Monitoring and Identifying Insulin-Like Growth Factor 1 Variants by Liquid Chromatography-High-Resolution Mass Spectrometry in a Clinical Laboratory.

Protein and peptide hormones often exist as sequence variants with different molecular mass. Monitoring these variants of different molecular mass by mass spectrometry using mass-to-charge (m/z) ratio that is indicative of the wild type may lead to inaccurate quantitative results. However, liquid chromatography-high-resolution mass spectrometry (LC-HRMS)-based techniques can capture these differences and provide an opportunity to resolve, or partially resolve, variant complexity. In this chapter, we describe a general approach for monitoring a set of peptide variants with similar m/z ratios and isotopic envelopes, but different in amino acid sequences. As an example, we use insulin-like growth factor-1 (IGF-1) to demonstrate a DNA database-guided approach to monitor protein variants by LC-HRMS in a clinical laboratory. The workflow is automated and therefore avoids manual calculations that are prone to human error. The method can also monitor multiple IGF-1 variants and discover new ones. It can also provide a profile of a patient's IGF-1 status and be used to explore genotype-phenotype relationships in IGF-1 variants.

High prevalence of subnormal testosterone in obese adolescent males: reversal with bariatric surgery.

OBJECTIVE: Obesity in adolescent males is associated with the lowering of total and free testosterone concentrations. Weight loss may increase testosterone concentrations. DESIGN AND METHODS: We evaluated the changes in sex hormones following bariatric surgery in 34 males (age range: 14.6-19.8 years) with obesity. These participants were part of a prospective multicenter study, Teen-Longitudinal Assessment of Bariatric Surgery. The participants were followed up for 5 years after surgery. Total testosterone, total estradiol, luteinizing hormone, follicle-stimulating hormone, sex hormone-binding globulin, C-reactive protein, insulin and glucose were measured at baseline, 6 months and annually thereafter. Free testosterone, free estradiol and HOMA2-IR were calculated. RESULTS: Study participants lost one-third of their body weight after bariatric surgery, with maximum weight loss achieved at 24 months for most participants. Free testosterone increased from 0.17 (95% CI: 0.13 to 0.20) at baseline to 0.34 (95% CI: 0.30 to 0.38) and 0.27 nmol/L (95% CI: 0.23 to 0.32) at 2 and 5 years (P < 0.001 for both), respectively. Total testosterone increased from 6.7 (95% CI: 4.7 to 8.8) at baseline to 17.6 (95% CI: 15.3 to 19.9) and 13.8 (95% CI: 11.0 to 16.5) nmol/L at 2 and 5 years (P < 0.001), respectively. Prior to surgery, 73% of the participants had subnormal free testosterone (<0.23 nmol/L). After 2 and 5 years, only 20 and 33%, respectively, had subnormal free testosterone concentrations. Weight regain was related to a fall in free testosterone concentrations. CONCLUSIONS: Bariatric surgery led to a robust increase in testosterone concentrations in adolescent males with severe obesity. Participants who regained weight had a decline in their testosterone concentrations.

Isotopic Peak Index, Relative Retention Time, and Tandem MS for Automated High Throughput IGF-1 Variants Identification in a Clinical Laboratory.

Measuring insulin-like growth factor-1 (IGF-1) is useful for assessing and managing growth-related disorders, such as acromegaly and growth hormone deficiency. High-resolution liquid chromatography-mass spectrometry (LC-MS) is used for measuring IGF-1 due to its molecular specificity, quantitative performance, well-characterized reference materials, and detailed age/sex-specific reference intervals. However, polymorphisms in the IGF1 gene may cause mass shifts in the polypeptide, which can impede quantitation and cause errors in clinical interpretation. We (1) developed a concept of "isotopic peak index", which allows simultaneous monitoring of 15 IGF-1 variants by using only four m/z ratios; (2) developed a "relative retention time" parameter that allows distinction of previously unresolved variants; and (3) utilized tandem mass spectrometry (MS/MS) to distinguish between the most common pair of variants: isobaric A67T and A70T. All methods were validated with DNA sequencing. This approach identified six variants from the ExAC database, P66A, A67S, S34N, A38 V, A67T, and A70T; two previously reported V44M and A67V variants; and discovered six unreported variants, Y31H, S33P, R50Q, R56K, T41I, and A62T. Major improvements in our workflow include enhanced automation, avoiding detailed manual calculations that are prone to human error, and the ability to monitor more, and discover new, IGF-1 variants. The workflow provides a profile of a patient's IGF-1 status and can be used to explore genotype-phenotype relationships in IGF-1 variants.

Association of Circulating Sex Hormones With Inflammation and Disease Severity in Patients With COVID-19.

