Göran I. Hansson

The BUME method: a novel automated chloroform-free 96-well total lipid extraction method for blood plasma

Lipid extraction from biological samples is a critical and often tedious preanalytical step in lipid research. Primarily on the basis of automation criteria, we have developed the BUME method, a novel chloroform-free total lipid extraction method for blood plasma compatible with standard 96-well robots. In only 60 min, 96 samples can be automatically extracted with lipid profiles of commonly analyzed lipid classes almost identically and with absolute recoveries similar or better to what is obtained using the chloroform-based reference method. Lipid recoveries were linear from 10–100 µl plasma for all investigated lipids using the developed extraction protocol. The BUME protocol includes an initial one-phase extraction of plasma into 300 µl butanol:methanol (BUME) mixture (3:1) followed by two-phase extraction into 300 µl heptane:ethyl acetate (3:1) using 300 µl 1% acetic acid as buffer. The lipids investigated included the most abundant plasma lipid classes (e.g., cholesterol ester, free cholesterol, triacylglycerol, phosphatidylcholine, and sphingomyelin) as well as less abundant but biologically important lipid classes, including ceramide, diacylglycerol, and lyso-phospholipids. This novel method has been successfully implemented in our laboratory and is now used daily. We conclude that the fully automated, high-throughput BUME method can replace chloroform-based methods, saving both human and environmental resources.

Hypoxia Increases LDL Oxidation and Expression of 15-Lipoxygenase-2 in Human Macrophages

Objective—Macrophage-mediated oxidation of low-density lipoprotein (LDL) by enzymes, such as the lipoxygenases, is considered of major importance for the formation of oxidized LDL during atherogenesis. Macrophages have been identified in hypoxic areas in atherosclerotic plaques. Methods and Results—To investigate the role of hypoxia in macrophage-mediated LDL oxidation, we incubated human monocyte-derived macrophages with LDL under normoxic (21% O2) or hypoxic (0% O2) conditions. The results showed that hypoxic macrophages oxidized LDL to a significantly higher extent than normoxic cells. Interestingly, the mRNA and protein expression of 15-lipoxygenase-2 (15-LOX-2) as well as the activity of this enzyme are elevated in macrophages incubated at hypoxia. Both the unspliced 15-LOX-2 and the spliced variant 15-LOX-2sv-a are found in macrophages. In addition, 15-LOX-2 was identified in carotid plaques in some macrophage-rich areas but was only expressed at low levels in nondiseased arteries. Conclusions—In summary, these observations show for the first time that 15-LOX-2 is expressed in hypoxic macrophages and in atherosclerotic plaques and suggest that 15-LOX-2 may be one of the factors involved in macrophage-mediated LDL oxidation at hypoxia.

Simultaneous assessment of lipid classes and bile acids in human intestinal fluid by solid-phase extraction and HPLC methods

The purpose of the study reported here was to develop a method for the determination of lipid classes in intestinal fluids, including bile acids (BAs). A solid-phase extraction (SPE) method using C18 and silica columns for the separation of BAs, phospholipids (PLs), and neutral lipids (NLs), including free fatty acids, has been developed and validated. Fed-state small intestinal fluid collected from humans was treated with orlistat to inhibit lipolysis and mixed with acetic acid and methanol before SPE to maximize lipid recoveries. BAs, PLs, and NLs were isolated using lipophilic and polar solvents to promote elution from the SPE columns. The different lipid classes were subsequently analyzed using three separately optimized HPLC methods with evaporative light-scattering detectors. High recoveries (>90%) of all lipids evaluated were observed, with low coefficients of variation (<5%). The HPLC methods developed were highly reproducible and allowed baseline separation of nearly all lipid classes investigated. In conclusion, these methods provide a means of lipid class analysis of NLs, PLs, and BAs in human fed-state small intestinal fluid, with potential use in other fluids from the intestinal tract and animals.

