Methods section. Homogenate proteins were separated on SDS-PAGE and immunostained for (A) SOD-1 protein and (B) corresponding activity was assessed; (C) GPx-1 and (D) corresponding activity and (E) p-P65 and actin as loading control; MCP-1 levels in (F) plasma and (G) renal tissues. Enzyme activity is expressed as a fold-change relative to the activity determined for the sham. Levels of MCP-1 were determined using a commercial EIA. Data are expressed as mean±SD; n=3 (SOD and GPx) or n=4 (p-p65 and MCP-1) independent experiments using renal and plasma samples from different animals. *Different to the sham; p<0.01. ^#Different to the RM group; p<0.05.

Gene expression for GPx-1 remained high in all glycerol-treated animals irrespective of Se supplementation (Table 3), and this resulted in weak increases in the renal content of GPx-1 protein in glycerol-treated animals as judged by Western blotting (Fig. 4C). By contrast, immunohistochemical assessment of GPx-1 (Supplementary Fig. S3B[i–iv]) showed similar levels of GPx-1 staining (localized to tubular epithelia and parietal epithelia lining Bowman's capsule) in all treatment groups. Unexpectedly, total GPx activity diminished after RM and, although Se supplementation improved tissue GPx activity, it remained below that determined in the sham (Fig. 4D). It is notable that Mb (present in the renal tissues after RM) also exhibits peroxidase-like activity (43) and this may interfere with ex vivo determinations of renal GPx activity relative to the sham samples that lack extracellular Mb.

Gene expression for the redox-sensitive transcription factor NFκB was unchanged 24 h after RM induction (Table 3). However, phosphorylation of the p65 subunit increased in response to RM (Fig. 4E) and this occurred concomitantly with enhanced gene expression of pro-inflammatory TNFα (Table 3) and increased levels of MCP-1 in both circulating plasma and renal tissues (Fig. 4F and 4G, respectively). Se supplementation decreased P65 phosphorylation, and at the highest dose, Se attenuated TNFα gene expression and MCP-1 accumulation in renal tissues, although plasma MCP-1 remained unaffected.

Neutrophil stimulation leads to the production of hypochlorous acid that oxidizes and chlorinates proteins including Mb (52). Monitoring the level of 3-chloro-tyrosine provides a useful index of tissue inflammation (26). Induction of experimental RM markedly increased the 3-chloro-tyrosine/tyrosine ratio as determined by quantitative mass spectrometry (Fig. 5A). Consistent with Se inhibiting inflammation, the 3-chloro-tyrosine/tyrosine ratio decreased significantly in Se supplemented animals and this reached significance at the highest Se dose administered (Fig. 5A).

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FIG. 5.

Impact of RM on the renal and plasma content of 3-chlroro-tyrosine. Animals were treated as described in the legend to Figure 1 before inducing experimental RM (except the sham). Animals were sacrificed, blood taken, and the kidneys harvested and homogenized. The total protein present in renal homogenates and isolated plasma were then precipitated and hydrolyzed to individual amino acids as described in the Materials and Methods section. Hydrolysates were then assessed for the content of 3-chlorotyrosine and unmodified tyrosine by quantitative mass spectrometry and finally expressed as (A) the ratio of 3-chlorotyrosine/tyrosine in the tissue. In some experiments, extracellular Mb was immunoprecipitated from the plasma and (B) the 3-chlorotyrosine/tyrosine ratio determined in the supernatant (RMIPS) and precipitate (RMIP). Data is expressed as a percentage of the maximum value measured in the RM group. Data represent mean±SD; n=4. *Different to the sham; p<0.01. ^#Different to the corresponding level in the RM group; p<0.05.

