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R L Czervionke

Publications and source records attributed to R L Czervionke.

15 recordsLinked to original sources
PubMed1986

Quantification of analyte and interferent by multipoint analysis.

We have investigated the application of multipoint kinetic curve-fitting methods to the determination of an analyte in the presence of a single interferent. Our model system for the analyte-interferent was creatinine-acetoacetate as determined with the kinetic Jaffé method. We examined the utility of the following multipoint approaches: simultaneous equations, multivariable linear regression, and iterative multivariable nonlinear regression. With appropriate restrictions, all approaches could detect acetoacetate interference and quantify both creatinine and acetoacetate. A two-stage linear regression approach was both versatile and computationally simple. Interferent was detected in the first stage, and both analyte and interferent were quantified in the second stage if the interferent was assumed known and an adequate fit of the model to the data was obtained. Using the two-stage linear regression model, we obtained results for 10 ketotic patients that correlated well with results by enzymatic methods for creatinine (r = 0.976) and acetoacetate (r = 0.995); we also demonstrated that creatinine could be quantified in the presence of the antibiotic cefoxitin.

Acetoacetates↗
PubMed1985

Effect of human plasma lipoproteins on prostacyclin production by cultured endothelial cells.

Prostacyclin (PGI2) production by bovine aortic or human umbilical vein endothelial cells increased when either human high density lipoproteins3 (HDL3) or low density lipoproteins (LDL) were added to a serum-free culture medium. At low concentrations and short incubation times, HDL3 produced more PGI2 than LDL, but LDL was just as effective as HDL3 in 18-hr incubations with high concentrations of lipoproteins. Neither lipoprotein was toxic to the cultures as assessed by [3H]leucine incorporation into cell protein. The stimulatory effect of HDL3 and LDL on PGI2 production decreased as growing cultures became confluent. Incubation with lipoproteins neither enhanced arachidonic acid release nor increased PGI2 formation when the cells were stimulated subsequently with ionophore A23187, indicating that the lipoproteins do not affect the intracellular processes involved in PGI2 production. The addition of albumin reduced the amount of PGI2 formation elicited by HDL3 or LDL. As compared with albumin-bound arachidonic acid, from 6- to 13-fold less PGI2 was produced during incubation with the lipoproteins. Furthermore, the amount of PGI2 formation elicited by the lipoproteins in 18 hr was 4-fold less than that produced during incubation with a fatty acid mixture containing only 5% arachidonic acid, and 3-fold less than when the cells were stimulated with the ionophore A23187 for 20 min. Taken together, our results indicate that human HDL and LDL contribute to endothelial PGI2 production only in a modest way and suggest that this process is not specific for either of these two plasma lipoproteins. In view of the greater participation of albumin-bound arachidonic acid in PGI2 production, plasma lipoproteins may not play as important a role in endothelial prostaglandin formation as has been suggested.

Animals↗
PubMed1983

Eicosapentaenoic acid and prostacyclin production by cultured human endothelial cells.

Human umbilical vein endothelial cells incorporate eicosapentaenoic acid (EPA) when this fatty acid is present in the culture medium. From 30 to 70% of the uptake remains as EPA, and much of the remainder is elongated to docosapentaenoic acid. All of the cellular glycerophospholipids become enriched with EPA and docosapentaenoic acid, with the largest increase in EPA occurring in the choline glycerophospholipids. When this fraction is enriched with EPA, it exhibits a large decrease in arachidonic acid content. Cultures exposed to tracer amounts of [1-14C]linolenic acid in 5% fetal bovine serum convert as much as 17% of the radioactivity to EPA. The conversion is reduced, however, in the presence of either 20% fetal bovine serum or 50 microM linolenic acid. Like arachidonic acid, some newly incorporated EPA was released from the endothelial cells when the cultures were exposed to thrombin. However, as compared with arachidonic acid, only very small amounts of EPA were converted to prostaglandins. Cultures enriched with EPA exhibited a 50 to 90% reduction in capacity to release prostacyclin (PGI2) when subsequently stimulated with thrombin, calcium ionophore A23187, or arachidonic acid. The degree of inhibition was dependent on the time of exposure to EPA and the EPA concentration, and it was not prevented by adding a reversible cyclooxygenase inhibitor, ibuprofen, during EPA supplementation. EPA appears to decrease the capacity of the endothelial cells to produce PGI2 in two ways: by reducing the arachidonic acid content of the cell phospholipid precursor pools and by acting as an inhibitor of prostaglandin production. These findings suggest that regimens designed to reduce platelet aggregation and thrombosis by EPA enrichment may also reduce the capacity of the endothelium to produce PGI2.

6-Ketoprostaglandin F1 alpha↗
PubMed1982

Role of the vascular endothelium.

