An isotope dilution liquid chromatography-tandem mass spectrometry candidate reference measurement procedure for aldosterone measurement in human plasma.

Accurate quantitation of aldosterone is clinically important in standardized testing for primary aldosteronism. The results are often variable when performed by clinical immunoassays. To standardize and ensure the accuracy of clinical systems, reference measurement procedures (RMPs) with higher metrological order are required. A simple and reliable isotope dilution LC-IDMS/MS-based measurement procedure for human plasma aldosterone has been developed. This method involved plasma spiked with a deuterium-labelled internal standard, equilibrated for 0.5 h, and extracted by liquid-liquid extraction (LLE) without derivatization. Aldosterone and its structural analogues were baseline separated with a C18-packed UHPLC column with gradient elution within 7 min. The signal intensity variability and measurement imprecision were reduced by bracketing calibration during plasma aldosterone value assignment. The limit of detection (LoD) was 19.4 pmol/L with a signal-to-noise ratio (S/N) > 3. The lowest limit of quantification (LLoQ) was 27.7 pmol/L (S/N > 10 and CV < 10.0%). LLE was performed with 1 mL of n-hexane/ethyl acetate (3:2, v/v), and the extraction recovery was determined to be 92.15 ± 3.54%. The imprecisions were ≤ 3.18% for samples at 124.8, 867.0, and 2628.5 pmol/L. The recoveries were 98.11-101.61%. The relative bias between this candidate RMP and the established RMP was 2.76-1.89%. The linearity response ranged from 27.7 to 2774.4 pmol/L with R2 = 0.999. The method performance met the requirements of RMPs (≤ 5% total CV and ≤ 3% bias). Furthermore, the developed method was applied to evaluate immunoassays through 41 patient sample comparisons. The calibration and measurement capability (CMC) of this method were also evaluated by measuring these samples. The candidate RMP can serve as an accurate reference baseline for routine methods and can be used for value assignment for reference materials. Selected ion chromatograms by LC-MS/MS using a C18 column for aldosterone and its structural analogues.

The blood copper isotopic composition is a prognostic indicator of the hepatic injury in Wilson disease.

Wilson disease (WD) is an autosomal recessive disorder of copper (Cu) metabolism. The gene responsible for WD, ATP7B, is involved in the cellular transport of Cu, and mutations in the ATP7B gene induce accumulation of Cu in the liver and ultimately in the brain. In a pilot study, the natural variations of copper stable isotope ratios (65Cu/63Cu) in the serum of WD patients have been shown to differ from that of healthy controls. In the present study, we challenged these first results by measuring the 65Cu/63Cu ratios in the blood of treated (n = 25), naïve patients (n = 11) and age matched healthy controls (n = 75). The results show that naïve patients and healthy controls exhibit undistinguishable 65Cu/63Cu ratios, implying that the Cu isotopic ratio cannot serve as a reliable diagnostic biomarker. The type of treatment (d-penicillamine vs. triethylenetetramine) does not affect the 65Cu/63Cu ratios in WD patients, which remain constant regardless of the type and duration of the treatment. In addition, the 65Cu/63Cu ratios do not vary in naïve patients after the onset of the treatment. However, the 65Cu/63Cu ratios decrease with the degree of liver fibrosis and the gradient of the phenotypic presentation, i.e. presymptomatic, hepatic and neurologic. To get insights into the mechanisms at work, we study the effects of the progress of the WD on the organism by measuring the Cu concentrations and the 65Cu/63Cu ratios in the liver, feces and plasma of 12 and 45 week old Atp7b-/- mice. The evolution of the 65Cu/63Cu ratios is marked by a decrease in all tissues. The results show that 63Cu accumulates in the liver preferentially to 65Cu due to the preferential cellular entry of 63Cu and the impairment of the 63Cu exit by ceruloplasmin. The hepatic accumulation of monovalent 63Cu+ is likely to fuel the production of free radicals, which is potentially an explanation of the pathogenicity of WD. Altogether, the results suggest that the blood 65Cu/63Cu ratio recapitulates WD progression and is a potential prognostic biomarker of WD.