Importance: Male sex is a risk factor for developing severe COVID-19 illness. It is not known whether sex hormones contribute to this predisposition. Objective: To investigate the association of concentrations of serum testosterone, estradiol, and insulinlike growth factor 1 (IGF-1, concentrations of which are regulated by sex hormone signaling) with COVID-19 severity. Design, Setting, and Participants: This prospective cohort study was conducted using serum samples collected from consecutive patients who presented from March through May 2020 to the Barnes Jewish Hospital in St Louis, Missouri, with COVID-19 (diagnosed using nasopharyngeal swabs). Exposures: Testosterone, estradiol, and IGF-1 concentrations were measured at the time of presentation (ie, day 0) and at days 3, 7, 14, and 28 after admission (if the patient remained hospitalized). Main Outcomes and Measures: Baseline hormone concentrations were compared among patients who had severe COVID-19 vs those with milder COVID-19 illness. RNA sequencing was performed on circulating mononuclear cells to understand the mechanistic association of altered circulating hormone concentrations with cellular signaling pathways. Results: Among 152 patients (90 [59.2%] men; 62 [40.8%] women; mean [SD] age, 63 [16] years), 143 patients (94.1%) were hospitalized. Among 66 men with severe COVID-19, median [interquartile range] testosterone concentrations were lower at day 0 (53 [18 to 114] ng/dL vs 151 [95 to 217] ng/dL; P = .01) and day 3 (19 [6 to 68] ng/dL vs 111 [49 to 274] ng/dL; P = .006) compared with 24 men with milder disease. Testosterone concentrations were inversely associated with concentrations of interleukin 6 (ß = -0.43; 95% CI, -0.52 to -0.17; P < .001), C-reactive protein (ß = -0.38; 95% CI, -0.78 to -0.16; P = .004), interleukin 1 receptor antagonist (ß = -0.29; 95% CI, -0.64 to -0.06; P = .02), hepatocyte growth factor (ß = -0.46; 95% CI, -0.69 to -0.25; P < .001), and interferon γ-inducible protein 10 (ß = -0.32; 95% CI, -0.62 to -0.10; P = .007). Estradiol and IGF-1 concentrations were not associated with COVID-19 severity in men. Testosterone, estradiol, and IGF-1 concentrations were similar in women with and without severe COVID-19. Gene set enrichment analysis revealed upregulated hormone signaling pathways in CD14+CD16- (ie, classical) monocytes and CD14-CD16+ (ie, nonclassical) monocytes in male patients with COVID-19 who needed intensive care unit treatment vs those who did not. Conclusions and Relevance: In this single-center cohort study of patients with COVID-19, lower testosterone concentrations during hospitalization were associated with increased disease severity and inflammation in men. Hormone signaling pathways in monocytes did not parallel serum hormone concentrations, and further investigation is required to understand their pathophysiologic association with COVID-19.

Discordance between mass spectrometry and immunometric IGF-1 assay in pituitary disease: a prospective study.

PURPOSE: Measuring IGF-1, a biomarker for GH activity, is critical to evaluating disordered hypothalamic-pituitary GH axis. Inconsistent IGF-1 measurements among different immunoassays are well documented. We switched from Immulite 2000 immunoassay to narrow-mass-extraction, high-resolution liquid chromatography mass-spectrometry (LC-MS) compliant with recent consensus recommendations on assay standardization. Comparability of these two assays in patients with pituitary disease in a clinical practice setting is not known. We sought to compare IGF-1 levels on Immulite 2000 and LC-MS in samples from naïve and treated patients with secretory and non-secretory pituitary masses. METHODS: We prospectively collected serum samples from 101 patients treated at the Cedars-Sinai Pituitary Center between February 2012 and March 2014. We intentionally recruited more patients with acromegaly or GH deficiency to ensure a clinically representative cohort. Samples were classified as in or out of the respective reference ranges. Bland-Altman analysis was used to assess agreement between assays. RESULTS: Twenty-four percent of samples were classified differently as below, in, or above range. Agreement between the assays was poor overall, with a significant bias for immunoassay reporting higher values than LC-MS. This pattern was also observed in patients with acromegaly and those with ≥ 2 pituitary hormone deficiencies. CONCLUSIONS: IGF-1 results may differ after switching from an older immunoassay to a consensus-compliant assay such as LC-MS. Clinicians should consider the potential impact of assay switching before altering treatment due to discrepant results, particularly in patients monitored over time, such as those with acromegaly and GH deficiency.

Serum vitamin D metabolites in colorectal cancer patients receiving cholecalciferol supplementation: correlation with polymorphisms in the vitamin D genes.

