R.S. Husain  ( Hamdard College of Medicine and Dentistry, Hamdard University, Karachi. )

Nitrous oxide is an anaesthetic gas used routinely in almost all cases requiring general anaesthesia. However prolonged exposure to nitrous oxide causes interference in vitamin B12 and folate metabolism thereby causing vitamin B12 and folate deficiency which results in megaloblastic anaemia, myeloneuropathies in the form of motor, sensory co-ordination and reflex defects and increased risk of abortion in females. Anaesthesiologists are repeatedly exposed to the patients expired gases in operation theatres. The aim of the study was to see whether chronic exposure to Nitrous oxide among anaesthesiologists causes vitamin BI2 deficiency .

Serum B12 levels of fifty doctors were measured. Informed consent was obtained from all participants and the study was approved by the ethical committee of Hamdard University Hospital Research Department.
Fifty physicians were recruited in the trial. They were divided into two study groups. Group I were practicing anaesthesiologists from Aga Khan University Hospital, Liaquat National Hospital, Abbasi Shaheed Hospital, Baqai Hospital and Hamdard University Hospital.
Group 1 were 25 anaesthesiologists working in operation theatres for more than five years and were using nitrous oxide in almost all cases where general anaesthesia was given . None were taking any Vitamin B12 supplements. All were using open or semi-open circuits for giving anaesthesia. Active scavenging system for exhaust gases was not present in any operation theatre.
Group 2 (control group) were 22 (non-anaesthesiologists) physicians working in the same institutions. Five ml of blood was drawn from each study participant. Vitamin B12 was done at the Aga Khan University Hospital Laboratory using Radioimmunoassay method.
Sensitivity of the test was high, i.e. 35 to 2400 pg/ml could be measured. The technician performing the test was blinded to the exposure of nitrous oxide among physicians.

A total of 179 patients were studied. Out of which 129 had nodular hyperplasia and 50 were suffering from carcinoma prostate. The average age of patients with nodular hyperplasia was 71.5 years (53-90 year), where as the average age of patients having carcinoma of prostate was 67.2 years (51-80 years).
The results of total PSA for all these patients showed that the mean value in both groups to be well apart. Calculation of 95% confidence interval (+2 SD = 4.93) reflected sinificant overlap between these two groups. The histogram of total PSA (Figure 1) showed that this overlap is considerable, particularly in the zone ranging from 5.0-20 ng/ml, consisting of majority of patients.
The results of percent free PSA showed that the mean values are more spaced out between these two subgroups and the 95% confidence interval (+2 SD = 10.12)

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Figure 1. Serum B-12 levels (pg/ml) in two combined samples.

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Figure 2. Anaesthetists.

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Figure 3. Non-anaesthetists.

250 to 290 pg/ml in the combined samples. The values were also plotted as histograms, as combined sample and separately for anaesthesiologists and the control group (Figures 1, 2 and 3).

Table 1. Age and sex distribution. Group Number Age Male Female I 25Anaesthesiologists 31-58years 6 19 II 22non-anaesthesiologists 32-56years 5 17

Serum B12 levels in pg/ml showed the theoretical normal distribution represented by the lines and the bars denoting individual readings in respective groups.The mean B12 level in anaesthesiologists was 231.0 pg /ml with a standard deviation of 105.11and in the control group 354.6 pg/ml with a standard deviation of 96.96.

Table 2. Stem and leaf plot showing serum B12 levels in pg/ml in the combined samples. Frequency Stem & leaf 7.00 1.0333334 8.00 1.57889999 3.00 2.223 10.00 2.5567778899 5.00 3 02334 6.00 3.666899 2.00 4.24 4.00 4.5679 2.00 5.44 Steam Widh: 100.0 each Leaf case(s)

The Box and Whisker plot (Figure 4) shows distribution of serum B12 (pg/ml) separately for the two sample groups. The median for anaesthesiologists was 200 pg/ml and for non-anaesthesiologists group above 350pg/ml. The boxes denote the central 50% cases showing that values in anaesthesiologists were below and the control group above 270 Pg/ml.

The Kolmogrov Smirnov test was applied with Lillieforts significance correction for test normality in the combined samples and the two samples separately. The test is based on the largest absolute difference between the observed and the expected cumulative distribution. The results indicated that deviation from normal distribution were non-significant. Thus parametric test could be applied to the data. To fulfill the assumption of equality of variances for two independent sample t-test. Levene's test was applied (as in table) which yielded non-significant values.

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Figure 4. Box and Whisker plots for two samples separately .

To test the null hypothesis that serum B12 levels in the two populations are same a t-test of independent samples was performed . The results indicated that the null hypothesis could be rejected (P<0.001).Thus it was concluded that the levels of serum B12 among anaesthesiologists were significantly lower than those of controls.

Vitamin B12 is a bound co-enzyme of methionine synthase and has a tetrapyrrole ring with a monovalent cobalt at the center. The cobalt functions as a methyl carrier in the transmethylation reaction. Nitrous oxide converts the cobalt from the monovalent form to the bivalent form. As a result the activity of methionine synthase is inhibited. Recovery is believed to require absorption of new unoxidised Vitamin B12 and synthesis of new apoenzyme. Thus gradually B12 deficiency sets in.1
Normal vitamin B12 values range from 200 pg/ml to 900 pg/ml. Occupational exposure to high levels of nitrous oxide has been associated with a higher incidence of spontaneous abortions2 and adverse effects on health.3 In another study it was found that anaesthesiologists complained of weakness and recurrent infections and had decreased peripheral blood leukocyte counts.4 Hazards of nitrous oxide have been reported even in patients who received it for a prolonged period of time.5
This study confirmed significantly lower levels of serum B12 in occupationally exposed anaesthesiologists as compared to normal controls.
There is no active scavenging system for removal of patients’ expired gases from the operation theatres. It is recommended that these be installed to overcome this occupational hazard but until such time that scavenging becomes available, anaesthesiologists should keep taking vitamin B12 supplements to prevent its deficiency. Although not determined in this study, but serum folic acid is likely to be low and should therefore be supplemented. It is suggested that all theatre personnel including surgeons should be checked for these deficiencies and alternative anaesthetic method not requiring nitrous oxide should be practiced.

We are grateful to Hakim Mohammed Said Shaheed, Chancellor, Hamdard University, for providing funds for measuring B12 levels, Professor Ahsan A Vahidy, MAHQ Biological Research Centre, University of Karachi, for statistical analysis. Professor Fauzia Anis Khan, Chairperson, Anaesthesiology, Aga Khan University Hospital, for her valuable guidance and all anaesthesiologists and non-anaesthesiologists who consented and provided samples for this study.

1. Bennett JC, Plum F. Mechanism of cobalamin deficiency In: Cecil textbook of medicine: 20th edition,W.B. Saunders, Philadelphia:1996, pp. 845-6.

2. Cohen EN, Brown BW, Bruce DL, et al. A survey of anesthetic health hazards among dentists: report of an American Society of Anesthesiologists adhoc committee on the effects of trace anesthetics on the health of operating room personnel. J Am Dent Assoc 1975; 90:1291.

3. Rowland AS, Baird DA, Weinberg CR, et el. Reduced fertility among women employed as dental assistants exposed to high levels of nitrous oxide. N Engl J Med 1992;327:9937. 4. Peric C. Nitrous oxide dangers in professionals. Anesthesia 1991; 46; 531-7.

5. Aamess JA, Berman JF, Rees GM, et al. Megaloblastic haemopoiesis in patients receiving Nitrous Oxide. Lancet 1978;5:339-42.