Pharmacokinetics of 10B-p-boronophenylalanine (BPA) in the blood and tumors in human patients: A critical review with special reference to tumor-to-blood (T/B) ratios using resected tumor samples.

We reviewed 10B concentration kinetics in the blood and tumors in human patients administered with BPA. The 10B concentration in the blood peaked at the end of intravenous infusion of BPA, followed by a biphasic-decreasing curve with half-lives for the first and second components of the curve being 0.7-3.7 and 7.2-12.0 h, respectively. The mean tumor-to-blood (T/B) ratio obtained from resected tumor samples was 3.40 ± 0.83 for melanoma and the ratio ranged from 1.4 to 4.7 for glioblastoma.

High-precision isotopic analysis of serum and whole blood Cu, Fe and Zn to assess possible homeostasis alterations due to bariatric surgery.

Bariatric surgery is an effective procedure to achieve weight loss in obese patients. However, homeostasis of essential metals may be disrupted as the main absorption site is bypassed. In this study, we determined Cu, Fe and Zn isotopic compositions in paired serum and whole blood samples of patients who underwent Roux-en-Y gastric bypass (RYGB) surgery for evaluation of longitudinal changes and their potential relation to mineral element concentrations and relevant clinical parameters used for monitoring the patient's condition. Samples from eight patients were collected pre-surgery and at 3, 6 and 12 months post-surgery. Multi-collector inductively coupled plasma-mass spectrometry (MC-ICP-MS) was used for high-precision isotope ratio measurements. Alterations in metal homeostasis related to bariatric surgery were reflected in the serum and whole blood Cu, Fe and Zn isotopic compositions. The serum and whole blood Cu became isotopically lighter (lower δ65Cu values) after bariatric surgery, reaching statistical significance at 6 months post-surgery (p < 0.05). The difference between the serum and the whole blood Zn isotopic composition increased after surgery, reaching significance from 6 months post-surgery onwards (p < 0.05). Those changes in Cu, Fe and Zn isotopic compositions were not accompanied by similar changes in their respective concentrations, making isotopic analysis more sensitive to physiological changes than elemental content. Furthermore, the Zn isotopic composition correlates with blood glycaemic and lipid parameters, while the Fe isotopic composition correlates with glycaemic parameters. Graphical Abstract.

BNCT induced immunomodulatory effects contribute to mammary tumor inhibition.

In the United States, breast cancer is one of the most common and the second leading cause of cancer-related death in women. Treatment modalities for mammary tumor are surgical removal of the tumor tissue followed by either chemotherapy or radiotherapy or both. Radiation therapy is a whole body irradiation regimen that suppresses the immune system leaving hosts susceptible to infection or secondary tumors. Boron neutron capture therapy (BNCT) in that regard is more selective, the cells that are mostly affected are those that are loaded with 109 or more 10B atoms. Previously, we have described that liposomal encapsulation of boron-rich compounds such as TAC and MAC deliver a high payload to the tumor tissue when injected intravenously. Here we report that liposome-mediated boron delivery to the tumor is inversely proportional to the size of the murine mammary (EMT-6) tumors. The plausible reason for the inverse ratio of boron and EMT-6 tumor size is the necrosis in these tumors, which is more prominent in the large tumors. The large tumors also have receding blood vessels contributing further to poor boron delivery to these tumors. We next report that the presence of boron in blood is essential for the effects of BNCT on EMT-6 tumor inhibition as direct injection of boron-rich liposomes did not provide any added advantage in inhibition of EMT-6 tumor in BALB/c mice following irradiation despite having a significantly higher amount of boron in the tumor tissue. BNCT reaction in PBMCs resulted in the modification of these cells to anti-tumor phenotype. In this study, we report the immunomodulatory effects of BNCT when boron-rich compounds are delivered systemically.

A new anion exchange purification method for Cu stable isotopes in blood samples.

