Within-day and between-day Reproducibility of Baroreflex Sensitivity in Healthy Adult Males.

Within-day and between-day reproducibility of supine and tilt baroreflex sensitivity were investigated utilising sequence and spectral indices in 46 healthy adult males employing 3 repeat measures; baseline, +60 min and +24 h. Reproducibility was assessed via the 95% limits of agreement and by the technical error of the measurement. For spectral parameters, the limits of agreement indicated same day was marginally better than between-day reproducibility. For sequence parameters, between-day had marginally better agreement than same-day reproducibility. Tilt markedly improved reproducibility across all outcome measures. Precision expressed by the technical error of the measurement for all spectral outcomes was good in both supine and tilt baroreflex sensitivity (<6%). Precision was lower, but acceptable, for sequence baroreflex sensitivity outcomes in both positions (<11%). Baroreflex sensitivity transfer gain provided the best agreement and reproducibility during supine and tilt conditions. These findings suggest time and spectral techniques may be employed to assess within-day and between-day baroreflex sensitivity changes in healthy individuals. The inclusion of a tilt manoeuvre may improve the reproducibility of the outcome measure, which may aid in the detection of modest baroreflex sensitivity changes in studies employing limited sample sizes.

A pilot study examining the effects of consuming a high-protein vs normal-protein breakfast on free-living glycemic control in overweight/obese 'breakfast skipping' adolescents.

To examine whether the daily consumption of normal-protein (NP) vs higher-protein (HP) breakfasts improve free-living glycemic control in overweight/obese, 'breakfast skipping' adolescents. Twenty-eight healthy, but overweight, teens (age: 19±1 year; BMI: 29.9±0.8 kg m(-2)) completed a 12-week randomized parallel-arm study in which the adolescents consumed either a 350 kcal NP breakfast (13 g protein) or HP breakfast (35 g protein). Pre- and post-study 24-h blood glucose measures were assessed using continuous glucose monitoring. Although no main effects of time or group were detected, time by group interactions were observed. Post hoc pairwise comparisons assessing the post-pre changes revealed that the daily consumption of the HP breakfasts tended to reduce the 24-h glucose variability (s.d.) vs NP (-0.17±0.09 vs +0.09±0.10 s.d.; P=0.06) and tended to reduce the time spent above the high glucose limit (-292±118 vs -24±80 min; P=0.09). The consumption of the HP breakfasts also reduced the 24-h maximal (peak) glucose response (-0.94±0.36 vs +0.30±0.18 mmol l(-1); P<0.01) and reduced postprandial glucose fluctuations (-0.88±0.44 vs +0.49±0.34 mmol l(-1); P<0.03) vs NP. These data suggest that the daily addition of a HP breakfast, containing 35 g of high-quality protein, has better efficacy at improving free-living glycemic control compared with a NP breakfast in overweight/obese, but otherwise healthy, 'breakfast skipping' adolescents.

Increased birth weight is associated with altered gene expression in neonatal foreskin.

Elevated birth weight is linked to glucose intolerance and obesity health-related complications later in life. No studies have examined if infant birth weight is associated with gene expression markers of obesity and inflammation in a tissue that comes directly from the infant following birth. We evaluated the association between birth weight and gene expression on fetal programming of obesity. Foreskin samples were collected following circumcision, and gene expression analyzed comparing the 15% greatest birth weight infants (n=7) v. the remainder of the cohort (n=40). Multivariate linear regression models were fit to relate expression levels on differentially expressed genes to birth weight group with adjustment for variables selected from a list of maternal and infant characteristics. Glucose transporter type 4 (GLUT4), insulin receptor substrate 2 (IRS2), leptin receptor (LEPR), lipoprotein lipase (LPL), low-density lipoprotein receptor-related protein 1 (LRP1), matrix metalloproteinase 2 (MMP2), plasminogen activator inhibitor-1 (PAI-1) and transcription factor 7-like 2 (TCF7L2) were significantly upregulated and histone deacetylase 1 (HDAC1) and thioredoxin (TXN) downregulated in the larger birth weight neonates v. CONTROLS: Multivariate modeling revealed that the estimated adjusted birth weight group difference exceeded one standard deviation of the expression level for eight of the 10 genes. Between 25 and 50% of variation in expression level was explained by multivariate modeling for eight of the 10 genes. Gene expression related to glycemic control, appetite/energy balance, obesity and inflammation were altered in tissue from babies with elevated birth weight, and these genes may provide important information regarding fetal programming in macrosomic babies.

