INTRODUCTION

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Severe congenital deficiency of ₁-antitrypsin (AAT) is associated with an increased risk of development of panacinar emphysema and is implicated in approximately 2% of cases of chronic obstructive pulmonary disease (COPD) (1). Human neutrophil elastase (HNE) is the main protease inhibited by AAT in vivo, and it has been postulated that the emphysema in severe AAT deficiency is due to the unchecked proteolytic activity of HNE in the lungs. Intravenous AAT supplementation is capable of increasing the serum and alveolar lining fluid AAT concentration to normal levels (4); however, it has not been demonstrated that this treatment can prevent the development or progression of emphysema.

The cross-link amino acid desmosine (DES) is derived from cross-linked elastin. It is not metabolized and is eliminated primarily via the urine. Urinary DES excretion is therefore a specific marker for degradation of cross-linked elastin, and has the potential to act as a biological marker of the processes believed to cause emphysema and to be a useful end point in treatment trials of antielastases such as AAT. We hypothesized that increased elastin degradation in the lungs of severely AAT-deficient persons with emphysema would result in increased DES excretion in the urine. We further hypothesized that if neutrophil elastase were the cause of the elastin degradation, intravenous supplementation therapy with a sufficient quantity of AAT would result in a decrease in elastin degradation and therefore in urinary DES excretion. In this study of 12 subjects with emphysema due to severe congenital deficiency of AAT, we compared urinary DES excretion during a 4-wk period prior to the start of AAT supplementation therapy and during the subsequent 8 wk of AAT supplementation. Our goal was to determine whether supplementation therapy reduces the rate of DES excretion and therefore of elastin degradation.

	METHODS

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Subjects

Potential subjects for this study were identified through the Italian National Registry for the Identification of AAT Deficiency (5), and were referred for participation in the study by their treating physician if that physician felt that AAT supplementation therapy was warranted. Eligible subjects included patients of either sex, aged 21 yr or older, current or former smokers, with protease inhibitor phenotype PiZ or other phenotype associated with blood AAT levels below 11 µM (80 mg/dl), and with spirometric evidence of obstructive lung disease. Subjects were excluded if they had end stage emphysema such that completion of the study protocol was in doubt, acute respiratory infection, a history of hypersensitivity to blood products, any contraindication to inoculation with recombinant hepatitis B vaccine, fluid retention with risk of cardiocirculatory overload, or treatment with other investigational drugs within 30 d prior to enrollment. Women who were pregnant or breastfeeding were excluded, as the physiological changes occurring during pregnancy and lactation might influence connective tissue metabolism.

Study Protocol

The study protocol was approved by the Ethics Committee of the local health authority of each hospital from which subjects were recruited. Each subject provided written informed consent. Prior to the initiation of the study protocol, each subject who was hepatitis B surface antigen negative underwent vaccination against hepatitis B. At entry, each subject underwent a standardized medical history and physical examination and spirometry before and after bronchodilator administration. Blood samples were obtained for measurement of AAT level, white blood cell count, erythrocyte sedimentation rate, and serum creatinine. After enrollment into the study, each subject underwent a run-in period of 4 wk followed by 8 wk of unblinded, open-label AAT supplementation via weekly intravenous infusions of human AAT (Prolastin; Bayer Corporation, West Haven, CT) 60 mg/ kg body weight. Prolastin is a sterile, stable, lyophilized preparation of purified human AAT, which is prepared from pooled human plasma of normal donors by fractionation of plasma proteins with the Cohn- Oncley method, and heat treated in solution at 60 ± 0.5° C for not less than 10 h. The specific activity of Prolastin is  0.35 mg functional AAT/mg protein. Serum AAT level was measured just prior to the second and fourth infusions in order to demonstrate adequate trough levels of AAT. A spot urine specimen was obtained from each subject at each of the 4 weekly visits during the run-in period, prior to each of the 8 weekly infusions during the treatment period, and 2 d after the infusion of Weeks 2 and 4, for measurement of DES. Cotinine was also measured in each urine specimen for validation of self-reported smoking status. Throughout the study period, subjects were seen weekly by the referring physician. At each visit, an interim history and examination were obtained to identify possible intercurrent respiratory illnesses. The eight infusions of AAT were performed during the weekly visits under the direct control of the physician.

