Thomas J. Bronzert

Gas Chromatography of Amino Acid Phenylthiohydantoins Vol.244,No. 20 often 240" but never above 270".The detector temperature was 280", and the carrier gas was argon.In all chromatograms, full scale deflection was 3 X lo-lo amps with a 5-mv recorder.Glass U or coiled columns employed throughout the study were silanized by treatment with 5% dichlorodimethylsilane in toluene (12).The columns were filled with this solution; after 10 min they were rinsed thoroughly, first with anhydrous methanol, then with acetone, and dried in air.Gas Chrom P (100 to 120 mesh) was obtained from Applied Science Laboratories, Inc., State College, Pennsylvania.Chromosorbs P and W (80 to 100 and 100 to 120 mesh) were purchased from Supelco, Inc., Bellefont, Pennsylvania.The supports were acid washed and silanized according to the procedure of Horning, VandenHeuvel, and Creech (12).Care was taken to prevent the further production of "fines" by avoiding excessive handling of the support.The silicone stationary phases, SE-30, OV-1, OV-17, OV-22, OV-210, XE-60, DC-560 (formerly F-60), &F-l, and the silylating reagents, dichlorodimethylsilane, N ,O-bis(trimethylsilyl)acetamide, and N , N-bis(trimethylsilyl)trifluoroacetamide were purchased from Supelco, Inc.The organo-silicone phase ECNSS-S and polyester HI-EFF-3BP were obtained from Applied Science Laboratories, Inc.The support was coated with different liquid phases according to the filtration procedure ( 12).The coating of mixed phase columns may be illustrated with reference to the "DXO" 2 column.Ten grams of DC-560 and 3 g of XE-60 were separately dissolved in acetone, and each volume was made up to 100 ml.Three grams of OV-22 were dissolved in warm chloroform, and the volume also made up to 100 ml.A mixture was made of 56 ml of the "10%" DC-560 phase, 22 ml of the "3%" XE-60 phase, and 22 ml of "3%" OV-22 phase.This was used to coat the support by the above-mentioned filtration method.Columns were uniformly packed by gentle tapping of the walls, but not with an electric vibrator which could damage the support.Glass wool plugs used in the columns were silanized by dipping in dichlorodimethylsilane solution (5% in toluene), washing in methanol and acetone, and drying at 80" or on a Buchner funnel at room temperature.Most amino acid phenylthiohydantoin derivatives were purchased from Mann Research Laboratories.Exceptions were the lysyl, tryptophanyl, histidyl, asparaginyl, glutaminyl, seryl, S-carboxymethylcysteinyl, and threonyl phenylthiohydantoins, which were synthesized by previously described procedures (13-15).The crystalline derivatives obtained gave sharp melting points at the expected temperatures.Seryl PTH and threonyl PTH were, in addition, shown to give single spots upon thin layer chromatography on silica gel, and had low ultraviolet absorption at 320 rnp, indicating the absence of significant amounts of the anhydroderivatives.Xtandard Solutions-Standard solutions of the phenylthiohydantoins, except the asparaginyl, glutaminyl, histidyl, and cysteic acid derivatives, were made up as separate solutions in ethyl acetate at concentrations of 0.1 to 1.0 mg per ml.They were stored at 4' in screw cap vials fitted with Teflon cap liners.The derivatives of asparagine, glutamine, and histidine were dissolved in methanol (1.0 to 2.0 mg per ml) and stored similarly.The cysteic acid compound was kept as a 1.0 mg per ml solution in distilled waterea 2 This designation refers to columns coated with mixtures of In the preparation of standard samples for gas chromatogra-

A method is described for the reliable, fast, and relatively inexpensive fractionation of plasma lipoproteins and quantitation of their cholesterol content. This procedure requires 350 microliter of plasma and can be completed within 3 h. Plasma lipoproteins (175 microliter of plasma) were prestained with Fat Red 7B and centrifuged (Beckman Airfuge) at plasma density (d = 1.006 kg/liter) and at a solvent density of 1.060 kg/liter, adjusted by adding solid KBr. Prestained centrifuged samples demonstrated the characteristic elevation of chylomicrons in phenotypes I and V, low-density lipoproteins of phenotype II, very-low-density lipoproteins in phenotype IV and V, and continuum of pink color throughout the centrifuge tube, diagnostic of the floating beta lipoprotein of type III. Centrifuged samples were separated into top and bottom fractions by aspiration. Cholesterol was quantitated with an enzymic oxygen-electrode analyzer (Beckman Cholesterol Analyzer). Correlation coefficients between cholesterol values for plasma from normal hyperlipidemic individuals obtained with the Beckman Analyzer vs. the Technicon AutoAnalyzer II and SMAC systems were 0.977 and 0.973, respectively.

The kinetics of the major apolipoproteins (apo) of plasma high density lipoproteins (HDL), apoA-I and apoA-II, were examined in a total of 44 individual tracer studies in 22 normal male and female subjects. Following the intravenous injection of radioiodinated HDL, the specific radioactivity decay of apoA-I within HDL (residence time, 5.07 +/- 1.53 days), as determined by column chromatography, was significantly (P < 0.01) faster than that of apoA-II (residence time, 5.96 +/- 1.84 days). The specific radioactivity decay of apoA-I within HDL when labeled on HDL or as apoA-I was found to be almost identical. Similar results were obtained for apoA-II. Analysis of simultaneous paired radiolabeled apoA-I and apoA-II studies revealed that the mean apoA-I plasma residence time (4.46 +/- 1.04 days) was significantly (P < 0.01) shorter than that for apoA-II (4.97 +/- 1.06 days). Females had significantly (P < 0.01) higher apoA-I plasma concentrations (124 +/- 24 mg/dl) and apoA-I synthesis rates (13.58 +/- 2.23 mg/kg. day) than did males (108 +/- 16 mg/dl, and 11.12 +/- 1.92 mg/kg. day, respectively). Plasma apoA-I levels were correlated with plasma apoA-I residence times, but not synthesis rates; and apoA-II concentrations were correlated only with apoA-II whole body residence times. ApoA-I and apoA-II plasma residence times were inversely correlated with plasma triglyceride levels. These data are consistent with the following concepts: 1) labeling of apoA-I and apoA-II as apolipoproteins or on HDL does not affect their specific radioactivity decay within HDL; 2) the mean residence time of apoA-I both in plasma and in HDL is significantly shorter than that of apoA-II; 3) the increased apoA-I levels seen in female subjects are due to increased apoA-I synthesis; and 4) the plasma apoA-I residence time, which is inversely correlated with plasma triglyceride levels, is an important determinant of apoA-I concentration in both males and females.-Schaefer, E. J., L. A. Zech, L. L. Jenkins, T. J. Bronzert, E. A. Rubalcaba, F. T. Lindgren, R. L. Aamodt, and H. B. Brewer, Jr. Human apolipoprotein A-I and A-II metabolism.