Trace analysis of platinum in blood and urine by ESI-MS-MS

A simple, rapid, and sensitive method has been developed for determination of platinum (Pt) in blood and urine by tandem mass spectrometry (MS-MS). Pt⁴⁺ in wet-ashed blood or acid-treated urine was complexed with diethyldithiocarbamate (DDC), extracted with isoamyl alcohol, and acidified with oxalic acid; a 1-μl aliquot of the isoamyl alcohol layer containing the Pt-DDC complex was directly injected into the MS-MS instrument without chromatographic separation. The quantitation was performed using selected reaction monitoring at m/z 491 of the product ion Pt(DDC)₂⁺, which was produced by collision-induced dissociation from the precursor ion Pt(DDC)₃⁺ at m/z 639. This method was validated for the analysis of blood and urine samples; the limits of quantitation were about 0.3 and 0.1 ng/ml for blood and urine, respectively, using only 10 μl of sample. The calibration curves for Pt in urine and blood showed linearity from 0.1 to 30 ng/ml. Because chromatographic separation is not required before MS-MS detection, the analysis can be completed in less than 10 min.     

Simultaneous determination of coinage metals, copper, silver, and gold in tissues using electrospray ionization tandem mass spectrometry

A rapid and sensitive mass spectrometric method was developed for the simultaneous determination of the coinage metals of copper (Cu), silver (Ag), and gold (Au). The metals in a wet-ashed tissue solution were complexed with diethyldithiocarbamate (DDC; C₄H₁₀NCS₂) and were extracted with isoamyl alcohol. After acidification of the extract with oxalic acid, metals were quantified using their product ions, Cu(DDCH)⁺, Ag(DDCH)⁺, and Au(DDCH)⁺ that derived from the precursor ions Cu(DDC)₂ ⁺, Ag(DDC)₂ ⁺, and Au(DDC)₂ ⁺, respectively, by electrospray ionization tandem mass spectrometry. The limits of detection were 0.6, 0.3, and 1 μg/l for Cu, Ag, and Au, and the quantification ranges were 2–100, 1–100, and 3–100 μg/l for Cu, Ag, and Au, respectively. Cu levels in spontaneously hypertensive osteogenic disorder rats at 6 weeks of age and 30 weeks of age were found to be 1.8 times and 5.1 times those of the normotensive osteogenic disorder rats, respectively, when using wet-ashed kidney solutions diluted 1000-fold.     

Determination of Urine Luck in urine using electrospray ionization tandem mass spectrometry

A simple, rapid and sensitive method using tandem mass spectrometry (MS-MS) has been developed for the determination of chromate Cr⁶⁺ in urine. Cr⁶⁺ is a substantial component of Urine Luck, which is used to conceal the presence of drugs in urine. Cr⁶⁺ was complexed with diethyldithiocarbamate (DDC) and extracted with isoamyl alcohol in the presence of citric acid. Then a 1-μl aliquot of isoamyl alcohol containing Cr-DDC complex was directly injected into an MS-MS instrument without chromatographic separation. The quantification was performed using selected reaction monitoring at m/z 363.8 of product ion CrO(DDC)₂ ⁺ obtained by collision-induced dissociation from the precursor ion, CrOH(DDC)₃ ⁺ at m/z 513.1. This method was validated with the analysis urine samples obtained from volunteers. A linear calibration curve could be obtained in the range of 0.18–100 ng/ml. The limits of detection and quantification of Cr⁶⁺ were 0.05 and 0.18 ng/ml, respectively, using only 10 μl of urine. Results could be obtained in less than 10 min for a sample. After oxidation of Cr³⁺ to Cr⁶⁺, near 100% recovery was confirmed using standard reference materials such as SRM 2670a (low level and high level) and SRM 1643e. The most outstanding advantage of this ESI-MS-MS method is that it gives excellent product ion mass spectra for identification of Cr⁶⁺.     

