Neutrophilic dermatoses.

Neutrophilic dermatoses (ND) are a group of inflammatory skin conditions characterized by a neutrophilic infiltrate on histopathology with no evidence of infection. ND are classified based upon the localization of neutrophils within the skin and clinical features. Recent findings suggest that ND are due to two main mechanisms: i) a polyclonal hereditary activation of the innate immune system (polygenic or monogenic); or ii) a clonal somatic activation of myeloid cells such as encountered in myelodysplastic syndrome or VEXAS syndrome. ND belong to internal medicine as a great number of patients with ND suffer from an underlying condition (such as hematological malignancy, inflammatory bowel disease, auto-immune and auto-inflammatory diseases). ND are diagnoses of exclusion and physicians should always consider differential diagnoses, particularly skin infections. Here, we review the pathophysiology and classification of the main ND (i.e., subcorneal pustular dermatosis (Sneddon-Wilkinson Disease) and Intercellular IgA dermatoses, aseptic pustulosis of the folds, Sweet syndrome, neutrophilic eccrine hidradenitis, pyoderma gangrenosum, erythema elevatum diutinum, neutrophilic urticarial dermatosis and neutrophilic panniculitis), their clinical and histopathological features, and we highlight the investigations that are useful to identify ND-associated diseases and to exclude the differential diagnoses.

Identification of IgG1 isotype phosphorylcholine antibodies for the treatment of inflammatory cardiovascular diseases.

BACKGROUND: Phosphorylcholine (PC) is an important pro-inflammatory damage-associated molecular pattern. Previous data have shown that natural IgM anti-PC protects against cardiovascular disease. We aimed to develop a monoclonal PC IgG antibody with anti-inflammatory and anti-atherosclerotic properties. METHODS: Using various techniques PC antibodies were validated and optimized. In vivo testing was performed in a femoral artery cuff model in ApoE3*Leiden mice. Safety studies are performed in rats and cynomolgus monkeys. RESULTS: A chimeric anti-PC (PC-mAb(T15), consisting of a human IgG1 Fc and a mouse T15/E06 Fab) was produced, and this was shown to bind specifically to epitopes in human atherosclerotic tissues. The cuff model results in rapid induction of inflammatory genes and altered expression of genes associated with ER stress and choline metabolism in the lesions. Treatment with PC-mAb(T15) reduced accelerated atherosclerosis via reduced expression of endoplasmic reticulum stress markers and CCL2 production. Recombinant anti-PC Fab fragments were identified by phage display and cloned into fully human IgG1 backbones creating a human monoclonal IgG1 anti-PC (PC-mAbs) that specifically bind PC, apoptotic cells and oxLDL. Based on preventing macrophage oxLDL uptake and CCL2 production, four monoclonal PC-mAbs were selected, which to various extent reduced vascular inflammation and lesion development. Additional optimization and validation of two PC-mAb antibodies resulted in selection of PC-mAb X19-A05, which inhibited accelerated atherosclerosis. Clinical grade production of this antibody (ATH3G10) significantly attenuated vascular inflammation and accelerated atherosclerosis and was tolerated in safety studies in rats and cynomolgus monkeys. CONCLUSIONS: Chimeric anti-PCs can prevent accelerated atherosclerosis by inhibiting vascular inflammation directly and through reduced macrophage oxLDL uptake resulting in decreased lesions. PC-mAb represents a novel strategy for cardiovascular disease prevention.

Lack of effect of acute repaglinide administration on postprandial lipaemia in patients with type 2 diabetes mellitus.

The effect of acute repaglinide administration (2 mg) on postprandial glycaemia and lipaemia has been examined in 20 subjects with type 2 diabetes mellitus. Each subject received in the morning, after a 12 to 14 h fast, a standard mixed meal (total energy 783 kcal), preceded by one tablet of 2 mg repaglinide or placebo. Chylomicrons and chylomicron-deficient plasma were prepared by ultracentrifugation. Triglyceride levels in CM fraction (CM-triglycerides) in total plasma as well as in CM-deficient plasma (non-CM-triglycerides) were determined. A significant reduction in postprandial glycaemia was observed after repaglinide administration compared to placebo ( p < 0.001). Plasma concentrations of total triglycerides, CM-triglycerides, non-CM-triglycerides, free fatty acids and the other plasma lipids measured, were not significantly different between the two phases of the study. It is concluded that, in contrast to sulphonylureas, acute repaglinide administration does not improve postprandial lipaemia in patients with type 2 diabetes.

