Improved selective ion monitoring mass-spectrometric assay for the determination of n,n-dimethyltryptamine in human blood utilizing capillary column gas chromatography

A gas—liquid chromatographic—mass spectrometric procedure is described for the assay of dimethyltryptamine (DMT) in whole blood. The use of a glass capillary column in combination with selective ion monitoring results in an assay with a high degree of specificity and sensitivity. 5-Methoxy-DMT is used as an internal standard and carrier in the isolation procedure. The superior resolving characteristics of the capillary column (as compared to previously employed packed columns) allows monitoring of the intense m/e 58 ion arising from the DMT side-chain. A sensitivity limit of 10 pg/ml blood is realized with a 10-ml blood sample.     

Molecular sieve chromatography of proteoglycans: a comparative analysis.

The separation of three major populations of proteoglycans (PG) by molecular sieve chromatography with either controlled-pore glass beads (CPG) or 1% agarose (A-150m) is compared. The resolving power and recovery on CPG or A-150m columns are comparable. However, CPG columns can be operated at a flow rate 50–100 times higher than those packed with 1% agarose. Chromatography on A-150m separates the purifled PG into three major peaks in a single run. Two sequential runs using material of two different pore sizes (CPG-10-1250, CPG-10-2500) are needed to obtain the same separation with CPG. CPG-10-1250 separates the ubiquitous peak (II) from the two peaks known to have an aggregate-subunit relationship. CPG-10-2500 resolves these two peaks allowing analysis of their interactions. Larger pore sized beads (CPG-10-2795) reveal size heterogeneity within the PG aggregate population.     

Determination of fatty acid compositions of Bacillus cereus and related bacteria: a rapid gas chromatographic method using a glass capillary column.

A rapid gas chromatographic method for the determination of fatty acid compositions of Bacillus cereus and related bacteria is presented. By the use of a free fatty acid phase-coated glass capillary column, the complete separation of fatty acids, including the branched ones, was achieved. The method enables a more distinct differentiation of Bacillus species than can be obtained with packed columns.     

Determination of fatty acid compositions of Bacillus cereus and related bacteria: a rapid gas chromatographic method using a glass capillary column.

A rapid gas chromatographic method for the determination of fatty acid compositions of Bacillus cereus and related bacteria is presented. By the use of a free fatty acid phase-coated glass capillary column, the complete separation of fatty acids, including the branched ones, was achieved. The method enables a more distinct differentiation of Bacillus species than can be obtained with packed columns.     

Glass capillary or fused-silica gas chromatography—mass spectrometry of several monosaccharides and related sugars: Improved resolution

The gas chromatographic separation of several monosaccharides and related sugars derivatized by methoxylation and trimethylsilylation reactions was optimized with glass capillary (SP-2250) and fused silica (SP-2100) columns. Individual sugars included aldoses, ketoses, polyols, acidic forms and N-acetylated amino sugars. Peaks were detected by selected ion monitoring (SIM). The fused silica column gave complete resolution of all peaks (two per hexose and one per hexitol) arising from glucose, galactose, mannose, fructose, sorbitol, mannitol and dulcitol. The resolution of these sugars with the glass capillary column was not as good, but full differentiation was possible on the basis of SIM. Because the fused silica column gave a better resolution of 33 sugars tested and was more easily installed than the glass capillary column, it was utilized for quantitative analysis. A deuterated algal sugar mixture used for quantitation by isotope dilution was found to contain glucose, galactose, mannose, xylose, arabinose, ribose and rhamnose. Full recoveries were obtained of various amounts of glucose, galactose, mannose, fructose and xylose added to human serum.     

