Journal oj Internal Medicine 1991 : 229: 49-54 ADONIS 095468209 100009

Serum erythropoietin levels in the anaemia of chronic disorders

J. CAMACHO, F. ARNALICH, A. F. ZAMORANO' & J. J. V A Z Q U E Z From the Department of Internal Medicine and the Haematology Unit. La Paz Hospital. Facultad Autonoma de Medicina. Madrid. S p i n

Abstract. Camacho J. Arnalich F. Zamorano AF. Vazquez JJ (Department of Internal Medicine and Haematology Unit. La Paz Hospital, Facultad Autcinoma de Medicina. Madrid, Spain). Serum erythropoietin levels in the anaemia of chronic disorders. journal ojlnternal Medicine 1 9 9 1 ; 229: 49-54.

Serum erythropoietin (S-EPO) levels were measured in 50 patients with anaemia of chronic disorders (ACD). classified into three groups according to their aetiology : inflammatory (n = 20). infectious (n = 15) and neoplastic (n = 15). The inflammatory group showed a higher mean S-EPO level (mean value+SEM, 69 f 11 mU ml-') than the neoplastic (43 5 mU ml-': P < 0.05) and infectious groups ( 2 7 + 4 mU ml-': P < 0.01). The S-EPO level in the inflammatory group also differed from that of 32 healthy controls ( 3 6 + 3 mU m1-I: P < 0.05). Fourteen patients with added iron deficiency (12 subjects from the inflammatory group) showed the highest S-EPO concentration (72 1 7 mU m1-l). Conversely, S-EPO levels were lower in febrile subjects (12 patients with infection and five with malignancy) than in non-febrile patients (28+4 mU ml-' vs. 55 + 7 mU ml-' : P < 0.01). In the infectious group, the logarithm of S-EPO correlated directly with the haemoglobin and haematocrit values. We conclude that differences in S-EPO concentration in ACD may be further related to the patient's iron stores and temperature. A decrease in EPO production may contribute to the pathogenesis of ACD secondary to infection.

Keywords: anaemia, erythropoietin, infection, inflammation, neoplasia.

Introduction The main underlying cause of anaemia of chronic disorders (ACD) is thought to be the failure of erythropoiesis to compensate for a slight shortening of red cell survival time [I , 21. In order to explain such a phenomenon, a defect in erythropoietin (EPO) secretion has been postulated, based on estimates performed using the polycythaemic mouse bioassay [3-71. However, contradictory results have been obtained in more recent studies using the radio- immunoassay technique ; some authors have con- firmed the above-mentioned hypothesis [8, 91, while others have considered the hormone response to be appropriate [ lo, 111. Such assays were performed on patients suffering from chronic inflammatory

Abbrevintiorrs: ACD = anaemia of chronic disorders. EPO = erythropoietin.

diseases, mainly rheumatoid arthritis (RA). At present, there is insufficient information available concerning other ACD-inducing processes.

The aim of this study was to determine whether the serum EPO level (S-EPO) differs in the three main ACD aetiological groups : those with chronic inflam- mation, infection and neoplasia. We have also investigated the relationship between S-EPO levels and other factors, such as iron metabolism, fever and consumptive syndrome.

Study population Fifty patients in whom ACD was diagnosed according to the published features [l, 21 were included in this study. They consisted of 20 male and 30 female subjects aged 16-75 years (mean age_+SD, 49 _+ 1 7 years).

Patients were classified into three aetiological

49

50 J. CAMACHO et al.

Table 1. Classification of ACD patients into three aetiological groups

No. of Sex Age* Croup patients (M/F) (years)

Inflammatory ( n = 20) 1/19 4 6 k 3 (16-73) Rheumatoid arthritis 20

Infectious (ti = 15) 9 /h 44k4 (22-74) Pulmonary tuberculosis 5 Purulent abscesses 3 Empyema 2 Bacterial endocarditis 2 Pyelonephritis 2 Brucellosis 1

Neoplastic (n = 15) 10/6 61 f 4 t (24-75) Hodgkin's disease 3 Non-Hodgkin's lymphoma 5 Carcinoma 7

Mean values+SEM are given: the range is shown in parentheses. t P c 0.01 with regard to the inflammatory and infectious groups (unpaired t-test).

groups : inflammatory, infectious and neoplastic, as shown in Table 1. In addition, three clinical sub- groups were considered.

