Relationships between geometry and kinematic characteristics in the temporomandibular joint.

Motions of the temporomandibular joint (TMJ) involve both translation and rotation; however, there may be substantial variations from one human to another, and these variations present significant difficulties when designing TMJ prostheses. The disc-condyle glides along the temporal bone and the condyle centre describe a curve that depends on the individual morphology. This study analyses disc-condyle rotatory and translatory displacements moving all along the temporal bone facets which are mainly composed of two areas: the articular tubercle slope (ATS) and the preglenoid plane separated by the articular tubercle crest. Displacements were quantified using 3D video analysis, and this technique was computer-assisted. From a population of 32 volunteers, we were able to establish a correlation between the kinematic characteristics of the joint and the disc-condyle trajectories. This study quantifies the geometrical characteristics of the ATS and their inter-individual variations, which are useful in TMJ prosthesis design.

HEXIM1 targets a repeated GAUC motif in the riboregulator of transcription 7SK and promotes base pair rearrangements.

7SK snRNA, an abundant RNA discovered in human nucleus, regulates transcription by RNA polymerase II (RNAPII). It sequesters and inhibits the transcription elongation factor P-TEFb which, by phosphorylation of RNAPII, switches transcription from initiation to processive elongation and relieves pauses of transcription. This regulation process depends on the association between 7SK and a HEXIM protein, neither isolated partner being able to inhibit P-TEFb alone. In this work, we used a combined NMR and biochemical approach to determine 7SK and HEXIM1 elements that define their binding properties. Our results demonstrate that a repeated GAUC motif located in the upper part of a hairpin on the 5'-end of 7SK is essential for specific HEXIM1 recognition. Binding of a peptide comprising the HEXIM Arginine Rich Motif (ARM) induces an opening of the GAUC motif and stabilization of an internal loop. A conserved proline-serine sequence in the middle of the ARM is shown to be essential for the binding specificity and the conformational change of the RNA. This work provides evidences for a recognition mechanism involving a first event of induced fit, suggesting that 7SK plasticity is involved in the transcription regulation.

Discrimination of objective kinematic characters in temporomandibular joint displacements.

Designing a temporomandibular joint (TMJ) total prosthesis requires the assessment of joint displacements for open/close movements. Current knowledge presents disc-condyle motions as involving both translation and rotation but there may be substantial variations from human to human. The aim of this study is to discriminate objective kinematic characters amongst thirty-two volunteers. The displacements are determined using 3D video analysis. The ratio between rotation and translation can be defined by introducing a coefficient. This coefficient varies relatively to the opening amplitude and presents the same dispersion rate whatever the variations. Then it allows to discriminate amongst volunteers, regardless of any jaw opening values. Three groups can be isolated relatively to three kinematic models: a translatory preponderant group, a common group and a rotatory preponderant group. All subjects in the first group present concomitant rotatory/translatory displacements up to maximal opening. The other two groups present variations due to different quasi-pure rotation phases at the end of the opening movement. These investigations will make it possible to establish a correlation between the kinematic characters and the disc-condyle trajectories. The disc-condyle glides along the temporal facet and the condyle centre describes the tubercular morphology. The temporal facet geometry, useful for the TMJ prosthesis design, will be studied in a next paper.

Application of packed capillary liquid chromatography/mass spectrometry with electrospray ionization to the study of the human biotransformation of the anti-emetic drug dolasetron.

Packed capillary liquid chromatography/mass spectrometry (LC/MS) using electrospray ionization (ESI) was used to study the human biotransformation of the anti-emetic drug dolasetron. Urine from subjects given a single 100 mg intravenous dose, containing 14C-labeled dolasetron (50 microCi), was de-salted and concentrated for LC/MS with minimal loss of radioactivity (97% recovery). Aliquots of the de-salted material were injected directly onto a C8 packed capillary column (25 cm x 0.32 mm i.d.) and eluted with an acetonitrile-water gradient, buffered with 1% acetic acid, at a flow rate of 2 microliters min-1. Five metabolites were detected by LC ESI-MS which, yielded molecular mass information but no fragmentation. The identity of each metabolite was confirmed in a subsequent analysis using product ion scans in conjunction with collisionally induced dissociation. Precursor ion scanning was also employed and did not reveal any new biotransformation products. In addition to defining the major routes of biotransformation, the data obtained were compared with a 14C radioprofile prepared in a separate experiment. Qualitative agreement in the two chromatographic profiles enabled the major clusters of radioactivity to be assigned to specific metabolites of dolasetron. An important observation in this comparison was that the signal obtained by ESI did not provide an accurate assessment of the quantity of each metabolite. This was especially true for acidic conjugates (i.e. glucuronides, sulfates), which in the case of dolasetron can exist as zwitterions (no net charge). The results demonstrate the power of packed capillary LC ESI-MS for use in drug biotransformation studies and suggest that caution should be exercised when interpreting relative metabolite abundances from ESI data in the absence of actual reference standards.