A possible explanation for the non-thrombogenic effect of the endothelium is the presence of prostacyclin, (PGI2), the potent inhibitor of platelet aggregation and adherence, which is produced and released by the endothelium in response to various stimuli. Removal of PGI2 from the endothelium did not increase baseline platelet adherence, but did increase thrombin-induced platelet adherence from 4 to 60%. Additions of exogenous PGI2 at low concentrations reversed the enhanced thrombin-induced platelet adherence under these conditions. Although it is unlikely that prostacyclin is the sole factor regulating platelet adherence to the endothelium, it appears to play a major role in the interaction of platelets with components of the blood vessel wall. Conditions which predispose to adherence of platelets to the vessel wall may involve entrapment of tumor cells and lead to metastasis formation. Whether prostacyclin and other factors involved in the non-thrombogenic character of the vascular endothelium provide a significant defense against attachment of tumor cells is not known, but the potential for such a primary or secondary role clearly exists. In our studies, prostacyclin did not appear to influence the adherence of Raji lymphoma cells to the endothelium. Additional studies are indicated to correlate adherence of tumor cells to the vascular wall with their potential for formation of metastatic lesions.

1-Methyl-3-isobutylxanthine↗
PubMed1982

Contribution of a platelet component to endothelial prostacyclin production despite inhibition of platelet cyclooxygenase activity.

Platelets aggregated with low concentrations of thrombin enhanced prostacyclin release from umbilical vein endothelium, which had an active cyclooxygenase. This result cannot be explained solely by an effect of thrombin on the endothelium or by transfer of the endoperoxide PGH2 from platelets to the endothelium. The quantity of prostacyclin released is sufficient to alter platelet adherence to umbilical vein endothelium in the presence of thrombin.

6-Ketoprostaglandin F1 alpha↗
PubMed1981

Role of the vascular endothelium.

The intact vascular endothelial surface is considered to be "non-thrombogenic", and blood platelets usually fail to adhere to ti. In this role, the endothelium serves to maintain the integrity of the vascular system by preventing the escape of blood and by preventing the build-up of solid thrombus within the vessel, which would compromise blood flow. A possible explanation for the non-thrombogenic effect of the endothelium is the presence of prostacyclin (PGI2), the potent inhibitor of platelet aggregation and adherence, which is produced and released by the endothelium in response to various stimuli. Removal of PGI2 from the endothelium by four different methods did not increase baseline platelet adherence, but did increase thrombin-induced platelet adherence from 4 to 60%. Addition of exogenous PGI2, at low concentrations reversed the enhanced thrombin-induced platelet adherence under these conditions. Although it is unlikely that prostacyclin is the sole factor regulating platelet adherence to the endothelium, it appears to play a major role in the interaction of platelets with components of the blood vessel wall.

Animals↗
PubMed1981

Comparative effect of ibuprofen on endothelial and platelet prostaglandin synthesis.

Ibuprofen (Motrin) decreases infarct size in animal models of myocardial infarction. Inasmuch as this effect might be related to the inhibitory effect of ibuprofen on platelet function, we have evaluated its effect on platelet and endothelial prostaglandin synthesis. Human umbilical vein endothelial cultures and washed human platelets were studied. Thromboxane (TX)A2 was determined by radioimmunoassay for its stable and product TXB2 and prostacyclin was determined by radioimmunoassay for its stable end product 6-keto-prostaglandin F1 alpha. At all concentrations of ibuprofen tested, platelet TXA2 and endothelial prostacyclin synthesis were inhibited to a similar degree. Unlike aspirin, the effect of ibuprofen on prostaglandin synthesis was rapidly reversible in both endothelial and platelet systems after removal of the drug. Ibuprofen was also shown to interfere with the irreversible inhibitory effect of aspirin, suggesting that ibuprofen and aspirin might compete for binding by the cyclooxygenase. Inhibition of endothelial and platelet prostaglandin synthesis was nearly complete with 100 microM concentrations of aspirin (100%) and ibuprofen (99%). Thrombin-stimulated platelet adherence to endothelial monolayers was related inversely to the amount of prostacyclin produced by the endothelium. Adherence was maximal at concentrations (100 microM) of ibuprofen and aspirin producing near complete inhibition of prostaglandin synthesis. Maximal adherence with both drugs was comparable (28 vs. 31%). Ibuprofen showed no preferential inhibitory effect on TXA2 synthesis and, at a 100 microM concentration, was as effective as aspirin in the systems studied.

6-Ketoprostaglandin F1 alpha↗
PubMed1980

Interaction of thrombin and platelets with the vascular endothelium.

Thrombin is a potent stimulus for prostacyclin (PGI2) release from the vascular endothelium. Treatment of the endothelium with high concentrations of aspirin to block PGI2 formation was associated with increased platelet adherence in a system employing thrombin and 51Cr-labeled platelets. Addition of exogenous PGI2 to the aspirin-treated endothelium restored platelet adherence to the low baseline values. After an initial exposure of the endothelium to thrombin, the cultured endothelium was unable to respond to a second thrombin stimulus with release of PGI2. During this refractory period, the absence of PGI2 was associated with increased platelet adherence. Thus thrombin, an active coagulant and thrombogenic substance, has the capability to release PGI2, the most potent inhibitor of platelet aggregation known to exist in vivo.

