Interaction of native alpha-cyclodextrin, beta-cyclodextrin and gamma-cyclodextrin and their hydroxypropyl derivatives with selected organic low molecular mass compounds at elevated and subambient temperature under RP-HPLC conditions.

The main focus of this study was to explore the capability of native alpha-cyclodextrin, beta-cyclodextrin and gamma-cyclodextrin and their hydroxypropyl derivatives for host-guest interaction with 7,8-dimethoxyflavone, selected steroids (estetrol, estriol, estradiol, estrone, testosterone, cortisone, hydrocortisone, progesterone and 17alpha-hydroxyprogesterone) and polycyclic aromatic hydrocarbons (toluene, naphthalene, 1,8-dimethylnaphthalene, 1-acenaphthenol, acenaphthylene and acenaphthene) under reversed-phase liquid-chromatography conditions. The study revealed that native cyclodextrins interact more efficiently with the analytes investigated than do their hydroxypropyl counterparts. In the low-temperature region, enormously high ratios were observed for polycyclic aromatic hydrocarbons, particularly 1,8-dimethylnaphthalene, acenaphthene and acenaphthylene chromatographed on a beta-cyclodextrin-modified mobile phase. In such a case, the retention times of the polycyclic aromatic hydrocarbons were strongly reduced (e.g. from 127 to 1.2 min for 1,8-dimethylnaphthalene) and were close to the hold-up time of the high-performance liquid chromatography (HPLC) system (0.7 min). Moreover, chiral separation of 1-acenaphthenol optical isomers was observed and the elution order of the enantiomers was determined. Within the steroids group, strong interaction was observed for estradiol and testosterone. The results of cluster analysis indicate that beta-cyclodextrin as well as gamma-cyclodextrin and its hydroxypropyl derivative can be most effective mobile-phase additives under reversed-phase HPLC conditions for 3D-shape-recognition-driven separation, performed at subambient and elevated temperatures, respectively.

Neoexpression of ABH and Lewis blood group antigens in human hepatocellular carcinomas.

Expression of blood group ABH, Lewis, and sialylated-Lea antigens in human hepatocellular carcinomas and the adjacent nontumorous liver tissues was investigated with the use of seven monoclonal antibodies against these carbohydrate determinants. Chromatogram antibody-binding assay and solid-phase enzyme immunoassay of the upper-phase neutral glycolipids revealed the tumor-associated expression of blood group A-active glycolipids incompatible with blood-type status of the patients, a blood group A-active glycolipid with mobility on thin-layer chromatography between the known 6- and 8-sugar blood group A-active glycolipids in human erythrocytes, blood group H-active glycolipids, and blocked synthesis of Lea-active glycolipids with or without concomitant accumulation of Leb-active glycolipids. Immunohistochemical analysis of the fixed tissues with the use of an avidin-biotin-peroxidase complex method revealed blood group antigens in biliary epithelial cells but not in parenchymal liver cells. However, hepatocellular carcinoma cells in some cases expressed H and Leb antigens. Although only type 1 chain H antigen was detected in biliary epithelial cells, both type 1 and type 2 chain H antigens were found in hepatocellular carcinoma cells.

Clinical significance of plasma vascular endothelial growth factor in gastrointestinal cancer.

