In vitro evaluation of the potential role of sulfite radical in morphine-associated histamine release

Emma M Gordon
BMC Pharmacology
Department of Pediatrics
Address: Division of Pediatric Pharmacology and Critical Care
Case Western Reserve University
ClevelandOhioUSA

Carolyn Myers
BMC Pharmacology
Department of Pediatrics
Address: Division of Pediatric Pharmacology and Critical Care
Case Western Reserve University
ClevelandOhioUSA

Jeffrey Blumer
BMC Pharmacology
Department of Pediatrics
Address: Division of Pediatric Pharmacology and Critical Care
Case Western Reserve University
ClevelandOhioUSA

In vitro evaluation of the potential role of sulfite radical in morphine-associated histamine release
10.1186/1471-2210-4-21BioMed Central Page 1 of 8 (page number not for citation purposes) Research article * Corresponding author
Background: Intravenous morphine use is associated with elevated histamine release leading to bronchoconstriction, edema and hemodynamic instability in some patients. This study evaluated the possibility that sulfite, which is present as a preservative in many morphine preparations, might contribute to histamine release in vitro.Results:The human mast cell line, HMC-1, was exposed to various morphine concentrations, in the absence of sulfite, under cell culture conditions. Clinically attained concentrations of morphine (0.018µg/ml and 0.45µg/ml) did not cause increased histamine release from mast cells. There was a significant increase in histamine release when the morphine concentration was increased by 1184fold (668µg/ml morphine). Histamine release from mast cells exposed to morphine and/or sulfite required the presence of prostaglandin H synthetase. Histamine release in experiments using sulfite-containing morphine solutions was not statistically different from that observed in morphine-only solutions.Conclusion: Sulfite in sulfite-containing morphine solutions, at concentrations seen clinically, is not responsible for histamine release in in vitro experiments of the human mast cell line, HMC-1. This does not preclude the fact that sulfite may lead to elevation of histamine levels in vivo.

Background

Morphine is among the most common analgesic agents used in the intensive care setting. It is usually administered parenterally as an intravenous infusion. Despite its efficacy, its use is associated with a number of hazards including respiratory depression [1], tolerance [2], physiological dependence [2] and abstinence upon discontinuation of treatment [2]. Less frequent, but perhaps more profound, is the bronchoconstriction, edema and hemodynamic instability seen in some patients following intravenous bolus doses of the drug [3][4][5][6][7]. These effects have been attributed to histamine release from mast cells by morphine [3][4][5][6][7].

A number of clinical studies have demonstrated an increase in plasma histamine concentration following intravenous morphine administration [3][4][5][6][7], but these studies generally fail to provide details concerning the formulation of the morphine employed. Most of the morphine used in both clinical practice and for clinical investigations contains sodium bisulfite as a preservative [8]. Until the mid 1980's sulfite preservatives were generally considered universally safe; however, their status was changed after the receipt by the FDA of more than 250 reports of serious and life-threatening reactions related to sulfite exposure through both diet and therapeutic agents [9,10].

In the body, sulfites are generally oxidized by the mitochondrial enzyme sulfite oxidase and non-toxic metabolites are excreted in the urine [11]. A small amount of the sulfite, either ingested or injected may also be metabolically activated by a number of other enzyme systems including xanthine oxidase [12], peroxidase [11] and prostaglandin H synthetase [13]. As an electron acceptor in these reactions or through electron transfer from transition metals such as copper and iron, sulfite may accept an electron from the resulting hydroxyl, superoxide or sulfate radicals [14] leading to the formation of sulfite radical.

Sulfite radicals are chemically reactive and have been implicated in lipid [15], protein [16] and nucleic acid [17] oxidation as well as neuronal injury [18]. They may stimulate neutrophils to release oxygen free radicals and augment the free radical response to other neutrophil activators [19]. In addition, their generation has been shown to contribute to oxidative stress resulting in sulfite toxicity and impaired B-cell function.

The potential generation of free radicals from sulfite preservatives may confound our understanding the association of morphine with histamine release in man. A number of studies have demonstrated that, when exposed to free radicals, mast cells may release histamine [20][21][22][23][24][25][26]. It is possible; therefore, that the histamine released following morphine infusion may be the result of the formation of sulfite radicals from the preservative rather than the morphine itself. This study evaluated the possibility that sulfite and its activation might contribute to histamine release by mast cells in vitro.

Results

Mast cell histamine release

Total histamine content of mast cells was calculated by freezing and thawing suspensions for 3 cycles. Cell suspensions containing 500,000 cells/ml released 1.27µg/ml (0.21) histamine, corresponding to 2.53 pg (0.41) histamine per mast cell.

Mast cells stimulated with 0.25µg/ml calcium ionophore released 63% (7.2) of total histamine.

Effect of sulfite-free morphine concentration on histamine release

The effect of histamine release from mast cells treated with both clinically attained concentrations of morphine and concentrations above those seen clinically was determined.

To chemically defined reaction mixtures, morphine was added at varying concentrations (18 ng/ml, 450 ng/ml, 6.68µg/ml and 668µg/ml). Enzymes causing free radical activation were not present. The blank sample contained no morphine. Reaction mixtures were incubated in a water bath at 37°C for 60 minutes. Experiments were performed in triplicate. Histamine release from samples was determined following derivitization with o-phthaldialdehyde and mercaptoethanol using high-performance liquid chromatography as detailed below. Histamine release from reaction mixtures was expressed as a % of total histamine present in the cell pellet.

Histamine release from reaction mixtures containing morphine at all concentrations was not statistically different (p > 0.1) from histamine release from blank samples ( Table 1).

To verify the time dependence of histamine release, a morphine concentration of 668 µg/ml was tested. Release was detectable as early as 5 minutes after the addition of drug and appeared to plateau between 40 and 60 minutes (Figure 1).

Effect of sulfite-containing morphine solutions on histamine release

To determine whether the addition of sodium bisulfite causes increased histamine release from mast cells, sodium bisulfite at a concentration of 0.1% of morphine concentration (the % of sulfite present in morphine most formulations) was added to the reaction mixtures described previously. Blank samples contained neither morphine nor sulfite. Specimens were incubated, dried, re-dissolved, derivitized and analyzed as above.

