Uncertainty of blank correction in isotope ratio measurement.

Blank correction for isotope ratio measurement on small amounts of substances is often limited by presence of a blank, with an apparent isotopic composition different from that of the sample. For isotope ratios, blank correction is commonly performed either by the regression method, which works without the need for estimation of the blank, or by the subtraction method. With the subtraction method, estimation of the amount and isotope delta of the blank is required, and these estimates could be obtained either by direct, semi-indirect, or indirect measurement. Previously given expressions for the standard uncertainties of indirectly measured blank amounts and blank isotope deltas did not account for covariance between input quantities. In the present work, a previously published data set was re-evaluated, with covariance terms properly included in the calculation of uncertainties. It was shown that covariance effects may yield a significant reduction in uncertainty estimates, both for blank quantities and for blank corrected results. For series measurements on a standard material, it was also shown that the distribution of individual corrected isotope delta values around the average value was approximately normal, with its standard deviation equal to the estimated standard uncertainty of the corrected values. In most cases, it was observed that the regression and subtraction methods yield approximately the same blank corrected average values and uncertainties, regardless of method selected for estimation of blank quantities.

Reduction of bias in static closed chamber measurement of delta13C in soil CO2 efflux.

The (13)C/(12)C ratio of soil CO(2) efflux (delta(e)) is an important parameter in studies of ecosystem C dynamics, where the accuracy of estimated C flux rates depends on the measurement uncertainty of delta(e). The static closed chamber method is frequently used in the determination of delta(e), where the soil CO(2) efflux is accumulated in the headspace of a chamber placed on top of the soil surface. However, it has recently been shown that the estimate of delta(e) obtained by using this method could be significantly biased, which potentially diminish the usefulness of delta(e) for field applications. Here, analytical and numerical models were used to express the bias in delta(e) as mathematical functions of three system parameters: chamber height (H), chamber radius (R(c)), and soil air-filled porosity (theta). These expressions allow optimization of chamber size to yield a bias, which is at a level suitable for each particular application of the method. The numerical model was further used to quantify the effects on the delta(e) bias from (i) various designs for sealing of the chamber to ground, and (ii) inclusion of the commonly used purging step for reduction of the initial headspace CO(2) concentration. The present modeling work provided insights into the effects on the delta(e) bias from retardation and partial chamber bypass of the soil CO(2) efflux. The results presented here supported the continued use of the static closed chamber method for the determination of delta(e), with improved control of the bias component of its measurement uncertainty.

Elevated serum levels of pancreatic secretory proteins in cigarette smokers after secretin stimulation.

The secretory pancreatic proteins in serum were analyzed in a group of cigarette smokers and a control group of nonsmokers before and after intravenous secretin stimulation. None of these persons had any signs of pancreatic disease. In the control group, serum total amylase activity, pancreatic isoamylase, cationic trypsinogen, and pancreatic secretory trypsin inhibitor concentrations varied within the normal range before and after secretin injection. In contrast, the concentrations of these pancreatic proteins in all the cigarette smokers elevated from normal to abnormally high serum concentrations after secretin stimulation. The results indicate a probable toxic effect of cigarette smoking on the exocrine pancreas.

Precision of measurements of physical workload during standardised manual handling. Part II: Inclinometry of head, upper back, neck and upper arms.

For measuring the physical exposure/workload in studies of work-related musculoskeletal disorders, direct measurements are valuable. However, the between-days and between-subjects variability, as well as the precision of the method per se, are not well known. In a laboratory, six women performed three standardised assembly tasks, all of them repeated on three different days. Triaxial inclinometers were applied to the head, upper back and upper arms. Between-days (within subjects) and between-subjects (within tasks) variance components were derived for the 10th, 50th and 90th percentiles of the angular and the angular velocity distributions, and for the proportion of time spent in predefined angular sectors. For percentiles of the angular distributions, the average between-days variability was 3.4 degrees , and the between-subjects variability 4.0 degrees . For proportion of time spent in angular sectors, the variability depended on the percentage of time spent in the sector; the relative variability was scattered and large, on average 103% between days and 56% between subjects. For the angular velocity percentiles, the average between-days variability was 7.9%, and the average between-subjects variability was 22%. The contribution of the measurement procedure per se to the between-days variability, i.e., the imprecision of the method, was small: less than 2 degrees for angles and 3% for angular velocity.

