The influence of chemotherapy on response of patients with hematologic malignancies to influenza vaccine. Bivalent influenza vaccine (containing antigens A/Victoria and A/New Jersey) was administered to 52 patients with hematologic malignancies, and pre- and postvaccination antibody titers to both antigens were determined by hemagglutination-inhibition. In comparison to healthy controls, mean antibody titer elevations were lower for both antigens in all disease groups, being significant (p less than 0.05) for A/Victoria in patients with non-Hodgkin's lymphoma, acute leukemia and lymphoproliferative diseases, and for A/New Jersey in patients with Hodgkin's and non-Hodgkin's lymphomas. In comparison to controls, significant depression of antibody response to both antigens was seen in patients on combination chemotherapy (p less than 0.0005), to a lesser extent in patients on daily single alkylating agent chemotherapy (p less than 0.05), while untreated patients did not differ significantly. Lymphopenia and depressed immunoglobulin levels were associated with a higher failure rate in eliciting "protective" greater than or equal to fourfold antibody titer increases. The findings suggest that patients with hematologic malignancies who are receiving chemotherapy at the time of vaccination are unlikely to attain seroconversion to protective antibody levels with influenza vaccine.


Meningiomas with conspicuous plasma cell-lymphocytic components: a report of five cases. Five meningeal neoplasms grossly resembling meningiomas but histologically containing meningothelial cells together with abundant plasma cells and lymphocytes are reported. These masses are regarded as meningiomas with extensive plasma cell--lymphocytic infiltrates.


Malignant melanoma producing serotonin.

Malignant melanoma producing serotonin. A case of malignant melanoma, the tumor of which seems to produce serotonin, is presented. Neither hyperserotoninemia nor carcinoid syndrome was observed in the clinical course. An autopsied sample of the metastatic lung tumor contained a significantly higher concentration (1.35 microgram/g wet weight tissue) of serotonin than that of normal lung (below 0.5 microgram wet wt.). Tryptophan hydroxylase activity, the key enzyme of the biosynthesis of serotonin, was substantial in the same tumor sample when assayed by a sensitive radioisotopic method, supporting the productivity of the amine of the tumor. The ability of melanoma to produce serotonin could be attributed to its neural crest origin.


The candida precipitin test in an immunosuppressed population. Ninety-one immunosuppressed, high-risk patients suspected of having invasive candidiasis were tested during their septic episode for the presence of candida precipitin (CP) antibody using the counterimmunoelectrophoresis (CIE) method. In 12 patients, systemic candidiasis was confirmed but only 6 of these (50%) had candida precipitins. In 14 "questionable" cases who died without a definitive diagnosis, 8 (57%) had precipitin antibody. Of the 65 patients who did not have serious candida infection, 20 (31%) had candida precipitins. In this population we found the presence of candida precipitins to be neither sensitive (50%) nor specific (69%) for the diagnosis of invasive candidiasis.


Stromomyoma of the uterus.

Stromomyoma of the uterus. We present a solitary uterine tumor that occurred in a 28-year-old woman who complained of vaginal bleeding. Grossly, the tumor was well circumscribed and composed of lobulated tissue that varied from yellow, tan and soft, to white and rubbery. At the light microscopic level, areas identical to those described and interpreted as sex-cord differentiation in a group of closely similar uterine tumors were found and obtained for electron microscopic study. Ultrastructurally, there were two basic types of cells, neither of which can be interpreted as sex-cord derivatives. Cells that closely resembled the predominant components of Clement and Scully's group I tumors were similar to the normal endometrial stromal cells. Cells that formed plexiform cords demonstrated features diagnostic of smooth muscle cells. Cellular contents in cells comprising the tubular structures resembling that interpreted as indistinguishable from that of a Sertoli-cell tumor (tubular androblastoma) were probably smooth muscle cells. We propose the term "Stromomyoma" to designate this peculiar uterine tumor. The similarity between our tumor and those reported in the literature raises the possibility that "uterine tumor resembling" ovarian sex-cord tumors" are actually stromomyomas.


Gross and histological types of early gastric carcinomas in relation to the acid-secreting area. The relation of gastric acid-secreting function to the gross and histological types and locations of carcinomas was examined in 53 patients with early gastric carcinomas. Studies were made by the endoscopic Congo red test developed in this clinic. Results indicated a close correlation between the gross and histological types of early gastric carcinoma and the extent of the acid-secreting area. In general, when the acid-secreting area was large, the carcinomas were ulcerated and histologically undifferentiated and they were chiefly located in nonacid-secreting areas adjacent to acid-secreting areas, or sometimes they were surrounded by acid-secreting areas. On the contrary, when there was little or no acid-secreting area the carcinomas were polyploid and histologically differentiated, and they were located in nonacid-secreting areas far from acid-secreting areas.


Lymphomatoid granulomatosis: a clinicopathologic study of 152 cases. A retrospective study of 152 cases of lymphomatoid granulomatosis (LYG) was undertaken to expand current knowledge about clinical manifestations and therapy, and to identify prognostic indicators in this disease. The major clinical and radiographic features and extra-pulmonary sites of involvement noted in previous studies were essentially confirmed in our study. Adverse prognostic factors included neurologic manifestations, and large numbers of atypical lymphoreticular cells within the pulmonary infiltrate. Unilateral chest lesions and large numbers of small lymphocytes and histiocytes within the infiltrate were associated with a better prognosis.  Malignant lymphoma involving lymph nodes developed in 12% of patients. Almost two-thirds of patients have died and the median survival was only 14 months. No particular mode of therapy was found to be satisfactory for LYG, although there was some suggestion that corticosteroids may be useful. Randomized prospective studies are necessary for definitive recommendations regarding therapy.


Endodermal variant of mature cystic teratoma of the ovary: report of a case. The clinical and pathological features of a case of mature monodermal cystic teratoma of the ovary in a five-year-old girl are reported. The tumor was composed purely of respiratory epithelium. This appears to be the first report of such a case.


Childhood cancer: nonmedical costs of the illness. The financial burden of cancer treatment is a major source of anxiety for the families of pediatric cancer patients. Parents of these patients report that nonmedical, out-of-pocket expenditures are the most troublesome because, unlike medical bills, nonmedical costs must be paid immediately and are rarely reimbursed. Data on nonmedical expenditures (transportation, food, lodging, clothing, family care, and miscellaneous) were collected from 70 patients' families for one-week periods at three-month intervals. Multiple regression analysis was carried out to ascertain the factors influencing costs. Factors contributing to nonmedical costs included level of care, the patient's Performance Status, distance from the treatment center, and family size. Thirty-two families reported loss of pay when one or both parents accompanied the child to the hospital. When this loss was combined with the nonmedical costs, the results indicated a serious strain on the family budget. For half the families surveyed, the total expenses plus loss of pay amounted to more than 25% of the weekly family income.


Combination chemotherapy in recurrent medulloblastoma. Five children with recurrent medulloblastomas were treated with Vincristine, BCNU, Methotrexate and Dexamethasone. All five patients responded to therapy. Two of the patients are alive and well 30 and 48 months later; one died of disease 19 months after first recurrence; one developed a further recurrence at 18 months but responded to additional chemotherapy and is alive; and the fifth patient developed a second recurrence after 28 months but has responded to repeat chemotherapy and radiotherapy and is now asymptomatic. These results support the concept that medulloblastomas are sensitive to chemotherapy and suggest that chemotherapy should be considered in cases of recurrent medulloblastomas. In addition, the importance of trials now in progress which employ both adjuvant chemotherapy and radiotherapy in newly diagnosed medulloblastomas is apparent.


Combination chemotherapy for advanced breast cancer utilizing vincristine, adriamycin, and cyclophosphamide (VAC). Thirty-two patients with advanced breast cancer were treated with a combination of vincristine (1 mg/m2, not exceeding 2 mg, administered intravenously on day 1), adriamycin (40 mg/m2 administered intravenously on day 1), and cyclophosphamide (200 mg/m2 administered orally for 4 days on days 3-6). Courses were repeated at 21-28-day intervals. The mean age of the patients was 57 years (range, 30-79 years) and 18 patients were postmenopausal. None of the patients had received prior chemotherapy although 15 had prior endocrine treatment. Objective response was observed in 23 (72%) of the 32 patients and 9 responses (28%) were complete. The median remission duration was estimated to be 22 months. Median survival has not been reached but exceeds 24 months with a median time of follow-up of 17 months. Toxicity was acceptable and included mild nausea, vomiting, alopecia, and paresthesias. Only one instance of serious infection and no instances of bleeding were observed. The addition of vincristine to combination chemotherapy with adriamycin and cyclophosphamide appears to prolong the remission duration and survival in patients with advanced breast cancer to a greater extent than is achieved with adriamycin and cyclophosphamide alone.


VP 16-213 (VP 16) and cyclophosphamide in the treatment of primitive lung cancer in phase M 1. Sixty patients suffering from primitive lung cancer in Phase M 1 were included in two successive chemotherapy Protocols, 30 patients to a group. The first of these (an attempt at cellular synchronization by combining VCR-Cyclophosphamide-Prednisone) is used as a historical group as compared to the second. In the second protocol the combination of VP 16.213 (100 mg/m2, po, day X 5 days) and cyclophosphamide (100 mg/m2, ev, day X 5 days) was attempted. The positive results obtained (more than 60% of the responses were above 50%) together with low toxicity was not accompanied by improvement of survival with respect to that observed in international medical literature. On the other hand, the number of cases studied--30 patients--was too low to permit a better evaluation of our results. Despite this fact, it seems appropriate to continue clinical investigation of the VP 16.213-Cyclophosphamide combination in lung cancer.


Restoration by levamisole of E-rosette formation and its abrogation by autochthonous serum from patients with bladder cancer. Incubation in vitro of lymphocytes from bladder cancer patients with levamisole (40 microgram/ml) resulted in a rise of the number of E-rosette forming cells from 39.2 +/- 11.8% to 57.5 +/- 11.3% (p less than 0.005). The same effect was observed when levamisole was administrated 150 mg/day for 3 days to the patients. The stimulatory effect of levamisole was abrogated when the lymphocytes were first incubated with levamisole and afterwards with 50% autochthonous serum from the patient. With more diluted serum concentrations (from 0.5% to 1%), the response decreased, and the response was not observed with allogeneic serum. When lymphocytes from healthy donors were incubated with 50% serum from bladder cancer patients, there were no significant changes in numbers of E-rosette forming cells. It was presumed that the suppression of E-rosette forming cells from the patients with bladder cancer was caused by blocking the receptor sites for sheep red blood cells by autochthonous serum components and that levamisole eliminated such substances from the cryptic sites on the surface of the lymphocytes.


