Structural and functional linkages between subunit interfaces in mammalian pyruvate kinase.

Mammalian pyruvate kinase (PK) is a four-domain enzyme that is active as a homo-tetramer. Tissue-specific isozymes of PK exhibit distinct levels of allosteric regulation. PK expressed in muscle tissue (M1-PK) shows hyperbolic steady-state kinetics, whereas PK expressed in kidney tissue (M2-PK) displays sigmoidal kinetics. Rabbit M1 and M2-PK are isozymes whose sequences differ in only 22 out of 530 residues per subunit, and these changes are localized in an inter-subunit interface. Previous studies have shown that a single amino acid mutation to M1-PK at either the Y (S402P) or Z (T340 M) subunit interface can confer a level of allosteric regulation that is intermediate to M1-PK and M2-PK. In an effort to elucidate the roles of the inter-subunit interaction in signal transmission and the functional/structural connectivity between these interfaces, the S402P mutant of M1-PK was crystallized and its structure resolved to 2.8 A. Although the overall S402P M1-PK structure is nearly identical with the wild-type structure within experimental error, significant differences in the conformation of the backbone are found at the site of mutation along the Y interface. In addition, there is a significant change along the Z interface, namely, a loss of an inter-subunit salt-bridge between Asp177 of domain B and Arg341 of domain A of the opposing subunit. Concurrent with the loss of the salt-bridge is an increase in the degree of rotational flexibility of domain B that constitutes the active site. Comparison of previous PK structures shows a correlation between an increase in this domain movement with the loss of the Asp177: Arg341 salt-bridge. These results identify the structural linkages between the Y and Z interfaces in regulating the interconversion of conformational states of rabbit M1-PK.

Individual responses to heparinization for extracorporeal circulation.

It has been proposed that wide variations in individual response to heparin lead to deficiencies in popular heparinization protocols for extracorporeal circulation (ECC). Thirty-nine patients undergoing open cardiac operations with ECC were anticoagulated with the heparinization protocol in use at St. Thomas' Hospital. The coagulation state was monitored with the blood activated recalcification time (BART) test. Wide variations in heparin dose-response and heparin activity-decay curves were observed. No patient was underheparinized, but many had markedly prolonged BART's. The total dose of heparin declined because BART monitoring allowed elimination of incremental heparin doses up to 180 minutes of ECC. Adequate reversal with protamine was achieved in all patients regardless of response to herparin. Alternative approaches for heparinization for ECC can be developed with the aid of rapid tests of the intraoperative coagulation state.

Modified ultrafiltration attenuates dilutional coagulopathy in pediatric open heart operations.

BACKGROUND: Extreme hemodilution caused by relatively large prime volumes required for cardiopulmonary bypass in infants causes a dilutional coagulopathy, characterized by low concentrations of fibrinogen and other circulating coagulation factors. Modified ultrafiltration results in hemoconcentration and is associated with decreases in postoperative bleeding and transfusion requirements in children. This study was undertaken to quantify the effect of modified ultrafiltration on concentrations of fibrinogen, plasma proteins, and platelets in infants and small children. METHODS: Twenty patients less than 15 kg were studied. Cardiopulmonary bypass circuits were primed with crystalloid solutions. Red blood cells were added during cardiopulmonary bypass for hematocrits less than 15%. Colloid solutions were not administered. Concentrations of fibrinogen, plasma proteins, and platelets, and hematocrit were measured before cardiopulmonary bypass, before modified ultrafiltration, and after modified ultrafiltration. RESULTS: Modified ultrafiltration was associated with significant (p < 0.001) increases in hematocrit (19% +/- 6% to 31% +/- 9%), fibrinogen (65 +/- 29 to 101 +/- 45 mg/dL), and total plasma proteins (2.7 +/- 0.3 to 4.9 +/- 0.7 g/dL), but no change (p = 0.129) in platelet count. CONCLUSIONS: We conclude that modified ultrafiltration significantly attenuates the dilutional coagulopathy associated with cardiopulmonary bypass in infants.

Cardiovascular effects of inhalation induction with isoflurane in infants.

