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PubMed1975

Effect of vasoactive agents on intestinal oxygen consumption and blood flow in dogs.

A comparison study of several vasoconstrictor and vasodilator agents was conducted measuring changes in intestinal blood flow and oxygen consumption during 10-min periods of intra-arterial infusion. Blood flow was measured in a branch of the superior mesenteric artery of anesthetized dogs with an electromagnetic blood flow meter, and the arteriovenous oxygen content difference across the gut segment was determined photometrically. Vasopressin (4 x 10(-3) and 7x 10(-4) U/kg-min) diminished blood flow 60 and 28% and reduced oxygen consumption 54 and 22%, respectively (all P less than 0.001). In a dose which did not lower blood flow, vasopressin still caused a decline in oxygen consumption (P less than 0.01). Epinephrine (5 x 10(-2) mug/kg-min) decreased blood flow 19% (P less than 0.001) but did not reduce oxygen consumption. After beta-adrenergic blockade, however, the same dose of epinephrine decreased blood flow 41% and oxygen consumption 33% (both P less than 0.001). Responses to angiotension II, calcium chloride, and prostaglandin F2alpha resembled effects of vasopressin rather than those of epinephrine, namely decreased blood flow and decreased oxygen consumption. The vasodilator agents, prostaglandin E1, is isoproterenol, and histamine, increased (P less than 0.001) both blood flow (130, 80, and 98%, respectively) and oxygen consumption (98, 64, and 70%, respectively). Vasopressin, angiotensin II, calcium chloride, and prostaglandin F2alpha appear to contract arteriolar and precapillary sphincteric smooth muscle indiscriminately to evoke both intestinal ischemia and hypoxia. Epinephrine is the exceptional constrictor in this case, producing diminished blood flow without a reduction in oxygen uptake.

Animals↗
PubMed1996

Calcium-activated chloride currents in primary cultures of rabbit distal convoluted tubule.

Chloride (Cl-) conductances were studied in primary cultures of rabbit distal convoluted tubule (very early distal "bright" convoluted tubule, DCTb) by the whole cell patch-clamp technique. We identified a Cl- current activated by 2 microM extracellular ionomycin. The kinetics of the macroscopic current were time dependent for depolarizing potentials with a slow developing component. The steady state current presented outward rectification, and the ion selectivity sequence was I- > Br- > > Cl > glutamate. The current was inhibited by 0.1 mM 5-nitro-2-(3-phenylpropyl-amino)benzoic acid, 1 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, and 1 mM diphenylamine-2-carboxylate. To identify the location of the Cl- conductance, 6-methoxy-N-(3-sulfopropyl)quinolinium fluorescence experiments were carried out in confluent cultures developed on collagen-coated permeable filters. Cl- removal from the apical solution induced a Cl- efflux that was stimulated by 10 microM forskolin. Forskolin had no effect on the basolateral Cl- permeability Cl- substitution in the basolateral solution induced an efflux stimulated by 2 microM ionomycin or 50 microM extracellular ATP Ionomycin had no effect on the apical Cl- fluxes. Thus cultured DCTb cells exhibit Ca(2+)-activated Cl- channels located in the basolateral membrane. This Cl- permeability was active at a resting membrane potential and could participate in the Cl- reabsorption across the DCTb in control conditions.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMedDate not supplied

[The anti-arrhythmia activity of quinazopyrine].

The antiarrhythmic activity of quinasopirine was studied in the experiments on rats with the use of models of calcium chloride- and aconitine-induced arrhythmias, disorders of cardiac rhythm in myocardial infarction produced by isadrine and pituitrin and also in the experiments on cats in arrhythmias caused by electric stimulation of the myocardium, postinfarction and reperfusion arrhythmias. Quinasopirine exhibits the antiarrhythmic effect being superior to that of anapriline and novocainamide in arrhythmias induced by calcium chloride and aconitine. In other types of the cardiac rhythm disorder its activity is comparable with that of ethmosine, obsidane and cordarone. Quinasopirine reduces automatism and contractile function of the myocardium.

Animals↗
PubMed1985

Do calories, osmolality, or calcium determine gastric emptying?

