Agonist-dependent failure of neutrophil function in diabetes correlates with extent of hyperglycemia.

Inexplicable controversies with regard to possible functional defects of neutrophilic polymorphonuclear leukocytes (PMNs) in diabetes persist. The purpose of the present study was to elucidate the relative effectiveness of several PMN agonists in stimulating lysosomal-enzyme secretion and leukotriene (LT) B(4) production by PMNs isolated from diabetic subjects. Formyl-methionyl-leucyl-phenylalanine (fMLP) and platelet-activating factor (PAF) induced significantly less lysosomal-enzyme secretion and LTB(4) production in diabetic-subject PMNs than in normal-subject PMNs. It is surprising that PMNs from these same diabetic subjects responded normally after stimulation with A23187, serum-opsonized zymosan, or phorbol myristate acetate. The in vitro responsiveness of PMNs stimulated with fMLP or PAF was inversely correlated with indices of in vivo glycemic control (fasting plasma glucose and glycated-hemoglobin levels). In combination, these results indicate that hyperglycemia is associated with sustained decreases in PMN function but only in response to agonists that initiate stimulus-response coupling via G-protein-coupled receptors. This agonist-selective reduction in PMN responsiveness may contribute to the compromised host defense associated with sustained hyperglycemia in diabetes.

Mass spectrometric analysis of platelet-activating factor after isolation by solid-phase extraction and direct derivatization with pentafluorobenzoic anhydride.

Platelet-activating factor is the term used to denote a class of extremely potent lipid mediators that consist predominantly of 1-O-alkyl- and 1-O-acyl-2-acetyl-sn-glycero-3-phosphocholines. A method has been devised for rapid isolation of these acetylated phospholipids by solid-phase extraction prior to direct derivatization with pentafluorobenzoic anhydride and analysis by gas chromatography (GC)/electron-capture mass spectrometry. Recovery through the entire method (lipid isolation, derivatization, and purification) typically ranged from 70% to 85%. Using the direct derivatization procedure described here, the practical limit of detection for each of the standard alkyl- and acyl-platelet-activating factor homologs was 1 fmol injected into the GC. Results from the application of the method to the analysis of alkyl and acyl homologs of platelet-activating factor isolated from stimulated human umbilical vein endothelial cells are presented, exhibiting excellent accuracy and precision for a wide range of tissue levels of this class of potent autacoids.

PAF, a putative mediator of oral inflammation.

PAF, or platelet-activating factor, is a family of structurally related phospholipids (1-O-alkyl/acyl/alkenyl-2-acetyl-sn-glycero-3-phosphocholine) which possesses a wide spectrum of potent pro-inflammatory actions. These phospholipids are synthesized by a diverse array of cells, including neutrophilic polymorphonuclear leukocytes (PMN), platelets, mast cells, monocytes/macrophages, vascular endothelial cells, and lymphocytes. PAF targets these and other cells via specific, G-protein-coupled receptors to initiate intracrine, autocrine, paracrine, and juxtacrine cell activation. Of importance, these unique acetylated phospholipids are frequently synthesized in concert with pro-inflammatory lipid mediators derived from arachidonic acid. Since PAF synergizes with these and other mediators to amplify the inflammatory response, it seems likely that PAF plays an integral, perhaps pivotal, role in acute and chronic inflammatory processes. PAF is present in the mixed saliva of dentate, but not edentulous, human subjects. The levels of PAF in mixed saliva or in gingival crevicular fluid and tissues are significantly increased during oral inflammatory conditions such as periodontitis and mucositis. Interestingly, the levels of salivary PAF correlate with the extent/severity of these oral diseases. These observations suggest that PAF may participate in pathophysiologic events during the course of oral inflammation. The availability of specific PAF receptor antagonists and human recombinant PAF-acetylhydrolase (PAF-AH), a plasma enzyme which rapidly destroys PAF, should provide clinical tools for the investigation of the role of PAF in these and other inflammatory disorders; and perhaps, ultimately, some of these reagents may prove to be therapeutically useful in the treatment and management of these conditions.

Alkyl-PAF and acyl-PAF human neutrophil priming for enhanced fMLP- and rC5a-induced superoxide anion production.

