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Folate deficiency in pulmonary tuberculosis: Relationship to treatment and to serum vitamin a and beta-carotene
288
Tubercle,
(1971),
52, 288
FOLATE DEFICIENCY IN PULMONARY TUBERCULOSIS: RELATIONSHIP TREATMENT AND TO SERUM VITAMIN A AND BETA-CAROTENE
TO
D. I. K. EVANS*and B. ATTOCK from Booth Hall and Monsall Group Laboratories. Manchester. SUMMARY
A group of 27 patients with pulmonary tuberculosis has been examined for evidence of any connection between the depression of red cell folate, serum folate, vitamin A and p-carotene, which are known findings in the disease. Serum and/or red cell folates were both reduced in 59 per cent of cases. Serum vitamin A was low in 51 per cent and @carotene was low in 33 per cent. There was no connection between folate deficiency and a low level of vitamin A or P-carotene; and patients treated for a longer period showed results no nearer normal than those treated for a shorter period. It is suggested that these blood abnormalities are intrinsic features of pulmonary tuberculosis and that in some cases a return to normal may be impaired by the drugs used in treatment.
Les auteurs essaient de mettre en evidence dans un groupe de 27 malades atteints de tuberculose pulmonaire, une relation quelconque entre la chute d’acide folique des globules rouges, celle du strum, ainsi que la diminution dans le strum de la vitamine A et du /3carotene, qui sont des signes connus de la maladie. Le taux d’acide folique de serum ou des globules rouges, ou bien des globules rouges seuls, ttait diminue dans 59 % des cas. Le taux serique de vitamine A Ctait bas chez 51% des malades et celui du P-carotene chez 33 %. 11n’y avait pas de relation entre la diminution de l’acide folique et un taux serique bas de la vitamine A ou du P-carotene; et les rtsultats trouves chez les malades trait& pendant longtemps n’etaient pas plus anormaux que ceux des malades trait& pendant un temps plus court. Les auteurs suggerent que ces taux sanguins anormaux sont intrinsequement lies a la tuberculose pulmonaire et que dans certains cas le retour a des taux normaux peut Ctre gene par les drogues utilistes dans le traitement.
RFXJMEN Se estudio un grupo de 27 pacientes tuberculosis buscando una explication de hechos conocidos en esta enfermedad coma son la disminucion de folatos en 10s hematies, folato serico, vitamina A y p-carotene. El folato serico y / o el de 10s hematles estaban reducidos en el 59 % de 10s cases. La vitamina A strica estaba descendida en el 51 ‘A y el p-carotene en el 33 ‘A. No habia rela*Requests for reprints should be. sent to: Dr D. I. K. Evans, Department Manchester, M9 2AA.
of Pathology, Charlestown Road, Blackley,
FOLATE
DEFICIENCY
289
cion entre la deficiencia en folatos y el nivel bajo de vitamina A 6 p-carotene; 10s pacientes con tratamientos prolongados mostraron las mismas alteraciones que 10s tratados por poco tiempo. Se sugiere que estas anomallas sanguineas son hechos intrlnsecos de la tuberculosis pulmonar y que en algunos cases la vuelta a la normalidad debe estar impedida por las drogas empleadas.
k%MMENFASSUNG
Eine Gruppe von 27 Patienten mit Lungentuberkulose wurde daraufhin untersucht, welcher Zusammenhang besteht zwischen den bei Tuberkulose bekannten Mangelzustiinden an Folslure (Folat) in Erythrozyten und im Serum sowie dem Vitamin A= und P-Karotin-Spiegel. Die FolsZiure war im Serum und/oder in den Erythrozyten in 59 % der Falle reduziert. Das Vitamin A war im Serum in 51% erniedrigt, das l3-Karotin in 33 %. Es gab keinen Zusammenhang zwischen einem Folsauremangel und einem niedrigen Spiegel von Vitamin A oder P-Karotin. Bei Patienten, die schon IHngere Zeit behandelt waren, hatten sich im Vergleich zu ktirzer behandelten die Werte nicht normalisiert. Es wird angenommen, daB diese Blutanomalien in enger Beziehung zur Lungentuberkulose stehen und da8 sie in manchen FHllen als Folge der Chemotherapie weitgehend behoben werden kiinnen. Introduction Low levels of folate are found in the serum and red cells of patients with tuberculosis (Roberts, Hoffbrand and Mollin, 1966; Markkanen and others, 1966; Klipstein, Berlinger and Reed, 1967; Cameron and Horne, 1971; Line and others, 1971) but the explanation for this abnormality is unknown (Chanarin, 1969). Similarly unexplained reductions of serum vitamin A, p-carotene, cholesterol and lipoproteins are also present (Straus and others, 1962; Prokopiev, 1965). All these abnormalities are also found in malabsorptive states. Malabsorption has been induced with antituberculous drugs (Akhtar, Crompton and Schonell, 1968; Coltart, 1969). PAS increases faecal fat excretion in normal subjects (Levine, 1968) and impairs absorption of vitamin B12 (Heinivaara and Palva, 1965). It is possible that the abnormalities of folate and fat metabolism are the result of treatment and not an intrinsic feature of the disease. We have studied serum and red cell folate levels, serum vitamin A and p-carotene, xylose tolerance tests and haematological indices in a group of patients with tuberculosis under treatment, in an attempt to elucidate the problem. Materials and Methods Twenty-seven patients (6 women and 21 men), aged from 20 to 73 years, were studied. All had active pulmonary tuberculosis with a positive culture for tubercle bacilli at the time of diagnosis. The drugs used were PAS.(23 cases), isoniazid (25 cases), streptomycin (11 cases), rifampicin (4 cases) and ethionamide (1 case). Standard haematological methods were used (Dacie and Lewis, 1968). Red cell folate was measured by the method of Hotfbrand,Newcombe and Mollin (1966) and serum folate by the method of Waters and Mollin (1961) in the laboratory of Dr. D. W. Dawson. Serum p-carotene and vitamin A were measured by the method of Neeld and Pearson (1963). In eighteen cases a xylose tolerance test was performed using a 5 g. dose and a 5-hour urine collection. Urinary xylose was measured by Goodhart and Kingston’s (1969) modification of the method of Roe and Rice (1948).
