Standard -20 °C freezer storage protocols may cause substantial plasma renin cryoactivation.

OBJECTIVES: To assess the appropriate preanalytical process for storage of plasma for renin concentration analysis. This study was initiated due to the wide variation in preanalytical handling of samples observed within our network, particularly with respect to freezing for longer term storage. METHODS: Pooled plasma from patient samples was analysed immediately post separation for renin concentration (n=30, concentration 4.0-204 mIU/L). Aliquots from these samples were frozen in a -20 °C freezer and then analysed, with the renin concentration compared to the respective baseline concentration. Comparisons were also made to: aliquots snap frozen using a dry ice/acetone bath, aliquots stored at room temperature, and aliquots stored at 4 °C. Subsequent experiments investigated the potential sources of cryoactivation observed in these initial studies. RESULTS: Substantial and highly variable cryoactivation was observed in samples frozen using a -20 °C freezer, with renin concentration increasing over 300% from baseline in some samples (median 21.3%). This cryoactivation could be prevented by snap freezing samples. Subsequent experiments determined that long term storage in a -20 °C freezer could prevent cryoactivation provided samples were initially frozen rapidly in a -70 °C freezer. Rapid defrosting of samples was not required to prevent cryoactivation. CONCLUSIONS: Standard -20 °C freezers may not be appropriate for freezing samples for renin analysis. Laboratories should consider snap freezing their samples using a -70 °C freezer or similar to avoid cryoactivation of renin.

Streamlined three step total vitamin C analysis by HILIC-UV for laboratory testing.

OBJECTIVES: In the clinical setting, the analysis and quantification of vitamin C (ascorbic acid) poses several challenges including analyte instability and poor retention by reverse phase HPLC systems. In this article we describe a rapid hydrophilic interaction chromatography ultraviolet method for the measurement of total vitamin C in plasma which overcomes these issues. METHODS: Ascorbic acid and the internal standard were separated under isocratic conditions using a Waters BEH-Amide column and a mobile phase containing 0.005 M potassium phosphate in 80% acetonitrile. RESULTS: The proposed method was validated and showed good precision (coefficient of variation <5%), accuracy (>99%), and analyte stability after extraction (>24 h). CONCLUSIONS: The simple sample preparation allows full automation and rapid analytical run times of the assay and is therefore suitable for a high-throughput clinical chromatography laboratory.

Hypoxia-Responsive Cobalt Complexes in Tumor Spheroids: Laser Ablation Inductively Coupled Plasma Mass Spectrometry and Magnetic Resonance Imaging Studies.

Dense tumors are resistant to conventional chemotherapies due to the unique tumor microenvironment characterized by hypoxic regions that promote cellular dormancy. Bioreductive drugs that are activated in response to this hypoxic environment are an attractive strategy for therapy with anticipated lower harmful side effects in normoxic healthy tissue. Cobalt bioreductive pro-drugs that selectively release toxic payloads upon reduction in hypoxic cells have shown great promise as anticancer agents. However, the bioreductive response in the tumor microenvironment must be better understood, as current techniques for monitoring bioreduction to Co(II) such as X-ray absorption near-edge structure and extended X-ray absorption fine structure provide limited information on speciation and require synchrotron radiation sources. Here, we present magnetic resonance imaging (MRI) as an accessible and powerful technique to monitor bioreduction by treating the cobalt complex as an MRI contrast agent and monitoring the change in water signal induced by reduction from diamagnetic Co(III) to paramagnetic Co(II). Cobalt pro-drugs built upon the tris(2-pyridylmethyl)amine ligand scaffold with varying charge were investigated for distribution and activity in a 3D tumor spheroid model by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) and MRI. In addition, paramagnetic 1H NMR spectroscopy of spheroids enabled determination of the speciation of activated Co(II)TPAx complexes. This study demonstrates the utility of MRI and associated spectroscopy techniques for understanding bioreductive cobalt pro-drugs in the tumor microenvironment and has broader implications for monitoring paramagnetic metal-based therapies.

The first human normative ranges and biomarker performance of dimethylguanidino valeric acid isoforms in fatty liver disease.