(a) Febrile state, defined as a temperature 2 38 O C

measured at least once during the previous 24 h. .These patients belonged to the infectious (1 2 cases) and neoplastic (5 cases) groups.

(b) Severe consumptive syndrome, defined as a weight loss 2 10% of the usual body weight of the patient, accompanied by marked asthenia and/or anorexia. Consumptive patients also belonged to the infectious (5 cases) and neo- plastic (10 cases) groups.

(c) Added iron deficiency (ID). including 14 patients whose bone marrow (BM) iron stores

were estimated to be scarce or low (Kath index 1 or 2) by the Pearls' staining technique. Twelve ID patients suffered from active KA. A11 patients in whom BM stores were absent (0 index) or the serum ferritin level was < 2 0 p g I-' were ex- cluded.

For inclusion in the study, the following criteria also had to be fulfilled: lack of analytical and histological data indicating neoplastic BM invasion, absence of hypoxaemia (arterial oxygen saturation =- 90%), renal failure (serum creatinine < 132 pmol I-'), hepatic failure, endocrinopathies (hypo- thyroidism. hypopituitarism and hyperparathyroid- ism), and no concomitant or recent therapy with iron, vitamins or potentially myelosuppressor drugs.

Table 2. Haematological parameters in the three ACD aetiological groups.

Methods A 10-ml sample of peripheral venous blood was obtained from each patient, following a 12-h over- night fast. The following analyses were performed on all samples: (a) blood count, using a Hemalog-6001 automatic counter: (b) serum iron and transferrin. transferrin iron saturation and serum ferritin ; and (c) serum erythropoietin level (S-EPO). The latter was measured in duplicate from 200-p1 samples, pre- viously frozen at - 2 0 "C, using a solid-phase enzyme-linked immunosorbent assay (ELISA) kit (EPO - EIA, JCL Clinical Research Corporation, Knox- ville, Tennessee, USA). Briefly, beads coated with (goat) anti-human EPO were incubated with test samples and appropriate standards and controls. The EPO standards were partially purified human urinary EPO preparations calibrated against the Second International Reference Preparation [ 121. EPO present in the samples binds to the solid phase.

Parameter

~

Inflamrna tory group (ti = 20)

Infectious Neoplastic group (11 = 15) group (n = 15)

Haemoglobin (g I-') 1 0 6 + 2 (76-126)

Haematocrit (I) 0 . 3 3 k 0 . 0 1 ( 0 . 2 6 4 . 3 8 )

Mean corpuscular volume (fl) 80k It (68-92)

Mean corpuscular haemoglobin (pg) 2 6 + 1 $ (2 1-29)

1 0 2 f 3 (88-122)

0.31 +0.01 ( 0 . 2 5 4 . 3 7 )

8 7 k 2 (76-97)

2 8 + 1 (22-34)

1 0 2 k 3 (84-1 16)

0.31 kO.01 ( 0 . 2 6 4 . 3 6 )

83 i - 2 ( 75-96)

2 7 + 1 (2 2-3 2 )

~ ~~ ~ ~

Mean valuesrf:SEM are given: the range is shown in parentheses. Differences were assessed by the unpaired 1-test. t P < 0.01 with regard to the infectious group. $ P c 0.05 with regard to the infectious group.

SERUM ERYTHROPOIETIN LEVELS I N ANAEMIA 51

Table 3. Iron metabolism parameters in the three ACD aetiological groups'

Parameter Inflammatory Infectious Neoplastic group (n = 20) group ( n = 1 5 ) group (n = 1 5 )

~ ~ ~ ~ ~

Serum iron (pmol I-') 5.8 k 0 . 4 6.1 k0.5 5 . 0 f 0 . 6

Serum transferrin (g 1-l) 2.14kO.lOt 1.61*0.09 1.63f0.12

Transferrin saturation (%) 13k1$ 17&1 1 4 + 4

Serum ferritin (pg I - ' ) 269k 130 5 8 j k 197 5 6 5 f 3 5 5

Bone marrow sideroblasts (%) 7 2 1 s 12*2 lo* 1

Bone marrow Rath index 2.2 f0.25 3.9 f 0.2 3 .5k0 .3

Mean valuesf SEM are given. Differences were assessed by the unpaired [-test. t P c 0.01 with regard to the infectious and neoplastic groups. $ P c 0.05 with regard to the infectious group.

