As the lipidomics field continues to advance, self-evaluation within the community is critical. Here, we performed an interlaboratory comparison exercise for lipidomics using Standard Reference Material (SRM) 1950-Metabolites in Frozen Human Plasma, a commercially available reference material. The interlaboratory study comprised 31 diverse laboratories, with each laboratory using a different lipidomics workflow. A total of 1,527 unique lipids were measured across all laboratories and consensus location estimates and associated uncertainties were determined for 339 of these lipids measured at the sum composition level by five or more participating laboratories. These evaluated lipids detected in SRM 1950 serve as community-wide benchmarks for intra- and interlaboratory quality control and method validation. These analyses were performed using nonstandardized laboratory-independent workflows. The consensus locations were also compared with a previous examination of SRM 1950 by the LIPID MAPS consortium. While the central theme of the interlaboratory study was to provide values to help harmonize lipids, lipid mediators, and precursor measurements across the community, it was also initiated to stimulate a discussion regarding areas in need of improvement.

The surface of the eye actively suppresses inflammation while maintaining a remarkable capacity for epithelial wound repair. Our understanding of mechanisms that balance inflammatory/reparative responses to provide effective host defense while preserving tissue function is limited, in particular, in the cornea. Lipoxin A 4 (LXA 4 ) and docosahexaenoic acid-derived neuroprotectin D1 (NPD1) are lipid autacoids formed by 12/15-lipoxygenase (LOX) pathways that exhibit anti-inflammatory and neuroprotective properties. Here, we demonstrate that mouse corneas generate endogenous LXA 4 and NPD1. 12/15-LOX (Alox15) and LXA 4 receptor mRNA expression as well as LXA 4 formation were abrogated by epithelial removal and restored during wound healing. Amplification of these pathways by topical treatment with LXA 4 or NPD1 (1 g) increased the rate of re-epithelialization (65-90%, n ‫؍‬ 6 -10, p < 0.03) and attenuated the sequelae of thermal injury. In contrast, the proinflammatory eicosanoids, LTB 4 and 12R-hydroxyeicosatrienoic acid, had no impact on corneal re-epithelialization. Epithelial removal induced a temporally defined influx of neutrophils into the stroma as well as formation of the proinflammatory chemokine KC. Topical treatment with LXA 4 and NPD1 significantly increased PMNs in the cornea while abrogating KC formation by 60%. More importantly, Alox15-deficient mice exhibited a defect in both corneal re-epithelialization and neutrophil recruitment that correlated with a 43% reduction in endogenous LXA 4 formation. Collectively, these results identify a novel action for the mouse 12/15-LOX (Alox15) and its products, LXA 4 and NPD1, in wound healing that is distinct from their well established anti-inflammatory properties.

Heme oxygenase (HO) is the rate-limiting enzyme in heme catabois and its activity is induced by many agents, including its substrate heme, heavy metals, UV radiation, and other injurious oxidant conditions. We examined the presence of several regulatory elements in the promoter region of the human HO-1 gene which could possibly account for its induction in response to diverse agents or influences. (11,12). Two HO isozymes, the products of two distinct genes, have been described (13,14). HO-1 is the inducible form which is ubiquitously distributed in mammalian tissues, whereas HO-2 is believed to be constitutively expressed, is not inducible by HO-1 inducers, and is present in tissues such as the brain and testis (14).One of the mechanisms by which hormones, growth factors, and other stimuli induce the expression of genes is by activating various transcription factors. This is a rapid process which frequently involves transcriptional or structural activation of the factor and allows its presence or transfer to the nucleus. These processes may be part of the mechanism by which various agents, including heme, increase HO expression and activity. Previous studies have shown the presence of AP-1-binding sequences and interleukin 6-, metal-, and heat-responsive elements in the HO-1 promoter region and have suggested the involvement of these nuclear factors in the regulation of several genes, including that encoding [13][14][15][16][17][18]. Erythropoietic cells are endowed with HO activity which is inducible by heme (6,7). We therefore used a human-derived erythroleukemic cell line, K562, to examine the presence of transcription factors which might be involved in heme-induced HO-1 expression and to determine whether binding sequences for these factors were present in the promoter region of the human HO-1 gene. tTo whom reprint requests should be addressed. MATERIALS AND METHODS 5987The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

these studies provides direct evidence that the inductive response of human HO to such injurious stimuli represents an important tissue adaptive mechanism for moderating the severity of cell damage produced by these blood components.

