Isotope-filtered nD NMR spectroscopy of complex mixtures to unravel the molecular structures of phenolic compounds in tagged soil organic matter.

Unravelling structures of molecules contained in complex, chromatographically inseparable mixtures is a challenging task. Due to the number of overlapping resonances in NMR spectra of these mixtures, unambiguous chemical shift correlations attributable to individual molecules cannot be achieved and thus their structure determination is elusive by this technique. Placing a tag carrying an NMR active nucleus onto a subset of molecules enables (i) to eliminate signals from the non-tagged molecules, and (ii) to obtain a set of correlated chemical shifts and coupling constants belonging to a single molecular type. This approach provides an opportunity for structure determination without the need for compound separation. Focusing on the most abundant functional groups of natural organic matter molecules, the carboxyl and hydroxyl groups were converted into esters and ethers, respectively by introducing (13)CH3O groups. A set of (13)C-filtered nD NMR experiments was designed yielding structures/structural motives of tagged molecules. The relative sensitivity of these experiments was compared and a step-by-step guide how to use these experiments to analyse the structures of methylated phenolics is provided. The methods are illustrated using an operational fraction of soil organic matter, fulvic acid isolated from a Scottish peat bog. Analysis of 33 structures identified in this sample revealed a correlation between the position of the methoxy cross-peaks in the (1)H, (13)C HSQC spectra and the compound type. This information enables profiling of phenolic compounds in natural organic matter without the need to acquire a full set of experiments described here or access to high field cryoprobe NMR spectrometers.

Investigation of extraction and clean-up procedures used in the quantification and stable isotopic characterisation of PAHs in contaminated urban soils.

Four different extraction methods, soxhlet, soxtherm, sonication and accelerated solvent extraction (ASE), were used to isolate the 16 priority pollutant PAHs from a certified reference soil (LGC 6140) and from a contaminated soil (BG CLR 17). Based on SIM-GC-MS results, all methods were found to give accurate and highly reproducible concentration data. There was, however, significant between-method and sometimes within-method variability in the stable carbon isotope signatures obtained for individual PAHs from the contaminated soil (BG CLR 17) using GC-C-IRMS. When two clean-up procedures, silica/dichloromethane and alumina/hexane/toluene, were used to remove co-extracted material, however, it was found that ASE gave the more consistent and reproducible stable carbon isotope data. These findings are likely to be of importance for the characterisation of natural and anthropogenic organic matter and, in particular, in source identification and apportionment studies.

The implications of integrated assessment and modelling studies for the future remediation of chromite ore processing residue disposal sites.

Chromite ore processing residue (COPR) waste from a former chromium chemical works (1830-1968) is still contaminating groundwater in Glasgow, Scotland, with carcinogenic hexavalent chromium, Cr(VI). An integrated analytical, experimental and modelling approach has identified and accounted for mineral phases and processes responsible for the retention and release of Cr(VI) under prevailing field conditions. Both the nature of mineral phase retention and the buffered high pH of the sites, however, militate against direct remediative treatment of the source material, for example by the application of generic methods (e.g. FeSO4) that have been successfully employed elsewhere for the reduction of Cr(VI) to Cr(III) in other matrices. The interception and treatment of groundwater to remove Cr(VI) and the capping of sites to reduce human exposure to airborne Cr(VI)-contaminated dust may well be more realistic and effective, at least in the short to medium term.

Characterization of Fe-humic complexes in an Fe-enriched biosolid by-product of water treatment.

The fertilizing potential of Fe-enriched biosolids has been attributed to Fe associations with humic substances contained therein. In this study, alkaline and near-neutral aqueous extractions of humic substances from an Fe-enriched biosolid were followed by gel chromatographic fractionation and characterization (CHNS elemental analysis; UV/visible and FTIR spectroscopy; FAAS analysis). The alkaline bulk humic extract had a strong fulvic character and Fe was predominantly associated with the higher molecular weight ( approximately 50000 Da) molecules, possibly including organic-coated Fe oxides from which Fe may be released more slowly. Under both near-neutral and alkaline conditions, associations with lower molecular weight humic molecules were also observed, indicative of the presence of Fe in more readily available forms. Thus the biosolid appears to have good short- and long-term fertilizing potential, particularly for alkaline, Fe-deficient soils.

