High accuracy isotope dilution analysis for the determination of ethanol using gas chromatography-combustion-isotope ratio mass spectrometry.

A procedure was established for the determination of ethanol in water samples by isotope dilution analysis. After spiking the sample with labelled [13C2]ethanol, it was analysed by gas chromatography-combustion-isotope ratio mass spectrometry. Results are reported for two certified reference materials and also ethanol solutions prepared for a CITAC (Co-operation on International Traceability in Analytical Chemistry) interlaboratory comparison. The certified reference materials were certified using the dichromate titration method at nominal levels of 80 and 200 mg per 100 mL. The CITAC solutions were prepared gravimetrically at nominal levels of 50, 80 and 200 mg per 100 mL. The results of the analysis agree well to within 0.5% of the gravimetric values of the different samples. The relative expanded standard uncertainties (with a coverage factor equal to 2) associated with the results varied between 0.18 and 0.37%, a range that encompassed the gravimetric values for the different samples. A complete uncertainty budget was also drawn up so that the different contributions could be identified and quantified. The main contributions were due to variations in the measured isotope amount ratios and a 'between' blend component introduced to quantify the contribution of factors such as the degree of matching of the isotope amount ratios between standards and samples used in the isotope dilution analysis.

The determination of lysergide (LSD) in urine by high-performance liquid chromatography-isotope dilution mass spectrometry (IDMS).

The use of isotope dilution mass spectrometry (IDMS) has been investigated for the forensic confirmation of lysergic acid diethylamide (LSD) in urine by LC-MS. The advantages of using a deuterated analog of LSD as an internal standard over methysergide are discussed. This study includes a comparison of the electrospray mass spectra of LSD, LSD-d3 and methysergide, and discusses the choice of suitable ions for use in selected ion monitoring (SIM) mode. An IDMS method is presented for the LC-MS confirmation of LSD in urine, with a limit of quantification (LOQ) of 0.5 ng/mL, reflecting the forensic requirement at this laboratory. Under some circumstances the LOQ can be improved to 0.1 ng/mL. This method is linear in the range tested (up to 10 ng/mL LSD in urine) and has been validated in terms of accuracy and precision.

Poor reproducibility of in-source collisional atmospheric pressure ionization mass spectra of toxicologically relevant drugs.

The purpose of the study was to examine the intra- and interlaboratory reproducibility of mass spectra obtained with liquid chromatography-atmospheric pressure ionization mass spectrometry (LC--API-MS) both in electrospray (ESI) and atmospheric pressure chemical ionization (APCI) modes. Toxicologically relevant drugs of different polarity were selected as test substances: morphine-6-glucuronide, 6-monoacetylmorphine, codeine, lysergic acid diethylamide, methylenedioxymethamphetamine. The study was performed in two laboratories using identical instruments and in one using a slightly different instrument. Basic instrument settings and mobile phase were identical in all laboratories. Mass spectra of drugs were taken at four collision energy voltages and using mobile phase of different composition (four concentration levels of acetonitrile and of ammonium formate buffer). The experiments demonstrated that mass spectra of given drugs, obtained in identical conditions with identical instruments, may show very different degrees of fragmentation. Mass spectra obtained with different instruments differed profoundly not only in the degree of fragmentation, but also different fragments and adducts were observed. Short-term intralaboratory reproducibility of mass spectra was satisfactory. On the other hand, the long-term experiments showed different degrees of fragmentation of APCI-generated mass spectra at nominally identical fragmentation energy. The changes in the composition of the mobile phase (concentration of organic modifier or buffer molarity) did not affect the reproducibility of fragmentation to any relevant degree. The study showed that the interlaboratory exchange and use of mass spectrum library, generated by single-quadrupole (LC--API-MS instruments, is hardly feasible at the moment, even under very carefully standardized conditions.

Determination of lysergide in urine by high-performance liquid chromatography combined with electrospray ionisation mass spectrometry.

A quantitative method which avoids derivatisation is described for the determination of lysergide (LSD) levels in urine. Sample preparation included addition of methysergide as an internal standard followed by solid-phase extraction. LSD was analysed on a system consisting of a C18 stationary phase and a mobile phase of 0.1 M acetate buffer pH 8.0-acetonitrile-triethylamine (75:25:0.25, v/v). LSD was detected by electrospray ionisation mass spectrometry with selected ion monitoring. The quantification limit was 0.5 ng/ml and the method was linear up to 10 ng/ml of LSD in urine.