Conventional and toxicogenomic assessment of the acute pulmonary damage induced by the instillation of Cardiff PM10 into the rat lung.

There is strong epidemiological evidence of association between PM10 (particulate matter with an aerodynamic diameter less than or equal to 10 microm) and adverse health outcomes including death and increased hospital admissions for cardiopulmonary conditions. Ambient PM10 surrogates such as diesel exhaust particles (DEP), a common component of UK PM10 have been shown to induce lung inflammation in both humans and rodents. To date, few studies have reported on the toxicological response of UK PM10 in experimental animals. This study examines the pulmonary toxicological responses in male Sprague Dawley rats following the intratracheal instillation of Cardiff urban PM10. A mild but significant change in lung permeability was observed in the lung post-instillation of a high (10 mg) dose of the whole PM10 as adjudged by increases in lung to body weight ratio and total acellular lavage protein. Such effects were less marked following instillation of a water-soluble fraction (80% of the total mass) but histological examination showed that lung capillaries were swollen in size with this treatment. In conclusion, conventional toxicological, histological and toxicogenomic studies have indicated that Cardiff PM10 exhibits low bioreactivity in the form of mild permeability changes. Differential gene expression was observed when the lung was treated with whole PM10, containing durable particles, in comparison with the water-soluble fraction of PM10 that was devoid of particles. Such changes were linked to different histopathological events within the lung.

Irreversible inhibition of Ca(2+)-independent phospholipase A2 by methyl arachidonyl fluorophosphonate.

Methyl arachidonyl fluorophosphonate (MAFP) has been recently reported to be a selective, active-site directed, irreversible inhibitor of the Group IV 85 kDa cytosolic phospholipase A2 (cPLA2). We have now shown that this compound also potently inhibits the Ca(2+)-independent cytosolic phospholipase A2 (iPLA2). MAFP inhibited iPLA2 in a concentration-dependent manner with half-maximal inhibition observed at 0.5 microM after a 5 min preincubation at 40 degrees C. This inhibition was not reversed upon extensive dilution of the enzyme into the assay mixture. Preincubation of iPLA2 with MAFP resulted in a linear, time-dependent inactivation of enzyme activity, and the enzyme was protected from inactivation by the reversible inhibitor PACOCF3. The ability of MAFP to inhibit the iPLA2 suggests that this enzyme proceeds through an acyl-enzyme intermediate as has been proposed for the cPLA2. Further testing indicated that MAFP did not inhibit the arachidonoyl-CoA synthetase, CoA-dependent acyltransferase, or CoA-independent transacylase activities from P388D1 cells. Thus, MAFP is not a general inhibitor for enzymes which act on arachidonoyl substrates. Instead, the inhibitor appears to show some selectivity for PLA2, although it does not discriminate between cPLA2 and iPLA2. Particular caution must be exercised to distinguish these activities if this inhibitor is used in intact cells.

Metal ion and salt effects on the phospholipase A2, lysophospholipase, and transacylase activities of human cytosolic phospholipase A2.

Human cytosolic phospholipase A2 (cPLA2) is an arachidonic acid specific enzyme which may play a role in arachidonic acid release, eicosanoid production, and signal transduction. The PLA2 activity of this enzyme is stimulated by microM levels of Ca2+. Using a pure recombinant enzyme, we have confirmed that cPLA2 is not absolutely dependent on Ca2+, since Sr2+, Ba2+ and Mn2+ also gave full enzyme activity. Heavy metals, in contrast, inhibited enzyme catalysis suggesting the involvement of an essential cysteine residue. In the absence of Ca2+, high salt concentrations overcame the requirement for divalent metals, indicating that Ca2+ is not required for PLA2 catalytic activity. cPLA2 also displays a lysophospholipase (lyso PLA) activity with lysophosphatidylcholine micelles as a substrate. Unlike the PLA2 activity, the lyso PLA activity toward these micelles is not stimulated by Ca2+. However, upon the addition of glycerol or Triton X-100 to the assay, Ca2+ activation is observed, indicating that substrate presentation can affect the apparent Ca2+ dependence. Glycerol was found to be a potent stimulator of lyso PLA activity and specific activities up to 50 mumol min-1 mg-1 were observed. In addition to the PLA2 and lyso PLA activities, we report that cPLA2 displays a relatively low, CoA-independent transacylase activity which produces phosphatidylcholine from lysophosphatidylcholine substrate. The observation of this novel transacylase activity is consistent with the formation of an acyl-enzyme intermediate.

