In vivo detection of antigen-specific CD8+ T cells by immuno-positron emission tomography.

The immune system's ability to recognize peptides on major histocompatibility molecules contributes to the eradication of cancers and pathogens. Tracking these responses in vivo could help evaluate the efficacy of immune interventions and improve mechanistic understanding of immune responses. For this purpose, we employ synTacs, which are dimeric major histocompatibility molecule scaffolds of defined composition. SynTacs, when labeled with positron-emitting isotopes, can noninvasively image antigen-specific CD8+ T cells in vivo. Using radiolabeled synTacs loaded with the appropriate peptides, we imaged human papillomavirus-specific CD8+ T cells by positron emission tomography in mice bearing human papillomavirus-positive tumors, as well as influenza A virus-specific CD8+ T cells in the lungs of influenza A virus-infected mice. It is thus possible to visualize antigen-specific CD8+ T-cell populations in vivo, which may serve prognostic and diagnostic roles.

Radioactive Transition Metals for Imaging and Therapy.

Nuclear medicine is composed of two complementary areas, imaging and therapy. Positron emission tomography (PET) and single-photon imaging, including single-photon emission computed tomography (SPECT), comprise the imaging component of nuclear medicine. These areas are distinct in that they exploit different nuclear decay processes and also different imaging technologies. In PET, images are created from the 511 keV photons produced when the positron emitted by a radionuclide encounters an electron and is annihilated. In contrast, in single-photon imaging, images are created from the γ rays (and occasionally X-rays) directly emitted by the nucleus. Therapeutic nuclear medicine uses particulate radiation such as Auger or conversion electrons or ß- or α particles. All three of these technologies are linked by the requirement that the radionuclide must be attached to a suitable vector that can deliver it to its target. It is imperative that the radionuclide remain attached to the vector before it is delivered to its target as well as after it reaches its target or else the resulting image (or therapeutic outcome) will not reflect the biological process of interest. Radiochemistry is at the core of this process, and radiometals offer radiopharmaceutical chemists a tremendous range of options with which to accomplish these goals. They also offer a wide range of options in terms of radionuclide half-lives and emission properties, providing the ability to carefully match the decay properties with the desired outcome. This Review provides an overview of some of the ways this can be accomplished as well as several historical examples of some of the limitations of earlier metalloradiopharmaceuticals and the ways that new technologies, primarily related to radionuclide production, have provided solutions to these problems.

Mitochondrial transplantation by intra-arterial injection for acute kidney injury.

Acute kidney injury is a common clinical disorder and one of the major causes of morbidity and mortality in the postoperative period. In this study, the safety and efficacy of autologous mitochondrial transplantation by intra-arterial injection for renal protection in a swine model of bilateral renal ischemia-reperfusion injury were investigated. Female Yorkshire pigs underwent percutaneous bilateral temporary occlusion of the renal arteries with balloon catheters. Following 60 min of ischemia, the balloon catheters were deflated and animals received either autologous mitochondria suspended in vehicle or vehicle alone, delivered as a single bolus to the renal arteries. The injected mitochondria were rapidly taken up by the kidney and were distributed throughout the tubular epithelium of the cortex and medulla. There were no safety-related issues detected with mitochondrial transplantation. Following 24 h of reperfusion, estimated glomerular filtration rate and urine output were significantly increased while serum creatinine and blood urea nitrogen were significantly decreased in swine that received mitochondria compared with those that received vehicle. Gross anatomy, histopathological analysis, acute tubular necrosis scoring, and transmission electron microscopy showed that the renal cortex of the vehicle-treated group had extensive coagulative necrosis of primarily proximal tubules, while the mitochondrial transplanted kidney showed only patchy mild acute tubular injury. Renal cortex IL-6 expression was significantly increased in vehicle-treated kidneys compared with the kidneys that received mitochondrial transplantation. These results demonstrate that mitochondrial transplantation by intra-arterial injection provides renal protection from ischemia-reperfusion injury, significantly enhancing renal function and reducing renal damage.

Mitochondrial transplantation enhances murine lung viability and recovery after ischemia-reperfusion injury.

