Molecular clocks and the human condition: approaching their characterization in human physiology and disease.

Molecular clockworks knit together diverse biological networks and compelling evidence from model systems infers their importance in metabolism, immunological and cardiovascular function. Despite this and the diurnal variation in many aspects of human physiology and the phenotypic expression of disease, our understanding of the role and importance of clock function and dysfunction in humans is modest. There are tantalizing hints of connection across the translational divide and some correlative evidence of gene variation and human disease but most of what we know derives from forced desynchrony protocols in controlled environments. We now have the ability to monitor quantitatively ex vivo or in vivo the genome, metabolome, proteome and microbiome of humans in the wild. Combining this capability, with the power of mobile telephony and the evolution of remote sensing, affords a new opportunity for deep phenotyping, including the characterization of diurnal behaviour and the assessment of the impact of the clock on approved drug function.

Vascular and upper gastrointestinal effects of non-steroidal anti-inflammatory drugs: meta-analyses of individual participant data from randomised trials.

BACKGROUND: The vascular and gastrointestinal effects of non-steroidal anti-inflammatory drugs (NSAIDs), including selective COX-2 inhibitors (coxibs) and traditional non-steroidal anti-inflammatory drugs (tNSAIDs), are not well characterised, particularly in patients at increased risk of vascular disease. We aimed to provide such information through meta-analyses of randomised trials. METHODS: We undertook meta-analyses of 280 trials of NSAIDs versus placebo (124,513 participants, 68,342 person-years) and 474 trials of one NSAID versus another NSAID (229,296 participants, 165,456 person-years). The main outcomes were major vascular events (non-fatal myocardial infarction, non-fatal stroke, or vascular death); major coronary events (non-fatal myocardial infarction or coronary death); stroke; mortality; heart failure; and upper gastrointestinal complications (perforation, obstruction, or bleed). FINDINGS: Major vascular events were increased by about a third by a coxib (rate ratio [RR] 1·37, 95% CI 1·14-1·66; p=0·0009) or diclofenac (1·41, 1·12-1·78; p=0·0036), chiefly due to an increase in major coronary events (coxibs 1·76, 1·31-2·37; p=0·0001; diclofenac 1·70, 1·19-2·41; p=0·0032). Ibuprofen also significantly increased major coronary events (2·22, 1·10-4·48; p=0·0253), but not major vascular events (1·44, 0·89-2·33). Compared with placebo, of 1000 patients allocated to a coxib or diclofenac for a year, three more had major vascular events, one of which was fatal. Naproxen did not significantly increase major vascular events (0·93, 0·69-1·27). Vascular death was increased significantly by coxibs (1·58, 99% CI 1·00-2·49; p=0·0103) and diclofenac (1·65, 0·95-2·85, p=0·0187), non-significantly by ibuprofen (1·90, 0·56-6·41; p=0·17), but not by naproxen (1·08, 0·48-2·47, p=0·80). The proportional effects on major vascular events were independent of baseline characteristics, including vascular risk. Heart failure risk was roughly doubled by all NSAIDs. All NSAID regimens increased upper gastrointestinal complications (coxibs 1·81, 1·17-2·81, p=0·0070; diclofenac 1·89, 1·16-3·09, p=0·0106; ibuprofen 3·97, 2·22-7·10, p<0·0001; and naproxen 4·22, 2·71-6·56, p<0·0001). INTERPRETATION: The vascular risks of high-dose diclofenac, and possibly ibuprofen, are comparable to coxibs, whereas high-dose naproxen is associated with less vascular risk than other NSAIDs. Although NSAIDs increase vascular and gastrointestinal risks, the size of these risks can be predicted, which could help guide clinical decision making. FUNDING: UK Medical Research Council and British Heart Foundation.

Biomarkers of oxidative stress study II: are oxidation products of lipids, proteins, and DNA markers of CCl4 poisoning?