Effects of dose and schedule of immune stimulant on efficacy of combination Corynebacterium parvum-cyclophosphamide treatment for a murine mammary adenocarcinoma. Certain variables which might influence the outcome of combining cytotoxic drug and immune stimulant therapy were studied to optimize the effectiveness of Corynebacterium parvum combined with cyclophosphamide (CY) as treatment for a murine mammary adenocarcinoma (CaD2). Optimal effects of combined C. parvum-CY treatment in the CaD2 system were obtained when 443 to 1400 microgram of this immune stimulant per mouse were injected 2 to 3 days after CY chemotherapy and when combination treatment was continued on a weekly basis. The most critical factors contributing to the effectiveness of combination treatment in this system were the dose of C. parvum and the treatment frequency. The interval between chemotherapy and immune stimulant therapy was less critical to the outcome of combination treatment. Combination treatment given once or weekly significantly decreased tumor size in comparison to single or weekly CY treatment. A single treatment with CY and C. parvum significantly improved the survival over mice given a single CY treatment, but weekly CY and C. parvum treatment did not increase the survival over mice, given weekly chemotherapy.


Transformation of mouse C3H/10T1/2 cells by single and fractionated doses of X-rays and fission-spectrum neutrons. A mouse embryo-derived cell line, C3H/10T1/2, was used to measure the frequency of in vitro neoplastic transformation induced by 50-kVp X-rays and by fission-spectrum neutrons from the JANUS reactor at the Argonne National Laboratory. The transformation frequency after single X-ray doses rises exponentially, reaching a plateau at about 3 X 10(-3) transformant/survivor. The induction curve following single neutron doses, while qualitatively similar, initially rises more steeply and levels off at a maximum of about 6 X 10(-3) transformant/survivor. For both radiations, transformation frequency varies with changes in the number of viable cells per dish, showing about a 10-fold decrease in transformation frequency when the number of viable cells per 90-mm dish was increased from about 300 to about 1000. Fractionation of a total X-ray dose of 700 rads results in an approximately 6-fold increase in survival and reduction in transformation frequency over a 16-hr interval. Fractionation of the total neutron dose of 378 rads has no effect upon cell survival, and transformation frequency declines by a factor of only about 1.7 at most over a 24-hr period. Cells derived from transformed foci formed fibrosarcomas when injected into appropriately treated mice.


Cytotoxic activity of 1-(o-chlorophenyl)-1-(p-chlorophenyl)-2,2-dichloroethane (mitotane) and its analogs on feminizing adrenal neoplastic cells in culture. Mitotane [1-(o-chlorophenyl)-1-(p-chlorophenyl)-2,2-dichloroethane], an antineoplastic agent for inoperable adrenal carcinoma, was studied for its cytotoxic activity on a clonal line of feminizing human adrenal neoplastic cells (Fang-8 cells) in culture. At concentrations higher than 1.68 X 10(-4) M, mitotane produced a dose-related toxic effect on the cells. The effect of the drug was specific to Fang-8 cells because the same treatment produced little or no toxicity on lines of rat pituitary GH3 cells and human skin fibrocytes. The effect of mitotane to Fang-8 cells was a reversible one. Electron microscopic pictures revealed that the drug was causing mitochondrial degeneration. In this testing system, several isomers and analogs of mitotane were found equally or more toxic than was mitotane itself. The dichloro- or trichloroethylene structure was essential for the cytotoxic activity while the chloro substituents on phenyl rings appeared to be unimportant. This system appears to be useful in elucidating the biochemical mechanism of mitotane action on adrenal cancer.


Determination of [3H]- and [14C]hematoporphyrin derivative distribution in malignant and normal tissue. The synthesis and tissue-localizing ability of [14C]- and [3H]hematoporphyrin derivative (HPD) in mice have been described. Tissue levels and distributions were the same for both radioactive compounds, indicating that in vivo tritium exchange did not occur with [3H]HPD. The amount of [14C]HPD or [3H]HPD which localized in the transplanted tumor tissue of mice at various times following i.p. injection (10 mg/kg) was higher than in skin or muscle tissue but was less than in liver, kidney, or spleen tissue. These results tend to disprove the generalization that HPD accumulates in malignant tissue to a higher degree than in all normal tissue. It is also reported that gross visualization of porphyrin fluorescence cannot be correlated with actual tissue concentrations of the dye.


Opposite effects of dextrans substituted with sulfhydryls or mercury on tumor growth. Macromolecular dextrans carrying substituents terminated by sulfhydryl groups or terminated by aromatic amines effectively inhibit the growth of a fibrosarcoma and of a mammary adenocarcinoma in a syngeneic mouse model. These compounds have no or very low toxicity to animals and are nontoxic to fibrosarcoma cells in vitro. Small-molecular-weight compounds carrying the same substituents as the above dextrans are without any effect on the growth of these tumors. A dextran substituted with mercury-containing side chains is growth promoting for the same fibrosarcoma in mice at doses which are nontoxic for these animals. However, the mercury-containing compound is toxic to fibrosarcoma cells in vitro. It is hypothesized that these nonpermeating macromolecules do not directly influence the tumor cells in animals but modulate the natural system of defense against tumors; cells of that system are stimulated or poisoned by the substituted dextrans.


Splenic granulopoiesis in mice following administration of cyclophosphamide. The adult mouse spleen retains granulocytic progenitor cells that are capable of proliferation and maturation. In mice given cyclophosphamide, spleen weight and cell content increased. In vitro granulocyte progenitor cells increased more than 300-fold in the spleen and remained elevated 14 days following cyclophosphamide. Proliferative and nonproliferative granulocytes were increased above controls until Days 21 and 28, respectively. In splenectomized mice, blood neutrophil recovery was delayed when compared to nonsplenectomized mice following cyclophosphamide. Although marrow cell number was similar in both groups, maximum marrow granulocyte progenitor cells were 2-fold greater in nonsplenectomized animals. These studies demonstrate that the murine spleen becomes a markedly granulopoietic organ and is a major contributor to recovery of granulocytes following cyclophosphamide. Splenic granulopoiesis should be taken into account in studies which measure toxicity of various agents in murine hematopoiesis.


Autoradiographic cytokinetics of colonic mucosal hyperplasia in mice. The cytokinetics of a naturally occurring hyperplastic disease of the colon in mice were determined by autoradiography and compared to kinetic changes seen by others in nonneoplastic and neoplastic colonic disease. Cell cycle parameters were determined using the fraction-labeled mitosis method. Labeling index, labeling pattern, and migration rates were also evaluated. During colonic hyperplasia, there was an increase in variation of DNA synthesis times, resulting in prolongation of the S phase. There also was prolongation of the total cell cycle time and G1 phase. In addition, labeling index was increased 2-fold, the proliferative zone was extended to include the entire crypt column and surface mucosa, and the migration rate was accelerated. These findings parallel the "atypical" cytokinetics found in human and murine neoplastic and preneoplastic disorders of the colon and may be a typical proliferative response of mucosa to a variety of stimuli.


Purification and partial characterization of nucleolar antigen-1 of the Novikoff hepatoma. A nucleolar chromatin antigen (NoAg-1) found in Novikoff hepatoma but not in normal liver has been purified to homogeneity as shown by two-dimensional gel electrophoresis. Initial purification of NoAg-1 was partially achieved by isolation of nucleolar chromatin and fractionation of its proteins by successive extraction with solutions of increasing salt concentration. Further purification of this antigen was achieved by affinity and hydroxylapatite chromatography. Although approximately 50% of the NoAg-1 antigen was in the 0.6 M NaCl extract of Novikoff nucleoli, it was less pure than in the 2 M NaCl:5 M urea extract which contained 25% of the NoAg-1 at a purity of 40%. The highly purified NoAg-1 had an approximate molecular weight of 60,000 and pl of 5.1; the yield of NoAg-1 was 0.22% of the total nucleolar proteins.


In vivo autoradiography and nitrosoheptamethyleneimine carcinogenesis in hamsters. Quantitative autoradiograms were made, in vivo, in European hamsters with the use of [14C]nitrosoheptamethyleneimine (260 muCi/animal; time between administration of nitrosamine and killing of animals, 6 hr). In this species, the lung is the principal target, and radioactivity was found in the Clara cells of the bronchial epithelium and in the nitrosoheptamethyleneimine-induced tumors which derive from these cells. Tumors are not induced in the liver, which can metabolize this compound, and labeling is found principally in the cytoplasm, whereas in the target cells there is a high degree of labeling in both the cytoplasm and the nuclei.


Photodynamic killing of retinoblastoma cells with hematoporphyrin and light. Hematoporphyrin and white light exerted a lethal effect on two cell lines of retinoblastoma cells. The lowest values of dye concentration and light exposure capable of killing an entire cell population were, respectively, 2 X 10(-5) M and 6 min exposure to an irradiance of 6.0 microwatts/sq mm. Cells exposed to light in the presence of the dye did not require lengthy incubation periods but were rapidly killed with increasing periods of light exposure. Cells washed free of the dye, however, required a minimum sensitizing period of 3.5 hr to achieve a value close to 100% cell death. An inhibitor of this photodynamic process was demonstrated in normal serum. When the concentration of either fetal calf serum, rabbit serum, or rabbit plasma was increased to 25% from a standard 10%, there was as much as a 100-fold greater requirement of hematoporphyrin concentration to produce the same lethal response. The suggested explanation for this phenomenon is a porphyrin-binding plasma factor, hemopexin, which is a natural beta-glycoprotein believed to be responsible for the transport of circulating porphyrins to the liver for their elimination. Retinoblastoma cells grow in suspension and thereby provide an excellent tool for study of photosensitive dyes, especially in the case of the rapidly growing Y79 cell line.


Fecal bile acids in two Japanese populations with different colon cancer risks. Some workers have associated fecal bile acids with colon cancer frequency. They suggest that the risk for colon cancer increases with a rise in the level of total and degraded fecal bile acids. The Japanese in Hawaii, who are at high risk for this cancer, had higher concentrations of deoxycholic acid (a degraded bile acid) in their fecal specimens than did the people in Akita, Japan, who are at low risk. However, the findings for the other bile acids were unremarkable or inconsistent. These data were suggestive, but not strongly supportive, of a relationship between fecal bile acid patterns and colon cancer risk.