The analysis of lysergide (LSD): the development of novel enzyme immunoassay and immunoaffinity extraction procedures together with an HPLC-MS confirmation procedure.

A forensic procedure for the screening and confirmation of the presence of lysergide (lysergic acid diethylamide, LSD) in urine is described together with the evaluation of a novel enzyme immunoassay (EIA) and immunoaffinity extraction procedure. Following initial screening using either an established radioimmunoassay (RIA) or a novel EIA procedure, a quantitative estimate is established using a conventional high performance liquid chromatography-fluorescence (HPLC) technique following solid phase extraction. Final confirmation and quantitation, without derivatization, is established using HPLC in combination with electrospray ionization (ESI) mass spectrometry using methysergide as an internal standard. The detection limit of LSD in urine is 0.5 ng/mL. A blind trial confirmed the validity of the results. The choice of internal standard is discussed. Consideration is given to the photosensitivity of LSD solutions. A study of potential interferants in the HPLC-MS confirmation of LSD is presented and shows that for the wide range of compounds studied, there are none that would interfere with this confirmation technique. A comparison is shown between solid phase and immunoaffinity extraction/clean up procedures, and between RIA and EIA screening procedures.

Combination versus single agent therapy in effecting complete therapeutic response in human bladder cancer: analysis of cisplatin and/or 5-fluorouracil in an in vivo survival model.

An in vivo study of cisplatin (CDDP) and 5-fluorouracil (5FU) cytotoxicity was performed using a multidose matrix with a human bladder transitional cell carcinoma xenograft tumor line (DU4284) tested by subrenal capsule assay in 154 nude mice (NM-SRCA). Statistical analysis of initial growth inhibition at 20 days and host survival demonstrates therapeutic, cooperative interaction. Toxic doses of either CDDP or 5FU alone as well as low-dose combinations provided modest or no survival benefit. The single dose of CDDP (7 mg/kg) and of 5FU (100 mg/kg) was best (by analysis of efficacy and toxicity) of those tested and caused > 97% initial regression. While 94% of controls incurred tumor deaths by 225 days, 75% treated at this dose were tumor free and likely cured. Our conclusions were: (a) NM-SRCA human xenograft testing is excellent for rapid in vivo screening of promising treatment strategies to evaluate for efficacy at acceptable toxicity, but confirmation of true therapeutic impact should be sought by correlating initial growth inhibition with host survival; (b) enhanced survival seen only when CDDP/5FU are used together (versus either single agent) supports the value of pursuing histiotype-specific screening of potentially synergistic drug combinations; and (c) of clinical relevance, human transitional cell carcinoma is now identified as a histiotype in which a therapeutic, cooperative interaction between CDDP/5FU has been demonstrated in vivo.

Establishment and characterization of a new human prostatic carcinoma cell line (DuPro-1).

A new human prostate adenocarcinoma cell line (DuPro-1) has been established from the athymic nude mouse supported xenograft DU5683. This was accomplished by embedding dispersed xenograft cells in 0.1 by 5.0 cm. spaghetti-like strands of Basement Membrane MATRIGEL [BMM (Collaborative Research, Inc.)], a unique technique facilitating the transition to tissue culture. Now passed over 30 times, the cells display anchorage and serum concentration independent growth with a doubling time of 22 to 24 hours. Cells exhibit pronounced morphological differences when grown on BMM coated culture dishes, assuming a pseudoglandular configuration, in contrast to typical homogeneous monolayer growth on plastic culture dishes. Light and electron microscopy show cohesive sheets of anaplastic epithelial cells, consistent with prostate carcinoma. Karyotypic analysis revealed all human chromosomes, near tetraploidy, 10 to 12 markers, and 3 to 4 X chromosomes, without a Y chromosome. Cells injected s.c. or embedded in BMM and implanted in the subrenal capsule space are equally tumorigenic in male and female athymic mice, suggesting that DuPro-1 cells are hormonally insensitive. Embedding cells in BMM may be useful in developing other tissue culture cell lines from neoplasms difficult to initiate in vitro. DuPro-1 should provide a valuable means to study the biology, immunology, and chemosensitivity of human prostate cancer.

Characterization of a new model of human prostatic cancer: the multicellular tumor spheroid.

