Flaxseed oil supplementation manipulates correlations between serum individual mol % free fatty acid levels and insulin resistance in type 2 diabetics. Insulin resistance and percent remaining pancreatic ß-cell function are unaffected.

OBJECTIVES: Elevated total serum free fatty acids (FFAs) concentrations have been suggested, controversially, to enhance insulin resistance and decrease percent remaining ß-cell function. However, concentrations of individual serum FFAs have never been published in terms of their relationship (correlation) to homeostatic model assessment-insulin resistance (HOMA-IR) and percent remaining ß-cell function (HOMA-%ß) in the type 2 diabetics (T2Ds). Alpha-linolenic acid consumption has a negative correlation with the insulin resistance, which in turn is negatively correlated with the remaining ß-cell function. The primary objective was to test the hypothesis that there would be different relationship (correlation) between the blood serum individual free FFA mol % levels and HOMA-IR and/or HOMA-%ß in T2D. The secondary objective was to test the hypothesis that flaxseed oil, previously being shown to be ineffective in the glycemic control in T2Ds, may alter these correlations in a statistically significant manner as well as HOMA-IR and/or HOMA-%ß. METHODS: Patients were recruited via a newspaper advertisement and two physicians have been employed. All the patients came to visit one and three months later for a second visit. At the second visit, the subjects were randomly assigned (double blind) to flaxseed or safflower oil treatment for three months, until the third visit. RESULTS: Different statistically significant correlations or trends towards among some serum individual free FFA mol % levels and HOMA-IR and HOMA-%ß, pre- and post-flaxseed and safflower oil supplementation were found. However, flaxseed oil had no impact on HOMA-IR or HOMA-%ß despite statistically significant alterations in correlations compared to baseline HOMA-IR. CONCLUSIONS: The obtained data indicate that high doses of flaxseed oil have no statistically significant effect on HOMA-IR or HOMA-%ß in T2Ds, probably due to the additive effects of negative and positive correlations.

Flaxseed lignan complex administration in older human type 2 diabetics manages central obesity and prothrombosis-an invitation to further investigation into polypharmacy reduction.

Aim. Animal and human study evidence supports the hypothesis that flaxseed lignan complex (FLC) at a dose of 600 mg secoisolariciresinol diglucoside (SDG)/day for three months would combat hyperglycaemia, dyslipidemia, blood pressure, central obesity, prothrombotic state, inflammation, and low density lipoprotein (LDL) oxidation. Methods. Sixteen type 2 diabetic patients completed this double-blind, randomised crossover placebo-controlled study. A univariate repeated measures analysis of covariance (significance P < 0.05) was followed by a mixed linear model effects analysis corrected for multiple comparisons (MCC). Results. Prior to MCC, FLC caused decreased fasting plasma glucose, A1c, inflammation (c-reactive protein (CRP) and interleukin-6 (IL-6)), and increased bleeding time. After correction for multiple comparisons, FLC induced a statistically significant increase in bleeding time and smaller waist circumference gain. No treatment effect occurred in the other variables before or after adjustment. Conclusions. It is concluded that FLC significantly increased bleeding time thus reducing the prothrombotic state, reduced central obesity gain as measured by waist circumference, and did not affect significantly the other dependent variables measured after adjustment for multiple comparisons. These findings, not yet published in human type 2 diabetes, suggest that this FLC dose over at least three months, may, subject to further investigation, reduce polypharmacy.

The molecular nature and consequences of lipoprotein (a)'s association with platelets.

Lipoprotein (a) (Lp (a)) may be pro-thrombotic in humans due to its apolipoprotein (a) (apo(a))-mediated decreases in fibrinolysis. Such decreased fibrinolysis arises putatively from interference with plasminogen conversion to plasmin due to the considerable homology between apolipoprotein (a) and plasminogen. However, in vitro, most studies have shown that human Lp (a) decreases agonist-stimulated platelet aggregation while in vivo it appears to decrease aggregation as implied by increased bleeding times with higher blood serum concentrations of Lp(a). Lp (a) binding to platelets mediated by apo (a) increases platelet intracellular c-AMP levels in resting platelets, and decreases platelet production of thromboxane A2 and fibrinogen binding to platelets all of which reduce platelet aggregation. One, though not the only, explanation of these conflicting data may be that Lp(a) self-regulates its interference with fibrinolysis by reducing platelet aggregation and platelet binding of fibrinogen and hence the degree of requirement for fibrinolysis. However, it is concluded more in vivo work needs to be done to fully understand whether, if at all, Lp(a) in varying concentrations and isoforms, favours reduced platelet aggregation or fibrinolysis.

