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Comparison of Urinary Desmosine Excretion in Patients with Chronic Obstructive Pulmonary Disease or Cystic Fibrosis
Pulmonary Pharmacology & Therapeutics (2000) 13, 175–180 doi:10.1006/pupt.2000.0245, available online at http://www.idealibrary.com on
PULMONARY PHARMACOLOGY & THERAPEUTICS
Comparison of Urinary Desmosine Excretion in Patients with Chronic Obstructive Pulmonary Disease or Cystic Fibrosis D. C. Bode, E. D. Pagani, W. R. Cumiskey, R. von Roemeling, L. Hamel, P. J. Silver Sterling Winthrop Pharmaceuticals Research Division Collegeville, Pennsylvania, USA
SUMMARY: Neutrophil elastase is involved in the pathogenesis of several pulmonary diseases; a strategy for monitoring in vivo elastase activity is to measure changes in biochemical markers. The objective of this study was to determine whether differences in the urinary excretion of the elastin crosslinks, desmosine and isodesmosine (which are unique amino acid products of elastase activity), could be discerned between groups of patients with chronic obstructive pulmonary disease (COPD) or cystic fibrosis (CF), and non-diseased, age-matched controls. Twenty-four-hour urine collections were analysed to eliminate variations in excretion throughout the day, and urine was collected on four separate days in 29–31 subjects/group to investigate the variability in desmosines excretion among the groups. Both sets of patient populations had significantly more variable desmosines readings (higher standard deviations) relative to their respective age-matched control group. The means for three adult groups (COPD, controls and a COPD-smoker subset) ranged from 28.4 to 35.5 pmol desmosines/mg creatinine and there were no differences among the groups. Values in children were higher: 55 pmol desmosines/mg creatinine in the non-CF children and 77 pmol desmosines/mg creatinine for the CF group (P<0.01 vs. age-matched controls). The results of this study show that urinary desmosines, as a surrogate marker for enhanced elastase activity, are more highly variant in both patient populations relative to age-matched controls, and an overall increase in the mean 2000 Academic Press value is further observed in patients with cystic fibrosis. KEY WORDS: Elastase, Desmosines, Cystic fibrosis, Chronic obstructive pulmonary disease.
directly related to elastin degradation.10–14 Desmosines and desmosine-containing small peptides are filtered completely by the kidney and are excreted in urine.15 Thus, urinary desmosine output can reflect the in vivo activity of elastase. Desmosines can be quantified by HPLC or radioimmunoassay. However, in some cases, the specificity of antibodies used in the latter method, along with interference from other urinary components, called into question the validity of this approach.16,17 HPLC, following acid hydrolysis to free desmosines from peptid fragments, can be laborious. However, we have previously published an improved method of sample preparation and HPLC analysis which can be used to analyse large numbers of samples with good recovery and reproducibility.18 The objective of the present study was to utilize this method to quantify and compare urinary desmosines output in two patient populations, children with cystic fibrosis and adults with COPD, which are purported to have high levels of elastase activity. Comparisons to age-matched controls in both populations have
INTRODUCTION Neutrophil elastase (E.C. 3.4.21.37) is a serine protease whose substrates include elastin, collagen and fibronectin. The enzyme is a target for pharmacological modulation as there is evidence for the involvement of this elastase in the pathogenesis of several diseases, including chronic obstructive pulmonary disease (COPD),1–4 cystic fibrosis,5–8 and adult respiratory distress syndrome.9 One strategy to determine the potential efficacy of elastase inhibitors in clinical trials is to measure biochemical markers of in vivo elastase activity. One of the most direct biochemical markers of in vivo elastin degradation is the amino acid desmosine and its isomer, isodesmosine (both hereafter referred to collectively as desmosines). These unique amino acids are only found in elastin, and their appearance is Author for correspondence: Dr Donald C. Bode, Rhone-Poulenc Rorer Research and Development, Lead Discovery Department, 500 Arcola Road, NW15, P.O. Box 5090, Collegeville, Pennsylvania 19426-0994, USA. 1094–5539/00/040175+06 $35.00/0
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been made. In addition, a subset of COPD patients that continue to smoke was analysed. Comparisons of the variability of 24-h urinary desmosines levels sampled on four different days throughout a 2-week period were made, with the hypothesis that the patient populations would show a higher variability than their age-matched non-disease counterparts. Also, the overall mean values for these groups were compared.