We evaluated hemodynamic changes during inhalation induction of isoflurane anesthesia in 60 healthy infants aged 5-26 weeks who were randomly divided into two groups of 30 patients each. In group 1 anesthesia was induced using isoflurane in concentrations that were increased to 3.5%. In group 2, 0.02 mg/kg of atropine was given intramuscularly before induction of anesthesia, as in group 1. In both groups, N2O (3 L/min) and O2 (2 L/min) were administered using a nonrebreathing system. Heart rate (HR) and blood pressure (BP) were recorded at 1-min intervals for 20 min. HR decreased 32% in group 1 and 20% in group 2; BP decreased 40% in group 1 and 38% in group 2. During isoflurane induction in infants, both HR and BP are depressed. Premedication with atropine minimizes the depression of HR, but does not affect the change in BP.

Oral transmucosal fentanyl citrate for preanesthetic medication of pediatric day surgery patients with and without droperidol as a prophylactic anti-emetic.

he safety and efficacy of oral transmucosal fentanyl citrate (OTFC) as a preanesthetic medication and the efficacy of droperidol as a prophylactic anti-emetic were evaluated in 100 children aged 2-8 yr undergoing general anesthesia for outpatient surgery. Patients were randomly assigned to one of four groups and managed in a double-blinded manner: 1) placebo lozenge 45 min preoperatively and placebo (normal saline) injected intravenously after induction of anesthesia; 2) placebo lozenge 45 min preoperatively and 50 micrograms/kg droperidol intravenously after induction; 3) 15-20 micrograms/kg OTFC lozenge 45 min preoperatively and placebo intravenously after induction; and 4) 15-20 micrograms/kg OTFC lozenge 45 min preoperatively and droperidol 50 micrograms/kg intravenously after induction. Anesthesia was induced and maintained with halothane and nitrous oxide in oxygen. Heart rate, respiratory rate, blood pressure, and hemoglobin oxygen saturation (SpO2) were monitored throughout the study. Scoring systems were used to evaluate sedation, anxiety, cooperation, and ease and quality of anesthetic induction. Emergence, recovery, and discharge times were recorded. Nausea, vomiting, and adverse effects were noted. Preoperatively, children receiving OTFC had significantly greater sedation, slower respiratory rates, lower SpO2, and less excitement during induction. Postoperative nausea and vomiting occurred significantly more frequently after OTFC than after placebo. Prophylactic droperidol did not significantly reduce the incidence of nausea and vomiting. The authors conclude that, in pediatric surgical outpatients, OTFC reliably induces preoperative sedation and facilitates inhalation induction of anesthesia, but it is associated with significant decreases in respiratory rate and SpO2 and a high incidence of postoperative nausea and vomiting that is not significantly reduced by prophylactic droperidol.

Elevated serum bromide concentrations following repeated halothane anaesthesia in a child.

A 20-month-old child received 25 brief halothane general anaesthetics over a five-week period to allow cranial irradiation treatments for a posterior fossa ependymoma. Personality change during the last week of the treatment protocol raised the question of possible bromide intoxication. Serum bromide concentrations, using a gold chloride assay technique, were monitored at that time, and at four- and six-week intervals thereafter. Serum bromide concentrations demonstrated a four-fold change during this period ranging from peak levels of 2.2 mEq.L-1 (176 micrograms.kg-1) during the fifth week of treatment decreasing to less than 0.5 mEq.L-1 (less than 40 micrograms.ml-1) six weeks following the end of treatments. This demonstrates the possibility for repetitive, short halothane exposures to result in elevations of serum bromide and the potential of bromide intoxication in paediatric neuro-oncology patients.

Oral atropine premedication in infants attenuates cardiovascular depression during halothane anesthesia.