Effects of various concentrations and combinations of prostaglandins on in vitro migration of frozen-thawed ram spermatozoa in cervical mucus of ewes

The aim of this investigation was to study the effects of various concentrations and combinations of prostaglandins (PG) on sperm migration in cervical mucus collected from estrous ewes. Semen from six rams was pooled and extended in Tes-tris-yolk-glycerol to 500 x 10(6) sperm per ml, cooled and held at 5 degrees C for three hours. Aliquots of the semen were supplemented with various concentrations of PGF(2alpha) or PGE(1), combinations of PGF(2alpha) + E(1), or PGE(1) + E(2) + E(3) + F(1alpha) + F(2alpha). The semen was filled into 0.25-ml French straws and immediately frozen in liquid nitrogen vapor. The semen was thawed in a 35 degrees C water bath for two minutes. Cervical mucus was collected from 30 ewes, pooled and filled in the capillary tubes. Frozen-thawed ram semen with PG added was brought into contact with cervical mucus, held at 37 degrees C and evaluated for sperm migration in the capillary tube at 15, 30, 60, 120, and 180 minutes under a phase-contrast microscope. At 180 minutes, sperm numbers were counted and sperm motility was graded at 20, 30, 40, 50, and 60-mm distances in the capillary tube. Analysis of the results showed that sperm migration distance increased with time, but there was no difference between the controls and samples with PG. At 180 minutes, sperm motility and number decreased, while distance of migration in the capillary tube increased. This sperm response was similar, whether the supplementation of semen was with any of several individual PGs, or a combination of two or five PGs.     

Gas chromatographic determination of the optical purities of amino acids using N-trifluoroacetyl menthyl esters

Nineteen α-amino acids and one β-amino acid were resolved as their N-TFA menthyl esters by gas-liquid chromatography. Neutral and basic amino acids were well resolved on some conventional packed columns. For the complete resolution of acidic amino acids, a capillary column was used. The results of the applications to some biochemical substances showed that this method is of value for practical usage. The mechanism of the resolution was also investigated and showed that the difference between the derivatives of the enantiomeric pairs in the ability to form intermolecular hydrogen bond contributes essentially to the resolution.     

Monolithic organic polymeric columns for capillary liquid chromatography and electrochromatography

This review briefly summarizes the present state of the preparation and use of capillary monolithic columns for liquid chromatography (LC) and electrochromatography (EC). Most important approaches to the preparation of monolithic stationary phases based on organic polymers are outlined and the properties of the monoliths obtained are compared with those of classical particulate phases. A few selected applications of monolithic columns are shown to demonstrate the most important advantages of monolithic capillary columns. It is concluded that both the monolithic and particulate capillary columns are important and that judicious choice of the type suitable for a particular application requires careful consideration of the purpose of the separation and the properties of the solutes to be separated. Monolithic columns are substantially younger than packed ones and thus will require further theoretical and experimental study to further improve their preparation and to enable reliable prediction of their properties and applicability; nevertheless, they are very promising for the future.     

Applications of monolithic silica capillary columns in proteomics

The use and applicability of silica based capillary monolithic reversed-phase columns in proteomic analysis has been evaluated by liquid chromatography-mass spectrometry (LC-MS). Chromatographic performance of the monolithic capillaries was evaluated with a tryptic digest of cytochrome C showing very good resolution and reproducibility in addition to the known advantages of a low pressure drop over a time period of 6 months. Monoliths were subsequently tested for their suitability to separate proteins and peptides from samples typically encountered in proteomic research such as in-gel digested tryptic peptide mixtures or fractions of proteolytically digested human serum. The monolithic capillaries also proved useful in the analysis of phospholipid species in bronchoalveolar lavage fluid. Compared to particle-filled conventional capillary columns, rapid and highly efficient separation of peptides and proteins was achieved using these bimodal pore size distribution columns, and good quality collision induced dissociation (CID) mass spectra were obtained on an ion trap mass spectrometer. These novel monolithic separation media are thus a promising addition to the methodological toolbox of proteomics research.     

Purification and properties of prostaglandin 9-ketoreductase from pig and human kidney. Identity with human carbonyl reductase.