METHODS Study design A clinical study was designed to determine whether differences in the urinary excretion of desmosines could be discerned between groups of patients with chronic obstructive pulmonary disease or cystic fibrosis and nondiseased, age-matched controls. The size of each group, approximately 30 subjects, was determined statistically, based on assumptions of a 30% difference in median values, 80% power in a twosided test, and equal variance in each group. Twentyfour-hour urine collections were analysed to eliminate variations in excretion throughout the day, and urine was collected on four separate days (Tuesday and Thursday of 2 consecutive weeks) to investigate the variability in desmosines excretion among the groups. Subject recruitment, evaluation, informed consent and clinical testing, as well as urine specimen collection, were coordinated by the Sterling Winthrop Pharmacology Study Unit, Albany, New York, USA. Each of the two control groups consisted of approximately 30 non-institutionalized, healthy subjects with normal respiratory function, who were agematched to the subjects in one of the disease groups. Exclusion criteria for the controls included a history of cancer, clinically significant gastrointestinal, cardiovascular, renal, hepatic, respiratory, endocrine, metabolic, hematopoietic, or atopic disorder, drug or alcohol abuse, use of a systemic prescription medication within 1 week, or prior participation in an investigational drug study within 4 weeks. The COPD group consisted of 30 subjects over 30 years of age with forced expiratory volume in 1 s (FEV1) less than 70%. The CF group consisted of 29 individuals aged eight to 18 who had been previously diagnosed with CF based on either genotyping or pilocarpine iontophoresis (with sweat chlorides >60 meq/l). Exclusion criteria for the disease groups were recent trauma or prior participation in an investigational drug study within 4 weeks. Within 3 weeks of entry into the study, a complete medical history (including concomitant medications) was taken, detailed instructions were given, and a 2-day dietary history was recorded.
Specimen collection and storage Urine samples (24-h) were collected from volunteer human subjects in plastic containers without a preservative. The urine collection for any given day consisted of all urine after the first morning urine of that day, including the first urine from the following morning. The container was stored at 4°C until it was received in our laboratory. Upon receipt, it was mixed well by inversion, and aliquots were removed for analysis of creatinine and desmosines. The aliquots were stored at −20°C. Processing of urine specimens Urine specimens were processed as reported previously.18 Briefly, aliquots were hydrolysed in 6 HCl at 92°C overnight. Contaminants were removed by two cycles of low-pressure chromatography on CF1 cellulose. Recoveries of desmosine and isodesmosine were determined by spiking a known amount of each analyte into four control urine specimens for simultaneous processing with unknowns. Analysis of desmosine and isodesmosine The residues from CF1 chromatography were analysed according to Cumiskey et al.18 HPLC on a C18 column was used to separate desmosine and isodesmosine, which were quantitated based on absorbance peak-heights in comparison with an external standard calibration curve. The derived values were corrected for recovery using spiked samples that were processed at the same time. Desmosine and isodesmosine concentrations were normalized for the concentration of creatinine in the same sample, to account for any variation in glomerular filtration rate within and between individual subjects. Thus, the values used for statistical comparisons were expressed as pmol/day per mg creatinine. Materials Desmosine and isodesmosine standards were from Elastin Products (Owensville, Missouri, USA); Nucleosil C18 analytical HPLC columns from Alltech Associates (Deerfield, Illinois, USA); WISP autosampler and Bondapak C18 precolumns from Waters Chromatography Division, Millipore (Milford, Massachusetts, USA); HPLC pumps from Rainin (Woburn, Massachusetts, USA); UV absorbance detector from Perkin Elmer (Norwalk, Connecticut, USA); CF1 cellulose from Whatman (Clifton, New Jersey, USA); Econo-Pac annd Poly-Prep columns and funnels for CF1 chromatography from Bio-Rad Laboratories (Richmond, California, USA); centrifuge tubes from Sarstedt (Newton, North Carolina, USA); analytical- or HPLC-grade chemicals from J. T. Baker
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Table 1 Demographics of COPD, cystic fibrosis, and age-matched counterpart groups. Group COPD COPD Smokers Controls (Adult) Cystic fibrosis Controls (Children)
Sex
Age (years)
Weight (kg)
Urinary creatinine (mg/kg per day)
28M/2F 8M/1F 17M/14F 15M/14F 14M/15F
64.63±1.9 61.11±3.2 64.94±1.9 11.48±0.52 11.48±0.54
81.4±3.3 82.9±5.1 76.3±2.2 37.1±2.2 41.3±2.7
14.0±0.85 13.6±2.2 16.1±0.76 17.9±0.84 21.6±1.4
Values for age, weight and urinary creatinine are the mean±SEM for the total number in each group.