To determine whether calories, osmolality, or calcium mediate gastric emptying we employed a standardized radioactive meal in 10 normal human volunteers. A variety of simple and complex sugars, medium-chain fatty acid (MCFA), pectin, and gluten were dissolved in water and ingested with the test meal. The studies were also performed with calcium chloride, EDTA, and an equimolar combination of these chemicals. Results of gastric emptying showed that incremental glucose produced an increase in emptying time with a tendency for emptying time to show a proportionally greater delay with increasing glucose concentrations. Fructose and polyhexose had similar effects to glucose. Pentoses (xylose and arabanose) markedly prolonged gastric emptying when compared with the same amount of glucose. The effect of sucrose and gluten on gastric emptying did not significantly differ from controls. Twenty-five grams MCFA had an effect similar to 50 g glucose. Pectin, a complex carbohydrate, produced a varied effect in different individuals. There was no obvious relationship between osmolality and gastric emptying. Calcium chloride and EDTA prolonged gastric emptying, but the equimolar combination gave values similar to controls. Our findings suggest 1) calories nor osmolality alone determine gastric emptying, 2) both calcium and calcium chelation with EDTA prolong gastric emptying, and 3) a specific food does not necessarily produce the same effect on gastric emptying in different individuals.

Adult↗
PubMed1979

Vitamin D dependence of in vivo calcium transport and mucosal calcium binding protein in rat large intestine.

Dependence of large intestinal calcium transport on vitamin D has been examined in vitro in colon only. The authors studied calcium fluxes in cecum and colon in vivo by perfusion with 1.6 mM calcium chloride in saline. Tracer 45Ca either was injected parenterally 24 hr before study or was added to the perfusates. For 8--10 wk after weaning, rats had been fed a rachitogenic diet; 48 and 24 hr before study, 50% of the animals were treated with 20,000 IU vitamin E2. In a separate set of animals, mucosal calcium binding protein was analyzed by the Chelex assay method. In comparison with vitamin D-deficient rats, the colon of vitamin D-treated rats showed higher lumen-to-plasma flux and lower plasma-to-lumen flux and net absorption instead of net secretion. In cecum, calcium transport was not significantly altered by vitamin D treatment. Mucosal calcium binding protein was higher in cecum than in colon in both groups and was higher in vitamin-D-treated than in vitamin D-deficient animals in both segments. The current study shows that in the rat colon calcium fluxes both into and out of the lumen as well as net transport are significantly by vitamin D treatment, but that cecal transport rates are not affected. In both cecum and colon, mucosal calcium binding protein increases with vitamin D treatment.

Animals↗
PubMed1992

Citrate anticoagulation and dialysate with reduced buffer content in chronic hemodialysis.

Anticoagulation with trisodium citrate (510 mmol/l) at a rate of 126 ml/h in combination with a calcium-free, 35 mmol/l acetate dialysate and i. v. supplementation of calcium chloride (350 mmol/l) at a rate of 22.5 ml/h has been performed in renal failure patients at risk of bleeding or actively bleeding. Short-term use-i. e. on the average four dialyses in 15 patients-showed no adverse effects or clotting phenomena. However, long-term use-i. e. at least four weeks and on average sixteen dialyses in six chronic hemodialysis patients-caused paresthesias and muscular cramps possibly due to the combination of insufficient calcium supplementation and metabolic alkalosis. To prevent metabolic alkalosis, the acetate content of the dialysate and the infusion rate of citrate were reduced to 29.5 mmol/l and 100 ml/h, respectively. To prevent clotting at this infusion rate, a calcium and magnesium-free dialysate was used and i. v. supplementation of calcium chloride (350 mmol/l) and magnesium chloride (250 mmol/l) was performed at rates of 30 ml/h and 15 ml/h, respectively. Six chronic renal failure patients were dialyzed with this regimen for an average of four months. There were no side effects or metabolic alkalosis noted. When an equimolar amount of bicarbonate replaced the acetate in the calcium and magnesium-free dialysate, side effects and metabolic alkalosis were seen within three dialyses. When the bicarbonate concentration in the dialysate was reduced to 25 mmol/l neither side effects nor metabolic alkalosis was observed. Thus citrate anticoagulation in patients on chronic hemodialysis can only be performed when the buffer content of the dialysate is reduced.

Acetates↗
PubMed1988

Renal effects of fenoldopam in refractory hypertension.

