Determination of regional ventilation and perfusion in the lung using xenon and computed tomography.

We propose a model to measure both regional ventilation (V) and perfusion (Q) in which the regional radiodensity (RD) in the lung during xenon (Xe) washin is a function of regional V (increasing RD) and Q (decreasing RD). We studied five anesthetized, paralyzed, mechanically ventilated, supine sheep. Four 2.5-mm-thick computed tomography (CT) images were simultaneously acquired immediately cephalad to the diaphragm at end inspiration for each breath during 3 min of Xe breathing. Observed changes in RD during Xe washin were used to determine regional V and Q. For 16 mm(3), Q displayed more variance than V: the coefficient of variance of Q (CV(Q)) = 1.58 +/- 0.23, the CV of V (CV(V)) = 0.46 +/- 0.07, and the ratio of CV(Q) to CV(V) = 3.5 +/- 1.1. CV(Q) (1.21 +/- 0.37) and the ratio of CV(Q) to CV(V) (2.4 +/- 1.2) were smaller at 1,000-mm(3) scale, but CV(V) (0.53 +/- 0.09) was not. V/Q distributions also displayed scale dependence: log SD of V and log SD of Q were 0.79 +/- 0.05 and 0.85 +/- 0.10 for 16-mm(3) and 0.69 +/- 0.20 and 0.67 +/- 0.10 for 1,000-mm(3) regions of lung, respectively. V and Q measurements made with CT and Xe also demonstrate vertically oriented and isogravitational heterogeneity, which are described using other methodologies. Sequential images acquired by CT during Xe breathing can be used to determine both regional V and Q noninvasively with high spatial resolution.     

Adhesion of dissociated mouse embryonic brain cells. A new method of quantitative evaluation

The adhesion of mouse embryonic brain cells was measured in a rotating chamber. A method is proposed for quantitative evaluation of adhesion kinetics. Dissociated cells were incubated in a planparallel chamber and pictures were taken between time 0-120 min. Film negatives were evaluated by computer--controlled scanning. Ten thousand individual data area obtained from one frame and 1,000 levels of absorbency are distinguished. A method is described which allows the discrimination of area, density and the shape of adhering cells. The influence of the dissociation procedure on cellular adhesion was studied. Short trypsinisation (0.025% trypsin for 5 min) followed by sieving was most favourable for adhesion. Mechanical sieving and dissociation with EGTA (Ca2+ chelator) gave less satisfactory results. Significantly diminished adhesion was observed after prolonged trypsinisation. If cells were incubated in media lacking Ca2+, adhesion was significantly inhibited. The kinetics of adhesion follows the curve of flocculation kinetics independently of the dissociation procedure and composition of the medium.     

Alterations in regional ventilation, perfusion, and shunt after smoke inhalation measured by PET.

Regional changes in ventilation and perfusion occurring in the early hours after smoke inhalation injury were evaluated through the use of positron emission tomography. Five lambs were imaged before and 1, 2, and 4 h after receiving 100 breaths of cotton smoke. Utilizing a recently developed model of (13)N tracer kinetics (3), we evaluated changes in ventilation, perfusion, shunt, and regional gas content in nondependent, middle, and dependent lung zones. The data demonstrated a progressive development of regional shunt in dependent (dorsal) regions in which perfusion remained the highest throughout the study. These findings, together with decreasing regional ventilation and fractional gas content in the dependent regions, correlated with decreasing arterial Pa(O(2)) values over the course of the study. A negative correlation between regional shunt fraction and regional gas content in dependent and middle regions suggests that shunt was caused by progressive alveolar derecruitment or flooding.     

Microsphere maps of regional blood flow and regional ventilation.

