Mathematical modeling for bioprocess optimization of a protein drug, uricase, production by Aspergillus welwitschiae strain 1-4.

Microbial uricase is effective protein drug used to treat hyperuricemia and its complications, including chronic gout, also in prophylaxis and treatment of tumor lysis and organ transplants hyperuricemia. Uricase is commonly used as diagnostic reagent in clinical analysis for quantification of uric acid in blood and other biological fluids. Also, it can be used as an additive in formulations of hair coloring agents. A newly isolated strain, Aspergillus sp. 1-4, was able to produce extracellular uricase on a medium containing uric acid as inducer. Phylogenetic analysis based on ITS region sequence analysis and phenotypic characteristics showed that Aspergillus sp. strain 1-4 is closely related to Aspergillus welwitschiae and its nucleotide sequence was deposited in the GenBank database and assigned sequence accession number MG323529. Statistical screening using Plackett-Burman design with 20 runs was applied to screen fifteen factors for their significance on uricase production by Aspergillus welwitschiae. Results of statistical analysis indicated that incubation time has the most significant positive effect on uricase production followed by yeast extract and inoculum size with the highest effect values of 13.48, 5.26 and 4.75; respectively. The interaction effects and optimal levels of these factors were evaluated using central composite design. The maximum uricase production was achieved at incubation time (5 days), yeast extract (2 g/L) and inoculum size (4 mL/50 mL medium) are the optimum levels for maximum uricase production (60.03 U/mL). After optimization, uricase production increased by 3.02-folds as compared with that obtained from the unoptimized medium (19.87 U/mL).

Identification of newly isolated Talaromyces pinophilus and statistical optimization of ß-glucosidase production under solid-state fermentation.

Fungi able to degrade agriculture wastes were isolated from different soil samples, rice straw, and compost; these isolates were screened for their ability to produce ß-glucosidase. The most active fungal isolate was identified as Talaromyces pinophilus strain EMOO 13-3. The Plackett-Burman design is used for identifying the significant variables that influence ß-glucosidase production under solid-state fermentation. Fifteen variables were examined for their significances on the production of ß-glucosidase in 20 experimental runs. Among the variables screened, moisture content, Tween 80, and (NH4)2SO4 had significant effects on ß-glucosidase production with confidence levels above 90% (p < 0.1). The optimal levels of these variables were further optimized using Box-Behnken statical design. As a result, the maximal ß-glucosidase activity is 3648.519 U g(-1), which is achieved at the following fermentation conditions: substrate amount 0.5 (g/250 mL flask), NaNO3 0.5 (%), KH2PO4 0.3 (%), KCl 0.02 (%), MgSO4 · 7H2O 0.01 (%), CaCl2 0.01 (%), yeast extract 0.07 (%), FeSO4 · 7H2O 0.0002 (%), Tween 80 0.02 (%), (NH4)2SO4 0.3 (%), pH 6.5, temperature 25°C, moisture content 1 (mL/g dry substrate), inoculum size 0.5 (mL/g dry substrate), and incubation period 5 days.

Optimization of ß-glucosidase production by Aspergillus terreus strain EMOO 6-4 using response surface methodology under solid-state fermentation.

Forty-two morphologically different fungal strains were isolated from different soil samples and agricultural wastes and screened for ß-glucosidase activity under solid-state fermentation. Eight species were chosen as the most active ß-glucosidase producers and were subjected to primary morphological identification. ß-Glucosidase was highly produced by Aspergillus terreus, which showed the highest activity, and was subjected to full identification using scanning electron microscopy and molecular identification. Initial screening of different variables affecting ß-glucosidase production was performed using Plackett-Burman design and the variables with statistically significant effects were identified. The optimal levels of the most significant variables with positive effect and the effect of their mutual interactions on ß-glucosidase production were determined using Box-Behnken design. Fifteen variables including temperature, pH, incubation time, inoculum size, moisture content, substrate concentration, NaNO3, KH2PO4, MgSO4 · 7H2O, KCl, CaCl2, yeast extract, FeSO4 · 7H2O, Tween 80, and (NH4)2SO4 were screened in 20 experimental runs. Among the 15 variables, NaNO3, KH2PO4 and Tween 80 were found as the most significant factors with positive effect on ß-glucosidase production. The Box-Behnken design was used for further optimization of these selected factors for better ß-glucosidase production. The maximum ß-glucosidase production was 4457.162 U g(-1).

