Ceruloplasmin and Transferrin Levels Are Altered in Serum and Bronchoalveolar Lavage Fluid of Patients with the Adult Respiratory Distress Syndrome 1- 3

JULIE A. KRSEK-STAPLES, RICHARD R. KEW, and ROBERT O. WEBSTER4

Introduction

Although the exact mechanism of acute lung injury in the adult respiratory distress syndrome (ARDS) is unknown, neutrophils are thought to participate in the process, in part by generating oxygenradicals (reviewed in reference 1). During the respiratory burst, neutrophils produce superoxide anion and hydrogen peroxide, which can then react with iron in the Haber-Weiss reaction to form hydroxyl radical. Hydroxyl radical has been implicated as an initiator of lipid peroxidation (2). However, other iron-dependent reactions form radicals that also may initiate lipid peroxidation (2). These reactions include decomposition of lipid peroxides by ferrous and ferric iron to produce alkoxy and peroxy radicals, and formation of ferryl radical by the interaction of ferrous iron with hydrogen peroxide. Oxygen radical formation by neutrophils can therefore lead to lipid peroxidation by several different irondependent reactions. Cellular components of the lung are susceptible to oxidant-induced injury. Neutrophil-derived oxygen species can damage pulmonary parenchymal cells(3) and endothelial cells (4, 5). Damage to endothelial cells resulting in increased vascular permeability has been measured in granulocyte-mediated lung injury in rabbits induced by phorbol myristate acetate (PMA) (6) and in oxidant-mediated lung injury in rats provoked by xanthine oxidase and xanthine (7). Peroxidation of lung lipids has been shown to occur as a result of lung injury induced by endotoxin (8) or complement activated by cobra venom factor (9). Furthermore, intracellular DNA strand breaks have been measured in human leukocytes (10) and in target cells (11) when the leukocytes are stimulated with PMA. Ceruloplasmin and transferrin may help limit oxidant-induced lung injury. Ceruloplasmin is an acute-phase protein

SUMMARY The respiratory burst of neutrophlls generates oxygen radicals that can result In lipid peroxldatlon and may contribute to acute lung Injury In the adult respiratory distress syndrome (ARDS). Because ceruloplasmin and transferrin are Inhibitors of lipid peroxldatlon and may play a role In regUlating tissue InJury, antigen levels of ceruloplasmin and transferrin and ceruloplasmin oxidase levels were measured In the serum and bronchoalveolar lavage fluid (BALF) of ARDS patients (n 28), patients at risk for ARDS (n = 22), and normal control subjects (n 45). Serum ceruloplasmin levels were similar In ARDS (mean ± SEM) (3.8 ± 0.3 IJ.M) and at-risk (3.3 ± 0.4 IJ.M) patients compared with control subjects (3.2 ± 0.2 IJ.M). Serum transferrin levels were decreased in ARDS (14.9 ± 1.7 IJ.M) and at-risk (20.4 ± 1.7 IJ.M) patients compared with normal control subjects (32.9 ± 1.2IJ.M), and serum transferrin levels correlated with serum unsaturated Iron binding capacity (UIBC). Ceruloplasmin was detected In only one of 38 normal BALF samples (0.002 ± 0.002 IJ.M) and two of 13at-risk BALF samples (0.15 ± 0.1 J.LM), yet It was present In 17of 23 ARDS BALF samples (0.9 ± 0.2IJ.M).Transferrin wes also Increased In ARDS BALF (5.4 ± 1.1 J.LM) compared with at-risk (0.7 ± 0.5 IJ.M) and normal (0.4 ± 0.1 J.LM) samples. Ceruloplasmin that was present In the BALF and serum samples had functional oxidase activity, and purified human ceruloplasmin Inhibited hydroxyl radical formation by phorbol myrlstate acetate (PMA)-stlmulated neutrophlls. Weconclude from this study that ceruloplasmin and transferrin, which are both present In ARDS BALF, may play a role In regulating oxldant-medlsted Inflammation In the lung.

=

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AM REV RESPIR DIS 1992; 145:1009-1015

with ferroxidase activity that oxidizes iron from Fe2+ to Fe3 +. The ferroxidase activity of ceruloplasmin plays an important role in its function as an antioxidant (12-14). By maintaining iron in an oxidized state, ceruloplasmin may prevent it from participating in the Haber-Weiss and other iron-dependent reactions that initiate lipid peroxidation (14). In addition, iron oxidized by ceruloplasmin can then be bound by transferrin and removed from the site of inflammation. In this way ceruloplasmin and transferrin together may regulate the iron-dependent reactions that cause lipid peroxidation and contribute to acute lung injury. Because of possible anti-inflammatory roles of ceruloplasmin and transferrin in lung injury, serum and bronchoalveolar lavage fluid (BALF) from ARDS patients and patients at risk for ARDS were analyzed for antigenic levelsof these two proteins. The results were compared with control serum and BALF from healthy donors. Serum and BALF ceruloplasmin were also tested for oxidase activity because of the potential inactivation of biologic

activity during inflammation. In addition, serum unsaturated iron binding capacity (VIBC) was measured as a marker of transferrin antioxidant activity (AOA)(15). To investigatefurther the role of ceruloplasmin in regulating oxidant production, the ability of ceruloplasmin to inhibit the formation of hydroxyl radical by PMA-stimulated neutrophils was also measured.

