一种测定总胆红素的新方法

[目的]建立一种快速、简便、稳定、高灵敏度，适应于血清、血浆测定的总胆红素的新方法。[方法]用聚氧乙烯月桂醚（Brij-35）作为加速剂使结合胆红素与未结合胆红素同重氮对氨基苯磺酸反应，形成偶氮胆红素。[结果]本试剂测定中的总胆红素，在样品加入试剂后5min显色达到终点，显色后最少可稳定3h不退色，试剂空白与水接近。反应成色后最大吸收峰在510nm，线形范围可达324nmol／L，回收率97．2％-102；4％。批内变异系数（CV）为0．58％，与复星长征总胆红素试剂相关性良好。Y=0．976X＋0．862r=0．999P〉0．05。[结论]本方法测定总胆红素具有操作快速、简便，结果稳定等优点，适用于血清及血浆总胆红素的手工测定及自动化分析。     

A new noninvasive method to determine central venous pressure

Knowledge of central venous pressure (CVP) is considered valuable in the assessment and treatment of various states of critical illness and injury. OBJECTIVES: We tested a noninvasive means of determining CVP (NICVP), by monitoring forearm volume changes in response to externally applied circumferential pressure to the upper arm veins. METHODS: Sixteen patients who were undergoing CVP monitoring as a part of their care had NICVP determined and compared with CVP. Volume changes were measured in the forearm with mercury-in-silastic strain gauge plethysmography. A pressure cuff is placed in the upper extremity. The cuff is inflated over 5s to a pressure above CVP but below diastolic arterial pressure (40 mmHg). This allows blood into the forearm but prevents venous return. After 45-60 s the cuff is rapidly deflated. NICVP was determined as the cuff pressure noted at the maximum derivative of the forearm volume decrease during deflation. NICVP was then compared to invasively measured CVP taken during the same period. RESULTS: A total of 48 trials (three per subject) were performed on 16 patients. The range of CVP recorded was 0-22 mmHg. The correlation between CVP and NICVP was 0.98 (95% CI: 0.95-0.98) (p<0.001). The bias between methods was 0.26 mmHg with the limits of agreement being 3.4 to -2.89 mmHg. When the average of three trials per patients was analysed the bias stayed at 0.26 mmHg but the limits of agreement improved to 2.54 and -2.03 mmHg. CONCLUSION: NICVP as determined in this study may be a clinically useful substitute for traditional CVP measurement and may offer a valid tool for early diagnosis and treatment of acute states in which knowledge of CVP would be helpful.     

A new method to determine postantibiotic effect and effects of subinhibitory antibiotic concentrations.

It has been shown that bacteria in a postantibiotic (PA) phase exposed to subinhibitory concentrations (sub-MICs) of antibiotics show a long delay before regrowth. This effect has been named the PA sub-MIC effect (PA SME). In the present study, we have used a new method to demonstrate this phenomenon. A computerized incubator for bacteria, Bioscreen C (Lab Systems, Helsinki, Finland), which incubates the bacteria, measures growth continuously by vertical photometry, processes the data, and provides a printout of the results was used. With this method, one may easily test several antibiotics against different bacteria for PA effects (PAEs), PA SMEs, and SMEs. In this study, the effects of benzylpenicillin against beta-hemolytic streptococci and pneumococci were examined. The bacteria were exposed to 2, 10, or 50x MIC for 2 h, washed and diluted, incubated in the Bioscreen C incubator, and then exposed to 0.1 to 0.9x MIC. The regrowth was monitored for 20 h. The PAE was calculated as the difference in the time required for the exposed and unexposed bacteria to grow to a defined point (A50) on the absorbance curve. A50 was defined as 50% of the maximum absorbance for the control cultures. The PA SMEs were calculated as the difference in the time required for the reexposed cultures and the unexposed controls to reach A50. The PAEs ranged between 0.6 and 3.2 h and varied little with the concentration used for the induction of the PAEs. At 0.2x MIC, the PA SMEs were 2 to 3 h longer than the PAEs. Higher sub-MICs increased this delay before regrowth. Most cultures exposed to sub-MICs alone were only slightly affected compared with the controls.     