There was no statistical difference in histamine release at any concentration of morphine with sulfite compared with blank samples (Table 1).

Reaction mixtures containing 668µg/ml morphine with 0.1% sodium bisulfite were incubated for 5, 10, 20, 40 and 60 minutes yielded results identical to those observed in the absence of sodium bisulfite (results not shown).

Effect of prostaglandin H synthetase on histamine release from mast cell reaction mixtures

Prostaglandin H synthestase may catalyze free radical formation [20]. The first experiment investigated its effect on mast cell histamine release in the absence of sulfite and morphine. This ensured that any increase in histamine release in subsequent experiments was not due to the effect of the enzyme only.

Prostaglandin H synthetase was added to reaction mixtures at a concentration of 25 mU. In reaction mixtures containing no drug, the enzyme was added to mast cell samples containing neither morphine nor sulfite. Controls contained no enzyme.

Reaction mixtures containing the enzyme alone (no sulfite or morphine) produced similar histamine release compared to blank samples (3.2% (0.7) of total and 2.9% (2.6) of total respectively). The presence of prostaglandin H synthetase had no effect.

The effect of clinically attained concentrations of morphine in the presence of prostaglandin H synthetase on mast cell histamine release was investigated. In vivo studies show histamine release at these concentrations of morphine [3][4][5][6][7]. Morphine sulfate was added to the mast cell solution at clinically attained concentrations of 450 ng/ml (a typical peak in vivo concentration following an intravenous morphine bolus) and 18 ng/ml (a typical steadystate concentration during a 20 µg/kg/hour morphine infusion) [27]. Mast cell histamine release from higher concentrations of morphine (668µg/ml and 6.68µg/ml) was also investigated.

Blank samples contained water instead of morphine. All samples contained 25 mU of prostaglandin H synthetase. Specimens were incubated, dried, re-dissolved, derivitized and analyzed as described below. Histamine release values are shown in Table 2. There was no statistical difference between histamine release from morphine samples at both clinically-attained concentrations (18 ng/ml (p = 0.87) and 450 ng/ml (p = 0.08)) and histamine release from blank samples. Histamine release from HMC-1 cells incubated with morphine 668µg/ml and 6.68µg/ml were significantly increased compared with blank samples (p = 0.03 at both concentrations). Results are shown in table 2. Maximum histamine release (16.1% of total (6.3)) occurred at the highest concentration of morphine (668µg/ml) (  Morphine concentration (mcg/ml) Histamine release (% of total release) thetase was investigated. This preservative is present in many morphine preparations and its effect on histamine release from mast cells is determined here. Morphine was added to the mast cell samples at concentrations of 668µg/ml, 6.68µg/ml, 450 ng/ml and 18 ng/ml. Sodium bisulfite was added to samples at a concentration of 0.1% of morphine concentration. Controls contained morphine only. Each sample also contained 25 mU of prostaglandin H synthetase. Specimens were incubated, dried, re-dissolved, derivitized and analyzed as described below. Reaction mixtures containing morphine 668µg/ml with 0.1% sodium bisulfite showed a statistically significant increase in histamine release compared with blank samples (p = 0.03). There was no statistical difference in histamine release between morphine only samples at all concentrations and samples containing morphine and 0.1% sodium bisulfite ( Table 2) (morphine 668µg/ml (p = 0.8), morphine 6.68µg/ml (p = 0.13), morphine 450 ng/ml (p = 0.89) and morphine 18 ng/ml (p = 0.56). Sulfite did not have any additive effect on histamine release from mast cells in the presence of morphine and prostaglandin H synthetase.

Studies looking at plasma histamine levels following morphine administration in humans have shown peak hista-mine levels within 5 minutes of administration [3,4,6]. This experiment investigated whether maximum histamine release occurred at a similar time in vitro in the presence of the enzyme prostaglandin H synthetase. HMC-1 cells were incubated at 37°C for 5, 10, 20, 30 and 60 minutes with morphine 668µg/ml, 0.1% sodium bisulfite and 25 mU prostaglandin H synthetase. The cells were centrifuged and the supernatant was examined for the presence of histamine and expressed as a % of total histamine. Histamine release was time dependent with a maximum effect after 1 hour of incubation ( Figure 2).

Interestingly, reaction mixtures containing a typical peak morphine concentration (450 ng/ml) showed a significant increase in histamine release in the presence of prostaglandin H synthetase compared with no enzyme. This was true for both sulfite-free (p = 0.003) and sulfitecontaining (p = 0.009) morphine solutions. This suggests that even at clinically-attained morphine concentrations, it is not only possible to cause the metabolic activation of sulfite, but also that of morphine.

The effect of sulfite alone (without morphine) on histamine release from mast cells was investigated. To 4 mast Table 1: Histamine release from HMC-1 cells stimulated with varying concentrations of morphine with and without sulfite Histamine release expressed as a percentage of release of histamine in comparison to control (SD). As a control, the HMC-1 cells were allowed to freeze and thaw for 3 cycles to release the remaining histamine from cells. Each value represents the mean (SD) of triplicate determinations.

Morphine concentration (µg/ml)

Histamine release (% total)* Sodium bisulfite    cell reaction mixtures, varying concentrations of sulfite (0.018 ng/ml, 0.45 ng/ml, 0.007µg/ml and 0.67µg/ml) were added. These concentrations of sulfite correspond to the concentration of sulfite present in morphine solutions of 18 ng/ml, 450 ng/ml, 6.6µg/ml and 668µg/ml. To each sample 25 mU prostaglandin H synthetase was added. The highest concentration of sulfite (0.67µg/ml) was incubated for 10, 20, 40 and 60 minutes to assess time dependence. The reaction mixtures were incubated, centrifuged and analyzed as described below.
+

Morphine concentration (µg/ml) Histamine release (% total)* Sodium bisulfite

In the presence of prostaglandin H synthetase, sulfite resulted in histamine release from mast cells. There was a significant increase in histamine release (9.7% (0.65) of total) in the reaction mixture containing 0.67µg/ml sulfite (p = 0.01). There was no statistically significant difference in histamine release compared with blank samples at all other concentrations of sulfite (0.018 ng/ml sulfite (2.8% (0.3) of total, p = 0.91), 0.45 ng/ml sulfite (1.93% (0.35) of total, p = 0.53) and 0.007µg/ml sulfite (4.5% (0.7) of total, p = 0.36) ( Table 3).