(Phillipsburg, New Jersey, USA); and creatinine assay reagents from Sigma Diagnostics (St Louis, Missouri, USA).
Statistical methods Significance of the variability observed in the four separate determinations of urinary desmosines was made by comparing the ratios of the standard deviations by an F-test for COPD patients and their age-matched controls, and cystic fibrosis patients and their age-matched controls. A P value Ζ0.05 was considered significant. Significance of the total mean urinary desmosines values between COPD, COPDsmoker, and age-matched controls was made via ANOVA; a Student’s t-test was used to compare the mean values of cystic fibrosis patients with their agematched controls. A P value Ζ0.05 was considered significant.
well as for the cystic fibrosis patients and their agematched controls, are shown in Figs 1 and 2 respectively. It is evident that both sets of patient populations have more variable desmosines readings (higher standard deviations) when sampled over a time frame as short as 2 weeks. A statistical comparison of these variations show significant differences in the magnitude of the standard deviations for both the COPD (PΖ0.01) and cystic fibrosis (PΖ0.005) populations relative to their respective age-matched controls. The overall mean±SE for all three groups (all readings combined) is shown in Fig. 3. Mean values for the three adult groups (COPD, COPD smokers, controls) ranged from 28.4 to 35.5 pmol desmosines/ mg creatinine and there were no differences among the groups. Values in children were higher: 55 pmol desmosines/mg creatinine in the non-cystic fibrosis children and 77 pmol desmosines/mg creatinine for the cystic fibrosis group (P<0.01 when compared with the age-matched non-cystic fibrosis group).
RESULTS DISCUSSION The demographics for these patient populations and their age-matched controls are shown in Table 1. The number of subjects per group ranged from 29 to 31, with the exception of the COPD smoker subset, which totalled nine patients. The other difference of note relates to the distribution of sex in the groups. There were approximately equal numbers of males and females tested in the cystic fibrosis group, their agematched control group, and the age-matched controls for the COPD group, while the COPD patient group itself had an overwhelming number of males. However, it should be noted that there is no reported difference in urinary desmosine levels related to gender, when normalized for creatinine clearance.13 The individual 24-h urinary desmosines values measured on four separate occasions, and the calculated standard deviations for these values, for COPD patients and their age-matched controls, as
In the present study, we have used an improved method of enrichment and HPLC quantification of urinary desmosines to study the activity of elastase in two patient populations where this enzyme may play a contributing role in the disease process. In both adults with COPD and children with cystic fibrosis, there was a significantly higher degree of variability in urinary desmosines concentration, when sampled four times over a 2-week period, than in age-matched controls. This suggests that, in both patient populations, neutrophil elastase activity surges from time to time, and is not as constant as that in age-matched controls. Intuitively this seems reasonable, and further suggests that treatment of either patient population with an elastase inhibitor could blunt these increases and normalize the fluctuations in activity. These results are consistent with the findings of Stone et al.8 and
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B Controls 110
100
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A COPD Patients 110
80 70 60 50 40 30
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Fig. 1 Individual values (panels A and B) and standard deviations (panels C and D) of 24-h urinary desmosines concentrations in COPD patients (n=30) and age-matched controls (n=31). Individual (panels A and B) 24-h desmosines (combined isodesmosine plus desmosine) concentrations were determined as described in the text for each subject on four different days over a 2-week period. Values are expressed as pmol desmosines/mg creatinine. In panels C and D, the range of standard deviation for these four determinations per subject are shown.
Starcher et al.19 Increased variability in urinary desmosines in individuals with pulmonary dysfunction was also reported by Pai et al,13 but this study examined only a few patients, did not compare mean values, and cystic fibrosis patients were not examined. Of further interest were the overall significant increase in the mean levels of desmosines in the cystic
fibrosis patients relative to their age-matched controls and the lack of differences in mean desmosines values among the COPD group, age-matched controls, and the smoker-COPD subgroup. Since a similar overall mean increase was not observed an COPD patients, this suggests that enhanced neutrophil elastase activity may be more important in cystic fibrosis patients, and
Urinary desmosines as markers of elastase
B Controls
250
250
200
200 pmol desmosines/mg creatinine
pmol desmosines/mg creatinine
A Cystic Fibrosis Patients
150
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510 520 Subject number
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1510 1520 Subject number
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80 Standard deviation (pmol des./mg creat.)