Circulating vascular endothelial growth factor (VEGF) was measured in gastric and colorectal cancer patients using an enzyme-linked immunosorbent assay (ELISA). Firstly, serum and plasma samples were collected from 20 normal controls to compare the values of VEGF and to determine which specimen type was most suitable for detecting circulating VEGF. Seventeen of 20 normal controls had plasma VEGF levels under the limit of detection (15 pg/ml) and the levels of the remaining three controls were 21, 22 and 38 pg/ml. In contrast, all serum samples indicated high levels of VEGF (mean 238 pg/ml), ranging from 44 to 450 pg/ml. In a time-course test of two normal controls serum VEGF values increased markedly between 30 and 60 min and remained high, whilst plasma VEGF values were low up to 480 min. Thus, plasma samples are more suitable for the measurement of circulating VEGF. Next, plasma VEGF levels were examined in 44 patients with gastric cancer (8 early, 7 advanced without remote metastasis and 29 metastatic), 13 with colorectal adenoma (2 with focal cancer) and 26 with colorectal carcinoma (8 advanced without metastasis and 18 metastatic) before treatment. An extremely high plasma concentration of VEGF was seen in some cancer patients with metastasis. To discriminate these patients, a cut-off level was determined, considering both the distribution of the sample concentration and the upper limit of 95% confidence area of VEGF in the cancer patients without metastasis. The cut-off value was 108 pg/ml and most cancer patients without metastases had VEGF levels below the cut-off value. In 11 of 29 metastatic gastric cancer patients (38%) and 9 of 18 metastatic colorectal cancer patients (50%), plasma VEGF levels were higher than the cut-off value. Survival was also analysed in the patients with metastasis. It was significantly longer in the patients with low VEGF levels (below the cut-off) than in those with high VEGF levels (logrank test, P = 0.042). 34 patients with metastasis (19 gastric cancer and 15 colorectal cancer) were treated with systemic chemotherapy, and their pretreatment levels of plasma VEGF and conventional tumour markers (CEA and CA19-9) were evaluated in relation to response. The response to chemotherapy was significantly higher in patients with low VEGF levels (< or = 108 pg/ml) than in those with high VEGF levels (P = 0.047). Such a difference was not observed with CEA/CA19-9. In conclusion, plasma VEGF is a useful marker for tumour metastasis and patient survival, and a possible predictive factor for the response of patients with gastrointestinal cancer to chemotherapy.

Prospective and randomized trial of lipiodol-transcatheter arterial chemoembolization for treatment of hepatocellular carcinoma: a comparison of epirubicin and doxorubicin (second cooperative study). The Cooperative Study Group for Liver Cancer Treatment of Japan.

A randomized, controlled clinical trial was conducted to compare the use of epirubicin (EPI) and doxorubicin (DOX) in Lipiodol (Laboratoire Guerbet, Roissy-Charles-de-Gaulle Cedex, France)-transcatheter arterial chemoembolization as a treatment of hepatocellular carcinoma. One hundred ninety-two hospitals participated, and 415 patients were enrolled in the study during the period between October 1989 and December 1990. The patients were randomly allocated to group A (EPI) or group B (DOX) by a centralized telephone registration. The actual doses of EPI and DOX were 72 mg/body and 48 mg/body, respectively. The 1-, 2-, and 3-year survival rates were, respectively, 69%, 44%, and 33% for group A and 73%, 54%, and 37% for group B. There were no statistically significant differences (P = .2296, log-rank test). When each group of patients was classified retrospectively into high-risk and low-risk subgroups based on the severity index calculated by the Cox regression model from the significant prognostic factors (the pretreatment tumor size, the pretreatment serum alpha-fetoprotein level, tumor encroachment, and Child's classification), the survival curve of the low-risk DOX subgroup was significantly superior to that of the low-risk EPI subgroup (P = .0182). However, there was no significant difference between the high-risk subgroups (P = .4606). The change in the serum alpha-fetoprotein level, the extent of Lipiodol accumulation in the tumor, and the extent of tumor reduction after the treatment did not show any significant differences between the groups. The white blood cell count in group B showed a tendency to decrease slightly more than in group A at 3 weeks after Lipiodol-transcatheter arterial chemoembolization. In conclusion, there was no statistically significant difference between the survival curves of the EPI and DOX groups in Lipiodol-transcatheter arterial embolization treatment of hepatocellular carcinoma.

Measurement of serum p53 protein in patients with small cell lung cancer and results of its clinicopathological evaluation.

Serum p53 protein levels were measured in 36 patients with small cell lung cancer (SCLC) and 35 patients with benign lung diseases in order to evaluate the relationship of these levels to clinicopathological features of SCLC. Serum levels of p53 protein were measured by an enzyme-linked immunosorbent assay, p53 protein level was 23.92 +/- 6.78 pg/ml in patients with SCLC, and similar to that (17.47 +/- 2.86 pg/ml) in patients with benign lung diseases. By the clinical stage of SCLC, the mean level of p53 protein was 16.68 +/- 4.62 pg/ml in 21 patients with limited disease, and lower than that in 15 patients with extensive disease (34.05 +/- 14.84 pg/ml) (P = 0.23). The levels of p53 protein were not correlated with age, smoking index, or presence of cancer history for patients with SCLC. However, immunohistochemical examination disclosed a mild correlation between the expression of p53 protein by SCLC tumor and p53 protein serum level (r = 0.45, P = 0.02). Two patients with SCLC had an elevated serum level of p53 protein (> 2 S.D. above the mean for benign lung diseases). However, measurement of p53 protein serum level was not found to be clinically useful for detection of SCLC.

