Long-range interactions in the dimer interface of ornithine decarboxylase are important for enzyme function.

Ornithine decarboxylase (ODC) is a pyridoxal 5'-phosphate dependent enzyme that catalyzes the first committed step in the biosynthesis of polyamines. ODC is a proven drug target for the treatment of African sleeping sickness. The enzyme is an obligate homodimer, and the two identical active sites are formed at the dimer interface. Alanine scanning mutagenesis of dimer interface residues in Trypanosoma brucei ODC was undertaken to determine the energetic contribution of these residues to subunit association. Twenty-three mutant enzymes were analyzed by analytical ultracentrifugation, and none of the mutations were found to cause a greater than 1 kcal/mol decrease in dimer stability. These data suggest that the energetics of the interaction may be distributed across the interface. Most significantly, many of the mutations had large effects (DeltaDeltaG kcat/Km > 2.5 kcal/mol) on the catalytic efficiency of the enzyme. Residues that affected activity included those in or near the substrate binding site but also a number of residues that are distant (15-20 A) from this site. These data provide evidence that long-range energetic coupling of interface residues to the active site is essential for enzyme function, even though structural changes upon ligand binding to wild-type ODC are limited to local conformational changes in the active site. The ODC dimer interface appears to be optimized for catalytic function and not for dimer stability. Thus, small molecules directed to the ODC interfaces could impact biological function without having to overcome the difficult energetic barrier of dissociating the interacting partners.

Isotope ratios and abundance sensitivity obtained with an inductively coupled plasma-time-of-flight mass spectrometer.

Isotope ratios and abundance sensitivities have been determined with an inductively coupled plasma-time-of-flight mass spectrometer (ICP-TOFMS). Abundance sensitivities are at least in the 10(6) range for low abundance ions that precede high abundance ions. Three methods of detection for isotope-ratio measurement have been compared. The three systems involve gated detection followed by analog integration, analog averaging, or ion counting. Gated ion counting offers excellent precision-between 0. 64 and 1. 00% relative standard deviation (RSD). These values approach those predicted from counting statistics and are comparable to those reported for other inductively coupled plasma-mass spectrometry (ICP-MS) instruments. In addition, a greater number of accumulated counts or longer analysis times would afford precisions of 0. 1% with stable gating electronics. The accuracy of the counting method is in the 1-10% range if no correction for mass bias is performed. However, this ion counting method suffers from a limited dynamic range due to pulse pileup. Constant-fraction discrimination gated integration and commercial boxcar averager techniques offer a broader dynamic range because of their analog nature, but the attainable RSD values are limited by drift in the detection systems and by the methods employed to calculate an accurate ratio. Overall, mass bias in the ICP-TOFMS is more severe than previous work in ICP-MS due primarily to detection system bias.

An inductively coupled plasma-time-of-flight mass spectrometer for elemental analysis. Part II: Direct current quadrupole lens system for improved performance.

An electrostatic quadrupole lens has been substituted for a cylindrical lens system used in the original inductively coupled plasma-time-of-flight mass spectrometer (ICP-TOFMS). With an improved vacuum system also installed, the cylindrical and quadrupole lenses are compared to each other and to the performance of the prototype ICP-TOFMS. The quadrupole lens requires no tradeoff between ion throughput and resolving power as was encountered with cylindrical lenses. The background noise in both ion-optical systems is within the same order of magnitude. Images of the ion beam formed by each ion-optical system have been obtained on a microchannel plate-phosphor screen. The quadrupole lens shows a higher ion-beam flux and produces a slitlike focus required in the orthogonal ICP-TOFMS instrument. Signal-to-noise ratios in the ICP-TOFMS can be improved by using a technique called pulsed-ion injection that is particularly convenient with the quadrupole lens. In this technique, one quadrupole electrode is pulsed to prevent ions from entering the extraction zone except when an ion packet is to be extracted for mass analysis. This technique significantly reduces the noise over continuous ion injection. In the orthogonal ICP-TOFMS with pulsed-ion injection, 0.5 frnol of analyte could be detected in 1.4 ms with a proper data acquisition system. Overall, the combination of a quadrupole lens and pulsed-ion injection may provide detection limits for the ICP-TOFMS that are competitive with those of quadrupole inductively coupled plasma-mass spectrometry instruments.

An inductively coupled plasma-time-of-flight mass spectrometer for elemental analysis. Part III: Analytical performance.

A time-of-flight mass spectrometer (TOFMS) was evaluated as a mass analyzer for inductively coupled plasma mass spectrometry (ICP-MS). The long-term drift of signals was in the range of 7-8% relative standard deviation, whereas the short-term precision was between 5 and 20%, somewhat worse than is typically reported for commercial ICP-MS instruments (5%). However, precision can be improved considerably in the TOFMS by ratioing isotopic peaks or through internal standardization, a consequence of its ability to extract all measured ions simultaneously from the inductively coupled plasma. This feature was demonstrated by monitoring the (206)Pb/(208)Pb ratio with boxcar averagers. In this ratioing mode, precision was improved to approximately 0. 5%. Detection limits were measured with two alternative signal processing systems: (1) discriminator-gated integration and (2) integration of digitized spectra. Both methods improved the signal-to-noise ratio by a factor of from 10 to 100, although detection limits were still 1-2 orders of magnitude poorer for most elements than from the best commercial ICP-MS instruments. The dynamic range of the discriminator-gated integration system is over 4 orders of magnitude, but can be extended to 10(6) with planned increases in primary ion-beam current, which is currently 10-100 times lower than is found in other instruments. Virtually simultaneous multielement and multiisotope analysis is possible for masses from (7)Li to (209)Bi with minimal mass bias and detection limits on the 0. 4-2-ppb level.

