Defining the limitations of transcutaneous bilirubin measurement in late preterm newborns.

OBJECTIVE: The objective of this study is to determine the impact of postnatal age on the bias between transcutaneous (TcB) and total serum bilirubin (TSB), and evaluate a TcB screening protocol. STUDY DESIGN: Preterm and term infants had paired TcB and TSB performed on days 1 to 3 of life; a subset of preterm infants had measurements on days 4 to 7. Sensitivity and specificity of TcB (plotted on an age-specific TSB nomogram) for prediction of high-intermediate (HIR) or high-risk TSB were calculated. RESULTS: Median TcB bias was 2.6 and 2.5 mg dl-1 for term and preterm infants in the first 3 days of life, respectively. However, median bias was 2.2 mg dl-1 for preterm infants at 4 to 7 days of life. TcB in preterm infants predicted HIR or high-risk TSB with 94% sensitivity and 56% specificity. CONCLUSION: TcB screening protocols developed for term infants can be used for late preterm infants in the first 3 days of life.

Correlation of cefpodoxime susceptibility with cephalothin and cefuroxime for urinary tract isolates.

This study attempted to determine whether cefuroxime was superior to cephalothin as a surrogate marker for cefpodoxime among urinary tract isolates. The MicroScan system (Siemens) was used to determine susceptibility for cephalothin and cefuroxime on consecutive cultures with a colony count of ≥50 000 organisms. Simultaneously, an Etest (bioMérieux) for cefpodoxime was conducted. The cefpodoxime interpretation was compared to that of the other two agents, and the categorical agreement was calculated, defined as the percentage of identical susceptibility interpretations. Cefuroxime (83 %) had a significantly higher categorical agreement than cephalothin (63 %) among 300 isolates (P<0.01). The major error rate was 16 % for cephalothin and 3 % for cefuroxime. The very major error rate was 7 % for cephalothin and 14 % for cefuroxime among the 14 cefpodoxime-resistant isolates. For Escherichia coli, the major error rates were 15 % and 1 % for cephalothin and cefuroxime, respectively. Very major error rates were 9 % for both agents. Cefuroxime was a better predictor of cefpodoxime susceptibility than cephalothin, and appears to be the preferred surrogate agent for the MicroScan system, particularly for E. coli.

Effect of universal neonatal transcutaneous bilirubin screening on blood draws for bilirubin analysis and phototherapy usage.

OBJECTIVE: To determine the effect of universal transcutaneous bilirubin (TcB) screening on total serum bilirubin (TSB) blood draws and phototherapy usage. STUDY DESIGN: The subjects were infants ≥ 36 weeks gestation. In period 1, TSB was ordered based on clinical factors. In period 2, all infants underwent predischarge TcB measurement; infants with adjusted TcB values in the high-intermediate or high-risk zones had TSB ordered. Data were extracted through chart review. RESULT: TSB measurements per 1000 infants decreased from 717 to 713 (P=0.008) between period 1 and 2, with more outpatient and less inpatient blood draws in period 2. Between periods 1 and 2, total phototherapy decreased from 59 to 39 per 1000 infants (P<0.0001), with less inpatient and more readmission phototherapy. CONCLUSION: Universal TcB screening was implemented without increasing total blood draws or phototherapy treatment; however, it was associated with a shift in blood draws and phototherapy usage from inpatients to outpatients.

Vancomycin MIC susceptibility testing of methicillin-susceptible and methicillin-resistant Staphylococcus aureus isolates: a comparison between Etest® and an automated testing method.

