Lenalidomide enhances MOR202-dependent macrophage-mediated effector functions via the vitamin D pathway.

Macrophages are key mediators of the therapeutic effects exerted by monoclonal antibodies, such as the anti-CD38 antibody MOR202, currently introduced in multiple myeloma (MM) therapy. Therefore, it is important to understand how antibody-mediated effector functions of myeloma-associated macrophages (MAMs) are regulated. Here, we focused on the effects of vitamin D, a known regulator of macrophage effector functions. Consequently, it was the aim of this study to assess whether modulation of the vitamin D pathway alters the tumoricidal activity of MAMs. Here, we demonstrate that MAMs display a defective vitamin D pathway with reduced expression level of CYP27B1 and limited tumoricidal activity which can be restored by the IMiD lenalidomide in vitro. Furthermore, our data indicate that the vitamin D pathway of MAMs from MM patients does recover during an IMiD-containing therapy shown by an improved MOR202-mediated cytotoxic activity of these MAMs against primary MM cells ex vivo. Here, the ex vivo cytotoxic activity could be further enhanced by vitamin D supplementation. These data suggest that vitamin D holds a key role for the effector functions of MAMs and that vitamin D supplementation in IMiD combination trials could further increase the therapeutic efficacy of anti-CD38 antibodies such as MOR202, which remains to be investigated in clinical studies.

Activation of Reactive MALDI Adduct Ions Enables Differentiation of Dihydroxylated Vitamin D Isomers.

Vitamin D compounds are secosteroids, which are best known for their role in bone health. More recent studies have shown that vitamin D metabolites and catabolites such as dihydroxylated species (e.g., 1,25- and 24,25-dihydroxyvitamin D3) play key roles in the pathologies of various diseases. Identification of these isomers by mass spectrometry is challenging and currently relies on liquid chromatography, as the isomers exhibit virtually identical product ion spectra under collision induced dissociation conditions. Here, we developed a simple MALDI-CID method that utilizes ion activation of reactive analyte/matrix adducts to distinguish isomeric dihydroxyvitamin D3 species, without the need for chromatography separation or chemical derivatization techniques. Specifically, reactive 1,5-diaminonaphthalene adducts of dihydroxyvitamin D3 compounds formed during MADI were activated and specific cleavages in the secosteroid's backbone structure were achieved that produced isomer-diagnostic fragment ions. Graphical Abstract ᅟ.

Quantification of the 3α and 3ß epimers of 25-hydroxyvitamin D3 in dried blood spots by LC-MS/MS using artificial whole blood calibration and chemical derivatization.

While the biological function of the 3α epimer of 25-hydroxyvitamin D3 (25(OH)D3) remains unknown, its presence needs to be accurately captured and separated from the main 3ß epimer, to avoid positive bias in vitamin D status analyses. Several recent LC-MS/MS assays for 25(OH)D3 successfully separate the 3α and 3ß epimers by chromatography. Unfortunately, none of the existing LC-MS/MS assays, which utilize dried blood spots (DBS) as sampling/storage vessels, is able to quantify the individual epimers. DBS are often used for analysis of infant blood, however, and these samples are particularly likely to contain significant levels of interfering 3α epimer. Furthermore, proper calibration of DBS samples is much more difficult to achieve than for liquid serum or plasma samples. We addressed this important issue by creating an artificial vitamin D-free whole blood for calibration and then quantified 3α- and 3ß-25(OH)D3 levels from DBS. After chemical derivatization, the vitamin D epimers were separated on a PFP column and concentrations determined by electrospray ionization LC-MS/MS on a triple quadrupole mass spectrometer. Calibration with artificial whole blood showed improved precision over standard addition (7.6 versus 31.5% RSD for 3ß-25(OH)D3). The limits of quantification for 3ß-25(OH)D3 and for 3α-25(OH)D3 were 1.0 and 0.1ng/mL, respectively. Excellent intra/interday precisions between 2.1 and 2.2% CV (intra) and 4.4-5.3% CV (inter) were established for 3ß-25(OH)D3 and 3α-25(OH)D3. For 3ß-25(OH)D3, only small concentration-independent bias and deviation of <3.3ng/mL were seen between serum LC-MS/MS and DBS-LC-MS/MS measurements; analyses of 3α-25(OH)D3 showed deviations of <0.8ng/mL in all experiments.

