Headspace gas chromatographic method for the measurement of difluoroethane in blood.

OBJECTIVES: To develop a gas chromatographic assay for the analysis of difluoroethane, a volatile substance, in blood and to determine assay characteristics including linearity, limit of quantitation, precision, and specificity. SETTING: Referral toxicology laboratory RESULTS: Difluoroethane, a colorless, odorless, highly flammable gas used as a refrigerant blend component and aerosol propellant, may be abused via inhalation. A headspace gas chromatographic procedure for the identification and quantitation of difluoroethane in blood is presented. A methanolic stock standard prepared from pure gaseous difluoroethane was used to prepare whole blood calibrators. Quantitation of difluoroethane was performed using a six-point calibration curve and an internal standard of 1-propanol. The assay is linear from 0 to 115 mg/L including a low calibrator at 4 mg/L, the limit of quantitation. Within-run coefficients of variation at mean concentrations of 13.8 mg/L and 38.5 mg/L were 5.8% and 6.8% respectively. Between-run coefficients of variation at mean concentrations of 15.9 mg/L and 45.7 mg/L were 13.4% and 9.8% respectively. Several volatile substances were tested as potential interfering compounds with propane having a retention time identical to that of difluoroethane. CONCLUSIONS: This method requires minimal sample preparation, is rapid and reproducible, can be modified for the quantitation of other volatiles, and could be automated using an automatic sampler/injector system.     

Ontogeny of β1- and β2-adrenergic receptors in rat cerebellum and cerebral cortex

The development of β₁- and β₂-adrenergic receptors was studied in rat cerebral cortex and cerebellum. In the cerebral cortex, which contains mostly β₁-adnergic receptors, total β-adrenergic receptor density increased sharply between postnatal days 10 and 21. The density of receptors remained fairly constant through 6 weeks of age and then subsequently declined. The proportion of β₁ and β₂ receptors was relatively constant throughout the development of the cerebral cortex. The development of the two receptor subtypes thus paralleled the development of total β-adrenergic receptors in the cerebral cortex.The ontogeny of β-adrenergic receptors in the cerebellum, which contains mainly β₂ receptors, was strikingly different from that observed in the cortex. Total cerebellar β receptor density exhibited a slow but steady increase from postnatal day 5 through day 42. The density of receptors then plateaued and remained constant until the animals were approximately 6 months of age. Unlike the results obtained in the cortex, the relative proportions of β₁ and β₂ receptors in the cerebellum changed markedly during development. Between postnatal days 8 and 13 approximately 18% of the receptors were of the β₁ subtype. This proportion steadily decreased with age, and in 3- and 6-month-old animals only approximately 2% of the receptors were of the β₁ subtype.The results demonstrate that two subtypes of β-adrenergic receptors can have different development patterns in the same brain area, and that a single receptor subtype can follow different development patterns in different brain regions. Possible correlations between the ontogeny of β₁ and β₂ receptors and various developmental events are discussed.     

immunoglobulin and histamine-sensitivity response of mice to live Bordetella pertussis

Mice injected intranasally (it.n.) and intraperitoneally (i.p.) with a nonlethal dose (2.5 x 10(5) colony-forming units) of live Bordetella pertussis were examined for 50 days for infection, respiratory tract immunoglobulins (Ig), changes in serum Ig, and histamine sensitivity. With mice infected it.n., respiratory infection markedly declined between day 20 and day 30. Ig classes (A, G(1), G(2a), G(2b), but no M), which had specificity for B. pertussis, were present in tracheobronchial wash (TBW) by day 15; by day 50, TBW immunodiffusion and immunoelectrophoretic precipitin bands were more intense. A sharp rise in serum IgA after day 30 was the only significant change relative to controls among the five serum Ig examined. A high degree of histamine sensitivity developed by day 15 to 20 and persisted for the 50 days. With mice inoculated i.p., no bacteria were recovered, no Ig or only traces were found in TBW and IgA only was specific, and no significant changes in the serum Ig relative to controls occurred. Histamine sensitivity developed somewhat more slowly and to a lesser degree than in it.n.-injected mice but persisted for the 50 days. A similar small number of killed bacteria (pertussis vaccine) injected it.n. or i.p. likewise induced slowly developing histamine sensitivity in contrast to published reports of 4 to 5 day peak sensitivity and decline following i.p. injection of 10(9) or more killed bacteria.     

