A novel method for detection of nitric oxide binding sites by using a chemiluminescence-headspace gas technique

Nitric oxide (NO) is important in many physiological, pharmacological, and pathological processes. According to current concepts, guanylyl cyclase is considered to be a receptor for NO in vascular and nonvascular smooth muscle and other tissues. Since there are no suitable radioisotopes of oxygen and nitrogen available for conventional radioligand-receptor binding studies for NO, a novel method was developed to identify NO binding site(s). A chemiluminescence-headspace gas assay was utilized to measure the sequestration of NO in biological systems, and this was used as an index of NO binding. In the present report, myoglobin (a hemoprotein, Mb) was used as a prototype macromolecule to develop the binding assay for subsequent application to studies of putative NO receptors. Solutions containing various concentrations of Mb were incubated with NO in sealed micro-Fernbach flasks at 37°C in an argon atmosphere for 30 min; NO remaining in the headspace gas was analyzed by means of the chemiluminescence assay. The magnitude of NO sequestration was dependent on Mb concentration, and 5 nM Mb was the lowest Mb concentration for which NO sequestration was measurable. Application of the method to the measurement of NO sequestration by bovine serum albumin (BSA) and pulmonary artery medial layer homogenate (BPA-M) revealed that the lowest BSA concentration at which NO sequestration was measurable was 1.6 μM, which was 320 times greater than that for Mb. Applicability of the method to address the question of putative NO receptors was indicated by significant NO sequestration after incubation with 20% (w/v) homogenate of BPA-M, which is responsive to NO and putative NO prodrugs.     

血清cTnI、CK-MB和Mb浓度动态变化对急性心肌梗死的诊断价值

目的 探讨血清心肌肌钙蛋白I(cTnI)、肌酸激酶同工酶(CK-MB)和肌红蛋白(Mb)浓度的动态变化对急性心肌梗死(AMI)的诊断价值.方法 采用ELISA法检测了40例AMI病人不同时间血清cTnI、CK-MB和Mb浓度的变化,同时对血清cTnI、CK-MB和Mb浓度进行了受试者工作特征曲线(ROC)分析.以40例健康查体者作对照.结果 AMI病人胸痛发作后不同时间的血清cTnI、CK-MB浓度明显高于对照组(F=72.679、109.768,q=3.049～27.652,P<0.01),且胸痛发作后不同时间浓度比较,差异有显著性(q=2.863～22.133,P<0.05、0.01);胸痛发作后3～6 h、1 d血清Mb浓度明显高于对照组、胸痛发作后3 d和7 d(F=17.729,q=6.175～8.688,P<0.01).且cTnI、CK-MB对诊断AMI的灵敏度、特异度、准确度、约登指数、阳性预测值(PPV)和阴性预测值(NPV)明显高于Mb.AMI病人胸痛发作后3～6 h血清CK-MB对诊断AMI的准确度高于cTnI和Mb,1 d、3 d血清CK-MB与cTnI诊断AMI的准确度相同,但优于Mb,而胸痛发作后7 d时cTnI对AMI诊断的准确度明显优于CK-MB.AMI病人胸痛发作后3～6 h和1 d时cTnI、 CK-MB的AUCROC高于Mb,7 d时cTnI的AUCROC高于CK-MB,且不同时间的cTnI和CK-MB的AUCROC均大于0.9.结论 血清cTnI、CK-MB和Mb浓度的动态变化对AMI具有明显的诊断价值和较高的准确度.     