Multicellular tumor spheroids (MTS) provide a closer in vitro correlate to in vivo malignancy than do conventional monolayer cultures; while simulating many parameters of in vivo growth, MTS systems provide those perquisites (i.e., experimental control, economy, expediency) associated with in vitro evaluation of preclinical therapeutic strategies. For these reasons, we exploited the proclivity of the highly metastatic human prostatic carcinoma subline I-LN-PC3-IA to spontaneously assume a spheroid morphology under routine culture conditions. I-LN spheroids demonstrate salient features described in other spheroid systems and exhibit histologic characteristics of human prostate carcinoma. Cells encompassed in the I-LN spheroid format demonstrated functional divergence from their monolayer counterparts with respect to immunoreactivity for prostatic acid phosphatase, positional dependence of prostate-restricted p40 antigen expression, and chemotherapeutic drug response. This new in vitro-in vivo transition model of human prostatic carcinoma should provide a valuable in vitro context to expediently evaluate in vivo correlates of oncolytic protocols on a malignancy that remains refractive to therapy.

Rationale for immunotoxin therapy of metastatic prostate carcinoma formatted as a multi-stage delivery system.

A family of triple hybridomas secreting hybrid monoclonal antibodies has been developed in our laboratory. The hybrid monoclonal antibodies exhibit bimolecular specificity towards both antigenic determinants on the prostate carcinoma cell surface as well as toxin or toxic moieties (ricin A chain and pokeweed antiviral protein). These hybrid antibodies, when bound univalently to their cognate toxin, constitute the primary reagents responsible for selective in vitro prostate carcinoma cell kill; oncolytic impact is achieved by binding of the hybrid antibody-toxin complex (primary hybrid immunotoxin) to a prostate carcinoma cell surface-expressed antigen by the remaining univalent binding site of the hybrid antibody, allowing access of the toxin to the cytosol by internalization of the hybrid antibody-toxin complex. Internalization by endocytosis of a hybrid antibody-toxic subunit has been strikingly enhanced by the use of secondary monoclonal antibody reagents alone or in conjunction with other biomodifier reagents. For example, use of a second monoclonal antibody specific for ricin A chain to which ricin B chain (binding subunit) is chemically coupled results in selective and synergistic cell kill of targeted cancer cells. In vitro studies involving temporally staggered exposure of the cells to the individual components (primary hybrid antibody, toxin, and secondary antitoxin monoclonal antibody biomodifier) have been performed in a manner allowing maintenance of cytotoxic efficacy. It is concluded that sequential administration of these immunotherapeutic components, individually nontoxic, is a feasible strategy to develop an effective immunotherapeutic treatment of human prostate carcinoma.

Hyperglycemia is not a poor prognostic sign in head-injured children.

Hyperglycemia among adult trauma patients with head injuries is a recognized phenomenon which has been shown to be associated with a poor prognosis when it takes the form of nonketotic hyperosmolar hyperglycemia. It is not known to what extent a similar phenomenon occurs in pediatric patients, although it is known that a child's physiologic response to injury, particularly to neurologic injury, is often different than an adult's. This study was undertaken to characterize the hyperglycemic response of children with closed head injury compared to children with a non-head injury, and to measure the extent to which the presence of hyperglycemia is associated with a poor outcome among children with severe head injury. Records for all children ages 2 to 12 years admitted to a major regional trauma center with closed head injury over a 6-year period were compared to the records of a control group of children hospitalized for a non-head injury. The hyperglycemic response was more common among those with head trauma, occurring in 40% compared to 5% of the controls (p less than 0.001); however, the level of hyperglycemia could not be associated with any indicator of outcome. The entity known as nonketotic hyperosmolar hyperglycemia did not occur in any of these patients. Apparently, the hyperglycemia associated with closed head injury in children is transient, does not need to be treated with insulin therapy, and in contrast to what has been demonstrated in adult trauma patients, does not predict patient outcome.

In vitro synergism between hybrid immunotoxins and chemotherapeutic drugs: relevance to immunotherapy of prostate carcinoma.