Transcatheter arterial embolization as a method of cisplatin-retention enhancement on the VX2 tumor uterus transplants

Purpose Enhanced cisplatin (Pt) retention using transcatheter arterial chemoembolization (TAE) with Gelfoam particles was studied in rabbit uterine tumors. Methods Ten rabbit uteri were inoculated with 5 X 10⁷ cells of VX2 carcinoma. Three to four weeks later cisplatin, 1 mg/kg, was injected, either with (TAE group) or without (IA group) being mixed with small Gelfoam particles, into the aortic bifurcation over 5 s. Blood and tissue concentration of cisplatin were determined. Results Slower arterial blood clearance of Pt was observed in the TAE group compared with the IA group, whereas the venous blood Pt clearance curves were similar for both groups. The uterine tumor Pt concentration at 80 min was found to be 2.52-fold higher after TAE compared with LA (p < 0.01). In the pelvic metastatic lymph nodes, the Pt concentration was 4.63 times higher after TAE than after IA (p < 0.01). Conclusion These data indicate that TAE is an effective means of increasing tissue concentration in uterine tumors.     

Combined gamma‐irradiation and subsequent cisplatin treatment in human squamous carcinoma cell lines sensitive and resistant to cisplatin

Purpose: To investigate the effects of combined radiation and subsequent cisplatin treatment on the human squamous carcinoma cell line SCC‐25 and its cisplatin‐resistant derivative SCC‐25/CP.

Materials and methods: SCC‐25 and SCC‐25/CP cells were treated with various gamma‐ray doses (5?cGy–7?Gy) followed 60?min later by cisplatin treatment and subsequently assayed for survival using a conventional colony assay. For SCC‐25, the subsequent cisplatin treatment was 0.1, 1, 10 and 20?µM for 1?h. For the more cisplatin‐resistant SCC‐25/CP cells, the subsequent cisplatin treatment was 10 and 50?µM for 1?h.

Results: The cisplatin‐resistant SCC‐25/CP cells were not cross‐resistant to gamma‐irradiation. Subsequent treatment with an LD₅₀ concentration of cisplatin (10 and 50?µM for SCC‐25 and SCC‐25/CP, respectively) resulted in radiosensitization for SCC‐25/CP but not for SCC‐25 cells. Gamma‐irradiation of SCC‐25/CP cells followed by treatment with 10 and 50?µM cisplatin for 1?h resulted in radiation survival curves displaying a significant low‐dose hypersensitive region followed by increased radioresistance at higher doses. A total of 10?µM cisplatin resulted in radiosensitization confined to the low‐dose region (0.05 and 0.25?Gy), whereas the higher cisplatin treatment of 50?µM resulted in the appearance of a hypersensitive region together with a reduction of the increased radioresistance region. In contrast, cisplatin treatment (0.1, 1, 10 and 20?µM for 1?h) of SCC‐25 cells had no significant effect on survival following 2.5 or 7.0?Gy and actually resulted in an increased low‐dose radiation survival (0.05, 0.25 and 1?Gy) when survival was corrected for cisplatin treatment (p<0.01 for all cisplatin concentrations tested).

Conclusions: The significant radiosensitization for SCC‐25/CP given subsequent treatment with 50?µM cisplatin indicates cisplatin can inhibit the increased radioresistance response in SCC‐25/CP cells. In contrast, the subsequent cisplatin treatment of SCC‐25 cells can enhance their survival following low radiation doses.     

Radiochemical synthesis and preliminary in vivo evaluation of new radioactive platinum complexes with carnosine