Oxidation products of lipids, proteins, and DNA in the blood, plasma, and urine of rats were measured as part of a comprehensive, multilaboratory validation study searching for noninvasive biomarkers of oxidative stress. This article is the second report of the nationwide Biomarkers of Oxidative Stress Study using acute CCl4 poisoning as a rodent model for oxidative stress. The time-dependent (2, 7, and 16 h) and dose-dependent (120 and 1200 mg/kg i.p.) effects of CCl4 on concentrations of lipid hydroperoxides, TBARS, malondialdehyde (MDA), isoprostanes, protein carbonyls, methionine sulfoxidation, tyrosine products, 8-hydroxy-2'-deoxyguanosine (8-OHdG), leukocyte DNA-MDA adducts, and DNA-strand breaks were investigated to determine whether the oxidative effects of CCl4 would result in increased generation of these oxidation products. Plasma concentrations of MDA and isoprostanes (both measured by GC-MS) and urinary concentrations of isoprostanes (measured with an immunoassay or LC/MS/MS) were increased in both low-dose and high-dose CCl4-treated rats at more than one time point. The other urinary markers (MDA and 8-OHdG) showed significant elevations with treatment under three of the four conditions tested. It is concluded that measurements of MDA and isoprostanes in plasma and urine as well as 8-OHdG in urine are potential candidates for general biomarkers of oxidative stress. All other products were not changed by CCl4 or showed fewer significant effects.

Enhanced monocyte expression of tissue factor by oxidative stress in patients with antiphospholipid antibodies: effect of antioxidant treatment.

In a first study, we performed a cross-sectional analysis of urinary excretion of isoprostanes, IPF(2alpha-III) and (VI), and monocyte tissue factor (TF) antigen and activity between 11 antiphospholipid (APL) antibody-positive patients and 13 APL negative subjects. In a second study, 11 APL positive patients were randomly supplemented either with (n = 6) or without (n = 5) antioxidants (vitamin E at 900 IU day(-1), vitamin C at 2000 mg day(-1)) for 6 weeks. In a third study, TF and superoxide anion were measured in human monocytes incubated with anti-beta(2) glycoprotein 1 (beta(2)GP(1)) or control IgG, either with or without vitamin E. APL-positive patients had higher values of isoprostanes (P < 0.05) and monocyte TF antigen (P = 0.001) and activity (P = 0.0001) than APL-negative subjects. Only in APL positive patients did monocyte TF antigen correlate significantly with IPF(2alpha-III) (rho 0.79; P < 0.003) and IPF(2alpha-VI) (rho = 0.87; P < 0.0001). In patients who received antioxidant supplementation, we found a significant decrease of isoprostanes (P < 0.05) and monocyte TF antigen (P < 0.01) and activity (P < 0.007). In vitro experiments demonstrated that anti-beta(2)GP(1) antibodies dose-dependently enhanced the monocyte production of the superoxide anion and TF, which were significantly inhibited by vitamin E. This study demonstrates that in APL-positive patients, oxidative stress contributes to activate the clotting system via over-expression of monocyte TF. We suggest that anti-beta(2)GP(1) antibodies could play a pivotal role by enhancing the monocyte production of oxygen free radicals.

Lipid peroxidation and platelet activation in murine atherosclerosis.

BACKGROUND: Lipid peroxidation and platelet activation are thought to be important contributors to the pathogenesis of atherosclerosis. The relevance of their interaction in vivo, however, is unknown. METHODS AND RESULTS: LDL receptor-deficient (LDLR(-/-)) mice on a high-fat diet developed extensive atherosclerosis and had increased urinary levels of 8,12-iso-isoprostane (iP) F(2alpha)-VI and 2,3-dinor-thromboxane (Tx) B(2), markers of in vivo lipid peroxidation and platelet activation, respectively. Vitamin E supplementation suppressed 8,12-iso-iPF(2alpha)-VI biosynthesis and reduced atherosclerosis (65%) without having a significant effect on lipid levels or TxB(2) biosynthesis. Addition of the platelet inhibitor indomethacin to vitamin E simultaneously suppressed 8,12-iso-iPF(2alpha)-VI and TxB(2), significantly reduced soluble intercellular adhesion molecule-1 and monocyte chemoattractant protein-1, and remarkably, further reduced atherosclerosis (80%). CONCLUSIONS: These results indicate that in vivo lipid peroxidation and platelet activation coexist in murine atherosclerosis and that lipid peroxidation does not contribute to platelet activation and reflects the oxidant component of the inflammatory response. Our findings suggest that oxidant stress and platelet activation represent 2 distinct therapeutic targets in atherogenesis. We propose that a combination of antioxidants and platelet inhibitors might be rationally evaluated in the prevention of progression of human atherosclerosis.