The importance of work organization on workload and musculoskeletal health--Grocery store work as a model.

We have evaluated the consequences of work organization on musculoskeletal health. Using a postal questionnaire, answered by 1600 female grocery store workers, their main work tasks were identified and four work groups were defined (cashier, picking, and delicatessen work, and a mixed group, who performed a mix of these tasks). The crude odds ratios (ORs) for neck/shoulder complaints were 1.5 (95% CI 1.0-2.2), 1.1 (0.7-1.5) and 1.6 (1.1-2.3), respectively, compared to mixed work. Adjusting for individual and psychosocial factors had no effect on these ORs. For elbows/hands, no significant differences were found. Technical measurements of the workload showed large differences between the work groups. Picking work was the most strenuous, while cashier work showed low loads. Quantitative measures of variation revealed for mixed work high between minutes variation and the highest between/within minutes variation. Combining work tasks with different physical exposure levels increases the variation and may reduce the risk of musculoskeletal complaints.

Isolation and immunochemical studies of dog trypsin.

Dog trypsin (EC 3.4.4.4) was isolated from dog pancreatic juice on SP-Sephadex C-50. The preparation was homogeneous on disc electrophoresis at pH 4.3. On agarose gel electrophoresis at pH 8.6, dog pancreas trypsinogen had the mobility of an alpha 2-globulin and trypsin the mobility of a beta-globulin. On gel filtration on Sephadex G-75 at pH 4.0, dog trypsin was eluted in the same fractions as bovine trypsin. It was inhibited by soybean trypsin inhibitor. Rabbit anti-dog trypsin inhibited the caseinolytic activity of bovine trypsin by about 60%.

Studies on the activation of canine trypsinogens in vitro.

The activation of canine anionic and cationic trypsinogen by enterokinase, trypsin, thrombin, plasmin and extracts from canine granulocytes were studied in vitro. Enterokinase activates both trypsinogens about 1000 times faster than trypsin. The enterokinase-catalyzed activation is not inhibited by the main serum protease inhibitors, alpha-macroglobulin and alpha 1-antitrypsin. alpha-Macroglobulin cannot inhibit the activation of the trypsinogens by trypsin but this reaction is completely inhibited by alpha 1-antitrypsin. The results are discussed in relation to the pathogenesis of acute pancreatitis.

An in vitro study of the influence of plasma protease inhibitors and aprotinin on trypsin-induced C3 cleavage in human serum.

Cleavage of native C3 in human serum following the addition of increasing amounts of human cationic trypsin was studied using an in vitro model. The cleavage was correlated with the degree of saturation of the plasma protease inhibitors alpha 2-macroglobulin and alpha 1-antitrypsin, and also with varying amounts of aprotinin (Trasylol). When alpha 2-macroglobulin reached 70% saturation, there was a prompt cleavage of most of the native C3 in spite of 90% free alpha 1-antitrypsin. The consumption of alpha 2-macroglobulin, and especially of alpha 1-antitrypsin, decreased with increasing concentrations of aprotinin. Aprotinin was needed in a concentration of 60 microM to block trypsin-induced C3 cleavage almost completely. This concentration is about 20 times higher than ever used clinically. One possible explanation of the poor clinical effect of aprotinin to date in human pancreatitis is the need for this high concentration.

Anionic and cationic dog trypsin. Isolation and partial characterization.