Alkyl-PAF induced two components of polymorphonuclear leukocyte (PMN) priming for enhanced fMLP- and rC5a-induced superoxide anion (O2-) production. Component A priming had a shallow, linear alkyl-PAF concentration-response slope (10 pM-1 nM), and component B priming had a significantly steeper concentration-response slope (1-100 nM alkyl-PAF). Whereas the extent of component B priming decayed significantly within 5-10 min after pretreatment of PMNs with alkyl-PAF, component A priming was completely stable. WEB 2086, a specific and potent PAF receptor antagonist, abolished component A priming when PMNs were simultaneously stimulated with alkyl-PAF and either fMLP or rC5a but only partially reduced component B priming. However, whereas WEB 2086 also obliterated component A priming when PMNs were pretreated with alkyl-PAF for 2.5, 5, or 10 min prior to fMLP stimulation, WEB 2086 had little or no inhibitory effect on component B priming. Paradoxically, WEB 2086 significantly augmented alkyl-PAF-induced component B priming for enhanced rC5a-induced PMN O2. production yet concomitantly obliterated component A priming. PMN priming by acyl-PAF (1 nm-1 micron) had characteristics identical to those of alkyl-PAF-induced component A priming. These studies suggest that there are at least two effector pathways modulating alkyl-PAF-induced PMN respiratory burst priming. They are also consistent with the notion that component A priming is initiated via high-affinity PAF receptors and component B priming is mediated through low-affinity PAF receptors; and whereas alkyl-PAF interacts with both high- and low-affinity PAF receptors, both acyl-PAF and WEB 2086 preferentially bind to the high-affinity PAF receptors.

Molecular heterogeneity of PAF in normal human mixed saliva: quantitative mass spectral analysis after direct derivatization of PAF with pentafluorobenzoic anhydride.

Platelet-activating factor (PAF), a family of phospholipid autacoids with potent pro-inflammatory activities, is present in saliva. The current study has quantitated various species of PAF isolated from normal human mixed saliva. Choline-containing, sn-2 acetylated phospholipids with sn-1 ether- or ester-linked fatty alcohol/acid moieties (alkyl-PAF or acyl-PAF, respectively) were evaluated after direct derivatization with pentafluorobenzoic (PFB) anhydride. Individual species of PFB-derivatized PAF were separated by gas chromatography prior to mass spectral analysis; quantitative estimates of six different species of PAF in saliva were made by comparison to corresponding authentic, synthetic PAF standards. In each saliva sample, all six species of PAF were readily detected by this facile procedure. The predominant PAF was 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine or 16:0-alkyl-PAF (0.75 +/- 0.09 pmol/ml saliva; mean +/- S.E.; n = 5) which represented only 30.4 +/- 1.5% of the total PAF. Substantial amounts of 18:1- and 18:0-alkyl-PAF and 16:0-acyl-PAF were also identified (0.52 +/- 0.07, 0.35 +/- 0.06, and 0.35 +/- 0.02 pmol/ml saliva, respectively). In summary, mass spectrometric analysis of PAF after direct derivatization with PFB anhydride has revealed that at least six different species of PAF are present in normal human mixed saliva. This structural diversity may represent an important aspect of homeostasis in the healthy oral cavity.

PAF molecular heterogeneity: pathobiological implications.

The existence and potential (patho)physiologic significance of PAF molecular heterogeneity can no longer be summarily dismissed or ignored. While significant advances in the chemistry of PAF have been made, the (patho)physiologic behaviors of most of the PAF molecular species of biologic origin await further study. This is because to date, investigators have studied the biologic activities of what was previously thought to be PAF, i.e., only 16:0- and 18:0-AGEPC. In view of the evidence presented in this review, a comprehensive investigation of the possible biologic relevance and significance of PAF molecular heterogeneity is warranted. Hopefully, such studies will be designed to elucidate the extent to which the various molecular species of PAF differ in their intrinsic in vitro and in vivo (patho)physiologic behaviors (agonistic, synergistic, and possibly antagonistic) and modes of action. Once this additional information has been derived, the pathobiologic relevance of this class of phospholipid autacoid may be better understood.

Differential electron capture mass spectral response of pentafluorobenzoyl derivatives of platelet activating factor alkyl chain homologs.

