Effect of DHA supplementation on DHA status and sperm motility in asthenozoospermic males.

The effects of supplementation with docosahexaenoic acid (DHA) on DHA levels in serum, seminal plasma, and sperm of asthenozoospermic men as well as on sperm motility were examined in a randomized, double-blind, placebo-controlled manner. Asthenozoospermic men (n = 28; < or =50% motility) were supplemented with 0, 400, or 800 mg DHA/d for 3 mon. Sperm motility and the fatty acid composition of serum, seminal plasma, and sperm phospholipid were determined before and after supplementation. In serum, DHA supplementation resulted in decreases in 22:4n-6 (-30% in the 800-mg DHA group only) and total n-6 (-6 and -12% in the 400- and 800-mg DHA groups, respectively) fatty acids. Increases were noted in DHA (71 and 131% in the 400- and 800-mg DHA groups, respectively), total n-3 fatty acids (42 and 67% in the 400- and 800-mg DHA groups, respectively), and the n-3/n-6 ratio (50 and 93% in the 400- and 800-mg DHA groups, respectively). In seminal plasma, DHA supplementation resulted in a decrease in 22:4n-6 (-31% in the 800-mg DHA group only) and an increase in the ratio of n-3 to n-6 (35 and 33% in the 400- and 800-mg DHA groups, respectively). There were insignificant increases in DHA and total n-3 fatty acids. In sperm, decreases were noted in 22:4n-6 (-37 and -31% in the 400- and 800-mg DHA groups, respectively). There were no other changes. There was no effect of DHA supplementation on sperm motility. The results show that dietary DHA supplementation results in increased serum--and possibly seminal plasma--phospholipid DHA levels, without affecting the incorporation of DHA into the spermatozoa phospholipid in asthenozoospermic men. This inability of DHA to be incorporated into sperm phospholipid is most likely responsible for the observed lack of effect of DHA supplementation on sperm motility.

Fatty acid analysis of blood plasma of patients with Alzheimer's disease, other types of dementia, and cognitive impairment.

Fatty acid differences, including docosahexaenoic acid (DHA; 22:6n-3) have been shown in the brains of Alzheimer's patients (AD) as compared with normal age-matched individuals. Furthermore, low serum DHA is a significant risk factor for the development of AD. The relative concentration of DHA and other fatty acids, however, in the plasma of AD patients compared with patients with other kinds of dementias (other dementias; OD), patients who are cognitively impaired but nondemented (CIND), or normal patients is not known. In this study we analyzed the total phospholipid, phosphatidylcholine (PC), phosphatidylethanolamine (PE), and lysophosphatidylcholine (lysoPC) fractions of plasma from patients diagnosed with AD, OD, or CIND and compared them with a group of elderly control subjects with normal cognitive functioning. Plasma phospholipid and PC levels of 20:5n-3, DHA, total n-3 fatty acids, and the n-3/n-6 ratio were lower in the AD, OD, and CIND groups. Plasma phospholipid 24:0 was lower in the AD, OD, and CIND groups as compared with the group of control patients, and total n-6 fatty acid levels were higher in the AD and CIND groups only. In the plasma PE fraction, levels of 20:5n-3, DHA, and the total n-3 fatty acid levels were significantly lower in the AD, OD, and CIND groups. DHA levels were lower in the lysoPC fraction of CIND individuals only. There were no other differences in the fatty acid compositions of the different phospholipid fractions. Therefore, in AD, OD, and CIND individuals, low levels of n-3 fatty acids in the plasma may be a risk factor for cognitive impairment and/or dementia. Interestingly, a decreased level of plasma DHA was not limited to the AD patients but appears to be common in cognitive impairment with aging.

Effect of supplementation with dietary seal oil on selected cardiovascular risk factors and hemostatic variables in healthy male subjects.

