Trypsin Inhibitory Capacity Research Articles

Cadmium-induced pulmonary injury in mouse: a relationship with serum antitrypsin activity.

Data on in vivo experiments on mice exposed to Cd with the attempt to correlate serum trypsin inhibitory capacity (TIC) changes with morphologic alterations is presented. The experimental data obtained indicate that cadmium chloride produced a significant reduction in the serum antitrypsin activity in mice. The data further indicates that the dimunition of serum TIC due to acute Cd exposure is a transient phenomenon but appears to produce lung damage rapidly as shown by morphologic examination. (JMT)

Serum phospholipase A2, immunoreactive trypsin, and trypsin inhibitors during human acute pancreatitis.

Serum immunoreactive trypsin and phospholipase A2 were analyzed at regular intervals in seven patients hospitalized as a result of acute hemorrhage pancreatitis. alpha 1-Antitrypsin and alpha 2-macroglobulin levels and trypsin-inhibitor capacity of serum were determined simultaneously. Serum trypsin concentrations were markedly raised in all patients. The levels of immunoreactive trypsin remained elevated for longer periods than those of urinary amylase. alpha 1-Antitrypsin and trypsin-inhibitor capacity were also significantly increased as compared with the post-illness values, but alpha 2-macroglobulin decreased considerably, reaching the lowest levels on the 5th day after admission. Consequently, phospholipase and trypsin are released to the circulation during hemorrhagic pancreatitis, but the increase in trypsin is compensated for by an increase in trypsin-inhibitor capacity of serum due to elevated alpha 1-antitrypsin levels. The decrease of alpha 2-macroglobulin in hemorrhagic pancreatitis was one of the most interesting findings, and it is proposed that this inhibitor may be consumed in the elimination of proteases through the reticuloendothelial system. The two patients who died had higher phospholipase values than those who recovered, but the prognosis could not be predicted from the values of the other measured variables.

Pancreatic Acinar Cell Function and Morphology in Rats Chronically Fed an Ethanol Diet

The aim of the present study was to determine the effect of prolonged ethanol intake on the morphology and protein metabolism in the rat pancreatic acinar cells. Weight-matched triplets of Sprague-Dawley rats were fed Lieber-DeCarli diet containing 5% (wt/vol) concentration of ethanol, isocaloric amounts of Lieber-DeCarli diet, or rat chow ad libitum for 6, 12, and 18 mo. In the ethanol-fed group, histologic studies by light microscopy showed absence of protein plugs in the pancreatic ducts and/or pancreatitis, but electron-microscopic evaluation revealed progressive accumulation of lipid droplets in acinar and ductal cells. No definite changes in the mitochondria and endoplasmic reticulum were noticed. Biochemical studies revealed increased specific activity of trypsinogen, chymotrypsinogen, and lipase, and decreased specific activity of amylase. Trypsin-inhibiting capacity was decreased in the tissue and in the medium in a progressive fashion. There was no increase in the secretion of total protein. These data show a complex and a nonparallel alteration of specific digestive enzymes and trypsin inhibitor in this model of chronic ethanol intoxication that may be of relevance to occurrence of pancreatitis.

Mouse plasma trypsin inhibitors. Isolation and characterization of alpha-1-antitrypsin and contrapsin, a novel trypsin inhibitor.