Interestingly, the extent of plasma protein chlorination equated to ∼7% of the available pool of protein tyrosine, well above levels previously reported. One possible explanation for increased protein chlorination is that administration of hypertonic glycerol to the hind leg muscle induces a local inflammatory response that generates chlorinated proteins, including chloro-Mb. Release of chloro-Mb into the circulation, combined with the rapid clearance of extracellular Mb to the kidney, may account in part for the 3-chloro-tyrosine/tyrosine ratio determined in the renal tissues after RM. To assess this possibility, we determined the levels of 3-chloro-tyrosine in plasma before and after immunoprecipitation of Mb. Overall, RM markedly increased plasma levels of 3-chloro-tyrosine (Fig. 5B). A comparison of 3-chloro-tyrosine content in the RM group before immunoprecipitation (RM), the residual supernatant (RMIPS), and the precipitate (RMIP) indicated that the level of 3-chloro-tyrosine in plasma decreased significantly by ∼70% after immunoprecipitation of Mb. Supplementation with Se at 5 ppm in the diet significantly decreased the plasma content of 3-chloro-tyrosine (Fig. 5B), consistent with the anti-inflammatory action of this essential micronutrient documented here (Fig. 4E–4G).

Kidney histology

Kidneys from the sham displayed normal tubular epithelia in the renal cortex, unobstructed tubules, and unremarkable glomeruli containing thin-walled capillaries within the glomerular tuft (Fig. 6). Consistent with RM inducing an increase in KIM-1, glycerol treatment yielded tubule epithelial necrosis, thickening and disruption/displacement of the tubular epithelium with evidence of cast material, irrespective of Se status (Fig. 6A, with 6B–6D). After RM, glomeruli appeared congested and the capillaries within the tuft were more difficult to identify due to the presence of relatively more nuclear staining than evident in the corresponding sham samples. The latter may indicate either cellular infiltration or mesangial proliferation stimulated by the elevated levels of renal MCP-1 and NFκB activation (Fig. 4G), which may be linked to the increase in the 3-chloro-tyrosine/tyrosine ratio determined in the corresponding renal homogenates (Fig. 5A).

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FIG. 6.

Changes to renal architecture after experimental RM. Animals were treated as described in the legend to Figure 1 before inducing experimental RM (except the sham). After 24 h, the animals were euthanized and samples of kidney were isolated, embedded, sectioned, and stained with PAS. Representative sections are shown for (A) Sham, (B) RM, (C) Se+, and (D) Se++ treated rats. Arrows in (A) highlight unremarkable glomeruli in the renal cortex with thin walled capillaries in the glomerular tuft (red arrow); intact tubule epithelia (black) that are adherent to the tubule wall (white) in Sham tissues. Arrows in (B–D) highlight tubular casts (yellow), congested glomeruli with increased nuclear staining (light blue), necrotic tubular epithelial cells with some showing displacement from tubule (orange). Images are representative of 3 microscopic regions from n=3–5 kidneys from different animals in the various treatment groups.

Angiotensin receptors

Gene expression of ATR I/II increased across all groups relative to the sham. This relative increase was ameliorated by Se supplementation; however, expression remained greater than that detected in the sham (Table 3). Parallel increase in immunoactive ATRI after RM was detected in the glomerular capillary network and in the epithelium lining the renal tubules in all groups after RM (Fig. 7). A similar pattern of enhanced protein expression was detected for ATRII (not shown), suggesting that upregulation of ATR I/II receptors facilitates the function of vasoactive AngII.

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FIG. 7.

Expression of Angiotensin II receptor 1 in renal tissues after experimental RM. Animals were treated as described in the legend to Figure 1 before inducing experimental RM (except the sham). After 24 h kidneys were harvested, fixed, embedded, sectioned, and stained. Panels show distribution of immunoactive ATRI accumulating in renal sections from (A) sham, (B) RM, (C) Se+, and (D) Se++ groups. Data are representative of several fields of view from n=3 independent kidneys.