The efficacy of oral atropine premedication in attenuation of the cardiovascular depression associated with halothane anesthesia has not been previously evaluated. A solution containing either oral atropine 0.04 mg/kg (HI), 0.02 mg/kg (LO), or a placebo (NO) was randomly administered to 36 infants 1-6 months old and 36 infants 7-15 months old 30-90 minutes before induction of anesthesia. The onset of action of atropine was approximately 25 minutes after administration as determined by a 15% increase in heart rate (HR) above baseline levels. Heart rate, systolic blood pressure (SBP), and mean arterial blood pressure (MAP) were then measured at 1-minute intervals starting just before induction of anesthesia and continuing until onset of surgical stimulation during anesthesia with halothane (up to 3%), nitrous oxide (60%), and oxygen (40%). In infants 1-6 months old, either dosage of oral atropine preserved HR and SBP as compared with placebo. In infants 7-15 months old, either dosage preserved HR but not SBP. The severity of hypotension was greatest in infants 1-6 months of age given placebos. No significant differences existed between oral atropine 0.04 mg/kg or 0.02 mg/kg in either age range. It is concluded that premedication with oral atropine 0.02 mg/kg is effective in attenuating the cardiovascular depression associated with halothane anesthesia in infants.

The negative dominant effects of T340M mutation on mammalian pyruvate kinase.

A fundamental issue in allosteric regulatory enzymes is the identification of pathways of signal transmission. Rabbit muscle and kidney pyruvate kinase isozymes are ideal to address this issue because these isozymes exhibit different enzymatic regulatory patterns, and the sequence differences between these isozymes have identified the amino acid residues that alter their kinetic behavior. In an earlier study, Cheng et al. (Cheng, X., Friesen, R. H. E., and Lee, J. C. (1996) J. Biol. Chem. 271, 6313-6321), reported the effects of a threonine to methionine mutation at residue 340 in the muscle isozyme. In this study, the same mutation was effected in the kidney isozyme. Qualitatively, the same negative effects are observed in both isozymes, namely a significant decrease in catalytic efficiency and decrease in apparent affinity for phosphoenolpyruvate but no change in affinity for ADP, and a decrease in responsiveness to the presence of effectors, be it activator or inhibitor. Because the diversity in the primary sequence between these two isozymes does not alter the negative impact of the T340M mutation, it can be concluded that this mutation exerts a dominant, negative effect. The negative effects of T340M mutation on the kinetic properties imply that there is communication between residue 340 and the active site. Residue 340 is located at the 1,4 subunit interface; however, a T340M mutation enhances the dimerization affinity along the 1,2 subunit interface. Thus, this study has identified a communication network among the active site, residue 340, and the 1,2 subunit interface.

End-tidal PCO2 monitoring via nasal cannulae in pediatric patients: accuracy and sources of error.

OBJECTIVE: To assess the correlation and accuracy of end-tidal PCO2 (PetCO2) sampled via nasal cannulae in pediatric patients by comparison to the criterion standard PaCO2, and to identify sources of error during PetCO2 monitoring via nasal cannulae. METHODS: PetCO2 was monitored continuously by sampling end-tidal gas through nasal cannulae that had been designed and manufactured for this purpose in spontaneously breathing children undergoing conscious or deep sedation during either cardiac catheterization (n = 43) or critical care (n = 54). When both the capnographic wave form and the PetCO2 value had been stable for at least 10 minutes, the PetCO2 value was recorded while blood was drawn from an indwelling arterial line for PaCO2 measurement. The effects of age, weight, respiratory rate, oxygen delivery system, airway obstruction, mouth breathing, and cyanotic heart disease were evaluated by linear regression analysis and calculation of absolute bias (PaCO2-PetCO2). RESULTS: Mouth breathing, airway obstruction, oxygen delivery through the ipsilateral nasal cannula, and cyanotic heart disease adversely affected accuracy. In patients without those factors, PetCO2 correlated well with PaCO2 (R2 = 0.994), and absolute bias was 3.0 +/- 1.8 mmHg. CONCLUSIONS: Several factors-some controllable and all recognizable-affect the accuracy of PetCO2 monitored via nasal cannulae in pediatric patients. When these factors are not present, PetCO2 correlates well with PaCO2 and appears to be a useful monitor of ventilatory status during conscious or deep sedation.

Changes in carbon dioxide tension and oxygen saturation during deep sedation for paediatric cardiac catheterization.