Measurement of Urinary Desmosine and Cotinine

Urine specimens were frozen at 20° C within 6 h of collection and sent to a central laboratory for measurement of DES using the method of isotope dilution and high-performance liquid chromatography (HPLC) previously described (6, 7). Briefly, [¹⁴C]DES (500 dpm) was added to each urine specimen. The specimen was then combined with an equal volume of 12 N HCl and refluxed under N₂ at 110° C to hydrolyze peptide bonds. The residue was dried and subjected to gel filtration steps in order to remove interfering contaminants and obtain fractions highly enriched in the relatively high-molecular-weight DES cross-link amino acid. DES was separated using an HPLC method that employs reverse-phase ion pairing on a C₁₈ column, and eluted DES was quantified by ultraviolet absorption spectroscopy. The eluant was collected and assessed for radioactivity in a liquid scintillation spectrometer, and the DES content of the sample determined using an isotope dilution calculation. Urine creatinine concentration was measured using a kit (Sigma Diagnostics, St. Louis, MO) based upon the Jaffe reaction (8), and DES content of the sample expressed as a ratio to creatinine content. Isodesmosine (IDES) was also measured in urine samples, assuming the same isotope dilution as that calculated for DES; because values for urine concentration of DES and IDES were highly correlated (r = 0.84, p < 0.001), only results for DES are presented. Urinary cotinine was measured in each urine specimen by radioimmunoassay to document that subjects were not currently smoking, and to assess extent of active or passive tobacco smoke exposure (9).

Analysis

All statistical analyses were performed using SAS software (SAS Institute, Cary, NC). Urinary excretion of DES during the run-in period was compared with DES excretion during the AAT supplementation period using a repeated measures analysis of variance. Analysis of covariance was used to adjust for potential confounding by changes in cigarette smoke exposure as measured by urinary cotinine concentration. The primary analysis included all urine specimens provided by all 12 subjects. Secondary analyses were performed excluding one subject for whom fewer than 75% of the expected samples were available for analysis, and excluding those specimens collected during periods of possible intercurrent respiratory infection or obstructive lung disease exacerbation, as determined by an increase in symptoms of cough or dyspnea.

	RESULTS

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Eight men and four women, all White, were enrolled in the study. Characteristics of the 12 subjects are shown in Table 1. The mean age was 54 (12) yr. One subject reported never smoking, whereas the others reported that they were former smokers, although measurement of urinary cotinine indicated that two subjects were current smokers throughout the course of the study (urinary cotinine persistently > 4 µM in each). Two others were exposed to significant levels of environmental tobacco smoke (urinary cotinine 0.2-0.7 µM). All subjects had chronic airflow obstruction and reduced diffusing capacity of the lung for CO DL_CO. Prior to treatment, all subjects had severely reduced AAT levels (17-69 mg/dl). The cause of AAT deficiency was PiZ protease inhibitor phenotype in 11 subjects; the other subject had an unidentified Pi phenotype, but severely reduced AAT level (baseline 37 mg/dl). Each subject completed 8 wk of AAT supplementation therapy according to protocol, with no adverse events noted. Serum AAT level measured 1 wk after the third weekly infusion of AAT was 111 (23) mg/dl, more than twice the baseline level (p < 0.001). All but one subject had trough AAT level > 80 mg/dl.

Mean urinary excretion of DES during the run-in period was 13.0 (5.0) µg/g creatinine. This is 73% above the mean DES excretion of 7.5 (1.4) µg/g creatinine, which we previously reported in 22 healthy nonsmokers, and slightly higher than that of 21 patients with COPD and a severity of lung function impairment similar to the subjects in the present study (7). During the 8-wk treatment period, the mean urinary DES excretion was unchanged at 13.0 (5.9) µg/g creatinine (p = 0.85) (Figure 1). There was no evidence of a trend toward lower rates of DES excretion over the 8 wk of the treatment period (Figure 2). These findings were not appreciably affected when the one subject who failed to adhere to all urine specimen collections was excluded from analysis. The two subjects who were current smokers, based on urinary cotinine concentration, had levels of urinary DES excretion (11.7 and 8.5 µg/g creatinine) that were somewhat below the group mean. The analysis of the effect of AAT supplementation on urinary DES excretion was not significantly affected by adjustment for urinary cotinine concentration.

View larger version (26K): [in this window] [in a new window]   Figure 1.   Urinary desmosine excretion during the 4-wk period prior to the initiation of AAT supplementation therapy, and during the 8-wk treatment period, by individual subject. The normal range for urinary desmosine excretion is shown in gray.

View larger version (16K): [in this window] [in a new window]   Figure 2.   Urinary desmosine excretion in the 12 subjects with severe AAT deficiency by week of study (mean value ± SE). The first infusion of AAT was given after collection of the Week 0 urine specimen, and continued weekly for 8 wk.