Spectral characteristics of speech at the ear: implications for amplification in children.

This study examined the long- and short-term spectral characteristics of speech simultaneously recorded at the ear and at a reference microphone position (30 cm at 0 degrees azimuth). Twenty adults and 26 children (2-4 years of age) with normal hearing were asked to produce 9 short sentences in a quiet environment. Long-term average speech spectra (LTASS) were calculated for the concatenated sentences, and short-term spectra were calculated for selected phonemes within the sentences (/m/, /n/, /s/, [see text], /f/, /a/, /u/, and /i/). Relative to the reference microphone position, the LTASS at the ear showed higher amplitudes for frequencies below 1 kHz and lower amplitudes for frequencies above 2 kHz for both groups. At both microphone positions, the short-term spectra of the children's phonemes revealed reduced amplitudes for /s/ and [see text] and for vowel energy above 2 kHz relative to the adults' phonemes. The results of this study suggest that, for listeners with hearing loss (a) the talker's own voice through a hearing instrument would contain lower overall energy at frequencies above 2 kHz relative to speech originating in front of the talker, (b) a child's own speech would contain even lower energy above 2 kHz because of adult-child differences in overall amplitude, and (c) frequency regions important to normal speech development (e.g., high-frequency energy in the phonemes /s/ and [see text]) may not be amplified sufficiently by many hearing instruments.     

Septic complications after cardiac catheterization and percutaneous transluminal coronary angioplasty

Septic complications after cardiac catheterization and percutaneous transluminal coronary artery angioplasty are distinctly uncommon. However, we have recently treated nine patients with sepsis and life-threatening complications after cardiac catheterization alone or after catheterization and subsequent percutaneous transluminal coronary angioplasty. The common denominator in all patients was either repeat puncturing of the ipsilateral femoral artery or leaving the femoral artery sheath in for 1 to 5 days after the procedure. Two patients died as a direct result of their septic complications. One death occurred in a patient in whom bacterial endocarditis with congestive heart failure developed, and the other patient had a large retroperitoneal hematoma that became secondarily infected. Infected aneurysms that were successfully treated developed in three patients. Our study suggests that colonization of the needle tract by skin flora predisposes to septic complications if repeat arterial punctures are required or if a femoral artery sheath is left in place for more than 24 hours. Patients in whom sepsis develops after these procedures should be initially treated with antibiotics effective against gram-positive organisms. CT scanning or angiography should be considered for patients with persistent sepsis, septic emboli, and abdominal or flank pain. Infected aneurysms require resection or ligation because of the propensity of these aneurysms to rupture.     

Vasopressin antagonist in nucleus tractus solitarius/vagal area reduces pressor and tachycardia responses to paraventricular nucleus stimulation in rats

In urethane-anesthetized rats, injections of 50 pmol of arginine-vasopressin (AVP) or thyrotropin-releasing hormone (TRH) into a lateral cerebral ventricle (i.c.v.) elicit short-latency increases in blood pressure. i.c.v. injection of 50 pmol of the AVP antagonist, d(CH2)5Tyr(Me)AVP, but not of the vehicle (artificial cerebrospinal fluid; a CSF), abolished the pressor action of i.c.v. AVP. The AVP antagonist did not antagonize the TRH-induced pressor responses. In another group of rats, a monopolar stainless-steel electrode was positioned stereotaxically in the paraventricular nucleus (PVN) and pressor responses were elicited by electrical stimulation of the PVN. Micro-injection of 1 nmol of the AVP antagonist, but not of aCSF alone, into the nucleus tractus solitarius/vagal area (NTS/VA), reduced PVN-stimulated pressor responses to 26 +/- 6% of control and stimulation-induced tachycardia to 37.3 +/- 9.0% of control. These studies indicate that the pressor and heart-rate responses to PVN stimulation may be mediated, in part, via AVP receptors in the NTS/VA.     