The most common cause of acute lung injury is ischemia-reperfusion injury (IRI), during which mitochondrial damage occurs. We have previously demonstrated that mitochondrial transplantation is an efficacious therapy to replace or augment mitochondria damaged by IRI, allowing for enhanced muscle viability and function in cardiac tissue. Here, we investigate the efficacy of mitochondrial transplantation in a murine lung IRI model using male C57BL/6J mice. Transient ischemia was induced by applying a microvascular clamp on the left hilum for 2 h. Upon reperfusion mice received either vehicle or vehicle-containing mitochondria either by vascular delivery (Mito V) through the pulmonary artery or by aerosol delivery (Mito Neb) via the trachea (nebulization). Sham control mice underwent thoracotomy without hilar clamping and were ventilated for 2 h before returning to the cage. After 24 h recovery, lung mechanics were assessed and lungs were collected for analysis. Our results demonstrated that at 24 h of reperfusion, dynamic compliance and inspiratory capacity were significantly increased and resistance, tissue damping, elastance, and peak inspiratory pressure (Mito V only) were significantly decreased (P < 0.05) in Mito groups as compared with their respective vehicle groups. Neutrophil infiltration, interstitial edema, and apoptosis were significantly decreased (P < 0.05) in Mito groups as compared with vehicles. No significant differences in cytokines and chemokines between groups were shown. All lung mechanics results in Mito groups except peak inspiratory pressure in Mito Neb showed no significant differences (P > 0.05) as compared with Sham. These results conclude that mitochondrial transplantation by vascular delivery or nebulization improves lung mechanics and decreases lung tissue injury.

The ionic charge of copper-64 complexes conjugated to an engineered antibody affects biodistribution.

The development of biomolecules as imaging probes requires radiolabeling methods that do not significantly influence their biodistribution. Sarcophagine (Sar) chelators form extremely stable complexes with copper and are therefore a promising option for labeling proteins with (64)Cu. However, initial studies using the first-generation sarcophagine bifunctional chelator SarAr to label the engineered antibody fragment ch14.18-ΔCH2 (MW 120 kDa) with (64)Cu showed high tracer retention in the kidneys, presumably because the high local positive charge on the Cu(II)-SarAr moiety resulted in increased binding of the labeled protein to the negatively charged basal cells of the glomerulus. To test this hypothesis, ch14.18-ΔCH2 was conjugated with a series of Sar derivatives of decreasing positive charge and three commonly used macrocyclic polyaza polycarboxylate (PAC) bifunctional chelators (BFC). The immunoconjugates were labeled with (64)Cu and injected into mice, and PET/CT images were obtained at 24 and 48 h postinjection (p.i.). At 48 h p.i., ex vivo biodistribution was assessed. In addition, to demonstrate the potential of metastasis detection using (64)Cu-labeled ch14.18-ΔCH2, a preclinical imaging study of intrahepatic neuroblastoma tumors was performed. Reducing the positive charge on the Sar chelators decreased kidney uptake of Cu-labeled ch14.18-ΔCH2 by more than 6-fold, from >45 to <6% ID/g, whereas the uptake in most other tissues, including liver, was relatively unchanged. However, despite this dramatic decrease, the renal uptake of the PAC BFCs was generally lower than that of the Sar derivatives, as was the liver uptake. Uptake of (64)Cu-labeled ch14.18-ΔCH2 in neuroblastoma hepatic metastases was detected using PET.

Convenient synthesis of 18F-radiolabeled R-(-)-N-n-propyl-2-(3-fluoropropanoxy-11-hydroxynoraporphine.

Aporphines are attractive candidates for imaging D2 receptor function because, as agonists rather than antagonists, they are selective for the receptor in the high affinity state. In contrast, D2 antagonists do not distinguish between the high and low affinity states, and in vitro data suggests that this distinction may be important in studying diseases characterized by D2 dysregulation, such as schizophrenia and Parkinson's disease. Accordingly, MCL-536 (R-(-)-N-n-propyl-2-(3-[(18)F]fluoropropanoxy-11-hydroxynoraporphine) was selected for labeling with (18)F based on in vitro data obtained for the non-radioactive ((19)F) compound. Fluorine-18-labeled MCL-536 was synthesized in 70% radiochemical yield, >99% radiochemical purity, and specific activity of 167 GBq/µmol (4.5 Ci/µmol) using p-toluenesulfonyl (tosyl) both as a novel protecting group for the phenol and a leaving group for the radiofluorination.

Systemically injected oxygen within rapidly dissolving microbubbles improves the outcomes of severe hypoxaemia in swine.

