Examination of structurally selective derivatization of vitamin D(3) analogues by electrospray mass spectrometry.

The structural specificity of vitamin D derivatization by PTAD (4-phenyl-1,2,4-triazoline-3,5-dione) was probed using synthetic analogues and ion trap mass spectrometry. EB 1089, a vitamin D(3) analogue which contains a second site for Diels--Alder cycloaddition on its side-chain, allowed the examination of derivatization modes and comparisons of ion fragment structures. The origins of a PTAD-vitamin D(3) ion fragment, commonly used in metabolite characterization and quantitation of vitamin D(3) analogues (m/z 314), were established; ion trap mass spectrometry revealed that the PTAD comprises a portion of this diagnostic fragment, and is not lost by a retro-Diels--Alder step. Furthermore, the unique structure of the EB 1089 side-chain also permits facile determination of its side-chain metabolism. Use of PTAD derivatization and detection of metabolite-specific ion fragments identify hydroxylation at the end of the EB 1089 sidechain. It is believed that the results from these studies provide a clearer understanding of the mass spectrometry of triazolinedione derivatives, not only in the specific case of EB 1089, but also in their application to other vitamin D compounds.

Isolation and identification of 1alpha-hydroxy-3-epi-vitamin D3, a potent suppressor of parathyroid hormone secretion.

Since our original demonstration of the metabolism of 1alpha,25(OH)2D3 into 1alpha,25(OH)2-3-epi-D3 in human keratinocytes, there have been several reports indicating that epimerization of the 3 hydroxyl group of vitamin D compounds is a common metabolic process. Recent studies reported the metabolism of 25OHD3 and 24(R),25(OH)2D3 into their respective C-3 epimers, indicating that the presence of 1alpha hydroxyl group is not necessary for the 3-epimerization of vitamin D compounds. To determine whether the presence of a 25 hydroxyl group is required for 3-epimerization of vitamin D compounds, we investigated the metabolism of 1alphaOHD3, a non-25 hydroxylated vitamin D compound, in rat osteosarcoma cells (ROS 17/2.8). We noted metabolism of 1alphaOHD3 into a less polar metabolite which was unequivocally identified as 1alphaOH-3-epi-D3 using the techniques of HPLC, GC/MS, and 1H-NMR analysis. We also identified 1alphaOH-3-epi-D3 as a circulating metabolite in rats treated with pharmacological concentrations of 1alphaOHD3. Thus, these results indicated that the presence of a 25 hydroxyl group is not required for 3-epimerization of vitamin D compounds. Furthermore, the results from the same studies also provided evidence to indicate that 1alphaOH-3-epi-D3, like 1alphaOHD3, is hydroxylated at C-25. We then evaluated the biological activities of 1alphaOH-3-epi-D3. Treatment of normal rats every other day for 7 days with 2.5 nmol/kg of 1alphaOH-3-epi-D3 did not raise serum calcium, while the same dose of 1alphaOHD3 increased serum calcium by 3.39 +/- 0.52 mg/dl. Interestingly, in the same rats which received 1alphaOH-3-epi-D3 we also noted a reduction in circulating PTH levels by 65 +/- 7%. This ability of 1alphaOH-3-epi-D3 to suppress PTH levels in normal rats without altering serum calcium was further tested in rats with reduced renal function. The results indicated that the ED50 of 1alphaOH-3-epi-D3 for suppression of PTH was only slightly higher than that of 1alpha,25(OH)2D3, but that the threshold dose of the development of hypercalcemia (total serum Ca > 10.5 mg/dl) was nearly 80 times higher. These findings indicate that 1alphaOH-3-epi-D3 is a highly selective vitamin D analog with tremendous potential for treatment of secondary hyperparathyroidism in chronic renal failure patients.

Characterization of oligosaccharide composition and structure by quadrupole ion trap mass spectrometry.

The use of electrospray ionization-quadrupole ion trap mass spectrometry for the characterization of linear oligosaccharides and N-linked protein oligosaccharide mixtures is described. Tandem mass spectrometry (MS/MS) experiments with orders higher than two offer a number of ways to enhance MS/MS spectra and to derive information not present in MS and MS2 spectra. Three such methods are presented in this paper. (a) Collisional activation of permethylated oligosaccharide molecular ions (MS2) as illustrated by maltoheptaose, produces abundant fragments from glycosidic bond cleavages which indicate composition and sequence, and weak cross-ring cleavage products which denote specific linkages within the oligosaccharide. Through the trapping and further dissociation of these fragments (MSn), cross-ring cleavage products can be confirmed and their relative abundances increased to facilitate interpretation. (b) The mechanisms of formation of two isobaric ions or ions isobaric with another ion's isotope peaks, such as those present in the MS2 spectrum of the ribonuclease B oligosaccharide GlcNAc2-Man5 can be independently established by separate MS3 experiments. (c) Ions in the MS2 spectrum, specific for individual components of an isobaric mixture, can be isolated and characterized by further stages of fragmentation. This is illustrated by two isobaric oligosaccharides from chicken ovalbumin of the composition HexNAc5Hex5. These findings indicate the utility of ion trap mass spectrometry towards the facile determination of oligosaccharide composition, sequence, branching and linkage, providing a wealth of structural information not obtainable by other individual methods of carbohydrate mass spectrometric analysis.

Electrospray ionization-ion trap mass spectrometry for structural analysis of complex N-linked glycoprotein oligosaccharides.

Electrospray ionization-ion trap mass spectrometry, with its capacity to perform multiple stages of fragmentation (MSn), is demonstrated as an effective method for the structural characterization of permethylated N-linked complex glycoprotein oligosaccharides. Complex glycan structural features, such as N-acetyllactosamine antenane, neuraminic acids, and nonreducing terminal GlcNAc monosaccharides, commonly suppress cross-ring and core saccharide cleavages in traditional MS/MS experiments. Using ion trap mass spectrometry, removal of these substituents permits determination of branching patterns and intersaccharide linkages by MS3 and MS4. Both sequence and linkage data are obtained for N-acetyllactosamine and sialyl-N-acetyllactosamine oligosaccharide antennae from biantennary glycans using MS3, and the location of a bisecting GlcNAc residue is also established after exposing the core pentasaccharide. Higher-order experiments further illustrate the potential of electrospray ionization-quadrupole ion trap mass spectrometry for carbohydrate analysis, as MS8 is used to produce significant and otherwise unobtainable branching information for an oligosaccharide from chicken ovalbumin. These studies constitute further evidence of the unique role that ion trap mass spectrometry can assume in the area of oligosaccharide analysis.