Application of cross-linking agents such as SATA and 2-iminothiolane (2-IT) for radiochemical synthesis of new radioactive Pt(II) and Pt(IV) complexes with carnosine was investigated.The mixed-ligand Pt(II)([¹²⁵I]Hist)(Carnosine) complex has been synthesized in a multi-step reaction. First, carnosine was modified by the attachment of SATA. After chromatographic purification, the conjugate was unprotected to form a reactive sulfhydryl functional group, and then the modified carnosine was substituted to PtCl₂[¹²⁵I]Hist complex. The Pt(II)(IT-[¹²⁵I]Carnosine) and Pt(IV)(IT-[¹³¹I]Carnosine) complexes were synthesized in a three-step reaction. First, carnosine was labeled with iodine radionuclide (¹²⁵I or ¹³¹I), followed by conjugation with 2-IT. The modified IT-[*I]Carnosine was complexed with tetrachloroplatinate or hexachloroplatinate. Comparative biodistribution studies were performed in normal Wistar rats and in Lewis rats with implanted (s.c.) rat pancreatic tumor cells (AR42J).The HPLC analysis showed a relatively fast formation of the new mixed-ligand Pt([¹²⁵I]Hist)(Carnosine) complex (yield ca. 50% after 20 h). Reaction of K₂PtCl₄ with [¹²⁵I]Carnosine modified by 2-IT proceeded rapidly and with a high yield (>95% after 2 h). The synthesis of the Pt(IV)IT-[*I]Carnosine complex was the slower reaction in comparison to the analogous synthesis of the Pt(II) complex (yield ca. 70% after 12 h), thus a purification step was necessary. The biodistribution study proved the in vivo stability of the newly synthesized complexes (a low accumulation in thyroid gland and in GIT) and showed that the conjugation of the modified carnosine changes significantly biodistribution scheme of the Pt complexes comparing to the reference Pt(II)[*I]Hist and Pt(IV)([*I]Hist)₂ complexes. The mixed-ligand complex was rapidly excreted in urine and revealed the highest accumulation in kidneys (>5%ID/g). A very high concentration in blood and in liver was observed for the Pt(II)(IT-[¹²⁵I]Carnosine) complex; however, at the same time the lowest concentration in kidneys was noted. Preliminary studies in the rat's tumor model indicated for this complex a favorable tumor to muscle ratio. In the case of Pt(IV)(IT-[*I]Carnosine) apart from ca. 12-times decrease of the liver accumulation, additional 4-times decrease of an accumulation in kidneys was observed in comparison to the Pt(IV)([*I]Hist)₂ complex.Our study showed that the short peptides can be efficiently substituted to the platinum core via the reactive sulfhydryl group introduced by SATA or 2-IT. The new radioactive platinum complexes with carnosine possess favorable biodistribution schemes, which make them potential candidates for radio-chemotherapeutical agents.     

Sensitive determination of arsenite and arsenate in plasma by electrospray ionization tandem mass spectrometry after chelate formation

Inorganic arsenite (As³⁺) and arsenate (As⁵⁺) are well-known poisons, and the toxicity of As³⁺ is about ten times that of As⁵⁺. In this study, a simple, rapid, and sensitive method was developed for As³⁺ in plasma using electrospray ionization (ESI) tandem mass spectrometry (MS-MS). After washing plasma with trichloroethylene (TCE), As³⁺ in the aqueous layer was reacted with pyrrolidinedithiocarbamate (PDC, C₄H₈NCSS_-), and the produced As(PDC)3 was extracted with methyl isobutyl ketone (MIBK); a 1-μl aliquot of the MIBK layer containing As(PDC)₃ was introduced into the MS-MS instrument in the direct-flow injection mode. Other arsenic compounds such as As⁵⁺, monomethyl arsonic acid, dimethyl arsinic acid, arsenobetaine, arsenocholine, and tetramethyl arsonium did not produce As(PDC)₃. Therefore, without liquid chromatographic separation, As³⁺ alone could be detected after washing with TCE followed by solvent extraction of As(PDC)₃ with MIBK. Thus, inorganic As⁵⁺ was reduced to As³⁺ with thiosulfate, and then the total inorganic As was quantifi ed as As³⁺; As⁵⁺could be calculated by subtracting As³⁺from the total inorganic As. The MS-MS quantification was performed by selected reaction monitoring using a peak at m/z 114 of a product ion (C₄H₈NCS)⁺ formed by collision-induced dissociation from the precursor ion As(PDC)₂ ⁺ at m/z 367. The mass spectral identification on MS-MS spectrum was possible even at 1 ng As³⁺/ml plasma. The calibration curve for As³⁺ showed linearity from 0.5 to 100 ng/ml plasma. The limits of detection by selected reaction monitoring were 0.3 ng/ml in water and 0.2 ng/ml in plasma. The analysis could be completed in less than 15 min, because chromatographic separation was not necessary before the MS-MS detection.     