The isotopic composition of iron, zinc, copper, and cadmium (δ56Fe, δ66Zn, δ65Cu, and δ114Cd) are novel and promising tools to study the metabolism and homeostasis of trace metals in the human body. Serum δ65Cu has been proposed as a potential tool for diagnosis of cancer in liquid biopsy, and other metals may have similar utility. However, accurate analysis of trace metal isotopes is challenging because of the difficulties in purifying the metals from biological samples. Here we developed a simple and rapid method for sequential purification of Cu, Fe, Zn, and Cd from a single blood plasma sample. By using a combination of 11 M acetic acid and 4 M HCl in the first steps of column chemistry on AG-MP1 resin, we dramatically improve the separation of Cu from matrix elements compared to previous methods which use concentrated HCl alone. Our new method achieves full recovery of Cu, Fe, Zn, and Cd to prevent column-induced isotope fractionation effects, and effectively separates analytes from the matrix in order to reduce polyatomic interferences during isotope analysis. Our methods were verified by the analysis of isotope standards, a whole blood reference material, and a preliminary sample set including five plasma samples from healthy individuals and five plasma samples from cancer patients. This new method simplifies preparation of blood samples for metal isotope analysis, accelerating multi-isotope approaches to medical studies and contributing to our understanding of the cycling of Fe, Zn, Cu, and Cd in the human body. Graphical abstract ᅟ.

Simultaneous measurement of total estradiol and testosterone in human serum by isotope dilution liquid chromatography tandem mass spectrometry.

Reliable measurement of total testosterone and estradiol is critical for their use as biomarkers of hormone-related disorders in patient care and translational research. We developed and validated a mass spectrometry method to simultaneously quantify these analytes in human serum without chemical derivatization. Serum is equilibrated with isotopic internal standards and treated with acidic buffer to release hormones from their binding proteins. Lipids are isolated and polar impurities are removed by two serial liquid-liquid extraction steps. Total testosterone and estradiol are measured using liquid chromatography tandem mass spectrometry (LC-MS/MS) in combination of positive and negative electrospray ionization modes. The method shows broad analytical measurement range for both testosterone 0.03-48.5 nM (0.75-1400 ng/dL) and estradiol 11.0-5138 pM (2.99-1400 pg/mL) and excellent agreement with certified reference materials (mean bias less than 2.1% to SRM 971, BCR 576, 577, and 578) and a high order reference method (mean bias 1.25% for testosterone and -0.84% for estradiol). The high accuracy of the method was monitored and certified by CDC Hormone Standardization (HoSt) Program for 2 years with mean bias -0.7% (95% CI -1.6% to 0.2%) for testosterone and 0.1% (95% CI -2.2% to 2.3%) for estradiol. The method precision over a 2-year period for quality control pools at low, medium, and high concentrations was 2.7-2.9% for testosterone and 3.3-5.3% for estradiol. With the consistently excellent accuracy and precision, this method is readily applicable for high-throughput clinical and epidemiological studies.

Simultaneous quantification of busulfan, clofarabine and F-ARA-A using isotope labelled standards and standard addition in plasma by LC-MS/MS for exposure monitoring in hematopoietic cell transplantation conditioning.

In allogeneic hematopoietic cell transplantation (HCT) it has been shown that over- or underexposure to conditioning agents have an impact on patient outcomes. Conditioning regimens combining busulfan (Bu) and fludarabine (Flu) with or without clofarabine (Clo) are gaining interest worldwide in HCT. To evaluate and possibly adjust full conditioning exposure a simultaneous analysis of Bu, F-ARA-A (active metabolite of Flu) and Clo in one analytical run would be of great interest. However, this is a chromatographical challenge due to the large structural differences of Bu compared to F-ARA-A and Clo. Furthermore, for the bioanalysis of drugs it is common to use stable isotope labelled standards (SILS). However, when SILS are unavailable (in case of Clo and F-ARA-A) or very expensive, standard addition may serve as an alternative to correct for recovery and matrix effects. This study describes a fast analytical method for the simultaneous analysing of Bu, Clo and F-ARA-A with liquid chromatography-tandem mass spectrometry (LC-MS/MS) including standard addition methodology using 604 spiked samples. First, the analytical method was validated in accordance with European Medicines Agency guidelines. The lower limits of quantification (LLOQ) were for Bu 10µg/L and for Clo and F-ARA-A 1µg/L, respectively. Variation coefficients of LLOQ were within 20% and for low medium and high controls were all within 15%. Comparison of Bu, Clo and F-ARA-A standard addition results correspond with those obtained with calibration standards in calf serum. In addition for Bu, results obtained by this study were compared with historical data analysed within TDM. In conclusion, an efficient method for the simultaneous quantification of Bu, Clo and F-ARA-A in plasma was developed. In addition, a robust and cost-effective method to correct for matrix interference by standard addition was established.