Dysfunctional endothelial nitric oxide biosynthesis in healthy smokers with impaired endothelium-dependent vasodilatation.

BACKGROUND: The mechanisms involved in the dysfunction of both endothelium-dependent vasodilatation (EDV) and NO biosynthesis related to smoking are unclear. In this study, EDV was assessed in healthy smokers and nonsmokers in vivo and, using serum from the same individuals, was related to the NO biosynthetic pathway in vitro. METHODS AND RESULTS: Flow-mediated EDV of the brachial artery was measured in 23 male patients (8 nonsmokers and 15 smokers). Serum was collected, added to confluent ( approximately 85%) monolayers of human umbilical vein endothelial cells (HUVECs), and incubated for 12 hours. Basal and substance P-stimulated NO production was measured. The HUVECs used for measuring basal NO production were lysed, and both endothelial NO synthase (eNOS) protein expression and eNOS activity were determined. EDV was lower in smokers compared with nonsmokers (P<0.001). HUVECs treated with serum from smokers compared with nonsmokers showed significantly lower basal (P<0.0001) and stimulated (P<0.02) NO production, higher eNOS expression (P<0.0001), but lower eNOS activity (P<0.004). There was a significant positive correlation between in vivo EDV and in vitro substance P-stimulated NO production (rho=0.57, P<0.01) and between basal NO production and eNOS activity (r=0.54, P<0.008) and a negative correlation between basal NO production and eNOS protein expression (r=-0.60, P<0.003). CONCLUIONS: This is the first study to combine an in vivo model with a near-physiological in vitro model to demonstrate an association between decreased NO production and reduced EDV. Cigarette smoking was associated with reduced EDV, NO generation, and eNOS activity in the presence of increased eNOS protein expression.

Monophosphoryl lipid A stimulated up-regulation of reactive oxygen intermediates in human monocytes in vitro.

The production of reactive oxygen and nitrogen intermediates is a common response to infectious challenge in vivo. These agents have been implicated in the modulation of cytokine responses and are produced in large amounts in response to endotoxins produced by a number of infectious agents. The antigen-presenting cell activation caused by these lipopolysacchardies (LPS) has been exploited in the use of these agents as adjuvants. In recent years, less-toxic derivatives have been sought. One such agent, monophosphoryl lipid A (MPL), has been used increasingly in vivo as an adjuvant and as a modulator of the inflammatory process. It is known that this agent modulates the inflammatory response and cytokine production. In addition, we have shown its effect on the production of reactive nitrogen intermediates. In this paper, we show that MPL stimulates the release of high levels of superoxide (O(2)(-)) and hydrogen peroxide (H(2)O(2)), the latter being greater than that seen with LPS and appearing to be related to the inability of MPL to stimulate catalase activity. When cells were pretreated with LPS or MPL and subsequently challenged with LPS, the production of O(2)(-) and H(2)O(2) was inhibited significantly by LPS and MPL. The concentration of MPL required to induce significant hyporesponsiveness to subsequent LPS challenge was 10 times lower than that of LPS. Hyporesponsiveness was greatest when induced by 10 microg/ml MPL, the same concentration that induced the maximum release of H(2)O(2) in primary stimulation. In addition, we have shown that following MPL pretreatment, LPS stimulation does not cause the loss of cytoplasmic IkappaBalpha, which occurs when human monocytes are cultured with LPS. From our results, we propose a model for the reduced toxicity of MPL.

The influence of reduced insulin sensitivity via short-term reductions in physical activity on cardiac baroreflex sensitivity during acute hyperglycemia.

Reduced insulin sensitivity and impaired glycemic control are among the consequences of physical inactivity and have been associated with reduced cardiac baroreflex sensitivity (BRS). However, the effect of reduced insulin sensitivity and acute hyperglycemia following glucose consumption on cardiac BRS in young, healthy subjects has not been well characterized. We hypothesized that a reduction in insulin sensitivity via reductions in physical activity would reduce cardiac BRS at rest and following an oral glucose tolerance test (OGTT). Nine recreationally active men (23 ± 1 yr; >10,000 steps/day) underwent 5 days of reduced daily physical activity (RA5) by refraining from planned exercise and reducing daily steps (<5,000 steps/day). Spontaneous cardiac BRS (sequence technique) was compared at rest and for 120 min following an OGTT at baseline and after RA5. A substudy (n = 8) was also performed to independently investigate the influence of elevated insulin alone on cardiac BRS using a 120-min hyperinsulinemic-euglycemic clamp. Insulin sensitivity (Matsuda index) was significantly reduced following RA5 (BL 9.2 ± 1.3 vs. RA5 6.4 ± 1.1, P < 0.001). Resting cardiac BRS was unaffected by RA5 and significantly reduced during the OGTT similarly at baseline and RA5 (baseline 0 min, 28 ± 4 vs. 120 min, 18 ± 4; RA5 0 min, 28 ± 4 vs. 120 min, 21 ± 3 ms/mmHg). Spontaneous cardiac BRS was also reduced during the hyperinsulinemic-euglycemic clamp (P < 0.05). Collectively, these data demonstrate that acute elevations in plasma glucose and insulin can impair spontaneous cardiac BRS in young, healthy subjects, and that reductions in cardiac BRS following acute hyperglycemia are unaffected by reduced insulin sensitivity via short-term reductions in physical activity.

