Measurement of Urinary Desmosine by Isotope Dilution and High Performance Liquid Chromatography Correlation between Elastase-induced Air-Space Enlargement in the Hamster and Elevation of Urinary Desmosine 1 - 4
PHILLIP J. STONE, JULIANNE BRYAN-RHADFI, EDGAR C. WCEY, DAVID E. CICCOLELLA GEORGE CROMBIE, BARBARA FARIS, GORDON L. SNIDER, and CARL FRANZBLAU
Introduction
It is believed that emphysema in smokers as wellas in persons deficient in alphaI-protease inhibitor is caused by elastic fiber destruction (1). The crosslink amino acids, desmosine (DES) and isodesmosine (IDES), are unique to elastin. Increased amounts of these marker amino acids have been reported in the urine of experimental animals treated intratracheally with elastase (2,3). However, investigators have not found elevated levelsof DES in the urine of smokers with chronic obstructive pulmonary disease as compared with normal smokers or nonsmokers (4, 5). Furthermore, urinary DES was similar in normal subjects, subjects with the homozygous (PiZZ) form of alphaI-protease inhibitor deficiency linked to emphysema, and subjects with interstitial lung diseases; nor were these markers elevated in PiZZ children when compared with age-matched control children (6). The above-mentioned studies were all done using a radioimmunoassay (RIA). Reports in the literature (7, 8) havesuggested that interfering substances in the urine may spuriously elevate DES values measured by RIA or enzyme-linked immunosorbent assay (ELISA). Weconsidered that such spurious elevation of urinary DES values might have masked differences between persons with and without emphysema. This led us to develop a newmethod of assaying DES and IDES in the urine. In our method most contaminants are separated from the DES and IDES by gel filtration. High performance liquid chromatography (HPLC) is used to separate DES and IDES; the eluted material is monitored by light absorption at 275 nm. An isotope dilution approach is used to quantify recovery of the marker amino acids and, thereby, to accurately calculate the 284
SUMMARY The accuracy of methods employed to measure the elastin-specific croaallnks, desmosine (DES) and Isodesmoslne (IDES), has been called Into question because contaminants In the urine may cause elevated values. In the present study urine samples were spiked with a known amount of [ 14C]DES and refluxed In 6 N HCI. Sephadex G-15chromatography of the hydrolyzed urine wes employed to remove contaminants. DES and IDES were quantified by high performance liquid chromatography (HPLC) as well as by amino acid analysis. The amount of Isotope recovered was used to determine losses during the overall procedure and the Isotope dilution to calculate the amounts of endogenous DES and IDES originally present In the urine. Because similar values were obtained by both methods, the more rapid HPLC method was used for II succeeding analyses. In one experiment, the DES amounts In urine collected from hamsters for 3 days after Intratracheal treatment with human neutrophil elastase (300 J.1g) or porcine pancreatic elastase (300 J.1g) were 0.212 ± 0.012(mean ± SEM, two measurements on a single pool) and 0.816 ± 0.005 (two measurements) J.1g per hamster per day, respectively. Urine from control hamsters had a mean value of 0.074 ± 0.008 (eight measurements) J.1g per hamster per day. The HNE- and PPE-treated hamaters had mean linear Intercept values of 119and 159% of control values, respectively, giving a positive correlation between Increase In airspace size and .Ievatlon of urinary DES. For eight normal men 25 to 68 yr of age who were never-smokers, mean ± SEM 24-h urinary values were 13.3 ± 2.3 J.1g DES and 11.3 ± 1.7 ~ IDES; DES/creatinine and IDES/creatinine ratios were 6.5 ± 0.7 ~ and 5.5 ± 0.4 J.1g per g creatinine, respectively. Values for subjects remained constant when measured on urine collected on different days. Hamster and human DES values are as much as 2D-fold and 1D-fold lower, respectively, than previously reported values. This assay shOUld prove useful In studying conditions when DES excretion may be elevated. AM REV RESPIR DIS 1111; 144:284-280
total DES and IDES present in the original sample. Methods
by liquid scintillation spectrometry. Appropriate quench-correction factors were used. The amino acid composition of the residue was similar to that reported by us earlier (11).
Preparation of {J4C}DES and f"CjIDES (14C]DES and (14C]lDES were isolated from neonatal rat smooth muscle cell cultures as previously described (9). Briefly, T-75 flasks containing l-wk-old first passage cells were pulsed for 24 h in the presence of serum by addition of [14C(U)]lysine (20 JjCi per flask) (New England Nuclear, Boston, MA), after which the medium was poured off and the cultures were refed. Five weeks later the cell layers were harvested by scraping and then homogenized. The elastin was isolated by hot alkali treatment (10). The residue was hydrolyzed in 6 N HCI at 110 0 C for 24 h in vacuo. An aliquot of the hydrolysate was loaded on a Beckman Model 119CL amino acid analyzer (Beckman Instruments, Fullerton, CA), and the eluted material was collected in 0.5min fractions and assessed for radioactivity
(Received in original form December 10, 1990) 1 From the Department of Biochemistry and the Pulmonary Center, Boston University School of Medicine, and the Pulmonary Section, Boston Veterans Administration Medical Center, Boston, Massachusetts. 1 Supported by Grant HL-19717 from the National Heart, Lung, and Blood Institute and by the Veterans Administration Research Service. 3 Presented in part at a meeting entitled "Pulmonary Emphysema: the Rationale for Therapeutic Intervention," sponsored by the New YorkAcademy of Sciences and the American Thoracic Society, Orlando, Florida, May 16-18, 1990. 4 Correspondence and requests for reprints should be addressed to Dr. Phillip J. Stone, Biochemistry Department, Boston University School of Medicine, 80 East Concord St., Boston, MA 02118.
