Influence of hemodilution of plasma proteins on erythrocyte aggregability: an in vivo study in patients undergoing cardiopulmonary bypass.

Erythrocyte aggregation is known to be affected by a number of factors including the concentration of various plasma proteins. This study was performed to examine the in vivo effect of hemodilution of plasma proteins on erythrocyte aggregation in patients undergoing cardiopulmonary bypass (CPB) surgery. Blood samples were taken before, during, and after operation from 40 coronary artery bypass grafting patients who were operated with CPB and concomitant hemodilution (CPB, n=20) and who without (nonCPB, n=20). Erythrocyte aggregation was determined with a LORCA aggregometer, during which all samples were standardized to a hematocrit level of 40%. Results showed that in the CPB patients the aggregation index (AI) dropped to 44% of its preoperative baseline level 5 minutes after the start of hemodilution (from 47.7+/-10.1 to 26.6+/-11.4, p<0.01). Meanwhile, plasma concentration of fibrinogen (Fb) dropped to 55%, haptoglobin to 85%, ceruloplasmin to 55%, and albumin to 67%. In the nonCPB patients, however, there was only a slight drop in AI and the concentrations of plasma proteins during the similar period of time. On postoperative day 1, AI was rebounded to 37.1+/-12.4 in CPB patients compared with 44.3+/-11.7 in nonCPB patients. At baseline, AI was correlated only with Fb. During CPB and hemodilution, AI was correlated not only with Fb but also with haptoglobin and ceruloplasmin. Postoperatively, significant correlationship was found between AI and Fb, CRP, haptoglobin, ceruloplasmin, as well as albumin. These results indicate that hemodilution of plasma proteins significantly reduces the aggregability of erythrocytes in patients undergoing CPB. Besides Fb, other plasma proteins also contribute to AI during the early postoperative period when patients are recovering from CPB surgery.

High-resolution small-angle x-ray diffraction study of long-range order in hard-sphere colloidal crystals.

The long-range order parameters in single crystals of hard colloidal spheres grown in sediments of colloid-polymer mixtures are determined using synchrotron small-angle x-ray diffraction with a resolution of 10(-6) of the wave vector. The interplanar positional order derived from the width of lattice reflections extends over at least 500 lattice planes. The lattice planes are orientationally correlated within approximately 0.1 degrees throughout the crystals, whereas the stacking of hexagonal planes remains random.

Mechanisms of phase separation and aggregation in colloid-polymer mixtures.

The final structure of a colloidal system is greatly influenced by the mechanisms by which phase separation and aggregation occur. The drive to phase separate can be altered in colloid-polymer mixtures (which phase separate due to the depletion interaction) by varying the polymer concentration. Here, we use small angle light scattering to follow the phase separation in such mixtures and analyze the results within a framework indicated by previous results from microscopy investigations. The mechanisms of diffusion-limited cluster aggregation, reaction-limited cluster aggregation, and nucleation and growth are found to provide good descriptions of the phase separation regimes. The growth rate in the nucleation and growth regime is shown to be dependent on the polymer concentration.

Direct observation of crystallization and aggregation in a phase-separating colloid-polymer suspension.

The depletion-induced phase separation in a mixture of colloidal particles (PMMA-latex) and nonadsorbing polymers [poly(styrene)] in a solvent (mixture of tetralin, cis-decalin, and carbon tetrachloride) was investigated in real space with confocal scanning laser microscopy in the initial, intermediate, and final stage. It was found that the kinetics and the morphology of the phase separation strongly depend on the polymer concentration, and thus on the strength of the depletion-induced attraction between the colloidal particles. At moderate polymer concentrations, crystallization of the PMMA particles is enhanced. At higher polymer concentrations, only aggregation is observed, resulting in amorphous sediments. The aggregation is diffusion-limited or reaction-limited, depending on the polymer concentration. Digital image processing was used to determine the dependence of the aggregation rate and the size of the clusters on the polymer concentration.

Compatibility of gelatin and dextran in aqueous solution.