Rapid analysis of antibiotic-containing mixtures from fermentation broths by using liquid chromatography-electrospray ionization-mass spectrometry and matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry.

A crucial step in the isolation of antibiotic substances is establishing whether or not the isolated material represents a new chemical entity. Because of the importance of molecular weight to this process-known as dereplication-mass spectrometry has traditionally played an active role. In this communication a strategy for utilizing liquid chromatography-mass spectrometry (LC/MS) for novelty assessment is described. Crude extracts (20-50 µg) are chromatographed by conventional bore high-performance liquid chromatography (1 mL/min) after which a postcolumn split to divert roughly one-tenth of the sample to the mass spectrometer for molecular weight determination by electrospray ionization (ESI) mass spectrometry. The majority of the effluent is sent to a UV detector and ultimately collected as 1-min fractions for biological testing. As a secondary confirmation of molecular weight, an aliquot of each fraction (< 5%) is taken for analysis by matrix-assisted laser desorption ionization (MALDI). The improved efficiency of this approach over more traditional schemes utilizing off-line fraction collection and conventional ionization methods can be explained by several factors. First, the superior sensitivity of ESI and MALDI means that less material is required for successful analysis. Second, on-line LC/MS optimizes the efficiency of sample transfer and saves both time and labor. Furthermore, the concentration dependence of ESI allows a majority of the material injected for LC/MS to be recovered for biological testing without compromising the signal available for molecular weight determination. As a validation of the above method, crude extracts containing two well-characterized antibiotics-teicoplanin and phenelfamycin-were examined. Results from these analyses are presented along with data from the analysis of a potent unknown antifungal sample.

Pharmacokinetics of MDL 63,246, a new semisynthetic glycopeptide antibiotic, in the rat.

Following intravenous administration in the rat, the concentration of MDL 63,246 in plasma was high and long-lasting. Concentrations fell with an apparent three-exponential decay. MDL 63,246 was distributed in a high volume and was cleared quite slowly. The pharmacokinetics of MDL 63,246 in the rat appear to be dose proportional in the dose range of 20 to 50 mg/kg of body weight. When administered subcutaneously, MDL 63,246 was slowly absorbed from the injection site, and the extent of availability was good, being 70.1%. MDL 63,246 was eliminated slowly by both renal and fecal excretion. MDL 63,246 is rapidly and extensively distributed into the tissues. At 0.5 h after drug administration, radioactivity was detected in all organs examined. At 24 h after administration, the total concentrations of radioactivity still increased in some organs which represent a slowly equilibrating compartment, but only the kidneys and liver showed a higher total concentration of radioactivity than plasma. Between 24 and 192 h after treatment, total concentrations of radioactivity decreased very slowly, and finally, apart from brain, all tissues showed higher concentrations than plasma, indicating a very high affinity of MDL 63,246 for tissues. The ratio of the concentration of radioactivity in blood to that in plasma ratio was 0.58, indicating that MDL 63,246 does not diffuse into erythrocytes and that binding to the erythrocyte membrane does not occur. All of these findings appear to correlate with the excellent in vitro and in vivo activities of the compound, suggesting that MDL 63,246 could be therapeutically efficacious at lower dosages and longer treatment intervals than those currently used for vancomycin and teicoplanin.

Preliminary investigation of glycosylated proteins by capillary electrophoresis and capillary electrophoresis/mass spectrometry using electrospray ionization and by matrix-assisted laser desorption ionization/time-to-flight mass spectrometry.