Systematically mapped samples cut from lungs previously labeled with intravascular and aerosol microspheres can be used to create high-resolution maps of regional perfusion and regional ventilation. With multiple radioactive or fluorescent microsphere labels available, this methodology can compare regional flow responses to different interventions without partial volume effects or registration errors that complicate interpretation of in vivo imaging measurements. Microsphere blood flow maps examined at different levels of spatial resolution have revealed that regional flow heterogeneity increases progressively down to an acinar level of scale. This pattern of scale-dependent heterogeneity is characteristic of a fractal distribution network, and it suggests that the anatomic configuration of the pulmonary vascular tree is the primary determinant of high-resolution regional flow heterogeneity. At approximately 2-cm(3) resolution, the large-scale gravitational gradients of blood flow per unit weight of alveolar tissue account for <5% of the overall flow heterogeneity. Furthermore, regional blood flow per gram of alveolar tissue remains relatively constant with different body positions, gravitational stresses, and exercise. Regional alveolar ventilation is accurately represented by the deposition of inhaled 1.0-microm fluorescent microsphere aerosols, at least down to the approximately 2-cm(3) level of scale. Analysis of these ventilation maps has revealed the same scale-dependent property of regional alveolar ventilation heterogeneity, with a strong correlation between ventilation and blood flow maintained at all levels of scale. The ventilation-perfusion (VA/Q) distributions obtained from microsphere flow maps of normal animals agree with simultaneously acquired multiple inert-gas elimination technique VA/Q distributions, but they underestimate gas-exchange impairment in diffuse lung injury.     

Effects of aesthetic abdominoplasty on abdominal wall perfusion: a quantitative evaluation

Abdominoplasty procedures involve a high risk of early complications, including hematomas, seromas, necrosis, and wound-healing problems. Their rationale is evident from the vascular anatomy of the abdominal wall, as traditional abdominoplasty includes a division of the main perforating vessels. No studies exist to quantitatively assess the consequences of abdominoplasty on the perfusion of the random pattern abdominal flap. To address this issue and quantify the influence of classic abdominoplasty on the perfusion of the abdominal skin, the authors performed a prospective clinical trial including 15 low-risk patients undergoing abdominoplasty for aesthetic purposes. Perfusion of the abdominal flap was measured intraoperatively using the technique of dynamic laser-fluorescence-videoangiography. In the region between the umbilicus and the transverse scar (zone 1), the increment of fluorescence (the slope of the intensity curve during inflow of the indocyanine green) was recorded and compared with the intensity curve of normal tissue that was not involved in surgery (thoracic wall). The results of the intraoperative indocyanine green perfusography showed a significant impairment of the vascular supply of zone 1 in all patients. The mean perfusion index in this region was 17.2 percent (range, 5 to 32 percent) of the perfusion of the surrounding skin that was not involved in surgery. The complication rate was 33 percent (five patients) and included two cases of hematoma and three cases of scar dehiscence with skin and/or fat necrosis. These data indicate that conventional abdominoplasty including extended undermining and division of the superficial and the deep arterial systems causes profound devascularization of the abdominal flap. This might explain the high incidence of complications following this procedure.     

Validation of a quantitative magnetic resonance method for measuring human body composition

Objective: To evaluate a novel quantitative magnetic resonance (QMR) methodology (EchoMRI‐AH, Echo Medical Systems) for measurement of whole‐body fat and lean mass in humans. Methods and Procedures: We have studied (i) the in vitro accuracy and precision by measuring 18 kg Canola oil with and without 9 kg water (ii) the accuracy and precision of measures of simulated fat mass changes in human subjects (n = 10) and (iii) QMR fat and lean mass measurements compared to those obtained using the established 4‐compartment (4‐C) model method (n = 30). Results: (i) QMR represented 18 kg of oil at 40°C as 17.1 kg fat and 1 kg lean while at 30°C 15.8 kg fat and 4.7 kg lean were reported. The s.d. of repeated estimates was 0.13 kg for fat and 0.23 kg for lean mass. Adding 9 kg of water reduced the fat estimates, increased misrepresentation of fat as lean, and degraded the precision. (ii) the simulated change in the fat mass of human volunteers was accurately represented, independently of added water. (iii) compared to the 4‐C model, QMR underestimated fat and over‐estimated lean mass. The extent of difference increased with body mass. The s.d. of repeated measurements increased with adiposity, from 0.25 kg (fat) and 0.51 kg (lean) with BMI <25 kg/m² to 0.43 kg and 0.81 kg respectively with BMI >30 kg/m². Discussion: EchoMRI‐AH prototype showed shortcomings in absolute accuracy and specificity of fat mass measures, but detected simulated body composition change accurately and with precision roughly three times better than current best measures. This methodology should reduce the study duration and cohort number needed to evaluate anti‐obesity interventions.     