Density and substrata are important in lung type II cell transdifferentiation in vitro.

Morphological techniques and metabolic cell marker assays were used to study the transdifferentiation of pulmonary type II epithelial cells to type I-like cells in vitro. In the lung this process is important during remodelling and alveolar repair. Type II cell phenotype was best maintained over eight days when densely packed cells were plated out on a commercially available extracellular matrix. Such cells retained type II cell characteristics (lamellar bodies, high activities of gamma glutamyl transpeptidase and alkaline phosphatase) but expressed low levels of rT1(40) a surface protein marker of type I cells. In contrast, low density cultures, irrespective of substratum, exhibited rapid cell spreading, loss of lamellar bodies, loss of type II cell enzyme markers and expressed high levels or rT1(40). Conditions have been described whereby the same isolate of type II cells can be used to produce differential epithelial phenotypes and use can be made of this for further characterisation or to investigate the effect of toxins on different lung cell types in vitro.

Assay strategies and methods for phospholipases.

Of the general considerations discussed, the two issues which are most important in choosing an assay are (1) what sensitivity is required to assay a particular enzyme and (2) whether the assay must be continuous. One can narrow the options further by considering substrate availability, enzyme specificity, assay convenience, or the presence of incompatible side reactions. In addition, the specific preference of a particular phospholipase for polar head group, micellar versus vesicular substrates, and anionic versus nonionic detergents may further restrict the options. Of the many assays described in this chapter, several have limited applicability or serious drawbacks and are not commonly employed. The most commonly used phospholipase assays are the radioactive TLC assay and the pH-stat assay. The TLC assay is probably the most accurate, sensitive assay available. These aspects often outweigh the disadvantages of being discontinuous, tedious, and expensive. The radioactive E. coli assay has become popular recently as an alternative to the TLC assay for the purification of the mammalian nonpancreatic phospholipases. The assay is less time consuming and less expensive than the TLC assay, but it is not appropriate when careful kinetics are required. Where less sensitivity is needed, or when a continuous assay is necessary, the pH-stat assay is often employed. With purified enzymes, when free thiol groups are not present, a spectrophotometric thiol assay can be used. This assay is approximately as sensitive as the pH-stat assay but is more convenient and more reproducible, although the substrate is not available commercially. Despite the many assay choices available, the search continues for a convenient, generally applicable assay that is both sensitive and continuous. The spectrophotometric SIBLINKS assay and some of the fluorescent assays show promise of filling this need.

Can toxicogenomics provide information on the bioreactivity of diesel exhaust particles?

Epidemiologists have linked increased cardio-respiratory hospital admissions, morbidity and mortality rates and increases in particulate matter with an aerodynamic diameter less than 10 microns (PM10) concentrations (Anderson et al., 1991). PM10 consist of a heterogeneous mixture of particles that include minerals, metal oxides, sea salt, biological components and soot. In urban locations, soot, especially ultrafine diesel exhaust particles (DEP), accounts for 20-80% by mass of the airborne PM10 arising from vehicular activities. In the experiment described here, control [NaCl] and 1.25 mg of DEP were instilled into rat lung and the responses assessed using changes in lung permeability, inflammation and epithelial cell markers in lavage fluid, with the addition of a new technique of gene expression profiling using macroarrays. The aim of the study was to use these macroarrays as a sensitive measurement of acute up- or down-regulation of genes that were taking place in the rat lung in response to the small instilled mass of DEP. DEP instillation caused a slight oedematous lung with no overt inflammation and ten out of a possible 207 (5%) rat stress genes were repeatedly changed in response to DEP instillation. Therefore, the conclusion from the macroarray analysis is in agreement with the conventional toxicology and suggest that DEP elicits a low bioreactive response in a healthy rat lung.

