Determination of leukotriene A(4) stabilization by S100A8/A9 proteins using mass spectrometry.

Leukotriene A(4) (LTA(4)) is the precursor for the formation of bioactive leukotrienes, but is highly susceptible to nonenzymatic hydrolysis. Although it is chemically reactive, LTA(4) participates in the process of transcellular metabolism, which requires the transfer of LTA(4) from one cell to another for the production of additional leukotrienes. Due to the susceptibility of LTA(4) to hydrolysis, various methods have been used to measure the half-life of LTA(4) in the presence of different proteins in efforts to understand how it is transported between cells. In this work, a new liquid chromatography mass spectrometry technique was developed to improve upon these previous assays that analyzed LTA(4) directly. The new technique derivatizes LTA(4) to stable compounds for analysis and removes the potential for sample decomposition between analytical runs. This assay was used in measuring the capabilities of the S100A8/A9 protein complex isolated from human neutrophils to stabilize LTA(4). It was determined that the S100A8/A9 protein complex protects LTA(4) from hydrolysis in a Ca(2+) dependent manner and increases LTA(4) half-life to in excess of 35 and 5 min at 4 degrees C and 37 degrees C, respectively.

Leukotrienes are a class of biologically active metabolites from arachidonic acid that play a role in various physiological processes. These compounds are biosynthesized within cells via a 5-lipoxygenase (5-LO) enzymatic pathway ( Scheme 1 ) ( 1-3 ). Leukotriene biosynthesis is initiated by the activation of cytosolic phospholipase A 2 , which in turn releases arachidonic acid from the sn -2 position of membrane phospholipids ( 4,5 ). The free arachidonic acid is then presented to 5-LO by 5-LO activating protein, FLAP ( 6,7 ). Within 5-LO, molecular oxygen is added to carbon-5 sibility remained that the LTA 4 could still undergo nonenzymatic hydrolysis despite the use of alkaline solvent conditions. In this report, a new method of measuring LTA 4 half-life is described that measured chemically stable ethoxy derivatives of ⌬ 6 -trans -LTB 4 and 5,6-diHETE using reversed phase liquid chromatography mass spectrometry (RP-LC/MS). In addition, the stabilization ability of the S100A8/A9 protein complex, which is found in large quantities within neutrophils, was analyzed.

LTA 4 half-life assay
For determination of LTA 4 stabilization, a solution (200 l) was made with the free acid of LTA 4 (5 M fi nal concentration), 1.7 M protein or stabilizing agent in 0.1 M phosphate buffer, pH 7.4. LTA 4 free acid was produced by hydrolysis of LTA 4 methyl ester using a 4:1 acetone:NaOH mixture as previously described ( 22 ). For conditions with Ca 2+ , a fi nal concentration of 60 M Ca 2+ was present from the addition of a stock CaCl 2 solution. At various times, 25 l aliquots were removed from the larger solution and the nonenzymatic hydrolyses were stopped by addition of 25 l of stimulation is released from the neutrophils to participate in transcellular metabolism (18). The susceptibility of LTA 4 to nonenzymatic hydrolysis indicates that some type of protection must be afforded to LTA 4 during its transport following synthesis for the transcellular processes.
Previous work has focused on assessing the ability of various proteins to stabilize LTA 4 against nonenzymatic hydrolysis. Serum albumin increases the half-life of LTA 4 to more than 20 min at 25°C at protein concentrations found in plasma ( 12 ). Fatty acid binding proteins were later studied and found to also increase LTA 4 half-life ( 19,20 ). However, neither of these proteins is found in large concentrations within neutrophils. The LTA 4 stabilizing protein in neutrophils is currently unknown, yet neutrophils are a major source of LTA 4 in transcellular production of leukotrienes. A possible protein of interest in neutrophils is the S100A8/A9 complex, which is a major arachidonate binding protein ( 21 ).
The LTA 4 half-life measurements in the previous studies were performed by measuring LTA 4 directly by reversed phase HPLC (RP-HPLC) with UV spectroscopy utilizing the unique chromophore of the triene epoxide. The pos-Scheme 2. Formation of stable products by nucleophilic addition to leukotriene A 4 . Scheme 1. Leukotriene synthesis pathway. samples contained 30 mM DTT and were heated at 95°C for 5 min. Coomassie staining was performed with Gel Code Blue stain (Pierce, Rockford, IL) following the directions from the manufacturer.
