Histidine as a dipolar eluent component in cation chromatography. II. Prediction of retention data for alkaline and alkaline-earth ions.

The utility of cation chromatography has been developed by the application of L-histidine as a multiprotic and dipolar (zwitterionic) eluent component. The method simplifies the cation analysis. The chromatographic characteristics of this system were studied in detail with a view to determining the selectivity and the mechanism by which the cations (Na+, K+, Mg2+, Ca2+) are retained. Complete separations were observed in the isocratic run over the eluent concentration range 3.0-6.0 mM at pH below 2.0. Sensitive detection was achieved using suppressed conductivity at the pH of isoelectric point of the histidine. Retention equations are derived for mono- and divalent cations eluted from ion-exchange separation column with multiple ionic eluents. The theory is based on the extension of ion-exchange equilibrium by protonation equilibria. The selectivity data for analyte and eluent species are deterrmined using the model from the experimental retention data by computer-assisted iterative calculations. The model was utilized to predict retention data. The results in three-dimensional retention surfaces together with species distribution graphs are presented.

Stage-related superoxide anion production of granulocytes of gynecologic cancer patients.

OBJECTIVE: To measure superoxide anion production of polymorphonuclear leukocytes (PMNLs) in 58 gynecologic cancer patients and compare to that of healthy controls. METHODS: PMNLs were separated from peripheral blood samples by Ficoll and subsequent Percoll gradient sedimentation. Baseline and phorbol-dibutyrate (100 nmol/l) stimulated superoxide anion production was measured spectrophotometrically as superoxide dismutase inhibitable reduction of ferricytochrome c (50 micromol/l) absorbance. Differences between the mean superoxide anion production of different patient groups and the control group were assessed by Student's t-test. RESULTS: The mean superoxide anion production of PMNLs of healthy controls was 1.855 nM/min/3 x 10(5) cells (SD=0.211 nM/min/3 x 10(5) cells). Superoxide anion production of gynecologic cancer patients and healthy controls varied in a wide range. PMNLs of patients had lower baseline and stimulated activity than those of healthy volunteers. The frequency of a mean superoxide production at least 2 x SD below the control showed a parallel increase with advancing stage. CONCLUSION: Granulocytes of gynecologic cancer patients have reduced capacity and inducibility of superoxide anion production already at an early stage of disease.

Age-dependent changes in transmembrane signalling: identification of G proteins in human lymphocytes and polymorphonuclear leukocytes.

In human neutrophils (PMNLs) we found that in the elderly IP3 formation was significantly decreased compared to that of young subjects. For FMLP receptor binding affinity and number no measurable differences occurred upon ageing, studying both the low or the high affinity receptors. The amount of ADP-ribosylated G proteins, catalysed by pertussis toxin (PT) or cholera toxin (CT), was significantly increased in PMNLs of the elderly. In lymphocytes, the PT-catalysed ADP ribosylation of G proteins was also increased with ageing, while the CT-catalysed ribosylation was decreased. The autoradiogram of [32P]ADP-ribosylated proteins by CT in lymphocytes of young individuals showed a major polypeptide of 40,000 M(r). In contrast, in lymphocytes of the elderly, the major polypeptide was 45,000 M(r). In PMNLs, CT labelled quite strongly the 45,000 M(r) band, mainly in the elderly. When PT was used, no age-related pattern changes could be demonstrated, while differences could be observed between the two types of cells. The use of antiserum P680 (G alpha common) showed no age-related pattern changes, while the intensity of the labelled proteins varies with age and cell type. The antiserum U46 (Go alpha) could identify in lymphocytes of young subjects two polypeptides 68,000 and 41,000 M(r). The prominent polypeptide in lymphocytes of the elderly was the 70,000 M(r) and no other polypeptides could be recognized. In PMNLs of young subjects the U46 and serum identified a range of species. In PMNLs of the elderly all these bands were weakly labelled. The present data indicate changes in the pattern and the quantity of G proteins in lymphocytes and PMNLs of elderly subjects.