Effects of vitamin E on lipid peroxidation in healthy persons.

CONTEXT: Oxidative stress may play a role in the development or exacerbation of many common diseases. However, results of prospective controlled trials of the effects of antioxidants such as vitamin E are contradictory. OBJECTIVE: To assess the effects of supplemental vitamin E on lipid peroxidation in vivo in healthy adults. DESIGN: Randomized, double-blind, placebo-controlled trial conducted March 1999 to June 2000. SETTING: A general clinical research center in a tertiary referral academic medical center. PARTICIPANTS: Thirty healthy men and women aged 18 to 60 years. INTERVENTIONS: Participants were randomly assigned to receive placebo or alpha-tocopherol dosages of 200, 400, 800, 1200, or 2000 IU/d for 8 weeks (n = 5 in each group), followed by an 8-week washout period. MAIN OUTCOME MEASURES: Three indices of lipid peroxidation, urinary 4-hydroxynonenal (4-HNE) and 2 isoprostanes, iPF(2alpha)-III and iPF(2alpha)-VI, measured by gas chromatography/mass spectrometry and compared among the 6 groups at baseline, 2, 4, 6, and 8 weeks, and 1, 3, and 8 weeks after discontinuation. RESULTS: Circulating vitamin E levels increased in a dose-dependent manner during the study. No significant effect of vitamin E on levels of urinary 4-HNE or either isoprostane was observed. Mean (SEM) baseline vs week 8 levels of iPF(2alpha)-III were 154 (20.1) vs 168 (22.3) pg/mg of creatinine for subjects taking placebo; 165 (19.6) vs 234 (30.1) pg/mg for those taking 200 IU/d of vitamin E; and 195 (26.7) vs 213 (40.6) pg/mg for subjects taking 2000 IU/d. Corresponding iPF(2alpha)-VI levels were 1.43 (0.6) vs 1.62 (0.4) ng/mg of creatinine for subjects taking placebo; 1.64 (0.3) vs 1.24 (0.8) ng/mg for those taking 200 IU/d of vitamin E; and 1.83 (0.3) vs 1.94 (0.9) ng/mg for those taking 2000 IU/d. Baseline vs week 8 levels of 4-HNE were 0.5 (0.04) vs 0.4 (0.05) ng/mg of creatinine for subjects taking placebo; 0.4 (0.06) vs 0.5 (0.02) ng/mg with 200 IU/d of vitamin E; and 0.2 (0.02) vs 0.2 (0.1) ng/mg with 2000 IU/d. CONCLUSIONS: Our results question the rationale for vitamin E supplementation in healthy individuals. Specific quantitative indices of oxidative stress in vivo should be considered as entry criteria and for dose selection in clinical trials of antioxidant drugs and vitamins in human disease.

Ongoing prothrombotic state in patients with antiphospholipid antibodies: a role for increased lipid peroxidation.

We measured the urinary excretion of Isoprostane F2alpha-III and Isoprostane-F2alpha-VI, two markers of in vivo lipid peroxidation, and the circulating levels of the prothrombin fragment F1+2, a marker of thrombin generation, in 18 antiphospholipid antibodies-positive patients, in 18 antiphospholipid antibodies-negative patients with systemic lupus erythematosus, and in 20 healthy subjects. Furthermore, 12 patients positive for antiphospholipid antibodies were treated with (n = 7) or without (n = 5) antioxidant vitamins (vitamin E at 900 IU/d and vitamin C at 2, 000 mg/d) for 4 weeks. Compared with antiphospholipid antibodies-negative patients, antiphospholipid antibodies-positive patients had higher urinary values of Isoprostane-F2alpha-III (P =. 0001), Isoprostane-F2alpha-VI (P =.006), and plasma levels of the prothrombin fragment F1+2 (P =.0001). In antiphospholipid-positive patients, F1+2 significantly correlated with Isoprostane-F2alpha-III (Rho =.56, P =.017) and Isoprostane-F2alpha-VI (Rho =.61, P =.008). After 4 weeks of supplementation with antioxidant vitamins, we found a significant decrease in F1+2 levels (P <.005) concomitantly with a significant reduction of both Isoprostane-F2alpha-III (P =.007) and Isoprostane-F2alpha-VI (P <.005). No change of these variables was observed in patients not receiving antioxidant treatment. This study suggests that lipid peroxidation might contribute to the activation of clotting system in patients positive for antiphospholipid antibodies.

