Glycosylphosphatidylinositol anchors of Plasmodium falciparum: molecular characterization and naturally elicited antibody response that may provide immunity to malaria pathogenesis.

Induction of proinflammatory cytokine responses by glycosylphosphatidylinositols (GPIs) of intraerythrocytic Plasmodium falciparum is believed to contribute to malaria pathogenesis. In this study, we purified the GPIs of P. falciparum to homogeneity and determined their structures by biochemical degradations and mass spectrometry. The parasite GPIs differ from those of the host in that they contain palmitic (major) and myristic (minor) acids at C-2 of inositol, predominantly C18:0 and C18:1 at sn-1 and sn-2, respectively, and do not contain additional phosphoethanolamine substitution in their core glycan structures. The purified parasite GPIs can induce tumor necrosis factor alpha release from macrophages. We also report a new finding that adults who have resistance to clinical malaria contain high levels of persistent anti-GPI antibodies, whereas susceptible children lack or have low levels of short-lived antibody response. Individuals who were not exposed to the malaria parasite completely lack anti-GPI antibodies. Absence of a persistent anti-GPI antibody response correlated with malaria-specific anemia and fever, suggesting that anti-GPI antibodies provide protection against clinical malaria. The antibodies are mainly directed against the acylated phosphoinositol portion of GPIs. These results are likely to be valuable in studies aimed at the evaluation of chemically defined structures for toxicity versus immunogenicity with implications for the development of GPI-based therapies or vaccines.

Occurrence of novel branched-chain fatty acids in Refsum's disease.

Two novel branched-chain fatty acids, which appear to be unsaturated analogs of phytanic acid, have been observed in sera and urine of patients with Refsum's disease. They occur in both phospholipids and neutral lipids, and have been isolated and characterized.

Bioanalytic applications of mass spectrometry.

Mass spectrometry has suddenly expanded out of research and assay laboratories into biology, medicine and therapeutics. Electrospray ionization and matrix-assisted laser desorption/ionization yield increased mass-range and sensitivity, leading to novel applications and sparking new analyzer designs, software, and robotics.

Kinetic equivalence of stable-isotope-labeled and unlabeled phenytoin.

Stable isotope labeling (SIL) of a drug results in a higher molecular weight than that of the unlabeled drug. SIL tracer doses can be quantitated separately from unlabeled drug by gas chromatography-mass spectrometry (GC-MS) without exposing the patient to radiation. The higher molecular weight of SIL drug could cause a higher energy of activation for (and slowing of) metabolic reactions ("isotope effect"). To evaluate possible isotope effect, three dogs and three men were infused with a mixture containing equal amounts of SIL (2-13C-1,3-15N2) and unlabeled phenytoin (PHT). Plasma and urine were collected at regular intervals. Concentrations of SIL and unlabeled PHT and HPPH (the major metabolite of PHT) were determined by GC-MS. Within each subject there was no trend for concentrations of SIL PHT or HPPH to be higher or lower than concentrations of their unlabeled analogs (greater than 0.20 to 0.90). There was no difference in the distribution and elimination half-lifes (t 1/2s), volume of distribution, volume of central compartment, or clearance of the two forms of PHT. Thus, no isotope effect was found.

Reliability of the toxic screen in drug overdose.

To determine the reliability of the laboratory in detecting drugs taken by overdosed patients, we evaluated laboratory performance on an unbiased sample of actual clinical specimens. Replicate serum and urine samples from a series of 20 consecutive clinically overdosed patients were sent to three commercial laboratories and one academic research laboratory for identification and quantification of intoxicating agents. All laboratories used the advance analytical techniques of gas chromatography-mass spectrometry. The results suggest that laboratories do not reliably identify drugs in the serum of overdosed patients, partly because of technical limitation, partly because of laboratory error, and possibly because of inadequate specimens. Drugs judged responsible for the overdose were identified in only 50% to 70% of the cases, depending on the laboratory. Reported concentrations sometimes varied over a 10-fold range.

The beta 1,6-GlcNAc transferase activity present in hog gastric mucosal microsomes catalyses site-specific branch formation on a long polylactosamine backbone.