290
EVANS
AND
ATTOC‘K
Results
Haemoglobin levels averaged 12.9 g. per 100 ml. in the group of 21 men (range 8.7 to 15.6) and 11.6 g. per 100 ml. in the 6 women (range 8.7 to 13.2). The average mean corpuscular volume (M.C.V.) for men was 90 p3 (range 79 to 101) and 87 p3 for women (range 76 to 102). Serum folate levels were below the lower limit of normal (which is taken as 3-O ng. per ml. in this laboratory) in 16 of the 27 patients (59 per cent). Red cell folate levels were likewise reduced below the lower normal limit of 100 ng. per ml. red cells in a similar number ofcases, although the two groups were not identical. Vitamin A levels were below the lower limit of normal (25 kg, per 100 ml.) in 14 of the 27 patients (51 per cent) and p-carotene levels were below the lower normal limit of 60 pg. per 100 ml. in 9 of the 27 patients (33 per cent). Xylose tolerance tests were performed on 18 cases. The urinary excretion of xylose was never less than 25 per cent of the ingested dose, i.e. all patients gave a normal result. Red cell and serum folates were each plotted against serum vitamin A and p-carotene. No correlation was seen: i.e. a low serum or red cell folate does not correlate with low vitamin A or $-carotene. (See Figure I ). +
&&
.I40
??
.I20
??
’
-20
60
2P
,
a 1yo
140
,
180
Red cell folate
Serum folate
70
. 70
60
40
0
?? o ??.
20
0
y'
O@ ??
0
() 0
0
0
0.
0
??
00
.a .40 4 .z b-030
0
0
??
50
<
0
30
;
0
e
0
4 -
0
0
??
60 0
5P
f
0
.20
0 e
-10
80
0-o (, 0 0
0
IO c
?? OO
0
I
1
,
2
6
,
4
,
20
5
Serum folate
,
60
0 *
IQ0
.
0 140 .
190
.
Red cell folate FIG. 1
Serum and red cell folates plotted against sewm vitamin A and p-carotene. No correlation is apparent. (Vitamin A and p-carotene in pg. per 100 ml.; serum and red cell folate in ng. per ml. The lower limit of normal is indicated by a line.)
291
FOLATE DEFICIENCY I.-MEAN TABLE AND
LEVEUOFSERUMVITAMINA, p-CARO~NE AND FOLATE
MEAN REDCELLFOLATES IN THIRTEEN PATIENTS ONTwo OCCASIONS
SEPARATED BY A MONTH. THE DIFFERENCES ARE NOT
Reading (i)
-___-
VitaminA S-carotene Serumfolate Red cell folate
SIGNIFICANT.
Reading (ii) -
_ / 30 pg. per 100 ml. ~ “,39~i.per;O0,ml. 91 ng. per ml.
26 pg. per 100 ml.
1 8: wg.per 100ml. i 2 9 ng. per ml. 108 ng. per ml.
Thirteen patients on whom serum and red cell folates, serum vitamin A and S-carotene had been estimated had these measurements repeated a month later. There was no significant change during this time (see table 1). Twenty-one patients who had been treated with PAS and isoniazid, with and without streptomycin, were studied in further detail. The median period of treatment for the group was nine weeks. We therefore compared the results from patients under treatment for less than nine weeks with those of patients tested after more than nine weeks’ treatment. The mean serum folate was low in both groups and showed no significant change; but the mean red cell folate in the first group was 105 ng. per ml. and fell to 73 ng. pet ml. in the second group, a significant difference (P ~0.05). The mean vitamin A levels were in the normal range and showed little difference in either group; and the S-carotene levels, likewise in the normal range, fell from 80 to 71 pg. per 100 ml , although this slight change was not significant. (Table II). Other indices measured in these two groups included haemoglobin levels, packed cell volume (P.C.V.), red cell count, mean corpuscular haemoglobin concentration (M.C.H.C.) and M.C.V., and nuclear lobe count of neutrophils including calculation of the number of cells with more than three and more than four lobes. (See Table II). Although haemoglobin levels and M.C.H.C. are higher in the group treated for the longer period, there is a slight increase of M.C.V. and of the percentage of neutrophils with more than 3 nuclear lobes. These are indirect signs of early folate deficiency, and while not statistically significant, correlate with the lower red cell folate. In order to check that dietary deficiency of folate was not the cause of these findings, we studied in parallel a group of old women in a geriatric ward, who were eating the same diet. They had low serum and red cell folates on admission, due to malnutrition. After a month on the hospital diet the mean serum folate levels rose from a subnormal 2.8 ng. per ml. to a low normal 3.4 ng. per ml. and the red cell folate levels rose slightly, but not significantly, from 87 to 90 ng. per ml. These results suggest that the hospital diet was providing sufficient folate. Discussion There is an undoubted abnormality in the levels of vitamin A in this group of tuberculous patients: 51 per cent of the total tested gave a figure below the accepted lower normal limit of 25 pg. per 100 ml. (mean of 28 pg. per 100 ml.). Similarly the S-carotene was reduced in 33 per cent of cases. The mean was 82 pg. per 100 ml. : the lower normal limit is 60 pg. per 100 ml. These results confirm Prokopiev’s (1965) figures, although all of his tuberculous patients had low carotene levels. Similarly, serum and red cell folates were low in 59 per cent of these patients: there was no improvement on a normal hospital diet. Indeed, red cell folates, far from being higher in the group treated for the longer period, were significantly lower. But red cell folate is taken as an index of tissue folate (Chanarin, 1969) and a fall in red cell folate must represent a fall in folate stores. It seems unlikely that the low serum folate is due to a shift in folate from the plasma compartment,
18
Mean
1 -
KM .I
Woman
2 4 7
9 13 15 15 18 20 48 50 58 80
-
No. of
vveeks on treatment
Treatedfor more than nine weeks Men C.T. A.T. A.M H.T. H.R. T.M. W.M. W.M. J.B. G.A.
