Impaired hemoglobin scavenging during an acute HIV-1 retroviral syndrome

Clinica Chimica Acta 411 (2010) 521–523

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Clinica Chimica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l i n c h i m

Impaired hemoglobin scavenging during an acute HIV-1 retroviral syndrome Joris R. Delanghe a,⁎, Jan Philippé a, Filip Moerman b, Marc L. De Buyzere a, Liesbeth L. Vynckier a, Alain G. Verstraete a, Dirk P. Vogelaers b, Linos Vandekerckhove b a b

Central Laboratory, Ghent University Hospital, De Pintelaan 185, B 9000 Gent, Belgium Department of Internal Medicine and Infectious Diseases, Ghent University Hospital, De Pintelaan 185, B 9000 Gent, Belgium

a r t i c l e

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Article history: Received 9 October 2009 Received in revised form 31 December 2009 Accepted 5 January 2010 Available online 13 January 2010 Keywords: Acute retroviral syndrome Hemoglobin scavenging CD 163 Haptoglobin Hemopexin

a b s t r a c t Background: We report a case of temporary impaired hemoglobin scavenging in a patient with an acute HIV1 retroviral syndrome. The patient was presented at the emergency department in a severe inflammatory state, mimicking bacterial sepsis and/or hemophagocytic syndrome. The serum showed a hemolytic aspect. In contrast, serum haptoglobin concentration was not decreased. Methods: The hemolysis index was determined and the visual absorbance spectroscopy spectrum of the serum was studied. α1 microglobulin and hemopexin concentrations were determined in serum. The presence of circulating hemoglobin:haptoglobin complexes in serum and the saturation of the haptoglobin were investigated using starch gel electrophoresis followed by peroxidase staining. CD163 expression on peripheral blood monocytes was analyzed using flow cytometry. Results: A temporarily impaired hemoglobin scavenging was documented by an increased hemolysis index, absence of decreased haptoglobin levels, presence of circulating hemoglobin:haptoglobin complexes in serum and decreased hemopexin and α1 microglobulin concentrations. Conclusions: A temporarily impaired hemoglobin scavenging was observed due to a transient CD163 pathway impairment following an acute HIV-1 retroviral syndrome. The patient improved clinically and biochemically after initiation of HIV-1 anti-retroviral therapy. The data suggest a transient HIV-1 mediated CD163 impairment, although a latent drug mediated block could not be ruled out completely. © 2010 Elsevier B.V. All rights reserved.

1. Introduction A 23-year old African male, returned from a 3 week holiday in Ghana with fever. He had unprotected sex during his stay. Four days before his return, the patient entered an outpatient clinic in Ghana with fever, myalgia and headache. A treatment with artemether, sulfadoxine– pyremethamine and ciprofloxacine was initiated. Upon arrival in Belgium (day 1), he was admitted to the emergency department of Ghent University hospital. Clinical examination showed no significant findings, except for a temperature of 38 °C. Upon admission, the patient's serum showed a pronounced hemolytic aspect (H-index: 168 corresponding to a serum free hemoglobin value of 1.68 g/l; reference values: b0.11 g/l) [1,2], in the absence of clinical and biochemical signs of hemolysis [3].

2. Methods HIV-1 viral load was assayed using an Ultrasensitive Amplicor HIV-1 Monitor Test v. 5.0; Roche Molecular Systems, Branchburg, NJ. Hemolytic index was assayed by photometry at 415 nm [1].Visible ⁎ Corresponding author. Tel.: +32 93322956; fax: +32 93324985. E-mail address: [email protected] (J.R. Delanghe). 0009-8981/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2010.01.006