MEASUREMENT OF DESMOSINE BY ISOTOPE DIWTION/HPLC
DES and IDES eluted 4 min apart (99 and 95 min, respectively). Four lysinecolor equivalents were used to calculate the amount of DES and IDES present. DES plus IDES composed 0.20070 of the amino acids present in the elastin. The specific radioactivity of DES and IDES were 776 and 836 cpm/nmol (80% efficiency) using Ultima Gold scintillation cocktail (Packard Instruments, Downers Grove, IL). These values include a 17% correction for the loss from both DES and IDES of four radiolabeled carbon atoms as carbon dioxide upon reaction with ninhydrin (12). Toisolate radiolabeled DES and IDES used in the isotope dilution procedure, larger aliquots of hydrolyzed rat smooth muscle cell elastin (4 mg) were loaded on the amino acid analyzer. The eluted material was not reacted with ninhydrin. Fractions were collected and assessed fOT radioactivity, and those containing P4C]DES or P4C]IDES were pooled. The specific radioactivity and purity of the pooled material was confirmed by loading aliquots on the amino acid analyzer. The eluted material was allowed to react with ninhydrin and assessed for radioactivity as above. Quantification of the DES and IDES by amino acid analysis (AAA) was confirmed using nonradioactive DES and IDES (gifts from Dr. Barry Starcher). The concentrations of DES and IDES in working solutions wereassessed spectrophotometrically (13). When 1.07 and 1.08 nmol of this DES and IDES, respectively, were loaded on the amino acid analyzer, the calculated amounts recovered were 1.05and 1.13 nmol.
High Performance Liquid Chromatography The paired-ion C 18 reversed-phase HPlC procedure used by Black and coworkers (14) for pyridinoline and deoxypyridinoline, lysinederived collagen crosslinks, was modified as follows. Urine was prefractionated in 1% acetic acid as described below and combined with an equal volume of 2x loading buffer up to 1 ml. This was loaded on a Varian model 5000 high performance liquid chromatograph equipped with a Vydac C 18 column (15 em long, 0.46-cm inner diameter, and 5-J.1m silica support with 300 A pore size). The column was run at a flow rate of 1 ml/min as follows. The first 2 min was run at 0% Solvent B; subsequently the percent of Solvent B was increased by 1% per min to 30010, and the concentration was increased to 70% B in the next 10 min and returned to 0% B in the next 8 min. Total cycle time was 52 min. Solvent A was 20 mM NH 4Cl at pH 3.5, containing 5 mM octyl sulfate (Aldrich Chemical Co.). Solvent B was 75% acetonitrile:25 % Solvent A, with the concentration of cetyl sulfate adjusted to 5 mM. The loading buffer was 100mM ammonium acetate/HCI at pH 3.5, containing 50 mM octyl sulfate. The column effluent was monitored for absorbance at 275 nm. One-minute fractions were collected for liquid scintillation spectrometry when appropriate.
285
Experiments in Hamsters In Experiment 1, hamsters (mean body mass, 132 ± 3 g) were maintained on standard 5001 Purina Rodent Laboratory Chow. Weightmatched groups of eight hamsters were anesthetized by inhalation of carbon dioxide and instilled intratracheally with 0.5 ml saline or 0.5 ml saline containing 300 J.1g human neutrophil elastase (HNE) purified and characterized as previously described (15). The hamsters were placed in metabolic cages, and urine was collected for 3 days after treatment. Sodium azide was added to each urine collection cup prior to collection. The cups were emptied at least daily,and the urine was stored at - 20° C. Fifty-six days after treatment, hamsters were anesthetized and exsanguinated. The lungs were inflation-fixed by injecting 5 ml of fixative (16). The lungs were excised, and the lung volume displacement was measured (17).Three transverse sections were cut from the left lung. Paraffin-embedded histologic sections were stained with hematoxylin-eosin and assessed for airspace enlargement by measuring the mean linear intercept (MLI) (16). In preparation for Experiment 2, hamster neutrophil granule extract (HANE) was prepared from neutrophil granules obtained by nitrogen cavitation (18) of neutrophils elicited into hamster lungs by intratracheal instillation of endotoxin. The details of the method will be published elsewhere. The extract was assessed for elastolytic activity with [3H]elastin (15, 19). In Experiment 2, 16 additional hamsters with a mean body mass of 152 ± 3 g were maintained on a Purina test diet with possible dietary sources of DES removed; fish meal, meat meal, bleachable fancy tallow, and dried whey were removed from standard 5001 Lab Chow. To balance the latter formula with fat and protein, corn oil and RP101 soy protein isolate were added, and the levels of ground corn and soybean mean wereincreased. These hamsters wereplaced into two weight-matched groups, anesthetized by carbon dioxide inhalation, and received 0.8 ml PBS (10hamsters) or PBS containing HANE with elastolytic activity equivalent to 250 J.1g HNE (six hamsters). Urine was collected for 3 days as described above. Fifty-six days after treatment, hamsters were anesthetized and studied as in Experiment 1. In Experiment 3, hamsters with a mean body mass of 130 ± 3 g were maintained on the test diet described above. One group of six hamsters remained untreated. 1\\'0 groups of six hamsters each were instilled with 0.5 ml saline containing 300 J.1g HNE or 300 J.1g porcine pancreatic elastase (PPE) purified and characterized as previously described (20).After 3 days of urine collection, hamsters were anesthetized and studied by lavaging the lungs three times with heparin-saline to remove exudate, inflation fixing the lungs, and processing the tissue as above for measurement of lung volume displacement and MLI.