For Western blotting, the proteins were electrolytically transferred to a membrane with a mixture of 25 mM Tris buffer, 192 mM glycine and 0.1% SDS and 20% methanol. Nonspecifi c binding of antibodies was blocked by washing the membrane with 5% powdered milk in water for 30 min. The membrane was then incubated with rabbit anti-S100A8 (calgranulin A) or anti-S100A9 (calgranulin B) antibody (Santa Cruz Biotechnologies, Santa Cruz, CA) at 1:1000 dilution overnight in 0.01 M PBS buffer, pH 7.4 containing 5% BSA at 4°C. After several washes in 20 mM Tris buffer with 0.5 M NaCl, 0.05% Tween 20 (3 × 15 min), the bound antibodies were detected by rinsing the membrane with HRP conjugated donkey anti-rabbit IgG antibody (Affi nity Bioreagents, Golden, CO) at 1:2500 in Tris buffer for 1 h. The membrane was then again washed 3 × 15 min with Tris buffer, 0.05% Tween 20. HRP labeled proteins were then detected using a Pierce ECL Western blotting kit.

Nucleophillic addition to LTA 4
The susceptibility of LTA 4 to nonenzymatic hydrolysis is due to the epoxide conjugated triene moiety. In aqueous conditions, the epoxide moiety can be protonated, which in turn leads to the opening of the epoxide to form a delocalized carbocation to which water can add to form stable 5,12 diHETEs and 5,6 diHETEs ( 11 ). The hydrolysis reaction is a typical nucleophillic addition of water. To form unique stable products at designated time points, a more reactive nucleophile was added to the reaction mixtures. The addition of 1M HCl in absolute ethanol provided another nucleophile to react with LTA 4 . The acidic conditions caused the rapid opening of the epoxide, which allowed the addition of ethanol to form unique ethoxy products that are similar to the nonenzymatic hydrolysis products of LTA 4 . These ethoxy products are chemically stable like the nonenzymatic products of LTA 4 hydrolysis and therefore remove the chemical liability inherent in LTA 4 during analysis.
The ethoxy derivatives were then analyzed by LC/MS and LC/MS/MS to determine whether the acidic ethanol that added to LTA 4 formed products truly similar to those produced through nonenzymatic hydrolysis. The ethoxy derivatives were separated using RP-HPLC into two distinct chromatogram peaks. LC/MS of both chromatogram peaks generated [M-H] − carboxylate anions at m/z 363.3. The CID of the [M-H] − ion of the less lipophilic peak in the RP-HPLC produced the spectrum shown in Fig. 1A . The CID of m/z 363.3 resulted in product ions of m/z 345 and a prominent m/z 317 that correspond to losses of water and ethanol, respectively, from the parent carboxylate ion. Additional product ions were seen at m/z 255, m/z 273, and m/z 299 that result from the additional neutral loss combinations of water, ethanol, and carbon dioxide from the parent carboxylate ion. The major fragment ion produced from the carboxylate ion was m/z 203. A similar minor product was seen in spectra analyses of 5,12 diHETE that 1M HCl in absolute ethanol. The reaction mixture was allowed to stand for 1 min, then adjusted to approximately pH 3 by addition of 1M NaOH, and fi nally diluted to a total volume of 150 l with water. An internal standard of 5-oxo-eicosa-6 E ,8 Z ,11 Z ,14 Z -tetraenoic acid (5-oxoETE), (20 ng), was then added. The individual samples were then prepared to remove proteins with solid phase extraction (SPE) cartridges (Strata C18-E, 100 mg/1ml; Phenomenex, Torrance, CA). The SPE cartridges were conditioned by running 1 ml of methanol and then 1 ml of water through each cartridge. After the samples were added to the cartridges, 2 ml of water was run through the columns before 1 ml of methanol was eluted and collected. The methanol was removed in vacu, and then 500 l of CH 2 Cl 2 was added to each sample, which usually contained residual water. The CH 2 Cl 2 layer was removed from the extractions and dried down under a N 2 stream. The ethoxy derivatives from LTA 4 were then dissolved in a 100 l of methanol:water (1:1) for LC/MS/MS analysis.

