Potential bias and mitigations when using stable isotope labeled parent drug as internal standard for LC-MS/MS quantitation of metabolites

In recent years, increasing emphasis has been placed on quantitative characterization of drug metabolites for better insight into the correlation between metabolite exposure and toxicological observations or pharmacological efficacy. One common strategy for metabolite quantitation is to adopt the stable isotope labeled (STIL) parent drug as the internal standard in an isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay. In the current work, we demonstrate this strategy could have a potential pitfall resulting in quantitation bias if the internal standard is subject to ion suppression from the co-eluting parent drug in the incurred samples. Propranolol and its metabolite 4-hydroxypropranolol were used as model compounds to demonstrate this phenomenon and to systematically evaluate different approaches to mitigate the issue, including atmospheric pressure chemical ionization (APCI) mode of ionization, increased internal standard concentration, quantitation without internal standard, the use of a structural analog as internal standard, and dilution of the samples. Case studies of metabolite quantitation in nonclinical and clinical studies in drug development were also included to demonstrate the importance of using an appropriate bioanalytical strategy for metabolite quantitation in the real world. We present that bias of metabolite concentrations could pose a potential for poor estimation of safety risk. A strategy for quantitation of metabolites in support of drug safety assessment is proposed.     

Relative quantitation of proteins in expressed prostatic secretion with a stable isotope labeled secretome standard

Expressed prostatic secretion (EPS) is a proximal fluid directly derived from the prostate and, in the case of prostate cancer (PCa), is hypothesized to contain a repertoire of cancer-relevant proteins. Quantitative analysis of the EPS proteome may enable identification of proteins with utility for PCa diagnosis and prognosis. The present investigation demonstrates selective quantitation of proteins in EPS samples from PCa patients using a stable isotope labeled proteome standard (SILAP) generated through the selective harvest of the "secretome" from the PC3 prostate cancer cell line grown in stable isotope labeled cell culture medium. This stable isotope labeled secretome was digested with trypsin and equivalently added to each EPS digest, after which the resultant mixtures were analyzed by liquid chromatography-tandem mass spectrometry for peptide identification and quantification. Relative quantification of endogenous EPS peptides was accomplished by comparison of reconstructed mass chromatograms to those of the chemically identical SILAP peptides. A total of 86 proteins were quantified from 263 peptides in all of the EPS samples, 38 of which were found to be relevant to PCa. This work demonstrates the feasibility of using a SILAP secretome standard to simultaneously quantify many PCa-relevant proteins in EPS samples.     

SELDI-TOF-MS determination of hepcidin in clinical samples using stable isotope labelled hepcidin as an internal standard

Background  

Hepcidin is a 25-residue peptide hormone crucial to iron homeostasis. It is essential to measure the concentration of hepcidin in cells, tissues and body fluids to understand its mechanisms and roles in physiology and pathophysiology. With a mass of 2791 Da hepcidin is readily detectable by mass spectrometry and LC-ESI, MALDI and SELDI have been used to estimate systemic hepcidin concentrations by analysing serum or urine. However, peak heights in mass spectra may not always reflect concentrations in samples due to competition during binding steps and variations in ionisation efficiency. Thus the purpose of this study was to develop a robust assay for measuring hepcidin using a stable isotope labelled hepcidin spiking approach in conjunction with SELDI-TOF-MS.     

Green fluorescent protein expression triggers proteome changes in breast cancer cells

Green fluorescent protein (GFP) is the most commonly used reporter of expression in cell biology despite evidence that it affects the cell physiology. The molecular mechanism of GFP-associated modifications has been largely unexplored. In this paper we investigated the proteome modifications following stable expression of GFP in breast cancer cells (MDA-MB-231). A combination of three different proteome analysis methods (2-DE, iTRAQ, label-free) was used to maximise proteome coverage. We found that GFP expression induces changes in expression of proteins that are associated with protein folding, cytoskeletal organisation and cellular immune response. In view of these findings, the use of GFP as a cell reporter should be carefully monitored.     