A phase I trial of monoclonal antibody M195 in acute myelogenous leukemia: specific bone marrow targeting and internalization of radionuclide.

Ten patients with myeloid leukemias were treated in a phase I trial with escalating doses of mouse monoclonal antibody (mAb) M195, reactive with CD33, a glycoprotein found on myeloid leukemia blasts and early hematopoietic progenitor cells but not on normal stem cells. M195 was trace-labeled with iodine-131 (131I) to allow detailed pharmacokinetic and dosimetric studies by serial sampling of blood and bone marrow and whole-body gamma-camera imaging. Total doses up to 76 mg were administered safely without immediate adverse effects. Absorption of M195 onto targets in vivo was demonstrated by biopsy, pharmacology, flow cytometry, and imaging; saturation of available sites occurred at doses greater than or equal to 5 mg/m2. The entire bone marrow was specifically and clearly imaged beginning within hours after injection; optimal imaging occurred at the lowest dose. Bone marrow biopsies demonstrated significant dose-related uptake of M195 as early as 1 hour after infusion in all patients, with the majority of the dose found in the marrow. Tumor regressions were not observed. An estimated 0.33 to 1.0 rad/mCi 131I was delivered to the whole body, 1.1 to 6.1 rad/mCi was delivered to the plasma, and up to 34 rad/mCi was delivered to the red marrow compartment. 131I-M195 was rapidly modulated, with a majority of the bound immunoglobulin G (IgG) being internalized into target cells in vivo. These data indicate that whole bone marrow ablative doses of 131I-M195 can be expected. The rapid, specific, and quantitative delivery to the bone marrow and the efficient internalization of M195 into target cells in vivo also suggest that the delivery of other isotopes such as auger or alpha emitters, toxins, or other biologically important molecules into either leukemia cells or normal hematopoietic progenitor cells may be feasible.

Dose-escalation trial of M195 labeled with iodine 131 for cytoreduction and marrow ablation in relapsed or refractory myeloid leukemias.

PURPOSE: Mouse monoclonal antibody (mAb) M195 (anti-CD33) is reactive with most myeloid leukemia cells, monocytes, and hematopoietic progenitors, but not with other hematopoietic cells or stem cells nor with nonhematopoietic human tissues. A therapeutic dose-escalation study of M195 labeled with iodine 131 was conducted in patients with relapsed or refractory myeloid leukemias. METHODS: Twenty-four patients (16 relapsed or refractory acute myeloid leukemias, five blastic myelodysplastic syndromes [MDS], two chemotherapy-related secondary leukemias, and one blastic chronic myelogenous leukemia [CML]), including seven who had failed to respond to prior bone marrow transplantation (BMT), received from 50 mCi/m2 to 210 mCi/m2 of 131I-M195 in divided doses. RESULTS: In 22 patients, whole-body gamma-imaging demonstrated marked uptake of antibody into all areas of bone marrow. Twenty-three patients (96%) demonstrated decreases in peripheral-blood cell counts, with decreased percentage of bone marrow blasts seen in 83% of cases. Eighty-nine percent of bone marrow biopsies examined quantitatively demonstrated substantial decreases in the number of blasts, with greater than 99% of blasts killed in some patients. The two cases that failed to demonstrate leukemic cytoreduction occurred in the first two dose levels. For 131I doses of 135 mCi/m2 or greater, pancytopenia was profound and lasted for at least 12 days. Eight patients had sufficient marrow cytoreduction to proceed to BMT. Three of these achieved marrow remission, one of 6+, and one of 9 months' duration. Two patients in blastic phase temporarily reverted to their original myelodysplastic states. Thirty-seven percent of assessable patients developed human anti-mouse antibody (HAMA). In two patients with HAMA who were re-treated, plasma 131I-M195 levels could not be maintained and no therapeutic effect resulted. Significant nonhematologic toxicity (hepatic) was seen in one patient and the maximum-tolerated dose (MTD) was not reached. CONCLUSION: These data suggest that safe leukemic cytoreduction can be achieved with 131I-M195 even in multiply relapsed or chemotherapy-refractory leukemias. This agent may be useful as part of a preparative regimen for BMT.