BACKGROUND: Vancomycin treatment failures and increased mortality have been reported in methicillin-resistant Staphylococcus aureus (MRSA) isolates with minimum inhibitory concentrations (MICs) >1 µg/mL. Most of this data utilized manual testing to determine the MIC. Recent vancomycin treatment guidelines do not specify the optimal testing method to define the MIC. METHODS: Over a twelve-month study period, we compared manual susceptibility testing by Etest® (AB Biodisk, Solna, Sweden) with automated testing by MicroScan Walk-Away® (Dade Behring, Inc., East Mississauga, Ontario) to determine the difference in the MICs among 383 sequential clinical S aureus isolates. RESULTS: Manual testing demonstrated MICs of 1.5 µg/mL or 2.0 µg/mL in 90% and 86% of MRSA and methicillin-sensitive Staphylococcus aureus (MSSA) isolates, respectively. Automated testing revealed MICs of 2.0 µg/mL for 56% and 54% of MRSA and MSSA isolates, respectively. The manual MIC test by Etest® was >1 µg/mL in 87% of MRSA isolates and 86% of methicillin-susceptible S aureus isolates in which the automated MIC result was 1 µg/mL. This same finding occurred in 94% (17/18) of S aureus isolates causing non-skin/skin structure infections. Among all subgroups of isolates, manual testing demonstrated statistically significant higher MICs compared to automated testing. CONCLUSIONS: MIC results generated by the Etest® consistently revealed a one dilution higher vancomycin MIC compared to MicroScan®. Automated MIC results of invasive MRSA isolates should be confirmed by manual Etest® to ensure identification of those isolates with vancomycin MICs >1µg/mL that are at risk for vancomycin treatment failure or increased mortality.

Results from a multicenter evaluation of the 4th generation Elecsys Troponin T assay.

BACKGROUND AND OBJECTIVE: Discrepancies between serum and heparin plasma samples have been described for many commercial troponin assays including the cardiac troponin T (cTnT) assay. Using the current 3rd generation Elecsys Troponin T immunoassay, heparin plasma cannot be recommended for the determination of cTnT due to systematic lower test results caused by a direct interference of the immunoassay by heparin. The purpose of the multicenter study was to evaluate the analytical performance of an improved 4th generation Elecsys Troponin T immunoassay with a special focus on the comparability of cTnT results determined in heparin plasma and serum. METHODS AND RESULTS: The multicenter evaluation was performed in 10 clinical laboratories according to a standardized protocol (Roche Diagnostics, Penzberg, Germany, Study No. B05P008). The Elecsys Troponin T immunoassay was performed on the Modular Analytics E170 and Elecsys 2010 systems. Intraassay imprecision (n = 21) and total imprecision (2 runs/d, 10 days, triplicate measurements) were evaluated using 2 commercial controls (Roche Diagnostics) and 6 different serum pools (cTnT: 0.0140 - 4.102 microg/L). Intraassay CVs ranged from 0.73 to 3.22%. Total imprecision CVs ranged from 3.61 to 35.45% (cTnT < 0.1 microg/L) and 1.82 to 9.09% (cTnT > 0.1 microg/L), respectively. The cut-off for myocardial necrosis was determined to be 0.03 microg/L using the 10% total imprecision CV criteria. Linearity was assessed by serial dilutions of 6 different serum samples using cTnT negative serum pools. Linearity was proven up to 21.3 microg/L (recoveries: 90% - 110%). Regression data of all comparison studies were calculated according to the method of Passing and Bablok. The method comparison between the 4th generation and the commercially available cTnT immunoassay showed highly similar results across the whole measuring range (0.01 - 25.0 microg/L): y = 1.024x -0.001, r = 0.998; n = 988. Using the commercially available cTnT reagent, the serum to heparin plasma comparison yielded a systematic bias to approximately 8% lower cTnT results in heparin plasma. However, suitable comparability was obtained using the 4th generation Elecsys cTnT assay. The regression analysis (serum vs. heparin plasma) across the studied measuring range (cTnT: 0.01 - 14 microg/L) yielded the following equation: y = 0.975x + 0.001; r = 0.986; n = 403. However, rare individual serum to matched heparin plasma samples still yielded poor comparability (deviation > 20%) using the 4th generation Elecsys Troponin T immunoassay. CONCLUSION: Our data confirm an excellent analytical performance of the improved troponin T immunoassay. Most importantly, no systematic bias between cTnT results determined in serum and heparin plasma was observed from data obtained in 7 evaluation sites. The performance of the 4th generation Elecsys Troponin T assay is therefore comparable to other commercially available troponin immunoassays. Further studies are necessary to investigate the cause of poor comparability of cTnT results in rare individual serum to matched heparin plasma samples.