Vitamin E reduces monocyte tissue factor expression in cirrhotic patients.

Clotting activation may occur in liver cirrhosis, but the pathophysiological mechanism has not been fully elucidated. Because a previous study demonstrated that lipid peroxidation is increased in cirrhosis, we analyzed whether there is a relationship between lipid peroxidation and clotting activation. Thirty cirrhotic patients (19 men and 11 women; age, 34 to 79 years) and 30 controls matched for sex and age were investigated. In all subjects, monocyte expression of tissue factor (TF) antigen and activity; plasma levels of prothrombin fragment 1+2 (F1+2), a marker of thrombin generation; and urinary excretion of Isoprostane-F2alpha-III, a marker of lipid peroxidation, were measured. Furthermore, the above-reported variables were re-evaluated after 30 days of treatment with standard therapy (n = 5) or standard therapy plus 300 mg vitamin E twice daily (n = 9). In addition, we analyzed in vitro if vitamin E (50 micromol/L) influenced monocyte TF expression and F1+2 generation. Cirrhotic patients had higher values of Isoprostane-F2alpha-III (P <. 0001), F1+2 (P <.0001), and monocyte TF antigen (P <.0001) and activity (P <.03) than controls. Isoprostane-F2alpha-III was significantly correlated with F1+2 (Rho = 0.85; P <.0001) and TF antigen (Rho = 0.95; P <.0001) and activity (Rho = 0.94; P <.0001). After vitamin E treatment, Isoprostane-F2alpha-III (P =.008), F1+2 (P <.008), and monocyte TF antigen (P =.012) and activity (P =.008) significantly decreased; no changes of these variables were detected in patients not receiving vitamin E. In vitro, vitamin E significantly reduced the expression of monocyte TF antigen (-52%; P =.001) and activity (-55%; P =.003), as well as F1+2 generation (-51%; P =.025). This study shows that vitamin E reduces both lipid peroxidation and clotting activation and suggests that lipid peroxidation may be an important mediator of clotting activation in liver cirrhosis.

Increased F2-isoprostanes in Alzheimer's disease: evidence for enhanced lipid peroxidation in vivo.

Alzheimer's disease (AD) includes a group of dementing neurodegenerative disorders that have diverse etiologies but the same hallmark brain lesions. Since oxidative stress may play a role in the pathogenesis of AD and isoprostanes are chemically stable peroxidation products of arachidonic acid, we measured both iPF2alpha-III and iPF2alpha -VI using gas chromatography-mass spectrometry in AD and control brains. The levels of both isoprostanes, but not of 6-keto PGF1alpha, an index of prostaglandin production, were markedly elevated in both frontal and temporal poles of AD brains compared to the corresponding cerebella. Levels were also elevated compared to corresponding areas of brains from patients who had died with schizophrenia or Parkinson's disease or from nonneuropsychiatric disorders. iPF2alpha -IV, but not iPF2alpha-III, levels were higher in ventricular CSF of AD brains relative to the non-AD brains. These data suggest that specific isoprostane analysis may reflect increased oxidative stress in AD.

Vitamin E suppresses isoprostane generation in vivo and reduces atherosclerosis in ApoE-deficient mice.

Oxidative modification of low density lipoprotein (LDL) has been implicated in atherogenesis. Evidence consistent with this hypothesis includes the presence of oxidized lipids in atherosclerotic lesions, the newly discovered biological properties conferred on LDL by oxidation and the acceleration of atherogenesis by in vivo delivery of the gene for 15-lipoxygenase, an oxidizing enzyme present in atherosclerotic lesions. However, it is still unknown whether oxidative stress actually coincides with the evolution of the disease or whether it is of functional relevance to atherogenesis in vivo. Isoprostanes are products of arachidonic acid catalyzed by free radicals, which reflect oxidative stress and lipid peroxidation in vivo. Elevation of tissue and urinary isoprostanes is characteristic of human atherosclerosis. Here, deficiency in apolipoprotein E in the mouse (apoE-/-) resulted in atherogenesis and an increase in iPF2alpha-VI, an F2-isoprostane, in urine, plasma and vascular tissue. Supplementation with vitamin E significantly reduced isoprostane generation, but had no effect on plasma cholesterol levels in apoE-/- mice. Aortic lesion areas and iPF2alpha-VI levels in the arterial wall were also reduced significantly by vitamin E. Our results indicate that oxidative stress is increased in the apoE-/- mouse, is of functional importance in the evolution of atherosclerosis and can be suppressed by oral administration of vitamin E.

