Dietary fish oil and olive oil supplementation in patients with rheumatoid arthritis. Clinical and immunologic effects.

Forty-nine patients with active rheumatoid arthritis completed a 24-week, prospective, double-blind, randomized study of dietary supplementation with 2 different dosages of fish oil and 1 dosage of olive oil. Clinical evaluations were performed at baseline and every 6 weeks thereafter, and immunologic variables were measured at baseline and after 24 weeks of study. The 3 groups of patients were matched for age, sex, disease severity, and use of disease-modifying antirheumatic drugs (DMARDs). Subjects continued receiving DMARDs and other background medications without change during the study. Twenty patients consumed daily dietary supplements of n3 fatty acids containing 27 mg/kg eicosapentaenoic acid (EPA) and 18 mg/kg docosahexaenoic acid (DHA) (low dose), 17 patients ingested 54 mg/kg EPA and 36 mg/kg DHA (high dose), and 12 patients ingested olive oil capsules containing 6.8 gm of oleic acid. Significant improvements from baseline in the number of tender joints were noted in the low-dose group at week 24 (P = 0.05) and in the high-dose group at week 18 (P = 0.04) and 24 (P = 0.02). Significant decreases from baseline in the number of swollen joints were noted in the low-dose group at weeks 12 (P = 0.003), 18 (P = 0.002), and 24 (P = 0.001) and in the high-dose group at weeks 12 (P = 0.0001), 18 (P = 0.008), and 24 (P = 0.02). A total of 5 of 45 clinical measures were significantly changed from baseline in the olive oil group, 8 of 45 in the low-dose fish oil group, and 21 of 45 in the high-dose fish oil group during the study (P = 0.0002). Neutrophil leukotriene B4 production decreased by 19% from baseline in the low-dose fish oil group (P = 0.0003) and 20% in the high-dose group (P = 0.03), while macrophage interleukin-1 production decreased by 38.5% in the olive oil group (P not significant), 40.6% in the low-dose group (P = 0.06), and 54.7% in the high-dose group (P = 0.0005). Tritiated thymidine incorporation in peripheral blood mononuclear cells after stimulation with concanavalin A increased significantly in all 3 groups after 24 weeks, compared with baseline values. We conclude that the clinical benefits of dietary supplementation with omega-3 fatty acids are more commonly observed in patients consuming higher dosages of fish oil for time intervals that are longer than those previously studied. Dietary supplementation with olive oil is also associated with certain changes in immune function, which require further investigation.

1,25-Dihydroxyvitamin D3-induced growth restriction of cultured epithelial cells derived from a murine hepatic tumor.

Recent evidence suggests that 1,25-dihydroxyvitamin D3 can accumulate in certain presumed non-target tissues, although the mechanism of action of the vitamin in such cells is not understood. Exposure of 77-1/3a mouse hepatic tumor cells, which derived from a non-target tissue of vitamin D action, to 1,25-dihydroxyvitamin D3 in chemically-defined serum-free medium resulted in a dose-dependent decline in cellular growth rate and maximal culture population density but did not adversely affect cell viability. Culture of 77-1/3a cells in defined medium containing 10(-7) or 10(-6) M 1,25-dihydroxyvitamin D3 for 150 hr reduced the growth rate to 64 and 50% of control values respectively. Albumin secretion was unaffected by 1,25-dihydroxyvitamin D3 exposure; in contrast, the cellular content of the proliferation-associated protein p35 was reduced by 39%, a decline similar in trend and degree to that observed in other tumor cells exposed to differentiation-inducing agents. It appears that 1,25-dihydroxyvitamin D3 regulates cellular p35 content (within a specific restricted range) as a consequence of proliferative perturbation, rather than differentiated status, of cultured hepatic tumor cells.

Lipoxygenase products induce neutrophil activation and increase endothelial permeability after thrombin-induced pulmonary microembolism.