Standard deviation (pmol des./mg creat.)
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70 60 50 40 30 20 10
70 60 50 40 30 20 10
0 500
510 520 Subject number
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0 1500
Fig. 2 Individual values (panels A and B) and standard deviations (panels C and D) of 24-h urinary desmosines concentrations in cystic fibrosis patients (n=29) and age-matched controls (n=29). Individual (panels A and B) 24-h desmosines (combined isodesmosine plus desmosine) concentrations were determined as described in the text for each subject on four different days over a 2-week period. Values are expressed as pmol desmosines/mg creatinine. In panels C and D, the range of standard deviation for these four determinations per subject are shown.
that this patient population might be more amenable to treatment with an elastase inhibitor. A limitation of the present study is the lack of a correlative measure of pulmonary function in these patients. It would be interesting to know whether, in a given patient, spikes in the excretion of desmosines
correlate with depressed ventilatory function. However, compliance for a study such as this is difficult, and pooling urine for 24-h collections does not allow analysis to determine the exact time of elastase flareup. In summary, the results of this study suggest that
pmol desmosines/mg creatinine
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D. C. Bode et al 100 90 80 70 60 50 40 30 20 10 0
(29) * (29) (30)
5.
(9) (31)
6.
COPD COPD A-M Smokers Control
CF
A-M Control
Fig. 3 Comparative mean (±SEM) desmosines concentrations for COPD, COPD-smoker, age-matched (A-M) children. The number of subjects in each group is indicated in parentheses above each bar. ∗PΖ0.05 when compared with A-M controls.
7. 8.
9.
urinary desmosines can be used as a surrogate marker for elastase activity in two patient populations, COPD adults and children with cystic fibrosis, where this enzyme has been implicated in the disease process. Increased variability over a 2-week period in the activity of this enzyme is evident in both populations, and an overall increase in the mean value is further observed in the cystic fibrosis group. As orally bioavailable inhibitors of neutrophil elastase are under development by several pharmaceutical companies, future studies are necessary to determine if this marker can be used to monitor the efficacy of an inhibitor, and, ultimately, to determine if inhibition of this enzyme can alter the pathogenesis and/or symptomatology of these diseases.
10. 11. 12. 13. 14.
15. 16.
REFERENCES 1. Snider G. L. Chronic obstructive pulmonary disease: risk factors, pathophysiology and pathogenesis. Ann Rev Med 1989; 40: 411–429. 2. Schriver E. E., Davidson J. M., Sutcliffe M. C., Swindell B. B., Bernard G. R. Comparison of elastin peptide concentrations in body fluids from healthy volunteers, smokers, and patients with chronic obstructive pulmonary disease. Am Rev Respir Dis 1992; 1445: 762–766. 3. Stone P. J., Gottlieb D. J., O’Connor G. T., Ciccolella D. E., Breuer R., Bryan-Rhadfi J., Shaw H. A., Franzblau C., Snider G. L. Elastin and collagen degradation products in urine of smokers with and without chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1995; 151: 952–959. 4. Luisetti M., Sturani C., Sella D., Madonini E., Galavotti V., Bruno G., Peona V., Kucich U., Dagnino G., Rosenbloom
17. 18. 19.