Resonance Raman spectroscopy: a new technology for tissue oxygenation monitoring

OBJECTIVE: To evaluate resonance Raman spectroscopy for the detection of changes in sublingual mucosal hemoglobin oxygen saturation (Smo2) in response to hemorrhage and resuscitation, and to compare Smo2 with other indicators of tissue oxygenation including central venous oxygen saturation (Scvo2), lactate, base excess, and shed blood volume. DESIGN: Prospective single group pilot study. SETTING: University laboratory. SUBJECTS: Five Sprague-Dawley rats. INTERVENTIONS: Animals were anesthetized and instrumented for measurement of arterial and central venous blood gases. Raman spectroscopy was performed using a krypton ion laser providing excitation at 406.7 nm (5 mW). A 1-mm2 region of the sublingual tongue surface was chosen for investigation. Animals were subjected to stepwise hemorrhage until approximately 50% of the blood volume was removed. At each hemorrhage and resuscitation interval, Raman spectroscopy was performed and corresponding arterial and central venous blood gas and lactate measurements were made. Smo2 was calculated as the ratio of the oxygenated heme spectral peak height to the sum of the oxy- and deoxyhemoglobin spectral peak heights. Raman spectroscopy-derived Smo2 measurements were compared with Scvo2 as well as with other indicators of oxygenation. MEASUREMENTS AND MAIN RESULTS: The mean difference between Smo2 and Scvo2 for all paired measurements was 5.8+/-11.7 absolute saturation points. Smo2 was significantly (p<.0001) correlated with Scvo2 (r=.80), lactate (r=-.78), base excess (r=.80), and shed blood volume (r=-.75). Smo2 and Scvo2 showed similar levels of precision for predicting elevated lactate and base deficit. CONCLUSIONS: These studies demonstrate the ability of Raman spectroscopy to noninvasively track microvascular hemoglobin oxygenation in tissue and favorably correlate with other important indicators of tissue oxygenation such as Scvo2, lactate, base deficit, and shed blood volume. The technique shows promise as a method to noninvasively monitor tissue oxygenation.     

A new video image analysis system to study red blood cell dynamics and oxygenation in capillary networks

OBJECTIVE: The authors present a Measurement and Analysis System for Capillary Oxygen Transport (MASCOT) to study red blood cell (RBC) dynamics and oxygenation in capillary networks. The system enables analysis of capillaries to study geometry and morphology and provides values for capillary parameters such as diameter and segment length. It also serves as an analysis tool for capillary RBC flow characteristics, including RBC velocity, lineal density, and supply rate. Furthermore, the system provides a means of determining the oxygen saturation of hemoglobin contained within RBCs, by analysis of synchronized videotapes containing images at two wavelengths, enabling the quantification of the oxygen content of individual RBCs. METHODS: Video recordings of RBC flow at two wavelengths, 420 nm (isosbestic) and 436 nm (oxygen sensitive), are made using a dual camera video microscopy system. The 420-nm recording is used to generate images based on the variance of light intensity fluctuations that help to identify capillaries in a given field of view that are in sharp focus and exhibit flow of individual RBCs separated by plasma gaps. A region of interest enclosing the desired capillary is defined and a fixed number of successive video frames at the two wavelengths are captured. Next a difference image is created, which delineates the RBC column, whose width is used to estimate the internal diameter of the capillary. The 420-nm images are also used to identify the location and centroid of each RBC within the capillary. A space-time image is generated to compute the average RBC velocity. Lineal density is calculated as the number of RBCs per unit length of a capillary segment. The mean optical density (OD) of each RBC is calculated at both wavelengths, and the average SO(2) for each cell is determined from OD(436)/OD(420). RESULTS AND CONCLUSIONS: MASCOT is a robust and flexible system that requires simple hardware, including a SGI workstation fitted with an audio-visual module, a VCR, and an oscilloscope. Since the new system provides information on an individual cell basis from entire capillary segments, the authors believe that results obtained using MASCOT will be more accurate than those obtained from previous systems. Due to its flexibility and ease of extension to other applications, MASCOT has the potential to be applied widely as an analysis tool for capillary oxygen transport measurements.     