Evaluation of triple quantum-filtered 23Na NMR spectroscopy in the in situ rat liver

Triple quantum (TQ)-filtered ²³Na NMR spectroscopy and the shift reagent, TmDOTP^5-, have been used to evaluate the contributions of intra- (Na_i⁺) and extracellular (Na_e⁺) sodium to the TQ-filtered signal in the rat liver, in situ. Na_e⁺ contributed significantly to the total TQ-filtered signal in live animals, and the intensity of this signal did not change postmortem. The TQ-filtered Na_i⁺ signal increased by approximately 380% over a period of 1 h postmortem, whereas the single quantum (SQ) Na_i⁺ increased by 90%. The constancy of the TQ-filtered Na_e⁺ signal indicates that changes in total TQ-filtered ²³Na signal intensity in liver (without a shift reagent) may accurately reflect changes in TQ-filtered Na_i⁺ signal intensity. The large percent increase in the TQ-filtered Na_i⁺ signal as compared to the SQ signal suggests that the fraction of Na_i⁺ interacting with macromolecules increases after death.     

��v��3 imaging can accurately distinguish between mature teratoma and necrosis in 18F-FDG-negative residual masses after treatment of non-seminomatous testicular cancer: a preclinical study

Purpose

We assessed whether imaging ??_v??₃ integrin could distinguish mature teratoma from necrosis in human non-seminomatous germ cell tumour (NSGCT) post-chemotherapy residual masses.

Methods

Human embryonal carcinoma xenografts (six/rat) were untreated (controls) or treated to form mature teratomas with low-dose cisplatin and all-trans retinoic acid (ATRA) over a period of 8?weeks. In another group, necrosis was induced in xenografts with high-dose cisplatin plus etoposide (two cycles).¹⁸F-Fluorodeoxyglucose (¹⁸F-FDG) small animal positron emission tomography (SA PET) imaging was performed in three rats (one control and two treated for 4 and 8?weeks with cisplatin+ATRA). Imaging of ??_v??₃ expression was performed in six rats bearing mature teratomas and two rats with necrotic lesions on a microSPECT/CT device after injection of the tracer [^99mTc]HYNIC-RGD [6-hydrazinonicotinic acid conjugated to cyclo(Arg-Gly-Asp-D-Phe-Lys)]. Correlative immunohistochemistry studies of human and mouse ??_v??₃ expression were performed.

Results

Cisplatin+ATRA induced differentiation of the xenografts. After 8?weeks, some glandular structures and mesenchymal cells were visible; in contrast, control tumours showed undifferentiated tissues. SA PET imaging showed that mature teratoma had very low avidity for ¹⁸F-FDG [mean standardised uptake value (SUV_mean)?=?0.48?±?0.05] compared to untreated embryonal carcinoma (SUV_mean?=?0.92?±?0.13) (p?=?0.005). ??_v??₃ imaging accurately distinguished mature teratoma (tumour to muscle ratio?=?4.29?±?1.57) from necrosis (tumour to muscle ratio?=?1.3?±?0.26) (p?=?0.0002). Immunohistochemistry studies showed that ??_v??₃ integrin expression was strong in the glandular structures of mature teratoma lesions and negative in host stroma.

Conclusion

Imaging ??_v??₃ integrin accurately distinguished mature teratoma from necrosis following cisplatin-based treatment in human NSGCT xenografts.     