J., Starcher B., Grassi C. MR889, a neutrophil elastase inhibitor, in patients with chronic obstructive pulmonary disease: a double-blind, randomized, placebo-controlled clinical trial. Eur Respir J 1996; 9: 1482–1486. Bruce M. C., Poncz L., Klinger, J. D., Stern R.C., Tomashefski J. F., Dearborn D. G. Biochemical and pathologic evidence for proteolytic destruction of lung connective tissue in cystic fibrosis. Am Rev Respir Dis 1985; 132: 529–535. O’Connor C. M., Gaffney K., Keane J., Southey A. Byrne N., O’Mahoney S., Fitzgerald M. X. Alpha 1-proteinase inhibitor, elastase activity, and lung disease severity in cystic fibrosis. Am Rev Respir Dis 1993; 148: 1665–1670. Meyer K. C., Zimmerman J. Neutrophil mediators, Pseudomonas, and pulmonary dysfunction in cystic fibrosis. J Lab Clin Med 1993; 121: 654–661. Stone P. J., Konstan M. W., Berger M., Dorkin H. L., Franzblau C., Snider G. L. Elastin and collagen degradation products in urine of patients with cystic fibrosis. Am J Respir Crit Care Med 1995; 152: 157–162. Tenholder M. F., Rajagopal K. R., Phillips Y. Y., Dillard T. A., Bennett L. L., Mundie T. G., Tellis C. J. Urinary desmosine excretion as a marker of lung injury in the adult respiratory distress syndrome. Chest 1991; 100: 1385–1390. Partridge S. M. Biosynthesis and nature of elastin structures. Fed Proc 1966; 25: 1023–1029. Starcher B. C., Goldstein R. A. Studies on the absorption of desmosine and isodesmosine. J Lab Clin Med 1979; 94: 848–852. Yu S. S., Ruthmeyer S. K., Shepard J. W. Digestion and absorption of radioactive elastins in rats. Proc Soc Exp Biol Med 1979; 161: 239–243. Pai V., Guz A., Phillips G. J., Cooke N. T., Hutchinson D. C., Tetley T. D. Urinary desmosine, elastolysis, and lung disease. Metabolism 1991; 40: 139–145. Stone P. J., Lucey E. C., Snider G. L., Franzblau C. Effect of diet on urinary excretion of desmosine and hydroxylysyl pyridinoline. Am J Respir Crit Care Med 1994; 149: 174–177. Goldstein R. A., Starcher B. C. Urinary excretion of elastin peptides containing desmosine after intratracheal injection of elastase in hamsters. J Clin Invest 1978; 61: 1286–1290. Stone P. J., Bryan-Rhadfi J., Lucey E. C., Ciccolella D. E., Crombie G., Faris B., Snider G. L., Franzblau C. Measurement of urinary desmosine by isotope dilution and high performance liquid chromatography. Correlation between elastase-induced airspace enlargement in the hamster and elevation of urinary desmosine. Am Rev Respir Dis 1991; 144: 284–290. Starcher B., Scott M. Fractionation of urine to allow desmosine analysis by radioimmunoassay. Ann Clin Biochem 1992; 29: 72–78. Cumiskey W. R., Pagani E. D., Bode D. C. Enrichment and analysis of desmosine and isodesmosine in biological fluids. J Chromatog B 1995; 668: 199–207. Starcher B., Green M., Scott M. Urinary desmosine as an indicator of acute pulmonary disease. Respiration 1995; 62: 252–257.
Date received: 15 May 2000. Date accepted: 8 June 2000.
PULMONARY PHARMACOLOGY & THERAPEUTICS
Comparison of Urinary Desmosine Excretion in Patients with Chronic Obstructive Pulmonary Disease or Cystic Fibrosis D. C. Bode, E. D. Pagani, W. R. Cumiskey, R. von Roemeling, L. Hamel, P. J. Silver Sterling Winthrop Pharmaceuticals Research Division Collegeville, Pennsylvania, USA
SUMMARY: Neutrophil elastase is involved in the pathogenesis of several pulmonary diseases; a strategy for monitoring in vivo elastase activity is to measure changes in biochemical markers. The objective of this study was to determine whether differences in the urinary excretion of the elastin crosslinks, desmosine and isodesmosine (which are unique amino acid products of elastase activity), could be discerned between groups of patients with chronic obstructive pulmonary disease (COPD) or cystic fibrosis (CF), and non-diseased, age-matched controls. Twenty-four-hour urine collections were analysed to eliminate variations in excretion throughout the day, and urine was collected on four separate days in 29–31 subjects/group to investigate the variability in desmosines excretion among the groups. Both sets of patient populations had significantly more variable desmosines readings (higher standard deviations) relative to their respective age-matched control group. The means for three adult groups (COPD, controls and a COPD-smoker subset) ranged from 28.4 to 35.5 pmol desmosines/mg creatinine and there were no differences among the groups. Values in children were higher: 55 pmol desmosines/mg creatinine in the non-CF children and 77 pmol desmosines/mg creatinine for the CF group (P<0.01 vs. age-matched controls). The results of this study show that urinary desmosines, as a surrogate marker for enhanced elastase activity, are more highly variant in both patient populations relative to age-matched controls, and an overall increase in the mean 2000 Academic Press value is further observed in patients with cystic fibrosis. KEY WORDS: Elastase, Desmosines, Cystic fibrosis, Chronic obstructive pulmonary disease.