Fenoldopam, a dopamine-1 (D1) agonist, was administered by a 6-h intravenous infusion to patients with refractory hypertension [diastolic blood pressure (DBP) greater than 115 mmHg while on triple therapy] in order to achieve a fall in DBP of 30 mmHg. The evolution of blood pressure, heart rate, glomerular filtration rate (GFR), renal plasma flow (RPF), urine volume, renal excretion of sodium, potassium, chloride, calcium, uric acid, phosphate, plasma renin activity (PRA), aldosterone and prolactin were evaluated. A significant fall in blood pressure (P less than 0.01) accompanied by an increase in heart rate (P less than 0.01) was attained after 30 min. GFR and RPF increased significantly (P less than 0.01) but the filtration fraction fell. Urine volume and urinary output of sodium, potassium, chloride, calcium, uric acid and phosphate increased markedly (P less than 0.01). Meanwhile, plasma potassium fell (P less than 0.01) and the hormonal parameters showed no significant change. We concluded that in refractory hypertension fenoldopam has potent renal and systemic vasodilatory properties through which blood pressure falls. The hypotensive effect of fenoldopam is also facilitated by its marked diuretic and natriuretic properties. The absence of variations of plasma prolactin confirm the D1 selectivity of fenoldopam and the lack of increase in PRA indicates that fenoldopam blocks the renin-angiotensin-aldosterone system.

Adult↗
PubMed1984

[Abnormal electrocardiographic changes induced by hypocalcemia secondary to extracorporeal circulation].

We report hereby the case of a 20-year-old woman, whose electrocardiogram showed unusual repolarization abnormalities 36 hours after open heart surgery. They were due to pure hypocalcemia (7,1 mg/100 ml) secondary to extracorporeal circulation, were sudden in onset, transient, variable within a few minutes, and immediately suppressed by intravenous calcium chloride. Some of the changes were similar to those already reported in literature (lengthening of QT and ST segments, changes in T wave voltage but not duration) whereas other were quite unusual i.e. the extreme variability and transientness of these changes, the marked ST level alterations and the presence of wide and diphasic U waves. Intravenous administration of 2 g of calcium chloride was followed by immediate and complete normalization of the electrocardiographic tracing.

Adult↗
PubMed2003

Suppressive effect of endogenous regucalcin on protein tyrosine phosphatase activity in the nucleus of rat brain: attenuation with increasing age.

The effect of endogenous regucalcin, which is a regulatory protein of Ca2+ signaling, on protein tyrosine phosphatase activity in the nucleus of brain tissues of young and aged rats was investigated. Phosphotyrosine was used as the substrate for assay of protein tyrosine phosphatase activity. Protein tyrosine phosphatase activity in the brain nucleus of young (5 weeks old) rats was significantly increased in the presence of calcium chloride (5-50 micro M) in the enzyme reaction mixture. The increase was completely blocked by the addition of trifluoperazine (10-50 micro M), an antagonist of calmodulin, indicating that the enzyme is activated by endogenous Ca2+/ calmodulin. The addition of regucalcin (10(-4)-10(-8) M) in the enzyme reaction mixture caused a significant decrease in protein tyrosine phosphatase activity in the absence or presence of calcium chloride (20 micro M). Brain nuclear protein tyrosine phosphatase activity was significantly raised in the presence of anti-regucalcin monoclonal antibody (10-50 ng/ml) in the enzyme reaction mixture. The increase was completely prevented by the addition of regucalcin (10(-6) M). In the brain nucleus of aged (50 weeks old) rats, protein tyrosine phosphatase activity was elevated significantly as compared with that of 5 weeks old rats. The effect of anti-regucalcin monoclonal antibody in increasing the enzyme activity in the brain nucleus of aged rats was seen in the presence of 50 ng/ml of the antibody. Such an effect was not found by the antibody of 10 and 25 ng/ml. Regucalcin protein in brain nucleus was detected by Western blot analysis. This level was significantly decreased by increasing age. The present study demonstrates that endogenous regucalcin plays a suppressive role in the regulation of protein tyrosine phosphatase activity in the nucleus of rat brain, and that this regulation is attenuated with increasing age.

Aging↗
PubMed2005

A new device for in vitro evaluation of thrombogenicity.

A device to measure the time of coagulation of whole blood has been designed in order to facilitate testing of thrombogenicity of biomaterials. The principle of operation of the apparatus is to record the time taken for a sphere to fall through a sample of blood. The coagulation time is defined here as the time from collection of the blood sample to coagulation, coagulation having deemed to have occurred when the sphere is prevented from falling by the presence of the fibrin-cell network. The device was tested with homogeneous fluids of different viscosity, milk containing different amounts of rennin and calcium chloride and non anti-coagulated whole blood obtained from five volunteers. Repeat measurements made with each homogeneous fluid show that the variance in the transit time of the sphere is small and consistent with small dispersion. In contrast, the onset of coagulation in milk and blood samples was readily detected. The clotting of milk was highly dependant on the concentration of rennin and calcium chloride. The coagulation time of blood samples from five individuals ranged from 23 to 33 min but simultaneous measurements of samples from the same individual in two identical devices agreed to within +/-1 min in all cases. This device may be easily adapted for use in studies to determine the thromboresistance of biomaterials where the onset of coagulation of whole blood in contact with different materials may be readily compared.

