Adhesive strength of autologous fibrin glue.

To establish an easy and rapid method for measuring the adhesive strength of fibrin glue and to clarify the factor(s) most affecting the strength, a study was made on the effect of the concentration of plasma components on the strength of cryoprecipitate (Cryo) prepared from a subject's own autologous plasma to be used as fibrin glue. The adhesive strength of the Cryo was measured with various supporting materials instead of animal skin using a tester of tension and compression. The results were as follows: (1) the strength of Cryo applied to ground flat glass (4 cm2) was significantly greater than that applied to clear glass, clear plastic, or smooth and flat wood chips; (2) the adhesive strength of Cryo depended on the concentration of thrombin with the optimal concentration being 50 units/ml; (3) the concentration of CaCl2 did not affect the adhesive strength of Cryo; (4) the adhesive reaction was dependent on the temperature and the adhesive strength more quickly reached a steady state at 37 degrees C than at lower temperature; (5) the adhesive strength was correlated well with the total concentration of fibrinogen and fibronectin. These results indicate that the adhesive strength of Cryo can be easily and quickly evaluated using a tester and ground glass with thrombin at 50 units/ml, and that the adhesive strength of Cryo can be predicted from the total concentration of fibrinogen and fibronectin.

Comparative study of autologous fibrin glues prepared by cryo-centrifugation, cryo-filtration, and ethanol precipitation methods.

To establish a speedy preparation method for the fibrinogen-rich fraction (FRF) from autologous plasma using fibrin glue, we compared the concentrations and yields of coagulation factors in FRF prepared by 3 methods. Human plasma from healthy volunteers was divided into 3 samples. Two samples were frozen at -20 degrees C in a freezer and defrosted in a 4 degrees C water bath. One sample of defrosted plasma was centrifuged and FRF was obtained (C method). Another sample of defrosted plasma was filtered and FRF was obtained (F method). The last sample was treated with cold ethanol(1/10) in a 4 degrees C water bath and FRF was obtained after centrifugation (E method). The concentrations of fibrinogen, fibronectin, factor XIII, and plasminogen in each obtained FRF were measured and yields were calculated. (1) The volume of FRF obtained by the E method was greater than that by the C method, but less than that by the F method. While the variation in volume obtained by the E method was the lowest among the 3 methods; (2) the concentrations of fibrinogen obtained by the E and C method were similar, but the yield from the E method was the highest; (3) the concentration and yield of fibronectin from the E and C method were similar and were greater than those by the F method; (4) the concentration and yield of factor XIII from the E method were significantly higher than those from the other methods; (5) the E method preparation time was about 1 h, the shortest among the 3 methods. These results indicate that high quality FRF from autologous plasma can be prepared easily and within 1 h by the E method.

Valproic acid elimination rate and urinary excretion of its glucuronide conjugate in patients with epilepsy.

We previously encountered a patient with epilepsy who exhibited rapid elimination of sustained-release valproic acid (VPA) administered at the dose of 2.8 g/d as a sodium salt. The purpose of this study was to clarify the relationship between the VPA elimination rate and the proportion of the dose excreted in urine as its glucuronide conjugate (VPA-G) in epileptic patients. Twenty-four-hour urine was collected from four epileptic patients who had taken VPA orally (age: 16-39 y, weight: 50-63 kg, dose: 1.0-2.8 g/d). VPA and its metabolites were detected by gas chromatography-mass spectrometry. The amounts of VPA, VPA-G, 3-keto VPA, and 3-OH VPA excreted in the 24-h urine were 1.8-13.2, 178-2158, 125-320, and 8.6-18.7 mg (converted into VPA), respectively, and 0.2-0.5, 20.5-88.7, 5.8-18.7, and 0.6-1.0% of the dose administered, respectively. The dose of VPA correlated well with the proportion of the dose excreted in urine as VPA-G in each patient, and the patients administered a high dose excreted a large amount of VPA-G in the urine. Thus, differences in the VPA-G production rate may be one of the major variable factors affecting the elimination of administered VPA. We also present a dynamic model of VPA in the kidney which may explain the VPA elimination phenomena in humans on the basis of the data obtained here regarding the concentrations of VPA and its metabolites in plasma and their urinary excretion levels.

Specificity of p-aminohippurate transport system in the OK kidney epithelial cell line.