Acute respiratory failure can cause profound hypoxaemia that leads to organ injury or death within minutes. When conventional interventions are ineffective, the intravenous administration of oxygen can rescue patients from severe hypoxaemia, but at the risk of microvascular obstruction and of toxicity of the carrier material. Here we describe polymeric microbubbles as carriers of high volumes of oxygen (350-500 ml of oxygen per litre of foam) that are stable in storage yet quickly dissolve following intravenous injection, reverting to their soluble and excretable molecular constituents. In swine with profound hypoxaemia owing to acute and temporary (12 min) upper-airway obstruction, the microbubble-mediated delivery of oxygen led to: the maintenance of critical oxygenation, lowered burdens of cardiac arrest, improved survival, and substantially improved neurologic and kidney function in surviving animals. Our findings underscore the importance of maintaining a critical threshold of oxygenation and the promise of injectable oxygen as a viable therapy in acute and temporary hypoxaemic crises.

Low-Fouling Zwitterionic Polymeric Colloids as Resuscitation Fluids for Hemorrhagic Shock.

Colloids, known as volume expanders, have been used as resuscitation fluids for hypovolemic shock for decades, as they increase plasma oncotic pressure and expand intravascular volume. However, recent studies show that commonly used synthetic colloids have adverse interactions with human biological systems. In this work, a low-fouling amine(N)-oxide-based zwitterionic polymer as an alternative volume expander with improved biocompatibility and efficacy is designed. It is demonstrated that the polymer possesses antifouling ability, resisting cell interaction and deposition in major organs, and is rapidly cleared via renal filtration and hepatic circulation, reducing the risk of long-term side effects. Furthermore, in vitro and in vivo studies show an absence of adverse effects on hemostasis or any acute safety risks. Finally, it is shown that, in a head-to-head comparison with existing colloids and plasma, the zwitterionic polymer serves as a more potent oncotic agent for restoring intravascular volume in a hemorrhagic shock model. The design of N-oxide-based zwitterionic polymers may lead to the development of alternative fluid therapies to treat hypovolemic shock and to improve fluid management in general.

The Non-Anhydrous, Minimally Basic Synthesis of the Dopamine D2 Agonist [18F]MCL-524.

The dopamine D2 agonist MCL-524 is selective for the D2 receptor in the high-affinity state (D2high), and, therefore, the PET analogue, [18F]MCL-524, may facilitate the elucidation of the role of D2high in disorders such as schizophrenia. However, the previously reported synthesis of [18F]MCL-524 proved difficult to replicate and was lacking experimental details. We therefore developed a new synthesis of [18F]MCL-524 using a "non-anhydrous, minimally basic" (NAMB) approach. In this method, [18F]F- is eluted from a small (10-12 mg) trap-and-release column with tetraethylammonium tosylate (2.37 mg) in 7:3 MeCN:H2O (0.1 mL), rather than the basic carbonate or bicarbonate solution that is most often used for [18F]F- recovery. The tosylated precursor (1 mg) in 0.9 mL anhydrous acetonitrile was added directly to the eluate, without azeotropic drying, and the solution was heated (150 °C/15 min). The catechol was then deprotected with the Lewis acid In(OTf)3 (10 equiv.; 150 °C/20 min). In contrast to deprotection with protic acids, Lewis-acid-based deprotection facilitated the efficient removal of byproducts by HPLC and eliminated the need for SPE extraction prior to HPLC purification. Using the NAMB approach, [18F]MCL-524 was obtained in 5-9% RCY (decay-corrected, n = 3), confirming the utility of this improved method for the multistep synthesis of [18F]MCL-524 and suggesting that it may applicable to the synthesis of other 18F-labeled radiotracers.

Detection and therapy of neuroblastoma minimal residual disease using [64/67Cu]Cu-SARTATE in a preclinical model of hepatic metastases.