In recent years, the biotechnological industry has emerged as the major source of new human therapeutic proteins. Although the great majority of these occur naturally as glycoproteins, it has been observed that glycosylation of the recombinantly produced proteins could be fundamental for their in vivo activity (e.g. tissue plasminogen activator, erythropoietin) or, on the contrary, insignificant (e.g. interleukin-1 receptor antagonist, gamma-interferon, granulocyte macrophage-colony-stimulating factor). The inherent heterogeneity of these complex biomolecules presents an exciting challenge in the analytical field for both their structural analysis and the development of suitable analytical methods to guarantee consistency of their production. Owing to this ever increasing therapeutic interest in proteins and glycoproteins, this paper compares the information provided by different analytical techniques (i.e. high-performance liquid chromatography, sodium dodecyl sulphate polyacrylamide gel electrophoresis, capillary electrophoresis, capillary electrophoresis/electrospray ionization mass spectrometry, matrix-assisted laser desorption/ionization time-of flight mass spectrometry and high-performance liquid chromatography/matrix-assisted laser desorption/ionization time-of flight mass spectrometry when used for the analysis of proteins and glycoproteins. For the sake of simplicity, reference standard proteins and glycoproteins were used as samples.

Determination of dolasetron and its reduced metabolite in human plasma by GC-MS and LC.

Both a GC-MS and an LC method have been developed for the simultaneous quantitation of dolasetron and reduced dolasetron in human plasma. The GC-MS method has been utilized in preliminary human pharmacokinetic studies of dolasetron mesylate. Selected ion monitoring was used in these initial studies to obtain the sensitivity and specificity required for quantitation. The GC-MS method has been used in the range of 1-120 ng ml-1 for dolasetron and 1-240 ng ml-1 for reduced dolasetron in plasma. The limit of quantitation for both compounds by GC-MS was 1 ng ml-1. Recently, an LC method has been utilized for quantitation of both compounds on a routine basis. This method utilizes essentially the same sample preparation procedure as the GC-MS method. The LC method has been used in the range of 5-200 ng ml-1 in plasma for dolasetron and reduced dolasetron. In addition, the relationship between the LC and GC-MS methods has been assessed using data obtained from human male volunteers following intravenous administration of 3.0 mg kg-1 of dolasetron mesylate monohydrate.

Evidence for a lysine-specific fragmentation in fast-atom bombardment mass spectra of peptides.

A fragmentation process observed for peptides that contain lysine, or other amino acids which possess a free amino group on their sidechain, is reported. The ions generated by this process are found 16 Da below the acylium-type B ions that result from fragmentation at the C-terminal side of lysine or other amine-containing residues in fast-atom bombardment (FAB) mass spectra. These ions, which are referred to as (B-16) ions, permit differentiation between the isobaric amino acids lysine and glutamine in peptide mass spectra. High resolution measurements indicate that (B-16) ions differ in composition from the corresponding B ions by the removal of one oxygen atom. Formation is believed to occur through a cyclization process initiated by nucleophilic attack by the free amino group of the lysine sidechain at the carbon of the acylium ion (B ion). A similar process initiated directly from the protonated peptide may also occur. Analogous cyclization processes are restricted for glutamine because this residue is comparatively less nucleophilic than lysine (i.e., amide vs amine). Although (B-16) ions have been detected under high energy collisionally induced dissociation, they are formed less readily than by FAB mass spectrometry. A mechanism consistent with this observation as well as other experimental evidence is presented to account for the formation of (B-16) ions.

Metabolism of the anticoagulant peptide, MDL 28,050, in rats.

Rats were each administered a 9 mg/kg iv bolus dose of a 3H-labeled decapeptide anticoagulant, MDL 28,050. Tritium was eliminated rapidly with approximately 50% of the dose recovered in urine within the first 6 hr. Renal excretion accounted for 68% of the dose, whereas fecal excretion accounted for 16% of the dose. Continuous flow fast atom bombardment mass spectrometry was used to identify the major urinary metabolites of MDL 28,050. Trace amounts of parent drug were found, and other biotransformation products indicated that hydrolysis had occurred at four peptide bonds. Two initial sites of hydrolysis were identified as 4I-5P and 6E-7E, which resulted in the peptide fragments Suc-Y-E-P-I-OH + P-E-E-A-Cha-E-OH and Suc-Y-E-P-I-P-E-OH + E-A-Cha-E-OH, respectively. Further metabolism of these fragments resulted in the N-terminal pentapeptide and the C-terminal dipeptide.

