British Journal ofHaematology, 1976,34, 575.
Serum ‘Uracil+Uridine’ Levels Before and After Vitamin B,, Therapy in Pernicious Anaemia T. E. PARRY AND J. A. BLACKMORE Department of Haematology, Llandough Hospital, Penarth, South GIamotgan Area Health Authority (Teaching) (Received 23 March 1976; acceptedfor publication 24. May 1976)
+
SUMMARY. The serum ‘uracil uridine’ levels, expressed as uracil, have been measured in 10 cases of pernicious anaemia both before and after treatment, and compared with the levels in 97 normal subjects. The mean pre-treatment value (8.82 pmol/l., range 6.0-12.0 pmolll.) differed significantly from that of the normal controls (15.7pmol/l., range 5.7-40.5pmolll., t = 8.8, Pco.001).This confirms the low serum uracil level previously reported in pernicious anaemia in relapse. The level rose progressively after treatment, reaching a maximum on the fourth day (mean 17.85 pmol/l,, range 9.3-23.4 pmol/l.). This was not significantly different from the mean of the normal control group. The difference between the pre- and post-treatment levels was significant on days 3, 4 and 5 (Pco.005, Pco.001 and Pc 0.005 respectively) and the rise preceded the reticulocyte response by 24 h. A further case was treated with physiological doses of vitamin BIZ( 2 pg daily for 6 d) and a similar rise in the serum uracil level noted. These results are not explained by any of the known functions of vitamin Bt 2 . They are, however, similar to the changes in the serum methionine levels previously reported in pernicious anaemia. The latter were readily explained by the known action of vitamin BIZ on ‘de novo’ methionine synthesis and it is suggested that the synthesis of uracil, like that of methionine, might be influenced by vitamin BIZin man. Failure to methylate deoxyuridine monophosphate (dUMP) to form deoxythymidine monophosphate (dTMP) has been regarded as the basic biochemical defect in megaloblastic anaemia but an attempt to confirm this by direct measurement of the serum uracil in patients with pernicious anaemia (PA) in relapse showed unexpectedly that the uracil concentration was significantly lower than in normal controls (Parry & Blackmore, 1976). It is therefore pertinent to establish whether or not the serum uracil level in this disease is influenced by vitamin Biz. This has now been measured both before and after treatment in 10cases of PA and the results are presented here. The effects of physiological doses of vitamin B i z were studied in a further patient (case 11). Correspondence:Dr T. E. Parry, Department of Haematology, Llaiidough Hospital, Penarth, Glam. CF6 IXX, Wales.
575
T. E. Parry a n d / . A. Blackmore
576
MATERIALS AND METHODS Serum vitamin B,, , folate and 'uracil+ uridine' (expressed as uracil) were all measured microbiologically, vitamin Bl using Lactobacillus leichrnannii, and folate and uracil as described previously (Parry & Blackmore, 1976). The clinical and haematological features (Table I) in all 11 cases were consistent with a diagnosis of PA in relapse. The serum uracil was assayed before treatment and daily after treatment for 5 d in eight cases and for 4 d in the remaining two. All venous samples for uracil assay and for a reticulocyte count were collected immediately before the patients received their midday meal. After the pre-treatment sample had been collected zoo pg of cyanocobalamin (Vitamin Biz) was given by intramuscular injection at 14.00hours the same day (day 0)to the first 10 cases with the exception of case 5 who received 1000 pg. The statistical analysis was carried out by Professor H. Campbell and Dr T. Khosla of the Welsh National School of Medicine. TABLE I
M M M
6 7 8 9
72 46 53 72 so 66 70 58 45
10
67
F M
11
63
F
I 2
3 4 5
F M F M M
1.72
6.0
I10
1.32
S.0 10.0
I39 104 I35
2.82 1.50
1.99 I .433 2.0
2.63 2.0
1.70 1.69
6.6 8.7 6.3 7.6 8.3 7.9 6.3 7.0
130
126 114 I12
I20 I00
123
Megaloblastic Megaloblastic Megaloblastic Megaloblastic Megaloblastic Megaloblastic Megaloblastic Megaloblastic Megaloblastic Megaloblastic Megaloblastic
6.8 12.6 5.2 11.3
3.5 4.5 7.6 11.0
4.6 7.8 7.0
RESULTS The uracil levels were compared with those previously reported in 97 normal control subjects (Parry & Blackmore, 1976). The mean pre-treatment value in the 10 cases was 8.82 pmol/l. (range 6.0-12.0 pmol/l.). The difference between this and the mean for the normal control group (1~.7pmol/l.,range 5.7-4o.spmol/l.) was highly significant(t = 8.8, P< 0.001). The low serum uracil level previously reported in PA in relapse was therefore confirmed. The uracil level rose progressively after treatment and was significantly higher than the pre-treatment level on the third, fourth and fifth post-treatment days (P< 0.005,P< 0.001 and P< 0.001 respectively). It reached its maximum on the fourth day (mean 17.85 pmol/l., range 9.3-23.4 pmol.). This was not significantly different from the mean of the normal control group. The serum uracil level was therefore subnormal in PA in relapse and rose to reach normal levels on the fourth post-treatment day. The rise preceded the reticulocyte response by 24 h. Vitamin BI2had no effect on the serum uracil level in four normal subjects
Serum Uracil and Vitamin BIZ Therapy
-4?
