Anomalous symptoms in mice injected with reverted tetanus toxoid.
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Animals↗
Biomedical subjects
A J Sheppard
Publications and source records attributed to A J Sheppard.
Comparison of the clearances of serum chylomicron triglycerides enriched with eicosapentaenoic acid or oleic acid.
Rat mesenteric lymph chylomicrons containing triglycerides enriched with either [14C]oleic acid (OA) or [14C]-eicosapentaenoic acid (EPA) were prepared by ultracentrifugation of lymph samples collected for 6 hr after a single duodenal infusion of an emulsion containing either fatty acid. These chylomicrons were injected into the jugular vein of recipient rats and, at various time intervals, blood was drawn and serum was assayed for radioactivity. In separate animals, serum lipoprotein fractions were separated by ultracentrifugation, and the redistribution of labeled fatty acid among circulating lipoproteins was determined by liquid scintillation spectrometry. When the early disappearance rates (10 min) of either total serum radioactivity or specifically the chylomicron fraction were compared, there were no differences between the groups receiving OA- or EPA-enriched chylomicrons. However, disappearance rates of EPA-enriched chylomicrons were slower than those of OA-enriched chylomicrons from 25 to 90 min. The small but significant differences in the disappearance rates for the longer time periods cannot be ascertained without further studies. At 5 min after injection of either type of chylomicron, the d less than 1.006 g/ml lipoprotein fraction of serum chylomicrons and very low density lipoproteins contained almost 90% of the original radioactivity. By 240 min, when less than 2% of the radioactivity remained, this radioactivity in the d less than 1.006 g/ml fraction was 43-46%, with concomitant increases in the low and high density lipoprotein fractions and in the lipoprotein-free serum.
Animals↗
In vitro clearance of chylomicron triglycerides containing (omega-3) eicosapentaenoate.
Rat mesenteric lymph chylomicrons, containing triglycerides enriched with either [14C]oleic acid or [14C]eicosapentaenoic acid, were prepared by ultracentrifugation of lymph samples collected for 6 h after a single duodenal infusion of an emulsion containing 0.3 mmol of either fatty acid. After determination of protein and of total fatty acid content and composition, enriched chylomicrons were suspended in Krebs-bicarbonate buffer. Non-working hearts were perfused in a recirculating system for 45 min using the enriched chylomicron preparations. At 15 min intervals during perfusion, the media were assayed for total radioactivity, 14CO2 and 14C-labeled fatty acids associated with triglycerides, unesterified fatty acids, phospholipids, mono- and diglycerides. After perfusion, the hearts were extracted and assayed for total lipid radioactivity and isotope distribution among heart lipid fractions. With this membrane-supported lipoprotein lipase system, clearances of chylomicron triglycerides containing either fatty acid were identical, as were the myocardial uptakes of the fatty acids and oxidations to 14CO2. Furthermore, except for a significantly greater incorporation of eicosapentaenoate into myocardial phospholipids, tissue isotope distributions of the two labeled fatty acids were also the same. These studies suggest that at least the initial phases of peripheral clearance of chylomicrons enriched in omega-3 fatty acids is as efficient as with those containing oleate.
Animals↗
Digestion, absorption and effects on cholesterol absorption of menhaden oil, fish oil concentrate and corn oil by rats.
Adult male rats were surgically provided with a drainage catheter in the left thoracic lymphatic channel and an indwelling duodenal catheter for constant infusion of physiological saline-5% glucose. After an overnight fast, animals were given a single duodenal dose of an aqueous emulsion containing one of the following: oleic acid, corn oil, menhaden oil or a fish oil concentrate (FOC) and [1,2-3H]cholesterol. Digestion and absorption were estimated by recovering the total fatty acids in the thoracic duct lymph over a 24-h collection period (after subtraction of the "baseline" endogenous fatty acids in the lymph). Cholesterol absorption in the thoracic duct lymph was significantly reduced (P less than 0.05) in the presence of menhaden oil or FOC compared to that in the presence of corn oil. With various fat feedings, the major increases in lymph fatty acids were directly related to the dietary fatty acid content. The relative amounts of eicosapentaenoic acid (EPA) and arachidonic acid (AA) in the thoracic lymph were influenced by the lipid content of the emulsion. The EPA/AA ratio in control, oleic acid and corn oil feedings ranged from 0.12 to 0.25. When marine oil was administered, the EPA/AA ratio was 0.78-0.98. The total amount of fatty acids found in the lymph after marine oil feeding was significantly less (P less than 0.01) than that found after corn oil feeding. The results suggested that the digestion and absorption of menhaden oil and FOC were decreased as compared with corn oil. The EPA/AA ratio was increased in the thoracic lymph after dietary fish oil feeding.
Animals↗
Affinity purification of tetanus toxin using polyclonal and monoclonal antibody immunoadsorbents.