The average daily consumption of seal oil by the Inuit people is approximately 8-9 g, yet there is very little information on the effect of seal oil consumption on cardiovascular disease risk factors. In this study, 19 healthy, normocholesterolemic subjects consumed 20 g of encapsulated seal oil containing eicosapentaenoic acid (EPA; 20:5n-3), docosahexaenoic acid (DHA; 22:6n-3), and docosapentaenoic acid (DPA; 22:5n-3) or 20 g of vegetable oil (control) per day for 42 days. Levels of selected cardiovascular and thrombotic risk factors as well as fatty acid profiles of serum phospholipid and nonesterified fatty acid (NEFA) were determined. EPA levels in serum phospholipid and NEFA increased by 4.3- and 2.7-fold, respectively, in the seal oil supplemented group. DHA levels rose 1.5- and 2.1-fold, respectively, and DPA levels rose 0.5- and 0.7-fold, respectively. Arachidonic acid (AA) levels dropped by 26% in both serum phospholipid and serum NEFA. There was a significant decrease in the ratio of n-6 to n-3 fatty acids in serum phospholipid from 7.2 to 2.1 and a significant increase in the ratio of EPA/AA in NEFA. Ingestion of seal oil raised the coagulant inhibitor, protein C, values by 7% and decreased plasma fibrinogen by 18%. No alterations in other hemostatic variables, including plasma activity of Factors VII, VIII, IX, and X and antithrombin, or in the concentrations of von Willebrand Factor, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglyceride, glucose, Apo A-1, or lipoprotein(a) were observed in either group. Other risk factors for cardiovascular disease, including hematocrit, white blood cell count, plasma viscosity, systolic and diastolic blood pressures, heart rate, and platelet aggregation after stimulation with ADP or collagen did not change. Our results indicate that seal oil supplementation in healthy, normocholesterolemic subjects decreased the n-6/n-3 ratio and increased EPA, DHA, and DPA and the ratio of EPA/AA and DHA/AA in the serum phospholipid and NEFA, while exhibiting a modest beneficial effect on fibrinogen and protein C levels.

Fatty acid analysis of blood serum, seminal plasma, and spermatozoa of normozoospermic vs. asthenozoospermic males.

Docosahexaenoic acid (DHA; 22:6n-3) is found in extremely high levels in human ejaculate with the majority occurring in the spermatozoa. However, the relative concentration of DHA and other fatty acids, in blood serum, seminal plasma, and spermatozoa of asthenozoospermic vs. normozoospermic individuals is not known. We analyzed the phospholipid fatty acid composition of blood serum, seminal plasma, and spermatozoa of normozoospermic men and asthenozoospermic men in order to determine if DHA levels, as well as the levels of other fatty acids, differed. The serum phospholipid DHA levels were similar in the two groups, suggesting similar intakes of dietary DHA. On the other hand, seminal plasma levels of DHA (3.0 vs. 3.7%) and total polyunsaturated fatty acids (PUFA) (11.8 vs. 13.5%) were significantly lower in asthenozoospermic vs. normozoospermic men, respectively, while 18:1 (19.0 vs. 16.8%) and monounsaturated fatty acids (MUFA) (24.2 vs. 21.7%) were significantly higher in the asthenozoospermic vs. the normozoospermic men. Spermatozoa from asthenozoospermic men had higher levels of 18:1, 20:0, 22:0, 22:1, and 24:0 than sperm from normozoospermic men, and lower levels of 18:0 and DHA (8.2 vs. 13.8%). Furthermore, total MUFA (19.3 vs. 16.5%) was higher and total PUFA (19.0 vs. 24.0%), n-3 fatty acids (9.3 vs. 14.6%), and the ratio of n-3 to n-6 fatty acids (1.0 vs. 1.6) were lower in the asthenozoospermic men. Therefore, in asthenozoospermic individuals, lower levels of DHA in the seminal plasma, but not in the blood serum, mimic the decreased concentrations of DHA in the spermatozoa. This suggests that the lower concentrations of spermatozoon DHA in these individuals are due not to dietary differences but to some type of metabolic difference in the asthenozoospermic men.