Arginyl-glycyl-aspartyl (RGD) epitope of human apolipoprotein (a) inhibits platelet aggregation by antagonizing the IIb subunit of the fibrinogen (GPIIb/IIIa) receptor.

An unknown epitope of apolipoprotein (a) antagonizes fibrinogen binding to agonist-stimulated platelet's fibrinogen (GPIIb/IIIa) receptor yielding lipoprotein (a) mediated decreased platelet aggregation. The purpose of this study was to test the hypothesis that human apolipoprotein (a)'s single arginyl-glycyl-aspartyl (RGD) epitope, unique to apolipoprotein (a) in lipoprotein (a) binds to the RGD binding motif on the IIb subunit of the GPIIb/IIIa receptor thus reducing platelet-bound fibrinogen and consequently decreasing agonist-stimulated platelet aggregation. Platelets (N=30 subjects) were prepared from fresh plasma, washed three times in Tyrode's buffer and stimulated using 10 microM ADP or 2 microg/ml collagen. Lipoprotein (a) was isolated from plasma using lectin affinity chromatography followed by ultracentrifugation. The peptide RGDS inhibited (125)I-labelled lipoprotein (a) binding to autologous platelets with IC-50's of 25.1+/-2.2 (mean+/-SEM) and 15.4+/-1.3 microM for collagen- and ADP-stimulation respectively. Further, RGDS reduced platelet binding of (125)I-labelled fibrinogen IC-50's of 35.5+/-3.2 (mean+/-SEM) and 20.7+/-2.2 microM for collagen- and ADP-stimulation respectively. The monoclonal antibody PAC-1, uniquely directed at the RGD binding motif on the IIb subunit on collagen- and ADP-stimulated platelets, inhibited binding of (125)I-labelled lipoprotein (a) with IC-50's of 6.4+/-0.7 and 2.5+/-2.2 microg/10(8) platelets for collagen- and ADP-stimulation respectively. Additionally, PAC-1 reduced platelet bound of (125)I-labelled fibrinogen with IC-50's of 9.0+/-1.4 and 4.1+/-2.2 microg/10(8) platelets for collagen- and ADP-stimulation respectively. In a dose-related fashion, a polyclonal antibody, specific for the RGD epitope on apolipoprotein (a), restored platelet aggregation to control levels, inhibited (125)I-labelled lipoprotein (a) binding, and increased (125)I-labelled fibrinogen by displacing lipoprotein (a) from the GPIIb/IIIa receptor. Thus a never before demonstrated aspect of the mechanism of lipoprotein (a)'s suggested novel role as an endogenous regulator of fibrinogen binding to collagen- and ADP-stimulated platelets has been shown. In conclusion, lipoprotein (a), via apolipoprotein (a)'s RGD epitope, binds to the RGD binding motif on the IIb protein of the GPIIb/IIIa receptor consequently reducing platelet-bound fibrinogen which results in decreased platelet aggregation.

Apolipoprotein (a) mediates the lipoprotein (a)-induced biphasic shift in human platelet cyclic AMP.

Lipoproteins are known to influence platelet cyclic adenosine monophosphate (c-AMP) levels. Lipoprotein (a) (Lp(a))'s impact on platelet c-AMP levels has never been assessed. Increasing levels of purified human Lp(a) (1-100) mg/dl were incubated with washed human platelets. Lp(a) concentrations of 1-25 mg/dl resulted an initial statistically significant increase of platelet c-AMP above basal levels and decreased collagen-stimulated platelet aggregation levels. Higher concentrations progressively returned the platelet c-AMP concentrations to basal levels accompanied by further decreases in platelet aggregation. Increasing concentrations of purified apolipoprotein (a) (apo(a)) also resulted in a similar biphasic c-AMP response while Lp(a) without apo(a) was without impact. One antibody directed against apo(a) in intact Lp(a) removed the biphasic c-AMP pattern and eliminated Lp(a) platelet aggregation. Antibodies directed against apo B in intact Lp(a) gave results similar to intact Lp(a) in terms of the biphasic response of c-AMP upon platelet exposure to increasing levels of Lp(a). It is concluded that apo(a) mediates the Lp(a)-induced biphasic response in platelet c-AMP as the result of platelet exposure to increasing levels of Lp(a). The biphasic response in c-AMP assists in platelet aggregation decreases up to a concentration of 25 mg/dl Lp(a), such assistance being lost at higher Lp(a) concentrations.