(Received in original form July J, J99J and in revisedform November 25, J991) I From the Departments of Internal Medicine, Microbiology, and Cell and Molecular Biology,St. Louis University School of Medicine, St. Louis, Missouri. 2 Supported by National Institutes of Health Grant Nos. HL 30752, HL 36275, and HL 07050. 3 Correspondence and requests for reprints should be addressed to Robert O. Webster, Ph.D.. Divisionof Pulmonology, St. Louis UniversityMedical Center, 3635 Vista at Grand Boulevard, PO Box 15250, St. Louis, MO 63110-0250. 4 Recipient of National Institutes of Health Research Career Development Award No. HL05142.

1009

1010

KRSEK-STAPLES, KEW, AND WEBSTER

TABLE 1 DATA FOR PATIENT GROUPS AND NORMAL CONTROLS

Age Mean ± SEM Range Sex Mortality n BALF cell counts and differentials Total cells per 111 Neutrophils, % Lymphocytes, % Macrophages, % Eosinophils, %

At-Risk

Normal

46 ± 4 20-82 18M/10F 14/28 28

54 ± 5 21-72 17M/5F 8/22 22

28 ± 1 21-43 22M/23F

928 63 6 31 0.3

Methods Patient Selection Patients in the intensive care units at St. Louis University Hospital and the Medical College of Virginia Hospital were selected as p~evI­ ously described (16).Three groups of subjects were studied: (1) healthy, normal nonsmokdeers (n = 45); (2) patients at high risk veloping ARDS (n = 22); and (3) patients with ARDS (n = 28). The age and gender distributions for these groups are shown in table 1. Risk factors included one or more of the following: sepsis syndrome, pulmonary aspiration, and nonthoracic trau~a and hypotension. Sepsis syndro~e required the presence of two of the following ~ntena: teJ?1perature> 390 C or < 360 C; penpheral white blood cell count < 3,000 or > 12,000 cells/ mm'; or positive blood culture for a commonly recognized pathogen or a strongly susp~cted source for systemic infection from which a known pathogen had been identified. In addition the following must also have been present: a deleterious systemic response to infection such as metabolic acidosis, systemic arterial hypotension with systolic blood pressu~ < 80 mm Hg for more than 2 h, or systemic vascular resistance < 800 dyn/s/cm'. Pulmonary aspiration required a witnessed event with or without suction of gastric contents from the trachea. Trauma and hypotension required acute nonthoracic ~rauma., including surgical operations, associated WIth blood loss and systemic systolic blood pressure < 80 mm Hg for more than 2 h or the requirement of vasopressor agents for longer than 2~. Patients were considered to have ARDS If all the following criteria were met: bilateral pulmonary infiltrates on chest radiograph~ mechanical ventilation, pulmonary capillary wedge pressure < 15 mm Hg, a total static pulmonary compliance < 50 ~lIcm ~zO, and a partial pressure of oxygen In artenal blood (Paoz) to fractional inspired oxygen conce~­ tration (Floz) ratio (Paoz/~.z) < 200 w?Ile the patient was receiving positive end-expiratory pressure from the mecha~ical ~en~ilator. Patients were considered at high nsk If they had one or more of the three risk factors but failed to meet the criteria for ARDS. Diagnosis distributions for the patient groups are shown in table 2. Patients with cancer,

or

ARDS

± ± ± ± ±

224 7 2 6 0.1

± ± ± ± o±

893 49 7 43

45

316 9 3 7

117 2 10 88 0.1

a

± ± ± ± ±

9 0.3 1 1 0.1

chronic renal failure, connective tissue diseases, primary bacterial pneumonia, diab~­ tes mellitus head trauma or subarachnoid bleeding, chronic interstitial lung disease, ~r cardiogenic shock were excluded from ~hIS study. Nonsmoking subjects were recruited to serve as the control group. Informed consent was obtained from either the patient when possible or from next of kin or legal guardian. This study was approved by the ~n­ stitutional review boards for human studies at both institutions.

Bronchoalveolar Lavage HAL was performed by a modification of a previously described procedure (17). In control subjects, a flexible fiberoptic bronc?oscope was introduced transnasally.followIng lidocaine anesthesia of the upper airway, trachea, and proximal mainstem bronchi. In ARDS and high-risk patients the bronchoscope was introduced through an indwelling endotracheal tube while they were undergoing mechanical ventilation. Lidocaine was not utilized in these subjects. The distal end of the bronchoscope was gently wedged into the lateral segment of the right middle lobe, ~d five 5o-ml aliquots of room temperature sterile 0.9070 NaCl solution wereinstilled through the bronchoscope channel and aspirated by applying gentle suction with the injecting syringe. Recovery of instilled fluid was approximately 50% in the two patient groups and 70070 in the control subjects. The fluid obtained was immediately placed at 4 0 C. Bronchoalveolar Lavage Processing HAL fluid was filtered through two layers of sterile gauze to remove mucus strands, and

TABLE 2 PATIENT DIAGNOSIS Number of Patients per Group

Aspiration Sepsis Hypotension Trauma

ARDS

At·Risk

6 14 3

8 8 3 3

5

the cellular fraction was removed via centrifugation (400 x g for 15 min at 4 0 C). The supernatant was removed and centrifuged (85,000 x g for 30 min at 4 0 C) to remove surfactant for use in other studies. The supernatant was then concentrated appr~xi­ mately to-fold under nitrogen pressure USIng a 5 kD mol wt cutoff (Diaflo YM 5 ultrafiltration) membranes (Amicon Corp., Danvers, MA). The concentrate was aliquoted and frozen (- 70 0 C) until used. Protein concentrations werecorrected to represent a IO-foldconcentration. Total cell counts of unprocessed lavage fluid were performed using a hemacytometer. Aliquots of cells were pelleted onto glass slides with a cytocentrifuge (Shandon Southern Instruments, Sewickley, PAl and then stained with Leukostat'", a modified Wright-Giemsa stain (Fisher Scientific, Pittsburgh, PAl. Differential counts w~r~ determined by counting 200 cells under OIl Immersion (x 1,000). Total and differential cell counts from normal and patient lavages are shown in table 1.