Blood Coagulation↗
PubMedDate not supplied

Ionotropic gelation: encapsulation of indomethacin in calcium alginate gel discs.

Ionotropic gelation by divalent metal interaction was employed as an approach to design a modified release multiple-unit oral drug-delivery system. This process was achieved by crosslinking an indomethacin-sodium alginate dispersion with calcium ions to induce the spontaneous formation of indomethacin-calcium alginate gel discs. A significant part in the validation of the integrity of the system, involved a preformulatory stage for the optimization of the curing conditions and potency determination of the gel discs. A three-phase approach was developed to establish the critical curing parameters. Since curing involved crosslinking of the sodium alginate with calcium ions, an optimal concentration of calcium chloride (phase one) and crosslinking reaction-time (phase two) had to be determined. Furthermore, the third phase involved the optimization of the air-drying time of the gel discs. In phases one and two, stabilization of in vitro drug-release characteristics was used as the marker of optimal crosslinking efficiency. Phase three was based on achieving fully dried gel discs by drying to constant weight at 21 degrees C under an extractor. The study revealed that optimal crosslinking efficiency was achieved in 1%w/v calcium chloride solution for 24 h and air-dried at 21 degrees C under an extractor for 48 h. The three solvent/solution systems investigated for their ability to liberate completely the drug from the matrix system were methanol, sodium citrate (1%w/v) and phosphate buffer pH 6.2. Phosphate buffer provided optimal drug removal, in addition to its ability to induce swelling of the calcium alginate gel discs. Furthermore, drug loading also increased with the use of increasing concentrations of sodium alginate in the formulations.

Administration, Oral↗
PubMed1998

Cardiac and vascular responses after monocrotaline-induced hypertrophy in rats.

In rats, monocrotaline causes pulmonary vascular damage leading to pulmonary hypertension, right ventricular hypertrophy, and eventually heart failure. This study determined the inotropic and chronotropic responses in isolated cardiac tissues from pulmonary hypertensive rats (single treatment with monocrotaline, 105 mg/kg) to noradrenaline, forskolin, EMD 57033 (calcium sensitizer), and calcium chloride. Further, vasoconstrictor responses to noradrenaline, 5-hydroxytryptamine (5-HT), and KCl were measured in isolated pulmonary artery and thoracic aortic rings. Marked right ventricular hypertrophy was evident 4 weeks after treatment; at 6 weeks, treated rats additionally showed symptoms of severe heart failure. Pulmonary hypertension led to marked increases in pulmonary artery responses to 5-HT and to decreases in positive inotropic responses in right ventricular papillary muscles to all compounds except calcium chloride. The development of heart failure maintained or increased these changes. Positive chronotropic responses were unchanged. In the right ventricle, beta1-adrenoceptor density decreased only in heart failure; beta2-adrenoceptor density was unchanged. The densities of both beta-adrenoceptor subtypes were decreased in the lungs but increased in the liver of pulmonary hypertensive rats. The functional changes in the failing human heart are similar to those in rats with monocrotaline-induced right ventricular hypertrophy. This may be a useful model to define adequate therapy in human right ventricular failure.

Animals↗
PubMed1994

Tachycardia heart failure alters rabbit aortic smooth muscle responsiveness to angiotensin II.