Women T.B. B.H. L.D.
Treatedfor less than nine weeks Men T.S. P.C. R.B. F.B. A.W. J.B. T.B. T.L.
-
244
1.2
2.0 3.9 2.1 3.7 2.4 1.1 1.1 3.2 2.1 4.0
-
73.18
32
54 133 80 52 59 63 100 88 71 73
104.64
2.56
84 51 114 197 96 118 125 122 59 114 71
_-
.-
Red cell folate ng.lml.
_L
5
31.73
21
57 53 43 22 7 18 16 41 22 49
2864
29 52 29
71.36
-
13.42
13.1
67
.-
15.4 14.3 15.5 14.2 12.5 13.1 9.0 11.7 13.5 15.3
11.99
112 95 60 87 59 92 57 25 40 91
19.55
.-
12.8 11.1 13.1
9.7 15.6 14.3 11.4 11.3 8.7 10.7 13.2
g. per 100 ml.
globin
iHaemo-
-
_-
_-
_-
-
of mean
41.55
41
47 42 44 46 40 41 29 36 44 47
38.73
4.62
4.00
5.56 4.84 5.09 4.56 4.01 4.89 360 3.96 5.10 5.19
4.34
3.79 4.27 4.18
37 36 41
__
3.83 4.99 4.94 4.59 4.29 4.00 4.18 4.72
90.36
102
85 87 87 101 100 84 81 91 86 90
89.09
98 84 98
84 90 93 89 91 85 79 89
M.C.
__ -
35 31 32
30 35 31 28 29 26 32 31
%
M. C. H. C.
32.27
32
33 34 35 31 31 32 31 32 31 33
30.91 _____
_-
v.
-
ISONIAZID.
count :<106/pI.
Red cell
AND
32 45 46 41 39 34 33 42
%
P.C. v.
* Omitted from calculation
-
_-
-
--
70 60 135
48 83 102 116 16 85 61 99
l.Lg/IM ml.
Wg./loo ml.
24 15 18 56 39 8 10 35
Serum pcarotene
--
-
Serum vit. A
_-
T
TABLE II.--RESULTS IN PATIENTSTREATED WITH PAS
3.0 2.2 3.2
1.8 2.2 3.0 3.2 0.9 1.7 2.1 4.9
ng./ml.
Serum folate
-
2.69
2.37
2.73 2.57 2.58 3.18 2.53 2.83 2.82 2.85 2.87 2.31
2.52
2.72 1.84 2.49
2.17 2.58 2.25 2.62 3.71* 2.13 2.76 2.50
Nuclear lobe count
L
--
%
22.36 __-.
9 ___.
19 20 24 33 13 36 27 24 31 10
15 __~
15 1 10
8 13 6 17 58* 6 22 9
over 3 lobes
- -_
--
-
2.55
1
0 4 3 6 1 3 4 3 3 0
0 0 1
6 2
1
1 0 0 2 25*
over 4 Iohes
; tl
$ z v)
m
FOLATE
DEFICIENCY
293
or to dilution by water retention, an explanation which has been proposed for the otherwise unexplained fall in plasma sodium and chloride which is also a feature of tuberculosis and some acute infections (Cantarow and Trumper, 1962; Searcy, 1969). The majority of these patients showed clinical improvement while under treatment in hospital. If the abnormalities reported here were a reflection of the severity of the disease, improvement in the clinical state should be reflected in a return to~normal of these tests. The lower red cell folate in the group treated for the longer period does not support the statement of Roberts, Hoffbrand and Mollin (1966) that folate deficiency is not related to the duration of treatment. A similar, though not statistically significant, fall in mean red cell folate after three months treatment was also described by Line and others (1971) in spite of a significant rise in serum folate at this time. These authors commented on the slow correction of folate deficiency in patients with tuberculosis. Xylose absorption was normal in every case, so there can be no generalised malabsorption in these cases. Levine (1968) tested normal subjects and showed that xylose absorption is only briefly affected by PAS, and only when a 12 g. dose is given. A 6 g. dose was without effect. Furthermore the drug did not affect vitamin A levels in these normal subjects at either dosage level although it increased excretion of faecal fat. These results are slightly at variance: vitamin A and p-carotene are fat-soluble, and any drug increasing fat excretion would be expected to increase excretion of these substances as well. But serum levels reflect a balance between input and output, and presumably dietary intake in the normal subjects was adequate to compensate for any possible increase of faecal excretion. Heinivaara and Palva (1965) demonstrated lower levels of serum B,, in patients treated with PAS. Levels fell with prolonged treatment, and Schilling tests before and after giving PAS showed a slight impairment of B,, absorption after PAS. It is clear that PAS can cause steatorrhoea; it was the presenting feature in the patient described by Coltart (1969) and there was impaired xylose absorption. Such patients may represent an exaggeration of a normal response. Six of 58 patients with tuberculosis studied by Cameron and Horne (1971) had impaired xylose absorption together with a serum folate of less than 3.0 ng. per ml.; and 50 per cent of their patients had either a low (less than 3.0 ng. per ml.) or intermediate (3-O to 4-5 ng. per ml.) serum folate. Six of their patients with folate deficiency had folate malabsorption, which they postulated might be due to treatment with PAS. This is an attractive hypothesis, but is not