Does Polyvinyl Alcohol Particle Size Change the Outcome of Prostatic Arterial Embolization for Benign Prostatic Hyperplasia? Results from a Single-Center Randomized Prospective Study

PurposeTo evaluate whether different polyvinyl alcohol (PVA) particle sizes change the outcome of prostatic arterial embolization (PAE) for benign prostatic hyperplasia (BPH).Materials and MethodsA randomized prospective study was undertaken in 80 patients (mean age, 63.9 y; range, 48–81 y) with symptomatic BPH undergoing PAE between May and December 2011. Forty patients underwent PAE with 100-µm (group A) and 200-µm PVA particles (group B). Visual analog scales were used to measure pain, and rates of adverse events were recorded. PAE outcomes were evaluated based on International Prostate Symptom Score (IPSS) and quality-of-life (QoL) questionnaires, prostate volume (PV), prostate-specific antigen (PSA) levels, and peak flow rate measurements at baseline and 6 months.ResultsNo differences between groups regarding baseline data, procedural details, or adverse events were noted. Mean pain scores were as follows: during embolization, 3.2 ± 2.97 (group A) versus 2.93 ± 3.28 (group B); after embolization, 0.10 ± 0.50 (group A) versus 0 (group B; P = .20); and the week after PAE, 0.85 ± 1.65 (group A) versus 0.87 ± 1.35 (group B; P = .96). Patients in group B had greater decreases in IPSS (3.64 points; P = .052) and QoL (0.57 points; P = .07). Patients in group A had a greater decrease in PV (8.75 cm³; P = .13) and PSA level (2.09 ng/mL; P < .001).ConclusionsNo significant differences were found in pain scores and adverse events between groups. Whereas PSA level and PV showed greater reductions after PAE with 100-µm PVA particles, clinical outcome was better with 200-µm particles.     

Electrolyte analysis of pleural effusion for discrimination between seawater and freshwater drowning in decomposed bodies

ObjectiveThe diagnosis of drowning is an important issue in forensic investigations. Moreover, discriminating between seawater and freshwater drowning is crucial to identify where the drowning occurred. The present study aimed to investigate electrolyte concentrations in pleural fluid in decomposed bodies in late postmortem intervals and derive cut-off values for the diagnosis of seawater and freshwater drowning.Study designData were collected from 44 seawater drowning cases, 60 freshwater drowning cases, and 30 non-drowning cases with pleural effusion which served as controls. The levels of sodium ion (Na⁺), potassium ion (K⁺), and chloride ion (Cl⁻) of pleural fluid were measured, and two indices were calculated: summation of Na⁺ and K⁺ levels (SUM _Na + K), and summation of Na⁺, K⁺, and Cl⁻ levels (SUM _{Na + K + Cl}). The means of the three ion concentrations and two indices significantly differed between the three groups (p < 0.0001).ResultsThe receiver operating characteristic analysis revealed that the sensitivity and specificity were both 1.000 for SUM _{Na + K + Cl} of 288.3 mEq/L between the seawater and control groups. The Na⁺ value of 109.0 mEq/L also had a high sensitivity of 0.977 and a specificity of 0.933 in the seawater and control groups. The sensitivity and specificity were 0.967 and 1.000, respectively, for SUM _Na + K of 123.2 mEq/L between the freshwater and control groups.ConclusionThe electrolyte concentrations in pleural effusion may be useful for the diagnosis of drowning in decomposed bodies with a longer postmortem interval.     

Quantitative determination of taxine B in body fluids by LC–MS–MS

A new specific and sensitive LC–MS–MS method for the detection of taxine B and isotaxine B, the main toxic pseudo-alkaloids from yew (Taxus sp.), in biological samples (blood, urine, gastric content) was developed. Biological samples were prepared for LC–MS–MS by means of solid-phase extraction (SPE) procedure and yielded a recovery of 86%. Chromatographic separation was achieved using an RP₁₈ column. Detection of taxine B and isotaxine B was performed using multiple reaction monitoring with m/z 584.2 as precursor ion, i.e. [M+H]⁺, of both isomers and m/z 194.3 and m/z 107.1 as product ions after collision-induced dissociation. Docetaxel was applied as internal standard. The method was fully validated for the analysis of blood samples. Linearity was proven in the range from 0.1–500 ng/g. The limit of detection and the limit of quantitation are 0.4 and 2 ng/g, respectively. The method was applied to the determination of taxine B and isotaxine B in four fatal cases (two humans, two horses) with suspected yew intoxication. Blood levels were 105, 168, 174 and 212 ng/g.     

