Kinetic modeling of hyperpolarized (13)C pyruvate metabolism in tumors using a measured arterial input function.

Mathematical models are required to estimate kinetic parameters of [1-(13)C] pyruvate-lactate interconversion from magnetic resonance spectroscopy data. One- or two-way exchange models utilizing a hypothetical approximation to the true arterial input function (AIF), (e.g. an ideal 'box-car' function) have been used previously. We present a method for direct measurement of the AIF in the rat. The hyperpolarized [1-(13)C] pyruvate signal was measured in arterial blood as it was continuously withdrawn through a small chamber. The measured signal was corrected for T1 relaxation of pyruvate, RF pulses and dispersion of blood in the chamber to allow for the estimation of the direct AIF. Using direct AIF, rather than the commonly used box-car AIF, provided realistic estimates of the rate constant of conversion of pyruvate to lactate, kpl, the rate constant of conversion of lactate to pyruvate klp, the clearance rate constant of pyruvate from blood to tissue, Kip, and the relaxation rate of lactate T1la. Since no lactate signal was present in blood, it was possible to use a simple precursor-product relationship, with the tumor tissue pyruvate time-course as the input for the lactate time-course. This provided a robust estimate of kpl, similar to that obtained using a directly measured AIF.

Investigation of protein induction in tumour vascular targeted strategies by MALDI MSI.

Characterising the protein signatures in tumours following vascular-targeted therapy will help determine both treatment response and resistance mechanisms. Here, mass spectrometry imaging and MS/MS with and without ion mobility separation have been used for this purpose in a mouse fibrosarcoma model following treatment with the tubulin-binding tumour vascular disrupting agent, combretastatin A-4-phosphate (CA-4-P). Characterisation of peptides after in situ tissue tryptic digestion was carried out using Matrix-Assisted Laser Desorption/Ionisation-Mass Spectrometry (MALDI-MS) and Matrix-Assisted Laser Desorption/Ionisation-Ion Mobility Separation-Mass Spectrometry Imaging (MALDI IMS-MSI) to observe the spatial distribution of peptides. Matrix-Assisted Laser Desorption/Ionisation-Ion Mobility Separation-Tandem Mass Spectrometry (MALDI-IMS-MS/MS) of peaks was performed to elucidate any pharmacological responses and potential biomarkers. By taking tumour samples at a number of time points after treatment gross changes in the tissue were indicated by changes in the signal levels of certain peptides. These were identified as arising from haemoglobin and indicated the disruption of the tumour vasculature. It was hoped that the use of PCA-DA would reveal more subtle changes taking place in the tumour samples however these are masked by the dominance of the changes in the haemoglobin signals.

An automatic algorithm for the segmentation and morphological analysis of microvessels in immunostained histological tumour sections.

A fully automatic segmentation and morphological analysis algorithm for the analysis of microvessels from CD31 immunostained histological tumour sections is presented. Development of the algorithm exploited the distinctive hues of stained vascular endothelial cells, cell nuclei and background, to provide the seeds for a 'region-growing' method for object segmentation in the 3D hue, saturation, value (HSV) colour model. The segmented objects, identified as microvessels by CD31 immunostaining, were post-processed with three morphological tasks: joining separate objects that were likely to belong to a single vessel, closing objects that had a narrow gap around their periphery, and splitting objects with multiple lumina into individual vessels. The automatic segmentation was validated against a hand-segmented set of 44 images from three different SW1222 human colorectal carcinomas xenografted into mice. 96.3 ± 0.9% of pixels were found to be correctly classified. Automated segmentation was carried out on a further 53 images from three histologically distinct mouse fibrosarcomas (MFs) for morphological comparison with the SW1222 tumours. Four morphometric measurements were calculated for each segmented vessel: vascular area (VA), ratio of lumen area to vascular area (lu/VA), eccentricity (e), and roundness (ro). In addition, the total vascular area relative to tumour tissue area (rVA) was calculated. lu/VA, e and ro were found to be significantly smaller in MF tumours than in SW1222 tumours (p < 0.05; unpaired t-test). The algorithm is available through the website http://www.caiman.org.uk where images can be uploaded, processed and sent back to users. The output from CAIMAN consists of the original image with boundaries of segmented vessels overlaid, the calculated parameters and a Matlab file, which contains the segmentation that the user can use to derive further results.