supported by the evidence of Klipstein, Berlinger and Reed (1967). They reported low serum folates in 15 of 29 tuberculous patients treated with isoniazid and cycloserine, but in only two of 55 patients taking isoniazid alone or with pyrazinamide, and in 4 of 24 patients taking PAS. Five of 24 patients having no chemotherapy at all also had a low serum folate. They postulated that cycloserine might be the responsible drug. We have shown that there is folate deficiency in these patients which does not appear to improve spontaneously while treatment is continued. Indirect tests for fat absorption remain low too, but these findings do not correlate with each other. The folate changes are not due to dietary deficiency yet do not improve in step with other evidence of response to treatment. Drugs may play a role in impairing this response; if so more than one drug may be responsible and several mechanisms may be involved, as there is no direct relationship between folate deficiency and vitamin A and p-carotene. Furthermore, these latter measurements were not affected by PAS in the normal subjects studied by Levine (1968) and it would appear unlikely that the low levels in tuberculosis are due to the drug alone. It seems more likely that these abnormalities are intrinsic features of pulmonary tuberculosis, but that in some cases a return to normal may be impaired by treatment. We are grateful to Dr D. W. Dawson for folate estimations and to Mrs Carol Bowman for statistical assistance. Thanks are expressed to Drs W. D. Anderson, M. O’Connor and F. W. A. Tumbull for permission to study their patients.
294
EVANS
AND
ATTOCK
REFERENCES AKtITAR, A. J., CROMPTON,G. K. & SCHONELL, M. E. (1968). Para-aminosalicylic acid as a cause of intestinal malabsorption. Tubercle, 49,328. CAMERON, S. J. & HORNE, N. W. (1971). The effect of tuberculosis and its treatment on erythropoiesis and folate activity. Tubercle, 52,37.
CAN~~RO~~~,A.& TRUMPER,M. (1962). Clinical Biochemistry, 6th edition, p. 303. W. B. Saunders, Philadelphia & CHANARIN,I: (1969). The Megaloblastic Anaemias. Blackwell, Oxford. COLTART,D. J. (1969). Malabsorption induced by para-aminosalicylate. British Medical Journal, 1,825. DACIE,J. V. & LEWIS,S. M. (1968). Practical Haematology, 4th edition, Churchill, London. GOODHART,J. M. & KINGSTON,G. R. (1969). Modification of the method for the estimation of xylose in urine. Journal of Clinical Pathology, 22,621.
HEINIVAARA,0. & PALVA,I. P. (1965). Malabsorption and deficiency of vitamin B,, caused by treatment with paraaminosalicylic acid. Acta Medica Scandinavica, 177, 337. HOFFBRAND,A. V., NEWCOMBE,B. F. A. & MOLLIN,D. L. (1966). Method of assay of red cell folate activity and the value of the assay as a test of folate deficiency. Journalof Clinical Pathology, 19,17. KLIPSTEIN.F. A.. BERLINGER.F. C. & REED. L. J. (1967). _ , Folate deficiency associated with drug therapy- for tuberculos~s. Bloo;z, 29,697.
LEVINE,R. A. (I 968). Steatorrhea induced by para-aminosalicylic acid. Annals of Internal Medicine, 68,1265. LINE, D. H., SEITANIDIS,B., MORGAN,J. 0. & HOFFBRAND,A. V. (1971). The effects of chemotherapy on iron, folate, and vitamin B12metabolism in tuberculosis. Quarterly Journal of Medicine, 40,331. MARKKANEN,T., LEVANTO,A., SALLINEN,V. & VIRTANEN,S. (1966). Folate deficiency during antituberculous (PASINH) medication. Experientia, 22,692. NEELD. J. B. Jr. & PEARSON.W. N. (1963). Macro- and micro-methods for the determination of vitamin A using trikuoracetic-acid. Journal of N&tion,‘79,454. PROKOPIEV,D. I. (1965). Vitamin A and carotene in the blood serum of patients with pulmonary tuberculosis. Problems of Tuberculosis, 43,46, (No. 8).
ROBERTS,P. D., HOFFBRAND,A. V. & MOLLIN, D. L. (1966). Iron and folate metabolism
in tuberculosis.
British
Medical Journal, 2, 198.
ROE, J. H. & RICE, E. W. (1948). A photometric method for the determination of free pentoses in animal tissues. Journal of Biological Chemistry, 173,507. SEARCY,R. L. (1969). Diagnostic Biochemistry, p. 476. McGraw-Hill Inc., New York and London. STRAUS,R., WURM, M., MYERS,W. E. & SALKIN,D. (1962). Distribution of serum lipids in tuberculous patients and its relation to clinical coronary heart disease. American Journal of Clinical Pathology, 37,339. WATERS,A. H. & MOLLIN, D. L. (1961). Studies on the folic acid activity of human serum. Journal of Clinical Pathology, 14,335.