Effect of alpha-stat versus pH-stat strategy on oxyhemoglobin dissociation and whole-body oxygen consumption during hypothermic cardiopulmonary bypass.

To determine whether alpha-stat or pH-stat strategy should be used, 20 patients undergoing coronary artery bypass grafting during moderate hypothermic hemodilutional cardiopulmonary bypass were studied. The carbon dioxide management during bypass was randomly done according to alpha-stat strategy in 10 patients (i.e., temperature-uncorrected PaCO2 was kept near 40 mm Hg and uncorrected pHa was kept at about 7.4) and according to pH-stat strategy in the other 10 patients (i.e., temperature-corrected PaCO2 was kept near 40 mm Hg and uncorrected pHa was kept at about 7.4). In both groups, when the central venous temperature was stable at 26.5 +/- 2.5 degrees C, the perfusion flow was altered sequentially from 2.4 to 1.8 and 1.2 L.min-1.m-2. The mixed venous oxyhemoglobin saturation at the different perfusion flows was monitored by the Oxy-Stat meter and was correlated with the corresponding mixed venous oxygen tension to construct an oxyhemoglobin dissociation curve. Also, the whole-body oxygen consumption at the different perfusion flows was computed. The whole-body oxygen consumption and the oxyhemoglobin dissociation were not significantly different between the alpha-stat and the pH-stat groups. In both groups, the dissociation curve is shifted to the left, but the oxygen consumption per unit time does not significantly change despite decreasing the perfusion flow from 2.4 to 1.2 L.min-1.m-2. The results suggest that oxygen delivery is not impaired during moderate hypothermic cardiopulmonary bypass independent of whether alpha-stat or pH-stat strategy is used.

Prediction of post-cardiopulmonary bypass cardiac output by venous oximetry.

The present study evaluates two equations for predicting the post-cardiopulmonary bypass cardiac output (CO) in 10 patients undergoing coronary artery bypass grafting. One equation is based on the relationship of CO with mixed venous oxygen saturation (SVO 2), while the second equation is based on the relationship with oxygen extraction (1 - SVO 2). Each patient served as his own control. During bypass, when the patients were normothermic and perfused with a pump flow of 2.4 L/min/m 2, the SVO 2 was monitored by an in-line Bentley oxystat Meter. Just before termination of bypass, the pump flow was decreased to 0.4 L/min/m 2 and the left atrial pressure was increased to 10-15 mmHg; the resulting SVO 2 was recorded. The post-bypass CO was predicted in every patient by the two equations. Immediately after weaning from bypass, the cardiac output was measured by thermodilution. The thermodilutional CO measurement was correlated with the CO predicted by the two equations. Correlation analysis suggests that CO prediction is more accurate and approaches the 1:1 ratio when the calculation of predicted CO is based on the relationship between cardiac output and oxygen extraction.

The hemodynamic effects of intravenous norepinephrine versus epinephrine and phenylephrine in patients with ischemic heart disease.

The hemodynamic effects of an intravenous bolus of norepinephrine 10 micrograms, phenylephrine 100 micrograms and epinephrine 10 micrograms were investigated in 30 patients scheduled for coronary artery bypass grafting. The hemodynamic changes following norepinephrine were similar to those achieved by phenylephrine. Both drugs increased the mean blood pressure and systemic vascular resistance without any significant change of cardiac output. In contrast, epinephrine increased the mean arterial pressure and cardiac output without a significant change of systemic vascular resistance. The results suggest that intravenous norepinephrine acts similar to phenylephrine as an alpha-adrenergic agonist, while epinephrine acts predominantly as a beta-adrenergic agonist.

Continuous venous oximetry during cardiopulmonary bypass: influence of temperature changes, perfusion flow, and hematocrit levels.