Renal kinase activity

Renal vasoconstriction is attributed to AngII binding to ATRI to enhance MAPK activity through kinase phosphorylation. MAPKinase activity increased significantly after RM and this was sensitive to MEK-1/2 inhibition. MAPKinase activity remained elevated in the Se+ group, but was decreased to similar levels determined for the sham in the Se++group (Fig. 8). Consistent with this documented increase in MAPK activity after RM, immunoactive p-p38 (Fig. 9A) and p-ERK1/2 (Fig. 9B) increased after RM and the proteins were distributed primarily in the tubule epithelia and glomeruli similar to ATRI/ATRII. The extent of p-p38 and p-ERK1/2 staining in renal tissues was largely unaffected by Se supplementation, even though ERK kinase activity was shown to be inhibited in the Se++ group.

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FIG. 8.

Assessment of MAPK activity determined in renal tissues after experimental RM. Animals were treated as described in the legend to Figure 1 before inducing experimental RM (except the sham). After 24 h, the animals were euthanized and samples of kidney were isolated, homogenized, and MAPK activity was determined in the absence or presence of the MEK inhibitor (U0126) as described in Methods section. Data represents mean±(SD); n=5. *Different to the sham; p<0.05. **Different to the RM group; p<0.05. ^#Different to the corresponding group in the absence of U0126; p<0.01.

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FIG. 9.

Immunoactive phosphorylated ERK1/2 and p38 in rat kidney after experimental RM. Animals were treated as described in the legend to Figure 1 before inducing experimental RM (except the sham). After 24 h, animals were euthanized, kidneys harvested, fixed, and prepared for immunohistochemical analysis, as described in Methods section. Representative images show immunoactive (A) phosphorylated p38 and (B) phosphorylated ERK1/2 determined in (i) sham, (ii) RM, (iii) Se+, and (iv) Se++ treatment groups (inset 40X magnification of corresponding glomeruli). Data representative of at least 3 fields of view from n=3 different kidneys.

Chemicals

Biochemicals were from Sigma Aldrich (Sydney, Australia), unless indicated otherwise. All chemicals were of the highest available quality. Antibodies for GPx-1, ATRI, and ATRII were from Abcam (Sydney, Australia), anti-SOD-1 (Sigma, MO) and anti-phospho ERK42/44 and p38 (Cell Signaling Technology, MA). The VECTASTAIN^® ABC kit was from Vector Laboratories (CA). All buffers were prepared with MilliQ^® Water.

Se supplementation

Male Sprague-Dawley rats (80–100 g) were from the Animal Resources Centre (Perth, Australia). Animals were acclimated for 2 weeks (body weights ranged 140 to 162 g), randomly assigned to groups, and provided a normal diet or sodium selenite supplemented diets ad libitum: 1 ppm (designated as Se+) or 5 ppm (Se++) prepared by Glen Forrest Specialty Feed (Perth, Western Australia). After 4-weeks of diet, animal weights did not differ significantly, although a trend to decreased weight was apparent in the Se++ group [normal chow 309±18 g (n=15); Se+ diet 292±17 g (n=9), and Se++ diet 285±13 g (n=6), mean±SD]. Studies were conducted according to Local Ethics guidelines that adhered to NIH Guidelines for the Care and Use of Laboratory Animals.

Experimental rhabdomyolysis

Animals were dehydrated for 18 h before experimental RM as described previously (14, 15). Animals assigned to RM and Se-supplemented groups were anesthetized with isoflurane (2% v/v in O_{2 (g)}, 1.5 l/min) then injected with 6 ml/kg of hypertonic glycerol (50% v/v in sterile saline) into each hind leg (this protocol yields RM with similar urinary Mb concentrations to levels determined after severe electrical burn in humans (32)). Animals in the sham group received an equal volume of sterile saline. Animals were then housed in metabolic cages with unrestricted access to chow/water and urine collected over 24 h.

Harvest of blood and organs

Animals were anesthetized with isoflurane, followed by i.p. injection of ketamine (50 mg/kg weight) and xylazine (10 mg/kg weight). A thoracotomy was performed to expose the heart, and blood was collected via cardiac puncture (8–10 ml in 100 IU-heparin) and plasma was immediately isolated and stored at −80°C. Next the kidneys and aortae were harvested, the left kidney was sectioned, and the ventral portion was immersed in Tissue-Tek OCT (ProSciTech, Qld, Australia) and stored at −80°C for preparation of frozen sections. The remaining sample was stored in 4% v/v formalin for histological analysis. The right kidney was snap frozen in liquid nitrogen and stored at −80°C for subsequent molecular/biochemical analyses.

