Scand J Haeniatol (1975) 14, 71-79
Folic Acid Deficiency in Sickle Cell Anaemia YONGK. LIU,M.D. Hematologic Research, Medical Service (Chief, L. G . Horan), Veterans Administration Hospital ( F D H ) and Department of Medicine (Chief, A. J . Bollet), Medical College of Georgia, Augusta, Georgia, U S A
Megaloblastic anaemia responding t o folic acid has been reported in a number of patients with sickle cell anaemia. The incidence of reduced serum and erythrocyte folate levels in such patients remains unclear, however. Serum and erythrocyte folate contents were measured microbiologically in patients with sickle cell anaemia as well as in two control groups with sickle cell trait or normal haemoglobin pattern. Low serum and erythrocyte folate values were significantly more common in 61 sickle-cell patients than in each of the two socio-economically-matchedcontrol groups including 61 subjects with Hb AS and 69 blacks with H b AA. There was no correlation between serum folate and haematocrit or reticulocyte count. The erythrocyte folate, measured before and after removal of reticulocytes, remains a reliable indicator of deficiency of folic acid in sickle-cell patients despite variable degrees of reticulocytes. Folate therapy resulted in higher haematocrits in three of four sickle-cell patients with low serum and low erythrocyte folate values, but in only one of 12 patients with low serum folate and normal erythrocyte folate. These findings are consistent with other observations that folate requirement may be increased in chronic haemolytic disorders and suggest that erythrocyte folate values should be obtained in sickle-cell patients suspected of having folate deficiency. K e y words: f o l k acid - sickle cell anaemia
Accepted for publication December 6, 1974 Correspondence to: Dr. Yong K. Liu, Section of Hematology-Oncology, Department of Medicine, University of Louisville School of Medicine, Health Sciences Center, Louisville, Kentucky 40202, USA
Megaloblastic anaemia due to folic acid deficiency has been reported in patients with chronic haemolytic anaemia (Chanarin 1969). As severe folate deficiency inter-
venes in such a patient, the high reticulocyte counts decline, the anaemia becomes more severe and the patient becomes more symptomatic. In the majority of reported
This investigation was supported by Grant LH-15158 from the National Institute of Health, U S . Public Health Service and by the Veterans Administration Hospital, Augusta, Georgia (MRIA 5280).
72
YONG K. LIU
cases with haemolytic anaemia and megaloblastic haematopoiesis, other complicating factors, including poor dietary intake, pregnancy and infection, were present. However, in two well-studied patients with sickle cell anaemia it was demonstrated that larger amounts of folic acid were required to elicit an adequate reticulocyte response (Lindenbaum & Klipstein 1963, Alperin 1967). These observations suggest that the minimal daily requirement of folic acid (Herbert 1962a) may be increased in patients with chronic haemolytic anaemia, presumably due to the accelerated rate of regeneration of blood. Megaloblastic anaemia responding to folic acid has been observed in a number of patients with sickle cell anaemia (Lindenbaum & Klipstein 1963, Alperin 1967). Megaloblastic arrest was reported to be the major cause of bone marrow failure and severe anaemia in children with this haemoglobinopathy (McIver & Went 1960). Despite the grave clinical consequences of severe folic acid deficiency in chronic haemolytic states, only limited information is available on the incidence of deficiency of this vitamin in patients with sickle cell anaemia. Experimental studies of nutritional folic acid deficiency in man (Herbert 1962) and observations in patients (Hornrand et a1 1966) suggest that reduced serum folate values frequently reflect reduced dietary folate intake and that low erythrocyte folate contents usually indicate clinically significant folate deficiency. In the present report, serum and erythrocyte folate levels were determined in a large group of sickle cell patients as well as in control subjects with sickle cell trait (Hb AS) and in blacks with normal haemoglobin pattern (Hb AA). Results from these studies indicate that low serum and red cell folate values occur more
commonly in patients with sickle cell anaemia than in the control groups.
