TlIROMI~OS1S
R IISlihRCll
Votumc
10,
Pngcs
337-317. Pcrgnmon
Press,
1977.Printccl inCt.ISritnin.
PRESENCE OF A LOW MOLECULAR WEIGHT PLASMA FIBRINOGEN STIMULATORAND INHIBITOR IN HUMAN URINE Stephen H. Wentland, Timothy H. Carlson, BruceD. Leonard, Demerick C. Fradl and E. B. Reeve University of Colorado 14dical Center Denver, Colorado 80262 USA
(Received 5.l..l.977.
Accepted by Editor M.I. Barnhart)
ABSTRACT When male human urine was fractionatedby butanol extraction and cation-exchangechromatography,a substance lowering plasma fibrinogen levels (in rabbits) was separated from a substance elevating fibrinogen levels. When the urine was fractionatedby ethanol precipitationand gel permeation chromatography,these fibrinogen elevating and depressing substanceswere shown to have low molecular ’ weight ( < 1000 daltons). In still other fractionationprocedures, the two substanceswere found to be interconvertible. This pair of elevating and depressing substancesmay be unreported regulatorsof fibrinogen levels, and may be part of a new hormonal system of regulation.
INTRODUCTION It is well known that injury (e.g. inflannnation, tissue damage) results in increased fibrinogen synthesis (1). This laboratoryhas been concerned with the regulation of fibrinogenmetabolism and earlier studies showed that pharmacologicaldoses (ca. 50 IU) of ACTH (2) and milligram doses of various prostaglandins (3, 4, 576) stimulate fibrinogen synthesis. Using ACTH, the kinetics of fibrinogen stimulationhave been studied (7). In addition to investigatingknown, well-defined compounds,we have searched biological fluids for other components that influence fibrinogenmetabolism. We now wish to report evidence for the presence of substances in male human urine which raise and lower plasma fibrinogenlevels in rabbits. A preliminary account of this work was reported elsewhere (8).
MATERIALS AND METHODS All chemicals used were reagent grade. Ultrafilterswere obtained from Amicon Corp., Lexington,MA, and all chromatographicresins were obtained
337
338
FIBRINOGEN SYNTHESIS REGULATORS
Vol.lO,No.3
from Bio-Rad Laboratories,Richmond, CA. Cellulose sheets for thin-layer chromatographywere obtained from Eastman Kodak, Rochester,NY. The assay used was described previously (7) and consists of dissolving the substance to be tested in 20 ml physiologicalsaline, and infusing the solution at a constant rate into the marginal ear vein of the rabbit over 3 hr. Blood was collected immediatelybefore and 24 hours after the infusion, and fibrinogen concentrations,$, in the plasma samples were measured by a radioisotopedilution technique. The 24 hour plasma fibrinogenconcentration was corrected to constant plasma volume to give the corrected plasma fibrinogen concentration,$124,as described elsewhere (9). A$, the change in plasma fibrinogen concentrationwas given by $24-$. where $. is initial plasma fibrinogen concentration. The average increase in plasma fibrinogen,resulting from the infusion of 25 saline controls was .?47 mglml, SD .222. Stimulating fractions were defined as those which gave fibrinogenincreases greater than 0.791 mg/ml (the mean control increase plus two standard deviations),while fibrinogen inhibiting fractionswere defined as those which gave fibrinogen decreases falling below -0.097 mg/ml (the mean control increase minus two standard deviations). Since bacteria and bacterial endotoxins have been shown to elevate fibrinogen levels (1, lo), care was taken to avoid contaminationin all procedures used. All heat-resistantapparatus was autoclaved and all other apparatus was washed well with sterile solutions before use. Buffers were prepared from autoclaved distilledwater, glacial acetic acid and concentrated ammonium hydroxide, and were found not to cause significantchanges in fibrinogen levels when lyophilizedsamples were inf6sed into rabbits. Human male urine was collected in sterile containersand processed within two hours after collectionby either of two methods: In the first method, the urine was first acidified with concentratedHCl to pH 1.5, then saturated with salt, and finally extracted with an equal volume of E-butanol in small portions. The butanol extracts were neutralizedwith concentrated sodium hydroxide, and then concentratedunder reduced pressure at 37O, yielding a light-to-darkbrown solid we call "butanol extracted material". In the second method, the urine was diluted with three volumes of ethanol (95%) at 5O, and the resultingmixture was stored at 4O for 24-48 hours. The precipitate,obtained by decantationand centrifugation,was then extracted with cold physiologicalsaline using a Waring blender. The resulting extract was continuouslydialyzed against cold tap water for three hours, and then lyophilized,yielding a tan solid we call "ethanol precipitated material". We reported a preliminary account of this method previously (11). Ultrafiltrationwas typically carried out in a 60 ml Amicon Ultrafiltration cell, using Amicon UM type filters. In order to remove the protective film of glycerol, the filters were soaked in sterile water for 3 hours, with replacementof the water every hour. To prevent bacterial contamination, the filters were stored in 6 M urea containing 0.1% sodium azide. Before use, they were washed well with buffer, and then flushed with 20 ml buffer. The material to be ultrafilteredwas dissolved in 50 ml buffer and passed through the filter under 50 psig nitrogen. Filtrationproceeded until the volume in the cell was reduced to 3 ml. Filtrationwas then stopped, giving a 3 ml retentate and a 47 ml filtrate. If the filtrate was to be further fractionatedby a filter with a lower molecular weight cutoff, it was then passed through the filter as above, giving a 3 ml retentate and a 45 ml filtrate.