横纹肌溶解致急性肾衰竭3例报告及文献复习

目的：探讨横纹肌溶解致急性肾衰竭的发病机制及有效的治疗方法。方法：分析报道典型的横纹肌溶解导致急性肾衰竭3例患者,并作文献复习。结果：患者均为男性,平均年龄33,3岁,2例大量饮酒（其中1例同时注射海洛因）后,另1例由于一氧化碳中毒。有长时间昏睡史,血压偏低。其中有1例患者的肾活检病理报告：急性肾小管坏死,肾小管内存在大量蛋白栓子;免疫组化证实为肌红蛋白。3例患者入院时均无尿,高钾血症,重度酸中毒,所以予以血液透析及对症治疗,痊愈出院。结论：虽然尿中检测到肌红蛋白是诊断横纹肌溶解的“金标准”,但我们认为肾穿刺活检免疫组化证明肾小管内肌红蛋白栓子可作为诊断横纹肌溶解引起急性肾小管坏死更直接证据。酗酒加注射毒品后引起肌溶解急性肾衰竭的病情较重且复杂,早期血液透析治疗可以减少死亡率。     

Mapping regional oxygenation and flow in pig hearts in vivo using near-infrared spectroscopic imaging

We used near-infrared spectroscopic (NIRS) imaging to assess the flow-dependence of both myocardial oxygenation and distribution of an intravascular tracer, indocyanine green (ICG). In open-chest pigs, nominal flow through the left anterior descending artery was reduced for 90 to 0 min (n = 6), 20 +/- 1 (n = 5) and 44% +/- 5% (n = 4) by variable occlusion and subsequently restored (to 219% +/- 71% at 45 min) for 120 min. Electrocardiogram-gated NIRS images of the heart were obtained using a CCD-array camera with a liquid crystal tunable filter, which acquired absorbance spectra in the range of 650-1050 nm for each of 256 x 256 pixels (0.4 x 0.4 mm each). Deoxy- and oxy-(hemoglobin (Hb) + myoglobin (Mb)) levels were determined independently by applying a spectral fitting algorithm to the spectra between 650 and 890 nm. Maps for oxygenation parameter (OP = oxy-(Hb + Mb)/deoxy-(Hb + Mb)) and oxygen saturation parameter (OSP) (oxy-(Hb + Mb)/total-(Hb + Mb)) were constructed. To visualize flow distribution, a bolus of ICG (8.3 mg/5 ml) was injected I.V. at each step of the protocol and gated images were acquired at 800 nm every second over 60 s period. The ratio of ICG wash-in velocity to equilibrium absorbance (V/DeltaA(tail)) was calculated. Changes in flow to 0%, 20%, 44%, 100% and 208% of baseline resulted in OP values of 1.46 +/- 0.25, 1.66 +/- 0.30, 2.22 +/- 0.45, 2.78 +/- 0.30 and 3.94 +/- 0.33 in the affected area. Flow rates of 20%, 44%, 100% and 220% of baseline corresponded to V/DeltaA(tail) values of 0.11 +/- 0.11, 0.39 +/- 0.13, 0.54 +/- 0.17 and 0.61 +/- 0.20, respectively. Thus, measurements of oxygenation and normalized ICG wash-in velocity correlated well with coronary flow, allowing for intraoperative optical assessment of the severity of regional ischemia.     

The diagnostic value of plasma myoglobin levels in the adult and fetus at-risk for Duchenne muscular dystrophy

In boys with Duchenne muscular dystrophy (DMD) plasma myoglobin levels remained approximately constant with age while creatine kinase (CK) activity progressively decreased. For carrier detection, plasma myoglobin level was found to be less reliable than CK activity. The myoglobin level was raised only in some of those subjects who also showed a raised CK activity and was normal in those with a normal CK activity. The myoglobin level in fetal muscle at 18-22 weeks gestational age was found to be low compared with adult skeletal muscle levels and, consequently, the myoglobin level in fetal plasma and in amniotic fluid was found to be negligible. It is concluded that measurement of myoglobin offers no advantage over CK for the investigation of any aspect of DMD.     