Fractal nature of regional ventilation distribution.

High-resolution measurements of pulmonary perfusion reveal substantial spatial heterogeneity that is fractally distributed. This observation led to the hypothesis that the vascular tree is the principal determinant of regional blood flow. Recent studies using aerosol deposition show similar ventilation heterogeneity that is closely correlated with perfusion. We hypothesize that ventilation has fractal characteristics similar to blood flow. We measured regional ventilation and perfusion with aerosolized and injected fluorescent microspheres in six anesthetized, mechanically ventilated pigs in both prone and supine postures. Adjacent regions were clustered into progressively larger groups. Coefficients of variation were calculated for each cluster size to determine fractal dimensions. At the smallest size lung piece, local ventilation and perfusion are highly correlated, with no significant difference between ventilation and perfusion heterogeneity. On average, the fractal dimension of ventilation is 1.16 in the prone posture and 1. 09 in the supine posture. Ventilation has fractal properties similar to perfusion. Efficient gas exchange is preserved, despite ventilation and perfusion heterogeneity, through close correlation. One potential explanation is the similar geometry of bronchial and vascular structures.     

Spatial correlation of regional pulmonary perfusion.

Despite the heterogeneous distribution of pulmonary blood flow, perfusion appears to be spatially ordered, with neighboring regions of lung having similar magnitudes of flow. This premise was tested by determining the spatial correlation of regional flow [rho(d)] as a function of distance (d) between regions. Regional pulmonary perfusion was measured in both supine and prone positions in seven anesthetized mechanically ventilated dogs with radiolabeled microspheres. After excision and drying, the lungs were cubed into pieces 1.2 cm on a side, with a three-dimensional coordinate assigned to each piece. The microsphere-determined flow to each piece was measured by radioactive counts, and rho(d) was calculated for all paired pieces within the same lobe. rho(d) was greatest for adjacent pieces (d = 1.2 cm) and decreased with increasing d, becoming negative at large distances in all dogs and positions. The spatial correlation of flow between adjacent pieces, rho(1.2 cm), was greater in the supine than in the prone position (0.66 vs. 0.72, P less than 0.05). The observations for each dog and position were fit to the equation rho(d) = d(a)+b.d+c, and the coefficients were used to compare rho(d) in the supine and prone positions. rho(d) differed in the two positions (P less than 0.05), with rho(d) falling off more rapidly with distance in the supine position. When trends in flow due to gravity were mathematically removed, differences between supine and prone positions were no longer observed. The spatial correlation of regional pulmonary perfusion was anisotropic in both supine and prone positions. The observation that regional pulmonary perfusion is highly correlated over large spatial distances has important implications for models of flow distribution.     

MIRA-SNuPE,a quantitative,multiplex method for measuring allele-specific DNA methylation

