Andrew S. Weiskopf

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 ions 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.

1α,25-Dihydroxy-3-epi-vitamin D3: In vivo metabolite of 1α,25-dihydroxyvitamin D3 in rats

We recently identified 1α,25‐dihydroxy‐3‐epi‐vitamin D3 as a major in vitro metabolite of 1α,25‐dihydroxyvitamin D3, produced in primary cultures of neonatal human keratinocytes. We now report the isolation of 1α,25‐dihydroxy‐3‐epi‐vitamin D3 from the serum of rats treated with pharmacological doses of 1α,25‐dihydroxyvitamin D3. 1α,25‐dihydroxy‐3‐epi‐vitamin D3 was identified through its co‐migration with synthetic 1α,25‐dihydroxy‐3‐epi‐vitamin D3 on both straight and reverse phase high performance liquid chromatography systems and by mass spectrometry. Along with 1α,25‐dihydroxy‐3‐epi‐vitamin D3, other previously known metabolites, namely, 1α,24(R),25‐trihydroxyvitamin D3, 1α,25‐dihydroxy‐24‐oxo‐vitamin D3 and 1α,25‐dihydroxyvitamin D3‐26,23‐lactone, were also identified. Thus, our study for the first time provides direct evidence to indicate that 1α,25‐dihydroxy‐3‐epi‐vitamin D3 is an in vivo metabolite of 1α,25‐dihydroxyvitamin D3 in rats.

Production of 1α,25-Dihydroxy-3-epi-vitamin D3 in two rat osteosarcoma cell lines (UMR 106 and ROS 17/2.8): existence of the C-3 epimerization pathway in ROS 17/2.8 cells in which the C-24 oxidation pathway is not expressed

The secosteroid hormone 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] is metabolized into calcitroic acid through the carbon 24 (C-24) oxidation pathway. It is now well established that the C-24 oxidation pathway plays an important role in the target tissue inactivation of 1alpha,25(OH)2D3. Recently, we reported that 1alpha,25(OH)2D3 is also metabolized into 1alpha,25-dihydroxy-3-epi-vitamin D3 [1alpha,25(OH)2-3-epi-D3] through the carbon 3 (C-3) epimerization pathway in human keratinocytes, human colon carcinoma cells (Caco-2), and bovine parathyroid cells. In a previous study, it was demonstrated that 1alpha,25(OH)2-3-epi-D3 when compared to 1alpha,25(OH)2D3 was less active in stimulating intestinal calcium absorption, calcium mobilization from bone, and induction of calbindin D28k. These findings suggest that the C-3 epimerization pathway, like the C-24 oxidation pathway, may play a role in the target tissue inactivation of 1alpha,25(OH)2D3. In this study, we determined the relationship between the C-24 oxidation and the C-3 epimerization pathways by investigating the metabolism of 1alpha,25(OH)2D3 in two rat osteosarcoma cell lines (UMR 106 and ROS 17/2.8). These two cell lines differ from each other in their ability to metabolize 1alpha,25(OH)2D3 through the C-24 oxidation pathway. It has been previously reported that the C-24 oxidation pathway is expressed only in UMR 106 cells but not in ROS 17/2.8 cells. The results of our present study provide new evidence that both cell lines possess the ability to metabolize 1alpha,25(OH)2D3 into 1alpha,25(OH)2-3-epi-D3 through the C-3 epimerization pathway. Our results also reconfirm the findings of previous studies indicating that UMR 106 cells are the only ones which express the C-24 oxidation pathway out of the two cell lines studied. Furthermore, this study reveals for the first time that the C-3 epimerization pathway may become an alternate metabolic pathway for the target tissue inactivation of 1alpha,25(OH)2D3 in some cells, such as ROS 17/2.8, in which the C-24 oxidation pathway is not expressed.