Mass spectrometric analysis of ethoxy, hydroxy derivatives and determination of LTA 4 half-life
An aliquot of each sample (80 l) was injected into a RP-HPLC system, and the 5-hydroxy, 12-ethoxy-⌬ 6 -trans -LTB 4 s (5-OH, 12-EtO-LTB 4 s) and the 5,6 ethoxy, hydroxyeicosatetraenoic acids [5,6-(EtO,OH)-ETEs] from LTA 4 were separated using a C18 column (Gemini, 2 × 150 mm, 5 m; Phenomenex) eluted at a fl ow rate of 200 l/min with a solvent system composition starting as 75% aqueous ammonium acetate (1 mM, pH 5.7, solvent A) and 25% methanol also 1 mM in ammonium acetate (solvent B). The starting solvent composition was held for 2 min after injection and then programmed to 90% solvent B in 6 min where it was held for 5 min prior to recycling to 25% solvent B. The 5-OH, 12-EtO-LTB 4 s and 5,6-(EtO,OH)-ETEs were monitored using the collision induced dissociation (CID) spectra m/z 363.4 → 317.2 and 5-oxoETE was followed using m/z 317.2 → 203.2. For determination of the half-life of LTA 4 in the presence of different stabilizing agents, the peak area ratio (5-OH, 12-EtO-LTB 4 s:5-oxoETE was determined from LC/MS/ MS chromatograms at the various time points. The natural logarithm of the peak area ratios were then plotted versus time. The rate of decay was determined as the slope of the linear regression of a logarithmic plot, which in turn was used to determine the half-life from the standard half-life equation: LTA 4 half-life = ln 2 /-(rate of decay).

Neutrophil cytosol and S100 proteins isolation
Human polymorphonuclear leukocytes (PMNs) were isolated from peripheral blood of healthy volunteers as described previously ( 23 ). Isolated PMNs were taken up in homogenization buffer (1.7 × 10 8 cells/ml). Homogenization buffer was prepared with 1 Complete mini tablet (Roche Applied Sciences, Indianapolis, IN) in 7 ml 0.2 M phosphate buffer with 0.1 M NaCl, pH 7.4. The ice-cooled cells were disrupted using a Sonics Vibracell VCX 600 sonicator (Newtown, CT) at 24% amplitude (4 × 15s). The homogenate was centrifuged at 10,000 g for 15 min at 4°C. The supernatant was then isolated and further centrifuged at 100,000 g for 1 h at 4°C. This supernatant was collected and used as the neutrophil cytosolic fraction. S100 proteins were isolated by purifying the cytosol fractions using fast-protein liquid chromatography (FPLC) with a Superdex 75 column (GE Healthcare, Piscataway, NJ). The elution solvent was 0.01 M PBS with 0.2 M NaCl, pH 7.4 at a fl ow rate of 1 ml/min.

Analysis of S100 proteins by Coomassie staining and immunostaining on Western blots
Cytosol fractions or purifi ed S100s were separated by SDS-PAGE gels (4-20% acrylamide gradient). For denatured gels, the and the more lipophilic peak was the 5,6-(EtO,OH)-ETEs.

Validation of the new assay
In order to validate the new derivatization assay, the ability of previously studied agents to stabilize LTA 4 was measured and then compared with previously reported values. The fi rst set of studies involved a 50% ethanol mixture in 0.1 M phosphate buffer at pH 7.4. Previous work had shown that the high organic composition of this mixture increased the half-life of LTA 4 to approximately 70 min at 4°C when measuring the LTA 4 directly by UV spectroscopy ( 12 ).
In this study, LTA 4 was placed in the 50% aqueous ethanol mixture and aliquots were removed at corresponding time points ( Figure 2 ). Acidic ethanol was immediately added to the aliquots to form ethoxy derivatives from the opening of the epoxide. After neutralizing the samples with NaOH, an internal standard of 5-oxoETE was added to each sample. The samples were then analyzed by RP-LC/ MS/MS while monitoring for the unique transitions m/z 363.3 → m/z 317.2 for the 5-OH, 12-EtO-LTB 4 s and arose from the dehydration involving the C-12 hydroxyl group to form the 5-oxoETE, then cleavage of the C-5,C-6 bond with loss of a neutral ketene ( 24 ).