We have recently determined dimethylguanidino valeric acid (DMGV) to be a novel biomarker of liver injury in non-alcoholic fatty liver disease (NAFLD) and an independent predictor of incident diabetes over a decade in advance. DMGV consists of two stereo-isomers, asymmetric dimethylguanidino valeric acid (ADGV) and symmetric dimethylguanidino valeric acid (SDGV). Here we report, for the first time, the upper limits of normal of both isomers in humans at the accepted 5.56% liver fat threshold for NAFLD, determined using in vivo magnetic resonance spectroscopy. We performed independent and blinded comparative analyses of ADGV and SDGV levels using two different liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods in (A) our laboratory, and (B) the New South Wales Chemical Pathology state laboratory, using unique columns, LC-MS/MS equipment, extraction protocols and normalisation approaches. Despite these differences, each laboratory reported consistent absolute concentrations across a range of liver fat percentages. We next determined the diagnostic performance of SDGV compared to ADGV in a cohort of 268 individuals with liver fat measurements. In derivation-validation analyses we determined rule-in/rule-out thresholds and the concentration of SDGV that provides optimal performance across sensitivity and specificity for the identification of NAFLD. In conclusion, we have herein determined for the first time the true human plasma reference range of both isoforms of an emerging novel biomarker of NAFLD, at the accepted upper normal threshold of liver fat. We have also identified that SDGV is the isoform with the best diagnostic performance and determined the optimal cut-point for its detection of NAFLD.

PCPro: a clinically accessible, circulating lipid biomarker signature for poor-prognosis metastatic prostate cancer.

BACKGROUND: Using comprehensive plasma lipidomic profiling from men with metastatic castration-resistant prostate cancer (mCRPC), we have previously identified a poor-prognostic lipid profile associated with shorter overall survival (OS). In order to translate this biomarker into the clinic, these men must be identifiable via a clinically accessible, regulatory-compliant assay. METHODS: A single regulatory-compliant liquid chromatography-mass spectrometry assay of candidate lipids was developed and tested on a mCRPC Discovery cohort of 105 men. Various risk-score Cox regression prognostic models of OS were built using the Discovery cohort. The model with the highest concordance index (PCPro) was chosen for validation and tested on an independent Validation cohort of 183 men. RESULTS: PCPro, the lipid biomarker, contains Cer(d18:1/18:0), Cer(d18:1/24:0), Cer(d18:1/24:1), triglycerides and total cholesterol. Within the Discovery and Validation cohorts, men who were PCPro positive had significantly shorter OS compared to those who were PCPro negative (Discovery: median OS 12.0 months vs 24.2 months, hazard ratio (HR) 3.75 [95% confidence interval (CI) 2.29-6.15], p < 0.001, Validation: median OS 13.0 months vs 25.7 months, HR = 2.13 [95% CI 1.46-3.12], p < 0.001). CONCLUSIONS: We have developed PCPro, a lipid biomarker assay capable of prospectively identifying men with mCRPC with a poor prognosis. Prospective clinical trials are required to determine if men who are PCPro positive will benefit from therapeutic agents targeting lipid metabolism.

Facile preparation of mono-, di- and mixed-carboxylato platinum(IV) complexes for versatile anticancer prodrug design.

Facile strategies were developed for the versatile functionalization of platinum(IV) axial sites, allowing for easy accessibility to unsymmetric mono- and mixed-carboxylato, as well as symmetric di-substituted platinum(IV) complexes. The first method involves the direct oxidation and carboxylation of the platinum(II) center using an appropriate peroxide and the carboxylate of choice to firstly yield a monocarboxylato monohydroxido platinum(IV) complex. This platinum(IV) intermediate can undergo further carboxylation to give rise to a mixed-carboxylato platinum(IV) complex. The second method involves the activation of the carboxylate of choice by a common carbodiimide coupling reagent, and its reaction with a dihydroxido platinum(IV) precursor to give the monocarboxylato platinum(IV) complex. Uronium salts can be employed to promote efficient dicarboxylation of the dihydroxido platinum(IV) precursor. Lastly, an axial azide pendant group was demonstrated to be suitable for orthogonal "click" conjugation reactions.

Design and validation of an LC-MS/MS method for simultaneous quantification of asymmetric dimethylguanidino valeric acid, asymmetric dimethylarginine and symmetric dimethylarginine in human plasma.