Vol.l.O,No.3
FIBRINOGEN SYNTHESIS REGULATORS
339
Gel-permeation resins (Bio-Gel P-Z) were equilibrated with buffer, separated from'fines, and autoclaved before use. Ion-exchange resins (CMcellulose, DRAE cellulose, Bio-Rex 70) were first washed with dilute acid and base, separated from fines, equilibrated with starting buffer, and then autoclaved. All chromatographic columns were flushed overnight with sterile buffers before being washed. Thin-layer chromatography was carried out using plastic-backed sheets of cellulose, which were eluted several times with sterile buffer before use. The plates were dried in a dust-free environment and then spotted with a concentrated solution of the sample, using a gentle stream of nitrogen to facilitate evaporation. The eluting solvent was the upper layer obtained by mixing 10 parts sterile H20, 10 parts n-butanol, and 2 parts glacial acetic acid, shaking, and letting the layers separate. After elution, the plate was cut into seven sections, the adsorbant in each section was scraped off the backing, and was then extracted 3 times with 5 ml 2.0 M ammonium acetate buffer In all the above steps sterile technique was used. The buffer extracts were finally lyophilized and assayed as described above. All solutions to be analyzed in the rabbit assay were prepared with sterile physiological saline solutions and were cultured to check for bacterial contamination. Since endotoxins have been shown to be pyrogenic, their presence or absence in active fractions was assayed using the pyrogen test described in the U.S. Pharmacopeia (12). This consists of rapidly injecting the material to be tested into the marginal ear vein of the rabbit, and observing the rises in temperature (if any) after the lst, Znd, and 3rd hour. If the sum of the three temperature rises was less than 40, the fraction was taken to be free of pyrogen.
RESULTS Ultrafiltration Studies Our first experiments involved the fractionation of the ethanol precipitated material on the basis of molecular weight. The ethanol precipitated material from 500 ml urine was dissolved in 0.1 M ammonium acetate buffer, pH 8.0 and filtered through a series of Amicon ultrafiltration membranes having different molecular weight cutoffs. The resulting fractions were lyophilized, dissolved in physiological saline and infused as described above. Results are shown in Table 1. Three fractions were obtained, the first containing substances with molecular weights greater than 10,000; the second, substances with molecular weights between 10,000 and 1,000; and the third, substances with molecular weights less than 1,000. When assayed, the first and third fractions showed stimulatory activity, while the second showed no activity at all. Both active fractions were tested for pyrogens. While the first fraction gave a positive response, the third fraction gave a negative response. Thus, we have obtained two stimulatory substances having very different molecular weights. While the substance of higher molecular weight may be contaminated with bacterial pyrogens, the substance of lower molecular weight is free from this kind of contamination. In subsequent runs the low molecular weight product was obtained, but not consistently. Several modifications of the ultrafiltration procedure
Vol.lO,No.3
FIBRINOGEN SYNTHESIS REGULATORS
340
TABLE I Ultrafiltrationof Ethanol PrecipitatedMaterial
Molecular Weight Cutoff of Ultrafilter
A$ (mdml)
Retentate
MW > 10,000
1.05
Retentate
MW >
1,000
.07
Filtrate
MW
R IISlihRCll
Votumc
10,
Pngcs
337-317. Pcrgnmon
Press,
1977.Printccl inCt.ISritnin.