PATIENTS AND CONTROLS STUDIED 61 patients, between 1 and 56 years of age, with H b SS and two control groups including 61 subjects, between 3 and 62 years of age, with Hb AS and 69 blacks, between 2 and 67 years of age, with H b AA were studied as outpatients in the Sickle Cell Clinic. The number of subjects below 12 years of age in each group was 27, 31 and 29, respectively. The number of females in each group was 24, 28 and 31, respectively. 6 subjects with H b AS and 4 subjects with H b AA were excluded from this study because of anaemia due to gastrointestinal bleeding, iron deficiency, renal disease or chronic infection. All subjects studied were from low income families, and the majority of controls with H b AS or H b AA were immediate members of patients’ families. None of the subjects were taking medications or folic acid supplements. Detailed dietary history disclosed no clear-cut difference in dietary intake between patients and controls. Clinical and haematological evaluations were performed two to three times each prior to folic acid therapy in all sickle cell patients at intervals of 8 to 12 weeks apart, and at least twice each in 24 patients at 6 to 12 weeks after folic acid therapy. None of the patients had experienced painful crisis or infection over a period of two weeks immediately prior to the clinic visit. METHODS Routine haematological tests were performed according to standard techniques (Cartwright 1968). Diagnoses of sickle cell anaemia and sickle cell trait (AS) were established by haemoglobin electrophoresis on starch gel (Efremov et al 1969). The presence of hypersegmentation of neutrophils in the peripheral blood smear was determined by the criteria recommended by Herbert (1965). The serum and erythrocyte folate levels were measured microbiologically using Lactobacillus casei as the assaying organism (Hoffbrand et al 1966, Herbert 1966). The normal ranges for serum and erythrocyte folate contents measured
FOLATE DEFICIENCY IN SICKLE CELL ANAEMIA
73
TABLE I Haernatological data
I
I
Haemoglobin pattern
I
ss
I
AA
61
AS 61
Haematocrit ( ~ 0 1 % ) mean range
25.2 17.0-34.0
38.6 34.5-46.5
38.8 33.5-46.5
Reticulocyte (%) mean range
13.0 1.4-29.2
1.5 0.4-2.4
1.4 0.5-2.6
5.8 1.3-33.0
7.2 2.6-19.0
7.9 1.7-19.6
31 (51)
16 (26.2)
19 (27.5)
521 108-1043
405 156-699
381 85-749
8 (13)
1 (1.6)
2 (2.9)
15 (24.6)
7 (11.4)
5 (9.2)
Total number of subjects
Serum folate (nglml) mean range number (%) of subjects with values < 5 Erythrocyte folate (ng/ml) mean range number (%) of subjects with values < 160 Number (%) of subjects with hypersegmented neutrophils
in our laboratory in a group of 41 normal subjects including physicians, technicians and medical students, are 7-16 ng/ml and 160460 nglml, respectively. A serum folate less than 5 ng/ml and an erythrocyte folate less than 160 ng/ml indicate definitely reduced values, whereas serum folate levels between 5.0 and 6.9 ng/ml are of indeterminate significance. Serum vitamin BIZlevels were determined by a modified radioisotope dilution method using haemoglobin-coated charcoal (Liu & Sullivan 1972). Reticulocytes were removed from whole blood by the method of Danon Pr Marikovsky (1964), i.e., by differential centrifugation of whole blood, from patients with sickle cell anaemia, suspended in a mixture of phthalate solutions with densities of 1086 or 1090. The reticulocyte-poor sediment was then reconstituted with autologous plasma. After mizing, a haematocnt and a reticulocyte count were determined and haemolysate prepared. The folate contents of the haemolysates prepared before and after the removal of reticulocytes were assayed in the same batch.