Mitochondrial superoxide production during oxalate-mediated oxidative stress in renal epithelial cells

Crystals of calcium oxalate monohydrate (COM) in the renal tubule form the basis of most kidney stones. Tubular dysfunction resulting from COM-cell interactions occurs by mechanism(s) that are incompletely understood. We examined the production of reactive oxygen intermediates (ROI) by proximal (LLC-PK1) and distal (MDCK) tubular epithelial cells after treatment with COM (25–250 μg/ml) to determine whether ROI, specifically superoxide (O₂^•−), production was activated, and whether it was sufficient to induce oxidative stress. Employing inhibitors of cytosolic and mitochondrial systems, the source of ROI production was investigated. In addition, intracellular glutathione (total and oxidized), energy status (ATP), and NADH were measured. COM treatment for 1–24 h increased O₂^•− production 3–6-fold as measured by both lucigenin chemiluminescence in permeabilized cells and dihydrorhodamine fluorescence in intact cells. Using selective inhibitors we found no evidence of cytosolic production. The use of mitochondrial probes, substrates, and inhibitors indicated that increased O₂^•− production originated from mitochondria. Treatment with COM decreased glutathione (total and redox state), indicating a sustained oxidative insult. An increase in NADH in COM-treated cells suggested this cofactor could be responsible for elevating O₂^•− generation. In conclusion, COM increased mitochondrial O₂^•− production by epithelial cells, with a subsequent depletion of antioxidant status. These changes may contribute to the reported cellular transformations during the development of renal calculi.     

Determination of phenelzine in human plasma with gas chromatography—mass spectrometry using an isotope labeled internal standard

A quantitative gas chromatographic—mass spectrometric assay was developed for the determination of phenelzine in human plasma. Phenelzine, in aqueous solution or in plasma reacts at room temperature with pentafluorobenzaldehyde to form quantitatively a hydrazone derivative. The derivative has good gas chromatographic characteristics. The assay utilizes selected ion monitoring in a gas chromatographic effluent, the molecular ion generated by electron impact ionization of phenelzine derivative. Phenelzine-d₇ was synthesized and used as an internal standard. The assay can measure 2 ng/ml of the drug with about 10% precision.The method was used for the determination of steady state levels of phenelzine in the plasma of patients taking a therapeutic dose of the drug.     

Determination of N-acetylneuraminic acid by gas chromatography-mass spectrometry with a stable isotope as internal standard

N-Acetylneuraminic acid was determined by gas chromatography-mass spectrometry using selected ion-monitoring technique with N-[²H₃]acetylneuraminic acid as an internal standard. M-COOTMS fragments at $\text{m}\text{z}$ 624 of trimethylsilyl derivatives of N-acetylneuraminic acid and at $\text{m}\text{z}$ 627 of that of the internal standard were used as monitoring ions. The standard curve obtained was linear in the range of over 10³, and the lower limit for quantitation was estimated to be a few hundred picograms. This method was used to measure total N-acetylneuraminic acid in the plasma of healthy humans and patients with lung cancer. The total N-acetylneuraminic acid level in the plasma was two to three times higher in the patients than in controls. A few hundred nanoliters of plasma was sufficient for the analysis. The mass fragmentogram of plasma gave a good signal/noise ratio, and measurements were very specific, accurate, and reproducible.     

Regulation of Bcl-2 protein expression during oxidative stress in neuronal and in endothelial cells.

The relationship between oxidative stress and Bcl-2 expression was investigated in two different experimental models of oxidative stress. Acute oxidative stress was assessed by measuring, with fluorescence microscopy and cytofluorimetry, the increase in fluorescence of the oxidation-sensitive probe dihydrorhodamine 123, both in retinal rod receptor cells exposed to bright light (0.32 mW/cm(2) for 15 minutes) and in human endothelial cells treated with the immunosuppressant cyclosporin A (200 microM for 21 h). In both cell types, acute oxidative stress reduced Bcl-2 expression and also caused a significant increase in the level of nucleosomes. Interestingly, chronic treatment with clinical concentrations of cyclosporin A (0.5-2.5 microM for 8 days) led to a significant increase in Bcl-2 expression, while nucleosomes were similar to control level. This suggests that up-regulation of Bcl-2 protein by low levels of oxidants may represent a critical factor in cellular adaptation to drug toxicity.     