Urine Sample Preparation The urine was thawed and centrifuged at 30,000 x g for 15 min to remove food material and other particulates. Pools of urine for each hamster treatment group were divided into portions representing approximately six hamster-days of urine, usually 15 to 30 ml. The samples were spiked with a known amount of P4C]DES and then stored at - 20° C. For analysis the spiked urine was combined with an equal volume of 12N HCl, and the sample was hydrolyzed by refluxing at 110° C under nitrogen for 24 h. The hydrolyzed sample was then dried under a stream of nitrogen gas, brought up to 10ml with 1% acetic acid, and divided in half; each aliquot was loaded on a 20-ml disposable column (Bio-Rad Laboratories, Richmond, CAl packed with Sephadex G-15 in 1% acetic acid. The early eluting fractions containing 14C radioactivity were collected, pooled, reduced to a volume of 1to 2 ml with a stream of nitrogen, loaded on a column 2.6 x 100em (Pharmacia Diagnostics, Piscataway, NJ) packed with Sephadex G-15 in 1% acetic acid, and run at room temperature. The column had been calibrated with bovine serum albumin and 3H20 to determine void volume (Vo) and total volume (Vt), respectively. Eluted fractions were assessed for radioactivity and absorbance at 280 nm. DES eluted with a distribution coefficient (Kav) of 0.26 before most of the 280om absorbing material. The column was flushed with 1% acetic acid until the effluent had no measurable absorbance at 280 nm. The column was washed between runs for as long as 5 days prior to addition of the next sample. Persistence of absorbance in the effluent beyond 5 days indicated the need to replace the Sephadex G-15. In early experiments column fractions containing 14C radioactivity were pooled; 50% of the pool was analyzed by AAA as described above and the remainder by HPlC. After these initial studies had validated the HPlC method for quantification of DES and IDES, individual column fractions were loaded on the HPLC. Human Urine Collection With informed consent and approval by our institutional review board, human urine from male volunteer laboratory workers (25 to 68 yr of age) who were never-smokers was collected for 24 h and stored in the presence of 0.02% sodium azide at 2° C. Volunteers were asked not to eat meat for 1 day before and during the urine collection. An aliquot of the urine was removed for measurement of creatinine using a kit (Sigma Diagnostics, St. Louis, MO). Other aliquots representing 10% by volume of the 24 h pool were stored at - 20° C after the addition of a known amount of [14C]DES. Aliquots representing as little as 7% or as much as 15% of the 24-h pool were also tried; similar results were obtained for DES and IDES in all instances. For analysis the sample was reduced in volume with a rotary evaporator by raising the tempera-
STONE, BRYAN-RHADFI, WCEY, CICCOLELLA, CROMBIE, FARIS, SNIDER, AND FRANZBLAU
286
ture of the sample to 40° C under reduced pressure. The residue was brought up to 20 ml with water, combined with 20 ml of 12 N HCI, hydrolyzed, and processed as above. In a separate group of experiments, other aliquots of hamster and human urine specimens were dialyzed three times in 6 L of 1070 acetic acid. The dialyzed samples were then spiked with [14C]DES, acid-hydrolyzed, and processed as above. This provided information on urinary DES peptide size.