69
RESULTS
Haematological data
Haematological data from patients with sickle cell anaemia and from controls with Hb AS or Hb AA are presented in Table I. Haematocrits between 31 and 34 vol% were observed in 8 sickle cell patients and reticulocyte counts below 5 % were present in 5. There was no difference in the average haematocrits and reticulocyte counts between the two control groups with Hb AS and H b AA. Haematocrit below 35 vol% was observed in only one control with Hb AS, who had a reticulocyte count of 1.8 %, a reduced serum folate value but a normal erythrocyte folate content. 5 blacks with H b AA had haematocrits between 33 and 34.5 ~ 0 1 % .2 of these 5 subjects had re-
74
YONG K.LIU TABLE I1 Relation of serum folate values with haernatocrit, reticulocyte count and erythrocyte folate in patients with sickle cell anaemia
I Number of subjects
Serum folate activity
31
30
Haematocrit ( ~ 0 1 % )
mean range
25.7 17-34
25.8 18-34
Reticulocytes (%)
mean range
11.3 1.4-26.9
14.1 1.7-29.2
Erythrocyte folate (ng/ml)
mean range
366 108-927
652 203-1043
duced serum folate values, hypersegmented neutrophils, and normal erythrocyte folate contents. The other 3 subjects had normal serum folate values. Hypersegmented neutrophils were observed significantly more often in patients with sickle cell anaemia than in control subjects with Hb AS or Hb AA (P < 0.01). The serum vitamin Biz values were normal in all patients and controls. Serum and erythrocyte folate levels
The mean serum folate level of the 61 patients with sickle cell anaemia was 5.8 ng/ ml (Table I). This was significantly lower than the mean values of 7.2 ng/ml and 7.9 ng/ml observed for the control subjects with Hb.s AS and AA, respectively (P < 0.005). Serum folate contents below the normal level (< 7 ng/ml) were found in 48 (78.7 %) sickle-cell patients, in 35 (57.3 %) controls with H b AS and in 40 (58 %) subjects with Hb AA. The incidence of definitely decreased serum folate values (< 5 ng/ml), observed in 51 % of patients with sickle cell anaemia, 26.2 % of controls with Hb AS and 27.5 % of subjects with
normal Hb pattern, was significantly higher in patients with sickle cell anaemia than in control subjects with Hb.s AS or AA (P < 0.01). There was no difference in the incidence of low serum folate levels between the control groups. The mean erythrocyte folate of the 61 sickle-cell patients was 521 ng/ml which was significantly higher (P < 0.005) than the values of the control groups. There was no significant difference in the average erythrocyte folate values between the two control groups. Low red cell folate levels (< 160 ng/ml) were observed in 8 sickle-cell patients, in only one control subject with H b AS and in two with Hb AA. An erythrocyte folate level greater than 660 ng/ml was observed in 19 patients with H b SS, in one subject with Hb AS and in two subjects with Hb AA. Relation of serum folate with haematocrit, reticulocyte count and red cell folate in patients with sickle cell anaemia
There was no difference in the mean haematocrits or mean reticulocyte counts between patients with low serum folate values and others who had serum folate activities of
FOLATE DEFICIENCY IN SICKLE CELL ANAEMIA
5 ng/ml or greater (Table 11). Furthermore, no correlation was observed between serum folate activity and haematocrit or reticulocyte count. The mean erythrocyte folate of the 30 patients with serum folate values of 5 ng/ml or greater was significantly higher than the mean value observed for the other 31 patients who had reduced serum folate contents (P < 0.01). There was no significant correlation between serum and red cell folate contents of the 61 patients with sickle cell anaemia (Figure 1). Influence o f reticulocyte count on erythrocyte folate content The red cell folate levels of the last 21 patients with sickle cell anaemia were determined both before and after the removal of the majority of reticulocytes from the
I
(33)
147
12
L
75
whole blood. The mean erythrocyte folate was 582 ng/ml and the mean reticulocyte count was 14.9 % in 12 patients with serum folate levels greater than 5 ng/ml (Table I11 - A). After the removal of reticulocytes, the mean red cell folate and reticulocyte count in these patients were 396 ng/ml and 4.1 %, respectively. A decrease in erythrocyte folate level greater than 20 % following the removal of reticulocytes was observed in 9 of these 12 patients. The mean erythrocyte folate was 233 ng/ml and the mean reticulocyte count was 13.6 % in another 9 patients whose serum folate levels were less than 5 ng/ml (Table I11 - B). After the removal of reticulocytes, the mean erythrocyte folate and mean reticulocyte count were 215 ng/ml and 4.8 %, respectively. Despite marked reduction in the number of reticulocytes, there was no significant decrease in the mean erythrocyte folate content. Furthermore a reduction in red cell folate greater than 20 % following removal of reticulocytes was only observed in two of these 9 patients. Of these 21 patients, a decrease of erythrocyte folate from a normal level of 252 ng/ml to a diagnostically low value of 145 ng/ml was only observed in one whose serum folate was 2.3 ng/ml and baseline reticulocyte count was 15.4 %.