Attitudes of women toward the gynecologic examination.

This study was undertaken to determine why patients feel uncomfortable during the gynecologic examination, to assess general knowledge about the procedures, and to find out what would make the patient feel better about the examination. Nine hundred seventy-seven women sampled in 14 different health care facilities in the Salt Lake area in Utah filled out a two-page, self-administered questionnaire while waiting for a medical appointment. Results show that women feel less comfortable during the pelvic examination than they do during the breast examination; physical discomfort of the pelvic examination is the reason most frequently cited. There were 77.1 percent who stated they would feel better about the examination if the physician told them what was going to happen. Over 70 percent wanted to know more about their female organs, normal sexual functions and emotions, and reasons and procedures for the pelvic examination; 68.3 percent felt that knowing more would make them more comfortable. There were 46.5 percent who thought the use of a mirror for the woman to observe the examination was a good idea. Only 28.9 percent knew the rectum was examined. This study shows that women need and want to be educated about the gynecologic examination.

Analysis of human synovial fluid phospholipase A2 on short chain phosphatidylcholine-mixed micelles: development of a spectrophotometric assay suitable for a microtiterplate reader.

The development of a reliable assay for human synovial fluid phospholipase A2 (HSF PLA2) is important for the kinetic characterization of the enzyme and for the identification of enzyme inhibitors. This enzyme behaves differently from other extracellular PLA2s in many standard phospholipase assays and is generally assayed using radiolabeled, autoclaved Escherichia coli as a substrate. We have now developed a nonradioactive, continuous, spectrophotometric assay for this enzyme that is adaptable for use with a microtiterplate reader and is suitable for screening enzyme inhibitors. The assay uses a thioester derivative of diheptanoyl phosphatidylcholine as a substrate, with which the enzyme displays a specific activity of about 25 mumol min-1 mg-1. The substrate concentration curve fits a Hill equation with an apparent Km of 500 microM and a Hill coefficient of two. The enzyme has a pH optimum of 7.5 in this assay and requires about 10 mM Ca2+ for maximal activity. The presence of 0.3 mM Triton X-100 was necessary to solubilize the substrate; however, higher concentrations of the detergent inhibited enzyme activity. Using this spectrophotometric assay, inhibition of HSF PLA2 by a thioether phosphonate phosphatidylethanolamine analog was observed with an IC50 of 18 microM.

Inhibition of venom phospholipases A2 by manoalide and manoalogue. Stoichiometry of incorporation.

We have previously described the irreversible inhibition of cobra venom phospholipase A2 (PLA2) by the marine natural product manoalide (MLD) (Lombardo, D., and Dennis, E. A. (1985) J. Biol. Chem. 260, 7234-7240) and by its synthetic analog, manoalogue (MLG) (Reynolds L. J., Morgan, B. P., Hite, G. A., Mihelich, E. D., and Dennis, E. A. (1988) J. Am. Chem. Soc. 110, 5172-5177). We have now made a direct comparison of the action of these two inhibitors on PLA2 from cobra, bee, and rattlesnake venoms and have found that MLG behaves kinetically similarly to MLD in all cases with only minor differences. The time courses of inactivation differ significantly between the three enzymes, however, with the inactivation of bee and rattlesnake PLAs2, occurring much faster than does the inactivation of the cobra venom enzyme. The enzymes also differ in their sensitivity to the presence of Ca2+ during the inactivation. Of the three enzymes, the most Ca(2+)-sensitive is the rattlesnake enzyme, which shows a much faster rate of inactivation in the presence of Ca2+ than in the presence of EGTA. However, the same rate of inactivation was also observed when the inhibitor Ba2+ was substituted for Ca2+, indicating that catalytic activity is not required for inactivation of the enzyme. To probe the mechanism of inactivation and to determine the stoichiometry of incorporation, we have synthesized 3H-labeled MLG and have found that inactivation of cobra PLA2 is accompanied by an incorporation of 3.8 mol of [3H]MLG/mol of enzyme. The same amount of 3H incorporation was observed when p-bromophenacyl bromide-inactivated PLA2 was incubated with [3H]MLG, again indicating that catalytic activity is not required for the reaction of PLA2 with MLG. All together, these results suggest that MLD and MLG are not suicide inhibitors of PLA2. A portion of the incorporated radioactivity was acid-labile, and dialysis of the radiolabeled PLA2 under acidic conditions resulted in a loss of about one-third of the enzyme-associated radioactivity, leaving 2.4 mol of [3H]MLG/mol of PLA2. In previous studies, amino acid analysis, which also included acid treatment, indicated that MLG-modified cobra phospholipase A2 contained 2.8 mol of Lys less than the native enzyme. Thus, 1 mol of [3H]MLG is incorporated per mol of Lys lost. The implications of this 1:1 stoichiometry of MLG to Lys on the mechanism of reaction of these inhibitors is discussed.