directly related to elastin degradation.10–14 Desmosines and desmosine-containing small peptides are filtered completely by the kidney and are excreted in urine.15 Thus, urinary desmosine output can reflect the in vivo activity of elastase. Desmosines can be quantified by HPLC or radioimmunoassay. However, in some cases, the specificity of antibodies used in the latter method, along with interference from other urinary components, called into question the validity of this approach.16,17 HPLC, following acid hydrolysis to free desmosines from peptid fragments, can be laborious. However, we have previously published an improved method of sample preparation and HPLC analysis which can be used to analyse large numbers of samples with good recovery and reproducibility.18 The objective of the present study was to utilize this method to quantify and compare urinary desmosines output in two patient populations, children with cystic fibrosis and adults with COPD, which are purported to have high levels of elastase activity. Comparisons to age-matched controls in both populations have
INTRODUCTION Neutrophil elastase (E.C. 3.4.21.37) is a serine protease whose substrates include elastin, collagen and fibronectin. The enzyme is a target for pharmacological modulation as there is evidence for the involvement of this elastase in the pathogenesis of several diseases, including chronic obstructive pulmonary disease (COPD),1–4 cystic fibrosis,5–8 and adult respiratory distress syndrome.9 One strategy to determine the potential efficacy of elastase inhibitors in clinical trials is to measure biochemical markers of in vivo elastase activity. One of the most direct biochemical markers of in vivo elastin degradation is the amino acid desmosine and its isomer, isodesmosine (both hereafter referred to collectively as desmosines). These unique amino acids are only found in elastin, and their appearance is Author for correspondence: Dr Donald C. Bode, Rhone-Poulenc Rorer Research and Development, Lead Discovery Department, 500 Arcola Road, NW15, P.O. Box 5090, Collegeville, Pennsylvania 19426-0994, USA. 1094–5539/00/040175+06 $35.00/0
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been made. In addition, a subset of COPD patients that continue to smoke was analysed. Comparisons of the variability of 24-h urinary desmosines levels sampled on four different days throughout a 2-week period were made, with the hypothesis that the patient populations would show a higher variability than their age-matched non-disease counterparts. Also, the overall mean values for these groups were compared.
METHODS Study design A clinical study was designed to determine whether differences in the urinary excretion of desmosines could be discerned between groups of patients with chronic obstructive pulmonary disease or cystic fibrosis and nondiseased, age-matched controls. The size of each group, approximately 30 subjects, was determined statistically, based on assumptions of a 30% difference in median values, 80% power in a twosided test, and equal variance in each group. Twentyfour-hour urine collections were analysed to eliminate variations in excretion throughout the day, and urine was collected on four separate days (Tuesday and Thursday of 2 consecutive weeks) to investigate the variability in desmosines excretion among the groups. Subject recruitment, evaluation, informed consent and clinical testing, as well as urine specimen collection, were coordinated by the Sterling Winthrop Pharmacology Study Unit, Albany, New York, USA. Each of the two control groups consisted of approximately 30 non-institutionalized, healthy subjects with normal respiratory function, who were agematched to the subjects in one of the disease groups. Exclusion criteria for the controls included a history of cancer, clinically significant gastrointestinal, cardiovascular, renal, hepatic, respiratory, endocrine, metabolic, hematopoietic, or atopic disorder, drug or alcohol abuse, use of a systemic prescription medication within 1 week, or prior participation in an investigational drug study within 4 weeks. The COPD group consisted of 30 subjects over 30 years of age with forced expiratory volume in 1 s (FEV1) less than 70%. The CF group consisted of 29 individuals aged eight to 18 who had been previously diagnosed with CF based on either genotyping or pilocarpine iontophoresis (with sweat chlorides >60 meq/l). Exclusion criteria for the disease groups were recent trauma or prior participation in an investigational drug study within 4 weeks. Within 3 weeks of entry into the study, a complete medical history (including concomitant medications) was taken, detailed instructions were given, and a 2-day dietary history was recorded.