The electron capture mass spectrometric response of pentafluorobenzoyl ester derivatives of platelet-activating factor alkyl chain homologs has been found to be inversely proportional to their alkyl chain length. This phenomenon was observed when either the gas chromatograph or direct insertion probe was utilized for sample introduction. A similar differential response was also obtained for a series of fatty alcohols, analyzed as pentafluorobenzoyl esters. These studies demonstrate that multiple standard curves must be prepared for analysis of platelet-activating factor and related lipids in order to obtain accurate quantification.

Radiation-induced increased platelet-activating factor activity in mixed saliva.

BACKGROUND: Platelet-activating factor (PAF), a family of structurally-related phospholipid mediators of inflammation, is present in normal human mixed saliva; however, its role in oral biology and the homeostasis of oral host defense mechanisms remains to be established. EXPERIMENTAL DESIGN: The current study was designed to evaluate the salivary levels of PAF in patients with oral mucositis that developed as a complication of head and neck irradiation for oral cancer. PAF activity was assessed in platelet bioassay and expressed relative to the activity of authentic PAF, 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (C16:0-AGEPC). RESULTS: A significant increase in salivary PAF levels was observed in patients with mucositis (47,032 +/- 12,731 C16:0-AGEPC fmole equivalents/ml of saliva, mean +/- SE, N = 7) as compared with normal subjects (5,568 +/- 1,135 C16:0-AGEPC fmole equivalents/ml of saliva, N = 27). Phospholipid fractionation of the PAF isolated from the saliva of patients with mucositis by reverse phase high performance liquid chromatography revealed a single peak of activity that corresponded with the elution profile of C16:0-AGEPC, the most biologically active molecular species of PAF. In contrast, the PAF isolated from normal human mixed saliva contained multiple molecular species of PAF. CONCLUSIONS: These results suggest that this potent phospholipid inflammatory mediator may play a role in the inflammation and tissue injury associated with mucositis resulting from radiation treatment for oral cancer.

Differential responsiveness of human neutrophils to the autocrine actions of 1-O-alkyl-homologs and 1-acyl analogs of platelet-activating factor.

The phlogistic actions of six molecular species of platelet-activating factor (PAF) (1-O-alkyl-PAF homologs, 16:0-, 18:0- and 18:1-alkyl-PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC) and their respective 1-acyl-PAF analog counterparts, 16:0-, 18:0- and 18:1-acyl-PAF, 1-acyl-2-acetyl-sn-glycero-3-phosphocholine (AGPC)) were assessed relative to five human neutrophilic polymorphonuclear leukocyte (PMN) functional responses: 1) lysosomal enzyme secretion; 2) specific desensitization to 16:0-AGEPC-induced lysosomal enzyme secretion; 3) O2- production; 4) chemotaxis; and 5) priming for enhanced O2- production. With respect to inducing lysozyme secretion, 18:0-AGEPC was 30- and 75-fold less potent than 16:0-AGEPC and 18:1-AGEPC, respectively, and was 25- and 40-fold less potent for inducing beta-glucuronidase secretion. 18:0-AGEPC was also 10-fold less active than 18:1- and 16:0-AGEPC for inducing O2- production. Thus, the rank order of potency of the alkyl-PAF homologs for inducing both lysosomal enzyme secretion and O2- production was 18:1- greater than or equal to 16:0- much greater than 18:0-AGEPC. In contrast, these three alkyl-PAF homologs had the same potency for desensitizing PMN to subsequent 16:0-AGEPC-induced lysosomal enzyme secretion and for priming PMN for augmented O2- production in response to FMLP or human recombinant C5a. Paradoxically, however, the rank order of potency of the alkyl-PAF homologs for effecting PMN chemotaxis was 18:0- greater than 18:1- much greater than 16:0-AGEPC. At concentrations as high as 1.0 microM, the acyl-PAF analogs did not initiate PMN lysosomal enzyme secretion, O2- production, or chemotaxis. However, the acyl-PAF analogs induced partial PMN desensitization to 16:0-AGEPC. A novel finding of potential (patho)-physiologic significance was the ability of acyl-PAF at nM concentrations to prime PMN for significantly enhanced O2- production after stimulation with FMLP or human recombinant C5a. The priming action of acyl-PAF was due to an increase in the rate as opposed to a prolongation of O2- production. The differing rank orders of potency of the alkyl-PAF homologs and acyl-PAF analogs for stimulating several physiologic responses of the same target cell, the human PMN, support the premise that there may be more than one PAF receptor subtype on the PMN and/or that differences in the biophysical properties of the various molecular species of PAF modulate their interaction with PAF receptor(s) linked to stimulus-response coupling.