Serum concentrations of cortisone and cortisol in premature infants.

To determine the relationship between biological active cortisol and its inert metabolite cortisone accurately in premature infants, serum cortisone and cortisol concentrations were measured by reversed-phase high-performance liquid chromatography (HPLC) in a group of 232 premature infants and in a control group of 127 children and 88 adults. In the control group, serum cortisone concentrations were greater than serum cortisol levels during the first 2 months after birth; cortisol levels were higher than cortisone levels after 2 months of age. However, in premature infants, serum cortisone concentrations were greater than serum cortisol levels even after the first 2 months, and total concentrations of cortisone and cortisol were equal to those in controls. Results were then analyzed according to the equivalent gestational age of premature infants. Cortisone was predominant in premature infants older than 32 weeks of equivalent gestational age, but cortisol was higher than cortisone from equivalent gestational age 24 to 31 weeks. These findings suggest that the ability of premature infants to secrete glucocorticoids resembled that of normal controls. Also, the fetal zone of the cortex, which is associated with a predominance of cortisone, remained functional in premature infants for a longer time than in control infants. Our findings that in premature infants cortisone was predominant compared with cortisol and the sum of cortisone and cortisol was equal to that in the controls indicate that cortisone cannot be disregarded whenever the cortisol level is estimated, although cortisone itself is recognized to be biologically inactive. Simultaneous measurement of serum cortisone and cortisol concentrations is important when adrenocortical function is being determined, especially in premature infants.

Clinicopathological study on combination therapy consisting of arterial infusion of lipiodol-dissolved SMANCS and transcatheter arterial embolization for hepatocellular carcinoma.

Combination therapy (LpTAE) consisting of arterial infusion of a lipophilic anticancer drug, SMANCS, dissolved in an oily lymphographic agent, lipiodol (LPD), and transcatheter arterial embolization (TAE) for hepatocellular carcinoma (HCC) was studied with special reference to the pathological findings. A total of 32 patients were subjected to surgical resection after LpTAE. The pattern of LPD deposition in the tumor was examined by CT scan (Lipiodol CT, LpCT) at 7 days and/or 1 month after LpTAE. The resected materials were examined radiographically with soft X-rays and histologically. LPD was deposited in tiny daughter nodules with a diameter of less than 5 mm and in tumor thrombi as well as in the main tumors, which showed necrotic change. Part of the LPD flowed out from the main tumor via the drainage vein and was deposited in the capsular invasion, resulting in necrosis. LPD accumulated almost exclusively within the blood spaces of trabecular-type HCC, creating a pattern corresponding to a cast of the tumor vessels, which showed prominent necrosis. On the other hand, LPD was not deposited in scirrhous, compact, or well-differentiated HCC, which showed little or no necrosis. It was demonstrated that LpCT images, which accurately depicted the existence and the extent of LPD deposition and necrosis in the tumor, were useful for precise evaluation of the therapeutic effect. Our findings indicate that LpTAE and LpCT are valuable for the diagnosis and treatment of HCC and should play a central role in systemic therapeutic approaches to this disease.

Treatment of hepatocellular carcinoma by transcatheter hepatic arterial injection of radioactive iodized oil solution.