Comparison of DES and IDES Recoveries A urine specimenfrom Volunteer 1wasspiked with 500 cpm each of P4C]DES and [14C]IDES and processed as above. Sephadex 0-15 fractions containing radioactivity were combined into two pools, early fractions containing the peak fraction and later fractions containing lesser amounts of radioactivity. Each pool was separately loaded on an amino acid analyzer, and the radioactivity eluting with DES and IDES was separately assessed and compared. Quantification of Nonradioactive DES Added to a Urine Sample Nonradioactive DES (4.2 nmol) was added to a urine sample that represented 10% by volume of a 24-h urine pool from Volunteer 2. The sample was then spiked with [14C]DES. The amount of DES in the sample was measured as above and corrected for the amount of endogenous DES present in the urine. Statistical Analysis Values given are the mean ± SEM. Statistical analyses involving two groups were carried out using the t test; differences among means of the other data were analyzed by analysis of variance (21). Probability values < 0.05 were considered significant. Results
Pooled fractions from the disposable columns contained more than 90070 of the 14C radioactivity, but only about 20070 of the 280-nm absorbing material that had been loaded on the columns. The pooled fractions were loaded on a Sephadex 0-15 column 2.6 x 100 em, The eluting fractions with 14C radioactivity contained less than 1% of the 280 nm absorbing material; the remaining 280 nm absorbing material eluted from the column in later fractions. A representative chromatogram for hydrolyzed [14C]DESspiked human urine eluting from the column 2.6 x 100em is shown in figure 1. The amount of radioactivity in 10070 of each fraction was used to help select fractions containing DES for assessment by HPLC. Overall the two gel exclusion steps removed more than 99.9% of the 280-nm absorbing material from the fractions containing 14C radioactivity.
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Without both chromatographic steps, material loaded on the amino acid analyzer or the HPLC produced high background absorbance so that IDES and DES were difficult to quantify. Indeed, as noted in the legend to figure 2, the amount of DES and IDES in the last two 0-15 fractions that contained 14C radioactivity were often difficult to quantify either by HPLC or AAA, because of interfering peaks from contaminants. More peaks were observed in those chromatograms than in chromatograms of earlier fractions. The HPLC chromatogram for 0-15 Fraction 60 is shown in figure 2. Separation of IDES and DES in the chromatogram was 0.6 min. By AAA, IDES and DES appeared 4 min apart as previously shown (9). With the [14C]DES standard (no urine added) there was a linear relation.ship between [14C]DES radioactivity recoveredand the maximal absorbance of the DES peak when [14C]DES standards were run on the HPLC (figure 3).
Comparison of IDES and DES Recoveries The following two experiments indicated that the percent recovery of IDES and DES were not different. In the first experiment, after spiking the urine specimen with 500 cpm of DES and 500 cpm of IDES, 147 cpm of DES and 137 cpm of IDES were recovered from the amino acid analyzer after loading the early Sephadex 0-15 fractions; 64 and 58 cpm, respectively, wererecovered from the late fractions or a total of 211 and 195 cpm
Fig. 2. HPLC chromatogram of Fraction 60 from a [14C]DE8-spiked sampleof hydrolyzed humanurinefrom Volunteer2. The full range of the y axis represents0.05 absorbanceunits at 275nm. Comparison with (l"C]IDES and [1"C]DES standardsindicatesthe presenceof 0.1313 nmoilDES at 23.1% solvent Band 0.1866 nmol DES (including 0.0593 nmol of [14C]DES) at 23.8% solvent B.The calculation of the 24-h values is given in a footnote to table 2. For Fractions 62 and 63, the amount of [14C]DES recovered was low, and the level of background absorbance interfered with the accurate determination of the DES and IDES peak heights.
for DES and IDES, respectively, or recoveries of 42 and 39070. Including the loss of radioactivity because of formation of 14C02 (17070) and the 150 cpm used to evaluate the radioactivity of eluted fractions, the overall recoveryexceeded 60070. In a second experiment, 153and 159cpm of DES and IDES, respectively, were recovered in the early fractions and 34 and 28 cpm in the later fractions. Similar relative recoveries of DES and IDES in the early and late fractions suggested that DES and IDES coeluted from the 0-15 column; if they had not coeluted, the early fractions would be relatively enriched in either DES or IDES.
Comparison of Results from AAA and HPLC in Hamsters To assess the accuracy of the HPLC determination of DES and IDES, 0-15 fractions containing radioactivity were pooled. One-halfof the pool wasassessed by AAA and the other half by HPLC. Urine for analysis was obtained from un-
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MEASUREMENT OF DESMOSINE BY ISOTOPE DIWTION/HPLC
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Fig. 5. Urinary DES (closed symbols) and IDES (open symbols) values for hamsters receiving saline (circles), hamster neutrophil granule extract (HANE, 250 J,1g human neutrophil elastase equivalents by [3HJelastin assay) (diamonds), and 300 J,1g human neutrophil elastase (squares) in Experiments 1 and 2. Hamsters were treated, their urine was collected for 3 days, and their lungs were studied for evidence of air-space enlargement 56 days after treatment. Note that saline values for both Experiments 1 and 2 are indicated; the DES values overlap.
