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Depression and tetrahydrobiopterin: The folate connection
Journal of Aflectiue Disorders, 16 (1989) 103-107 Elsevier
103
JAD 00600
Depression and tetrahydrobiopterin: A. Coppen
the folate connection
‘, C. Swade ‘, S.A. Jones *, R.A. Armstrong and R.J. Leeming 3
2, J.A. Blair 2
’ MRC Neuropsychiatry Research Laboratory, West Park Hospital, Epsom KT19 8PB, U.K., ’ Molecular Sciences Department, Aston University, Aston Triangle, Birmingham B4 7ET, U.K. and ’ Haematology Department, General Hospital, Steelhowe Lane, Birmingham B4 6NH. U.K. (Received 9 April 1986) (Revision received 2 June 1988) (Accepted 29 June 1988)
Total biopterin, neopterin and creatinine were measured in spot urine samples from affective disorder patients on lithium therapy and control subjects. Folk acid was also measured in plasma in a sample of the patients. The mean neopterin : biopterin ratio was significantly higher in the 76 patients (3.2 f 0.5) than in the 61 controls (1.8 f 0.1). In female patients biopterin levels were significantly lower than in controls. In the control groups there was a significant correlation between the molar concentration of neopterin and biopterin. No such correlation was found in the patients. These data indicate that tetrahydrobiopterin (BH,) biosynthesis is reduced in this group. A significant positive correlation was found between plasma folate and urinary biopterin. It is suggested that folate deficiency may impair the synthesis of BH,, a cofactor essential for the synthesis of 5-HT and other monoamines that are involved in the pathogenesis of affective disorders. Key woruk Tetrahydrobiopterin;
Neopterin;
Depression;
Introduction Tetrahydrobiopterin (BH,) is the cofactor for the hydroxylation of phenylalanine and tryptophan in a rate-limiting step in the biosynthesis of dopamine, noradrenaline and 5-hydroxytryptamine (5HT) (Levitt et al., 1965). Cellular levels of BH, are maintained by a combination of dihydropteridine reductase
Address for correspondence: Dr. A. Coppen, MRC Neuropsychiatry Research Laboratory, West Park Hospital, Epsom KT19 8PB, U.K. 0165-0327/89/$03.50
Folic acid; Monoamines
(DHPR) activity and de novo synthesis. In the synthesis of BH,, guanosine triphosphate (GTP) is converted to dihydroneopterin triphosphate (NTPH,) (Blau and Niederweiser, 1983), and then by a series of tetrahydro intermediates to BH, (Switcher&o and Brown, 1985). BH, and dihydrobiopterin are excreted in human urine. Hydrolysis of NTPH, yields dihydroneopterin which is excreted in the urine along with its oxidation product neopterin. Measurement of the total biopterins and total neopterins in urine can be used to study changes in BH, metabolism. Reduced folates are essential to man for a variety of one-carbon transfer reactions in amino
0 1989 Elsevier Science Publishers B.V. (Biomedical Division)
104
acid and purine biosynthesis and degradation. The current recommended dietary allowance for adult man is 300 pg folate per day (Herbert, 1977), with serum folate levels becoming depleted below an intake of 50 pg/day (Herbert, 1962). Lack of folate causes megaloblastic anaemia and neurological and neuropsychiatric disorders. Major neurological symptoms include difficulty in walking, restless legs, and degeneration of the spinal cord which is responsive to folate therapy (Lever et al., 1986). Minor neurological signs are subjective complaints of lassitude, lack of concentration, weight loss and depression (Botez et al., 1979a). Blood folate levels have been reported to be low in psychiatric patients, especially those suffering from a depressive illness (Reynolds et al., 1970; Carney and Sheffield, 1978; Thornton and Thornton, 1978). Leeming and colleagues (1982a) showed a connection between folate and BH, in that folic acid in the form of 5-methyltetrahydrofolate increases the biosynthesis of BH,. According to the monoamine hypothesis of affective disorders, depression is due to a deficiency of 5-HT or noradrenaline or of both these monoamines (Coppen, 1967; Van Praag, 1982). BH, is required for the synthesis of 5-HT and noradrenaline. BH, synthesis in brain preparations from depressed patients has been found to be lower than in control subjects (Blair et al., 1984). Furthermore, administration of BH, to depressed patients may alleviate their symptoms (Curtius et al., 1983; Fleischhacker et al., 1985). A significant increase in serum biopterin has been found in patients receiving tricyclic antidepressant therapy and lithium (Leeming et al., 1982b). We have carried out a study to compare the urinary excretion of biopterin and neopterin by depressive patients on prophylactic lithium therapy and by control subjects. In addition, to investigate the possible link between folic acid and BH, we measured plasma folate concentration in a group of female patients. Patients and methods Fifty-one female and 25 male patients (48 unipolar and 28 bipolar patients) who had been attending a lithium clinic for periods of l-17 years were studied. Their ages ranged from 26 to 83