Docosahexaenoic acid and docosapentanoic acid incorporation into human platelets after 24 and 72 hours: inhibitory effects on platelet reactivity.

Short-term in vitro platelet membrane lipid enrichment studies and feeding trials of human subjects with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have shown a decreased reactivity in the platelet response to collagen. In this study, exogenous albumin-bound n-3 polyunsaturated fatty acids (PUFAs), namely EPA, DHA and docosapentanoic acid (DPA) were added to platelet suspensions and maintained at 22 degrees C for 24 and 72 hours. Subsequently, the aggregation response to agonist stimulation and the morphological appearance of the platelets were evaluated. A significant enrichment of platelet phospholipids (PL) in n-3 fatty acids occurred upon incubation with n-3 PUFAs in vitro, which was accompanied by a decrease in the aggregation response to collagen and preservation of platelet morphology compared with non-supplemented control platelet preparations. The inhibitory effect of the n-3 PUFAs appeared to be surface mediated in the case of DHA and DPA because the platelet response to agonist returned when the fatty acids were removed by washing. The platelet aggregation response after storage at 22 degrees C was also evaluated in platelet suspensions collected from healthy individuals before and after 42 days of dietary supplementation with seal oil, rich in DPA and DHA. Unlike the in vitro supplementation, in vivo modification and enrichment of platelet PLs by ingestion of seal oil did not appear to improve platelet function during storage relative to the placebo group.

Supplementation with quercetin markedly increases plasma quercetin concentration without effect on selected risk factors for heart disease in healthy subjects.

The purpose of this double-blind study was to investigate the influence of adding a quercetin-containing supplement to the diet on plasma quercetin status, serum/platelet fatty acid levels and risk factors for heart disease. Healthy men and women with cholesterol levels of 4.0-7.2 mmol/L, consumed four capsules daily of either a quercetin-containing supplement (1.0 g quercetin/d) or rice flour placebo for 28 d. Quercetin intakes were approximately 50-fold greater than the dietary intakes associated with lower coronary heart disease mortality on the basis of epidemiologic studies. Subjects consuming quercetin-containing capsules had plasma quercetin concentrations approximately 23-fold higher than those of subjects consuming the control capsules. Quercetin supplementation did not modify serum total, LDL or HDL cholesterol or triglyceride levels. There were also no alterations of other cardiovascular disease or thrombogenic risk factors, including platelet aggregation, platelet thromboxane B2 production, blood pressure or resting heart rate. Furthermore, there was no effect on the levels of (n-6) or (n-3) polyunsaturated fatty acids in serum or platelet phospholipids. In conclusion, supplementation with quercetin-containing capsules markedly enhanced the plasma quercetin concentration but had no effect on other cardiovascular or thrombogenic risk factors.

Interleukin-1 beta induction of c-fos and collagenase expression in articular chondrocytes: involvement of reactive oxygen species.

Interleukin-1 beta (IL-1) is implicated in cartilage destruction in arthritis through promotion of matrix metalloproteinase production. Upregulation of collagenase gene expression by IL-1 is known to require the transactivators Fos and Jun. Recently, reactive oxygen species (ROS) have been suggested to act as intracellular signaling molecules mediating the biological effects of cytokines. Here, we demonstrated ROS production by IL-1-stimulated bovine chondrocytes and that neutralizing ROS activity by the potent antioxidant, N-acetylcysteine, or inhibiting endogenous ROS production by diphenyleneiodonium (DPI), significantly attenuated IL-1-induced c-fos and collagenase gene expression. The inhibitory effect of DPI implicates enzymes such as NADPH oxidase in the endogenous production of ROS. Chondrocytes were also found to produce nitric oxide (NO) upon IL-1 stimulation. That NO may mediate part of the inducing effects of IL-1 was supported by the observation that L-NG-monomethylarginine, a NO synthase inhibitor, partially inhibited IL-1-regulated collagenase expression. Moreover, treatment of chondrocytes with the NO-producing agent, S-nitroso-N-acetylpenicillamine, was sufficient to induce collagenase mRNA levels. In summary, our results suggest that ROS released in response to IL-1 may function as second messengers transducing extracellular stimuli to their targets in the nucleus, leading to augmentation of gene expression.