Determination of terfenadine and terfenadine acid metabolite in plasma using solid-phase extraction and high-performance liquid chromatography with fluorescence detection.

This work describes the methodology for the analysis of terfenadine and the acid metabolite of terfenadine in plasma using high-performance liquid chromatography. The use of solid-phase extraction allows the use of robotic or manual sample preparation for the efficient clean-up of terfenadine and terfenadine acid metabolite from plasma. Additional selectivity is obtained through the use of fluorescence detection. For terfenadine, the validated quantitation range of this method is 10.0-84.2 ng/ml with coefficients of variation of 5.7-30%. For terfenadine acid metabolite, the validated quantitation range of this method is 8.2-500 ng/ml with coefficients of variation of 4.1-24%.

Incorporation of tandem mass spectrometric detection to the analysis of peptide mixtures by continuous flow fast atom bombardment mass spectrometry.

The ability to acquire structurally informative daughter ion spectra for individual peptides undergoing separation and analysis by continuous flow fast atom bombardment (CF FAB) is demonstrated. To illustrate the potential of this methodology, tryptic and chymotryptic digests of the 29-residue peptide glucagon were analyzed by CF FAB using mass spectrometric and tandem mass spectrometric detection in consecutive analyses. Daughter ion spectra were recorded using B/E linked scans for the major hydrolysis products observed by liquid chromatography/mass spectrometry. The peptide mixtures were separated by gradient capillary high-performance liquid chromatography with the FAB matrix being added post-column using a coaxial flow interface between the column and flow probe. The entire effluent (3 microl min(-1)) was sampled by the mass spectrometer. Results obtained using less than 300 pmol of digested glucagon indicated several advantages to tandem mass spectrometric detection including the ability to confirm identities for products of enzymatic digestion and the potential use of this method for tandem sequence analysis of peptide mixtures.

Determination of the metabolites of terfenadine in human urine by thermospray liquid chromatography-mass spectrometry.

Thermospray liquid chromatography-mass spectrometry (TSP LC-MS) was used to determine human urinary metabolites of terfenadine after oral administration of terfenadine tablets. In addition to the two previously identified major metabolites, azacyclonol (MDL 4829) and the 'acid' metabolite (MDL 16,455), three additional metabolites were also detected. One of the additional metabolites was identified as the 'alcohol' metabolite (MDL 17,523) and the other two were proposed to be an 'aldehyde' and a 'ketone-acid' metabolites from their TSP mass spectra. The results of this study demonstrated that TSP LC-MS is a useful technique for the study of terfenadine biotransformation.

Interfacing microbore and capillary liquid chromatography to continuous-flow fast atom bombardment mass spectrometry for the analysis of glycopeptides.

This work describes a system to interface either microbore or packed capillary gradient liquid chromatography (LC) to fast atom bombardment mass spectrometry (FAB-MS). The interface incorporates on-line ultraviolet detection and post-column matrix addition to enable independent optimization of both LC and FAB-MS. The glycopeptide antibiotic teicoplanin was chosen as a model system because this group of compounds places severe demands on the chromatographic separation and is difficult to analyze by FAB-MS. For both microbore and capillary LC, high-quality mass spectra of the major components in teicoplanin were obtained; however, the increased sensitivity of the capillary system allowed spectra to be obtained at low picomole concentrations. The sensitivity and ease of use make capillary LC the preferred system for use in LC-FAB-MS.

Curtatoxins. Neurotoxic insecticidal polypeptides isolated from the funnel-web spider Hololena curta.

Three polypeptide neurotoxins (curtatoxins) were isolated from the venom of the spider Hololena curta by reverse-phase high performance liquid chromatography, gel permeation, and ion-exchange chromatography. The purified toxins induced an immediate paralysis in the cricket Acheta domestica that resulted in desiccation and death of the insect within 24-48 h (LD50 congruent to 4-20 micrograms/g); this toxic effect is consistent with irreversible presynaptic neuromuscular blockade. Curtatoxins are a class of sequence-related, cysteine-rich, carboxyl-terminal amidated polypeptides of 36 to 38 amino acid residues. The cysteine residues are conserved at identical sequence positions among these polypeptides and form 4 intramolecular disulfide bonds. Hydropathy calculations show that the toxins have an internal hydrophobic region flanked by hydrophilic and oppositely charged amino- and carboxyl-terminal ends. By analogy to other cysteine-rich arthropod venom proteins, the folded structure of the curtatoxins is likely important for their target specificity and mode of action at the neuromuscular junction.

