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EFFECT OF NITROUS OXIDE ON PLACENTAL METHIONINE SYNTHASE ACTIVITY
Br. J. Anaesth. (1986), 58, 524-527
EFFECT OF NITROUS OXIDE ON PLACENTAL METHIONINE SYNTHASE ACTIVITY M. J. LANDON AND V. J. TOOTHILL
PATIENTS, MATERIALS AND METHODS
All the patients were apparently normal healthy women who all delivered a healthy, full term, singleton infant after a wholly uneventful pregnancy. Placentae were collected from 11 patients who had been delivered by lower segment Caesarean section (LSCS). Caesarean sections were all elective procedures performed for the indications listed in table I. Brief clinical details of these patients, together with the anaesthetic agent and drugs used during the operation, are given in table II. For comparison, 20 placentae were collected after normal, routine vaginal deliveries during M. J. LANDON, PH.D.; VALERIE J. TOOTHIIX, M.I.BIOL.;
Div-
ision of Perinatal Medicine, MRC Clinical Research Centre, Watford Road, Harrow, Middlesex, HA1 3UJ.
SUMMARY Methionine synthase activity was measured in 11 placentae after Caesarean section during which nitrous oxide had been used as an anaesthetic agent, and compared with that from 20 placentae after normal vaginal delivery with no exposure to nitrous oxide. There was no significant difference in enzyme activity between the two series.
which i.v. pethidine was used for analgesia, if necessary (that is, nitrous oxide was not used). Placentae were processed within minutes of delivery. Each was shredded to a coarse mince which was then stirred well. An aliquot was thoroughly rinsed with ice-cold phosphate-buffered saline to remove as much adherent blood as possible and 10 g homogenized in 10 ml of phosphate buffer 0.1 mol litre"1 (pH 7.4). After centrifugationat 1500 g for 10 min, the supernatant was removed and stored at — 20 °C before assay within 1 week. The assay of methionine synthase was based on the method of Kamely, Littlefield and Erbe (1973). Preliminary studies established that the optimum reaction mixture for the placental TABLE I. Reasons for elective Caesarean section Patient 1
2 3 4 5 6 7 8 9 10 11
Reason for elective section Elderly primigravida, breech presentation Maternal idiopathic thrombocytopenia Breech presentation, previous section CPD (small pelvic outlet, large baby) Two previous sections for CPD Big baby - non-engaged breech at term Previous section, borderline pelvis, failed forceps Two previous sections for CPD Previous LSCS Primigravid breech presentation CPD (contracted pelvis, large baby)
Downloaded from http://bja.oxfordjournals.org/ at University of Exeter on July 21, 2015
There is now clear evidence that nitrous oxide, by inactivating vitamin B12, may interfere with certain transmethylation reactions and, consequently, DNA synthesis (Nunn, 1984). The inhibition of methionine synthase is a sensitive indicator of vitamin B u inactivation and a recent study in rats by Baden, Serra and Mazze (1984) has shown that fetal hepatic methionine synthase activity is decreased by 74 % after 30 min exposure of the mother to 50 % nitrous oxide. Although there is some evidence that the inactivation of vitamin B l t in man is somewhat slower than in rodents (Kano et al., 1981, 1984; Koblin et al., 1982), a study in man comparable to that of Baden, Serra and Mazze (1984) would be ethically and practically impossible. Therefore, we measured methionine synthase activity in placental tissue collected after operative deliveries during which nitrous oxide was used as an anaesthetic agent.