Effect of supplementation with different doses of DHA on the levels of circulating DHA as non-esterified fatty acid in subjects of Asian Indian background.

There is evidence to indicate that the high rates of coronary heart disease and myocardial infarction amongst Indians of Asian descent may be partly related to circulating nonesterified fatty acids (NEFA). As docosahexaenoic acid (DHA,22:6n-3) in NEFA form has been found to exhibit anti-platelet aggregatory and anti-arrhythmic potential in vitro, the effect of supplementary DHA was examined in healthy subjects of Asian Indian background. Furthermore, time- and dose-dependent changes in absolute levels of DHA as NEFA or phospholipid (PL) were compared. The subjects consumed 8 capsules daily of placebo (DHA-free) or low DHA (0.75 g/day)or high DHA (1.50 g/day) over 6 wks. Fasting blood samples were drawn at days 0, 21, and 42 for analysis of serum lipid/lipoprotein composition. No significant effect of DHA supplementation on the levels of serum lipid/lipoproteins (including Lp[a]) or blood pressure was found. However, the DHA level in serum phospholipid rose by 167% overall with low-dose supplementation (from 2.4-6.4 mol%) but only by an additional 23% upon doubling the dose from 0.75 g to 1.50 g/day. Furthermore, after 6 weeks of supplementation with 0.75 g or 1.5 g DHA/day, absolute concentrations of DHA as PL were not significantly different from the corresponding 3-week values. Interestingly, the absolute concentrations of serum DHA as NEFA showed a marked rise with low-dose supplementation (by 212% overall, from 2.4 to 7.5 microM) and a further 70% rise (to 12.7 microM) upon doubling the supplementation from 0.75 to 1.50 g/day. As well, the 6-week concentrations (DHA-NEFA) were significantly different than the corresponding 3-week values at both dose levels. Elevation of circulating DHA-NEFA levels via DHA supplementation, as shown herein, to concentrations that exhibit anti-thrombotic and anti-arrhythmic potential in vitro needs to be extended to trials where clinical end-points are determined.

Dietary docosahexaenoic acid as a source of eicosapentaenoic acid in vegetarians and omnivores.

The utilization of dietary docosahexaenoic acid (DHA; 22:6n-3) as a source of eicosapentaenoic acid (EPA; 20:5n-3) via retroconversion was investigated in both vegetarians and omnivores. For this purpose, an EPA-free preparation of DHA was given as a daily supplement (1.62 g DHA) over a period of 6 wk. The dietary supplement provided for a marked increase in DHA levels in both serum phospholipid (from 2.1 to 7.1 mol% in vegetarians and 2.2 to 7.6 mol% in omnivores) and platelet phospholipid (from 1.1 to 3.4 mol% in vegetarians and 1.4 to 3.9 mol% in omnivores). EPA levels rose to a significant but much lesser extent, while 20:4n-6, 22:5n-6, and 22:5n-3 all decreased. Based on the serum phospholipid data, the retroconversion of DHA to EPA in vivo was estimated to be 9.4% overall with no significant difference between omnivores and vegetarians.

Supplementation with an algae source of docosahexaenoic acid increases (n-3) fatty acid status and alters selected risk factors for heart disease in vegetarian subjects.