Time (minutes) % of total histamine release

The time-dependent experiment showed histamine release of 8% of total (3.3) at 10 minutes incubation, 7.7% of total (2.2) at 20 minutes, 10.5% of total (6.1) at 40 minutes and 9.7% of total (0.65) at 60 minutes.

Discussion

Our experiments showed similar histamine release from the mast cell line HMC-1 stimulated with morphine with or without 0.1% sodium bisulfite. Maximum histamine release was 16.1% of total in morphine only samples and 17.6% of total in morphine with 0.1% sodium bisulfite samples. This percentage is similar to other studies of histamine release in the human mast cell line HMC-1, where maximum release was 20-30% [28][29][30]. In our study, the concentrations of morphine and sulfite required to achieve maximum histamine release were significantly higher than concentrations seen clinically. Reaction mixtures containing sulfite only also caused significant histamine release at high concentrations. Blank samples, containing neither morphine nor sulfite, did not cause a significant increase in histamine release ( Table 2). These results suggest that at high concentrations, both sulfite and morphine could be responsible for elevated histamine release from mast cells. In addition to the formation of the sulfite radical from sulfite, it is possible that carboncentered or nitrogen-centered free radicals are being generated from the metabolic activation of morphine [20], thereby inducing mast cell histamine release.

HMC-1 cells contain histamine and release their histamine content in response to various exogenous stimuli [28][29][30]. Our study showed no evidence of elevated histamine release from mast cells when stimulated with morphine and/or sulfite at concentrations seen clinically (both peak and steady-state). Although HMC-1 exhibits a phenotype similar to that of human mast cells [31], it is possible that immature cells (cell lines) are less sensitive than mature (primary) cells [28]. The model used is an artificial system and may not represent physiological conditions. In addition, maximum histamine release occurred after a one hour incubation. This is significantly different from the in vivo situation, where maximum release usually occurs within 5 minutes of morphine administration, and suggests that we were not able to simulate exactly the human environment where histamine release occurs. It is likely that although the sulfite radical is formed rapidly in vitro, it may take some time for its effects to become evident [32].

Metabolic activation of sodium bisulfite may occur through a variety of mechanisms. We investigated a number of enzyme systems and our final model used prostaglandin H synthetase. Other enzyme systems (horseradish peroxidase/ hydrogen peroxide and hypoxanthine/ xanthine oxidase) did release histamine in our model (results not shown), but they produced excess background interference in the chromatograms and made identification of peaks difficult. Activation of sulfite following morphine administration may not occur through this mechanism, however, and is another possible flaw in our aim to construct a model similar to the one that exists in vivo.

Morphine has been shown to cause significant histamine release in vivo, but it has not been investigated whether sulfite, in the form of the sulfite radical, is the cause of this release. Our study finds that sulfite in sulfite-containing morphine solutions, at clinically attained concentrations, is not responsible for histamine release in in vitro experiments of the mast cell line HMC-1, but this does not preclude the fact that sulfite may lead to marked elevation in histamine levels in vivo. The release of a significant amount of histamine with either sulfite or morphine at concentrations higher than those seen clinically suggests that both substances may be responsible for histamine release from mast cells. Further clinical research studies need to be performed to examine this possibility.

Conclusions

Our study finds that the sulfite in sulfite-containing morphine solutions, at concentrations seen clinically is not responsible for histamine release in in vitro experiments of the mast cell line, HMC-1, but this does not preclude the fact that sulfite may lead to marked elevation in histamine levels in vivo. Our experiments suggest that at high concentrations, both morphine and sulfite are responsible for histamine release from mast cells. Further in vivo research needs to be performed to examine this possibility.

Methods

Preparation of HMC-1 cells

Human leukemic mast cells, HMC-1 [33] were kindly provided by J. Butterfield, Rochester, Minnesota. This cell line shows many characteristics of immature mast cells. They have been shown to release histamine following stimulation [28][29][30] Cells were carried in 75 cm 2 vented tissue culture flasks (BD Falcon, Franklin Lakes, NJ) containing Iscoves modified Dulbecco's medium (Sigma, St.Louis, MO) with 10% iron-supplemented calf serum (Hyclone, Logan, Utah) and monothioglycerol 1.2 mM (Sigma, St.Louis, MO). Incubations were conducted in a humidified atmosphere at 5% CO 2 and 37°C. Cells were passed weekly and fed only when necessary (evidence of color change in media).

To pass cells, the cell suspension was placed in a 50 ml polypropylene centrifuge tube (BD Falcon, Franklin Lakes, NJ) and centrifuged at 250 × g at 4°C for 10 minutes. The culture medium was discarded and the cells were re-suspended in fresh medium. Cell density was maintained at 500,000 cells/ml.

Chemicals

Reagent grade O-phthaldialdehyde (OPA), mercaptoethanol, dihydrogen sodium phosphate, disodium EDTA, sodium bisulfite, morphine sulfate, hydrogen peroxide, xanthine oxidase, boric acid and phosphate buffered saline (PBS) were purchased from Sigma (St.Louis, MO, USA). Methanol (HPLC grade), acetonitrile (HPLC grade) and sodium hydroxide were from Fisher Scientific (Hampton, NH). Phosphate buffered saline (PBS) pH 7.4, contained 120 mmol/L NaCl, 2.7 mmol/L KCl and 10 mmol/L phosphate. Horseradish peroxidase was purchased from Cooper Biomedical. Prostaglandin H synthetase was purchased from Calbiochem (Darmstadt, Germany).

Determination of histamine release

Mast cell suspensions, containing approximately 500,000 mast cells were, aliquoted into 1 ml samples. These were rinsed 4 times with 4 ml of PBS, pH 7.4. Cells were then re-suspended in 1 ml PBS. Reaction mixtures were supplemented with morphine, sulfite and prostaglandin H synthase at this stage according to experimental design, unless otherwise specified. Reaction mixtures were incubated for 60 minutes in a water bath at 37°C. Reactions were stopped by centrifugation at 250 × g for 4 minutes at 4°C and placed on ice. Then 200µl of the supernatant was collected and dried under nitrogen at 40°C.