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treated, saline-treated, HNE- and PPEtreated animals in Experiments 1 and 3. The values for DES derived by these two methods are compared in figure 4 (top panel); values for IDES are compared in figure 4 (bottom panel). It can be seen from the equations of the regression lines that values derived by HPLC were generally within 3OJo ofthose derived by AAA. After this finding, samples were assessed by HPLC because it is more rapid than AAA. Values for Hamster Samples With instillation of elastase into hamsters a positive correlation was found between elevation of urinary DES and airspace enlargement (MLI). Urine from control hamsters in Experiments 1 and 2 contained 0.077 ± 0.004 (n = 2) and 0.077 ± 0.010 (n = 2) ug DES per hamster per day (figure 5). Hamsters receiving 300 ug of HNE or HANE with elastolytic activity equivalent to 250 ~g of HNE had a 116and 30OJo increase, respectively, in
Fig. 6. Urinary DES (closed symbols) and IDES (open symbols) values for control hamsters (circles), 300 IJ.g human neutrophil elastase (HNE) (squares), or 300 J,1g porcine pancreatic elastase (PPE) (triangles) in Experiment 3. Hamsters were treated, their urine was collected for 3 days, and respiratory airspace enlargement was estimated by measuring the mean linear intercept.
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PHILLIP J. STONE, JULIANNE BRYAN-RHADFI, EDGAR C. WCEY, DAVID E. CICCOLELLA GEORGE CROMBIE, BARBARA FARIS, GORDON L. SNIDER, and CARL FRANZBLAU
Introduction
It is believed that emphysema in smokers as wellas in persons deficient in alphaI-protease inhibitor is caused by elastic fiber destruction (1). The crosslink amino acids, desmosine (DES) and isodesmosine (IDES), are unique to elastin. Increased amounts of these marker amino acids have been reported in the urine of experimental animals treated intratracheally with elastase (2,3). However, investigators have not found elevated levelsof DES in the urine of smokers with chronic obstructive pulmonary disease as compared with normal smokers or nonsmokers (4, 5). Furthermore, urinary DES was similar in normal subjects, subjects with the homozygous (PiZZ) form of alphaI-protease inhibitor deficiency linked to emphysema, and subjects with interstitial lung diseases; nor were these markers elevated in PiZZ children when compared with age-matched control children (6). The above-mentioned studies were all done using a radioimmunoassay (RIA). Reports in the literature (7, 8) havesuggested that interfering substances in the urine may spuriously elevate DES values measured by RIA or enzyme-linked immunosorbent assay (ELISA). Weconsidered that such spurious elevation of urinary DES values might have masked differences between persons with and without emphysema. This led us to develop a newmethod of assaying DES and IDES in the urine. In our method most contaminants are separated from the DES and IDES by gel filtration. High performance liquid chromatography (HPLC) is used to separate DES and IDES; the eluted material is monitored by light absorption at 275 nm. An isotope dilution approach is used to quantify recovery of the marker amino acids and, thereby, to accurately calculate the 284
SUMMARY The accuracy of methods employed to measure the elastin-specific croaallnks, desmosine (DES) and Isodesmoslne (IDES), has been called Into question because contaminants In the urine may cause elevated values. In the present study urine samples were spiked with a known amount of [ 14C]DES and refluxed In 6 N HCI. Sephadex G-15chromatography of the hydrolyzed urine wes employed to remove contaminants. DES and IDES were quantified by high performance liquid chromatography (HPLC) as well as by amino acid analysis. The amount of Isotope recovered was used to determine losses during the overall procedure and the Isotope dilution to calculate the amounts of endogenous DES and IDES originally present In the urine. Because similar values were obtained by both methods, the more rapid HPLC method was used for II succeeding analyses. In one experiment, the DES amounts In urine collected from hamsters for 3 days after Intratracheal treatment with human neutrophil elastase (300 J.1g) or porcine pancreatic elastase (300 J.1g) were 0.212 ± 0.012(mean ± SEM, two measurements on a single pool) and 0.816 ± 0.005 (two measurements) J.1g per hamster per day, respectively. Urine from control hamsters had a mean value of 0.074 ± 0.008 (eight measurements) J.1g per hamster per day. The HNE- and PPE-treated hamaters had mean linear Intercept values of 119and 159% of control values, respectively, giving a positive correlation between Increase In airspace size and .Ievatlon of urinary DES. For eight normal men 25 to 68 yr of age who were never-smokers, mean ± SEM 24-h urinary values were 13.3 ± 2.3 J.1g DES and 11.3 ± 1.7 ~ IDES; DES/creatinine and IDES/creatinine ratios were 6.5 ± 0.7 ~ and 5.5 ± 0.4 J.1g per g creatinine, respectively. Values for subjects remained constant when measured on urine collected on different days. Hamster and human DES values are as much as 2D-fold and 1D-fold lower, respectively, than previously reported values. This assay shOUld prove useful In studying conditions when DES excretion may be elevated. AM REV RESPIR DIS 1111; 144:284-280
total DES and IDES present in the original sample. Methods
by liquid scintillation spectrometry. Appropriate quench-correction factors were used. The amino acid composition of the residue was similar to that reported by us earlier (11).