We find that the beta 1,6-GlcNAc transferase activity present in hog gastric mucosal microsomes converts the linear pentasaccharide GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc (1) in a site-specific way to the branch-bearing hexasaccharide GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc (2). The product is a positional isomer of GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc (3), reportedly formed from 1 by another polylactosamine beta 1,6-GlcNAc transferase activity present in human serum (Leppänen et al., Biochemistry, 30 (1991) 9287). Combined use of the two kinds of activities gave in the present experiments the heptasaccharide GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc (4), in which one of the branches occupies the position of the branch in 2 and the other the position of the branch in 3.

Enzyme-assisted synthesis of a bivalent high-affinity dodecasaccharide inhibitor of mouse gamete adhesion. The length of the chains carrying distal alpha 1,3-bonded galactose residues is critical.

Proposing to study the molecular mechanisms of mouse gamete adhesion with the aid of high affinity adhesion inhibitors of saccharide nature, we report here the enzymatic synthesis of a bivalent oligosaccharide Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3(Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc (4), consisting of two long arms that link together two distal alpha 1,3-galactose residues. Binding data reported elsewhere (E. Litscher et al., Biochemistry, 1995, 34, 4662-4669) show that 4 is a high affinity inhibitor of mouse gamete adhesion in vitro (IC50 = 9 microM), while a related octasaccharide Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3(Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc, consisting of two short arms is of very low inhibitory activity. The data highlight the importance of the two alpha-galactose residues of 4, and the length of the sugar chains joining them.

Improved enzymatic synthesis of a highly potent oligosaccharide antagonist of L-selectin.

The polylactosamine sLex beta1-3'(sLex beta1-6')LacNAc beta1-3'(sLex beta1-6')LacNAc beta1-3'(sLex beta1-6')LacNAc (7) (where sLex is Neu5Ac alpha2-3Gal beta1-4(Fuc alpha1-3)GlcNAc and LacNAc is Gal beta1-4GlcNAc) is a nanomolar L-selectin antagonist and therefore a potential anti-inflammatory agent (Renkonen et al. (1997) Glycobiology, 7, 453). Here we describe an improved synthesis of 7. The octasaccharide LacNAc beta1-3'LacNAc beta1-3'LacNAc beta1-3'LacNAc (4) was converted into the triply branched undecasaccharide LacNAc beta1-3'(GlcNAc beta1-6')LacNAc beta1-3'(GlcNAc beta1-6')LacNAc beta1-3'(GlcNAc beta1-6')LacNAc (5) by incubation with UDP-GlcNAc and the midchain beta1,6-GlcNAc transferase activity of rat serum. Glycan 5 was enzymatically beta1,4-galactosylated to LacNAc beta1-3'(LacNAc beta1-6')LacNAc beta1-3'(LacNAc beta1-6')LacNAc beta1-3'(LacNAc beta1-6')LacNAc (6). Combined with the enzymatic conversion of 6 to 7 (Renkonen et al., loc. cit.) and the available chemical synthesis of 4, our data improve the availability of 7 for full assessment of its anti-inflammatory properties.

Tegument galactosylceramides of the cestode Spirometra mansonoides.

The brush border-like surface of the tegument of the adult and the plerocercoid larva of a pseudophyllidean cestode, Spirometra mansonoides, has been shown to contain hydroxylated galactosylceramides. D-Galactosyl-N-(2-D-hydroxyoctadecanoyl)-D-phytosphingosine, D-galactosyl-N-(2-D-hydroxyoctadecanoyl)-D-dihydrosphingosine and D-galactosyl-N-(octadecanoyl)-D-phytosphingosine were identified as major glycosphingolipids in a tegumental plasma membrane fraction with associated microtriches, by combinations of chromatography (column, high performance thin-layer, gas-liquid), mass spectrometry (electron impact, field desorption, fast atom bombardment, collisionally induced decomposition) and proton nuclear magnetic resonance spectrometry. Galactosylceramides with hydroxylated long chain bases and fatty acids are known to occur in some eukaryotic microbes and in cells of vertebrate tissues exposed to plasma membrane destabilizing environments. This has led to a proposal that the capacity of hydroxylated ceramide moieties for intermolecular hydrogen bonding among themselves and with phosphoglycerides acts to stabilize the plasma membrane. Saturated fatty acyl groups in the ceramides would enhance stabilization by their orderly packing in the lipid bilayer. Consequently, the presence of such hydroxylated galactosylceramides in the tegument surface of S. mansonoides may contribute to the maintenance of its normal barrier properties in the face of the varied environmental insults encountered by the cestode in its life-cycle.

