Use of Breath Carbon Monoxide Measurements to Assess Erythrocyte Survival in Subjects with Chronic Diseases

Benjamin L. Mitlyng, Jasvinder A. Singh, Julie K. Furne, John Ruddy, and Michael D. Levitt* 

Department of Medicine, Minneapolis Veterans Affairs Medical Center, 1 Veterans Drive, Minneapolis, Minnesota

Anemia is very common in patients with chronic diseases. To determine the role of in-creased red blood cell (RBC) turnover in such subjects, we estimated RBC survival in three groups of chronically ill patients using a simple technique in which RBC life span is esti-mated via measurements of breath carbon monoxide concentration. The study groups con-sisted of subjects with: (1) osteoarthritis, (2) rheumatoid arthritis, and (3) anemia who were hospitalized for treatment of a variety of chronic illnesses. None of the anemic subjects had evidence of hemorrhage, a deficiency state, or a marrow abnormality to account for their reduced hemoglobin concentration. Subjects with osteoarthritis had a mean RBC life span (127 ± 25 days) that did not differ significantly from normal (122 ± 23 days). In contrast, RBC life span was significantly reduced (P < 0.001) in both the rheumatoid arthritis subjects (90 ± 15 days) and the anemic, hospitalized patients (87 ± 33 days). The hemoglobin concentration of the rheumatoid patients was near normal (13.5 ± 1.5 g/dl), indicating that the marrow was compensating for the reduced RBC life span, whereas no such compensation was apparent in the anemic, chronically ill subjects. We conclude that a modest (approximately 25%) reduction in RBC life span commonly occurs in patients with chronic disease, and this reduction becomes clinically relevant in subjects whose marrow cannot respond with increased RBC output. Am. J. Hematol. 81:432–438, 2006. CVV 2006 Wiley-Liss, Inc.

Key words: erythrocyte survival; anemia of chronic disease; carbon monoxide

INTRODUCTION

Chronically ill subjects frequently have an anemia that cannot be attributed to deficiency states, blood loss, hemolytic disorders, or cytological abnormalities of the marrow. When such patients suffer from chronic infectious, inflammatory, or neoplastic conditions, they are said to have anemia of chronic disease (ACD)[1,2]. Although excluded from the strict definition of ACD [2], a similar type of anemia occurs in chronically ill patients with a variety of other conditions including cardiac, pulmonary, renal, and endocrine diseases.

While the primary cause of the anemia in ACD is thought to be a cytokine-mediated inhibition of erythropoiesis [3–5], this problem would be aggra-vated by excessively rapid red blood cell (RBC) turnover. Red cell survival studies in patients with chronic diseases seemingly have been limited to two studies [6,7] that used Fe59 kinetics to assess RBC life span in patients with rheumatoid arthritis. We have developed and validated a simple, rapid, non-invasive technique to assess RBC survival based on measurement of the concentration of carbon mon-oxide (CO) in exhaled air [8,9]. In the present study, we employed this technique to assess RBC survival in subjects with rheumatoid and osteoarthritis as well as a chronically ill group of subjects with an anemia characteristic of ACD.

Research supported by funds Minnesota Veterans Research Institute.

*Correspondence to: Michael D. Levitt, Mpls. VAMC (151), 1 Veterans Drive, Minneapolis, MN 55417.

E-mail: Levit015@umn.edu

Received for publication 11 August 2005; Accepted 17 January 2006

Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ajh.20644

METHODS

Subjects

Erythrocyte survival measurements were obtained in 64 nonsmoking patients at the Minneapolis Veter-ans Affairs Medical Center (Mpls. VAMC). The arthritis group was ambulatory and consisted of 21 subjects (50–81 years old, 19 male, 2 female) with rheumatoid arthritis (RA) and 18 subjects (45–82 years old, 16 male, 2 female) with osteoarthritis (OA). These subjects were selected randomly from the clinic population based on their willingness to participate in the study, independent of the normal-ity of their hemoglobin concentration. The diagnosis of RA was made by a Mpls. VAMC rheumatologist using standard criteria [10]. Data concerning drug therapy, status of joint inflammation (flare versus stable disease as determined by a rheumatologist on the day of enrollment), erythrocyte sedimentation rate, and C-reactive protein were obtained on each RA subject. The OA group had clinical and radio-logical evidence typical of osteoarthritis involving primarily the hips and/or knees. None of the sub-jects had a history of heart failure, hepatic, renal, or pulmonary disease, recent hemorrhage, or transfu-sions within the past 3 months. In addition, we studied a group of chronically ill subjects consisting of 25 male hospitalized anemic patients selected because they had a: (1) hemoglobin < 12 g/dl; (2) normal or elevated serum ferritin, and (3) normal mean corpuscular volume (MCV). Exclusion criteria included a recent history of bleeding or transfusion and lung disease leading to carbon dioxide (CO₂) retention. These patients suffered from multiple con-ditions including urinary or pulmonary infections, diabetes mellitus, cholecystitis, coronary disease, mild renal insufficiency, and various forms of malig-nancy. Eleven of these 25 subjects died within 1 year of the time of study. The study was approved by the Institutional Review Board of the Minneapolis VAMC and was carried out according to the Decla-ration of Helsinki. Informed consent was obtained from all patients and controls.