Cardiac functional and structural alterations induced by endotoxin in rats: importance of platelet-activating factor

OBJECTIVE: In this study, we evaluated the time course of the alterations in left ventricular (LV) dimensions, LV wall thickness, and LV systolic function in rats with endotoxemia by using echocardiography as well as myocardial histopathologic assessments. Our second goal was to examine whether pretreatment with a platelet-activating factor (PAF) antagonist would ameliorate the lipopolysaccharide (LPS)-induced cardiovascular collapse during the early phase. DESIGN: A prospective, controlled, in vivo animal laboratory study. SETTING: Research laboratory at a university. SUBJECTS: Male, Wistar rats (8-9 wks old; n = 83). INTERVENTIONS: In pentobarbital-anesthetized rats, the right carotid artery was cannulated to measure the arterial blood pressure and to sample blood. The right jugular vein also was catheterized for the administration of drugs. LPS (2 mg/kg) derived from Klebsiella pneumoniae or physiologic saline was administered in the presence or absence of pretreatment with TCV-309, a specific potent PAF antagonist. Echocardiographic studies were performed with an 8- to 13-MHz transducer. MEASUREMENTS AND MAIN RESULTS: LPS administration immediately induced progressive hypotension. The maximal hypotensive response was observed at 10 mins after LPS infusion with mean arterial pressure decreasing from 119 +/- 2 to 56 +/- 3 mm Hg (p < .001). LV end-diastolic internal dimensions decreased from 6.4 +/- 0.1 to 3.1 +/- 0.1 mm (p < .001) at 30 mins after LPS and remained significantly reduced compared with control rats. LV end-systolic dimensions also decreased dramatically from 3.5 +/- 0.2 to 0.5 +/- 0.1 mm (p < .001) at 30 mins after LPS and remained significantly reduced throughout the experiment. LV fractional shortening increased from 45 +/- 1% to 84 +/- 2% (p < .001) at 30 mins after LPS and remained elevated compared with control rats. LV wall thickness increased strikingly from 15 mins until 2 hrs after LPS infusion. Pathologic studies demonstrated marked congestion of capillaries and mild edema in the LV myocardium. The hematocrit increased after the administration of LPS. LPS markedly increased sympathetic tone as demonstrated by the elevation of plasma concentrations of epinephrine and norepinephrine. There was no elevation of concentrations of nitrite and nitrate. Pretreatment with TCV-309, a specific potent PAF antagonist, reduced LPS-induced hypotension and attenuated LV functional and structural changes. TCV-309 administration reduced the LPS-induced adrenergic activation and hemoconcentration. CONCLUSIONS: The hypotension that occurred during the initial phase of LPS-induced shock was accompanied by LV functional and structural alterations. The marked increase in LV wall thickness can be ascribed to the congestion of capillaries and edema in the LV myocardium. Pretreatment with a PAF antagonist reduced LPS-induced alterations. PAF may play a pivotal role during the initial phase of LPS-induced cardiovascular responses.     

Diabetes incidence and long-term exposure to air pollution: a cohort study

OBJECTIVE

Animal and cross-sectional epidemiological studies suggest a link between air pollution and diabetes, whereas the limited prospective data show mixed results. We studied the association between long-term exposure to traffic-related air pollution and incidence of diabetes.

RESEARCH DESIGN AND METHODS

We followed 57,053 participants of the Danish Diet, Cancer, and Health cohort in the Danish National Diabetes Register between baseline (1993–1997) and 27 June 2006. We estimated the mean levels of nitrogen dioxide (NO₂) at the residential addresses of the cohort participants since 1971 and modeled the association between NO₂ and diabetes incidence with a Cox regression model, separately for two definitions of diabetes: all cases and a more strict definition where unconfirmed cases were excluded.

RESULTS

Over a mean follow-up of 9.7 years of 51,818 eligible subjects, there were 4,040 (7.8%) incident diabetes cases in total and 2,877 (5.5%) with confirmed diagnoses. Air pollution was not associated with all diabetes cases (hazard ratio 1.00 [95% CI 0.97–1.04] per interquartile range of 4.9 μg/m³ mean NO₂ levels since 1971), but a borderline statistically significant association was detected with confirmed cases of diabetes (1.04 [1.00–1.08]). Among confirmed diabetes cases, effects were significantly enhanced in nonsmokers (1.12 [1.05–1.20]) and physically active people (1.10 [1.03–1.16]).