The investigation of radiopharmaceutical components by fast atom bombardment mass spectrometry: the identification of Tc-HIDA and the epimers of Tc-CO2DADS.

The nature of two technetium-labeled radiopharmaceutical components has been established by means of fast-atom-bombardment mass spectrometry (FABMS) in combination with carrier-added (CA) and no-carrier-added (NCA) reversed-phase high-pressure liquid chromatography (HPLC). Negative-ion FABMS was used to determine that the epimers of Tc-CO2DADS are the oxo[N,N'-(1-carboxyethylene)-bis-(2-mercaptoacetimido)]technetate(V) ions; positive-ion FABMS showed that Tc-HIDA is bis[N-(2,6-dimethylphenyl-carbamoylmethyliminodiaceto]technetate(III).

Chemical and in vivo studies of the anion oxo[N,N'-ethylenebis(2-mercaptoacetimido)]Technetate(V).

Studies of the anionic coordination complex 99Tc-oxo[N,N'-ethylene-bis(2-mercaptoacetimido)]technetate(V) ([TcO(ema)]-) are described. Syntheses performed both at carrier levels (10(-5)M) and with no carrier added (less than 10(-8)M) indicate that the complex is formed virtually quantitatively from pertechnetate ion over this range. Tissue distributions in normal rats are similar at both concentrations up to one hour after administration. It has been shown--using a combination of high-pressure liquid chromatography and field-desorption mass spectrometry--that the anion is excreted unchanged into both urine and bile. The effectiveness of this N2S2 donor set in sequestering Tc-99m, and the in vivo stability of the resulting complex, suggest that modified chelates of this structural class could provide a series of useful diagnostic agents.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry of underivatized and permethylated gangliosides.

Underivatized and permethylated gangliosides have been studied by the matrix-assisted laser desorption (MALO) ionization technique. The samples investigated included commercially available and highly purified gangliosides from the human brain containing up to five sialic acid residues. Several permethylated gangliosides have also been studied, and MALD has proven successful in analyzing multicomponent mixtures of glycolipids with different fatty acyl residues. During the studies a variety of matrix and wavelength combinations have been tested in both the positive and negative ion modes. The best results have been obtained with the matrices 2,5-dihyd roxybenzoic acid, 4-hydrazinobenzoic acid, 1,5-diaminonaphthalene, and 6-aza-2-thiothymine. Negative ion mass spectra of the underivatized gangliosides have always been of better quality than the positive ion mass spectra; exhibiting better signal-to-noise ratio, better resolution, less fragmentation, and less adduct formation with Na(+) and K(+). With increasing number of sialic acid substituents the molecular ion region became less and less resolvable leading to broadened peaks even in the negative ion mode. Fragmentation could frequently be observed in the negative ion mode, and it was pronounced in the positive ion mode. The major fragmentation pathways corresponded to loss of sialyl groupts) and to decarboxylation of one of the sialyl residues. For underivatized gangliosides the typical sample amount used was 10-20 pmol, Permethylation led to a significant improvement in sensitivity (two orders of magnitude); the detection limit of permethylated gangliosides was about 10 fmol. The higher stability of the permethylated compounds was indicated by the fact that positive ion mass spectra exhibited only a marginal extent of fragmentation.

Matrix-assisted laser desorption ionization mass spectrometry with 2-(4-hydroxyphenylazo)benzoic acid matrix.