A second more lipophilic peak was seen in the RP-HPLC chromatogram of the ethoxy derivatives also with a [M-H] − ion of m/z 363.3. The CID of this peak is shown in Fig. 1B . The CID of the precursor ion m/z 363.3 resulted in the formation of many of the same product ions seen in the less lipophilic peak described above. Distinct peaks of m/z 345 and m/z 317 were formed from the neutral loss of water and ethanol. In addition, product ions of m/z 255, m/z 273, and m/z 299 were again seen that are the result of various neutral loss combinations of water, ethanol, and carbon dioxide from the parent carboxylate ion. The major m/z 203 fragment was also seen in this peak; however, it was not the major fragment ion. The predominant product ion was m/z 115. In LC/MS/MS scans of 5,6-diHETEs, the major fragmentation ion was also m/z 115 that comes from the cleavage of the C-5,C-6 carbon bond with transfer of the hydrogen atom from the 5-hydroxy group to C-8 ( 24 ). Based on the fragmentation patterns from the two ethoxy derivative peaks, the two derivatives separated in the same manner as the nonenzymatic hydrolysis peaks of LTA 4 . The less lipophilic peak was the 5-OH, 12-EtO-LTB 4 s  were similar to the values measured using UV absorbance to monitor LTA 4 directly.

LTA 4 stabilization by S100A8/A9 protein complex isolated from PMNs
The new LC/MS/MS assay was then used to measure the ability of the S100A8/A9 protein complex isolated from PMNs to stabilize LTA 4 . Previous reports indicated that the S100A8/A9 complex is the major fatty acid binding protein within neutrophils ( 21 ). The protein complex has been shown to have a high affi nity for arachidonic acid ( 25 ). In this work, the ability of the S100 complex to increase the half-life of LTA 4 is measured in the same manner as the work performed with BSA as the stabilizing protein.
First, the S100A8/A9 protein complex had to be isolated from human PMNs. Approximately 200 × 10 6 PMNs were obtained from a blood draw for the protein isolations. After disrupting the cells, a series of centrifugations were used to isolate a solution of the cytosolic proteins. The proteins were then separated by FPLC size exclusion chromatography using an isocratic mobile phase while collecting fractions every 2 min. The fractions were then analyzed by immunoassays to determine which fractions contained the S100A8 and S100A9 proteins. Individual Western blots were performed using antibodies for S100A8 and S100A9 ( Fig. 4A ). From the analysis, it was obvious the S100 proteins were found primarily in two isolated fractions from the protein separation. The purity of the protein in the fractions was assessed by Coomassie gel staining of the FPLC fractions 7 and 8 that were shown to contain the S100 proteins by Western blotting ( Fig. 4B ). From the gel, the individual monomers of S100A8 and S100A9 were visible as distinct bands. The S100A8 monomer had a 5,6-(EtO,OH)-ETEs and m/z 317.2 → m/z 203.2 for 5-oxo-ETE. The LC/MS/MS chromatograms from monitoring the half-life of LTA 4 in the presence of 50% aqueous ethanol revealed two peaks for the 5-OH, 12-EtO-LTB 4 s and 5,6-(EtO,OH)-ETEs (shown as the solid lines and the internal standard shown as the dotted line in Fig. 2 ). The peak area from the 5-OH, 12-EtO-LTB 4 s ion transition was then ratioed to that from the 5-oxoETE ion transition. The natural logarithm of ion transition for the 5,OH,12-EtO-LTB 4 s was then plotted versus time to determine the rate of LTA 4 decay via hydrolysis ( Fig. 3 ). Using this technique, the half-life of LTA 4 in the presence of 50% aqueous ethanol was determined to be 68 ± 4 min (SEM, n = 6) whereas the measured half-life in PBS under the assay conditions employed was 5 ± 1 min ( Fig. 3 ). In separate experiments, the signal for the 5-OH, 12-EtO-LTB 4 s ( m/z 363.3 → 317.2) as well as the signal from the more abundant product ion ( m/z 363.3 → 203) were used to defi ne the half-life of LTA 4 and compared. As expected, the signal (and corresponding response factor) for the later transition was higher, but the calculated half-life of LTA 4 was the same within experimental error (± 4 min). Furthermore, using the less abundant hydrolysis product 5,6-(EtO,OH)-ETE observed at the longer retention time yielded the same estimated halflife (± 1 min) for LTA 4 stabilized by ethanol.
