Oleic acid content is responsible for the reduction in blood pressure induced by olive oil.

Numerous studies have shown that high olive oil intake reduces blood pressure (BP). These positive effects of olive oil have frequently been ascribed to its minor components, such as alpha-tocopherol, polyphenols, and other phenolic compounds that are not present in other oils. However, in this study we demonstrate that the hypotensive effect of olive oil is caused by its high oleic acid (OA) content (approximately 70-80%). We propose that olive oil intake increases OA levels in membranes, which regulates membrane lipid structure (H(II) phase propensity) in such a way as to control G protein-mediated signaling, causing a reduction in BP. This effect is in part caused by its regulatory action on G protein-associated cascades that regulate adenylyl cyclase and phospholipase C. In turn, the OA analogues, elaidic and stearic acids, had no hypotensive activity, indicating that the molecular mechanisms that link membrane lipid structure and BP regulation are very specific. Similarly, soybean oil (with low OA content) did not reduce BP. This study demonstrates that olive oil induces its hypotensive effects through the action of OA.

Docosahexaenoic acid-containing phospholipid molecular species in brains of vertebrates.

The fatty acid composition of phospholipids and the contents of docosahexaenoic acid (DHA)-containing diacyl phosphatidylcholine and diacyl phosphatidylethanolamine molecular species were determined from brains of five fresh-water fish species from a boreal region adapted to 5 degrees C, five fresh-water fish species from a temperate region acclimated to 5 degrees C, five fresh-water fish species from a temperate region acclimated to 20 degrees C, and three fresh water fish species from a subtropic region adapted to 25-26 degrees C, as well as six mammalian species and seven bird species. There was little difference in DHA levels of fish brains from the different thermal environments; mammalian and bird brain phospholipids contained a few percentage points less DHA than those of the fish investigated. Molecular species of 22:6/22:6, 22:6/20:5, 22:6/20:4, 16:0/22:6, 18:0/22:6, and 18:1/22:6 were identified from all brain probes, and 16:0/22:6, 18:0/22:6, and 18:1/22:6 were the dominating species. Cold-water fish brains were rich in 18:1/22:6 diacyl phosphatidylethanolamine (and, to a lesser degree, in diacyl phosphatidylcholine), and its level decreased with increasing environmental/body temperature. The ratio of 18:0/22:6 to 16:0/22:6 phosphatidylcholine and phosphatidylethanolamine was inversely related to body temperature. Phospholipid vesicles from brains of cold-acclimated fish were more fluid, as assessed by using a 1, 6-diphenyl-1,3,5-hexatriene fluorescent probe, than those from bird brains, but the fluidities were almost equal at the respective body temperatures. It is concluded that the relative amounts of these molecular species and their ratios to each other are the major factors contributing to the maintenance of proper fluidity relationships throughout the evolutionary chain as well as helping to maintain important brain functions such as signal transduction and membrane permeability.

Involvement of phospholipid molecular species in controlling structural order of vertebrate brain synaptic membranes during thermal evolution.

Fluorescence anisotropy parameter of [p-(6-phenyl)-1,3,5-hexatrienyl]phenyl-propionic acid (DPH-PA) and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) embedded in synaptic plasma membranes prepared from brains of cold (5 degrees C) and warm (22 degrees C) adapted fish (Cyprinus carpio L.), rat (Rattus norvegicus) and bird (Branta canadensis), was studied. Fatty acid composition of total lipids as well as molecular species composition of diacyl phosphatidylcholines and phosphatidylethanolamines was also determined. The amount of long-chain polyunsaturated fatty acids decreased with increasing body temperature. There was a near-complete compensation of membrane structural order for environmental/body temperature over the evolutionary scale as seen by DPH-PA. Using TMA-DPH, the compensation was partial with rat and bird. Since DPH-PA and TMA-DPH differ in their charges, it is proposed, that the former reported membrane regions rich in cationic or zwitterionic (neutral) phospholipids and the latter, membrane regions rich in negatively charged phospholipids in the synaptic plasma membranes. Many different molecular species (20-25) of diacyl phosphatidylcholines and diacyl phosphatidylethanolamines were identified. The level of 16:0/22:6 phosphatidylcholine decreased while disaturated phosphatidylcholines increased with increase of environmental/body temperature from the fish through the bird. Level of 1-monoenoic, 2-polyenoic phosphatidylethanolamines also decreased with an increase in environmental/body temperature. Experiments using vesicles made of mixed synthetic phosphatidylcholine vesicles (16:0/16:0, 16:0/18:1, 16:0/22:6 in various proportions) showed that increase in disaturated phosphatidylcholine species does not explain the observed complete adjustment of membrane structural order in synaptic plasma membranes. Change in level of 1-monoenoic, 2-polyenoic phosphatidylethanolamines might be one of the factors involved in controlling the biophysical properties of the membrane according to the temperature.

L-ascorbyl-2-sulfate alleviates Atlantic salmon scurvy.