Nuclear ultrastructure of epithelial cell lines derived from human carcinomas and nonmalignant tissues. The nuclear ultrastructure of sixteen human epithelial cell lines has been characterized in detail by transmission electron microscopy. The cell lines were derived from normal tissues, nonmalignant tissues of cancerous organs, primary carcinomas, and metastatic carcinomas. Every cell section on a grid containing a clearly defined nucleus and nucleolus was scored blindly utilizing a checklist of markers. The goal of these studies was to determine whether any ultrastructural markers consistently distinguished the different stages of malignant progression represented among the lines. Nuclear bodies and perichromatin granules were found in all lines derived from cancer and were not observed in any nonmalignant lines. Nuclear envelope dilation was seen in all lines derived from cancerours organs as well as from malignant tissues but not in any lines derived from normal tissue. Margination of chromatin, irregularity of nuclear outline, redistribution of nucleolar components, and margination were expressed slightly by the normal lines, to variable degrees by the lines derived from cancerous organs, and to a much greater extent by all lines derived from malignant tissues. No differences were found between lines derived from primary carcinomas and those derived from metastatic specimens. There were no ultrastructural differences comparing subconfluent and confluent cells or cells at different passage levels. In addition, the nuclear ultrastructure of a malignant line in culture was similar to that of a tumor induced by those cells in an immunosuppressed mouse.


Pharmacokinetics and disposition of 3-deazauridine in humans. 3-Deazauridine (3-DAU) pharmacology was studied in 20 patients who received the drug by rapid or continuous infusion. In 8 studies, the plasma clearance of 3-DAU after rapid administration was biphasic, with an average terminal t1/2 of 4.4 hr and an extrapolated volume of distribution of 0.57 liter/kg. After 5-day continuous infusion of 3-DAU, the plasma clearance was also biphasic, with an average terminal t1/2 of 21.3 hr and an extrapolated volume of distribution of 18.8 liter/kg. 2,4-Dihydroxypyridine, the aglycone of 3-DAU, was observed in plasma but not in urine of patients receiving the drug by rapid infusion. The urinary excretion of 3-DAU was low, only 7.8% 24 hr after rapid infusion and 7.2% up to 4 days after continuous infusion. Tissue distribution of 3-DAU was determined from autopsy samples of 2 patients. Not only were high levels of 3-DAU detected in the tissues studied, but 3-DAU triphosphate, the active metabolite of 3-DAU, was present in brain, lung, and liver.


Comparison of the sensitivities of human, canine, and murine hematopoietic precursor cells to adriamycin and N-trifluoroacetyladriamycin-14-valerate. The sensitivities of leukocyte-committed stem cells of human, dog, and mouse bone marrow to an 18-hr exposure to Adriamycin (ADR) and N-trifluoroacetyladriamycin-14-valerate (AD32) were compared using the agar diffusion chamber assay. The survival of in situ mouse marrow stem cells following the drugs was also measured. The in vivo doses required to produce 37% survival of hematopoietic precursor cells for human, dog, mouse, and in situ mouse marrow stem cells for ADR were 9.5, 3.5, 8, and 8 mg/kg, respectively, while those for AD32 were 22, 17, 37, and 40 mg/kg. Mouse bone marrow cellularity was reduced to 50% by drug doses producing 3 and 7% stem cell survival. AD32 given i.p. to mice was less effective than i.v. drug in producing marrow stem cell death, reduction of marrow cellularity, or acute lethality. Although more AD32 than ADR was required to kill marrow stem cells from each species and canine marrow stem cells were the most sensitive to either drug, human stem cells were relatively more sensitive to AD32 than were mouse stem cells. The reverse was true for ADR. Granulocytopenia may be an important limiting toxicity in the clinical use of AD32.


Effect of hyperbaric oxygen on the cytotoxicity of adriamycin and nitrogen mustard in cultured Burkitt's lymphoma cells. The effects of hyperbaric oxygen (HPO) exposure on the cytotoxicity of Adriamycin (ADM) and nitrogen mustard have been examined in Burkitt's lymphoma cells (P3J) in vitro. Exposure of cells to 3 atmospheres of pressure HPO for 2 hr produces inhibition of DNA synthesis and mitosis. Simultaneous exposure to HPO and ADM results in decreased cytotoxicity compared to drug treatment alone. However, exposure to ADM (0.15 microgram/ml) 2 to 8 hr before or after HPO produces an increase in drug effect. There is no potentiation of lower ADM concentrations. Cytotoxicity is increased when cells are exposed to HPO during, before, or after exposure to nitrogen mustard (NSC 762) (0.15 and 0.25 microgram/ml). Lower nitrogen mustard (NSC 762) concentrations are not potentiated. HPO potentiation of antineoplastic agents appears to depend upon the agent studied, the concentration of the agent, and the scheduling of the drug and HPO exposure.


A methodological approach to the prediction of anticancer drug effect in humans. Tumor cells from animals and humans were treated with drugs under tissue culture conditions. Tumor cells from the sensitive L1210 model were studied first. A dose-response curve was derived between drug exposure and subsequent cytotoxicity in L1210. The concentration of drug and duration of exposure were factors critical to the subsequent development of in vitro cytotoxicity. The in vitro dosage which effected 50% leukemic cell death in L1210 cells correlated with reported in vivo drug levels. Other tumor models and human neoplastic cells were studied at this dosage level. A good correlation was noted in these studies between the in vivo responsiveness and the in vitro chemotherapy results in both animals and humans. It was suggested by these results that it may be possible to predict cancericidal drug activity for individual neoplasms by assaying the tumor cells in vitro for drug sensitivity.


Stimulation of ultraviolet-induced carcinogenesis by 1,3-Bis(2-chloroethyl)-1-nitrosourea. The influence of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) on ultraviolet-induced carcinogenesis was examined in hairless mouse skin in vivo. Noncarcinogenic amounts of topically applied BCNU and carcinogenic levels of UVB energy were utilized in the study. The applications of BCNU significantly accelerated the appearance and growth of the cutaneous tumors in this study. Thus, the BCNU acted as either a promoting or a cocarcinogenic agent for ultraviolet-induced cancer formation. Although the mechanism of this effect has not been established, avoidance of extensive sun exposure would probably be prudent when topical BCNU is being utilized therapeutically.


Inhibitory effect of growth hormone on Gross passage A virus-induced thymus leukemia in C3Hf mice. Administration of ovine growth hormone to young C3Hf mice inhibited Gross passage A virus-induced leukemogenesis as manifested by a delayed onset and a lower incidence of thymus leukemia. These results can be interpreted that growth hormone inhibited thymus-dependent leukemogenesis perhaps through thymotrophic influences which prevented or delayed the thymus involution believed to be essential for leukemia change. In female but not in male Gross passage A virus-infected mice, development of a thymus-independent leukemia appeared to be promoted by growth hormone.


Effects of N-trifluoroacetyladriamycin-14-valerate and related agents on DNA strand damage and thymidine incorporation in CCRF-CEM cells. N-Trifluoroacetyladriamycin-14-valerate (AD32) and N-trifluoroacetyladriamycin (AD41) have been compared with Adriamycin and daunorubicin in CCRF-CEM human lymphoblastic cells. All four agents produce DNA damage, as measured by alkaline unwinding methods. Thymidine incorporation, a measure of DNA synthesis, is inhibited by AD32 and AD41 and completely blocked by the other drugs. AD32 and AD41 inhibit growth of the cells after exposure for 2 hr but are considerably less potent than is either Adriamycin or daunorubicin. In drug uptake studies, cellular levels after 2-hr incubations are lower for AD32 than for Adriamycin, and both are severalfold lower than are those of daunorubicin. The results indicate that AD32 and AD41 (or their metabolites) have DNA-related effects similar to those of Adriamycin and daunorubicin. These common actions may be the proximal cytotoxic events for the four agents.


1,2-Dimethylhydrazine-induced methylation of DNA bases in various rat organs and the effect of pretreatment with disulfiram. The extent and persistence of methylated purines were determined in DNA of various rat organs following a single s.c. injection of 1,2-di[14C]methylhydrazine. Maximum alkylation of purine bases occurred within 12 hr, with highest concentrations in liver, followed by colon, ileum, and kidney. Over a period of 3 days, O6-methylguanine was removed much more slowly from colon, the principal target organ for carcinogenesis, than from ileum or liver DNA. Dietary pretreatment of rats with disulfiram is known to prevent 1,2-dimethylhydrazine-induced colon carcinogenesis and was found to reduce DNA alkylation to less than 1% of that detected in animals treated with 1,2- di[14C]methylhydrazine alone.


In vivo tumor discrimination in a rat by proton nuclear magnetic resonance imaging. Transverse cross-sectional images which represent the spatial distribution of the proton nuclear magnetic resonance signal in a live Wistar rat have been produced by the multiple-sensitive-point method. The images form a chronological series demonstrating the detection and development of a D23 hepatoma in the abdomen. Discrimination of the tumor is by contrast with the surrounding tissue and is attributed to the elevated water content and relaxation times of the  former.


DNA repair in xeroderma pigmentosum cells treated with combinations of ultraviolet radiation and N-acetoxy-2-acetylaminofluorene. We used three techniques to examine excision repair in human cells treated with ultraviolet radiation, N-acetoxy-2-acetylaminofluorene, and a combination of the two. The three techniques gave similar results. Two types of human cells were used: (a) excision repair proficient (normal human fibroblasts and xeroderma pigmentosum variants); and (b) excision repair deficient (xeroderma pigmentosum C, D, and E). Saturation doses were determined and used for combined treatments with both agents. We observed two patterns of repair: (a) in repair-proficient cells total repair was additive; and (b) in repair-deficient cells total repair was much less than additive (usually less than that repair was much less than additive (usually less than that observed for separate treatments) and N-acetoxy-2-acetylaminofluorene inhibited excision of pyrimidine dimers. We conclude that, in the first group of cells, pathways for repair of ultraviolet radiation- and N-acetoxy-2-acetylaminofluorene-induced lesions are not identical and, in the second group of cells, there is an inhibitory effect exerted by major or minor products of each agent on the repair enzyme(s) of the other.


Dihydrofolate reductase in primary brain tumors, cell cultures of central nervous system origin, and normal brain during fetal and neonatal growth. Dihydrofolate reductase (DHFR) was measured during the development in rats of brain tumors induced following inoculation with avian sarcoma virus. Increasing activity of this enzyme in brain was correlated with the course of primary brain tumor growth. The specific activities of DHFR in primary human brain tumor tissues were comparable to those found in avian sarcoma virus-induced brain tumors in rats. Specific activities of DHFR in cell cultures derived from human and rat primary intracranial gliomas and sarcomas were up to 6 times those found in adult rat liver. The presence of DHFR in neoplasms of central nervous system origin is relevant to the development of folate antagonists which, unlike methotrexate, can readily cross the blood-brain barrier. In normal developing rat brain, DHFR specific activity was high in embryos at 19 days of gestation and declined thereafter, until at 20 days after birth the activity was very low. The methotrexate titration assay was used to measure enzyme levels in the brains of fetal and newborn rats, and good correlation with the spectrophotometric assay was observed. The pattern was different in liver, showing maximum activity 11 days after birth and retaining high activity in adult liver. Both the cofactor requirement and the sensitivity to methotrexate indicate that the enzyme in the brain is DHFR.