Haematological data in sickle-cell patients with low erythrocyte folate
2
I
I
I
I
200
400
600
800
I
1,000
Erythrocyte Folate (ng/ml)
Figure 1. Lack of significant correlation between serum and erythrocyte folate levels in 61 patients with sickle cell anaemia.
Only 8 patients had significantly reduced red cell folate and two of them were children (Table IV). All 8 had low serum folate levels and hypersegmentation of neutrophils. The haematwrits averaged 25.4 % which was. similar to the mean value observed for all sickle-cell patients, but the average reticulocyte count of these 8 patients was significantly lower (P < 0.31). The bone
76
YONG K. LIU TABLE 111 The erythrocyte folate content before and after the removal of reticulocytes in sickle-cell patients with (A) serum folate greater than 5 ng/ml and (B) serum folate less than 5 ng/ml
(vO1%)
Serum folate (ng/ml)
1 2 3 4 5 6 7 8 9 10 11 12
23 20 24 23 20 27 28 20 30 24 26 29
5.7 6.6 33.0 6.3 6.6 8.9 7.7 5.3 7.1 5.1 9.0 5.7
Mean Range
24.5 20-30
13 14 15 16 17 18 19 20 21
Haematocrit
Patient
(A)
(B)
Mean Range
Erythrocyte folate (WmU
Reticulocyte (%)
I
I
After
Before
15.5 19.5 11.2 8.9 15.6 12.6 16.2 18.4 16.3 7.3 29.6 7.5
4.0 5 .O 2.6 4.3 4.1 3.8 6.2 0.4 3.7 3.5 7.2 4.3
575 774 940 872 794 752 498 449 411 374 319 225
431 470 513 610 612 356 416 362 208 214 290 214
8.9 5.1-33
14.9 7.3-29.6
4.1 0.4-7.2
582 225-940
396 214612
27 24 28 21 25.5 25 26 24 26
4.3 4.4 3.6 2.8 2.3 4.7 3.8 4.7 2.3
14.9 13.8 25.8 4.1 15.4 23.9 8.5 6.9 9.1
5.8 4.2 6.2 1.2 4.4 9.9 4.0 3.7 3.9
225 268 335 108 252 25 1 260 250 150
263 256 304 122 145 256 263 204 119
25.1 20-29
3.7 2.3-4.3
13.6 4.5-25.8
4.8 1.2-9.9
233 108-335
215 122-304
marrow, examined in two patients, revealed moderate megaloblastic erythropoiesis. There was no distinct difference in clinical findings, including dietary intake and frequency of painful crises and infection, in these 8 patients as compared with the whole group. Efect of folk acid therapy
24 patients with sickle cell anaemia have thus far been treated as outpatients with folic acid (1 mg/day) continuously for a period of 3-12 months. There was no haematological response in 8 patients whose serum folate ranged between 5.1 ng/ml and 33 ng/ml and red cell folate ranged between
Before
After
311 ng/ml and 1043 ng/ml. In the other 16 patients the serum folate levels were less than 5 ng/ml (range 1.3-4.9 ng/ml) and the red cell folate ranged between 108 ng/ml and 584 ng/ml, with low values (
Folic Acid Deficiency in Sickle Cell Anaemia YONGK. LIU,M.D. Hematologic Research, Medical Service (Chief, L. G . Horan), Veterans Administration Hospital ( F D H ) and Department of Medicine (Chief, A. J . Bollet), Medical College of Georgia, Augusta, Georgia, U S A
Megaloblastic anaemia responding t o folic acid has been reported in a number of patients with sickle cell anaemia. The incidence of reduced serum and erythrocyte folate levels in such patients remains unclear, however. Serum and erythrocyte folate contents were measured microbiologically in patients with sickle cell anaemia as well as in two control groups with sickle cell trait or normal haemoglobin pattern. Low serum and erythrocyte folate values were significantly more common in 61 sickle-cell patients than in each of the two socio-economically-matchedcontrol groups including 61 subjects with Hb AS and 69 blacks with H b AA. There was no correlation