Bruce J. Holub

Anthocyanin metabolites in human urine and serum

In the present study we investigated the metabolic conversion of cyanidin glycosides in human subjects using solid-phase extraction,HPLC-diode array detector, MS, GC, and enzymic techniques. Volunteers consumed approximately 20 g chokeberry extract containing 1.3 g cyanidin 3-glycosides (899 mg cyanidin 3-galactoside, 321 mg cyanidin 3-arabinoside, 51 mg cyanidin 3-xyloside and 50 mg cyanidin 3-glucoside). Blood samples were drawn at 0, 0.5, 1, and 2 h post-consumption of the extract. Urine samples were also collected at 0, 4-5,and 22-24h. We have confirmed that human subjects have the capacity to metabolise cyanidin 3-glycosides, as we observed at least ten individual anthocyanin metabolites in the urine and serum. Average concentrations of anthocyanins and anthocyanin metabolites in the urine reached levels of 17.9 (range 14.9-20.9) l.mol/l within 5 h post-consumption and persisted in 24h urine samples at levels of 12.1 (range 11.1-13.0) nmol/l. In addition, average total levels of anthocyanins and anthocyanin metabolites detected in the serum were observed at 5917 (range 197.3-986.1) nmol/ within 2h post-consumption. Cyanidin 3-galactoside accounted for 55.4% (9.9(range 7-2-12-6) l.mol/) and 66.0% (390.6 (range 119.4-661-9) nmol V) of the detected anthocyanins in the urine and serum samples,respectively. The metabolites were identified as glucuronide conjugates, as well as methylated and oxidised derivatives of cyanidin 3-galactoside and cyanidin glucuronide. Conjugation probably affects the biological activity of anthocyanins and these metabolic products are likely in part responsible for the reported health benefits associated with the consumption of anthocyanins.

THE EFFECT OF WILD BLUEBERRY (VACCINIUM ANGUSTIFOLIUM) CONSUMPTION ON POSTPRANDIAL SERUM ANTIOXIDANT STATUS IN HUMAN SUBJECTS

The aim of the present study was to determine whether the consumption of wild blueberries (Vaccinium angustifolium), a concentrated source of non-nutritive antioxidant phytochemicals, would enhance postprandial serum antioxidant status in healthy human subjects. A single-blinded crossover study was performed in a group of eight middle-aged male subjects (38-54 years). Subjects consumed a high-fat meal and a control supplement followed 1 week later by the same high-fat meal supplemented with 100.0 g freeze-dried wild blueberry powder. Upon brachial vein catheterization, fasting and postprandial serum samples were taken sequentially and analysed for lipids and glucose and for serum antioxidant status. Serum antioxidant status was determined using the oxygen radical absorbance capacity (ORAC) assay and the total antioxidant status (TAS) assay. The wild-blueberry treatment was associated with a significant treatment effect as determined by the ORAC assay (water-soluble fraction ORAC(perchloric acid (PCA)), P=0.04). Significant increases in serum antioxidant status above the controls were observed at 1 h (ORAC(PCA) (8.5 % greater), P=0.02; TAS (4.5 % greater), P=0.05), and 4 h (ORAC(total) (15.0 % greater), P=0.009; ORAC(acetone) (16.0 % greater), P=0.007) post-consumption of the high-fat meal. In conclusion, the consumption of wild blueberries, a food source with high in vitro antioxidant properties, is associated with a diet-induced increase in ex vivo serum antioxidant status. It has been suggested that increasing the antioxidant status of serum may result in the reduced risk of many chronic degenerative diseases.

Alteration and recovery of bleeding times, platelet aggregation and fatty acid composition of individual phospholipids in platelets of human subjects receiving a supplement of cod-liver oil

The effect of supplementation with cod-liver oil containing eicosapentaenoic acid (EPA), 20∶5ω3, on bleeding times, thrombin-induced platelet aggregation, platelet protein, platelet cholesterol, and the level and fatty acid composition of individual phospholipids in the platelets of human subjects was determined. Measurement of these parameters was conducted before the subjects received the supplement (day 0), after they received the supplement for 14 days (day 14), and 14 days after the supplement was terminated (day 28) so as to monitor recovery. The mean bleeding times exhibited a marked increase (by 81%) with supplementation and returned to near basal (day 0) values within 14 days after the supplement was terminated. Cod-liver oil supplementation significantly reduced thrombin-induced platelet aggregation with a partial recovery being exhibited by day 28. The content of phospholipid, cholesterol and protein (μg/109 platelets) was not significantly different (P>0.05) when isolated from the subjects at day 0, 14 and 28, as neither were the composition of individual phospholipids [phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI) and sphingomyelin (SPH)] given as % of total phospholipid. However, the fatty acid compositions of all platelet phospholipids were altered significantly by the fish oil supplement. In PC, EPA rose from 0.3 to 2.9% of total fatty acids and docosahexaenoate from 0.7 to 1.8% concomitant with a drop in arachidonate (from 14.1 to 9.6%) and linoleate (from 10.2 to 7.9%); these levels approached basal levels 14 days after supplementation was terminated. The highest percentage of EPA with supplementation was found in PE (4.3%), while the arachidonate fell from 38.8 to 30.5%, with low percentages of EPA occurring in PS (0.7%) and PI (0.5%). The level of 24∶1 in SPH increased significantly (from 17.8 to 24.8) with supplementation and reverted to basal values by day 28. These results suggest a close relationship of the observed fatty acid changes in individual platelet phospholipids to the altered hematological parameters and platelet-vessel wall interactions produced by cod-liver oil supplementation.