We examined the mechanism of the neutrophil (PMN)-dependent increase in pulmonary vascular permeability to protein after thrombin-induced pulmonary microembolism. Humoral factors that activate PMNs after thrombin-induced pulmonary microembolism were characterized in pulmonary lymph obtained from unanesthetized sheep challenged with intravenous infusion of alpha-thrombin. Time-dependent increases in PMN migration, aggregation, and superoxide anion (O2-) generation were induced by the pulmonary lymph obtained within 20 minutes after thrombin infusion. The pulmonary lymph neutrophil activating factors present in ether extracts of lymph had retention times of leukotriene B4 (LTB4) and monohydroxyeicosatetraenoic acids (HETEs) by high-performance liquid chromatography. The postthrombin lymph samples containing the LTB4 and HETEs increased PMN O2- generation and endothelial monolayer permeability to 125I-albumin in the presence of PMNs layered on the endothelial monolayers. Control lymph samples replete with LTB4, 5-HETE, and 15-HETE induced increases in PMN O2- generation and endothelial monolayer permeability to 125I-albumin in the presence of PMNs layered on the endothelial monolayers. Maximal increases in PMN O2- production and endothelial permeability occurred when LTB4, 5-HETE, and 15-HETE were coincubated with PMNs, indicating a synergistic action of these mediators in inducing PMN activation. Endothelial monolayer permeability to 125I-albumin did not increase with postthrombin lymph samples obtained after pretreatment with the 5-lipoxygenase inhibitor, L-651,392. The results indicate that lipoxygenase products generated in the lungs after thrombin-induced microembolism contribute to increased endothelial permeability secondary to PMN activation.

Biosynthesis of 20,20,20-trifluoroleukotriene B4 from 20,20,20-trifluoroarachidonic acid: a metabolically stable analog of leukotriene B4 and its application to a study of stimulation of leukotriene B4 synthesis by immunoglobulin G1,2.

Leukotriene B4 is rapidly metabolized through omega-oxidation, preventing its detection when it is produced under certain biological conditions. To investigate leukotriene B4 production in various physiological conditions, analogs of arachidonic acid which are converted to metabolically stable analogs of leukotriene B4 would be useful. We have synthesized 20,20,20-trifluoroarachidonic acid by the cis-selective Wittig reaction of the C12-C20 fragment with phosphonium salt. 20,20,20-trifluoroarachidonic acid was transformed into 20,20,20-trifluoroleukotriene B4 when incubated with human neutrophils in the presence of the calcium ionophore A23187. The product was identified by uv absorption spectrophotometry, gas chromatography-mass spectrometry, and coelution on high-performance liquid chromatography with 20,20,20-trifluoroleukotriene B4, which was enantioselectively synthesized by the reaction of the fluorine-containing C11-C20 fragment with the C1-C10 phosphonate. The fluorinated leukotriene B4 demonstrated as much chemotactic activity on human neutrophils as natural leukotriene B4 and was metabolically stable when incubated with human neutrophils, probably by blocking omega-oxidation. Also, enzymes catalyzing the transformation of arachidonic acid (AA) into leukotriene B4 did not discriminate the fluorinated precursors from the natural, nonfluorinated AA, thus 20-F3-AA is a valid analog of AA to be used in the study of AA metabolism. When 50 microM of the fluorinated acid was incubated with neutrophils stimulated with heat-aggregated human immunoglobulin G, a significant amount of fluorinated leukotriene B4 (4.3 ng/10(6) cells/40 min, at most) was formed in a dose-dependent manner while little leukotriene B4 was detected with incubation with 50 microM arachidonic acid, probably due to omega-oxidation of the product, leukotriene B4. 20,20,20-Trifluoroarachidonic acid appears to be a useful tool for studying the capacity of leukotriene B4 synthesis in various biological systems while long-lasting 20,20,20-trifluoroleukotriene B4 would serve as an excellent analog of leukotriene B4 in pharmacological studies to understand functions of leukotrienes B4.

In vivo effects of 1,25-dihydroxyvitamin D3 and minerals on synthesis of leukotriene B4 and 5-hydroxyeicosatetraenoic acid by rat peritoneal neutrophils.

We investigated the in vivo effects of 1,25-dihydroxyvitamin D3, and serum calcium and inorganic phosphorus concentrations on productions of leukotriene B4 and 5-hydroxyeicosatetraenoic acid by glycogen stimulated peritoneal neutrophils of the rat. Neutrophils were incubated with ionophore A23187 for 5 min and the incubation medium was subjected to a reverse-phase high performance liquid chromatography for quantitation. Productions of these lipoxygenase metabolites by neutrophils from vitamin D depleted rats were decreased compared to the vitamin D sufficient levels. This difference was not observed when arachidonic acid was added to the incubation medium. When serum calcium concentrations of rats were altered by dietary calcium levels but 1,25-dihydroxyvitamin D3 was administered to those rats, the difference in serum calcium concentration did not show any effect on the production of leukotriene B4. In contrast, neutrophils from 1,25-dihydroxyvitamin D3 sufficient but phosphorus deficient rats still demonstrated decreased leukotriene B4 production. This reduction was corrected by exogenous arachidonic acid. The data suggest that 1,25-dihydroxyvitamin D3 or cellular calcium translocation mediated by the hormone, and serum inorganic phosphorus independent of 1,25-dihydroxyvitamin D3 affect arachidonic acid release, thus productions of leukotriene B4 and 5-hydroxyeicosatetraenoic acid.