Cooperative effects of potassium, magnesium, and magnesium-ADP on the release of Escherichia coli dihydrofolate reductase from the chaperonin GroEL.

Previous investigation has shown that at 22 degrees C and in the presence of the chaperonin GroEL, the slowest step in the refolding of Escherichia coli dihydrofolate reductase (EcDHFR) reflects release of a late folding intermediate from the cavity of GroEL (Clark AC, Frieden C, 1997, J Mol Biol 268:512-525). In this paper, we investigate the effects of potassium, magnesium, and MgADP on the release of the EcDHFR late folding intermediate from GroEL. The data demonstrate that GroEL consists of at least two conformational states, with apparent rate constants for EcDHFR release that differ by four- to fivefold. In the absence of potassium, magnesium, and ADP, approximately 80-90% of GroEL resides in the form with the faster rate of release. Magnesium and potassium both shift the distribution of GroEL forms toward the form with the slower release rate, though cooperativity for the magnesium-induced transition is observed only in the presence of potassium. MgADP at low concentrations (0-50 microM) shifts the distribution of GroEL forms toward the form with the faster release rate, and this effect is also potassium dependent. Nearly identical results were obtained with a GroEL mutant that forms only a single ring, demonstrating that these effects occur within a single toroid of GroEL. In the presence of saturating magnesium, potassium, and MgADP, the apparent rate constant for the release of EcDHFR from wild-type GroEL at 22 degrees C reaches a limiting value of 0.014 s(-1). For the single ring mutant of GroEL, the rate of EcDHFR release under the same conditions reaches a limiting value of 0.024 s(-1), suggesting that inter-ring negative cooperativity exists for MgADP-induced substrate release. The data suggest that MgADP preferentially binds to one conformation of GroEL, that with the faster apparent rate constant for EcDHFR release, and induces a conformational change leading to more rapid release of substrate protein.

Different anesthetic sensitivities of skeletal and cardiac isoforms of the Ca-ATPase.

We have previously shown that low levels of the volatile anesthetic halothane activate the Ca-ATPase in skeletal sarcoplasmic reticulum (SR), but inhibit the Ca-ATPase in cardiac SR. In this study, we ask whether the differential inhibition is due to (a) the presence of the regulatory protein phospholamban in cardiac SR, (b) different lipid environments in skeletal and cardiac SR, or (c) the different Ca-ATPase isoforms present in the two tissues. By expressing skeletal (SERCA 1) and cardiac (SERCA 2a) isoforms of the Ca-ATPase in Sf21 insect cell organelles, we found that differential anesthetic effects in skeletal and cardiac SR are due to differential sensitivities of the SERCA 1 and SERCA 2a isoforms to anesthetics. Low levels of halothane inhibit the SERCA 2a isoform of the Ca-ATPase, and have little effect on the SERCA 1 isoform. The biochemical mechanism of halothane inhibition involves stabilization of E2 conformations of the Ca-ATPase, suggesting direct anesthetic interaction with the ATPase. This study establishes a biochemical model for the mechanism of action of an anesthetic on a membrane protein, and should lead to the identification of anesthetic binding sites on the SERCA 1 and SERCA 2a isoforms of the Ca-ATPase.

Mechanisms of dopamine and dobutamine interference in biochemical tests that use peroxide and peroxidase to generate chromophore.

Dopamine and dobutamine have recently been shown to produce a negative interference in several biochemical tests that use peroxide and peroxidase to generate a chromophore. To define the chemical mechanism of this interference, we examined the effects of dopamine and dobutamine in various peroxidase-based biochemical tests. Dopamine interfered stoichiometrically with peroxidase-based tests that use 4-aminophenazone to form chromophore but interfered little in those that use other compounds to generate chromophore. Dopamine reacts with 4-aminophenazone in the presence of peroxide and peroxidase to form a novel quinone-imine dye, with a smaller absorptivity than the chromophore formed in the absence of dopamine. The smaller absorptivity of this novel chromophore results in negative interference by reducing the total absorbance at the wavelength used to measure analyte. In contrast, dobutamine interfered stoichiometrically with all peroxidase-based tests studied, regardless of whether 4-aminophenazone was used to form the chromophore. Dobutamine was rapidly oxidized by peroxide in the presence of peroxidase, thus depleting the peroxide necessary to generate chromophore. Dopamine and dobutamine demonstrate two distinct general mechanisms of interference in peroxidase-based biochemical tests.

