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Anesth Analg 2001;92:927-929
© 2001 International Anesthesia Research Society

ANESTHETIC PHARMACOLOGY

The In Vitro Effects of Ketamine at Large Concentrations Can Be Attributed to a Nonspecific Cytostatic Effect

Eli Lewis, MSc*, Boris Rogachev, MD*, Gadi Shaked, MD{dagger}, and Amos Douvdevani, PhD*

Department of *Nephrology and {dagger}Department B of General Surgery and Trauma Service, Soroka University Medical Center, Ben-Gurion University of the Negev, Faculty of Health Sciences, Beer-Sheva, Israel

Address correspondence to Dr. Amos Douvdevani, Nephrology Laboratory, Soroka Medical Center, PO Box 151, Beer-Sheva 84101, Israel. Address e-mail to amosd{at}bgumail.bgu.ac.il Reprints will not be available from the author.


   Introduction
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Methods
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 Discussion
 References
 
Ketamine seems to promote a favorable outcome for critically ill patients, including those with septic shock (1). It has been proposed that the nonanesthetic effects of ketamine may be attributed to antiinflammatory mechanisms. This hypothesis has been supported by in vivo findings, such as decreased levels of interleukin (IL)-6 in patients treated with a single IV small dose of ketamine during postoperative stress (2) or during abdominal hysterectomy (3). Notably, the doses that produce the antiinflammatory effects in these studies reach a plasma concentration of 0.1 µg/mL, whereas the plasma ketamine concentration required for anesthesia is 0.6–2.0 µg/mL (1,4,5).

In vitro attempts to uncover the antiinflammatory mechanisms of ketamine revealed widespread inhibitory activities, but they used large ketamine concentrations. For example, inhibition of tumor necrosis factor-{alpha} and nitric oxide production in lipopolysaccharide (LPS)-stimulated mouse-activated macrophagelike cells was attained at ketamine concentrations of 7.1–142.6 µg/mL (6); inhibition of action and production of nitric oxide synthase in LPS-treated rat alveolar macrophages, at 2.38 µg/mL ketamine concentration (7); and inhibition of IL-6, IL-8, and tumor necrosis factor-{alpha} production by LPS-stimulated human whole blood, at ketamine concentrations of 100–500 µg/mL (8).

We believe that the in vitro responses with large concentrations of ketamine are misleading. To verify this, we sought to clarify whether these large concentrations of ketamine exert a broad nonspecific cytostatic effect that includes both the arrest of cell proliferation and a blockade of cytokine production. We introduced varying concentrations of ketamine to both growth factor-dependent and independent human and mouse cell cultures and examined cell proliferation. Our data indicate that large doses of ketamine arrest cytokine production and cell proliferation in a strikingly similar pattern.


   Methods
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 Introduction
 Methods
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This study was approved by our institutional human investigation committee. A written informed consent was obtained from all subjects.

We measured IL-6 production in human LPS-stimulated whole blood according to the protocol reported by Kawasaki et al. (8). Briefly, blood was obtained from healthy volunteers and diluted in RPMI medium containing 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin. LPS (10 ng/mL) and ketamine (25–500 µg/mL; Parke-Davis, Hampshire, UK) were added to 1 mL diluted blood. After 6 h of incubation at 37°C in an atmosphere of 5% CO2 and 95% air, blood was centrifuged at 700g for 10 min. The supernatant was examined for IL-6 levels by commercial ELISA (R&D Systems, Minneapolis, MN), according to the manufacturer’s instructions.

Human primary tubular epithelial cells (TEC) were prepared from normal cortical tissues of hypernephrotic kidneys as previously described (9). Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Hypaque (Sigma Israel Chemicals Ltd., Rehovot, Israel) density gradient centrifugation of heparinized peripheral blood obtained from three healthy donors. The mouse fibroblast cell line (L-cells) and the mouse T-cell line (CTLD) were obtained from the American tissue culture cell collection.