525
NITROUS OXIDE AND PLACENTAL METHIONINE SYNTHASE
TABLE II. Outcome of pregnancy and drugs used during elective Caesarean section. NR = not recorded. * Methohexitone not thiopentone; t sodium citrate not Mist. Mag. Trisil. %50% nitrous oxide was used except in patients 10 and 3, when 75% was used and in patient 9 the % was not recorded Patient
Patient 1 2 3 4
5 6 7 8 9 10 11
Parity
Gestation (weeks)
Infant sex
Birthweight (kg)
Placenta] weight (kg)
31 29 29 25 34 30 28 28 34 26 26
1 1 2 1 3 2 2 3 2 1 1
38 37 39 39 39 38 41 39 40 39 40
Boy Boy Boy Girl Girl Boy Girl Girl Girl Boy Boy
2.78 2.16 3.4 3.58 2.9 2.81 3.42 3.16 3.38 2.9 3.88
0.58 0.41 0.53 0.62 0.56 0.52 0.78 0.62 0.7 0.5 0.67
Mist. Mag. Trisil (ml) 20 NR 30 none 30 30
30+
20 NR 20 30
Thiopentone (mg)
Suxamethonium (mg)
Alcuronium (mg)
Pancuronium (mg)
Exposure to N t O$ (min)
Placental methionine synthase activity (nmol h" 1 / mg protein)
250 250 250 100* 300 300 250 275 350 350 250
75 100 100 100 100 75 100 100 100 100 100
10 + 2 5 NR — 12.5 15 15 10 — 15 15 —
— — 5 — — — — 5 — — 3
15 20 15 22 19 17 17 18 15 13 18
1.85 L.9 :2.05 L.95 L.85 1.70 L.4 1.5 2.75 2.7 1.1
enzyme should contain: potassium phosphate buffer 20 umol, pH 7.4; s-adenosylmethionine 50 nmol; cyanocobalamin 10 nmol; 2-mercaptoethanol 30 nmol; DL-homocysteine 50 nmol; DL-N-5 methyltetrahydrofolate 100 nmol ( r e labelled supplied by Amersham International, specific activity 1 uCi/100 nmol); placental extract 50 jxl. After incubation in the dark for 1 h at 37 °C, the reaction was stopped by adding 0.8 ml of ice-cold water. The uC-labelled product was separated from unreacted substrate by ion exchange chromatography using mini columns packed with 0.5-ml equivalent volume of Dowex 1 x 8-400 (Chloride form) and counted in Bray's scintillation cocktail with appropriate quench correction. Protein content of the placental extract was measured as described by Lowry and colleagues (1951) and the results expressed as nmol of product formed per milligram of protein per hour (nmol h"V m 8 protein).
Using this method the reaction was linear throughout the incubation period and the amount of product formed was directly proportional to the volume of placental extract used. Enzyme activity was stable for up to 1 week when stored as a buffered extract at — 20 °C, but not as whole tissue or unextracted mince. Removal of contaminating blood was important as there was evidence that highly coloured extracts gave lower specific activities. Hoffstee and Lineweaver-Burke plots indicated that the placental enzyme had a Km of about 80 umol and a Vmax of 1.75 nmol h~'/mg protein.
RESULTS
There was no statistically significant difference between the methionine synthase activity of placentae delivered after LSCS and that of the controls (fig. 1).
Downloaded from http://bja.oxfordjournals.org/ at University of Exeter on July 21, 2015
1 2 3 4 5 6 7 8 9 10 11
Age (yr)
BRITISH JOURNAL OF ANAESTHESIA
526
t • ••
SI 8*
1! §5
NVD (Controls)
LSCS (with N 2 0)
FIG. 1. Placenta! methionine lynthase activity. Horizontal line represents the mean for each group. NVD = normal vaginal delivery; LSCS •= lower segment Caesarean section.
DISCUSSION
The average value of 1.7 nmol h~1/mg protein is similar to that reported for adult tissue, but rather less than one-half that of fetal liver (Gaull et al., 1973), in contrast to the rat, in which fetal activity is slightly lower than the maternal value (Baden, Serra and Mazze, 1984). This study adds to the evidence that there is a significant species difference in the inactivation of vitamin Bj, by nitrous oxide. In rodents, hepatic methionine synthase activity is decreased to less than 30% of control after only 30 min exposure to 50% or 70% nitrous oxide (Deacon et al., 1980; Koblin et al., 1981; Baden, Serra and Mazze, 1984). In contrast, studies on human tissue suggest a half-time for inactivation of between 1.5 and 6 h (Kano et al., 1981; Koblin et al., 1982). Furthermore, serum methionine and s-adenosyl-
ACKNOWLEDGEMENTS We thank the staff of the Delivery Suite at Northwick Park Hospital for their help in the collection of the placentae and Dr J. F. Nunn for advice and encouragement.