Electrospray ionization for analysis of platelet-activating factor.

Platelet-activating factor (PAF) was analyzed by electrospray-ionization mass spectrometry (ESI-MS) using a single quadrupole mass spectrometer. The positive-ion spectrum was dominated by an ion corresponding to a sodiated molecule when a low potential difference between the capillary exit (nozzle) and the skimmer was employed, but when the capillary exit voltage was increased, fragmentation of PAF was observed. Initial fragmentation involved the loss of the elements of trimethylamine from the sodiated molecule to yield [M+Na-59]+. An intense ion at m/z 147, generated by the loss of trimethylamine from the sodiated phosphocholine portion of the molecule was also detected, along with a lower intensity ion at m/z 184 which is representative of a protonated phosphocholine moiety. With negative-ion detection the major molecular species was [M+Cl]-. Interpretation of the mass spectral fragments was verified by ESI tandem mass spectrometry on a triple-quadrupole tandem mass spectrometer.

Isotopic exchange during derivatization of platelet activating factor for gas chromatography-mass spectrometry.

One approach to the quantitative analysis of platelet activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycerol-3-phosphocholine; also referred to as AGEPC, alkyl glyceryl ether phosphocholine) is hydrolytic removal of the phosphocholine group and conversion to an electron-capturing derivative for gas chromatography-negative ion mass spectrometry. [2H3]Acetyl-AGEPC has been commonly employed as an internal standard. When 1-hexadecyl-2-[2H3]acetyl glycerol (obtained by enzymatic hydrolysis of [2H3]-C16:0 AGEPC) is treated with pentafluorobenzoyl chloride at 120 degrees C, the resulting 3-pentafluorobenzoate derivative shows extensive loss of the deuterium label. This exchange is evidently acid-catalyzed since derivatization of 1-hexadecyl-2-acetyl glycerol under the same conditions in the presence of a trace of 2HCl results in the incorporation of up to three deuterium atoms. Isotope exchange can be avoided if the reaction is carried out at low temperature in the presence of base. Direct derivatization of [2H3]-C16:0 AGEPC by treatment with pentafluorobenzoyl chloride or heptafluorobutyric anhydride also results in loss of the deuterium label. The use of [13C2]-C16:0 AGEPC as an internal standard is recommended for rigorous quantitative analysis.

Novel mass spectral fragmentation of heptafluorobutyryl derivatives of acyl analogues of platelet-activating factor.

Direct derivatization of the acyl analogue of platelet-activating factor (acyl.PAF) with heptafluorobutyric anhydride results in replacement of the phosphocholine moiety with a heptafluorobutyryl (HFB) group. Electron capture (EC) mass spectrometric analysis of this compound that makes use of negative ion detection along with subsequent accurate mass measurement and tandem mass spectrometry studies revealed that in addition to expected fragmentation due to losses of elements of HF, ketene, and/or acetic acid, there is a rearrangement reaction between the HFB group and the subsequent on carbon-2 of the glycerol backbone. For 2-acetyl isomers, this fragmentation yields a characteristic ion at m/z 237; for 1-acetyl isomers, the analogous ion is observed at [M-135](-), along with a corresponding carboxylate anion. The use of the HFB derivative is invaluable for analysis of PAF homologues and analogues because it provides detailed structural information in combination with the high sensitivity of a gas chromatography combined with EC-mass spectrometry assay.

Analysis of platelet-activating factor by GC-MS after direct derivatization with pentafluorobenzoyl chloride and heptafluorobutyric anhydride.

Parallel analysis of platelet-activating factor (PAF) using chemical ionization gas chromatography-mass spectrometry after direct derivatization with pentafluorobenzoyl chloride (PFB) and heptafluorobutyric anhydride (HFB) provides a facile and highly sensitive means for detecting and elucidating the structure of the numerous alkyl-chain homologs of this acetylated phospholipid autacoid. In the present study, the PFB derivative was used for initial electron capture negative ion chemical ionization analysis of PAF candidate molecules in human PMN extracts of unknown composition. Subsequent pulsed positive ion/electron capture negative ion chemical ionization evaluation of the HFB derivative furnished a measure of the molecular weight from [MH]+ and yielded the required structural information from characteristic negative ions, in particular [M-(2HF + ketene)]- and [M-(HF + acetic acid)]-. These procedures easily permitted confirmation of the presence of C16:0-, C17:0-, C18:0-, and C18:1-AGEPC (acetyl glyceryl ether phosphocholine) in extracts of stimulated human PMN and also demonstrated that the C17:0- homolog was comprised of both straight-chain and branch-chain varieties.