absorbance spectroscopy of the serum was carried out on a Beckman DU-70 photometer. Serum hemopexin, α-1 microglobulin, serum hemoglobin and haptoglobin were assayed using immunonephelometry [2,4,5]. Analysis of circulating haptoglobin complexes, haptoglobin phenotype and haptoglobin saturation was carried out using starch gel electrophoresis of the serum followed by peroxidase staining [4]. Serum arthemeter concentration was assayed using gas chromatography coupled to mass spectrometry analysis (lower limit of detection of the assay: 6 ng/ml). CD163 expression on peripheral blood monocytes was analyzed using flow cytometry. 3. Results 3.1. Routine laboratory findings The patient's biochemical tests are shown in Table 1. The malaria antigen test and thin smear were both negative. Despite a negative HIV-1 ELISA in recent history, an HIV-test (Inno-Lia, Innogenetics) upon admission revealed a weak but clearly apparent gp41 band and a weak but positively scored p24 signal. On day 6, the CD3/CD4 count was very low (85 cells/µl). HIV-1 viral load was 66,000,000 copies/ml indicating an acute HIV-1 retroviral syndrome or at least acute HIV-1 seroconversion. The patient was initiated on day 7 on a highly active anti-retroviral therapy (HAART) regimen: emtricitabine 200 mg qd,

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Table 1 Selected laboratory parameters upon admission and during post-hospital period. Parameter, units (reference values)

Hemoglobin, mmol/l (8.26–10.99) Haptoglobin, g/l (0.3–2.0) Hemopexin, g/l (0.4–1.5) Total bilirubin, µmol/l (5.1–20.5) Alpha 1 microglobulin, mg/l (30–80) Aspartate transaminase, U/l (0–37) Alanine transaminase, U/l (7–40) Creatine kinase, U/l (90–195) Serum creatinine, µmol/l (64–103) T-helper lymphocytes (CD3+/CD4+), cells/µl (300–1400) HIV-1 viral load (copies/ml)

Time point (day 7: start HAART) Day 1

Day 6

Day 16

9.44 3.4 0.1 12.0 94

6.21 0.9 0.0 17.1 45

5.84 1.5 0.8 15.4 27

425 160 5137 174 –

163 129 3901 92 85

67 40 186 95 –

–

66×106 27,100

Day 33 6.89 1.7 0.9 15.4 25

Day 70 9.00 2.0 1.0 15.5 42

34 46 190 88 662

29 28 325 81 738

4960

1920

Day 1: hospital admission.

tenofovir 245 mg qd, and lopinavir 200 mg/ritonavir 50 mg 2 tablet bid. The clinical syndrome of sepsis resolved upon initiation of HAART. 3.2. Hemolysis testing Upon admission, total and direct bilirubin values were within the reference range. Lactate dehydrogenase activity in serum was 4121 U/l (ref. values: 231–462 U/l). Serum myoglobin value was 180 µg/l (ref. values: b50 µg/l), which is due to rhabdomyolyis which is a part of the acute retroviral syndrome [6]. Red blood cell count was 4.0×1012/l. Reticulocyte count was 0.32%. Surprisingly, serum haptoglobin concentration was 3.37 g/l (reference values: 0.3–2.0 g/l). In contrast, serum hemopexin was virtually absent (ref. values: 0.4–1.5 g/l). C-reactive protein was only moderately increased (maximum value: 13 mg/l). Urinalysis demonstrated presence of limited hemoglobinuria without hematuria. 3.3. Hemoglobin scavenging analysis As the hemolytic aspect of the patient's serum could not be explained by hemolysis, an impaired macrophage specific CD 163 hemoglobin: haptoglobin scavenging pathway was suspected. Subsequently these remarkable biochemical findings were elucidated. Fig. 1 summarizes the evolution of some laboratory parameters during the patient's hospitalization: the temporary impairment of the CD 163 scavenging pathway (evidenced by the combined increase of serum hemolytic index and (free+ complexed) haptoglobin and the depletion of hemopexin and

Fig. 1. Evolution of key parameters during the first days following admission. Haptoglobin (sum of free and complexed haptoglobin), hemopexin and hemolytic index (absorption at 415 nm).

Fig. 2. Visible absorbance spectroscopy of the serum sample obtained upon admission showed an atypical absorption spectrum with slightly shifted absorption maxima at 407 nm with minor absorption bands at 537 and 573 nm. The shifted spectrum suggests peroxidation of the hemoglobin.