BACKGROUND: A major challenge to the long-term success of neuroblastoma therapy is widespread metastases that survive initial therapy as minimal residual disease (MRD). The SSTR2 receptor is expressed by most neuroblastoma tumors making it an attractive target for molecularly targeted radionuclide therapy. SARTATE consists of octreotate, which targets the SSTR2 receptor, conjugated to MeCOSar, a bifunctional chelator with high affinity for copper. Cu-SARTATE offers the potential to both detect and treat neuroblastoma MRD by using [64Cu]Cu-SARTATE to detect and monitor the disease and [67Cu]Cu-SARTATE as the companion therapeutic agent. In the present study, we tested this theranostic pair in a preclinical model of neuroblastoma MRD. An intrahepatic model of metastatic neuroblastoma was established using IMR32 cells in nude mice. The biodistribution of [64Cu]Cu-SARTATE was measured using small-animal PET and ex vivo tissue analysis. Survival studies were carried out using the same model: mice (6-8 mice/group) were given single doses of saline, or 9.25 MBq (250 µCi), or 18.5 MBq (500 µCi) of [67Cu]Cu-SARTATE at either 2 or 4 weeks after tumor cell inoculation. RESULTS: PET imaging and ex vivo biodistribution confirmed tumor uptake of [64Cu]Cu-SARTATE and rapid clearance from other tissues. The major clearance tissues were the kidneys (15.6 ± 5.8% IA/g at 24 h post-injection, 11.5 ± 2.8% IA/g at 48 h, n = 3/4). Autoradiography and histological analysis confirmed [64Cu]Cu-SARTATE uptake in viable, SSTR2-positive tumor regions with mean tumor uptakes of 14.1-25.0% IA/g at 24 h. [67Cu]Cu-SARTATE therapy was effective when started 2 weeks after tumor cell inoculation, extending survival by an average of 13 days (30%) compared with the untreated group (mean survival of control group 43.0 ± 8.1 days vs. 55.6 ± 9.1 days for the treated group; p = 0.012). No significant therapeutic effect was observed when [67Cu]Cu-SARTATE was started 4 weeks after tumor cell inoculation, when the tumors would have been larger (control group 14.6 ± 8.5 days; 9.25 MBq group 9.5 ± 1.6 days; 18.5 MBq group 15.6 ± 4.1 days; p = 0.064). CONCLUSIONS: Clinical experiences of peptide-receptor radionuclide therapy for metastatic disease have been encouraging. This study demonstrates the potential for a theranostic approach using [64/67Cu]Cu-SARTATE for the detection and treatment of SSTR2-positive neuroblastoma MRD.

Conformation-sensitive targeting of lipid nanoparticles for RNA therapeutics.

The successful in vivo implementation of gene expression modulation strategies relies on effective, non-immunogenic delivery vehicles. Lipid nanoparticles are one of the most advanced non-viral clinically approved nucleic-acid delivery systems. Yet lipid nanoparticles accumulate naturally in liver cells upon intravenous administration, and hence, there is an urgent need to enhance uptake by other cell types. Here we use a conformation-sensitive targeting strategy to achieve in vivo gene silencing in a selective subset of leukocytes and show potential therapeutic applications in a murine model of colitis. In particular, by targeting the high-affinity conformation of α4ß7 integrin, which is a hallmark of inflammatory gut-homing leukocytes, we silenced interferon-γ in the gut, resulting in an improved therapeutic outcome in experimental colitis. The lipid nanoparticles did not induce adverse immune activation or liver toxicity. These results suggest that our lipid nanoparticle targeting strategy might be applied for selective delivery of payloads to other conformation-sensitive targets.

Synthesis and Characterization of a Tetramethyl Furanone Functionalized Diiminedioxime, A Potential Ligand for Cu Radiopharmaceuticals, and its Copper(II) and Nickel(II) Complexes.

As part of our on-going effort to develop (64)Cu-based radiopharmaceuticals for PET (positron emission tomography) imaging of multidrug resistance in cancer, we prepared a tetramethylfuranone-functionalized diiminedioxime ligand, TMFPreH (TMFPreH = 4-[3-(4-Hydroxyimino-2,2,5,5-dimethyl-dihydro-furan-3-ylideneamino)-propylimino]-2,2,5,5-tetramethyl-dihydro-furan-3-one oxime) and its Cu(II) and Ni(II) complexes. When the copper(II) complex was prepared from Cu(ClO(4))(2) in ethanol, it was isolated as a Cu(II)-bridged dimer, but when it was prepared from Cu(OAc)(2) and heated in acetone, an unusual example of an acetone adduct of the ligand is formed by reduction of one of the imine double bonds by the solvent. The Ni(II) complex is square pyramidal with the perchlorate counterion at the apex.