Alkyl chain homologs of platelet-activating factor and their effects on the mammalian heart.

Platelet-activating factor (PAF; AGEPC) is a potent negative inotropic and coronary-vasoconstricting agent. Minor structural alterations in the PAF molecule are known to greatly affect its biological activity; thus, we have investigated the effects of selected synthetic saturated and unsaturated alkyl chain PAF homologs on the isolated guinea pig heart. The rank order of potency for the negative inotropic effect was C16:0- greater than C18:1- greater than beef-heart AGEPC greater than C15:0- greater than C18:0- greater than C14:0-AGEPC; the rank order for the coronary-vasoconstricting effect was C16:0- approximately C18:1- approximately beef-heart AGEPC greater than C15:0- greater than C18:0- approximately C14:0-AGEPC. With the exception of C16:0- and C18:1-AGEPC, the relative potencies for the cardiac and coronary effects of the alkyl chain AGEPC homologs did not correlate well with their relative potencies in stimulating rabbit platelets and human neutrophils. The differences in the rank order of potency for these AGEPC homologs suggest the presence of species and/or target cell PAF receptor heterogeneity.

A novel method for the analysis of platelet-activating factor: direct derivatization of glycerophospholipids.

A novel, facile, and sensitive method for the quantitative and complete structure-proof analysis of platelet-activating factor (PAF) and other glycerophospholipids is described. 1-O-Alkyl/acyl-2-acyl-3-glycerophospholipids were treated with heptafluorobutyric anhydride in a one-step reaction to yield 1-O-alkyl/acyl-2-acyl-3-heptafluorobutyroyl-sn-glycerols as gas-liquid chromatography (GLC)-compatible derivatives. Furthermore, the components of the polar head group were also analyzed from the aqueous extract of the same reaction mixture as t-butyldimethylsilyl derivatives. Thus, this new method eliminates the need for phospholipase C treatment and subsequent purification procedures. Moreover, the direct derivatization of PAF homologs and analogs with hepatofluorobutyric anhydride does not result in positional isomerization of the product, providing increased specificity for gas-liquid chromatography-mass spectrometric (MS) analysis. It has also been shown that the heptafluorobutyroyl (HFB) derivative can easily be converted to the respective t-butyldimethylsilyl analog in a one-step reaction using t-butyldimethylsilyl chloride/imidazole reagent. Analogous to the formation of heptafluorobutyroyl derivatives, PAF also was reacted with pentafluorobenzoyl chloride to generate the pentafluorobenzoyl derivative. Therefore, this method has wide applicability for the formation of GLC-compatible derivatives of various glycerophospholipids. Our successful HFB derivatization and GLC-MS detection of subnanogram quantities of PAF indicate that this analytical procedure will greatly facilitate complete and quantitative identification of each of the molecular species of biologically derived PAF.

Complement and neutrophil activation in the pathogenesis of ischemic myocardial injury.

