Scand J Haematol(l983) 30, 125-129

Folic Acid Deficiency in (3-Thalassaemia Heterozygotes

F. TROITA,4G. MENEGALE,4A. R. CAVALLINI’ & A. P1FFANELL13
G . CASTALD1,l B. BAGNI,*
Istituto di Semeiotica Medica, Istituto di Fisica, Istituto di Radiologia, Universita di Ferrara
and
Divisione di Reumatologia and Servizio di Fisica Sanitaria, Arcispedale
Sant’Anna di Ferrara, Italy

Plasma and red cell folate mean contents have been found to be significantly lower in 41
symptom-free P-thalassaemia heterozygotes than in 21 controls. Such decreases must be
considered as effects of an increased folate utilization caused by the enhanced total, both
effective and ineffective, erythropoiesis.
Since no close correlation has been found between packed cell volume and plasma or red
blood cell folate levels, it seems that in ‘healthy’ P-thalassaemia subjects, the degree of
anaemia is not influenced by folate body reserves. However, the frequent finding of reduced
red cell folate contents suggests that further folate imbalance might lead to clinically
significant degrees of folate deficiency.

Key words: anaemia - folk acid deficiency - thalassaemia

Accepted for publication July 20, 1982

Correspondence to: Dr. Giuseppe Castaldi, Istituto di Semeiotica Medica, Universita di Ferrara.
Via Savonarola 9 , 44100 Ferrara, Italy

Anaemia of varying degree is a quite com-
mon finding in heterozygous P-thalassaemia
(Knox-MacAuley et a1 1973, Pootrakul et al
1973, Castaldi et a1 1974, Mazza et al 1976,
Galanello et a1 1979). Quantitative mea-
surements of the erythropoietic activity in
carriers have demonstrated a marked in-
crease of total, effective and ineffective ery-
thropoiesis and a variably shortened red
cell lifespan (Rotoli 1976, Cazzola et al
1979). Their extent seems to indicate that
both intramedullary and peripheral haemo-
lysis play a role in the production of the
anaemia. Since the requirement for folic
acid rises sharply in haemolytic conditions
associated with chronic hyperactivity of the
bone marrow (Chanarin et a1 1959, Jandl &
Greenberg 1959, Delamore et al 1961, Lin-
denbaum & Klipstein 1963, Pearson &
Cobb 1964, Komminos et a1 1965, Pavlic &
Bouroncle 1965), fl-thalassaemia hetero-
zygotes should demonstrate some degree of
folate-deficiency .
In the present report, plasma and ery-
throcyte folate levels were determined in a
group of P-thalassaemia heterozygotes and
in a group of healthy subjects with normal
haemoglobin pattern. Results from these
studies indicate that low plasma and red
cell folate values occur more commonly in
126 CASTALDI, BAGNI, TROTTA, MENEGALE et al

the (3-thalassaemia group than in the con-

RESULTS
trol group.
Table 1 shows the mean value and ranges of
plasma and red-cell folate which were found
in the P-thalassaemia heterozygotes and in
MATERIAL AND METHODS
normal controls. In the normal controls, the
We studied 21 normal subjects recruited from healthy mean plasma folate was 8.1 nmol/l (extreme
laboratory personnel and medical technology students
(average age 26 years, range 18 to 46 years, 10 male and
values: 2.5-15.7) and the mean red-cell
11 female) and 41 symptom-free P-thalassaemia hetero- folate was 1429 nmol/l (extreme values:
zygotes (average age 29 years, range 16 to 59 years, 21 354-3076). In the (3-thalassaemia hetero-
male and 20 female). zygotes, the mean plasma folate was 5.0
The diagnosis of heterozygous P-thalassaemia was nmol/l (extreme values: 0.2-9.3) and the
established on the basis of the following criteria (Sil-
vestroni 8~ Bianco 1975): (a) abnormal red cell mor-
mean red cell folate 808 nmol/l (extreme
phology (poikilocytosis, microcytosis, target cells, baso- values: 70-1946). In the latter both plasma
philic stippling); (b) decreased osmotic red cell fragility; and red cell folate levels were significantly
(c) increased Hb A2 level (>3.5%). Subjects with lower than the corresponding mean values
normal red cell morphology and red cell fragility and
in the normal subjects (P < 0.01). The
with Hb A2 level below 3 % were considered normal
controls.
plasma and red cell folate values were
None of the subjects were taking medications or folic under the lower limits of our normal ranges
acid supplements. in 18 and in 16 of the (3-thalassaemia hetero-
The plasma and red cell folate levels were measured zygotes, respectively.
by radiometric assay using the method of Rothenberg No correlation was found between PCV,
et al (1972) as modified by Mincey et al (1973) and
subsequently by Bagni et a1 (1975). Following a 12 h
utilized as a criterion of anaemia (Com-
overnight fast, the blood specimens were drawn, be- mittee on Iron Deficiency 1968), and either
tween 8 and 9 a.m., by venipuncture without tour- plasma or red blood cell folate levels in
niquet, from subjects who had been sitting for 5 min. (3-thalassaemia heterozygotes (r = 0.27 and
Triplicate measurement was carried out for both plasma
0.13, respectively).
and red cell folate levels in all subjects. Since the
triplicate values were not significantly different, the
mean values from the triplicate measurements were
used for subsequent elaboration. Internal quality con-
trol parameters were: intra-assay coefficient of varia- DISCUSSION
tion (CV) 10 %, and between-assay CV 8 %. Red cell Our study evidenced a significant decrease
folate levels were calculated according to the following
formula:
of both mean plasma and red cell folate
levels in (3-thalassaemia carriers, compared
WBF - P F (1 - PCV X 0.861100) to healthy controls.
RCF =
PCV x 0.86/100
In (3-thalassaemia heterozygotes, de-
where RCF = red-cell folate, WBF = whole blood
creased serum folate contents have been
folate, PF = plasma folate, PCV = packed cell volume,
0.86 = correction coefficient for trapped plasma frac- previously reported (Gyftaki et a1 1973,
tion. Bernasconi et a1 1974). Nevertheless no
Routine haematological tests were performed by conclusive evidence for the presence of a
standard techniques (Cartwright !968). folate deficiency has been provided by
Significance of differences between the means were
these studies, since serum or plasma folate
determined by the Student t-test.
concentrations change rapidly following in-
 FOLIC ACID IN fi-THALASSAEMIA TRAIT 127