Serum lipoprotein(a) concentrations and apolipoprotein(a) isoforms: association with the severity of clinical presentation in patients with coronary heart disease.

OBJECTIVE: The aim of this study was to investigate the possible associations between lipoprotein(a) [Lp(a)] concentrations or apolipoprotein(a) isoforms and the mode of clinical presentation of coronary heart disease (CHD) (acute thrombotic event or not). METHODS: A total of 131 CHD patients and 71 age- and gender-matched individuals without known CAD (free of symptoms of heart disease) were enrolled in the study. CHD patients were classified into patients with a history of an acute coronary syndrome (ACS, n=94) and patients with stable angina (SA, n=37). Lp(a) levels were measured with an ELISA method, whereas apolipoprotein(a) isoform analysis was performed (in all patients and 33 controls) by electrophoresis in 1.5% SDS-agarose gels followed by immunoblotting. Isoform size was expressed as the number of kringle 4 (K4) repeats. RESULTS: ACS patients had higher Lp(a) plasma levels 121.9 (0.8-84.1) mg/dl] and a greater proportion of elevated (> or = 30 mg/dl) Lp(a) concentrations (25.5%) compared with SA patients [9.2 (0.8-50.5) mg/dl, P < 0.01 and 10.8%, P < 0.05] and controls [8.0 (0.8-55.0) mg/dl, P < 0.01 and 11.2%, P < 0.05], while there were no differences between SA patients and controls. The median apolipoprotein(a)-isoform size was 26 K4. In 17 (10%) patients we could not detect any apolipoprotein(a) isoform bands by immunoblotting. ACS patients had a higher proportion of isoforms < 26 K4 (low molecular weight) than SA patients (56/85 vs. 12/33, P < 0.005) and controls (10/29, P < 0.005). CONCLUSIONS: CAD patients with a history of ACS have higher Lp(a) plasma levels and a significantly higher proportion of low molecular weight apolipoprotein(a) isoforms compared with patients with SA or to controls.

N-linked glycosylation of macrophage-derived PAF-AH is a major determinant of enzyme association with plasma HDL.

Human plasma PAF-AH (platelet-activating factor-acetylhydrolase) is a Ca(2)+-independent phospholipase A2 of hematopoietic origin associated with LDL and HDL; it degrades PAF and oxidizes phospholipids. We show that human macrophages synthesize PAF-AH as a premedial Golgi precursor containing high mannose N-linked glycans. Secreted PAF-AH possesses a molecular mass of approximately 55 kDa and contains mature N-linked glycans. Secreted PAF-AH activity (90 +/- 4% of the total) bound to a wheat germ lectin column and could be eluted with N-acetylglucosamine, whereas digestion with N-acetylneuraminidase II completely abolished enzyme absorption. Tunicamycin significantly reduced cell-associated PAF-AH activity and inhibited enzyme secretion; but it did not alter the ratio of secreted to cell-associated enzyme (1.8 at 6 h and 3.1 at 24 h), suggesting that glycosylation is not essential for PAF-AH secretion. Digestion of cell-associated PAF-AH or secreted PAF-AH with peptide N-glycosidase F affected neither catalytic activity nor its resistance to proteolysis with trypsin or proteinase K; in addition, it did not affect PAF-AH association with LDL, but significantly increased its association with HDL. We suggest that macrophage-derived PAF-AH contains heterogeneous asparagine-conjugated sugar chain(s) involving sialic acid, which hinders its association with HDL but does not influence the secretion, catalytic activity, or resistance of PAF-AH to proteases.

Glibenclamide improves postprandial hypertriglyceridaemia in type 2 diabetic patients by reducing chylomicrons but not the very low-density lipoprotein subfraction levels.