Guidelines for the welfare and use of animals in cancer research.

Animal experiments remain essential to understand the fundamental mechanisms underpinning malignancy and to discover improved methods to prevent, diagnose and treat cancer. Excellent standards of animal care are fully consistent with the conduct of high quality cancer research. Here we provide updated guidelines on the welfare and use of animals in cancer research. All experiments should incorporate the 3Rs: replacement, reduction and refinement. Focusing on animal welfare, we present recommendations on all aspects of cancer research, including: study design, statistics and pilot studies; choice of tumour models (e.g., genetically engineered, orthotopic and metastatic); therapy (including drugs and radiation); imaging (covering techniques, anaesthesia and restraint); humane endpoints (including tumour burden and site); and publication of best practice.

Inhibition of nitric oxide synthase induces a selective reduction in tumor blood flow that is reversible with L-arginine.

The effect of i.v. administration of the nitric oxide synthase (NOS) inhibitor N(omega)-nitro-L-arginine (L-NNA) on tumor blood flow compared with normal tissue blood flow was studied in anesthetized BD9 rats bearing subcutaneous P22 carcinosarcomas. Blood flow was measured by the tissue uptake of radiolabeled iodoantipyrine. The reversibility of blood flow changes was tested by subsequent administration of L-arginine, the natural substrate for NOS. The effect of L-NNA was compared to that of the imidazolineoxyl N-oxide C-PTIO, a carboxyl derivative of 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide and a nitric oxide scavenger. Drug-induced changes in mean arterial blood pressure (MABP) were monitored and used to calculate relative drug-induced changes in tissue vascular resistance. Heart rate was measured from blood pressure traces. L-NNA significantly decreased heart rate and increased MABP in a dose-dependent manner. Significant dose-dependent reductions in blood flow with L-NNA were observed in tumor, skeletal muscle, spleen, and skin overlying the tumor. No significant effect was found for normal skin, brain, heart, kidney, and small intestine. At 1 mg/kg, the effect of L-NNA was selective for the tumor, with a significant decrease in tumor blood flow to 0.45 of the control level and no significant effect in any of the normal tissues. Higher doses did not produce any further reduction in tumor blood flow, presumably due to an increase in tumor perfusion pressure arising from the increase in MABP at these doses. Vascular resistance was increased to some extent in all of the tissues studied but, overall, was greatest in the tumor. At 1 mg/kg, there was a 2-2.5-fold increase in tumor vascular resistance but no significant increase in any of the normal tissues. At the highest dose used (10 mg/kg), the increases in vascular resistance in the skeletal muscle and spleen were equivalent to that in the tumor. Administration of L-arginine 15 min after L-NNA completely reversed the decrease in tumor blood flow observed for 1 mg/kg L-NNA alone. In contrast to the effect of L-NNA, constant i.v. infusion of C-PTIO had no effect on tumor or normal tissue blood flow. These results indicate that nitric oxide is important for maintaining a vasodilatory tone in tumors and that inhibition of NOS may provide a means for enhancing therapeutic regimens that would benefit from a selective reduction in tumor blood flow.

Limitations of the reporter green fluorescent protein under simulated tumor conditions.

This paper reports a detailed analysis of the effect of low oxygen conditions (hypoxia) on the reporter green fluorescent protein (GFP). It questions the feasibility of using GFP for gene expression studies under tumor conditions. Hypoxia is a characteristic of both experimental and clinical tumors. Several important factors are pointed out which need to be considered when using GFP as reporter gene. GFP fluorescence is the final product of a long and complex pathway involving transcription, translation, and posttranslational modifications. All of these steps may be affected by the availability of oxygen. We show specifically that cellular GFP fluorescence decreased with reduced oxygenation, anoxia virtually eliminated fluorescence and protein levels, and fluorescence recovery after anoxia required 5-10 h of reoxygenation. In conclusion, GFP appears to be a good marker gene to study location or movement of proteins or cells but should be used with great caution as a reporter of gene expression under tumor conditions.