REFERENCES Baden, J. M., Serra, M., and Mazze, R. L. (1984). Inhibition of fetal methionine 3ynthase by nitrous oxide. Br. J. Anaesth., 56, 523. Deacon, R., Lumb, M., Chanarin, I., Minty, B., Halsey, M. J., and Nunn, J. F. (1980). Inactivation ofmethionine synthetase by N t O. Eur. J. Biochem., 104, 419. Gaull, G. E., von Berg, W., Raiha, N. C. R., and Sturman, J. A. (1973). Development of methyltransferase activities of human fetal tissues. Pcdiatr. Res., 7, 527. Kamely, D., Littlefield, J. W., and Erbe, R. W. (1973). Regulation of s-methyltetrahydrofolate:homocysteine methyltransferase activity by methionine, vitamin B l t and folate in cultured baby hamster kidney cells. Proc. Natl Acad. Set. U.S^i., 70, 2585. Kano, Y., Sakamoto, S., Sakuraya, K., Kubota, T., Hida, K., Suda, K., and Takaku, F. (1981). Effect of nitrous oxide on human bone marrow cells and its synergistic effect with methionine and methotrexate on functional folate deficiency. Can. Ret., 41, 4698. Taguchi, H., Muira, Y., and Takaku, F. (1984). Effects of leucovorin and methylcobalamin with N,O anesthesia. J. Lab. Clin. Mtd., 104, 711. Koblin, D. D., Waskell, L., Watson, J. E., Stokstad, E. L. R., and Eger, E. I. (1982). Nitrous oxide inactivates methionine synthetase in human liver. Anetth. Analg., 61, 75.
Downloaded from http://bja.oxfordjournals.org/ at University of Exeter on July 21, 2015
methionine are unchanged after exposure of patients to 70% nitrous oxide for an average of 85 min (Nunn et al., 1985), and deoxyuridine synthesis in bone marrow is unaffected by exposing patients to 66 % nitrous oxide for up to 4 h (Kano et al., 1984). These differences between the human and the rodent enzyme are unexplained, but could be attributable to minor differences in the amino acid sequence of the apoenzyme which would alter the accessibility of the vitamin B1S cobalt to nitrous oxide. The present data clearly show that human placental methionine synthase activity is unaffected by nitrous oxide, and also suggest that this transmethylation reaction, in common with others (van der Ploeg and Flavell, 1980; Welsch, Wenger and Stedman, 1981) is not an important function of the placenta. In these circumstances it cannot be assumed that the fetal liver will be unaffected, but it seems improbable that any inhibition would occur in fetal tissue and there appears to be no contraindication, attributable to its interaction with fetal vitamin B12, to the use of nitrous oxide during Caesarean section of normal duration.
NITROUS OXIDE AND PLACENTAL METHIONINE SYNTHASE Koblin, D. D., Watson, J. E., Deady, J. E., Stokstad, E. L. R., andEger,E. I. (1981). Inactivation of methionine synthetase by N,O in mice. Anathesiology, 54, 318. Lowry, O. H., Rosenbrough, N. J., FarT, A. L., and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193, 265. Nunn, J. F. (1984). Interaction of nitrous oxide and vitamin B l r T.I.P.S., 5, 225. Sharer, M., Bottiglieri, T., and Rossiter, J. (1985). Plasma
527
methionine concentrations during elective surgery and nitrous oxide, Br. J. Anaath., 57, 342P. van der Ploeg, L. H. T., and FlaveU, R. A. (1980). D N A methylation in the human y8P-globin locus in erythroid and nonerythroid tissue. Cell, 19, 947. Welsch, F., Wenger, W. C , and Stedman, D. B. (1981). Choline metabolism in placenta: evidence for the biosynthesis of phosphatidylcholine in microsomes via the methylation pathway. Placenta, 2, 211.