The purpose of this observational study was to determine whether hypercarbia or oxygen desaturation occurred during our current regimens of deep sedation or general anaesthesia of infants and children undergoing cardiac catheterization. Data were gathered prospectively from 50 consecutive infants and children aged 4 months to 12 years undergoing cardiac catheterization. Several anaesthetists used the following regimens, which were not randomized: 1) propofol. 1.5-2.0 mg.kg-1 and fentanyl 1 microgram.kg-1 IV over 2 min for induction, followed by propofol infusion of 100-150 micrograms.kg-1.min-1; 2) fentanyl 2-3 micrograms.kg-1 and midazolam 0.1-0.2 mg.kg-1 IV over 10-15 min; 3) ketamine 8 mg.kg-1 IM, or 4) same as regimens 1 or 2, plus pancuronium, intubation and controlled ventilation. Regimens 1, 2, and 3 were associated with spontaneous ventilation through the natural airway. End-tidal carbon dioxide tension (PetCO2), SpO2, and respiratory rate were monitored for 60 min. The three regimens employing spontaneous ventilation through the natural airway were associated with both statistically and clinically significant increases in PetCO2 and decreases in SpO2. This raises the possibility that acute exacerbation of PAP and PVR may occur in pulmonary hypertensive patients. In contrast, PetCO2 and SpO2 did not change significantly from baseline in the controlled ventilation group.

Indirect measurement of blood pressure in neonates and infants utilizing an automatic noninvasive oscillometric monitor.

An indirect automatic blood pressure (BP) monitor utilizing the oscillometric principle and a microprocessor is now available for use in infants. This device was evaluated by comparing its systolic BP determinations with simultaneous Doppler systolic BP determinations in neonates and infants undergoing general anesthesia. Five determinations each were made in 125 babies who were grouped according to age. Correlation coefficients of the paired BP determinations were: in 27 premature neonates, r = 0.82; in 24 term neonates, r = 0.93; in 47 infants aged 5 to 13 weeks, r = 0.92; and in 27 infants aged 14 to 26 weeks, r = 0.94. The device was also evaluated by comparing its BP determinations with those of indwelling arterial lines present in 20 additional infants (five determinations each). Correlation coefficients were 0.96 for systolic determinations and 0.94 for diastolic determinations. No morbidity or significant technical problems were noted. It was concluded that the device is an accurate, safe, and easy noninvasive method for monitoring BP in infants.

Cardiovascular depression during halothane anesthesia in infants: study of three induction techniques.

Hemodynamic changes during three techniques for induction of halothane anesthesia were studied in 90 healthy infants aged 5 to 26 weeks who were randomly divided into three groups of 30 patients each. In group I, anesthesia was induced using halothane in concentrations that were increased to 3%. In group II, atropine, 0.02 mg/kg, was given intramuscularly before induction, followed by induction as in group I. In group III, halothane in concentrations that were increased to 1.25% was followed by intramuscular succinylcholine, 2 mg/kg, 90 seconds after the start of induction. In all three groups N2O 3 L/min and O2 2 L/min were employed using a non-rebreathing system. Heart rate (HR) and blood pressure (BP) were recorded at 1-minute intervals for 20 minutes. HR decreased 30% in group I, 18% in group II (p less than 0.01), and 29% in group III. BP decreased 50% in group I (p less than 0.01), 34% in group II, and 33% in group III. During halothane induction in infants, bradycardia can be minimized with preoperative atropine; the degree of hypotension can be diminished with either preoperative atropine or the use of intramuscular succinylcholine combined with lower concentrations of halothane.

Cardiovascular changes in preterm neonates receiving isoflurane, halothane, fentanyl, and ketamine.

Hemodynamic changes during four anesthetic techniques were studied in 80 preterm neonates. Atropine, 0.02 mg/kg, and pancuronium, 0.1 mg/kg, were given intravenously to all patients, who were ventilated with oxygen and air. Each group of 20 patients then received 0.75% isoflurane, 0.5% halothane, 20 micrograms/kg fentanyl, or 2 mg/kg ketamine. Heart rate (HR), systolic blood pressure (SAP), and mean blood pressure (MAP) were recorded at 1-min intervals until surgical stimulation. HR remained at or above control level in all groups. Statistically significant decreases (P less than 0.01) in SAP and MAP occurred following administration of each anesthetic. SAP decreased 30% during isoflurane administration, 25% during halothane, 21% following fentanyl, and 16% following ketamine. Clinically important decreases (25% or greater) in SAP were observed in some patients in each group, but the incidence was significantly less in patients receiving ketamine (P less than 0.02). The covariables of conceptual age, postnatal age, weight, urine specific gravity, hematocrit, and presence of patent ductus arteriosus did not have statistically significant effects on SAP and MAP changes. The authors conclude that SAP and MAP decrease significantly during each of the anesthetic techniques studied and that clinically important decreases in SAP occur less frequently during the technique using ketamine.