Asymmetric dimethylguanidino valeric acid (ADGV), asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) are three arginine metabolites which have utility in the assessment of cardiovascular disease, renal disease and non-alcoholic fatty liver disease (NAFLD). Translation of these research metabolomic markers into routine clinical use requires the development of robust assays with appropriately assessed preanalytical variables and traceable clinical reference intervals. A hydrophilic interaction liquid chromatography (HILIC) tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of ADGV, ADMA and SDMA was developed. Sample stability and collection conditions were scrutinised to determine any preanalytical factors that could affect quantification under routine laboratory conditions. Patient samples from 120 males and 120 females were used to derive preliminary reference intervals. All three analytes were quantifiable in human plasma using unique MS/MS transitions. The analytes were stable for up to a week once separated from red cells, though reduced stability was observed upon extraction of the analytes from plasma. The assay was linear for concentration of ADGV between 1.6 nmol/L and 200 nmol/L and for ADMA and SDMA between 0.1 µmol/L and 4.0 µmol/L. The accuracy for all analytes was 97-103% and interday and intraday imprecisions (coefficients of variation) were less than 10%. ADGV concentrations were noted to be lower in the female reference population when compared to males. The analytical method shows excellent performance and is sufficiently robust to be used in the clinical investigation of cardiovascular disease and NAFLD.

Overcoming enzalutamide resistance in metastatic prostate cancer by targeting sphingosine kinase.

BACKGROUND: Intrinsic resistance to androgen receptor signalling inhibitors (ARSI) occurs in 20-30% of men with metastatic castration-resistant prostate cancer (mCRPC). Ceramide metabolism may have a role in ARSI resistance. Our study's aim is to investigate the association of the ceramide-sphingosine-1-phosphate (ceramide-S1P) signalling axis with ARSI resistance in mCRPC. METHODS: Lipidomic analysis (∼700 lipids) was performed on plasma collected from 132 men with mCRPC, before commencing enzalutamide or abiraterone. AR gene aberrations in 77 of these men were identified by deep sequencing of circulating tumour DNA. Associations between circulating lipids, radiological progression-free survival (rPFS) and overall survival (OS) were examined by Cox regression. Inhibition of ceramide-S1P signalling with sphingosine kinase (SPHK) inhibitors (PF-543 and ABC294640) on enzalutamide efficacy was investigated with in vitro assays, and transcriptomic and lipidomic analyses of prostate cancer (PC) cell lines (LNCaP, C42B, 22Rv1). FINDINGS: Men with elevated circulating ceramide levels had shorter rPFS (HR=2·3, 95% CI=1·5-3·6, p = 0·0004) and shorter OS (HR=2·3, 95% CI=1·4-36, p = 0·0005). The combined presence of an AR aberration with elevated ceramide levels conferred a worse prognosis than the presence of only one or none of these characteristics (median rPFS time = 3·9 vs 8·3 vs 17·7 months; median OS time = 8·9 vs 19·8 vs 34·4 months). SPHK inhibitors enhanced enzalutamide efficacy in PC cell lines. Transcriptomic and lipidomic analyses indicated that enzalutamide combined with SPHK inhibition enhanced PC cell death by SREBP-induced lipotoxicity. INTERPRETATION: Ceramide-S1P signalling promotes ARSI resistance, which can be reversed with SPHK inhibitors. FUNDING: None.

In vitro and in vivo evaluation of bifunctional bisthiosemicarbazone 64Cu-complexes for the positron emission tomography imaging of hypoxia.

The copper(II) bisthiosemicarbazonato complex, copper-diacetyl-bis(N4-methylthiosemicarbazonate) (Cu-ATSM), has been used clinically as a positron emission tomography (PET) tracer for the delineation of hypoxia. Six novel, asymmetric bis(thiosemicarbazones) derived from diacetyl-2-(4-N-methyl-3-thiosemicarbazone)-3-(4-N-amino-3-thiosemicarbazone) (H2ATSM/A), one of which contained a nitroimidazole functionality, were radiolabeled with 64Cu (t1/2=12.7 h, beta+=19.3%). In vitro studies were performed on three of the compounds using EMT6 mammary carcinoma cells under hypoxic and normoxic conditions. All compounds displayed rapid cellular association and appreciable hypoxic selectivity with increased uptake under normoxic and hypoxic conditions when compared to 64Cu-ATSM. Biodistribution and small animal PET imaging studies were then carried out in vivo using two compounds in EMT6 tumor-bearing mice. The compounds showed high tumor uptake, but also substantial accumulation in the liver. These complexes demonstrate that H 2ATSM/A represents a novel and versatile synthetic platform that can be utilized to provide hypoxic cell selectivity through functionalization of the bisthiosemicarbazonate group.