AIM: There are scarce data dealing with the degree of postprandial lipaemia after sulphonylurea administration. The aim of this study was to examine the effect of acute glibenclamide administration on postprandial lipaemia in Type 2 diabetic patients. METHODS: Eight randomly selected Type 2 diabetic individuals, aged 43-65 years (mean, 54 years), who had never received any anti-diabetic drug, were included in the study. Each patient was given a 485 kcal mixed meal (45% fat, 40% carbohydrate and 15% protein) twice on separate days after an overnight fast: once with placebo and once with 5 mg glibenclamide, per os, in a random order. The two tests were performed with an interval of 7 days. Venous blood samples were drawn just before and 2 h, 4 h and 6 h after meal consumption. Total triglyceride levels in plasma, in chylomicrons (CM), in CM-deficient plasma, in very low-density lipoprotein (VLDL) subfractions (VLDL-1, VLDL-2) and in intermediate-density lipoprotein (IDL) were determined. Free fatty acid (FFA) and total cholesterol levels in plasma, as well as high-density lipoprotein (HDL) cholesterol and low-density lipoprotein (LDL) cholesterol levels in CM-deficient plasma, were also measured. Finally, serum glucose, insulin and C-peptide concentrations were measured in each sample. RESULTS: As expected there was a significant decrease in postprandial glycaemia after glibenclamide administration compared to placebo (mean area under the curve values: AUC = 53.3 +/- 18.2 and 69.1 +/- 21.6 mm/h, P = 0.00009). In addition, the mean AUC values of insulin and C-peptide were significantly greater after drug administration. The AUC values of total plasma triglyceride and of CM triglyceride following glibenclamide administration were significantly lower compared to placebo, while the AUC values of postprandial triglyceride in CM-deficient plasma and of postprandial triglyceride in VLDL-1, VLDL-2 and IDL were not different after drug administration compared to placebo. Finally, no significant differences were noted in the AUC values of total cholesterol, LDL cholesterol, HDL cholesterol and plasma FFA levels after glibenclamide administration. CONCLUSIONS: These results demonstrate that glibenclamide administration improves postprandial hypertriglyceridaemia acutely by reducing postprandial triglycerides of intestinal origin.

Evaluation of five methods for determining low-density lipoprotein cholesterol (LDL-C) in hemodialysis patients(1).

OBJECTIVES: Current recommendations for the management of dyslipidemia are largely based on the concentration of LDL-C. Most clinical laboratories estimate the concentration of LDL-C by the recommended routine method, the equation of Friedewald, in specimens from fasting subjects and with TG concentrations < 4.52 mmol/L. Because of the limitations of the Friedewald calculation, direct methods for an accurate quantification of LDL-C are needed. DESIGN AND METHODS: In the present study we evaluated the accuracy of the following 5 different procedures for LDL-C in 98 patients on hemodialysis: the Friedewald equation, where LDL-C is calculated from HDL-C, measured either by the precipitation procedure with dextran sulfate-Mg(2+) (Method 1), or by a direct HDL-C assay (Method 2), the Direct LDL assay (Method 3), the homogeneous N-geneous LDL assay (Method 4) and the calculated LDL-C values deriving from the ApoB based equation: 0.41TC - 0.32TG + 1.70ApoB - 0.27, (Clin Chem 1997;43:808-815) (Method 5). RESULTS: All five LDL-C methods were found to be in good agreement with ultracentrifugation/dextran sulfate-Mg(2+) precipitation with the coefficients of correlation of the assays to ranging between 0.93-0.95. However, significant differences in the mean values and biases vs. the reference method were observed. The Friedewald equation and the Direct assay were less affected by high LDL-C levels, and they presented higher sensitivity and higher negative predictive value. The N-geneous assay and the ApoB derived calculation were less affected by high triglyceride levels, and they presented higher specificity and higher positive predictive value. At the diagnostic LDL-C level of 3.37 mmol/L, both Friedewald calculations correctly classified 82/92 patients; Direct assay 86/98; N-geneous assay 88/98; and ApoB derived calculation 88/98. At the diagnostic LDL-C level of 2.98 mmol/L, Friedewald calculations (Method 1 and Method 2) correctly classified 82/92 and 81/92 patients, respectively; Direct assay (LDL-3) 87/98; N-geneous assay (LDL-4) 91/98; and ApoB derived calculation (LDL-5) 91/98. CONCLUSIONS: Among hemodialysis patients, who commonly present "average" LDL-C concentrations and high TG levels, the N-geneous assay and the apoB derived calculation seem to yield more acceptable results for the estimation of LDL-C.

LDL subfractions in patients with myocardial infarction: effect of smoking and beta-blocker treatment.

The aim of the present case-control study was to estimate, by density gradient ultracentrifugation, LDL heterogeneity in myocardial infarction, and to evaluate the effect of smoking and beta-blocker treatment on LDL subfraction profile. Our results show that patients who survive myocardial infarction have an abundance of small, dense LDL in their plasma, compared with controls. Patients who were on beta-blockers and those who smoked showed a more atherogenic LDL subfraction profile than the rest. In patients on beta-blocker treatment, the proportion of LDL3 was positively correlated with triglyceride concentration and body mass index. Dense LDL predominates in patients irrespective of smoking or beta-blocker treatment. The relative risk, calculated by logistic regression as the odds ratio of high LDL3, was 7.5 (95% confidence interval 2.5-22.1) and was not significantly influenced when smoking, beta-blocker treatment, triglycerides or the other parameters of the study were included in the statistical model.