Characterization of messenger RNA for fructose 1,6-bisphosphate aldolase A isozyme of rat ascites hepatoma AH 7974 cells. The messenger activity for fructose 1,6-bisphosphate aldolase (EC4.1.2.13) (aldolase) A isozyme has been characterized in the polysome- or the messenger RNA-directed, protein-synthesizing system using the pH 5 fraction of rat liver or wheat germ extracts, respectively. The subunit of aldolase A synthesized in vitro was detected by immunoprecipitation with anti-aldolase A antibody raised in chickens followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The synthesis of the enzyme depended on the addition of polysomes or polyadenylate-containing RNA of rat ascites hepatoma AH 7974 cells which show a complete shift of aldolase isozyme to type A, whereas polysomes of adult rat liver were inactive. The messenger activity for aldolase A was present exclusively on free polysomes but absent on membrane-bound polysomes and in the soluble supernatant fraction of AH 7974 cells. The size of aldolase A messenger RNA determined by formamide-containing sucrose density gradient centrifugation was approximately 5.8 X 10(5) daltons corresponding to 1650 nucleotides. Taking into account the number of amino acid residues in the aldolase A subunit, approximately 400 nucleotides correspond to the noncoding region of aldolase A messenger RNA.


Sensitivity of human granulopoiesis in vitro to adriamycin before and after exposure in vivo. The present studies were performed to determine whether the sensitivity of a normal human granulopoiesis to Adriamycin changes after repeated exposure to the drug in vivo. A single-layer agar culture for the in vitro growth of granulocytic colonies from committed stem cells was used as the assay system. The in vitro sensitivity of the human granulopoiesis to different doses of Adriamycin was determined from the reduction in colony incidence (a) following exposure of marrow cells to Adriamycin for 30 min before initiation of cultures and (b) in the presence of Adriamycin during the total culture period. Using both test systems, the sensitivity of granulopoiesis in vitro remained unchanged after patients had received six courses of chemotherapy incorporating Adriamycin. Sensitivity to Adriamycin appeared to increase by Day 4 after chemotherapy, probably as a result of the increased proliferative activity of granulopoietic precursor cells at this time. The data thus fail to provide evidence that a granulopoietic subpopulation with increased resistance to the cytotoxic effects of Adriamycin emerges after repeated in vivo exposure to the drug.


Presence and cell growth-related variations of glycogen in human colorectal adenocarcinoma cell lines in culture. The presence and kinetics of intracellular glycogen levels were studied, in relationship to cell growth, in asynchronous and in synchronized cultures of four human colorectal adenocarcinoma cell lines (HT-29, HRT-18, SW-480, and Caco-2). The results show that a specific pattern of glycogen accumulation occurs during the process of cell growth of the studied cell lines. The kinetics of glycogen accumulation in asynchronous cultures were similar from one cell line to another and were characterized by a low amount in the exponential phase of growth, followed by a 3- to 4-fold increase in the stationary phase. The quantities found in either phase were specific for each cell line. The maximum values found in Caco-2, HRT-18, HT-29, and SW-480 cells were, respectively, 258.5 +/- 6.9 (S.D.), 88.9 +/- 2.6, 87.5 +/- 3, and 17.5 +/- 1.8 microgram of glycogen per mg of proteins. The kinetics of glycogen accumulation during the cell cycle was also studied in synchronized cultures of HT-29 and HRT-18 cell lines. Both cell lines exhibited a common pattern of low glycogen quantities during S, G2, and M followed by an increase beginning with G1 and peaking (2.5 to 3 times the initial values) in the middle of this phase. This was followed by a symmetrical decrease in the second half of G1.


Influence of oil concentration on the efficacy of tumor regression by emulsified components of mycobacteria. The influence of mineral oil content on the antitumor activity of emulsified Bacillus Calmette-Guérin cell walls (CW) was studied in mice each with an established transplant of a syngeneic fibrosarcoma. Animals received intratumoral injections of CW (0.02 to 0.6 mg/mouse) emulsified in 1 to 9% oil. The number of animals in which tumor regressed completely depended on the concentration of oil in the emulsion. The methanol extraction residue (MER) of Bacillus Calmette-Guérin administered intratumorally in oil in water emulsion was also immunotherapeutically active. Neither CW nor MER was active when given as aqueous suspensions without oil. Guinea pigs with transplanted hepatomas growing in their skin and with tumor cells in the draining lymph node could be cured of malignant disease by a single intralesional injection of CW or MER (1 mg per guinea pig) if these agents were emulsified in oil (3.3%) and water. Aqueous suspensions of CW and MER were not therapeutically active in the guinea pig.


Effect of thymidine on the sensitivity of cultured mouse tumor cells to 1-beta-D-arabinofuranosylcytosine. Assays of thirteen cell lines, derived from mouse lymphomas, myelomas, myeloid tumors, and a mastocytoma, for sensitivity to growth inhibition by 1-beta-D-arabinofuranosylcytosine (ara-C) revealed a spectrum between the most and least sensitive which differed 100-fold from each other. An inverse correlation between sensitivity and cellular deoxycytidine 5'-triphosphate (dCTP) content was found, and this suggested that sensitivity of cells might be increased if the dCTP content was lowered during cell exposure to ara-C. Previous work has shown that thymidine treatment of cells lowers their dCTP content, and the effect of thymidine on the sensitivity of six of the cell lines to ara-C was therefore measured. Concentrations of thymidine below those inhibitory to cell growth by themselves caused an increase in ara-C sensitivity by up to 3-fold in 4 cell lines in which thymidine causes a depression in dCTP content but not in 2 myeloid lines in which the dCTP content was found not to be depressed by the same thymidine treatment. The results confirm an important role for dCTP in determining cellular sensitivity to ara-C. The finding that the sensitivity of 2 lymphoma cell lines to ara-C could be increased by concentrations of thymidine in the region of 10 micrometer, which are attainable clinically in humans, suggests that a combination of ara-C with thymidine might be useful in the treatment of some human tumors.


Monitoring of antitumor immunity in patients with larynx cancer by tube leukocyte adherence inhibition assay. The presence of specific antitumor immunity was examined by the tube leukocyte adherence inhibition (LAI) assay in patients with carcinomas of the larynx. Peripheral blood leukocytes (PBL) from patients with larynx cancer gave positive reactions in the LAI assay with the antigen prepared from the carcinoma of the larynx but not with antigen from normal larynx tissue. Adherence of PBL from normal donors did not differ significantly irrespective of whether antigens from tumor or normal larynx tissue were used. With a panel of tumor extracts tested, PBL of some patients reacted to only one antigen, wheras PBL of other patients reacted to several antigens. LAI reactivity disappeared after surgery, reappeared at 4 weeks after surgery, and then declined when patients were tumor free. These results show that the tube LAI assay may be of value in diagnosing larynx cancer and in following up its treatment.


Cellular antitumor immune response in women with risk factors for breast cancer. The leukocyte adherence inhibition technique was used to study cell-mediated immunoactivity to breast adenocarcinoma. In a group of 74 patients with untreated breast cancer in Stages I and II, 69% showed a positive response, while 48% of the 25 patients in Stages III and IV had reactive leukocytes. Among 43 control persons, only 2 women showed positive responses. In a group of 161 women with benign breast diseases, 24% showed a positive reaction against breast carcinoma extract. The percentage of positive responses was higher than the average of women with benign disease among those who had a mother or a sister with breast cancer, who had previously had benign breast lumps removed, who had microcalcifications in their breasts, or for whom an increased intraductal epithelial proliferation was found in their biopsies. Twenty-eight women with benign breast diseases and two or more of the above factors were assigned to the high-risk group, in which 43% had reactive leukocytes. Of the 50 women with one risk factor, 34% showed a positive response, while of the 83 women in the low-risk group, with none of the above factors, only 11% had a positive reaction. The results suggest that the leukocyte adherence inhibition test may be used to identify groups of women who have a high risk for breast cancer.


Influence of aging on left ventricular hemodynamics and stiffness in beagles. We studied the influence of aging on the contractile performance, stiffness, and contraction time of the canine left ventricle. Eight young (27 +/- 2.5 months, mean +/- SE) and seven old (128 +/- 20.5 months) beagles were placed on complete cardiopulmonary bypass, the arterial pressure was adjusted to 80 mm Hg, and the heart contracted isovolumically at a paced rate of 120 beats/min. Diastolic pressure-volume curves were established for each unpaced left ventricle at the beginning of each experiment, and the volume at the knee of the curve was used during the subsequent data collection when the heart was paced. Stiffness was measured with a sinusoidal forcing function, which imposed a sinusoidal displacement of 1 ml at 20 Hz into a balloon placed in the left ventricle. In each ventricle, stiffness was related linearly to pressure during the cardiac cycle, and was greater for any given pressure in the older beagles. Contraction duration was prolonged in the older dogs. In an additional seven old beagles during right heart bypass time, and duration of contraction were longer than in seven young beagles. Aging of the beagle heart is associated with an increase in left ventricular systolic and diastolic stiffness and prolonged duration of contraction.


Cellular mechanisms of normal growth in the mammalian heart. I. Qualitative and quantitative features of ventricular architecture in the dog from birth to five months of age. This paper describes the qualitative and quantitative composition of dog myocardium over the first 5 months of life. The quantitative composition of dog right and left ventricle over this period does not vary. A stereological analysis of electron micrographs representing 32,000 micron2 of tissue surface revealed that 79% of the heart is made up of myofibers, whereas 21% is extracellular space. Twenty-eight percent of the extracellular compartment by volume is vasculature (tissue was preserved by immersion rather than vascular perfusion); 72% is occupied by nonvascular elements and "empty" space. In contrast to the remarkable constancy of quantitative composition of the whole myocardium, myocyte shape and dimensions and the arrangement of intercellular connections vary dramatically over the age period studied. In early postnatal life, the morphology of blood vessels, many of which have completely partitioned lumina, also changes significantly.