Combretastatin A-4, an agent that displays potent and selective toxicity toward tumor vasculature.

Selective induction of vascular damage within tumors represents an emerging approach to cancer treatment. Histological studies have shown that several tubulin-binding agents can induce vascular damage within tumors but only at doses approximating the maximum tolerated dose, which has limited their clinical applicability. In this study, we show that the combretastatin A-4 prodrug induces vascular shutdown within tumors at doses less than one-tenth of the maximum tolerated dose. In vitro studies indicate that a short drug exposure results in profound long-term antiproliferative/cytotoxic effects against proliferating endothelial cells but not cells that are quiescent prior to and during drug exposure. Vascular shutdown, within experimental and human breast cancer models in vivo following systemic drug administration, was demonstrated with a reduction in functional vascular volume of 93% at 6 h following drug administration and persisted over the next 12 h, with corresponding histology consistent with hemorrhagic necrosis resulting from vascular damage. These actions against tumor vasculature and the broad therapeutic window demonstrate the clinical potential of these drugs and warrant further study to elucidate the mechanisms responsible for the antivascular effects of combretastatin A-4.

Mechanisms associated with tumor vascular shut-down induced by combretastatin A-4 phosphate: intravital microscopy and measurement of vascular permeability.

The tumor vascular effects of the tubulin destabilizing agent disodium combretastatinA-4 3-O-phosphate (CA-4-P) were investigated in the rat P22 tumor growing in a dorsal skin flap window chamber implanted into BD9 rats. CA-4-P is in clinical trial as a tumor vascular targeting agent. In animal tumors, it can cause the shut-down of blood flow, leading to extensive tumor cell necrosis. However, the mechanisms leading to vascular shut-down are still unknown. Tumor vascular effects were visualized and monitored on-line before and after the administration of two doses of CA-4-P (30 and 100 mg/kg) using intravital microscopy. The combined effect of CA-4-P and systemic nitric oxide synthase (NOS) inhibition using N(omega)-nitro-L-arginine (L-NNA) was also assessed, because this combination has been shown previously to have a potentiating effect. The early effect of CA-4-P on tumor vascular permeability to albumin was determined to assess whether this could be involved in the mechanism of action of the drug. Tumor blood flow reduction was extremely rapid after CA-4-P treatment, with red cell velocity decreasing throughout the observation period and dropping to <5% of the starting value by 1 h. NOS inhibition alone caused a 50% decrease in red cell velocity, and the combined treatment of CA-4-P and NOS inhibition was approximately additive. The mechanism of blood flow reduction was very different for NOS inhibition and CA-4-P. That of NOS inhibition could be explained by a decrease in vessel diameter, which was most profound on the arteriolar side of the tumor circulation. In contrast, the effects of CA-4-P resembled an acute inflammatory reaction resulting in a visible loss of a large proportion of the smallest blood vessels. There was some return of visible vasculature at 1 h after treatment, but the blood in these vessels was static or nearly so, and many of the vessels were distended. The hematocrit within larger draining tumor venules tended to increase at early times after CA-4-P, suggesting fluid loss from the blood. The stacking of red cells to form rouleaux was also a common feature, coincident with slowing of blood flow; and these two factors would lead to an increase in viscous resistance to blood flow. Tumor vascular permeability to albumin was increased to approximately 160% of control values at 1 and 10 min after treatment. This could lead to an early decrease in tumor blood flow via an imbalance between intravascular and tissue pressures and/or an increase in blood viscosity as a result of increased hematocrit. These results suggest a mechanism of action of CA-4-P in vivo. Combination of CA-4-P with a NOS inhibitor has an additive effect, which it may be possible to exploit therapeutically.