The trypsin-inhibiting capacity of male adult mouse plasma was several-fold greater than that of the corresponding human plasma. Two major trypsin inhibitors were isolated from mouse plasma in an apparently homogeneous state. One seems to be homologous to alpha-1-antitrypsin isolated from several mammalian species, while the other, tentatively named contrapsin, does not correspond to any of the known plasma protease inhibitors that have been well characterized in human or other animals. When human plasma was fractionated in exactly the same fashion, no inhibitor corresponding to contrapsin was detected, suggesting the absence of this inhibitor in human plasma. No cross-reactivity was observed either between contrapsin and antiserum specific for alpha-1-antitrypsin or between alpha-1-antitrypsin and antiserum specific for contrapsin. alpha-1-Antitrypsin inhibited three serine proteases tested, i.e. porcine elastase, bovine trypsin, and chymotrypsin, whereas contrapsin inhibited only trypsin, but not the other two. The two inhibitors, however, shared a number of other properties in common. They were monomeric glycoproteins having total carbohydrate content of 9.6% in alpha-1-antitrypsin (Mr = 53,000) and 15.1% in contrapsin (Mr = 55,000). They had similar amino acid and carbohydrate compositions. They inhibited bovine beta-trypsin by forming stable complexes. They showed similar sex differences in blood concentrations. Serum levels of alpha-1-antitrypsin and contrapsin in males were 5.2 +/- 0.6 and 2.8 +/- 0.2 mg/ml, respectively, whereas those in females were 3.5 +/- 0.2 and 2.1 +/- 0.4 mg/ml, respectively.

Turkey serum trypsin inhibitors: Description and characterization

1. 1. Turkey serum trypsin inhibitors were studied on whole and chromatographically fractionated normal turkey serum using both quantitative (trypsin inhibitory capacity measurement) and qualitative (antitryptic activity detection methods) determinations, coupled to electrophoretic and isoelectrophoretic studies. 2. 2. Five proteins with trypsin inhibitory activity were described, the most important ones being α 2 and β-globulins with a multibanded pattern revealed by isoelectric focusing. 3. 3. Trypsin inhibitory capacity assays, performed on individual sera, as well as isoelectric focusing studies, failed to find any quantitative and/or qualitative deficiency of these antiproteases. 4. 4. Evidence is given that round heart disease in turkeys is not related to serum trypsin inhibitor deficiency.

Isolation and characterization of goat serum alpha 1-globulin protease inhibitors.

α1-Globulin-type protease inhibitors were isolated from goat serum by two methods, namely preparative isoelectric focusing and preparative electrophoresis in polyacrylamide gel. The fractions obtained by the first method showed varying isoprotein compositions by analytical isoelectric focusing. Sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE) revealed the presence of one protein in the fractions with the same velocity of migration as purified human α1-antitrypsin and a second protein with a slightly higher migration velocity. The ratios of trypsin-inhibiting to chymotrypsin-inhibiting capacities in all the fractions were the same and both inhibitors were stable upon storage. The reaction of the inhibitors with trypsin and chymotrypsin was also demonstrated by analytical isoelectric focusing.The fractions obtained by preparative gel electrophoresis (the second method) contained the same proteins but their proportions varied widely in different fractions as demonstrated by analytical electrofocusing in the presence of urea and by SDS–PAGE. The early fractions, which consisted predominantly of α1-antitrypsin, showed a high inhibiting capacity for trypsin and none or only negligible capacity for chymotrypsin. Conversely, in the late fractions, the proportions of the proteins and inhibiting capacities were reversed. At 4 °C the trypsin-inhibiting capacity was stable for weeks but the chymotrypsin-inhibiting capacity of the preparation rapidly decreased.These observations indicate that the inhibition of proteases by goat α1-globulins is due to at least two closely associated but distinguishable proteins. One of these, corresponding to human α1-antitrypsin, would have an appreciable capacity to inhibit trypsin, but unlike the latter, little or no capacity for chymotrypsin inhibition. The inhibition of chymotrypsin is due to the second, unidentified α1-globulin.

Clinical symptoms and results of skin test, RAST and bronchial provocation test in thirty-three papain workers: evidence for strong immunogenic potency and clinically relevant 'proteolytic effects of airborne papain'.