Histamine in the supernatant was measured using derivitization with OPA and mercaptoethanol (ME) as described [34]. Dried samples were re-dissolved in 100µl of derivitisation buffer (0.4 M boric acid adjusted to pH 9.5 with 1 M sodium hydroxide) and then derivitized with 20 µL of a freshly made 1:1 mixture of OPA (3.8 mM in methanol) and ME (2.5 ml/L in methanol) and analyzed within 30 minutes. The derivitized samples were used for chromatography.

The response of HMC-1 cells to the stimulant calcium ionophore was investigated to assess the releasability of HMC-1 cells. 0.25µg/ml calcium ionophore was added to mast cell suspensions. Reaction mixtures were incubated and analyzed as above.

Recovery of total histamine from cells

To extract the remaining histamine from the original sample, the mast cell pellet in suspension was frozen and thawed 3 times. It was then centrifuged at 250 × g for 4 minutes and 200µl of the supernatant was dried under nitrogen as described previously. Samples were re-dissolved, derivitized and analyzed as above. Histamine release during morphine incubations was expressed as a percentage of total histamine release.

Chromatography system

Histamine was measured by high-performance liquid chromatography using a Varian model 5500 chromatograph. The mobile phase was 0.1 M dihydrogen sodium phosphate and 1 mM disodium EDTA with 16% methanol and 14% acetonitrile adjusted to pH 6.4. The flow rate was 0.8 ml/minute.

Separations were performed using a 15 cm × 4.6 mm, Ace 5 C18 reversed-phase cartridge column (Advanced Chromatography technologies, UK). A Zorbax C8 guard column with a 5 micron, 4.6 × 12.5 mm analytical guardcolumn cartridge (Agilent technologies, Palo Alto, CA) was used. Fluorescence detection was used with the fluorimeter (Varian 9070) set at an excitation of 348 nm and an emission of 444 nm (determined by running individual excitation and emission spectra on the fluorometer).

Chromatographic conditions involved the use of a mobile phase gradient to optimize peak separation. A linear gradient starting at 90% mobile phase, 10% acetonitrile and ending at 80% mobile phase, 20% acetonitrile took place over 20 minutes. The column was washed by increasing the acetonitrile to 30% by 22 minutes and by 23 minutes the proportions were returned to the original values. The length of each run was 30 minutes.

Data analysis

Values are expressed as a mean +/-SD of 3 experiments. One-way analysis of variance was used to determine the significance of the difference between groups. Results were considered significantly different when p < 0.05.

Figure 1 )
1. The effect of sodium bisulfite 0.1% in varying concentrations of morphine in the presence of prostaglandin H syn-Concentration-effect of morphine in the presence of prostaglandin Hsynthetase on histamine release from the mast cell line HMC-1 (mean of 3 experiments) Figure 1 Concentration-effect of morphine in the presence of prostaglandin Hsynthetase on histamine release from the mast cell line HMC-1 (mean of 3 experiments).

2 :
2Histamine release from HMC-1 cells stimulated with varying concentrations of morphine with and without sulfite in the presence of prostaglandin H synthetase. Histamine release expressed as a percentage of release of histamine in comparison to control (SD). As a control, the HMC-1 cells were allowed to freeze and thaw for 3 cycles to release the remaining histamine from cells. Each value represents the mean (SD) of triplicate determinations.

Time response effects of morphine 668 µg/ml in the presence of 0.1% Na bisulfite and 25 mU prostaglandin H synthetase on histamine release in HMC-1 cells (mean of 3 experiments) Figure 2 Time response effects of morphine 668 µg/ml in the presence of 0.1% Na bisulfite and 25 mU prostaglandin H synthetase on histamine release in HMC-1 cells (mean of 3 experiments).

3 :
3Histamine release from HMC-1 cells stimulated with varying concentrations of sulfite-only solutions in the presence of prostaglandin H synthetase. Histamine release expressed as a percentage of release of histamine in comparison to control (SD). As a control, the HMC-1 cells were allowed to freeze and thaw for 3 cycles to release the remaining histamine from cells. Each value represents the mean (SD) of triplicate determinations.Sulfite concentration (ng/ml)Histamine release (% total)

Table

Table

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AcknowledgementsThe authors wish to thank Carolyn Myers, Ph.D. and Anita Pettigrew, MS for their assistance in assay development and Mary Thomas for her expert assistance in the preparation of the manuscript. This work was supported in part by a NIH Pediatric Pharmacology Research Units grant HD-31323-09.Authors' contributionsEG participated in experiments and drafted the manuscript. JB conceived of the study and participated in its design. CM suggested the methodology of experiments. All authors read and approved the final manuscript.
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Histamine release by free radicals: paracetamol-induced histamine release from rat peritoneal mast cells after in vitro activation by monooxygenase. E Masini, M Lodovici, R Fantozzi, S Brunelleschi, A Conti, P F Mannaioni, Agents Actions. 18Masini E, Lodovici M, Fantozzi R, Brunelleschi S, Conti A, Mannaioni PF: Histamine release by free radicals: paracetamol-induced histamine release from rat peritoneal mast cells after in vitro activation by monooxygenase. Agents Actions 1986, 18:85-88.