Prostaglandin 9-ketoreductase (PG-9-KR) was purified from pig kidney to homogeneity, as judged by SDS/PAGE using an improved procedure. The enzyme is pro-S stereoselective with regard to hydrogen transfer from NADPH with prostaglandin E2 as substrate and reduces its 9-keto group with approximately 90% stereoselectivity to form prostaglandin F2 alpha. Approximately 8% of the prostaglandin F formed has the beta-configuration. In addition to catalyzing the interconversion of prostaglandin E2 to F2 alpha, PG-9-KR also oxidizes prostaglandin E2, F2 alpha and D2 to their corresponding, biologically inactive, 15-keto metabolites. Incubation of PG-9-KR with prostaglandin F2 alpha and NAD+ leads to the preferential formation of 15-keto prostaglandin F2 alpha rather than prostaglandin E2. This suggests that the prostaglandin E2/prostaglandin F2 alpha ratio is not determined by the NADP+/NADPH redox couple. The enzyme also reduces various other carbonyl compounds (e.g. 9,10-phenanthrenequinone) with high efficiency. The catalytic properties measured for PG-9-KR suggest that its in vivo function is unlikely to be to catalyze formation of prostaglandin F2 alpha. The monomeric enzyme has a molecular mass of 32 kDa and exists as four isoforms, as judged by isoelectric focusing. PG-9-KR contains 1.9 mol Zn2+/mol enzyme and no other cofactors. Human kidney PG-9-KR was also purified to homogeneity. The human enzyme has a molecular mass of 34 kDa and also exists as four isoforms. Polyclonal antibodies raised against pig kidney PG-9-KR cross-react with human kidney PG-9-KR and also with human brain carbonyl reductase, as demonstrated by Western blot analysis. Sequence data of tryptic peptides from pig kidney PG-9-KR show greater than 90% identity with human placenta carbonyl reductase. From comparison of several properties (catalytical, structural and immunological properties), it is concluded that PG-9-KR and carbonyl reductase are identical enzymes.     

Simultaneous extraction and separation of trace amines of biological interest

A method is described for the simultaneous extraction and separation of the trace amines 2-phenylethylamine, m-tyramine, p-tyramine, p-octopamine, normetanephrine, and 3-methoxytyramine. The method involves acetylation in aqueous solution, specific hydrolysis of phenolic acetate groups, derivatization with trifluoroacetic anhydride and analysis on a gas chromatograph equipped with an electron-capture detector. Analyses utilizing both packed glass columns and glass capillary columns are described.The method possesses the potential for quantitative as well as qualitative analysis, with one or more of the following amines employed as internal standards: benzylamine, 3-phenylpropylamine, tranylcypromine, and 2-(4-chlorophenyl)ethylamine.     

A central role for the armadillo protein plakoglobin in the autoimmune disease pemphigus vulgaris

In pemphigus vulgaris (PV), autoantibody binding to desmoglein (Dsg) 3 induces loss of intercellular adhesion in skin and mucous membranes. Two hypotheses are currently favored to explain the underlying molecular mechanisms: (a) disruption of adhesion through steric hindrance, and (b) interference of desmosomal cadherin-bound antibody with intracellular events, which we speculated to involve plakoglobin. To investigate the second hypothesis we established keratinocyte cultures from plakoglobin knockout (PG-/-) embryos and PG+/+ control mice. Although both cell types exhibited desmosomal cadherin-mediated adhesion during calcium-induced differentiation and bound PV immunoglobin (IgG) at their cell surface, only PG+/+ keratinocytes responded with keratin retraction and loss of adhesion. When full-length plakoglobin was reintroduced into PG-/- cells, responsiveness to PV IgG was restored. Moreover, in these cells like in PG+/+ keratinocytes, PV IgG binding severely affected the linear distribution of plakoglobin at the plasma membrane. Taken together, the establishment of an in vitro model using PG+/+ and PG-/- keratinocytes allowed us (a) to exclude the steric hindrance only hypothesis, and (b) to demonstrate for the first time that plakoglobin plays a central role in PV, a finding that will provide a novel direction for investigations of the molecular mechanisms leading to PV, and on the function of plakoglobin in differentiating keratinocytes.     

Estimation of plasma free fatty acids as the trimethylsilyl (TMS) esters

Quantitative estimates of free fatty acids in total lipid extracts of plasma were obtained by glc on nonpolar columns following trimethylsilylation. The presence of other lipid esters in the reaction mixture had no effect upon the yield of the trimethylsilyl (TMS) esters or upon their resolution on the glc column. Routine quantitations by gas-liquid chromatography (glc) were obtained on 2 ft × 1/8 in. o.d. stainless steel columns packed with 3% OV-1 on 100–120 mesh Gas Chrom Q by means of temperature programming in the range 175–350°C with tridecanoin as internal standard. High resolution glc of the TMS esters of fatty acids was done on a 6 ft × 1/8 in. o.d. glass column packed with 1% SE-30 on Gas Chrom Q. In both instances the fatty acids were resolved on the basis of carbon number and by the presence or absence of double bonds. On gas chromatography/mass spectrometry (GC/MS), TMS esters of fatty acids were shown to yield proportionally greater amounts of high mass fragments, including the parent ions, than their methyl or ethyl esters, which has special advantages for the detection and characterization of polyunsaturated fatty acids.     