Collection of Exhaled and Atmospheric Air

Alveolar air samples were obtained as described previously [9]. In brief, immediately upon awakening in the morning subjects closed their nares with a nose pinch, inhaled normally, placed the mouthpiece of a breath collection apparatus (AlveoSampler; Quintron Instruments, Milwaukee, WI) in their mouths, and sealed their lips around the mouth-piece. After a timed 20-s period of breath holding, the subjects exhaled into the collection system, which automatically discards the first 500 ml (dead space) and directs subsequent alveolar air into a self-sealing foil bag. After collection of a second, duplicate breath sample, an atmospheric sample from the bedroom was aspirated into a 20-ml syringe, and the syringe was sealed with a stopcock. The samples of the arthritic subjects were self-col-lected in their home and delivered to the laboratory, either directly or via the mail. Preliminary studies showed that the concentration of CO in the foil bag and syringe decreased by <3% during the up to 2-day period required for mail delivery.

Gas Analysis

To ensure that breath samples represented alveo-lar air, exhaled samples were analyzed for CO₂ con-centration via an infrared analyzer (CAPSTAR-100; CWE, Ardmore, PA). Data for the rare sample (6 of 128) that contained <5% CO₂ were not used. Concentrations of CO were determined by gas chro-matography using an instrument equipped with a

400-ml gas sampling valve, a column (6 ft * 0.125 inch) packed with molecular sieve 5A, and a reduc-tion detector (RGD2; Trace Analytical, Menlo Park, CA). The oven temperature was 1108C, and nitro-gen was used as the carrier gas (40 ml/min). The CO concentration of the unknowns was determined via reference to peak areas of standards of known concentrations. The means of the results of the duplicate CO measurements of alveolar air were determined for use in subsequent calculations. The precision of the CO assay was 0.3% (coefficient of variation) at a CO concentration of 5.2 ppm.

Hematological Measurements

Reticulocyte percentage was determined via a Coulter GEN-S apparatus (32,000 cells counted) and serum iron, transferrin, and ferritin via an auto-analyzer technique (Architect, Abbott Laboratories, Abbott Park, IL).

Calculation of RBC Life Span

Atmospheric CO concentration was subtracted from the alveolar concentration to obtain the sub-ject’s ‘‘endogenous alveolar CO concentration’’ [9]. The estimation of RBC life span (days) from the endogenous CO concentration (ppm) and hemoglo-bin concentration (g/dl) has been extensively dis-cussed and is calculated according to the equation (9)

RBC life span = (4)[Hb] (22,400)(blood vol)/

    0.7(endog PCO) (64,400) (1,440)(alv vent),

where 22,400 is milliliters of CO per mole, 4 is moles of CO released per mole of hemoglobin; 0.7 is the fraction of VCO derived from hemoglobin turn-over (0.3 of the CO is the estimated production from non-hemoglobin sources and ineffective eryth-ropoiesis); endogenous PCO is alveolar PCO minus atmospheric PCO (in parts per million); 64,400 is the molecular weight of hemoglobin; 1,440 is minutes per day. Because blood volume and resting alveolar ventilation vary with body size and both have roughly similar magnitude if blood volume is expressed as milliliters and alveolar ventilation as milliliters per minute, these two values cancel out. When [Hb] is in units of grams per milliliter and endogenous PCO is in parts per million, Eq. (1) reduces to the simple expression

Erythrocyte life span (days)

= 1380 [Hb]/Endogenous PCO.

Statistical Analyses

Results are expressed as means ± standard devia-tion (SD).

The various hematological indicies of patients with rheumatoid arthritis and osteoarthritis and those of chronically ill patients were compared using ANOVA. Correlation of RBC survival with percent-age reticulocytes and hemoglobin level was calcu-lated using Pearson’s correlation coefficient. A P value < 0.05 was considered statistically significant.