One anionic and one cationic dog trypsin have been identified in dog pancreatic juice and isolated. The dog pancreatic juice contained 1.4 mg/ml of anionic trypsinogen and 1.0 mg/ml of cationic trypsinogen, respectively. The conversion of the trypsinogens to active enzymes is autocatalytic with the formation of active enzymes with higher isoelectric points than the zymogens. Although the amino acid compositions of the two trypsins were similar to those of other mammalian trypsins, a significant difference in content of acidic and basic amino acids, respectively, was found between the two dog trypsins. A high extent of cross-inhibition of the dog trypsins and of bovine trypsin by antibodies to each of the dog trypsins indicated immunological cross-reaction between the various trypsins despite lack of demonstrable cross-precipitation in gels. The calculated s(20,w)0 value for both dog trypsins was 3.3 S. The Km value of anionic trypsin for benzoyl-D,L-arginine-p-nitroanilide' HCl was found to be similar to that of cationic trypsin and to that of bovine cationic trypsin. Soybean trypsin inhibitor, chicken ovomucoid, porcine and bovine secretory inhibitors are potent inhibitors of anionic and cationic dog trypsin.

Isolation and partial characterization of the pancreatic secretory trypsin inhibitor in the rat.

Isolation in a 55% yield of the low molecular weight pancreatic secretory trypsin inhibitor was achieved by gel filtration of an acid extract of whole inactive rat pancreas juice on Sephadex G-50 at pH 2.5 followed by desalting and ion-exchange chromatography on SP Sephadex C-50 at pH 4.5. Two distinct chromatographic fractions were obtained, labeled fraction 1 and 2. Fractions 1 and 2 showed three, respectively two, distinct closely migrating cationic bands on gel electrophoresis in barbital buffer, pH 8.6. Each fraction demonstrated one band on polyacrylamide disc electrophoresis at pH 4.6. The inhibitor is homogenous on gel filtration and on the basis of its stoichiometry with active site titrated rat anionic trypsin. Its molecular weight is approx. 6024. The amino acid composition is included. Rat pancreatic secretory trypsin inhibitor is trypsin-specific and interacts on a 1:1 molar basis with rat trypsin. It is a good inhibitor of bovine trypsin but a poor inhibitor of human cationic trypsin and its binding to trypsin is reversible by acidification. Like other inhibitors of this sort, it is present in about 0.1-0.2% of the total protein content of the juice, and normally exists in its free form. A simple procedure for the production of antiserum to the inhibitor which is a poor antigen is also described.

Interactions in vitro and in vivo between rat serum protease inhibitors and anodal and cathodal rat trypsin and chymotrypsin.

Reaction mixtures of increasing amounts of the pancreatic homologous proteases, anodal and cathodal chymotrypsin and trypsin, respectively, and normal rat serum were analyzed by immunoelectrophoretic methods in order to determine their distribution on serum protease inhibitors. This paper concerns three proteins occurring in normal serum and capable of binding protease viz. alpha1-macroglobulin, alpha1-antitrypsin and alpha1-inhibitor 3. The distribution of the enzymes among these protease inhibitors differed significantly from one protease to another. The distribution of the proteases among the serum protease inhibitors following intravenous injection of 125I-labelled proteases corresponded to that in vitro. Complexes formed with alpha1-macroglobulin and alpha1-inhibitor 3 were quickly eliminated irrespective of the enzyme species used, whereas those formed with alpha1-antitrypsin persisted much longer in the circulation.

The in vitro interactions of rat pancreatic elastase and normal and inflammatory ray serum.

The partition of labelled rat pancreatic elastase (EC 3.4.21.11) between the different protease inhibitors of rat plasma was studied at different levels of saturation of the inhibitors of rat plasma was studied at different levels of saturation of the inhibitor capacity of plasma with the enzyme. The reaction mixtures were analysed by immunoelectrophoretic methods utilizing specific antisera against the different inhibitors and by gel filtration on Sephadex G-200. Rat serum was shown to contain four elastase binding proteins. alpha 1-antitrypsin, alpha 1-macroglobulin and alpha 2-acute phase protein and alpha 1-inhibitor 3 which exhibits immunologic cross-reaction with human inter-alpha-trypsin inhibitor and is of similar molecular weight. With minute amounts of labelled elastase the partition among the binding protein was alpha 1-macroglobulin 60%, alpha 1-antitrypsin 24% and alpha 1-I3 16%. The 60% value of alpha 1-M bound radioactivity in normal serum corresponds to the sum of alpha 1-M and alpha 2-AP labelling in inflammatory serum.