Functional characterization of the ocular prostaglandin f2alpha (PGF2alpha) receptor. Activation by the isoprostane, 12-iso-PGF2alpha.

Prostaglandin F2alpha (PGF2alpha) is a product of cyclooxygenase-catalyzed metabolism of arachidonic acid. Recently, PGF2alpha analogs have been hypothesized to reduce intraocular pressure via relaxation of the ciliary muscle. To investigate the molecular basis of PGF2alpha receptor (FP) activation in the eye, we cloned the FP from a human ciliary body (hcb) cDNA library. The open reading frame of the hcb-FP cDNA was identical to the uterine FP cDNA. The hcb-FP appeared to be predominantly membrane-localized, as visualized by an FP-specific peptide antibody, and coupled to inositol phosphate formation when stably expressed in HEK 293 cells. Interestingly, the hcb-FP could also be activated by the F2 isoprostane, 12-iso-PGF2alpha, in addition to its cognate ligand, PGF2alpha. 12-iso-PGF2alpha was less potent (EC50 = 5 microM) than PGF2alpha (EC50 = 10 nM) in generating inositol phosphates via the hcb-FP in HEK 293 cells. Both ligands also stimulated mitogenesis in NIH 3T3 cells. Although 12-iso-PGF2alpha caused a dose-dependent activation of the FP, it failed to activate the recombinant human prostacyclin receptor and caused only minimal activation of the thromboxane receptor isoforms stably expressed in HEK 293 cells. Four additional F2 isoprostanes, 8-iso-PGF2alpha, IPF2alpha-I, IPF2alpha-III, and 9beta,11beta-PGF2, caused trivial, or no, activation of the FP. Consistent with these observations, only PGF2alpha and 12-iso-PGF2alpha caused rapid homologous desensitization of FP and also exhibited cross-desensitization, with PGF2alpha resulting in a maximum of approximately 60% desensitization. The human FP may thus be activated specifically, by the free radical-catalyzed F2 isoprostane, 12-iso-PGF2alpha, in addition to the cyclooxygenase product, PGF2alpha. Incidental receptor activation by isoprostanes may complement the actions of PGF2alpha in clinical syndromes where oxidant stress and augmented prostaglandin biosynthesis coincide.

The human thromboxane A2 receptor alpha isoform (TP alpha) functionally couples to the G proteins Gq and G11 in vivo and is activated by the isoprostane 8-epi prostaglandin F2 alpha.

To establish whether the thromboxane A2 (TXA2) receptor (TP) functionally couples to the Gq family of heterotrimeric G proteins in vivo, we have coexpressed the cDNAs coding for the human platelet/placental TP alpha isoform (TP alpha) and the alpha subunits of Gq or G11 in human embryonic kidney (HEK) 293 cells. TP activation in response to ligand stimulation was monitored by analyzing mobilization of intracellular calcium (Ca++i) in FURA2/AM-loaded transfected HEK 293 and in platelets. Second, we wished to examine the possible interaction of the isoprostane 8-epi prostaglandin F2 alpha with the TP alpha, in transfected HEK 293 cells and with the TPs expressed in platelets. Thus both the prostaglandin endoperoxide/TXA2 analog (U46619) and the 8-epi PGF2 alpha were utilized as ligand probes of TP alpha activation. The results demonstrate that each ligand induced elevations of Ca++i levels in HEK 293 cells, cotransfected with either the TP alpha and G alpha q or the TP alpha and G alpha 11, and also in platelets. Initial stimulation of these cells with U46619 or 8-epi PGF 2 alpha desensitized a subsequent rise in [Ca++]i in response to U46619 or 8-epi PGF 2 alpha, respectively. Moreover, prestimulation with U46619 desensitized a subsequent rise in Ca++i concentration in response to 8-epi PGF 2 alpha, and vice versa. These responses were blocked by the TP antagonist SQ29,548 in both cell types. In contrast, prestimulation of the transfected HEK 293 cells or platelets with thrombin did not desensitize a subsequent rise in [Ca++]i in response to U46619 or 8-epi PGF 2 alpha. After stimulation with either U46619 or 8-epi PGF 2 alpha, no significant rise in Ca++i levels was observed in HEK 293 cells transfected with the TP alpha receptor only or in control cells transfected with the vector pCMV5. These results demonstrate that the TP alpha isoform functionally couples with either Gq or G11 in vivo, whether activated by a PG/TXA2 analog or by the F2 isoprostane 8-epi PGF2 alpha.

