Development of metal-ion containing catalysts for the decomposition of phosphorothioate esters.

The widespread use of phosphorothioate esters as agricultural pesticides, chemical weapons and mechanistic probes in enzymology has sparked interest in the reactivity of these thio-substituted analogues of phosphate esters. In this brief account, we summarize the recent developments in our understanding of the mechanisms of hydrolysis (and solvolysis in methanol) of phosphorothioates containing a sulfur atom in the bridging and/or non-bridging position. A small number of highly efficient catalytic systems containing the metal ions La(III), Pd(II), Cu(II) and Zn(II) have been developed to promote the degradation of the various classes of phosphorothioate esters. The mechanisms of the base promoted solvolytic reactions in water and methanol and those of the metal catalyzed cleavage are presented, as well as a discussion of the energetics of the catalytic processes and other salient features. The aim of this review is to provide the reader with a contemporary physical organic description of phosphorothioate ester cleavage. This article is part of a Special Issue entitled: Chemistry and mechanism of phosphatases, diesterases and triesterases.

Catalysis by desolvation: the catalytic prowess of SAM-dependent halide-alkylating enzymes.

In the biological fixation of halide ions, several enzymes have been found to catalyze alkyl transfer from S-adenosylmethionine to halide ions. It proves possible to measure the rates of reaction of the trimethylsulfonium ion with I(-), Br(-), Cl(-), F(-), HO(-), and H2O in water at elevated temperatures. Comparison of the resulting second-order rate constants, extrapolated to 25 °C, with the values of k(cat)/K(m) reported for fluorinase and chlorinase indicates that these enzymes enhance the rates of alkyl halide formation by factors of 2 × 10(15)- and 1 × 10(17)-fold, respectively. These rate enhancements, achieved without the assistance of cofactors, metal ions, or general acid-base catalysis, are the largest that have been reported for an enzyme that acts on two substrates.

Catalysis by a de novo zinc-mediated protein interface: implications for natural enzyme evolution and rational enzyme engineering.

Here we show that a recent computationally designed zinc-mediated protein interface is serendipitously capable of catalyzing carboxyester and phosphoester hydrolysis. Although the original motivation was to design a de novo zinc-mediated protein-protein interaction (called MID1-zinc), we observed in the homodimer crystal structure a small cleft and open zinc coordination site. We investigated if the cleft and zinc site at the designed interface were sufficient for formation of a primitive active site that can perform hydrolysis. MID1-zinc hydrolyzes 4-nitrophenyl acetate with a rate acceleration of 10(5) and a k(cat)/K(M) of 630 M(-1) s(-1) and 4-nitrophenyl phosphate with a rate acceleration of 10(4) and a k(cat)/K(M) of 14 M(-1) s(-1). These rate accelerations by an unoptimized active site highlight the catalytic power of zinc and suggest that the clefts formed by protein-protein interactions are well-suited for creating enzyme active sites. This discovery has implications for protein evolution and engineering: from an evolutionary perspective, three-coordinated zinc at a homodimer interface cleft represents a simple evolutionary path to nascent enzymatic activity; from a protein engineering perspective, future efforts in de novo design of enzyme active sites may benefit from exploring clefts at protein interfaces for active site placement.

Catalytic proficiency: the extreme case of S-O cleaving sulfatases.