Outcome after normal exercise echocardiography and predictors of subsequent cardiac events: follow-up of 1,325 patients.

OBJECTIVES: This study sought to examine the outcome of a large group of patients after normal exercise echocardiography and to identify potential predictors of subsequent cardiac events. BACKGROUND: Earlier studies suggested that prognosis after normal exercise echocardiography is favorable, with a low subsequent cardiac event rate. These studies involved a small number of patients and did not have sufficient statistical power to stratify risk. METHODS: The outcomes of 1,325 patients who had normal exercise echocardiograms were examined. End points were overall and cardiac event-free survival. Cardiac events were defined as cardiac death, nonfatal myocardial infarction and coronary revascularization. Patient characteristics were analyzed in relation to time to first cardiac event in a univariate and multivariate manner to determine which, if any, were associated with an increased hazard of subsequent cardiac events. RESULTS: Overall survival of the study group was significantly better than that of an age- and gender-matched group obtained from life tables (p < 0.0001). The cardiac event-free survival rates at 1, 2 and 3 years were 99.2%, 97.8% and 97.4%, respectively. The cardiac event rate per person-year of follow-up was 0.9%. Subgroups with an intermediate or high pretest probability of having coronary artery disease also had low cardiac event rates. Multivariate predictors of subsequent cardiac events were angina during treadmill exercise testing (risk ratio [RR] 4.1, 95% confidence interval [CI] 1.5 to 11.0), low work load (defined as < 7 metabolic equivalents [METs] for men and < 5 METs for women; RR 3.2, 95% CI 1.4 to 7.6), echocardiographic left ventricular hypertrophy (RR 2.6, 95% CI 1.1 to 6.3) and advancing age (RR 1.04/year, 95% CI 1.0 to 1.1). CONCLUSIONS: The outcome after normal exercise echocardiography is excellent. Subgroups with an intermediate or high pretest probability of having coronary artery disease also have a favorable prognosis after a normal exercise echocardiogram. Characteristics predictive of subsequent cardiac events (i.e., patient age, work load, angina during exercise testing and echocardiographic left ventricular hypertrophy) should be considered in the clinical interpretation of a normal exercise echocardiogram.

Phospholamban-dependent effects of C12E8 on calcium transport and molecular dynamics in cardiac sarcoplasmic reticulum.

We have studied the effects of the nonionic detergent C12E8 on Ca-ATPase enzymatic activity and oligomeric state (detected by time-resolved phosphorescence anisotropy, TPA) in skeletal and cardiac sarcoplasmic reticulum (SR). In skeletal, SR, C12E8 inhibits the CA-ATPase, both at high (micromolar and above) and low (submicromolar) Ca. In cardiac SR, C12E8 inhibits at high Ca but activates at low Ca. Thus C12E8 activates enzymatic activity only in cardiac SR and only under conditions (submicromolar Ca) where phospholamban (PLB) regulates (inhibits) the enzyme [Lu, Y.-Z., & Kirchberger, M.A. (1994) Biochemistry 33, 5056-5062]. TPA of skeletal SR at low and high Ca demonstrates that C12E8 induces aggregation of ATPase monomers and small oligomers. C12E8 also aggregates the Ca-ATPase in cardiac SR at high Ca. In cardiac SR at low Ca, the Ca-ATPase is already highly aggregated, and C12E8 partially dissociates these aggregates. Thus the TPA results provide a simple physical explanation for the functional effects: C12E8 inhibits the ATPase when it aggregates the enzyme (skeletal SR at high and low Ca; cardiac SR at high Ca), and the detergent activates when it dissociates ATPase oligomers (cardiac SR at low Ca). C12E8 stabilizes the E2P conformation of the Ca-ATPase with respect to the E2 conformation, and this stabilization is PLB-dependent. Both the physical and functional effects of C12E8 on the Ca-ATPase are PLB-dependent, with C12E8 reversing the effects of PLB. The results provide insight into the mechanism by which PLB regulates the Ca-ATPase in cardiac SR.