Tetanus toxin has been immunopurified on immunoadsorbent columns derived from equine polyclonal antitoxin coupled to cyanogen bromide-activated Sepharose CL4B. Desorption of bound toxin in active form was achieved only when the immunoadsorbent was mixed with Sephadex G15 and this mixture overlaid on a further volume of Sephadex G15. With equine antibody, 64% of adsorbed toxin was recovered with a specific activity of 2400 limiting flocculation units (Lf)/mg protein N (1.2 X 10(8) minimum lethal doses (MLD)/mg protein N). Similarly prepared immunoadsorbent derived from murine monoclonal antitoxin of low affinity had improved desorption with less acidic desorbents, without the requirement for Sephadex G15; greater than 80% of adsorbed toxin was recovered with a specific activity of 3000 Lf/mg protein N (1.6 X 10(8) MLD/mg protein N).
Animals↗
Intestinal absorption and lipoprotein transport of (omega-3) eicosapentaenoic acid.
Adult male rats were surgically provided with a drainage catheter in the left thoracic lymphatic channel and an indwelling gastric catheter for constant infusion of saline-glucose. After an overnight fast, animals were given a single gastric dose of an aqueous emulsion containing one of the following 1-14C-labeled fatty acids: oleic, arachidonic or eicosapentaenoic acid, and [1,2-3H]cholesterol. Absorption was estimated by the appearance of radioactivity in lymph over a 24-h collection period, and the lymph lipoprotein distributions and lipoprotein lipid distribution were determined in the 24-h samples. Although there were apparent differences in the rates of eicosapentaenoic and arachidonic acid absorption, the overall appearance (after 24 h) of these acids in lymph was quantitatively equivalent to that of oleate. Furthermore, cholesterol absorption from each fatty acid medium was quantitatively similar. The distributions of each fatty acid among major lymph lipoproteins were similar with 93-95% recovered in chylomicrons and very low density lipoprotein (VLDL) fractions. Furthermore in these lipoproteins, 85-91% of each of the three fatty acids was recovered as triglycerides. With both polyunsaturated fatty acids, there was greater incorporation into phospholipids and their precursor partial glycerides than with oleate. These studies suggest that unesterified eicosapentaenoic acid is absorbed efficiently into the lymphatic system, and has lymph lipoprotein and lipid distribution comparable to those of the more typical mono- and polyunsaturated fatty acids.
Animals↗
Detection of vegetable oil adulteration in ice cream.
This study was to demonstrate the application of various analytical methods to the detection, identification, and quantitation of vegetable oil adulteration of ice cream. Total fat content, sterols, long- and short-chain fatty acids, vitamin E, Reichert-Meissl values, and Polenske values were measured in ice cream. All methods except total fat determination were capable of detecting vegetable oil adulteration. Sterol determination was the most effective and versatile measurement because it provided information not only on the detection and extent of adulteration but also on the possible identity of the adulterant.
Chromatography, Gas↗
Production and characterization of monoclonal antibodies to tetanus toxin.
Monoclonal antibodies against tetanus toxin were produced to obtain highly specific antisera. Ten hybridoma cell lines producing monoclonal antibodies were derived from the fusion of rat myeloma cells and spleen cells from rats immunized with tetanus toxoid. Eight produced monoclonal antibodies specific for determinants on toxin and toxoid, whereas two were specific only for determinants on the toxoid. The antibodies produced by hybridomas were characterized by determination of the class of light and heavy chain components, epitope specificity, toxin neutralization, and subunit specificity. All of the antibodies contained kappa light chain, eight contained the gamma 1 heavy chain, and the remaining two contained the gamma 2a heavy chain. Five distinct epitopes were indicated by competition assay of paired monoclonal antibodies, and 4 of the 10 monoclonal antibodies neutralized the in vivo activity of tetanus toxin. The four neutralizing monoclonal antibodies and one other were specific for the C fragment of the heavy chain of the toxin molecule.
Animals↗
Enzymatic determination of cholesterol in egg yolk.
The quantitative determination of cholesterol in egg yolk by using an enzymatic test kit is described. Cholesterol in the egg yolk is extracted with other lipid components by methylene chloride-methanol (2 + 1) and is enzymatically determined after saponification of the lipid extract. The method is relatively rapid, simple, and accurate and gives results which agree with those obtained by using a gas-liquid chromatographic (GLC) method. The mean cholesterol content of egg yolk determined by the enzymatic and GLC methods was 1237 and 1240 mg/100 g, respectively.
Cholesterol Oxidase↗
High pressure liquid chromatographic determination of cholesterol in foods.