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

Since our original demonstration of the metabolism of 1α,25(OH)2D3 into 1α,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 1α 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 1αOHD3, a non‐25 hydroxylated vitamin D compound, in rat osteosarcoma cells (ROS 17/2.8). We noted metabolism of 1αOHD3 into a less polar metabolite which was unequivocally identified as 1αOH‐3‐epi‐D3 using the techniques of HPLC, GC/MS, and 1H‐NMR analysis. We also identified 1αOH‐3‐epi‐D3 as a circulating metabolite in rats treated with pharmacological concentrations of 1αOHD3. 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 1αOH‐3‐epi‐D3, like 1αOHD3, is hydroxylated at C‐25. We then evaluated the biological activities of 1αOH‐3‐epi‐D3. Treatment of normal rats every other day for 7 days with 2.5 nmol/kg of 1αOH‐3‐epi‐D3 did not raise serum calcium, while the same dose of 1αOHD3 increased serum calcium by 3.39 ± 0.52 mg/dl. Interestingly, in the same rats which received 1αOH‐3‐epi‐D3 we also noted a reduction in circulating PTH levels by 65 ± 7%. This ability of 1αOH‐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 1αOH‐3‐epi‐D3 for suppression of PTH was only slightly higher than that of 1α,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 1αOH‐3‐epi‐D3 is a highly selective vitamin D analog with tremendous potential for treatment of secondary hyperparathyroidism in chronic renal failure patients.

Enhanced biological activity of 1α,25-dihydroxy-20-epi-vitamin D3, the C-20 epimer of 1α,25-dihydroxyvitamin D3, is in part due to its metabolism into stable intermediary metabolites with significant biological activity

1alpha,25-dihydroxy-20-epi-vitamin D3 (1alpha,25(OH)2-20-epi-D3), the C-20 epimer of the natural hormone 1alpha,25(OH)2D3, is several fold more potent than the natural hormone in inhibiting cell growth and inducing cell differentiation. At present, the various mechanisms responsible for the enhanced biological activities of this unique vitamin D3 analog are not fully understood. In our present study we compared the target tissue metabolism of 1alpha,25(OH)2D3 with that of 1alpha,25(OH)2-20-epi-D3 using the technique of isolated perfused rat kidney. The results indicated that the C-24 oxidation pathway plays a major role in the metabolism of both compounds in the rat kidney. However, it was noted that the concentrations of two of the intermediary metabolites of 1alpha,25(OH)2-20-epi-D3, namely, 1alpha,24(R),25(OH)3-20-epi-D3 and 1alpha,25(OH)2-24-oxo-20-epi-D3 in the kidney perfusate, exceeded the concentrations of the corresponding intermediary metabolites of 1alpha,25(OH)2D3. Furthermore, 1alpha,25(OH)2-24-oxo-20-epi-D3 induces the conformation of the vitamin D receptor similar to that induced by its parent analog and is nearly as potent as its parent in inducing transactivation of a gene construct containing the human osteocalcin vitamin D-responsive element. We conclude that 1alpha,25(OH)2-20-epi-D3 by itself is not metabolically stable when compared to 1alpha,25(OH)2D3, but it acquires its metabolic stability because of the reduced rate of catabolism of its intermediary metabolites. Furthermore, 1alpha,25(OH)2-24-oxo-20-epi-D3, the stable bioactive intermediary metabolite plays a significant role in generating the enhanced biological activities ascribed to 1alpha,25(OH)2-20-epi-D3.

Tissue specific metabolism of 1α,25‐dihydroxy‐20‐epi‐vitamin D3 into new metabolites with significant biological activity: Studies in rat osteosarcoma cells (UMR 106 and ROS 17/2.8)