Cellular mechanisms of normal growth in the mammalian heart. II. A quantitative and qualitative comparison between the right and left ventricular myocytes in the dog from birth to five months of age. This paper describes and contrasts the changes in myocytes taken from the right and left ventricules of dog heart over the first 5 months of life. The development of the two populations of cells differs in important respects: sarcomeric volume, the proportion of the myocyte occupied by the contractile apparatus, and the volume and surface area of the transverse tubular system all were greater on the average in the left than in the right ventricle (P less than 0.001). Other intracellular structures also changed significantly as development progressed, but did so in both chambers; the surface area and surface-to-volume (s/v) ratio of both right and left ventricular myocytes increased with age (P less than 0.001) as did mitochondrial and mitochondrial-myocyte volume (P less than 0.001). The surface area (P less than 0.01) and the s/v ratio of the mitochondria (P less than 0.001) also increased with development. Nuclear and nuclear myocyte volume grew smaller with age in both chambers (P less than 0.002), as did the surface area (P less than 0.001) and the s/v ratio of the nucleus (P less than 0.01). Not only does myocyte composition change quantitatively, striking changes in intracellular architecture and the appearance and arrangement of intracellular organelles occur during postnatal life.


Supraventricular pacemaker underdrive in the absence of sinus nodal influences in the conscious dog. The ability to reduce and maintain heart rate by electrical stimulation (underdrive) was tested in three groups of chronically instrumented dogs: sinoatrial node intact, ectopic atrial pacemaker produced by injection of rapidly hardening latex into the sinoatrial nodal artery, and idioventricular pacemaker produced by injection of formalin into the atrioventricular node. In the conscious unsedated state, underdrive of sinoatrial or idioventricular pacemakers resulted in competition between driven and intrinsic foci. However, the cycle length of ectopic atrial pacemakers could be increased by 148.4 +/- 30.7 msec (P less than 0.001) and maintained at that value. Cessation of underdrive resulted in a period of pacemaker suppression similar to that produced following overdrive. Single premature stimuli produced marked cycle length prolongations in dogs with ectopic atrial foci. Total autonomic blockade had no significant effect on the production of underdrive. Thus, the results of the present experiments provide evidence for maintained capture of cardiac pacemakers at rates significantly below intrinsic control and indicate that underdrive varies as a function of pacemaker site. Underdrive may explain the failure of junctional escape in the presence of slower atrial rhythms.


Ontogenetic increase of the maximal rate of rise of the chick embryonic heart action potential. Relationship to voltage, time, and tetrodotoxin. The maximal rate of rise (Vmax) of the embryonic chick ventricular action potential increased about 2-fold from 112 +/- 6 V/sec and 113 +/- 5 V/sec on the 4th and 6th incubation days, respectively, to 217 +/- 7 V/sec on the 12th incubation day. Neither the steady state inactivation (hinfinity) of Vmax, the temperature-dependent shift of hinfinity, nor the time constant for recovery (tauRec) of Vmax changed significantly when Vmax had doubled. Inhibition of Vmax by tetrodotoxin (TTX) was the same in hearts from the 4th, 6th, and 18th incubation days. The relation between Vmax and TTX concentration could be described by a one-to-one binding curve with an apparent dissociation constant of 2 X 10(-8) M. The increase of Vmax, a valid measure of the early inward Na+ current (iNa) during the rising phase of the cardiac action potential, can be attributed to an elevated maximal Na+ conductance (-gNa) during ontogenesis. These results indicate that the physicochemical properties of the gNa unit responsible for the rapid rising phase of the action potential and of the TTX binding site associated with it may remain constant during a period of embryonic development when -gNa increased significantly.


Acetylcholine antagonism of the electrophysiological effects of isoproterenol on canine cardiac Purkinje fibers. The purpose of these experiments was to determine whether or not acetylcholine modulated the electrophysiological effects of isoproterenol on canine cardiac Purkinje fibers. Conventional microelectrode techniques were used. Predictably, isoproterenol produced shortening of action potential duration; acetylcholine significantly blunted this effect of isoproterenol. Isoproterenol restored excitability to fibers exposed to 22 mM potassium solutions, and acetylcholine abolished this isoproterenol-restored excitability. Both of these antagonistic effects of acetylcholine were blocked by atropine. Acetylcholine alone did not affect action potential duration in polarized fibers or excitability in potassium-depolarized fibers. Furthermore, acetylcholine had no effect on the decrease in action potential duration induced by premature electrical stimulation or by acetylstrophanthidin administration, or on excitability of fibers exposed to a zero sodium, high calcium superfusant. These data demonstrate a direct cellular basis for cholinergic antagonism of the electrophysiological effects of beta-adrenergic stimulation of canine cardiac specialized intraventricular conducting tissue.


Variables predictive of survival in patients with coronary disease. Selection by univariate and multivariate analyses from the clinical, electrocardiographic, exercise, arteriographic, and quantitative angiographic evaluations. A progression of univariate followed by multivariate analyses was applied to 46 variables selected from the clinical examination, exercise test, coronary arteriography, and quantitative angiographic assessment of left ventricular function in patients with coronary disease to determine those variables most predictive of survival. For the  733 medically treated patients, the final Cox's regression analysis showed that the left ventricular ejection fraction was most predictive of survival, followed by age, number of vessels with stenosis(es) greater than or equal to 70%, and ventricular arrhythmia on the resting electrocardiogram. For the 1870 surgically treated patients, ventricular arrhythmia on the resting electrocardiogram was most predictive of survival followed by ejection fraction, heart murmur, left main coronary artery stenosis greater than or equal to 50%, and use of diuretic agents.


Evidence from a nonrandomized study that coronary surgery prolongs survival in patients with two-vessel coronary disease. Within the larger Seattle Heart Watch arteriography registry, surgically treated patients non randomly selected for direct myocardial revascularization were matched to medically treated patients such that each of the 287 pairs was identical in seven variables (ejection fraction, ventricular arrhythmia on resting electrocardiogram, age, heart murmur, stenosis of left main coronary artery greater than or equal to 50%, number of vessels with stenosis greater than or equal to 70%, and use of diuretics) previously demonstrated to be independently predictive of survival. Actuarial survival analyses based on cardiovascular deaths (average follow-up 3.5 years) indicate improved survival for the entire surgical matched pair cohort (p = 0.008) and for the surgically treated subgroup with two-vessel disease (p = 0.0002) when compared to the medical cohort. These results were confirmed by examination of the entire arteriography registry (n = 1524) in which these seven variables were known, using Cox's model for survival analysis. This multivariate, statistical technique indicated that the surgical mode of therapy was significantly predictive of improved survival in surgically treated patients for the entire registry (p = 0.008) and for the subgroup with two-vessel disease (p = 0.0005).


An ultrasound Doppler technique for the noninvasive determination of the pressure gradient in the Björk-Shiley mitral valve. The accuracy in determining the pressure gradient in the Björk-Shiley mitral implant from noninvasive ultrasound Doppler data was explored in nine adult patients. Manometric and ultrasound data were collectd simultaneously, and identical diastolic periods were used to compare the manometric gradient (delta Pm) with the gradient obtained from ultrasound data (delta Pu). In the nine patients the mean diastolic value of delta Pm ranged from 2-12.5 mm Hg and the difference between the mean diastolic values of delta Pm and delta Pu was 0.3 +/- 1.0 mm Hg(SD). The results of the investigation indicated that the method is accurate and reliable in the nonivasive determination of the mean diastolic gradient in the Björk-Shiley mitral implant.


Recurrent sustained ventricular tachycardia. 4. Pleomorphism. Two or more morphologically distinct ventricular tachycardias were observed during electrophysiologic study in 14 patients with chronic sustained ventricular tachycardia. Nine of these patients had clinical ventricular tachycardia with multiple morphologies. During the study 13 patients manifested both right bundle branch block (RBBB) and left bundle branch block (LBBB) morphologies. The remaining patient had RBBB with both right and left axis deviation. Changing morphologies were observed spontaneously in four patients and could be produced in all 14 by ventricular stimulation. In 12 patients both RBBB and LBBB originated in the left ventricle, and in 11 of these patients, from within a left ventricular aneurysm. Diastolic fragmented activity representing reentry was unchanged during both morphologies in four patients and during one morphology in five patients. Epicardial mapping confirmed the aneurysm as the site of origin of multiform ventricular tachycardias in two patients. Our data suggest that 1) ventricular tachycardia is frequently pleomorphic; 2) multiple morphologies usually represent variable exit sites and/or ventricular activation during the same tachycardia; and 3) there is a frequent association of pleomorphic ventricular tachycardia with left ventricle aneurysm.


Subxiphoid cross-sectional echocardiography in infants and children with congenital heart disease. We developed anatomically related cross-sectional echocardiographic views to image the heart from the subxiphoid area in 100 children and infants with various forms of congenital heart disease studied prospectively before cardiac catheterization. Wide-angle cross-sectional views were achieved using a mechanical sector scanner in a scan plane oriented parallel to a line between the patient's shoulders (a coronal plane) showing the equivalent anatomy of an anteroposterior angiogram. The subxiphoid technique appeared to be better than chest wall-based imaging in demonstrating obstructive lesions in the proximal portion of the right ventricular outflow tract (19 patients), i.e., the subpulmonic area, which is often at the narrow edge of the sector and behind the transducer artifact in chest wall studies. The subxiphoid technique was also useful for imaging the interatrial and interventricular septae; in subxiphoid views, as opposed to four-chamber apical views, there was significantly less false septal dropout and atrial septal defects (19 patients) as well as ventricular septal aneurysms (seven patients) were easily imaged. Finally, the subxiphoid orientation provided more adequate imaging in patients with discrete diaphragmatic subaortic stenosis (four patients), even when the diaphragm was just beneath the aortic valve. Subxiphoid cross-sectional echocardiography is an easily understood anatomical format for imaging cross-sectional anatomy in congenital heart disease and is a valuable adjunct to cross-sectional echocardiography from the chest wall.


Pulmonary vascular disease in transposition of the great vessels and intact ventricular septum. Eight of 135 (6%) children with d-transposition of the great vessels and with intact ventricular septum and no patent ductus arteriosus had evidence of progressive pulmonary vascular disease. Seven of 101 (7%) patients for whom histologic data was available, had Heath-Edwards grades IV or V pulmonary vascular disease, six had grade II, and 88 had either normal or grade I findings. One of 34 patients for whom histologic data was not available had hemodynamic evidence of pulmonary vascular disease at cardiac catheterization after the Mustard operation. When infants younger than 3 months old were excluded, eight of 85 (9%) had advanced pulmonary vascular disease. Twenty-three patients had microthrombi in their pulmonary arteries and arterioles, and in one patient thrombi were observed before the development of pulmonary vascular disease. Clinically unrecognized pulmonary microthrombi are suggested as a possible etiologic agent in the development of pulmonary vascular disease in patients with transposition of the great vessels. Progressive pulmonary vascular disease can first be discovered after the Mustard operation, even in patients without preoperative evidence of pulmonary hypertension or elevated pulmonary vascular resistance.