Cytochrome P-450-dependent metabolites of arachidonic acid (AA) increased in the kidneys of young, spontaneously hypertensive rats (SHRs) during the period of rapid elevation of blood pressure (BP) but not in adult SHRs or in Wistar Kyoto rats (WKYs) with normal BP. Treatment of SHRs and WKYs with stannous chloride (SnCl2), which selectively depletes renal cytochrome P-450, restored BP to normal, coincident with a natriuresis, in young but not in adult SHRs and did not affect either BP or sodium excretion in WKYs. Depletion of renal cytochrome P-450 was associated with decreased generation of these AA metabolites only in young SHRs. The antihypertensive effect of SnCl2 in young SHRs was greatly reduced by prevention of its cytochrome P-450-depleting action.

Abstract-Epidemiological evidence suggests a role for sex-dependent mechanisms in the pathophysiology of hypertension. It has been shown that 5␣-dihydrotestosterone (DHT) administration (56 mg/kg of body weight per day IP for 14 days) increases blood pressure, cytochrome P450 4A expression, and 20-hydroxyeicosatetraenoic acid synthesis in rats.We examined whether increased vascular 20-hydroxyeicosatetraenoic acid synthesis underlies endothelial dysfunction and hypertension in DHT-treated male Sprague-Dawley rats by using HET0016, a selective cytochrome P450 4A inhibitor. Coadministration of HET0016 (10 mg/kg per day IP for 14 days) to DHT-treated rats markedly reduced DHT-induced interlobar arterial production of 20-hydroxyeicosatetraenoic acid (14.3Ϯ1.5 versus 1.5Ϯ0.5 ng/mg of protein per hour; PϽ0.05), superoxide anion (246Ϯ47 versus 31Ϯ8 cpm/g of protein), and the levels of gp91-phox, p47-phox, and 3-nitrosylated proteins. Moreover, the maximal relaxing response to acetylcholine in phenylephrinepreconstricted renal interlobar arteries from DHT-treated rats (42.8Ϯ4.8%) significantly (PϽ0.05) increased in the presence of HET0016 (81.5Ϯ10.8%). Importantly, the administration of HET0016 negated DHT-induced hypertension; systolic blood pressure was reduced from 146Ϯ2 mm Hg in DHT-treated rats to 130Ϯ1 mm Hg ( The synthesis of 20-HETE is catalyzed primarily by enzymes of the cytochrome P450 (CYP) 4A family. 6,7 CYP4A proteins are present in vascular tissues and show distinct distribution along the vascular tree. 8 Suppression and overexpression of CYP4A proteins in small arteries and arterioles decreases and increases, respectively, vascular reactivity and myogenic tone 7,9,10 ; these effects can be reversed by the addition of 20-HETE or inhibition of its synthesis.CYP4A and 20-HETE synthesis have been linked to hypertension in numerous experimental models. In the spontaneously hypertensive rat, depletion or inhibition of CYP4A activity lowers blood pressure (BP). 11,12 Inhibition of vascular 20-HETE synthesis by intravenous administration of CYP4A1 or CYP4A2 antisense oligonucleotides decreases BP in normotensive and hypertensive rats, 6,7 whereas transduction with adenoviruses expressing the CYP4A2 protein increases vascular CYP4A expression and 20-HETE levels and augments BP. 13 A role for androgens in promoting elevation of BP is well recognized 14 and, according to recent studies, such a role may rely on increased synthesis of vascular 20-HETE. Hence, mice deficient in cyp4a14 (the mouse homologue of CYP4A2) displayed androgen-sensitive hypertension, which was reversed by castration. 15 In these mice, cyp4a12 expression (the mouse homologue of CYP4A8) is elevated and so is renal microsomal 20-HETE synthesis. Similarly, androgeninduced hypertension in rats treated with 5␣-dihydrotestosterone (DHT) has been associated with increased CYP4A8 expression and renal vascular 20-HETE synthesis. 16 The mechanisms by which 20-HETE promotes hypertension are primarily linked to its ability to sensitize constrictor responsi...