Plasma and Lp(a)-associated PAF-acetylhydrolase activity in uremic patients undergoing different dialysis procedures.

UNLABELLED: Plasma and Lp(a)-associated PAF-acetylhydrolase activity in uremic patients undergoing different dialysis procedures. BACKGROUND: Platelet-activating factor (PAF) is a potent inflammatory mediator associated with several physiopathological conditions, including renal diseases. PAF is degraded to the inactive metabolite lyso-PAF by PAF-acetylhydrolase (PAF-AH), which is considered as a potent anti-inflammatory and anti-atherogenic enzyme associated with lipoproteins. In this study, we evaluated the plasma- and lipoprotein(a) [Lp(a)]-associated PAF-AH activity in relationship to plasma lipid parameters and Lp(a) isoform size in patients with mild/moderate chronic renal failure (CRF), as well as in hemodialysis (HD) and chronic ambulatory peritoneal dialysis (CAPD) patients. METHODS: We studied 74 patients undergoing maintenance HD, 44 patients undergoing CAPD, 56 patients with mild/moderate CRF, and 98 healthy subjects whose lipid profile, as well as plasma and high-density lipoprotein (HDL)-associated PAF-AH activity, was determined. Moreover, the effect of Lp(a) plasma levels on the distribution of PAF-AH among plasma lipoproteins, as well as the specific activity and kinetic properties of PAF-AH on two different Lp(a) isoforms, was measured in each studied group. RESULTS: The plasma PAF-AH activity in all studied groups was significantly higher than in controls, and the increase was more profound in CAPD patients. The HDL-associated PAF-AH activity, expressed per milliliter of plasma, was similar among all studied groups; however, when it was expressed as either per milligrams of HDL cholesterol or per milligrams of plasma apolipoprotein (apo) AI, the PAF-AH activity was significantly higher in all patient groups compared with controls. All patient groups had significantly elevated plasma Lp(a) levels, which altered the distribution of PAF-AH among the plasma lipoproteins compared with that observed in subjects with very low plasma Lp(a) levels (<8 mg/dl). Additionally, in each studied group, the specific activity as well as the apparent Km and Vmax values of the 19K4 apo(a) isoform were significantly higher (P < 0.01) compared with the values of the 23K4 isoform. However, the specific activity, as well as the Km and Vmax values on either the 19K4 apo(a) isoform or the 23K4 isoform, was significantly higher in CAPD patients compared with the other three groups. CONCLUSIONS: Plasma PAF-AH activity is increased in uremic patients. This elevation is more profound in CAPD patients, who also exhibit a more atherogenic lipid profile and more pronounced alterations in the specific activity and the kinetic constants of Lp(a)-associated PAF-AH.

The effect of moxonidine on plasma lipid profile and on LDL subclass distribution.

Moxonidine is a new antihypertensive agent whose mechanism of action appears to involve specific stimulation of imidazoline receptors resulting in an inhibition of the activity of the central and peripheral sympathetic nervous system. The drug seems to behave neutrally with respect to plasma lipid parameters. However, there are no data on the effects of moxonidine on the low-density lipoprotein (LDL) subclass pattern or on the LDL oxidation susceptibility, both of which are known to play a prominent role in the pathogenesis of atherosclerosis. Thus, we undertook the present study to examine the influence of moxonidine on the LDL subspecies profile and their susceptibility to copper-induced oxidative modification in 20 hypertensive patients (11 men, 9 women) aged 38-61 years. Moxonidine administered at a dose of 0.4 mg daily for 8 weeks produced a significant decrease in both systolic and diastolic blood pressure (from 147 +/- 10 to 131 +/- 11 mm Hg, P < 0.001, and from 98 +/- 4.5 to 86 +/- 5 mm Hg, P < 0.001, respectively). No significant change in plasma lipid profile was observed after moxonidine administration. Additionally, no change in the susceptibility of LDL subclasses to copper-induced oxidative modification was noticed. Finally, drug therapy was not followed by any change in either LDL phenotype or in mass and composition of the three LDL subfractions. We conclude, that unlike other antihypertensive drugs, such as beta-blockers which may predispose to expression of a relatively atherogenic lipoprotein subclass pattern, moxonidine does not affect either plasma lipid parameters or lipoprotein composition.

Apolipoprotein(a) phenotypes and lipoprotein(a) concentrations in patients with renal failure.

