Sharon L. Burnard

Incorporation and effects of dietary eicosapentaenoate (20:5(n-3)) on plasma and erythrocyte lipids of the marmoset following dietary supplementation with differing levels of linoleic acid.

The effect of dietary eicosapentaenoic acid (EPA, 20:5(n-3), as the ethyl ester) on plasma lipid levels and the incorporation of EPA into erythrocyte and plasma lipids were investigated in the marmoset monkey. Marmosets were fed high mixed-fat diets (14.5% total fat) supplemented with or without 0.8% EPA for 30 weeks. Markedly elevated plasma cholesterol (16.4 mmol/l) was induced by an atherogenic-type diet but with EPA supplementation, plasma cholesterol increased to only 6.6 mmol/l. Plasma triacylglycerol levels were not elevated with an atherogenic type diet. Substantial EPA incorporation was evident for plasma phospholipid, triacylglycerol and cholesterol ester fractions. The proportion of docosapentaenoic acid (22:5(n-3)) but not docosahexaenoic acid (22:6(n-3)) was also elevated in these plasma lipid fractions. Greatest incorporation of EPA occurred when it was administered with an atherogenic type diet having a P:M:S (polyunsaturated:monounsaturated:saturated) fatty acid ratio of about 0.2:0.6:1.0 in comparison to the control diet of 1.0:1.0:1.0. Incorporation of EPA and 22:5(n-3)) into erythrocyte phospholipids was also apparent and this was at the expense of linoleic acid (18:2(n-6)). These results in the marmoset highlight both the cholesterol-lowering properties of EPA and the extent of its incorporation into plasma lipids and erythrocyte membrane phospholipids with far greater incorporation occurring when the level of dietary linoleic acid was reduced.

The effect of dietary supplementation with eicosapentaenoic acid on the phospholipid and fatty acid composition of erythrocytes of marmoset

Adult male marmoset monkeys were fed eicosapentaenoic acid (20∶5n−3) as the ethyl ester in diets containing either 32% (reference diet, no added cholesterol) or 7% (atherogenic diet with 0.2% added cholesterol) linoleic acid (18∶2n−6) for 30 wk. No changes were seen in the level of phosphatidylcholine (PC) or phosphatidylethanolamine (PE) but minor changes were observed in both the sphingomyelin (SPM) and phosphatidylinositol plus phosphatidylserine (PI+PS) fractions of erythrocyte lipids. The extent of total n−3 fatty acid incorporation into membrane lipids was higher in atherogenic diets (polyunsaturated/monounsaturated/saturated (P/M/S) ratio 0.2∶0.6∶1.0) than reference diets (P/M/S ratio 1∶1∶1) and this was true for both PE (33.4±1.0%vs 24.3±1.1%) and PC (9.3±0.5%vs 4.9±0.3%). Although suitable controls for cholesterol effects were not included in the study, earlier results obtained with marmosets lead us to believe such effects were probably small. Regardless of basic diet (atherogenic, reference), 20∶5n−3 was preferentially incorporated into PE (10.8±0.2%, 6.0±0.02%) while smaller amounts were incorporated into PC (6.9±0.4%, 3.2±0.2%). The major n−3 polyunsaturated fatty acid found in PE in response to dietary 20∶5n−3 was the elongation metabolite 22∶5n−3 in both the atherogenic (17.7±0.7%) and reference (14.3±1.0%) dietary groups; 22∶6n−3 levels were less affected by diet (4.7±0.3% and 3.9±0.2%, respectively). The results can be interpreted to indicate an inverse relationship between the amount of dietary 18∶2n−6 and incorporation of 20∶5n−3 into erythrocyte membrane phospholipids regardless of whether the major dietary n−3 fatty acid was α-linolenate (18∶3n−3) or 20∶5n−3. This interpretation is supported by theoretical calculations.

Molecular Size Fractions of Bay Leaf (Laurus nobilis) Exhibit Differentiated Regulation of Colorectal Cancer Cell Growth In Vitro

Numerous in vitro studies using solvent or aqueous extracts of raw dietary plant material have demonstrated modulation of colon cancer cell growth and apoptosis and effects on immune and nonimmune pathways of inflammation. We have developed a generic, 3-staged food-compatible process involving heating for conversion of dietary plants into food ingredients and report results on potential colon cancer-regulating properties of processed forms of Bay leaf (Laurus nobilis). In vitro studies demonstrated inhibition of cancer cell growth by processed Bay leaf products in HT-29, HCT-116, Caco-2, and SW-480 human cancer cell lines, which were accompanied by variable levels of elevated apoptosis. Bay leaf also exerted moderate inhibition of cycloxygenase 2 and 5 lipoxygenase enzymatic activity. In addition, these extracts significantly downregulated interferon-γ production in T helper Type 1-stimulated whole blood from healthy donors. Furthermore, size fractionation of the extracts revealed that antiproliferative and proapoptotic activities were associated with low mass (primarily polyphenolics and essential oils) and high mass (primarily proteins including polyphenol oxidase) chemical classes, respectively. Bay leaf exerted in vitro bioactivity that might be relevant to protecting against early events in sporadic colorectal cancer, with potential for further optimization of bioactivity by size-based fractionation.