As benchmarks for judging the catalytic power of sulfate monoesterases, we sought to determine the rates of spontaneous hydrolysis of unactivated alkyl sulfate monoesters by S-O bond cleavage. Neopentyl sulfate proved to be unsuitable for this purpose, since it was found to undergo hydrolysis by a C-O bond cleaving mechanism with rearrangement of its carbon skeleton. Instead, we examined the temperature dependence of the spontaneous hydrolyses of aryl sulfate monoesters, which proceed by S-O cleavage. Extrapolation of a Bronsted plot [log(k(25)(N)) = (-1.81 ± 0.09) pK(a)(LG) + (3.6 ± 0.7)] based on the rate constants at 25 °C for hydrolysis of a series of sulfate monoesters to a pK(a)(LG) value of 16.1, typical of an aliphatic alcohol, yields k(25)(N) = 3 × 10(-26) s(-1). Comparison of that value with established k(cat) values of bacterial sulfatases indicates that these enzymes produce rate enhancements (k(cat)/k(uncat)) of up to 2 × 10(26)-fold for the hydrolysis of sulfate monoesters. These rate enhancements surpass by several orders of magnitude the ~10(21)-fold rate enhancements that are generated by phosphohydrolases, the most powerful biological catalysts previously known. The hydrolytic rates of phosphate and sulfate monoesters are compared directly, and the misleading impression that the two classes of ester are of similar reactivity is dispelled.

Proton-in-flight mechanism for the spontaneous hydrolysis of N-methyl O-phenyl sulfamate: implications for the design of steroid sulfatase inhibitors.

The hydrolysis of N-methyl O-phenyl sulfamate (1) has been studied as a model for steroid sulfatase inhibitors such as Coumate, 667 Coumate, and EMATE. At neutral pH, simulating physiological conditions, hydrolysis of 1 involves an intramolecular proton transfer from nitrogen to the bridging oxygen atom of the leaving group. Remarkably, this proton transfer is estimated to accelerate the decomposition of 1 by a factor of 10(11). Examination of existing kinetic data reveals that the sulfatase PaAstA catalyzes the hydrolysis of sulfamate esters with catalytic rate accelerations of ~10(4), whereas the catalytic rate acceleration generated by the enzyme for its cognate substrate is on the order of ~10(15). Rate constants for hydrolysis of a wide range of sulfuryl esters, ArOSO(2)X(-), are shown to be correlated by a two-parameter equation based on pK(a)(ArOH) and pK(a)(ArOSO2XH).

Hydrolysis of N-alkyl sulfamates and the catalytic efficiency of an S-N cleaving sulfamidase.

The final step in the degradation of heparin sulfate involves the enzymatic hydrolysis of its 2-sulfamido groups. To evaluate the power of the corresponding sulfamidases as catalysts, we examined the reaction of N-neopentyl sulfamate at elevated temperatures and found it to undergo specific acid catalyzed hydrolysis even at alkaline pH. A rate constant of 10(-16) s(-1) was calculated using the Eyring equation for water attack on the N-protonated species at pH 7, 25 °C. As a model for the pH neutral reaction, a rate constant for hydroxide attack on (CH(3))(3)CCH(2)N(+)H(2)SO(3)(-) at pH 7, 25 °C was calculated to be 10(-19) s(-1). The corresponding rate enhancement (k(cat)/k(non)) produced by the N-sulfamidase of F. heparinum is approximately 10(16)-fold, which is somewhat larger than those generated by most hydrolytic enzymes but considerably smaller than those generated by S-O cleaving sulfatases.

Study on the transesterification of methyl aryl phosphorothioates in methanol promoted by Cd(II), Mn(II), and a synthetic Pd(II) complex.