Effects of rosuvastatin on cardiovascular morphology and function in an ApoE knockout mouse model of atherosclerosis

This study investigated the effects of rosuvastatin on plaque progression and in vivo coronary artery function in apolipoprotein E-knockout (ApoE-KO) mice, using noninvasive high-resolution ultrasound techniques. Eight-week-old male ApoE-KO mice (n = 20) were fed a high-fat diet with or without rosuvastatin (10 micromol.kg(-1).day(-1)) for 16 wk. When compared with control, rosuvastatin reduced total cholesterol levels (P < 0.05) and caused significant retardation of lesion progression in the brachiocephalic artery, as visualized in vivo using an ultrasound biomicroscope (P < 0.05). Histological analysis confirmed the reduction of brachiocephalic atherosclerosis and also revealed an increase in collagen content in the statin-treated group (P < 0.05). Coronary volumetric flow was measured by simultaneous recording of Doppler velocity signals and left coronary artery morphology before and during adenosine infusion. The hyperemic flow in response to adenosine was significantly greater in left coronary artery following 16 wk of rosuvastatin treatment (P < 0.001), whereas the baseline flow was similar in both groups. In conclusion, rosuvastatin reduced brachiocephalic artery atherosclerotic plaques in ApoE-KO mice. Coronary artery function assessed using recently developed in vivo ultrasound-based protocols, also improved.

High-dose aspirin in dogs increases vascular resistance with limited additional anti-platelet effect when combined with potent P2Y12 inhibition.

INTRODUCTION With the arrival of the potent P2Y12 antagonists, ticagrelor and prasugrel, the need for co-treatment with aspirin in acute coronary syndromes must be re-examined. This study assessed whether high-dose aspirin: a) provides additional anti-platelet efficacy, assessed in vivo and ex vivo, when combined with P2Y12 inhibition; and/or b) has a negative effect on vascular function. MATERIALS AND METHODS Using an anaesthetized dog model of thrombosis, the effects of aspirin (50mg/kg) in addition to clopidogrel and ticagrelor were evaluated at two levels of P2Y12 inhibition, maximal (≥96%) and sub-maximal (~80%), as assessed by ex vivo ADP-induced whole blood impedence aggregometry. RESULTS In the absence of aspirin, maximal and sub-maximal P2Y12 inhibition inhibited arachidonic acid-induced platelet aggregation by approximately 80% and 24%, respectively, without affecting platelet TXA2 formation. During maximal P2Y12 inhibition, aspirin provided less additional inhibition of ex vivo arachidonic acid- and collagen-induced platelet aggregation, as compared with sub-maximal P2Y12 inhibition, without additional anti-thrombotic effect in vivo. Aspirin significantly decreased in vivo PGI2 production (27%) and increased vascular resistance (16%), independently of P2Y12 antagonism. CONCLUSION In the dog, P2Y12 antagonists inhibit TXA2-mediated platelet-aggregation independently of aspirin. Aspirin provides less additional anti-platelet effects during maximal compared with sub-maximal P2Y12 inhibition but increases vascular resistance.

Pharmacological PPARα activation markedly alters plasma turnover of the amino acids glycine, serine and arginine in the rat.

The current study extends previously reported PPARα agonist WY 14,643 (30 µmol/kg/day for 4 weeks) effects on circulating amino acid concentrations in rats fed a 48% saturated fat diet. Steady-state tracer experiments were used to examine in vivo kinetic mechanisms underlying altered plasma serine, glycine and arginine levels. Urinary urea and creatinine excretion were measured to assess whole-body amino acid catabolism. WY 14,643 treated animals demonstrated reduced efficiency to convert food consumed to body weight gain while liver weight was increased compared to controls. WY 14,643 raised total amino acid concentration (38%), largely explained by glycine, serine and threonine increases. 3H-glycine, 14C-serine and 14C-arginine tracer studies revealed elevated rates of appearance (Ra) for glycine (45.5±5.8 versus 17.4±2.7 µmol/kg/min) and serine (21.0±1.4 versus 12.0±1.0) in WY 14,643 versus control. Arginine was substantially decreased (−62%) in plasma with estimated Ra reduced from 3.1±0.3 to 1.2±0.2 µmol/kg/min in control versus WY 14,643. Nitrogen excretion over 24 hours was unaltered. Hepatic arginase activity was substantially decreased by WY 14,643 treatment. In conclusion, PPARα agonism potently alters metabolism of several specific amino acids in the rat. The changes in circulating levels of serine, glycine and arginine reflected altered fluxes into the plasma rather than changes in clearance or catabolism. This suggests that PPARα has an important role in modulating serine, glycine and arginine de novo synthesis.