Cells were seeded in a flat-bottomed 96-well plate; TEC (5 x 103 cells per well), in M199 medium; and PBMC (50 x 103 cells per well), CTLD (5 x 103 cells per well), and mouse fibroblasts (103 cells per well), in RPMI medium. Both types of medium contained 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, and 50µM ß-mercaptoethanol. PBMC proliferation was induced by phytohemagglutinin (PHA) (0.2%; Difco Laboratories, Detroit, MI), and CTLD proliferation was induced by IL-2 (50 U/mL). Cells were treated with medium or ketamine (25–500 µg/mL). At 48 h, TEC, CTLD, and mouse fibroblasts’ relative cell proliferation was measured by XTT colorimetric assay (Biological Industries, Beit Haemek, Israel), according to the manufacturer’s instructions. This is an in vitro assay for determining numbers of viable cells in proliferation and cytotoxicity studies, on the basis of the bioreduction of the tetrazolium compound XTT by viable cells. PBMC proliferation was determined by XTT assay at 72 h. The reliability of the XTT assay in the presence of ketamine was confirmed by manual cell count.

Statistical analysis was performed with Pearson correlation and one-way analysis of variance. A value of P < 0.05 was considered significant. Results are presented as mean ± SEM.


   Results
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Discussion
 References
 
Introduction of LPS to human whole blood induced IL-6 secretion that was inhibited by ketamine at a 50% inhibitory concentration (IC50) of approximately 100 µg/mL ( Fig. 1). The proliferation of PHA-stimulated human PBMC and the primary culture of human TEC was also inhibited by ketamine (IC50 50 µg/mL and IC50 200 µg/mL, respectively) ( Fig. 2). This pattern of inhibition was also observed while introducing ketamine to a mouse T-cell line (IC50 200 µg/mL) and a mouse fibroblast cell line (IC50 350 µg/mL) (data not shown). The inhibition of IL-6 secretion correlated with the inhibition of proliferation of PBMC (r = 0.81, P = 0.0055) ( Fig. 3) and to the inhibition of proliferation of CTLD (r = 0.92, P < 0.005).



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  		Figure 1. The effect of ketamine on interleukin (IL)-6 production. Whole blood from healthy volunteers was diluted fivefold in RPMI medium. Lipopolysaccharide (LPS; 10 ng/mL) and the indicated ketamine concentrations were added to 1 mL diluted blood. After 6 h of incubation, the supernatant was examined for IL-6 levels by specific enzyme-linked immunoassay. The histogram indicates the level of IL-6 secretion from unstimulated blood. This experiment is representative of three independent experiments. Error bars indicate SEM. **P < 0.005 (one-way analysis of variance).

 


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  		Figure 2. The effect of ketamine on cell proliferation. A, Primary cultures of human renal tubular epithelial cells (TEC); B, human peripheral blood mononuclear cells (PBMC) stimulated by phytohemagglutinin were seeded in a 96-well plate in medium containing increasing concentrations of ketamine. At 48 h (TEC) or 72 h (PBMC), cell number was evaluated by XTT assay. Relative cell number is represented by percentage of ketamine-untreated cells. The histogram in graph A represents the level of proliferation of TEC in the presence of cycloheximide (CHX), a potent protein synthesis inhibitor. Ketamine 50% inhibitory concentration is &sim;200 µg/mL for TEC and &sim;50 µg/mL for PBMC. This experiment is representative of three independent experiments performed on PBMC from three healthy volunteers. ***P < 0.001 (one-way analysis of variance).

 


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  		Figure 3. Correlation between ketamine inhibition of interleukin (IL)-6 production and human peripheral blood mononuclear cells (PBMC) number. The data presented in Figures 1 and 2A were analyzed by linear regression to demonstrate the correlation between the inhibition of increasing doses of ketamine on IL-6 levels and the relative number of phytohemagglutinin-stimulated PBMCs.

 

   Discussion
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 Introduction
 Methods
 Results
 Discussion
References
 
Studies involving the administration of ketamine to patients and animal models have demonstrated an antiinflammatory activity at a plasma concentration of 0.1 µg/mL (2,3). At this concentration, ketamine has no anesthetic effect (1,4,5). Despite these findings, various in vitro studies have examined the antiinflammatory activity of ketamine at concentrations 10- to 1000-fold larger. These studies, without exception, show inhibitory activities to ketamine (6–8,10,11).