Medical applications of Cu, Zn, and S isotope effects.

This review examines recent applications of stable copper, zinc and sulfur isotopes to medical cases and notably cancer. The distribution of the natural stable isotopes of a particular element among coexisting molecular species varies as a function of the bond strength, the ionic charge, and the coordination, and it also changes with kinetics. Ab initio calculations show that compounds in which a metal binds to oxygen- (sulfate, phosphate, lactate) and nitrogen-bearing moieties (histidine) favor heavy isotopes, whereas bonds with sulfur (cysteine, methionine) favor light isotopes. Oxidized cations (e.g., Cu(ii)) and low coordination numbers are expected to favor heavy isotopes relative to their reduced counterparts (Cu(i)) and high coordination numbers. Here we discuss the first observations of Cu, Zn, and S isotopic variations, three elements closely related along multiple biological pathways, with emphasis on serum samples of healthy volunteers and of cancer patients. It was found that heavy isotopes of Zn and to an even greater extent Cu are enriched in erythrocytes relative to serum, while the difference is small for sulfur. Isotopic variations related to age and sex are relatively small. The 65Cu/63Cu ratio in the serum of patients with colon, breast, and liver cancer is conspicuously low relative to healthy subjects. The characteristic time over which Cu isotopes may change with disease progression (a few weeks) is consistent with both the turnover time of the element and albumin half-life. A parallel effect on sulfur isotopes is detected in a few un-medicated patients. Copper in liver tumor tissue is isotopically heavy. In contrast, Zn in breast cancer tumors is isotopically lighter than in healthy breast tissue. 66Zn/64Zn is very similar in the serum of cancer patients and in controls. Possible reasons for Cu isotope variations may be related to the cytosolic storage of Cu lactate (Warburg effect), release of intracellular copper from cysteine clusters (metallothionein), or the hepatocellular and biosynthetic dysfunction of the liver. We suggest that Cu isotope metallomics will help evaluate the homeostasis of this element during patient treatment, notably by chelates and blockers of Cu trafficking, and understand the many biochemical pathways in which this element is essential.

Cu isotopic signature in blood serum of liver transplant patients: a follow-up study.

End-stage liver disease (ESLD) is life-threatening and liver transplantation (LTx) is the definitive treatment with good outcomes. Given the essential role of hepatocytes in Cu homeostasis, the potential of the serum Cu isotopic composition for monitoring a patient's condition post-LTx was evaluated. For this purpose, high-precision Cu isotopic analysis of blood serum of ESLD patients pre- and post-LTx was accomplished via multi-collector ICP-mass spectrometry (MC-ICP-MS). The Cu isotopic composition of the ESLD patients was fractionated in favour of the lighter isotope (by about -0.50‰). Post-LTx, a generalized normalization of the Cu isotopic composition was observed for the patients with normal liver function, while it remained light when this condition was not reached. A strong decrease in the δ(65)Cu value a longer term post-LTx seems to indicate the recurrence of liver failure or cancer. The observed trend in favour of the heavier Cu isotopic composition post-LTx seems to be related with the restored biosynthetic capacity of the liver, the restored hepatic metabolism and/or the restored biliary secretion pathways. Thus, Cu isotopic analysis could be a valuable tool for the follow-up of liver transplant patients and for establishing the potential recurrence of liver failure.

Copper isotope effect in serum of cancer patients. A pilot study.