The new assay was then used to measure the stabilization of LTA 4 in the presence of a protein, specifi cally BSA. Previous work had shown the capability of 5 mg/ml human albumin to increase the half-life of LTA 4 at 25°C to approximately 19 ± 2.5 min ( 12 ). In this study, LTA 4 was added to a 5 mg/ml mixture of BSA in 0.1 M PBS buffer, pH 7.4. At various intervals, aliquots were removed and the remaining LTA 4 was derivatized with acidic ethanol. LC/MS/MS analysis was performed as described above to determine the half-life in the presence of BSA. With the new assay, 5 mg/ml BSA was determined to increase the half-life of LTA 4 to 18 ± 3 min at 25°C (SEM, n = 6) (Data not shown). The half-lives measured with this new assay  lized by binding (saturating concentrations of S100 proteins were not employed) and therefore, unbound LTA 4 would be immediately hydrolyzed ( Fig. 3 ) and only bound LTA 4 would be hydrolyzed based on rate of leaving the protein complex.
The LTA 4 stabilization was observed not only at lower temperatures. If the reaction mixtures were heated to 37°C, the half-life of LTA 4 was considerably shorter. In the absence of Ca 2+ , the half-life of LTA 4 in PBS buffer and 1.7 M S100A8/A9 protein was measured as approximately 1.5 min. The inclusion of Ca 2+ with 1.7 M S100A8/A9 protein increased the half-life of LTA 4 to over 5 min ( Fig.  5B ). This increase in LTA 4 half-life would be a reasonable amount of time for possible transport of LTA 4 during transcellular metabolism pathways.

DISCUSSION
The detailed mechanism by which leukotriene transcellular metabolism occurs is still undetermined. One of the unresolved questions is how LTA 4 is stabilized long enough for transport between donor and acceptor cells for the production of leukotrienes. Previous studies have shown that LTA 4 is susceptible to nonenzymatic hydrolysis. At 37°C, the half-life of LTA 4 has been reported as approximately 3 s in PBS buffer, pH 7.4 ( 12 ). Therefore, it is generally assumed that the reactive molecule is bound to a stabilizing protein. Several studies have focused on identifying these potential stabilizing proteins. Albumin was the fi rst protein identifi ed that was capable of increasing the half-life of LTA 4 to the order of minutes ( 12 ). Subsequent work found various fatty acid binding proteins that were also capable of stabilizing LTA 4 ( 19,20 ). molecular weight of 10.8 kDa and the S100A9 monomer a mass of 13.2 kDa ( 26 ). The reducing conditions of the gel and presence of DTT caused the separation of the heterodimeric S100A8/A9 complex typically found within unstimulated PMNs.
With the protein complex isolated, the half-life of LTA 4 in the presence of various concentrations of the S100A8/ A9 complex was measured using the LC/MS/MS assay. The ability of the S100A8/A9 complex to bind arachidonic acid is known to be a Ca 2+ dependent process ( 27 ). The S100A8/A9 heterodimer is the normal state of S100A8/A9 proteins in nonstimulated PMNs and is unable to bind fatty acids ( 25 ). However, in the presence of Ca 2+ , two S100A8/A9 heterodimers associate to form a heterotetrameric complex that possesses fatty acid binding abilities. In this study, the half-life of LTA 4 was measured with both Ca 2+ present and absent from the protein mixtures. Because the S100A8/A9 proteins used in this study were isolated from the cytosol of nonstimulated human neutrophils, little endogenous arachidonic acid would be present in the partially purifi ed protein to compete with added LTA 4 due to the poor fatty acid binding properties of the S100A8/A9 proteins in the presence of low Ca 2+ ion concentrations ( 25 ) and the low levels of free arachidonate present in the neutrophil cytosol ( 23 ). For the experiments with Ca 2+ , the isolated S100 proteins were incubated with Ca 2+ for 30 min prior to the addition of LTA 4 to allow for the formation of the heterotetrameric complex. Following the incubation, LTA 4 was added to the protein mixture for half-life studies at 4°C and 37°C. At progressing times, aliquots were removed and reacted with acidic ethanol to form ethoxy derivatives in the same manner as BSA described earlier. The LC/MS/MS analyses of the ethoxy derivatives demonstrated the stabilizing effect of the S100A8/A9 protein complex toward LTA 4 , particularly in the presence of Ca 2+ ( Fig. 5 ). The half-life of LTA 4 was measured at both 4°C and the more biologically relevant 37°C. The S100A8/A9 complex showed a concentration and Ca 2+ dependent stabilization of LTA 4 . At 4°C, LTA 4 (5 M) had a half-life of 5 min in PBS buffer with no stabilizing proteins. The inclusion of S100 proteins at concentrations of 2.6 M and 1.7 M in the absence of Ca 2+ demonstrated minimum stabilization of LTA 4 (5 M) in comparison to buffer alone. A 40 g/ ml mixture of the isolated cytosolic proteins, which contained approximately 40-45% S100 proteins, also showed little stabilization of LTA 4 in the absence of Ca 2+ . However, a significant increase in the ability of the S100A8/A9 protein to stabilize LTA 4 was seen with the addition of an excess of Ca 2+ to the reaction mixtures. In a concentration-dependent manner, a S100A8/A9 mixture at a concentration of 2.6 M and 1.7 M increased the half-life of LTA 4 (5 M) to 37 min and 22 min, respectively, showing the dependency of the binding protein concentration on LTA 4 stabilization as observed previously for fatty acid binding proteins ( 19 ). Even the isolated 40 g/ml cytosolic protein mixture increased the LTA 4 halflife to 13 min ( Fig. 5A ). In protein stabilization studies, it was likely that only a fraction of the added LTA 4 was stabi- substituent from both the 5,12 and 5,6 products. Although this was not the most predominant fragmentation pathway for either product, it did provide a method to ensure the products measured came only from the LTA 4 derivatization. If greater sensitivity were required in future work, the precursor/product ions m/z 363.3 → m/z 203.2 and m/z 363.3 → m/z 115.0 could be monitored simultaneously for the 5-OH, 12-EtO-LTB 4 s and 5,6-(EtO,OH)-ETEs, respectively.