between serum folate and haematocrit or reticulocyte count. The erythrocyte folate, measured before and after removal of reticulocytes, remains a reliable indicator of deficiency of folic acid in sickle-cell patients despite variable degrees of reticulocytes. Folate therapy resulted in higher haematocrits in three of four sickle-cell patients with low serum and low erythrocyte folate values, but in only one of 12 patients with low serum folate and normal erythrocyte folate. These findings are consistent with other observations that folate requirement may be increased in chronic haemolytic disorders and suggest that erythrocyte folate values should be obtained in sickle-cell patients suspected of having folate deficiency. K e y words: f o l k acid - sickle cell anaemia
Accepted for publication December 6, 1974 Correspondence to: Dr. Yong K. Liu, Section of Hematology-Oncology, Department of Medicine, University of Louisville School of Medicine, Health Sciences Center, Louisville, Kentucky 40202, USA
Megaloblastic anaemia due to folic acid deficiency has been reported in patients with chronic haemolytic anaemia (Chanarin 1969). As severe folate deficiency inter-
venes in such a patient, the high reticulocyte counts decline, the anaemia becomes more severe and the patient becomes more symptomatic. In the majority of reported
This investigation was supported by Grant LH-15158 from the National Institute of Health, U S . Public Health Service and by the Veterans Administration Hospital, Augusta, Georgia (MRIA 5280).
72
YONG K. LIU
cases with haemolytic anaemia and megaloblastic haematopoiesis, other complicating factors, including poor dietary intake, pregnancy and infection, were present. However, in two well-studied patients with sickle cell anaemia it was demonstrated that larger amounts of folic acid were required to elicit an adequate reticulocyte response (Lindenbaum & Klipstein 1963, Alperin 1967). These observations suggest that the minimal daily requirement of folic acid (Herbert 1962a) may be increased in patients with chronic haemolytic anaemia, presumably due to the accelerated rate of regeneration of blood. Megaloblastic anaemia responding to folic acid has been observed in a number of patients with sickle cell anaemia (Lindenbaum & Klipstein 1963, Alperin 1967). Megaloblastic arrest was reported to be the major cause of bone marrow failure and severe anaemia in children with this haemoglobinopathy (McIver & Went 1960). Despite the grave clinical consequences of severe folic acid deficiency in chronic haemolytic states, only limited information is available on the incidence of deficiency of this vitamin in patients with sickle cell anaemia. Experimental studies of nutritional folic acid deficiency in man (Herbert 1962) and observations in patients (Hornrand et a1 1966) suggest that reduced serum folate values frequently reflect reduced dietary folate intake and that low erythrocyte folate contents usually indicate clinically significant folate deficiency. In the present report, serum and erythrocyte folate levels were determined in a large group of sickle cell patients as well as in control subjects with sickle cell trait (Hb AS) and in blacks with normal haemoglobin pattern (Hb AA). Results from these studies indicate that low serum and red cell folate values occur more
commonly in patients with sickle cell anaemia than in the control groups.