Blood Collection and Processing Blood samples from patients were collected from indwelling pulmonary artery catheters; those from normal control subjects by venipuncture. The blood samples were allowed to clot at room temperature in 10 ml sterile serum separator tubes (Corvac; Sherwood Medical, St. Louis, MO). Following clot formation the blood was centrifuged (1,500 x g for min at 4 0 C). The serum was aliquoted and stored at - 70 0 C until used.

i5

Total Protein Measurement The Lowry method (18) was used to quantitate total protein in the HALF samples. Some samples were diluted from 1:to to 1:500 so that absorbances would be within the range of the standard curve (10 to 350 mg/L) constructed from bovine serum albumin (Pierce, Rockford, IL). A commercial kit (Stanbio, San Antonio, TX) based on the biuret method for protein determination was used to measure the total serum protein. The range of detection was 2 to 100 mg/ml. Quantitation of Ceruloplasmin and Transferrin Radial immunodiffusion (19) was used to measure ceruloplasmin and transferrin levels in serum and HALE Nephelometric grade antihuman ceruloplasmin and antihuman transferrin (Atlantic Antibodies, Scarborough, ME) were added to phosph~te-buffered saline (PBS), pH 7.4, and mixed WIthan equal volume of 2% Seakem ME agarose (FMC Bioproducts, Rockland, ME) in PBS. The amount of antigen in each sample was calculated with a standard curve of a calibrated human serum reference (Atlantic Antibodies). Because of differences in antigen. concentration in serum versus HALF and In serum of normal versus ARDS samples, separate standard curves were established for low, medium, and high concentrations of antigen. The lower limit of detection for ceruloplas-

1011

CERULOPLASMIN AND TRANSFERRIN IN ARDS

min and transferrin was IS mg/L. Data are presented as micromolars using a molecular weight of 80,000 for transferrin and 132,000 for ceruloplasmin.

Ceruloplasmin Oxidase Activity Ceruloplasmin oxidase activity wasmeasured using o-dianisidine dihydrochloride (Sigma Chemical Company, St. Louis, MO) as a substrate. Serum or concentrated BALF (50 j.11) was assayed at 5 and 15min in 750 j.11 of 0.1 M acetate buffer, pH 5.0, and 200 j.11 of7.88 mM o-dianisidine dihydrochloride at 30 0 C as previously described (20). The reaction was stopped by adding 9 M sulfuric acid (Fisher Scientific) and the absorbance read at 540 nm. Oxidase activity was calculated in international units as follows: (A 15min - Asmin) x 6.25 x 100 = U/L (21). Human ceruloplasmin (Sigma Chemical Co.) and ceruloplasmin heated to 1000 C for 10min were used as positive and negative controls, respectively. This assay was linear to 4 ug/ml of ceruloplasmin. Hydrogen peroxide (Fisher Scientific) wasalso used as a control to determine if hydrogen peroxide that may be present in the ARDS BALF samples would interfere with the measurement of ceruloplasmin oxidase activity.

Purification of Human Ceruloplasmin Ceruloplasmin was purified by a modified chromatography procedure (22). Four units of blood (450 ml each) from healthy donors were drawn into a 1/10 volume of sodium citrate (3.8OJo) and EDTA (10 mM). After centrifugation and removal of cells, protease inhibitors were added to give the following final concentrations: 152 j.1M s-aminocaproic acid (Sigma Chemical Company), I mM phenylmethylsulfonylfluoride (PMSF) (Sigma Chemical Company), and 20 j.1M leupeptin (Calbiochem Corp., San Diego, CAl. The plasma (approximately I L) was diluted with three volumes of double-deionized, distilled water to decrease the conductivity to 3 mS. The diluted plasma was applied to a 5 x 60 ern DEAE-Sephacel~ (Pharmacia LKB Biotechnology, Inc., Piscataway, NJ) anionexchange column equilibrated with 70 mM sodium phosphate buffer, pH 7.4, at a flow rate of 2 ml/min. The column was then washed with the same buffer and 25-ml fractions collected until the absorbance at 280 nm returned to background. Ceruloplasmin was eluted with a 2-L gradient of 0 to 0.4 M NaCI in the starting buffer at a flow rate of 2.5 ml/min. Fractions containing ceruloplasmin were identified by a gradient profile that included conductivity measurements and absorbance measurements at 280 and 610 nm. Ceruloplasmin-containing fractions wereconfirmed by Ouchterlony assay. These fractions were pooled and dialyzed against 30 mM sodium acetate buffer containing 0.1 M NaCI, pH 5.0. Albumin was removed by applying the pooled ceruloplasmin fractions to a 2.5 x 60 em blue Sepharosef CL-6B column (Pharmacia LKB Biotechnology) equilibrated in the same buffer. The flow rate for both the application and elution of ceruloplasmin

was 1.0ml/min. The column was washed and ceruloplasmin was eluted with a 1,600-ml gradient of 0.1 to 2.0 M NaCI in 30 mM sodium acetate buffer, pH 5.0. Ceruloplasmincontaining fractions were pooled and concentrated under nitrogen pressure using 30 kD mol wt cutoff (Diaflo YM 30 ultrafiltration) membranes (Amicon Corp.). They were then dialyzed against 50 mM sodium acetate buffer containing 0.1 M NaCI, pH 5.5, and frozen at - 70 0 C. Analysis of the final ceruloplasmin preparation by electrophoresis on a 7.5 to 15OJo sodium dodecyl sulfate (SDS)-polyacrylamide gel followed by immunoblotting revealed three major bands identified as ceruloplasmin, as recently described (23). These three bands comprised greater than 90% of the total protein on the gel by densitometry.