Decreased in vitro formation of AGEs with extraneal solution compared to dextrose-containing peritoneal dialysis solutions.

Extraneal peritoneal dialysis (PD) solution (Baxter Healthcare, Deerfield, Illinois, U.S.A.) contains glucose polymer (icodextrin) as an osmotic agent in place of dextrose. We investigated the ability of Extraneal to form advanced glycation end products (AGEs) in vitro compared to standard PD solutions containing dextrose. Extraneal, Dianeal PD-2 [1.5%, 2.5%, or 4.25% dextrose (Baxter Healthcare)], or phosphate buffered saline (PBS) were incubated for 45 days with human serum albumin (HSA) or type IV collagen. AGE formation was measured by spectrofluorometry using excitation at 350 nm and emission at 430 nm. Solutions were also incubated with collagen affixed to plastic, simulating matrix collagen in the peritoneal membrane. In addition, AGE formation was assessed using icodextrin metabolites (maltose, maltotriose, and maltotetraose) at concentrations normally found in the plasma of patients treated using icodextrin. For PD solutions incubated with albumin, the relative order of AGE formation was: 4.25% dextrose > 2.5% dextrose > 1.5% dextrose > Extraneal. For incubations with collagen (in solution or affixed to plastic), AGE formation was greatest for 4.25% dextrose, intermediate for Extraneal and 2.5% dextrose, and lowest for 1.5% dextrose. Incubation of icodextrin metabolites with albumin for 45 days did not result in appreciable AGE formation. These results confirm that solutions containing icodextrin result in less in vitro AGE formation than do high dextrose solutions. The results also suggest that Extraneal may lead to improved solution biocompatibility in vivo.

Lipoprotein (a) reduces platelet aggregation via apo(a)-mediated decreases in thromboxane A(2)production.

Agonist (collagen- or ADP-)-stimulated platelet aggregation and thromboxane B(2) (T X B(2) ) production was reduced in human whole blood (WB) and washed platelets (WP) that were co-incubated with lipoprotein (a)[Lp(a)] at levels of 25, 50 and 100 mg % but not at 5 mg % relative to a baseline concentration of 1 mg %. Significant decreases in agonist-stimulated aggregation and T X B(2) levels were seen with 5, 25, 50 and 100 mg %purified apo (a) that was co-incubated with WB and WP relative to a baseline concentration of 1 mg %. Purified Lp(a) that was free of apo(a) [Lp(a)(-)], at concentrations of 5, 25, 50 and 100 mg %, that were co-incubated with WB and WP, had no impact on agonist-induced platelet aggregation and T X B(2) production relative to a baseline level of 1 mg %. A monoclonal antibody (Mab) (3B1) against apo(a) blocked Lp(a)-mediated reduction in platelet aggregation and T X B(2) concentrations in WB and WP that were stimulated by either agonist. Various Mabs against apoB failed to affect an Lp(a)-induced reduction in WB and WP aggregation or T X B(2) levels in response to either agonist. These results strongly suggest that Lp(a)-induced decreases in collagen or ADP stimulated platelet aggregation and T X B(2) production are mediated by apo(a).

Human platelets have cholesteryl ester hydrolytic activity resulting in esterification of [1-14C]oleate to individual phospholipids of platelets.

Human platelet cholesteryl ester hydrolytic (CEH) activity was determined toward cholesteryl [1-14C]oleate resulting in esterification of [1-14C]oleate to individual platelet phospholipids: choline-containing phospholipids (PC); ethanolamine-containing phospholipids (PE); phosphatidylserine (PS); phosphatidylinositol (PI); and sphingomyelin (SPH). Liberation of [1-14C]oleate and esterification of [1-14C]oleate to platelet phospholipids was enhanced by 100 nM iloprost (a stable analogue of prostacyclin that increases platelet cyclic adenosine monophosphate (c-AMP)), inhibited by 30 microM H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide)) (a specific c-AMP dependent protein kinase (CADPK) inhibitor) and 500 microM 2',5' dideoxyadenosine (DDA) (an inhibitor of iloprost-induced rise in platelet c-AMP), but unaffected by 150 mM choloroquine diphosphate. These observations suggest that the CEH activity is mediated by a CADPK phosphorylation of an enzyme with the phosphorylated state representing the active form of the enzyme and that the CEH activity is extralysosomal.