The fi rst two systems tested with the new LC/MS/MS assay were two that had been previously used in the initial measurements of LTA 4 half-lives. The LTA 4 half-life in 50% aqueous ethanol was measured at 76 ± 5 min, which corresponded well with the previously measured value of approximately 70 min in the same system ( 12 ). The halflife of LTA 4 was measured as 18 ± 3 min in 5 mg/ml BSA at 25°C. This compared well to the value of 19 ± 2.5 min measured previously ( 12 ).
Because the results of the new LC/MS/MS assay matched well with previously reported LTA 4 half-lives under different conditions, it was used in an effort to determine the stabilizing protein found within stimulated PMNs. The only fatty acid binding protein that has been reported to be expressed by the human PMNs is the S100A8/A9 complex ( 21 ). S100A8/A9 proteins constitute approximately 40-45% of the cytosolic proteins within PMNs ( 28 ). Upon stimulation of PMNs and monocytes, the protein complex has been shown to move to the plasma membrane and secreted (29)(30)(31). This protein complex is found as a heterodimer in nonactivated PMNs; however, in the presence of Ca 2+ from stimulated neutrophils, two of the S100A8/A9 heterodimers associate to form a heterotetrameric complex. Studies with [ 3 H]labeled arachidonic acid have demonstrated that only the heterotetrameric complex is capable of binding fatty acids ( 25 ). The S100 complex shows a high specifi city toward polyunsaturated fatty acids and the greatest affi nity for arachidonic acid with a K d of 0.13 M.
The S100 proteins were isolated using a simple size exclusion chromatography technique that was shown to provide the proteins with good purity as demonstrated by Coomassie gel staining. Next, the ability of the purifi ed proteins to stabilize LTA 4 was measured. These experiments indicated that the S100A8/A9 complex stabilized LTA 4 in a protein concentration and Ca 2+ dependent manner. At both 4°C and 37°C, the S100 complex (1.7 M) provided only slight protection of LTA 4 was compared with 0.1 M PBS, pH 7.4 buffer. However, if the S100 proteins were incubated with Ca 2+ for 30 min prior to their use in stabilizing LTA 4 , a signifi cant increase in the half-life of LTA 4 was seen. This stabilization was shown to be protein concentration dependent as demonstrated with the LTA 4 half-life increases with the two purifi ed S100 samples as well as the more dilute PMN cytosolic mixture at 4°C. Even at the more biologically relevant temperature of 37°C, the S100 proteins were able to increase the half-life of LTA 4 to in excess of 5 min. This seems a reasonable amount of time for LTA 4 to potentially be passed from a donor PMN to an acceptor cell for use in transcellular metabolism.
These previous studies relied upon the analysis of the LTA 4 molecule itself using RP-HPLC. The aqueous solvent system was kept suffi ciently alkaline to reduce the rate of LTA 4 hydrolysis. However, even under these conditions, hydrolysis of LTA 4 could still occur. The studies of albumin and fatty acid binding proteins relied on unique HPLC retention time and UV spectrophotometry to monitor LTA 4 utilizing the distinct chromophore at 280 nm from the epoxide triene moiety ( 12,19,20 ). In the study with fatty acid binding proteins, LC/MS/MS analysis was also used to both specifi cally monitor LTA 4 and to increase the sensitivity of the assay in relation to the UV analysis ( 20 ). Although mass spectrometry successfully increased the sensitivity in specifi c analysis of LTA 4 , it still suffered from the potential drawback of additional LTA 4 hydrolysis in the RP-HPLC solvents during runs and in the times awaiting analyses.