RESULTS

Hematological Values

Table I summarizes the results of hematological values obtained in the patients expressed as means± 1 SD. The mean hemoglobin concentration of the RA patients and the OA subjects was not signifi-cantly different. The ferritin concentrations of the RA subjects (obtained on 15 of the 21 subjects) were within normal limits. The mean reticulocyte percen-tages of the two groups of subjects were not signifi-cantly different and fell within the limits of normal (0.6–2.1%). The chronically ill, anemic patients had a mean hemoglobin concentration of 9.8 ± 1.1. While 66% of these subjects had a low serum iron concentration, none had an elevated iron-binding capacity. Each of these subjects had a normal to ele-vated serum ferritin and a normal MCV (inclusion criteria). The mean reticulocyte percentage of these subjects was normal but ranged widely with 3 and 5 of the subjects, respectively, having values falling below and above normal limits for this test.

RBC Life Span

Subjects with arthritis. Figure 1 shows the RBC sur-vivals of 42 previously studied healthy controls (using the present technique) with that of the sub-jects in each of the three study groups. The RBC life span of patients with RA (90 ± 15 days) was signif-icantly less (P < 0.001) than that of either the healthy controls (122 ± 23 days) or the OA subjects (128 ± 26 days). No significant differences in RBC survival were observed among the RA patients when analyzed on the basis of various therapies (metho-trexate, sulfasalazine, prednisone) versus no therapy; acute flare of joint disease versus stable disease; and level of C-reactive protein or erythrocyte sedimenta-tion rate. Figure 2 shows plots of RBC life span ver-sus the reticulocyte percentage and hemoglobin con-centration for RA patients. The inverse correlation between RBC survival and reticulocyte percentage was statistically significant. The correlation between hemoglobin concentration and RBC survival was not significant. The RBC life span of OA subjects did not differ significantly from that of healthy con-trols, and there was no significant correlation be-tween RBC life span versus reticulocyte percentage or hemoglobin concentration in these subjects (data not shown).

Chronically ill, anemic subjects. The mean RBC sur-vival of these subjects (87 ± 33 days) was signifi-cantly less (P < 0.001) than that of the healthy con-trols (see Figure 1). As shown in Figure 3, the RBC survival of these subjects did not significantly corre-late with their reticulocyte percentage, while there was a trend (r = 0.34, P = 0.10) toward a significant correlation between RBC survival and hemoglobin concentration. The degree of renal insufficiency, as indicated by creatinine concentration, did not signif-icantly correlate with either the hemoglobin concen-tration (r =-0.19) or RBC survival (r=-0.12).

DISCUSSION

Under steady-state conditions, a subject’s hemo-globin concentration reflects the balance between hemoglobin delivery to and removal from the circu-lation. The major problem in ACD has been assumed to be inadequate hemoglobin production; however, the anemia resulting from defective RBC production would be aggravated by reduced RBC survival. Knowledge concerning the role of RBC turnover in disease states has been limited by the lack of a simple, rapid, noninvasive means of assess-ing RBC life span. The only technique employed with any frequency to quantitate RBC survival in the clinical situation involves labeling circulating RBCs (usually with ⁵¹Cr), followed by measure-ments of disappearance of the label from the circu-lation. Since the normal ½ time of such labeled cells is about 27 days, a single RBC life span measure-ment requires venesections over a multiweek period. While measurements of RBC creatine [11] and HbA1c concentrations [12] have been touted as sim-ple indicators of RBC survival, there has been lim-ited study of the validity of these measurements.

In the present study, we utilized measurements of breath CO concentration to estimate RBC turnover. This technique is based on the concept that virtually all CO produced in the body is derived from the a-methene carbon of heme, which is released during cleavage of the heme molecule by heme oxygenase [13,14]. Since the bulk of heme turnover reflects hemoglobin destruction, and all CO released from this process is excreted via the lungs, the breath CO concentration attributable to this endogenous pro-duction (as opposed to the environment) provides a measure of the rate of hemoglobin breakdown. The assumptions underlying this technique have been extensively discussed in previous publications [9,15], and RBC life spans obtained with this technique were shown to correlate with values obtained with ⁵¹Cr-tagged RBCs and fecal bile pigment excretion [8]. In contrast to other methods, the CO technique is noninvasive, simple, and provides near-real time measurements of the rate of hemolysis. However, this technique cannot be employed in smokers or subjects with severe pulmonary dysfunction, and ineffective erythropoiesis will be reflected as a decrease in RBC survival. This technique yields a mean value for RBC survival for healthy subjects of 122 ± 23 (1 SD) days. While it is believed that the RBC survival of healthy subjects is almost always about 120 days, results obtained with several differ-ent techniques using ⁵⁹Fe kinetics [6] or bilirubin production [16] have also found appreciable individ-ual variability in RBC survival.