The Arachidonate 15-Lipoxygenase Enzyme Product 15-HETE Is Present in Heart Tissue from Patients with Ischemic Heart Disease and Enhances Clot Formation.

Ischemic heart disease is a major cause of death and morbidity and the search for novel therapeutic targets is still required. We have previously shown that the enzyme arachidonate 15 lipoxygenase (ALOX15), which catalyzes the conversion of arachidonic acid to 15-hydroxy eicosatetraenoic acid (15-HETE), is highly expressed in ischemic heart tissue, but its role in the pathogenesis of ischemic heart disease is unclear. Here we showed that expression of ALOX15, but not ALOX12 or ALOX15B, was increased in ischemic versus non-ischemic human heart biopsy samples. A similar ALOX expression pattern was found in hypoxic human cardiomyocytes and cardiac endothelial cells. We also showed that levels of 15-HETE were significantly higher in ischemic versus non-ischemic human heart biopsy samples and showed a tendency to increase in serum from the patients with ischemic heart disease. Moreover, hypoxia increased the production of 15-HETE levels from human cardiomyocytes and cardiac endothelial cells. The hypoxia-induced increase in 15-HETE levels from human cardiomyocytes was inhibited by the ALOX15 inhibitor baicalein. Finally, by using intrinsic rotational thromboelastometry, we showed that human whole blood clotted faster in the presence of 15-HETE. In summary, we propose that increased ALOX15 expression in heart tissue under ischemic conditions may lead to increased production of 15-HETE, potentially contributing to thrombosis.

Baseline Anandamide Levels and Body Weight Impact the Weight Loss Effect of CB1 Receptor Antagonism in Male Rats

The individual weight loss response to obesity treatment is diverse. Here we test the hypothesis that the weight loss response to the CB1 receptor antagonist rimonabant is influenced by endogenous levels of receptor agonists. We show that baseline anandamide levels and body weight independently contribute to predict the treatment response to rimonabant in rodents, demonstrating that addition of biomarkers related to mode of action is relevant for a personalized health care approach to obesity treatment.

Activation of the 12/15 lipoxygenase pathway accompanies metabolic decline in db/db pre-diabetic mice

The 12-lipoxygenase (12LO) pathway is a promising target to reduce islet dysfunction, adipose tissue (AT) inflammation and insulin resistance. Optimal pre-clinical models for the investigation of selective12LO inhibitors in this context have not yet been identified. The objective of this study was to characterize the time course of 12LO isoform expression and metabolite production in pancreatic islets and AT of C57BLKS/J-db/db obese diabetic mouse in a pre-diabetic state in order to establish a suitable therapeutic window for intervention with selective lipoxygenase inhibitors. Mice have 2 major 12LO isoforms -the leukocyte type (12/15LO) and the platelet type (p12LO) and both are expressed in islets and AT. We found a sharp increase in protein expression of 12/15LO in the pancreatic islets of 10-week old db-/- mice compared to 8- week old counterparts. Immunohistochemistry showed that the increase in islet 12/15LO parallels a decline in islet number. Analysis of 12- and 15-hydroperoxytetraeicosanoid acids (HETE)s showed a 2-3 fold increase especially in 12(S)-HETE that mirrored the increase in 12/15LO expression in islets. Analysis of AT and stromal vascular fraction (SVF) showed a significant increase of platelet 12LO gene expression along with 12- and 15- HETEs. The data demonstrate that the db/db mouse is a suitable model for investigation of 12/15LO inhibitors in the development of inflammatory mediated type 2 diabetes, with a narrow window of therapeutic intervention prior to 8 weeks of age.