Isolation of Human Neutrophi/s Heparinized (10 U/ml) blood was obtained from healthy donors by venipuncture. Erythrocytes were sedimented using 6% dextran T-500 (Pharmacia LKB Biotechnology, Inc.) in normal saline. Density gradient centrifugation employing Lymphoprepf (Accurate Chemical and Scientific Corp., Westbury, NY) was utilized to isolate the neutrophils. Contaminating erythrocytes were removed by hypotonic lysis. The leukocytes were resuspended in PBS containing 0.1% glucose and were> 94% neutrophils and> 97% viable as determined by trypan blue exclusion.

Deoxyribose Degradation Assay The ability of ceruloplasmin to inhibit hydroxyl radical production by PMA-stimulated neutrophils was measured using a modified procedure (24) in which neutrophils (2 x 106 ) were added to a reaction mixture containing: 0.5 ml of 20 mM deoxyribose (Sigma Chemical Co.), 0.01 ml freshly prepared FeCl 3 or FeS04: EDTA mixture (10 mM FeCh or FeS04: 2 mM EDTA), and 0.28 ml PBS with 0.1% glucose. To the reaction tubes was added 500 ug/ml of (1) ceruloplasmin, (2) bovine serum albumin, which was essentially fatty acid free (Sigma Chemical Company), or (3) human IgG, which was purified as previously described (25). The reaction was then initiated by addition of PMA (Sigma Chemical Company) at a final concentration of 10 ng/ml in dimethylsulfoxide. The cells were then incubated 30 min at 37 0 C and the reaction stopped by centrifugation (15,600 x g for 2 min). Cell-free supernatants (0.8 ml) were mixed with 0.5 mI each of 1% 2-thiobarbituric acid (Sigma Chemical Company) and 2.8% trichloroacetic acid (Fisher Scientific). After boiling for 20 min, samples were centrifuged (850 x g for 10 min) to remove precipitated protein, and the absorbances of the supernatants measured at 532 nm.

Distribution Coefficient The distribution coefficient (DC) of ceruloplasmin or transferrin was calculated to determine the concentration of these proteins in the BALF relative to their concentration

in the serum. This calculation has been previously described (26, 27), and a modification of the formula was used as follows: DC

=

XBALP/TPBALP Xserum/TPserum

where X = the concentration of ceruloplasmin or transferrin in serum or BALF, and TP = the total protein concentration in serum or BALE A value of 1.0 indicates that the individual protein constitutes an equal percentage of the serum and BALF total proteins.

Transferrin Unsaturated Iron Binding Capacity in Serum Samples The unsaturated iron binding capacity (DIBC) of serum transferrin was measured using a commercial diagnostic kit (Sigma Chemical Company). Ferrous iron is added to the serum and binds to transferrin at unsaturated iron binding sites. The remaining unbound iron is measured spectrophotometrically by a reaction with ferrozine. The difference between the amount of unbound iron and the total amount added to the serum is the UIBC. Serum samples and reagent volumes were proportionally decreased to give a final reaction volume of I ml instead of 3 ml recommended by the commercial protocol. Transferrin UIBC in concentrated BALF samples was below the sensitivity of this assay and was not reported.

Statistics Data are presented as the mean ± SEM. For analysis of the patient data, normal probability plots were used to assess whether each set of data had a normal distribution. Because most of the data were not normally distributed, nonparametric statistical analysis was performed. For multiple comparisons of three groups, the Kruskal-Wallis test was applied, followed by an additional test by Dunn (28) to determine differences between groups. The Mann-Whitney U test was used to compare two groups. This analysis was done for both the comparison of the distribution coefficients of matched serum and BALF samples and for the comparison of normal serum and BALF with ARDS serum and BALF in the matched sample data. The deoxyribose degradation data was analyzed using analysis of variance (ANOVA) followed by a NewmanKeuls test.

Results

Ceruloplasmin, Transferrin, and Protein Levels in Serum Serum ceruloplasmin levelswere not significantly increased in either ARDS patients (3.8 ± 0.3 JlM) or at-risk patients (3.3 ± 0.4 JlM) compared with normal individuals (3.2 ± 0.2 JlM) (figure tal. Serum transferrin levels, however, were significantly decreased in ARDS (14.9 ± 1.7 JlM) and at-risk (20.4 ± 1.7 JlM) patients compared with normal individu-

KRSEK·STAPLES, KEW, AND WEBSTER

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0 ARDS Fig. 1. Ceruloplasmin(a) andtransferrin (b) levelsin the serum of ARDS (n = 14),at-risk (n = 10),and normal control (n .. 19)samples. Radial immunodiffusion was used to measure ceruloplasmin and transferrin. Solid lines represent the mean in each group.

als (32.9 ± 1.2 ~M) (figure lb). As in previous studies (26), we found that total serum protein levels were about 1.5fold lower in ARDS (46 ± 2 mg/ml) and at-risk patients (48 ± 3 mg/ml) than in control subjects (75 ± 1 mg/ml). When this overall decrease in total serum proteins in the patient groups was taken into account by expressing ceruloplasmin and transferrin as a percentage of total serum protein, ceruloplasmin comprised a significantly greater percentage of total serum protein in both ARDS (1.1 ± 0.1070) and at-risk patients (0.9 ± 0.1%) compared with normal individuals (0.6 ± 0.0070). This increase was nearly twofold higher in ARDS serum than control serum. Serum transferrin comprised a significantly smaller percentage of total serum protein in ARDS patients (2.5 ± 0.2070) compared with at-risk patients (3.5 ± 0.2070) and normal individuals (3.5 ± 0.1070).