Effect of dietary α-linolenic acid and its ratio to linoleic acid on platelet and plasma fatty acids and thrombogenesis

The effect of dietary α-linolenic acid (18∶3n−3) and its ratio to linoleic acid (18∶2n−6) on platelet and plasma phospholipid (PL) fatty acid patterns and prostanoid production were studied in normolipidemic men. The study consisted of two 42-d phases. Each was divided into a 6-d pre-experimental period, during which a mixed fat diet was fed, and two 18-d experimental periods, during which a mixture of sunflower and olive oil [low 18∶3n−3 content, high 18∶2/18∶3 ratio (LO-HI diet)], soybean oil (intermediate 18∶3n−3 content, intermediate 18∶2/18∶3 ratio), canola oil (intermediate 18∶3n−3 content, low 18∶2/18∶3 ratio) and a mixture of sunflower, olive and flax oil [high 18∶3n−3 content, low 18∶2/18∶3 ratio (HI-LO diet)] provided 77% of the fat (26% of the energy) in the diet. The 18∶3n−3 content and the 18∶2/18∶3 ratio of the experimental diets were: 0.8%, 27.4; 6.5%, 6.9; 6.6%, 3.0; and 13.4%, 2.7, respectively. There were appreciable differences in the fatty acid composition of platelet and plasma PLs. Nevertheless, 18∶1n−9, 18∶2n−6 and 18∶3n−3 levels in PL reflected the fatty acid composition of the diets, although very little 18∶3n−3 was incorporated into PL. Both the level of 18∶3n−3 in the diet and the 18∶2/18∶3 ratio were important in influencing the levels of longer chain n−3 fatty acid, especially 20∶5n−3, in platelet and plasma PL. Production of 6-keto-PGF1α was significantly (P<0.05) higher following the HI-LO diet than the LO-HI diet although dietary fat source had no effect on bleeding time or thromboxane B2 production. The present study showed that both the level of 18∶3n−3 in the diet and its ratio to 18∶2n−6 were important in influencing long-chain n−3 fatty acid levels in platelet and plasma PL and that prostanoid production coincided with the diet-induced differences in PL fatty acid patterns.

Omega-3 fatty acids and risk of cognitive impairment and dementia.

It has been suggested that the dietary intake of omega-3 polyunsaturated fatty acids could be inversely related to the risk of dementia and cognitive decline. This analysis examined the association between plasma concentration of omega-3 polyunsaturated fatty acids and prevalence and incidence of cognitive impairment and dementia. Data are reported on subjects 65 years or older who had a complete clinical evaluation at the first two waves (1991-1992 and 1996-1997) of the Canadian Study of Health and Aging. Main outcome measures were cognitive impairment and dementia by mean relative plasma concentrations of fatty acids in the phospholipid fraction at baseline. Results were adjusted for age, sex, education, smoking, alcohol intake, body mass index, history of cardiovascular disease, and apolipoprotein E e4 genotype. In the cross-sectional analysis, no significant difference in omega-3 polyunsaturated fatty acid concentrations was observed between controls and both prevalent cases of cognitive impairment and dementia. In the prospective analysis, a higher eicosapentaenoic acid (p < 0.01) concentration was found in cognitively impaired cases compared to controls while higher docosahexaenoic acid (p < 0.07), omega-3 (p < 0.04) and total polyunsaturated fatty acid (p < 0.03) concentrations were found in dementia cases. These findings do not support the hypothesis that omega-3 polyunsaturated fatty acids play a protective role in cognitive function and dementia.

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.

Elevated levels and altered fatty acid composition of plasma lysophosphatidylcholine(lysoPC) in ovarian cancer patients.

Lysophosphatidylcholine (lysoPC), a product of phosphatidylcholine (PC) hydrolysis via phospholipase A activity, has been proposed to activate cells from a number of lineages. Here, we demonstrate that lysoPC levels are significantly elevated (by 43% overall, relative to normal controls) in the plasma of ovarian cancer patients. This does not appear to be common to all cancers as 5 out of 6 leukemia patients tested had markedly lower (less than one‐half of normal) plasma lysoPC. In the plasma of ovarian cancer patients, the percentages of palmitoyl‐ and stearoyl‐lysoPC species were significantly higher, whereas oleoyl and particularly linoleoyl‐lysoPC were significantly lower than in control subjects. The molar ratios of lysoPC/PC and palmitoyl‐lysoPC/linoleoyl‐lysoPC were also significantly elevated in the plasma of ovarian cancer patients compared with those of control subjects. Furthermore, the calculated value of plasma (lysoPC/PC) × (palmitoyl‐lysoPC/linoleoyl‐lysoPC) was markedly higher in patients compared with controls. Int. J. Cancer, 71:31–34, 1997.

