Stephen A. Brose

LC/MS/MS method for analysis of E2 series prostaglandins and isoprostanes

15-series prostaglandins (PGE2s) and isoprostanes (isoPGE2s) are robust biomarkers of oxidative stress, possess potent biological activity, and may be derived through cyclooxygenase or free radical pathways. Thus, their quantification is critical in understanding many biological processes where PG, isoPG, or oxidative stress are involved. LC/MS/MS methods allow a highly selective, sensitive, simultaneous analysis for prostanoids without derivatization. However, the LC/MS/MS methods currently used do not allow for simultaneous separation of the major brain PGE2/D2 and isoPGE2 without derivatization and multiple HPLC separations. The developed LC/MS/MS method allows for the major brain PGE2/PGD2/isoPGE2 such as PGE2, entPGE2, 8-isoPGE2, 11β-PGE2, PGD2, and 15(R)-PGD2 to be separated and quantified without derivatization. The method was validated by analyzing free and esterified isoPGE2 in mouse brains fixed with head-focused microwave irradiation before or after global ischemia. Using the developed method, we report for the first time the esterified isoPGE2 levels in brain tissue under basal conditions and upon global ischemia and demonstrate a nonreleasable pool of esterified isoPG upon ischemia. In addition, we demonstrated that PGE2s found esterified in the sn-2 position in phospholipids are derived from a free radical nonenzymatic pathway under basal conditions. Our method for brain PG analysis provides a high level of selectivity to detect changes in brain PG and isoPG mass under both basal and pathological conditions.

Baking Reduces Prostaglandin, Resolvin, and Hydroxy-Fatty Acid Content of Farm-Raised Atlantic Salmon (Salmo salar)

The consumption of seafood enriched in n-3 polyunsaturated fatty acids (PUFA) is associated with a decreased risk of cardiovascular disease. Several n-3 oxidation products from eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (22:6n-3) have known protective effects in the vasculature. It is not known whether the consumption of cooked seafood enriched in n-3 PUFA causes appreciable consumption of lipid oxidation products. We tested the hypothesis that baking Atlantic salmon (Salmo salar) increases the level of n-3 and n-6 PUFA oxidation products over raw salmon. We measured the contents of several monohydroxy-fatty acids (MHFA), prostanoids, and resolvins. Our data demonstrate that baking did not change the overall total levels of MHFA. However, baking resulted in selective regioisomeric loss of hydroxy fatty acids from arachidonic acid (20:4n-6) and EPA, while significantly increasing hydroxyl-linoleic acid levels. The contents of prostanoids and resolvins were reduced several-fold with baking. The inclusion of a coating on the salmon prior to baking reduced the loss of some MHFA but had no effect on prostanoid losses incurred by baking. Baking did not decrease n-3 PUFA contents, indicating that baking of salmon is an acceptable means of preparation that does not alter the potential health benefits of high n-3 seafood consumption. The extent to which the levels of MHFA, prostanoids, and resolvins in the raw or baked fish have physiologic consequence for humans needs to be determined.

LC/MS/MS method for analysis of E₂ series prostaglandins and isoprostanes.

15-series prostaglandins (PGE2s) and isoprostanes (isoPGE2s) are robust biomarkers of oxidative stress, possess potent biological activity, and may be derived through cyclooxygenase or free radical pathways. Thus, their quantification is critical in understanding many biological processes where PG, isoPG, or oxidative stress are involved. LC/MS/MS methods allow a highly selective, sensitive, simultaneous analysis for prostanoids without derivatization. However, the LC/MS/MS methods currently used do not allow for simultaneous separation of the major brain PGE2/D2 and isoPGE2 without derivatization and multiple HPLC separations. The developed LC/MS/MS method allows for the major brain PGE2/PGD2/isoPGE2 such as PGE2, entPGE2, 8-isoPGE2, 11β-PGE2, PGD2, and 15(R)-PGD2 to be separated and quantified without derivatization. The method was validated by analyzing free and esterified isoPGE2 in mouse brains fixed with head-focused microwave irradiation before or after global ischemia. Using the developed method, we report for the first time the esterified isoPGE2 levels in brain tissue under basal conditions and upon global ischemia and demonstrate a nonreleasable pool of esterified isoPG upon ischemia. In addition, we demonstrated that PGE2s found esterified in the sn-2 position in phospholipids are derived from a free radical nonenzymatic pathway under basal conditions. Our method for brain PG analysis provides a high level of selectivity to detect changes in brain PG and isoPG mass under both basal and pathological conditions.

Fatty acid biosynthesis from glutamate and glutamine is specifically induced in neuronal cells under hypoxia.