Ceruloplasmin, Transferrin, and Protein Levels in BALF Ceruloplasmin was detected in 17 of 23 ARDS and two of 13at-risk BALF sam-

At Risk

Normal

Fig. 2. Ceruloplasmin (a) and transferrin (b) levels in the BALF of ARDS (n .. 23), at-risk (n .. 13),and normal control (n .. 38) samples.Radial immunodiffusion was used to measure ceruloplasmin and transferrin in the 1o-foldconcentrated BALFsamples. Solid lines represent the mean in each group.

ples, but only one of 38 normal individuals had detectable amounts of ceruloplasmin in the BALF (figure 2a). BALF ceruloplasmin levels in ARDS (0.92 ± 0.2 ~M) and at-risk patients (0.15 ± 0.1 ~M) were significantly increased compared with those in normal individuals (0.002 ± 0.002 IlM). Transferrin levels inARDS BALF (5.4 ± 1.1 ~M)werealso significantly greater than in at-risk patients (0.7 ± 0.5 IlM) or normal control subjects (0.4 ± 0.1 IlM). As previously observed (16, 26), total lavage protein levels were increased in ARDS patients (20 ± 3 mg/ml) versus at-risk patients (5 ± 3 mg/ml) and control subjects (1 ± 0.1 mg/ml). When ceruloplasmin was analyzed as a percentage of the total BALF protein, there was a significantly greater percentage of ceruloplasmin in the BALF of ARDS patients (0.5 ± 0.1070) compared with at-risk patients (0.1 ± 0.0070) and normal individuals (0.01 ± 0.01070). As a percentage of total BALF protein, transferrin was not significantly different in normal (2.9 ± 0.3070) versus ARDS (2.0 ± 0.2070) groups but was significantly lower in the at-risk group (0.7 ± 0.2070).

Ceruloplasmin Oxidase Activity Ceruloplasmin oxidase activity correlated (r = 0.9) with immunoreactive levels in the normal, at-risk, and ARDS serum samples and ARDS BALF samples (figure 3). The ceruloplasmin immunologic and oxidase activities wereeither too low or nondetectable in the at-risk and control BALF samples, so these groups were not included in this correlation. Hydrogen peroxide has been detected in concentrations of approximately 21lM in the expired breath of ARDS patients (29)and could also oxidize the o-dianisidine substrate in the ceruloplasmin oxidase assay.Therefore, H 1 0 2 from 0.5 IlM to 1 M was added to determine the amount of H 1 0 1 that would cause measurable oxidation of o-dianisidine and interfere with the determination of ceruloplasmin oxidase activity. Only concentrations greater than 10mM oxidized o-dianisidine dihydrochloride (data not shown). Because these concentrations represent more than I,OOO-fold those found in the expired breath of ARDS patients, hydrogen peroxide interference from the ARDS BALF in this assay is highly unlikely. Distribution Coefficients in Matched Serum and BALF Samples To evaluate the ceruloplasmin and transferrin composition of serum compared with BALF, a subset of matched samples from normal individuals and ARDS patients werecompared (figure 4). Tocalculate distribution coefficients, these data are expressed as a percentage of total protein. Ceruloplasmin constituted 0.03 ± 0.03070 of the total BALF protein and 0.6 ± 0.04070 of the total serum protein in the normal samples. A significantly greater percentage of ARDS BALF (0.5 ± 0.2070) and ARDS serum (1.1 ± 0.1070) was comprised by ceruloplasmin (figure 4a). Transferrin constituted nearly the same percentage of total protein in both the BALF (3.3 ± 0.3070) and serum (3.5 ± 0.1070) of normal samples. BALF and serum from ARDSlpatients were also similar in transferrin composition (1.7 ± 0.2 versus 2.4 ± 0.3%) but were significantly decreased compared with normal BALF and serum (figure 4b). Distribution coefficients computed from matched ARDS and control samples revealed an altered distribution of ceruloplasmin but not transferrin in ARDS patients. Normal individuals had a ceruloplasmin DC of 0.04 ± 0.04 because it was undetectable in all but one BALE However, ARDS patients had a higher ceruloplasmin DC (0.46 ± 0.13) because their