α1 microglobulin) lasts more than 1 week, slowly returning to normal values (Table 1). Visible absorbance spectroscopy of the serum sample obtained upon admission showed an atypical absorption spectrum with slightly shifted absorption maxima at 407 nm with minor bands at 537 and 573 nm, suggesting peroxidation of the hemoglobin (Fig. 2). This was confirmed by starch electrophoresis of the same serum sample, followed by peroxidase staining [3]. Electrophoresis demonstrated that the patient had circulating complexes of Hp 2-2 phenotype with hemoglobin in the serum (Fig. 3). In vitro supplementation of hemoglobin to the patient's serum (obtained upon admission) did not result in a further increase of hemoglobin binding by the circulating haptoglobin, confirming the saturation of the circulating haptoglobin pool (Fig. 3). Upon admission, the observed ratio of hemoglobin:haptoglobin in serum corresponded with the stoichiometric ratio of saturated hemoglobin:haptoglobin complexes. These data suggest an impaired CD163 hemoglobin scavenging pathway [3]. Following the start-up of HAART and the withdrawal of artemether, the haptoglobin values and the red pigmentation of the serum values gradually disappeared with a common half-life of 2.2 days. Concomitantly, serum hemopexin levels slowly returned to normal. Hemoglobin electrophoresis showed absence of hemoglobinopathy.

Fig. 3. Starch gel electrophoresis followed by peroxidase staining. Lane 1 shows the presence of hemoglobin–haptoglobin complexes (Hb–Hp) in the patient's serum (sample obtained upon admission). Hemoglobin supplementation of the patient's serum (lane 2) does not increase the amount of Hb–Hp complexes; the presence of free Hp indicates saturation of the patient's hemoglobin binding capacity. For comparison, patterns of a Hp 1-1 phenotype (lane 3) and a Hp 2-1 phenotype (lane 4) are shown (the latter samples have been hemoglobin supplemented).

J.R. Delanghe et al. / Clinica Chimica Acta 411 (2010) 521–523

Upon admission, CD163 expression on peripheral blood monocytes was analyzed using flow cytometry which demonstrated on a slightly lower than normal expression of the CD 163 molecules. At the time corresponding with the maximal concentration of the circulating haptoglobin:hemoglobin complexes (day 1), gas chromatography coupled to mass spectrometry analysis of serum could not detect any traces of artemether (lower limit of detection of the assay: 6 ng/ml) in the patient's serum. 4. Discussion We report a patient with an otherwise unexplained inflammatory febrile syndrome, with organ dysfunction (renal, hepatic, muscular), in whom the pattern of biochemical abnormalities indicated a rare and recently recognised pathological condition, namely inhibition of hemoglobin scavenging. This physiological mechanism prevents oxidative and NO-scavenging toxic effects of hemoglobin. The macrophage specific CD 163 hemoglobin:haptoglobin scavenging pathway plays a major role in removing hemoglobin following hemolysis. The accumulation of haptoglobin:hemoglobin complexes in the patient's serum suggests an impaired CD163 hemoglobin:haptoglobin scavenging pathway [3,7]. Under physiological conditions, haptoglobin:hemoglobin complexes are instantly cleared from plasma with a half-life time of b10 min [4,8]. The accumulation of these complexes, together with the acute phase driven upregulation of haptoglobin explains the paradox between the hemolytic aspect of the serum and the increased haptoglobin values. Since hemopexin and α1 microglobulin have a much lower affinity for heme than the affinity of haptoglobin towards hemoglobin, serum hemopexin and α1 microglobulin values only tend to decrease when circulating haptoglobin has been depleted [5,8,9]. Apparently, the CD91-mediated hemopexin–heme scavenging pathway [8] was exhausted which resulted in a complete depletion of the circulating hemopexin because of the limited heme binding capacity of this second line defense protein. Following restoration of the major hemoglobin scavenging pathway, the hemopexin concentrations slowly recovered (Table 1). This unique case report deals with an unusual condition due to a temporary inhibition of the CD163 hemoglobin scavenger pathway. Impaired hemoglobin scavenging has recently been described in patients treated with gemtuzumab ozogamicin [3]. The coincidence of the impaired hemoglobin scavenging with the artemether treatment theoretically would suggest that the reported findings were induced as a possible side effect of the drug. The artemether metabolite artesiminine has a known affinity for heme containing proteins such as hemoglobin [10]. However, the level of artemether was below the level of detection (6 ng/ml), making this hypothesis unlikely. Recently, it was shown that the CD163 receptor [11] also functions as a macrophage receptor for bacteria. The scavenger receptor CD163 also