Preclinical imaging characteristics and quantification of Platinum-195m SPECT

Aims

In vivo biodistribution imaging of platinum-based compounds may allow better patient selection for treatment with chemo(radio)therapy. Radiolabeling with Platinum-195m (^195mPt) allows SPECT imaging, without altering the chemical structure or biological activity of the compound. We have assessed the feasibility of ^195mPt SPECT imaging in mice, with the aim to determine the image quality and accuracy of quantification for current preclinical imaging equipment.

Methods

Enriched (>96%) ¹⁹⁴Pt was irradiated in the High Flux Reactor (HFR) in Petten, The Netherlands (NRG). A 0.05 M HCl ^195mPt-solution with a specific activity of 33 MBq/mg was obtained. Image quality was assessed for the NanoSPECT/CT (Bioscan Inc., Washington DC, USA) and U-SPECT⁺/CT (MILabs BV, Utrecht, the Netherlands) scanners. A radioactivity-filled rod phantom (rod diameter 0.85-1.7 mm) filled with 1 MBq ^195mPt was scanned with different acquisition durations (10-120 min). Four healthy mice were injected intravenously with 3-4 MBq ^195mPt. Mouse images were acquired with the NanoSPECT for 120 min at 0, 2, 4, or 24 h after injection. Organs were delineated to quantify ^195mPt concentrations. Immediately after scanning, the mice were sacrificed, and the platinum concentration was determined in organs using a gamma counter and graphite furnace – atomic absorption spectroscopy (GF-AAS) as reference standards.

Results

A 30-min acquisition of the phantom provided visually adequate image quality for both scanners. The smallest visible rods were 0.95 mm in diameter on the NanoSPECT and 0.85 mm in diameter on the U-SPECT⁺. The image quality in mice was visually adequate. Uptake was seen in the kidneys with excretion to the bladder, and in the liver, blood, and intestine. No uptake was seen in the brain. The Spearman correlation between SPECT and gamma counter was 0.92, between SPECT and GF-AAS it was 0.84, and between GF-AAS and gamma counter it was0.97 (all p?<?0.0001).

Conclusion

Preclinical ^195mPt SPECT is feasible with acceptable tracer doses and acquisition times, and provides good image quality and accurate signal quantification.

     

Effects of Irradiation on the Mediterranean Meal Moth,Ephestia Kuehniella Zeller,Cultured on 89Sr-spiked Food

Summary

Ephestia kuehniella Zeller was cultured on corn-meal spiked with ⁸⁹Sr at concentrations of 0, 0·1, 0·3, 1·0, 3·0 and 5·0 µc/g of food.

As the environmental radiation was increased, the adults which were subjected throughout their life-cycle produced progressively fewer progeny.

5·0 µc ⁸⁹Sr/g food approaches the critical level which will inhibit population development.

Life-span of the adults was not influenced by the experimental conditions.

Delayed development occurred at all isotopic concentrations employed.

The radiostrontium-content of females at the end of their life-cycle was half that of males. Twenty-four hours after eclosion, the radioactivity of the female moth was equal to, or greater than, that of corresponding males. Reproductive metabolism is offered in explanation.

At the two highest culture levels (3 and 5 µc ⁸⁹Sr/g), the increased number of males (homogametic sex) over females (heterogametic sex) is consistent with the radiation genetic effect of induced recessive lethals. No selective radiation effect upon the sexual capacity of gametes of the F₁ was indicated, since the subsequent F₂ sex ratio was 1 : 1.     

