Conversion of α-linolenic acid to long-chain omega-3 fatty acid derivatives and alterations of HDL density subfractions and plasma lipids with dietary polyunsaturated fatty acids in Monk parrots (Myiopsitta monachus).

The effect of α-linolenic acid from a flaxseed (FLX)-enriched diet on plasma lipid and fatty acid metabolism and possible atherosclerosis risk factors was studied in Monk parrots (Myiopsitta monachus). Twenty-four Monk parrots were randomly assigned to diets containing either 10% ground SUNs or 10% ground FLXs. Feed intake was calculated daily. Blood samples, body condition scores and body weights were obtained at -5 weeks, day 0, 7, 14, 28, 42 and 70. Plasma samples were analysed for total cholesterol, free cholesterol, triacylglycerols and lipoproteins. Phospholipid subfraction fatty acid profiles were determined. By day 70, the FLX group had significantly higher plasma phospholipid fatty acids including 18:3n-3 (α-linolenic acid), 20:5n-3 (eicosapentaenoic acid) and 22:6n-3 (docosahexaenoic acid). The sunflower group had significantly higher plasma phospholipid levels of 20:4n-6 (arachidonic acid). By day 70, the high-density lipoprotein (HDL) peak shifted resulting in significantly different HDL peak densities between the two experimental groups (1.097 g/ml FLX group and 1.095 g/ml SUN group, p = 0.028). The plasma fatty acid results indicate that Monk parrots can readily convert α-linolenic acid to the long-chain omega-3 derivatives including docosahexaenoic acid and reduce 20:4n-6 accumulation in plasma phospholipids. The reason for a shift in the HDL peak density is unknown at this time.

Californium-252 plasma desorption mass spectroscopy.

We have shown that 252Cf-PDMS is capable of producing mass spectra of quasi-molecular ions for a wide variety of compounds, including amino acids, moderately large peptides, nucleotides, and natural products. Positive and negative ion mass spectra can be obtained, and in many cases quasi-molecular ions are observed in both. The method is nondestructive, as only a relatively few molecules are used and samples can be recovered after the measurement is made. Fragmentation patterns are obtained which can yield structure information. The present sensitivity of the method is at the nanogram level and there are possibilities for reducing this to picograms. The mass resolution is sufficient to give elemental identification up to mass 500. This may be extended to higher masses with improved time-of-flight techniques. There are indications that 252Cf-PDMS may extend the mass range of molecules that can be studied to as high as 3000 or more.

Small molecules as substrates for adsorption/desorption in (252)CF plasma desorption mass spectrometry.

Department of Chemistry, Texas A & M University, College Station, Texas, USA We present a new approach to substrate selection for californium-252 plasma desorption mass spectrometry ((252)Cf_PDMS) in which small volatile molecules that are water insoluble are used as matrices in place of the polymeric substrates used in previous studies. The desirable features of analyte adsorption are combined with the concept of using a volatile matrix to reduce the level of internal excitation of a desorbed analyte and to assist in ionization during the desorption process. Derivatives of anthracene were found to meet these requirements and to perform satisfactorily as substrates in (252)Cf-PDMS. Spectra were obtained for bovine insulin (m I z 5734) adsorbed onto 9-anthroic acid and 2-aminoanthracene and compared with spectra using a nitrocellulose substrate. Sharper peaks and lower backgrounds are observed when the 9-anthroic acid matrix is used, indicating reduced levels of internal excitation and initial kinetic energy for the desorbed molecular ion of insulin. A comparison of the performance of 9-anthroic acid and 2-aminoanthracene shows the influence of substrate functional groups on desorbed protein yields. Finally, the versatility of the small-molecule matrix concept is discussed with respect to selection of a range of functionality, solubility, and hydrophilicity.

Fragmentation analysis of bradykinin by (252)cf-plasma desorption mass spectrometry.

The (252)Cf-plasma desorption (PO) mass spectrum of the nonapeptide bradykinin was studied in detail with particular emphasis on the fragmentation pattern resulting from fast metastable decay of the molecular ion. N-acetyl and O-methyl derivatives of bradykinin were used as mass shift species for assignment of N-terminal and C-terminal fragment ions. Thirty-seven sequence-specific fragment ions were identified, including those that are due to cleavage of peptide backbone as well as side chain bonds (i.e., dn, vn, wn fragment ions). The degree of fragmentation correlates with what has been observed by fast atom bombardment tandem mass spectrometry using hi~h energy collisional activation. The high degree of excitation of bradykinin molecules in (252)Cf-POMS is undoubtedly due to the special nature of excitation intrinsic to the (252)Cf_PD process where electronic excitation occurs with a very high probability due to the high electron and photon flux surrounding the nuclear fission fragment track.These results offer further evidence that (252)Cf_PDMS, in addition to providing molecular weight information, can be used to sequence peptides without the need for a second stage of molecular excitation.