In vitro and in vivo evaluations of a hydrophilic 64Cu-bis(thiosemicarbazonato)-glucose conjugate for hypoxia imaging.

UNLABELLED: A water-soluble glucose conjugate of the hypoxia tracer 64Cu-diacetyl-bis(N4-methylthiosemicarbazone) (64Cu-ATSM) was synthesized and radiolabeled (64Cu-ATSE/A-G). Here we report our initial biological experiments with 64Cu-ATSE/A-G and compare the results with those obtained for 64Cu-ATSM and 18F-FDG. METHODS: The uptake of 64Cu-ATSE/A-G and 64Cu-ATSM into HeLa cells in vitro was investigated at a range of dissolved oxygen concentrations representing normoxia, hypoxia, and anoxia. Small-animal PET with 64Cu-ATSE/A-G was performed in male BDIX rats implanted with P22 syngeneic carcinosarcomas. Images of 64Cu-ATSM and 18F-FDG were obtained in the same model for comparison. RESULTS: 64CuATSE/A-G showed oxygen concentration-dependent uptake in vitro and, under anoxic conditions, showed slightly lower levels of cellular uptake than 64Cu-ATSM; uptake levels under hypoxic conditions were also lower. Whereas the normoxic uptake of 64Cu-ATSM increased linearly over time, 64Cu-ATSE/A-G uptake remained at low levels over the entire time course. In the PET study, 64CuATSE/A-G showed good tumor uptake and a biodistribution pattern substantially different from that of each of the controls. In marked contrast to the findings for 64Cu-ATSM, renal clearance and accumulation in the bladder were observed. 64Cu-ATSE/A-G did not display the characteristic brain and heart uptake of 18F-FDG. CONCLUSION: The in vitro cell uptake studies demonstrated that 64Cu-ATSE/A-G retained hypoxia selectivity and had improved characteristics when compared with 64Cu-ATSM. The in vivo PET results indicated a difference in the excretion pathways, with a shift from primarily hepatointestinal for 64Cu-ATSM to partially renal with 64Cu-ATSE/A-G. This finding is consistent with the hydrophilic nature of the glucose conjugate. A comparison with 18F-FDG PET results revealed that 64Cu-ATSE/A-G was not a surrogate for glucose metabolism. We have demonstrated that our method for the modification of Cu-bis(thiosemicarbazonato) complexes allows their biodistribution to be modified without negating their hypoxia selectivity or tumor uptake properties.

Clinical imaging of hypoxia: Current status and future directions.

Tissue hypoxia is a key feature of many important causes of morbidity and mortality. In pathologies such as stroke, peripheral vascular disease and ischaemic heart disease, hypoxia is largely a consequence of low blood flow induced ischaemia, hence perfusion imaging is often used as a surrogate for hypoxia to guide clinical diagnosis and treatment. Importantly, ischaemia and hypoxia are not synonymous conditions as it is not universally true that well perfused tissues are normoxic or that poorly perfused tissues are hypoxic. In pathologies such as cancer, for instance, perfusion imaging and oxygen concentration are less well correlated, and oxygen concentration is independently correlated to radiotherapy response and overall treatment outcomes. In addition, the progression of many diseases is intricately related to maladaptive responses to the hypoxia itself. Thus there is potentially great clinical and scientific utility in direct measurements of tissue oxygenation. Despite this, imaging assessment of hypoxia in patients is rarely performed in clinical settings. This review summarises some of the current methods used to clinically evaluate hypoxia, the barriers to the routine use of these methods and the newer agents and techniques being explored for the assessment of hypoxia in pathological processes.

XANES investigation of the Co oxidation state in solution and in cancer cells treated with Co(III) complexes.

