BritishJournal of Haematofogy, 1976,34, 567.
Serum ‘Uracil+Uridine’ Levels in Pernicious Anaemia T. E. PARRY AND J. A. BLACKMORE Department of Haernatology, Llandough Hospital, Penarth, South Glamorgan Area Health Authority (Teaching) (Received 23 March 1976; accepted for publication 24 M a y 1976)
SUMMARY.The serum ‘uracil+ uridine’ level, expressed as uracil, has been measured in 21 cases of vitamin B,, deficiency, in which the serum folate was normal, and compared with the level in 97 normal subjects. The level in the vitamin Blz deficient group (11.9 pmolll.) was significantly lower than in the controls (15.7pmol/l., P< 0.005). Nine of the former were complicated by systemic illness but the clinical and haematological features in the remaining 12 were consistent with the diagnosis of pernicious anaemia in relapse. The serum uracil level in this group was even lower (10.21pmol/l., lk0.01). This finding is unexpected in view of the generally accepted indirect role of vitamin B,, in the methylation of deoxyuridine monophosphate to deoxythymidine monophosphate. Reasons are given for not accepting these results as reflecting the main biochemical lesion in vitamin B12 deficiency. Although they do not give direct support to an impairment in the methylation of deoxyuridine monophosphate, they do not exclude it as they test only one possible metabolic pathway and moreover they could represent the result of more than one action ofvitamin B,, on uracil metabolism. They do show, however, that some aspect of uracil metabolism other than methylation is affected in vitamin Blz deficiency in man. Impaired methylation of deoxyuridine monophosphate (dUMP) to form deoxythymidine monophosphate (dTMP) has been regarded as the basic biochemical defect in megaloblastic anaemia (Beck, 1964; Parry, 1966; Waxman et al, 1970). This reaction has also been regarded as the rate limiting reaction in DNA synthesis (Chanarin, 1969; H o a r a n d , 1972). The enzyme system involved (thymidylate synthetase) has as its active component 5,10methylene tetrahydrofolic acid which donates the carbon atom as well as two of the three hydrogen atoms to the methyl group and is itself oxidized to dihydrofolic acid (Wahba & Friedkin, 1961; Pastore & Friedkin, 1962). The reaction is completely independent of vitamin B I Z(Wahba & Friedkin, 1962). To perpetuate the reaction the 5,ro-methylene tetrahydrofolic acid has to be reconstituted and this proceeds in two stages : (a) the reduction of dihydrofolic acid to tetrahydrofolic acid by the enzyme dihydrofolic reductase, and (b) the conversion of tetrahydrofolic acid to 5,Io-methylene tetrahydrofolic acid by the enzyme serine hydroxymethylase, the CH, group being transferred from the position of serine (Waxman et a!, 1970). The supply of tetrahydrofolic acid to the above cycle is augmented by the demethylation of 5-methyl tetrahydrofolic acid, the methyl group being transferred to Correspondence: Dr T. E. Parry, Department of Haematology, Llandough Hospital, Penarth, Glam. CF6 IXX, Wales.
567
T.E. Parry and I. A. Blackmore
568
homocysteine to form methionine. This is the only one of the above reactions which is known to be vitamin B, as well as folate dependent. The blocking of this reaction, with the accumulation of 5-methyl tetrahydrofolic acid-the methyl folate trap-is considered to be the basis of the megaloblastic anaemia of vitamin B,, deficiency (Waxman et a!, 1969; Hoffbrand, 1972; Lavoie et a!, 1974). The abnormally low serum methionine level encountered in pernicious anaemia in relapse which is returned to normal by both vitamin Blz and folate therapy (Parry, 1969) is consistent with the above hypothesis and it would be interesting if similar changes in the serum levels of thymine and uracil could be demonstrated in megaloblastic anaemia. If thymine is present in serum its level is too low to be measured by methods currently available but uracil is present in measurable quantities (Parry & Blackmore, 1974), and this has been measured in 21 cases of pure vitamin B1, deficiency.