Downloaded from http://bja.oxfordjournals.org/ at University of Exeter on July 21, 2015
EFFECT OF NITROUS OXIDE ON PLACENTAL METHIONINE SYNTHASE ACTIVITY M. J. LANDON AND V. J. TOOTHILL
PATIENTS, MATERIALS AND METHODS
All the patients were apparently normal healthy women who all delivered a healthy, full term, singleton infant after a wholly uneventful pregnancy. Placentae were collected from 11 patients who had been delivered by lower segment Caesarean section (LSCS). Caesarean sections were all elective procedures performed for the indications listed in table I. Brief clinical details of these patients, together with the anaesthetic agent and drugs used during the operation, are given in table II. For comparison, 20 placentae were collected after normal, routine vaginal deliveries during M. J. LANDON, PH.D.; VALERIE J. TOOTHIIX, M.I.BIOL.;
Div-
ision of Perinatal Medicine, MRC Clinical Research Centre, Watford Road, Harrow, Middlesex, HA1 3UJ.
SUMMARY Methionine synthase activity was measured in 11 placentae after Caesarean section during which nitrous oxide had been used as an anaesthetic agent, and compared with that from 20 placentae after normal vaginal delivery with no exposure to nitrous oxide. There was no significant difference in enzyme activity between the two series.
which i.v. pethidine was used for analgesia, if necessary (that is, nitrous oxide was not used). Placentae were processed within minutes of delivery. Each was shredded to a coarse mince which was then stirred well. An aliquot was thoroughly rinsed with ice-cold phosphate-buffered saline to remove as much adherent blood as possible and 10 g homogenized in 10 ml of phosphate buffer 0.1 mol litre"1 (pH 7.4). After centrifugationat 1500 g for 10 min, the supernatant was removed and stored at — 20 °C before assay within 1 week. The assay of methionine synthase was based on the method of Kamely, Littlefield and Erbe (1973). Preliminary studies established that the optimum reaction mixture for the placental TABLE I. Reasons for elective Caesarean section Patient 1
2 3 4 5 6 7 8 9 10 11
Reason for elective section Elderly primigravida, breech presentation Maternal idiopathic thrombocytopenia Breech presentation, previous section CPD (small pelvic outlet, large baby) Two previous sections for CPD Big baby - non-engaged breech at term Previous section, borderline pelvis, failed forceps Two previous sections for CPD Previous LSCS Primigravid breech presentation CPD (contracted pelvis, large baby)
Downloaded from http://bja.oxfordjournals.org/ at University of Exeter on July 21, 2015
There is now clear evidence that nitrous oxide, by inactivating vitamin B12, may interfere with certain transmethylation reactions and, consequently, DNA synthesis (Nunn, 1984). The inhibition of methionine synthase is a sensitive indicator of vitamin B u inactivation and a recent study in rats by Baden, Serra and Mazze (1984) has shown that fetal hepatic methionine synthase activity is decreased by 74 % after 30 min exposure of the mother to 50 % nitrous oxide. Although there is some evidence that the inactivation of vitamin B l t in man is somewhat slower than in rodents (Kano et al., 1981, 1984; Koblin et al., 1982), a study in man comparable to that of Baden, Serra and Mazze (1984) would be ethically and practically impossible. Therefore, we measured methionine synthase activity in placental tissue collected after operative deliveries during which nitrous oxide was used as an anaesthetic agent.