39K NMR measurement of intracellular potassium during ischemia in the perfused guinea pig heart

The hyperfine shift reagent, TmDOTP^5?, was used to resolve the ³⁹K NMR resonances of intra- (K_i⁺) and extracellular (K_e⁺) potassium in isolated, perfused guinea pig hearts. [K_i⁺] as measured by ³⁹K NMR was 25.9 ± 10.3 mM, compared with 114.4 ± 10.8 mM as measured by atomic absorption spectros-copy (AAS) using TmD0TP^5- as a marker of extracellular space. Thus, only approximately 23% of intracellular potassium was detected by ³⁹K NMR using our experimental conditions. The area of the K_i⁺ signal increased during early ischemia then returned to baseline levels during reperfusion. In an effort to learn more about the K_i⁺ not detected by ³⁹K NMR, hearts were perfused with a Rb⁺-enriched, K⁺ -depleted buffer for an extended period. This resulted in loss of the entire ³⁹K NMR signal, and K_i⁺, as measured by AAS, decreased from ～60 to ～6 to 7 μmol/g wet weight. When K⁺-depleted hearts were subjected to global ischemia, a small ³⁹K NMR signal reappeared, suggesting that at least a portion of the nonexchangeable K_i⁺ becomes detectable by NMR during ischemia. This newly visible K⁺ signal subsequently dissipated during reperfusion of ischemic hearts. We conclude that ischemia induces changes in the NMR visibility of ³⁹K in perfused guinea pig hearts.     

低剂量半身照射对恶性肿瘤患者免疫功能的影响

目的 评价低剂量辐射对恶性肿瘤患者免疫功能的影响。方法 将20例患者(其中非何杰金氏淋巴瘤13例、小细胞肺癌7例)随机分为两组:HBR组和RR组。其中HBR组10例患者采用常规放疗(RR)加低剂量半身照射,低剂量半身照射方法为10 cGy/次,2次/周,总剂量为100 cGy,每次均间隔6～8 h再行常规放疗;RR组10例患者只予以常规放疗。采用流式细胞术(FCM)双标法检测HBR与RR组在放疗前、中、后外周血淋巴细胞CD₄、CD₈、CD₂₅和CD₅₆等指标的变化。结果 照射后RR组患者的CD₄⁺ CD₈⁺降低(P<0.05),HBR组患者的CD₄⁺升高(P<0.05),CD₂₅⁺和CD₅₆⁺分子表达均明显地增加(P<0.05),放疗前和放疗后CD₈⁺均低(P<0.05),放疗中(P<0.05)和放疗后(P<0.01)CD₄⁺ CD₈⁺均高。结论 低剂量半身照射可增强机体的免疫功能。     

Lethal and mutagenic interactions between γ-rays,cisplatin and etoposide at the cellular and molecular levels

Purpose:?We analysed the lethal and mutagenic interactions between γ-rays, cisplatin (Pt) and etoposide (E), three agents used in tumour chemoradiotherapy. Corresponding results at cellular and molecular levels could provide additional elements on involved mechanisms and, on antitumour activity and toxicity in combined cancer treatments.

Materials and methods:?The yeast Saccharomyces cerevisiae SC7K(lys2–3) (auxotrophic for lysine) was used as eukaryotic model. Exponential growing cells were exposed to the mentioned agents, as single and combined treatments. Lethal and mutation interaction equations were determined as a function of doses according to quantitative models. DNA double-strand breaks were evaluated immediately after treatments, through pulsed-field electrophoresis and laser densitometry.

Results:?All three agents induced significant mutant frequency. The γ?+Pt + E combination determined maximal lethal and mutagenic synergism, followed by γ?+?Pt and γ?+?E combinations. Meanwhile, Pt?+?E combination showed lethal additivity and very low mutagenic synergism. Pt?+?E double combination determined moderate DNA degradation. DNA degradation after γ-exposure, was similar to that of γ?+?Pt, γ?+?E and γ?+?Pt?+?E combinations.

Conclusions:?Synergistic lethal and mutagenic interactions indicate crosstalk between non-homologous end joining, homologous recombination and postreplicative repair pathways. Pt?+?E additivity indicate independence of involved repair pathways. Furthermore, the quantification of interactive events may be an additional suitable tool in tumour therapy planning.     

Porphyrin-mediated boron neutron capture therapy: evaluation of the reactions of skin and central nervous system

Purpose: Recently, various boronated porphyrins have been shown to preferentially target a variety of tumour types. Of the different porphyrins evaluated, copper tetra-phenyl-carboranyl porphyrin (CuTCPH) is a strong candidate for future preclinical evaluation. In the present study, the responses of two critical normal tissues, skin and central nervous system (CNS), to boron neutron capture (BNC) irradiation in the presence of this porphyrin were evaluated.