XANES spectroscopy has been used to investigate whether it is possible to determine the oxidation state and coordination environment of Co complexes following treatment of cancer cells with Co(III) or Co(II) complexes. Our results show that the variation of the XANES with coordination geometry make it impossible to do this in a completely reliable way which is in contrast to the situation for platinum and chromium. It was established that the XANES spectrum obtained from cells treated with [Co(diNOsar)]Br(3) remained unchanged with respect to its XANES spectrum obtained in solution, demonstrating that the [Co(diNOsar)]Br(3) complex remained intact after 24h in cellular media (diNOsar=1,8-dinitro-3,6,10,13,16,19-hexaazabicyclo[6.6.6]eicosane). In contrast, the XANES spectra obtained from cells treated with Na[Co(acac)(3)] and [Co(acac)(3)] differed from the XANES spectra of the respective complexes obtained in solution, indicating a change in co-ordination environment for both complexes upon uptake in cells. The similarity of these spectra suggests that appearance of this XANES can be used as an indication of loss of the carrier ligands, a useful indicator in the study of hypoxia selective complexes. The results obtained for Na[Co(acac)(3)] and [Co(acac)(3)] are consistent with the intracellular coordination of cobalt(III) to sulfur ligands upon cellular uptake.

Cobalt complexes with tripodal ligands: implications for the design of drug chaperones.

Extensive research is currently being conducted into metal complexes that can selectively deliver cytotoxins to hypoxic regions in tumours. The development of pharmacologically suitable agents requires an understanding of appropriate ligand-metal systems for chaperoning cytotoxins. In this study, cobalt complexes with tripodal tren (tris-(2-aminoethyl)amine) and tpa (tris-(2-pyridylmethyl)amine) ligands were prepared with ancillary hydroxamic acid, ß-diketone and catechol ligands and several parameters, including: pK(a), reduction potential and cytotoxicity were investigated. Fluorescence studies demonstrated that only tpa complexes with ß-diketones showed any reduction by ascorbate in situ and similarly, cellular cytotoxicity results demonstrated that ligation to cobalt masked the cytotoxicity of the ancillary groups in all complexes except the tpa diketone derivative [Co(naac)tpa](ClO(4))(2) (naac = 1-methyl-3-(2-naphthyl)propane-1,3-dione). Additionally, it was shown that the hydroxamic acid complexes could be isolated in both the hydroxamate and hydroximate form and the pK(a) values (5.3-8.5) reveal that the reversible protonation/deprotonation of the complexes occurs at physiologically relevant pHs. These results have clear implications for the future design of prodrugs using cobalt moieties as chaperones, providing a basis for the design of cobalt complexes that are both more readily reduced and more readily taken up by cells in hypoxic and acidic environments.

Nitroimidazole conjugates of bis(thiosemicarbazonato)64Cu(II) - Potential combination agents for the PET imaging of hypoxia.

Combination agents comprising two different pharmacophores with the same biological target have the potential to show additive or synergistic activity. Bis(thiosemicarbazonato)copper(II) complexes (e.g. (64)Cu-ATSM) and nitroimidazoles (e.g. (18)F-MISO) are classes of tracer used for the delineation of tumor hypoxia by positron emission tomography (PET). Three nitroimidazole-bis(thiosemicarbazonato)copper(II) conjugates were produced in order to investigate their potential as combination hypoxia imaging agents. Two were derived from the known bifunctional bis(thiosemicarbazone) H(2)ATSM/A and the third from the new precursor diacetyl-2-(4-N-methyl-3-thiosemicarbazone)-3-(4-N-ethylamino-3-thiosemicarbazone) - H(2)ATSM/en. Oxygen-dependent uptake studies were performed using the (64)Cu radiolabelled complexes in EMT6 carcinoma cells. All the complexes displayed appreciable hypoxia selectivity, with the nitroimidazole conjugates displaying greater selectivity than a simple propyl derivative used as a control. Participation of the nitroimidazole group in the trapping mechanism is indicated by the increased hypoxic uptake of the 2- vs. the 4-substituted (64)Cu-ATSM/A derivatives. The 2-nitroimidazole derivative of (64)Cu-ATSM/en demonstrated superior hypoxia selectivity to (64)Cu-ATSM over the range of oxygen concentrations tested. Biodistribution of the radiolabelled 2-nitroimidazole conjugates was carried out in EMT6 tumor-bearing mice. The complexes showed significantly different uptake trends in comparison to each other and previously studied Cu-ATSM derivatives. Uptake of the Cu-ATSM/en conjugate in non-target organs was considerably lower than for derivatives based on Cu-ATSM/A.