The effects of heart failure on the responsiveness of aortic smooth muscle tissue to various vasoactive agents were examined. Heart failure was induced in rabbits by sustained rapid ventricular pacing (400 beats/min) for 6-7 wk. After the rabbits were killed, strips of thoracic aorta were prepared and mounted in tissue baths. Responsiveness of these aortic strips to potassium depolarization and cumulative additions of vasoactive agents was determined. Aortic strips from control and tachycardia heart failure (THF) rabbits developed similar maximum forces to stimulation by potassium depolarization (0.77 +/- 0.08 vs. 0.96 +/- 0.16 kg/cm2), calcium chloride (0.71 +/- 0.09 vs. 0.88 +/- 0.06 kg/cm2), and phenylephrine (0.90 +/- 0.08 vs. 1.14 +/- 0.09 kg/cm2). The maximum relaxation to isoproterenol was also unaffected by THF (0.08 +/- 0.02 vs. 0.07 +/- 0.01 kg/cm2). In contrast, the maximum response of aortic strips from THF rabbits to angiotensin II was significantly lower than control (0.37 +/- 0.07 vs. 0.069 +/- 0.09 kg/cm2). With regard to aortic smooth muscle sensitivity, no differences in the concentrations at which 50% of the maximal response is achieved (EC50) were observed between THF and control strips for calcium chloride (0.10 +/- 0.01 vs. 0.16 +/- 0.04 mM), isoproterenol (51.5 +/- 13.5 vs. 51.0 +/- 5.4 nM), and phenylephrine (65.2 +/- 12.3 vs. 92.5 +/- 18.0 nM). However, THF was associated with a significant increase in the EC50 value for angiotensin II response (2.03 +/- 0.25 vs. 0.58 +/- 0.05 nM). These results demonstrate that THF is associated with a specific and significant reduction in the sensitivity and maximal responsiveness of aortic smooth muscle to angiotensin II.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin II↗
PubMedDate not supplied

Gellan-based scleral implants of indomethacin: in vitro and in vivo evaluation.

Film-type scleral implants of indomethacin with gellan gum were prepared by solvent casting and evaluated for uniformities of thickness, weight, drug content, and surface pH. The effect of plasticizers like glycerol, propylene glycol (PG), and polyethylene glycol 200, and 400 on the void volume of free gellan films (placebo) was calculated from the water content of the films. The drug release from the prepared implants was determined using a static dissolution set-up developed and optimized in our laboratory. Based on the results of the void volume and initial drug release studies, glycerol and PG were selected as the plasticizers for the gellan-based implants. The morphology of the drug-free films (containing 10% and 40% of PG) and the drug-loaded films (before and after dissolution and crosslinked) was studied using scanning electron microscopy. Further, the effect of plasticizer concentration, gellan concentration, effect of crosslinking technique, and duration of crosslinking using calcium chloride on in vitro drug release characteristics were evaluated. Selected batches of the implants were subjected to pharmacodynamic studies, after scleral placement, in uveitis-induced (intravitreal injection of bovine serum albumin 50 microg/ml) rabbit eyes. The release of indomethacin from the prepared implants followed matrix diffusion kinetics with diffusion co-efficient (n) values ranging between 0.358 to 0.708 and seemed to depend on both gellan and plasticizer concentration. Surface crosslinking with 10% calcium chloride for 8 hr retarded drug release (1.42 times less than noncrosslinked implant) and was optimum. The pharmacodynamic studies showed a marked improvement in the various clinical parameters (congestion, keratitis, flare, clot, aqueous cells, and synechias) in the implanted eye compared with the control eye in the rabbits. The scleral implants survived up to 3 weeks in vivo.

Animals↗
PubMed1996

Oral sodium phosphate versus sulfate-free polyethylene glycol electrolyte lavage solution in outpatient preparation for colonoscopy: a prospective comparison.

BACKGROUND: Biochemical abnormalities induced by oral sodium phosphate and the risk of cardiac arrhythmias as potential sequelae have yet to be investigated. METHODS: We studied 98 outpatients scheduled to undergo diagnostic colonoscopy and prospectively randomized them to receive oral sodium phosphate or sulfate-free polyethylene glycol electrolyte lavage solution (SF-PEG-ELS) as recommended by the manufacturers. RESULTS: Forty-nine patients received sodium phosphate and 49 received SF-PEG-ELS. There was no significant difference in tolerance or quality of preparation as judged by blinded endoscopists. Significant changes in serum sodium, potassium, chloride, calcium, ionized calcium, and inorganic phosphorus levels were noted following sodium phosphate preparation when compared to values before preparation. A significantly greater number of patients who received sodium phosphate preparation had serum potassium and ionized calcium levels that fell into the abnormal range. Ambulatory electrocardiogram monitors placed 24 hours before the preparation and removed after colonoscopy showed no increase in ventricular premature contractions or other serious arrhythmias in either group during preparation or colonoscopy. CONCLUSIONS: (1) Sodium phosphate and SF-PEG-ELS are equally well tolerated and effective in preparation for outpatient colonoscopy, and (2) sodium phosphate preparation at the recommended dose causes significant alterations in serum sodium, potassium, chloride, calcium, ionized calcium, and phosphorus levels.

Adult↗
PubMed2001

Radionuclide fluxes at a plant manufacturing dicalcium phosphate for domestic animals.