Heme oxygenase (HO) and cytochrome P450 (P450)-derived epoxyeicosatrienoic acids (EETs) participate in vascular protection, and recent studies suggest these two systems are functionally linked. We examined the consequences of HO deficiency on P450-derived EETs with regard to body weight, adiposity, insulin resistance, blood pressure, and vascular function in HO-2-null mice. The HO-2-null mice were obese, displayed insulin resistance, and had high blood pressure. HO-2 deficiency was associated with decreases in cyp2c expression, EET levels, HO-1 expression, and HO activity and with an increase in superoxide production and an impairment in the relaxing response to acetylcholine. In addition, HO-2-null mice exhibited increases in serum levels of tumor necrosis factor (TNF)-␣ and macrophage chemoattractant protein (MCP)-1 and a decrease in serum adiponectin levels. Treatment of HO-2-null mice with a dual-activity EET agonist/soluble epoxide hydrolase inhibitor increased renal and vascular EET levels and HO-1 expression, lowered blood pressure, prevented body weight gain, increased insulin sensitivity, reduced subcutaneous and visceral fat, and decreased serum TNF-␣ and MCP-1, while increasing adiponectin and restoring the relaxing responses to acetylcholine. The decrease in cyp2c expression and EETs levels in HO-2-null mice underscores the importance of the HO system in the regulation of epoxygenase levels and suggests that protection against obesityinduced cardiovascular complications requires interplay between these two systems. A deficiency in one of these protective systems may contribute to the adverse manifestations associated with the clinical progression of the metabolic syndrome.

Recent studies have indicated that renal arteries can produce 20-hydroxyeicosatetraenoic acid (20 -HETE) and suggest the potential involvement of a P450 metabolite of arachidonic acid in the myogenic activation of canine renal arteries. In the present study, the effects of 20-HETE on isolated canine renal arcuate arteries were studied. Administration of 20-HETE to the bath or the lumen at concentrations of 0.01-1 ,uM produced a graded reduction in the diameter of these vessels. In Received December 17, 1991; accepted September 29, 1992. strictor factor8 and can be attenuated by inhibitors of protein kinase C.9,10 These findings suggest that local paracrine factors may play a role in the myogenic response; however, the nature of these substances is unknown.Recently, arachidonic acid has been reported to enhance the vasoconstrictor response of canine renal arcuate arteries to elevations in transmural pressure."1 This effect was potentiated by indomethacin and blocked by inhibitors of cytochrome P450, suggesting that an endogenous P450 metabolite of arachidonic acid modulates the myogenic response. Microsomes prepared from canine renal arcuate arteries produced a P450 metabolite of arachidonic acid that coelutes with 20-hydroxyeicosatetraenoic acid (20-HETE)."1 Since 20-HETE is a potent vasoconstrictor of the rat aorta,12 it potentially could be an endogenous P450 metabolite of arachidonic acid that modulates the myogenic response.The purpose of the present study was to characterize the effects of 20-HETE on vascular diameter and membrane potential in isolated canine renal arcuate arteries to determine whether it could play a role in the modulation of the myogenic response by arachidonic acid and P450 inhibitors previously observed in these vessels." The effects of 20-HETE on potassium channel activity and intracellular calcium concentration in vascular smooth muscle cells isolated from these arteries by guest on May 7, 2018