An interlaboratory comparison of methods used to assess antioxidant potentials.

Many analytical methods are used to measure the antioxidative activity of substances yet little is known about the comparability of the test results between laboratories. After an initial evaluation of a broad range of methods conducted by one laboratory, the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, the trolox equivalent antioxidant capacity (TEAC) assay, the lipid assay (or 2,2'-azobis(2-aminepropane) (ABAP) assay) and the thiobarbituric acid (TBA) assay were selected to be evaluated in the interlaboratory study. The antioxidative potentials of trolox, tocopherol, lipochroman-6, ascorbic acid, 4-methyl-brenzcatechin, and/or 3,5-di-tert-butyl-4-hydroxytoluene (BHT) were assessed using each of the methods. These methods were then evaluated in respect of their reproducibility and classification properties. Based on the results of this study, the DPPH assay followed by the TEAC assay yielded the best results based on reproducibility and sensitivity both within one laboratory and between laboratories. The results of the interlaboratory study were then compared with the single center results obtained from the commercially available photochemolumiescence (PCL) kit. To assess the transferability of chemical data to biological systems, they were also compared with the single center results obtained using the cell-based Dichlorodihydrofluoresceine (DCFH) assay.

Dose-dependent access of murine anti-epidermal growth factor receptor monoclonal antibody to tumor cells in patients with advanced laryngeal and hypopharyngeal carcinoma.

The murine IgG2a monoclonal antibody (mAb) EMD 55900 was produced against the human epidermoid carcinoma cell line A431, with binding occurring to the polypeptide chain of the external domain of human epidermal growth factor receptor (EGF-R). In the present clinical study, 12 patients with advanced squamous cell carcinoma of the larynx or hypopharynx received a single dose of either 20, 100 or 400 mg EMD 55900 3 days prior to laryngectomy and neck dissection. Clinical signs and laboratory parameters of toxicity, development of human anti-mouse antibodies, and mAb plasma concentrations were monitored. In tumor specimens studied from primary tumors and lymph node metastases, expression of EGF-R, distribution of EMD 55900, and occupation of EGF-R by EMD 55900 (double staining) were determined by immunohistochemistry. Single-dose administration of EMD 55900 was very well tolerated in all patients, and good (100 mg) to excellent (400 mg) homogeneous binding of mAb to EGF-R was obtained in the advanced laryngeal and hypopharyngeal carcinomas studied.

EGFR and PCNA expression in oral squamous cell carcinomas--a valuable tool in estimating the patient's prognosis.

We investigated 100 cases of oral squamous cell carcinomas immunohistologically with respect to the expression of the epidermal growth factor receptor (EGFR) and the proliferating cell nuclear antigen (PCNA). The results were correlated with a new malignancy grading of the invasive tumour areas and the clinical outcome of the patients to estimate the individual prognosis. In conclusion, the amount of antigen expression of both antigens increases with the increasing grade of malignancy of the oral squamous cell carcinoma. Furthermore, there is a statistically significant correlation between the amount of antigen expression and the patient's prognosis. An overexpression of EGFR and PCNA is associated with a short survival of the patient. Both antigens detect relevant tumour biological parameters and are worthy factors in estimating the individual prognosis in patients suffering from oral squamous cell carcinomas.

A comparative study on proliferation, macromolecular synthesis and energy metabolism of in vitro-grown Ehrlich ascites tumor cells in the presence of glucosone, galactosone and methylglyoxal.