Rhenium-186-labeled hydroxyethylidene diphosphonate dosimetry and dosing guidelines for the palliation of skeletal metastases from androgen-independent prostate cancer.

Rhenium-186 (tin)-labeled hydroxyethylidene diphosphonate (186Re-labeled HEDP) was evaluated in 27 men with progressive androgen-independent prostate cancer and bone metastases. Administered activities ranged from 1251 to 4336 MBq (33.8-117.2 mCi). The primary objectives were to assess tumor targeting, normal organ dosimetry, and safety. Antitumor effects were assessed by posttherapy changes in prostate-specific antigen and, when present, palliation of pain. Whole-body kinetics, blood and kidney clearance, skeletal dose, marrow dose, and urinary excretion of the isotope were assessed. Targeting of skeletal disease was observed over the period of quantification (4-168 h). Radiation doses to whole body, bladder, and kidney were well tolerated. The dose-limiting toxicity was myelosuppression (grade III) at 4107 MBq (111 mCi) and grade II at 296 MBq (80 mCi). Probe clearance (whole body) and urinary excretion measurements were highly correlated. Of the six patients treated at the highest dosage schedules (three at 1510 MBq/m2 and three at 1665 MBq/m2), three showed a posttherapy decline in prostate-specific antigen of 50% or more. The declines were not sustained. The determination of total activity retained at 24 h, as well as an estimate of marrow dose, correlated with the amount of myelosuppression observed. These results suggest that a single 24-h measurement of retained activity would allow individualized dosing and an improved therapeutic index relative to fixed dosing schema. Repetitive dosing is required to increase palliation.

Phase I/II radioimmunotherapy trial with iodine-131-labeled monoclonal antibody G250 in metastatic renal cell carcinoma.

This Phase I/II radioimmunotherapy study was carried out to determine the maximum tolerated dose (MTD) and therapeutic potential of 131I-G250. Thirty-three patients with measurable metastatic renal cell carcinoma were treated. Groups of at least three patients received escalating amounts of 1311I (30, 45, 60, 75, and 90 mCi/m2) labeled to 10 mg of mouse monoclonal antibody G250, administered as a single i.v. infusion. Fifteen patients were studied at the MTD of activity. No patient had received prior significant radiotherapy; one had received prior G250. Whole-body scintigrams and single-photon emission computed tomography images were obtained in all patients. There was targeting of radioactivity to all known tumor sites that were > or =2 cm. Reversible liver function test abnormalities were observed in the majority of patients (27 of 33 patients). There was no correlation between the amount of 131I administered or hepatic absorbed radiation dose (median, 0.073 Gy/mCi) and the extent or nature of hepatic toxicity. Two of the first six patients at 90 mCi/m2 had grade > or =3 thrombocytopenia; the MTD was determined to be 90 mCi/m2 131I. Hematological toxicity was correlated with whole-body absorbed radiation dose. All patients developed human antimouse antibodies within 4 weeks posttherapy; retreatment was, therefore, not possible. Seventeen of 33 evaluable patients had stable disease. There were no major responses. On the basis of external imaging, 131I-labeled mouse monoclonal antibody G250 showed excellent localization to all tumors that were > or =2 cm. Seventeen of 33 patients had stable disease, with tumor shrinkage observed in two patients. Antibody immunogenicity restricted therapy to a single infusion. Studies with a nonimmunogenic G250 antibody are warranted.