I
I
I
I
I
I
I
2
3
4
5
6
577
Cays
FIG I. Difference between pre- (day 0) and post-treatment serum uracil values ( 0 ) and reticulocyte count (0) in 10cases ofpernicious anaemia given a therapeutic dose ofvitamin B,, on day o compared with four normal subjects (A) given a similar dose of vitamin B I 2 (zoo pg i.m., case 5 received ZOO0 pg).
(Fig I). It was noteworthy also that the range of the pretreatment values was fairly narrow as was reflected by the low standard deviation of 1.8 which differs significantly from the standard deviation of 5.26 in the control group (F = 8.7, P< 0.005).This confirms the previously reported observation that the serum uracil values in PA in relapse form a compact group, the mean value of which is significantly lower than normal. Following treatment, however, the range widens and the standard deviation increases to reach approximately those of the normal controls on the third, fourth and fifth post-treatment days (SD 4.4,4.7 and 4.6 respectively). These results were obtained after the administration of a fairly large therapeutic dose of vitamin Biz. Such a dose might have secondary effects which might mask the primary action of the vitamin. The action of a physiological dose of vitamin BIZwas therefore determined in a further case (case 11) and Fig 2 shows the uracil level in this case treated with 2 pg vitamin BIZdaily for 6 d. Readings in this case were continued for 8 post-treatment days. The uracil levels fluctuated widely after the fourth day reaching as high as 40.8 pmol/l. on the eighth post-treatment day. This was 4 SDs above the mean level of the control group. DISCUSSION
A subnormal serum uracil level in PA in relapse which returns to normal or above under the influence of vitamin B, 2 , even in physiological doses,is the exact antithesis of what would be expected if the methylation of dUMP to form dTMP was the only metabolic block in vitamin B, deficiency. The possible bearing that the serum ‘uracil+ uridine’ level might have on ‘de novo’ dTMP synthesis has been discussed previously as also has the significance of the low
578
T. E. Parry and]. A. Blackmore 2pg Vitomin Biz daily
DOYS
FIG 2. Difference between pre- (day 0) and post-treatment serum uracil levels (-) and actual reticulocyte count (- - - -) in a single case (case I I ) of pernicious anaemia treated with physiological doses of vitamin B12 (2 fig daily for 6 d).
serum uracil levels encountered in PA in relapse (Parry & Blackmore, 1976). These, although statistically significant, were still of the same order as those encountered in half of the group of 97 normal subjects in whom the serum vitamin B,, level was normal and it was therefore concluded that the low serum uracil encountered in PA in relapse did not represent the main biochemical lesion in vitamin BIZ deficiency. It follows, therefore, that the return of the serum uracil level to normal after the administration of vitamin BIZreported here, although statistically highly significant, does not represent the main action of vitamin BIZin PA. Only two vitamin B,, dependent reactions have been demonstrated in man, the methylnialonyl-coenzyme A mutase reaction (White, 1962; Cox & White, 1962) and methionine synthesis (Parry, 1969). Ribonucleotide reductase is vitamin B, dependent in Lactobacillus leichnzannii but not so in Escherichia coli (Weissbach & Taylor 1968; Metz et al, 1968). It has Lecn postulated that it is vitamin BIZdependent also in man (Beck, 1968) but this has not been confirmed. The other known actions of vitamin B,Z (reviewed by Smith, 1965; Weisbach & Taylor, 1968; Silber & Moldow, 1970) have been described in bacteria only. None of these will explain the findings reported here nor do they suggest that vitamin B I Z has any role in pyrimidine synthesis.