Development and validation of a liquid chromatographic-tandem mass spectrometric method for determination of piperaquine in plasma stable isotope labeled internal standard does not always compensate for matrix effects

A bioanalytical method for the analysis of piperaquine in human plasma using off-line solid-phase extraction and liquid chromatography coupled to positive tandem mass spectroscopy has been developed and validated. It was found that a mobile phase with high pH (i.e. 10) led to better sensitivity than mobile phase combinations with low pH (i.e. 2.5-4.5) despite the use of positive electrospray and a basic analyte. The method was validated according to published FDA guidelines and showed excellent performance. The within-day and between-day precisions expressed as R.S.D., were lower than 7% at all tested concentrations (4.5, 20, 400 and 500ng/mL) and below 10% at the lower limit of quantification (LLOQ) (1.5ng/mL). The calibration range was 1.5-500ng/mL with a limit of detection (LOD) at 0.38ng/mL. Validation of over-curve samples ensured that it would be possible with dilution if samples went outside the calibration range. Matrix effects were thoroughly evaluated both graphically and quantitatively. Matrix effects originating from the sample clean-up (i.e. solid-phase extraction) procedure rather than the plasma background were responsible for the ion suppression seen in this study. Salts remaining from the buffers used in the solid-phase extraction suppressed the signals for both piperaquine and its deuterated internal standard. This had no effect on the quantification of piperaquine. Triethylamine residues remaining after evaporation of the solid-phase extraction eluate were found to suppress the signals for piperaquine and its deuterated internal standard differently. It was found that this could lead to an underestimation of the true concentration with 50% despite the use of a deuterated internal standard.     

Differential gene expression in normal and transformed human mammary epithelial cells in response to oxidative stress

Oxidative stress plays a key role in breast carcinogenesis. To investigate whether normal and malignant breast epithelial cells differ in their responses to oxidative stress, we examined the global gene expression profiles of three cell types, representing cancer progression from a normal to a malignant stage, under oxidative stress. Normal human mammary epithelial cells (HMECs), an immortalized cell line (HMLER-1), and a tumorigenic cell line (HMLER-5) were exposed to increased levels of reactive oxygen species (ROS) by treatment with glucose oxidase. Functional analysis of the metabolic pathways enriched with differentially expressed genes demonstrated that normal and malignant breast epithelial cells diverge substantially in their response to oxidative stress. Whereas normal cells exhibit the up-regulation of antioxidant mechanisms, cancer cells are unresponsive to the ROS insult. However, the gene expression response of normal HMECs under oxidative stress is comparable to that of the malignant cells under normal conditions, indicating that altered redox status is persistent in breast cancer cells, which makes them resistant to increased generation of ROS. We discuss some of the possible adaptation mechanisms of breast cancer cells under persistent oxidative stress that differentiate them from normal mammary epithelial cells as regards the response to acute oxidative stress.     

Indole-3-acetic acid biosynthesis in Lemna gibba studied using stable isotope labeled anthranilate and tryptophan

IAA biosynthesis in many plants, including Lemna gibba, has been shown to involve at least two different pathways, one from tryptophan and a tryptophan-independent route. To study the kinetics of IAA biosynthesis in Lemna, we simultaneously measured the incorporation of label from [15N]-anthranilate and [2H5]-tryptophan into IAA by Lemna plants in short term feeding studies. The data show that label from anthranilate rapidly goes into IAA and tryptophan. Labeling of the IAA pool by [15N]-anthranilate slightly precedes labeling of the tryptophan pool, confirming that more than one route to IAA exists in these plants. Longer term feeding studies (5–25 h) suggest that exogenous tryptophan is used preferentially to label IAA as compared to tryptophan made by the plant. This is indicated by the fact that the IAA pool was more enriched than the tryptophan pool in [2H5]-label, but less enriched than the tryptophan pool in [15N] (which comes about by de novo synthesis of tryptophan from [15N]-anthranilate by the plant).     

Identification of the oxidative stress proteome in the brain

The redox proteomics technique normally combines two-dimensional gel electrophoresis, mass spectrometry, and protein databases to analyze the cell proteome from various samples, thereby leading to the identification of specific targets of oxidative modification. Oxidative stress that occurs because of increased levels of reactive oxygen species and reactive nitrogen species can target most biomolecules, consequently leading to altered physiological function of the cells. Redox proteomics has identified oxidatively modified protein targets in various pathological conditions, consequently providing insight into the pathways involved in the pathogenesis of these conditions. This approach also can be used to identify possible protective mechanisms to prevent or delay these disorders.     

