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Domoic acid (DA) is a potent toxin produced by bloom-forming phytoplankton in the genus Pseudo-nitzschia, which is responsible for causing amnesic shellfish poisoning (ASP) in humans. ASP symptoms include vomiting, diarrhea, and in more severe cases confusion, loss of memory, disorientation, and even coma or death. This paper describes the development and validation of a rapid, sensitive, enzyme linked immunosorbent assay test kit for detecting DA using a monoclonal antibody. The assay gives equivalent results to those obtained using standard high performance liquid chromatography, fluorenylmethoxycarbonyl high performance liquid chromatography, or liquid chromatography-mass spectrometry methods. It has a linear range from 0.1-3 ppb and was used successfully to measure DA in razor clams, mussels, scallops, and phytoplankton. The assay requires approximately 1.5 h to complete and has a standard 96-well format where each strip of eight wells is removable and can be stored at 4°C until needed. The first two wells of each strip serve as an internal control eliminating the need to run a standard curve. This allows as few as 3 or as many as 36 duplicate samples to be run at a time enabling real-time sample processing and limiting degradation of DA, which can occur during storage. There was minimal cross-reactivity in this assay with glutamine, glutamic acid, kainic acid, epi-or iso-DA. This accurate, rapid, cost-effective, assay offers environmental managers and public health officials an effective tool for monitoring DA concentrations in environment samples.

Plasma lipid profiles were determined in two inbred strains of mice, C57BR/cdJ and CBA/J, fed either a normal chow or an atherogenic diet for a 15-week period, starting at 10 weeks of age. On the chow diet, the C57BR/cdJ had significantly higher mean free cholesterol, esterified cholesterol, and total lipid values, and a significantly lower mean phosphatidylcholine/free cholesterol ratio than the CBA/J mice. On the atherogenic diet, the C57BR/cdJ had significantly higher mean levels for all lipid classes, except triacylglycerols, than the CBA/J mice. The mean plasma free cholesterol and esterified cholesterol levels of the C57BR/cdJ were four times greater than those of the CBA/J strain on the atherogenic diet. The mean plasma phosphatidylcholine/free cholesterol ratio of the C57BR/cdJ mice on the high cholesterol diet was 0.87 compared to 1.91 for CBA/J mice. These plasma lipid changes were associated with a marked development of atheromatous deposits in the wall of the aortic sinus of the C57BR/cdJ compared to the CBA/J animals. The phosphatidylcholine/free cholesterol ratios of the liver lipids of both strains decreased from 2.5-2.7 on the chow diet to 1.0-1.1 on the high cholesterol diet. It is suggested that a plasma phosphatidylcholine/free cholesterol ratio less than 1 represents a supersaturation of the vascular system and the vessel wall with cholesterol, which leads to a destabilization of the plasma membranes of the endothelial and smooth muscle cells, and an infiltration of the vessel wall by the plasma lipids. (Arteriosclerosis 3:389-397, July/August 1983) T he pathogenesis of atherosclerosis is believed to depend on complex interactions of the constituents of the blood and the hemodynamic stresses acting on the endothelial cells lining the vessel wall, on the smooth muscle cells in the media, and on the supporting connective tissue components within the vessel wall itself. The accumulation of lipid within the vessel wall that results in the development of the initial fatty atheromatous lesion in humans is usually preceded by hypercholesterolemia and hyperlipoFrom the Banting and Best

Downloaded from LIPOPROTEIN LEVELS IN ATHEROSCLEROSIS-SUSCEPTIBLE MICEBreckenridge et al. 257 Methods AnimalsMale mice of the C57BR/cdJ (susceptible) and CBA/J (resistant) strains were purchased from the Jackson Laboratories, Bar Harbor, Maine, when they were 6 weeks old and were placed on Purina Laboratory Chow (containing 4.5% crude fat) and water ad libitum. At 10 weeks of age, mice of both strains were divided into treatment and control groups. The treatment groups were started 5 on a diet consisting of 70% atherogenic (high fat, high cholesterol) food and 30% Purina Laboratory Chow for 2 days, then a 80% atherogenic and 20% chow diet for 4 days, followed by a 90% atherogenic and 10% chow diet for various periods of time until sacrificed. The animals accepted the diet readily. The corresponding control groups were maintained for the same periods of time on Purina Laboratory Chow. Lipoproteln IsolationThe animals were fasted overnight and sacrificed by using intraperitoneal sodium pentobarbital anesthesia (0.02 ml/g body weight). Whole blood (0.5 to 1.0 ml per animal) was removed from the inferior vena cava, was mixed with solid ethylenediaminetetraacetic acid (EDTA, 1.2 mg/ml blood) and was immediately chilled on ice. For the preparation of lipoproteins, blood samples were pooled separately from 16 C57BR/cdJ mice on the 90% atherogenic, 10% chow diet, from 19 C57BR/cdJ mice on the chow diet, and from 20 CBA/J mice on each of the atherogenic and the chow diets. Lipoprotein fractions were obtained by ultracentrifugation 6 at selected densities in a Beckman rotor (40:3) as follows: VLDL (d < 1.006) at saline density for 18 hours at 40,000 rpm; IDL at 1.006 > d < 1.019 for 20 hours at 40,000 rpm; LDL at 1.019 > d < 1.063 for 20 hours at 40,000 rpm, and HDL at 1.063 > d < 1.21 for 48 hours at 40,000 rpm. Lipoproteins were separated by agarose gel electrophoresis as described elsewhere. 7 Isolated lipoproteins were also characterized by gel filtration on Beckman TSK columns as described previously.8 Apoprotein patterns were determined by isoelectric focusing after delipidation with ethanol-diethyl ether and solubilization of the apoproteins in the urea.9 Alternatively, the apoproteins were solubilized in 1% sodium dodecyl sulfate containing dithioerythritol and were resolved with sodium dodecyl sulfate polyacrylamide gel electrophoresis by using 4% to 30% gradient polyacrylamide gels and 0.2 M Tris glycine buffer (pH 8.3) containing 0.1% sodium dodecyl sulfate. Total Lipld Profile DeterminationThe total lipid profiles of the various lipoprotein fractions were determined as previously described. 10 " For this purpose, a neutral lipid extract was prepared as follows. An aliquot of the fraction equivalent to about 0.2 ml (0.5 ml of the solution) of plasma was added to a Teflon-lined, screw-cap centrifuge tube containing 0.2 to 0.4 ml phospholipase C (C. welchii, Sigma Chemical Company, St. Louis, Missouri) in 4 ml of 17.5 mM Tris buffer (pH 7.3) along with 1.3 ml of 1% CaCI 2 and 1 ml of diethyl ether. The mixture...

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