A high pressure liquid chromatographic method that uses the benzoate ester of cholesterol has been developed for the measurement of cholesterol in foods. A 300 x 3.9 mm id muBondapak C18 column and a variable wavelength ultraviolet detector are used with a 100% methanol mobile phase. Amounts as low as 10 ng cholesterol benzoate can be detected. The results from the analyses of a variety of foods are given and compared with results from gas-liquid chromatographic analyses. The comparison of the 2 chromatographic techniques is favorable.
Cholesterol↗
Evidence for an immunological relationship between Streptococcus mutans and human cardiac tissue.
Two-dimensional immunoelectrophoresis, indirect immunofluorescence, and radioimmunoassay were used to demonstrate that antisera from rabbits immunized with some strains of Streptococcus mutans contain antibodies that cross-react with human cardiac tissue. These rabbits were sensitized to a shocking dose of human heart antigen, and anaphylactic deaths were sometimes produced. Myocarditis was also a result of the immunization procedure. Data obtained with all five techniques were comparable. Cross-reactivity could be associated with three antigens designated ID, IF, and HL. Antigens ID and IF were major immunogens of S. mutans Ingbritt, but HL antibodies were produced only after hyperimmunization. Corss-reactivity was of an immunological nature and not the result of nonspecific factors such as bacterial Fc reactive components or antibody elicited to growth medium constituents. These findings support the hypothesis that immunization with S. mutans can induce autoimmune reactions and indicate that antigens must be selected with caution before formulating any dental caries vaccine.
Anaphylaxis↗
Effect of dietary Lunaria oil on chick growth and organ lipid content.
An experiment was conducted to determine the effects of feeding Lunaria oil to chicks. A deit containing Lunaria oil produced poorer growth and lower feed conversion than one containing corn oil. White Plymouth Rock chicks had apparent digestibility values of 89.9% and 55.8% for corn and Lunaria oil, respectively. Gas-liquid chromatographic analysis of heart, liver, and body cavity lipids (fat) showed that more erucic and nervonic acids accumulated in the fat of the birds fed Lunaria oil than in the fat of those fed corn oil.
Animals↗
Collaborative study of a GLC method for vitamin E.
The official GLC method of the Association of Official Analytical Chemists (AOAC) for determining vitamin E was modified and collaboratively studied for the National Formulary (NF). The internal standard hexadecyl hexadecanoate (cetyl palmitate) was substituted for the dotriacontane used in the AOAC method, and some other minor changes were made. Eleven samples, representing all types of NF formulations and NF bulk materials, were analyzed by 11 laboratories. The coefficients of variation of the reproducibility and repeatability were 4.5 and 2.4%, respectively, for all laboratories and samples. The values were 3.4 and 1.6%, respectively, when the one laboratory statistically determined to be an outlier was excluded. The coefficients of variation of reproducibility and repeatability for alpha-tocopheryl acid succinate were 2.1 and 1.5%, respectively. All of these values lie within the 5% limit required by the NF.
Analysis of Variance↗
Comparison of total fat, fatty acids, cholesterol, and other sterols in mayonnaise and imitation mayonnaise.
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Cholesterol, Dietary↗
Comparison of various methods for the extraction of total lipids, fatty acids, cholesterol, and other sterols from food products.
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Cholesterol↗
Butyl ester preparation for gas-liquid chromatographic determination of fatty acids in butter.
Butter oil is extracted from butter with hexane, using a rolling boil technique. The resulting 10% butter oil in hexane is converted to butyl esters, using butanol in place of methanol as specified in the official final action method, 28.063-28.067. A water wash and centrifuge technique is used to remove butanol from the n-hexane-butyl ester solution. Conversions to butyl esters are quantitative by the boron trifluoride, sulfuric acid, and sodium butoxide procedures. When methyl ester methods are used, there are serious losses of the short chain volatile and water-soluble acids, making the methyl ester procedures questionable for determining the authenticity of milk fat.
Butter↗
Gas-liquid chromatographic determination of cholesterol and other sterols in foods.
A gas-liquid chromatographic (GLC) method, using the butyryl esters of sterols, has been developed for the measurement of cholesterol, stigmasterol, sitosterol, and campesterol in foods. An immobile phase of 1% SE-30 coated on 100-120 mesh Gas-Chrom Q packed in a 6 inch X 4 mm id glass column operated at 255 degrees C was the most satisfactory of 7 column packings evaluated. Extraction with chloroform-methanol gave 98.7% recovery with a coefficient of variation of 1.8% for cholesterol added to a variety of foods. When cholesteryl palmitate was added to vegetable oil and the butyryl derivative was prepared, followed by GLC analysis, the recovery was 99.3% with a coefficient of variation of 0.9%. Amounts as low as 1 mg/100 g food can be detected with a precision of 2.5%. The results of the analysis of a variety of foods for cholesterol, campesterol, sitosterol, and stigmasterol are given.
Cholesterol↗
Programmed temperature gas chromatographic analysis of esters of fatty acids.
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Butter↗