We have studied a phosphate rock plant which produces dicalcium phosphate (DCP), used as a source of calcium and phosphorus for domestic animals. A by-product in the manufacturing process is calcium chloride which is used in the oil industry, the food industry and as road-salt. The objectives of our study were to describe the fluxes of radionuclides from the 238U decay series and to estimate the radiation doses to workers at the plant. The radionuclides in the phosphate rock were found to be in secular radioactive equilibrium with 238U, with an average activity concentration of 837 Bq kg-1. Separation and concentration processes were observed at different stages in the plant. Most of the 226Ra was found in the calcium chloride, while the major part of the 238U, about 950 Bq kg-1, was found in the dicalcium phosphate. The annual occupational effective dose to the workers was found to be below the 1 mSv limit recommended by ICRP (1991a) for the public. This study has shown a good example of an important non-nuclear industry with a high input of natural radionuclides with several conceivable pathways to man.

Animal Feed↗
PubMed2001

226Ra bioavailability to plants at the Urgeiriça uranium mill tailings site.

Large amounts of solid wastes (tailings) resulting from the exploitation and treatment of uranium ore at the Urgeiriça mine (north of Portugal) have been accumulated in dams (tailing ponds). To reduce the dispersion of natural radionuclides into the environment, some dams were revegetated with eucalyptus (Eucalyptus globolus) and pines (Pinus pinea). Besides these plants, some shrubs (Cytisus spp.) are growing in some of the dams. The objective of this study is to determine the 226Ra bioavailability from uranium mill tailings by quantifying the total and available fraction of radium in the tailings and to estimate its transfer to plants growing on the tailing piles. Plant and tailing samples were randomly collected and the activity concentration of 226Ra in plants (aerial part and roots) and tailings was measured by gamma-spectrometry. The exchangeable fraction of radium in tailings was quantified using one single step extraction with 1 mol dm-3 ammonium acetate (pH = 7) or 1 mol dm-3 calcium chloride solutions. The results obtained for 226Ra uptake by plants show that 226Ra concentration ratios for eucalyptus and pines decrease at low 226Ra concentrations in the tailings and appear relatively constant at higher radium concentrations. For shrubs, the concentration ratios increase at higher 226Ra solid waste concentrations approaching a saturation value. Percentage values of 16.0 +/- 8.3 and 12.9 +/- 8.9, for the fraction of radium extracted from the tailings, using 1 mol dm-3 ammonium acetate or calcium chloride solutions, respectively, were obtained. The 226Ra concentration ratios determined on the basis of exchangeable radium are one order of magnitude higher than those based on total radium. It can be concluded that, at a 95% confidence level, more consistent 226Ra concentration ratios were obtained when calculated on the basis of available radium than when total radium was considered, for all the dams.

Biological Availability↗
PubMed1992

Purification of sufficiently gamma-carboxylated recombinant protein C and its derivatives. Calcium-dependent affinity shift in immunoaffinity and ion-exchange chromatography.

Protein C, which is an important anti-thrombotic factor in the blood coagulation cascade, undergoes several post-translational modifications. gamma-Carboxylation on nine glutamic acid residues at the N-terminal region of the light chain [gamma-carboxylated glutamic acid (Gla) domain] is considered to be critical for full anti-clotting activity. It is also known that when recombinant protein C is expressed in animal cells this particular modification is often lost. We were successful in preparing a monoclonal antibody (PC01) which distinguishes the sufficiently gamma-carboxylated protein from the rest by its specific affinity for the Ca(2+)-induced conformational change of the former, and thereby developed a simple process of purifying sufficiently gamma-carboxylated protein C. Culture supernatant of Chinese hamster ovary cell transformants was first applied to Q-Sepharose and recombinant protein C was partially purified. It was then loaded onto a PC01 affinity column in the presence of 5 mM calcium chloride. Sufficiently gamma-carboxylated protein C was retained while insufficient-carboxylated protein C quickly passed through. The former was eluted with 5 mM EDTA efficiently and with high purity, contained eight Gla units per molecule, and had similar anti-clotting activity. The flow-through was relatively impure protein C which contained five Gla units per molecule and showed limited anti-clotting activity. We extended the application of the Ca(2+)-induced conformational change to conventional ion-exchange chromatography. The sufficiently gamma-carboxylated protein C was found to elute earlier in the salt gradient from an anion-exchange column in the presence of 5 mM calcium chloride being fully separated from the insufficiently carboxylated protein C.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