Isotopic characterisation of the historical lead deposition record at Glensaugh, an organic-rich, upland catchment in rural N.E. Scotland.

As part of a wider investigation of the biogeochemistry and fate of Pb deposited from the atmosphere at Glensaugh, a rural upland catchment in N.E. Scotland, the concentration and isotopic composition of Pb were determined in four thinly sectioned monolith cores (25 cm) of peat collected at altitudes of 426--434 m from different faces of Thorter Hill and in a series of 21 10-cm unsectioned cores of peat and organic-rich soil along a transect from near the top (434 m) to the bottom (224 m) of the catchment. Depth profiles of Pb concentration and (206)Pb/(207)Pb ratio were similar for the longer cores. Subsurface Pb maxima (238--489 mg kg(-1)) typically occurred below (206)Pb/(207)Pb minima (1.123-1.134). One core was (210)Pb-dated and had a fairly constant (206)Pb/(207)Pb value of 1.170 from mid-19th century to ca. 1930, followed by a decline (attributable to the increasing influence of Australian Pb of much lower (206)Pb/(207)Pb ratio) to 1.134 by the early 1990s, and then a rapid increase to 1.160 by 2002, after the phased withdrawal of leaded petrol. The fluxes of Pb increased from 15 mg m(-2) year(-1) in the late 19th century to a peak of 60 mg m(-2) year(-1) ca. 1960, before declining steadily to 3.6 mg m(-2) year(-1) by the beginning of the 21st century. Some 40% of the anthropogenic Pb in the core had been deposited prior to 1900. The mean anthropogenic Pb inventory of the four longer cores was 7.4+/-1.5 g m(-2), of which approximately 70% occurred in the top 10 cm, in good agreement with the inventories of the shorter cores collected above 400 m. These inventories are higher than those of the industrial central belt of Scotland, probably because of enhanced deposition at altitude. This is consistent with the derived average (210)Pb flux of 198+/-11 Bq m(-2) year(-1), which is twice that of typical UK (210)Pb deposition and the rainfall for the site. The past deposition of Pb at Glensaugh, including that from sources (e.g., smelting, coal combustion) other than leaded petrol, has clearly been considerable. Even since the introduction of leaded petrol ca. 1930, car-exhaust emissions may have accounted for no more than 35% of the Pb deposited.

Krypton-79m: a new radionuclide for applications in nuclear medicine.

Krypton-79m emits 130-keV gamma rays in 27 +/- 1% of its disintegrations and decays with a half-life of 50 +/- 3 sec. It is generated readily by bombarding nearly saturated aqueous solutions of bromide salts, or bromoform, with 14-MeV protons. The 79mKr is swept out continuously as it is produced by bubbling helium upward through the liquids. Up to 200 mCi per I are obtained of the resulting mixture of gases. The 79mKr + helium is mixed with about five volumes of air and then driven continuously through a small-bore tube to an Anger scintillation camera located approximately 200 yards away. The rate of flow is adjusted so that the amounts of 13-sec 81mKr and of 35-hr 79Kr are inconsequential at the time and point of use. When the gases are inhaled, good images of the lungs are obtained with an Anger scintillation camera. The trachea and bronchi commonly are revealed also.

Xenon-127m: a new radionuclide for applications in nuclear medicine.

Xenon-127m (127mXe) emits two gamma rays in cascade, with half-life of 69.2 +/- 0.9 sec. The first has the energy of 172.5 keV, and is emitted from the nucleus in 38% of the decays. The second gamma ray has the energy of 124.8 keV and is emitted from the nucleus in 69% of the disintegrations. Together they furnish 107 easily collimated gamma rays per 100 decays. Xenon-127m is generated readily by bombarding nearly saturated aqueous solutions of sodium or potassium iodide with 14-MeV protons. The 127mXe is swept out continuously, as it is produced, by bubbling helium upward through the solutions. Up to approximately 100 mci/l are obtained from the resulting mixture of gases. The 127mXe + helium is admixed with about five volumes of air (or oxygen) and then driven continuously to a scintillation camera located approximately 200 yd distant. When the mixture of gases is inhaled, high quality images of the lungs are obtained by means of an Anger scintillation camera.