Cholecalciferol (D(3)) supplementation results in variable increases in serum 25(OH)D(3) levels, however, the influence of genetic polymorphisms on these variable responses is unclear. We measured serum 25(OH)D(3), 24,25(OH)(2)D(3), 1,25(OH)2D(3) and VDBP levels in 50 colorectal cancer (CRC) patients before and during 2,000 IU daily oral D(3) supplementation for six months and in 263 archived CRC serum samples. Serum PTH levels and PBMC 24-OHase activity were also measured during D(3) supplementation. TagSNPs in CYP2R1, CYP27A1, CYP27B1, CYP24A1, VDR, and GC genes were genotyped in all patients, and the association between these SNPs and serum vitamin D(3) metabolites levels before and after D(3) supplementation was analyzed. The mean baseline serum 25(OH)D(3) level was less than 32 ng/mL in 65 % of the 313 CRC patients. In the 50 patients receiving D(3) supplementation, serum levels of 25(OH)D(3) increased (p = 0.008), PTH decreased (p = 0.036) and 24,25(OH)(2)D(3), 1,25(OH)(2)D(3), VDBP levels and PBMC 24-OHase activity were unchanged. GC SNP rs222016 was associated with high 25(OH)D(3) and 1,25(OH)(2)D(3) levels at baseline while rs4588 and rs2282679 were associated with lower 25(OH)D(3) and 1,25(OH)(2)D(3) levels both before and after D(3) supplementation. CYP2R1 rs12794714 and rs10500804 SNPs were significantly associated with low 25(OH)D(3) levels after supplementation but not with baseline 25(OH)D(3). Our results show that D(3) supplementation increased 25(OH)D(3) levels in all patients. GC rs4588 and rs2283679 SNPs were associated with increased risk of vitamin D(3) insufficiency and suboptimal increase in 25(OH)D(3) levels after D(3) supplementation. Individuals with these genotypes may require higher D(3) supplementation doses to achieve vitamin D(3) sufficiency.

Pharmacokinetics, pharmacodynamics and metabolism of a novel anticancer agent for prostate cancer.

The objective of these studies was to examine the murine pharmacokinetics, pharmacodynamics and metabolism of (3-(1H-indol-2-yl)phenyl)(1H-indol-2-yl)methanone (Indole 15), a novel tubulin inhibitor for the treatment of cancer. We developed HPLC and LC/MS/MS assays to quantitate Indole 15 and characterize its metabolites in vivo. Pharmacokinetic studies were performed after intravenous (IV), oral (PO) and subcutaneous (SC) administration of 10 mg/kg doses to male ICR mice. Urine and fecal samples were also collected over a 72-h period to identify metabolites. Pharmacodynamic studies were conducted by monitoring the tumor size change during a period of two weeks in PC-3 tumor bearing mice after daily IV administration of Indole 15 at doses of 0, 10, 50, 100 and 150 mg/kg. The pooled plasma concentration data after administration via different dose routes was simultaneously fitted by a two-compartmental model. Indole 15 was moderately well absorbed after PO and SC administration, with a bioavailable fraction of 0.27 and 0.72, respectively. The steady state volume distribution (Vss) and clearance (CL) were estimated to be 7.0 l/kg and 4.36 l/h/kg, respectively. The mean data of PC-3 tumor growth in mice was fitted well by a transduction model using fixed plasma pharmacokinetics as a driving function. Analysis of the metabolites in mice indicated that the compound undergoes extensive oxidative metabolism with subsequent sulfation. These studies demonstrate that favorable pharmacokinetic and pharmacodynamic properties of Indole 15 offer hope for achieving pharmacological activity in early clinical trials.

Pharmacokinetics, biodistribution and metabolism of a novel selective androgen receptor modulator designed for prostate cancer imaging.

Knowledge of the presence and extent of disease plays a major role in clinical management of prostate cancer, as it provides meaningful information as to which therapy to choose and who might benefit from this therapy. The wide expression of androgen receptor (AR) in primary and metastatic prostate tumors offers a cellular target for receptor-mediated imaging of prostate cancer. In our previous study, a non-steroidal AR ligand, S-26 [S-3-(4-fluorophenoxy)-2-hydroxy-2-methyl-N-(4-cyano-3-iodophenyl)-propionamide] showed promising in vitro pharmacological properties as an AR-mediated imaging agent, with high AR binding affinity and AR specificity. The overall goal of this study was to characterize the in vivo metabolic and biodistribution profile of S-26 in rats. Non-compartmental pharmacokinetic analysis of S-26 in rat plasma showed that clearance (CL), volume of distribution (Vd(ss)), and half-life (T(1/2)) of S-26 were 0.30 + or - 0.07 l/h/kg, 1.44 + or - 0.33 l/kg, and 4 h, respectively, after intravenous (i.v.) administration. Dose proportionality (1, 10 and 30 mg/kg) studies suggested that the pharmacokinetics of S-26 are dose-independent. The plasma concentrations of all 3 doses were further simultaneously fitted with a two-compartmental model and the results were similar to those obtained from non-compartmental analysis. Biodistribution studies using (125)I-labeled S-26 indicated that it did not specifically target AR-rich tissue (e.g. prostate). A substantial amount of radioactivity recovered from thyroid gland indicated the release of free iodine. In metabolism studies, unchanged S-26 and its metabolites were detected in rat urine and fecal samples. Oxidation, de-iodination, hydrolysis, and sulfate conjugation were the major metabolic pathways of S-26 in rats, with de-iodination representing a unique metabolic pathway of S-26 among other selective androgen receptor modulators. In conclusion, the extensive plasma clearance and de-iodination of S-26 likely contribute to its lack of AR tissue selectivity in vivo. Future studies using metabolically stable ligands with less lipophilicity and higher AR binding affinity may represent a promising and rational approach for AR-mediated imaging.