Diagnosis and outpatient management of congestive heart failure.

Congestive heart failure causes substantial patient morbidity and mortality in the United States. Symptoms and physical findings can be helpful in diagnosis but have limited sensitivity and specificity. Objective measurement of ventricular function is essential in virtually all patients in whom a diagnosis of heart failure is suspected. Reversible causes of heart failure must be sought. Outpatient management includes education and counseling, emphasis on and assessment of compliance with diet, and pharmacologic treatment. Angiotensin-converting enzyme inhibitors are the mainstay of treatment but are underused, and maximal doses are not given apparently because of concern about side effects. Diuretic therapy should be administered only as needed to manage fluid overload. Calcium channel blockers are relatively contraindicated in patients with impaired ventricular function. Patient follow-up should be guided by the results of the medical history and physical examination. Routine serial testing of ventricular function and exercise performance is discouraged.

A continuous spectrophotometric assay for simultaneous measurement of calcium uptake and ATP hydrolysis in sarcoplasmic reticulum.

A continuous, spectrophotometric assay to simultaneously measure Ca uptake and ATP hydrolysis has been developed, in order to assess the function of the Ca-ATPase in skeletal and cardiac sarcoplasmic reticulum (SR) vesicles. The absorbance of Fura Red was measured continuously at 490 nm, in EGTA-buffered solutions containing initial free ionized calcium concentrations of 300 nM, 500 nM, 790 nM, and 2 microM, during assays of oxalate-facilitated or phosphate-facilitated active calcium uptake in skeletal SR. Simultaneous measurement of ATP hydrolysis during the measurement of phosphate-facilitated Ca uptake was accomplished by measuring the disappearance of NADH at 340 nm, coupled to the hydrolysis of ATP by an enzyme-linked, continuous ATPase assay. This new method, unlike the standard 45Ca-filtration assay, measures calcium uptake in real time and eliminates the need for radioactivity. Moreover, the rates of calcium uptake and ATP hydrolysis are measured simultaneously, allowing the direct quantitative comparison of the two parameters. This assay will facilitate the characterization of Ca-ATPase function and malfunction in skeletal and cardiac SR and advances the methodology for comparison of normal and physically, chemically, or biologically altered Ca-ATPase.

Anesthetics alter the physical and functional properties of the Ca-ATPase in cardiac sarcoplasmic reticulum.

We have studied the effects of the local anesthetic lidocaine, and the general anesthetic halothane, on the function and oligomeric state of the CA-ATPase in cardiac sarcoplasmic reticulum (SR). Oligomeric changes were detected by time-resolved phosphorescence anisotropy (TPA). Lidocaine inhibited and aggregated the Ca-ATPase in cardiac SR. Micromolar calcium or 0.5 M lithium chloride protected against lidocaine-induced inhibition, indicating that electrostatic interactions are essential to lidocaine inhibition of the Ca-ATPase. The phospholamban (PLB) antibody 2D12, which mimics PLB phosphorylation, had no effect on lidocaine inhibition of the Ca-ATPase in cardiac SR. Inhibition and aggregation of the Ca-ATPase in cardiac SR occurred at lower concentrations of lidocaine than necessary to inhibit and aggregate the Ca-ATPase in skeletal SR, suggesting that the cardiac isoform of the enzyme has a higher affinity for lidocaine. Halothane inhibited and aggregated the Ca-ATPase in cardiac SR. Both inhibition and aggregation of the Ca-ATPase by halothane were much greater in the presence of PLB antibody or when PLB was phosphorylated, indicating a protective effect of PLB on halothane-induced inhibition and aggregation. The effects of halothane on cardiac SR are opposite from the effects of halothane observed in skeletal SR, where halothane activates and dissociates the Ca-ATPase. These results underscore the crucial role of protein-protein interactions on Ca-ATPase regulation and anesthetic perturbation of cardiac SR.

Halothane and cyclopiazonic acid modulate Ca-ATPase oligomeric state and function in sarcoplasmic reticulum.