Modeling and dosimetry of monoclonal antibody M195 (anti-CD33) in acute myelogenous leukemia.

Individual patient response to radioimmunotherapy is influenced by each patient's tumor burden, antibody clearance kinetics and the antibody-antigen interaction. In hematologic malignancies, wherein antibody access to tumor-cell associated antigen is rapid, mathematical modeling may provide a quantitative basis for assessing the impact of patient variability on a particular therapeutic protocol. Compartmental modeling analysis of antibody pharmacokinetics from a Phase I trial of 131I-labeled monoclonal antibody, M195 (anti-CD33), was used to estimate tumor burden in cases of acute myelogenous leukemia and the absorbed dose in liver, spleen and red marrow. The suitability of a nonlinear, two-compartment model for simulating M195 distribution in leukemia patients was evaluated by comparing model predictions with patient measurements. The results demonstrate that for directly accessible, hematologically distributed tumor cells, a two-compartment model fits observed patient biodistribution data and may provide information regarding both total tumor burden and tumor burden in the liver, spleen and red marrow. The model also provides biodistribution information for absorbed dose calculations to tissues that are not directly sampled. Such information is important in determining the optimum therapeutic dose of radiolabeled antibody for a given patient.

Image registration of SPECT and CT images using an external fiduciary band and three-dimensional surface fitting in metastatic thyroid cancer.

Image registration of 131I SPECT with CT scans was performed in a patient with metastatic thyroid carcinoma using an external fiduciary band and a three-dimensional surface-fitting algorithim. Areas of metastatic disease taking up 131I were accurately localized to the liver, lungs and vertebral bodies; providing information that could not be obtained by planar or SPECT images alone. Based on these findings, further invasive diagnostic procedures were not performed, therefore considerably altering management in this patient. This approach to image registration has immediate clinical utility in the registration and interpretation of SPECT studies with corresponding CT or MRI scans.

Clinical validation of SPECT and CT/MRI image registration in radiolabeled monoclonal antibody studies of colorectal carcinoma.

UNLABELLED: Registration methods combine the anatomic localizing ability of CT or MRI with SPECT images of radiolabeled monoclonal antibodies (Mabs), allowing the accurate staging of patients prior to surgery or following treatment. METHODS: Twenty-four patients (15 males and 9 females, mean age 55 yr, range 29-70 yr) were studied with this technique. Ten patients had suspected colorectal cancer recurrence and were infused with 10 mCi of 131I-CC49 prior to staging laparotomy. Fourteen patients treated in a Phase I radioimmunotherapy study with 131I-CC49 were also studied. All patients underwent SPECT imaging of the abdomen and pelvis 5-7 days following infusion of Mab. RESULTS: Phantom studies demonstrated a 3.6-mm surface fitting mean accuracy of datasets for the liver and 1.8 mm for an intrahepatic tumor. In the presurgical group, SPECT and CT/MRI registration allowed more accurate identification of uptake abnormal sites. Areas of metastatic disease > 1 cm confirmed at surgery were found in six of nine patients with liver lesions and in two patients with extrahepatic (including one patient with pelvic) disease. In patients imaged following radioimmunotherapy, all lesions > 1.5 cm seen on CT/MRI were identified, and activity distribution in tumor and normal tissue could be more accurately assessed. CONCLUSIONS: Routine registration of SPECT and CT/MRI images is feasible and allows more accurate anatomic assessment of sites of abnormal uptake in radiolabeled Mab studies.

Three-dimensional dosimetry for radioimmunotherapy treatment planning.

