Use of glucose-lowering drugs in Hungary between 2008 and 2017: the increasing use of novel glucose-lowering drug groups.

AIMS: To analyse glucose-lowering drug utilization, focusing on the novel glucose-lowering drug groups dipeptidyl peptidase-4 inhibitors, glucagon-like peptide-1 receptor agonists and sodium-glucose co-transporter-2 inhibitors, and the financial burden they entail. METHODS: Crude reimbursed national drug utilization and expenditure data for the entire population of Hungary were obtained from the National Health Insurance Fund for the study period: 2008 to 2017. Data were analysed using the WHO's Anatomical Therapeutic Chemical Classification/defined daily dose system and were expressed in defined daily dose per 1000 inhabitants per day. RESULTS: Total glucose-lowering drug consumption in Hungary showed an 18% increase over the study period, reaching 74.7 defined daily doses per 1000 inhabitants per day, while novel glucose-lowering drug use increased to 11.7 defined daily doses per 1000 inhabitants per day (16% of total glucose-lowering drug use) by 2017. Dipeptidyl-peptidase 4 inhibitor consumption grew to 7.4 defined daily doses per 1000 inhabitants per day by 2017. The most widely used dipeptidyl-peptidase 4 inhibitor was sitagliptin. Glucagon-like peptide-1 receptor agonists were used the least, but by 2017 rose to 1.5 defined daily doses per 1000 inhabitants per day, led by liraglutide. Sodium-glucose co-transporter-2 inhibitors appeared in the utilization data in 2014 and their consumption, mainly empagliflozin, reached 2.8 defined daily doses per 1000 inhabitants per day by 2017. The total expenditure on glucose-lowering drugs increased 94% between 2008 and 2017, and the total cost of novel glucose-lowering drug utilization comprised 44% of the total glucose-lowering drug expenditure in 2017. CONCLUSIONS: Both the use of and the financial burden posed by novel glucose-lowering drugs in Hungary increased steadily between 2008 and 2017. This increase is expected to continue.

Clinical, radiological, and flow-related risk factors for growth of untreated, unruptured intracranial aneurysms.

BACKGROUND AND PURPOSE: Unruptured intracranial aneurysms are frequently followed to monitor aneurysm growth. We studied the yield of follow-up imaging and analyzed risk factors for aneurysm growth. METHODS: We included patients with untreated, unruptured intracranial aneurysms and ≥6 months of follow-up imaging from 2 large prospectively collected databases. We assessed the proportion of patients with aneurysm growth and performed univariable and multivariable Cox regression analyses to calculate hazard ratios with corresponding 95% confidence intervals (CI) for clinical and radiological risk factors for aneurysm growth. We repeated these analyses for the subset of small (<7 mm) aneurysms. RESULTS: Fifty-seven (12%) of 468 aneurysms in 363 patients grew during a median follow-up of 2.1 years (total follow-up, 1372 patient-years). In multivariable analysis, hazard ratios for aneurysm growth were as follows: 1.1 (95% CI, 1.0-1.2) per each additional mm of initial aneurysm size; 2.7 (95% CI, 1.2-6.4) for dome > neck ratio; 2.1 (95% CI, 0.9-4.9) for location in the posterior circulation; and 2.0 (95% CI, 0.8-4.8) for multilobarity. In the subset of aneurysms <7 mm, 37 of 403 (9%) enlarged. In multivariable analysis, hazard ratios for aneurysm growth were 1.1 (95% CI, 0.8-1.5) per each additional mm of initial aneurysm size, 2.2 (95% CI, 1.0-4.8) for smoking, 2.9 (95% CI, 1.0-8.5) for multilobarity, 2.4 (95% CI, 1.0-5.8) for dome/neck ratio, and 2.0 (95% CI, 0.6-7.0) for location in the posterior circulation. CONCLUSIONS: Initial aneurysm size, dome/neck ratio, and multilobarity are risk factors for aneurysm growth. Cessation of smoking is pivotal because smoking is a modifiable risk factor for growth of small aneurysms.

PHASES Score for Prediction of Intracranial Aneurysm Growth.