Cultured prostate carcinoma cells incubated in the presence of a novel hybrid immunotoxin and ricin A chain exhibited synergy with the chemotherapeutic drugs vinblastine, methotrexate, and bleomycin. No cooperative effect was noted with adriamycin. Under conditions where individual components of immunotoxin or chemotherapeutic drug mixtures were nontoxic or minimally toxic the immunotoxin-drug mixture exhibited marked impact on 14C amino acid incorporation into prostate carcinoma cells. Analysis of drug-treated cells by flow cytometry indicated that cells exposed to vinblastine and bleomycin bound hybrid immunotoxin antibody to a greater extent than cells not exposed to these drugs. Adriamycin did not exhibit synergistic cytotoxicity with hybrid immunotoxin. Also, adriamycin did not enhance antibody binding as evaluated by flow cytometry. The fact that hybrid monoclonal antibody-ricin A chain (HIT-RAC) conjugates inhibited uptake of 14C amino acids 3 to 10-fold within 48 h of incubation with target cells and that this inhibition was further increased 2 to 3-fold in conjunction with three out of four chemotherapeutic drugs tested may be attributed to the unique cytotoxicity imposed by the hybrid immunotoxins. The RAC moiety is not chemically coupled to antibody but instead occupies one of the antigen-combining sites of the molecule. In this manner, RAC is closely juxtaposed to the cell membrane of the target cell and is anchored in this position via binding of the remaining antigen-combining site to p40 prostate restricted antigen.

Metastatic phenotype of human prostate tumor cells in athymic nude mice: alteration by exposure to ethyl methanesulfonate and "reversion" by 5-azacytidine.

The human prostate tumor subline 1-LN-PC-3-1A (1-LN) is reproducibly metastatic in adult athymic nude mice. Cells surviving a brief in vitro exposure to ethyl methanesulfonate (EMS) exhibited a profound decrease in capacity for experimental lung metastasis in nude mice. Thirty days after EMS treatment, 1 X 10(6) uncloned EMS-treated 1-LN cells (1-LN-EMS-10) were injected IV into groups of 6 to 8-week-old male athymic nude mice (BALB/cAnBOM). A median of 8.5 colonies/lung was observed among 20 1-LN-EMS-10-injected mice, which was significantly different from the median of 51 colonies/lung produced among 14 1-LN-injected mice (P = 0.0002). This altered phenotype remained stable during 150 days of continuous culture. However, the 1-LN-EMS-10 cells were tumorigenic in 10/10 nude mice injected SC. Single lung tumor colonies recovered from 1-LN-EMS-10-injected mice and reinjected IV into nude mice produced medians of 32-63 colonies/lung. The altered metastatic phenotype resulting from treatment of 1-LN with EMS was reversed by exposure to a noncytotoxic dose of 5-azacytidine, but unaffected by a second exposure to EMS. Collectively these data demonstrate that the metastatic phenotype of these human tumor cells in athymic nude mice can be heritably altered by in vitro exposure to EMS and 5-azacytidine. Analysis of the mechanisms underlying these phenotypic changes may provide insight into parts of the complex process of tumor cell evolution.

Evidence for a novel hybrid immunotoxin recognizing ricin A-chain by one antigen-combining site and a prostate-restricted antigen by the remaining antigen-combining site: potential for immunotherapy.

We have used cell fusion technology to produce second-generation hybridomas which secrete a population of murine monoclonal antibodies (MABs), a portion of which are bifunctional antibodies. The bifunctional hybrid MABs produced are capable of recognizing ricin A-chain (RAC) via one antigen-combining site and a prostate-restricted antigen via the other antigen-combining site of the IgG molecule. The second-generation hybridoma described in this report resulted from the fusion of spleen cells from mice immunized with purified RAC to hybridoma cells which secrete prostate-directed alpha Pro 15 monoclonal antibody. We have demonstrated that the MAB population secreted by the second-generation hybridoma can be physicochemically separated into distinct populations exhibiting differential binding to the cultured prostatic carcinoma cell surface and to RAC immobilized in a solid phase; specifically, a subset of the monoclonal antibody population which exhibits high binding to both prostatic carcinoma cells and to solid-phase RAC can be enriched by physicochemical methods. Binding of hybrid immunotoxin (HIT) MAB population to RAC can be quantitatively reduced by prior adsorption of the antibody population with prostate carcinoma cells; conversely, hybrid MAB binding to prostate carcinoma cells can be quantitatively reduced by prior adsorption with RAC. The biologic impact of the HIT has been evaluated by the ability of the HIT-RAC conjugate to reduce the uptake of 14C-amino acids into cellular protein. This effect is selective, since HIT-RAC conjugates do not exert an effect on labeled amino acid uptake by a cell line that does not express the target antigen recognized by the prostate-directed component of the hybrid MAB. Furthermore, depression of labeled amino acid uptake by prostate carcinoma cells exhibits a stoichiometric relationship with respect to both the concentration of HIT-MAB and to RAC to which the cells are exposed. The biologic impact of HIT-RAC conjugates on prostate carcinoma cells is enhanced markedly in the presence of lysosomotropic amines.

