2.4.1 Enzyme Immunoassay o f Serum Leukotrienes and Prostaglandins
Oxidation of arachidonic acid via cyclooxygenase or lipoxygenase results in the production of vasoactive eicosanoids such as the thromboxanes, prostacyclin, prostaglandins and leukotrienes. Cyclooxygenase and lipoxygenase activity occurs in a wide variety o f cells, which in the kidney include mesangial cells, endothelial cells, mTAT cells, collecting duct cells and inflammatory cells. Assays of serum leukotriene b4 (LTB4, a potent vasoconstrictor and chemoattractant), thromboxane b2 (TxB], a breakdown product of TxA2, a potent vasoconstrictor) and 6-keto- prostaglandin Fia (6-k-PGFia, a breakdown product o f prostacyclin, a potent vasodilator) were made by enzyme immunoassay (ELISA) using commercially available enzyme|mmunoassay systems.
The principle of enzyme immuoassay depends on competition between unlabelled eicosanoid in the serum sample and a fixed quantity of peroxidase-labelled ligand for a limited number of binding sites on a specific antibody. The amount of peroxidase-labelled ligand bound by the antibody is therefore inversely proportional to the concentration of added unlabelled ligand. The peroxidase ligand bound to the antibody is immobilized on polystyrene microtitre wells precoated with a second antibody. Unbound ligand can therefore be removed from the well by means o f simple washing procedures. The amount of peroxidase labelled eicosanoid bound to the antibody is determined by the addition o f a substrate such as tetramethylbenzidine/hydrogen peroxide. The reaction is stopped by acidification (1 M sulphuric acid), and the resultant colour read at 450 nm in a microtitre plate photometer. The concentration o f unlabelled eicosanoid in the sample is determined by interpolation from a standard curve.
Ch a p t e r 2 Ma t e r i a l sa n d Me t h o d s
Serum samples (0.35 ml) were taken from the renal vein o f anaesthetized rabbits after 10, 30, 60, 120, 240 and 360 min of reperfiision, snap frozen in liquid nitrogen and stored at -20®C until required. On thawing, samples were purified by a triple phase wash (with 5 ml redistilled water, 5 ml ethanol (10% v/v) and 5 ml hexane) on C2 reverse phase chromatography columns, eluted with methyl formate, ev^orated to dryness and reconstituted in 0.35 ml phosphate buffered saline (assay buffer). ELISA assays were then carried out as described by Amersham. The average optical density (OD) was calculated for each set o f duplicate wells. The percentage o f bound ligand for each standard and sample was then calculated according to the following relationship:
%B/Bq = (standard or sample OD - non specific binding OD) x 100 (Bo OD - non specific binding OD)
where Bq is the OD in the zero standard wells. The standard curve was constructed by plotting B/Bq as a function o f the log eicosanoid concentration (in pg) per well. The pg/well value of each sample was read directly from the standard curve and multiplied by 20 to give pg/ml of semm.
2.4.2 Determination o f Lipid Peroxidation in Rabbit Kidneys
Oxygen derived free radical attack can lead to the propagation o f membrane lipid peroxidation of polyunsaturated fatty acids (Halliwell and Gutteridge, 1984). Lipid radicals are formed which can break down via further interaction with molecular oxygen to form lipid hydroperoxides and eventually a plethora of low molecular weight products. The susceptibility o f renal homogenates to lipid peroxidation was determined after incubation o f homogenates at 37^C in open vessels with mechanical shaking for 60 min by fluorescent measurement o f two such low molecular weight markers of lipid peroxidation.
Nephrectomies were performed as described in Section 2.2.3. Kidneys were flushed with 40 ml HCA, and stored as described above at 0-2*^C for the required time (0-72 h). After the storage period, kidneys were dissected into cortex and medulla, suspended (5% w/v) in phosphate- buffered saline (40 mM KH2PO4 : K2HPO4; pH 7.4) and homogenized using a Potter-Elvehjem homogenizer. The protein content o f homogenates (stored at -70^C) was determined later by the method o f Lowry et a l, (1951) using bovine serum albumin as the standard (see Section 2.4.3). The two markers o f lipid peroxidation determined were:
(i) Lipid-soluble Schijfs base formation (Bidlack and T^pel, 1973). Duplicate aliquots (1 ml) were removed after 0 and 60 min o f incubation at 37®C, the lipids extracted into 4 ml chloroform : methanol (2 ; 1) in tubes pre-washed with chloroform/methanol to remove possible contamination, vortexed and centrifuged in a bench-top centrifuge for 10 min at 3000 rpm. The lower chloroform layer was monitored for a fluorescence maximum between 400 and 450 nm (usually 417 nm) when excited at 360 nm on a Perkin-Elmer LS 50 spectrofluorimeter standardized against a fluorescent polymer block. Readings were corrected by those obtained from PBS buffer alone. The change in fluorescence intensity following the incubation period was taken to indicate tissue susceptibility to lipid peroxidation.