P c 0.001 with regard to the infectious group, and P c 0.01 with regard to the neoplastic group.

Goat anti-human EPO conjugated with alkaline phosphatase is incubated with'the previously washed beads and, if EPO is present in the test sample, the conjugate binds to the EPO on the beads. Further addition of an enzyme substrate (paranitrophenyl- phosphate) results in the development of a coloured product, the intensity of which is measured with a spectrophotometer at 4 1 0 nm. The assay variability obtained in our laboratory using samples at low (mean value 12 mU ml-l), intermediate (mean value 32 mU ml-I) and high (mean value 106 mU ml-I) concentrations (n = 10) was less than 10% (intra- assay) and 12% (inter-assay). The minimum detec- tion limit was 3 mU ml-'. The S-EPO levels were also compared to those obtained from 32 healthy controls, 15 male and 17 female subjects, of similar age to the patient population (43 k 1 7 years; range, 19-75 years).

The statistical analysis was performed using the BMDP program. The differences in mean values between the patient groups and subgroups and the controls were assessed by the unpaired t-test. The correlation between variables was measured by means of Pearson's correlation coefficient. The level of statistical significance was taken to be P < 0.05.

Results The haematological and iron-related parameters corresponding to the three ACD aetiological groups are shown in Tables 2 and 3.

The mean haemoglobin and haematocrit values in the patients presenting with fever, consumptive syndrome or added ID did not differ significantly from those in the patients who presented no such findings (data not shown). Furthermore, 'there were no

P < 0.05 n

Control fn=,32)

PCQ.05 - PCO.01 P C 0.05

Fig. 1 . Serum erythropoietin (EPO) concentration in the healthy controls and in the three ACD aetiological groups. Columns represent mean values minus SEM.

differences with regard to the iron metabolism parameters, except in the ACD subgroup with ID (data not shown).

The S-EPO level in the inflammatory group (mean value +_ SEM 69 +_ 1 1 mU m1-I) was higher than that in the infectious ( 2 7 f 4 mU ml-I; P < 0.01) and neoplastic (43 k 5 mU rnl-I : P < 0.05) groups and the healthy controls (36 k 3 mU ml-': P < 0.05) (Fig. 1). In addition, the mean hormone concentra- tion was higher in the neoplastic than in the infectious group (P < 0.05).

52 J. C A M A C H O et al.

Table 4. Serum erythropoietin levels (S-EPO) in the three ACD clinical subgroups.

Subgroup S-EPO (mu m1-l) P-value

Fevert Present ( 1 1 = 17) 2 8 k 4 < 0.01 Absent (n = 33) 5 5 * 7

Present (n = 15) 3 6 + 5 Absent (n = 35) 5 2 + 8

Present ( n = 14) 7 2 k 7 41 _+4

Consumptive syndrome$

NS

Added iron deficiency5

NSYI Absent (n = 36)

Mean values+SEM are shown. Differences were assessed by the unpaired t-test. NS denotes no statistical significance. t Delined as a temperature 2 38 'C measured at least once during the last 2 4 h. $ Defined as a weight loss 2 10% of the usual body weight of the patient, accompanied by marked asthenia and/or anorexia. 5 When bone marrow iron stores were estimated as scarce or low (Rath index 1 or 2). 11 P < 0.05 comparing the log,, of S-EPO values.

-- L 2[ E 3 E - c + .-

e 8 1.5

0 W

c

s a

s i B 1

I

00

-

0 0

' & 0 0 m )o

o n o -

0. 8 0 0

0 0

0 0

0

I I 1 I I 75 I00 125

Hoemoglobin ( g 1 I I I I

0.25 0.30 0.35 Hoemotocrit (1)

Fig. 2. Correlation between the log,, of serum erythropoietin (log EPO) and the haemoglobin (0 ) ( r = 0.706. P < 0.01) and haematocrit (0) ( r = 0 . 5 7 9 . P < 0.05) in the ACD infectious group (n = 15).

As shown in Table 4. the febrile patients had a lower S-EPO value than the non-febrile subjects. On the other hand. the subgroup of patients with added ID displayed the highest mean hormone level of all anaemic subjects : the difference with respect to the non-ID patients reached statistical significance (P < 0.05), comparing the log,, of S-EPO (log S-EPO).