Methanol solutions containing Cd(II), Mn(II), and a palladacycle, (dimethanol bis(N,N-dimethylbenzylamine-2C,N)palladium(II) (3), are shown to promote the methanolytic transesterification of O-methyl O-4-nitrophenyl phosphorothioate (2b) at 25 °C with impressive rate accelerations of 10(6)-10(11) over the background methoxide promoted reaction. A detailed mechanistic investigation of the methanolytic cleavage of 2a-d having various leaving group aryl substitutions, and particularly the 4-nitrophenyl derivative (2b), catalyzed by Pd-complex 3 is presented. Plots of k(obs) versus palladacycle [3] demonstrate strong saturation binding to form 2b:3. Numerical fits of the kinetic data to a universal binding equation provide binding constants, K(b), and first order catalytic rate constants for the methanolysis reaction of the 2b:3 complex (k(cat)) which, when corrected for buffer effects, give corrected (k(cat)(corr)) rate constants. A sigmoidal shaped plot of log(k(cat)(corr)) versus (s)(s)pH (in methanol) for the cleavage of 2b displays a broad (s)(s)pH independent region from 5.6 ≤ (s)(s)pH ≤ 10 with a k(minimum) = (1.45 ± 0.24) × 10(-2) s(-1) and a [lyoxide] dependent wing plateauing above a kinetically determined (s)(s)pK(a) of 12.71 ± 0.17 to give a k(maximum) = 7.1 ± 1.7 s(-1). Brønsted plots were constructed for reaction of 2a-d at (s)(s)pH 8.7 and 14.1, corresponding to reaction in the midpoints of the low and high (s)(s)pH plateaus. The Brønsted coefficients (ß(LG)) are computed as -0.01 ± 0.03 and -0.86 ± 0.004 at low and high (s)(s)pH, respectively. In the low (s)(s)pH plateau, and under conditions of saturating 3, a solvent deuterium kinetic isotope effect of k(H)/k(D) = 1.17 ± 0.08 is observed; activation parameters (ΔH(Pd)(++) = 14.0 ± 0.6 kcal/mol and ΔS(Pd)(++)= -20 ± 2 cal/mol·K) were obtained for the 3-catalyzed cleavage reaction of 2b. Possible mechanisms are discussed for the reactions catalyzed by 3 at low and high sspH. This catalytic system is shown to promote the methanolytic cleavage of O,O-dimethyl phosphorothioate in CD3OD, producing (CD3O)2P═O(S(-)) with a half time for reaction of 34 min.

A study of the kinetics of La3+-promoted methanolysis of S-aryl methylphosphonothioates: possible methodology for decontamination of EA 2192, the toxic byproduct of VX hydrolysis.

The kinetics of the La3+-catalyzed methanolysis of a series of S-aryl methylphosphonothioates (4a-e, phenyl substituents = 3,5-dichloro, 4-chloro, 4-fluoro, 4-H, 4-methoxy) were studied at 25 °C with s(s)pH control. The reaction involves saturation binding of the anionic substrates to dimeric La3+/methoxide catalysts formulated as La2(3+)(-OCH3)x, where x = 2-5 depending on the solution s(s)pH. Cleavage of the La3+-bound methylphosphonothioates is fast, ranging from 5 × 10(-3) s(-1) to 5.5 × 10-(5) s(-1) for substrates 4a-e at a s(s)pH of 8.4 and 1.6 × 10(-1) s(-1) to 4 × 10(-3) s(-1) at a s(s)pH of 11.7. The rate accelerations for the methanolysis of substrates 4a-e, relative to their background methoxide-promoted reactions, average 7 × 10(10) and 1.5 × 10(9), respectively, at s(s)pH's of 8.4 and 11.7. The catalytic system is predicted to cleave EA 2192 (S-2(N,N-di-iso-propylaminoethyl)methylphosphonothioate), a toxic byproduct of the hydrolysis of VX, with a t1/2 between 4 and 8 min at a s(s)pH of 8.4, and 27 min at a s(s)pH of 11.7.

Mechanistic and computational study of a palladacycle-catalyzed decomposition of a series of neutral phosphorothioate triesters in methanol.