BACKGROUND AND PURPOSE: Growth of an intracranial aneurysm occurs in around 10% of patients at 2-year follow-up imaging and may be associated with aneurysm rupture. We investigated whether PHASES, a score providing absolute risks of aneurysm rupture based on 6 easily retrievable risk factors, also predicts aneurysm growth. METHODS: In a multicenter cohort of patients with unruptured intracranial aneurysms and follow-up imaging with computed tomography angiography or magnetic resonance angiography, we performed univariable and multivariable Cox regression analyses for the predictors of the PHASES score at baseline, with aneurysm growth as outcome. We calculated hazard ratios and corresponding 95% confidence intervals (CI), with the PHASES score as continuous variable and after division into quartiles. RESULTS: We included 557 patients with 734 unruptured aneurysms. Eighty-nine (12%) aneurysms in 87 patients showed growth during a median follow-up of 2.7 patient-years (range 0.5-10.8). Per point increase in PHASES score, hazard ratio for aneurysm growth was 1.32 (95% CI, 1.22-1.43). With the lowest quartile of the PHASES score (0-1) as reference, hazard ratios were for the second (PHASES 2-3) 1.07 (95% CI, 0.49-2.32), the third (PHASES 4) 2.29 (95% CI, 1.05-4.95), and the fourth quartile (PHASES 5-14) 2.85 (95% CI, 1.43-5.67). CONCLUSIONS: Higher PHASES scores were associated with an increased risk of aneurysm growth. Because higher PHASES scores also predict aneurysm rupture, our findings suggest that aneurysm growth can be used as surrogate outcome measure of aneurysm rupture in follow-up studies on risk prediction or interventions aimed to reduce the risk of rupture.

Difference in aneurysm characteristics between ruptured and unruptured aneurysms in patients with multiple intracranial aneurysms.

BACKGROUND AND PURPOSE: Prediction of the risk of rupture of unruptured intracranial aneurysms is mainly based on aneurysm size and location. Previous studies identified features of aneurysm shape and flow angles as additional risk factors for aneurysm rupture, but these studies were at risk for confounding by patient-specific risk factors such as hypertension and age. In this study, we avoided this risk by comparing characteristics of ruptured and unruptured aneurysms in patients with both aneurysmal subarachnoid hemorrhage and multiple intracranial aneurysms. METHODS: We included patients with aneurysmal subarachnoid hemorrhage and multiple aneurysms who presented to our hospital between 2003 and 2013. We identified the ruptured aneurysm based on bleeding pattern on head computed tomography or surgical findings. Aneurysm characteristics (size, location, shape, aspect ratio [neck-to-dome length/neck-width], flow angles, sidewall or bifurcation type, and contact with bone) were evaluated on computed tomographic angiograms. We calculated odds ratios with 95% confidence intervals with conditional univariable logistic regression analysis. Analyses were repeated after adjustment for aneurysm size and location. RESULTS: The largest aneurysm had not ruptured in 36 (29%) of the 124 included patients with 302 aneurysms. Odds ratios for aspect ratio≥1.3 was 3.3 (95% confidence intervals [1.3-8.4]) and odds ratios for irregular shape was 3.0 (95% confidence intervals [1.0-8.8]), both after adjustment for aneurysm size and location. CONCLUSIONS: Aspect ratio≥1.3 and irregular shape are associated with aneurysm rupture independent of aneurysm size and location, and independent of patient characteristics. Additional studies need to assess to what extent these factors increase the risks of rupture of small aneurysms in absolute terms.

Differences in risk factors according to the site of intracranial aneurysms.