Combretastatin A-4 phosphate as a tumor vascular-targeting agent: early effects in tumors and normal tissues.

The potential for tumor vascular-targeting by using the tubulin destabilizing agent disodium combretastatin A-4 3-0-phosphate (CA-4-P) was assessed in a rat system. This approach aims to shut down the established tumor vasculature, leading to the development of extensive tumor cell necrosis. The early vascular effects of CA-4-P were assessed in the s.c. implanted P22 carcinosarcoma and in a range of normal tissues. Blood flow was measured by the uptake of radiolabeled iodoantipyrine, and quantitative autoradiography was used to measure spatial heterogeneity of blood flow in tumor sections. CA-4-P (100 mg/kg i.p.) caused a significant increase in mean arterial blood pressure at 1 and 6 h after treatment and a very large decrease in tumor blood flow, which-by 6 h-was reduced approximately 100-fold. The spleen was the most affected normal tissue with a 7-fold reduction in blood flow at 6 h. Calculations of vascular resistance revealed some vascular changes in the heart and kidney for which there were no significant changes in blood flow. Quantitative autoradiography showed that CA-4-P increased the spatial heterogeneity in tumor blood flow. The drug affected peripheral tumor regions less than central regions. Administration of CA-4-P (30 mg/kg) in the presence of the nitric oxide synthase inhibitor, N(omega)-nitro-L-arginine methyl ester, potentiated the effect of CA-4-P in tumor tissue. The combination increased tumor vascular resistance 300-fold compared with less than 7-fold for any of the normal tissues. This shows that tissue production of nitric oxide protects against the damaging vascular effects of CA-4-P. Significant changes in tumor vascular resistance could also be obtained in isolated tumor perfusions using a cell-free perfusate, although the changes were much less than those observed in vivo. This shows that the action of CA-4-P includes mechanisms other than those involving red cell viscosity, intravascular coagulation, and neutrophil adhesion. The uptake of CA-4-P and combretastatin A-4 (CA-4) was more efficient in tumor than in skeletal muscle tissue and dephosphorylation of CA-4-P to CA-4 was faster in the former. These results are promising for the use of CA-4-P as a tumor vascular-targeting agent.

Longitudinal imaging of the ageing mouse.

Several non-invasive imaging techniques are used to investigate the effect of pathologies and treatments over time in mouse models. Each preclinical in vivo technique provides longitudinal and quantitative measurements of changes in tissues and organs, which are fundamental for the evaluation of alterations in phenotype due to pathologies, interventions and treatments. However, it is still unclear how these imaging modalities can be used to study ageing with mice models. Almost all age related pathologies in mice such as osteoporosis, arthritis, diabetes, cancer, thrombi, dementia, to name a few, can be imaged in vivo by at least one longitudinal imaging modality. These measurements are the basis for quantification of treatment effects in the development phase of a novel treatment prior to its clinical testing. Furthermore, the non-invasive nature of such investigations allows the assessment of different tissue and organ phenotypes in the same animal and over time, providing the opportunity to study the dysfunction of multiple tissues associated with the ageing process. This review paper aims to provide an overview of the applications of the most commonly used in vivo imaging modalities used in mouse studies: micro-computed-tomography, preclinical magnetic-resonance-imaging, preclinical positron-emission-tomography, preclinical single photon emission computed tomography, ultrasound, intravital microscopy, and whole body optical imaging.

Horseradish peroxidase-mediated gene therapy: choice of prodrugs in oxic and anoxic tumor conditions.