Modulation of oxidant stress in vivo in chronic cigarette smokers.

BACKGROUND: Free radical-induced oxidative damage is thought to be involved in the pathogenesis of diseases associated with cigarette smoking. We examined the production of 8-epi-prostaglandin (PG) F2 alpha, a stable product of lipid peroxidation in vivo, and its modulation by aspirin and antioxidant vitamins in chronic cigarette smokers. METHODS AND RESULTS: We performed the following studies: (1) a cross-sectional comparison of smokers and control subjects, (2) an examination of the dose-response relationship, (3) an exploration of the effect of smoking cessation (3 weeks) and nicotine patch supplementation, (4) the effect of aspirin consumption, and (5) the effects of 5 days' dosing with vitamin E (100 and 800 U), vitamin C (2 g), and their combination. 8-epi-PGF2 alpha excretion (in pmol/mmol, mean +/- SEM) was 176.5+/-30.6 in heavy smokers, 92.7+/-4.8 (P<.05) in moderate smokers, and 54.1+/-2.7 (P<.005) in nonsmokers. Urinary levels fell from 145.5+/-24.9 to 114.6+/-27.1 (week 2, P<.05) and 112.6+/-24.9 (week 3, P<.05) on cessation of smoking. Aspirin treatment failed to suppress urinary levels of 8-epi-PGF2 alpha despite a significant reduction in urinary 11-dehydro-TxB2 production and suppression of 8-epi-PGF2 alpha and TxB2 in serum. Vitamin C (pre, 194.6+/-40.9; post, 137.2+/-34.1; P<.05) and a combination of vitamin C and E (pre, 171.0+/-39.8; post, 133.5+/-29.6 P<.05) suppressed urinary 8-epi-PGF2 alpha, whereas vitamin E alone had no effect. CONCLUSIONS: Urinary 8-epi-PGF2 alpha may represent a noninvasive, quantitative index of oxidant stress in vivo. Elevated levels of 8-epi-PGF2 alpha in smokers may be modulated by quitting cigarettes and switching to nicotine patches or by antioxidant vitamin therapy.

Effects of dietary fat content, saturated fatty acids, and fish oil on eicosanoid production and hemostatic parameters in normal men.

Populations that consume a diet rich in marine lipids have been reported to have a lower risk of coronary heart disease. However, some Western population groups with a high fish consumption continue to suffer elevated rates of coronary heart disease. Many of these individuals consume a diet rich in saturated fats in addition to the fish. To examine these possible dietary interactions we fed six healthy men diets that contained two levels of saturated fat (5% and 19% of energy). During 3-week periods the study subjects were given diets with a low-(25% of energy) and high-(39% of energy) fat content with and without inclusion of n-3 polyunsaturated or monounsaturated fatty acids (2% of energy). The effects of the n-3 fatty acids on the principal plasma lipid fractions were similar regardless of the saturated fat intake. Platelet function, as measured by the skin bleeding time, was inhibited when n-3 fatty acids were added to the low saturated-fat diet. In vivo thromboxane A2 production as assessed by urinary metabolites also declined (p < 0.01) during supplementation with n-3 fatty acids to a low-fat diet. Prostacyclin production were reduced on a low-fat diet compared to a high-fat diet regardless of supplementation with n-3 fatty acids. N-3 fatty acids stimulated the synthesis of modest amounts of thromboxane A3 and prostacyclin I3, on both the low and high saturated-fat diets. These studies showed that the effects of eicosapentaenoic and docosahexaenoic acids on platelet and vascular function and eicosanoid production are modulated by the content of saturated fatty acids in the diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Interruption of vascular thrombus formation and vascular lesion formation by dietary n-3 fatty acids in fish oil in nonhuman primates.