BACKGROUND AND PURPOSE: Several risk factors for aneurysmal subarachnoid haemorrhage have been identified but it is not known whether some sites of aneurysms are linked to a specific risk factor. In a series of patients with aneurysmal subarachnoid haemorrhage, we compared risk factors according to the site of the ruptured aneurysm at the circle of Willis. METHODS: From our prospectively collected database of patients with aneurysmal subarachnoid haemorrhage admitted to our hospital between 2003 and 2007, we retrieved 304 patients with saccular aneurysms on the anterior communicating artery, middle cerebral artery, posterior communicating artery, basilar artery and vertebral artery. Risk factors (age, gender, smoking, no or excessive alcohol intake, hypertension and familial preponderance) were assessed per aneurysm location and compared with the anterior communicating artery as reference. We calculated odds ratios (ORs) and corresponding 95% CI. RESULTS: In comparison with aneurysms at the anterior communicating artery, those at the middle cerebral artery were less associated with age >55 years (OR 0.4; 95% CI 0.2 to 0.8), those at the posterior communicating artery were less associated with male gender (OR 0.4; 95% CI 0.2 to 0.9) and those at the basilar artery were more associated with no alcohol consumption (OR 5.8; 95% CI 1.1 to 29.9). CONCLUSION: Risk factors differ according to the site of aneurysm. This heterogeneity should be kept in mind in studies on the aetiology of aneurysms, such as genetic studies.

Anosmia after coiling of ruptured aneurysms: prevalence, prognosis, and risk factors.

BACKGROUND AND PURPOSE: Anosmia occurs frequently in patients with subarachnoid hemorrhage (SAH) from a ruptured aneurysm treated with clipping. We analyzed prevalence, prognosis, and potential risk factors for anosmia after coiling for SAH. METHODS: We interviewed all patients who resumed independent living after SAH treated with coiling between 1997 and 2007. We assessed by means of logistic regression analyses whether risk of anosmia was influenced by site of the ruptured aneurysm, neurological condition on admission, amount of extravasated blood, hydrocephalus, and treatment for hydrocephalus. RESULTS: Of 197 patients, 35 (18%; 95%CI:12 to 23) experienced anosmia. Anosmia had improved in 23 (66%) of them; in 20 the recovery had been complete after a median period of 6 weeks (SD +/-6.5). Intraventricular hemorrhage was a risk factor for anosmia (OR 2.4; 95%CI:1.0 to 5.9). Anterior aneurysm location (OR 1.1; 95%CI:0.5 to 2.3) and high amount of extravasated blood (OR 0.9; 95%CI:0.4 to 2.1) were not related to anosmia. CONCLUSIONS: Anosmia occurs after coiling in 1 of every 6 SAH patients, but has a good prognosis in most patients. The cause of anosmia after coiling for ruptured aneurysms remains elusive; severity of the initial hemorrhage or long lasting hydrocephalus may be contributing factors.

Risk of subarachnoid haemorrhage according to number of affected relatives: a population based case-control study.

Relatives of patients with aneurysmal subarachnoid haemorrhage (SAH) have an increased risk of this type of stroke. In a population-based study, we analysed individualized risks of SAH according to the number of affected first-degree relatives. We retrieved all patients diagnosed with SAH in 2001-05 from the Swedish Inpatient Register. For each of the 5,282 patients, we identified five controls (n = 26,402) through the nationwide Register of Total Population. Through the Multi-generation Register, we retrieved all first-degree relatives for patients and controls and checked whether these 130,373 relatives had been diagnosed with SAH. By means of conditional logistic regression, we calculated odds ratios with corresponding 95% confidence intervals (95% CI) for the risk of SAH according to the number of affected relatives, and to the gender, age and type of kinship of the patient and affected relative. The odds ratio of SAH for individuals with one affected first-degree relative was 2.15 (95% CI 1.77-2.59). For individuals with two affected first-degree relatives, the odds ratio was 51.0 (95% CI 8.56-1117). Gender, age and type of kinship did not influence the risk for individuals with one or more affected relatives. The risk of SAH is slightly increased in the cases with one, but strongly increased in cases with two or more affected first-degree relatives. The latter strongly increased risk corresponds to a considerable absolute life-time risk of SAH and underscores the need to consider screening for aneurysms in these individuals.

Copper-induced peroxidation of phosphatidylserine-containing liposomes is inhibited by nanomolar concentrations of specific antioxidants.