The use of thermospray liquid chromatography/tandem mass spectrometry for the class identification and structural verification of phytoestrogens in soy protein preparations.

The thermospray mass spectra of the phytoestrogens have intense protonated molecular ions but contain few or no ions indicative of structure. Tandem mass spectrometry (MS/MS) was used to obtain daughter ion spectra containing ions unique to the different structural characteristics of each phytoestrogen subclass and was used both to confirm identification and propose structures for unknowns. In addition to unique daughter ion spectra, MS/MS was used as a class identifier to detect phytoestrogens through the neutral loss of 56 (due to consecutive losses of CO) that is common to all members of this family. Several sources of soy protein were investigated to confirm the presence or absence of phytoestrogens. In one preparation investigated, daidzein and genistein were detected as well as an unknown phytoestrogen of the Biochanin A subclass. This unknown has been tentatively identified as 6,7-dihydroxy-4'-methoxyisoflavone using its daughter ion spectrum.

Fast atom bombardment mass spectrometric investigation of in vitro degradation within the disulfide-linked core of atrial natriuretic factor.

The use of high-performance liquid chromatography and fast atom bombardment mass spectrometry are shown to be an efficient combination for investigating protease-mediated digestion of synthetic analogs of the peptide hormone ANF (atrial natriuretic factor). As examples of the reported methodology, rANF5-23-NH2 and rANF7-23-NH2 were digested with the endopeptidase thermolysin. These truncated analogs were selected to investigate metabolism within the disulfide-linked core of ANF, particularly at the Cys7-Phe8 bond. While this position was the site of initial hydrolysis for rANF5-23-NH2 (t1/2 = 0.5 min), the Cys7-Phe8 bond remained intact for all observed degradation products of rANF7-23-NH2 (t1/2 = 16 min). These findings suggest that improved stability towards endopeptidase-mediated core hydrolysis may be conferred to analogs of ANF by removal of the first six residues from the N-terminus.

Thermospray high-performance liquid chromatographic-mass spectrometric characterization of biological macromolecules. I. Analysis of acid hydrolysate of peptides.

A method for the routine analysis of phenylthiocarbamyl (PTC) amino acids by thermospray high-performance liquid chromatography-mass spectrometry (TSP LC-MS) is described. Data were acquired on a small dedicated TSP LC-MS system in which the temperature of the vaporizer and ion source block were optimized. PTC-amino acids exhibited unique TSP mass spectra containing sufficient fragment ions to determine structural data. Therefore, using this method the amino acids contained in the acid hydrolysates of unique and modified peptides were able to be positively identified. Additionally, the amino acid composition of peptides as determined by TSP LC-MS in the selected ion monitoring mode corresponded well with the theoretical value. The detection limits for the PTC-amino acids were at the low picomole level.

Identification in man of urinary metabolites of a new bronchodilator, MDL 257, using triple stage quadrupole mass spectrometry-mass spectrometry.

The structure elucidation of drug metabolites directly from urine by tandem mass spectrometry (MS/MS) for a new bronchodilator is described. When urine samples from human subjects dosed with 400 mg of MDL 257 were examined by MS/MS, three major urinary metabolites previously characterized in animal studies were confirmed and two previously unsuspected metabolites were identified. Using the operational modes of a triple stage quadrupole mass spectrometer, it is possible both to detect and to identify possible metabolites. Since the pure drug and its metabolites often contain common structural daughter ions, the parent spectra of these common daughter ions should contain some or all of the molecular ions of possible metabolites. Daughter spectra of these suspected molecular ions were obtained and the resulting daughter spectra were interpreted for structural information of suspected metabolites. This study confirms the utility of MS/MS to do rapid metabolic profiling and identification directly from complex samples such as urine, with minimal time for sample preparation and analysis. This technique can provide unique and complimentary data when combined with the more classical approaches such as HPLC profiling, isolation, and off-line spectroscopy.