(mean 58.6 k 1.3) years. All patients were receiving lithium carbonate (Priadel) once daily at night in doses to achieve plasma lithium levels 12 h later of 0.4-0.8 (mean 0.56 _t 0.02) mmol/l. The affective morbidity of these outpatients varied, but none were severely depressed or manic. Fifty-seven patients were not receiving any additional psychotropic medication, but 21 were receiving benzodiazepines as anxiolytics or for night-time sedation. The control subjects, 32 women and 28 men aged between 23 and 80 (mean 51.8 + 1.8) years, were all in good physical health, and none had a history of psychiatric illness. Urine samples were taken at random between 9:00 and 11:OO a.m., and stored at -20°C until assayed. Plasma samples for folate determination were collected from 17 unipolar female patients. Total biopterin (tetrahydrobiopterin, dihydrobiopterin and biopterin) and total neopterin (dihydroneopterin and neopterin) were measured by the method of Fukushima and Nixon (1980). Creatinine was measured by calorimetric Jaffe determination, and urinary free cortisol and plasma folate by radioimmunoassay.
Results The urine samples from the patients were less concentrated due to lithium-induced polyuria and had significantly lower concentrations of creatinine than the samples from the control subjects. Allowance for the variation in concentrating ability was made by expressing the urinary levels of biopterin, neopterin and cortisol as .~mol/mol creatinine. There were no significant differences between unipolar and bipolar patients in any of the variates, so the results for the two diagnostic categories were combined. The female patients were significantly older than the other three groups, but there were no significant correlations between age and biopterin, neopterin, cortisol or creatinine in any of the groups. There was, however, a significant correlation between age and the neopterin : biopterin ratio (after angular transformation) in the male controls (r = -t-0.39, P < 0.05). In the female controls the correlation did not reach significance (r = + 0.32, P = 0.07). There was no correlation between age
105 TABLE
1
URINARY EXCRETION OF BIOPTERIN MAINTAINED ON LITHIUM THERAPY Group
n
AND
Age (years)
NEOPTERIN
IN CONTROL
Biopterin
28 25 33 51
53.5 54.8 50.9 60.4
f 2.1 + 2.2 rf: 2.6 + 1.7 *
AND
Neopterin
(mol X 10-S/mmol Male controls Male patients Female controls Female patients
SUBJECTS
36.0 + 2.9 32.4 + 2.9 52.4+4.2 ** 4O.Ok3.1
6.4 *** 10.3 9.5 14.8
Results are expressed as mean f SEM. * Significantly older than male controls, male patients, both P c 0.05, and female controls, P -Z0.002. ** Significantly higher than male controls, P i 0.0025, male patients, P < 0.00025, and female patients, *** Significantly lower than female controls and female patients, both P < 0.005. + Significantly higher than male controls, P c 0.025, and female controls. P < 0.05.
and the neopterin : biopterin ratio in either male or female patients. The female controls excreted significantly more biopterin and neopterin than the male controls (Table 1). The female patients excreted significantly less biopterin than the female controls. The ratio of neopterin to biopterin was significantly higher in female patients than female controls. As the neopterin : biopterin ratio was very similar in male and female controls, and in male and female patients, the results for the two sexes were combined. The mean neopterin : biopterin ratio was significantly higher in the 76 patients (3.21 f 0.49) than in the 61 controls (1.78 f 0.10; P -e 0.01 after angular transformation and using Satterthwaite’s approximation), indicating that the patients have an abnormality in their synthesis of tetrahydrobiopterin. There was a significant correlation between the urinary concentrations of biopterin and neopterin in both the male controls (r = +0.67, P -c 0.001) and the female controls (r = +0.67, P < 0.001). There was no significant correlation between biopterin and neopterin excretion in either the female patients (r = + 0.10) or the male patients (r = -O.Ol), which also suggests an abnormality in tetrahydrobiopterin synthesis in the patients. The excretion of cortisol was similar in all groups, indicating that the altered pattern of excretion of biopterin and neopterin observed in the patients is not due to changes in the activity of the adrenal cortex. There were no significant correlations between plasma lithium concentration and
PATIENTS
Neopterin Biopterin
creatinine) 60.0+ 71.1* 93.9f 106.9 +
DEPRESSIVE
1.69rtO.12 2.99 f 0.70 1.86&0.15 3.3240.64 +
P < 0.02.