A ferrokinetic technique was used to assess RBC survival in two previous studies of anemic, non-iron-deficient RA patients, i.e., patients assumed to be suffering from ACD. These studies found mean reductions in RBC survival of 21% (normal:114 days, ACD: 90 days) [6] and 17% (normal 98 days, ACD: 81 days) [7]. In the present study, we com-pared the RBC life span of subjects with RA with that of subjects with OA, an arthritic condition with a minimal systemic inflammatory component. While the prevalence of anemia in RA is said to be between 33 and 75% [17], most of our subjects had normal or near-normal hemoglobin levels (mean:13.5 ± 0.34 g/dl). Presumably, these subjects had less active RA than patients investigated in pre-vious reports. Nevertheless, the RBC survival (90 ± 15 days) of our RA subjects was significantly reduced (P < 0.001) relative to that of healthy con-trols (122 ± 23 days) and OA subjects (128 ± 26 days). The failure of the hemoglobin of the RA sub-jects to fall commensurately with the roughly 25%reduction in RBC survival indicates that the bone marrow was responding with an increased RBC out-put. This concept is supported by the finding of a significant inverse correlation between RBC survival and reticulocyte percentage (Figure 2).

The mean RBC life span (87 ± 33 days) of the anemic, chronically ill subjects was significantly less (P < 0.001) than that of the healthy controls or that of the OA subjects (P < 0.001) and comparable to that observed in RA. In contrast to the situation with the RA patients, there was no significant corre-lation between RBC survival and reticulocyte per-centage (Figure 3), suggesting that the erythropoietic response was not closely linked to RBC life span.

Previous studies reported that RBCs of subjects with ACD survived normally following transfusion into healthy subjects, whereas the life span of RBCs from healthy subjects was reduced following trans-fusion into ACD subjects [1,18]. Thus, the exces-sively rapid RBC turnover has been attributed to extracorpuscular factors. Normally, aging of RBCs is associated with decreased deformablity, decreased hemoglobin content, lower antioxidant activity, and increased concentrations of oxidized lipids and pro-teins [19–21]. Such alterations are thought to expose epitopes that are recognized by naturally occurring antibodies with resultant phagocytosis of the senes-cent RBCs. The elevated levels in ACD of various pro-inflammatory cytokines such as IL-1, TNF-a, and TGF-b are thought to induce similar senescent type changes in RBCs [22,23], with a resultant pre-mature removal of these cells from the circulation.

If stimulated by sufficient erythropoietin, a nor-mally responding marrow could enhance RBC output to counter the roughly 25% decrease in RBC life span observed in the RA subjects and the chronically ill, anemic patients investigated in this study. The near normal hemoglobin levels observed in the RA subjects indicated that the marrow was compensating for the increased RBC turnover, suggesting that some factor, presumably cytokines, was altering RBC sur-vival to a greater extent than RBC production.

The appreciable reduction in hemoglobin concen-tration in the anemic, chronically ill subjects indicates that the increased RBC turnover of these subjects was not accompanied by a compensatory output of erythrocytes. The defective erythropoiesis of ACD has been attributed to the ability of pro-inflammatory cytokines to inhibit a variety of factors including erythropoietin release [24,25], iron transfer from stor-age areas to marrow [26,27], and proliferation of erythropoietic precursors in the marrow [4,28,29].

The clinical importance of the reduced RBC life span in the anemia of the chronically ill patients is not clear. Correction of the observed hemoglobin levels (mean: 9.8 ± 1.1 g/dl) to that expected with a normal RBC life span (122 days) raised the observed hemoglobin to a theoretical value of 12.5 ± 2.7 g/dl. However, if rapid RBC turnover was the major determinant of the reduced hemoglobin concentration, one would expect a strong positive correlation between hemoglobin level and RBC life span rather than the weak correlation (r = 0.32, P = 0.10) shown in Figure 3. The lack of a strong correlation indicates that while the reduced life span played a role in the anemic process, the degree of a sub-ject’s anemia is largely related to the inadequacy of the erythropoietic response.

It has been hypothesized that the seemingly defec-tive hematopoiesis of ACD might not be a pathologi-cal process but a physiological response to decreased tissue O2 utilization that allows for diversion of eryth-ropoietic substrates to other metabolic pathways [30]. However, increased RBC turnover is wasteful of eryth-ropoietic substrates, and the modestly reduced RBC life span observed in our patients presumably reflects a pathological process that attains clinical significance when the marrow is unable to respond with an in-creased output of erythrocytes.

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