Leon C. Boyd

Antioxidant properties of individual phospholipids in a salmon oil model system

The antioxidant properties of phospholipids (PL) in a refined salmon oil model system were measured by determining changes in the 2-thiobarbituric acid number and decreases in the ratio of docosahexaenoic acid (DHA)/palmitic acid (22:6/16:0) of a fish oil system incubated at 180°C for up to 3 h. The more phosphatidylcholine (PC) added to the oil system, the higher the oxidative stability obtained. The order of effectiveness of commercial phospholipids in inhibiting oxidation and the loss of polyunsaturated fatty acids was as follows: sphingomyelin (SPH)=lysophosphatidylcholine (LPC)=phosphatidylcholine (PC)=phosphatidylethanolamine (PE)>phosphatidylserine (PS)>phosphatidylinositol (PI)>phosphatidylglycerol (PG)>control salmon oil. Nitrogen containing PL, including PE, PC, LPC and SPH, were equally effective in exerting greater antioxidant properties than PS, PG and PI. The inverse relationship observed between the oxidation index (C22:6/C16:0) and color intensity for treatments following 2 h of heating suggests that Maillard-type reaction products may have contributed to the oxidative stability of PL-supplemented fish oils.

A rapid method for determining the oxidation of n-3 fatty acids

The stability of unsaturated fatty acids to oxidation was monitored by following gas chromatographic (GC) analyses of headspace volatiles in comparison to changes in polyunsaturated fatty acids (PUFA) and increases in malonaldehydevia the 2-thiobarbituric (TBA) assay. Pure standards of linoleic acid (Lo) and n-3 fatty acids [eicosapentaenoic (EPA) and docosahexaenoic acid (DHA)] were added to headspace vials, equilibrated in air for 10 min, followed by heating at 80°C in teflon-capped vials for different time intervals. Headspace analysis showed increases in acetaldehyde, propenal, and propanal, corresponding to the oxidation of n-3 fatty acids, whereas hexanal production corresponded to losses of linoleic acid. The analysis of propanal by GC-headspace after only five minutes of heating appeared to be the most effective method of monitoring the oxidation of n-3 fatty acids, as indicated by correlations between TBA values and loss of PUFA. The oxidation of Lo, EPA and DHA appeared to be a function of the number of double bonds. Correlations between PUFA depletion, TBA values and volatile formation indicate that under the prescribed conditions of this experiment, GC-headspace analysis of propanal and pentane/hexanal is an excellent method for following the oxidation of selected n-3 fatty acids and linoleic acid.

Effects of phospholipids on lipid oxidation of a salmon oil model system

Total lipid (TL), neutral lipid (NL), and phospholipid (PL) fractions were extracted from bluefish (Pomatomus saltatrix) white and dark muscle with skin. The effects of each fraction on the oxidative stability of a refined salmon oil model system was measured by monitoring changes in the 2-thiobarbituric acid assay and decreases in the ratio of docosahexaenoic acid (DHA) to palmitic acid (C22:6/C16:0) following incubation at 55°C or 180°C. Phospholipid fractions at 2.5% and 5.0% (wt/wt) of oil improved the oxidative stability of oils incubated at both temperatures compared to controls, TL- and NL-supplemented oils at similar concentrations. Phospholipid fractions exhibiting antioxidant properties contained an average of 34% DHA as compared to only 15% in the NL and TL fractions.

Elevated prothrombin level and shortened clotting times in subjects with type 2 diabetes

The partial thromboplastin time [1] and its successor, the activated partial thromboplastin time (aPTT) [2], have long been used to detect coagulation factor deficiencies and monitor replacement therapy in patients at risk of bleeding. More recently, a correlation has been reported between short aPTT values and the risk of thrombosis [3,4], as well as the risk of recurrence in patients who have already suffered a thromboembolic event [5]. High factor (F)VIII or IX levels can contribute, but are not the only determinants of a short aPTT [6]. An elevated level of prothrombin has also been associated with thrombosis [7], and elevated levels of prothrombin lead to increased thrombin generation in an in vitro model of hemostasis [8]. Thus, it seems likely that elevated prothrombin levels could contribute both to thrombotic risk and to a shortening of the aPTT. Insulin resistance and type 2 diabetes mellitus (DM) are associated with an increased risk of atherothrombotic events [9], as well. In order to determine whether the levels of prothrombin or other plasma clotting factors might play a role in the thrombotic tendency in diabetes, we assayed clotting times, and prothrombin, antithrombin, fibrinogen and plasminogen activator inhibitor-1 (PAI-1) levels in subjects with or without type 2 DM. The prothrombin time (PT) and aPTT assays were performed in the Durham Veterans Affairs Medical Center Clinical Hematology Laboratory on an STA analyzer (Diagnostica Stago, Asnières, France) using Neoplastin (ISI 1.3) and PTT Automate reagents, respectively. The reference range for the PT International Normalized Ratio (INR) is 0.89–1.20 and for the aPTT it is 23.5–35.1 s. Levels of prothrombin, fibrinogen and PAI-1 were determined by ELISA. The subjects were the first 40 of 81 subjects recruited into a trial on the effects of muscadine grape juice on parameters related to glycemic control and cardiovascular risk [10]. This study was conducted under a protocol approved by the Institutional Review Board of North Carolina State University. The details of this study, including subject characterization have been published previously [10]. Blood samples were collected before and after a period during which some of the subjects consumed wine or grape juice. Blood samples collected before the dietary intervention were used to assess coagulation parameters. Subjects were provisionally classified as DM based on their report of having been so diagnosed by a physician. They were subsequently reclassified based on fasting glucose, insulin and glycated hemoglobin (HbA1C) levels. Selfclassification into DM or control groups was verified for all except three subjects, who were excluded from the analysis because the fasting glucose, insulin and HbA1C levels did not allow them to be unambiguously classified. Data from a total of 10 male and 9 female controls, and 8 male and 10 female diabetic subjects were available for analysis. Informed consent was obtained from all participants. We found that DM subjects had shorter values than controls for the aPTT (25.6 ± 3.7 vs. 29.3 ± 3.4 s; P 1⁄4 0.006) and PT assays (11.3 ± 0.5 vs. 11.9 ± 0.6 s; P 1⁄4 0.005). While the differences were small in absolute terms, they were highly statistically significant. In addition, the distribution of the values was different in controls and diabetics. Only the distributions of aPTT values are shown in the top panel of Fig. 1, but the distribution of PT values looked very similar. In addition, male controls tended to have a shorter mean aPTT than females, but the difference did not reach statistical significance. PT values were not different for male and female control subjects. DM subjects also had significantly higher prothrombin levels than controls (1.8 ± 1.2 vs. 1.0 ± 0.3 units mL; P 1⁄4 0.04), and male controls had higher prothrombin levels than female controls (1.22 ± 0.48 vs. 0.83 ± 0.29 units mL; P 1⁄4 0.03). There was also a striking tailing of prothrombin values to the high side of the distribution curve in the diabetic subjects, as shown in the lower panel of Fig. 1. In the diabetic subjects there was a significant inverse correlation between the prothrombin level and the PT and aPTT (r 1⁄4 )0.4 for females and )0.55 for males), which was not found in the control groups. This suggests that the elevated prothrombin level in diabetics is an important contributor to the shortened clotting times. There was no significant difference in fibrinogen, antithrombin or PAI-1 levels between the groups. We believe that the difference in prothrombin level could be responsible for shortening of both the PT and the aPTT. However, because the PT clots so much more rapidly than the Correspondence: Maureane Hoffman, Laboratory Service (113), Durham Veterans Affairs Medical Center, 508 Fulton St, Durham, NC 27705, USA. Tel.: +1 919 286 6925; fax: +1 919 286 6828; e-mail: maureane@ med.unc.edu