biopterin, neopterin, or cortisol in either male or female patients. The administration of additional medication did not make any significant change in any of the variates measured. In the 17 female unipolar patients for whom plasma folate concentrations were available, a significant correlation was found between plasma folate and urinary biopterin concentration (r = + 0.63, P < 0.01). Discussion Various groups have used the measurement of urinary pterins as a diagnostic tool in various disease states. Blair and co-workers (1984) reported a decrease in the excretion of biopterin in a small group of depressive patients, and a decreased BH, synthesis in post-mortem brain samples from patients with a history of severe depression. However, Duch and colleagues (1984) reported an increased excretion of biopterins in severely depressed patients, although the small number of patients examined after recovery had a reduced biopterin output. Tetrahydrobiopterin is present in the adrenal cortex and since there is an increase in cortisol excretion in acute depression (Milln et al., 1981), which may be accompanied by an increased BH, excretion, this may mask a central deficiency of BH,. In the present study we found a decreased output of biopterin by depressive patients maintained on prophylactic lithium therapy. Animal studies in the rat indicate that administration of
106
lithium as either an acute or a chronic dose does not reduce brain biopterins or DHPR. Lithium may increase total brain biopterins (Jones, to be published) and plasma biopterins (Leeming et al., 1982b). Therefore, it is assumed that the lowered urinary biopterin seen in depressed patients is not iatrogenic due to lithium. All the patients were euthymic at the time of testing, and none were receiving any additional antidepressant medication. The reduced excretion of biopterin is probably a reflection of a BH, deficit in the brain, and lends support to the suggested administration of BH, as an antidepressant (Curtius et al., 1983; Fleischhacker et al., 1985). The BH, deficit in depressive patients is probably due to an abnormality in the synthesis of BH, from dihydroneopterin triphosphate, as the ratio of urinary neopterin to biopterin is significantly raised in patients compared with control subjects. Also, in the controls there is a highly significant correlation between urinary levels of biopterin and neopterin, while there is no such correlation in the depressive patients. The finding of a significant correlation between plasma folate and urinary biopterin is of considerable interest because of the well-documented association between plasma folate and depression. In a recent survey in a group of affective disorder patients treated with lithium prophylactically, it was found that residual affective morbidity showed a negative correlation with plasma folate (Coppen and Abou-Saleh, 1982). In a double-blind controlled trial in these patients, it was found that small doses (200 pg daily) of folate reduced the affective morbidity in these patients (Coppen et al., 1986). The relationship between affective morbidity, BH,, folate and 5-HT is clearly complex. There is good evidence for 5-HT being a factor in the pathogenesis of affective disorders (Coppen and Wood, 1982). There is also evidence that rats kept on a folate-deficient diet have significantly decreased brain 5-HT content (Botez et al., 1979b). Hamon and colleagues (1986) have recently shown that BH, biosynthesis in rat brains is significantly increased by the addition of 5methyltetrahydrofolate (5-MeTHF), a finding similar to that shown by Leeming and colleagues (Leeming et al., 1982a). No significant increase was found in normal human temporal lobe, but