In this work, a new assay was developed for measuring the ability of different reagents and proteins to stabilize LTA 4 . The new method utilized LC/MS/MS; therefore, it had higher sensitivity and specifi city associated with mass spectrometry over absorbance spectroscopy. The advantage of this new technique over the previous mass spectrometric assay was rapid conversion of LTA 4 into chemically stable 5-OH, 12-EtO-LTB 4 s and 5,6-(EtO,OH)-ETEs by an acidic ethanol mixture that were then analyzed. Precedence for the derivatization of LTA 4 with an acidic alcohol was used in the initial identifi cation of the structure of LTA 4 ( 11 ).
The nucleophilic addition of acidic ethanol opened the epoxide ring of LTA 4 to produce derivatives that were similar to nonenzymatic hydrolysis products. Under RP-HPLC conditions, the 5-OH, 12-EtO-LTB 4 s eluted before the 5,6-(EtO,OH)-ETEs, which was the same elution order as the ⌬ 6 -trans -LTB 4 s and 5,6-diHETEs. LC/MS/MS analysis of the products displayed spectra with similar fragmentation patterns as nonenzymatic 5,12-and 5,6-diHETES ( 24 ). Both sets of regioisomers from the ethoxy derivatives and nonenzymatic products had fragment ions resulting from various neutral loss combinations of ethanol, water, and carbon dioxide. The most abundant fragment from the 5,6 products of the derivatization and nonenzymatic hydrolysis were m/z 115, which likely resulted from a chargeremote fragmentation of the C-5,C-6 bond. The 5,12 derivatives and hydrolysis products also had similar fragmentation patterns, but the most abundant fragments differed. With ⌬ 6 -trans -LTB 4 s, the major LC/MS/MS fragment was m/z 195 that resulted from a charge-driven fragmentation of the C-11, C-12 bond. The 5-OH, 12-EtO-LTB 4 s had a major fragment of m/z 203 without any formation of an m/z 195. In the CID spectra of ⌬ 6 -trans -LTB 4 s, a minor m/z 203 fragment was previously described ( 24 ).
Once the products from the LTA 4 derivatization were identifi ed by RP-LC/MS/MS, the next step was to incorporate the derivatization technique into a LC/MS/MS assay for determining LTA 4 half-lives. The mass fragmentation chosen for monitoring both the 5-OH, 12-EtO-LTB 4 s and the 5,6-(EtO,OH)-ETEs were m/z 363.3 → m/z 317.2 because these were unique ions from the loss of the ethoxy One matter of concern with the values obtained from the experiments with the S100 proteins was the measured half-life of LTA 4 being approximately 1 min in PBS at 37°C in comparison to the previously reported half-life of approximately 3s under these conditions ( 12 ). This original value was extrapolated from a series of experiments with different percentages of aqueous ethanol. Although this original value was only from extrapolation, it is believed the difference in this experiment comes from a difference in the reaction set-up. The LTA 4 that was added to the reaction mixtures was in a (4:1) acetone:0.25 M NaOH mixture. The reaction mixtures used in the experiments described here were only several hundred l of solution. It is believed that the addition of even the small quantities of the LTA 4 mixture into these mixtures was probably overloading the buffering capacity of the PBS, thereby increasing its pH. The more alkaline buffer then stabilized LTA 4 to some extent. The original experiments with UV spectroscopy used larger quantities of solutions for their experiments so the potential of overrunning the buffering capacity of the solutions was reduced.
This new LC/MS/MS technique is advantageous over previously used methods. The primary advantage of this new assay is the ability to perform the half-life experiments with low quantities of proteins and LTA 4 due to the high sensitivity and selectivity associated with LC/MS/MS. Thereby, it should permit the studies of additional proteins and reagents. The discovery that the highly abundant S100A8/A9 in the human neutrophil is capable of stabilizing LTA 4 suggests that these proteins and formation of their complex may play an important role in transcellular biosynthesis of leukotrienes in vivo, mediated by the human neutrophil.