Similarly, in the whole patient sample the log S-EPO correlated with the serum transferrin level ( r = 0.439, P < 0.01), its saturation (r = -0.409. P < 0.01), the serum ferritin level ( r = -0.442, P < 0.01) and the percentage of BM sideroblasts ( r = -0.326. P < 0.05).

In the infectious group, the log S-EPO correlated directly with the haemoglobin and haematocrit values (Fig. 2). but no correlation was found in the other two groups, or in the ACD patients as a whole (data not shown).

Discussion The most important findings of this study are as

(a) Anaemic patients suffering from active RA (ACD inflammatory group) had higher S-EPO levels than those with ACD secondary to in- fection or malignancy, and the HC group.

(b) The maximum S-EPO level was observed in the ID subgroup.

(c) Febrile patients had a lower hormone con- centration than those without fever.

(d) In the ACD infectious group, the log S-EPO correlated directly with the haemoglobin and haematocrit values.

The S-EPO determination was performed by an ELISA method using polyclonal anti-human EPO antibodies. Although radioimmunoassays (RIAs) are the current standard procedure for measuring EPO levels in biological fluids, several disadvantages of this method [ 13, 141 have led to the development of new improved ELISAS for EPO, using highly specific monoclonal antibodies [15, 161. However, the ac- curacy and sensitivity of the ELISA performed in our laboratory were comparable with those of RIA and monoclonal antibody-based ELISA. We therefore con- sider that the S-EPO results obtained in the present study are reliable.

Since both haemoglobin and haematocrit values were similar in all groups and subgroups of ACD patients, other factors must be considered in order to explain the observed differences in S-EPO levels. In this context, the direct correlation between log S-EPO and BM iron stores might account, in part, for the higher hormone level in the inflammatory group and the maximum concentration observed in patients with added ID. The prevalence of ID within this group (60%) was similar to that of previous study samples of patiehts with RA [17-191. Our results are

follows.

SERUM ERYTHROPOIETIN LEVELS IN ANAEMIA 53

in agreement with those of other authors who have reported higher S-EPO levels in patients suffering from simple iron deficiency anaemia than in those with genuine ACD of similar intensity [7, 201.

The S-EPO concentration in the neoplastic group and in patients with consumptive syndrome (66% suffered from malignancy) did not differ from that of the healthy controls. This finding may be due to the presence of a nutritional deficiency, since previous authors have proposed the hypothesis that protein malnutrition, a frequently observed complication in malignancy, may cause a decrease in EPO production [21. 221. It is possible that the observed differences in neoplastic patients with regard to S-EPO levels [7,23, 241 may be due, in part, to differences in the degree of nutritional deficiency.

The lowest S-EPO concentration was observed in the infectious group and the febrile patients (70% belonged to this group). The correlation between haemoglobin and haematocrit values and the log S- EPO suggests that ACD secondary to infection may, to a large extent, be due to inhibition of EPO production. This hypothesis is supported by the observations of Udupa and Lipschitz who, by means of endotoxin injection, induced a hypoproliferating erythroid state in rats, which subsided following exogenous EPO administration [2 51.

It is conceivable that such hypothetical inhibition of EPO production may be related to extensive release of interleukin-1 (E-1) or tumour necrosis factor (TNF), since both monokines mediate fever and other components of the acute phase response [26, 271, and have also been found in high concentrations in the serum of febrile [28] and infectious [29] patients. Further studies are needed to elucidate the possible relationship between IL-1 or TNF and EPO production in different ACD-inducing processes and, in general, with the complex pathogenicity of such anaemia.

In summary. we have observed that the S-EPO level differed significantly in the three main aetio- logies of ACD: chronic inflammation, infection and neoplasia. However, such differences may also be attributed to other factors, such as the level of BM iron deposits, and the possible coexistence of systemic manifestations of the acute-phase response.

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Received 1 7 March 1990. accepted 30 May 1990.

Corresponderice : lost Camacho Siles. MD. Departamento de Medicina Interna. Fdificio de Maternidad. planta 10". Hospital La Paz. Paseo de la Castellana No 26 1 , 28046 Madrid, Esparia.