The purpose of this double-blind study was to investigate the influence of dietary supplementation with an algae source of docosahexaenoic acid [DHA; 22:6(n-3)], devoid of any eicosapentaenoic acid [EPA; 20:5(n-3)], on serum/platelet DHA status, the estimated retroconversion of DHA to EPA, and risk factors for heart disease in vegetarian subjects. Healthy vegetarians (12 male, 12 female) consumed nine capsules daily of either DHA (1.62 g/d) or corn oil for 6 wk. Consumption of DHA capsules increased DHA levels in serum phospholipid by 246% (from 2.4 to 8.3 g/100 g fatty acids) and in platelet phospholipid by 225% (from 1.2 to 3.9 g/100 g fatty acids). EPA levels increased in serum phospholipid by 117% (from 0.57 to 1.3 g/100 g fatty acids) and in platelet phospholipid by 176% (0.21 to 0.58 g/100 g fatty acids) via metabolic retroconversion; the estimated extent of DHA retroconversion to EPA was 11.3 and 12.0%, based on the serum and platelet analyses, respectively. Arachidonic acid [AA; 20:4(n-6)] levels in serum and platelet phospholipids decreased moderately during the trial period (DHA group) as did both docosapentaenoic acids [22:5(n-6) and 22:5(n-3)]. Although no significant changes were found in the total and LDL-cholesterol levels with DHA supplementation, the total cholesterol:HDL-cholesterol ratio showed a moderate decrease over time as did the LDL-cholesterol:HDL-cholesterol ratio and serum triglyceride concentrations. DHA supplementation did not alter the various thrombogenic factors measured. In conclusion, DHA supplementation markedly enhanced the DHA status (of serum and platelets), provided for the formation of substantial EPA, and lowered the total and LDL-cholesterol:HDL-cholesterol ratios.

Effect of interleukin 1, lipopolysaccharide and phorbol esters on phospholipase D activity in chondrocytes.

Phorbol 12-myristate 13-acetate (PMA), interleukin-1 (IL-1) and lipopolysaccharide (LPS) induce similar responses in a variety of cell types, including chondrocytes. These responses include the release of arachidonic acid (AA) and the production of prostaglandin E2 (PGE2). Although PMA is known to stimulate phospholipase D (PLD) activity in most cells, it is not known whether LPS and IL-1 also stimulate PLD activity, or whether PLD activity contributes to AA liberation and PGE2 production in chondrocytes. In the present study we compared the effect of IL-1, LPS and protein kinase C (PKC) activators (PMA), mezerein, phorbol dibutyrate on PGE2 synthesis and PLD activity in articular chondrocytes. Although IL-1, LPS and PKC activators stimulate PGE2 synthesis, only the PKC activators stimulated PLD activity. The PKC inhibitor, staurosporine, as well as PKC downregulation, were both found to inhibit PMA-induced PLD activity without inhibiting other PMA-induced effects in chondrocytes. Our data suggest that although chondrocytes contain a PKC-regulated PLD activity, this is not a possible mechanism by which IL-1 or LPS stimulate early events in these cells.

Interleukin 1 and phorbol 12-myristate 13-acetate induce collagenase and PGE2 production through a PKC-independent mechanism in chondrocytes.

In the present study we demonstrate that interleukin 1 (IL 1) and phorbol 12-myristate 13-acetate (PMA) stimulate collagenase production by bovine chondrocytes in monolayer culture. Since it has been well established that PMA stimulates protein kinase C (PKC), we examined whether IL 1 and PMA also stimulate PKC in chondrocytes. In agreement with other studies, PMA induced the translocation of PKC, reflecting PKC activation by PMA. In contrast, IL 1 did not induce the translocation of PKC. Both IL 1 and PMA stimulated the release of [14C]arachidonic acid from chondrocyte phospholipids, suggesting that both agents stimulate phospholipase A2 (PLA2). Concomitantly, IL 1 and PMA also induced a pronounced increase in the production of PGE2. Pre-incubation of chondrocytes with staurosporine, a PKC inhibitor, did not affect the stimulation of collagenase production by IL 1 and only minimally that induced by PMA. Similarly, high concentrations of staurosporine did not inhibit prostaglandin E2 (PGE2) production induced by IL 1 or PMA. These data show that IL 1 and PMA stimulate the PLA2 pathway and collagenase production, however, these processes can occur in the absence of PKC activation.