Tubercle,
(1971),
52, 288
FOLATE DEFICIENCY IN PULMONARY TUBERCULOSIS: RELATIONSHIP TREATMENT AND TO SERUM VITAMIN A AND BETA-CAROTENE
TO
D. I. K. EVANS*and B. ATTOCK from Booth Hall and Monsall Group Laboratories. Manchester. SUMMARY
A group of 27 patients with pulmonary tuberculosis has been examined for evidence of any connection between the depression of red cell folate, serum folate, vitamin A and p-carotene, which are known findings in the disease. Serum and/or red cell folates were both reduced in 59 per cent of cases. Serum vitamin A was low in 51 per cent and @carotene was low in 33 per cent. There was no connection between folate deficiency and a low level of vitamin A or P-carotene; and patients treated for a longer period showed results no nearer normal than those treated for a shorter period. It is suggested that these blood abnormalities are intrinsic features of pulmonary tuberculosis and that in some cases a return to normal may be impaired by the drugs used in treatment.
Les auteurs essaient de mettre en evidence dans un groupe de 27 malades atteints de tuberculose pulmonaire, une relation quelconque entre la chute d’acide folique des globules rouges, celle du strum, ainsi que la diminution dans le strum de la vitamine A et du /3carotene, qui sont des signes connus de la maladie. Le taux d’acide folique de serum ou des globules rouges, ou bien des globules rouges seuls, ttait diminue dans 59 % des cas. Le taux serique de vitamine A Ctait bas chez 51% des malades et celui du P-carotene chez 33 %. 11n’y avait pas de relation entre la diminution de l’acide folique et un taux serique bas de la vitamine A ou du P-carotene; et les rtsultats trouves chez les malades trait& pendant longtemps n’etaient pas plus anormaux que ceux des malades trait& pendant un temps plus court. Les auteurs suggerent que ces taux sanguins anormaux sont intrinsequement lies a la tuberculose pulmonaire et que dans certains cas le retour a des taux normaux peut Ctre gene par les drogues utilistes dans le traitement.
RFXJMEN Se estudio un grupo de 27 pacientes tuberculosis buscando una explication de hechos conocidos en esta enfermedad coma son la disminucion de folatos en 10s hematies, folato serico, vitamina A y p-carotene. El folato serico y / o el de 10s hematles estaban reducidos en el 59 % de 10s cases. La vitamina A strica estaba descendida en el 51 ‘A y el p-carotene en el 33 ‘A. No habia rela*Requests for reprints should be. sent to: Dr D. I. K. Evans, Department Manchester, M9 2AA.
of Pathology, Charlestown Road, Blackley,
FOLATE
DEFICIENCY
289
cion entre la deficiencia en folatos y el nivel bajo de vitamina A 6 p-carotene; 10s pacientes con tratamientos prolongados mostraron las mismas alteraciones que 10s tratados por poco tiempo. Se sugiere que estas anomallas sanguineas son hechos intrlnsecos de la tuberculosis pulmonar y que en algunos cases la vuelta a la normalidad debe estar impedida por las drogas empleadas.
k%MMENFASSUNG
Eine Gruppe von 27 Patienten mit Lungentuberkulose wurde daraufhin untersucht, welcher Zusammenhang besteht zwischen den bei Tuberkulose bekannten Mangelzustiinden an Folslure (Folat) in Erythrozyten und im Serum sowie dem Vitamin A= und P-Karotin-Spiegel. Die FolsZiure war im Serum und/oder in den Erythrozyten in 59 % der Falle reduziert. Das Vitamin A war im Serum in 51% erniedrigt, das l3-Karotin in 33 %. Es gab keinen Zusammenhang zwischen einem Folsauremangel und einem niedrigen Spiegel von Vitamin A oder P-Karotin. Bei Patienten, die schon IHngere Zeit behandelt waren, hatten sich im Vergleich zu ktirzer behandelten die Werte nicht normalisiert. Es wird angenommen, daB diese Blutanomalien in enger Beziehung zur Lungentuberkulose stehen und da8 sie in manchen FHllen als Folge der Chemotherapie weitgehend behoben werden kiinnen. Introduction Low levels of folate are found in the serum and red cells of patients with tuberculosis (Roberts, Hoffbrand and Mollin, 1966; Markkanen and others, 1966; Klipstein, Berlinger and Reed, 1967; Cameron and Horne, 1971; Line and others, 1971) but the explanation for this abnormality is unknown (Chanarin, 1969). Similarly unexplained reductions of serum vitamin A, p-carotene, cholesterol and lipoproteins are also present (Straus and others, 1962; Prokopiev, 1965). All these abnormalities are also found in malabsorptive states. Malabsorption has been induced with antituberculous drugs (Akhtar, Crompton and Schonell, 1968; Coltart, 1969). PAS increases faecal fat excretion in normal subjects (Levine, 1968) and impairs absorption of vitamin B12 (Heinivaara and Palva, 1965). It is possible that the abnormalities of folate and fat metabolism are the result of treatment and not an intrinsic feature of the disease. We have studied serum and red cell folate levels, serum vitamin A and p-carotene, xylose tolerance tests and haematological indices in a group of patients with tuberculosis under treatment, in an attempt to elucidate the problem. Materials and Methods Twenty-seven patients (6 women and 21 men), aged from 20 to 73 years, were studied. All had active pulmonary tuberculosis with a positive culture for tubercle bacilli at the time of diagnosis. The drugs used were PAS.(23 cases), isoniazid (25 cases), streptomycin (11 cases), rifampicin (4 cases) and ethionamide (1 case). Standard haematological methods were used (Dacie and Lewis, 1968). Red cell folate was measured by the method of Hotfbrand,Newcombe and Mollin (1966) and serum folate by the method of Waters and Mollin (1961) in the laboratory of Dr. D. W. Dawson. Serum p-carotene and vitamin A were measured by the method of Neeld and Pearson (1963). In eighteen cases a xylose tolerance test was performed using a 5 g. dose and a 5-hour urine collection. Urinary xylose was measured by Goodhart and Kingston’s (1969) modification of the method of Roe and Rice (1948).