Complement depletion with cobra venom factor (CVF) before coronary artery ligation has been previously shown to reduce subsequent ischemic myocardial tissue injury in the baboon; however, whether complement depletion after the initiation of acute myocardial ischemia affords similar myocardial preservation is not known. Both complement depletion with CVF or the administration of certain nonsteroidal anti-inflammatory drugs, including ibuprofen, are thought to decrease myocardial infarct size by reducing polymorphonuclear leukocytic (PMN) infiltration; nevertheless, complement activation also could alter tissue injury by PMN-independent actions. Thus, the relative effects of CVF administered after coronary artery ligation on the subsequent development of myocardial tissue injury were assessed in a baboon myocardial infarction model. The animals were randomized into three treatment groups (n = 6): either CVF (125 units/kg) or saline was given 30 minutes after coronary artery ligation, and ibuprofen (12.5 mg/kg) was administered 30 minutes and 4 hours after ligation. The extent of ischemic myocardial injury was assessed 24 hours later. Relative to saline-treated baboons, both CVF and ibuprofen reduced PMN infiltration (36 +/- 4 vs. 24 +/- 4 and 24 +/- 4 PMN/mm2, respectively; mean +/- SEM) and histological evidence of transmural myocardial infarction (100% vs. 47% and 53%, respectively) in electrocardiographically designated, expected infarct sites. In both saline- and ibuprofen-treated animals, there was extensive localization of C4, C3, and C5 in all infarct sites; in contrast, there was only C4 localization in the CVF-treated baboons. When expected infarct sites were assessed for creatine kinase content as an indicator of tissue injury, there was significantly less epicardial and endocardial creatine kinase depletion in the CVF-treated animals (31.7 +/- 5.6% and 39.3 +/- 4.8%) than in the saline-treated animals (54.1 +/- 5.4% and 59.0 +/- 4.7%; p = 0.012 and 0.011, respectively). The percent creatine kinase depletion in the ibuprofen-treated animals was intermediate between the two other groups. These results suggest that depletion of complement after coronary ligation has beneficial effects in reducing tissue injury that cannot be explained solely on the basis of reducing PMN infiltration into the ischemic myocardium.

Platelet-activating factor in the rabbit uterus during early pregnancy.

Platelet-activating factor (PAF) concentrations were low in the non-pregnant, oestrous uterus (mean +/- s.e.m.: 2.2 +/- 1.2 pmol/g, n = 3). However, uterine PAF increased dramatically during pregnancy to a maximum of 37.8 +/- 4.90 pmol/g (n = 7) on Day 5. By Day 7, PAF concentrations in the uteri of pregnant rabbits had returned to levels similar to those found at oestrus. In contrast, uterine PAF in pseudopregnant rabbits peaked at 30.6 +/- 2.8 pmol/g (n = 8) on Day 4, declined to 20.5 +/- 2.4 pmol/g (n = 8) on Day 5 and then remained at that concentration through Day 7. Uterine PAF co-migrated with synthetic PAF (1-O-hexadecyl-2-acetyl-sn-glycero-phosphocholine) in both thin-layer and normal-phase high-performance liquid chromatography. PAF activity in the uterus during pregnancy and pseudopregnancy was found almost exclusively in the endometrium; little or no PAF was found in myometrium, uterine flushings or blastocysts. While no PAF was detected in blastocysts on Days 5 and 6 of pregnancy, the presence of the embryo appears to modulate biosynthesis and/or degradation of PAF by the uterus, since PAF decreased significantly in uterine tissue apposed to the implanting embryo (but not in similar areas between such attachment sites). Increased concentrations of PAF in the preimplantation rabbit uterus followed by a dramatic decrease on the day of blastocyst attachment suggest that this potent inflammatory autacoid may play a vital role in implantation.

Ontogeny of the responsiveness to intravenous platelet-activating factor.

Neonatal and young animals fail to develop antigen-induced, lethal, systemic anaphylactic reactions. Recent evidence has documented that, in the adult rabbit, an unusual phospholipid autacoid, platelet-activating factor, induces almost all of the physiologic events associated with IgE-induced anaphylaxis. Thus, in the present study, the intravascular alterations after intravenous infusion of synthetic platelet-activating factor (1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine; AGEPC) into young rabbits were examined. In comparison to 13-week-old, adult rabbits, the intravascular infusion of greater than 1.0 micrograms/kg AGEPC was not lethal in rabbits of 8 weeks of age or less. In dose-response studies, the amount of AGEPC required to induce a lethal response in 50% of the animals tested (LD50) was found to inversely correlate with age. In contrast, AGEPC-induced platelet aggregation in vitro was not affected by the age of the donor animal. Consistent with age-independent platelet responsiveness in vitro, AGEPC-induced thrombocytopenia and intravascular accumulation of platelet factor 4 and thromboxane B2 were also unaffected by animal age. Neutropenia and basopenia, as well as platelet and neutrophil sequestration in the pulmonary microvasculature after intravenous AGEPC infusion also were similarly unaffected by animal age. Although the mechanisms which modulate the profound and lethal physiologic responses following AGEPC infusion in the adult rabbit remain to be established, the current study clearly documents an age-dependent acquisition of systemic physiologic sensitivity to AGEPC and/or other mediators released as a result of intravascular AGEPC administration.