This study was performed in 11 patients undergoing cardiac surgery during cardiopulmonary bypass (CPB). A Bentley-10 oxygenator (American Bentley, Irvine, CA) was used during bypass, and 1,500 mL of Ringer's solution was used to prime the oxygenator. A perfusion flow of 2.4 L/min/m2 was used, and an equivalent flow of 100% oxygen was added to the oxygenator. The mixed venous oxygen saturation (SvO2) was monitored by continuous in-line venous oximetry using the Bentley Oxy-Stat Meter. Body temperature and SvO2 measurements were made at the venous blood port of the oxygenator. The authors investigated the correlation between SvO2 and changes of body temperature, perfusion flow, and hematocrit values. SvO2 correlated inversely, in a linear fashion, with the body temperature. The SvO2 was markedly decreased after rewarming, and was significantly affected during normothermia by changes in perfusion flow rates and hematocrit levels. An adequate SvO2 was found when the flow was greater than 2.4 L/min/m2 and the hematocrit was greater than 20%. In-line oximetry can help to optimize perfusion during CPB and to detect episodes of desaturation.

Action of adrenergic agonists on resistance v capacitance vessels during cardiopulmonary bypass.

The peripheral vascular effects of three different adrenergic agonists were investigated in 13 patients undergoing valve replacement during cardiopulmonary bypass (CPB). The venous reservoir (RV) and mean arterial pressure (MAP) were used as indices of the changes in venous capacitance and arterial resistance, respectively, produced by the adrenergic agonists. Isoproterenol, a pure beta-adrenergic agonist, decreased both MAP and RV. Norepinephrine (NE), which activates both alpha 1- and alpha 2-receptors in humans, increased both MAP and RV, while phenylephrine (PH) a selective alpha 1-adrenergic agonist, increased only MAP with no significant change in RV. It is concluded that in humans during hypothermic CPB, beta-agonists dilate both the resistance and capacitance vessels, selective alpha 1-adrenergic agonists preferentially constrict the resistance vessels, and non-selective alpha 1- and alpha 2-adrenergic agonists constrict both the resistance and capacitance vessels.

Effects of preoperative calcium channel and beta-adrenergic blockade on the vascular response to adrenergic agonists during cardiopulmonary bypass.

The present report compares the effects of isoproterenol (ISO), norepinephrine (NE), and phenylephrine (PH) on the mean arterial pressure (MAP) and reservoir volume (RV) during cardiopulmonary (CPB) in 16 patients undergoing coronary artery bypass grafting (CABG) who were treated preoperatively with oral nifedipine (10 to 40 mg, three times a day) and propranolol (40 to 60 mg, three times a day). The changes of RV and MAP were used as indices of the changes in venous capacitance and arterial resistance, respectively, produced by the adrenergic agonists. ISO, a beta-adrenergic agonist, decreased both MAP and RV. NE, which activates both alpha 1- and alpha 2- adrenoceptors, increased both MAP and RV, while PH, a selective alpha 1-adrenergic agonist, increased only MAP with no significant change in RV. The changes are qualitatively similar to those previously achieved in patients undergoing valve replacement who did not receive any blocker preoperatively. However, the decrease of MAP by ISO and its increase by NE were significantly less in the CABG group. It is concluded that preoperative preparation of patients undergoing CABG with beta-adrenergic blockers and calcium channel blockers can modify the effect of ISO and NE on the peripheral resistance, but they have no significant effect on the action of PH at the doses selected in this study.

Verapamil pretreatment before aortic cross-clamping in patients undergoing coronary artery bypass graft.

In 12 of 22 patients with coronary artery disease undergoing elective coronary artery bypass graft (CABG), verapamil (0.075 mg/kg body weight) pretreatment was given on initiation of cardiopulmonary bypass (CPB) before aortic cross-clamping (ACC), whereas no verapamil was used in 10 control patients. The volume of cardioplegia solution required to achieve and maintain cardiac asystole during ACC while on CPB was not significantly different in the two groups. After the release of ACC, the energy of direct current countershock required for defibrillation, the incidence of heart block, and the need for pacemaker were not significantly different. However, defibrillation was more readily achieved by a single countershock in the verapamil group than in the control group. Also, the incidence of ST segment changes after defibrillation was significantly lower in the verapamil group, suggesting that verapamil pretreatment before ACC may potentiate the myocardial preservation achieved by the cardioplegia, and hence may decrease the incidence of ischemic changes during the critical reperfusion period.