Kinetics of the inhibition of leukocyte elastase by the bronchial inhibitor.

The rate constant for the association between human leukocyte elastase (EC 3.4.21.11) and human bronchial inhibitor has been determined by competition experiments with alpha 1-proteinase inhibitor. This constant (1.1.10(7) M(-1) . s(-1)) is 6-times lower than that for the association of leukocyte elastase and alpha 1-proteinase inhibitor. The latter inhibitor is able to dissociate the leukocyte elastase-bronchial inhibitor complex with a rate constant 1.3.10(-4) s-1. The equilibrium dissociation constant Ki of the complex is 1.2.10(-11) M. The physiopathological significance of these constants is discussed.

Leukocyte activation in atherosclerosis: correlation with risk factors.

Leukocytes have been implicated in the development of atherosclerotic vascular diseases, and numerous abnormalities of leukocytes in conjunction with atherosclerosis have been reported. The aim of this study of middle-aged asymptomatic subjects with early atherosclerosis was to determine whether a relationship exists between the levels of plasma markers of leukocyte activation, i.e. cytokines and proteases and risk factors for atherosclerosis or the degree of atherosclerotic disease. Using ELISAs we measured the plasma levels of neutrophil gelatinase-associated lipocalin (NGAL), neutrophil protease 4 (NP4) as markers for neutrophil activation, tumor necrosis factor alpha (TNF) and soluble TNF receptor-1 (sTNFR-1) as markers of monocyte/macrophage activation in 156 subjects with asymptomatic carotid artery plaque detected at ultrasound examination. Plasma TNF and sTNFR-1 levels were found to correlate with systolic blood pressure (r = 0.32, P < 0.04 and r = 0.22, P < 0.05, respectively). plasma NGAL level to correlate with diastolic blood pressure (r = 0.22; P < 0.005), the plasma levels of sTNFR-1 and NGAL to correlate with age (r = 0.28, P < 0.001 and r = 0.20, P < 0.05, respectively). As compared with non-smokers (n = 112), smokers (n = 43) had higher plasma levels of TNF (2.9 vs. 1.4 microg/l; P < 0.02) and of NP4 (27.5 vs. 23.4 microg/l; P < 0.05). The plasma NGAL level was higher in hypertensive women (n = 7) than in normotensive women (n = 85) (109 vs. 87 microg/l; P < 0.05). We thus demonstrated that, in subjects with asymptomatic early atherosclerosis, the plasma levels of markers of systemic leukocyte activation were correlated with age and blood pressure, and were higher in smokers and hypertensives. These results support the hypothesized relationship between the level of systemic leukocyte activation and risk factors for atherosclerotic vascular disease.

Varying occurrence of gastroduodenal immunoreactive pancreatic secretory trypsin inhibitor.

Operative specimens from various parts of gastroduodenal mucosa were analysed for immunoreactive pancreatic secretory trypsin inhibitor (PSTI) using a peroxidase-antiperoxidase method. Normal gastric mucosa exhibited a varying degree of PSTI immunoreactivity, which was more pronounced in the foveolar cells of gastric mucosa of fundus type than in the non-pepsinogen producing antrum-pyloric mucosa. With the exception of metaplastic Paneth cells and some goblet cells, the intracellular content of PSTI was low in gastric mucosa with intestinal metaplasia. These findings may indicate that a PSTI immunoreactive substance has a role in the normal defence of the gastric mucosa.

Pancreatic endoproteases and pancreatic secretory trypsin inhibitor immunoreactivity in human Paneth cells.