As urine specimens were collected prior to each AAT infusion, and thus at the trough of serum AAT concentration, we also assessed the rate of DES excretion in each subject 2 d following infusion during Weeks 2 and 4 after the start of therapy. The mean urinary DES excretion in these "peak" specimens was 11.8 (4.4) µg/g creatinine, which was slightly lower than the 13.2 (6.6) µg/g creatinine DES excretion in the preceding "trough" specimens, although this difference was of borderline statistical significance (p = 0.06).

There were 12 episodes of acute respiratory infection or exacerbation of obstructive lung disease occurring in five subjects, as evidenced by a change in clinical status consisting of an increase from the preceding visit in cough, sputum production, or dyspnea, as determined by the referring physician at the time of each weekly visit. Excluding these 12 specimens had no appreciable effect on the above analyses.

The change in rate of DES excretion between the run-in and AAT supplementation periods was not significantly related to age, sex, baseline lung function, white blood cell (WBC) count, erythrocyte sedimentation rate, symptom severity, baseline AAT level, or change in AAT level with supplementation.

	DISCUSSION

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Excess elastin degradation by endogenous proteases such as HNE is believed to play an important role in the pathogenesis of emphysema (10). This hypothesis is particularly compelling for emphysema resulting from severe congenital deficiency of AAT, in which the unopposed activity of HNE is presumed to allow an increased rate of degradation of lung elastin and other connective tissues. Desmosine, a cross-link amino acid present only in mature elastin, is excreted in the urine as a constituent of the peptides produced by elastin degradation. Measurement of urinary DES excretion should therefore provide an accurate measure of total body elastin degradation. An increased rate of DES excretion has been demonstrated in healthy smokers compared with nonsmokers, and in COPD patients compared with both of these groups (7). Even in apparently healthy smokers, DES excretion is higher in those experiencing rapid decline in lung function than in those without rapid decline of lung function (11). These findings support the concept that DES, a biomarker for lung parenchyma destruction, can provide insights into the pathogenesis of emphysema and also might rapidly provide evidence of treatment efficacy in studies of therapy for destructive lung diseases such as emphysema.

In the present study of AAT supplementation therapy in 12 patients with severe AAT deficiency and documented obstructive lung disease, we found that the rate of elastin degradation, assessed by mean level of urinary DES excretion prior to initiation of therapy, was 73% higher than in healthy nonsmokers. The level of urinary DES excretion was similar to that previously reported in patients with usual, smoking-related COPD, being intermediate between COPD patients who had quit smoking and those who continued to smoke (7). This observation provides confirmatory evidence that the rate of elastin degradation is increased in persons with emphysema due to AAT deficiency; however, we were unable to demonstrate a reduction in the rate of elastin degradation during 8 wk of AAT supplementation therapy.

There are several potential explanations for this unexpected finding. Inadequate study power is unlikely to explain the lack of effect of AAT supplementation on DES excretion. Given the standard deviation for change in DES excretion observed in this study, the sample of 12 subjects had 90% power to detect a change in DES excretion rate of 1.2 µg/g creatinine using the method described by Cohen for power calculations in paired t tests (12). This is less than one-fourth of the amount by which DES excretion in the subjects of this study is increased over the mean normal value, and less than half of the amount by which DES excretion is increased over the mean value we previously reported for formerly smoking COPD patients (7).

Measurement of urinary excretion of elastin degradation products or DES by immunoassays may be inaccurate due to the presence of cross-reacting substances in the urine, and studies using such assays have generated conflicting results regarding rates of elastin degradation in COPD (7). The technique of preparatory chromatography, HPLC, and isotope dilution employed in the present study for measurement of DES overcomes the problem of cross-reacting substances and this assay has successfully identified increased DES excretion in relation to smoking and COPD (7, 11). It is therefore unlikely that inaccurate measurement of DES is a cause of the negative findings of this study.

As subjects are clinically stable at the time of recruitment into the study, the likelihood of a disease exacerbation may be greater during the 8-wk treatment period than during the 4-wk run-in period. Because acute respiratory infection or exacerbation of obstructive lung disease might increase the rate of elastin degradation, all analyses were repeated after excluding specimens collected at the time of a possible infection or exacerbation, with no appreciable effect on the results. Thus, it is unlikely that intercurrent infection or disease exacerbation explains the observed results.

Although urinary DES excretion is a specific marker of elastin degradation, it is not specific as to the tissue of origin of the elastin degraded. The normal rate of lung elastin turnover accounts for approximately 19% of urinary DES (6). It is therefore possible that substantial increases in lung elastin degradation might account for only small increases in urinary DES excretion. Because of the demonstrable emphysema in our subjects, we believe the site of the excess elastin degradation is most likely the lung.