Copper-induced peroxidation of liposomal palmitoyllinoleoyl-phosphatidylcholine (PLPC) is inhibited by alpha-tocopherol at micromolar concentrations. In our previous study we found that when the liposomes contain phosphatidylserine (PS), nanomolar concentrations of Toc were sufficient to inhibit peroxidation. In an attempt to gain understanding of the origin of this extreme antioxidative potency, we tested the antioxidative potency of 36 additional antioxidants and the dependence of their potency on the presence of PS in the liposomes. The results of these studies reveal that only 11 of the tested antioxidants possess similar antioxidative potency to that of Toc. These include trolox, butylated hydroxytoluene (BHT), curcumin, nordihydroguaiaretic acid (NDGA), diethylstilbestrol (DES), 2 of the 13 tested flavonoids (luteolin and 7,3',4'-trihydroxyflavone; T-414), alpha-naphthol, 1,5-, 1,6- and 1,7-dihydroxynaphthalenes (DHNs). Propyl gallate (PG), methyl syringate, rosmarinic acid, resveratrol, other flavonoids, as well as beta-naphthol, 1,2-, 1,3-, 1,4-, 2,3-, 2,6-, and 2,7-DHNs were either moderately antioxidative or pro-oxidative. For liposomes made of PLPC (250 microM) and PS (25 microM) the "lag" preceding copper-induced peroxidation (5 microM copper) was doubled upon addition of 30-130nM of the "super-active" antioxidants. We propose that the mechanism responsible for the extreme antioxidative potency against copper-induced peroxidation in PS-containing liposomes involves replenishment of the antioxidant in a ternary PS-copper-antioxidant complex. Based on structure-activity relationship of the 37 tested antioxidants, the "super-antioxidative potency" is attributed to the recycling of relatively stable semiquinone or semiquinone-like radicals.

Oxidation of liposomal cholesterol and its effect on phospholipid peroxidation.

Lipid peroxidation is believed to play an important role in the pathogenesis of many diseases. Much research has therefore been devoted to peroxidation of different lipids in biomembranes and in model systems (liposomes) of different compositions. Yet, in spite of the relative simplicity of the liposomes, the existing literature is insufficient to reach definite conclusions regarding basic questions including the susceptibility of cholesterol to oxidation, its effect on the peroxidation of polyunsaturated phospholipids such as palmitoyllinoleoylphosphatidylcholine (PLPC) and how cholesterol influences the effect of water-soluble antioxidants such as urate on the peroxidation. The aim of the present study was to clarify these issues. Its major findings are that: (i) AAPH-induced peroxidation of cholesterol is slow and independent of the peroxidation of PLPC. In turn, AAPH-induced peroxidation of PLPC is not affected by cholesterol, independent of the presence of urate in the system. (ii) Cholesterol is not susceptible to copper-induced oxidation, but its inclusion in PLPC liposomes affects the peroxidation of PLPC, slowing down the initial stage of oxidation but promoting later stages. (iii) Addition of urate accelerates copper-induced peroxidation of PLPC in the absence of cholesterol, whereas in cholesterol-containing liposomes it inhibits PLPC oxidation. We attribute the complexity of the observed kinetics to the known cholesterol-induced rigidization of liquid crystalline bilayers.

Carrying the FecB (Booroola) mutation is associated with lower birth weight and slower post-weaning growth rate for lambs, as well as a lighter mature bodyweight for ewes.

The present study was conducted in an Assaf flock in which the FecB (Booroola) mutation was segregated to determine whether the FecB mutation affects birthweight and the pre- and post-weaning growth rate of ewe lambs, as well as the mature bodyweight of ewes. Significant differences (P = 0.01) in birthweight (mean +/- s.e.m.) were found between BB ewe lambs (4.03 +/- 0.08 kg) and B+ and ++ ewe lambs (4.16 +/- 0.04 and 4.32 +/- 0.07 kg, respectively), which themselves did not differ significantly (P > 0.05). An FecB-associated maternal effect on the birthweight of ewe lambs was also detected, with the birthweight of lambs born to BB mothers (3.93 +/- 0.08 kg) being significantly (P < 0.0001) different from the birthweight of lambs born to B+ and ++ mothers (4.26 +/- 0.04 and 4.33 +/- 0.07 kg, respectively), which did not differ significantly. The genotypes of the lambs did not affect their preweaning growth rate. However, the post-weaning growth rate of ewe BB lambs (274 +/- 5 g day(-1)) was significantly (P = 0.05) different from the similar (P > 0.05) post-weaning growth rates of B+ and ++ lambs (284 +/- 3 and 290 +/- 4 g day(-1), respectively). The genotype at the FecB locus also affected the mature bodyweight of ewes, with that of BB ewes (67.3 +/- 1.4 kg) being significantly (P < 0.001) different from the similar mature bodyweight of B+ and ++ ewes (70.8 +/- 1.1 and 70.1 +/- 1.7 kg, respectively).