The temperature-composition phase diagrams of aqueous solutions of gelatin and dextran, which show liquid/liquid phase segregation, were explored at temperatures above the gelation temperature of gelatin. The compositions of the coexisting phases were found to show practically no dependence on temperature between 40 and 80 degrees C. Also, the total polymer concentration at which phase separation occurred was found to be nearly independent of temperature. These observations suggest an entropy-driven phase separation. An explanation in terms of depletion, reversible clustering, and subsequent transient network formation of gelatin at temperatures well above the temperature of gelation is suggested. Phase separation is found to be accompanied by strong fractionation of the molar mass distribution in the two phases.

In vivo manipulation of L1210 cell cycle phase distribution with alpha-difluoromethylornithine, 4-amidinoindan-1-one 2'-amidinohydrazone and N1-acetylspermine.

We investigated whether the in vivo growth inhibitory effect of the combination of 4-amidinoindan-1-one 2'-amidinohydrazone (CGP 48664A) and alpha-difluoromethylornithine (DFMO) is reversible by treatment with N1-acetylspermine (N1-acSp). DBA-2 mice were inoculated with 10(5) L1210 cell i.p. on day 0. From day 1 they received 2.50 mg CGP 48664A/kg i.p. once daily and 500 mg DFMO/kg i.p. twice daily. On day 5 they received 3 x 2500 nmol N1-acSp i.p. with 15-min intervals. L1210 cell numbers, S-phase percentage and polyamine contents, and liver and spleen polyamine contents were monitored in the following 48 h. Four days treatment with CGP 48664A/DFMO reduced L1210 cell numbers, S-phase, and spermidine. N1-acSp treatment increased L1210 spermidine from < or = 8 h and percentage S-phase from 12 h. Maxima for spermidine and S-phase were reached at < or = 8 and 18 h, respectively. These were below levels of untreated controls. Decreases were noted from 12 and 18 h, respectively. N1-acSp was detectable in L1210 from 0-18 h. Liver spermidine was decreased by CGP 48664A/DFMO. After N1-acSp treatment, liver N1-acSp and N1-acSd increased from < or = 8 h, reached maxima at < or = 8 and 10 h, respectively, and were undetectable from 15 h. We conclude that the in vivo growth inhibitory effect of CGP 48664A/DFMO is reversible by N1-acSp treatment. The liver is probably involved in N1-acSp terminal catabolism. The effect of the polyamine depletion-repletion scheme on S-phase cell numbers may be much more profound than present estimates from 5-bromo-2'-deoxyuridine incorporation.

In vitro manipulation of L1210 cell cycle kinetics with 4-amidinoindan-1-one 2'-amidinohydrazone, alpha-difluoromethylornithine and N1-acetylspermine.

We investigated whether in vitro L1210 growth inhibition by alpha-difluoromethylornithine (DFMO; 740 microM) and 4-amidinoindan-1-one 2'-amidinohydrazone (CGP 48664A; 1.7 microM) is reversible with N1-acetylspermine (N1-acSp). Influences of N1-acSp dose (1-100 microM), time (0-12 h at 100 microM), aminoguanidine (AG, 1 mM) and cell numbers (at 1 microM N1-acSp) on percentage S-phase, polyamine contents and viability were determined. DFMO/CGP 48664A decreased percentage S-phase from 58 to 26%, decreased spermidine (Sd) and spermine (Sp) contents 3-fold, but did not affect viability. With increasing N1-acSp dose, S-phase percentage and Sd contents increased concomitantly, reaching plateau values that were comparable with those of untreated controls. S-phase and Sd content increased from 4-6 h after N1-acSp administration, reaching plateau values from 11 and 6 h, respectively. N1-acSp content was dose dependent and increased linearly to reach plateau values from 8 h. AG did not affect any of these parameters. Addition of 1 microM N1-acSp to decreasing numbers of DFMO/CGP 48664A-treated cells caused increasing S-phase percentage, Sd and N1-acSp contents. We conclude that cell cycle kinetics of cultured L1210 cells can be manipulated by the induction of growth inhibition with DFMO/CGP 48664A and its subsequent abolishment with N1-acSp. N1-acSp accumulation rate and its subsequent conversion to Sd is relatively slow compared with intracellular Sd needs. The data support the notion that Sd is the most important polyamine for growth.

Oral administration of deuterium-labelled polyamines to sucking rat pups: luminal uptake, metabolic fate and effects on gastrointestinal maturation.