Duplicate lots of 150 Atlantic salmon (Salmo salar), average weight 0.5 g, were fed NRC diet H-440 base containing L-ascorbic acid (C1) or L-ascorbyl-2-sulfate (C2S); or L-ascorbyl-2-monophosphate (C2MP): at 0 or 100 mg C1; 50, 100, 300 mg C2S; or 50, 100 mg C2MP per kg dry diet in 12 degrees C freshwater tanks. After 12 weeks, negative controls (no vitamin C) exhibited reduced growth, scoliosis, lordosis, and petechial hemorrhages typical of fish scurvy. All other lots grew normally. Four 100-fish lots of scorbutic salmon, average weight 3.3 g, were placed on recovery diets of 0, 50, or 300 mg C2S, or 100 mg C2MP per kg dry diet. After 5 weeks, fish fed either level of C2S intake had recovered and resumed growth. Negative controls continued to develop acute scurvy. The 41 survivors in this no-vitamin-C group all had advanced scurvy, whereas all fish in both C2S-fed recovery groups appeared normal. Tissue assays for C vitamers disclosed normal levels of C1 and C2S in the recovery groups. All other test treatment lots containing C1, C2S, or C2MP had fish with normal appearance and no significant differences in growth response for the 17-week test period. C2S at 50 mg or more per kg diet as the sole vitamin C source promoted normal growth in young Atlantic salmon for more than 20-fold increase in weight.

Role of phospholipid molecular species in maintaining lipid membrane structure in response to temperature.

The compositions and physical states of the liver phospholipids of fish and phospholipids of shrimps adapted to relatively constant but radically different temperatures were investigated. There were no measurable differences in their gross fatty acid compositions of phospholipids from the species obtained from identical temperature. Saturated-to-unsaturated fatty acid ratio did not show any convincing difference. Docosahexaenoic acid (22:6) did not seem to participate in the process of adaptation. Cold adaptation was coincidental with oleic acid (18:1) accumulation, preferentially in the phosphatidylethanolamine. In the experiment with rats, it has been found that the fish oil fed rats showed a similarity in their liver phospholipid fatty acid composition like fish liver phospholipids. Determination of molecular species composition of phosphatidylcholine and phosphatidylethanolamine revealed a 4- to 5-fold and 10-fold increase in the level of 18:1/22:6 and 18:1/20:5 species respectively in favor of cold adaptation. Phospholipids from cold-adapted species showed a more fluid structure than that of warm-adapted species near the C-2 segment of the bilayer, but not in the deeper regions. Phospholipids from the rat livers did not show any change irrespective of diet. Phosphatidylcholines from rat liver, fish liver or shrimps did not show any difference among them in their fluidity irrespective of their environmental conditions or diet. An appropriate combination (75:25) of phosphatidylcholine from rat liver with phosphatidylethanolamines from cold adapted species showed a drastic fluidization at the C-2 segment, in comparison with their phosphatidylcholines.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular and structural composition of phospholipid membranes in livers of marine and freshwater fish in relation to temperature.

The compositions and physical states of the liver phospholipids of marine and freshwater fish adapted to relatively constant but radically different temperatures were investigated. Fish adapted to low temperature (5-10 degrees C) accumulated more unsaturated fatty acids than those in a warm (25-27 degrees C) environment. There were no measurable differences in the gross fatty acid compositions of the total liver phospholipids from identical thermal environments. Docosahexaenoic acid (22:6) did not seem to participate in the process of adaptation. Cold adaptation was coincidental with oleic acid (18:1) accumulation, preferentially in the phosphatidylethanolamine. Determination of the molecular species composition of phosphatidylethanolamine revealed a 2- to 3-fold and 10-fold increase in the level of 18:1/22:6 and 18:1/20:5 species, respectively. ESR spectroscopy revealed a 7-10% compensation in the ordering state of native phospholipids with temperature. Combination of 16:0/22:6 phosphatidylcholine with phosphatidylethanolamines of cold-adapted marine fish showed a drastic fluidization near the C-2 segment of the bilayer, but not in the deeper regions. An appropriate combination (75:25) of phosphatidylcholines from warmth-adapted marine fish with phosphatidylethanolamines from cold-adapted marine fish mimicked a 100% adaptational efficacy in the C-2 segment as compared with the phosphatidylethanolamines of warmth-adapted marine fish. A specific role of 18:1/22:6 phosphatidylethanolamine in controlling membrane structure and physical state with thermal adaptation is proposed.

Separation of three commercial forms of vitamin C (ascorbic acid, ascorbic-2-sulfate and ascorbate-2-polyphosphate) by HPLC.

A modification of an existing separation technique by this laboratory is described for the separation and quantification of the three commercially available forms of ascorbic acid. The technique has the potential for identifying the various metabolic and degradation products resulting from vitamins C2 and C3 metabolism. A microwave technique is used for tissue heat denaturation and extraction.

Brain ascorbate depletion as a response to stress.