Orthogonal 18F and 64Cu labelling of functionalised bis(thiosemicarbazonato) complexes.

The synthesis of three pairs of orthogonally labelled fluorinated Cu bis(thiosemicarbazonato) complexes is presented. These are the first examples of (18)F-labelled Cu(II)-complexes designed to serve as new hypoxia selective PET tracers and as mechanistic probes to study the mode of action of this class of markers. In vitro evaluation revealed that the fluorinated Cu-complex derived from amide coupling is suitable for in vivo work.

Cellular uptake and distribution of cobalt complexes of fluorescent ligands.

The development of complexes that allow the monitoring of the release and distribution of fluorescent models of anticancer drugs initially bound to cobalt(III) moieties is reported. Strong quenching of fluorescence upon ligation to cobalt(III) was observed for both the carboxylate- and the hydroximate-bound fluorophores as was the partial return of fluorescence following addition of ascorbate and cysteine. The extent of the increase in the fluorescence intensity observed following addition of these potential reductants is indicative of the fluorophore being displaced from the complex by the action of ascorbate or cysteine, by ligand exchange. The cellular distribution of the fluorescence revealed that coordination to cobalt can dramatically alter the subcellular distribution of a bound fluorophore. This work shows that fluorescence can be an effective means of monitoring these agents in cells, and of determining their sites of activation. The results also reveal that the cytotoxicity of such agents correlates with their uptake and distribution patterns and that these are influenced by the types of ligands attached to the complex.

The exocyclic functionalisation of bis(thiosemicarbazonate) complexes of zinc and copper: the synthesis of monomeric and dimeric species.

This paper reports the synthesis of bimetallic zinc thiosemicarbazone complexes with rigid aromatic linkers, using either 1,3- or 1,4- benzenediamines or 1,3- or 1,4- benzenedialdehydes as the basis of the linking groups. Non-rigid aliphatic diamines and dialdehydes were also used to link the zinc chelating units. Reaction of a bis(thiosemicarbazone) with a pendant NHNH(2) group with monoaldehydes or ketones gives a range of monomeric complexes with exocylic imine groups bearing a range of substituents. The zinc complexes can be quantitatively and rapidly transmetallated to the corresponding copper complexes and this route or direct reaction with the free ligand can be used to radiolabel the monomeric species with (64)Cu. In vivo and in vitro studies of one of the (64)Cu imine complexes shows substantial hypoxic selectivity and high tumour uptake in a murine model.

Functionalized bis(thiosemicarbazonato) complexes of zinc and copper: synthetic platforms toward site-specific radiopharmaceuticals.

Two new types of unsymmetrical bis(thiosemicarbazone) proligands and their neutral zinc(II) and copper(II) complexes have been synthesized. These bifunctional ligands both chelate the metal ions and provide pendent amino groups that can be readily functionalized with biologically active molecules. Functionalization has been demonstrated by the synthesis of three water-soluble glucose conjugates of the new zinc(II) bis(thiosemicarbazonato) complexes, and their copper(II) analogues have been prepared in aqueous solution via transmetalation. A range of techniques including NMR, electron paramagnetic resonance, cyclic voltammetry, high-performance liquid chromatography (HPLC), UV/vis, and fluorescence emission spectroscopy have been used to characterize the complexes. Four compounds, including two zinc(II) complexes, have been characterized by X-ray crystallography. The connectivity and conformation of the glucose conjugates have been assigned by NMR spectroscopy. Time-dependent density functional theory calculations have been used to assign the electronic transitions of the copper(II) bis(thiosemicarbazonato) chromophore. Two copper-64-radiolabeled complexes, including one glucose conjugate, have been prepared and characterized using radio-HPLC, and transmetalation is shown to be a viable method for radiolabeling compounds with copper radionuclides. Preliminary cell washout studies have been performed under normoxic conditions, and the uptake and intracellular distribution have been studied using confocal fluorescence microscopy.