Relationship between coronary flow and adenosine release during severe and mild hypoxia in the isolated perfused rat heart with special reference to time-course change

Summary The contribution of endogenous adenosine to coronary vasodilation induced by global myocardial hypoxia was examined. In isolated rat hearts perfused by means of Langendorff's technique, the relationship between chronological changes in coronary flow and adenosine release during hypoxia was analyzed. The oxygenation level of myoglobin (MbO₂), myocardial oxygen uptake, lactate release, and left ventricular pressure (LVP) was also measured. Adenosine was determined by radio-immunoassay, and the MbO₂ levels by the optical method. Severe hypoxia (20% O₂+75% N₂+5% CO₂) increased coronary flow, adenosine release, and lactate release and decreased both myocardial oxygen uptake and LVP. Mild hypoxia (50% O₂+45%N₂+5%CO₂) also increased coronary flow, adenosine release, and lactate release, while it affected neither myocardial oxygen uptake nor LVP. These results suggest that the oxygen supply is compensated by an increase in coronary flow in mild hypoxia, whereas this does not occur in severe hypoxia. Changes in MbO₂ were the reverse of those in coronary flow during severe hypoxia, confirming that a decrease in intracellular oxygen correlates well with an increase in coronary flow. The pattern of changes in adenosine release, however, was not identical with that in coronary flow in severe and mild hypoxia, indicating that there is no significant relationship between coronary flow and adenosine release in either severe or mild hypoxic hearts. These findings suggest that adenosine is not the only metabolic mediator of regulation of coronary flow in hypoxic hearts.     

Determination of local myoglobin concentration in the guinea pig heart

Summary A method is described for calculating local myoglobin concentration in hemoglobin-free perfused guinea-pig heart by measuring the decrease of tissueP _o2 during stop of perfusion. The myoglobin concentration in ca. 10 nanogram of tissue varies between 0.106 and 0.676 g-% of the wet weight. The mean value of 0.373 g-% (standard deviation of ±0.138) agrees very well with the values obtained with chemical methods by other authors. The great variation of our values is interpreted as an inhomogeneous distribution of the local myoglobin concentration.Part of the results have been reported during the Workshop on Oxygen Transport in Tissue, July 19–22, 1971 in Dortmund and at the 39. Tagung der Deutschen Physiologischen Gesellschaft April 12–13, 1972 in Erlangen.     

The diffusion coefficient of myoglobin in muscle homogenate

Summary One layer of muscle homogenate rich in myoglobin (heart muscle of the rat) and three layers of muscle homogenate poor in myoglobin (skeletal muscle of the rat) were arranged one upon the other, than reseparated after a certain time. According to the concentration change of myoglobin in the layers the diffusion coefficient of myoglobin is 1.5×10^–7 cm²/sec at 20° C and 2.7×10^–7 cm²/sec at 37° C. From this result it was calculated that the facilitated O₂ diffusion on the basis of myoglobin diffusion amounts, at 37° C, to the free O₂ diffusion at about 3 mm Hg partial pressure difference when myoglobin concentration is 2×10^–7 mol (3 mg) per g wet muscle.     

烧伤患者血清肌红蛋白水平的变化及其意义

目的探讨血清肌红蛋白(Mb)在烧伤患者血清中的动态变化及其意义。方法将2012年5月至2013年4月于我院住院治疗的烧伤患者(烧伤面积>15%)61例设为观察组,并根据烧伤面积的不同进一步分为三组,分别对三组患者伤后第1、3、7天血清Mb、肌酸激酶(CK)、C-反应蛋白(CRP)、白细胞计数(WBC)及中性粒细胞百分比(NET%)进行检测,同法测定50名健康体检者血清相应指标作为对照组。同时动态观察烧伤患者病情变化情况,其中并发多器官功能障碍综合征(MODS)者9例。结果烧伤组伤后第1天与对照组比较,血清Mb、CK、CRP、WBC及NET%含量差异均有统计学意义(P<0.05);随烧伤面积及采集时间的不同,血清中Mb、CK和CRP比较有显著差异(P<0.05);烧伤并发MODS组血清Mb和CK水平显著高于非MODS组。结论 Mb、CK和CRP的动态变化与烧伤患者病情严重程度、变化趋势及预后具有良好相关性,对MODS的早期诊断、治疗有重要意义,与其他炎性反应指标(CRP、WBC、NET%)联合监测对烧伤患者的救治具有较高临床价值。