5-MeTHF was shown to stimulate BH, synthesis in a brain preparation from a child who died from methyltetrahydrofolate reductase deficiency (Hamon et al., 1986). Tetrahydrofolate (Leeming et al., 1982a) and 5-formyltetrahydrofolate (Smith et al., 1985) have also been shown to result in clinical improvement in children with DHPR deficiency leading to reduced BH, levels. The present paper shows an association between plasma folate and BH, which is the cofactor for tryptophan hydroxylation, the rate limiting step in the synthesis of 5-HT. Links are thus shown between folates, BH, and depression, and further investigation should be made to establish if the administration of folate will restore BH, metabolism to normal in these patients. References Blair, J.A., Morar, C., Hamon, C.G.B., Barford, P.A., Pheasant, A.E., Whitbum, S.B., Leeming, R.J., Reynolds, G.P. and Copper, A. (1984) Tetrahydrobiopterin metabolism in depression. Lancet ii, 163. Blau, N. and Niederwieser, A. (1983) GTP cyclohydrolase I assay in human and rat liver using HPLC of neopterin phosphates and guanosine nucleotides. Anal. B&hem. 128, 446452. Botez, MI., Botez, T., Leveille, J., Bielman, P. and Cadotte, M. (1979a) In: M.I. Botez and E.H. Reynolds (Ids.), Folic Acid in Neurology, Psychiatry and Internal Medicine. Raven Press, New York, NY. Botez, M.I., Young, S.M., Bachevalier, J. and Gauthier, S. (1979b) Folate deficiency and decreased brain 5-hydroxytryptamine synthesis in man and rat. Nature 278,, 182-183. Camey, M.W.P. and Sheffield, B.F. (1978) Serum folic acid and B,, in 272 psychiatric patients. Psychol. Med. 8, 139-144. Coppen, A. (1967) Biochemistry of affective disorders. Br. J. Psychiatry 113, 1237-1264. Coppen, A. and Abou-Saleh, M.T. (1982) Plasma folate and affective morbidity during long-term lithium therapy. Br. J. Psychiatry 141, 87-89. Coppen, A. and Wood, K. (1982) 5Hydroxytryptamine in the pathogenesis of affective disorders. In: B.T. Ho, J.C. Schoolar and E. Usdin (Eds.), Serotonin in Biological Psychiatry. Raven Press, New York, NY, pp. 249-258. Coppen, A., Chaudhury, S. and Swade, C. (1986) Folic acid enhances lithium prophylaxis. J. Affect. Disord. 10, 9-13. Curtius, H.C., Niederwieser, A., Levine, R.A., Lovenberg, W., Woggon, B. and Angst, J. (1983) Successful treatment of depression with tetrahydrobiopterin. Lancet i, 657-658. Duch, D.S., Woolf, J.H., Nichol, C.A., Davidson, J.R. and Garbutt, J.C. (1984) Urinary excretion of biopterin and neopterin in psychiatric disorders. Psychiatr. Res. 11, 83-80.
107 Fleischhacker, W.W., Mease, U. and Schubert, H. (1985) Reevaluation of antidepressant effect of tetrahydrobiopterin. Lancet ii, 387. Fukushima, T. and Nixon, J.C. (1980) Analysis of reduced forms of biopterin in biological tissues and fluids. Anal. B&hem. 102, 176-188. Hamon, C.G.B., Blair, J.A. and Barford, P.A. (1986) The effect of tetrahydrofolate on tetrahydrobiopterin metabolism. J. Ment. Defic. Res. 30, 170-183. Herbert, V. (1962) Minimal daily adult folate requirement. Arch. Int. Med. 110, 649. Herbert, V. (1977) In: H.P. Broquist, C.E. Butterworth and C. Wagner (Eds.), Folic Acid. National Academy of Sciences, Washington, DC, pp. 277-293. Leeming, R.J., Harpey, J.P., Brown, S.M. and Blair, J.A. (1982a) Tetrahydrofolate and hydroxycobalamin in the management of dihydropteridine reductase deficiency. J. Ment. Defic. Res. 26, 21-25. Leeming, R.J., Blair, J.A. and Walters, J. (1982b) Serum dihydrobiopterin levels in patients on tricyclic antidepressants. Psychol. Med. 12, 191-192. Lever, E.G., Elwas, R.D.C., Williams, A. and Reynolds, E.H. (1986) Subacute combined degeneration of the cord due to folate deficiency: response to methylfolate treatment. J. Neurol. Neurosurg. Psychiatry 49, 1203-1209.
Levitt, M., Spector, S., Sjoerdsman, P. and Udenfried, S. (1965) Elucidation of the rate-limiting step in norepinephrine biosynthesis in the perfused guinea pig heart. J. Pharmacol. Exp. Ther. 148, l-8. Milln, P., Bishop, M. and Coppen, A. (1981) Urinary free cortisol and clinical classification of depressive illness. Psychol. Med. 11, 643-645. Reynolds, E.H., Preece, J.M., Bailey, J. and Coppen, A. (1970) Folate deficiency in depressive illness. Br. J. Psychiatry 117, 287-292. Smith, I., Hyland, K., Kendall, B. and Leeming, R.J. (1985) Clinical role of pteridine therapy in tetrahydrobiopterin deficiency. J. Inherit. Met. Dis. 8, 39-45. Switchenko, A. and Brown, G. (1985) The enzymatic conversion of dihydroneopterin triphosphate to L-pyruvoyltetrahydrobiopterin, an intermediate in the biosynthesis of other pterins in Drosophila melanogaster. J. Biol. Chem. 260, 2945-2951. Thornton, W.E. and Thornton, B.P. (1978) Folic acid, mental function and dietary habits. J. Clin. Psychiatry 39, 315-322. Van Praag, H.M. (1982) Neurotransmitters and CNS disease: depression. Lancet ii, 1259-1283.