The methanolytic cleavage of a series of O,O-dimethyl O-aryl phosphorothioates (1a−g) catalyzed by a C,N-palladacycle, (2-[N,N-dimethylamino(methyl)phenyl]-C1,N)(pyridine) palladium(II) triflate (3), at 25 °C and sspH 11.7 in methanol is reported, along with data for the methanolytic cleavage of 1a−g. The methoxide reaction gives a linear log k2−OMe vs sspKa (phenol leaving group) Brønsted plot having a gradient of ßlg = −0.47 ± 0.03, suggesting about 34% cleavage of the P−OAr bond in the transition state. On the other hand, the 3-catalyzed cleavage of 1 gives a Brønsted plot with a downward break at sspKa (phenol) 13, signifying a change in the rate-limiting step in the catalyzed reaction, with the two wings having ßlg values of 0.0 ± 0.03 and −1.93 ± 0.06. The rate-limiting step for good substrates with low leaving group sspKa values is proposed to be substrate/pyridine exchange on the palladacycle, while for substrates with poor leaving groups, the rate-limiting step is a chemical one with extensive cleavage of the P−OAr bond. DFT calculations support this process and also identify two intermediates, namely, one where substrate/pyridine interchange has occurred to give the palladacycle coordinated to substrate through the S═P linkage and to methoxide (6) and another where intramolecular methoxide attack has occurred on the P═S unit to give a five-coordinate phosphorane (7) doubly coordinated to Pd via the S− and through a bridging methoxide linked to P and Pd. Attempts to identify the existence of the phosphorane by 31P NMR in a d4-methanol solution containing 10 mM each of 3, trimethyl phosphorothioate (a very slow cleaving substrate), and methoxide proved unsuccessful, instead showing that the phosphorothioate was slowly converted to trimethyl phosphate, with the palladacycle decomposing to Pd0 and free pyridine. These results provide the first reported example where a palladacycle-promoted solvolysis reaction exhibits a break in the Brønsted plot signifying at least one intermediate, while the DFT calculations provide further insight into a more complex mechanism involving two intermediates.

On the question of stepwise vs. concerted cleavage of RNA models promoted by a synthetic dinuclear Zn(II) complex in methanol: implementation of a noncleavable phosphonate probe.

To address the question of concerted versus a stepwise reaction mechanisms for the cyclization of the 2-hydroxypropyl aryl and alkyl RNA models (1a-k) promoted by dinuclear Zn(II) complex (4) at (s)spH 9.8 and 25 degrees C, the non-cleavable O-hydroxypropyl phenylphosphonate analogues 6a and 6b were subjected to the catalytic reaction in methanol. These phosphonates did not undergo isomerization in the study, the only observable methanolysis reaction being release of 1,2-propanediol and the formation of O-methyl phenylphosphonate. The observed first order rate constants for methanolysis promoted by 4 are k(obs)(6a) = (1.47 +/- 0.09) x 10(-4) s(-1) and k(obs)(6b) = (2.08 +/- 0.09) x 10(-6) s(-1), respectively. The rates of methanolysis of a series of O-aryl phenylphosphonates (8a-f) in the presence of increasing [4] were analyzed to provide binding constants, Kb, and the catalytic rate constant, kcat(max), for the unimolecular decomposition of the 8:4 Michaelis complex. A Brønsted plot of the log (k(cat)(max)) vs. sspKa(phenol) (acidity constant of the conjugate acid of the leaving group in methanol) was fitted to a linear regression of log kcat(max) = (-0.80 +/- 0.07)(s)spKa + (10.2 +/- 1.0) which includes the datum for 6a. The datum for 6b, which reacts approximately 70-fold slower, falls significantly below the linear correlation. The data provide additional evidence consistent with a concerted cyclization of RNA models 1a-k promoted by 4.

Leaving group assistance in the La3+-catalyzed cleavage of dimethyl (o-methoxycarbonyl)aryl phosphate triesters in methanol.

