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Is Nitrous Oxide Safe for Bone Marrow Harvest?

Lederhaas, George MD; Brock-Utne, John G. MD; Negrin, Robert S. MD; Riley, Edward MD; Brodsky, Jay B. MD

General Article

Patients with non-Hodgkins lymphoma undergoing autologous bone marrow harvest were studied in a prospective, randomized fashion. All patients received a general anesthetic consisting of intravenous thiopental, fentanyl, and vecuronium and were ventilated with oxygen and isoflurane. Group I (19) patients also were ventilated with nitrous oxide (70%) whereas patients in Group II (19) did not receive nitrous oxide. Bone marrow samples were obtained at the beginning and end of the harvest. Viability of bone marrow mononuclear cells was assessed with a colony-forming unit-granulocyte macrophage (CFU-GM) assay, CFU-GM growth is a marker for myeloid progenitor cells and is dependent on intact deoxyribonucleic acid synthesis. Rate of neutrophil engraftment after autologous bone marrow transplantation was also studied. Both groups of patients were statistically similar in age, weight, anesthetic duration, CFU-GM counts at both sample draws, and the time for successful engraftment. There appears to be no difference in bone marrow viability as assayed by both CFU-GM colony growth and engraftment in human bone marrow exposed to a general anesthetic with nitrous oxide.

(Anesth Analg 1995;80:770-2)

Departments of Anesthesiology (Lederhaas, Brock-Utne, Riley, Brodsky) and Internal Medicine, Bone Marrow Transplant Program, Stanford University School of Medicine, Stanford, California (Negrin).

Accepted for publication November 8, 1994.

Address correspondence to John G. Brock-Utne, MD, Room H3580, Department of Anesthesia, Stanford University Medical Center, Stanford, CA 94305.

Nitrous oxide inactivates vitamin B12 which is an essential coenzyme for methionine synthetase [1]. This enzyme facilitates the conversion of homocystine and methyltetrahydrofolate to methionine and tetrahydrofolate. Both these latter compounds are essential for deoxyribonucleic acid (DNA) synthesis. Thus, nitrous oxide's interference with folate metabolism can lead to an impairment of DNA synthesis [2]. Patients with non-Hodgkins lymphoma undergoing general anesthesia for autologous bone marrow harvest were studied prospectively to analyze the effects of nitrous oxide exposure on growth of hematopoietic precursor cells and time to successful transplantation.

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After institutional approval and informed, written consent, 38 patients with non-Hodgkins lymphoma undergoing autologous bone marrow harvest under general anesthesia were studied prospectively. Patients were prepared for transplantation with cyclophosphamide 100 mg/kg on Day 2, etoposide 60 mg/kg on Day 4, and either carmustine 15 mg/kg on Day 6, or fractionated total body irradiation (1200 rads) from Days 5 to 8. Patients were randomly designated to receive a general anesthetic consisting of intravenous thiopental, fentanyl, vecuronium, and oxygen and isoflurane. Patients in Group I (n = 19) were also ventilated with 70% nitrous oxide whereas patients in Group II (n = 19) were not exposed to nitrous oxide.

Bone marrow samples for our study were taken at the beginning (Sample I) and end of the harvest (Sample II). The time difference between the samples was termed "anesthetic duration."

Marrow viability was assessed with a colony-forming unit-granulocyte macrophage (CFU-GM) assay. CFU-GM growth is dependent on intact DNA synthesis [3]. This test studies bone marrow mononuclear cells (2 times 105 cells/mL) depleted of monocytes and cultured in Iscoves modified Dulbecco's media (GIBCC, Grand Island, New York) with 15% fetal calf serum (Hyclone, Logan, UT), 0.9% bovine serum albumin (Armour Pharmaceutical Co., Tarrytown, NY), 50 micro mol/L 2 mEq, 1% penicillin/streptomycin, 1% l-glutamine, and methylcellulose (final concentration, 1.1%). Placental conditioned medium was added to cultures as a source of colony-stimulating factors at a final concentration of 15%. Cultures were plated in 0.25-mL volumes in Mark II tissue culture plates (Corning-Costar, Cambridge, MA). After 10 days of incubation in humidified 5% CO2 at 37 degrees C, the cultures were examined under aninverted microscope and CFU-GM colonies were scored visually.

Bone marrow transplantation was performed as previously described [4]. The time between transplantation and myeloid recovery to absolute neutrophil counts of >or=to500/mm3 and >or=to1000/mm3 was also measured. Statistical analysis was performed using paired and unpaired Student's t-test where appropriate.

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The two patient groups were similar in regard to age, weight, and anesthetic duration Table 1. There were 13 males and 6 females in Group I and 12 males and 7 females in Group II.

Table 1

Table 1

Results are shown in Figure 1 and Figure 2. Large SDS indicate a large scatter. There are no statistical differences between Groups I and II in CFU-GM 105 count and absolute neutrophil count recovery of >or=to500/mm3 or >or=to1000/mm (3). There was no obvious adverse effect in the patients exposed to high clinical concentrations of nitrous oxide compared to patients receiving an identical general anesthetic but without nitrous oxide. All patients underwent successful treatment and left the hospital feeling well, except one who died of complications related to her underlying disease.

Figure 1

Figure 1

Figure 2

Figure 2

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Concern has been raised as to the safety of nitrous oxide for general anesthesia. In controlled animal studies, nitrous oxide causes depression of bone marrow function, and has additional toxic effects on the immune, reproductive, and nervous systems [5]. Extrapolation from animal studies suggests that nitrous oxide should be avoided in certain clinical situations where rapid DNA synthesis occurs, such as bone marrow harvest. The CFU-GM assay has been used to study the growth of precursor cells of the myeloid lineage [3]. Colonies are derived from a single progenitor cell and, therefore, require multiple cell divisions. Although growth of CFU-GM does not rigorously correlate with hematopoietic stem cell activity and engraftment, this assay has been used by most laboratories to assess viability of bone marrow mononuclear cells [6]. Based on the CFU-GM assay, this preliminary study showed no difference in bone marrow viability between patients who received nitrous oxide and those who did not. The results we obtained in this study are somewhat different from the results we published in 1992 [7]. However, we believe the differences are presentational rather than significant. In another study, Carmel et al. [8] document the potential effects of nitrous oxide after a relatively short exposure. The assays used in that report were not used in our study. Instead we used the CFU-GM assay and hematologic engraftment as end-points. Although the variability in CFU-GM assays are wide, we chose to use a change in CFU-GM growth before and after exposure to air or nitrous oxide as the in vitro measurement. The lack of difference in both CFU-GM growth and engraftment supports our conclusion that exposure to nitrous oxide for the brief periods of time required for bone marrow harvesting has no clinically apparent effects.

As a measure of the time to successful transplantation, the interval between bone marrow aspiration until the absolute neutrophil counts were >or=to500/mm (3) and >or=to1000/mm3 was used. This study demonstrated no statistical differences between the two groups in the time required for successful engraftment. Our results are similar to Fausel et al. [9] who found that nitrous oxide during bone marrow harvest had no significant effect on the number of days to engraftment in autologous bone marrow transplantation.

In conclusion, the present study failed to demonstrate that short exposure (approximately 1 h) to nitrous oxide in high clinical concentrations (70%) has any detrimental effects on CFU-GM growth or rates of neutrofil engraftment.

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