Systematic characterization of nuclear proteome during apoptosis: a quantitative proteomic study by differential extraction and stable isotope labeling

Identification and characterization of the nuclear proteome is important for detailed understanding of multiple signaling events in eukaryotic cells. Toward this goal, we extensively characterized the nuclear proteome of human T leukemia cells by sequential extraction of nuclear proteins with different physicochemical properties using three buffer conditions. This large scale proteomic study also tested the feasibility and technical challenges associated with stable isotope labeling by amino acids in cell culture (SILAC) to uncover quantitative changes during apoptosis. Analyzing proteins from three nuclear fractions extracted from naive and apoptotic cells generated 780,530 MS/MS spectra that were used for database searching using the SEQUEST algorithm. This analysis resulted in the identification and quantification of 1,174 putative nuclear proteins. A number of known nuclear proteins involved in apoptosis as well as novel proteins not known to be part of the nuclear apoptotic machinery were identified and quantified. Consistent with SILAC-based quantifications, immunofluorescence staining of nucleus, mitochondria, and some associated proteins from both organelles revealed a dynamic recruitment of mitochondria into nuclear invaginations during apoptosis.     

Analysis of chicken serum proteome and differential protein expression during development in single-comb White Leghorn hens

Serum is believed to harbor thousands of distinct proteins that are either actively secreted or leak from various blood cells or tissues. Exploring protein composition in serum may accelerate the discovery of novel protein biomarkers for specific economic traits in livestock species. This study analyzed serum protein composition to establish a 2-DE reference map, and monitored protein dynamics of single-comb White Leghorn hens at 8, 19 and 23 weeks after hatching. A total of 119 CBB-stained and 315 silver-stained serum protein spots were analyzed by MALDI-TOF MS. Of these, 98 CBB-stained and 94 silver-stained protein spots were significantly matched to existing chicken proteins. The identified spots represented 30 distinctive proteins in the serum of laying hens. To compare protein expression during development, expression levels of 47 protein spots were quantified by relative spot volume with Melanie 3 software. Ten protein spots increased and 3 protein spots decreased as hen age increased. Previous research has suggested that some of these proteins play critical roles in egg production. The differentially expressed proteins with unknown identities will be valuable candidates for further explorations of their roles in egg production of laying hens.     

Multidrug-resistance-associated protein plays a protective role in menadione-induced oxidative stress in endothelial cells

AimsMenadione, a redox-cycling quinone known to cause oxidative stress, binds to reduced glutathione (GSH) to form glutathione S-conjugate. Glutathione S-conjugates efflux is often mediated by multidrug-resistance-associated protein (MRP). We investigated the effect of a transporter inhibitor, MK571 (3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid), on menadione-induced oxidative stress in bovine aortic endothelial cells (BAECs).Main methodsBAECs were treated with menadione and MK571, and cell viability was measured. Modulation of intracellular GSH levels was performed with buthionine sulfoximine and GSH ethyl ester treatments. Intracellular superoxide was estimated by dihydroethidium oxidation using fluorescence microscopy or flow cytometry. Expression of MRP was determined by flow cytometry using phycoerythrin-conjugated anti-MRP monoclonal antibody.Key findingsIntracellular GSH depletion by buthionine sulfoximine promoted the loss of viability of BAECs exposed to menadione. Exogenous GSH, which does not permeate the cell membrane, or GSH ethyl ester protected BAECs against the loss of viability induced by menadione. The results suggest that GSH binds to menadione outside the cells as well as inside. Pretreatment of BAECs with MK571 dramatically increased intracellular levels of superoxide generated from menadione, indicating that menadione may accumulate in the intracellular milieu. Finally, we found that MK571 aggravated menadione-induced toxicity in BAECs and that MRP levels were increased in menadione-treated cells.SignificanceWe conclude that MRP plays a vital role in protecting BAECs against menadione-induced oxidative stress, presumably due to its ability to transport glutathione S-conjugate.