We have previously proposed the plant enzyme horseradish peroxidase (HRP) and the plant hormone indole-3-acetic acid (IAA) as an enzyme/prodrug combination for cancer gene therapy. In the current study, we evaluated the potential of HRP/IAA for gene-directed enzyme/prodrug therapy in three human tumor cell lines (T24 bladder carcinoma, MCF-7 breast adenocarcinoma, and FaDu nasopharyngeal squamous carcinoma) and one endothelial cell line (HMEC-1). The action of 10 IAA analogues in combination with HRP was studied in vitro in normoxic conditions as well as in the extreme tumor conditions of anoxia. Compounds characterized by prompt normoxic or anoxic cytotoxic activation and high HRP transfectant killing or selectivity were identified. Some variations were observed in the response of cells of different origin, with IAA, 1-Me-IAA, and 5-Br-IAA representing the most promising candidates for HRP gene therapy. In particular, 5-Br-IAA showed a very prompt and selective activation in anoxia. A strong bystander effect was produced by activated IAA and analogues because 70-90% cell kill was obtained when only 5% of the cells expressed the HRP enzyme. These results indicate that HRP/IAA represents an effective system for enzyme/prodrug-based anticancer approaches, and further improvements could be achieved by the use of novel IAA derivatives.

Development of a novel enzyme/prodrug combination for gene therapy of cancer: horseradish peroxidase/indole-3-acetic acid.

This paper demonstrates the potential for utilizing the plant enzyme, horseradish peroxidase (HRP), in a gene-directed enzyme prodrug therapy context. Human T24 bladder carcinoma cells transfected with a mammalian expression vector containing the HRP cDNA were selectively sensitized to the nontoxic plant hormone, indole-3-acetic acid (IAA). The HRP/IAA-induced cell kill was effective in normoxic and anoxic conditions. The activated drug is a long-lived species able to cross cell membranes, and cell contact appears not to be required for a bystander effect to take place. These preliminary results suggest that the delivery of the HRP gene to human tumors followed by IAA treatment may provide a novel cancer gene-directed enzyme prodrug therapy approach, with potential to target hypoxic cells.

Modification of tumor blood flow: current status and future directions.

Suboptimal drug distribution and hypoxia, which can contribute to treatment failure, are a direct consequence of the spatial and temporal heterogeneity in perfusion that occurs in solid tumors. Therefore, improvements in tumor blood flow have wide-ranging therapeutic importance. Paradoxically, controlled decreases in tumor blood flow can also be exploited and, if permanent, induce extensive tumor cell death on their own. We review the current knowledge of the factors controlling tumor blood flow with emphasis on the roles of the endogeneous vasodilator nitric oxide and the endogenous vasoconstrictor endothelin-1. The potential importance and application of approaches that irreversibly damage vascular function, so-called vascular targeting, are also discussed. Emphasis is given to the drug-based approaches to vascular targeting that are now entering clinical evaluation. There is no doubt that increased understanding of the processes that determine blood flow in tumors, coupled with the availability of techniques to monitor blood flow noninvasively in the clinic, will enable strategies for selectively modifying tumor blood flow to be transferred from the laboratory to the clinical setting.

Hypoxia modulated gene expression: angiogenesis, metastasis and therapeutic exploitation.

Tumour hypoxia is the result of an imbalance in oxygen supply and demand. It is an adverse prognostic indicator in cancer as it modulates tumour progression and treatment. Many genes controlling tumour biology are oxygen regulated, and new ones are constantly added to the growing list of hypoxia-induced genes. Of specific importance are hypoxia-responsive transcription factors, as they can modulate the expression of numerous different genes. Similarly, growth factors which govern the formation of new blood vessels or which control blood flow are vitally important for both the maintenance of the primary tumour and metastases at distant sites. The purpose of this review is to present an update of selected issues regarding hypoxia-inducible gene expression and how this affects prognosis, angiogenesis and metastasis. It will conclude by discussing gene therapy as one possible means of exploiting tumour hypoxia for the treatment of cancer.

The comparative effects of the NOS inhibitor, Nomega-nitro-L-arginine, and the haemoxygenase inhibitor, zinc protoporphyrin IX, on tumour blood flow.