Improving compliance with therapeutic regimens in hypertensive patients in a community health center. A 1-year, randomized study was conducted to test the possibility of improving compliance with therapeutic regimens in hypertensives by means of certain simple arrangements. Patients were given written treatment instructions concerning hypertension, a personal blood-pressure follow-up card, and, for those who failed to attend their blood-pressure check-up, an invitation for a new check-up. Using matched pairs, 202 Finnish hypertensives were randomly allocated either to an ordinary or a reorganized treatment group. By means of the latter system, patient compliance could be significantly (p less than 0.01) improved: Only 4% of the patients in this group dropped out of treatment, compared with 19% in the ordinary treatment group. By the end of the year, blood pressure had been lowered by at least 10% in 95% of the patients in the reorganized group and in 78% of those in the ordinary group (p less than 0.01). This was achieved in approximately 60% of cases using chlorthalidone alone.


Alterations in lung volume and pulmonary function in relation to hemodynamic changes in acute myocardial infarction. To characterize the changes in lung volumes after acute myocardial infarction (AMI), and the relationship of these changes to other alterations in lung function which correlate with the severity of pulmonary vascular congestion, we made measurements of pulmonary hemodynamics, lung volume, closing volume, frequency dependence of total pulmonary resistance to forced oscillation, and arterial PO2 in 18 subjects with AMI. The most consistent finding was reduced lung volume which correlated with the severity of pulmonary diastolic hypertension. Frequency dependence of resistance showed a small but significant correlation with pulmonary hemodynamics. Closing volume measurements by the resident gas method in nine subjects was not related to hemodynamics. Follow-up studies at the time of hospital discharge revealed a significant return toward normal for arterial PO2, all lung volumes, and total pulmonary resistance at 9 Hz. Based on measurements in healthy subjects, the reduced lung volume after AMI may explain the changes in resistance. In acute and follow-up studies the degree of lung volume reduction and the severity of hypoxemia were strongly correlated.


Coronary artery spasm during exercise in patients with variant angina. Seven patients with typical variant angina without coronary stenoses greater than 50% developed angina and ST-segment elevation during treadmill exercise testing. In all cases the ST-segment elevation occurred in the same leads during exercise testing as during spontaneous attacks at rest. Five of the patients had developed spontaneous coronary spasm during coronary arteriography, in each case in the artery corresponding to the site of ST-segment elevation. In five patients, thallium was injected during the exercise test during which angina and ST-segment elevation occurred. In each case, a large perfusion defect not present at rest was found in the zone corresponding to the site of ST-segment elevation. These findings suggest that coronary artery spasm may occur during exercise in patients with variant angina.


Congenital abnormalities of the conduction system in two patients with tachyarrhythmias. Serial sections of the conduction system (CS) were performed in two patients with recurrent tachyarrhythmias. Case 1, a 34-year-old female who had dual atrioventricular (AV) nodal pathways with recurrent paroxysmal supraventricular tachycardia, committed suicide. Autopsy revealed an abnormally formed atrial septum with insertion of eustachian valve on the AV part of the pars membranacea. The intercuspid portion of the pars membranacea was muscular. The AV node was located adjacent to the membranous part of the ventricular septum rather than the central fibrous body. In addition, there was an accessory anterior AV node on the parietal wall of the right atrium. Case 2, a 13-year-old boy with history of recurrent ventricular tachycardia, died suddenly. CS revealed a right-sided, markedly septated bundle. The first part of right bundle branch was divided into three parts, which later joined together. Both cases showed fatty infiltration of the atrial septum, more than normal for the age of the patients. The relationship of the recurrent tachyarrhythmias to the congenital abnormalities in the CS in the two cases and the fatty infiltration is reviewed.


Enzymic assay for lecithin in amniotic fluid. We describe an enzymic method for determining lecithin in amniotic fluid. Phospholipase D from savoy cabbage is used to liberate choline from lecithin, and the liberated choline is oxidized to betaine and hydrogen peroxide by choline oxidase from Arthrobacter globiformis. The hydrogen peroxide forms a colored complex with phenol and 4-aminoantipyrene in the presence of horseradish peroxidase, and this color is measured spectrophotometrically. The method is relatively easy, precise, and accurate, requires less time and sample than other methods, and offers a reliable alternative to the lecithin/sphingomyelin ratio techniques for assessing fetal lung maturity.


Radioimmunoassay for pantothenic acid in blood and other tissues. We describe a radioimmunoassay for pantothenic acid in biological tissues. D-Pantothenic acid was conjugated with bovine serum albumin by use of a bromoacetyl derivative of pantothenic acid, and antibody to this antigen was raised by injecting it into the foot pads of rabbits. For the radioimmunoassay, a 100-fold dilution of the resulting antiserum was incubated with radiolabeled pantothentic acid. The antibodies were precipitated and dissolved, and the radioactivity of the solution was measured in a liquid scintillation counter. Between 5 and 125 ng of pantothenic acid can be detected in 75 muL of tissue extract. Validation included recovery and precision studies, parallelism with tissue extracts, and competitive binding studies. Results of the radioimmunoassay and those of microbiological assay with use of Lactobacillus plantarum correlated well (r = 0.80).


Interaction between human IgG and human creatine kinase isoenzyme-1 in serum: a route for the intravascular catabolism of creatine kinase-1? In vitro incubation, at 37 degrees C, of human creatine kinase isoenzyme-1 (isoenzyme BB) and human immunoglobulin G in a buffer results in the formation of a complex of high relative molecular mass (Mr approximately 825,000), which contains both proteins. This complex also forms in vitro if creatine kinase isoenzyme-1 is incubated with fresh human serum. The creatine kinase activity of the complex obtained from either incubation is extremely labile, even in the presence of a chelating agent and a thioglycerol. We present evidence for the existence of this complex in the sera of patients who have detectable serum creatine kinase isoenzyme-1 activity. Sera with high activities of creatine kinase isoenzyme-2 do not appear to have this complex. We therefore speculate that complexing of creatine kinase isoenzyme-1 with serum immunoglobulin G may be a pathway of enzyme degradation.


Absorbance measurement in cuvettes lying longitudinal to the light beam. Measurement in cuvettes with their longitudinal dimension parallel to the light beam, while under the influence of a centrifugal force, has many advantages that are not immediately apparent. This survey summarizes the advantages in comparison to cuvettes standing with their long axis perpendicular to the light beam: in the latter the pathlength is fixed by the geometry, but if the cuvette is lying longitudinal to the light beam the pathlength is given by the filling volume. Because of the dependence of the pathlength on the filling (reaction) volume when cuvettes are lying longitudinal to the light beam, the calculation formulas show that the calculation factors, both for enzyme and substrate determination, become independent of the reagent volume form. This is demonstrated by simple equations and confirmed in practice by use of the (Cobas) Bio centrifugal analyzer in assays for glucose and aspartate aminotransferase.


Detergent-containing glucose oxidase reagent for use with the Beckman glucose analyzer. We describe a modified glucose oxidase reagent for use in the polarographic determination of glucose with the Beckman "Glucose Analyzer." The reagent contains 1 mL/L of a surfactant (Brij-35 250 g/L solution) as the wetting agent instead of glycerol. Precipitation of components associated with the formulation recommended by Fischl et al. [Clin. Chem. 21, 760 (1975)] does not occur with this reagent. It can be used immediately after preparation. When compared to analytical performance of the commercially prepared reagent, the precision was unchanged by the modified reagent, but the upper limit of accurate response was diminished (7.5 g/L vs. 6.7 g/L). The modified reagent is less expensive than are commercially prepared reagents.


Discrete modular system for automation of radioimmunoassay of serum choriomammotropin. We describe a discrete automated radioimmunoassay system for determining choriomammotropin (placental lactogen) in human serum. With the present system it can be measured in as many as 37 unknown sera (50 muL) and three quality-control sera, in duplicate, within 1.5 h. The time required for sample preparation, incubation (15 min), and separation of free and bound radioactivities (a 150 mL/L polyethylene glycol solution is superior to a twofold volume of absolute ethanol) is less than 45 min. The remaining time required is for counting and data processing. Intra-assay precision is 4.6% (CV). The modular approach endows the instrumentation with much flexibility, and consequently is suitable for automation of a wide range of assay protocols.


Aqueous primary standard for use in measuring cholesterol by the cholesterol oxidase method. An aqueous primary standard is needed for measuring cholesterol by enzymatic procedures. Sodium deoxycholate solubilizes cholesterol in water. Crystalline cholesterol (1.00, 3.00, 4.00, and 5.00 g/L), added to a solution containing 150 g of this compound per liter of a 9 g/L saline solution, was measured by a cholesterol oxidase procedure, with a centrifugal analyzer. The solubilizer did not interfere. When compared to an isopropanol-based commercial cholesterol standard, the cholesterol standard in solubilizer showed excellent correlation (r = 0.986; m = 0.989). Day-to-day variation for the mixture during nine days was small (CV, 2.9% at 100 g/L, 3.7% at 3.00 g/L, and 1.8% at 4.00 g/L). Linearity was maintained up to 5.00 g/L. The cholesterol concentration in four reference sera so analyzed maintained CV's of less than 4%. The viscosity of the mixture was similar to that of serum. The standard mixtures, stored at room temperature for 360 days, remained stable. The solubilized cholesterol standard is shown to be suitable for use in the enzymatic procedure.


A further comparison of radioassay results for serum and plasma. We evaluated results for serum vs. plasma for 12 selected radioassay procedures. Blood samples were collected with heparin, oxalate-fluoride, or ethylenediaminetetraacetate as anticoagulant and compared with the corresponding serum samples. In combination with a previous report [Kubasik, N.P., and Sine, H.E., Clin. Chem. 24 137 (1978)] we have now tested a total of 25 different analytes, using 31 manufacturers' kits. Differences were again demonstrated between serum and plasma that may be of sufficient magnitude to alter clinical interpretation of the results. Assays also demonstrated differences based on the procedure used.


Removal of contaminating substances before gas-liquid-chromatographic determination of aldosterone in urine. This paper deals with removal of contaminants before gas-chromatographic determination of aldosterone in urine. Urine is incubated with beta-glucuronidase, which hydrolyzes all beta-glucuronides except aldosterone-18-glucuronide. The contaminants (the aglycones released and other methylene chloride-soluble substances) are extracted with methylene chloride. Solvolysis of the aqueous phase liberates aldosterone from aldosterone-18-glucuronide, which then is extracted with methylene chloride and oxidized by use of periodic acid. The resulting lactone can be easily separated by one-dimensional thin-layer chromatography and determined by gas-liquid chromatography.