Patients with renal failure have an increased incidence of atherosclerotic disease. Numerous studies have shown that these patients show increased serum lipoprotein(a) [Lp(a)] concentrations compared with the control population. However, variable alleles at the apolipoprotein(a) [apo(a)] gene locus determine to a large extent the Lp(a) concentration in the general population. We therefore undertook the present study to evaluate apo(a) phenotypes and Lp(a) serum concentrations in a large number of patients with renal disease. Seventy-nine patients treated by hemodialysis (HD), 47 patients treated by continuous ambulatory peritoneal dialysis (CAPD), 68 patients with mild/moderate chronic renal failure (CRF) and serum creatinine levels of 1.8 to 8 mg/dL, and 73 healthy controls were studied. All patients showed significantly elevated median serum Lp(a) concentrations in comparison with controls: HD patients, 15.7 mg/dL (P < 0.01); CAPD patients, 20 mg/dL (P < 0. 005); CRF patients, 15.1 mg/dL (P < 0.01) versus controls, 7 mg/dL. The greater Lp(a) values in all groups were not explained by differences in isoform frequencies, whereas their increase was apo(a)-type specific. Thus, patients in all groups with high-molecular-weight (HMW) apo(a) isoforms showed a significant elevation of Lp(a) levels, whereas serum Lp(a) concentrations in patients with low-molecular-weight (LMW) isoforms were not significantly different from controls, except for CAPD patients, who presented increased serum Lp(a) concentrations. We conclude that in patients with renal failure, even of mild/moderate degree, as well as in patients with end-stage renal disease undergoing HD or CAPD, elevated Lp(a) concentrations are mainly observed in those with HMW apo(a) phenotypes.

Association of the inflammatory state in active juvenile rheumatoid arthritis with hypo-high-density lipoproteinemia and reduced lipoprotein-associated platelet-activating factor acetylhydrolase activity.

OBJECTIVE: To investigate the relationship between the quantitative and qualitative abnormalities of apolipoprotein B (Apo B)- and Apo A-I-containing lipoproteins and between lipoprotein-associated platelet-activating factor acetylhydrolase (PAF-AH) activity in patients with juvenile rheumatoid arthritis (JRA) as a function of the inflammatory state. METHODS: Twenty-six JRA patients and 22 age- and sex-matched control subjects with normal lipid levels participated in the study. Fourteen patients had active disease, and 12 had inactive disease. Plasma lipoproteins were fractionated by gradient ultracentrifugation into 9 subfractions, and their chemical composition and mass were determined. The PAF-AH activity associated with lipoprotein subfractions and the activity in plasma were also measured. RESULTS: Patients with active JRA had significantly lower plasma total cholesterol and high-density lipoprotein (HDL) cholesterol levels as compared with controls, due to the decrease in the mass of both the HDL2 and HDL3 subfractions. Patients with active JRA also had higher plasma triglyceride levels, mainly due to the higher triglyceride content of the very low-density lipoprotein plus the intermediate-density lipoprotein subfraction. The plasma PAF-AH activity in patients with active JRA was lower than that in controls, mainly due to the decrease in PAF-AH activity associated with the intermediate and dense low-density lipoprotein subclasses. The lipid abnormalities and the reduction in plasma PAF-AH activity were significantly correlated with plasma C-reactive protein levels and were not observed in patients with inactive JRA. CONCLUSION: This is the first study to show that patients with active JRA exhibit low levels of HDL2 and HDL3 and are deficient in plasma PAF-AH activity. These alterations suggest that active JRA is associated with partial loss of the antiinflammatory activity of plasma Apo B- and Apo A-I-containing lipoproteins.

Increased platelet reactivity to the aggregatory effect of platelet activating factor, in vitro, in patients with heterozygous familial hypercholesterolaemia.