Normal and metaplastic gastrointestinal mucosa obtained at surgical resection were studied by light microscopy, using the unlabelled antibody enzyme method for immunohistochemical staining of lysozyme, pancreatic endoproteases, and pancreatic secretory trypsin inhibitor (PSTI). Paneth cells in the mucosa of normal small intestine, gastric mucosa with intestinal metaplasia, and colonic metaplastic mucosa were found to contain anionic trypsin, cationic trypsin, lysozyme, and PSTI immunoreactivity, but not chymotrypsin and elastase immunoreactivity. Normal gastric and colonic mucosa and some goblet cells in the small intestine showed positive PSTI immunoreactivity but no endoprotease immunoreactivity. The presence of immunoreactive trypsin and immunoreactive PSTI in the Paneth cells, which are of secretory type, probably indicates an important extrapancreatic source of these proteins rather than a storage of endocytosed material.

Immunohistochemical demonstration of pancreatic secretory trypsin inhibitor in normal and neoplastic colonic mucosa.

Specimens of normal and neoplastic colonic mucosa from 52 patients were analysed by immunohistochemistry using a monospecific polyclonal antiserum against human pancreatic secretory trypsin inhibitor (PSTI). In normal colonic mucosa PSTI was found in the goblet cells in the basal parts of the crypts. In adenomas of tubular, villous, and tubulo-villous types PSTI was also found in the upper parts of the polyps, usually occurring in the regeneration zone. There was a more intense staining reaction in polyps with increased atypia. Carcinomas of different types and of various grades of differentiation and of in situ type did not contain PSTI. These findings indicate that PSTI could be a marker for adenomatous rather than carcinomatous epithelium in the colon. Furthermore, the absence of the inhibitor in malignant cells might facilitate tissue invasion by malignant cells because of deficient protease inhibition.

Quantification of pancreatic secretory trypsin inhibitor in colonic carcinoma and normal adjacent colonic mucosa.

AIMS: To measure the content of immunoreactive human pancreatic secretory trypsin inhibitor (irPSTI) in colonic carcinoma and adjacent normal colonic mucosa. METHODS: From a stable hybridoma cell line producing monoclonal antibodies specific for human PSTI, a specific enzyme linked immunosorbent assay (ELISA) for human PSTI was developed. In a precipitation assay system these antibodies bound human PSTI in a dose-dependent manner. The specimens were obtained from resectional surgery. RESULTS: The content of irPSTI was 19.9 micrograms/g protein (0.55 micrograms/g tissue wet weight) in colonic carcinoma. In adjacent normal colonic mucosa 43.6 micrograms/g protein (1.12 micrograms/g tissue wet weight) was shown. CONCLUSIONS: The enzymatic degradation of surrounding tissue necessary for tumour cell invasion could be facilitated by this relative deficit of the inhibitor in infiltrative carcinoma.

Collagenase and elastase released during peritonitis are complexed by plasma protease inhibitors.

Peritoneal fluids from patients with diffuse peritonitis secondary to perforation of the appendix contained large quantities of collagenase and elastase. The enzymes, which existed in the form of complexes with plasma protease inhibitors, probably had been released from the granulocytes. The two enzymes had linked almost half of the alpha 1-antitrypsin and four fifths of the alpha 2-macroglobulin in the fluid. Evidence that regional defense against protease had failed was obtained by finding the C3 component of complement completely converted. Toxic peptides probably had been released. Recognition of plasma protease inhibitors as an important part of the regional defense against protease also suggested use for therapy. We lavaged the peritoneum postoperatively with fluid that did not contain plasma inhibitors but with volumes large enough to eliminate the accumulation of enzymes for the granulocyte.

Granulocyte collagenase and elastase and the plasma protease inhibitors in human pus.

Granulocyte collagenase and elastase was demonstrated in human pus using specific antisera. The enzymes were complexed by alpha1-antitrypsin and alpha2-macroglobulin but also were present as free proteases. All purulent exudates showed free elastolytic and collagenolytic activity.