CONCLUSIONS

Long-term exposure to traffic-related air pollution may contribute to the development of diabetes, especially in individuals with a healthy lifestyle, nonsmokers, and physically active individuals.The prevalence and incidence of type 2 diabetes are rising rapidly in both the developed and developing world, presenting one of the greatest contributors to the global burden of the disease (1). The diabetes epidemic is in large part attributable to established causes related to modern lifestyle including obesity, physical inactivity, and the growing aging populations (1). Environmental exposures linked to industrialization and urbanization, such as air pollution, have not been considered risk factors for diabetes until recently (2). In the U.S., the prevalence of diabetes correlated with the release of toxicants into the air (3), whereas diabetic people appeared more vulnerable than nondiabetic people to cardiovascular health effects associated with exposure to air pollution (4). Diabetes and cardiovascular diseases share many risk factors, and diabetic people are at a highly increased risk for heart or circulatory disorders (5). The central biological mechanisms of air pollution damage to the heart and blood vessels include inflammation (6), which is also believed to be involved in the promotion of insulin resistance and type 2 diabetes (7). An enhanced association between air pollution and inflammation, endothelial dysfunction, prothrombotic changes, and altered heart rate variability was found in diabetic people (6). A plausible biological mechanism of air pollution promoting diabetes was provided by Sun et al. (8), showing that exposure to particulate air pollution caused increased blood glucose, inflammation in adipose tissue, and insulin resistance in high-fat diet–fed mice. Recent study confirmed that prolonged exposure to air pollution leads to insulin resistance and impaired glucose tolerance in rats and that this association is not limited to high-fat diet rats (9). Epidemiological evidence is sparse. Short-term exposure to air pollution was linked to exacerbations of diabetes leading to death (10–14) and hospitalizations (13). Prevalence of diabetes was linked to air pollution (14,15). Two prospective diabetes studies investigated the link with air pollution, with one reporting significant associations among a small number (n = 87) of women (16) and another failing to detect association in two large cohorts, except with a single traffic proximity proxy in women (17). Limited and mixed evidence precludes conclusions about causality between air pollution and diabetes and merits more study.We studied the association between traffic-related air pollution levels at the residence and the risk for diabetes in an elderly Danish cohort and tested for an effect modification by lifestyle, education, and comorbid conditions.     

Tissue factor generation by human mononuclear cells: effects of endotoxin and dissociation of tissue factor generation from mitogenic response.

The effects of the presence of endotoxin in a mononuclear cell culture system have been assessed. Endotoxin was shown to be mitogenic for human peripheral blood lymphocytes and capable of stimulating the generation of tissue factor. Concentrations of endotoxin, found to contaminate many commercial mitogens and antigens, activated mononuclear cells in a time-dependent manner. Generation of tissue factor was detected in cultures harvested from 2 to 72 hours following stimulation with endotoxin. Dose-response curves relating concentrations of endotoxin to mononuclear cell stimulation were determined; as little as 0.001 microng/ml. of E. coli endotoxin was capable of stimulating the generation of tissue factor in the cell cultures. The mitogenic effect of endotoxin was modest, however, and appeared to be unrelated to the ability of endotoxin to active tissue factor. Inhibition of DNA synthesis in the cell cultures by cytosine arabinoside or nonlethal irradiation failed to impair the generation of tissue factor. Endotoxin contamination of various reagents used in cell culture was evaluated with the Limulus assay, which detected as little as 1 X 10(-4) microng/ml. of endotoxin. Endotoxin was detected in preparations of phytohemagglutinin, purified protein derivative of the tubercle bacillus, mumps vaccine, tetanus toxoid, concanavalin A, and pokeweed mitogen. Because of the broad implications of contamination by endotoxin of various reagents, we assessed the specificity of the Limulus assay for the detection of endotoxin in the lectin, concanavalin A, and determined that the reaction was specific for endotoxin. Contamination by endotoxin of mononuclear cell culture systems should be considered as a possible factor in the production of various biological effects attributed to some commonly used mitogens and antigens.     