525
NITROUS OXIDE AND PLACENTAL METHIONINE SYNTHASE
TABLE II. Outcome of pregnancy and drugs used during elective Caesarean section. NR = not recorded. * Methohexitone not thiopentone; t sodium citrate not Mist. Mag. Trisil. %50% nitrous oxide was used except in patients 10 and 3, when 75% was used and in patient 9 the % was not recorded Patient
Patient 1 2 3 4
5 6 7 8 9 10 11
Parity
Gestation (weeks)
Infant sex
Birthweight (kg)
Placenta] weight (kg)
31 29 29 25 34 30 28 28 34 26 26
1 1 2 1 3 2 2 3 2 1 1
38 37 39 39 39 38 41 39 40 39 40
Boy Boy Boy Girl Girl Boy Girl Girl Girl Boy Boy
2.78 2.16 3.4 3.58 2.9 2.81 3.42 3.16 3.38 2.9 3.88
0.58 0.41 0.53 0.62 0.56 0.52 0.78 0.62 0.7 0.5 0.67
Mist. Mag. Trisil (ml) 20 NR 30 none 30 30
30+
20 NR 20 30
Thiopentone (mg)
Suxamethonium (mg)
Alcuronium (mg)
Pancuronium (mg)
Exposure to N t O$ (min)
Placental methionine synthase activity (nmol h" 1 / mg protein)
250 250 250 100* 300 300 250 275 350 350 250
75 100 100 100 100 75 100 100 100 100 100
10 + 2 5 NR — 12.5 15 15 10 — 15 15 —
— — 5 — — — — 5 — — 3
15 20 15 22 19 17 17 18 15 13 18
1.85 L.9 :2.05 L.95 L.85 1.70 L.4 1.5 2.75 2.7 1.1
enzyme should contain: potassium phosphate buffer 20 umol, pH 7.4; s-adenosylmethionine 50 nmol; cyanocobalamin 10 nmol; 2-mercaptoethanol 30 nmol; DL-homocysteine 50 nmol; DL-N-5 methyltetrahydrofolate 100 nmol ( r e labelled supplied by Amersham International, specific activity 1 uCi/100 nmol); placental extract 50 jxl. After incubation in the dark for 1 h at 37 °C, the reaction was stopped by adding 0.8 ml of ice-cold water. The uC-labelled product was separated from unreacted substrate by ion exchange chromatography using mini columns packed with 0.5-ml equivalent volume of Dowex 1 x 8-400 (Chloride form) and counted in Bray's scintillation cocktail with appropriate quench correction. Protein content of the placental extract was measured as described by Lowry and colleagues (1951) and the results expressed as nmol of product formed per milligram of protein per hour (nmol h"V m 8 protein).
Using this method the reaction was linear throughout the incubation period and the amount of product formed was directly proportional to the volume of placental extract used. Enzyme activity was stable for up to 1 week when stored as a buffered extract at — 20 °C, but not as whole tissue or unextracted mince. Removal of contaminating blood was important as there was evidence that highly coloured extracts gave lower specific activities. Hoffstee and Lineweaver-Burke plots indicated that the placental enzyme had a Km of about 80 umol and a Vmax of 1.75 nmol h~'/mg protein.
RESULTS
There was no statistically significant difference between the methionine synthase activity of placentae delivered after LSCS and that of the controls (fig. 1).
Downloaded from http://bja.oxfordjournals.org/ at University of Exeter on July 21, 2015
1 2 3 4 5 6 7 8 9 10 11
Age (yr)
BRITISH JOURNAL OF ANAESTHESIA
526
t • ••
SI 8*
1! §5
NVD (Controls)
LSCS (with N 2 0)
FIG. 1. Placenta! methionine lynthase activity. Horizontal line represents the mean for each group. NVD = normal vaginal delivery; LSCS •= lower segment Caesarean section.
DISCUSSION
The average value of 1.7 nmol h~1/mg protein is similar to that reported for adult tissue, but rather less than one-half that of fetal liver (Gaull et al., 1973), in contrast to the rat, in which fetal activity is slightly lower than the maternal value (Baden, Serra and Mazze, 1984). This study adds to the evidence that there is a significant species difference in the inactivation of vitamin Bj, by nitrous oxide. In rodents, hepatic methionine synthase activity is decreased to less than 30% of control after only 30 min exposure to 50% or 70% nitrous oxide (Deacon et al., 1980; Koblin et al., 1981; Baden, Serra and Mazze, 1984). In contrast, studies on human tissue suggest a half-time for inactivation of between 1.5 and 6 h (Kano et al., 1981; Koblin et al., 1982). Furthermore, serum methionine and s-adenosyl-
ACKNOWLEDGEMENTS We thank the staff of the Delivery Suite at Northwick Park Hospital for their help in the collection of the placentae and Dr J. F. Nunn for advice and encouragement.