5-methyl-C (5mC) and 5-hydroxymethyl-C (5hmC) are epigenetic marks with well-known and putative roles in gene regulation, respectively. These two DNA covalent modifications cannot be distinguished by bisulfite sequencing or restriction digestion, the standard methods of 5mC detection. The methylated CpG island recovery assay (MIRA), however, specifically detects 5mC but not 5hmC. We further developed MIRA for the analysis of allele-specific CpG methylation at differentially methylated regions (DMRs) of imprinted genes. MIRA specifically distinguished between the parental alleles by capturing the paternally methylated H19/Igf2 DMR and maternally methylated KvDMR1 in mouse embryo fibroblasts (MEFs) carrying paternal and maternal duplication of mouse distal Chr7, respectively. MIRA in combination with multiplex single nucleotide primer extension (SNuPE) assays specifically captured the methylated parental allele from normal cells at a set of maternally and paternally methylated DMRs. The assay correctly recognized aberrant biallelic methylation in a case of loss of imprinting. The MIRA-SNuPE assays revealed that placenta exhibited less DNA methylation bias at DMRs compared to yolk sac, amnion, brain, heart, kidney, liver and muscle. This method should be useful for the analysis of allele-specific methylation events related to genomic imprinting, X chromosome inactivation and for verifying and screening haplotype-associated methylation differences in the human population.Key words: epigenetics, imprinting, DMR, MIRA, MBD, DNA methylation, SNuPE     

Pulmonary regional ventilation. C15O2 single breath method. New technical improvements

After a single inhalation of C15O2 labelled air, from residual volume to total lung capacity, followed by a few seconds apnea, it is possible to record in four or six lung zones the regional activity changes on a time basis. A first fast rise in activity is seen during the inhalation phase, corresponding to the ventilation in the region of interest (ROI). During the apnea, a slower decreasing component is observed in the different ROI, proportional to the regional blood flow, as almost instantaneously C15O2 is converted to labelled water. After the pioneer work of West & Dollery (1962) very few papers appeared on the subject despite technical improvements as, for instance, coincidence counting of radioactive events. We thus decided to improve the reproducibility and accuracy of the method which were of such a poor quality that no individual measurements were possible in the past. Were of critical importance: a careful positioning of the patients between the counters, a faster electronics improving true coincidence counting. In six healthy volunteers (age 22-35) the values obtained for regional ventilation with the C15O2 single breath were compared to the values given by the inhalation of a poorly soluble gas (Nitrogen13). The correlation between the two sets of measurements is high (r = 0.975). The slope of the regression line is 0.859, ordinate at origin is 2.35. This reveals a systematic error. A mathematical model was therefore developed to take into account the "washout" occurring during the constitution of the ventilation peak, and to correct its value.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hydrolytic enzyme substrates. II. A new method for measuring periodate

This report describes an accurate and sensitive method for quantitatively measuring periodate concentration. The substances used to determine periodate are 4(p-nitrophenoxy)1,2-butanediol and 4(2,4-dinitrophenoxy)1,2-butanediol. These substances are readily oxidized by periodate yielding β-nitrophenoxy aldehydes which undergoes a facile β-elimination in base to yield the colored nitrophenolate ion. The concentration of the nitrophenolate ion is thus equivalent to the concentration of periodate. This report documents the validity of this reaction as an analytical method. The method was shown to be capable of accurately measuring periodate in concentrations as low as 10^?8M. Its value in biochemical analyses was demonstrated by quantitatively measuring the amount of periodate used to oxidize small quantities of adenosine 5′-phosphate, d-arabitol and d-glucose. Its accuracy, sensitivity and ease of use was shown by its utility in estimating the molecular weight of yeast transfer RNA using about 6 A₂₆₀ units of this material.     

一种新的稀释定量方法在标本检测中的应用

我们使用注射器装入小金属棒在磁力搅拌器上作连续定量稀释方法与传播的吸管或移液器稀释方法,用于含菌量大的粪便标本及含菌量较小的烧伤病人创面和血液标本进行标本细菌的定量分析,结果证实两种方法结果较准确,重复性好,且无统计学差异。     

Statistical evaluation of a new method to detect carriers of phenylketonuria.

40 positive heterozygotes and 43 controls were loaded with 200 mg phenylalanine per kilogram body weight. The aromatic acids excreted 2 hrs after the loading were quantified by gaschromatography. The amounts of mandelic acid (MA), 2-hydroxyphenylacetic acid (2HOPAA) and phenylpyruvic acid (PPA) were used for a discriminatory analysis. The MA concentration alone gives a better discrimination than the statistical analysis.