The catalytic methanolysis of a series of dimethyl aryl phosphate triesters where the aryl groups contain an o-methoxycarbonyl (o-CO2Me) substituent (4a-i) was studied at 25 degrees C in methanol containing La3+ at various concentrations and (s)(s)pH. Determination of the second-order rate constant for La3+(2)-catalyzed cleavage of substrate 4a (dimethyl (o-methoxycarbonyl)phenyl phosphate) as a function of (s)(s)pH was assessed in terms of a speciation diagram that showed that the process was catalyzed by La3+(2)(-OCH3)x dimers, where x = 1-5, that exhibit only a 5-fold difference in activity between all the species. The second-order catalytic rate constants (k2(La)) for the catalyzed methanolysis of 4a-i at (s)(s)pH 8.7 fit a Brønsted relationship of log k2(La) = (-0.82 +/- 0.11)(s)(s)pKa(lg) + (11.61 +/- 1.48), where the gradient is shallower than that determined for a series of dimethyl aryl phosphates that do not contain the o-CO2Me substituent, log k2(La) = (-1.25 +/- 0.06)(s)(s)pKa(lg) + (16.23 +/- 0.75). Two main observations are that (1) the o-CO2Me group preferentially accelerates the cleavage of the phosphate triesters with poor leaving groups relative to those with good leaving groups and (2) it provides an increase in cleavage rate relative to those of comparable substrates that do not have that functional group, e.g., k2(La)(dimethyl o-(methoxycarbonyl)phenyl phosphate)/k2(La)(dimethyl phenyl phosphate) = 60. Activation parameters for the La3+(2)-catalyzed methanolysis of 4a and dimethyl 4-nitrophenyl phosphate show respective DeltaH(double dagger) (DeltaS(double dagger)) values of 3.3 kcal/mol (-47 cal/mol x K) and 0.7 kcal/mol (-46.5 cal/mol x K). The data are analyzed in terms of a concerted reaction where the catalytic complex (La3+(2)(-OCH3)(x-1)) binds to the three components of a rather loose transition state composed of a nucleophile CH3O-, a nucleofuge -OAr, and a central (RO)2P(2+)-O(-) in a way that provides leaving group assistance to the departing aryloxy group.

Dinuclear Zn(II) complex promotes cleavage and isomerization of 2-hydroxypropyl alkyl phosphates by a common cyclic phosphate intermediate.

The kinetics and cleavage products of 2-hydroxypropyl p-nitrophenyl phosphate were determined in methanol containing the di-Zn(II) complex of bis-1,3-N1,N1'-(1,5,9-triazacyclododecyl)propane (4). Time-dependent 1H NMR spectra of the reaction mixture at sspH 9.8 +/- 0.1 show that the catalytic reaction proceeds via a cyclic phosphate (4-methylethylene phosphate, 2) that is subsequently cleaved into a kinetic mixture of two isomeric products, 2-hydroxypropyl methyl phosphate (3) and 1-hydroxypropan-2-yl methyl phosphate (3a), in a 29/71 ratio. In the presence of 4, the kinetic mixture of 3/3a is transformed into a thermodynamic mixture of 72/28 3/3a. The time-dependent 1H NMR spectra of 4 and a 22/78 mixture of 3/3a in CD3OH show that the formation of the thermodynamic mixture occurs on the same time scale as replacement of the P-OCH3 group of the 3/3a starting materials with OCD3. Detailed kinetic studies indicate that the dominant process for loss of the OCH3 group and equilibration of 3/3a is via a 4-catalyzed process where each of the isomers cyclizes to methylethylene phosphate (2), which subsequently reforms the 3/3a thermodynamic mixture. The kcatmax for 4-catalyzed cyclization of 3 and three other 2-hydroxypropyl O-alkyl phosphates (alkyl = CF3CH2- (6a), CH2FCH2- (6b), and CH3CH2- (6c)) has been determined, and the Brønsted plot comprising the log kcatmax vs leaving group sspKa that includes several previously studied 2-hydroxypropyl aryl phosphates is linear, following the expression log kcatmax = (-0.85 +/- 0.02) sspKa + (12.8 +/- 0.4). The betalg value of -0.85 suggests that the catalyzed cleavage of the P-OAr/OR bond has progressed to about 45% in the transition state. The combined results are analyzed in terms of two possible processes involving either a concerted reaction leading to the cyclic phosphate 2 from which the thermodynamic mixture of 3/3a is formed or a stepwise one involving a transient phosphorane whose predominant fate is to eliminate methoxide and proceed to 2 rather than partitioning between 3, 3a, and 2.