A pulsed ion deflection system for background reduction in (252)CF-plasma desorption mass spectrometry.

The use of an electrostatic particle guide (EPG) for background reduction in a time-of-flight mass spectrometer is described. Operating with reverse polarity, the EPG deflects ions radially from the beam axis, separating the ionic and neutral components of the beam. Use of the deflection EPG in a synchronized pulsed mode with a barrier disk aligned in the center of the beam axis eliminates up to 80% of the spectral background in the molecular ion region of an insulin spectrum obtained by (252)Cf-plasma desorption mass spectrometry. The background eliminated is due to the neutral products of metastable fragmentation and to uncorrelated events. Although peak intensities are reduced when the pulsed deflection EPG system is used, the reduction in background achieved is greater, resulting in an overall improvement in peak-to-background ratios of up to a factor of three. A new large-area stop detector designed for use with the pulsed deflection EPG is described. The new hybrid detector, which utilizes the combination of a large (75-mm active diameter) microchannel plate (MCP) and a scintillation detector, provides greater sensitivity for high-mass ions than a conventional MCP chevron detector.

A new method for electrostatic ion deflection.

A new approach to electrostatic ion deflection is described where an electrostatic particle guide (EPG) operating with reversed polarity is used to deflect ions in a cylindrical geometry about the axis of a time-of-flight mass spectrometer. The method has advantages over the standard parallel-plate deflector geometry in that it is more effective in deflecting ion beams that have a significant radial velocity component. The device is being used in (252)cs Cf plasma desorption mass spectrometry ((252)cs Cf -PDMS) experiments in an on/off mode to record neutral particle spectra and in a synchronized pulsed mode to reduce the magnitude of the uncorrelated background in the time-of-flight spectrum. Radial distribution functions have been measured for various EPG voltages. Its use as a background suppression technique is demonstrated by using the (252)cs Cf-PDMS spectrum of insulin. (28-36).

Characterization of lipoprotein a by capillary zone electrophoresis.

Lipoprotein a [Lp(a)] has been recognized as a significant marker for premature coronary heart disease (CHD). In this paper, we present the results of Lp(a) analysis based on capillary zone electrophoresis (CZE). CZE separation of Lp(a) and its reduced species, lipoprotein a- [Lp(a-)] and apolipoprotein a [apo(a)], was accomplished using 50 mM borate buffer containing 3.5 mM sodium dodecyl sulfate (SDS) and 20% (v/v) acetonitrile (ACN). Low density lipoprotein (LDL) and high density lipoprotein (HDL) were separated under the same buffer conditions. The electrophoretic mobilities of both Lp(a) and Lp(a-) were found to be different from that of LDL. Benzyl alcohol (BA) and methanol (MeOH) were used as electroosmotic flow markers. BA molecules associated with Lp(a-) and LDL to enhance their UV absorbance, but did not change their effective electrophoretic mobilities. Our results show that CE is a very efficient and effective technique for lipoprotein analysis.

Fast C18 solid-phase desalting/delipidation of the human serum apolipoproteins for matrix-assisted laser desorption ionization and electrospray ionization mass spectrometric analysis.

A new method for the delipidation of human serum lipoproteins involving the use of a reversed-phase C18 solid-phase extraction (SPE) cartridge is introduced for use with matrix-assisted laser desorption ionization and electrospray ionization mass spectrometry. This method is compared with two other methods of lipoprotein delipidation. The SPE method of delipidation produces a higher and more reproducible protein yield than the conventional liquid-liquid methanol-diethyl ether delipidation technique. Furthermore, the SPE method implements a fast, sequential, desalting and delipidation of the lipoproteins for subsequent mass spectrometric analysis providing high quality spectra.

Serum apolipoproteins.

The apolipoproteins associated with serum lipoprotein particles give structural stability as well as regulatory control in lipid metabolism. The development of atherosclerosis is linked to dysfunction in lipid metabolism, and the serum lipoproteins are directly involved, either through the action of their apolipoprotein components or in combination with the lipids sequestered in these particles. Consequently, quantitative features of the apolipoproteins (apos), serum levels, distribution within the lipoprotein population, and the appearance of isoforms, are potential markers for cardiovascular disease risk that are being added to the lipid profile for more effective cardiovascular risk screening (1). Currently, apo quantitation is being carried out using immunoassay or gel electrophoresis for separation and staining with image analysis for quantitation (2). Capillary electrophoresis has the potential for higher resolution, greater specificity, speed, and automation, coupled with on-line detection for more accurate and precise quantitation.

Quantitative and qualitative studies of gut flora in striped bass from estuarine and coastal marine environments.

Examination of the intestinal contents of 130 striped bass (Morone saxatilis) collected from the Hudson River and Long Island Sound during May to October 1981 showed that opportunistic fish pathogens--especially Aeromonas hydrophila--predominated in samples from both locations. Other isolates from both groups of striped bass included Vibrio, pseudomonads, flavo-bacteria, Alcaligenes, and enterics. Small numbers of Micrococcus, Bacillus, Corynebacterium, and Acinetobacter were also isolated. Total numbers of bacteria in the intestines were 100 to 1,000 times higher in striped bass from the Hudson River than in those from Long Island Sound.