Aspirin↗
PubMed1979

Inhibition of prostacyclin by treatment of endothelium with aspirin. Correlation with platelet adherence.

Aspirin treatment of cultured endothelial cells from the umbilical vein increased the adherence of 51Cr-platelets when thrombin was present. If the cyclooxygenase activity of endothelium was inhibited by aspirin, as it is in the platelet, reduction of endogenous prostacyclin (PGI2) production could have been responsible. By correlating thrombin-induced adherence of platelets to endothelial monolayers with PGI2 release (as measured by radioimmunoassay for 6-keto-prostaglandin FI1 alpha [6-keto-PGF1 alpha]), we have demonstrated an inverse relationship between platelet adherence and PGI2 levels. Untreated endothelial monolayers exposed to thrombin and platelets resulted in 4% platelet adherence and 107 nM 6-keto-PGF1 alpha. With 0.1 mM aspirin treatment, which is known to block platelet cyclooxygenase, adherence was 5% and 6-keto-PGF1 alpha decreased to 45 nM. Increasing the aspirin concentration to 1 mM resulted in 44% adherence and less than 3 nM 6-keto-PGF1 alpha. When 25 nM exogenous PGI2 was added to 1 mM aspirin-treated endothelium, adherence returned to 5%. The increase in thrombin-induced platelet adherence to 1 mM aspirin-treated monolayers was reversed 2 h after removal of the aspirin solution. 6-Keto-PGF1 alpha returned to 37% of the untreated monolayer value. Recovery from the aspirin effect did not occur when cycloheximide, an inhibitor of protein synthesis, was present during the 2-h period.

Aspirin↗
PubMed1978

Effect of aspirin on thrombin-induced adherence of platelets to cultured cells from the blood vessel wall.

An in vitro method was used to detect adherence of (51)Cr-labeled platelets to monolayers of cultured human endothelial, fibroblast, and smooth muscle cells. Washed platelets did not adhere to untreated or aspirin-treated endothelial monolayers in the absence of thrombin. In contrast, thrombin-induced platelet aggregates adhered to all of the monolayers but adherence to endothelium was significantly less than to the other cells. Additional evidence for adherence of platelets to the endothelium was provided by scanning and transmission electron microscopy. Thrombin-induced platelet adherence to endothelium was inhibited by hirudin. Platelet adherence induced by thrombin was enhanced significantly by treatment of the endothelial monolayer with 1-2 mM aspirin. This increase in adherence was seen even when aspirin-treated platelets were used; adherence values approached those seen with fibroblasts and smooth muscle cells. An aspirin concentration of 0.1 mM was sufficient to block thrombin-induced malonaldehyde production in platelets but it did not interfere with the inhibitory effect of the endothelium against platelet adherence. The effect of aspirin on the endothelium was temporary and inhibitory activity of the endothelium was restored 1 h after aspirin had been removed from the incubation system. The ability of thrombin to cause adherence of platelets to undamaged endothelium, and the potential for aspirin to enhance this adherence have implications for mechanisms which operate in platelet interaction with the blood vessel wall.

Aspirin↗
PubMedDate not supplied

Arachidonic acid availability and prostacyclin production by cultured human endothelial cells.

When human umbilical vein endothelial cultures were grown in the presence of supplemental arachidonic acid, the cell phospholipids became enriched with arachidonic acid. Prostacyclin (PGI2) accumulated in the medium during supplementation with arachidonic acid. The capacity of these enriched cultures to produce PGI2 when subsequently incubated with either arachidonic acid or thrombin was reduced by as much as 90%, but release of arachidonic acid from the cell lipids in response to thrombin stimulation was not inhibited. Refractory cultures completely recovered the capacity to form PGI2 within 18 hours after removal of the medium containing supplemental arachidonic acid. However, recovery was prevented by cycloheximide. When enrichment with arachidonic acid was done in the presence of ibuprofen, a reversible cyclooxygenase inhibitor, PGI2 did not accumulate in the medium during supplementation, and the subsequent capacity of the cultures to produce PGI2 in response to thrombin increased by 70% to 240%. By contrast, the capacity of these supplemented cultures to convert added arachidonic acid to PGI2 did not increase. Therefore, the enhancement in thrombin-stimulated PGI2 production when the cultures are supplemented with arachidonic acid probably is due to the larger amount of arachidonic acid available in the intracellular lipid substrate pools, rather than to an activation of the PGI2 synthetic pathway. These findings suggest that changes in the arachidonic acid content of the endothelial cell lipids may modulate the capacity of the endothelium to produce PGI2 in response to stimulation.

Arachidonic Acid↗