Insertion selectivity of antimicrobial peptide protegrin-1 into lipid monolayers: Effect of head group electrostatics and tail group packing

The ability to selectively target the harmful microbial membrane over that of the host cell is one of the most important characteristics of the antimicrobial peptides (AMPs). This selectivity strongly depends on the chemical and structural properties of the lipids that make up the cell membrane. A systematic study of the initial membrane selectivity of protegrin-1 (PG-1), a β-sheet AMP, was performed using Langmuir monolayers. Constant pressure insertion assay was used to quantify the amount of PG-1 insertion and fluorescence microscopy was employed to observe the effect of PG-1 on lipid ordering. Charge and packing properties of the monolayer were altered by using lipids with different head groups, substituting saturated with unsaturated lipid tail group(s) and incorporating spacer molecules. PG-1 inserted most readily into anionic films composed of phosphatidylglycerol (PG) and lipid A, consistent with its high selectivity for microbial membranes. It also discriminated between zwitteranionic phospholipids, inserting more readily into phosphatidylcholine (PC) monolayers than those composed of phosphatidylethanolamine, potentially explaining why PG-1 is hemolytic for PC-rich human erythrocytes and not for the PE-rich erythrocytes of ruminants. Increased packing density of the monolayer by increased surface pressure, increased tail group saturation or incorporation of dihydrocholesterol diminishes the insertion of PG-1. Fluorescence microscopy shows that lipid packing is disordered upon PG-1 insertion. However, the presence of PG-1 can still affect lipid morphology even with no observed PG-1 insertion. These results show the important role that lipid composition of the cell membrane plays in the activity of AMPs.     

Covalent immobilization of proteins and peptides for solid-phase sequencing using prepacked capillary columns

The use of prepacked capillary columns for immobilizing proteins and peptides for solid-phase Edman degradation is described. Capillary tubes with an internal volume of about 30 microliters are filled with glass beads bearing isothiocyanato groups (DITC-glass), aminophenyl groups (AP-glass), or aminoethylaminopropyl groups (AEAP-glass) and are sealed with porous plugs. Proteins or peptides in appropriate buffers are introduced into the columns by capillary action and are covalently coupled to the glass beads, either by reaction of lysine side-chain amino groups with DITC-glass, by carbodi-imide-mediated reaction of carboxyl groups with AP-glass, or by reaction of homoserine lactone groups with AEAP-glass. Optimization of attachment conditions is described. The capillary columns are loaded into the sequencer and, when sequencing has been completed, are discarded. This technique greatly simplifies polypeptide immobilization and is suitable for microsequencing (less than 50-1000 pmol) or macrosequencing (1-50 nmol).     

Mechanisms of plakoglobin-dependent adhesion: desmosome-specific functions in assembly and regulation by epidermal growth factor receptor

Plakoglobin (PG) is a member of the Armadillo family of adhesion/signaling proteins that can be incorporated into both adherens junctions and desmosomes. Loss of PG results in defects in the mechanical integrity of heart and skin and decreased adhesive strength in keratinocyte cultures established from the skin of PG knock-out (PG-/-) mice, the latter of which cannot be compensated for by overexpressing the closely related beta-catenin. In this study, we examined the mechanisms of PG-regulated adhesion in murine keratinocytes. Biochemical and morphological analyses indicated that junctional incorporation of desmosomal, but not adherens junction, components was impaired in PG-/- cells compared with PG+/- controls. Re-expression of PG, but not beta-catenin, in PG-/- cells largely reversed these effects, indicating a key role for PG in desmosome assembly. Epidermal growth factor (EGF) receptor activation resulted in Tyr phosphorylation of PG, which was accompanied by a loss of desmoplakin from desmosomes and decreased adhesive strength following 18-h EGF treatment. Importantly, introduction of a phosphorylation-deficient PG mutant into PG null cells prevented the EGF receptor-dependent loss of desmoplakin from junctions, attenuating the effects of long term EGF treatment on cell adhesion. Therefore, PG is essential for maintaining and regulating adhesive strength in keratinocytes largely through its contributions to desmosome assembly and structure. As a target for modulation by EGF, regulation of PG-dependent adhesion may play an important role during wound healing and tumor metastasis.     