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Clinical Study Postoperative Adiponectin Levels in Pediatric Patients Undergoing Open Heart Surgery
Hindawi Publishing CorporationCopyright Hindawi Publishing Corporation

A Thaler
Pediatric Intensive Care Department
Chaim Sheba Medical Center
Safra Children's Hospital
52621Tel-HashomerIsrael

Sackler School of Medicine
Tel-Aviv University
39040Tel-AvivIsrael

H Kanety
Sackler School of Medicine
Tel-Aviv University
39040Tel-AvivIsrael

Institute of Endocrinology
Chaim Sheba Medical Center
Safra Children's Hospital
Tel-Hashomer
52621Israel

T Avni
Pediatric Intensive Care Department
Chaim Sheba Medical Center
Safra Children's Hospital
52621Tel-HashomerIsrael

Sackler School of Medicine
Tel-Aviv University
39040Tel-AvivIsrael

D Mishali
Sackler School of Medicine
Tel-Aviv University
39040Tel-AvivIsrael

Department of Cardiothoracic Surgery
Chaim Sheba Medical Center
Safra Children's Hospital
52621Tel-HashomerIsrael

R Hemi
Sackler School of Medicine
Tel-Aviv University
39040Tel-AvivIsrael

Institute of Endocrinology
Chaim Sheba Medical Center
Safra Children's Hospital
Tel-Hashomer
52621Israel

E Yissaschar
Sackler School of Medicine
Tel-Aviv University
39040Tel-AvivIsrael

Institute of Endocrinology
Chaim Sheba Medical Center
Safra Children's Hospital
Tel-Hashomer
52621Israel

C Pariente
Sackler School of Medicine
Tel-Aviv University
39040Tel-AvivIsrael

Institute of Endocrinology
Chaim Sheba Medical Center
Safra Children's Hospital
Tel-Hashomer
52621Israel

G Paret
Pediatric Intensive Care Department
Chaim Sheba Medical Center
Safra Children's Hospital
52621Tel-HashomerIsrael

Sackler School of Medicine
Tel-Aviv University
39040Tel-AvivIsrael

D Modan-Moses
Sackler School of Medicine
Tel-Aviv University
39040Tel-AvivIsrael

Pediatric Endocrinology and Diabetes Unit
Chaim Sheba Medical Center
The Edmond and Lily Safra Children's Hospital
52621Tel-HashomerIsrael

Clinical Study Postoperative Adiponectin Levels in Pediatric Patients Undergoing Open Heart Surgery

BioMed Research International
Hindawi Publishing Corporation201310.1155/2013/408680Received 15 April 2013; Revised 5 September 2013; Accepted 6 September 2013Correspondence should be addressed to D. Modan-Moses; Academic Editor: Rei Shibata
Background. Adipose tissue is an important endocrine organ that secretes cytokines, including adiponectin, levels of which are negatively correlated with the severity of the inflammatory process. Aim. To assess the time course of adiponectin levels following open heart surgery with cardiopulmonary bypass and its correlation with early postoperative outcomes. Materials and Methods. Blood samples were obtained from 24 children undergoing cardiac surgery and analyzed for adiponectin, C-reactive protein, and other inflammatory markers. Results. Baseline adiponectin levels were negatively correlated with patients' preoperative weight and age. Postoperative adiponectin levels decreased compared to baseline ( = 0.01) and correlated negatively with duration of cardiopulmonary bypass ( = −0.438, = 0.037), length of stay in the pediatric intensive care unit ( = −0.457, = 0.025), and the inotropic score ( = −0.471, = 0.02). Adiponectin levels were positively correlated with sVCAM 1 levels; however, there was no correlation between adiponectin levels and sP selectin, tPA, MCP1, and sCD40. Conclusions. The inflammatory response after open heart surgery with cardiopulmonary bypass is associated with a reduction in adiponectin levels. Prolonged or more complicated surgery induced a more substantial inflammatory process characterized by a significant reduction in adiponectin levels over time and a delayed return to baseline levels.

Introduction

Open heart surgery (OHS) involving cardiopulmonary bypass (CPB) triggers a well-recognized systemic inflammatory response. This response is thought to be due to endothelial cell injury, neutrophil activation, increased synthesis of multiple cytokines, upregulation of adhesion molecules, initiation of the coagulation cascade, and free radical formation, possibly leading to severe postoperative complications [1].

Adipose tissue has been demonstrated to be an important endocrine organ which secretes cytokines and regulatory factors. Adiponectin, the most abundant adipose-derived protein in humans, has significant anti-inflammatory and insulin-sensitizing effects. In contrast to other adipocytokines, adiponectin levels are inversely correlated with body fat mass [2]. Adiponectin levels are elevated at birth, increase from birth to the first month of life, and decrease over time until adulthood [3]. Adiponectin circulates in three distinct multimers: trimers (low molecular weight, LMW), hexamers (middle molecular weight, MMW), and larger multimers of 12 to 18 subunits (high molecular weight, HMM). The amount and distribution of these molecular forms can determine the activity of adiponectin in different tissue [4].

Adiponectin inhibits macrophage activation through the NF-B pathway [5] resulting in markedly decreased uptake of oxidized low-density lipoproteins and foam cell formation 2 BioMed Research International [6], reduces the production and activity of tumor necrosis factor (TNF-), and inhibits (IL-6) production [7]. It is known to exert antiapoptotic effects and to prevent myocardial apoptosis in response to myocardial ischemia reperfusion injury [8].

Plasma adiponectin levels are decreased in critically ill patients during the acute phase of their illness [9]. In contrast, adiponectin levels are elevated and positively correlated with inflammatory markers in chronic inflammatory diseases that are unrelated to increased adiposity [10].

In the current study, we sought to establish the pattern of changes in adiponectin levels in the context of the inflammatory response in pediatric patients with congenital heart defects undergoing OHS with CPB, focusing on the immediate postoperative period. We related the observed values to clinical outcome and to postoperative changes in other inflammatory markers.

Materials and Methods

Patients. 24 consecutive children who underwent OHS

with CPB for the correction of congenital heart defects were enrolled. Exclusion criteria included need for inotropic support or mechanical ventilation prior to surgery, recent cardiac arrest, sepsis, bout of severe hypotension or infection within one week of surgery, and weight of less than 2 kg. None of the patients received glucocorticoid therapy before enrollment. Preoperative informed consent was obtained from the parents of all children. The study was approved by the Chaim Sheba Medical Center ethics committee and the Israeli Ministry of Health.

Intraoperative Management.