Specimen collection and storage Urine samples (24-h) were collected from volunteer human subjects in plastic containers without a preservative. The urine collection for any given day consisted of all urine after the first morning urine of that day, including the first urine from the following morning. The container was stored at 4°C until it was received in our laboratory. Upon receipt, it was mixed well by inversion, and aliquots were removed for analysis of creatinine and desmosines. The aliquots were stored at −20°C. Processing of urine specimens Urine specimens were processed as reported previously.18 Briefly, aliquots were hydrolysed in 6 HCl at 92°C overnight. Contaminants were removed by two cycles of low-pressure chromatography on CF1 cellulose. Recoveries of desmosine and isodesmosine were determined by spiking a known amount of each analyte into four control urine specimens for simultaneous processing with unknowns. Analysis of desmosine and isodesmosine The residues from CF1 chromatography were analysed according to Cumiskey et al.18 HPLC on a C18 column was used to separate desmosine and isodesmosine, which were quantitated based on absorbance peak-heights in comparison with an external standard calibration curve. The derived values were corrected for recovery using spiked samples that were processed at the same time. Desmosine and isodesmosine concentrations were normalized for the concentration of creatinine in the same sample, to account for any variation in glomerular filtration rate within and between individual subjects. Thus, the values used for statistical comparisons were expressed as pmol/day per mg creatinine. Materials Desmosine and isodesmosine standards were from Elastin Products (Owensville, Missouri, USA); Nucleosil C18 analytical HPLC columns from Alltech Associates (Deerfield, Illinois, USA); WISP autosampler and Bondapak C18 precolumns from Waters Chromatography Division, Millipore (Milford, Massachusetts, USA); HPLC pumps from Rainin (Woburn, Massachusetts, USA); UV absorbance detector from Perkin Elmer (Norwalk, Connecticut, USA); CF1 cellulose from Whatman (Clifton, New Jersey, USA); Econo-Pac annd Poly-Prep columns and funnels for CF1 chromatography from Bio-Rad Laboratories (Richmond, California, USA); centrifuge tubes from Sarstedt (Newton, North Carolina, USA); analytical- or HPLC-grade chemicals from J. T. Baker
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Table 1 Demographics of COPD, cystic fibrosis, and age-matched counterpart groups. Group COPD COPD Smokers Controls (Adult) Cystic fibrosis Controls (Children)
Sex
Age (years)
Weight (kg)
Urinary creatinine (mg/kg per day)
28M/2F 8M/1F 17M/14F 15M/14F 14M/15F
64.63±1.9 61.11±3.2 64.94±1.9 11.48±0.52 11.48±0.54
81.4±3.3 82.9±5.1 76.3±2.2 37.1±2.2 41.3±2.7
14.0±0.85 13.6±2.2 16.1±0.76 17.9±0.84 21.6±1.4
Values for age, weight and urinary creatinine are the mean±SEM for the total number in each group.
(Phillipsburg, New Jersey, USA); and creatinine assay reagents from Sigma Diagnostics (St Louis, Missouri, USA).
Statistical methods Significance of the variability observed in the four separate determinations of urinary desmosines was made by comparing the ratios of the standard deviations by an F-test for COPD patients and their age-matched controls, and cystic fibrosis patients and their age-matched controls. A P value Ζ0.05 was considered significant. Significance of the total mean urinary desmosines values between COPD, COPDsmoker, and age-matched controls was made via ANOVA; a Student’s t-test was used to compare the mean values of cystic fibrosis patients with their agematched controls. A P value Ζ0.05 was considered significant.
well as for the cystic fibrosis patients and their agematched controls, are shown in Figs 1 and 2 respectively. It is evident that both sets of patient populations have more variable desmosines readings (higher standard deviations) when sampled over a time frame as short as 2 weeks. A statistical comparison of these variations show significant differences in the magnitude of the standard deviations for both the COPD (PΖ0.01) and cystic fibrosis (PΖ0.005) populations relative to their respective age-matched controls. The overall mean±SE for all three groups (all readings combined) is shown in Fig. 3. Mean values for the three adult groups (COPD, COPD smokers, controls) ranged from 28.4 to 35.5 pmol desmosines/ mg creatinine and there were no differences among the groups. Values in children were higher: 55 pmol desmosines/mg creatinine in the non-cystic fibrosis children and 77 pmol desmosines/mg creatinine for the cystic fibrosis group (P<0.01 when compared with the age-matched non-cystic fibrosis group).