PATIENTS AND CONTROLS STUDIED 61 patients, between 1 and 56 years of age, with H b SS and two control groups including 61 subjects, between 3 and 62 years of age, with Hb AS and 69 blacks, between 2 and 67 years of age, with H b AA were studied as outpatients in the Sickle Cell Clinic. The number of subjects below 12 years of age in each group was 27, 31 and 29, respectively. The number of females in each group was 24, 28 and 31, respectively. 6 subjects with H b AS and 4 subjects with H b AA were excluded from this study because of anaemia due to gastrointestinal bleeding, iron deficiency, renal disease or chronic infection. All subjects studied were from low income families, and the majority of controls with H b AS or H b AA were immediate members of patients’ families. None of the subjects were taking medications or folic acid supplements. Detailed dietary history disclosed no clear-cut difference in dietary intake between patients and controls. Clinical and haematological evaluations were performed two to three times each prior to folic acid therapy in all sickle cell patients at intervals of 8 to 12 weeks apart, and at least twice each in 24 patients at 6 to 12 weeks after folic acid therapy. None of the patients had experienced painful crisis or infection over a period of two weeks immediately prior to the clinic visit. METHODS Routine haematological tests were performed according to standard techniques (Cartwright 1968). Diagnoses of sickle cell anaemia and sickle cell trait (AS) were established by haemoglobin electrophoresis on starch gel (Efremov et al 1969). The presence of hypersegmentation of neutrophils in the peripheral blood smear was determined by the criteria recommended by Herbert (1965). The serum and erythrocyte folate levels were measured microbiologically using Lactobacillus casei as the assaying organism (Hoffbrand et al 1966, Herbert 1966). The normal ranges for serum and erythrocyte folate contents measured
FOLATE DEFICIENCY IN SICKLE CELL ANAEMIA
73
TABLE I Haernatological data
I
I
Haemoglobin pattern
I
ss
I
AA
61
AS 61
Haematocrit ( ~ 0 1 % ) mean range
25.2 17.0-34.0
38.6 34.5-46.5
38.8 33.5-46.5
Reticulocyte (%) mean range
13.0 1.4-29.2
1.5 0.4-2.4
1.4 0.5-2.6
5.8 1.3-33.0
7.2 2.6-19.0
7.9 1.7-19.6
31 (51)
16 (26.2)
19 (27.5)
521 108-1043
405 156-699
381 85-749
8 (13)
1 (1.6)
2 (2.9)
15 (24.6)
7 (11.4)
5 (9.2)
Total number of subjects
Serum folate (nglml) mean range number (%) of subjects with values < 5 Erythrocyte folate (ng/ml) mean range number (%) of subjects with values < 160 Number (%) of subjects with hypersegmented neutrophils
in our laboratory in a group of 41 normal subjects including physicians, technicians and medical students, are 7-16 ng/ml and 160460 nglml, respectively. A serum folate less than 5 ng/ml and an erythrocyte folate less than 160 ng/ml indicate definitely reduced values, whereas serum folate levels between 5.0 and 6.9 ng/ml are of indeterminate significance. Serum vitamin BIZlevels were determined by a modified radioisotope dilution method using haemoglobin-coated charcoal (Liu & Sullivan 1972). Reticulocytes were removed from whole blood by the method of Danon Pr Marikovsky (1964), i.e., by differential centrifugation of whole blood, from patients with sickle cell anaemia, suspended in a mixture of phthalate solutions with densities of 1086 or 1090. The reticulocyte-poor sediment was then reconstituted with autologous plasma. After mizing, a haematocnt and a reticulocyte count were determined and haemolysate prepared. The folate contents of the haemolysates prepared before and after the removal of reticulocytes were assayed in the same batch.