103
JAD 00600
Depression and tetrahydrobiopterin: A. Coppen
the folate connection
‘, C. Swade ‘, S.A. Jones *, R.A. Armstrong and R.J. Leeming 3
2, J.A. Blair 2
’ MRC Neuropsychiatry Research Laboratory, West Park Hospital, Epsom KT19 8PB, U.K., ’ Molecular Sciences Department, Aston University, Aston Triangle, Birmingham B4 7ET, U.K. and ’ Haematology Department, General Hospital, Steelhowe Lane, Birmingham B4 6NH. U.K. (Received 9 April 1986) (Revision received 2 June 1988) (Accepted 29 June 1988)
Total biopterin, neopterin and creatinine were measured in spot urine samples from affective disorder patients on lithium therapy and control subjects. Folk acid was also measured in plasma in a sample of the patients. The mean neopterin : biopterin ratio was significantly higher in the 76 patients (3.2 f 0.5) than in the 61 controls (1.8 f 0.1). In female patients biopterin levels were significantly lower than in controls. In the control groups there was a significant correlation between the molar concentration of neopterin and biopterin. No such correlation was found in the patients. These data indicate that tetrahydrobiopterin (BH,) biosynthesis is reduced in this group. A significant positive correlation was found between plasma folate and urinary biopterin. It is suggested that folate deficiency may impair the synthesis of BH,, a cofactor essential for the synthesis of 5-HT and other monoamines that are involved in the pathogenesis of affective disorders. Key woruk Tetrahydrobiopterin;
Neopterin;
Depression;
Introduction Tetrahydrobiopterin (BH,) is the cofactor for the hydroxylation of phenylalanine and tryptophan in a rate-limiting step in the biosynthesis of dopamine, noradrenaline and 5-hydroxytryptamine (5HT) (Levitt et al., 1965). Cellular levels of BH, are maintained by a combination of dihydropteridine reductase
Address for correspondence: Dr. A. Coppen, MRC Neuropsychiatry Research Laboratory, West Park Hospital, Epsom KT19 8PB, U.K. 0165-0327/89/$03.50
Folic acid; Monoamines
(DHPR) activity and de novo synthesis. In the synthesis of BH,, guanosine triphosphate (GTP) is converted to dihydroneopterin triphosphate (NTPH,) (Blau and Niederweiser, 1983), and then by a series of tetrahydro intermediates to BH, (Switcher&o and Brown, 1985). BH, and dihydrobiopterin are excreted in human urine. Hydrolysis of NTPH, yields dihydroneopterin which is excreted in the urine along with its oxidation product neopterin. Measurement of the total biopterins and total neopterins in urine can be used to study changes in BH, metabolism. Reduced folates are essential to man for a variety of one-carbon transfer reactions in amino
0 1989 Elsevier Science Publishers B.V. (Biomedical Division)
104
acid and purine biosynthesis and degradation. The current recommended dietary allowance for adult man is 300 pg folate per day (Herbert, 1977), with serum folate levels becoming depleted below an intake of 50 pg/day (Herbert, 1962). Lack of folate causes megaloblastic anaemia and neurological and neuropsychiatric disorders. Major neurological symptoms include difficulty in walking, restless legs, and degeneration of the spinal cord which is responsive to folate therapy (Lever et al., 1986). Minor neurological signs are subjective complaints of lassitude, lack of concentration, weight loss and depression (Botez et al., 1979a). Blood folate levels have been reported to be low in psychiatric patients, especially those suffering from a depressive illness (Reynolds et al., 1970; Carney and Sheffield, 1978; Thornton and Thornton, 1978). Leeming and colleagues (1982a) showed a connection between folate and BH, in that folic acid in the form of 5-methyltetrahydrofolate increases the biosynthesis of BH,. According to the monoamine hypothesis of affective disorders, depression is due to a deficiency of 5-HT or noradrenaline or of both these monoamines (Coppen, 1967; Van Praag, 1982). BH, is required for the synthesis of 5-HT and noradrenaline. BH, synthesis in brain preparations from depressed patients has been found to be lower than in control subjects (Blair et al., 1984). Furthermore, administration of BH, to depressed patients may alleviate their symptoms (Curtius et al., 1983; Fleischhacker et al., 1985). A significant increase in serum biopterin has been found in patients receiving tricyclic antidepressant therapy and lithium (Leeming et al., 1982b). We have carried out a study to compare the urinary excretion of biopterin and neopterin by depressive patients on prophylactic lithium therapy and by control subjects. In addition, to investigate the possible link between folic acid and BH, we measured plasma folate concentration in a group of female patients. Patients and methods Fifty-one female and 25 male patients (48 unipolar and 28 bipolar patients) who had been attending a lithium clinic for periods of l-17 years were studied. Their ages ranged from 26 to 83