Hypoxia is involved in many neuronal and non‐neuronal diseases, and defining the mechanisms for tissue adaptation to hypoxia is critical for the understanding and treatment of these diseases. One mechanism for tissue adaptation to hypoxia is increased glutamine and/or glutamate (Gln/Glu) utilization. To address this mechanism, we determined incorporation of Gln/Glu and other lipogenic substrates into lipids and fatty acids in both primary neurons and a neuronal cell line under normoxic and hypoxic conditions and compared this to non‐neuronal primary cells and non‐neuronal cell lines. Incorporation of Gln/Glu into total lipids was dramatically and specifically increased under hypoxia in neuronal cells including both primary (2.0‐ and 3.0‐fold for Gln and Glu, respectively) and immortalized cultures (3.5‐ and 8.0‐fold for Gln and Glu, respectively), and 90% to 97% of this increase was accounted for by incorporation into fatty acids (FA) depending upon substrate and cell type. All other non‐neuronal cells tested demonstrated decreased or unchanged FA synthesis from Gln/Glu under hypoxia. Consistent with these data, total FA mass was also increased in neuronal cells under hypoxia that was mainly accounted for by the increase in saturated and monounsaturated FA with carbon length from 14 to 24. Incorporation of FA synthesized from Gln/Glu was increased in all major lipid classes including cholesteryl esters, triacylglycerols, diacylglycerols, free FA, and phospholipids, with the highest rate of incorporation into triacylglycerols. These results indicate that increased FA biosynthesis from Gln/Glu followed by esterification may be a neuronal specific pathway for adaptation to hypoxia.

Oxidation Sensitive Nociception Involved in Endometriosis Associated Pain

Abstract Endometriosis is a disease characterized by the growth of endometrial tissue outside the uterus and is associated with chronic pelvic pain. Peritoneal fluid (PF) of women with endometriosis is a dynamic milieu and is rich in inflammatory markers, pain-inducing prostaglandins prostaglandin E2 and prostaglandin F2&agr;, and lipid peroxides; and the endometriotic tissue is innervated with nociceptors. Our clinical study showed that the abundance of oxidatively modified lipoproteins in the PF of women with endometriosis and the ability of antioxidant supplementation to alleviate endometriosis-associated pain. We hypothesized that oxidatively modified lipoproteins present in the PF are the major source of nociceptive molecules that play a key role in endometriosis-associated pain. In this study, PF obtained from women with endometriosis or control women were used for (1) the detection of lipoprotein-derived oxidation-sensitive pain molecules, (2) the ability of such molecules to induce nociception, and (3) the ability of antioxidants to suppress this nociception. LC–MS/MS showed the generation of eicosanoids by oxidized-lipoproteins to be similar to that seen in the PF. Oxidatively modified lipoproteins induced hypothermia (intracerebroventricular) in CD-1 mice and nociception in the Hargreaves paw withdrawal latency assay in Sprague-Dawley rats. Antioxidants, vitamin E and N-acetylcysteine, and the nonsteroidal anti-inflammatory drug indomethacin suppressed the pain-inducing ability of oxidatively modified lipoproteins. Treatment of human endometrial cells with oxidatively modified lipoproteins or PF from women with endometriosis showed upregulation of similar genes belonging to opioid and inflammatory pathways. Our finding that oxidatively modified lipoproteins can induce nociception has a broader impact not only on the treatment of endometriosis-associated pain but also on other diseases associated with chronic pain.

Amyloid precursor protein modulates macrophage phenotype and diet-dependent weight gain

It is well known that mutations in the gene coding for amyloid precursor protein are responsible for autosomal dominant forms of Alzheimer’s disease. Proteolytic processing of the protein leads to a number of metabolites including the amyloid beta peptide. Although brain amyloid precursor protein expression and amyloid beta production are associated with the pathophysiology of Alzheimer’s disease, it is clear that amyloid precursor protein is expressed in numerous cell types and tissues. Here we demonstrate that amyloid precursor protein is involved in regulating the phenotype of both adipocytes and peripheral macrophages and is required for high fat diet-dependent weight gain in mice. These data suggest that functions of this protein include modulation of the peripheral immune system and lipid metabolism. This biology may have relevance not only to the pathophysiology of Alzheimer’s disease but also diet-associated obesity.

Eicosanoid post-mortem induction in kidney tissue is prevented by microwave irradiation

Previously, we, and others, have demonstrated a rapid and significant post-mortem increase in brain prostanoid (PG) levels analyzed without microwave fixation, and this is not the result of PG trapping or destruction in microwave-irradiated brain tissue. In the present study, we demonstrate a dramatic increase in kidney eicosanoid levels when analyzed without microwave fixation which was mainly accounted for by the 142-, 81-, and 62-fold increase in medullary 6-ketoPGF1α, PGE2, and PGF2α, levels, respectively, while PGD2 and TXB2 levels were increased ~7-fold. Whole kidney and cortex PG were also significantly increased in non-microwaved tissue, but at lesser extent. Arachidonic acid and the lipoxygenase products hydroxyeicosatetraenoic acids (HETE) were also induced in whole kidney, cortex, and medulla 1.5- to 5.5-fold depending upon tissue and metabolite. Cyclooxygenase inhibition with indomethacin decreased PG mass in non-microwaved tissue to basal levels, however HETE and arachidonic acid were not decreased. These data demonstrate the critical importance of kidney tissue fixation to limiting artifacts during kidney eicosanoid analysis.