In-Cell Western assay: a new approach to visualize tissue factor in human monocytes

BACKGROUND: Tissue factor (TF) is an integral membrane protein essential for the initiation of the extrinsic pathway of hemostasis. A precise understanding of the TF regulation is still limited and dependent on the availability of methodological tools. Here, we describe a new approach for assessing TF amounts in human mononuclear cells (MNCs) by using the whole blood experimental conditions. AIM: In order to study TF antigen levels in human MNCs, we applied a quantitative immunostaining technique-- in-cell Western (ICW) assay using an Odyssey Infrared Imaging System. METHODS AND RESULTS: The ICW assay of TF in resting or lipopolysaccharide (LPS)-stimulated human MNCs was performed. Several sample preparation conditions were tested, namely the plating of MNCs prior to immunostaining, paraformaldehyde fixation, and an adequate cell number was used in the assay. By the use of recombinant human TF standards, it was possible, for the first time, to measure TF amounts in LPS-stimulated MNCs as 0.09 +/- 0.02 ng and 0.43 +/- 0.15 ng 10(-6) cells of surface and total TF, respectively. The concentrations of TF in resting MNCs, however, were below the detection limit. CONCLUSIONS: A novel TF ICW assay is a reproducible, time- and cost-saving method, which could become useful for studies in the fields of physiology and pathophysiology of human hemostasis.     

Membrane microdialysis: Evaluation of a new method to assess splanchnic tissue metabolism

OBJECTIVE: Measuring peritoneal lactate concentrations could be useful for detecting splanchnic hypoperfusion. The aims of this study were to evaluate the properties of a new membrane-based microdialyzer in vitro and to assess the ability of the dialyzer to detect a clinically relevant decrease in splanchnic blood flow in vivo. DESIGN: A membrane-based microdialyzer was first validated in vitro. The same device was tested afterward in a randomized, controlled animal experiment. SETTING: University experimental research laboratory. SUBJECTS: Twenty-four Landrace pigs of both genders. INTERVENTIONS: In vitro: Membrane microdialyzers were kept in warmed sodium lactate baths with lactate concentrations between 2 and 8 mmol/L for 10-120 mins, and microdialysis lactate concentrations were measured repeatedly (210 measurements). In vivo: An extracorporeal shunt with blood reservoir and roller pump was inserted between the proximal and distal abdominal aorta, and a microdialyzer was inserted intraperitoneally. In 12 animals, total splanchnic blood flow (measured by transit time ultrasound) was reduced by a median 43% (range, 13% to 72%) by activating the shunt; 12 animals served as controls. MEASUREMENTS AND MAIN RESULTS: In vitro: The fractional lactate recovery was 0.59 (0.32-0.83) after 60 mins and 0.82 (0.71-0.87) after 90 mins, with no further increase thereafter. At 60 and 90 mins, the fractional recovery was independent of the lactate concentration. In vivo: Abdominal blood flow reduction resulted in an increase in peritoneal microdialysis lactate concentration from 1.7 (0.3-3.8) mmol/L to 2.8 (1.3-6.2) mmol/L (p = .006). At the same time, mesenteric venous-arterial lactate gradient increased from 0.1 (-0.2-0.8) mmol/L to 0.3 (-0.3 -1.8) mmol/L (p = .032), and mesenteric venous-arterial Pco2 gradients increased from 12 (8-19) torr to 21 (11-54) torr (p = .005). CONCLUSIONS: Peritoneal membrane microdialysis provides a method for the assessment of splanchnic ischemia, with potential for clinical application.     

A simple liquid-chromatographic method applied to determine caffeine in plasma and tissues

In this simple, reliable assay for quantifying caffeine in plasma and tissues, methylxanthines are first partly purified from plasma and acid extracts of tissue by passage through solid-phase columns. The ease of this extraction method permits a relatively large number of samples to be processed daily. Quantification is by reversed-phase high-pressure liquid chromatography (mobile phase: acetic acid/acetonitrile/water, 2/6/92 by vol). Caffeine is eluted in 20 min. The reliability of this method allows its automation. This method has been adapted to measure caffeine in brain and kidney extracts and in as little as 10 microL of plasma. After 10 days of oral administration of caffeine (1 g/L, in drinking water) to six rats, the mean (+/- SEM) concentrations of caffeine in plasma, brain, and kidney were 18.6 +/- 6.0 micrograms/mL, 16.2 +/- 1.5 micrograms/g, and 18.9 +/- 2.0 micrograms/g, respectively. Correlations were linear between concentrations of caffeine in plasma and brain (r = 0.86) and between concentrations in plasma and kidney (r = 0.91). This method should be useful in studying the effects and mechanisms of actions of methylxanthines.     