CERULOPLASMIN AND TRANSFERRIN IN ARDS

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Deoxyribose Degradation Assay Transferrin Unsaturated Iron Binding The inhibition of hydroxyl radical forCapacity in Serum Samples mation by ceruloplasmin in the presence Transferrin UIBC was measured in of PMA-stimulated neutrophils was meaARDS (n = 6), at-risk (n = 8), and nor- sured by the degradation of deoxyribose. mal (n = 7) serum samples as an index This reaction was dependent on the vaof potential transferrin antioxidant ac- lence state of the iron present in the mixtivity. Transferrin levelswere significantly ture. When the ferric form of iron (FeCI3) decreased in ARDS serum (14.5 ± 2.8 was used, inhibition by 500 ug/ml of J.1M) compared with at-risk (20.7 ± 2.1 ceruloplasmin (26 ± 2070) was not sigJ.1M) and normal (34.4 ± 2.2 J.1M) indi- nificantly different than the same conviduals (table 3). Although there was also centration of BSA (24 ± 3070) or IgG (24 ± 2OJo)(datanotshown). However,when a trend of decreasing transferrin UIBC in the ARDS (17.1 ± 7.1 J.1M) and at-risk the ferrous form of iron (FeS04 ) was (21.5 ± 3.6 J.1M) patients compared with used, ceruloplasmin inhibition of deoxynormal control subjects (38.1 ± 6.9 J.1M), ribose degradation (45 ± 7%) was twothese values did not reach statistical sig- fold greater than the inhibition by BSA nificance. Because transferrin levels and (21 ± 3070) or IgG (19 + 3070) (Figure UIBC were lower in patient than in nor- 5). Ceruloplasmin heated to 100° C for mal samples, correlation coefficients 10 min before addition to the assay inwere calculated for each group. Transfer- hibited less than the control proteins (darin UIBC correlated with the amount of ta not shown). transferrin in the serum samples of Discussion ARDS patients (r = 0.97), at-risk patients (r = 0.57), and normal controls The antioxidant effect of ceruloplasmin (r = 0.95) (table 3). The r value for the and transferrin has been measured in norat-risk group was much lower than the mal serum (15, 30) and may be imporARDS and normal controls because there tant in regulating tissue injury during inwerethree samples that had slightly high- flammatory conditions, such as ARDS. er UIBC relative to the transferrin levels. Ceruloplasmin acts as an acute-phase When calculated without these three protein with an increase of two- to threesamples, ther value for the at-risk group fold during inflammation (31). Conversewas 0.92. ly, transferrin is referred to as an "an-

TABLE 3

AROS

Fig. 4. Ceruloplasmin (a) and transferrin (b) in the matched serum and BALF samples from control subjects (n 12) and ARDS patients (n 9). Transferrin and ceruloplasmin are calculated as a percentageof the total protein in each sample.The serum and BALF samples were from the same donor or patient in each group.

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Fig. 3. Correlation of ceruloplasmin oxidase activity to immunoreactivelevels in control serum(solidcircles)(n = 19), at-risk serum (solid triangles)(n = 10), ARDS serum (solid squares)(n = 14), and ARDS BALF (+) (n = 23). r value = 0.9. Ceruloplasmin oxidase activity was measured using o-dianisidine dihydrochloride as a substrate.

i

SERUM TRANSFERRIN AND UIBC LEVELS

Transferrin, 11M UIBC,I1M r Value n

ARDS

At-Risk

Normal

14.5 ± 2.8 17.1 ± 7.1 0.97 6

20.7 ± 2.1 21.5 ± 3.6 0.57 8

34.4 ± 2.2* 38.1 ± 6.9 0.95 7

• p < 0.01 for normal versus ARDS transferrin.

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ti-acute-phase" protein with an actual decrease in serum levels during inflammation (32). Therefore, serum levels of ceruloplasmin and transferrin vary in opposite directions during inflammation. Serum transferrin was significantly decreased in ARDS and at-risk patients in this study. However, serum ceruloplasmin was not significantly increased in these patient groups. The lack of an increase in serum ceruloplasmin may be explained because of an overall decrease in the total serum protein of ARDS patients and at-risk patients compared with normal control subjects. If this decrease in total serum protein is taken into account and ceruloplasmin is expressed as a percentage of total protein, a significant increase in ARDS and at-risk serum samples is observed. In contrast, serum transferrin remains significantly decreased in ARDS serum samples when analyzed as a percentage of total serum protein. Increased ceruloplasmin and transferrin levels were measured in the BALF of ARDS patients in this study, similar to those previously reported by others (26). However, ceruloplasmin was detectable in only one of the normal BALF samples, which is in contrast to other studies (26, 27) in which very low ceruloplasmin levels « 0.5 ug/ml) were detected in normal BALE A difference in the concentration of the BALF samples may account for this discrepancy because the assays used here were similar in sensitivity. In both of the previous studies the BALF samples wereconcentrated at least 200-fold before radial immunodiffusion was performed, but the BALF samples in our study were only concentrated 10fold. In contrast to a lack of ceruloplasmin oxidase activity in lung epitheliallining fluid (33), ceruloplasmin oxidase activity in ARDS BALF samples correlated significantly with the immunologic activity (figure 3). This biologically active ceruloplasmin in the ARDS BALF could presumably interact with the transferrin and provide additional antioxidant protection in the lungs of ARDS patients. We also found that ARDS BALF had an increase in total protein, confirming previous studies (16,26). The protein increase may be due to a loss of integrity of the lung epithelial lining in ARDS patients, which results in a bulk flow of plasma proteins into alveoli (26). Thus, larger molecular weight proteins become less restricted from the lung during ARDS. Distribution coefficients measure the proportion of proteins in the lung relative to plasma and reflect the degree of