Inflammatory events after fibrin microembolization. Alterations in alveolar macrophage and neutrophil function.

We performed bronchoalveolar lavage (BAL) 0.5 to 24 h after thrombin-induced pulmonary microembolization in spontaneously breathing sheep to examine the inflammatory events that occur after pulmonary intravascular coagulation. Neutrophil alveolitis was evident as early as 0.5 h after microembolization and was maximal at 4 h (4.9 +/- 1.5% neutrophils of total BAL cells at baseline versus 26.2 +/- 2.8% at 4 h post-thrombin). Neutrophils obtained both at baseline (isolated from peripheral blood) and at 0.5 to 24 h after thrombin (isolated from BAL) did not demonstrate significant basal production of superoxide anion (O2-) and produced similar amounts of O2- upon challenge with phorbol myristate acetate (PMA) 200 micrograms/ml. The basal O2- production by alveolar macrophages was also not increased. However, alveolar macrophages recovered after fibrin microembolization produced greater amounts of O2- (29.1 +/- 6.3 nm O2-/10(6) cells at 0.5 h) after challenge with PMA compared with alveolar macrophages recovered prior to embolization (10.6 +/- 1.6 nm O2-/10(6) cells baseline), suggesting that thrombin-induced microembolization primes alveolar macrophages and enhances their O2- generation. Neutrophil chemotactic activity was detected in BAL fluid at 0.5 h post-microembolization and reached a peak level at 2 h. Alveolar macrophages were a source of the chemotactic activity since conditioned medium obtained from 2-h post-thrombin macrophages induced neutrophil chemotaxis, whereas baseline cells did not. The addition of the thrombin to macrophages did not result in the generation of chemotactic activity from baseline macrophages, indicating that macrophages were activated during the process of intravascular coagulation rather than by thrombin per se. Post-thrombin BAL fluid also stimulated O2- generation from sheep neutrophils.(ABSTRACT TRUNCATED AT 250 WORDS)

Fish-oil fatty acid supplementation in active rheumatoid arthritis. A double-blinded, controlled, crossover study.

STUDY OBJECTIVE: to determine the efficacy of fish-oil dietary supplements in active rheumatoid arthritis and their effect on neutrophil leukotriene levels. DESIGN: nonrandomized, double-blinded, placebo-controlled, crossover trial with 14-week treatment periods and 4-week washout periods. SETTING: academic medical center, referral-based rheumatology clinic. PATIENTS: forty volunteers with active, definite, or classical rheumatoid arthritis. Five patients dropped out, and two were removed for noncompliance. INTERVENTIONS: treatment with nonsteroidal anti-inflammatory drugs, slow-acting antirheumatic drugs, and prednisone was continued. Twenty-one patients began with a daily dosage of 2.7 g of eicosapaentanic acid and 1.8 g of docosahexenoic acid given in 15 MAX-EPA capsules (R.P. Scherer, Clearwater, Florida), and 19 began with identical-appearing placebos. The background diet was unchanged. MEASUREMENTS AND MAIN RESULTS: the following results favored fish oil placebo after 14 weeks: mean time to onset of fatigue improved by 156 minutes (95% confidence interval, 1.2 to 311.0 minutes), and number of tender joints decreased by 3.5 (95% Cl, -6.0 to -1.0). Other clinical measures favored fish oil as well but did reach statistical significance. Neutrophil leukotriene B4 production was correlated with the decrease in number of tender joints (Spearman rank correlation r=0.53; p less than 0.05). There were no statistically significant differences in hemoglobin level, sedimentation rate, or presence of rheumatoid factor or in patient-reported adverse effects. An effect from the fish oil persisted beyond the 4-week washout period. CONCLUSIONS: fish-oil ingestion results in subjective alleviation of active rheumatoid arthritis and reduction in neutrophil leukotriene B4 production. Further studies are needed to elucidate mechanisms of action and optimal dose and duration of fish-oil supplementation.

Quadruple injection of hypothalamic peptides to evaluate pituitary function in normal subjects.