Fatty Acids, Diet, and Body Indices of Type II Diabetic American Whites and Blacks and Ghanaians

OBJECTIVE This research was designed to study the diet, lipid profile, and metabolic and body indices of type II diabetic and non-diabetic subjects among American white and black and Ghanaian populations. METHODS Fifty-one type II diabetic and non-diabetic volunteers were recruited through medical clinics. Data collected included food intake and anthropometric measurement. Blood samples were taken for glucose and serum lipid analyses. Serum non-esterified fatty acids, very low-density lipoproteins, low-density lipoproteins, high-density lipoproteins, total cholesterol, and triacylglycerols levels were measured. RESULTS The Ghanaian subjects had lower body mass indexes than did the American white and black subjects (P < 0.01), although they recorded the highest carbohydrate intake. Dietary fat intake was not significantly correlated with body fat level or body mass index among the different observational groups. The serum ratio of saturated to polyunsaturated fat was higher in all diabetics than in controls and higher in Ghanaians than in Americans. Total cholesterol, triacylglycerols, and lipoproteins were within normal ranges for diabetic and non-diabetic subjects. The ratio of total cholesterol to high-density lipoprotein cholesterol was slightly elevated among the white diabetics (P < 0.05). CONCLUSIONS The data showed a higher metabolism of carbohydrate for energy in the Ghanaian group than in the other groups. In addition, fat metabolism may differ between Americans and Ghanaians. For many variables, black Americans were more similar to white Americans than to Ghanaians. These observations imply that cultural factors may contribute more than ethnic origin to the etiology of diabetes.

Can anthropometric measurements and diet analysis serve as useful tools to determine risk factors for insulin-resistant diabetes type 2 among white and black Americans?

OBJECTIVE Central obesity is implicated in the development of insulin resistance by increasing insulin demand and eventually leading to hyperinsulinemia. Anthropometric measurements have been helpful in determining the risk factors in developing diabetes mellitus type 2. In this study we investigated whether anthropometric measurements differ among diabetics of different races. We also evaluated whether nutrient intake of these individuals was related to anthropometric measurement changes. METHODS Subjects were recruited from four groups: white control (n = 10), black control (n = 10), white diabetic (n = 5), and black diabetic (n = 10). The diabetic subjects had type 2 diabetes with insulin resistance on insulin monotherapy (age and sex matched). The following determinations were made: diet analysis, body mass index (kg/m(2)), the ratio of waist (umbilical level) to hip (maximum at buttocks) circumference, the ratio of waist to thigh (mid-thigh), and body fat percentage. RESULTS The micronutrient consumption was fairly similar in all groups with the exception of vitamin A (greatest consumption in the white control group, P < 0.05; and the lowest consumption in the black control group, P < 0.05). The data also suggested that central obesity (greatest waist-to-hip ratio) was present in the individuals with type 2 diabetes. The higher total fat, including saturated, monounsaturated, polyunsaturated, and cholesterol, intake in the diabetic groups were observed. CONCLUSION The type of fat consumed may be as important as the total fat consumption in the development of insulin resistance. The diet analysis can provide valuable information about the dietary habits of an individual and the possible causes of metabolic problems leading to a disease state. However, genetic factors must be considered when looking at diabetes incidence in different ethnic groups. For example, even though the black diabetic group consumed less fat in comparison with the other groups, their body fat percentages were higher. Therefore, we cannot conclude that high fat intake is primarily responsible for increased body fat percentage. Although anthropometric measurements are a useful tool in risk assessment, researchers should consider anatomic differences among different racial groups as covariables. Diet analysis when used in conjunction with anthropometric measurements can serve as a useful tool to detect whether metabolic alterations are related to dietary habits.