Expression of foreign genes in chicks by hydrodynamics-based naked plasmid transfer in vivo.

The study of gene function in vivo is considered one of the top achievements of modern biology, inasmuch as it provides tools to study gene function in the context of the whole animal. In chickens, techniques of DNA-mediated gene transfer are less advanced than in other animal or livestock models, and remain a significant challenge. The study presented here is the first to show that a hydrodynamics-based gene-transfer technique, originally developed for naked DNA transfer in mice, can be applied to chickens. Rapid injection of naked plasmids containing expression cassettes into the jugular vein of 6- to 10-day-old chicks resulted in specific expression of the transgenes. A CMV promoter-driven luciferase reporter gene was expressed at significant levels in the liver during the first 3 days post-injection with lower levels also detected in the kidney. Significantly, all injected birds showed detectable levels of luciferase expression. Similarly, injection of a plasmid containing the secreted human coagulation factor IX (hFIX) gene under the control of human alpha-1-anti-trypsin promoter resulted in detectable levels of the hFIX in the plasma during the first 2 days post-injection. The method described herein has the potential for a quick and simple route for gain and loss-of function experiments in chicken liver and kidney, as well as for studying systemic effects of secreted proteins and hormones.

Short exposure to actinomycin D induces "reversible" translocation of protein B23 as well as "reversible" inhibition of cell growth and RNA synthesis in HeLa cells.

HeLa cells were grown in medium containing various amounts of actinomycin D for various times. Cellular localization of protein B23 was detected using an immunofluorescence technique. Bright nucleolar fluorescence was observed in untreated cells. A shifting of nucleolar to nuclear fluorescence was observed with increasing doses of actinomycin D and longer incubation times. The degree of translocation of protein B23 from nucleoli to nucleoplasm is dependent on the amount of the drug used and the duration of incubation. Short exposure (0.5 h) of HeLa cells to actinomycin D (0.01-0.25 microgram/ml) induced "reversible" translocation of protein B23, inhibition of cell growth, and RNA synthesis. A majority of cells (greater than 75%) treated with actinomycin D (0.01-0.25 microgram/ml) for 0.5 h still retained bright nucleolar fluorescence. A shifting of nucleolar to nuclear fluorescence as well as inhibition of cell growth and RNA synthesis were observed within 6 h after the removal of the drug. However, at the extended periods (greater than 24 h) after drug removal, RNA synthesis and cell growth resumed at the normal rate, and protein B23 relocated from nucleoplasm to nucleoli. This is in contrast to the results obtained from the experiments using higher doses (1 microgram/ml; 0.5 h) or longer (0.25 microgram/ml; 2 h) exposure of HeLa cells to actinomycin D, which induced irreversible B23 translocation as well as irreversible inhibition of cell growth and RNA synthesis. These results indicated that actinomycin D can be a reversible inhibitor depending on the drug extracellular concentrations and exposure times. Our results also indicated that "B23 translocation" is closely associated with states of cell growth and inhibition of RNA synthesis. "B23 translocation" may therefore be a simple and rapid method for assessing the inhibition of cell growth in response to antitumor therapy.

Cholesterol precipitation from cholesterol-supersaturated bile models.