Seventeen out of thirty-three workers who have been exposed to airborne papain at their place of work regularly developed asthmatic symptoms such as shortness of breath, wheezing, coughing, sneezing, rhinorrhea and conjunctival irritation upon contact with this proteolytic enzyme. Investigations by RAST, skin test and bronchial provocation test proved IgE-mediated hypersensitivity to papain in fourteen symptomatic workers. Ten of these were in a screening investigation involving twenty-nine of the thirty-three workers; i.e. the incidence of IgE-mediated sensitization was 34.5% of this group. Bronchial provocation of as little as 0.001--0.5 mg of papain was shown to elicit immediate or dual asthmatic reactions in all eight tested workers with RAST values greater than 3 u/ml. On the other hand, inhalation of 0.5 mg of papain did not cause any remarkable change in non-exposed asthmatics. Occupation-related blood-stained nasal secretions and/or cutaneous flare reactions in all four heavily-exposed papain workers, of whom three had negative skin test and RAST results, suggest a direct effect of the proteolytically active enzyme on human tissue. There was a significant elevation (P less than 0.001) of serum trypsin inhibitory capacity in papain workers which seems to depend on the degree of exposure. No significant differences between symptomatic and asymptomatic workers in alpha 1-antitrypsin (Pi) phenotype subtypes were found.

Protease inhibitor levels in porcine mammary secretions.

The total trypsin inhibiting capacity (TIC), and the content of the individual protease inhibitors, both the specific colostrum protease inhibitor (SCTI) and the serum-type inhibitors, i.e., alpha 2-macroglobulin f and s, inter-alpha-trypsin inhibitor, alpha 2-antitrypsin and alpha 1-protease inhibitor, were quantified in porcine mammary secretions throughout lactation using immunoprecipitation methods. In addition, albumin, of serum origin, and beta-lactoglobulin, a milk-specific protein, were quantified and used as references. In presuckled colostrum, high levels of TIC, SCTI, albumin and beta-lactoglobulin were found, while the serum-type inhibitors appeared in amounts related to their molecular weights ranging from 3-6% of the adult serum levels for the macroglobulins to 42% for alpha 1-protease inhibitor. These parameters all decreased during lactation. The levels of TIC and SCTI especially decreased during the first days of lactation, and SCTI disappeared from milk after 5-7 days. Mature milk showed low TIC levels, about 0.1 IU/ml, which originated from the low amounts of the serum-type inhibitors (1-4% of the adult serum level). The levels of both alpha 2-macroglobulin f and s decreased less than albumin did in sow milk, while the other inhibitors decreased more or on the same order as albumin. A considerable variation in the amounts of the protease inhibitors between individuals and also between different teat secretions of the same sow, especially in colostrum, was evident. The possible physiological functions of the protease inhibitors in the mammary gland of the sow and in the gastrointestinal tract of her nursing offspring are discussed.

A model of decreased functional alpha-1-proteinase inhibitor. Pulmonary pathology of dogs exposed to chloramine T.

The objective of this study was to develop an animal model representative of chronic human alpha-1-proteinase inhibitor deficiency. Eight dogs were treated with a mild oxidizing agent, chloramine T, with varying regimens for 3--27 wk. The capacity of the serum to inhibit both trypsin and elastase was examined and found to respond differently. Although immunologically determined levels of protease inhibitor did not change, the ability of serum to inhibit elastase in an in vitro assay decreased in direct response to chloramine T treatment. The trypsin inhibitory capacity was less affected. Emphysemalike alterations in lung morphology were observable when histologic sections were evaluated both subjectively and objectively by mean linear intercept measurements. The data suggest that this model parallels the emphysema associated with the genetic alpha-1-proteinase inhibitor deficiency in man.

Trypsin-inhibitors in mastitic milk and colostrum: correlation between trypsin-inhibitor capacity, bovine serum albumin and somatic cell contents.

The trypsin-inhibitor capacity of bovine milk was seen to increase in mastitis. This capacity showed good correlation with the California Mastitis Test score, somatic cell count (Counter method) and bovine serum albumin (BSA) content (radial immunodiffusion). The elevated antitrypsin level proved to be a safer indicator of mastitis than BSA alone. Using gel-filtration chromatography, the major trypsin-inhibitor activity of mastitic milk and plasma was eluted with BSA, indicating a similar mol. wt (70 000). This fraction showed electrophoretic polymorphism. A large molecular weight fraction cross-reacted immunologically with human alpha 2-macroglobulin. The antitrypsin activity in mastitic milk was clearly different from the principal activity in colostrum, which had a mol. wt of about 13 000.