Influence of dodecyl sulfate ions on the electrophoretic mobilities of lipoprotein particles measured by HPCE.

The authors have developed a method for profiling plasma lipoproteins based on differences in their surface chemical properties using HPCE. Low-density lipoprotein (LDL) and high-density lipoprotein (HDL) were used as model compounds representing components of the lipoprotein population with high surface polarity (HDL) and low surface polarity (LDL). Using an uncoated fused-silica capillary in the HPCE measurement, the effective electrophoretic mobilities (mu eff) of HDL and LDL at high pH were found to be nearly identical in different buffer systems and ionic strengths. Both HDL and LDL particles have a high affinity for dodecyl sulfate anions (DS-), increasing the mu eff for both particles by almost a factor of two but not giving significantly different mu eff values. Decreasing the polarity of the solution by the addition of acetonitrile resulted in a partitioning of the DS- ions between the solvent and lipoprotein particles. The more hydrophobic LDL particles retain a larger fraction of DS- ions than do HDL particles. Under these conditions, the LDL particles have a significantly higher charge/volume ratio than do HDL particles and a large difference in mu eff values is achieved. The conditions for maximizing the mu eff difference were found by studying the influence of the concentration of the DS- ions and percent acetonitrile of the mu eff of LDL and HDL at high pH.

252Californium plasma desorption mass spectrometry.

The mass spectrometry of involatile molecules presents problems not encountered when the molecule can be volatilized and subsequently ionized in the gas phase. The production of ions from a condensed phase is sensitively dependent on the properties of the matrix. The presence of impurities in the sample can totally quench molecular ion formation even though the sample contains a high percentage of the molecule of interest. One of the methods used for the mass spectrometry of involatile molecules is 252Californium plasma desorption mass spectrometry. The basic principles of this method will be presented with examples of its application that include the identification of involatile marine toxins (including the red tide toxins). We shall also introduce a new variation in mass spectrometry, a study of time-dependent effects, a technique that can be used to follow dynamic changes as a result of chemical reactions occurring in the sample at room temperature. This is possible in 252Californium plasma desorption mass spectrometry because the sample is conserved during the analysis.

Fragmentation of proteins in the 13- to 29-kDa mass range observed by 252Cf-plasma desorption mass spectrometry.

Results are presented on the observation of sequence-specific fragmentation of proteins in the 13- to 29-kDa mass range (ribonuclease A, papain, and proteinase K) by 252Cf-plasma desorption mass spectrometry. For these proteins, extensive N-terminal an, cn + 2, and dn fragment ions are observed with mass accuracies approaching 200 ppm for the most prominent fragment ions. The fragmentation pattern of these proteins shows the same fundamental behavior (arginine-directed, charge-remote fragmentation) that is observed in the fragmentation of small peptides, indicating that similar processes are occurring when large proteins and small peptides fragment in plasma desorption mass spectrometry.

A novel mass spectrometric procedure to rapidly determine the partial structure of heparin fragments.

The molecular weight and degree of sulfation has been obtained for di-, tetra- and hexasaccharide fragments of heparin obtained by enzymatic depolymerization of porcine mucosal heparin. The sodium salt form of the sulfated oligosaccharide is adsorbed onto an immobilized cationic surfactant film which is inserted directly into the mass spectrometer. Analyses are routinely obtained on 25-50 microgram samples in less than an hour. This approach provides rapid confirmatory structural information that is complementary to existing methodologies.

Heterogeneity of bacterial antigenic lipooligosaccharides determined by californium-252 plasma desorption mass spectrometry.

Californium-252 plasma desorption mass spectrometric analysis of complex lipooligosaccharide antigens from Mycobacterium kansasii has provided molecular weight information. From the spectra obtained and from other data it can be concluded that these lipooligosaccharides generally contain three 2,4-dimethyltetradecanoyl groups, and considerable heterogeneity in the attached acyl functions is apparent.

Self-assembled chlorophyll a systems as studied by californium-252 plasma desorption mass spectroscopy.

Self-assembled chlorophyll a and pheophytin a systems in thin solid films have been studied by (252)Cf plasma desorption mass spectrometry (PDMS). The (252)Cf-PDMS spectra of these films show monomer cation and anion molecular ions, ions of molecular aggregates, and positive and negative ion fragmentation patterns arising from the loss of various aliphatic side chains from the chlorin ring. Chlorophyll a films cast from dry carbon tetrachloride solution, in which chlorophyll a is known to occur as the dimer, produced an abundant dimer ion. The highest degree of chlorophyll a self-assembly was observed in chlorophyll a films cast from n-octane solutions. Oligomer ions extending upwards in size to the heptamer were detected in this system.