REFERENCES Baden, J. M., Serra, M., and Mazze, R. L. (1984). Inhibition of fetal methionine 3ynthase by nitrous oxide. Br. J. Anaesth., 56, 523. Deacon, R., Lumb, M., Chanarin, I., Minty, B., Halsey, M. J., and Nunn, J. F. (1980). Inactivation ofmethionine synthetase by N t O. Eur. J. Biochem., 104, 419. Gaull, G. E., von Berg, W., Raiha, N. C. R., and Sturman, J. A. (1973). Development of methyltransferase activities of human fetal tissues. Pcdiatr. Res., 7, 527. Kamely, D., Littlefield, J. W., and Erbe, R. W. (1973). Regulation of s-methyltetrahydrofolate:homocysteine methyltransferase activity by methionine, vitamin B l t and folate in cultured baby hamster kidney cells. Proc. Natl Acad. Set. U.S^i., 70, 2585. Kano, Y., Sakamoto, S., Sakuraya, K., Kubota, T., Hida, K., Suda, K., and Takaku, F. (1981). Effect of nitrous oxide on human bone marrow cells and its synergistic effect with methionine and methotrexate on functional folate deficiency. Can. Ret., 41, 4698. Taguchi, H., Muira, Y., and Takaku, F. (1984). Effects of leucovorin and methylcobalamin with N,O anesthesia. J. Lab. Clin. Mtd., 104, 711. Koblin, D. D., Waskell, L., Watson, J. E., Stokstad, E. L. R., and Eger, E. I. (1982). Nitrous oxide inactivates methionine synthetase in human liver. Anetth. Analg., 61, 75.
Downloaded from http://bja.oxfordjournals.org/ at University of Exeter on July 21, 2015
methionine are unchanged after exposure of patients to 70% nitrous oxide for an average of 85 min (Nunn et al., 1985), and deoxyuridine synthesis in bone marrow is unaffected by exposing patients to 66 % nitrous oxide for up to 4 h (Kano et al., 1984). These differences between the human and the rodent enzyme are unexplained, but could be attributable to minor differences in the amino acid sequence of the apoenzyme which would alter the accessibility of the vitamin B1S cobalt to nitrous oxide. The present data clearly show that human placental methionine synthase activity is unaffected by nitrous oxide, and also suggest that this transmethylation reaction, in common with others (van der Ploeg and Flavell, 1980; Welsch, Wenger and Stedman, 1981) is not an important function of the placenta. In these circumstances it cannot be assumed that the fetal liver will be unaffected, but it seems improbable that any inhibition would occur in fetal tissue and there appears to be no contraindication, attributable to its interaction with fetal vitamin B12, to the use of nitrous oxide during Caesarean section of normal duration.
NITROUS OXIDE AND PLACENTAL METHIONINE SYNTHASE Koblin, D. D., Watson, J. E., Deady, J. E., Stokstad, E. L. R., andEger,E. I. (1981). Inactivation of methionine synthetase by N,O in mice. Anathesiology, 54, 318. Lowry, O. H., Rosenbrough, N. J., FarT, A. L., and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193, 265. Nunn, J. F. (1984). Interaction of nitrous oxide and vitamin B l r T.I.P.S., 5, 225. Sharer, M., Bottiglieri, T., and Rossiter, J. (1985). Plasma
527
methionine concentrations during elective surgery and nitrous oxide, Br. J. Anaath., 57, 342P. van der Ploeg, L. H. T., and FlaveU, R. A. (1980). D N A methylation in the human y8P-globin locus in erythroid and nonerythroid tissue. Cell, 19, 947. Welsch, F., Wenger, W. C , and Stedman, D. B. (1981). Choline metabolism in placenta: evidence for the biosynthesis of phosphatidylcholine in microsomes via the methylation pathway. Placenta, 2, 211.
Downloaded from http://bja.oxfordjournals.org/ at University of Exeter on July 21, 2015