Non-physiological amounts of oral polyamines have been reported to induce precocious gut maturation in rat pups. The aim of the present study was to investigate organ distribution and metabolic fate of orally administered stable-isotopically labelled polyamines in rat pups. Pups received tetradeuterium-labelled putrescine (Pu-d4; 3 mumol), spermidine (Sd-d4; 5 mumol), spermine (Sp-d4; 3 mumol), or physiological saline twice daily on postnatal days 7-10 or 12-15. They were killed on days 10 and 15. We determined activities of ileal lactase (EC 3.2.1.23), maltase (EC 3.2.1.20), sucrase (EC 3.2.1.48) and diamine oxidase (EC 1.4.3.6) and established villus and crypt lengths. Polyamines and their labelling percentages in organs were determined by GC and mass fragmentography. Treatments did not affect growth rate, but caused lower weights of liver, kidneys and heart. Maltase activity increased, lactase decreased, whereas sucrase and diamine oxidase did not change. Villus and crypt lengths increased. Organ polyamine pools were labelled to different extents. Irrespective of the orally administered polyamine, all organs contained Pu-d4, SD-d4 and Sp-d4. Administered Pu-d4 and Sd-d4 were recovered mainly as Sd-d4, whereas Sp-d4 was recovered as Sp-d4 and Sd-d4. Total polyamines in a caecum, colon and erythrocytes increased, but increases were only to a minor extent with regard to labelled polyamines. Our data confirm precocious gut maturation by exogenous polyamines. Putrescine appears to be limiting factor. The exogenous polyamines were distributed among all investigated organs. They are not only used for the synthesis of higher polyamines, but also retroconverted to their precursors. Changes in erythrocyte polyamine contents suggest precocious stimulation of erythropoiesis.

Simultaneous determination of polyamines, N-acetylated polyamines and the polyamine analogues BE-3-3-3 and BE-4-4-4-4 by capillary gas chromatography with nitrogen-phosphorus detection.

We describe a method for the profiling of polyamines, N-acetylated polyamines and the polyamine analogues N1,N11-bis(ethyl)norspermine (BE-3-3-3) and 1,19-bis(ethylamino)-5,10,15-triazanonadecane (BE-4-4-4-4) in L1210 murine leukaemia cells by capillary gas chromatography with nitrogen-phosphorus detection. The method makes use of four intemal standards. Prepurification comprises deproteinization, isolation with Sep-Pak silica at pH 9.0, conversion to heptafluorobutyryl derivatives and postderivatization organic fluid extraction. Within- and between-series precisions (given as CV.s) for analysis of 1-2x10(6) cells were: putrescine 5.5 and 29.4%; spermidine 1.6 and 7.1%; and spermine 3.2 and 7.6%, respectively. Recoveries relative to the respective internal standards, were in the 70.6-104.7% range. Accuracy and precision of measurements of BE-4-4-4-4 can probably be improved by the introduction of a separate pentamine internal standard. We conclude that the method can be used for studying the effect of BE-3-3-3 and BE-4-4-4-4, and possibly their metabolites, on polyamine homeostasis (biosynthesis, retroconversion, transport, terminal catabolism) and polyamine function.

4-Amidinoindan-1-one 2'-amidinohydrazone (CGP 48664A) exerts in vitro growth inhibitory effects that are not only related to S-adenosylmethionine decarboxylase (SAMdc) inhibition.

The competitive S-adenosylmethionine decarboxylase (SAMdc; EC 4.1.1.50) inhibitor 4-amidinoindan-1-one 2'-amidinohydrazone (CGP 48664A) inhibits growth more effectively than the irreversible SAMdc inhibitor 5'-[[(Z)-4-amino-2-butenyl]methylamino]-5'-deoxyadenosine (AbeAdo), while having similar effects on polyamine contents. We hypothesized that growth inhibition by CGP 48664A is not merely accomplished by SAMdc inhibition. Concentration-related growth inhibitory effects of AbeAdo, CGP 48664A and methylglyoxal bis(guanylhydrazone) (MGBG) were investigated in L1210 cells that were additionally exposed to 10 microM AbeAdo. This concentration causes maximal growth inhibition, profound SAMdc inhibition and plateau polyamine contents. Almost complete inhibition of functional SAMdc activity by 10 microM AbeAdo was confirmed by demonstration of poor conversion of tetradeuterated spermidine to tetradeuterated spermine by gas chromatography-mass spectrometry. Increasing AbeAdo did not affect L1210 cell numbers, viability, nor polyamine contents. MGBG proved highly toxic. CGP 48664A did not affect L1210 polyamine contents, but cell numbers and viability decreased dose-dependently to 50% and 70% of control, respectively. We conclude that CGP 48664A inhibits L1210 growth not only through SAMdc inhibition, but also by an as yet poorly understood second effect with higher IC50. The alleged second effect of CGP 48664A appears important for its potent antitumor effect.