Effect of B-ring substitution pattern on binding mode of propionamide selective androgen receptor modulators.

Selective androgen receptor modulators (SARMs) are essentially prostate sparing androgens, which provide therapeutic potential in osteoporosis, male hormone replacement, and muscle wasting. Herein we report crystal structures of the androgen receptor (AR) ligand-binding domain (LBD) complexed to a series of potent synthetic nonsteroidal SARMs with a substituted pendant arene referred to as the B-ring. We found that hydrophilic B-ring para-substituted analogs exhibit an additional region of hydrogen bonding not seen with steroidal compounds and that multiple halogen substitutions affect the B-ring conformation and aromatic interactions with Trp741. This information elucidates interactions important for high AR binding affinity and provides new insight for structure-based drug design.

Crystal structure of the T877A human androgen receptor ligand-binding domain complexed to cyproterone acetate provides insight for ligand-induced conformational changes and structure-based drug design.

Cyproterone acetate (CPA) is a steroidal antiandrogen used clinically in the treatment of prostate cancer. Compared with steroidal agonists for the androgen receptor (AR) (e.g. dihydrotestosterone, R1881), CPA is bulkier in structure and therefore seemingly incompatible with the binding pockets observed in currently available x-ray crystal structures of the AR ligand-binding domain (LBD). We solved the x-ray crystal structure of the human AR LBD bound to CPA at 1.8A in the T877A variant, a mutation known to increase the agonist activity of CPA and therefore facilitate purification and crystal formation of the receptor.drug complex. The structure demonstrates that bulk from the 17alpha-acetate group of CPA induces movement of the Leu-701 side chain, which results in partial unfolding of the C-terminal end of helix 11 and displacement of the loop between helices 11 and 12 in comparison to all other AR LBD crystal structures published to date. This structural alteration leads to an expansion of the AR binding cavity to include an additional pocket bordered by Leu-701, Leu-704, Ser-778, Met-780, Phe-876, and Leu-880. Further, we found that CPA invokes transcriptional activation in the L701A AR at low nanomolar concentrations similar to the T877A mutant. Analogous mutations in the glucocorticoid receptor (GR) and progesterone receptor were constructed, and we found that CPA was also converted into a potent agonist in the M560A GR. Altogether, these data offer information for structure-based drug design, elucidate flexible regions of the AR LBD, and provide insight as to how CPA antagonizes the AR and GR.

Favorable effects of weak acids on negative-ion electrospray ionization mass spectrometry.

Despite widespread use in pharmacokinetic, drug metabolism, and pesticide residue studies, little is known about the factors governing response during reversed-phase liquid chromatography coupled with negative-ion electrospray ionization (ESI(-)) mass spectrometry. We examined the effects of various mobile-phase modifiers on the ESI(-) response of four selective androgen receptor modulators using a postcolumn infusion system. Acetic, propionic, and butyric acid improved the ESI(-) responses of analytes to varying extents at low concentrations. Formic acid suppressed ionization, as did neutral salts (ammonium formate, ammonium acetate) and bases (ammonium hydroxide, triethylamine) under most conditions. Two modifiers (2,2,2-trifluoroethanol, formaldehyde) that produce anions with high gas-phase proton affinity increased ESI(-) responses. However, the concentrations of these modifiers required to enhance ESI(-) response were higher than that of acidic modifiers, which is a phenomenon likely related to their low pK(a) values. 2,2,2-Trifluoroethanol increased response of more hydrophobic compounds but decreased response of a more hydrophilic compound. Formaldehyde improved response of all the compounds, especially the hydrophilic compound with lower surface activity. In summary, these results suggest that an ideal ESI(-) modifier should provide cations that can be easily electrochemically reduced and produce anions with small molecular volume and high gas-phase proton affinity.