Induction and maintenance of anesthesia were carried out in a standard manner and consisted of weight-related dosages of fentanyl, midazolam, and pancuronium bromide. Routine operations were performed using a roller pump and membrane oxygenator. A single dose of cefazolin 0.5 mg/kg and methylprednisolone 30 mg/kg was administered. Five minutes prior to being connected to the Cobe Century 2000 CPB (Cobe Cardiovascular Inc, Arvada, CO, USA), patients were given 300 IU/kg bovine heparin in order to achieve an activated clotting time of 400-500 seconds. Surface body cooling was instituted with low ambient room temperature and a cooling mattress. The aorta was cross-clamped and a cold crystalloid antegrade cardioplegia solution (4 ∘ C) was infused through the aortic root in order to bring the body temperature to 25-27 ∘ C. Plegisol cardioplegia solution (Abbott Laboratories, North Chicago, IL, USA) was used for the first 17 CPBs, and Custodiol cardioplegia solution (Koehler Chemie, Alsbach-Haenlein, Germany) for the remaining 7. No modified ultrafiltration or leukocyte depletion techniques were employed for any of the patients during this study.

Collection of Blood Samples.

Serial blood samples were collected from arterial or venous lines 24 hours prior to surgery, after anesthesia induction, 1 and 2 hours while under CPB, upon arrival to the pediatric intensive care unit (PICU), and 6, 12, 24, and 96 hours afterwards. Blood was stored for 20 minutes at 4-8 ∘ C, centrifuged for 15 minutes at 2700 rpm, separated, and frozen at −70 ∘ C until use.

Total serum adiponectin was measured using human adiponectin radioimmunoassay (Millipore, St. Charles, MO, USA) [11]. The sensitivity of the assay was 1 g/L, and the within and between assay variations were 6.9% and 9.3%, respectively. CRP levels were determined with a hsCRP kit (Olympus, AU2700 immunoanalyzer). The within and between assay variations were 1.10% and 1.83%, respectively. sP selectin, tissue-type plasminogen activator (tPA), monocyte chemoattractant protein-1 (MCP1), soluble CD40 (sCD0), and soluble vascular cell adhesion molecule 1 (sVCAM1) levels were measured using the Human Chemokines 6plex FlowCytomix Multiplex kit (Bender MedSystems Inc).

The distribution of adiponectin multimers in serum samples was analyzed under nonreducing and nondenaturing conditions [11]. The relative proportion of each multimer to total adiponectin was calculated by dividing band densitometry of the multimer by total density in each lane.

Selected Patient Data.

Demographic data, medical history, and relevant laboratory parameters were recorded preoperatively. Each child's vital signs and laboratory results were monitored continuously while in the PICU. These included arterial blood gas, complete blood count, blood chemistry, duration of ventilation and stay at the PICU, rectal temperature, blood pressure, pulse, blood product infusion, use of intravenous solutions, and the inotropic score (IS) which was calculated as described by Wernovsky et al. [12].

Statistical

Analysis. All parameters are expressed as mean values ± SD. CRP is expressed as median (25th and 75th percentiles). Analysis of variance (ANOVA) with repeated measures was applied for the testing of the time effect on adiponectin, log CRP, length of ventilation, difference between cardioplegia solutions, differences between cyanotic and acyanotic heart defects, IS, duration of operation, and length of stay at the PICU. Analysis of covariance (ANCOVA) with age as covariate was applied for the testing of time effect on adiponectin depending on the length of time on ventilation and length of stay in the PICU. Pearson correlations were used to test linear association between adiponectin and length of stay at the PICU, hours of ventilation, IS, weight, age, sVCAM1, and duration of surgery. A value of less than 0.05 was considered significant. In addition, we separated our study group into two subgroups according to the median stay in the PICU in days ( = 5), hours ventilated ( = 22), and minutes on CPB ( = 72) and compared adiponectin levels between these two subgroups.

Results

Twenty-four children, aged 1 month-11 years, met the study inclusion criteria. Patients' characteristics are summarized in Table 1. Individual patients' data is presented in Table 2. The surgical and clinical outcomes were favorable with the exception of one fatality. Table 2 and Figure 1 depict the changes in serum adiponectin levels that occurred over the study period. Adiponectin levels were highest before surgery, declining significantly ( = 0.01) from the time of induction of anesthesia up to 6 hours after the child's arrival to the PICU. Adiponectin levels at 96 hours after the arrival to the PICU were still significantly lower ( = 0.006) than those obtained at baseline. These observations held true when adiponectin levels were presented as percent of baseline ( Figure 2).

Adiponectin Levels.

Anthropometric Data.

Baseline adiponectin negatively correlated with weight ( = −0.419, = 0.042) and age ( = −0.545, = 0.006). There were no gender-based differences in adiponectin levels. Adiponectin levels at induction and sVCAM 1 levels at 12 and 24 hours after arrival at the PICU correlated positively ( = 0.01, = 0.006) as did the adiponectin levels at 12 hours after arrival to the PICU and sVCAM 1 levels at 12 and 24 hours after arrival to the PICU ( = 0.017, = 0.036) (Figure 3). Moreover, there was a positive correlation between sVCAM 1 at 12 hours after arrival to the PICU and adiponectin levels from the induction of anesthesia and up to 24 hours after arrival to the PICU ( = 0.013, = 0.035, = 0.044, = 0.17, and = 0.044, resp.).

Inflammatory Markers

No correlations were noted between adiponectin levels and the following parameters: white blood cell count, platelets, lactate, troponin, length of cross-clamp time, brand of cardioplegia used (Plegisol versus Custodiol) or type of cardiac defect (cyanotic versus acyanotic), sP selectin, tPA, MCP1, and sCD40. Next, we divided the patients into two subgroups according to group median in each of the severity indicators and compared adiponectin levels between them. Adiponectin levels were significantly lower in the longer ventilation subgroup at arrival to the PICU and up to 24 hours afterwards ( = 0.029). No significant differences were noted after dividing the cohort based on median length of CPB and length of stay at the PICU. Finally, grouping the patients according to the use of inotropic agents revealed no significant difference between the patients who did not need inotropic assistance and those who did.

Higher preoperative adiponectin levels were associated with a longer length of stay in the PICU ( < 0.001) and longer length of time on ventilation ( < 0.001). Figure 5 depicts the changes in the molecular weight isoform ratio over time.

Molecular Forms of Adiponectin.

We assessed the relative distribution of the three molecular isoforms of adiponectin at the 8 time points in 8 patients (4 boys and 4 girls) in order to exclude the possibility that an alteration in the concentration of one of adiponectin's isoforms was responsible for the change in total adiponectin levels. The relative distribution of the three isoforms was preserved at all time points and changes in the relative distribution of the three fractions were not significant over time.