A rapid oxygen exchange on prostaglandins in plasma represents plasma esterase activity that is inhibited by diethylumbelliferyl phosphate with high affinity

RATIONALE Fatty acids (FA) labeled with (18) O at the carboxyl group, including oxidized species (FA(18) O), are a useful, low-cost, and easy to prepare tool for quantitative and qualitative mass spectrometry (MS) analysis in biological systems. In addition, they are used to trace the fate of FAs in metabolic pathways including FA re-esterification and lipid remodeling pathways. Although a rapid (18) O exchange on FA(18) O in biological systems has been reported, the mechanism contributing to (18) O exchange has not been fully evaluated. This gap in knowledge limits the use of FA(18) O as a standard for MS and complicates data interpretation for FA metabolism in biological systems. METHODS In the present study we have addressed a number of possible mechanisms for a rapid (18) O exchange on prostaglandin E(2) (PGE(2) ) using rat plasma as a model. High-performance liquid chromatography coupled with electrospray ionization triple quadrupole MS in the multiple reaction monitoring mode was used for quantification. RESULTS The major mechanism for a rapid (18) O exchange on PGE(2) (18) O in rat plasma is PGE(2) processing with esterases, while FA re-esterification and non-enzymatic mechanisms do not significantly contribute to this phenomenon. In addition, we report a highly effective inhibition of (18) O exchange with diethylumbelliferyl phosphate that can be used to stabilize FA(18) O in biological samples. CONCLUSIONS These data indicate the necessity to consider esterase activity when FA(18) O are used to study FA metabolism, and the importance of esterase activity inhibition when FA(18) O are used as internal standards for MS analysis in biological systems. In addition, the results provide a rational for the development of new approaches to study esterase activities and affinity towards modified FA.

Effects of cooking techniques on fatty acid and oxylipin content of farmed rainbow trout (Oncorhynchus mykiss)

Abstract The aim of this study was to investigate the effect of various cooking techniques on the fatty acid and oxylipin content of farmed rainbow trout. Rainbow trout is an excellent source of long‐chain omega‐3 (n‐3) polyunsaturated fatty acids (PUFA) which have beneficial health effects. Fillets of 2‐year‐old farmed rainbow trout were baked, broiled, microwaved, or pan‐fried in corn (CO), canola (CaO), peanut (PO), or high oleic sunflower oil (HOSO). Fatty acids and oxidized lipids were extracted from these samples and their respective raw fillet samples. Fatty acid content was determined using gas chromatography and oxylipin content by mass spectroscopy. The values obtained from each cooking method were compared to those obtained from the respective raw fillets using paired t tests. PUFA content was not altered when samples were baked, broiled, microwaved, or pan‐fried in CO or CaO. Pan‐frying in PO reduced α‐linolenic acid (18:3n‐3), eicosadienoic acid (20:2n‐6), and dihomo‐γ‐linolenic acid (20:3n‐6), while pan‐frying in HOSO reduced 18:3n‐3, eicosapentaenoic acid (20:5n‐3), docosapentaenoic acid (22:5n‐3), docosahexaenoic acid (22:6n‐3), linoleic acid (18:2n‐6), 18:3n‐6, 20:2n‐6, 20:3n‐6, docosatrienoic acid (22:2n‐6), and adrenic acid (22:4n‐6) compared to raw fish. Cooking decreased the omega‐6 (n‐6) PUFA‐derived oxylipins, but caused no change in 20:5n‐3 or 22:6n‐3‐derived oxylipins of the fillets. In conclusion, pan‐frying was the only cooking method to alter the fatty acid content of the fillets, while observed changes in oxylipin content varied by cooking method. As the physiological impact of oxylipins is currently unknown, these results suggest that the cooking methods which optimize the consumption of n‐3 PUFA from rainbow trout are baking, broiling, microwaving, or pan‐frying in CO, CaO, or PO.

Inhibition of the Serotonin Transporter Is Altered by Metabolites of Selective Serotonin and Norepinephrine Reuptake Inhibitors and Represents a Caution to Acute or Chronic Treatment Paradigms

Previous studies of transgenic mice carrying a single isoleucine to methionine substitution (I172M) in the serotonin transporter (SERT) demonstrated a loss of sensitivity to multiple antidepressants (ADs) at SERT. However, the ability of AD metabolites to antagonize SERT was not assessed. Here, we evaluated the selectivity and potency of these metabolites for inhibition of SERT in mouse brain-derived synaptosomes and blood platelets from wild-type (I172 mSERT) and the antidepressant-insensitive mouse M172 mSERT. The metabolites norfluoxetine and desmethylsertraline lost the selectivity demonstrated by the parent compounds for inhibition of wild-type mSERT over M172 mSERT, whereas desvenlafaxine and desmethylcitalopram retained selectivity. Furthermore, we show that the metabolite desmethylcitalopram accumulates in the brain and that the metabolites desmethylcitalopram, norfluoxetine, and desvenlafaxine inhibit serotonin uptake in wild-type mSERT at potencies similar to those of their parent compounds, suggesting that metabolites may play a role in effects observed following AD administration in wild-type and M172 mice.