Absorbed-dose calculations for radioimmunotherapy are generally based on tracer imaging studies of the labeled antibody. Such calculations yield estimates of the average dose to normal and target tissues assuming idealized geometries for both the radioactivity source volume and the target volume. This work describes a methodology that integrates functional information obtained from SPECT or PET with anatomical information from CT or MRI. These imaging modalities are used to define the actual shape and position of the radioactivity source volume relative to the patient's anatomy. This information is then used to calculate the spatially varying absorbed dose, depicted in "colorwash" superimposed on the anatomical imaging study. By accounting for individual uptake characteristics of a particular tumor and/or normal tissue volume and superimposing resulting absorbed-dose distribution over patient anatomy, this approach provides a patient-specific assessment of the target-to-surrounding normal tissue absorbed-dose ratio. Such information is particularly important in a treatment planning approach to radioimmunotherapy, wherein a therapeutic administration of antibody is preceded by a tracer imaging study to assess therapeutic benefit.

Bone marrow dosimetry: regional variability of marrow-localizing antibody.

UNLABELLED: In radiolabeled antibody therapy, imaging and biopsy-based methods are used to estimate marrow activity concentration when the administered antibody localizes to the marrow. Absorbed dose estimates obtained using such measurements may be subject to large variability due to the potential for regional differences in marrow activity concentration. This variability was examined in ten patients with leukemia after administration of 131I-labeled HuM195 antibody. METHODS: Regions of interest were drawn around the head and neck of the humerus and femur (both sides) and around lumbar vertebra 3 (L3) and 4 (L4) on a series of planar images collected at multiple times postadministration of the antibody. A single exponential fit to each attenuation-corrected, time-activity curve was obtained to estimate clearance half-life and the back-extrapolated percent injected activity. RESULTS: The activity concentration in the femoral head and neck (mean and s.d. = 0.04 +/- 0.02 %ID/g) was not significantly different than that measured in L3 and L4 (0.06 +/- 0.02% ID/g) but was not significantly lower than the concentrations measured in the humeral head and neck regions (0.07 +/- 0.03 %ID/g, p < 0.05). Although half-life estimates differing by more than a factor of 2 were observed in half-life between regions overall. S-factors were used for individual marrow regions to determine the mean absorbed dose to marrow in the femoral and humeral heads and the lumbar vertebrae (L3 and L4) which were 0.66 +/- 0.3, 1.0 +/- 0.3 and 2.2 +/- 0.5 mGy/MBq (2.4, 3.8 and 8.3 rad/mCi), respectively. CONCLUSION: A single value is generally quoted for the absorbed dose delivered to the red marrow following marrow-localizing radiolabeled antibody administration. These results suggest that the regional marrow dose may differ significantly from the mean.

PET scanning of iodine-124-3F9 as an approach to tumor dosimetry during treatment planning for radioimmunotherapy in a child with neuroblastoma.

A patient with advanced neuroblastoma who had failed chemotherapy presented with a large abdominal mass and virtually total bone marrow replacement by tumor on repeated marrow biopsies. She was considered a candidate for a Phase I 131I-3F8 radioimmunotherapy trial, (MSKCC 89-141A). As a potential aid to treatment planning, a test dose of 124I-3F8 was injected and the patient was imaged over the 72 hr postinjection using two BGO based PET scanners of different designs. Time activity curves were obtained, and the cumulated activity concentration of radiolabeled 3F8 in tumor was determined. Based on MIRD, an estimated radiation absorbed dose for 131I-3F8 was 7.55 rad/mCi, in the most antibody avid lesions. Because of low uptake and unfavorable dosimetry in some bulky tumor sites, it was decided not to treat the patient with radiolabeled antibody. Positron emission tomography of 124I-labeled antibodies can be used to measure cumulated activity or residence time in tumor for more accurate estimates of radiation absorbed tumor dose from radioiodinated antibodies and can help guide management decisions in patients who are candidates for radioimmunotherapy.

Quantitative imaging of iodine-124 with PET.