A novel matrix substance, 2-(4-hydroxyphenylazo) benzoic acid, or HABA, has been found to be very advantageous for matrix-assisted ultraviolet laser desorption ionization mass spectrometry. This compound has been successfully used for the desorption of peptides, proteins, and glycoproteins up to approximately 250 kDa. For these materials, the most abundant analyte-related peaks correspond to [M + H](+) ions and multiply protonated molecules. Comparisons with sinapic acid, 2,5-dihydroxybenzoic acid, and α-cyano-4-hydroxycinnamic acid indicate that the new matrix provides comparable sensitivity for peptides and smaller proteins but results in better sensitivity for larger proteins and glycoproteins in protein mixtures. Other matrices discriminate against the higher mass components in these cases. Somewhat reduced mass resolution has been found for smaller proteins, but for larger proteins and glycoproteins the best mass resolution can often be obtained with the new matrix. For other classes of compounds that form ions predominantly via cation attachment, at least as good sensitivity and even better resolution have been obtained. Derivatized glycolipids and synthetic polymers have been studied in detail. For the analysis of many synthetic polymers, the best performance in terms of sensitivity and mass resolution has been observed with HABA matrix. Mass resolution was higher for cation adducts than for the protonated peptide molecules in the same mass range. The new matrix exhibits greatly extended (in time) analyte ion production and reproducibility. Owing to the uniform sample surface with this matrix, barely any spatial variation of the ion signal could be observed. In addition, many hundreds of single-shot mass spectra could be accumulated from the same spot, even for larger proteins.

Detection of transthyretin variants using immunoprecipitation and matrix-assisted laser desorption/ionization bioreactive probes: a clinical application of mass spectrometry.

In our continuing efforts to develop mass spectrometry-based methods for transthyretin (TTR) variant detection and characterization, we have sought to use matrix-assisted laser desorption/ionization (MALDI) bioreactive probes incorporating immobilized trypsin for screening purposes. These devices show good diagnostic potential as a clinical screening tool to detect amino acid substitutions in TTR. MALDI probes allow the on-probe generation of tryptic digests. The subsequent mass analysis of the on-probe digest yields the peptide map. The inherent advantages of this method include considerably reduced digestion times (minutes vs. hours), absence of autolysis products, minimized sample handling, and hence minimal sample loss. A further advantage is that the opportunity for loss of hydrophobic peptides is reduced because no sample transfer occurs. The method can be applied as a preliminary screen for TTR variants where TTR is isolated from patient serum through immunoprecipitation. This method should also be applicable to other proteins and suitable for automation.

A new transthyretin variant (Ser23Asn) associated with familial amyloidosis in a Portuguese patient.

The detection and characterization of a new transthyretin (ATTR) variant, Ser23Asn, associated with cardiomyopathy in a Portuguese patient with familial amyloidosis is described. Isoelectric focusing (IEF) of serum from the propositus demonstrated heterozygosity for the presence of wild type and variant ATTR. A combination of mass spectrometric (MS) analyses, including electrospray ionization mass spectrometry (ESI MS), high performance liquid chromatography (HPLC)/ESI MS and matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) performed on the serum-derived TTR were used to identify and locate the amino acid replacement in the variant protein. Genetic mutation analysis by DNA sequencing and allele-specific PCR confirmed this finding.

A new amyloidogenic transthyretin variant (Val122Ala) found in a compound heterozygous patient.

A new TTR variant, Val122Ala, was characterized in an individual who carried the Gly6Ser polymorphism on the opposite allele. The main clinical feature of this familial transthyretin amyloidosis (ATTR) variant is extensive cardiomyopathy. The detection and characterization of the variant were performed using a combination of isoelectric focusing (IEF), restriction fragment length polymorphism (RFLP), immunoprecipitation, electrospray ionization mass spectrometry (ESIMS), HPLC (high performance liquid chromatography)/ESIMS, and matrix-assisted laser desorption/ionization mass spectrometry (MALDIMS). The results were confirmed by DNA analysis. The propositus has a brother who carries the new variant but not the polymorphism.

Fundamentals of present-day mass spectrometry.