Vasopressin autoreceptors and nitric oxide-dependent glutamate release are required for somatodendritic vasopressin release from rat magnocellular neuroendocrine cells responding to osmotic stimuli

Magnocellular neuroendocrine cells of the supraoptic nucleus (SON) release vasopressin (VP) systemically and locally during osmotic challenge. Although both central VP and nitric oxide (NO) release appear to reduce osmotically stimulated systemic VP release, it is unknown whether they interact locally in the SON to enhance somatodendritic release of VP, a phenomenon believed to regulate systemic VP release. In this study, we examined the contribution of VP receptor subtypes and NO to local VP release from the rat SON elicited by systemic injection of 3.5 m saline. Treatment of SON punches with VP receptor antagonists decreased osmotically stimulated intranuclear VP release. Similarly, blockade of NO production, or addition of NO scavengers, reduced stimulated VP, glutamate, and aspartate release, suggesting that local NO production and activity are critical for osmotically induced intranuclear VP and excitatory amino acid release. An increase in endogenous NO release from SON punches in response to hyperosmolality was confirmed by enzymatic NO assay. Consistent with enhanced glutamate and VP release from stimulated rat SON punches, the ionotropic glutamate receptor blocker kynurenate decreased stimulated local VP release without affecting NO release. These data suggest that NO enhances local VP release in part by facilitating local release of glutamate/aspartate and that glutamate receptor activity is required for the stimulation of local VP release by osmotic challenge. Collectively, these results suggest that local VP receptors, NO, and glutamatergic signaling mediate the amplification of intranuclear VP release during hyperosmolality and may contribute to efficient, but not exhaustive, systemic release of VP during osmoregulatory challenge.     

Aggressive versus moderate lipid-lowering therapy in postmenopausal women with hypercholesterolemia: Rationale and design of the Beyond Endorsed Lipid Lowering with EBT Scanning (BELLES) trial

BACKGROUND: Electron beam tomography (EBT) is a noninvasive technique that allows the study of the entire coronary artery tree during a brief imaging session without the injection of any contrast media. Atherosclerosis is identified vicariously through the visualization of coronary calcific deposits. Quantitative assessments of calcium burden, such as calcium volume scores, have been shown to be a useful means to assess treatment-related changes in the extent of atherosclerotic plaques. Historically, the elderly female population has received less medical recognition regarding the risk and severity of coronary heart disease (CHD). METHODS: In the BELLES (Beyond Endorsed Lipid Lowering with EBT Scanning) trial, the presence of asymptomatic CHD in 600 postmenopausal women will be assessed by EBT. In this 1-year, multicenter, randomized, double-blind, parallel-group study, aggressive lipid-lowering treatment will be compared with moderate lipid-lowering treatment in postmenopausal women with hypercholesterolemia. The hypothesis we will test is that aggressive lipid-lowering therapy with 80 mg/d atorvastatin can produce greater reductions in atherosclerotic plaque burden as assessed by volumetric calcium scores than a moderate treatment with 40 mg/d pravastatin. The primary outcome measure will be the percent change from baseline in total CVS determined by EBT at 12 months. CONCLUSIONS: The results of the BELLES trial will help assess the actual incidence of CHD in postmenopausal women and the relative ability of two different lipid-lowering therapies to halt its progression.