Factors influencing phenotypic diversity of human prostate carcinoma cells metastasizing in athymic nude mice.

Three related human prostate carcinoma cell lines, PC-3, 1-LN-PC-3-1A (1-LN), and 1-LN-PC-3-1A clone 4 (clone 4) were compared in terms of relative metastatic capacity in adult and young male nude mice. Only 1-LN produced lung metastases after intravenous injection into both 6- to 8-week-old and 4-week-old nude mice, as well as in mice injected intraperitoneally. The extent of the phenotypic diversity exhibited by these human prostate tumor lines was influenced by inherent dissemination ability, age of the host, and route of injection. These lines provide a useful system for the analysis of the biology of human tumor metastasis in nude mice.

1,10-Phenanthroline reversibility inhibits proliferation of two human prostate carcinoma cell lines (PC-3 and DU145).

The metal chelator 1,10-phenanthroline reversibly inhibits proliferation of two human prostate carcinoma cell lines, PC-3 and DU145. Inhibition is dose-dependent, and persists for several days after removal of the chelator. The ability to induce reversible quiescence in human prostate carcinoma cells in vitro may facilitate the study of their growth cycle biochemistry.

Characterization of prostate-tissue-directed monoclonal antibody, alpha-Pro 13.

The alpha-Pro 13-secreting hybridoma was produced by immunizing mice with an equal mixture of PC-3, DU145, and LNCaP established prostatic carcinoma cell lines. The specificity of alpha-Pro 13 monoclonal antibody was evaluated by the criteria of differential binding to cultured cells; differential binding to extracts of malignant prostate, nonmalignant prostate, and malignant and nonmalignant tissues of various histiotypes in solid phase radioimmunoassay; and by immunoperoxidase staining of primary surgical tissues of varied histiotypes. The data generated by multiple assay investigation indicate that alpha-Pro 13 exhibits preferential binding to the ductal epithelium of prostate tissue; immunoperoxidase evaluation indicates a considerable heterogeneity of staining of ductal epithelial cells. The most prevalent cross-reactivity of alpha-Pro 13 monoclonal antibody with non-prostate tissue occurs with blood vessel endothelium of restricted tissues. Electrophoretic analysis of immunoprecipitates from radioiodinated prostatic tumor extracts indicates that the molecule recognized by alpha-Pro 13 is of 120,000 dalton apparent nonreduced molecular weight. Under reducing conditions, the antigen (p40) consists of a major component of 40,000 dalton apparent MW and a minor component of 17,000 dalton MW. p40 has an isoelectric point of 3.5-4.5. The antigen is intrinsically stable on the PC-3 cell surface; its release into spent culture medium is negligible. p40 is also stable upon complexation with alpha-Pro 13 antibody in that it is not shed from the cell surface as an immune complex nor is it endocytosed to any extent as an immune complex.

The effect of the intake of a nitrosatable drug on the nitrosamine levels in human urine.

Experiments are described that demonstrate the presence of an N-nitroso compound in non-infected and infected human urine. An increase in the concentration of N-nitrosodimethylamine in the urine was usually associated with the administration of oxytetracycline.

An examination of human blood for the presence of volatile nitrosamines.

Human blood was examined for the presence of volatile nitrosamines. Nitrosamines were detected by chemiluminescence and mass spectrometry after separation from blood by distillation and solvent extraction. N-nitrosodimethylamine was detected in all but one of 51 blood samples taken from 23 different people, at concentrations from the detection limit (0.1 microgram/litre) to 1.4 microgram/litre with a mean concentration of 0.5 microgram/litre. N-Nitrosodiethylamine was detected in 11 samples, the detection limit being 0.1 microgram/litre. No other volatile nitrosamines were detected. After a test meal of bacon, spinach, bread and beer, the concentration of N-nitrosodimethylamine increased. There was no appreciable difference between the nitrosamine concentrations in the blood of laboratory workers and in the blood of other people. Salivary nitrite concentrations measured semi-quantitatively concurrently with blood sampling varied considerably but showed no apparent correlation with blood nitrosamine levels. Measurements in rabbits given a continuous infusion of N-nitrosodimethylamine gave a clearance rate approximately equal to the blood flow through the liver and a volume of distribution of 1.2 litre/kg body weight. By applying these results to man, the body burden after the meal was calculated as 40-50 microgram. This is substantially higher than the estimated weekly intake of volatile nitrosamines from food.