BACKGROUND: Because of discrepant claims regarding the relative biological effects of n-3 fatty acids (n-3FAs), we have concurrently measured the effects of dietary n-3FAs on blood and vascular lipid composition, hemostatic function, blood thrombotic responses, vascular thrombus formation, and vascular lesion formation in baboons. METHODS AND RESULTS: Dietary n-3FAs displaced n-6FAs in plasma, platelets, blood vessels, and corresponding urinary eicosanoid metabolites (p < 0.01 in all cases) within weeks after initiation of a semipurified diet containing 1 g/kg per day n-3FA-ethyl ester concentrate (composed of two thirds eicosapentanoic acid and one third docosahexanoic acid). Coincidentally, platelet hemostatic function became minimally impaired (template bleeding times prolonged from 4.3 +/- 0.5 minutes to 7.6 +/- 1.3 minutes, p = 0.039); concentrations of collagen producing half-maximal platelet aggregation increased (from 6.4 +/- 2.1 to 8.5 +/- 2.5 micrograms/mL, p = 0.045); and tissue factor expression by endotoxin-stimulated blood monocytes fell (from 6.5 +/- 1.2 to 1.7 +/- 0.14 mU/10(6) cells, p < 0.005). Dietary n-3FAs decreased deposition of platelets onto thrombogenic segments of Dacron vascular graft incorporated into chronic exteriorized femoral arteriovenous (AV) shunts, a thrombotic process resistant to the effects of both aspirin and heparin (111In-labeled platelet deposition decreased from 14.1 +/- 1.4 x 10(9) platelets/5-cm segment at 40-60 minutes with occlusion to 7.5 +/- 0.8 x 10(9) platelets/5-cm segment without occlusion; p < 0.001). Platelet deposition onto segments of endarterectomized homologous normal aorta in the AV shunts of n-3FA-treated animals was similarly reduced (from 4.4 +/- 0.9 to 1.8 +/- 0.4 x 10(9) platelets; p < 0.01). Dietary n-3FAs interrupted vascular thrombus formation at sites of surgical carotid endarterectomy (platelet deposition, 1.5 +/- 0.4 versus 4.4 +/- 1.0 x 10(9) platelets in untreated controls; p < 0.001). Moreover, endarterectomized aortic segments (EASs) from n-3FA-treated donors exhibited little capacity to induce thrombus formation when tested in the AV shunts of control recipient animals (0.24 +/- 0.10 versus 4.4 +/- 0.90 x 10(9) platelets). However, in the converse crossover experiments, EASs from control animals actively accumulated platelets when studied in the AV shunts of n-3FA-treated animals (1.8 +/- 0.4 x 10(9) platelets; p < 0.01 versus n-3FA-treated EASs in shunts of normal animals). Dietary n-3FAs also abolished vascular lesion formation at sites of carotid endarterectomy 6 weeks after surgery (cross-sectional area of neointima 0.048 +/- 0.031 mm2 compared with 0.428 +/- 0.104 mm2 in control arteries; p = 0.010). CONCLUSIONS: In nonhuman primates, dietary n-3FAs in high doses eliminate both vascular thrombus formation and vascular lesion formation after mechanical vascular injury while largely sparing hemostatic function and modestly reducing blood thrombotic responses. These effects are attributed to selective n-3FA-dependent alterations in cellular membrane functions.

Effects of fish oil supplementation in the third trimester of pregnancy on prostacyclin and thromboxane production.