Seruni Uracil and Vitamin B,, Therapy
5 79
The changes in the serum uracil level in PA reported here are remarkably similar to those previously described in the serum methionine level, assayed under similar conditions, in this disease (Parry, 1969). The pre-treatment levels of both methionine and uracil are significantly lower than normal in PA, both return to normal values following specific therapy and the rise in each case is highly significant (P< 0.001in each instance). The rise in serum methionine preceded that of uracil by at least 24 h and both precede the rise in the reticulocyte count. The change in the methionine levels were readily explained by the known action of vitamin B, on the demethylation of 5-methyltetrahydropteroylglutamic acid the methyl group being transferred to homocysteine to form methionine, i.e. by the direct action of vitamin B on ‘de YZOVO’ methionine synthesis. The close parallelism in the behaviour of the two metabolites, both before and after treatment, suggests, but does not prove, that the synthesis of uracil, Iike that of methionine, might be influenced by vitamin B12 in man. This hypothesis is supported by the submaximal response to uracil obtained in PA (Vilter et al, 1950) a response which was, however, considerably inferior to that produced by thymine (Spies et al, 1946). Even so, that a response, albeit a submaximal one, was obtained, together with the subnormal pre-treatment uracil levels encountered in this disease, suggests that uracil synthesis is defective in PA in relapse and together with the hitherto unrecognized action of vitamin B,, reported here, supports the hypothesis that vitamin B,, has a role in uracil synthesis in man. In both instances, however, it is clear that this role is a subsidiary one and does not represent the main biochemical lesion in vitamin B, deficiency.
, ,
,
ACKNOWLEDGMENTS
We wish to thank our physician colleagues, Dr D. A. Williams, Dr Byron Evans, Dr A. J. Thomas and Dr Paul Smith, for making patients under their care available to us, Professor H. Campbell and Dr T. Khosla for the statistical analysis, Dr J. L. Withey for the folate assays and Mr David Llewellyn for the photographs. REFERENCES BECK,W.S. (1968) Deoxyribonucleotidesynthesis and the role of vitamin B,, in erythropoiesis. Vitamins and Hormones, 26, 413. Cox, E.V. & WHITE,A.M. (1962) Methylmalonic acid excretion: an index of vitamin-BIZdeficiency. Lancet, ii, 853. MEW, J., KELLY,A., SWETT, V.C., WAXMAN, S. & HERBERT, V. (1968) Deranged DNA synthesis by bone marrow from vitamin B , ,-deficient humans. British Journal ofHaematology, 14, 575. PARRY, T.E. (1969) Serum valine and methioninelevels in pernicious anaemia under treatment. British Jorrrnal ~ H a e m a t o l o g y 16, , 221. PARRY,T.E. & BLACKMORE, J.A. (1976) Serum ‘uracil + uridine’ levels in pernicious anaemia.Brirish Journal 4Haemafology, 34, 567. SILBER, R.& MOLDOW, C.F. (1970) The biochemistry of B12-mediatedreactions in man. AmericanJorimal of Medicine, 48, 549.
SMITH, E.L. (1965) Vitainin B,,, pp 153-169. Methuen, London. SPIES, T.D., FROMMEYER, W.S., VILTER,C.F. & ENGLISH,A. (1946) Antianaemic properties of thymine. Blood, I, 185. VILTER, R.W., HARIGAN, D., MUELLER, J.F., JAROLD, T., VILTER, C.F., HAWKINS, V. & SEAMAN, A. (1950) Studies on the relationships of vitamin B I Z ,folic acid, thymine, uracil and methyl group donors in persons with pernicious anaemia and related niegaloblastic anaemia. Blood, 5 , 695. WEISSBACH, H. & TAYLOR, R.T. (1968) The mctabolic role of vitamin BIZ. Vitamins and Hormones, 26, 395. WHITE,A.M. (1962) Vitamin BIZdeficiency and the excretion of methylinalonic acid by the human. Biochemical Journal, 84,4 1 ~ .