Ascorbate-depleted rainbow trout were fed a single dose of L-1-14C-ascorbic acid, then held in metabolism chambers for four consecutive five-day periods. Tissue samples were analyzed for 14C, ascorbate, and ascorbate-2-sulfate. Brain ascorbate showed a long turnover time following a very slow uptake. The loss of brain ascorbate after five days in metabolism chambers was highly significant (p less than 0.001) when compared with similar dosed fish not chambered. A brain ascorbate pool which does not exchange with the body pool is proposed. Possible mechanisms are discussed and a kinetic model is suggested.

Utilization of ascorbate-2-sulfate in fish.

Although most vertebrate animals synthesize L-ascorbic acid (C1), some animal species lack the ability to produce L-gulonolactone oxidase and are thus dependent upon a dietary source of vitamin C. Fish are unique among this latter group in that they store a chemically stable form of vitamin C and appear to metabolize this compound differently from other vitamin C-requiring organisms. Ascorbate-2-sulfate (C2) contributes to total body stores of ascorbate, but the commonly used assays for ascorbate concentrations in tissues and body fluids do not generally measure C2. An HPLC assay distinguishes between and measures both C1 and C2. Modification of the less exact but commonly used DNPH method can provide adequate data to estimate total vitamers C, C1, and (by difference) C2. Since vitamin C is a required component of feed for salmonids, catfish, eels, shrimp and carp, use of C2 in feed formulation would provide a bioavailable form of ascorbate which is heat and water stable at pH 4-13.

Distribution of ascorbate-2-sulfate and distribution, half-life and turnover rates of [1-14C]ascorbic acid in rainbow trout.

Rainbow trout (250 g) were maintained at 15 degrees C for 3 months on a low ascorbic acid diet, given [1-14C]ascorbic acid by gavage, then fed the NAS/NRC requirement 12 times per week. Total urine, fecal water and branchial water were collected daily from five fish placed in metabolism chambers for four successive 5-day periods. Tissue samples were analyzed for 14C, ascorbic acid (C1) and ascorbate-2-sulfate sulfate (C2). Excretion analysis indicated t1/2 = 42 days. After 20 days, the feeding schedule was changed to 3 times per week. Fish fed 14C were sampled after 1, 2, 3 and 4 months. The half-life in each organ except brain was inversely proportional to the dietary level of ascorbate. Concentrations of C1 and C2 in the various tissues reflected dietary intake of vitamin C. Total C (CT = C1 + C2) levels were maintained in the liver even with the low vitamin C diet. Estimates of body pool for C1 are 27-29 mg/kg. At the higher ascorbate intake CT was 92-114 mg/kg, but decreased by 34% at the lower feeding rate to 51-62 mg/kg. Data indicate that there are two or more body pools that include a store of C2, which is readily interconverted in metabolizing tissues to and from C1. Since air and water stable C2 is antiscorbutic for fish, it is the preferred form of ascorbate for fish feeds.

Ascorbic acid sulfate sulfohydrolase (C2 sulfatase): the modulator of cellular levels of L-ascorbic acid in rainbow trout.

The enzyme L-ascorbic acid 2-sulfate sulfohydrolase (C2 sulfatase) was purified from rainbow trout liver. The enzyme catalyzes the hydrolysis of L-ascorbic acid 2-sulfate and has a pH optimum at 6.0. It has a molecular weight of about 117,500 at pH 5.0 and is inhibited by a number of sulfhydryl blocking agents including L-ascorbic acid. C2 sulfatase activity was observed in most metabolic organs of rainbow trout. These findings suggest that the physiologic role of the enzyme is to maintain adequate cellular concentrations of L-ascorbic acid in the fish. The activity of the enzyme is controlled by L-ascorbic acid through feedback inhibition. Comparison of kinetic constants and inhibition patterns suggests that C2 sulfatase is structurally identical to human arylsulfatase A. However, unlike C2 sulfatase, human arylsulfatase A may not be involved in ascorbate metabolism. Its physiologic substrate is reported to be cerebroside-3-sulfate, not L-ascorbic acid 2-sulfate. A scheme is proposed to account for the functional divergence of these two structurally identical enzymes.

Metabolism of ascorbic acid and ascorbic-2-sulfate in man and the subhuman primate.

Man does not catabolize ascorbate to CO2, whereas the monkey does catabolize ascorbate and ascorbate sulfate to CO2 when these compounds are given orally. However, it takes the same length of time to produce frank scurvy in both man and the monkey, thus indicating that the comparative storage, rate of use, and mode of metabolism of ascorbate is similar in both species. Preliminary feeding and isotope studies conducted on monkeys are in agreement with the fact that only a small amount of labeled ascorbate or ascorbate sulfate equilibrated with body stores. These results are in agreement with published ascorbic acid requirements of 10 mg/kg body weight. In our experiments, 250 mg/day had to be fed to a 10-kg monkey to completely clear all signs of scurvy and return blood ascorbate levels to normal. Ascorbic acid administered intravenously to monkeys appears to equilibrate completely with the ascorbate pool(s). Ascorbate sulfate was found to be a urinary metabolite of both ascorbic-1-14C acid and ascorbic-6-14C acid fed orally to monkeys.