PURPOSE: To determine the relative effects of inhibiting nitric oxide synthase (NOS) and haemoxygenase (HO) on blood flow to the rat P22 carcinosarcoma. METHODS AND MATERIALS: HO is the enzyme responsible for in vivo production of carbon monoxide (CO). The vascular effects of zinc protoporphyrin IX (ZnPP), a competitive inhibitor of HO, were compared with those of copper protoporphyrin IX (CuPP), a poor inhibitor of HO, in isolated ex vivo perfusions of the P22 tumour and in intact tumour-bearing rats. In ex vivo perfusions, tumour vascular resistance was calculated from measurements of perfusion pressure at a known flow rate. In intact animals, blood flow to tumour and normal tissues was calculated using a radiotracer uptake method. The effects of ZnPP were compared with those of the NOS inhibitor, N(omega)-nitro-L-arginine (L-NNA), and the combination of the two drugs. RESULTS: HO activity in the P22 tumour was reduced by 50% following administration of either ZnPP or CuPP directly to ex vivo perfused tumours, suggesting an indirect effect on the enzyme. Enzyme inhibition was not associated with any significant vasoactive effect. Neither ZnPP nor CuPP, at a dose of 45 micromol x kg(-1) administered i.p., inhibited tumour HO in vivo. However, they did significantly decrease tumour blood flow to 60-70% of control, with similar effects in skin and brain. Skeletal muscle blood flow was increased to 150% of control. L-NNA decreased both tumour and skeletal muscle blood flow to around 40% of control. These differences suggest that the nonspecific effects of ZnPP and CuPP were not mediated by NOS inhibition. The combination of ZnPP and L-NNA improved the selective reduction in tumour blood flow achieved with either agent alone. CONCLUSION: This suggests that the HO/CO pathway does not play a major vasodilatory role in this tumour. However, ZnPP and CuPP could be useful for inducing a relatively selective decrease in tumour blood flow via mechanisms unrelated to HO inhibition, especially when combined with NOS inhibition.

Phosphorus-31 magnetic resonance spectroscopy and blood perfusion of the RIF-1 tumor following X-irradiation.

Phosphorus-31 magnetic resonance spectra were obtained from the RIF-1 tumor in C3H mice before and up to 2 days after various doses of X rays. Parallel studies were performed to measure relative changes in tumor blood perfusion using [14C]iodo-antipyrine and changes in % tumor necrosis using Chalkley's method. Tumor ratios of phosphocreatine to inorganic phosphate (PCr/Pi) and nucleotide triphosphates to inorganic phosphate (NTP/Pi) as well as pH as measured by 31P-MRS increased significantly at most time points after irradiation with doses of 5, 10, and 20 Gy. Tumor blood perfusion was found to significantly improve after a dose of 20 Gy but not after a dose of 2 Gy. Percent tumor necrosis increased to about 3 times its control level at 1 day after a dose of 20 Gy and then declined to about twice its control value at 2 days. The magnitude of the changes in the 31P-MRS parameters makes it unlikely that any of them are entirely due to radiation-induced changes in the radiobiologically hypoxic fraction of these tumors. Changes in the necrotic fraction did not appear to influence the tumor spectra. However, the observed improvement in tumor blood perfusion may have resulted in an increase in oxidative phosphorylation of the whole tumor population as well as a clearance of inorganic phosphate and acid metabolites, so that 31P-MRS changes may indirectly reflect changes in tumor blood perfusion.

Blood flow and blood volume in a transplanted rat fibrosarcoma: comparison with various normal tissues.

Blood flow measurements following i.v. infusion of iodo-antipyrine labelled with 14C(14C-IAP) and blood volume measurements following i.v. injection of 125I human serum albumin and 51Cr-labelled red blood cells were made in a transplanted rat fibrosarcoma for comparison with various normal tissues. The tumour-blood partition co-efficient for 14C-IAP was found to be 0.79 +/- 0.07 which is similar to most of the normal tissues studied. The solubility of 14C-IAP in plasma was found to be higher than that in whole blood. Blood flow to tumours less than 1000 mm3 was found to be 17.9 +/- 4.0 ml blood 100 g tissue-1.min-1. These values were considered to be primarily measurements of nutritive flow. Blood in the tumours was found to occupy around 1% of the tissue space which was similar to that found for normal muscle and skin. There was no direct correlation between % blood volume and blood flow for the different tissues studied. The haematocrit of blood contained in tumour tissue was calculated to be significantly lower than that of blood contained in the normal tissues. It was suspected that permeability of tumour blood vessel walls to 125I-HSA could have accounted for this difference.