The isotope effect describes mass-dependent variations of natural isotope abundances for a particular element. In this pilot study, we measured the (65)Cu/(63)Cu ratios in the serums of 20 breast and 8 colorectal cancer patients, which correspond to, respectively, 90 and 49 samples taken at different times with molecular biomarker documentation. Copper isotope compositions were determined by multiple-collector inductively coupled plasma mass spectrometry (MC-ICP-MS). When compared with the literature data from a control group of 50 healthy blood donors, abundances of Cu isotopes predict mortality in the colorectal cancer group with a probability p = 0.018. For the breast cancer patients and the group of control women the probability goes down to p = 0.0006 and the AUC under the ROC curve is 0.75. Most patients considered in this preliminary study and with serum δ(65)Cu lower than the threshold value of -0.35‰ (per mil) did not survive. As a marker, a drop in δ(65)Cu precedes molecular biomarkers by several months. The observed decrease of δ(65)Cu in the serum of cancer patients is assigned to the extensive oxidative chelation of copper by cytosolic lactate. The potential of Cu isotope variability as a new diagnostic tool for breast and colorectal cancer seems strong. Shifts in Cu isotope compositions fingerprint cytosolic Cu chelation by lactate mono- and bidentates. This simple scheme provides a straightforward explanation for isotopically light Cu in the serum and isotopically heavy Cu in cancer cells: Cu(+) escaping chelation by lactate and excreted into the blood stream is isotopically light. Low δ(65)Cu values in serum therefore reveal the strength of lactate production by the Warburg effect.

(10)B-NMR determination of (10)B-BPA, (10)B-BPA-fructose complex and total (10)B in blood for BNCT.

First spontaneous, noninvasive determination method of (10)B-BPA, (10)B-BPA-fructose complex, and total (10)B in blood is described. (10)B-NMR measurement with 100,000 FT accumulation enables us to obtain the result within 100min/sample. The detection limits for the simultaneous analysis were 3ppm, 3ppm and 6ppm for (10)B-BPA, (10)B-BPA-fructose complex and total (10)B respectively in this study. By this method, we can actually discuss behavior of the (10)B-BPA-fructose complex in blood.

Selenium metabolism in rats with long-term ingestion of Se-methylselenocysteine using enriched stable isotopes.

Se-methylselenocysteine (MeSeCys) is not only a selenium (Se) supplement but also a more promising precursor of an anti-tumor drug containing Se than selenomethionine, which is currently used as Se supplement. In this study, the metabolism of MeSeCys labeled with an Se isotope, 82Se, in rats depleted of endogenous natural abundance isotopes with another Se isotope, 78Se, was traced for 21 days when MeSeCys was continuously and perorally ingested at a supplemental dose. The tracer experiment was performed with our improved method that utilized an inductively coupled plasma-deuterium reaction-mass spectrometer. The substitution of endogenous Se with a single isotope, 78Se, facilitated the detection of exogenous labeled Se. Exogenous Se in the form of MeSeCys preferably accumulated and/or assimilated in the liver, kidneys and testes with long-term ingestion of MeSeCys and was utilized for the synthesis of selenoproteins, i.e., extracellular and cellular glutathione peroxidases and selenoprotein P. Meanwhile, intact MeSeCys was not excreted into urine although trimethylselenonium was detected in addition to selenosugar. The results suggest that MeSeCys was transformed into selenide via methylselenol by beta-lyase. Consequently, it is surmised that MeSeCys is a precursor of methylselenol under long-term ingestion.

Boron biodistribution study in colorectal liver metastases patients in Argentina.

Ex-situ BNCT for multifocal unresectable liver metastases employing whole or partial autograft techniques requires knowledge of boron concentrations in healthy liver and metastases following perfusion and immersion in Wisconsin solution (W), the procedure employed for organ preservation during ex-situ irradiation. Measurements of boron concentration in blood, liver and metastases following an intravenous infusion of BPA-F in five colorectal liver metastases patients scheduled for surgery were performed. Tissue samples were evaluated for boron content pre and post perfusion and immersion in W. Complementary histological studies were performed. The data showed a dose-dependent BPA uptake in liver, a boron concentration ratio liver/blood close to 1 and a wide spread in the metastases/liver concentration ratios in the range 0.8-3.6, partially attributable to histological variations between samples. Based on the boron concentrations and dose considerations (liver < or =15 Gy-Eq and tumor> or =40 Gy-Eq) at the RA-3 thermal neutron facility (mean flux of about (6+/-1) x 10(9) n cm(-2)s(-1)), ex-situ treatment of liver metastases at RA-3 would be feasible.