(mean 58.6 k 1.3) years. All patients were receiving lithium carbonate (Priadel) once daily at night in doses to achieve plasma lithium levels 12 h later of 0.4-0.8 (mean 0.56 _t 0.02) mmol/l. The affective morbidity of these outpatients varied, but none were severely depressed or manic. Fifty-seven patients were not receiving any additional psychotropic medication, but 21 were receiving benzodiazepines as anxiolytics or for night-time sedation. The control subjects, 32 women and 28 men aged between 23 and 80 (mean 51.8 + 1.8) years, were all in good physical health, and none had a history of psychiatric illness. Urine samples were taken at random between 9:00 and 11:OO a.m., and stored at -20°C until assayed. Plasma samples for folate determination were collected from 17 unipolar female patients. Total biopterin (tetrahydrobiopterin, dihydrobiopterin and biopterin) and total neopterin (dihydroneopterin and neopterin) were measured by the method of Fukushima and Nixon (1980). Creatinine was measured by calorimetric Jaffe determination, and urinary free cortisol and plasma folate by radioimmunoassay.
Results The urine samples from the patients were less concentrated due to lithium-induced polyuria and had significantly lower concentrations of creatinine than the samples from the control subjects. Allowance for the variation in concentrating ability was made by expressing the urinary levels of biopterin, neopterin and cortisol as .~mol/mol creatinine. There were no significant differences between unipolar and bipolar patients in any of the variates, so the results for the two diagnostic categories were combined. The female patients were significantly older than the other three groups, but there were no significant correlations between age and biopterin, neopterin, cortisol or creatinine in any of the groups. There was, however, a significant correlation between age and the neopterin : biopterin ratio (after angular transformation) in the male controls (r = -t-0.39, P < 0.05). In the female controls the correlation did not reach significance (r = + 0.32, P = 0.07). There was no correlation between age
105 TABLE
1
URINARY EXCRETION OF BIOPTERIN MAINTAINED ON LITHIUM THERAPY Group
n
AND
Age (years)
NEOPTERIN
IN CONTROL
Biopterin
28 25 33 51
53.5 54.8 50.9 60.4
f 2.1 + 2.2 rf: 2.6 + 1.7 *
AND
Neopterin
(mol X 10-S/mmol Male controls Male patients Female controls Female patients
SUBJECTS
36.0 + 2.9 32.4 + 2.9 52.4+4.2 ** 4O.Ok3.1
6.4 *** 10.3 9.5 14.8
Results are expressed as mean f SEM. * Significantly older than male controls, male patients, both P c 0.05, and female controls, P -Z0.002. ** Significantly higher than male controls, P i 0.0025, male patients, P < 0.00025, and female patients, *** Significantly lower than female controls and female patients, both P < 0.005. + Significantly higher than male controls, P c 0.025, and female controls. P < 0.05.
and the neopterin : biopterin ratio in either male or female patients. The female controls excreted significantly more biopterin and neopterin than the male controls (Table 1). The female patients excreted significantly less biopterin than the female controls. The ratio of neopterin to biopterin was significantly higher in female patients than female controls. As the neopterin : biopterin ratio was very similar in male and female controls, and in male and female patients, the results for the two sexes were combined. The mean neopterin : biopterin ratio was significantly higher in the 76 patients (3.21 f 0.49) than in the 61 controls (1.78 f 0.10; P -e 0.01 after angular transformation and using Satterthwaite’s approximation), indicating that the patients have an abnormality in their synthesis of tetrahydrobiopterin. There was a significant correlation between the urinary concentrations of biopterin and neopterin in both the male controls (r = +0.67, P -c 0.001) and the female controls (r = +0.67, P < 0.001). There was no significant correlation between biopterin and neopterin excretion in either the female patients (r = + 0.10) or the male patients (r = -O.Ol), which also suggests an abnormality in tetrahydrobiopterin synthesis in the patients. The excretion of cortisol was similar in all groups, indicating that the altered pattern of excretion of biopterin and neopterin observed in the patients is not due to changes in the activity of the adrenal cortex. There were no significant correlations between plasma lithium concentration and
PATIENTS
Neopterin Biopterin
creatinine) 60.0+ 71.1* 93.9f 106.9 +
DEPRESSIVE
1.69rtO.12 2.99 f 0.70 1.86&0.15 3.3240.64 +
P < 0.02.