Differential reactivity in the processing of [p-(halomethyl)benzoyl] formates by benzoylformate decarboxylase, a thiamin pyrophosphate dependent enzyme.

A series of [p-(halomethyl)benzoyl]formates have been investigated as substrates for benzoylformate decarboxylase. These analogues vary from acting as normal substrates to acting as potent competitive inhibitors. The fluoro analogue is a substrate with Km (190 microM) and turnover number (20 s-1) similar to those of benzoylformate (Km = 340 microM; 81 s-1). The bromo analogue is a competitive inhibitor (Ki = 0.3 microM) and exhibits processing to eliminate bromide and form (p-methylbenzoyl)thiamin pyrophosphate. This modified cofactor hydrolyzes to form the p-methylbenzoate in quantitative yield. The chloro analogue [Km(app) = 21 microM] partitions between these two pathways such that 0.6% of the analogue ultimately forms p-methylbenzoate. These data are consistent with the interpretation that the leaving group potential of the halogen determines the enzymic fate of the analogue and that the potent inhibition observed for the bromo analogue is due to covalent modification of the cofactor.

Activation, inhibition, and regiospecificity of the lysophospholipase activity of the 85-kDa group IV cytosolic phospholipase A2.

The 85-kDa Group IV calcium-dependent cytosolic phospholipase A2 (cPLA2) catalyzes the hydrolysis of palmitoylglycero-3-phosphocholine to palmitic acid and glycero-3-phosphocholine. Palmitoylglycero-3-phosphocholine exists as a 9:1 equilibrium mixture of the sn-1 and sn-2 isomers, with the fatty acid predominately at the sn-1 position. We have monitored this reaction by 31P NMR to determine which palmitoylglycero-3-phosphocholine isomer is processed by cPLA2. When both lysophospholipid isomers are present in a 1:1 mixture under conditions in which acyl migration is minimized, cPLA2 rapidly consumes both isomers. However, 1-palmitoylglycero-3-phosphocholine is consumed seven times faster than the 2-palmitoylglycero-3-phosphocholine isomer. We have previously reported that this lysophospholipase reaction is accelerated in the presence of glycerol. We now find that this apparent increase in activity is accounted for, in part, by glycerol acting as an alternative acceptor for the cleaved fatty acid, as is the case for this enzyme's phospholipase A2 (PLA2) activity. In contrast, dioleoylglycerol, which accelerates the PLA2 activity, does not act as an acceptor in either the lysophospholipase or the PLA2 reaction, but can affect enzyme activities by altering substrate presentation. We also show that a known inhibitor of the PLA2 activity of cPLA2 is able to inhibit its lysophospholipase activity with a similar IC50 to its PLA2 activity. However, the effect of inhibitors is dependent on the manner in which they are presented to the enzyme.

1-Hexadecyl-2-arachidonoylthio-2-deoxy-sn-glycero-3-phosphorylcholine as a substrate for the microtiterplate assay of human cytosolic phospholipase A2.

Human cytosolic phospholipase A2 (cPLA2) is an 85-kDa protein which displays a preference for arachidonoyl phospholipids as substrates. This substrate preference and the assay characteristics of the enzyme are quite different from those of the smaller, more well-studied extracellular PLA2s. We now report the development of a nonradioactive, spectrophotometric, microtiterplate assay for human cPLA2 using a novel synthetic thio-phospholipid analog as a substrate. This substrate is a phosphatidylcholine derivative with an arachidonoylthioester in the sn-2 position and an alkyl-ether in the sn-1 position. The use of an sn-1 alkyl-ether in the substrate ensures that the assay will only measure PLA2 activity and will not be complicated by the metabolism of the lysophospholipid product by the enzyme's lysophospholipase activity. cPLA2 is assayed at pH 7.4 and 37 degrees C with a mixed micellar substrate consisting of 2 mM thio-phospholipid and 4 mM Triton X-100 in 30% glycerol. Under these conditions, the assay is fairly linear for over 1 h.