Biomonitoring of 37 trace elements in blood samples from inhabitants of northern Germany by ICP-MS.

The trace elements Ag, As, Au, B, Ba, Be, Bi, Cd, Ce, Co, Cs, Cu, Ga, Hf, Hg, In, La, Mn, Mo, Ni, Pb, Pd, Rb, Rh, Ru, Sb, Se, Sn, Sr, Te, Th, Tl, U, V, W, Y and Zr were determined in 130 human blood samples from occupationally non-exposed volunteers living in the greater area of Bremen in northern Germany. The blood samples were collected in lithium heparin monovettes developed for trace metal determination and were analysed by inductively coupled plasma mass spectrometry (ICP-MS) with an octopole-based collision/reaction cell. For sample introduction into the ICP, the blood samples were diluted 1/10 (V/V) with a 0.1% Triton-X-100 and 0.5% (V/V) ammonia solution. The method validation of our developed routine method is described for all 37 elements and results about internal and external quality assurance are discussed. Information on exposure conditions of all human subjects were collected by questionnaire-based interviews, including smoking habits, seafood consumption and the type of dental alloys in the teeth. Mean values, geometric mean values, ranges and selected percentiles of all elemental concentrations in human blood are presented, which helps toxicologists and clinical chemists planning research about exposition to metals and health effects caused by exposition to metals.

'The measurement of thermal neutron flux depression for determining the concentration of boron in blood'--suggestions as to further development of the method.

Suggestions about determining the concentration of 10B in blood via the thermal neutron flux depression measurement (NFDM) are made. The use of a measuring set-up consisting of a 252Cf neutron source, polyethylene moderator and a slim BF3 counter surrounded by an annular sample is examined. It is shown experimentally that using 6 ml samples and the source emitting 1.4 x 10(7) neutrons s(-1), one can determine the concentration of 10B in water at the level of 10 ppm with a statistical precision of 10% in about 20 min. Monte Carlo simulations performed with the use of MCNP-4C code revealed a potential for further improvements of the NFDM technique both in respect of the sample volume and counting period.

Development and application of a simple routine method for the determination of selenium in serum by octopole reaction system ICPMS.

The aim of the study was to develop an inductively coupled plasma mass spectrometry (ICPMS) method for robust and simple routine determination of selenium in serum. Polyatomic interferences on 76Se, 77Se, and 78Se were removed by applying an octopole reaction system ICPMS with the reaction cell pressurized with H2 gas. We developed a novel simple optimization routine for the H2 gas flow based on a signal-to-noise ratio (SNR) calculation of the selenium signal measured in a single selenium standard. The optimum H2 flow was 2.9 mL min-1. The selenium content in serum was determined after a 50-fold dilution with 0.16 M HNO3 and quantified by using addition calibration and gallium as an internal standard. The method detection limit was 0.10 microg L-1 for 76Se and 78Se and 0.13 microg L-1 for 77Se. Human serum samples from a case-control study investigating if selenium was associated with risk of colorectal adenoma were analyzed. The average selenium concentration for the control group (n=768) was 137.1 microg L-1 and the range was 73.4-305.5 microg L-1. The within-batch repeatability (a batch is ten samples) estimated from 182 replicate analyses was 6.3% coefficient of variation (CV), whereas the between-batch repeatability was 7.4% CV estimated from 361 replicates between batches. The method accuracy was evaluated by analysis of a human serum certified reference material (Seronorm Serum level II, Sero A/S, Norway). There was a fairly good agreement between the measured average of 145+/-3 microg L-1 (n=36) and the certified value of 136+/-9 microg L-1. In addition the method was successfully applied for analysis of zinc serum concentrations without further optimization. For the Seronorm certified reference material a value of 911+/-75 microg L-1 (n=31) for zinc was obtained, which corresponds well to the certified zinc value of 920+/-60 microg L-1.