Positron Emission Tomography Detects In Vivo Expression of Disialoganglioside GD2 in Mouse Models of Primary and Metastatic Osteosarcoma.

The cell membrane glycolipid GD2 is expressed by multiple solid tumors, including 88% of osteosarcomas and 98% of neuroblastomas. However, osteosarcomas are highly heterogeneous, with many tumors exhibiting GD2 expression on <50% of the individual cells, while some tumors are essentially GD2-negative. Anti-GD2 immunotherapy is the current standard of care for high-risk neuroblastoma, but its application to recurrent osteosarcomas, for which no effective therapies exist, has been extremely limited. This is, in part, because the standard assays to measure GD2 expression in these heterogeneous tumors are not quantitative and are subject to tissue availability and sampling bias. To address these limitations, we evaluated a novel, sensitive radiotracer [64Cu]Cu-Bn-NOTA-hu14.18K322A to detect GD2 expression in osteosarcomas (six patient-derived xenografts and one cell line) in vivo using positron emission tomography (PET). Tumor uptake of the radiolabeled, humanized anti-GD2 antibody [64Cu]Cu-Bn-NOTA-hu14.18K322A was 7-fold higher in modestly GD2-expressing osteosarcomas (32% GD2-positive cells) than in a GD2-negative tumor (9.8% vs. 1.3% of the injected dose per cc, respectively). This radiotracer also identified lesions as small as 29 mm3 in a 34% GD2-positive model of metastatic osteosarcoma of the lung. Radiolabeled antibody accumulation in patient-derived xenografts correlated with GD2 expression as measured by flow cytometry (Pearson r = 0.88, P = 0.01), distinguishing moderately GD2-expressing osteosarcomas (32%-69% GD2-positive cells) from high GD2 expressors (>99%, P < 0.05). These results support the utility of GD2 imaging with PET to measure GD2 expression in osteosarcoma and thus maximize the clinical impact of anti-GD2 immunotherapy. SIGNIFICANCE: In situ assessment of all GD2-positive osteosarcoma sites with a novel PET radiotracer could significantly impact anti-GD2 immunotherapy patient selection and enable noninvasive probing of correlations between target expression and therapeutic response.

Mitochondrial transplantation prolongs cold ischemia time in murine heart transplantation.

BACKGROUND: Cold ischemia time (CIT) causes ischemia‒reperfusion injury to the mitochondria and detrimentally effects myocardial function and tissue viability. Mitochondrial transplantation replaces damaged mitochondria and enhances myocardial function and tissue viability. Herein we investigated the efficacy of mitochondrial transplantation in enhancing graft function and viability after prolonged CIT. METHODS: Heterotopic heart transplantation was performed in C57BL/6J mice. Upon heart harvesting from C57BL/6J donors, 0.5 ml of either mitochondria (1 × 108 in respiration buffer; mitochondria group) or respiration buffer (vehicle group) was delivered antegrade to the coronary arteries via injection to the coronary ostium. The hearts were excised and preserved for 29 ± 0.3 hours in cold saline (4°C). The hearts were then heterotopically transplanted. A second injection of either mitochondria (1 × 108) or respiration buffer (vehicle) was delivered antegrade to the coronary arteries 5 minutes after transplantation. Grafts were analyzed for 24 hours. Beating score, graft function, and tissue injury were measured. RESULTS: Beating score, calculated ejection fraction, and shortening fraction were significantly enhanced (p < 0.05), whereas necrosis and neutrophil infiltration were significantly decreased (p < 0.05) in the mitochondria group as compared with the vehicle group at 24 hours of reperfusion. Transmission electron microscopy showed the presence of contraction bands in vehicle but not in mitochondria grafts. CONCLUSIONS: Mitochondrial transplantation prolongs CIT to 29 hours in the murine heart transplantation model, significantly enhances graft function, and decreases graft tissue injury. Mitochondrial transplantation may provide a means to reduce graft failure and improve transplantation outcomes after prolonged CIT.

A Novel Biological Strategy for Myocardial Protection by Intracoronary Delivery of Mitochondria: Safety and Efficacy.