290
EVANS
AND
ATTOC‘K
Results
Haemoglobin levels averaged 12.9 g. per 100 ml. in the group of 21 men (range 8.7 to 15.6) and 11.6 g. per 100 ml. in the 6 women (range 8.7 to 13.2). The average mean corpuscular volume (M.C.V.) for men was 90 p3 (range 79 to 101) and 87 p3 for women (range 76 to 102). Serum folate levels were below the lower limit of normal (which is taken as 3-O ng. per ml. in this laboratory) in 16 of the 27 patients (59 per cent). Red cell folate levels were likewise reduced below the lower normal limit of 100 ng. per ml. red cells in a similar number ofcases, although the two groups were not identical. Vitamin A levels were below the lower limit of normal (25 kg, per 100 ml.) in 14 of the 27 patients (51 per cent) and p-carotene levels were below the lower normal limit of 60 pg. per 100 ml. in 9 of the 27 patients (33 per cent). Xylose tolerance tests were performed on 18 cases. The urinary excretion of xylose was never less than 25 per cent of the ingested dose, i.e. all patients gave a normal result. Red cell and serum folates were each plotted against serum vitamin A and p-carotene. No correlation was seen: i.e. a low serum or red cell folate does not correlate with low vitamin A or $-carotene. (See Figure I ). +
&&
.I40
??
.I20
??
’
-20
60
2P
,
a 1yo
140
,
180
Red cell folate
Serum folate
70
. 70
60
40
0
?? o ??.
20
0
y'
O@ ??
0
() 0
0
0
0.
0
??
00
.a .40 4 .z b-030
0
0
??
50
<
0
30
;
0
e
0
4 -
0
0
??
60 0
5P
f
0
.20
0 e
-10
80
0-o (, 0 0
0
IO c
?? OO
0
I
1
,
2
6
,
4
,
20
5
Serum folate
,
60
0 *
IQ0
.
0 140 .
190
.
Red cell folate FIG. 1
Serum and red cell folates plotted against sewm vitamin A and p-carotene. No correlation is apparent. (Vitamin A and p-carotene in pg. per 100 ml.; serum and red cell folate in ng. per ml. The lower limit of normal is indicated by a line.)
291
FOLATE DEFICIENCY I.-MEAN TABLE AND
LEVEUOFSERUMVITAMINA, p-CARO~NE AND FOLATE
MEAN REDCELLFOLATES IN THIRTEEN PATIENTS ONTwo OCCASIONS
SEPARATED BY A MONTH. THE DIFFERENCES ARE NOT
Reading (i)
-___-
VitaminA S-carotene Serumfolate Red cell folate
SIGNIFICANT.
Reading (ii) -
_ / 30 pg. per 100 ml. ~ “,39~i.per;O0,ml. 91 ng. per ml.
26 pg. per 100 ml.
1 8: wg.per 100ml. i 2 9 ng. per ml. 108 ng. per ml.
Thirteen patients on whom serum and red cell folates, serum vitamin A and S-carotene had been estimated had these measurements repeated a month later. There was no significant change during this time (see table 1). Twenty-one patients who had been treated with PAS and isoniazid, with and without streptomycin, were studied in further detail. The median period of treatment for the group was nine weeks. We therefore compared the results from patients under treatment for less than nine weeks with those of patients tested after more than nine weeks’ treatment. The mean serum folate was low in both groups and showed no significant change; but the mean red cell folate in the first group was 105 ng. per ml. and fell to 73 ng. pet ml. in the second group, a significant difference (P ~0.05). The mean vitamin A levels were in the normal range and showed little difference in either group; and the S-carotene levels, likewise in the normal range, fell from 80 to 71 pg. per 100 ml , although this slight change was not significant. (Table II). Other indices measured in these two groups included haemoglobin levels, packed cell volume (P.C.V.), red cell count, mean corpuscular haemoglobin concentration (M.C.H.C.) and M.C.V., and nuclear lobe count of neutrophils including calculation of the number of cells with more than three and more than four lobes. (See Table II). Although haemoglobin levels and M.C.H.C. are higher in the group treated for the longer period, there is a slight increase of M.C.V. and of the percentage of neutrophils with more than 3 nuclear lobes. These are indirect signs of early folate deficiency, and while not statistically significant, correlate with the lower red cell folate. In order to check that dietary deficiency of folate was not the cause of these findings, we studied in parallel a group of old women in a geriatric ward, who were eating the same diet. They had low serum and red cell folates on admission, due to malnutrition. After a month on the hospital diet the mean serum folate levels rose from a subnormal 2.8 ng. per ml. to a low normal 3.4 ng. per ml. and the red cell folate levels rose slightly, but not significantly, from 87 to 90 ng. per ml. These results suggest that the hospital diet was providing sufficient folate. Discussion There is an undoubted abnormality in the levels of vitamin A in this group of tuberculous patients: 51 per cent of the total tested gave a figure below the accepted lower normal limit of 25 pg. per 100 ml. (mean of 28 pg. per 100 ml.). Similarly the S-carotene was reduced in 33 per cent of cases. The mean was 82 pg. per 100 ml. : the lower normal limit is 60 pg. per 100 ml. These results confirm Prokopiev’s (1965) figures, although all of his tuberculous patients had low carotene levels. Similarly, serum and red cell folates were low in 59 per cent of these patients: there was no improvement on a normal hospital diet. Indeed, red cell folates, far from being higher in the group treated for the longer period, were significantly lower. But red cell folate is taken as an index of tissue folate (Chanarin, 1969) and a fall in red cell folate must represent a fall in folate stores. It seems unlikely that the low serum folate is due to a shift in folate from the plasma compartment,
18
Mean
1 -
KM .I
Woman
2 4 7
9 13 15 15 18 20 48 50 58 80
-
No. of
vveeks on treatment
Treatedfor more than nine weeks Men C.T. A.T. A.M H.T. H.R. T.M. W.M. W.M. J.B. G.A.