Estrogen-stimulated uptake of alpha 1-protease inhibitor and other plasma proteins by the mouse uterus.

Administration of a single low dose of estradiol to the immature female mouse resulted in a rapid increase in uterine trypsin inhibitory capacity. The increase was apparent within 1 h, reached a maximum in 3--4 h, and returned to base line after 18 h. No corresponding increases were observed in liver or heart. The properties of the uterine inhibitor were found to be essentially the same as those of plasma alpha 1-protease inhibitor (alpha 1-PI). When 125I-labeled mouse plasma alpha 1-PI was given iv to immature mice the administration of estradiol caused a specific stimulation of uterine uptake of the labeled protein that closely matched the estrogen-stimulated increase in uterine trypsin inhibitory capacity. Half-maximal stimulation of uptake occurred at a dose of 0.15 microgram estradiol/animal. After 3 h 97% of the labeled alpha 1-PI taken up by the uterus was in the soluble (105,000 x g) fraction of which 50% was in the lumen. Estradiol also stimulated the uptake of soybean trypsin inhibitor, bovine serum albumin, porcine fibrinogen, and human alpha 2-macroglobulin. Under conditions where puromycin blocked the synthesis of an estrogen-stimulated uterine-specific hydrolase, puromycin had no effect on the estrogen-stimulated uptake of either mouse alpha 1-PI or mouse albumin. These results suggest that the estrogen-stimulated uptake of plasma proteins by the uterus does not require new protein synthesis.

The coagulation system in septic newborns.

Coagulation factors were examined in 48 newborns with spesis caused mainly by Klebsiella enterobacteriaceae and E. coli. The first examination revealed increased mean values of fibrinogen, antiplasmin, trypsin inhibitory capacity, factors II and X. Thrombocytes, plasminogen, antithrombin III, alpha 2-macroglobulin and factor V were reduced on average. Serial examinations brought to light turbulent dynamics of the parameters. In most cases the fibrinogen level increased first together with factors II, V and X, and with antiplasmin, and followed by antithrombin III and alpha 2-macroglobulin after several days. Trypsin inhibitory capacity decreases progressively, starting from extremely high levels or after an initial rapid increase. Thrombocytopenia is the last to correct. Close correlations between the fibrinogen level and factors II and X in the initial values, as well as in the course of the disease, point to the dynamics of these components being similar. These findings are reason to believe the pathogenetic importance of the disseminated intravascular coagulation in many cases. Rapid overproduction of components takes place after disseminated intravascular coagulation in sepsis; overproduction has its limits in production capacity and thrombocytopoiesis is the weakest link in newborns. Certain parameters are of low diagnostic significance. Sepsis in newborns is well characterized by the discrepancy between thrombocytopenia and high fibrinogen levels, and thus by the difference between fibrinogen level and thrombocyte count. The value of this criterion in separating healthy newborns and those with respiratory distress syndrome from newborns with sepsis has been proved.

Anti-inflammatory action of progesterone and its possible mode of action in rats

The effect of progesterone on carrageenin-induced paw edema was studied in rats. Repeated injections of progesterone (1 mg/kg body weight) were given subcutaneously every 12 hr; the swelling of the paw edema was significantly inhibited by five, seven and nine injections of progesterone, whereas three injections of progesterone caused a significant inhibition at 1 hr only after carrageenin injection into the hind paw. α 1 Macroglobulin ( α 1 M) was purified by gel filtration on Sephadex G-200 and DEAE-cellulose column chromatography from the pooled sera of rats injected repeatedly with progesterone (1 mg/kg body weight) or the vehicle (sesame oil). The trypsin-inhibiting capacity of the α 1 M fraction from the progesterone-treated group was about twice as high as that from the control group. The anti-inflammatory action of the purified α 1 M from the serum of the progesterone-treated rats was studied; the swelling of carrageenin-induced paw edema was significantly inhibited for 1–2 hr after carrageenin injection, and this effect became progressively less pronounced over 3–4 hr when a single intravenous injection of the purified α 1 M fraction (19 mg protein/rat) was given immediately before the carrageenin injection into the hind paw. A granuloma pouch was induced by the injection of a 2% carrageenin solution into a pre-formed air pouch on the back of rats and the purified α 1 M (70 mg protein/kg body weight) was injected into the air pouch immediately after the carrageenin injection, with the result that a single injection of the purified α 1 M significantly inhibited the formation of granulation tissue on day 4 after the carrageenin injection. These results suggest that progesterone exerts its anti-inflammatory action through an increased protease-inhibiting activity of α 1 M, although anti-inflammatory action of the purified α 1 M from vehicle-treated rat serum, alone, was not examined.