After 12 days of culture, VX2 carcinoma cells were inoculated into the liver of 16 rabbits; 14 days later, 131I-labeled iodized oil ([131I]-Lp) suspended in lipiodol was injected into the hepatic artery. Selective accumulation of the contrast material in the tumor for an extended time was evident on X-rays and hepatic scintiphotographs. The antitumor effect was remarkable. [131I]-Lp agents warrant further examination for their clinical usefulness. Internal radiation therapy by transcatheter hepatic arterial injection of [131I]-Lp (group A) was evaluated in 9 patients with hepatocellular carcinoma (HCC, tumor stage III or IV) associated with liver cirrhosis (LC) and compared with combination therapy of Lp-TAE (group B) in 18 patients with HCC (tumor stage III or IV) associated with LC. In group A, serum AFP levels dropped rapidly in eight of the nine patients who had an elevated initial level of more than 500 ng/ml. The average reduction in tumor size was 50% in eight cases as determined by computed tomography. Histological examination of one resected liver specimen at 3 months after the third injection of [131I]-Lp revealed microscopic features highly suggestive of a radiation effect in the [131I]-Lp-containing area. The 1-year survival value for patients with HCC was estimated at 49.0% using the Kaplan-Meier method. The survival of patients treated with internal radiation therapy tended to be better than that of those treated with Lp-TAE (P = 0.119).

Evaluation of endoscopic injection sclerotherapy with and without simultaneous ligation for the treatment of esophageal varices.

For more effective and simple endoscopic injection sclerotherapy (EIS) for esophageal varices, we developed an EIS procedure with ligation (EISL) that is non-invasive, in which EIS and endoscopic variceal ligation (EVL) are performed simultaneously. In this study, we compared EISL and EIS in a randomized sample of patients (n = 14 for each procedure). For EISL, EVL was performed, including the injection site, after the injection of 5% ethanolamine oleate with iopamidol (EOI) into a varix. The mean number of treatment sessions required for eradication of esophageal varices was 2.3+/-0.5 for EISL and 3.9+/-0.8 for EIS (P < 0.001); the mean number of treatment sites was 6.2+/-2.2 for EISL and 14.0+/-5.0 for EIS (P < 0.001); the mean total amount of EOI used was 13.8+/-5.2ml for EISL and 26.3+/-9.8ml for EIS (P < 0.001). There were no significant differences in rates of recurrence of varices or in bleeding between the two groups. For EISL, fewer treatment sessions and less sclerosant were sufficient, probably because the sclerosants were more effective due to the blockage of variceal blood flow by the ligation. This method should provide a novel modification of EIS.

Circulating vascular endothelial growth factor (VEGF) is a possible tumor marker for metastasis in human hepatocellular carcinoma.

Vascular endothelial growth factor (VEGF) is closely related to angiogenesis in various human cancers. However, little is known of its circulating levels in hepatocellular carcinoma (HCC). We examined circulating VEGF levels in chronic liver disease to assess their clinical significance. Plasma VEGF concentrations were determined, by enzyme immunoassay, in patients with chronic hepatitis (CH; n = 36), liver cirrhosis (LC; n = 77), and HCC (n = 86) for a cross-sectional study. Plasma VEGF levels in healthy controls (n = 20) and CH, LC, and HCC patients were 17.7 +/- 5.4 (mean +/- SD), 30.6 +/- 22.8, 34.4 +/- 27.0, and 51.1 +/- 71.9 pg/ml, respectively. The levels were significantly elevated in the HCC group, compared with the control, CH, and LC groups. Plasma VEGF levels in stage I, II, III, IVA, and IVB HCC patients were 27.6 +/- 16.1, 26.5 +/- 13.7, 35.8 +/- 15.3, 45.4 +/- 39.4, and 103.1 +/- 123.2 pg/ml, respectively. The stage IVB patients with remote metastasis showed significantly marked elevation compared with the patients at the other stages. Platelet numbers were weakly correlated with plasma VEGF levels in the HCC group. Plasma VEGF level was highly elevated in patients with HCC, particularly those with metastatic disease. We consider that plasma VEGF is a possible tumor marker for metastasis of HCC. Circulating VEGF may be derived mainly from the large burden of tumor cells, and partly from platelets activated by the vascular invasion of HCC cells.

Clinical significance of cortisone and cortisone/cortisol ratio in evaluating children with adrenal diseases.