Women T.B. B.H. L.D.
Treatedfor less than nine weeks Men T.S. P.C. R.B. F.B. A.W. J.B. T.B. T.L.
-
244
1.2
2.0 3.9 2.1 3.7 2.4 1.1 1.1 3.2 2.1 4.0
-
73.18
32
54 133 80 52 59 63 100 88 71 73
104.64
2.56
84 51 114 197 96 118 125 122 59 114 71
_-
.-
Red cell folate ng.lml.
_L
5
31.73
21
57 53 43 22 7 18 16 41 22 49
2864
29 52 29
71.36
-
13.42
13.1
67
.-
15.4 14.3 15.5 14.2 12.5 13.1 9.0 11.7 13.5 15.3
11.99
112 95 60 87 59 92 57 25 40 91
19.55
.-
12.8 11.1 13.1
9.7 15.6 14.3 11.4 11.3 8.7 10.7 13.2
g. per 100 ml.
globin
iHaemo-
-
_-
_-
_-
-
of mean
41.55
41
47 42 44 46 40 41 29 36 44 47
38.73
4.62
4.00
5.56 4.84 5.09 4.56 4.01 4.89 360 3.96 5.10 5.19
4.34
3.79 4.27 4.18
37 36 41
__
3.83 4.99 4.94 4.59 4.29 4.00 4.18 4.72
90.36
102
85 87 87 101 100 84 81 91 86 90
89.09
98 84 98
84 90 93 89 91 85 79 89
M.C.
__ -
35 31 32
30 35 31 28 29 26 32 31
%
M. C. H. C.
32.27
32
33 34 35 31 31 32 31 32 31 33
30.91 _____
_-
v.
-
ISONIAZID.
count :<106/pI.
Red cell
AND
32 45 46 41 39 34 33 42
%
P.C. v.
* Omitted from calculation
-
_-
-
--
70 60 135
48 83 102 116 16 85 61 99
l.Lg/IM ml.
Wg./loo ml.
24 15 18 56 39 8 10 35
Serum pcarotene
--
-
Serum vit. A
_-
T
TABLE II.--RESULTS IN PATIENTSTREATED WITH PAS
3.0 2.2 3.2
1.8 2.2 3.0 3.2 0.9 1.7 2.1 4.9
ng./ml.
Serum folate
-
2.69
2.37
2.73 2.57 2.58 3.18 2.53 2.83 2.82 2.85 2.87 2.31
2.52
2.72 1.84 2.49
2.17 2.58 2.25 2.62 3.71* 2.13 2.76 2.50
Nuclear lobe count
L
--
%
22.36 __-.
9 ___.
19 20 24 33 13 36 27 24 31 10
15 __~
15 1 10
8 13 6 17 58* 6 22 9
over 3 lobes
- -_
--
-
2.55
1
0 4 3 6 1 3 4 3 3 0
0 0 1
6 2
1
1 0 0 2 25*
over 4 Iohes
; tl
$ z v)
m
FOLATE
DEFICIENCY
293
or to dilution by water retention, an explanation which has been proposed for the otherwise unexplained fall in plasma sodium and chloride which is also a feature of tuberculosis and some acute infections (Cantarow and Trumper, 1962; Searcy, 1969). The majority of these patients showed clinical improvement while under treatment in hospital. If the abnormalities reported here were a reflection of the severity of the disease, improvement in the clinical state should be reflected in a return to~normal of these tests. The lower red cell folate in the group treated for the longer period does not support the statement of Roberts, Hoffbrand and Mollin (1966) that folate deficiency is not related to the duration of treatment. A similar, though not statistically significant, fall in mean red cell folate after three months treatment was also described by Line and others (1971) in spite of a significant rise in serum folate at this time. These authors commented on the slow correction of folate deficiency in patients with tuberculosis. Xylose absorption was normal in every case, so there can be no generalised malabsorption in these cases. Levine (1968) tested normal subjects and showed that xylose absorption is only briefly affected by PAS, and only when a 12 g. dose is given. A 6 g. dose was without effect. Furthermore the drug did not affect vitamin A levels in these normal subjects at either dosage level although it increased excretion of faecal fat. These results are slightly at variance: vitamin A and p-carotene are fat-soluble, and any drug increasing fat excretion would be expected to increase excretion of these substances as well. But serum levels reflect a balance between input and output, and presumably dietary intake in the normal subjects was adequate to compensate for any possible increase of faecal excretion. Heinivaara and Palva (1965) demonstrated lower levels of serum B,, in patients treated with PAS. Levels fell with prolonged treatment, and Schilling tests before and after giving PAS showed a slight impairment of B,, absorption after PAS. It is clear that PAS can cause steatorrhoea; it was the presenting feature in the patient described by Coltart (1969) and there was impaired xylose absorption. Such patients may represent an exaggeration of a normal response. Six of 58 patients with tuberculosis studied by Cameron and Horne (1971) had impaired xylose absorption together with a serum folate of less than 3.0 ng. per ml.; and 50 per cent of their patients had either a low (less than 3.0 ng. per ml.) or intermediate (3-O to 4-5 ng. per ml.) serum folate. Six of their patients with folate deficiency had folate malabsorption, which they postulated might be due to treatment with PAS. This is an attractive hypothesis, but is not