X-ray fluorescence microspectroscopy

Where required, kidneys were cryo-sectioned (30 μm) and mounted on to silicon nitride windows as described elsewhere (12), vacuum-sealed, and stored at 4°C prior to XFM. Elemental distribution maps of kidney sections were recorded on the X-ray fluorescence microspectroscopy (XFM) microprobe-beamline (39) at the Australian Synchrotron (Victoria, Australia). A monochromatic 12.75 keV X-ray beam was focused (to a spot size of about 6×2.5 μm) using a pair of horizontally- and vertically-focusing Kirkpatrick-Baez mirrors, and the fluorescence signal was collected using a single element silicon drift energy dispersive detector (Vortex EX, SII Nanotechnology, Northridge, CA), for 1 s per spatial point at a step size of 10 μm. As areas of interest were identified within the XFM images, higher resolution images were obtained from fluorescence signals collected for 2 s per spatial point at step size 2 μm.

Spectra for each spatial point was fit to Gaussians, modified by the addition of a step function and a tailing function to describe incomplete charge collection and other detector artifacts (19). Elemental contents (counts/s/pixel) were extracted from the fitted images. Further analysis was performed using MAPS software (61). Elemental content (counts/s/pixel) was normalized by X-ray scatter (counts/s/pixel) as a proxy for tissue thickness and therefore, the relative elemental contents are reported as dimensionless quantities.

Preparation of tissue homogenate

Intact frozen kidneys were thawed, diced, snap frozen in liquid nitrogen, pulverized to a fine powder, and homogenized for use in lipid analysis, enzyme activity assays, and gene response studies, as described previously (23, 32). Homogenate protein content was measured using the BCA assay (48).

Tissue and plasma lipid profile

Renal levels of UC, TOH, C18:2, C20:4, and CE-O(O)H were determined by high-performance liquid chromatography (48, 64). F₂-isoprostanes (nonenzymatic oxidation of arachidonic acid) were determined with an immunoassay kit (Cayman, Ann Arbor, MI; detection limit ∼12 pmol/mg protein). All samples were saponified (10 M NaOH), neutralized (1 M HCl), centrifuged (3060 g), and the supernatant collected for isoprostane determinations. Finally, results were normalized to total homogenate protein.

Assessment of tissue cGMP

Isolated aortae or renal tissues were thawed and homogenized in buffer containing the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (100 μM, Sigma, MO) (23). Tissue cGMP was determined with a commercial ELISA kit (Cayman Chemical, MI) and normalized against renal homogenate protein.

Tissue gene response

Total RNA was extracted from renal homogenates with a kit (GeneElute; Sigma Aldrich, Sydney, Australia) and cDNA was constructed using BioScript Reverse Transcriptase (Bioline, Sydney, Australia) using a PCR-MasterCycler (Eppendorf, Sydney, Australia) (23, 32). Gene expression was determined by q-PCR, quantified by the comparative Ct method, normalized against β-actin, and expressed as fold-change relative to the sham using primer sequences shown in Table 4.

Renal antioxidant enzyme activity

Enzyme activity was determined using renal homogenates, and normalized against total protein (BCA Assay; Sigma, Sydney, Australia). The homogenized tissue was centrifuged (5000 g, 5 min) and the supernatant further clarified (5000 g, 5 min) and collected for enzyme activity assays. Total SOD activity was measured as described previously (2). Glutathione peroxidase activity was analyzed by monitoring NADPH consumption with H₂O₂ as the cofactor (63).

Western blot analysis

Renal homogenates were normalized to total protein, loaded onto SDS-PAGE, then transferred to nitrocellulose for Western blotting. Antibodies against GPx-1 (Sigma, Australia), β-actin (loading control; Sigma) and p-(serine 276)P65 (Signalway, MD) were employed.