Suggested isosbestic wavelength calibration in clinical analyses. I recommend the use of isosbestic points for conveniently checking the wavelength scale of spectrophotometers in the ultraviolet and visible regions. Colorimetric pH indicators, hemoglobin derivatives, and other radiation-absorbing substances that are convertible into stable isomers of different absorption spectra provide a means for calibrating many different wavelengths by comparing the absorptivities of these isomers in equimolar solutions. The method requires no special precautions and results are independent of substance concentration and temperature between 4 and 45 degrees C. Isosbestic calibration may be important for (e.g.) coenzyme-dependent dehydrogenase activity determinations and in quality assurance programs.


Improved determination of serum theophylline by gas chromatography with use of a nitrogen-phosphorus detector. We describe a method for the 7-min determination of theophylline in less than 100 muL of serum. The procedure requires no centrifugation or solvent evaporation after extraction. Butylation is done on the gas-chromatographic column by injecting the serum extract followed by a butylating mixture which contains 1-iodobutane as the alkylating agent. The method is precise, accurate, and free of interference. Results correlate well with those by ultraviolet spectrophotometry.


Quality of sweat test performance in the diagnosis of cystic fibrosis. The sweat test, correctly performed, appears to be reliable, but results were found to be unreliable about half the time in 84 small community hospitals assessed. The reasons are inexperienced and untrained technicians, the infrequency of test performance, the use of unstandardized equipment, and the lack of appropriate standards. It behooves all clinical laboratory directors to re-examine and standardize their procedures and use the recommendations of the Cystic Fibrosis Foundation. Physicians should be made aware of the current situation and send their patients only to laboratories that perform the test reliably.


Comparison of three iodination methods for human somatotropin. Human somatotropin (growth hormone) iodinated by use of lactoperoxidase or Chloramine T added stoichiometrically has been compared with hormone iodinated by conventional Chloramine T method. The three preparations were indistinguishable, both in their stability to storage and in their suitability for use in radioimmunoassay. Degradation of tracer, as measured by decreasing precipitability with trichloroacetic acid, was found to be an unsatisfactory measure of immunoreactivity, because aggregates of high relative molecular mass formed during storage at--15 or--80 degrees C are less immunoreactive.


Improved method of purifying some radiolabeled glycopeptide hormones. We describe a simplified method for purifying radiolabeled glycopeptide hormones (thyrotropin, lutropin, follitropin, and human choriogonadotropin), which results in preparations of over 95% immunoreactivity. This method is based on competitive elution from columns of the plant lectin, concanavalin A, and is applicable to glycopeptide hormones derived from several species.


Radioimmunoassay for chlorpromazine in plasma. A radioimmunoassay for chlorpromazine in plasma is described. The antiserum was obtained by immunizing rabbits with a conjugate of bovine serum albumin and N-(2-carboxyethyl)desmethylchlorpromazine. It is specific for chlorpromazine and its minor active metabolite, N-desmethylchlorpromazine. Other known active or inactive chlorpromazine metabolites and other psychotropic drugs tested do not cross react with the antiserum. Less than 34 pg of the drug can be detected in 200 muL of plasma. As many as 100 samples can be processed in a day by one technician. Concentrations of chlorpromazine can be measured in 200-muL samples of plasma collected as late as 48 h after a single oral 25-mg dose.


Gas-chromatographic determination of mephenytoin and desmethylmephenytoin, after off-column alkylation. We describe a gas-liquid chromatographic method for determining mephenytoin and its active metabolite, desmethylmephenytoin, in human serum. 5-Methyl-5-phenylhydantoin is used as the internal standard. The method involves extraction of the drugs by adsorption onto charcoal and off-column derivatization to their pentyl derivatives. Peak height and concentration are linearly related and the day-to-day CV for therapeutic concentration is about 2 to 6%. No interferences by endogenous compounds or drugs commonly used for seizure control have been encountered.


Performance of the Du Pont aca ammonia method. We evaluated the performance of the Du Pont aca ammonia procedure with regard to (a) linearity, (b) precision, (c) interferences, and (d) effect of anticoagulants. Linearity extends to 2,000 mumol/L. The SD of the method was essentially constant (2 to 3 mumol/L) and independent of the NH3 concentration. Hemoglobin, bilirubin, and lipemia do not interfere. Either EDTA or heparin is suitable as anticoagulant. Recovery of NH3 added to plasma samples averaged 102% (range: 97--107%). We established normal values by measuring NH3 in 188 plasma samples from apparently healthy individuals. The 95% confidence range is from 10 to 35 mumol/L. The aca ammonia method compares very well with that of Kingsley and Tager but correlates less strongly with that of Reinhold and Chung. We describe a protein-based solution with stable NH3 concentration that is suitable as a control material.


New technique for solid-phase immunoassay: application to hepatitis B surface antigen. I describe a new technique for improving the sensitivity of the solid-phase "sandwich" assay, by using the through-passage receptacle in a novel flow-communication device. The technique allows a large volume of serum to flow through the antibody-coated receptacle repeatedly during the incubation and is thus termed the "flow-through large-volume incubation" method. Binding of 125I-labeled hepatitis B surface antigen to its corresponding antibody on a solid-phase by this method was more rapid and persistent than binding by the conventional method. When the method was applied to the first incubation of the sandwich assay, the test for the antigen was rendered four-, eight-, and 32-fold as sensitive as an accepted third-generation test for the antigen, by incubating 5-mL volumes of serum at (a) room temperature for 18 h, (b) 45 degrees C for 8 h, or (c) room temperature for seven days, respectively.


Immobilized enzymes in continuous-flow analysis. Glucose oxidase, uricase, and urease were immobilized on the interior surface of activated polyamide tubing. The shelf-life of such enzyme bearing tubes was at least six months. The tubes were used for continuous-flow analysis of glucose, uric acid, and urea with conventional systems and with hybrid micro-scale systems in which modules of different manufacture were combined. The length of enzyme-bearing tube required for each system was ascertained empirically. Each tube could be used for several thousand assays, but glucose oxidase-bearing tubes were more stable than urease- or uricase-bearing tubes. Results for patients' samples correlated well with results obtained by accepted methods.


Stability of dried blood spots on paper, as used in screening neonates for hypothyroidism. Thyroxine values for dried blood spots on paper may be low if the paper has been long exposed to high ambient temperature and (or) humidity and (or) to sunlight. Low values are associated with poor extractability of the red color from the spots. We recommend appropriate precautions for such samples.


Determination of amniotic fluid phospholipids by thin-layer chromatography, with use of a hydrogen flame ionization detector. We describe a new specific method for measuring lecithin, sphingomyelin, and phosphatidylglycerol in amniotic fluid by thin-layer chromatography, with use of a hydrogen flame ionization detector. After extraction and acetone precipitation to isolate surface-active lecithin, the phospholipids are separated on a thin rod of refractory and chemically stable material, having an outer coating of a bonded, sintered partition medium. Two solvent systems are used to develop the chromatograms, chloroform/methanol/water to separate lecithin and sphingomyelin, tetrahydrofuran/methylal/methanol for phosphatidylglycerol. Then the rod is passed through an hydrogen flame; the resulting ions produced generate a current, which is amplified and fed to a potentiometric recorder. The height of integral curves was proportional to the area under each peak. For quantitation we used an internal standard (lysolecithin in the first system. phosphatidyldimethylethanolamine in the second). The method requires less than 5 mL of amniotic fluid; results are available within 6 h.


On-line continuous potentiometric measurement of potassium concentration in whole blood during open-heart surgery. We describe a flow-through system with an ion-selective electrode for measurement of blood potassium ion concentration, continuously and on-line off the extracorporeal blood circulation in an operating theater during human open-heart surgery. Comparison measurements were made with the SMA flame photometer (blood plasma) and an Orion SS 30 sodium/potassium analyzer (whole blood). The potassium concentration values obtained with the flow-through system agree well with the ones determined with the flame photometer. The time delay of the measurement with the flow-through system was relatively long (2 min) but delays of only 10--20 s seem feasible. Short time delays can deepen insight and simplify rational treatment under surgery conditions.


Iodinated digoxin derivatives with improved reactivity and stability, for use in radioimmunoassay. We report the synthesis and properties of a new digoxin derivative, prepared by reacting cyanogen bromide-activated digoxin with tyramine. The derivative is extremely stable, and its monoiodinated form can be isolated in 85--90% purity by a single thin-layer chromatographic step. [125I]Tyramine-digoxin is immunologically stable for at least four months (immunoreactivities, 90% or greater throughout), with a shelf life of about six months. Nonspecific binding of less than 10% was observed for several batches during this time.


Spironolactone-associated digoxin radioimmunoassay interference. Apparent digoxin was measured in the serum of 21 patients receiving spironolactone and in 21 controls, by use of a sequential-saturation 3H-radioimmunoassay (RIA) and an equilibrium 125I-RIA. No patient had been given digoxin for at least four weeks. "Digoxin" values in the former group were significantly (p less than 0.05) higher than in the control group, and often were in or near the "therapeutic" range by the equilibrium 125I-RIA, but not by the sequential-saturation 3H-RIA. Canrenone (a major active metabolite of spironolactone) in the serum of the former group was measured by a newly developed liquid-chromatographic technique and correlated (r = 0.73) with "digoxin" concentrations by the 125I-RIA. However, external addition of canrenone to control serum in comparable concentrations did not cause appreciable "digoxin" values by the 125I-RIA. These findings suggest that other metabolites of spironolactone are responsible for the assay interference, the degree of which appears to depend on antibody specificity. Therefore, assay specificity should be established in clinical laboratories by using digoxin-free serum from patients ingesting spironolactone, and not by using spironolactone- or canrenone-fortified digoxin-free serum.


A liquid-chromatographic analysis for ritalinic acid [alpha-phenyl-alpha-(2-piperidyl) acetic acid] in serum. We describe a "high-performance" liquid-chromatographic assay for measuring ritalinic acid in 300 muL of serum. The procedure includes adsorption of ritalinic acid and internal standard (alpha,alpha-dimethyl-beta-emthylsuccinimide) from serum onto charcoal, from which both compounds are eluted with methanol. The methanol extract is evaporated, the residue dissolved in a 93/7 (by volume) mixture of potassium phosphate buffer (20 mmol/L, pH 3.8) and acetonitrile, and an aliquot of this solution is chromatographed. We used a mu-Bondapak C-18 column with the above-mentioned buffer system as mobile phase, a flow rate of 2.0 mL/min, and a column temperature of 40 degrees C. Ritalinic acid and internal standard are detected by their absorbance at 192 nm and quantitated by measuring peak heights. The procedure allows for the reliable analysis for ritalinic acid in serum at concentrations greater than 50 microgram/L. Analytical recoveries were greater than 73% and the method affords good day-to-day precision (CV, less than 12%).