69
RESULTS
Haematological data
Haematological data from patients with sickle cell anaemia and from controls with Hb AS or Hb AA are presented in Table I. Haematocrits between 31 and 34 vol% were observed in 8 sickle cell patients and reticulocyte counts below 5 % were present in 5. There was no difference in the average haematocrits and reticulocyte counts between the two control groups with Hb AS and H b AA. Haematocrit below 35 vol% was observed in only one control with Hb AS, who had a reticulocyte count of 1.8 %, a reduced serum folate value but a normal erythrocyte folate content. 5 blacks with H b AA had haematocrits between 33 and 34.5 ~ 0 1 % .2 of these 5 subjects had re-
74
YONG K.LIU TABLE I1 Relation of serum folate values with haernatocrit, reticulocyte count and erythrocyte folate in patients with sickle cell anaemia
I Number of subjects
Serum folate activity
31
30
Haematocrit ( ~ 0 1 % )
mean range
25.7 17-34
25.8 18-34
Reticulocytes (%)
mean range
11.3 1.4-26.9
14.1 1.7-29.2
Erythrocyte folate (ng/ml)
mean range
366 108-927
652 203-1043
duced serum folate values, hypersegmented neutrophils, and normal erythrocyte folate contents. The other 3 subjects had normal serum folate values. Hypersegmented neutrophils were observed significantly more often in patients with sickle cell anaemia than in control subjects with Hb AS or Hb AA (P < 0.01). The serum vitamin Biz values were normal in all patients and controls. Serum and erythrocyte folate levels
The mean serum folate level of the 61 patients with sickle cell anaemia was 5.8 ng/ ml (Table I). This was significantly lower than the mean values of 7.2 ng/ml and 7.9 ng/ml observed for the control subjects with Hb.s AS and AA, respectively (P < 0.005). Serum folate contents below the normal level (< 7 ng/ml) were found in 48 (78.7 %) sickle-cell patients, in 35 (57.3 %) controls with H b AS and in 40 (58 %) subjects with Hb AA. The incidence of definitely decreased serum folate values (< 5 ng/ml), observed in 51 % of patients with sickle cell anaemia, 26.2 % of controls with Hb AS and 27.5 % of subjects with
normal Hb pattern, was significantly higher in patients with sickle cell anaemia than in control subjects with Hb.s AS or AA (P < 0.01). There was no difference in the incidence of low serum folate levels between the control groups. The mean erythrocyte folate of the 61 sickle-cell patients was 521 ng/ml which was significantly higher (P < 0.005) than the values of the control groups. There was no significant difference in the average erythrocyte folate values between the two control groups. Low red cell folate levels (< 160 ng/ml) were observed in 8 sickle-cell patients, in only one control subject with H b AS and in two with Hb AA. An erythrocyte folate level greater than 660 ng/ml was observed in 19 patients with H b SS, in one subject with Hb AS and in two subjects with Hb AA. Relation of serum folate with haematocrit, reticulocyte count and red cell folate in patients with sickle cell anaemia
There was no difference in the mean haematocrits or mean reticulocyte counts between patients with low serum folate values and others who had serum folate activities of
FOLATE DEFICIENCY IN SICKLE CELL ANAEMIA
5 ng/ml or greater (Table 11). Furthermore, no correlation was observed between serum folate activity and haematocrit or reticulocyte count. The mean erythrocyte folate of the 30 patients with serum folate values of 5 ng/ml or greater was significantly higher than the mean value observed for the other 31 patients who had reduced serum folate contents (P < 0.01). There was no significant correlation between serum and red cell folate contents of the 61 patients with sickle cell anaemia (Figure 1). Influence o f reticulocyte count on erythrocyte folate content The red cell folate levels of the last 21 patients with sickle cell anaemia were determined both before and after the removal of the majority of reticulocytes from the
I
(33)
147
12
L
75
whole blood. The mean erythrocyte folate was 582 ng/ml and the mean reticulocyte count was 14.9 % in 12 patients with serum folate levels greater than 5 ng/ml (Table I11 - A). After the removal of reticulocytes, the mean red cell folate and reticulocyte count in these patients were 396 ng/ml and 4.1 %, respectively. A decrease in erythrocyte folate level greater than 20 % following the removal of reticulocytes was observed in 9 of these 12 patients. The mean erythrocyte folate was 233 ng/ml and the mean reticulocyte count was 13.6 % in another 9 patients whose serum folate levels were less than 5 ng/ml (Table I11 - B). After the removal of reticulocytes, the mean erythrocyte folate and mean reticulocyte count were 215 ng/ml and 4.8 %, respectively. Despite marked reduction in the number of reticulocytes, there was no significant decrease in the mean erythrocyte folate content. Furthermore a reduction in red cell folate greater than 20 % following removal of reticulocytes was only observed in two of these 9 patients. Of these 21 patients, a decrease of erythrocyte folate from a normal level of 252 ng/ml to a diagnostically low value of 145 ng/ml was only observed in one whose serum folate was 2.3 ng/ml and baseline reticulocyte count was 15.4 %.