Patients with familial hypercholesterolaemia (FH) present with high plasma total- and low density lipoprotein (LDL)-cholesterol levels and develop premature and often severe atherosclerosis. Elevations of total- and LDL cholesterol levels are not only related to an increased risk of atherosclerosis, but may also exert prothrombotic effects via platelet activation leading to acute coronary events. In the present work, the platelet response to the aggregatory effect of platelet-activating factor (PAF) in relation to the plasma PAF-acetylhydrolase (PAF-AH) activity and to their lipidemic profile was studied in 20 heterozygous FH patients. The PAF EC(50) aggregation values in the patient group were significantly decreased ( P < 0.03) compared with the control group (19.5 5.2 nM and 30.4 7.2 nM, respectively). Moreover, the maximal percentage of aggregation to 100 nM PAF was significantly increased in the patient group compared with controls (26.5 8.2% vs 15.2 3.1%, respectively, P < 0.03). Both platelet aggregation parameters were correlated to the plasma total- and LDL-cholesterol levels, as well as to the apolipoprotein B (apo B) levels. The maximal percentage of aggregation to 10 microM ADP was also significantly increased in the patient group compared with controls (51.5 10.3% vs 32.4 9.0%, respectively, P < 0.02) but was not correlated to any plasma lipid parameter. The total plasma PAF-AH activity in the heterozygous FH patients was significantly higher compared with controls (109.8 15.9 nmol/ml per min vs 68.4 18.0 nmol/ml per min, respectively, P < 0.0001), whereas the HDL-associated PAF-AH activity did not differ significantly between the two groups. Our results suggest that the increased aggregatory response of platelets to PAF despite the significantly higher plasma PAF-AH activity, could be an important factor contributing to the higher atherogenicity and incidence of acute coronary events observed in patients with heterozygous FH.

Increased activity of platelet-activating factor acetylhydrolase in low-density lipoprotein subfractions induces enhanced lysophosphatidylcholine production during oxidation in patients with heterozygous familial hypercholesterolaemia.

Patients with heterozygous familial hypercholesterolaemia (FH) have elevated plasma concentrations of low-density lipoprotein (LDL) and develop premature atherosclerosis. There is increasing evidence that oxidative modification of LDL is important for the pathogenesis of atherosclerosis, and the LDL-associated platelet-activating factor acetylhydrolase (PAF-AH) seems to play a key role in LDL oxidation by hydrolysing the oxidized phospholipids of phosphatidylcholine (PC) and producing lysophosphatidylcholine (lyso-PC). We measured the total serum and high-density lipoprotein (HDL) levels of PAF-AH activity and studied the distribution of PAF-AH activity among three LDL subfractions isolated by gradient ultracentrifugation in 15 patients with heterozygous FH and 13 normolipidaemic control subjects. We also determined the lyso-PC production in each LDL subfraction during Cu2(+)-induced oxidation in vitro. The total serum PAF-AH activity in heterozygous FH patients was significantly higher than in control subjects, whereas the HDL-associated PAF-AH activity, expressed as a percentage of total serum PAF-AH activity, was significantly lower in the FH patients than in control subjects (13.9 +/- 6.6% vs. 30.6 +/- 4.4%, P < 0.001). Among the LDL subfractions, the PAF-AH activity in both normolipidaemic control subjects and FH patients, expressed as nmol mg-1 protein min-1, was significantly higher in the LDL3 subfraction (33.1 +/- 4.8 and 53.4 +/- 11.5 respectively) than in the LDL2 (18.6 +/- 5.3 and 26.8 +/- 10.4 respectively, P < 0.0001 for both comparisons) and LDL1 subfractions (5.1 +/- 1.5 and 7.8 +/- 2.6, respectively, P < 0.0001 for both comparisons). Additionally, the enzyme activity in each LDL subfraction of the heterozygous FH patients was significantly higher than in control subjects (P < 0.02 for LDL1, P < 0.03 for LDL2 and P < 0.0001 for LDL3). No difference was observed in the susceptibility to oxidation of each LDL subfraction among the heterozygous FH patients and the normolipidaemic control subjects. During oxidation, the PAF-AH activity decreased, whereas the lyso-PC levels significantly increased in all subfractions of both groups. The lyso-PC/sphingomyelin molar ratio in each LDL subfraction of the FH patients 3 h after the onset of the oxidation was significantly higher than in control subjects [0.38 +/- 0.05 and 0.27 +/- 0.04, respectively, for LDL1 (P < 0.006), 0.47 +/- 0.08 and 0.39 +/- 0.03, respectively, for LDL2 (P < 0.04), 0.55 +/- 0.11 and 0.42 +/- 0.06, respectively, for LDL3 (P < 0.02)]. Our results show that heterozygous FH patients exhibit higher PAF-AH activity than control subjects in all LDL subfractions, resulting in higher lyso-PC production during oxidation, which confers on these subfractions higher biological potency. This phenomenon, in combination with the diminished anti-atherogenic and antioxidant capability of HDL in these patients due to the relatively low HDL-cholesterol levels compared with LDL-cholesterol levels and, consequently, the relatively low HDL-associated PAF-AH activity, could contribute to the higher atherogenicity and incidence of coronary artery disease observed in FH patients.