UNLABELLED: PET is potentially very useful for the accurate in vivo quantitation of time-varying biological distributions of radiolabeled antibodies over several days. The short half-lives of most commonly used positron-emitting nuclides make them unsuitable for this purpose. Iodine-124 is a positron emitter with a half-life of 4.2 days and appropriate chemical properties. It has not been widely used because of a complex decay scheme including several high energy gamma rays. However, measurements made under realistic conditions on several different PET scanners have shown that satisfactory imaging and quantitation can be achieved. METHODS: Whole-body and head-optimized scanners with different detectors (discrete BGO, block BGO and BaF2 time-of-flight), different septa and different correction schemes were used. Measurements of resolution, quantitative linearity and the ability to quantitatively image spheres of different sizes and activities in different background activities were made using phantoms. RESULTS: Compared with conventional PET nuclides, resolution and quantitation were only slightly degraded. Sphere detectability was also only slightly worse if imaging time was increased to compensate for the lower positron abundance. CONCLUSION: Quantitative imaging with 124I appears to be possible under realistic conditions with various PET scanners.

Thallium-201 myocardial scintigraphy: improved sensitivity, specificity and predictive accuracy by application of a statistical image analysis algorithm.

Exercise and 3 hour delayed redistribution thallium-201 myocardial perfusion imaging was performed in 107 patients including 87 patients with documented coronary artery disease and 20 patients with normal coronary arteriograms. A computer algorithm for statistical analysis and redisplay of the analog scintillation data was developed to augment the visual analysis of the standard analog image. The basic algorithm identified the myocardial area (pixel) with the most absolute counts and developed an appropriate standard deviation range from a table. All pixels in the area of the myocardium containing absolute counts below three standard deviation ranges (or approximately 6 standard deviations) were deleted from the final image. The remaining pixels were redisplayed in a digitized bimodal format and the image photographed. Two experienced observers compared visual analysis of the analog image alone and the analog image in conjunction with the computer-analyzed image for sensitivity, specificity and predictive accuracy in the detection of perfusion defects in patients with coronary artery disease and in normal subjects. Sensitivity in patients with coronary artery disease and in normal subjects. Sensitivity in patients with coronary artery disease for the analog scintillation image alone was 79 percent (69 of 87) and with computer analysis 95 percent (83 of 87). Specificity in the patients with no coronary disease was 100 percent (20 of 20) for both techniques. The predictive accuracy of the test was 83 percent (89 of 107) for the analog image alone and 96 percent (103 of 107) for the two images combined. It is concluded that use of a computer statistical analysis algorithm of thallium-201 analog myocardial perfusion images improves the accuracy of detection of perfusion defects in patients with coronary artery disease.

Estradiol and progesterone production by cultured granulosa cells cryopreserved from in vitro fertilization patients.

Gonadotropin-stimulated steroidogenesis was studied in cultured human granulosa-lutein cells obtained from patients undergoing procedures for in vitro fertilization. The impact of cryopreservation on cell function in vitro was studied. Granulosa cells obtained from in vitro fertilization patients were cultured in serum-supplemented medium or cryopreserved at -135 degrees C for 2-22 months. Fresh (unfrozen) cells (10(5) produced estradiol at a rate of 1320 pmol/l (over 72 h) and progesterone at about 2500 nmol/l. Estradiol production by either fresh or cryopreserved granulosa cells in culture was unaffected by physiological concentrations of follicle-stimulating hormone (7 IU/l). Adding testosterone (10(-7) mol/l) to the medium increased estradiol secretion approximately sixfold. In contrast, progesterone production was not affected by follicle-stimulating hormone or testosterone. No significant differences were observed in cultures of cryopreserved granulosa cells compared to cultures of unfrozen cells with respect to estradiol secretion, the effects of follicle-stimulating hormone or testosterone on estradiol secretion, or progesterone production. Progesterone production by fresh and cryopreserved cells was stimulated by human chorionic gonadotropin. These data indicate that cryopreservation offers the potential to facilitate prospective studies utilizing large numbers of human granulosa-lutein cells in culture.