Serum ‘Uracil+Uridine’ Levels Before and After Vitamin B,, Therapy in Pernicious Anaemia T. E. PARRY AND J. A. BLACKMORE Department of Haematology, Llandough Hospital, Penarth, South GIamotgan Area Health Authority (Teaching) (Received 23 March 1976; acceptedfor publication 24. May 1976)
+
SUMMARY. The serum ‘uracil uridine’ levels, expressed as uracil, have been measured in 10 cases of pernicious anaemia both before and after treatment, and compared with the levels in 97 normal subjects. The mean pre-treatment value (8.82 pmol/l., range 6.0-12.0 pmolll.) differed significantly from that of the normal controls (15.7pmol/l., range 5.7-40.5pmolll., t = 8.8, Pco.001).This confirms the low serum uracil level previously reported in pernicious anaemia in relapse. The level rose progressively after treatment, reaching a maximum on the fourth day (mean 17.85 pmol/l,, range 9.3-23.4 pmol/l.). This was not significantly different from the mean of the normal control group. The difference between the pre- and post-treatment levels was significant on days 3, 4 and 5 (Pco.005, Pco.001 and Pc 0.005 respectively) and the rise preceded the reticulocyte response by 24 h. A further case was treated with physiological doses of vitamin BIZ( 2 pg daily for 6 d) and a similar rise in the serum uracil level noted. These results are not explained by any of the known functions of vitamin Bt 2 . They are, however, similar to the changes in the serum methionine levels previously reported in pernicious anaemia. The latter were readily explained by the known action of vitamin BIZ on ‘de novo’ methionine synthesis and it is suggested that the synthesis of uracil, like that of methionine, might be influenced by vitamin BIZin man. Failure to methylate deoxyuridine monophosphate (dUMP) to form deoxythymidine monophosphate (dTMP) has been regarded as the basic biochemical defect in megaloblastic anaemia but an attempt to confirm this by direct measurement of the serum uracil in patients with pernicious anaemia (PA) in relapse showed unexpectedly that the uracil concentration was significantly lower than in normal controls (Parry & Blackmore, 1976). It is therefore pertinent to establish whether or not the serum uracil level in this disease is influenced by vitamin Biz. This has now been measured both before and after treatment in 10cases of PA and the results are presented here. The effects of physiological doses of vitamin B i z were studied in a further patient (case 11). Correspondence:Dr T. E. Parry, Department of Haematology, Llaiidough Hospital, Penarth, Glam. CF6 IXX, Wales.
575
T. E. Parry a n d / . A. Blackmore
576
MATERIALS AND METHODS Serum vitamin B,, , folate and 'uracil+ uridine' (expressed as uracil) were all measured microbiologically, vitamin Bl using Lactobacillus leichrnannii, and folate and uracil as described previously (Parry & Blackmore, 1976). The clinical and haematological features (Table I) in all 11 cases were consistent with a diagnosis of PA in relapse. The serum uracil was assayed before treatment and daily after treatment for 5 d in eight cases and for 4 d in the remaining two. All venous samples for uracil assay and for a reticulocyte count were collected immediately before the patients received their midday meal. After the pre-treatment sample had been collected zoo pg of cyanocobalamin (Vitamin Biz) was given by intramuscular injection at 14.00hours the same day (day 0)to the first 10 cases with the exception of case 5 who received 1000 pg. The statistical analysis was carried out by Professor H. Campbell and Dr T. Khosla of the Welsh National School of Medicine. TABLE I
M M M
6 7 8 9
72 46 53 72 so 66 70 58 45
10
67
F M
11
63
F
I 2
3 4 5
F M F M M
1.72
6.0
I10
1.32
S.0 10.0
I39 104 I35
2.82 1.50
1.99 I .433 2.0
2.63 2.0
1.70 1.69
6.6 8.7 6.3 7.6 8.3 7.9 6.3 7.0
130
126 114 I12
I20 I00
123
Megaloblastic Megaloblastic Megaloblastic Megaloblastic Megaloblastic Megaloblastic Megaloblastic Megaloblastic Megaloblastic Megaloblastic Megaloblastic
6.8 12.6 5.2 11.3
3.5 4.5 7.6 11.0
4.6 7.8 7.0
RESULTS The uracil levels were compared with those previously reported in 97 normal control subjects (Parry & Blackmore, 1976). The mean pre-treatment value in the 10 cases was 8.82 pmol/l. (range 6.0-12.0 pmol/l.). The difference between this and the mean for the normal control group (1~.7pmol/l.,range 5.7-4o.spmol/l.) was highly significant(t = 8.8, P< 0.001). The low serum uracil level previously reported in PA in relapse was therefore confirmed. The uracil level rose progressively after treatment and was significantly higher than the pre-treatment level on the third, fourth and fifth post-treatment days (P< 0.005,P< 0.001 and P< 0.001 respectively). It reached its maximum on the fourth day (mean 17.85 pmol/l., range 9.3-23.4 pmol.). This was not significantly different from the mean of the normal control group. The serum uracil level was therefore subnormal in PA in relapse and rose to reach normal levels on the fourth post-treatment day. The rise preceded the reticulocyte response by 24 h. Vitamin BI2had no effect on the serum uracil level in four normal subjects
Serum Uracil and Vitamin BIZ Therapy
-4?