Changes in anterior fontanel pressure in preterm neonates during tracheal intubation.

Anterior fontanel pressure (AFP), a noninvasive indicator of intracranial pressure (ICP), was monitored during tracheal intubation in two groups of preterm neonates without neurologic disease. Anterior fontanel pressure was monitored and recorded continuously with a Ladd AFP monitor. Systolic and mean blood pressures were recorded at 1-min intervals. In group 1 (n = 6) patients, 0.02 mg/kg intravenous atropine was administered and awake intubation was performed. Group 2 (n = 6) patients received 0.02 mg/kg intravenous atropine and 0.1 mg/kg pancuronium and one of four anesthetics--0.75% isoflurane, 0.5% halothane, 20 micrograms/kg fentanyl, or 2 mg/kg ketamine--with intubation after 10 min of mask ventilation. In group 1, AFP increased from 7.7 cm H2O to 23.8 cm H2O (P less than 0.05); the mean increase in AFP was 197%. Anterior fontanel pressure did not change significantly in group 2. Significant increases in AFP may increase the risk of intraventricular hemorrhage in preterm neonates. The present data indicate that indirectly measured ICP increases significantly during awake tracheal intubation in preterm neonates and that this increase can be prevented by prior administration of pancuronium and a general anesthetic.

Haemodynamic depression by halothane is age-related in paediatric patients.

The hypothesis that young infants are more sensitive to the haemodynamic depressant effects of halothane compared with older children was tested. One hundred and sixty unpremedicated, ASA physical status I or II paediatric patients without cardiac or pulmonary disease were divided into five age groups: term neonates, 1-6 months, 6-24 months, 2-6 years and 6-12 years. Anaesthetic induction was achieved with halothane in oxygen and air via mask. Vecuronium 0.1 mg.kg-1 was administered intravenously. During normocapnic manual ventilation by mask, endtidal halothane concentration was maintained at either 2xage-specific MAC (Method I) or 1.7% (Method II) in 20 patients in each age group for 10 min. In both Method I and Method II, systolic and mean blood pressure of term neonates and infants aged 1-6 months decreased significantly (P < 0.01) compared with other age groups. The results of this study demonstrate that neonates and young infants are more susceptible to haemodynamic depression during halothane anaesthesia than are older children, confirming clinical experience.

Effect of preanesthetic medication on carbon dioxide tension in children with congenital heart disease.

Hypercarbia during the postoperative period following repair of congenital heart defects in children has been associated with acute pulmonary hypertension. Because decreases in respiratory rate (RR) and digital pulse oximetry (SpO2) have been observed after preanesthetic medication of similar children, it is possible that hypercarbia and pulmonary hypertension may be unappreciated risks in premedicated children during the preoperative period. As the first step in addressing this question, changes in transcutaneous and end-tidal PCO2 (PtcCO2 and PetCO2) were examined after preanesthetic medication of children prior to cardiac surgery. Forty-four children were randomly assigned to receive either intramuscular morphine, 0.2 mg/kg, and scopolamine, 0.01 mg/kg, or oral midazolam, 0.75 mg/kg, 1 hour before anesthetic induction. PtcCO2, PetCO2, SpO2, RR, and sedation score were monitored. Significant sedation occurred after both premedication regimens. Following morphine/scopolamine, PtcCO2 increased from 36 +/- 4 (mean +/- SD) to 43 +/- 6 mmHg (P < 0.01), PetCO2 increased from 35 +/- 3 to 40 +/- 5 mmHg (P < 0.01), SpO2 decreased from 93 +/- 2 to 91 +/- 4% (P < 0.01), and RR decreased from 30 +/- 10 to 24 +/- 7 breaths/minute (P < 0.01). After midazolam, PtcCO2 increased from 35 +/- 4 to 40 +/- 6 mmHg (P < 0.01), PetCO2 increased from 34 +/- 5 to 39 +/- 3 mmHg (P < 0.01), SpO2 decreased from 93 +/- 6 to 90 +/- 7% (P < 0.01), and RR decreased from 33 +/- 13 to 30 +/- 13 breaths/minute (P < 0.01). Clinically significant increases in PtcCO2 (> 45 mmHg) occurred in nine patients, including five with pulmonary hypertension.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of conserved residues on the regulation of rabbit muscle pyruvate kinase.