Cilazapril and Dietary Gamma-Linolenic Acid Prevent the Deficit in Sciatic Nerve Conduction Velocity in the Streptozotocin Diabetic Rat

Young adult male Hooded Wistar rats were rendered diabetic by administration of streptozotocin and maintained for 5 weeks on a diet containing either 6% olive oil as the total source of fat (OO diet), or purified gamma-linolenic acid (GLA) at a concentration of 0.5% with the remaining 5.5% provided by olive oil (GLA diet). Rats were treated with the angiotensin converting inhibitor, cilazapril, administered in the drinking water at a dose of 20 mg kg-1 body weight day-1. For the OO diet groups, sciatic nerve conduction velocity (NCV) in diabetic rats was reduced by 32% (p < 0.01) in comparison with nondiabetic (vehicle-treated) rats and 27.5% (p < 0.05) in comparison with diabetic rats treated with cilazapril. Diabetic, cilazapril-treated rats showed no reduction in NCV. For the nondiabetic, diabetic, and diabetic plus cilazapril groups fed GLA, the NCV was not significantly different, indicating that dietary GLA also prevented the deficit in the NCV induced by the diabetic state. Analysis of the sciatic nerve endoneurial phospholipid fatty acids revealed a significant reduction in the proportion of GLA and an elevation in the proportion of linoleic acid in the diabetic groups compared with the nondiabetic groups and this was independent of the cilazapril treatment or the dietary lipid supplement. Sciatic nerve myo-inositol content was unaltered while mannose, fructose, glucose, and sorbitol levels were elevated in the diabetic groups and these changes were independent of the cilazapril treatment or the dietary lipid supplement. These results indicate that in the rat, cilazapril treatment or dietary GLA, at the doses tested, are effective in preventing the deficit in the NCV induced by diabetes.

Stimulation of human cheek cell Na+/H+ antiporter activity by saliva and salivary electrolytes: amplification by nigericin

Proton-dependent, ethylisopropylamiloride (EIPA)-sensitive Na+ uptake (Na+/H+ antiporter) studies were performed to examine if saliva, and ionophores which alter cellular electrolyte balance, could influence the activity of the cheek cell Na+/H+ antiporter. Using the standard conditions of 1 mmol/1 Na+, and a 65:1 (inside:outside) proton gradient in the assay, the uniport ionophores valinomycin (K+) and gramicidin (Na+) increased EIPA-sensitive Na+ uptake by 177% (p < 0.01) and 227% (p < 0.01), respectively. The dual antiporter ionophore nigericin (K+-H+) increased EIPA-sensitive Na+ uptake by 654% (p < 0.01), with maximal Na+ uptake achieved by 1 min and at an ionophore concentration of 50 μmol/l, with an EC50 value 6.4 μmol/l. Preincubation of cheek cells with saliva or the low molecular weight (MW) components of saliva (saliva activating factors, SAF) for 2 h at 37°C, also significantly stimulated EIPA-sensitive Na+ uptake. This stimulation could be mimicked by pre-incubation with 25 mmol/l KCl or K+-phosphate buffer. Pre-incubating cheek cells with SAF and the inclusion of 20 μmol/1 nigericin in the assay, produced maximum EIPA-sensitive Na+ uptake. After pre-incubation with water, 25 mmol/1 K+-phosphate or SAF, with nigericin in all assays, the initial rate of proton-gradient dependent, EIPA-sensitive Na+ uptake was saturable with respect to external Na+ with Km values of 0.9, 1.7, and 1.8 mmol/l, and Vmax values of 13.4, 25.8, and 31.1 nmol/mg protein/30 sec, respectively. With 20 μmol/1 nigericin in the assay, Na+ uptake was inhibited by either increasing the [K+]o in the assay, with an ID50 of 3 mmol/l. These results indicate that nigericin can facilitate K+i exchange for H+o and the attending re-acidification of the cheek cell amplifies IINa+ uptake via the Na+/H+ antiporter. The degree of stimulation of proton-dependent, EIPA-sensitive Na+ uptake is therefore dependent, in part, on the intracellular K+i.