PiPclifton: a new α1-antitrypsin allele in an American Negro family*

Serum specimens from eight females and two males representing three generations of an American Negro family exhibited an α1-antitrypsin phenotype that we labelled MPclifton because of its electrophoretic mobility. The...

Successful spleno-renal shunt and splenectomy in two patients with alpha-1-antitrypsin deficiency

Recent evidence suggests that the cirrhosis of alpha-1-antitrypsin deficiency is not invariably fatal as it was previously thought. Portal hypertension is often the major determinant of survival. The few reports of porta-systemic venous anastomosis in this disorder have shown poor results or uncertain outcome. Thus, doubts exist as to whether porta-systemic shunts should be performed in alpha-1-antitrypsin deficiency. Two patients with alpha-1-antitrypsin deficiency (PiZZ) and associated portal hypertension, cirrhosis, and hypersplenism underwent splenorenal shunt and splenectomy 8 yr ago, and both have done well. One of the patients has chronic severe headaches, diarrhea, exudative enteropathy, sinusitis, and hematuria, all uncommon in alpha-1-antitrypsin deficiency but possibly related to the antienzyme deficiency. She also has a higher trypsin inhibitory capacity than is generally reported in ZZ individuals. Based on the experience with these 2 patients, it appears that alpha-1-antitrypsin deficiency with cirrhosis is not a valid contraindication to the performance of a portasystemic shunt.

PiZpratt: a new alpha 1-antitrypsin allele in an American Negro family.

A rare alpha 1-antitrypsin phenotype was detected in serum of individuals belonging to three generations of an American Negro family. Following the recommended guidelines of nomenclature, we labeled the new Pi type Zpratt; the corresponding allele is PiZpratt. Alpha 1-antitrypsin concentration and trypsin inhibitory capacity are normal in MZpratt serum.

Familial temperature sensitive alpha 1 protease inhibitor (m1anaheim)

An α 1 protease inhibitor which is sensitive to increased temperatures and which migrates in the same position as the normal M1 phenotype, has been identified. The sensitivity of the trypsin inhibitory capacity is an inherited characteristic, which is enhanced by acid conditions, as well as conditions during isoelectric focusing. Loss of the serum trypsin inhibitory capacity is not parallel to that of the serum elastase capacity, suggesting that the inhibitory sites for these pro teases are oriented differently within the α 1 protease inhibitor molecule.

Purification and characterization of a glycoprotein, from normal human liver, which has the same molecular weight and chemical properties as plasma [formula omitted

An α 1- mantitrypsin-like material has been purified to homogeneity from the soluble fraction of normal human liver by procedures adapted from those employed for plasma α 1- antrypsin . The liver material, in contrast to a previous report 1 has the same molecular weight as the corresponding normal plasma α 1- antrypsin . The subunit structure, immunoelectrophoretic and immunological properties of the liver glycoprotein are identical to those of normal plasma α 1- antrypsin . Amino acid and carbohydrate compositions of the liver material are similar to those of α 1- antrypsin obtained from the plasma. The α 1- antrypsin-like material has also been isolated and purified from the microsomal fraction of liver It has the same molecular weight and immunological properties as glycoprotein obtained from the cytosol. Although inhibitors of lysosmal proteases were added during the homogenization of the liver, the purified glycoprotein is devoid of trypsin-inhibitory capacity. The loss of inhibitory activity could be due to extensive cellular autolysis before autopsy.