biopterin, neopterin, or cortisol in either male or female patients. The administration of additional medication did not make any significant change in any of the variates measured. In the 17 female unipolar patients for whom plasma folate concentrations were available, a significant correlation was found between plasma folate and urinary biopterin concentration (r = + 0.63, P < 0.01). Discussion Various groups have used the measurement of urinary pterins as a diagnostic tool in various disease states. Blair and co-workers (1984) reported a decrease in the excretion of biopterin in a small group of depressive patients, and a decreased BH, synthesis in post-mortem brain samples from patients with a history of severe depression. However, Duch and colleagues (1984) reported an increased excretion of biopterins in severely depressed patients, although the small number of patients examined after recovery had a reduced biopterin output. Tetrahydrobiopterin is present in the adrenal cortex and since there is an increase in cortisol excretion in acute depression (Milln et al., 1981), which may be accompanied by an increased BH, excretion, this may mask a central deficiency of BH,. In the present study we found a decreased output of biopterin by depressive patients maintained on prophylactic lithium therapy. Animal studies in the rat indicate that administration of
106
lithium as either an acute or a chronic dose does not reduce brain biopterins or DHPR. Lithium may increase total brain biopterins (Jones, to be published) and plasma biopterins (Leeming et al., 1982b). Therefore, it is assumed that the lowered urinary biopterin seen in depressed patients is not iatrogenic due to lithium. All the patients were euthymic at the time of testing, and none were receiving any additional antidepressant medication. The reduced excretion of biopterin is probably a reflection of a BH, deficit in the brain, and lends support to the suggested administration of BH, as an antidepressant (Curtius et al., 1983; Fleischhacker et al., 1985). The BH, deficit in depressive patients is probably due to an abnormality in the synthesis of BH, from dihydroneopterin triphosphate, as the ratio of urinary neopterin to biopterin is significantly raised in patients compared with control subjects. Also, in the controls there is a highly significant correlation between urinary levels of biopterin and neopterin, while there is no such correlation in the depressive patients. The finding of a significant correlation between plasma folate and urinary biopterin is of considerable interest because of the well-documented association between plasma folate and depression. In a recent survey in a group of affective disorder patients treated with lithium prophylactically, it was found that residual affective morbidity showed a negative correlation with plasma folate (Coppen and Abou-Saleh, 1982). In a double-blind controlled trial in these patients, it was found that small doses (200 pg daily) of folate reduced the affective morbidity in these patients (Coppen et al., 1986). The relationship between affective morbidity, BH,, folate and 5-HT is clearly complex. There is good evidence for 5-HT being a factor in the pathogenesis of affective disorders (Coppen and Wood, 1982). There is also evidence that rats kept on a folate-deficient diet have significantly decreased brain 5-HT content (Botez et al., 1979b). Hamon and colleagues (1986) have recently shown that BH, biosynthesis in rat brains is significantly increased by the addition of 5methyltetrahydrofolate (5-MeTHF), a finding similar to that shown by Leeming and colleagues (Leeming et al., 1982a). No significant increase was found in normal human temporal lobe, but