Preparation of {J4C}DES and f"CjIDES (14C]DES and (14C]lDES were isolated from neonatal rat smooth muscle cell cultures as previously described (9). Briefly, T-75 flasks containing l-wk-old first passage cells were pulsed for 24 h in the presence of serum by addition of [14C(U)]lysine (20 JjCi per flask) (New England Nuclear, Boston, MA), after which the medium was poured off and the cultures were refed. Five weeks later the cell layers were harvested by scraping and then homogenized. The elastin was isolated by hot alkali treatment (10). The residue was hydrolyzed in 6 N HCI at 110 0 C for 24 h in vacuo. An aliquot of the hydrolysate was loaded on a Beckman Model 119CL amino acid analyzer (Beckman Instruments, Fullerton, CA), and the eluted material was collected in 0.5min fractions and assessed for radioactivity
(Received in original form December 10, 1990) 1 From the Department of Biochemistry and the Pulmonary Center, Boston University School of Medicine, and the Pulmonary Section, Boston Veterans Administration Medical Center, Boston, Massachusetts. 1 Supported by Grant HL-19717 from the National Heart, Lung, and Blood Institute and by the Veterans Administration Research Service. 3 Presented in part at a meeting entitled "Pulmonary Emphysema: the Rationale for Therapeutic Intervention," sponsored by the New YorkAcademy of Sciences and the American Thoracic Society, Orlando, Florida, May 16-18, 1990. 4 Correspondence and requests for reprints should be addressed to Dr. Phillip J. Stone, Biochemistry Department, Boston University School of Medicine, 80 East Concord St., Boston, MA 02118.
MEASUREMENT OF DESMOSINE BY ISOTOPE DIWTION/HPLC
DES and IDES eluted 4 min apart (99 and 95 min, respectively). Four lysinecolor equivalents were used to calculate the amount of DES and IDES present. DES plus IDES composed 0.20070 of the amino acids present in the elastin. The specific radioactivity of DES and IDES were 776 and 836 cpm/nmol (80% efficiency) using Ultima Gold scintillation cocktail (Packard Instruments, Downers Grove, IL). These values include a 17% correction for the loss from both DES and IDES of four radiolabeled carbon atoms as carbon dioxide upon reaction with ninhydrin (12). Toisolate radiolabeled DES and IDES used in the isotope dilution procedure, larger aliquots of hydrolyzed rat smooth muscle cell elastin (4 mg) were loaded on the amino acid analyzer. The eluted material was not reacted with ninhydrin. Fractions were collected and assessed fOT radioactivity, and those containing P4C]DES or P4C]IDES were pooled. The specific radioactivity and purity of the pooled material was confirmed by loading aliquots on the amino acid analyzer. The eluted material was allowed to react with ninhydrin and assessed for radioactivity as above. Quantification of the DES and IDES by amino acid analysis (AAA) was confirmed using nonradioactive DES and IDES (gifts from Dr. Barry Starcher). The concentrations of DES and IDES in working solutions wereassessed spectrophotometrically (13). When 1.07 and 1.08 nmol of this DES and IDES, respectively, were loaded on the amino acid analyzer, the calculated amounts recovered were 1.05and 1.13 nmol.
High Performance Liquid Chromatography The paired-ion C 18 reversed-phase HPlC procedure used by Black and coworkers (14) for pyridinoline and deoxypyridinoline, lysinederived collagen crosslinks, was modified as follows. Urine was prefractionated in 1% acetic acid as described below and combined with an equal volume of 2x loading buffer up to 1 ml. This was loaded on a Varian model 5000 high performance liquid chromatograph equipped with a Vydac C 18 column (15 em long, 0.46-cm inner diameter, and 5-J.1m silica support with 300 A pore size). The column was run at a flow rate of 1 ml/min as follows. The first 2 min was run at 0% Solvent B; subsequently the percent of Solvent B was increased by 1% per min to 30010, and the concentration was increased to 70% B in the next 10 min and returned to 0% B in the next 8 min. Total cycle time was 52 min. Solvent A was 20 mM NH 4Cl at pH 3.5, containing 5 mM octyl sulfate (Aldrich Chemical Co.). Solvent B was 75% acetonitrile:25 % Solvent A, with the concentration of cetyl sulfate adjusted to 5 mM. The loading buffer was 100mM ammonium acetate/HCI at pH 3.5, containing 50 mM octyl sulfate. The column effluent was monitored for absorbance at 275 nm. One-minute fractions were collected for liquid scintillation spectrometry when appropriate.