Bile-model systems containing cholesterol (CH), phosphatidylcholine (PC) and sodium cholate (NaC) at concentrations similar to those found in supersaturated human gall bladder bile ([CH]/[PC] = 0.60 +/- 0.01; CH saturation index, CSI = 1.58 +/- 0.03) were prepared by mixing PC-CH vesicles with NaC micellar solutions. Following mixing, the dispersion became transparent and gave rise to high resolution 1H-NMR spectra typical of mixed micellar systems. Cryo-transmission electron micrographs of specimens vitrified at that stage support the conclusion that the vesicles had become completely micellized. Following micellization, the metastable (cholesterol-supersaturated) bile-models spontaneously underwent a series of reorganizational steps: first, cholesterol-rich vesicles with a [CH]/[PC] ratio of 1.57 +/- 0.69 were formed, in co-existence with a mixed micellar system with [CH]/[PC] = 0.43 +/- 0.01 and CSI = 1.12 +/- 0.03. The resultant cholesterol-rich vesicles subsequently aggregated and cholesterol crystals of varying sizes and shapes appeared within the aggregates: needle-like structures were first observed, followed by clusters of those crystals and of helical crystals. Eventually, typical plate-like cholesterol crystals appeared, at which time some of the PC returned to the non-particulate (isotropic) phase. Consequently, the system contained cholesterol crystals coexisting with mixed micelles, whose composition was close to the limit of saturation (CSI = 1.08). These findings confirm the sequence of events preceding the appearance of cholesterol crystals, as previously proposed in our less detailed studies ((1990) Hepatology 12, 149S) and support the relevance of the morphologically similar results of Konikoff et al. ((1992) J. Clin. Invest. 90, 1155) obtained in a very dilute supersaturated bile-model.

Physico-chemical characterization of Intralipid emulsions.

Fat emulsions containing soy triacylglycerols (100-300 g/l) and egg-yolk phospholipids (12 g/l) are often used for intravenous feeding. Previous studies have shown that these emulsions contain chylomicron-like emulsion particles of diameters of 300-400 nm and excess phospholipids aggregated as vesicles (liposomes), which remain in the infranatant upon floatation of the emulsion particles by ultracentrifugation. This work is devoted to the characterization of the commercial lipid emulsions commonly denoted Intralipids, with special emphasis on the presently ill-defined liposomes. The lipid particles composing commercial lipid emulsions (10%, 20% and 30% Intralipids, Kabivitrum Nutrition) were characterized by the combined use of physical and chemical methods. Each of the emulsions was fractionated by ultracentrifugation in saline into a 'cream' layer which floats to the top of the dispersion upon ultracentrifugation and a relatively transparent infranatant. The cream layer contains large emulsion particles of diameters ranging from 300 to 400 nm, in agreement with theoretical considerations based on their chemical composition as determined by chemical analysis. The infranatants contain about 1 g/l triacylglycerols in addition to phospholipids (from 7.2 g/l in 10% Intralipid to 2.4 g/l in 30% Intralipid) in the form of smaller particles of 70-100 nm diameter. Cryo-transmission electron microscopy shows that the infranatants contain vesicles (mostly unilamellar) at the side of residual small emulsion particles. This conclusion is also consistent with the distribution of phospholipids between outer and inner lamellae, as determined by 31P-NMR.

The effect of albumin on copper-induced LDL oxidation.

In an attempt to gain deeper understanding of the mechanism or mechanisms responsible for the protective effect of serum albumin against Cu(2+)-induced peroxidation of low density lipoprotein (LDL), we have examined the influence of the concentrations of bovine serum albumin (BSA), Cu2+ and LDL on the kinetics of peroxidation. Since the common method of monitoring the oxidation by continuous recording of the absorbance of conjugated dienes at 234 nm cannot be used at high BSA-concentrations because of the intensive absorption of BSA, we have monitored the time-dependent increase of absorbance at 245 nm. At this wavelength, conjugated dienes absorb intensely, whereas the background absorbance of BSA is low. Using this method, as well as the TBARS assay for determination of malondialdehyde, over a large range of BSA concentrations, we show that in many cases the influence of BSA on the kinetics of oxidation can be compensated for by increasing the concentration of copper. This reconciles the apparent contradiction between previously published data. Detailed studies of the kinetic profiles obtained under different conditions indicate that binding of Cu2+ to albumin plays the major role in its protective effect while other mechanisms contribute much less than copper binding. This conclusion is consistent with the less pronounced effect of BSA on the oxidation induced by the free radical generator AAPH. It is also shown that the copper-albumin complex is capable of inducing LDL oxidation, although the kinetics of the latter process is very different from that of copper-induced oxidation. Nevertheless, when compared to copper induced oxidation at similar concentration of the oxidation-promotor, the kinetics of oxidation induced by copper-albumin complex is very different and is consistent with a tocopherol mediated peroxidation, characteristic under low radical flux. Similar kinetics was observed for copper-induced oxidation only at much lower copper concentrations.