Mass spectral analysis offers much to the field of clinical pharmacology: specificity, sensitivity, reliability, and potential for the structural determination of unknown compounds, even when they are present at very low concentrations as part of complex mixtures. In order to employ mass spectrometry effectively, the researcher or analyst must acquire an understanding of the fundamental processes involved in the technique and some overview of the strengths and limitations of the different options now available. This brief summary is presented as an introduction to the field and should provide the impetus for further reading by the interested investigator.

Pharmacokinetic equivalence of stable-isotope-labeled and unlabeled drugs. Phenobarbital in man.

Stable isotope labeling of drugs has been used in human metabolism studies because it eliminates the risk of radiation exposure accompanying use of radioactive tracers. The labeled drug can be measured by gas-chromatographic mass spectrometry (GCMS). However, if reliable pharmacokinetic data are to be obtained, one has to be certain the rate of metabolism of labeled and unlabeled drug is the same, i.e., there is no kinetic isotope effect. To evaluate this for phenobarbital (PB), three humans were infused with a 1:1 mixture of phenobarbital and 1,3-15N2-2-13C-PB. Serum was collected at regular intervals. Concentrations of labeled and unlabeled phenobarbital were determined by GCMS. Within each subject, there was no trend for concentrations of labeled phenobarbital to be higher or lower than concentrations of unlabeled phenobarbital (P greater than 0.90 for all three subjects). There was no difference in the zero time intercepts, distribution and elimination time constants and half-lives, volumes of distribution and central compartment, or clearance of the two forms of phenobarbital. Thus, no isotope effect was found. Published data on other labeled drugs and the likelihood of encountering an isotope effect based on type of isotope and its location in the molecule are discussed.

Novel neuropeptide Y processing in human cerebrospinal fluid from depressed patients.

In vitro processing of neuropeptide Y (NPY) in cerebrospinal fluid (CSF) of patients with depression was monitored by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Single peptide bonds in NPY were cleaved to yield N- or C-terminal fragments. Multiple cleavage to form internal peptides was unimportant. Degradation rates varied between individuals, whereas the product distributions were fairly constant. Other peptides did not evidence such proteolysis. MALDI-TOF MS will facilitate extensive investigations of NPY processing that could provide the basis for clinical assays and illuminate the pathophysiology related to depression.

Stereochemical effects on the mass spectrometric behavior of native and derivatized trisaccharide isomers: comparisons with results from molecular modeling.

Differentiation of oligosaccharide isomers by mass spectrometry (MS) is a challenging task. For native, permethylated and peracetylated trisaccharides, matrix-assisted laser desorption/ionization time-of-flight (MALDI/TOF) MS and liquid secondary ionization (LSI) MS experiments can produce complementary results that are useful for molecular mass and sugar sequence determination and isomer differentiation. Linear MALDI/TOF-MS analysis of native and derivatized oligosaccharides usually produces cationized molecular ions. Characterization by LSI-MS and tandem mass spectrometry (LSI-MS/MS) typically may yield only low-abundance protonated molecular ions but produces dominant B-type ions by elimination of ROH (R = Me, Ac) from the C-1 position at the reducing end and distinctive sequence-related fragments. Results for four milk trisaccharides, two neutral (fucosyllactoses) and two sialylated (sialyllactoses), are presented to demonstrate the utility of microscale permethylation and gas-to-solid phase peracetylation for high sensitivity structural elucidation. For the pairs of carbohydrates investigated in this study by LSI-MS, LSI-MS/MS and linear MALDI/TOF-MS, the fragmentation patterns of the native, permethylated and peracetylated isomer pairs are shown to differ markedly as a consequence of their limited dissimilarities. In addition, the tendency of sialylated carbohydrates to form lactones upon peracetylation has been exploited to take advantage of the variation in the extent of lactonization with orientation of the sialic acid moiety relative to the adjacent sugar rings. Lactone formation is favored for 3'-sialyllactose compared with its 6'-isomer; Hyperchem was employed to indicate the relative stabilities of the molecular and fragment ions and to visualize the molecules in 3D (rather than to obtain absolute conformational energy values). The relative conformational energies of lactonized and non-lactonized ions were calculated using the Hyperchem software; their values support the trends observed by MS.