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appears to be a key entry mediator for the porcine reproductive and respiratory syndrome virus [12]. In the present case, the patient presented with a Hp 2-2 phenotype, which is rather rare in West-Africa [3]. Hp 2-2 has a multimeric character and a higher affinity for CD163 than the other Hp phenotypes. HIV viral load has been linked to haptoglobin [13,14]. Especially, HIV-1 infected patients carrying the Hp 2-2 phenotype show a worse prognosis, which is reflected by a more rapid rate of viral replication (in the absence of antiviral treatment) [13]. Our data do show a clinical case of impaired hemoglobin scavenging during acute HIV seroconversion, with resolving of both the clinical and biochemical findings upon initiation of HAART. A temporary blocking of the CD163 pathway by alternative ligands is assumed to be the cause of the impaired hemoglobin scavenging in this patient. 5. Conclusions The combination of an increased hemolytic index and increased serum haptoglobin values in the absence of either in vitro hemolysis or hemoglobin-based blood substitutes, is the key element which should render clinicians aware of impaired hemoglobin scavenging as a cause of an unexplained inflammatory syndrome. References [1] Provasek D, Jay D. Biochromatic determination of hemoglobin in Hitachi 717. Clin Chem 1992;38:942 [Abstract]. [2] Lammers M, Gressner AM. Immunonephelometric quantification of free haemoglobin. J Clin Chem Clin Biochem 1987;25:363–7. [3] Maniecki MB, Hasle H, Friis-Hansen L, et al. Impaired CD163-mediated hemoglobinscavenging and severe toxic symptoms in patients treated with gemtuzumab ozogamicin. Blood 2008;112:1510–4. [4] Langlois MR, Delanghe JR. Biological and clinical significance of haptoglobin polymorphism in humans. Clin Chem 1996;42:1589–600. [5] Penders J, Delanghe JR. Alpha 1-microglobulin: clinical laboratory aspects and applications. Clin Chim Acta 2004;346:107–18. [6] Douvoyiannis M, Litman N. Acute encephalopathy and multi-organ involvement with rhabdomyolysis during primary HIV infection. Int J Infect Dis 2009;13:e299–304. [7] Kristiansen M, Graversen JH, Jacobsen C, et al. Identification of the haemoglobin scavenger receptor. Nature 2001;409:198–201. [8] Delanghe JR, Langlois MR. Hemopexin: a review of biological aspects and the role in laboratory medicine. Clin Chim Acta 2001;312:13–23. [9] Hvidberg V, Maniecki MB, Jacobsen C, Højrup P, Møller HJ, Moestrup SK. Identification of the receptor scavenging hemopexin–heme complexes. Blood 2005;106:2572–9. [10] Ponmee N, Chuchue T, Wilairat P, Yuthavong Y, Kamchonwongpaisan S. Artemisinin effectiveness in erythrocytes is reduced by heme and heme-containing proteins. Biochem Pharmacol 2007;74:153–60. [11] Fabriek BO, van Bruggen R, Deng DM, et al. The macrophage scavenger receptor CD163 functions as an innate immune sensor for bacteria. Blood 2009;113:887–92. [12] Van Gorp H, Van Breedam W, Van Doorsselaere J, Delputte PL, Nauwynck HJ. Identification of the CD163 protein domains involved in infection of the porcine reproductive and respiratory syndrome virus. J Virol Dec 23 2009 [Electronic publication ahead of print]. [13] Delanghe JR, Langlois MR, Boelaert JR, et al. Haptoglobin polymorphism, iron metabolism and mortality in HIV infection. AIDS 1998;12:1027–32. [14] Delanghe J, Langlois M. Haptoglobin polymorphism and body iron stores. Clin Chem Lab Med 2002;40:212–6.