RESULTS DISCUSSION The demographics for these patient populations and their age-matched controls are shown in Table 1. The number of subjects per group ranged from 29 to 31, with the exception of the COPD smoker subset, which totalled nine patients. The other difference of note relates to the distribution of sex in the groups. There were approximately equal numbers of males and females tested in the cystic fibrosis group, their agematched control group, and the age-matched controls for the COPD group, while the COPD patient group itself had an overwhelming number of males. However, it should be noted that there is no reported difference in urinary desmosine levels related to gender, when normalized for creatinine clearance.13 The individual 24-h urinary desmosines values measured on four separate occasions, and the calculated standard deviations for these values, for COPD patients and their age-matched controls, as
In the present study, we have used an improved method of enrichment and HPLC quantification of urinary desmosines to study the activity of elastase in two patient populations where this enzyme may play a contributing role in the disease process. In both adults with COPD and children with cystic fibrosis, there was a significantly higher degree of variability in urinary desmosines concentration, when sampled four times over a 2-week period, than in age-matched controls. This suggests that, in both patient populations, neutrophil elastase activity surges from time to time, and is not as constant as that in age-matched controls. Intuitively this seems reasonable, and further suggests that treatment of either patient population with an elastase inhibitor could blunt these increases and normalize the fluctuations in activity. These results are consistent with the findings of Stone et al.8 and
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B Controls 110
100
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Fig. 1 Individual values (panels A and B) and standard deviations (panels C and D) of 24-h urinary desmosines concentrations in COPD patients (n=30) and age-matched controls (n=31). Individual (panels A and B) 24-h desmosines (combined isodesmosine plus desmosine) concentrations were determined as described in the text for each subject on four different days over a 2-week period. Values are expressed as pmol desmosines/mg creatinine. In panels C and D, the range of standard deviation for these four determinations per subject are shown.
Starcher et al.19 Increased variability in urinary desmosines in individuals with pulmonary dysfunction was also reported by Pai et al,13 but this study examined only a few patients, did not compare mean values, and cystic fibrosis patients were not examined. Of further interest were the overall significant increase in the mean levels of desmosines in the cystic
fibrosis patients relative to their age-matched controls and the lack of differences in mean desmosines values among the COPD group, age-matched controls, and the smoker-COPD subgroup. Since a similar overall mean increase was not observed an COPD patients, this suggests that enhanced neutrophil elastase activity may be more important in cystic fibrosis patients, and
Urinary desmosines as markers of elastase
B Controls
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Standard deviation (pmol des./mg creat.)
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0 500
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Fig. 2 Individual values (panels A and B) and standard deviations (panels C and D) of 24-h urinary desmosines concentrations in cystic fibrosis patients (n=29) and age-matched controls (n=29). Individual (panels A and B) 24-h desmosines (combined isodesmosine plus desmosine) concentrations were determined as described in the text for each subject on four different days over a 2-week period. Values are expressed as pmol desmosines/mg creatinine. In panels C and D, the range of standard deviation for these four determinations per subject are shown.
that this patient population might be more amenable to treatment with an elastase inhibitor. A limitation of the present study is the lack of a correlative measure of pulmonary function in these patients. It would be interesting to know whether, in a given patient, spikes in the excretion of desmosines
correlate with depressed ventilatory function. However, compliance for a study such as this is difficult, and pooling urine for 24-h collections does not allow analysis to determine the exact time of elastase flareup. In summary, the results of this study suggest that
pmol desmosines/mg creatinine
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D. C. Bode et al 100 90 80 70 60 50 40 30 20 10 0
(29) * (29) (30)
5.
(9) (31)
6.
COPD COPD A-M Smokers Control
CF
A-M Control
Fig. 3 Comparative mean (±SEM) desmosines concentrations for COPD, COPD-smoker, age-matched (A-M) children. The number of subjects in each group is indicated in parentheses above each bar. ∗PΖ0.05 when compared with A-M controls.
7. 8.
9.
urinary desmosines can be used as a surrogate marker for elastase activity in two patient populations, COPD adults and children with cystic fibrosis, where this enzyme has been implicated in the disease process. Increased variability over a 2-week period in the activity of this enzyme is evident in both populations, and an overall increase in the mean value is further observed in the cystic fibrosis group. As orally bioavailable inhibitors of neutrophil elastase are under development by several pharmaceutical companies, future studies are necessary to determine if this marker can be used to monitor the efficacy of an inhibitor, and, ultimately, to determine if inhibition of this enzyme can alter the pathogenesis and/or symptomatology of these diseases.
10. 11. 12. 13. 14.
15. 16.
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Date received: 15 May 2000. Date accepted: 8 June 2000.