KRSEK-8TAPLES, KEW, AND WEBSTER

restriction of specific proteins from the lung. The relative changes in ARDS and normal distribution coefficients observed here are in agreement with those mentioned earlier (26). The transferrin DC was similar in ARDS and normal individuals because transferrin is relatively small (mol wt 80,000 D) and is not restricted from normal lungs. Therefore, the increase in permeability during ARDS does not affect the DC of transferrin. The significant increase in the ARDS ceruloplasmin DC indicates that ceruloplasmin, with a larger molecular weight (l32,OOO D) than transferrin, becomes less restricted from the lung during ARDS. The antioxidant activity. (AOA) of ceruloplasmin is a result of its ferroxidase activity. Likewise, the UIBC of transferrin is responsible for its AOA. This has been demonstrated by abolishing ceruloplasmin AOA through inhibition of its ferroxidase activity with sodium azide and eliminating transferrin AOA by preloading with ferric iron (30). Although on a molar basis serum ceruloplasmin has Io-fold greater AOA than transferrin (15), both proteins contribute to overall serum AOA because serum transferrin levels (micromolars) are approximately 10-foldgreater than ceruloplasmin. The contribution to overall serum AOA has beencalculated to be about 240/0 by ceruloplasmin and about 36% by transferrin (15, 30). Because of the importance of ceruloplasmin oxidase activity and transferrin UIBC in serum AOA, these activities were measured in the serum samples in this study. The ceruloplasmin oxidase activity and transferrin UIBC correlated with immunologic levels of these proteins in both patient and normal samples (figure 3 and table 3). In addition, transferrin concentrations were greater than ceruloplasmin in all three groups, which compensates for the lower AOA of transferrin. Serum transferrin concentrations were 10-, four-, and sixfold greater than ceruloplasmin in normal, ARDS, and at-risk samples, respectively. Higher levels of transferrin compared with ceruloplasmin were observed in patient serum samples despite the decrease in serum transferrin levels in patient samples compared with those from normal individuals. The correlation of UIBC with these transferrin levels suggests that transferrin has the ability to bind iron that is oxidized by ceruloplasmin, thereby protecting against ironcatalyzed oxidative injury. Ceruloplasmin has been shown to scavenge superoxide anion in the xan-

thine oxidase-hypoxanthine or xanthine oxidase-acetaldehyde chemically generated superoxide anion systems (34). In contrast, ceruloplasmin did not decrease the amount of superoxide anion produced by PMA-stimulated human neutrophils (Krsek-Staples, unpublished data). Because of these findings and the proposal that hydroxyl radical production by neutrophils may contribute to lung injury (35), the role of ceruloplasmin in preventing hydroxyl radical formation was investigated. The inhibition of purified human ceruloplasmin on hydroxyl radical formation by PMAstimulated neutrophils (figure 5) suggests a role for ceruloplasmin in regulating iron-dependent oxygen radical formation. This inhibition was dependent on the form of iron present. Significant inhibition of hydroxyl radical production compared with control proteins occurred only when the ferrous form of iron was used in the reaction mixture. This observation is consistent with the idea that ceruloplasmin oxidizes the ferrous form of the iron to prevent it from participating in the Haber-Weiss reaction, thus decreasing the amount of hydroxyl radical formed. Albumin has been proposed to act as a serum antioxidant in some oxidant-generating systems but does not usually inhibit iron-dependent hydroxyl radical formation (36).Albumin and IgG may havebeen weaklyinhibitory because they could act as site-specific targets for hydroxyl radical formation and compete with deoxyribose in the assay mixture. The ability of ceruloplasmin to inhibit the formation of hydroxyl radicals by PMA-stimulated neutrophils, along with an increase in biologically active ceruloplasmin and the presence of transferrin in the ARDS lung, suggeststhat these two proteins may act together in regulating oxidant-mediatedinflammation in ARDS. Acknowledgment The authors thank Dr. A. A. Fowler, Department of Medicine, Medical College of Virginia, and Dr. Thomas M. Hyers, St. Louis University Medical Center, for generously providing BALF and serum samples, and Ms. Sally Tricomi and Ms. Patty Dettenmeier for technical assistance. References 1. Repine JE, Bowman GM, late RM. Neutrophils and lung edema. Chest 1982; 818:478-508. 2. Halliwell B, Gutteridge JMC. The importance of free radicals and catalytic metal ions in human diseases. Mol Aspects Med 1985; 8:89-193. 3. Martin WJ II, Gadek JE, Hunninghake GW, Crystal RG. Oxidant injury of lung parenchymal cells. J Clin Invest 1981; 68:1277-88.

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4. Sacks T, Moldow CF, Craddock PR, Bowers TK, Jacob HS. Oxygen radicals mediate endothelial cell damage by complement-stimulated granulocytes. An in vitro model of immune vascular damage. J Clin Invest 1978; 61:1161-7. 5. Weiss SJ, Young J, LoBuglio AF, Slivka A, Nimeh NF. Role of hydrogen peroxide in neutrophilmediated destruction of cultured endothelial cells. J Clin Invest 1981; 68:714-21. 6. Shasby DM, Vanbenthuysen KM, late RM, Shasby SS, McMurtry I, Repine JE. Granulocytes mediate acute edematous lung injury in rabbits and in isolated rabbit lungs perfused with phorbol myristate acetate: role of oxygen radicals. Am Rev Respir Dis 1982; 125:443-7. 7. Johnson KJ, Fantone JC III, Kaplan J, Ward PA. In vivo damage of rat lungs by oxygen metabolites. J Clin Invest 1981; 67:983-93. 8. Demling R, LaLonde C. Relationship between lung injury and lung lipid peroxidation caused by recurrent endotoxemia. Am Rev Respir Dis 1989; 139:1118-24. 9. Ward PA, Till GO, Hatherill JR, Annesley TM, Kunkel RG. Systemic complement activation, lung injury, and products of lipid peroxidation. J Clin Invest 1985; 76:517-27. 10. Birnboim HC, Kanabus-Kaminska M. The production of DNA strand breaks in human leukocytes by superoxide anion may involve a metabolic process. Proc Natl Acad Sci USA 1985; 82:6820-4. 11. Schraufstatter I, Hyslop PA, Jackson JH, Cochrane CG. Oxidant-induced DNA damage of target cells. J Clin Invest 1988; 82:1040-50. 12. Al-Timimi DJ, Dormandy TL. The inhibition of lipid autoxidation by human caeruloplasmin. Biochem J 1977; 168:283-8. 13. Yamashoji S, Kajimoto G. Antioxidant effect of caeruloplasmin on microsomal lipid peroxidation. FEBS Lett 1983; 152:168-70. 14. Gutteridge JMC, Stocks J. Caeruloplasmin: physiological and pathological perspectives. CRC Crit Rev Clin Lab Sci 1981; 14:257-329. 15. Galdston M, Feldman JG, Levytska V, Magnusson B. Antioxidant activity of serum ceruloplas-