(ii) Thiobarbituric acid reactive material (TBA-RM) formation (Suematsu and Abe, 1982). Duplicate aliquots (1 ml) were removed after 0 and 60 min o f incubation at 37®C. These aliquots were then incubated at 95^C with 100 pi sodium dodecyl sulphate (SDS), 150 pi phosphotungstic acid (PTA), 0.5 ml thiobarbituric acid (TBA) and 1 ml hydrochloric acid (1 M). The SDS was added to the homogenate to solubilize the fat in the renal tissue. Lipids were isolated by precipitation with semm protein using the phosphotungstic acid-hydrochloric acid system. After the incubation period, samples were allowed to cool before addition of 2.5 ml butan-l-ol, then vortexed and centrifuged in a bench-top centrifuge for 10 min at 3000 rpm. TBA reactivity was determined in the upper butanol phase by monitoring fluorescence at 553 nm when excitated at 515 nm. The change in fluorescence intensity following the incubation period was taken to indicate tissue susceptibility to lipid peroxidation. A standard curve was constmcted using a series o f different concentrations o f malondialdehyde (MDA) tetraethylacetal and results were expressed as nmol MDA equivalents/mg protein/h.
2.4.3 Protein Determination
Lipid peroxidation results were corrected for variability in the protein content of homogenates (Lowry et al.,, 1951). Stored homogenates were re-homogenized and diluted 20-fold in PBS buffer. Duplicate aliquots (200 |xl) were then added to 3 ml of a reaction mixture containing potassium sodium-taitrate (0.02%), CUSO4 (0.01%) and Na2C0 3 (2%) in 0.1 M NaOH. After incubating at room temperature for 30 min, 300 pi of Folin and Ciocalteu's phenol reagent (2 M) was added, and the tubes were vortexed and incubated at room temperature for exactly 30 min before reading at 650 nm on a Uvicon 81 OP spectrophotometer. The protein concentration was
Ch a p t e r 2 m a t e r i a l sa n d Me t h o d s
interpolated from a standard curve constructed from a series o f concentrations o f bovine serum albumin (0 -2 0 0 mg protein) subjected to the same procedure.
2.4.4 Measurements o f Serum Urea and Creatinine
Blood samples (2 ml) were taken from the marginal ear vein (swabbed with xylene to dilate the vein) o f rabbits which had been allowed to recover after autografting, using a heparinized syringe and a 23-gauge needle immediately after completion o f the autograft operation and subsequently on days 1 to 4, 6 and 10. Samples were immediately centrifuged in a bench-top centrifuge at 1000 x g and stored at -20®C until required for assay o f urea and creatinine.
(i) Serum urea concentrations were measured by the enzymatic colorimetric method described by Fawcett and Scott (1960), using a commercially available kit (Boehringer Mannheim). The principle depends on cleavage of urea with urease (Berthelot's reaction):
urea-2H2 0 urease - -► 2NH4^ + COg^
Ammonium ions (NH4 ) react with phenol and hypochlorite to give a coloured complex.
Serum samples were first diluted 20-fold in isotonic saline and mixed thoroughly. Duplicate aliquots (200 p,l) were incubated for 10 min at 37®C with 100 |il urease (10 U ml) in 50 mM phosphate buffer, prior to addition of 5 ml phenol (0.106 M) and 5 ml sodium hypochlorite (11 mM) and incubated for a further 15 min at 37®C. The absorbance at 550 nm was read in a Uvicon 81 OP spectrophotometer against a distilled water blank, and the concentration o f urea calculated from the equation:
c (mmol/L) = 10 x Abs sample Abs standard
in which the standard was urea (0.5 mM).
(ii) Serum creatinine concentrations were measured by the colorimetric method described by Bartels (1971), using a commercially available kit (Boehringer Maimheim). Creatinine forms a coloured complex with picrate in alkaline medium (Jaffe's reaction). The assay principle depends on measurement of the rate o f formation o f the complex.
Serum samples (250 pi) were first deproteinized by addition o f 250 pi sodium chloride (0.9%) and 500 pi trichloroacetic acid (1.2 M). After vortexing and centrifuging at 10,000 G for 2 min, duplicate samples (500 pi) o f the pure supernatant were added to an equal volume of a 1:1 v/v mixture o f picric acid (35 nM) and sodium hydroxide (1.6 mM). The samples were mixed and incubated at 25®C in an Eppendorf 5320 thermostat for 20 min, and the absorbance at 520 nm measured in a Uvicon 81 OP spectrophotometer against a distilled water blank. The creatinine concentration was calculated from the formula:
c (mmol/1) = 177 X Abs sample Abs standard
in which the standard was creatinine (177 pmol/1).