Intracoronary injection of basic fibroblast growth factor enhances angiogenesis in infarcted swine myocardium.

OBJECTIVES: This study was performed to examine the effect of intracoronary exogenous basic fibroblast growth factor (bFGF) on angiogenesis in infarcted myocardial regions. BACKGROUND: Exogenous bFGF is a potent promoter of angiogenesis. Little information is available on its effect on myocardial angiogenesis. METHODS: Myocardial infarction was induced in 10 pigs by intracoronary injection of microscopic beads. Four pigs served as a control group; in six pigs slow-release bFGF was delivered by the beads. Cardiac performance was evaluated by repeated echocardiographic measurement and angiogenesis was evaluated by immunohistochemical studies 14 days later. RESULTS: As compared with control pigs, pigs treated with bFGF had higher microvessel counts (mean +/- SEM) in both viable tissue (141 +/- 27 per field vs. 39 +/- 4, p = 0.01) and nonviable tissue (329 +/- 26 per field vs. 95 +/- 7, p < 0.001) within the infarct area. No significant differences in total regional left ventricular wall motion were noted between the two groups throughout the 14-day study period. CONCLUSIONS: In the swine, direct intracoronary application of bFGF to infarcted myocardium enhances myocardial neovascularization within 2 weeks.

Modulation of the reversibility of actinomycin D cytotoxicity in HeLa cells by verapamil.

Actinomycin D treatment (0.001-0.005 micrograms/ml; 0.5-24 h) induced a dose and time response shifting of nucleolar to nuclear fluorescence. In the presence of verapamil, cells were more responsive to actinomycin D. Translocation of protein B23 occurred with lower doses of actinomycin D and in shorter incubation times in the presence of verapamil. Short exposure (0.5 h) of HeLa cells to actinomycin D (0.05-0.25 micrograms/ml) induced 'reversible' translocation of protein B23 as well as 'reversible' inhibition of cell growth and RNA synthesis. Verapamil (5 microM) included in the cell culture after removal of actinomycin D inhibited the recoveries of cell growth, RNA synthesis as well as the corresponding relocalization of protein B23 from the nucleoplasm to nucleoli. These results indicate that verapamil can potentiate the antiproliferating activity of actinomycin D by inhibiting reversibility of its cytotoxicity and suggest clinical application.

Precipitation of calcium palmitate from bile salt-containing dispersions.

Addition of calcium chloride to mixed micellar systems composed of sodium salts of palmitic acid and high concentrations of different bile acids results in precipitation of Ca(palmitate)2 only when the palmitate concentration exceeds a critical value, which is dependent on the concentrations of Ca2+, Na+ and bile salt, and on the type of bile salt used. All these dependencies, as well as the complex and interrelated effects of the various parameters on the kinetics of Ca(palmitate)2 precipitation are consistent with the following mechanism: (i) calcium binds to palmitate-bile salt mixed micelles and promotes their aggregation, at a rate governed by the concentration ratio between bound calcium and micelles (here denoted "binding ratio"). (ii) Ca(palmitate)2 precipitation occurs within the aggregate of micelles only if those micelles include sufficient amounts of Ca2+ and palmitate to allow for the formation of large enough crystal units of Ca(palmitate)2 which can serve as nucleation "seeds". Both the concentrations of micelles and Na+ have dual effects on the rate of precipitation. Increasing micelle concentration, by itself, accelerates aggregation but at the same time leads to a decrease of the binding ratio, thus reducing the rate of precipitation. Na+ which reduces the binding ratio through competitive binding also reduces the surface charge, thus assisting micelle aggregation. Our model also explains the facilitation of precipitation observed when phosphatidylcholine is contained in the palmitate-bile salt mixed micelles and the inhibitory effect of the water soluble bovine serum albumin.