Cortisone is derived from the peripheral metabolism of cortisol and lacks biological activity. The rapid interconversion between cortisol and cortisone has been well established. The altered equilibrium between these steroids may regulate glucocorticoid activity in various tissues. We evaluated the serum levels of cortisol and cortisone, and the cortisone/cortisol ratio in ten children with adrenal diseases using reversed-phase high performance liquid chromatography. Children with hypoadrenalism exhibited a greater decrease in cortisol as compared with cortisone. Cortisone levels were higher than cortisol levels, and the cortisone/ cortisol ratio exceeded 1.0. Children with adrenal cancer exhibited normal or high values of cortisol, whereas cortisone levels were decreased and the cortisone/cortisol ratio was decreased to nearly zero. The ratio returned to normal after the excision of adrenal tumors. The simultaneous evaluation of cortisol, cortisone, and cortisone/cortisol ratio provides a clinical clue of adrenal diseases.

Fullerenes separation with monomeric type C30 stationary phase in high-performance liquid chromatography.

The temperature effect on the separation of fullerenes in LC was examined using monomeric type C30, C18 and C8 alkyl bonded stationary phases. It appears that the C30 phase exhibits superior separation ability for fullerenes. It is observed that the maximum retention temperature of fullerenes on the C30 phase is around 20 degrees C. A strong correlation between the changes in NMR spectra and the retention behavior of the solutes was found. The interpretation of the retention behavior of fullerenes on the alkyl bonded stationary phases, including the behavior in subambient temperature, is discussed using the information obtained by CP-MAS solid-state NMR spectroscopy and LC.

Analysis of pesticides in environmental water samples by solid-phase micro-extraction-high-performance liquid chromatography.

Solid-phase micro-extraction (SPME) followed by high-performance liquid chromatography (LC) has been applied to analyze pesticides in water samples. A device interfaces SPME to the LC injection process by solvent extracting analytes from the fiber and then introducing the solvent into the LC injector. LC analysis with UV detection was performed first with a conventional column (4.6 mm I.D.). To enhance efficiency of SPME, three extraction conditions, stirring, temperature and salt concentration, were optimized. Subsequently, semi-microcolumn LC (1.5 mm I.D.) was evaluated for this method, giving lower detection limits and less solvent consumption. Detection limits were within Japanese regulatory limits for drinking water. The SPME-LC method was applied to real-world environmental water samples.

Comparison of separation behavior of benzodiazepines in packed capillary electrochromatography and open-tubular capillary electrochromatography.

Packed column capillary electrochromatography (CEC), open-tubular CEC and microcolum liquid chromatography (LC) using a cholesteryl silica bonded phase have been studied to compare the retention behavior for benzodiazepines. It has been found that packed column CEC gives better resolution, faster analysis time than microcolumn LC for benzodiazepines maintaining similar selectivity except for some solutes which are charged species under the separation conditions. However, open-tubular CEC gave different selectivities to a larger extent for charged benzodiazepines from that which should be produced by the chromatographic properties of the cholesteryl silica phase. Charged species migration times are mainly influenced by electrophoretic mobility rather than the chromatographic interactions.

The quantitative structure-activity relationship approach to the mutagenicity of N-nitrosomethylaniline compounds.

In order to clarify the correlations between the substituent properties and the mutagenicity of N-nitrosomethylaniline compounds, the quantitative structure-activity relationship approach has been applied. The results indicate that the property and the shape of the para-substituent groups influence the mutagenic potency, although this has not yet been verified for the meta-substituted compounds.

Fibrous stationary phase in capillary electrochromatography.

Capillary electrochromatography (CEC) using fibrous cellulose acetate (CA) stationary phase was investigated. The advantage of this fiber-packed column is relatively easy preparation process compared with other conventional CEC columns, such as particle-packed and wall-coated capillaries. CA fibers are manually packed into a capillary with two guide liners and fixed with a frit at the column inlet. The separation characteristics of this column were investigated using n-alkyl p-hydroxybenzoates (parabens) as the sample probe. It has been demonstrated that the use of a short column length and a specially designed tee-connector as the injection device should make the separation performance and efficiency much higher on the fiber-packed columns. Sufficient separation between methyl and n-butylparabens is obtained on the 5-cm-packed column and linear relationships between the injection time and the peak area are observed. Bubble formation is not encountered during the analysis.

Solid phase micro extraction coupled with semi-microcolumn high performance liquid chromatography for the analysis of benzodiazepines in human urine.