Experimental investigation into the mechanism of the epoxidation of aldehydes with sulfur ylides.

A mechanistic study of the epoxidation of aldehydes with sulfur ylides has been carried out. The DeltaG++ of the reaction was determined to be 22.2 kcal/mol at 298 K. A 13C kinetic isotope effect was determined to be 1.026 for the carbonyl carbon of benzaldehyde. A secondary deuterium isotope effect was determined to be 0.93 for the aldehydic hydrogen atom of benzaldehyde. Substituent effects on reaction rate were studied, and a Hammett rho of +2.50 was found.

Experimental demonstration of base-catalyzed interconversion of isomeric betaine intermediates in the Corey-Chaykovsky epoxidation.

The collapse of hydroxysulfonium salts has been examined as a model for the epoxidation of aldehydes. The anti diastereomer reacted with retention of stereochemistry and no crossover, while the syn diastereomer gave crossover products along with cis and trans epoxides. Deprotonation and reprotonation on the carbon of the alpha-hydroxy sulfonium ylide is presumed responsible for production of the trans epoxide. This reaction pathway has been proposed to explain losses of enantioselectivity but never directly observed.

Lower limb orthopaedic surgery results in changes to coagulation and non-specific inflammatory biomarkers, including selective clinical outcome measures.

BACKGROUND: With an aging society and raised expectations, joint replacement surgery is likely to increase significantly in the future. The development of postoperative complications following joint replacement surgery (for example, infection, systemic inflammatory response syndrome and deep vein thrombosis) is also likely to increase. Despite considerable progress in orthopaedic surgery, comparing a range of biological markers with the ultimate aim of monitoring or predicting postoperative complications has not yet been extensively researched. The aim of this clinical pilot study was to test the hypothesis that lower limb orthopaedic surgery results in changes to coagulation, non-specific markers of inflammation (primary objective) and selective clinical outcome measures (secondary objective). METHODS: Test subjects were scheduled for elective total hip replacement (THR) or total knee replacement (TKR) orthopaedic surgery due to osteoarthritis (n = 10). Platelet counts and D-dimer concentrations were measured to assess any changes to coagulation function. C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) were measured as markers of non-specific inflammation. Patients were monitored regularly to assess for any signs of postoperative complications, including blood transfusions, oedema (knee swelling), wound infection, pain and fever. RESULTS: THR and TKR orthopaedic surgery resulted in similar changes of coagulation and non-specific inflammatory biomarkers, suggestive of increased coagulation and inflammatory reactions postoperatively. Specifically, THR and TKR surgery resulted in an increase in platelet (P = 0.013, THR) and D-dimer (P = 0.009, TKR) concentrations. Evidence of increased inflammation was demonstrated by an increase in CRP and ESR (P ≤ 0.05, THR and TKR). Four patients received blood transfusions (two THR and two TKR patients), with maximal oedema, pain and aural temperatures peaking between days 1 and 3 postoperatively, for both THR and TKR surgery. None of the patients developed postoperative infections. CONCLUSIONS: The most noticeable changes in biological markers occur during days 1 to 3 postoperatively for both THR and TKR surgery, and these may have an effect on such postoperative clinical outcomes as oedema, pyrexia and pain. This study may assist in understanding the postoperative course following lower limb orthopaedic surgery, and may help clinicians in planning postoperative management and patient care.

Tourniquet-applied upper limb orthopaedic surgery results in increased inflammation and changes to leukocyte, coagulation and endothelial markers.