Mitochondrial dysfunction is the determinant insult of ischemia-reperfusion injury. Autologous mitochondrial transplantation involves supplying one's healthy mitochondria to the ischemic region harboring damaged mitochondria. The authors used in vivo swine to show that mitochondrial transplantation in the heart by intracoronary delivery is safe, with specific distribution to the heart, and results in significant increase in coronary blood flow, which requires intact mitochondrial viability, adenosine triphosphate production, and, in part, the activation of vascular KIR channels. Intracoronary mitochondrial delivery after temporary regional ischemia significantly improved myocardial function, perfusion, and infarct size. The authors concluded that intracoronary delivery of mitochondria is safe and efficacious therapy for myocardial ischemia-reperfusion injury.

Skeletal PET with 18F-fluoride: applying new technology to an old tracer.

Although (18)F-labeled NaF was the first widely used agent for skeletal scintigraphy, it quickly fell into disuse after the introduction of (99m)Tc-labeled bone-imaging agents. Recent comparative studies have demonstrated that (18)F-fluoride PET is more accurate than (99m)Tc-diphosphonate SPECT for identifying both malignant and benign lesions of the skeleton. Combining (18)F-fluoride PET with other imaging, such as CT, can improve the specificity and overall accuracy of skeletal (18)F-fluoride PET and probably will become the routine clinical practice for (18)F-fluoride PET. Although (18)F-labeled NaF and (99m)Tc-diphosphonate have a similar patient dosimetry, (18)F-fluoride PET offers shorter study times (typically less than 1 h), resulting in a more efficient workflow, improved patient convenience, and faster turnarounds of reports to the referring physicians. With the widespread availability of PET scanners and the improved logistics for the delivery of (18)F radiopharmaceuticals, prior limitations to the routine use of (18)F-fluoride bone imaging have largely been overcome. The favorable imaging performance and the clinical utility of (18)F-fluoride PET, compared with (99m)Tc-diphosphonate scintigraphy, support the reconsideration of (18)F-fluoride as a routine bone-imaging agent.

Molecular imaging in nanomedicine - A developmental tool and a clinical necessity.

The development of nanomedicines presents the potential to deliver more potent drugs targeted more specifically to the site(s) of disease than is currently achievable. While encouraging results have been achieved, including at the clinical level, significant challenges and opportunities for development remain, both in terms of further developing the technology and in understanding the underlying biology. Given the lessons learned regarding variations in nanomedicine delivery to different tumor types and between different patients with the same tumor type, this is an area of drug development that, rather than simply benefiting from a patient-specific approach, actually demands it. The only way that this distribution information can be obtained is through imaging, and this requires labeling of the nanomedicine to enable detection outside the body. In this review, we describe recent advances in the labeling of nanomedicines, how imaging studies are guiding nanomedicine development, and the role of imaging in the future development of nanomedicines.

A Sensitive Method for the Measurement of Copper at Trace Levels Using an HPLC-Based Assay.

BACKGROUND: Measurement of trace metal contamination is critical in the production of radiometals, such as 64Cu, for protein labeling. ICP-MS provides these data with high sensitivity and high specificity, but at high (instrument) cost. TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11- tetraacetic acid) titration provides high sensitivity at low cost but with low specificity. A method that allowed the measurement of trace metals with high sensitivity but also at relatively low cost would, therefore, be very useful in the development of new radiometal production methods. OBJECTIVE: The goal of this project was to develop an analytical method for copper that uses readily available laboratory equipment while minimally achieving low ppm sensitivity. METHOD: The metal-chelating macrocycle 2-(4-aminobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7, 10-tetraacetic acid (DOTA) was coupled to fluorescein to produce a molecule that combines high UV absorbance and high quantum yield with the ability to chelate a wide range of transition metals. The fluorescein-DOTA was mixed with Cu(II) samples at low ppm concentrations, and the samples were analyzed by reversed-phase HPLC. RESULTS: Copper chelation by the DOTA moiety decreased its overall charge, leading to a delayed elution from a C18 HPLC column. The absorbance signal of the fluorescein-DOTA-Cu(II) peak (453 nm) linearly correlated with the copper concentration allowing measurement of Cu(II) down to 1.25 ppm. Furthermore, using fluorescence detection (521 nm) the detection limit was reduced by almost three orders of magnitude, to 2.5 ppb (p<0.05). CONCLUSION: Using a fluorescent dye (fluorescein) coupled to a macrocyclic chelator (DOTA) and an HPLC equipped with a standard UV detector is it possible to measure Cu at ppm concentrations, the Cu concentration observed in typical samples of 64Cu.