Human platelets have cholesteryl ester hydrolytic activity toward plasma high density lipoproteins.

Human platelet cholesteryl ester hydrolytic (CEH) activity was determined toward high density lipoprotein (HDL) labelled with cholesteryl [1-(14)C] oleate resulting in esterilkation of [l-(14)C] oleate to platelet phospholipid. The observed CEH activity was enhanced by 100 nM prostacyclin (PGI(2)), inhibited by 500 µM 2', 3' dideoxyadenosine (DDA), but unaffected by 100 mM chloroquine diphosphate. The CEH activity may represent a mechanism for delivery of other unsaturated fatty acids from HDL to platelets with subsequent modification of the fatty acid composition of platelet phospholipids and potential modification of platelet reactivity.

Genetic analysis of a polymorphism in the human apolipoprotein A-I gene promoter: effect on plasma HDL-cholesterol levels.

Previous studies have indicated that a G to A substitution at position -76 in the gene encoding apolipoprotein A-I (apoA-I) confers increased plasma high density lipoprotein cholesterol (HDL-C). Increased HDL-C may be a direct consequence of the A allele, or may reflect the action of a locus in linkage disequilibrium with the A allele. To elucidate this question, we examined the effect of this polymorphism in a large sample (n = 409) of unrelated Caucasians and their nuclear families (n = 22). To eliminate the confounding effects of hypertriglyceridemia, individuals with triglyceride levels greater than 150 mg/dl were excluded from the study. ApoA-I genotype was determined by polymerase chain reaction (PCR) amplification of genomic DNA and restriction digestion with Msp I. Individuals were grouped by genotype (GG, GA or AA) and mean adjusted HDL levels of the three groups were compared by one-way analysis of variance. Our analysis indicates that HDL-C levels do not vary by genotype, and no gene dosage effect is apparent in men or in women. Analysis of 22 informative Caucasian nuclear families showed no significant difference between individuals with the A allele and their GG siblings. These data suggest that polymorphism at -76 in the apoA-I gene does not directly affect HDL levels. Therefore, the increased HDL-C levels reported in other populations must reflect linkage disequilibrium between the A allele and a putative HDL-raising allele. As we find no evidence for association between the A allele and high HDL levels, this putative allele must occur at a low frequency in the population sampled in this study.

The effect of borage oil consumption on the composition of individual phospholipids in human platelets.

The effect of supplementation with borage oil containing gamma-linolenic acid (GLA, 18:3n-6) on the levels and fatty acid compositions of individual human platelet phospholipids was evaluated. For this purpose, male volunteers were given an average daily intake of 5.23 g of GLA (as borage oil) for 42 days, after which the supplement was withdrawn for an additional 42-day period. No significant differences were found in the relative amounts of the choline phospholipids (PC), ethanolamine phospholipids (PE), phosphatidylserine (PS), phosphatidylinositol (PI), and sphingomyelin (SPH) at days 0, 22, 43, 64, and 85. However, marked differences were observed in the fatty acid compositions of all the phospholipids including a marked, and reversible, rise in the level of dihomo-gamma-linolenic acid (DGLA, 20:3n-6), without a significant elevation in arachidonic acid (AA, 20:4n-6) and decreases in n-3 polyunsaturated fatty acids. In the case of PC, a net rise in DGLA of 1.8 mol% was observed by day 22 (from 2.1 to 3.9 mol%). The DGLA/AA ratios at day 43 exhibited considerable variability across phospholipids with PC greater than PS greater than PE = PI; the PC, PE, PS, and PI accounted for 67.6, 16.7, 12.9, and 2.6%, respectively, of the total DGLA in platelet phospholipids. Interestingly, despite the lack of DGLA in SPH, this phospholipid exhibited a marked enrichment in nervonic acid (NA, 24:1n-9) from 16.2 to 24.7 mol% upon borage oil consumption. The observed alterations may represent biochemical strategies for adaptation to dietary fatty acid modifications and the regulation of platelet membrane functioning.