285
Experiments in Hamsters In Experiment 1, hamsters (mean body mass, 132 ± 3 g) were maintained on standard 5001 Purina Rodent Laboratory Chow. Weightmatched groups of eight hamsters were anesthetized by inhalation of carbon dioxide and instilled intratracheally with 0.5 ml saline or 0.5 ml saline containing 300 J.1g human neutrophil elastase (HNE) purified and characterized as previously described (15). The hamsters were placed in metabolic cages, and urine was collected for 3 days after treatment. Sodium azide was added to each urine collection cup prior to collection. The cups were emptied at least daily,and the urine was stored at - 20° C. Fifty-six days after treatment, hamsters were anesthetized and exsanguinated. The lungs were inflation-fixed by injecting 5 ml of fixative (16). The lungs were excised, and the lung volume displacement was measured (17).Three transverse sections were cut from the left lung. Paraffin-embedded histologic sections were stained with hematoxylin-eosin and assessed for airspace enlargement by measuring the mean linear intercept (MLI) (16). In preparation for Experiment 2, hamster neutrophil granule extract (HANE) was prepared from neutrophil granules obtained by nitrogen cavitation (18) of neutrophils elicited into hamster lungs by intratracheal instillation of endotoxin. The details of the method will be published elsewhere. The extract was assessed for elastolytic activity with [3H]elastin (15, 19). In Experiment 2, 16 additional hamsters with a mean body mass of 152 ± 3 g were maintained on a Purina test diet with possible dietary sources of DES removed; fish meal, meat meal, bleachable fancy tallow, and dried whey were removed from standard 5001 Lab Chow. To balance the latter formula with fat and protein, corn oil and RP101 soy protein isolate were added, and the levels of ground corn and soybean mean wereincreased. These hamsters wereplaced into two weight-matched groups, anesthetized by carbon dioxide inhalation, and received 0.8 ml PBS (10hamsters) or PBS containing HANE with elastolytic activity equivalent to 250 J.1g HNE (six hamsters). Urine was collected for 3 days as described above. Fifty-six days after treatment, hamsters were anesthetized and studied as in Experiment 1. In Experiment 3, hamsters with a mean body mass of 130 ± 3 g were maintained on the test diet described above. One group of six hamsters remained untreated. 1\\'0 groups of six hamsters each were instilled with 0.5 ml saline containing 300 J.1g HNE or 300 J.1g porcine pancreatic elastase (PPE) purified and characterized as previously described (20).After 3 days of urine collection, hamsters were anesthetized and studied by lavaging the lungs three times with heparin-saline to remove exudate, inflation fixing the lungs, and processing the tissue as above for measurement of lung volume displacement and MLI.
Urine Sample Preparation The urine was thawed and centrifuged at 30,000 x g for 15 min to remove food material and other particulates. Pools of urine for each hamster treatment group were divided into portions representing approximately six hamster-days of urine, usually 15 to 30 ml. The samples were spiked with a known amount of P4C]DES and then stored at - 20° C. For analysis the spiked urine was combined with an equal volume of 12N HCl, and the sample was hydrolyzed by refluxing at 110° C under nitrogen for 24 h. The hydrolyzed sample was then dried under a stream of nitrogen gas, brought up to 10ml with 1% acetic acid, and divided in half; each aliquot was loaded on a 20-ml disposable column (Bio-Rad Laboratories, Richmond, CAl packed with Sephadex G-15 in 1% acetic acid. The early eluting fractions containing 14C radioactivity were collected, pooled, reduced to a volume of 1to 2 ml with a stream of nitrogen, loaded on a column 2.6 x 100em (Pharmacia Diagnostics, Piscataway, NJ) packed with Sephadex G-15 in 1% acetic acid, and run at room temperature. The column had been calibrated with bovine serum albumin and 3H20 to determine void volume (Vo) and total volume (Vt), respectively. Eluted fractions were assessed for radioactivity and absorbance at 280 nm. DES eluted with a distribution coefficient (Kav) of 0.26 before most of the 280om absorbing material. The column was flushed with 1% acetic acid until the effluent had no measurable absorbance at 280 nm. The column was washed between runs for as long as 5 days prior to addition of the next sample. Persistence of absorbance in the effluent beyond 5 days indicated the need to replace the Sephadex G-15. In early experiments column fractions containing 14C radioactivity were pooled; 50% of the pool was analyzed by AAA as described above and the remainder by HPlC. After these initial studies had validated the HPlC method for quantification of DES and IDES, individual column fractions were loaded on the HPLC. Human Urine Collection With informed consent and approval by our institutional review board, human urine from male volunteer laboratory workers (25 to 68 yr of age) who were never-smokers was collected for 24 h and stored in the presence of 0.02% sodium azide at 2° C. Volunteers were asked not to eat meat for 1 day before and during the urine collection. An aliquot of the urine was removed for measurement of creatinine using a kit (Sigma Diagnostics, St. Louis, MO). Other aliquots representing 10% by volume of the 24 h pool were stored at - 20° C after the addition of a known amount of [14C]DES. Aliquots representing as little as 7% or as much as 15% of the 24-h pool were also tried; similar results were obtained for DES and IDES in all instances. For analysis the sample was reduced in volume with a rotary evaporator by raising the tempera-
STONE, BRYAN-RHADFI, WCEY, CICCOLELLA, CROMBIE, FARIS, SNIDER, AND FRANZBLAU
286
ture of the sample to 40° C under reduced pressure. The residue was brought up to 20 ml with water, combined with 20 ml of 12 N HCI, hydrolyzed, and processed as above. In a separate group of experiments, other aliquots of hamster and human urine specimens were dialyzed three times in 6 L of 1070 acetic acid. The dialyzed samples were then spiked with [14C]DES, acid-hydrolyzed, and processed as above. This provided information on urinary DES peptide size.