supported by the evidence of Klipstein, Berlinger and Reed (1967). They reported low serum folates in 15 of 29 tuberculous patients treated with isoniazid and cycloserine, but in only two of 55 patients taking isoniazid alone or with pyrazinamide, and in 4 of 24 patients taking PAS. Five of 24 patients having no chemotherapy at all also had a low serum folate. They postulated that cycloserine might be the responsible drug. We have shown that there is folate deficiency in these patients which does not appear to improve spontaneously while treatment is continued. Indirect tests for fat absorption remain low too, but these findings do not correlate with each other. The folate changes are not due to dietary deficiency yet do not improve in step with other evidence of response to treatment. Drugs may play a role in impairing this response; if so more than one drug may be responsible and several mechanisms may be involved, as there is no direct relationship between folate deficiency and vitamin A and p-carotene. Furthermore, these latter measurements were not affected by PAS in the normal subjects studied by Levine (1968) and it would appear unlikely that the low levels in tuberculosis are due to the drug alone. It seems more likely that these abnormalities are intrinsic features of pulmonary tuberculosis, but that in some cases a return to normal may be impaired by treatment. We are grateful to Dr D. W. Dawson for folate estimations and to Mrs Carol Bowman for statistical assistance. Thanks are expressed to Drs W. D. Anderson, M. O’Connor and F. W. A. Tumbull for permission to study their patients.
294
EVANS
AND
ATTOCK
REFERENCES AKtITAR, A. J., CROMPTON,G. K. & SCHONELL, M. E. (1968). Para-aminosalicylic acid as a cause of intestinal malabsorption. Tubercle, 49,328. CAMERON, S. J. & HORNE, N. W. (1971). The effect of tuberculosis and its treatment on erythropoiesis and folate activity. Tubercle, 52,37.
CAN~~RO~~~,A.& TRUMPER,M. (1962). Clinical Biochemistry, 6th edition, p. 303. W. B. Saunders, Philadelphia & CHANARIN,I: (1969). The Megaloblastic Anaemias. Blackwell, Oxford. COLTART,D. J. (1969). Malabsorption induced by para-aminosalicylate. British Medical Journal, 1,825. DACIE,J. V. & LEWIS,S. M. (1968). Practical Haematology, 4th edition, Churchill, London. GOODHART,J. M. & KINGSTON,G. R. (1969). Modification of the method for the estimation of xylose in urine. Journal of Clinical Pathology, 22,621.
HEINIVAARA,0. & PALVA,I. P. (1965). Malabsorption and deficiency of vitamin B,, caused by treatment with paraaminosalicylic acid. Acta Medica Scandinavica, 177, 337. HOFFBRAND,A. V., NEWCOMBE,B. F. A. & MOLLIN,D. L. (1966). Method of assay of red cell folate activity and the value of the assay as a test of folate deficiency. Journalof Clinical Pathology, 19,17. KLIPSTEIN.F. A.. BERLINGER.F. C. & REED. L. J. (1967). _ , Folate deficiency associated with drug therapy- for tuberculos~s. Bloo;z, 29,697.
LEVINE,R. A. (I 968). Steatorrhea induced by para-aminosalicylic acid. Annals of Internal Medicine, 68,1265. LINE, D. H., SEITANIDIS,B., MORGAN,J. 0. & HOFFBRAND,A. V. (1971). The effects of chemotherapy on iron, folate, and vitamin B12metabolism in tuberculosis. Quarterly Journal of Medicine, 40,331. MARKKANEN,T., LEVANTO,A., SALLINEN,V. & VIRTANEN,S. (1966). Folate deficiency during antituberculous (PASINH) medication. Experientia, 22,692. NEELD. J. B. Jr. & PEARSON.W. N. (1963). Macro- and micro-methods for the determination of vitamin A using trikuoracetic-acid. Journal of N&tion,‘79,454. PROKOPIEV,D. I. (1965). Vitamin A and carotene in the blood serum of patients with pulmonary tuberculosis. Problems of Tuberculosis, 43,46, (No. 8).
ROBERTS,P. D., HOFFBRAND,A. V. & MOLLIN, D. L. (1966). Iron and folate metabolism
in tuberculosis.
British
Medical Journal, 2, 198.
ROE, J. H. & RICE, E. W. (1948). A photometric method for the determination of free pentoses in animal tissues. Journal of Biological Chemistry, 173,507. SEARCY,R. L. (1969). Diagnostic Biochemistry, p. 476. McGraw-Hill Inc., New York and London. STRAUS,R., WURM, M., MYERS,W. E. & SALKIN,D. (1962). Distribution of serum lipids in tuberculous patients and its relation to clinical coronary heart disease. American Journal of Clinical Pathology, 37,339. WATERS,A. H. & MOLLIN, D. L. (1961). Studies on the folic acid activity of human serum. Journal of Clinical Pathology, 14,335.