SPME/semi-microcolumn HPLC (SPME/LC) was investigated to analyze benzodiazepines in human urine samples. SPME conditions such as extraction time, extraction temperature, salt concentration and pH of matrix, flush volume and desorption time were optimized by extracting various drugs from a prepared water matrix. Combination of adding saturated salts to the matrix and controlling pH ranged from neutral to weakly alkaline conditions makes the increase of extraction efficiency. Under optimal condition SPME/LC is more sensitive than direct HPLC analysis without the SPME process. The limits of detection (LODs) was several ppb level and the relative standard deviation (RSD) was < 15% when human urine samples were analyzed by this analytical system. The system is very useful and is enough to assay benzodiazepines in a human urine sample without tedious and complex analytical procedures. In this paper the applicability of SPME/LC to the analysis of benzodiazepines in human urine samples was reported. In addition, the extension to the evaluation of SPME/LC/MS system was also described.

Determination of bis(amidinohydrazones) by micellar electrokinetic capillary chromatography.

A micellar electrokinetic capillary chromatography method was developed for the separation and determination of aliphatic congeners of bis(amidinohydrazones) in standard solution. Eight bis(amidinohydrazones) could be determined in less than 15 min at an applied voltage of 22 kV, using 0.05 M sodium phosphate as buffer (pH 7.0) together with 1 mM N-cetyl-N,N,N-trimethylammonium bromide. Hydrostatic sample injection was employed. The method exhibited good repeatability and a linear range of 2.5-100 micrograms ml-1. A detection limit of 1 micrograms ml-1 was achieved. The method also allows the determination of bis(amidinohydrazones) in human serum samples.

Large round cell granulomas induced by murine sarcoma virus (Moloney) in mouse brains.

The effect of murine sarcoma virus of Moloney strain on central nervous system was examined morphologically in Swiss mice of different age. A single intracranial inoculation of cell-free virus solution resulted in the induction of characteristic intracerebral granulomas in 82.8% of the newborn to 5 day-old group, in 71.4% of the 6 to 10 day-old group, and in 68.0% of the 11 to 20 day-old group. The mean latency periods to tumor recognition were 16.5, 21.1, and 33.5 days, respectively. The granuloma consisted of inflammatory cell infilrations, reactive gliosis, and richly developed blood vessels. The lesions consistently contained numerous characteristic large round cells. In cases of long-survival, the findings included reparative changes, such as extensive gliosis, withdrawal of inflammation, and a decrease in the numbers of large round cells and blood vessels. These lesions were tentatively designated as "large round cell granuloma." The early foci of the granoloma were composed of proliferating glial cells and large round cells at the subependymal regions. Electron microscopically these large round cells had abundant intracytoplasmic fibrils quite similar to gliofibrils. Numerous C-type virus particles were present in the intercellular nad perivascular spaces, and occasionally budded from cell membranes of the large round cells and vascular endothelia. The large round cells were considered to be reactive astrocytes activated by biral infection. It was conclided that MSV-M was not a sarcomogenic but a granulomogenic virus in mice. Control animals showed no pathological changes.

Molecular-dynamics simulation for liquid chromatographic interactions: effect of mobile phase composition.

A molecular-dynamics simulation method has been applied to investigate the influence of the mobile-phase composition on the retention of solutes in HPLC. The distribution profiles of the distance between two atoms in ODS ligands were constructed to characterize the conformation of ODS ligand molecules. The distinct difference of ODS conformation is observed by comparing molecular models consisting of solvent molecules at each solvent composition. The distribution profiles of the distance between the mobile-phase solvent molecules and ODS ligand molecules were also constructed to characterize the distribution of the solvent molecules at each composition. In all distribution profiles, the difference in the distribution due to a change in the solvent compositions was very clearly found, and the facts seem to be very reasonable. The distribution profiles of the distance between the solute, n-propylbenzene, and the terminal carbon atom in the ODS ligand, and between the solute and the silicon atom in the ODS ligand have been also constructed to see the distribution of the solutes in the separation system. The calculated solute distribution in the ODS-methanol/water system is very consistent with the actual chromatographic retention behaviors.