PAF-acetylhydrolase activity of Lp(a) before and during Cu(2+)-induced oxidative modification in vitro.

In human plasma with no detectable lipoprotein (a) (Lp(a)) levels, platelet-activating factor acetylhydrolase (PAF-AH) is associated with low density lipoprotein (LDL) and high density lipoprotein (HDL) with a distribution of 70 and 30%, respectively. We used a density gradient ultracentrifugation procedure to study the distribution of PAF-AH among lipoproteins in plasma containing Lp(a). Lp(a) was migrated as a broad band in the density region of d = 1.050-1.100 g/ml, independently of its isoform size. In plasma with Lp(a) levels 30-40 mg/dl or 80-100 mg/dl the PAF-AH activity migrated in this density region was 4 or 9% higher as compared to plasma having Lp(a) levels < 8 mg/dl (P < 0.05 or P < 0.02, respectively). Enrichment of plasma with the dense LDL5 subfraction, significantly increased the enzyme activity distributed in this density region. The physicochemical properties of the Lp(a)-associated PAF-AH activity were similar to those reported for the LDL-associated enzyme. However, the kinetic constants in small Lp(a) isoforms were significantly higher compared to large ones. Isoform F had apparent Km = 117 +/- 9 mumol/l and Vmax = 94 +/- 5 nmol/mg protein per min, and isoform S2/S3 had apparent Km = 36 +/- 9 mumol/l and Vmax = 25 +/- 5 nmol/mg protein per min. Removal of apolipoprotein (a) (apo(a)) from Lp(a) by reductive cleavage with dithiothreitol, slightly affected the amount of PAF-AH existing on Lp(a) since, only 15 +/- 5% of the total enzyme activity dissociated from its particle after density gradient ultracentrifugation. During Cu(2+)-induced Lp(a) oxidation, the PAF-AH activity decreased from 10.90 +/- 2.30 nmol/mg per min to 2.57 +/- 0.56 nmol/mg per min 4 h after the initiation of the oxidation (P < 0.001). The apparent Km of the enzyme remained essentially unchanged during oxidation, whereas Vmax was significantly decreased from 58.6 +/- 7.8 nmol/mg protein per min to 38.2 +/- 8.7 nmol/mg protein per min (P < 0.03). An extensive hydrolysis of the endogenous phosphatidylcholine (PC) to lysophosphatidylcholine (lyso-PC) was observed during Lp(a) oxidation, since the Lyso-PC/sphingomyelin molar ratio at the end of oxidation (0.55 +/- 0.09) was significantly higher than that before oxidation (0.19 +/- 0.01, P < 0.001). Our results show that the existence of Lp(a) in plasma alters the distribution of PAF-AH among the other lipoproteins. Apo(a) seems to affect the association of the enzyme with Lp(a) but does not bind itself to PAF-AH. During Lp(a) oxidation, the PAF-AH activity decreases whereas an extensive hydrolysis of the endogenous PC to Lyso-PC is observed which is possibly due to the PAF-AH activity.

PAF-degrading acetylhydrolase is preferentially associated with dense LDL and VHDL-1 in human plasma. Catalytic characteristics and relation to the monocyte-derived enzyme.