Kidney histology

Kidney sections were de-paraffinized, rehydrated, and thin sections (5 μm) were treated with periodic acid and stained in Schiff's reagent, counterstained with Mayer's hematoxylin, dehydrated, and mounted with DPX solution, as described previously (41).

Immunohistochemistry

Thin sections (5 μm) were cut and mounted onto SuperFrost Plus slides for staining using a Dako Auto-immunostainer. Sections were de-paraffinized, blocked with 3% v/v albumin in TBS-T and 5% v/v goat serum, then incubated with the appropriate primary antibody (dilution 1:200 v/v, 60 min). Controls were incubated with TBS-T (negative control). All sections were washed and incubated with a biotin-conjugated secondary antibody (1:250 v/v, 30 min, Vectastain ABC). Samples treated with secondary antibody alone or TBS-T acted as negative controls. Sections were washed, incubated with streptavidin-conjugated HRP (1:250 v/v, 30 min, Vectastain ABC) and visualized with DAB (DAKO, Sydney Australia). Sections were counterstained with Mayer's hematoxylin, dehydrated, and mounted using DPX solution (DAKO). Images were captured with an Olympus Microscope using a digital camera (DP Controller; v2.2.1.227) and converted to TIFF for handling with MS PowerPoint (2008, v7).

Inflammatory marker CCL2/MCP-1

The pro-inflammatory chemokine MCP-1 was analyzed in samples of plasma and renal homogenates using a commercial AlphaLISA® CCL2 / MCP1 Kit, following the manufacturer's instructions and using a Pherastar Microplate Reader with AlphaScreen standard settings.

Markers for acute kidney injury

Kinase activity assay

An ADP-Glo™ Kinase Assay (Promega, WI) was used to analyze MAPK activity in renal homogenates using phospholipase A2 (a specific substrate for ERK). Where required, MAPK activity was inhibited by addition of the MEK1/2 inhibitor (U0126–IC50 of 72 nM for MEK1 and 58 nM for MEK2; Sigma). Plates were read using a Pherastar Microplate Reader and exported to Microsoft Excel (v7) for further manipulation.

Assessment of renal 3-chloro-tyrosine levels

Renal homogenates were delipidated and precipitated by the addition of 3% w/v sodium deoxycholate and 50% w/v trichloroacetic acid prior to centrifugation at (6000 g×5 min). Pellets were washed with acetone, centrifuged (6000 g×5 min) to remove acetone, dried under a stream of N_2(g), resuspended in 6 M methanesulfonic acid containing 0.2% tryptamine, and finally hydrolyzed under vacuum at 110°C for ∼12 h, as described previously (27).

Detection and quantification of 3-chlorotyrosine (3-Cl-Tyr) was performed by quantitative mass spectrometry (54). Briefly, 3-chloro-[13C9,15N] tyrosine and [15N]-tyrosine (final concentration 1.5 nmol) were added to each sample as an internal standards before hydrolysis, using the protocol outlined above. The hydrolysates were purified using solid-phase extraction columns (Supelco, Sydney, Australia) that were activated with 100% methanol before preconditioning (2×2 ml) using 0.1% TFA/H₂O. Samples were loaded and washed on the columns with 2 ml of 0.1% v/v TFA/H₂O and eluted with 80% v/v methanol/H₂O, dried under vacuum at 60°C, and re-dissolved in 100 μl of 0.1% v/v formic acid. Unmodified tyrosine and 3-Cl-Tyr were detected using an Agilent LC-MS and quantified using an external calibration curve that was constructed with authentic Tyr and 3-Cl-tyr and their corresponding isotopically labeled forms (0–500 pmol). The peak area for each Tyr residue relative to its internal standard was then employed in determinations of the ratio of the 3-Cl-tyr/Tyr ratio.

Statistical analysis

Statistical analysis was performed using GraphPad Prism (Version 5.00 for Windows), and data were evaluated using one-way ANOVA with Newman-Keuls post-hoc test for multiple comparisons; significance was accepted at the 95% confidence level (p<0.05).