A cDNA encoding the complete rabbit muscle pyruvate kinase isozyme (RMPK) was cloned using the method of rapid amplification of cDNA ends. The sequence encodes a polypeptide chain of 530 amino acids which differs in three amino acid residues from a sequence reported by Larsen et al. (Larsen, T.M., Laughlin, T., Holden, H.M., Rayment, L, and Reed, G.H. (1994) Biochemistry 33, 6301-6309). Glu233-Gln234 and Ala400 were identified instead of Asp233-Glu234 and Ser400, respectively. The recombinant RMPK was overexpressed in the Escherichia coli JM 105 cells. Purified recombinant pyruvate kinase displayed identical physical and enzymatic properties as the authentic enzyme. Three point mutants of RMPK were constructed using site-directed mutagenesis. Like the wild type RMPK, sedimentation, and CD spectroscopic studies show that purified RI 19C and T340M are tetrameric proteins with similar secondary and tertiary structures. Mutant R119C enzyme exhibits 0.6% of the value of k(cat) and an order of magnitude decrease in the apparent affinity for ADP as compared to the wild type PK. The overall response to inhibitor and activator, Phe and FBP, respectively, were not affected by the R119C mutation. The T340M mutant enzyme is only half as active as the wild type PK. T340M is more susceptible to inhibition by Phe but apparently is not responsive to the activator FBP. The kinetic behavior of the Q377K mutant enzyme is in between that of the R119C and T340M mutants exhibiting 5% of the wild type enzymatic activity and an enhanced sensitivity to the inhibitor, Phe, while maintaining the same responsiveness to FBP and apparent affinities for substrates. The significant decrease in activity in all three mutants mimics the exact consequences of the same mutations in human erythrocyte PK from hemolytic anemia patients. Thus, this study demonstrates not only the effects of these conserved residues in the regulatory properties of mammalian PK. but also that the observed effects are most likely applicable to all isozymic forms of PK.

Quaternary structure of the lactose transport protein of Streptococcus thermophilus in the detergent-solubilized and membrane-reconstituted state.

The quaternary structure of LacS, the lactose transporter of Streptococcus thermophilus, has been determined for the detergent-solubilized and the membrane-reconstituted state of the protein. The quaternary structure of the n-dodecyl-beta-d-maltoside-solubilized state was studied using a combination of sedimentation velocity and equilibrium centrifugation analysis. From these measurements it followed that the detergent-solubilized LacS undergoes reversible self-association with a monomer to dimer mode of association. The association constants were 5.4 +/- 3.6 and 4.4 +/- 1.0 ml mg(-1) as determined from the velocity and equilibrium sedimentation measurements, respectively. The experiments did not indicate significant changes in the shape of the protein-detergent complex or the amount of detergent bound in going from the monomeric to dimeric state of LacS. Importantly, a single Cys mutant of LacS is labeled by 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid in a substrate-dependent manner, indicating that the detergent-solubilized protein exhibits ligand binding activity. The quaternary structure of membrane-reconstituted LacS was determined by freeze-fracture electron microscopy analysis. Recent developments in the analysis of freeze-fracture images (Eskandari, S. P., Wright, E. M., Freman, M., Starace, D. M., and Zampighi, G. A. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 11235-11240) allowed us to directly correlate the cross-sectional area of the transmembrane segment to a dimeric state of the functionally membrane-reconstituted LacS protein. The cross-sectional area of the LacS protein was calibrated using the membrane-reconstituted transmembrane domain of the mannitol transporter enzyme II, an intramembrane particle for which the cross-sectional area was obtained from maps of two-dimensional crystals. The consequences of the determined quaternary structure for the transport function and regulation of LacS are discussed.