In human plasma, platelet activating factor (PAF)-degrading acetylhydrolase (acetylhydrolase) is principally transported in association with LDLs and HDLs; this enzyme hydrolyzes PAF and short-chain forms of oxidized phosphatidylcholine, transforming them into lyso-PAF and lysophosphatidylcholine, respectively. We have examined the distribution, catalytic characteristics, and transfer of acetylhydrolase activity among plasma lipoprotein subspecies separated by isopycnic density gradient ultracentrifugation; the possibility that the plasma enzyme may be partially derived from adherent monocytes has also been evaluated. In normolipidemic subjects with Lp(a) levels < 0.1 mg/mL, acetylhydrolase was associated preferentially with small, dense LDL particles (LDL-5; d = 1.050 to 1.063 g/mL) and with the very-high-density lipoprotein-1 subfraction (VHDL-1; d = 1.156 to 1.179 g/mL), representing 23.9 +/- 1.7% and 20.6 +/- 3.2%, respectively, of total plasma activity. The apparent Km values for PAF of the enzyme associated with such lipoproteins were 89.7 +/- 23.4 and 34.8 +/- 4.5 mumol/L for LDL-5 and VHDL-1, respectively: indeed, the Km value for LDL-5 was some 10-fold higher than that of the light LDL-1, LDL-2, and LDL-3 subspecies, whereas the Km of VHDL-1 was some twofold greater than those of the HDL-2 and HDL-3 subspecies. Furthermore, when expressed on the basis of unit plasma volume, the Vmax of the acetylhydrolase associated with LDL-5 was some 150-fold greater than that in LDL-1 (d = 1.019 to 1.023 g/mL). No significant differences in the pH dependence of enzyme activity or in sensitivity to protease inactivation, sulfydryl reagents, the serine protease inhibitor Pefabloc, or the PAF antagonist CV 3988 could be detected between apo B-containing and apo A-I-containing lipoprotein particle subspecies. Incubation of LDL-1 (Km = 8.4 +/- 2.6 mumol/L) and LDL-2 (d = 1.023 to 1.029 g/mL; Km = 8.4 +/- 3.3 mumol/L) subspecies with LDL-5, in which acetylhydrolase had been inactivated by pretreatment with Pefabloc, demonstrated preferential transfer of acetylhydrolase to LDL-5. Acetylhydrolase transferred to LDL-5 from the light LDL subspecies exhibited a Km of 9.4 +/- 2.2 mumol/L, a value characteristic of the particle donors. Finally, acetylhydrolase (Km = 23.4 +/- 7.6 mumol/L) released by adherent human monocytes in culture was found to bind preferentially to small, dense LDL subspecies upon incubation of Pefabloc-inactivated plasma with monocyte supernatant.(ABSTRACT TRUNCATED AT 400 WORDS)

Distribution of PAF-acetylhydrolase activity in human plasma low-density lipoprotein subfractions.

The distribution of PAF-acetylhydrolase (PAF-AH) activity in 3 LDL subfractions prepared by density gradient ultracentrifugation as well as the rate of phosphatidylcholine (PC) hydrolysis during oxidation was studied. PAF-AH activity, measured before oxidation, was much higher in LDL3 subfraction (28.4 +/- 8.6 nmol/mg per min) comparing to LDL2 (14.1 +/- 5.8 nmol/mg per min), and to LDL1, 8.7 +/- 3.7 nmol/mg per min. During oxidation, the enzyme activity was continuously decreased and this phenomenon was more pronounced in LDL1. PC hydrolysis was studied measuring the lyso-PC production expressed as lyso-PC/Sph molar ratio. Before oxidation, the lyso-PC/Sph molar ratio, did not differ significantly among the LDL subfractions, whereas, 4 h after the onset of oxidation, it was significantly higher in LDL2 and LDL3 subfractions (0.42 +/- 0.12 and 0.45 +/- 0.10, respectively), comparing to LDL1 (0.29 +/- 0.06). Our results show that the distribution of PAF-AH activity in LDL subfractions is heterogeneous (mainly distributed in LDL2 and LDL3 subfractions) and it is positively correlated with higher lyso-PC production in those subfractions during oxidation. The contribution of this phenomenon to the enhanced susceptibility to oxidation as well as to the higher atherogenicity of the dense LDL subfractions is under investigation.

Platelet-activating factor formation during oxidative modification of low-density lipoprotein when PAF-acetylhydrolase has been inactivated.

A PAF aggregating activity corresponding to 427 +/- 91, 668 +/- 111 and 1319 +/- 217 pg/mg protein was detected when LDL was preincubated at pH 3.5 or with 4 mM PMSF or both for 30 min (treatments that inactivate PAF-AH) and then oxidized with 20 microM Cu2+ at 37 degrees C for 24 h. This molecule was characterized as PAF by its chromatographic behavior on TLC and other established methods and was further characterized as 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (C16: PAF) by its retention time on reverse phase HPLC and by fast atom bombardment-mass spectroscopy. Native LDL incubated under non oxidizing conditions, even when PAF-AH has been inactivated, or oxidized in the absence of PAF-AH inactivating agents or after pretreatment with 0.5 mM pBPB, does not produce detectable amounts of PAF. The kinetics of PAF formation in relation to PAF-AH activity, show that the apparent rate of PAF formation as well as its total amount depends on both the existence of oxidative conditions and the remaining PAF-AH activity the first hours following the onset of oxidation. Peroxidation of the phosphatidylcholine (PC) content of native LDL produces PAF-like aggregating activity much lower than that produced when intact LDL is oxidized and is not inhibited by BN 52021 as effectively as PAF produced by LDL peroxidation. Our results provide evidence that C16: PAF is formed during LDL peroxidation when PAF-AH has been inactivated and it does not result as a product of peroxidation of the LDL-PC content.