Comparison of DES and IDES Recoveries A urine specimenfrom Volunteer 1wasspiked with 500 cpm each of P4C]DES and [14C]IDES and processed as above. Sephadex 0-15 fractions containing radioactivity were combined into two pools, early fractions containing the peak fraction and later fractions containing lesser amounts of radioactivity. Each pool was separately loaded on an amino acid analyzer, and the radioactivity eluting with DES and IDES was separately assessed and compared. Quantification of Nonradioactive DES Added to a Urine Sample Nonradioactive DES (4.2 nmol) was added to a urine sample that represented 10% by volume of a 24-h urine pool from Volunteer 2. The sample was then spiked with [14C]DES. The amount of DES in the sample was measured as above and corrected for the amount of endogenous DES present in the urine. Statistical Analysis Values given are the mean ± SEM. Statistical analyses involving two groups were carried out using the t test; differences among means of the other data were analyzed by analysis of variance (21). Probability values < 0.05 were considered significant. Results
Pooled fractions from the disposable columns contained more than 90070 of the 14C radioactivity, but only about 20070 of the 280-nm absorbing material that had been loaded on the columns. The pooled fractions were loaded on a Sephadex 0-15 column 2.6 x 100 em, The eluting fractions with 14C radioactivity contained less than 1% of the 280 nm absorbing material; the remaining 280 nm absorbing material eluted from the column in later fractions. A representative chromatogram for hydrolyzed [14C]DESspiked human urine eluting from the column 2.6 x 100em is shown in figure 1. The amount of radioactivity in 10070 of each fraction was used to help select fractions containing DES for assessment by HPLC. Overall the two gel exclusion steps removed more than 99.9% of the 280-nm absorbing material from the fractions containing 14C radioactivity.
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Without both chromatographic steps, material loaded on the amino acid analyzer or the HPLC produced high background absorbance so that IDES and DES were difficult to quantify. Indeed, as noted in the legend to figure 2, the amount of DES and IDES in the last two 0-15 fractions that contained 14C radioactivity were often difficult to quantify either by HPLC or AAA, because of interfering peaks from contaminants. More peaks were observed in those chromatograms than in chromatograms of earlier fractions. The HPLC chromatogram for 0-15 Fraction 60 is shown in figure 2. Separation of IDES and DES in the chromatogram was 0.6 min. By AAA, IDES and DES appeared 4 min apart as previously shown (9). With the [14C]DES standard (no urine added) there was a linear relation.ship between [14C]DES radioactivity recoveredand the maximal absorbance of the DES peak when [14C]DES standards were run on the HPLC (figure 3).
Comparison of IDES and DES Recoveries The following two experiments indicated that the percent recovery of IDES and DES were not different. In the first experiment, after spiking the urine specimen with 500 cpm of DES and 500 cpm of IDES, 147 cpm of DES and 137 cpm of IDES were recovered from the amino acid analyzer after loading the early Sephadex 0-15 fractions; 64 and 58 cpm, respectively, wererecovered from the late fractions or a total of 211 and 195 cpm
Fig. 2. HPLC chromatogram of Fraction 60 from a [14C]DE8-spiked sampleof hydrolyzed humanurinefrom Volunteer2. The full range of the y axis represents0.05 absorbanceunits at 275nm. Comparison with (l"C]IDES and [1"C]DES standardsindicatesthe presenceof 0.1313 nmoilDES at 23.1% solvent Band 0.1866 nmol DES (including 0.0593 nmol of [14C]DES) at 23.8% solvent B.The calculation of the 24-h values is given in a footnote to table 2. For Fractions 62 and 63, the amount of [14C]DES recovered was low, and the level of background absorbance interfered with the accurate determination of the DES and IDES peak heights.
for DES and IDES, respectively, or recoveries of 42 and 39070. Including the loss of radioactivity because of formation of 14C02 (17070) and the 150 cpm used to evaluate the radioactivity of eluted fractions, the overall recoveryexceeded 60070. In a second experiment, 153and 159cpm of DES and IDES, respectively, were recovered in the early fractions and 34 and 28 cpm in the later fractions. Similar relative recoveries of DES and IDES in the early and late fractions suggested that DES and IDES coeluted from the 0-15 column; if they had not coeluted, the early fractions would be relatively enriched in either DES or IDES.
Comparison of Results from AAA and HPLC in Hamsters To assess the accuracy of the HPLC determination of DES and IDES, 0-15 fractions containing radioactivity were pooled. One-halfof the pool wasassessed by AAA and the other half by HPLC. Urine for analysis was obtained from un-
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Fig. 5. Urinary DES (closed symbols) and IDES (open symbols) values for hamsters receiving saline (circles), hamster neutrophil granule extract (HANE, 250 J,1g human neutrophil elastase equivalents by [3HJelastin assay) (diamonds), and 300 J,1g human neutrophil elastase (squares) in Experiments 1 and 2. Hamsters were treated, their urine was collected for 3 days, and their lungs were studied for evidence of air-space enlargement 56 days after treatment. Note that saline values for both Experiments 1 and 2 are indicated; the DES values overlap.
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