OBJECTIVE: Disturbance in thromboxane and prostacyclin biosynthesis has been observed in preeclampsia. We studied whether fish oil supplementation in late pregnancy interferes with maternal and fetal production of thromboxane A2 and prostacyclin I2. STUDY DESIGN: Forty-seven women in the thirtieth week of pregnancy were randomly assigned in a ratio of 2:1:1 to receive fish oil (2.7 gm of n-3 fatty acid per day [Pikasol], or either olive oil or no oil supplementation as controls. Metabolites of thromboxane A2 and A3 and of prostacyclin I2 and I3 were quantified by mass spectrometry methods in serum and urine, respectively. Maternal serum and urine were sampled at baseline, in the thirty-third and thirty-seventh weeks of pregnancy. Fetal serum was sampled at delivery. RESULTS: At the thirty-seventh week the mean concentrations of the eicosapentaenoic-derived metabolites, thromboxane B3 and prostacyclin I3, was twofold to threefold higher (p < 0.001) in the group receiving fish oil compared with combined control groups. There were no significant effects of fish oil on the prostacyclin I2 metabolite, although there was a trend toward a reduction in thromboxane B2 in this group. In umbilical cord blood the mean concentration of thromboxane B2 was lowest in the group receiving fish oil (p = 0.03). CONCLUSIONS: Fish oil was metabolized to the eicosapentaenoic acid-derived eicosanoids thromboxane A3 and prostacyclin I3 in pregnant women. Correspondingly, analog products of arachidonic acid tended to be depressed. It remains to be established whether these biochemical effects will prove beneficial in the prevention or treatment of preeclampsia and intrauterine growth retardation.

Suppression of eicosanoid biosynthesis during coronary angioplasty by fish oil and aspirin.

BACKGROUND: Percutaneous transluminal coronary angioplasty (PTCA) is an acute, localized stimulus to platelet and vascular function. Periprocedural cardiovascular complications are reduced by moderate-dose aspirin (ASA), presumably due to inhibition of thromboxane (TX) A2. METHODS AND RESULTS: Excretion of TXA2 and prostacyclin (PGI2) metabolites in urine increased during PTCA. Pretreatment for 3 days with either moderate- (325 mg/day) or low-dose (80 mg/day) ASA inhibited the increase in both eicosanoids. Pretreatment for 3 weeks with fish oil (10 g/day) only partially suppressed TXA2. Formation of trienoic eicosanoids and accumulation of omega-3 fatty acids in platelet membranes confirmed fish oil ingestion. Although basal PGI2 was not inhibited, the PTCA-related increment was suppressed. CONCLUSIONS: PTCA results in an acute, transient alteration of eicosanoid biosynthesis consistent with accelerated platelet-vascular interactions. Pretreatment for 3 days with moderate or low doses of ASA suppresses TXA to a similar extent during PTCA, and their effects on acute cardiovascular complications of this procedure are likely to be comparable. It is unlikely that even prolonged pretreatment with fish oil can substitute for the platelet inhibitory action of ASA during PTCA. Suppression of PGI2 may contribute to the residual acute periprocedural complication rate in patients taking ASA.

The antihypertensive effects of fish oil. A controlled study of polyunsaturated fatty acid supplements in essential hypertension.

Both n-3 and n-6 polyunsaturated fats have been suggested to lower blood pressure, an effect ascribed to altered biosynthesis of eicosanoids. To test these hypotheses, we studied blood pressure and eicosanoid production during supplementation of dietary fat for four weeks in 32 men with mild essential hypertension. Supplementation was preceded and followed by four-week run-in and recovery periods. Groups of eight subjects received either 10 ml or 50 ml of fish oil (3 or 15 g of n-3 fatty acids) daily, 50 ml of safflower oil (39 g of n-6 fatty acids), or 50 ml of a mixture of oils that approximated the types of fat present in the American diet. The biosynthesis of eicosanoids was assessed by the measurement of urinary metabolites. Blood pressure decreased in the men who received the high dose of fish oil (systolic pressure by a mean of 6.5 mm Hg [P less than 0.03] and diastolic pressure by 4.4 mm Hg [P less than 0.015]), but not in the other groups. Although the formation of vasodilatory prostacyclins (prostaglandins I2 and I3) increased initially, this increase was not maintained as blood pressure fell. The level of thromboxane A2 metabolites fell; metabolites of thromboxane A3 were detected in the groups receiving fish oil. The formation of prostaglandin E2 increased during supplementation with safflower oil and tended to decrease with fish oil; no prostaglandin E3 metabolite was detected. Our data indicate that high doses of fish oil can reduce blood pressure in men with essential hypertension. However, the clinical usefulness and safety of fish oil in the treatment of hypertension will require further study.