min and transferrin available iron-binding capacity in smokers and nonsmokers. Am Rev Respir Dis 1987; 135:783-7. 16. Fowler AA, Hyers TM, Fisher BJ, Bechard DE, Centor RM, Webster RO. The adult respiratory distress syndrome. Cell populations and soluble mediators in the air spaces of patients at high risk. Am Rev Respir Dis 1987; 136:1225-31. 17. Hunninghake GW, Gadek JE, Sawanami 0, Ferrans VJ, Crystal RG. Inflammatory and immune processes in the human lung in health and disease: evaluation by bronchoalveolar lavage. Am J Pathol 1979; 97:149-206. 18. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chern 1951; 193:267-75. 19. Johnson AM. Immunoprecipitation in gels. In: Rose NP, Friedman H, Fahey JL, eds. Manual of clinical laboratory immunology. 3rd ed. Washington, DC: American Society for Microbiology, 1986; 17-24. 20. Schosinsky KH, Lehmann HP, Beeler MF. Measurement of ceruloplasmin from its oxidase activity in serum by use of o-dianisidine dihydrochloride. Clin Chern 1974; 20:1556-63. 21. Lehmann HP, Schosinsky KH, Beeler ME Standardization of serum ceruloplasmin concentrations in international enzyme units with o-dianisidine dihydrochloride as substrate. Clin Chern 1974; 20:1564-7. 22. Arnaud P, Gianazza E, Miribel L. Ceruloplasmin. Methods Enzymol 1988; 163:441-52. 23. Sato M, Schilsky ML, Stockert RJ, Morell AG, Sternlieb I. Detection of multiple forms of human ceruloplasmin. A novel M, 200,000 form. J BioI Chern 1990; 265:2533-7. 24. Greenwald RA, Rush SW, Moak SA, Weitz Z. Conversion of superoxide generated by polymorphonuclear leukocytes to hydroxyl radical: a direct spectrophotometric detection system based on degradation of deoxyribose. Free Radic BioI Med 1989; 6:385-92. 25. Webster RO, Lawrence DA. Antigenic modulation of the cytophilic binding of guinea-pig IgG and IgM antibodies to homologous macrophages.

Immunology 1979; 36:659-70. 26. Holter JF, Weiland JE, Pacht ER, Gadek JE, Davis WB. Protein permeability in the adult respiratory distress syndrome. Loss of size selectivity of the alveolar epithelium. J Clin Invest 1986; 78:1513-22. 27. Bell DY, Haseman JA, Spock A, Mclennan G, Hook GER. Plasma proteins of the bronchoalveolar surface of the lungs of smokers and nonsmokers. Am Rev Respir Dis 1981; 124:72-9. 28. Dunn OJ. Multiple comparisons using rank sums. Technometrics 1964; 6:241-52. 29. Baldwin SR, Grum CM, Boxer LA, Simon RH, Ketai LH, Devall LJ. Oxidant activity in expired breath of patients with adult respiratory distress syndrome. Lancet 1986; 1:J1-4. 30. Pacht ER, Davis WB. Decreased ceruloplasmin ferroxidase activity in cigarette smokers. J Lab Clin Med 1988; 111:661-8. 31. Markowitz H, Gubler CJ, Mahoney JP, Cartwright GE, Wintrobe MM. Studies on copper metabolism. XIV. Copper, ceruloplasmin and oxidase activity in sera of normal human subjects, pregnant women, and patients with infection, hepatolenticular degeneration and the nephrotic syndrome. J Clin Invest 1955; 34:1498-508. 32. Brown EB. Transferrin: physiology and function in iron transport. In: Iron metabolism, 51st ed. Ciba Foundation Symposium. Amsterdam: Elsevier, 1977; 125-38. 33. Pacht ER, Davis WB. Role of transferrin and ceruloplasmin in antioxidant activity of lung epithelial lining fluid. J Appl Physiol 1988; 64(5): 2092-9. 34. Goldstein 1M, Kaplan HB, Edelson HS, Weissmann G. Ceruloplasmin: a scavenger of superoxide anion radicals. J Bioi Chern 1979; 254:4040-5. 35. Ward PA, Warren JS, Johnson KJ. Oxygen radicals, inflammation and tissue injury. Free Radic Bioi Med 1988; 5:403-8. 36. Halliwell B. Albumin - an important extracellular antioxidant? Biochem Pharmacol 1988; 37: 569-71.

Ceruloplasmin and transferrin levels are altered in serum and bronchoalveolar lavage fluid of patients with the adult respiratory distress syndrome.

The respiratory burst of neutrophils generates oxygen radicals that can result in lipid peroxidation and may contribute to acute lung injury in the ad...
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