Gas—liquid chromatography of isobutyl ester,N(O)-heptafluorobutyrate derivatives of amino acids on a glass capillary column for quantitative separation in clinical biology

A gas chromatographic method adapted to routine analysis has been developed for quantitative separation on glass capillary columns for free proteic and other known amino acids normally or abnormally found in physiological fluids. The procedure involves ion-exchange chromatography and isobutyl ester, N(O)-heptafluorobutyrate derivatization of free plasma and urine amino acid samples. Derivatized components were ascertained by combined gas chromatography—mass spectrometry. The use of glass for the capillary column is mandatory to achieve qualitative and quantitative analysis of the known occurring amino acids in urine and small plasma samples. Quantitative analysis of several types of human amino acid disorders are presented.     

Insertion selectivity of antimicrobial peptide protegrin-1 into lipid monolayers: effect of head group electrostatics and tail group packing

The ability to selectively target the harmful microbial membrane over that of the host cell is one of the most important characteristics of the antimicrobial peptides (AMPs). This selectivity strongly depends on the chemical and structural properties of the lipids that make up the cell membrane. A systematic study of the initial membrane selectivity of protegrin-1 (PG-1), a beta-sheet AMP, was performed using Langmuir monolayers. Constant pressure insertion assay was used to quantify the amount of PG-1 insertion and fluorescence microscopy was employed to observe the effect of PG-1 on lipid ordering. Charge and packing properties of the monolayer were altered by using lipids with different head groups, substituting saturated with unsaturated lipid tail group(s) and incorporating spacer molecules. PG-1 inserted most readily into anionic films composed of phosphatidylglycerol (PG) and lipid A, consistent with its high selectivity for microbial membranes. It also discriminated between zwitteranionic phospholipids, inserting more readily into phosphatidylcholine (PC) monolayers than those composed of phosphatidylethanolamine, potentially explaining why PG-1 is hemolytic for PC-rich human erythrocytes and not for the PE-rich erythrocytes of ruminants. Increased packing density of the monolayer by increased surface pressure, increased tail group saturation or incorporation of dihydrocholesterol diminishes the insertion of PG-1. Fluorescence microscopy shows that lipid packing is disordered upon PG-1 insertion. However, the presence of PG-1 can still affect lipid morphology even with no observed PG-1 insertion. These results show the important role that lipid composition of the cell membrane plays in the activity of AMPs.     

Effect of Heat Treatment on the Development of Polygalacturonase Activity in Tomato Fruit during Ripening

When mature green tomato fruits are stored at 22?C for 30 days,they ripen normally and soften, but if they are kept at 33?Cfor 15 days (heat treatment), then stored at 22?C they do notsoften. The effect of heat treatment on the development of polygalacturonase(PG, EC 3.2.1.15 [EC] ) activities in tomato fruits during storagetherefore was studied. When mature green tomato fruits werestored at 22?C, PG activity, which had not been detectable inthe fruits, appeared as the color changed and increased dramaticallythereafter. PG activity, however, did not appear during heattreatment. When heat-treated fruits were transferred to 22?C,PG activity appeared after a 6-day lag period and increasedduring the next 30 days at 22?C to about 15% of the value detectedin ripe tomato fruits. The PG in ripe tomato fruits was composed of two isoenzymesthat had different mol wts. A high molecular form (PG-1, molwt 100K) appeared during the early stage of ripening and a lowmolecular form (PG-2, mol wt 44K) a little later. PG-2 increasedvigorously during ripening and eventually accounted for mostof the enzyme activity in the ripe fruits. Only a single isoenzyme(Y-PG, mol wt 100K), however, was detected in heat-treated tomatofruits stored at 22?C for 30 days. PG-1 and Y-PG gave the samemol wt on Sephacryl S-200 gel nitration, but could be separatedby Toyopearl HW-55 F gel filtration. (Received October 31, 1983; Accepted February 20, 1984)