Haematological data in sickle-cell patients with low erythrocyte folate
2
I
I
I
I
200
400
600
800
I
1,000
Erythrocyte Folate (ng/ml)
Figure 1. Lack of significant correlation between serum and erythrocyte folate levels in 61 patients with sickle cell anaemia.
Only 8 patients had significantly reduced red cell folate and two of them were children (Table IV). All 8 had low serum folate levels and hypersegmentation of neutrophils. The haematwrits averaged 25.4 % which was. similar to the mean value observed for all sickle-cell patients, but the average reticulocyte count of these 8 patients was significantly lower (P < 0.31). The bone
76
YONG K. LIU TABLE 111 The erythrocyte folate content before and after the removal of reticulocytes in sickle-cell patients with (A) serum folate greater than 5 ng/ml and (B) serum folate less than 5 ng/ml
(vO1%)
Serum folate (ng/ml)
1 2 3 4 5 6 7 8 9 10 11 12
23 20 24 23 20 27 28 20 30 24 26 29
5.7 6.6 33.0 6.3 6.6 8.9 7.7 5.3 7.1 5.1 9.0 5.7
Mean Range
24.5 20-30
13 14 15 16 17 18 19 20 21
Haematocrit
Patient
(A)
(B)
Mean Range
Erythrocyte folate (WmU
Reticulocyte (%)
I
I
After
Before
15.5 19.5 11.2 8.9 15.6 12.6 16.2 18.4 16.3 7.3 29.6 7.5
4.0 5 .O 2.6 4.3 4.1 3.8 6.2 0.4 3.7 3.5 7.2 4.3
575 774 940 872 794 752 498 449 411 374 319 225
431 470 513 610 612 356 416 362 208 214 290 214
8.9 5.1-33
14.9 7.3-29.6
4.1 0.4-7.2
582 225-940
396 214612
27 24 28 21 25.5 25 26 24 26
4.3 4.4 3.6 2.8 2.3 4.7 3.8 4.7 2.3
14.9 13.8 25.8 4.1 15.4 23.9 8.5 6.9 9.1
5.8 4.2 6.2 1.2 4.4 9.9 4.0 3.7 3.9
225 268 335 108 252 25 1 260 250 150
263 256 304 122 145 256 263 204 119
25.1 20-29
3.7 2.3-4.3
13.6 4.5-25.8
4.8 1.2-9.9
233 108-335
215 122-304
marrow, examined in two patients, revealed moderate megaloblastic erythropoiesis. There was no distinct difference in clinical findings, including dietary intake and frequency of painful crises and infection, in these 8 patients as compared with the whole group. Efect of folk acid therapy
24 patients with sickle cell anaemia have thus far been treated as outpatients with folic acid (1 mg/day) continuously for a period of 3-12 months. There was no haematological response in 8 patients whose serum folate ranged between 5.1 ng/ml and 33 ng/ml and red cell folate ranged between
Before
After
311 ng/ml and 1043 ng/ml. In the other 16 patients the serum folate levels were less than 5 ng/ml (range 1.3-4.9 ng/ml) and the red cell folate ranged between 108 ng/ml and 584 ng/ml, with low values (