A new integrated method for tissue extracellular vesicle enrichment and proteome profiling

Extracellular vesicles (EVs) are membranous vesicles released by cells that carry a number of biologically important components such as lipids, proteins, and mRNAs. EVs can mediate cancer cell migration, invasion, angiogenesis, and cell survival, greatly contributing to cell-to-cell communication in the tumor microenvironment. Additionally, EVs have been found to have diagnostic and prognostic significance in various cancers. However, the direct isolation of pure EVs remains challenging, especially from tissue samples. Currently available EV isolation approaches, e.g., ultracentrifugation, are time-consuming, instrumental dependent, and have a low recovery rate with limited purity. It is urgent to develop rapid and efficient methods for enriching tissue EVs for biological and clinical studies. Here, we developed a novel isolation approach for tissue EVs using an extraction kit combined with TiO₂ microspheres (kit-TiO₂). The EVs were first precipitated from the tissue fluid using a precipitation agent and then further enriched using microspheres based on the specific interaction between TiO₂ and the phosphate groups on the lipid bilayer of the EVs. Kit-TiO₂ approach led to improved purity and enrichment efficiency of the isolated EVs, as demonstrated by western blot and proteomic analysis, compared with previously reported methods. A total of 1966 protein groups were identified from the tissue EVs. We compared the proteomic profiles of the liver tissue EVs from healthy and hepatocellular carcinoma (HCC) bearing-mice. Twenty-five significantly upregulated and 75 downregulated protein groups were found in the HCC EVs. Among the differentially expressed proteins, Atic, Copa, Cont3, Me1, Anxa3, Fth1, Anxa5, Phb1, Acaa2, ATPD, and Glud1 were reported to be highly relevant to HCC. This novel isolation strategy has provided a powerful tool for enriching EVs directly from tissues, and may be applied in biomarker discovery and drug screening of HCC.

Extracellular vesicles were successfully extracted from mouse liver tissue using kit combined with TiO₂.     

A new and specific enzyme-linked immunosorbent assay for the detection of C3 nephritic factor

C3 nephritic factor (C3 NeF) was measured by assessing its capacity to form complex with C3 and B using an enzyme-linked immunosorbent assay (ELISA). Incubation of C3 NeF with normal human serum in the presence of MgEGTA resulted in a dose-dependent increase of C3-B-IgG complex. No complex was formed in EDTA. The C3 NeF titer estimated in this way was in good accordance with those reported previously by other indirect methods.     

A simple method to determine body segment masses in vivo: reliability,accuracy and sensitivity analysis

OBJECTIVE: To show that force plates can be used to quickly acquire subject-specific segment mass data. DESIGN: In vivo measurements were performed on subjects belonging to three populations: female varsity swimmers, female varsity volleyball players, and male college students. Segmental masses were measured using a force plate technique, and were compared with published data. BACKGROUND: Patients from populations for which data from the literature are not applicable (e.g. pathological, aging females, obese, children, etc.) would benefit from a direct measure of inertial parameters for accurate joint moment calculations. METHODS: Eight female varsity volleyball players, 17 female varsity swimmers, and 10 male college students were measured anthropometrically. They then lay on a board placed on a force plate and the center of pressure was recorded while the subjects adopted various prescribed limb positions. Their limb masses were subsequently calculated from the center of pressure data given estimated center of mass locations. RESULTS: The method was highly reproducible with an average reliability coefficient of 0.83 and yielded results similar to those of published methods. Significantly different mass distributions were found between the two female populations tested (P<0.025). CONCLUSIONS: The method can quickly provide subject-specific limb segment mass information. RELEVANCE: Measuring subjects' segment masses individualizes clinical assessments and may be necessary for those from special populations to avoid erroneous biomechanical conclusions.     

Speckle contrast optical tomography: A new method for deep tissue three-dimensional tomography of blood flow

A novel tomographic method based on the laser speckle contrast, speckle contrast optical tomography (SCOT) is introduced that allows us to reconstruct three dimensional distribution of blood flow in deep tissues. This method is analogous to the diffuse optical tomography (DOT) but for deep tissue blood flow. We develop a reconstruction algorithm based on first Born approximation to generate three dimensional distribution of flow using the experimental data obtained from tissue simulating phantoms.OCIS codes: (170.3880) Medical and biological imaging, (110.3010) Image reconstruction techniques, (110.6150) Speckle imaging, (110.6955) Tomographic imaging