,
MATERIALS AND METHODS Both serum vitamin B,, and folate were assayed microbiologically, the former with Euglena gracilis and the latter with Lactobacillus casei as test organism. Serum uracil was likewise assayed microbiologically by the method of Merrifield & Dunn (1948), as modified by Parry & Blackmore (1974).The growth of the test organism (Streptococcus h i s , National Collection of Industrial Bacteria No. 10769) is supported by both uracil and uridine equally and the method therefore measures ‘uracil+ uridine’ together. The results are expressed in terms of uracil. The serum ‘uracil+ uridine’level has been measured in 21 cases ofpure vitamin B12 deficiency in whom the serum folate level was within normal limits. Eleven sera were obtained from patients under observation in hospital; the remainder from a vitamin B,, laboratory. The only data of the latter group known at the time of analysis were the levels of the serum vitamin B12 and folate but the clinical and laboratory findings of each patient were subsequently examined. The serum ‘uracil+ uridine’ levels of 97 normal subjects were examined at the same time. These varied in age from 19 months to 86 years and included normal subjects matched for age and sex with each case of vitamin B12 deficiency and the pairs assayed together. Although the ‘uracil+ uridine’ level showed some variation with age in a larger series of 144 normal subjects-including cord blood from six full term infants-previously reported (Parry & Blackmore, 1974) such a variation was not seen in the present series. The entire group of 97 normal subjects was therefore taken as the control group. The statistical analysis was carried out by Professor H. Campbell and Dr T. Khosla of the Welsh National School of Medicine. RESULTS
,
The mean serum ‘uracil+ uridine’ level was significantly lower in the vitamin B, deficient group(11.88pmol/l.) than in the normal controls (15.7pmol/l., P
Serum ‘Uracil+Uridine’ Levels in Pernicious Anaemia T. E. PARRY AND J. A. BLACKMORE Department of Haernatology, Llandough Hospital, Penarth, South Glamorgan Area Health Authority (Teaching) (Received 23 March 1976; accepted for publication 24 M a y 1976)
SUMMARY.The serum ‘uracil+ uridine’ level, expressed as uracil, has been measured in 21 cases of vitamin B,, deficiency, in which the serum folate was normal, and compared with the level in 97 normal subjects. The level in the vitamin Blz deficient group (11.9 pmolll.) was significantly lower than in the controls (15.7pmol/l., P< 0.005). Nine of the former were complicated by systemic illness but the clinical and haematological features in the remaining 12 were consistent with the diagnosis of pernicious anaemia in relapse. The serum uracil level in this group was even lower (10.21pmol/l., lk0.01). This finding is unexpected in view of the generally accepted indirect role of vitamin B,, in the methylation of deoxyuridine monophosphate to deoxythymidine monophosphate. Reasons are given for not accepting these results as reflecting the main biochemical lesion in vitamin B12 deficiency. Although they do not give direct support to an impairment in the methylation of deoxyuridine monophosphate, they do not exclude it as they test only one possible metabolic pathway and moreover they could represent the result of more than one action ofvitamin B,, on uracil metabolism. They do show, however, that some aspect of uracil metabolism other than methylation is affected in vitamin Blz deficiency in man. Impaired methylation of deoxyuridine monophosphate (dUMP) to form deoxythymidine monophosphate (dTMP) has been regarded as the basic biochemical defect in megaloblastic anaemia (Beck, 1964; Parry, 1966; Waxman et al, 1970). This reaction has also been regarded as the rate limiting reaction in DNA synthesis (Chanarin, 1969; H o a r a n d , 1972). The enzyme system involved (thymidylate synthetase) has as its active component 5,10methylene tetrahydrofolic acid which donates the carbon atom as well as two of the three hydrogen atoms to the methyl group and is itself oxidized to dihydrofolic acid (Wahba & Friedkin, 1961; Pastore & Friedkin, 1962). The reaction is completely independent of vitamin B I Z(Wahba & Friedkin, 1962). To perpetuate the reaction the 5,ro-methylene tetrahydrofolic acid has to be reconstituted and this proceeds in two stages : (a) the reduction of dihydrofolic acid to tetrahydrofolic acid by the enzyme dihydrofolic reductase, and (b) the conversion of tetrahydrofolic acid to 5,Io-methylene tetrahydrofolic acid by the enzyme serine hydroxymethylase, the CH, group being transferred from the position of serine (Waxman et a!, 1970). The supply of tetrahydrofolic acid to the above cycle is augmented by the demethylation of 5-methyl tetrahydrofolic acid, the methyl group being transferred to Correspondence: Dr T. E. Parry, Department of Haematology, Llandough Hospital, Penarth, Glam. CF6 IXX, Wales.
567
T.E. Parry and I. A. Blackmore
568
homocysteine to form methionine. This is the only one of the above reactions which is known to be vitamin B, as well as folate dependent. The blocking of this reaction, with the accumulation of 5-methyl tetrahydrofolic acid-the methyl folate trap-is considered to be the basis of the megaloblastic anaemia of vitamin B,, deficiency (Waxman et a!, 1969; Hoffbrand, 1972; Lavoie et a!, 1974). The abnormally low serum methionine level encountered in pernicious anaemia in relapse which is returned to normal by both vitamin Blz and folate therapy (Parry, 1969) is consistent with the above hypothesis and it would be interesting if similar changes in the serum levels of thymine and uracil could be demonstrated in megaloblastic anaemia. If thymine is present in serum its level is too low to be measured by methods currently available but uracil is present in measurable quantities (Parry & Blackmore, 1974), and this has been measured in 21 cases of pure vitamin B1, deficiency.
,
MATERIALS AND METHODS Both serum vitamin B,, and folate were assayed microbiologically, the former with Euglena gracilis and the latter with Lactobacillus casei as test organism. Serum uracil was likewise assayed microbiologically by the method of Merrifield & Dunn (1948), as modified by Parry & Blackmore (1974).The growth of the test organism (Streptococcus h i s , National Collection of Industrial Bacteria No. 10769) is supported by both uracil and uridine equally and the method therefore measures ‘uracil+ uridine’ together. The results are expressed in terms of uracil. The serum ‘uracil+ uridine’level has been measured in 21 cases ofpure vitamin B12 deficiency in whom the serum folate level was within normal limits. Eleven sera were obtained from patients under observation in hospital; the remainder from a vitamin B,, laboratory. The only data of the latter group known at the time of analysis were the levels of the serum vitamin B12 and folate but the clinical and laboratory findings of each patient were subsequently examined. The serum ‘uracil+ uridine’ levels of 97 normal subjects were examined at the same time. These varied in age from 19 months to 86 years and included normal subjects matched for age and sex with each case of vitamin B12 deficiency and the pairs assayed together. Although the ‘uracil+ uridine’ level showed some variation with age in a larger series of 144 normal subjects-including cord blood from six full term infants-previously reported (Parry & Blackmore, 1974) such a variation was not seen in the present series. The entire group of 97 normal subjects was therefore taken as the control group. The statistical analysis was carried out by Professor H. Campbell and Dr T. Khosla of the Welsh National School of Medicine. RESULTS
,
The mean serum ‘uracil+ uridine’ level was significantly lower in the vitamin B, deficient group(11.88pmol/l.) than in the normal controls (15.7pmol/l., P