The blood levels of AAT achieved in the present study, even measured at their nadir just prior to the weekly AAT infusion, were well above the level at which emphysema risk is thought to be increased (13), although it is possible that the level of 80 mg/dl thought to be protective against development of emphysema in severe AAT deficiency is too low to accomplish this therapeutic goal. The protective efficacy of this level has never been empirically demonstrated. It may also be that 8 wk of therapy is insufficient to see an effect on connective tissue degradation, although no trend is apparent over the 8 wk of treatment.

It has been demonstrated that renal sequestration may act as a capacitor, dampening the rate at which changes in blood levels of elastin degradation products are reflected in urinary desmosine excretion (14). In that study, desmosine was elevated in kidney extracts 3 d after the intratracheal instillation of elastase, but had returned to baseline levels by 7 d. An earlier study in healthy human volunteers found that following ingestion of a large bolus of elastin from calf ligamentum nuchae there was a 10-fold increase in urinary excretion of desmosine between the second and third days after ingestion (15). Although urinary levels of desmosine remained elevated above baseline values 10 d after ingestion, they were not distinguishable from baseline values by 8 wk after ingestion. Moreover, we have previously demonstrated diurnal variation in desmosine excretion in women, despite possible damping of the effect by renal sequestration, using specimens collected approximately 12 h apart (16). These findings suggest that although renal sequestration of desmosine-containing peptides may delay the change in urinary desmosine excretion following a change in elastin degradation rate, the 8-wk study period should have been sufficient to observe a change in elastin degradation rate, had one occurred.

The mean rate of urinary DES excretion measured 2 d following the AAT infusion was 11% lower than that measured just prior to the AAT infusion. Although this modest effect is of borderline significance, and is likely due to random variation in DES excretion, it is plausible that an effect of AAT supplementation on elastin degradation is seen only at blood levels substantially above those present 1 wk after infusion. As AAT is normally produced in the liver and reaches the lungs via circulating blood, it is unlikely that inadequate lung penetrance of AAT explains the lack of effect on elastin degradation. There is some evidence, however, that AAT can be produced locally by lung epithelial cells and alveolar macrophages, and that the rate of AAT production by these cells increases in response to inflammatory mediators (17, 18). Therefore, the possibility that restoration of normal circulating levels of AAT might result in lung parenchymal AAT levels that are insufficient to protect against inflammatory lung injury must be considered.

The change in rate of DES excretion between the run-in and treatment periods was not significantly related to age, sex, baseline lung function, WBC count, erythrocyte sedimentation rate, symptom severity, baseline AAT level, or change in AAT level with supplementation. The small number of subjects makes it unlikely, however, that we would identify small or moderate effects of these variables on change in DES excretion with AAT supplementation. We are therefore unable to assess whether some patients might benefit more than others from AAT supplementation. For example, as cigarette smoking leads to an increased burden of neutrophils, and consequently of HNE, in the lung, it is possible that AAT supplementation may be of greater benefit to current than to former smokers. Our sample of two current smokers is too small to shed light on this possibility.

The findings of this study suggest that at least in the case of former smokers (75% of the present study population) with established emphysema due to AAT deficiency, short-term replacement of AAT does not inhibit ongoing elastin degradation. This study was based on the hypothesis that HNE is the major elastase degrading lung elastin in COPD due to AAT deficiency. It is possible that other elastases such as macrophage metalloelastase, which is not inhibited by AAT, may play an important role in the development and progression of emphysema (19). Another possibility is that while AAT deficiency may promote emphysema both through unopposed action of HNE and through a permissive effect on lung inflammation (13), once the inflammatory processes that cause emphysema are well established, neither AAT deficiency nor neutrophil elastase may be necessary for maintenance of an elastolytic state. If this is the case, then AAT supplementation earlier in the course of the pulmonary disease might be more effective.

In summary, using a biochemical assay of urinary excretion of DES, we have found that patients with emphysema due to severe AAT deficiency have an increased rate of elastin degradation. We find no evidence that the rate of elastin degradation is reduced by short-term AAT supplementation using a conventional weekly dosing regimen. Whether this reflects a lack of efficacy of AAT supplementation in the treatment of emphysema due to AAT deficiency is uncertain; however, given the personal and financial cost of AAT supplementation therapy, there is clearly a need for prospective trials of AAT replacement therapy that assess clinical and physiological measures of efficacy.