In this study, we sought to establish whether previously described inhibitory effects of ketamine at large concentrations could be attributed to nonspecific cytostatic effects. We have demonstrated that large concentrations of ketamine exert a broad, nonspecific cytostatic effect. This effect includes failure to proliferate, as well as the previously reported failure to produce cytokines (8). This was established by observing the inhibition of proliferation of human PTEC and of human PHA-stimulated PBMC. Additionally, a mouse T-cell line and a mouse fibroblast cell line exhibited the same pattern of inhibition under ketamine treatment (data not shown).

Several reports have attempted to exclude the presence of cytotoxic effects by performing trypan-blue staining within the first few hours of ketamine treatment (8). However, trypan-blue staining is indicative of cell death associated with membrane leakage and does not reflect cytostatic events in intact, viable cells. Although this assay excludes a cytotoxic activity, it cannot eliminate a cytostatic effect.

We conclude that reported in vitro inhibitory effects of ketamine at large concentrations are misleading. Because of the cytostatic activity exerted by large ketamine concentrations on a variety of cell types and cell functions, we recommend that the valuable antiinflammatory effects of ketamine be examined by using concentrations that do not exceed the upper anesthetic plasma concentrations of 2.0 µg/mL.


   Footnotes
 

Implications: Previous studies investigating the antiinflammatory mechanisms of ketamine exposed cells to ketamine in concentrations 10- to 1000-fold larger than plasma ketamine therapeutic levels. We demonstrate that large ketamine concentrations induce concentration-dependent cell cytostasis and urge that the cytostatic effect of ketamine be considered in future experiments.


   References
Top
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Reves JG, Glass PSA. Nonbarbiturate intravenous anesthetics. In: Miller RD, ed. Anesthesia. Vol 1. 3rd ed. New York: Churchill Livingstone, 1990: 243–79.
  2. Roytblat L, Talmor D, Rachinsky M, et al. Ketamine attenuates the interleukin-6 response after cardiopulmonary bypass. Anesth Analg 1998; 87: 266–71.[Abstract/Free Full Text]
  3. Roytblat L, Roy-Shapira A, Greemberg L, et al. Preoperative low dose ketamine reduces serum interleukin-6 response after abdominal hysterectomy. Pain Clin 1996; 9: 327–34.
  4. Chang T, Glazko AJ. A gas chromatographic assay for ketamine in human plasma. Anesthesiology 1972; 36: 401–4.[Web of Science][Medline]
  5. White PF, Way WL, Trevor AJ. Ketamine: its pharmacology and therapeutic uses. Anesthesiology 1982; 56: 119–36.[Web of Science][Medline]
  6. Shimaoka M, Iida T, Ohara A, et al. Ketamine inhibits nitric oxide production in mouse-activated macrophage-like cells. Br J Anaesth 1996; 77: 238–42.[Abstract/Free Full Text]
  7. Li CY, Chou TC, Wong CS, et al. Ketamine inhibits nitric oxide synthase in lipopolysaccharide-treated rat alveolar macrophages. Can J Anaesth 1997; 44: 989–95.[Web of Science][Medline]
  8. Kawasaki T, Ogata M, Kawasaki C, et al. Ketamine suppresses proinflammatory cytokine production in human whole blood in vitro. Anesth Analg 1999; 89: 665–9.[Abstract/Free Full Text]
  9. Detrisac C, Sens M, Garvin A, et al. Tissue culture of human kidney epithelial cells of proximal tubule origin. Kidney Int 1984; 25: 383–90.[Web of Science][Medline]
  10. Kanmura Y, Kajikuri J, Itoh T, et al. Effects of ketamine on contraction and synthesis of inositol 1,4,5-trisphosphate in smooth muscle of the rabbit mesenteric artery. Anesthesiology 1993; 79: 571–9.[Web of Science][Medline]
  11. Shakunaga K, Kojima S, Jomura K, et al. Ketamine suppresses the production and release of endothelin 1 from cultured bovine endothelial cells. Anesth Analg 1998; 86: 1098–102.[Abstract]
Accepted for publication December 13, 2000.




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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins with the assistance of Stanford University Libraries' HighWire Press®. Copyright 2006 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press