In a recent study, we investigated the metabolism of 1α,25‐dihydroxy‐20‐epi‐vitamin D3 (1α,25(OH)2‐20‐epi‐D3), a potent synthetic vitamin D3 analog in the isolated perfused rat kidney and proposed that the enhanced biological activity of 1α,25(OH)2‐20‐epi‐D3 is in part due to its metabolism into stable bioactive intermediary metabolites derived via the C‐24 oxidation pathway (Siu‐Caldera et al. [ 1999 ] J. Steroid. Biochem. Mol. Biol. 71:111–121). It is now well established that 1α,25(OH)2D3 and its analogs are metabolized in target tissues not only via the C‐24 oxidation pathway but also via the C‐3 epimerization pathway. As the perfused rat kidney does not express the C‐3 epimerization pathway, we could not identify other possible bioactive metabolites of 1α,25(OH)2‐20‐epi‐D3 such as 1α,25(OH)2‐20‐epi‐3‐epi‐D3, derived via the C‐3 epimerization pathway. Therefore, we studied the metabolism of 1α,25(OH)2‐20‐epi‐D3 in rat osteosarcoma cells (UMR 106) which express both the C‐24 oxidation and the C‐3 epimerization pathways. Our results indicate that 1α,25(OH)2‐20‐epi‐D3 is metabolized in UMR 106 cells into several metabolites which included not only the previously known metabolites of the C‐24 oxidation pathway but also three new metabolites which were labeled as metabolites X, Y1, and Y2. Metabolite X was unequivocally identified as 1α,25(OH)2‐20‐epi‐3‐epi‐D3. Eventhough definite structure identification of the metabolites, Y1 and Y2 was not achieved in our present study, we determined that the metabolite Y1 is produced from 1α,25(OH)2‐20‐epi‐D3 and the metabolite Y2 is produced from 1α,25(OH)2‐20‐epi‐3‐epi‐D3. We also noted the production of both 1α,25(OH)2‐20‐epi‐3‐epi‐D3 and the two metabolites Y1 and Y2 in different rat osteosarcoma cells (ROS 17/2.8) which express only the C‐3 epimerization pathway but not the C‐24 oxidation pathway. Furthermore, we investigated the metabolism of 1α,25(OH)2‐20‐epi‐D3 in the isolated perfused rat kidney in an earlier study. The results of this study indicated that the rat kidney unlike rat osteosarcoma cells did not produce either 1α,25(OH)2‐20‐epi‐3‐epi‐D3 or the metabolites Y1 and Y2. Thus, it appears that the metabolites Y1 and Y2, like 1α,25(OH)2‐20‐epi‐3‐epi‐D3, are produced only in specific tissues. Preliminary biological activity of each new metabolite is assessed by measuring its ability to generate VDR‐mediated gene transcription. 1α,25(OH)2‐20‐epi‐3‐epi‐D3 was found to be almost equipotent to 1α,25(OH)2‐20‐epi‐D3 while the metabolites, Y1 and Y2 were found to be less active. The metabolite Y1 when compared to the metabolite Y2 has higher biological activity and its potency is almost equal to 1α,25(OH)2D3. In summary, we report for the first time tissue specific metabolism of 1α,25(OH)2‐20‐epi‐D3 into several bioactive metabolites which are derived not only via the previously established C‐24 oxidation and C‐3 epimerization pathways but also via a new pathway. J. Cell. Biochem. 82: 599–609, 2001.

Metabolism of selective 20-epi-vitamin D3 analogs in rat osteosarcoma UMR-106 cells: Isolation and identification of four novel C-1 fatty acid esters of 1α,25-dihydroxy-16-ene-20-epi-vitamin D3

&NA; Analogs of 1&agr;,25‐dihydroxyvitamin D3 (S1) with 20‐epi modification (20‐epi analogs) possess unique biological properties. We previously reported that 1&agr;,25‐dihydroxy‐20‐epi‐vitamin D3 (S2), the basic 20‐epi analog is metabolized into less polar metabolites (LPMs) in rat osteosarcoma cells (UMR‐106) but not in a perfused rat kidney. Furthermore, we also noted that only selective 20‐epi analogs are metabolized into LPMs. For example, 1&agr;,25‐dihydroxy‐16‐ene‐20‐epi‐vitamin D3 (S4), but not 1&agr;,25‐dihydroxy‐16‐ene‐23‐yne‐20‐epi‐vitamin D3 (S5) is metabolized into LPMs. In spite of these novel findings, the unequivocal identification of LPMs has not been achieved to date. We report here on a thorough investigation of the metabolism of S4 in UMR‐106 cells and isolated two major LPMs produced directly from the substrate S4 itself and two minor LPMs produced from 3‐epi‐S4, a metabolite of S4 produced through C‐3 epimerization pathway. Using GC/MS, ESI‐MS and 1H NMR analysis, we identified all the four LPMs of S4 as 25‐hydroxy‐16‐ene‐20‐epi‐vitamin D3‐1‐stearate and 25‐hydroxy‐16‐ene‐20‐epi‐vitamin D3‐1‐oleate and their respective C‐3 epimers. We report here for the first time the elucidation of a novel pathway of metabolism in UMR‐106 cells in which both 1&agr;,25(OH)2‐16‐ene‐20‐epi‐D3 and 1&agr;,25(OH)2‐16‐ene‐20‐epi‐3‐epi‐D3 undergo C‐1 esterification into stearic and oleic acid esters. Graphical abstract Figure. No caption available. HighlightsA novel pathway of 20‐epi‐3‐epi analogs of vitamin D3 in UMR‐106 cells.C‐1 esterification of both 16‐ene‐20‐epi‐D3 and 16‐ene‐20‐epi‐3‐epi‐D3.GC–MS, ESI‐MS and NMR confirm esterification into stearic and oleic acid esters.The modifications may alter the biological activity of synthetic VD3 analogs.