PURPOSE: During this pilot clinical study, patients scheduled for elective tourniquet-applied upper limb orthopaedic surgery were recruited to investigate the effects of surgery on various biological markers (n = 10 patients). METHODS: Three venous blood samples were collected from the arm at the ante-cubital fossa, at baseline (pre-operatively), 5 and 15 minutes after reperfusion (post-operatively). Neutrophil and monocyte leukocyte sub-populations were isolated by density gradient centrifugation techniques. Leukocyte activation was investigated by measuring the cell surface expression of CD62L (L-selectin), CD11b (Mac-1) and the intracellular production of hydrogen peroxide (H2O2), via flow cytometry. C-reactive protein (CRP) was measured using a clinical chemistry analyser. Plasma concentrations of protein C and von Willebrand factor (vWF) were measured using enzyme-linked fluorescent assays (ELFA). RESULTS: Following tourniquet-applied upper limb orthopaedic surgery, there was a decrease in neutrophil CD62L expression (p = 0.001), an increase in CD11b expression and in the intracellular production of H2O2 by neutrophils and monocytes (p<0.05). An increase in CRP concentration (p<0.001), a decrease in protein C concentration (p = 0.004), with a trend towards elevated vWF levels (p = 0.232) were also observed during this time. CONCLUSIONS: Conventionally, patients undergoing orthopaedic surgery have been monitored in the peri-operative period by means of CRP, which is a non-specific marker of inflammation. This test cannot differentiate between inflammation due to current or pre-existing disease processes and the development of ischaemia-reperfusion injury surgery. The findings from this study suggest that markers such as CD11b, protein C and H2O2 may provide alternative ways of assessing leukocyte and coagulation activation peri-operatively. It is proposed that by allowing orthopaedic surgeons access to laboratory markers such as CD11b, protein C and H2O2, an accurate assessment of the extent of inflammation due to surgery per se could be made.

Total hip and knee replacement surgery results in changes in leukocyte and endothelial markers.

BACKGROUND: It is estimated that over 8 million people in the United Kingdom suffer from osteoarthritis. These patients may require orthopaedic surgical intervention to help alleviate their clinical condition. Investigations presented here was to test the hypothesis that total hip replacement (THR) and total knee replacement (TKR) orthopaedic surgery result in changes to leukocyte and endothelial markers thus increasing inflammatory reactions postoperatively. METHODS: During this 'pilot study', ten test subjects were all scheduled for THR or TKR elective surgery due to osteoarthritis. Leukocyte concentrations were measured using an automated full blood count analyser. Leukocyte CD11b (Mac-1) and CD62L cell surface expression, intracellular production of H(2)O(2 )and elastase were measured as markers of leukocyte function. Von Willebrand factor (vWF) and soluble intercellular adhesion molecule-1 (sICAM-1) were measured as markers of endothelial activation. RESULTS: The results obtained during this study demonstrate that THR and TKR orthopaedic surgery result in similar changes of leukocyte and endothelial markers, suggestive of increased inflammatory reactions postoperatively. Specifically, THR and TKR surgery resulted in a leukocytosis, this being demonstrated by an increase in the total leukocyte concentration following surgery. Evidence of leukocyte activation was demonstrated by a decrease in CD62L expression and an increase in CD11b expression by neutrophils and monocytes respectively. An increase in the intracellular H(2)O(2 )production by neutrophils and monocytes and in the leukocyte elastase concentrations was also evident of leukocyte activation following orthopaedic surgery. With respect to endothelial activation, increases in vWF and sICAM-1 concentrations were demonstrated following surgery. CONCLUSION: In general it appeared that most of the leukocyte and endothelial markers measured during these studies peaked between days 1-3 postoperatively. It is proposed that by allowing orthopaedic surgeons access to alternative laboratory markers such as CD11b, H(2)O(2 )and elastase, CD62L, vWF and sICAM-1, an accurate assessment of the extent of inflammation due to surgery per se could be made. Ultimately, the leukocyte and endothelial markers assessed during this investigation may have a role in monitoring potential infectious complications that can occur during the postoperative period.