I
I
I
I
I
I
I
2
3
4
5
6
577
Cays
FIG I. Difference between pre- (day 0) and post-treatment serum uracil values ( 0 ) and reticulocyte count (0) in 10cases ofpernicious anaemia given a therapeutic dose ofvitamin B,, on day o compared with four normal subjects (A) given a similar dose of vitamin B I 2 (zoo pg i.m., case 5 received ZOO0 pg).
(Fig I). It was noteworthy also that the range of the pretreatment values was fairly narrow as was reflected by the low standard deviation of 1.8 which differs significantly from the standard deviation of 5.26 in the control group (F = 8.7, P< 0.005).This confirms the previously reported observation that the serum uracil values in PA in relapse form a compact group, the mean value of which is significantly lower than normal. Following treatment, however, the range widens and the standard deviation increases to reach approximately those of the normal controls on the third, fourth and fifth post-treatment days (SD 4.4,4.7 and 4.6 respectively). These results were obtained after the administration of a fairly large therapeutic dose of vitamin Biz. Such a dose might have secondary effects which might mask the primary action of the vitamin. The action of a physiological dose of vitamin BIZwas therefore determined in a further case (case 11) and Fig 2 shows the uracil level in this case treated with 2 pg vitamin BIZdaily for 6 d. Readings in this case were continued for 8 post-treatment days. The uracil levels fluctuated widely after the fourth day reaching as high as 40.8 pmol/l. on the eighth post-treatment day. This was 4 SDs above the mean level of the control group. DISCUSSION
A subnormal serum uracil level in PA in relapse which returns to normal or above under the influence of vitamin B, 2 , even in physiological doses,is the exact antithesis of what would be expected if the methylation of dUMP to form dTMP was the only metabolic block in vitamin B, deficiency. The possible bearing that the serum ‘uracil+ uridine’ level might have on ‘de novo’ dTMP synthesis has been discussed previously as also has the significance of the low
578
T. E. Parry and]. A. Blackmore 2pg Vitomin Biz daily
DOYS
FIG 2. Difference between pre- (day 0) and post-treatment serum uracil levels (-) and actual reticulocyte count (- - - -) in a single case (case I I ) of pernicious anaemia treated with physiological doses of vitamin B12 (2 fig daily for 6 d).
serum uracil levels encountered in PA in relapse (Parry & Blackmore, 1976). These, although statistically significant, were still of the same order as those encountered in half of the group of 97 normal subjects in whom the serum vitamin B,, level was normal and it was therefore concluded that the low serum uracil encountered in PA in relapse did not represent the main biochemical lesion in vitamin BIZ deficiency. It follows, therefore, that the return of the serum uracil level to normal after the administration of vitamin BIZreported here, although statistically highly significant, does not represent the main action of vitamin BIZin PA. Only two vitamin B,, dependent reactions have been demonstrated in man, the methylnialonyl-coenzyme A mutase reaction (White, 1962; Cox & White, 1962) and methionine synthesis (Parry, 1969). Ribonucleotide reductase is vitamin B, dependent in Lactobacillus leichnzannii but not so in Escherichia coli (Weissbach & Taylor 1968; Metz et al, 1968). It has Lecn postulated that it is vitamin BIZdependent also in man (Beck, 1968) but this has not been confirmed. The other known actions of vitamin B,Z (reviewed by Smith, 1965; Weisbach & Taylor, 1968; Silber & Moldow, 1970) have been described in bacteria only. None of these will explain the findings reported here nor do they suggest that vitamin B I Z has any role in pyrimidine synthesis.