Substrate specificity of the p-aminohippurate (PAH) transport system was investigated in the OK kidney epithelial cells. PAH uptake by OK cells from the basal side was inhibited by beta-lactam antibiotics such as benzylpenicillin (PCG) and cefazolin. The inhibition of PAH uptake by PCG was competitive and the Ki value was calculated as 108.8 microM. Transcellular transport of PCG across OK cell monolayers occurred unidirectionally from the basal to apical side, and transcellular transport and basolateral uptake were inhibited by PAH, probenecid and beta-lactam antibiotics. The basolateral uptake of cefazolin and cefotiam was also inhibited by PAH and probenecid. The basolateral uptake of PAH and PCG were not affected by aliphatic dicarboxylates with 3 or 4 carbon atoms, but were strongly inhibited by those with 5 or 6 carbon atoms. The inhibitory effect became weaker for a longer dicarboxylate with 7 carbon atoms, then increased again with increasing number of carbon atoms. Such a pattern of inhibition by dicarboxylates is essentially the same with that observed in rat renal proximal tubules in situ. These findings suggest that the PAH transport system in OK cells has a substrate specificity similar to that in rat renal proximal tubules, which is involved in the active secretion of various organic anions including drugs.

Differential sensitivity of organic anion transporters in rat renal brush-border membrane to diethyl pyrocarbonate.

The effect of various chemical modifiers on p-aminohippurate (PAH) uptake by a potential-sensitive system and by an anion exchanger was studied in rat renal brush-border membrane vesicles. Among various chemical modifiers, diethyl pyrocarbonate (DEPC) selectively inhibited potential-sensitive PAH uptake but not the uptake by the anion exchanger. The inhibitory effect of DEPC on potential-sensitive PAH uptake was not due to the facilitated dissipation of membrane potential, which was evidenced by the studies with a potential-sensitive fluorescence dye diS-C3(5). The potential-sensitive PAH uptake was inhibited by DEPC in a concentration-dependent manner, and kinetic analysis showed that the decreased uptake of PAH in DEPC-treated vesicles was due to the decrease of Vmax. The inhibition of the PAH uptake was protected by the presence of organic anions during the DEPC treatment. These findings indicate that PAH transport by the potential-sensitive system and by the anion exchanger is mediated by structurally distinct transporters. Amino acid residue(s) modified by DEPC, most likely a histidine residue, should play an important role in the potential-sensitive transport of PAH in rat renal brush-border membrane.

p-Aminohippurate transport in apical and basolateral membranes of the OK kidney epithelial cells.

p-Aminohippurate (PAH) transport in apical and basolateral membranes of OK cells was studied to characterize each membrane process and to determine which process is important for the vectorial transport of PAH across the OK cell monolayers. PAH uptake from the basal side into OK cells was much higher than from the apical side, and was inhibited almost completely by anion transport inhibitors (probenecid and furosemide) and low temperature. On the other hand, PAH efflux from OK cells to the apical side was higher than to the basal side. The efflux across the apical membrane also was inhibited by the transport inhibitors and low temperature. The uptake of PAH across the basolateral membrane was inhibited by dicarboxylates such as alpha-ketoglutarate, and kinetic analysis showed that the inhibition was a mixed-type. These findings suggest that PAH transport in apical and basolateral membranes of OK cells is a specifically mediated process, and both processes contribute to the vectorial transport of PAH across the cell monolayers. PAH/dicarboxylate exchange may be involved in PAH transport in the basolateral membrane of OK cells.

Transport of organic anion in the OK kidney epithelial cell line.

Transport of an organic anion, p-aminohippurate (PAH), was investigated in an established cell line derived from the American opossum kidney (OK). Transepithelial transport and accumulation of PAH were measured in OK cells cultured on permeable supports. Specifically mediated transcellular transport of PAH across OK cell monolayers occurred only from basal to apical side, and the accumulation of PAH in the cells was much larger from basal side than from apical side. Both the transcellular transport of PAH from basal to apical side and the accumulation from basal side were temperature dependent, saturable, and inhibited by organic anions such as probenecid but not by organic cations. In contrast, such a specific transport of PAH was not observed in LLC-PK1, another epithelial cell line derived from pig kidney. These findings suggest that OK cells can transport PAH by the organic anion transport system unidirectionally from basal to apical side across the cell monolayers and that OK cells might be a useful model for the study of organic anion secretion in the renal proximal tubules.