We have studied the effects of cyclopiazonic acid (CPA) and halothane on the enzymatic activity, oligomeric state, and conformational equilibrium of the Ca-ATPase in skeletal muscle sarcoplasmic reticulum (SR). CPA is a potent inhibitor of Ca-ATPase activity, and this inhibition is competitive with respect to ATP concentration. Time-resolved phosphorescence anisotropy was used to detect the fraction of Ca-ATPase monomers, dimers, and larger aggregates in the absence and presence of CPA. CPA increased the fraction of dimers and larger aggregates of the Ca-ATPase. Addition of halothane to SR, or detergent solubilization of the Ca-ATPase, increased the apparent KI of CPA inhibition, and increased the fraction of Ca-ATPase present as monomers. CPA stabilized the E2 conformational state of the Ca-ATPase relative to the E1 and E2-P states, as measured by fluorescein 5-isothiocyanate fluorescence and enzyme phosphorylation from inorganic phosphate. E2-P formation in the presence of CPA was partially restored by halothane and solubilization. We conclude that CPA inhibits the Ca-ATPase in part by overstabilizing dimers or small oligomers of the Ca-ATPase, which is correlated with stabilization of the E2 conformation of the enzyme.

Restrictive cardiomyopathy associated with the eosinophilia-myalgia syndrome.

OBJECTIVE: We report the first case of restrictive cardiomyopathy occurring in a patient with the eosinophilia-myalgia syndrome. DESIGN: In this article, we discuss the various clinical manifestations of the eosinophilia-myalgia syndrome. MATERIAL AND METHODS: In a 46-year-old woman with the eosinophilia-myalgia syndrome, orthopnea, chronic persistent edema, and severe dyspnea on exertion developed 2 years after she had discontinued use of L-tryptophan. Doppler echocardiography showed ventricular filling confined to early diastole and no atrial filling during ventricular systole--the Doppler hallmarks of restrictive disease. Right-sided cardiac catheterization revealed that the pulmonary wedge pressure equaled the pulmonary artery diastolic pressure and the mean right atrial pressure. A myocardial biopsy specimen showed dense endocardial fibrosis. Special immunofluorescent stains for eosinophilic granule major basic protein showed substantial deposition along the endocardial myocardial interface, an indication that eosinophils were present some time in the past. RESULTS: A follow-up telephone call 14 months after the patient's initial assessment at the Mayo Clinic revealed that she had class III symptoms of congestive heart failure. She was receiving high doses of three diuretics daily, and her condition had improved considerably since her first examination at our institution. CONCLUSION: Restrictive cardiomyopathy may occur in the setting of the eosinophilia-myalgia syndrome and should be considered in patients with this disease in whom exertional dyspnea and peripheral edema occur.

Molecular mechanism of Ca-ATPase activation by halothane in sarcoplasmic reticulum.

We have studied the molecular mechanism of Ca-ATPase activation in sarcoplasmic reticulum (SR) by the volatile anesthetic halothane. Using time-resolved phosphorescence anisotropy, we determined the rotational correlation times and mole fractions of different oligomeric states of the enzyme, as a function of halothane and temperature. Lipid fluidity was measured independently, using EPR of spin-labeled lipids. At 4 and 7 degrees C, the principal effects of halothane were to increase the activity of the Ca-ATPase and to promote the formation of monomers and dimers of the enzyme from larger aggregates. At higher temperatures (up to 25 degrees C), halothane activated the enzyme, but to a lesser extent than observed at lower temperatures. While the functional effects of halothane were temperature dependent, the effects of halothane on lipid fluidity and protein aggregation state were similar at all temperatures tested. We conclude that at low temperatures Ca-ATPase activity is dominated by aggregation state, so halothane activates the enzyme primarily by promoting the formation of monomers and dimers of the enzyme from larger aggregates. At higher temperatures, the activity of the enzyme is dominated by lipid fluidity, so halothane activates the enzyme by increasing the lipid fluidity. The physical mechanism of Ca-ATPase activation, dominated by aggregation state at low temperature and lipid fluidity at higher temperature, provides an explanation for the break in the Arrhenius plot of Ca-ATPase activity (in the absence of halothane) at approximately 20 degrees C.