Direct aqueous measurement of 25-hydroxyvitamin D levels in a cellular environment by LC-MS/MS using the novel chemical derivatization reagent MDBP.

Vitamin D measurements in biological fluids by mass spectrometry are challenging at very low concentration levels. As a result, chemical derivatization is often employed to enhance the ionization properties of low abundant vitamin D compounds. Cookson-type reagents such as 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) or similar derivatives work well but require careful, water-free experimental conditions, as traces of water inactivate the reagent and inhibit or stop the derivatization reactions, thus making quantitative measurements in aqueous samples impossible. We describe a novel electrospray liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for determining 25-hydroxyvitamin D3 (25(OH)D3) directly in aqueous cellular systems using a new derivatization reagent, the ionic liquid 12-(maleimidyl)dodecyl-tri-n-butylphosphonium bromide (MDBP). The proof-of-concept for the MDBP assay was demonstrated by measuring the levels of 25(OH)D3 in four different human cell types, namely T cells, helper T cells, B cells, and macrophages. In addition to the ability to determine the levels of 25(OH)D3 directly in aqueous samples, the cellular integrity was maintained in our application. We show the time-dependent uptake of 25(OH)D3 into the investigated cells to demonstrate the applicability of the new label. Furthermore, the MDBP derivatization technique may be equally useful in imaging mass spectrometry, where it could be used for response enhancements of spatially localized vitamin D metabolites on wet tissue surfaces, without destroying the integrity of the tissue surface. Graphical Abstract MDBP labelling of 25-hydroxyvitamin D in the extracellular space.

Chemotyping the distribution of vitamin D metabolites in human serum.

Most studies examining the relationships between vitamin D and disease or health focus on the main 25-hydroxyvitamin D3 (25(OH)D3) metabolite, thus potentially overlooking contributions and dynamic effects of other vitamin D metabolites, the crucial roles of several of which have been previously demonstrated. The ideal assay would determine all relevant high and low-abundant vitamin D species simultaneously. We describe a sensitive quantitative assay for determining the chemotypes of vitamin D metabolites from serum after derivatisation and ultra-high performance liquid chromatography-electrospray ionisation-tandem mass spectrometry (UHPLC-ESI-MS/MS). We performed a validation according to the 'FDA Guidance for Industry Bioanalytical Method Validation'. The proof-of-concept of the method was then demonstrated by following the metabolite concentrations in patients with chronic liver diseases (CLD) during the course of a vitamin D supplementation study. The new quantitative profiling assay provided highly sensitive, precise and accurate chemotypes of the vitamin D metabolic process rather than the usually determined 25(OH)D3 concentrations.

Mass spectrometric profiling of vitamin D metabolites beyond 25-hydroxyvitamin D.

BACKGROUND: The frequency of measurements of vitamin D in the human population has significantly increased over the last decade because vitamin D has now been linked to many diseases, in addition to its established role in bone health. Usually, serum 25-hydroxyvitamin D concentrations are measured to assess the vitamin D status of individuals. Unfortunately, many studies investigating links between vitamin D and disease also use only this single metabolite. Intricate correlations with other vitamin D metabolites or dynamic effects of downstream metabolites may therefore be overlooked. Fortunately, powerful LC-MS/MS approaches have recently become available that can simultaneously quantify the concentrations of multiple vitamin D metabolites. These approaches are challenging, however, because of inherent instrumental problems with detection of vitamin D compounds and the low concentrations of the metabolites in biological fluids. CONTENT: This review summarizes recent mass spectrometry assays for the quantitative measurement of multiple vitamin D metabolites and their application in clinical research, with a particular focus on the low-abundance downstream metabolic species generated after the initial hydroxylation to 25-hydroxyvitamin D. SUMMARY: To study the pathobiological effects and function of vitamin D metabolites in disease, in particular in low-abundance species beyond 25-hydroxyvitamin D, we need to know their concentrations. Although detection of these vitamin D species is challenging, a number of recent mass spectrometry assays have successfully demonstrated that LC-MS/MS methods can quantify multiple vitamin D compounds over a wide dynamic range individually or as part of multimetabolite assays.