An inductively coupled plasma-time-of-flight mass spectrometer for elemental analysis. Part I: Optimization and characteristics.

An inductively coupled plasma-time-of-flight mass spectrometer (ICP-TOFMS) has been constructed and evaluated for elemental analysis. The instrument produces analog spectra similar to those from quadrupole inductively coupled plasma mass spectrometers. The large abundance of Ar ions is deflected away from the microchannel plate detector to reduce detector dead time and space-charge complications. The ICP-TOFMS, operated in a linear (nonreflecting) mode, currently has a resolving power of 500 (full width at half maximum). Present ion optics employed in the instrument require a trade-off between signal-to-noise ratio and resolving power. In addition, mass-dependent kinetic energies in the supersonic beam created in the ICP mass spectrometer interface cause a mass bias in the right-angle TOFMS because the ions must be steered to the detector to compensate for their velocity in the supersonic beam direction. In the current design the sampling duty cycle is only approximately 3%, thereby limiting sensitivity. However, positive potentials applied to the right-angle extraction region can increase sensitivity by a factor of 2-4 by slowing down the ions that enter the extraction zone. The transmission efficiency of the TOFMS is approximately 20% and is limited by divergence of the ion packet in the drift tube.

Interpleural analgesia for the treatment of severe cancer pain in terminally ill patients.

Interpleural analgesia was used to alleviate acute, severe exacerbations of chronic pain unrelieved by pharmacologic therapy in ten terminally ill cancer patients. Pain from metastatic disease to the neck, arms, chest, brachial plexus, thorax, or abdomen was effectively eliminated between 7 hr and 40 days in nine patients, who died with minimal or no pain. The technique was performed primarily using bupivacaine. No side effects were detected. Interpleural analgesia appears to be effective in rapidly controlling acute exacerbations of cancer pain in terminally ill patients. Moreover, it may also be a suitable therapy for moribund patients when used as a continuous-infusion technique.

Bilateral pneumothoraces from a malfunctioning resuscitation valve.

Disposable resuscitation valves appear as relatively simple devices used to ventilate patients during both emergent situations and transport from the operating room to the intensive care unit. We report a case of a nonfunctional disposable resuscitation valve that resulted in barotrauma and bilateral pneumothoraces. A routine check for proper function of these valves before use in critically ill patients may help to eliminate such cases.

A comparison of postoperative epidural analgesia between patients with chronic cancer taking high doses of oral opioids versus opioid-naive patients.

Our study evaluated 116 surgical patients with cancer who received postoperative epidural analgesia with bupivacaine (BUP) (0.1%) and morphine (MS) (0.01%) during 5 days after epidural-light general anesthesia. Patients in group I (n = 17) were taking opioids in doses larger than 50 mg of morphine daily for 3 mo or more, whereas patients in group II (n = 99) were opioid-naive. Postoperative epidural infusions were started at 10 mL.h-1 for group I and 5 mL.h-1 for group II. All patients were evaluated every 6 h for pain, withdrawal, and overdosing. Dynamic pain scores were kept below 4/10 by titrating infusions and/or giving intravenous (IV) MS 4 mg every hour as needed. Fifteen patients were taking opioids for 3-6 mo and the remainder for more than 6 mo. Mean oral MS preoperative usage for group I was 183 mg (90-360 mg range). All patients experienced adequate analgesia. Group I required more epidural (137 vs 44 mg) and IV (48 vs 10 mg) MS and had a longer requirement for analgesic therapy (9 vs 3 days) when compared with group II. Daily epidural and IV MS usage were always more for group I by two- to threefold. No patient experienced respiratory depression or opioid withdrawal during the hospitalization. Thus, epidural BUP-MS appears to provide adequate postoperative analgesia while preventing withdrawal in opioid-dependent patients, if three times the normal dosage and duration of therapy are employed.

Pleural phenol therapy for the treatment of chronic esophageal cancer pain.

BACKGROUND AND OBJECTIVES: Pleural phenol was used successfully to alleviate terminal pain associated with metastatic esophageal cancer in a 43-year-old man. METHODS: The patient first was given 20 ml 0.5% bupivacaine for diagnostic purposes through an interpleural catheter to control a severe aching pain throughout the T5-8 dermatomes. Seven days later, 10 ml 6% phenol suspended in 0.5% bupivacaine was injected and produced analgesia for two days. A second injection using 18 ml 10% phenol provided adequate analgesia for the next four weeks until the patient's death. Two weeks after the lower thoracic pain was relieved, the patient had complained of a new right-sided pain in the region of C7-T2, which was treated successfully with 10 ml 10% phenol. RESULTS: During the course of this therapy, his sustained-release morphine consumption diminished from 540 mg to 90 mg despite his terminal disease. Post mortem examination and histologic studies revealed no macroscopic or microscopic changes to the viscera, spinal cord, or sympathetic chain. CONCLUSIONS: Repeated doses of phenol suspended in bupivacaine provide adequate analgesia for terminal cancer pain when administered interpleurally. No pathologic changes were detected despite a total of 3400 mg phenol delivered into the right thoracic cavity.

Adolescent addiction. Assessment and identification.

Adolescents who are addicted to drugs present unique assessment and diagnostic problems. The primary drugs used by adolescents are alcohol, marijuana, and cocaine, respectively. The adolescent who uses drugs in a regular and frequent manner is affected more rapidly than is the adult who uses in the same way and for the same length of time. The social and emotional skills usually acquired during this period of rapid growth often are not learned when the adolescent is abusing drugs, and this results in psychosocial stunting of development. Drugs and alcohol have been implicated in teenage traffic accidents, school drop out rates, promiscuity, crime, and suicide. Assessing adolescent drug use and potential addiction is not difficult once good history and examination techniques are acquired. This article presents techniques for assessing and identifying addicted adolescents.