Determination of serum aminotransferases: activation by pyridoxal-5'-phosphate in relation to substrate concentration. To investigate the activation of aspartate- and alanine aminotransferases by pyridoxal-5'-phosphate, we determined the enzymatic activity in serum in two different ways: (a) Preincubation of the serum alone or the serum with pyridoxal-5'-phosphate and starting the reaction by the addition of the serum sample or the serum sample + coenzyme, respectively. (b) Preincubation of the serum or the serum with pyridoxal-5'-phosphate in the reaction medium and starting the reaction by adding 2-oxoglutarate. There are only small differences in activities of both aminotransferases determined according to these two different methods. The stimulation by pyridoxal-5'-phosphate is also of the same order, when both methods are compared. Further, these enzymatic activities were measured with use of various concentrations of substrates. From our experiments we conclude that the degree of stimulation of the apoenzyme of the two enzymes is independent of which way the enzymatic reaction is carried out or the substrate concentration, except that aspartate aminotransferase activity is more stimulated by the coenzyme at higher 2-oxoglutarate concentrations.


Blood cyanide and thiocyanate concentrations after administration of sodium nitroprusside as hypotensive agent in neurosurgery. In a group of 50 neurosurgical patients receiving sodium nitroprusside as a hypotensive agent, in doses of 50--2131 microgram/kg body weight, whole blood cyanide was estimated before infusion of drug, during infusion, immediately after infusion, and 1 and 3 h later. Cyanide values increased significantly, reaching the maximum in a sample taken immediately after infusion (mean value, 333; highest value, 1800 microgram/liter). Values correlated closely with both dose and rate of infusion. Blood thiocyanate, estimated before, immediately after, and 3 h after the infusion, showed no significant changes in thiocyanate concentration. In any case, no signs or symptoms of cyanide or thiocyanate poisoning were observed. We conclude that sodium nitroprusside, in the proper dose and given at the proper rate, does not precipitate cyanide poisoning.


Microviscosity of amniotic fluid phospholipids, and its importance in determining fetal lung maturity. Fluorescence polarization measurements of microviscosity (apparent viscosity within the hydrophobic center of lipid bilayers) of amniotic fluid correlate well with lecithin/sphingomyelin ratios determined by thin-layer chromatography. In addition to lecithin, phosphatidylglycerol and phosphatidylinositol are important for determining fetal lung maturity, but the lecithin/sphingomyelin ratio gives no information concerning these other phospholipids. The microviscosity of sphingomyelin significantly exceeded that of lecithin over the temperature range 25--37 degrees C; values for phosphatidylglycerol, phosphatidylinositol, and phosphatidylserine were lower. Phosphatidylglycerol, phosphatidylinositol, and phosphatidylserine, added individually, significantly decreased the microviscosity of dispersions with lecithin/sphingomyelin ratios corresponding either to fetal lung immaturity or maturity. Phosphatidylglycerol caused the greatest decrease in both. Mixtures of the three phospholipids in the proportions found in term amniotic fluid decreased the microviscosity of fluids with either mature or immature lecithin/sphingomyelin ratios by 23--27%. When each was present in the proportion found in tracheal aspirate (twice that of term amniotic fluid), the decreases uere 46--50%. This technique quickly and precisely indicates not only fetal lung maturity but also the presence of important phospholipids other than lecithin and sphingomyelin.


Quantitation of urinary porphyrins by use of second-derivative spectroscopy. We describe a simple, rapid procedure for measuring porphyrins in urine. After acidification of a urine specimen with hydrochloric acid, the second derivative of the absorption spectrum is recorded in the region of the Soret band maximum. The amplitude of the deflection is linearly related to porphyrin concentration, and the point at which the second derivative minimum occurs provides an estimate of the ratio of uroporphyrin to coproporphyrin in the sample. Nonspecific interference is not significant and reproducibility is excellent. Porphyrin excretion was studied in 24-h urine collections from men and non-pregnant as well as pregnant women. Median daily porphyrin excretion (nmol) was: men, 65.0; non-pregnant women, 60.1; and third-trimester pregnant women, 175.0. Distribution of these data was significantly skewed; upper and lower 2.5 percentile limits are given.


Comparison of liquid- and gas-liquid chromatographic assays of 5-fluorouracil in plasma. A liquid-chromatographic assay of 5-fluorouracil in plasma is described. Advantages of this procedure over gas chromatography are the simpler extraction procedure, elimination of the need for a derivitization step with silylating agents, and a 20-fold greater sensitivity. The minimum detectable concentration of 5-fluorouracil in plasma is 25 microgram/L. The enhanced sensitivity enabled measurement of the concentrations of 5-fluorouracil found in plasms of patients receiving continuous intravenous infusions of the drug; such concentrations are generally unmeasurable by gas-chromatographic methods. We compared liquid-chromatographic and gas-chromatographic measurements on 36 plasma samples obtained from patients after rapid intravenous injection of 5-fluorouracil. The resulting correlation coefficient was 0.97, with a regression slope of 0.94. The liquid-chromatographic method is free of interference from other cytotoxic agents, anti-emetics, and folate derivatives that are frequently combined with 5-fluorouracil therapy.


Erythrocyte protoporphyrin/heme ratio in the assessment of iron status. The protoporphyrinemia of iron deficiency is well recognized. Clinically, information on the protoporphyrin/heme molar ratio in whole blood offers certain advantages over protoporphyrin measurement alone. A procedure for determining this ratio is reported. Protoporphyrin is extracted, solubilized, and measured fluorometrically. Heme (as hemin chloride) is precipitated with the blood proteins, the precipitate is dissolved in an alkaline/pyridine solvent, and the resulting bispyridine ferriprotoporphyrin is measured spectrophotometrically. The molar ratio of these two metabolites correlates well with values for plasma ferritin, plasma iron, transferrin saturation, hemoglobin, and hematocrit. In some cases the ratio increases detectably while the other variables, especially hematocrit and hemoglobin, remain normal. Evidently it is a more sensitive index to iron status. For healthy men and women, the mean ratio is 16.0 (SD, 5.3). The mean + 3 SD, or a ratio of 32, is distinctly abnormal, as shown by a confirmatory test. We validated the test by surveying routine blood specimens obtained from several population groups.


Improved radioimmunoassay of urinary estriol. We report a rapid double-antibody radioimmunoassay for urinary estriol. Advantages over other current methods include: (a) 30-min hydrolysis; (b) total incubation time, 55 min; (c) assay unaffected by urinary glucose; (d) no degradation of estriol evident during hydrolysis; (e) superior (85%) analytical recovery of estriol conjugates; (f) linear standard curve by logit-log extrapolation; (g) good correlation (r = 0.83) with total estrogen determination by a generally accepted colorimetric method; (h) only 20 muL of urine required; and (i) the detection range is 1.9 to 100.5 mg/24-h urine.


Dual-isotope separation technique for radioassay. Precision of calculated results affected by degree of separation. The dual-isotope separation technique for radioassay is reviewed. An expression is derived describing the effect of counting errors on the precision of the calculated bound count rate in the dual-isotope technique. This mathematic model is tested in experiments using sodium iothalamate (I-125) as a marker in the Phadebas radiosorbent assay of cobalamin (Co-57). The coefficient of variation of results calculated on the basis of the dual-isotope technique is shown to be dependent on the amount of supernatant removed. The conclusion is that relatively large amounts of the supernatant must be removed before counting if the dual-isotope technique is to give acceptable results. The experimental model is proposed as a simple test of the suitability of a projected dual-isotope system.


Dual-isotope separation technique for radioassay of cobalamin. Cobalamin is assayed by a dual-isotope separation method using sodium [125I]iothalamate as a marker. Two systems are used: one in which the incompletely-separated bound fraction is counted and compared with the single-isotope method in which the bound fraction is separated by washing (Phadebas radiosorbent assay); and one in which an aliquot of the free fraction is counted. In the dual-isotope method counting bound fractions, about 97% of the supernatant is removed by pouring from silicone fluid separators. The results for serum samples obtained using dual- and single-isotope methods were similar (between run coefficients of variation 5--7%). Experimental errors were smaller in the dual-isotope method. A factor in the kit standards, presumably the absence of proteins, was found to affect the separation technique, resulting in relatively large experimental errors for standards in the single-isotope method. Washing the solid phase in the single-isotope method apparently resulted in a loss of bound isotope. In the dual-isotope method counting free fractions reasonable precision was obtained (coefficient of variation of serum samples 6.6%) even though only about 56% of the supernatant (free fraction) was counted.


[Microdetermination of lead and cadmium in blood and urine by graphite furnace atomic absorption spectrophotometry (author's transl)]. A microtechnique is described for determination of lead and cadmium in blood (0.1 ml) and in urine (0.2 ml) using extraction into methyl isobutyl-ketone after chelation by ammonium pyrrolidine dithiocarbamate. Mineralization, precipitation of proteins or addition of hemolysing reagents are not necessary. Blood is hemolysed by addition of deionised water. pH of urine is fixed by a pH 5 buffer. This microtechnique is simple, rapid, reproducible and suitable for routine analysis. In healthy subjects it gives values comparable to data of other workers: blood 158 microgram/l lead (s = 40, n = 18); 0.7 microgram/l cadmium (s = 0.3, n = 10); urine 16 microgram/l lead (s = 9.4, n = 10); 0.5 microgram/l cadmium (s = 0.3, n = 10).


Release of hypoxanthine from and enzyme depletion in rat heart cell cultures deprived of oxygen and metabolic substrates. In a monolayer cultures of neonatal rat heart cells deprived of oxygen and metabolic substrates we measured (1) the release of hypoxanthine, a product of ATP catabolism, and (2) enzyme depletion with creatine phosphokinase (CPK) and alpha-hydroxybutyrate dehydrogenase (alpha-HBDH). Taking samples each hour up to 7 h of anoxia we were able to demonstrate that hypoxanthine release precedes cellular enzyme depletion. The release rate of hypoxanthine is maximal in the second hour of anoxia and the depletion rate of CPK and alpha-HBDH is maximal in the fourth hour. These results suggest that for early diagnosis of damage to heart cells due to deprivation of oxygen and metabolic substrates the measurement of hypoxanthine release is preferred to that of enzyme release.