Development of an Animal Model of Functional Alpha1-Antiprotease Deficiency

Acanine model of functional alpha1-antiprotease (A1-AP) deficiency has been developed using intravenous administration of chloramine-T (CT), a mild oxidizing agent to which A1-AP is particularly sensitive. The dose was 300 mg in saline solution three times a week. Precautions were taken to minimize possible toxic side effects of hemolytic anemia and methemoglobulinernia by treatment with methylene blue and vitamin E. Serum samples, taken regularly over a six-week period, were tested for elastase inhibitory capacity (EIC), trypsin inhibitory capacity (TIC) and total immunologically active A1-AP. Bronchial lavage, using fiberoptic bronchoscopy, was performed twice on each animal and analyzed for EIC, TIC and differential cell count. The serum EIC was reduced to 30 percent of control values within one week after CT treatment was initiated and remained reduced over the remaining six weeks of CT exposure. The TIC was only marginally affected, being reduced to 80 percent of normal. The unique response of the EIC suggested a specific binding site of elastase to A1-AP which was markedly affected by CT and a trypsin binding site which was relatively unaffected. These findings were supported by in vitro studies. The total serum A1-AP remained normal as judged by rocket immunoelectrophoresis. The alteration in EIC was reflected in the lavage fluid even though the immunologically active A1-AP was comparable to a control animal. Cessation of CT treatment for ten days caused restoration of normal TIC and EIC levels in the serum. Differential count of the lavage fluid showed an increase of lymphocytes and a decrease of macrophages as compared to the control lavages. Morphologic examination of lungs from the CT treated dogs showed considerable dilatation of the terminal airways with focal destruction of alveolar septa. Therefore, this animal model suggests that loss of the elastase inhibitory site on Ai-AP can lead to spontaneous development of pulmonary changes associated with emphysema and may be a satisfactory model for study of the human A1-AP deficiency state.

Danazol-Induced Elevation of Serum Alpha1-Antitrypsin in Individuals with Severe Deficiency of this Antiprotease

The concept of a central role for endogenous, neutrophil-associated proteases in the pathogenesis of pulmonary emphysema resulted from recognition of the association of panacinar emphysema with the hereditary deficiency of the serum antiprotease, alpha1-antitrypsin (α1AT). Those α1AT phenotypes at greatest risk are those associated with serum α1AT levels <40 percent of normal levels (PiZ, PiMDuarteZ, and PiSZ). To assess the capacity of a potent anabolic agent to increase serum α1AT levels in these three at-risk groups, eight individuals with pronounced serum deficiency of α1AT (six PiZ, one PiMDuarteZ, one PiSZ) were studied during a 30-day therapeutic trial of danazol (600 mg, po, qd). The six PiZ and one PiMDuarteZ individual had evidence of advanced pulmonary emphysema (mean ± SEM, percent predicted: VC 63 ± 10, TLC 93 ± 8, FEV1 26 ± 5, RV/TLC 150 ± 20, DLco 32 ± 3) and a mean age of 47. The 19-year-old patient with PiSZ was asymptomatic and had normal physiology. During the danazol trial, mean serum α1AT levels of the six PiZ individuals increased from pretreatment levels of 32 ± 4 to 30-day levels of 44 ± 2 mg/dl (P < 0.04). Serum at AT levels in the PiMDuarteZ patient increased from 20 to 37 mg/dl, and in the PiSZ individual from 83 to 160 mg/dl. Bidirectional immunoelectrophoresis revealed that the serum α1AT increment in all patients occurred only in the α1 region; there was no evidence of protease-antiprotease complex formation. Isoelectric focusing and crossed antigen-antibody electrophoresis demonstrated no significant danazol-induced alterations in the micro-heterogeneity of the serum α1AT. Danazol increased the functional trypsin inhibitory capacity of serum in these patients by a mean of 64 ± 15 percent. These findings demonstrate that danazol increases functional and antigenic serum levels of α1AT in patients with severe α1AT deficiency. Although long term efficacy and toxicity studies need to be done, danazol therapy may represent one approach to bringing α1AT deficient individuals closer to the threshold for protease-antiprotease homeostasis.