Materials and methods: Standard models for the skin and spinal cord of adult male Fischer 344 rats were used. CuTCPH was administered by intravenous infusion at a dose of 200 mg kg^{m 1} body weight, over 48 h. The thermal beam at the Brookhaven Medical Research Reactor was used for the BNC irradiations. The 20-mm diameter irradiation field, for both the skin and the spinal cord, was located on the mid-dorsal line of the neck. Dose-response data were fitted using probit analysis and the doses required to produce a 50% incidence rate of early and late skin changes or myeloparesis (ED 50 - SE) were calculated from these curves.

Results: Biodistribution studies indicated very low levels of boron (<3 µ;g g^{m 1}) in the blood 3 days after the administration of CuTCPH. This was the time point selected for radiation exposure in the radiobiological studies. Levels of boron in the CNS were also low (2.8 - 0.6 µ;g g^{m 1}) after 3 days. However, the concentration of boron in the skin was considerably higher at 22.7 - 2.6 µ;g g^{m 1}. Single radiation exposures were carried out using a thermal neutron beam. The impact of CuTCPH-mediated BNC irradiation on the normal skin and CNS at therapeutically effective exposure times was minimal. This was primarily due to the very low blood boron levels (from CuTCPH) at the time of irradiation. Analysis of the relevant dose-effect data gave compound biological effectiveness factors of about 1.8 for skin (moist desquamation) and about 4.4 for spinal cord (myeloparesis) for CuTCPH. These values were based on the BNC radiation doses to tissues calculated using the blood boron levels at the time of irradiation.

Conclusions: CuTCPH-mediated BNC irradiation will not cause significant damage to skin and CNS at clinically relevant radiation doses provided that blood boron levels are low at the time of radiation exposure.     

Determination of aconitine in body fluids by LC-MS-MS

A very sensitive and specific method was developed for the determination of aconitine, the main toxic alkaloid from plants of the genus Aconitum L., in biological samples. The method comprised solid-phase extraction using mixed-mode C₈ cation exchange columns followed by liquid chromatography-tandem mass spectrometry (LC-MS-MS). Chromatographic separation was achieved with a RP₈ column. Detection of aconitine was achieved using electrospray in the positive ionisation mode and quantification was performed using multiple reaction monitoring with m/z 646.4 as precursor ion, i.e. [M+H]⁺ of aconitine and m/z 586.5, m/z 526.4 and m/z 368.4 as product ions after collision-induced dissociation. The method was fully validated for the analysis of blood samples: the limit of detection and the limit of quantitation were 0.1 ng/g and 0.5 ng/g, respectively. Within the linear calibration range of 0.5–25 ng/g, analytical recovery was 79.9%. In two fatal cases with suspected aconite intoxication, aconitine could be detected in blood samples at concentrations of 10.0 and 12.1 ng/g. In one case, aconitine could also be detected in the stomach content (3 ng/g) and in the other in the urine (180 ng/ml).     

1.5Gy X射线全身照射对小鼠胸腺细胞CD、CD4、CD8分子表达及

采用单克隆抗体免疫荧光技术,流式细胞术检测,观察了1.5GyX射线单次全身照射(剂量率0.286Gy/min)对小鼠胸腺细胞CD₃分子表达的影响,及胸腺细胞亚群和细胞周期的变化。结果表明,中等剂量照射后,胸腺细胞CD₃分子的百分率明显增高,胸腺细胞CD₃阳性细胞比例为对照组的177.61%.同时,胸腺中CD₄⁺及CD₈⁺细胞比例亦显着增加,分别为对照的202.5%及165.87%.然而,胸腺中CD₄⁺、CD₈⁺细胞的比例显着降低,为对照组的68.28%,与此同时,胸腺细胞周期中S期细胞比例明显降低,为对照组的55.14%.