PRESENCE OF A LOW MOLECULAR WEIGHT PLASMA FIBRINOGEN STIMULATORAND INHIBITOR IN HUMAN URINE Stephen H. Wentland, Timothy H. Carlson, BruceD. Leonard, Demerick C. Fradl and E. B. Reeve University of Colorado 14dical Center Denver, Colorado 80262 USA
(Received 5.l..l.977.
Accepted by Editor M.I. Barnhart)
ABSTRACT When male human urine was fractionatedby butanol extraction and cation-exchangechromatography,a substance lowering plasma fibrinogen levels (in rabbits) was separated from a substance elevating fibrinogen levels. When the urine was fractionatedby ethanol precipitationand gel permeation chromatography,these fibrinogen elevating and depressing substanceswere shown to have low molecular ’ weight ( < 1000 daltons). In still other fractionationprocedures, the two substanceswere found to be interconvertible. This pair of elevating and depressing substancesmay be unreported regulatorsof fibrinogen levels, and may be part of a new hormonal system of regulation.
INTRODUCTION It is well known that injury (e.g. inflannnation, tissue damage) results in increased fibrinogen synthesis (1). This laboratoryhas been concerned with the regulation of fibrinogenmetabolism and earlier studies showed that pharmacologicaldoses (ca. 50 IU) of ACTH (2) and milligram doses of various prostaglandins (3, 4, 576) stimulate fibrinogen synthesis. Using ACTH, the kinetics of fibrinogen stimulationhave been studied (7). In addition to investigatingknown, well-defined compounds,we have searched biological fluids for other components that influence fibrinogenmetabolism. We now wish to report evidence for the presence of substances in male human urine which raise and lower plasma fibrinogenlevels in rabbits. A preliminary account of this work was reported elsewhere (8).
MATERIALS AND METHODS All chemicals used were reagent grade. Ultrafilterswere obtained from Amicon Corp., Lexington,MA, and all chromatographicresins were obtained
337
338
FIBRINOGEN SYNTHESIS REGULATORS
Vol.lO,No.3
from Bio-Rad Laboratories,Richmond, CA. Cellulose sheets for thin-layer chromatographywere obtained from Eastman Kodak, Rochester,NY. The assay used was described previously (7) and consists of dissolving the substance to be tested in 20 ml physiologicalsaline, and infusing the solution at a constant rate into the marginal ear vein of the rabbit over 3 hr. Blood was collected immediatelybefore and 24 hours after the infusion, and fibrinogen concentrations,$, in the plasma samples were measured by a radioisotopedilution technique. The 24 hour plasma fibrinogenconcentration was corrected to constant plasma volume to give the corrected plasma fibrinogen concentration,$124,as described elsewhere (9). A$, the change in plasma fibrinogen concentrationwas given by $24-$. where $. is initial plasma fibrinogen concentration. The average increase in plasma fibrinogen,resulting from the infusion of 25 saline controls was .?47 mglml, SD .222. Stimulating fractions were defined as those which gave fibrinogenincreases greater than 0.791 mg/ml (the mean control increase plus two standard deviations),while fibrinogen inhibiting fractionswere defined as those which gave fibrinogen decreases falling below -0.097 mg/ml (the mean control increase minus two standard deviations). Since bacteria and bacterial endotoxins have been shown to elevate fibrinogen levels (1, lo), care was taken to avoid contaminationin all procedures used. All heat-resistantapparatus was autoclaved and all other apparatus was washed well with sterile solutions before use. Buffers were prepared from autoclaved distilledwater, glacial acetic acid and concentrated ammonium hydroxide, and were found not to cause significantchanges in fibrinogen levels when lyophilizedsamples were inf6sed into rabbits. Human male urine was collected in sterile containersand processed within two hours after collectionby either of two methods: In the first method, the urine was first acidified with concentratedHCl to pH 1.5, then saturated with salt, and finally extracted with an equal volume of E-butanol in small portions. The butanol extracts were neutralizedwith concentrated sodium hydroxide, and then concentratedunder reduced pressure at 37O, yielding a light-to-darkbrown solid we call "butanol extracted material". In the second method, the urine was diluted with three volumes of ethanol (95%) at 5O, and the resultingmixture was stored at 4O for 24-48 hours. The precipitate,obtained by decantationand centrifugation,was then extracted with cold physiologicalsaline using a Waring blender. The resulting extract was continuouslydialyzed against cold tap water for three hours, and then lyophilized,yielding a tan solid we call "ethanol precipitated material". We reported a preliminary account of this method previously (11). Ultrafiltrationwas typically carried out in a 60 ml Amicon Ultrafiltration cell, using Amicon UM type filters. In order to remove the protective film of glycerol, the filters were soaked in sterile water for 3 hours, with replacementof the water every hour. To prevent bacterial contamination, the filters were stored in 6 M urea containing 0.1% sodium azide. Before use, they were washed well with buffer, and then flushed with 20 ml buffer. The material to be ultrafilteredwas dissolved in 50 ml buffer and passed through the filter under 50 psig nitrogen. Filtrationproceeded until the volume in the cell was reduced to 3 ml. Filtrationwas then stopped, giving a 3 ml retentate and a 47 ml filtrate. If the filtrate was to be further fractionatedby a filter with a lower molecular weight cutoff, it was then passed through the filter as above, giving a 3 ml retentate and a 45 ml filtrate.