The energy metabolism of RIF-1 tumours following hydralazine.

Phosphorus-31 Magnetic Resonance Spectroscopy (MRS) was used to observe the effect of two doses of the vasodilator hydralazine on the energy status of RIF-1 tumours. An intravenous dose of 5 mg/kg hydralazine reduced the high energy phosphate metabolites PCr and ATP, lowered pHMRS and raised the levels of inorganic phosphate of tumours within 20 min of administering the drug. The levels of high energy metabolites continued to decrease for at least 24 h. Normal muscle spectra obtained up to 1 h after drug administration remained unchanged. An intravenous dose of 0.5 mg/kg hydralazine also reduced NTP/Pi and PCr/Pi levels of tumours up to at least 5 h after drug administration, but the effect was smaller than for the higher dose. Blood flow measurements and measurements of systemic blood pressure demonstrated that 5 mg/kg of hydralazine produced a reduction in both systemic blood pressure and tumour blood flow relative to most normal tissues investigated. It is concluded that the changes in the P-31 MRS spectra of tumours were due to a reduction in tumour vascular perfusion following administration of hydralazine.

Measurement of tumor oxygenation: in vivo comparison of a luminescence fiber-optic sensor and a polarographic electrode in the p22 tumor.

Hypoxia is important in tumor biology and therapy. This study compared the novel luminescence fiber-optic OxyLite sensor with the Eppendorf polarographic electrode in measuring tumor oxygenation. Using the relatively well-oxygenated P22 tumor, oxygen measurements were made with both instruments in the same individual tumors. In 24 air-breathing animals, pooled electrode pO(2) readings lay in a range over twice that of sensor pO(2(5min)) values (-3.2 to 80 mm Hg and -0.1 to 34.8 mm Hg, respectively). However, there was no significant difference between the means +/- 2 SE of the median pO(2) values recorded by each instrument (11.0 +/- 3.3 and 8.1 +/- 1.9 mm Hg, for the electrode and sensor respectively, P = 0.07). In a group of 12 animals treated with carbon monoxide inhalation to induce tumor hypoxia, there was a small but significant difference between the means +/- 2 SE of the median pO(2) values reported by the electrode and sensor (1.7 +/- 0.9 and 2.9 +/- 0.7 mm Hg, respectively, P = 0.009). A variable degree of disparity was seen on comparison of pairs of median pO(2) values from individual tumors in both air-breathing and carbon monoxide-breathing animals. Despite the differences between the sets of readings made with each instrument from individual tumors, we have shown that the two instruments provide comparable assessments of tumor oxygenation in groups of tumors, over the range of median pO(2) values of 0.6 to 28.1 mm Hg.

Nitric oxide in biological fluids: analysis of nitrite and nitrate by high-performance ion chromatography.

The analysis of nitric oxide-derived nitrite and nitrate ions in biological fluids represents a proven strategy for determining nitric oxide participation in a diverse range of physiological and pathophysiological processes in vivo. In this article we describe a versatile method for the simultaneous measurement of NO2- and NO3- anions in both plasma and isolated tumour models based on anion-exchange chromatography with spectrophotometric detection (214 nm). This method compares well with the capillary electrophoresis technique, exhibiting an equivalent sensitivity for NO2-/NO3- anions and short run-times, i.e. not greater than 4 min. Comparisons are also made with two alternative but less satisfactory methods which employ ion-exchange or reversed-phase ion-pair chromatography with conductimetric as well as spectrophotometric detection. Technical problems associated with each method, particularly those arising from nitrate contamination, have been addressed.