Lipid oxidation in unfractionated serum and plasma.

In an attempt to develop an assay for the susceptibility of plasma lipids to oxidation, we have studied the kinetics of copper-induced oxidation in diluted serum and plasma prepared with different anticoagulants (heparin, citrate and EDTA) by monitoring the absorbance of oxidation-products at several wavelengths. These studies revealed the complex and interrelated effects of the water-soluble antioxidant ascorbic acid, citrate and chloride ions on the kinetics of copper-induced oxidation of plasma lipids. Specifically, the onset of oxidation induced by copper-citrate chelates is only slightly affected by chloride ions and is accelerated upon increasing the copper concentration. By contrast, in the absence of citrate, the lag preceding oxidation in diluted serum or plasma (but not the maximal rate of oxidation) depends markedly on the chloride concentration in the diluting medium. In the absence of Cl-, the lag preceding oxidation is a decreasing saturable function of copper concentration, whereas in a normal phosphate-buffered saline solution (PBS), the lag shows a biphasic dependence on copper concentration such that at copper concentrations above 10-30 microM (depending on the extent of plasma dilution), increasing the concentration of copper results in prolongation of the lag. This dependence of copper-induced oxidation on the concentration of copper is not observed for dialyzed serum unless ascorbic acid is added. Our interpretation of these results is that water-soluble reductants and chloride ions act synergistically to stabilize Cu+, on the expense of Cu2+. Quenching of free radicals by Cu+ may be responsible for the prolongation of the lag at high copper concentrations, with no reduction of the maximal rate of oxidation. In spite of the complex dependencies described above, spectrophotometric monitoring of the kinetics of oxidation of plasma lipids, under 'optimized conditions' (50-fold diluted serum, in PBS containing 720 microM sodium citrate and 100 microM copper), agrees with independent measurements of the consumption of polyunsaturated fatty acids. Hence, the spectroscopic method may become useful for evaluation of the susceptibility of plasma lipids to oxidation. This possibility, however, has yet to be elucidated through investigations of the correlation between the susceptibility of serum lipids to copper-induced oxidation in vitro and clinical factors of significance.

On the solubility of calcium deoxycholate: kinetics of precipitation and the effect of conjugated bile salts and lecithin.

In view of the low solubility of calcium deoxycholate and the possible induction of cholesterol precipitation in the gallbladder by calcium insoluble salts, we find it of interest to study the precipitation of calcium deoxycholate and its dependence on other bile components. The findings of these studies were as follows: (i) Precipitation of calcium deoxycholate from mixtures of calcium chloride and monomeric deoxycholate (at concentrations below the critical micelle concentration (CMC] is very slow even at relatively high CaCl2 concentrations (more than 20 days at 50 mM CaCl2). (ii) At higher deoxycholic acid (DOC) concentrations, precipitation of micellar DOC is faster and requires much lower calcium chloride concentrations. For any given calcium concentration, the rate of precipitation is maximal at an optimal DOC concentration. In solutions containing 150 mM NaCl, the maximal rate of precipitation occurs at about 10 mM DOC, almost independent of Ca2+ concentration. At lower ionic strength (10 mM NaCl), the optimal DOC concentration is 30 mM. These observations suggest that the most important factors in determining the rate of Ca(DOC)2 precipitation are (a) the ratio between calcium ions bound to the surface of a DOC micelle, and the [DOC] (the Ca2+/DOC binding ratio) and (b) the concentration of DOC micelles. (iii) In the presence of conjugated deoxycholates, the crystallization of calcium deoxycholate is inhibited. Phosphatidylcholine has a similar, although smaller, inhibitory effect. Upon precipitation of calcium deoxycholate from a mixed micellar system containing sodium deoxycholate, phosphatidylcholine and cholesterol, the latter two components spontaneously form vesicles. The anti-nucleating effect of PC and conjugated bile salts is explained in terms of "poisoning" of the crystallization process. In view of the latter results we conclude that under normal conditions calcium deoxycholate is not likely to precipitate in the gallbladder.