A new approach to determine tumor-to-blood 10B concentration ratios from the clinical outcome of a BNCT treatment.

A new approach to determine the tumor-to-blood (10)B concentration ratio in boron neutron capture therapy (BNCT) is introduced. It is a statistical method, which uses maximum likelihood estimation on the clinical outcome of a BNCT treatment. Its performance is shown in a clinical case of cutaneous multiple nodular melanomas. The calculations involve a detailed dosimetry analysis, the determination of tumor control probabilities for the different nodules, the maximum likelihood estimation itself, and a parametric bootstrap to obtain confidence intervals for the tumor-to-blood ratio. The obtained ratio is 3.05 +/- 0.46 with a 95%-confidence interval. These results are consistent with those found in literature. Moreover, a single patient with multiple nodules proves enough to get statistically relevant results. The proposed method does not involve surgery and can be performed after a BNCT treatment without being invasive for the patient.

Biodistribution of 10B in a rat liver tumor model following intra-arterial administration of sodium borocaptate (BSH)/degradable starch microspheres (DSM) emulsion.

We reported that intra-arterial administration of borocaptate sodium (BSH)/lipiodol emulsion provided selectively high (10)B concentrations (approximately 200 ppm 6 h after administration) in experimental liver tumors. In the present study, we investigated the pharmacokinetics of BSH following intra-arterial administration of BSH with other embolizing agent, degradable starch microspheres (DSM). The (10)B concentration in the tumor at 1 h after administration of BSH with DSM was 231 ppm. At 6 h, the (10)B concentration in the tumor in BSH with DSM group was 81.5 ppm. The (10)B concentration in the liver at 1 h after administration of BSH with DSM was 184 ppm. At 6 h, the(10)B concentration in the liver in BSH with DSM group was 78 ppm. The tumor/liver (10)B concentration ratios (T/L ratio) in the "BSH+DSM" group were significantly smaller than those in the "BSH+lipiodol" group at 1 h (1.4 vs. 3.6) and 6h (1.1 vs. 14.9). BSH/DSM-boron neutron capture therapy (BNCT) was not suitable for treatment of multiple liver tumors due to the low T/L (10)B concentration ratio. However, the high (10)B accumulation in the liver tumors following intra-arterial administration of BSH/DSM emulsion suggests that BSH/DSM-BNCT has the potential for application to malignant tumors in other sites.

Delivery of (10)boron to oral squamous cell carcinoma using boronophenylalanine and borocaptate sodium for boron neutron capture therapy.

Boron neutron capture therapy (BNCT) is a unique radiation therapy in which boron compounds are trapped into tumor cells. To determine the biodistribution of boronophenylalanine (BPA) in nude mice carrying oral squamous cell carcinoma (SCC), BPA was administered at a dose of 250 mg/kg body weight intraperitoneally. Two hours later, (10)B concentration in the tumor was 15.96 ppm and tumor/blood, tumor/tongue, tumor/skin and tumor/bone (10)B concentration ratios were 6.44, 4.19, 4.68 and 4.56, respectively. Two hours after the administration of borocaptate sodium (BSH) at a dose of 75 mg/kg body weight, (10)B concentration in the tumor was 3.61 ppm, and tumor/blood, tumor/tongue, tumor/skin and tumor/bone (10)B concentration ratios were 0.77, 1.05, 0.60 and 0.59, respectively. When cultured oral SCC cells were incubated with BPA or BSH for 2 h and then exposed to thermal neutrons, the proportion of survival cells that were capable of forming cell colonies decreased exponentially, depending on (10)B concentration. BPA-mediated BNCT was more efficient than BSH-mediated BNCT. Addition of boron compounds in the cell suspension during neutron irradiation enhanced the cell-killing effect of the neutrons. These results indicate that BPA is more selectively incorporated into human oral SCC as compared with normal oral tissues, and that both extra- and intra-cellular BPA contribute to the cell-killing effect of BNCT. BPA may be a useful boron carrier for BNCT in the treatment of advanced oral SCC.