Vol.l.O,No.3
FIBRINOGEN SYNTHESIS REGULATORS
339
Gel-permeation resins (Bio-Gel P-Z) were equilibrated with buffer, separated from'fines, and autoclaved before use. Ion-exchange resins (CMcellulose, DRAE cellulose, Bio-Rex 70) were first washed with dilute acid and base, separated from fines, equilibrated with starting buffer, and then autoclaved. All chromatographic columns were flushed overnight with sterile buffers before being washed. Thin-layer chromatography was carried out using plastic-backed sheets of cellulose, which were eluted several times with sterile buffer before use. The plates were dried in a dust-free environment and then spotted with a concentrated solution of the sample, using a gentle stream of nitrogen to facilitate evaporation. The eluting solvent was the upper layer obtained by mixing 10 parts sterile H20, 10 parts n-butanol, and 2 parts glacial acetic acid, shaking, and letting the layers separate. After elution, the plate was cut into seven sections, the adsorbant in each section was scraped off the backing, and was then extracted 3 times with 5 ml 2.0 M ammonium acetate buffer In all the above steps sterile technique was used. The buffer extracts were finally lyophilized and assayed as described above. All solutions to be analyzed in the rabbit assay were prepared with sterile physiological saline solutions and were cultured to check for bacterial contamination. Since endotoxins have been shown to be pyrogenic, their presence or absence in active fractions was assayed using the pyrogen test described in the U.S. Pharmacopeia (12). This consists of rapidly injecting the material to be tested into the marginal ear vein of the rabbit, and observing the rises in temperature (if any) after the lst, Znd, and 3rd hour. If the sum of the three temperature rises was less than 40, the fraction was taken to be free of pyrogen.
RESULTS Ultrafiltration Studies Our first experiments involved the fractionation of the ethanol precipitated material on the basis of molecular weight. The ethanol precipitated material from 500 ml urine was dissolved in 0.1 M ammonium acetate buffer, pH 8.0 and filtered through a series of Amicon ultrafiltration membranes having different molecular weight cutoffs. The resulting fractions were lyophilized, dissolved in physiological saline and infused as described above. Results are shown in Table 1. Three fractions were obtained, the first containing substances with molecular weights greater than 10,000; the second, substances with molecular weights between 10,000 and 1,000; and the third, substances with molecular weights less than 1,000. When assayed, the first and third fractions showed stimulatory activity, while the second showed no activity at all. Both active fractions were tested for pyrogens. While the first fraction gave a positive response, the third fraction gave a negative response. Thus, we have obtained two stimulatory substances having very different molecular weights. While the substance of higher molecular weight may be contaminated with bacterial pyrogens, the substance of lower molecular weight is free from this kind of contamination. In subsequent runs the low molecular weight product was obtained, but not consistently. Several modifications of the ultrafiltration procedure
Vol.lO,No.3
FIBRINOGEN SYNTHESIS REGULATORS
340
TABLE I Ultrafiltrationof Ethanol PrecipitatedMaterial
Molecular Weight Cutoff of Ultrafilter
A$ (mdml)
Retentate
MW > 10,000
1.05
Retentate
MW >
1,000
.07
Filtrate
MW