Proliferation of in vitro grown Ehrlich ascites tumor cells is completely inhibited by 0.2-0.4 mM methylglyoxal and 1-2 mM glucosone or galactosone without severely affecting viability (dye exclusion test); no phase-specific arrest of cell growth is observed. Incorporation of [14C] thymidine into the acid-insoluble fraction of the cells decreases within a few minutes to less than 50% of that in controls in the presence of 0.4 mM methylglyoxal, and 2 mM glucosone or galactosone causes a comparable inhibition of DNA synthesis after 2 h or 4 h, respectively. The action of 0.4 mM methylglyoxal inhibits incorporation of [14C] leucine within a few minutes by more than 70%, while 2 mM glucosone and galactosone are significantly less effective (50%-60% inhibition after 12 h). While methylglyoxal and galactosone do not severely affect lactate production of the cells, 2 mM glucosone reduces glycolysis by 60%-70%; ATP/ADP ratios did not fall below 3.5 in the presence of the inhibitors (controls 4-6). It is suggested that the reaction potentialities of the oxaldehyde function of the inhibitors play an important role in their growth-inhibitory activity, besides exerting a specific effect on hexokinase (glucosone) and UTP-trapping activity.

Uridylate trapping, induction of UTP deficiency, and stimulation of pyrimidine synthesis de novo by D-galactosone.

d-Galactosone (d-lyxo-2-hexosulose) is phosphorylated and metabolized to the uridine diphosphate derivative in AS-30D hepatoma cells and rat liver. These reactions were catalysed in vitro by galactokinase and hexose-1-phosphate uridylyltransferase. Nucleotide analyses by high-performance liquid chromatography and enzymic assays revealed that this galactose analogue interferes with cellular pyrimidine nucleotide metabolism leading to a deficiency of UTP. [(14)C]Uridine labelling of hepatoma cells indicated a division of [(14)C]uridylate from UTP into UDP-galactosone; the latter was formed at a rate of more than 1.7mmolxh(-1)x(kg AS-30D or liver wet wt.)(-1). As a consequence of UTP deficiency, d-galactosone (1mmol/1 or 1mmol/kg body wt.) strongly enhanced the rate of pyrimidine synthesis de novo as evidenced by incorporation of (14)CO(2) into uridylate and by an expansion of the uridylate pool. This resulted in a doubling of the total acid-soluble uridylate pool within 70min in the hepatoma cells and within 110min in rat liver. Combined treatment of hepatoma cells with d-galactosone and N-(phosphonoacetyl)-l-aspartate, an inhibitor of aspartate carbamoyltransferase, prevented the expansion of the uridylate pool and led to a synergistic reduction of UTP to 10% of the content in control cells. Hepatic UTP deficiency was selective with respect to other nucleotide 5'-triphosphates but was associated with reduced contents of UDP-glucose, UDP-glucuronate, and UDP-N-acetylhexosamines. Isolation of the UDP derivative of d-galactosone revealed an extremely alkali-labile UDP-sugar, probably an isomerization product of UDP-galactosone, that was degraded by elimination of UDP with a half-life of 45min at pH7.5 and 37 degrees C. The instability of UDP-galactosone may contribute in vivo to limit the time period of severe uridine phosphate deficiency in addition to the compensatory role of pyrimidine synthesis de novo. During the initial time period, however, d-galactosone is effective as a powerful uridylate-trapping sugar analogue.

The effect of glucosone on the proliferation and energy metabolism of in vitro grown Ehrlich ascites tumor cells.

1) Proliferation and energy metabolism of in vitro growth Ehrlich ascites tumor (EAT) cells in the presence of glucosone, (D-arabino-3.4.5.6-tetrahydroxy-2-oxo-hexanal) a competitive inhibitor of hexokinase, were studied. 2) Proliferation of the cells was completely inhibited by 2 mM glucosone without severely affecting viability (dye exclusion test). No phase specific arrest of cell growth was observed. 3) Incorporation of [14C]thymidine into an acid insoluble fraction of the cells decreases to 5% of the control within 8-10 h. Incorporation of [14C]leucine begins to slow down immediately after treatment with glucosone. 4) The inhibitor (2 mM) reduces the lactate production of the cells by 60%, respiration by about 20%; the ATP/ADP ratio slows down from 4.75 to 3.5. 5) The total inhibition of cell proliferation by 2 mM glucosone cannot be explained exclusively by inhibition of hexokinase activity and impairment of energy metabolism. Because of a lack of specificity, glucosone is not a suitable inhibitor for studies on the relationship between hexokinase activity and cell proliferation of tumor cells.