5-MeTHF was shown to stimulate BH, synthesis in a brain preparation from a child who died from methyltetrahydrofolate reductase deficiency (Hamon et al., 1986). Tetrahydrofolate (Leeming et al., 1982a) and 5-formyltetrahydrofolate (Smith et al., 1985) have also been shown to result in clinical improvement in children with DHPR deficiency leading to reduced BH, levels. The present paper shows an association between plasma folate and BH, which is the cofactor for tryptophan hydroxylation, the rate limiting step in the synthesis of 5-HT. Links are thus shown between folates, BH, and depression, and further investigation should be made to establish if the administration of folate will restore BH, metabolism to normal in these patients. References Blair, J.A., Morar, C., Hamon, C.G.B., Barford, P.A., Pheasant, A.E., Whitbum, S.B., Leeming, R.J., Reynolds, G.P. and Copper, A. (1984) Tetrahydrobiopterin metabolism in depression. Lancet ii, 163. Blau, N. and Niederwieser, A. (1983) GTP cyclohydrolase I assay in human and rat liver using HPLC of neopterin phosphates and guanosine nucleotides. Anal. B&hem. 128, 446452. Botez, MI., Botez, T., Leveille, J., Bielman, P. and Cadotte, M. (1979a) In: M.I. Botez and E.H. Reynolds (Ids.), Folic Acid in Neurology, Psychiatry and Internal Medicine. Raven Press, New York, NY. Botez, M.I., Young, S.M., Bachevalier, J. and Gauthier, S. (1979b) Folate deficiency and decreased brain 5-hydroxytryptamine synthesis in man and rat. Nature 278,, 182-183. Camey, M.W.P. and Sheffield, B.F. (1978) Serum folic acid and B,, in 272 psychiatric patients. Psychol. Med. 8, 139-144. Coppen, A. (1967) Biochemistry of affective disorders. Br. J. Psychiatry 113, 1237-1264. Coppen, A. and Abou-Saleh, M.T. (1982) Plasma folate and affective morbidity during long-term lithium therapy. Br. J. Psychiatry 141, 87-89. Coppen, A. and Wood, K. (1982) 5Hydroxytryptamine in the pathogenesis of affective disorders. In: B.T. Ho, J.C. Schoolar and E. Usdin (Eds.), Serotonin in Biological Psychiatry. Raven Press, New York, NY, pp. 249-258. Coppen, A., Chaudhury, S. and Swade, C. (1986) Folic acid enhances lithium prophylaxis. J. Affect. Disord. 10, 9-13. Curtius, H.C., Niederwieser, A., Levine, R.A., Lovenberg, W., Woggon, B. and Angst, J. (1983) Successful treatment of depression with tetrahydrobiopterin. Lancet i, 657-658. Duch, D.S., Woolf, J.H., Nichol, C.A., Davidson, J.R. and Garbutt, J.C. (1984) Urinary excretion of biopterin and neopterin in psychiatric disorders. Psychiatr. Res. 11, 83-80.
107 Fleischhacker, W.W., Mease, U. and Schubert, H. (1985) Reevaluation of antidepressant effect of tetrahydrobiopterin. Lancet ii, 387. Fukushima, T. and Nixon, J.C. (1980) Analysis of reduced forms of biopterin in biological tissues and fluids. Anal. B&hem. 102, 176-188. Hamon, C.G.B., Blair, J.A. and Barford, P.A. (1986) The effect of tetrahydrofolate on tetrahydrobiopterin metabolism. J. Ment. Defic. Res. 30, 170-183. Herbert, V. (1962) Minimal daily adult folate requirement. Arch. Int. Med. 110, 649. Herbert, V. (1977) In: H.P. Broquist, C.E. Butterworth and C. Wagner (Eds.), Folic Acid. National Academy of Sciences, Washington, DC, pp. 277-293. Leeming, R.J., Harpey, J.P., Brown, S.M. and Blair, J.A. (1982a) Tetrahydrofolate and hydroxycobalamin in the management of dihydropteridine reductase deficiency. J. Ment. Defic. Res. 26, 21-25. Leeming, R.J., Blair, J.A. and Walters, J. (1982b) Serum dihydrobiopterin levels in patients on tricyclic antidepressants. Psychol. Med. 12, 191-192. Lever, E.G., Elwas, R.D.C., Williams, A. and Reynolds, E.H. (1986) Subacute combined degeneration of the cord due to folate deficiency: response to methylfolate treatment. J. Neurol. Neurosurg. Psychiatry 49, 1203-1209.
Levitt, M., Spector, S., Sjoerdsman, P. and Udenfried, S. (1965) Elucidation of the rate-limiting step in norepinephrine biosynthesis in the perfused guinea pig heart. J. Pharmacol. Exp. Ther. 148, l-8. Milln, P., Bishop, M. and Coppen, A. (1981) Urinary free cortisol and clinical classification of depressive illness. Psychol. Med. 11, 643-645. Reynolds, E.H., Preece, J.M., Bailey, J. and Coppen, A. (1970) Folate deficiency in depressive illness. Br. J. Psychiatry 117, 287-292. Smith, I., Hyland, K., Kendall, B. and Leeming, R.J. (1985) Clinical role of pteridine therapy in tetrahydrobiopterin deficiency. J. Inherit. Met. Dis. 8, 39-45. Switchenko, A. and Brown, G. (1985) The enzymatic conversion of dihydroneopterin triphosphate to L-pyruvoyltetrahydrobiopterin, an intermediate in the biosynthesis of other pterins in Drosophila melanogaster. J. Biol. Chem. 260, 2945-2951. Thornton, W.E. and Thornton, B.P. (1978) Folic acid, mental function and dietary habits. J. Clin. Psychiatry 39, 315-322. Van Praag, H.M. (1982) Neurotransmitters and CNS disease: depression. Lancet ii, 1259-1283.