A single intravenous injection of four hypothalamic releasing hormones-corticotropin-, growth hormone-, gonadotropin- and thyrotropin-releasing hormones-was administered to normal subjects. Except for the plasma adrenocorticotropic hormone (ACTH) level, a statistically significant increase in all anterior pituitary hormone levels occurred. Transient flushing was the only consistent side effect. In the same persons, results were compared with those obtained with insulin-induced hypoglycemia and a single-dose overnight metyrapone test. Growth hormone and cortisol responses to insulin-induced hypoglycemia were similar but prolactin increment was less than that obtained by the peptide injection. ACTH increments from both tests were substantially less than those obtained by the overnight metyrapone test. We conclude that pituitary function can be effectively studied in normal subjects by the combination of a metyrapone test with a triple bolus of growth hormone-, thytropin- and gonadotropin-releasing hormones, but not by a quadruple bolus of the hypothalamic peptides. Compared with insulin-induced hypoglycemia, this approach yields more information with fewer side effects.

Decreased leukotriene B4 synthesis by polymorphonuclear leukocytes from male patients with diabetes mellitus.

Leukotriene B4 (LTB4) synthesis and release by polymorphonuclear leukocytes (PMNL) from 17 male patients with type I diabetes mellitus was decreased (407.1 +/- 55.6 vs 709.4 +/- 62.7 ng/30 X 10(6) cells/10 min, mean +/- SE, p less than 0.001) as compared with 17 normal subjects matched for sex and age. There was an inverse correlation (r = -0.50, p less than 0.04) between LTB4 synthesis and plasma glucose concentration in the diabetic patients. The same correlation (r = -0.71, p less than 0.01) was observed in three diabetic patients studied weekly for one month. Because LTB4 is a potent chemoattractant and stimulates PMNL function, the defect in its synthesis may contribute to the predisposition to infections in the diabetic patient.

A previously unidentified leukotriene produced by human polymorphonuclear leukocytes.

A previously unidentified leukotriene was isolated from incubations of human polymorphonuclear leukocytes with ionophore A23187. The compound eluted between LTB4 and 20-carboxy LTB4 on SP-HPLC and between the two 6-trans isomers of LTB4 on RP-HPLC. Ultraviolet spectroscopy revealed three absorption bands at 258 nm, 268 nm and 278 nm. 1802 was incorporated by the OH at both C5 and C12. Oxidative ozonolysis indicated the presence of 5S, 12S configuration. The structure of the newly identified leukotriene is 5S, 12S-dihydroxy-icosatetraenoic acid. Stereochemistry of the double bonds and biologic activity were not investigated.

Dissociation of E prostaglandin effects on liver glycogenolysis and cyclic AMP levels.

Some metabolic effects of prostaglandins have been related to their alteration of adenosine-3',5'-monophosphate (cyclic AMP) metabolism in different tissues. Prostaglandins E1 and E2 stimulate liver adenylate cyclase in vitro, but conflicting reports have been made about metabolic changes caused by E prostaglandins in hepatic tissue. We have attempted to resolve these issues by comparing the effects of PGE1 with those of glucagon using broken-cell homogenates, intact hepatocytes, liver slices and perfused liver. Prostaglandin E1 (PGE1) increased cyclic AMP in liver slices and in perfused liver without increasing glycogenolysis, but PGE1 had no discernible effect on carbohydrate or cyclic AMP metabolism in isolated hepatocytes. Glucagon caused predictable increases in cyclic AMP and glycogenolysis using hepatocytes, liver slices or perfused liver. These data can be explained by the absence of PGE effects on cyclic AMP metabolism in hepatocytes. The concentration of E prostaglandins (PGEs) increased 1.75-fold during incubations (37 degrees C) of hepatocyte suspensions, but cyclic AMP remained constant. Addition of exogenous arachidonate and indomethacin to cell suspensions increased and decreased PGEs, respectively, but cyclic AMP and glycogen metabolism were unchanged. Arachidonate and indomethacin likewise did not alter glucagon-stimulated glycogenolysis or cyclic AMP biosynthesis. The production of E prostaglandins and cyclic AMP appears to be unrelated in hepatocytes.

Leukotrienes produced by incubation of dihomo-delta-linolenic acid with human polymorphonuclear leukocytes.

Two new leukotrienes, 8,15-dihydroxy-9,11,13-icosatrienoic acid (8,15-LTB3) and 14,15-dihydroxy-8,10,12-icosatrienoic acid (14,15-LTB3) were identified in incubations of human polymorphonuclear leukocytes with dihomo-delta-linolenic acid (8,11,14-icosatrienoic acid). The yield of these compounds was very low (0.5% of the total radioactivity) and did not increase with indomethacin alone or in combination with ionophore A-23187. Stereochemistry and biologic activity of the two leukotrienes remain to be established.