Seruni Uracil and Vitamin B,, Therapy
5 79
The changes in the serum uracil level in PA reported here are remarkably similar to those previously described in the serum methionine level, assayed under similar conditions, in this disease (Parry, 1969). The pre-treatment levels of both methionine and uracil are significantly lower than normal in PA, both return to normal values following specific therapy and the rise in each case is highly significant (P< 0.001in each instance). The rise in serum methionine preceded that of uracil by at least 24 h and both precede the rise in the reticulocyte count. The change in the methionine levels were readily explained by the known action of vitamin B, on the demethylation of 5-methyltetrahydropteroylglutamic acid the methyl group being transferred to homocysteine to form methionine, i.e. by the direct action of vitamin B on ‘de YZOVO’ methionine synthesis. The close parallelism in the behaviour of the two metabolites, both before and after treatment, suggests, but does not prove, that the synthesis of uracil, Iike that of methionine, might be influenced by vitamin B12 in man. This hypothesis is supported by the submaximal response to uracil obtained in PA (Vilter et al, 1950) a response which was, however, considerably inferior to that produced by thymine (Spies et al, 1946). Even so, that a response, albeit a submaximal one, was obtained, together with the subnormal pre-treatment uracil levels encountered in this disease, suggests that uracil synthesis is defective in PA in relapse and together with the hitherto unrecognized action of vitamin B,, reported here, supports the hypothesis that vitamin B,, has a role in uracil synthesis in man. In both instances, however, it is clear that this role is a subsidiary one and does not represent the main biochemical lesion in vitamin B, deficiency.
, ,
,
ACKNOWLEDGMENTS
We wish to thank our physician colleagues, Dr D. A. Williams, Dr Byron Evans, Dr A. J. Thomas and Dr Paul Smith, for making patients under their care available to us, Professor H. Campbell and Dr T. Khosla for the statistical analysis, Dr J. L. Withey for the folate assays and Mr David Llewellyn for the photographs. REFERENCES BECK,W.S. (1968) Deoxyribonucleotidesynthesis and the role of vitamin B,, in erythropoiesis. Vitamins and Hormones, 26, 413. Cox, E.V. & WHITE,A.M. (1962) Methylmalonic acid excretion: an index of vitamin-BIZdeficiency. Lancet, ii, 853. MEW, J., KELLY,A., SWETT, V.C., WAXMAN, S. & HERBERT, V. (1968) Deranged DNA synthesis by bone marrow from vitamin B , ,-deficient humans. British Journal ofHaematology, 14, 575. PARRY, T.E. (1969) Serum valine and methioninelevels in pernicious anaemia under treatment. British Jorrrnal ~ H a e m a t o l o g y 16, , 221. PARRY,T.E. & BLACKMORE, J.A. (1976) Serum ‘uracil + uridine’ levels in pernicious anaemia.Brirish Journal 4Haemafology, 34, 567. SILBER, R.& MOLDOW, C.F. (1970) The biochemistry of B12-mediatedreactions in man. AmericanJorimal of Medicine, 48, 549.
SMITH, E.L. (1965) Vitainin B,,, pp 153-169. Methuen, London. SPIES, T.D., FROMMEYER, W.S., VILTER,C.F. & ENGLISH,A. (1946) Antianaemic properties of thymine. Blood, I, 185. VILTER, R.W., HARIGAN, D., MUELLER, J.F., JAROLD, T., VILTER, C.F., HAWKINS, V. & SEAMAN, A. (1950) Studies on the relationships of vitamin B I Z ,folic acid, thymine, uracil and methyl group donors in persons with pernicious anaemia and related niegaloblastic anaemia. Blood, 5 , 695. WEISSBACH, H. & TAYLOR, R.T. (1968) The mctabolic role of vitamin BIZ. Vitamins and Hormones, 26, 395. WHITE,A.M. (1962) Vitamin BIZdeficiency and the excretion of methylinalonic acid by the human. Biochemical Journal, 84,4 1 ~ .