Cardiac membrane lipid composition and adenylate cyclase activity following dietary eicosapentaenoic acid supplementation in the marmoset monkey

Abstract The effects of dietary eicosapentaenoic acid (20:5(n-3)) on cardiac membrane phospholipid fatty acid composition and membrane-associated adenylate cyclase activity were investigated in the marmoset monkey. A 20:5(n-3) concentrate (as the ethyl ester) was tested with high fat diets formulated to give a polyunsaturated:monounsaturated:saturated (P:M:S) fatty acid ratio of either 1:1:1 (control diet, no added cholesterol) or 0.1:0.6:1.0 (atherogenic-type diet, 0.2% cholesterol) that were fed for 30 weeks. The various dietary lipid treatments did not alter the proportions of the major phospholipids present in cardiac membranes. Both 20:5(n-3)-supplemented diets resulted in an increase in the proportions of 20:5(n-3) and 22:5(n-3), but not 22:6(n-3) in membrane phospholipids and a decrease in the proportion of 18:2(n-6) and 20:4(n-6). Greater incorporation of the n-3 polyunsaturated fatty acids occurred when the level of dietary linoleic acid was reduced. These changes in fatty acid profiles were evident to a greater extent in phosphatidylcholine and phosphatidylethanolamine in comparison with cardiolipin and sphingomyelin. These diets did not influence catecholamine-stimulated adenylate cyclase activity in terms of sensitivity or responsiveness to isoproterenol. The response of adenylate cyclase to other agonists such as epinephrine, norepinephrine, sodium fluoride, and forskolin was also unaffected by the dietary lipid treatments. The results clearly show that there is extensive incorporation of dietary 20:5(n-3) and its elongation product 22:5(n-3) into cardiac membrane phosphatidylcholine and phosphatidylethanolamine. However, unlike the reported effect of altered cardiac membrane cholesterol status, alterations in cardiac membrane phospholipid fatty acid composition alone appear not to affect membrane-associated adenylate cyclase activity in the marmoset monkey.

Depressed Cheek Cell Sodium Transport in Human Hypertension

Na+ transport activity was measured in cheek cells from untreated hypertensive subjects and age-matched normotensive controls identified from a blood pressure screening program. Cheek cells were isolated by a simple mouth wash procedure and Na+ transport activity was measured as the proton-dependent uptake of 22Na+ using a rapid filtration assay. The rate of Na+ uptake was about 45% lower in hypertensive subjects and this difference persisted in a follow up study 2 years later involving those subjects who remained untreated for their hypertension. The proton independent Na+ uptake was also reduced by about 46% in the hypertensive group. The increase in the rate of cheek cell Na+ transport with increasing transcellular proton gradient values was also significantly lower in hypertensive subjects. The reduced cheek cell Na+ transport observed in hypertensive subjects may indicate decreased activity of the Na+/H+ antiporter and/or changes in the ion permeability properties of the cheek cell plasma membrane in the hypertensive state. This novel assay provides a biochemically based method for discriminating between normotensive and hypertensive subjects and makes use of tissue which can be obtained in a relatively non-invasive manner.

The Relationship Between Salivary Growth Factors, Electrolytes and Abnormal Sodium Transport in Human Hypertension

We have previously shown cheek cell Na+/H+ antiporter activity to be reduced in human hypertensives. We have now examined the relationship between abnormal antiporter activity and a variety of salivary factors. Total protein concentration and amylase activity were higher in hypertensives, but salivary flow rate and epidermal growth factor, transforming growth factor-alpha, calcium, and magnesium concentrations were not significantly different between hypertensives and normotensives. The lowered cheek cell Na+/H+ antiporter activity in those hypertensives with diastolic BP greater than 95 mmHg was accompanied by lowered salivary Na+/H+ ratios. In borderline hypertensives (diastolic BP between 90 and 95 mmHg), the Na+/H+ ratio was reduced to a similar extent to that seen in those hypertensives with a diastolic BP above 95 mmHg, however the cheek cell antiporter activity was not reduced, suggesting that these two differences are not related in a simple fashion in all hypertensives. It is concluded that it is unlikely that differences in salivary growth factors explain the lowered cheek cell Na+/H+ antiporter activity observed in human hypertension. Our findings indicate that salivary electrolyte composition may be related to cheek cell Na+/H+ antiporter activity and these parameters may be altered in hypertension.

Genetic factors associated with altered sodium transport in human hypertension: a twin study.

1. Na+/H+ antiporter/exchange activity (NHE) in human cheek epithelial cells was assessed in 288 female twins and siblings. The genetic contribution of factors to NHE activity was assessed in 128 matched twin pairs (76 monozygotic (MZ); 52 dizygotic (DZ)).