The molecular species composition of individual diacyl phospholipids in human platelets

The molecular species composition of the individual diacyl phospholipids was determined in human platelets. The 1-acyl (16:0, 18:0, etc.) homologues of the various 2-acyl (16:0, 18:1, 18:2, 20:4, etc) species of the phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol were assessed by the use of thin-layer and gas-liquid chromatography in combination with specific lipases for establishing the positional distribution of the fatty acyl chains and generating the diacylglycerol derivatives. A marked heterogeneity was found in the complement of individual molecular species associated with the different platelet phospholipids. The 1-16:0 2-18:1 species predominated in the phosphatidylcholine (21% of total) but contributed only 5%, 2%, and trace amounts to the ethanolamine, inositol, and serine phospholipids, respectively. The 1-stearoyl 2-arachidonoyl species was by far the most prevalent one in the diacyl phosphatidylethanolamine (47% of total) and phosphatidylinositol (71% of total) but represented only 10% of the phosphatidylcholine. The 1-18:0 2-18:1 (37%) plus 1-18:0 2-20:4 (41%) together contributed 78% to the total phosphatidylserine. Interestingly, the 1-stearoyl 2-arachidonyl phosphatidylinositol represented only 6 mol% of the total diacyl phospholipid in human platelets. Of the total mixed 1-acyl 2-20:4 species in human platelets, 31, 35, 17 and 17% was found in the phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol, respectively. These results indicate that the mechanisms involved in the release of arachidonate for prostaglandin and thromboxane synthesis would need to possess a profound degree of selectivity if any single molecular species of a given platelet phospholipid were the source of the released arachidonic acid.

Clinical Trial to Evaluate Omega-3 Fatty Acids and Alternate Day Prednisone in Patients with IgA Nephropathy: Report from the Southwest Pediatric Nephrology Study Group

This randomized, placebo-controlled, double-blind trial evaluated the role of prednisone and omega 3 fatty acids (O3FA) in patients with IgA nephropathy. Entry criteria were (1) biopsy-proven IgA nephropathy, (2) estimated GFR > or = 50 ml/min per 1.73 m2, and (3) moderate to severe proteinuria. Thirty-three patients were randomly assigned to receive prednisone 60 mg/m2 every other day for 3 mo, then 40 mg/m2 every other day for 9 mo, then 30 mg/m2 every other day for 12 mo (prednisone group); 32 were randomly assigned to receive O3FA 4 g/d for 2 yr (1.88 g eicosapentaenoic acid, 1.48 g docosahexaenoic acid; O3FA group); and 31 were randomly assigned to receive placebo (placebo group). Most (73%) patients completed 2 yr of treatment. Randomly assigned patients who were hypertensive were given enalapril 2.5 to 40 mg/d. The primary end point was time to failure, defined as estimated GFR <60% of baseline. An overall significance level of 0.10 was used. The three groups were comparable at baseline except that the O3FA group had higher urine protein to creatinine (UP/C) ratios than the placebo group (P = 0.003). Neither treatment group showed benefit over the placebo group with respect to time to failure, with 14 patient failures overall (two in the prednisone group, eight in the O3FA group, and four in the placebo group). The primary factor associated with time to failure was higher baseline UP/C ratios (P = 0.009). Superiority of prednisone or O3FA over placebo in slowing progression of renal disease was not demonstrated in this study. However, the relatively short follow-up period, inequality of baseline UP/C ratios, and small numbers of patients precludes definitive conclusions.

Diacylglycerol lipase pathway is a minor source of released arachidonic acid in thrombin-stimulated human platelets

It has been postulated that the diacylglycerol lipase pathway is a predominant source of the free arachidonic acid which is released from phospholipids upon the exposure of human platelets to thrombin. The amount of released arachidonic acid and other fatty acids in thrombin-stimulated platelets was determined in the presence of BW755C, the cyclooxygenase/lipoxygenase inhibitor, and in relation to phosphatidylinositol degradation and phosphatidic acid formation. A stearic acid:arachidonic acid molar ratio approaching unity would be expected in the free fatty acid fraction if the latter pathway were a major source of released arachidonic acid. Our results indicate that the diacylglycerol lipase pathway contributes a maximum of 3-4 nmol of arachidonic acid/2 X 10(9) platelets or 12-15% of the total arachidonic acid released (25.8 nmol/2 X 10(9) platelets) upon exposure to thrombin (2 units/ml) for 4 min. Trifluoperazine inhibited most of the thrombin-dependent free arachidonic acid release but only 15% of the absolute loss of arachidonic acid from phosphatidylinositol. Therefore, we conclude that the diacylglycerol lipase pathway represents only a minor source of the free arachidonic acid that is released upon thrombin stimulation of human platelets.