Discussion

In the current study we longitudinally measured adiponectin levels and correlated them to clinical outcomes in pediatric patients undergoing OHS with CPB, a procedure which is expected to induce acute illness and inflammation [1]. Postoperative adiponectin levels were reduced over time compared to baseline levels. Similar results were recently observed by Caselli et al.; however, these authors measured adiponectin levels 3 days after surgery rather than during the immediate postoperative period as we did [14]. One possible explanation for the postoperative decline of adiponectin could be a reduction in adiponectin transcription due to the inflammatory process and the release of proinflammatory cytokines and endogenous cortisol. TNF-, known to surge after CPB [15], suppresses the transcription of adiponectin in adipocytes [16]. In agreement with this hypothesis, Witasp  Points of blood draw the circulation due to the inflammatory process [18]. We established that changes in the secretion of the molecular forms of adiponectin were not responsible for the alterations in the total adiponectin levels, as we noted no change in the ratio of different adiponectin isoforms. Interestingly, adiponectin levels in our study began to rise faster than previously reported after the onset of a critical illness [9]; that is, levels had already started to increase 12 hours after the arrival to the PICU.

Patients with a longer length of stay in the PICU and those with longer duration of ventilation had significantly higher preoperative levels of adiponectin and significantly lower adiponectin levels upon arrival to the PICU, even when age was taken into consideration. There is some evidence that patients who develop postoperative complications may be identified preoperatively based on the hypothesis that the primed immune system amplifies the immune response to cardiac surgical trauma. Bocsi et al. demonstrated elevated levels of the percentage of leukocytes that are neutrophils, the complement components C3 and C5 as well as TNFone day before surgery in patients that had a complicated   postoperative course [19]. The amount of inflammatory markers released in critical care situations appears to be influenced genetically [20]. Thus, the host response to cardiac surgery is likely a result of interactions of the surgical procedure and innate patient characteristics which may have a genetic basis with carriage of specific functional polymorphic alleles in genes encoding for proteins involved in inflammation and vascular regulation, such as adiponectin.

Children with a more complicated course, as indicated by longer postoperative ventilation duration, had significantly lower postoperative adiponectin concentrations. Similarly, we observed negative correlations between adiponectin levels and clinical indicators of a more severe inflammatory process [19]-length of CPB, length of stay at the PICU, and IS. These findings support the notion that adiponectin participates in the inflammatory process that is set into motion after OHS and CPB.

CRP, an acute phase reactant and a marker of the inflammatory process, has been shown to decrease immediately after OHS with CPB and to be elevated up to 5 days afterwards [21]. CRP levels negatively correlate with adiponectin levels in healthy men, obese women, and diabetic patients [22]. In the current study, the usual negative correlation between CRP and adiponectin was abolished following CPB. Adiponectin levels fell together with a slight drop in CRP levels and returned to near basal levels at the point when CRP was at its peak, demonstrating a trend for a positive correlation between the two 24 hours after arrival to the PICU. These results are in accordance with those found by Venkatesh et al. [20] and might indicate that adiponectin is a sensitive inflammatory marker for both the onset and the termination of the inflammatory process. The lack of the expected negative correlation between adiponectin and CRP could, however, be attributed to the time points of blood assessments that were chosen in this study.

We demonstrated a positive correlation between sVCAM-1 at 12 and 24 hours after arrival at the PICU and adiponectin levels up to 24 hours after arrival to the PICU. VCAM-1 is an endothelial ligand for integrins expressed on leukocytes and platelets, with the function of facilitation of endothelial adhesion of circulating leukocytes. The endothelial expression of VCAM-1 is increased in response to inflammatory cytokines, and the soluble ectodomain of VCAM-1 (sVCAM-1) is proteolytically shed from the cell surface into the circulation upon endothelial activation and injury. Elevated plasma sVCAM-1 level was reported in many diseases, including coronary and peripheral atherosclerosis, diabetes mellitus, systemic lupus erythematosus, and Crohn's disease, and it is now considered as a marker for endothelial injury under inflammatory processes [23]. There are contradictory reports regarding the association between adiponectin and sVCAM-1; adiponectin was found to suppress the adhesion of monocytes to endothelial cells by decreasing the TNF--mediated expression of sVCAM-1, thus reducing atherosclerosis [24]. On the other hand, Vaverkova et al. demonstrated a significant independent positive association of adiponectin with sVCAM-1 in patients at risk for coronary vascular disease [25]. We found no correlation between adiponectin and other inflammatory markers, such as white blood count, platelet count, sP selectin, MCP-1, sCD 40, or tPA.

Several limitations of this study should be considered. The reduction of adiponectin levels could be due to the pulse dose of methylprednisolone (30 mg/kg) which was administered in theater, in accordance with current practice [26]. The effects of glucocorticoids on the expression and secretion of adiponectin remain controversial. Dexamethasone has been shown to reduce adiponectin gene expression in murine adipocytes, and glucocorticoids were demonstrated to negatively regulate adiponectin mRNA in human adipose tissue [27]. A decrease of about 25% in adiponectin levels was noted by Fallo et al. when administering 25 mg of hydrocortisone to adults, lasting up to 120 minutes [27], while Uchida et al.

demonstrated an increase in adiponectin after 3 days of steroid pulse therapy in patients with IgA nephropathy [28]. These differences could be related to adaptive mechanisms compensating for glucocorticoid-induced reduction in insulin sensitivity [29]. The fact that longer duration of stay at the PICU, longer duration of ventilation, and higher IS were correlated with lower adiponectin levels does not support the assumption that steroid therapy was solely responsible for the reduction in adiponectin levels, which lasted up to 96 hours after methylprednisolone administration, beyond the effect noted by Fallo et al. [27]. This fact further supports the role of inflammation in reducing adiponectin levels, rather than the effect of the single dose of methylprednisolone which was administered at the beginning of surgery.