Estimation of 24-hour polyamine intake from mature human milk.

It has been suggested that milk polyamines stimulate GI tract proliferation and maturation in newborns. We determined human milk polyamine concentrations and estimated 24-h outputs on days 16 +/- 4 (n = 98), 44 +/- 3 (n = 97) and 91 +/- 6 (n = 25) after delivery. Median concentrations in micromolars were, respectively, putrescine 0.77, 0.63, and 0.63; spermidine 4.54, 3.07, and 2.73; spermine 3.76, 2.90, and 2.22; and total polyamines 9.82, 6.83, and 5.71. Concentrations of spermidine, spermine, and total polyamines decreased during the observation period. Putrescine, spermidine, and spermine milk/maternal plasma ratios were estimated to be 16-19, 14-24, and 44-75, respectively. It would appear that milk polyamines are derived from the high polyamine contents in the mammary gland and that they may be important in infant nutrition.

In vivo effects of 4-amidinoindan-1-one 2'-amidinohydrazone (CGP 48664A) and alpha-difluoromethylornithine (DFMO) on L1210 growth, cell-cycle phase distribution and polyamine contents.

We studied the in vivo effects of 4-amidinoindan-1-one 2'-amidinohydrazone (CGP 48664A), alpha-difluoromethylornithine (DFMO) and a combination of CGP 48664A-DFMO on tumor growth, cell-cycle phase distribution and polyamine contents. DBA-2 mice were inoculated i.p. with 10(5) L1210 cells on day 0, treated i.p. on days 1-4 and killed on day 5. As compared to controls, CGP 48664A, DFMO and the CGP 48664A-DFMO combination reduced L1210 cell numbers by 33, 43 and 85%, respectively. CGP 48664A did not affect cell-cycle phase distribution. DFMO and the CGP 48664A-DFMO combination caused a moderate and a heavy accumulation in G0/G1- and G2/M-phases, respectively. Compared with controls, the CGP 48664A-DFMO combination reduced putrescine, spermidine and total polyamines, but did not affect spermine. Compared with CGP 48664A, the CGP 48664A-DFMO combination caused lower putrescine and total polyamines, higher spermine, but no change in spermidine. Compared with DFMO, the CGP 48664A-DFMO combination caused higher putrescine and spermidine, lower spermine, but no change in total polyamine levels. We conclude that CGP 48664A potentiates the cystostatic effect of DFMO in vivo. The resulting growth inhibition is accompanied by an accumulation in G0/G1- and G2/M-phases and a reduction of putrescine and spermidine. The data suggest that perturbed polyamine composition rather than reduced spermidine or total polyamine pool size causes a profound growth inhibition.

In vivo growth inhibition of L1210 leukemia by 4-amidinoindan-1-one 2'-amidinohydrazone (CGP 48664A), a new inhibitor of S-adenosylmethionine decarboxylase.

We studied the in vivo growth-inhibitory effect of the new S-adenosylmethionine decarboxylase inhibitor 4-amidinoindan-1-one 2'-amidinohydrazone (CGP 48664A). L1210-bearing DBA-2 mice were treated with increasing CGP 48664A doses from 1 day after i.p. L1210 cell inoculation. Treatment was continued for 4 days, after which all mice were killed. CGP 48664A caused dose-related exponential decreases of L1210 cell numbers and spermidine and spermine contents. Putrescine contents increased exponentially. Polyamine changes in spleen and liver were less profound. L1210 growth inhibition was not accompanied by changes in cell cycle phase distribution. It is concluded that CGP 48664A is an effective inhibitor of S-adenosylmethionine decarboxylase but that CGP 48664A-induced changes in intracellular polyamine compositions are not necessarily the cause of growth inhibition.