A reevaluation of the chemotactic potency of leukotriene B4 (LTB4).

The chemotactic potency of leukotriene B4 (LTB4) was reevaluated based on an improved purification procedure which combines reversed phase and straight phase high pressure liquid chromatography (RP-HPLC and SP-HPLC). Socalled LTB4 isomer III prepared by RP-HPLC contains two double oxygenated 5,12-dihydroxy acids in addition to LTB4. On a molar basis, the chemotactic activity of LTB4 repurified by SP-HPLC was far greater than that of the other two 5,12-dihydroxy acids and comparable to that of formyl-methionyl-leucyl-phenylalanine (fMLP). The chemotactic activity of LTB4 isomer III is dependent upon the relative concentrations of the double oxygenated 5,12-dihydroxy acids and LTB4. Further purification of peak III by SP-HPLC is required before assessing the biologic activity of LTB4.

Evaluation of the hypothalamic hypophyseal adrenal axis in patients receiving long-term hemodialysis.

Several alterations are present in the hypothalamic hypophyseal regulation of many hormones in patients with chronic renal failure. Evaluation of the hypothalamic hypophyseal adrenal axis in these groups of patients demonstrated normal levels of plasma cortisol. Dexamethasone suppression is abnormal after administration of 1 mg of oral dexamethasone, but normal after 3 mg. Dexamethasone blood levels were lower than the control after administration of 1 mg of oral dexamethasone. A dexamethasone metabolic clearance showed a similar half-life between the patients and controls. Oral absorption study showed poor absorption of the drug. Therefore, there is a problem of gastrointestinal absorption producing the abnormal dexamethasone suppression test in patients with renal failure. Results of metyrapone tests were normal. Corticotropin stimulation tests elicited a normal response. Insulin-induced hypoglycemia does not produce an increment in plasma cortisol or adrenocorticotropic hormone levels.

A novel leukotriene produced by stimulation of leukocytes with formylmethionylleucylphenylalanine.

Stimulation of human polymorphonuclear leukocytes with the chemotactic peptide formylmethionylleucylphenylalanine led to the formation of a novel leukotriene: 5(S),12(R)-dihydroxy-6,8,10,14-eicosatetraen-1,20-dioic acid. This dihydroxydicarboxylic acid is derived from omega-oxidation of 5(S),12(R),dihydroxy-6,8,10,14-eicosatetradienoic acid (leukotriene B4). The intermediate 5(S),12(R),20-trihydroxy-6,8,10,14-eicosatetraenoic acid was also isolated from these incubations. The two metabolites of leukotriene B4 exhibit chemotactic properties for human polymorphonuclear leukocytes but are less active in this respect than the parent compound.

OKY-1581: a selective inhibitor of thromboxane synthesis in vivo and in vitro.

OKY-1581 is an effective inhibitor of thromboxane synthesis in vivo and in vitro. The generation of thromboxane B2 (TxB2), prostaglandin E (PGE) and prostaglandin F (PGF) was measured following clotting and during platelet aggregation induced by collagen. The presence of OKY 1581 either in vivo or in vitro caused a reduction in TxB2 generation during clotting and platelet aggregation with a concomitant increase in PGE and PGF. The effect could be observed two hours after oral or subcutaneous administration of 5 to 100 mg per rabbit and lasted for 24 to 48 hours. The reduction in TxB2 was not accompanied by an inhibition of clotting or platelet aggregation. OKY-1581 appears to be a suitable agent for studying the role of TxB2 in atherosclerosis.

Spurious overestimation of plasma cortisol in patients with chronic renal failure.

Plasma cortisol concentrations in patients with chronic renal failure were measured by five different RIAs, four commercial methods, and an in-house method after paper chromatography. It was observed that cortisol concentrations of the same samples measured by the various methods did not agree. The difference was attributed to cross-reactivity of the antisera with steroids and glucuronide conjugates which circulate at high concentrations in patients with chronic renal failure. With two of the methods, values were higher in unextracted plasma (P less than 0.001) than after paper chromatography (in-house procedure). The values obtained with the other two methods were not significantly different from those obtained by the postpaper chromatography method for baseline and postdexamethasone samples. Plasma cortisol values after methyrapone administration were much higher when measured by any of the four commercial assays than after paper chromatography. A simple dichloromethane extraction improved the results from one of the methods. Laboratories are encouraged to assess carefully the behavior of cortisol antisera before using them in the assay of plasma cortisol concentrations in patients with chronic renal failure.