Conclusions

We have demonstrated that a complicated postoperative course is associated with significantly lower postoperative adiponectin concentrations. Our data contributes to the accumulating evidence showing that adipose tissue is an active organ secreting multiple adipocytokines, which may have an impact on numerous pathways of response to critical illness. Overall, our findings raise the possibility that adiponectin may play a part in the response to CPB. The significance of this phenomenon, however, remains to be elucidated, and further efforts in basic research to account for the mechanisms beyond the clinical observation of low adiponectin levels in children following OHS are warranted.

3. 4 .
4Severity of the Postoperative Course. Using the wellestablished severity indicators, duration of CPB, duration of mechanical ventilation, length of stay at the PICU and IS [13], we found negative correlations between the duration of CPB and adiponectin levels at the end of surgery and at 6 hours after arrival to the PICU ( = −0.438, = 0.037 and = −0.488, = 0.021, resp.). There was also a negative correlation between adiponectin levels and log of length of stay in the PICU, starting from the time of arrival to the PICU ( = −0.457, = 0.025) (Figure 4) and up to 12 hours afterwards ( = −0.495, = 0.016). IS and adiponectin levels correlated negatively from the time of arrival to the PICU ( = −0.471, = 0.02) and up to 24 hours afterwards, ( = −0.453, = 0.03). No correlations were demonstrated between adiponectin levels and length of mechanical ventilation. Again, these observations remained unchanged when adiponectin was presented as percent of baseline levels (data not shown). Delta adiponectin levels (i.e., the difference between the levels 24 hours preoperation and at the time of arrival to the ICU) were positively correlated with length of CPB ( = 0.515, = 0.007) and with the inotropic score ( = −0.394, = 0.047), but not with the length of stay at the PICU.

Figure 1 :Figure 2 :Figure 3 :
123Time course of adiponectin serum concentrations in patients undergoing cardiopulmonary bypass (CPB). PICU = pediatric intensive care unit. been a decrease in adiponectin secretion from adipose tissue, or rapid degradation and clearance of Time course of adiponectin serum concentrations as percentage of baseline in patients undergoing cardiopulmonary bypass (CPB). PICU: pediatric intensive care unit. The correlation between adiponectin levels and sVCAM 1 levels 12 hours after arrival to the PICU.

Figure 4 :
4The correlation between adiponectin levels upon arrival to the PICU and length of stay in the PICU.

Figure 5 :
5Time course of adiponectin isoforms/total adiponectin ratio in the study group.

Table 1 :
1Characteristics of the study patients. CPB: cardiopulmonary bypass; PICU: pediatric intensive care unit.Mean ± SD
Range

Age (m)
20.08 ± 32.45 (1-132)

Sex (male/female)
12/12

Weight (kg)
8.87 ± 6.75
(2.7-28)
Type of surgery

Ventricular septal defect
3

Atrial septal defect
1

Tetralogy of Fallot
6

Dextrotransposition of the great arteries
5
Total anomalous pulmonary venous
connection
2

Pulmonic atresia
3

Discrete subaortic stenosis
2

Other
2

CPB duration (min)
93.56 ± 52.43 (30-197)

Cross-clamp duration (min)
59.30 ± 48.24 (0-159)

PICU stay (days)
8.6 ± 9.08
(1-46)
Postoperative ventilation (hours)
92.02 ± 220.86 (0-1100)

88, and 33.14 mg/L, 23.46, 62.43, resp.). Log transformation was performed in order to obtain normal distribution. At 24 hours after arrival to the PICU, log CRP and adiponectin demonstrated a trend of correlation ( = 0.41, = 0.06); at this point adiponectin levels began to rise, while CRP levels were at their peak.. CRP levels were evaluated at
baseline (median 25th and 75th percentiles) (1.94 mg/L, 0.99,
3.39), upon arrival to the PICU (1.99 mg/L, 0.98, 2.91), and
24 and 96 hours after arrival to the PICU (33.3 mg/L, 22.91,
53.

Table 2 :
2Adiponectin levels and weight Z-scores.Patient
number
Patient sex
Age
(months)

Normative
adiponectin levels

Baseline
Adiponectin

Adiponectin
at PICU

Adiponectin
after 96 hours
Weight (Kg)
Weight
Z-score

1
0
60
9.3
17.8
7.4
12.5
14
−2.07

2
0
1
11.2
32.7
10.6
24.8
3
−2.03

3
1
12
14.3
23.8
12.2
18.1
7.6
−2.85

4
0
37
11.2
27.5
15.5
16.6
11.7
−1.66

5
1
98
7.6
24.9
12.5
12.2
18.6
−2.73

6
1
132
7.8
16.9
12.9
15.9
28
−1.49

7
0
12
11.2
19.9
10
8
8.55
−1.01

8
1
1
26.7
26
9.7
17.5
2.7
−2.61

9
0
16
11.2
48.1
32.3
58
7.25
−3.83

10
1
1
26.7
44.1
17.9
35.9
3.4
−1.62

11
0
18
11.2
30
20.7
25
13.5
−1.87

12
1
12
14.3
31
16.8
24.2
7.58
−2.87

13
0
1
26.1
53.9
6
18.2
3.79
−0.69

14
0
18
11.2
31.5
17.6
20.9
10.1
−0.81

15
0
11
11.2
27.2
22.4
33.3
6.8
−2.82

16
1
2
26.7
43.3
28.9
26.3
4.2
−1.52

17
1
1
26.7
49.2
29
36.4
3.67
−1.22

18
1
10
14.3
46.1
20.5
28.6
8.8
−0.84

19
0
4
26.1
32.1
21
24.7
4.3
−2.61

20
0
3
26.1
38.8
15
27.4
3
−3.86

21
1
1
26.7
38.8
24.1
13.6
25
−2.88

22
1
3
26.7
32.2
9
21.4
5
−1.39

23
1
6
26.7
45.2
22.6
42.2
6.25
−1.98

24
0
9
11.2
24
19.8
No data
6.19
−2.82

et al. have recently demonstrated a significant decrease in adiponectin mRNA in adipose tissue during major abdominal surgery [17]. Alternatively, there may have50

45

40

35

30

25

20

15

10

5

0

Adiponectin ( g/mL)

Day before
Induction

CPB
+ 1
PICU

PICU
+ 12
PICU
+ 6
PICU
+ 24
PICU
+ 96

Authors' ContributionG. Paret and D. Modan-Moses contributed equally to the study.
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