TABLE 1
Plasma and red cell folate levels in normal and (3-thalassaemic subjects

Plasma folate (nmol/l) Red Cell folate (nmolll)

Normal &thal Normal fl-thal

Cases ( n ) 21 41 21 41

MeantSD 8.1 t 3 . 4 5.0 2 3.0 1 4 2 9 T 1 0 31 808: 57 8

(pc0.01) (pc0.01)

E x t r e m e values 2.5-1 5 . 7 0.2-9.3 354-3076 70-19 46

gestion or cessation of 'folic acid intake.
Therefore, even if reduced serum or plasma
folic acid contents may be common in 0-
thalassaemia heterozygotes, quantification
of red blood cell folate is required to con-
firm true tissue folate deficiency.
The significance decrease of the mean
red cell folate levels observed in our 0-
thalassaemia carriers seems to better dem-
onstrate the presence of a folate deficiency,
since folate gains entry into the red cell only
at the stage of the marrow erythroblast, but
neither enters nor leaves mature red cells in
the circulation (Herbert & Zalusky 1962).
The lower red cell folate levels in b-
thalassaemia heterozygotes can be brought
about by a deficient folate intake or by an
increased folate utilization. Since the socio-
economic status of our P-thalassaemic sub-
jects was similar to that of the controls, the
latter hypothesis is likely to be true. As a
matter of fact, the anaemia of P-thalassae-
mia trait is characterized by 2 main patho-
genetic mechanisms (Cazzola et a1 1979),
brought about by the well-known imbalance
in synthesis of CI and non-a chains (Vigi et a1
1969): (1) increased erythropoietic activity
with variable degrees of ineffective erythro-
poiesis; (2) variably shortened red cell life-
span. These are the very same mechanisms
which can produce folic acid deficiency in
some forms of chronic haemolytic anaemia
associated with persistent hyperactivity of
the bone marrow, such as, e.g., thalassae-
mia major (Jandl & Greenberg 1959, Robin-
son & Watson 1963) and sickle cell anaemia
(Pearson & Cobb 1964, Liu 1975).
T o the above mentioned possible mech-
anisms producing anaemia in fi-thalassaemia
heterozygotes, a relative deficiency of folate
might be added, but no close correlation
was found between PCV and either plasma
or red blood cell folate level in our P-thalas-
saemic subjects. Thus, the present study
shows that, despite many adult P-thalassae-
mic subjects of both sexes being folate-defi-
cient or on the verge of folate deficiency, in
P-thalassaemia erythroid steady state, the
degree of anaemia is not influenced by
folate body reserves. However, the fre-
quently reduced erythrocyte folate contents
suggest that a further imbalance in folate
128 CASTALDI, BAGNI, TROTTA, MENEGALE et a1
intake, absorption or metabolism may clin-
ically produce significant degrees of folate
deficiency in P-thalassaemia trait.
Finally, it is well-known that in hetero-
zygous P-thalassaemia, patterns of severe
anaemia due to folate deficiency may be
observed, especially when folate require-
ments are increased, as in pregnancy
(Meital et a1 1961) and occasionally in some
adults (Arlotti et a1 1967, Malacarne et a1
1974). Thus, Weatherall (1965) suggests
giving thalassaemic subjects folic acid sup-
plements in order to prevent heavy anae-
mia, and moreover Vatanavicharn et a1
(1979) prescribe routinely folate, 5 mg/d, to
thalassaemic patients (with P-thalassaemia/
H b E disease or with Hb H disease), espe-
cially to those who are severely anaemic.
Since there are several reports in the liter-
ature describing megaloblastic anaemia due
to iatrogenic folate deficiency, we now pre-
scribe with caution and under frequent
haematological controls, drugs interacting
with folate absorption or metabolism in fi-
thalassaemia heterozygotes.
REFERENCES
Arlotti 0, Soffritti E & Catalfamo G (1967) La pato-
logia d’organo del talassemico eterozigote. Riv Patol
Clin Sper 8,585-613.
Bagni B, Malavasi F, Codech L & Cavallini A R
(1975) Folate and vitamin B12 radiometric assay. Ric
Clin Lab 5,363-69.
Bernasconi C, Pontiggia P & Lazzarino M (1974) La
carenza di acido folico, latente o conclamata, nelle
emoglobinopatie. Aspetti diagnostici e terapeutici.
Min Med 65, 3207-14.
Cartwright G E (1968) Diagnostic Laboratory Hema-
tology, 4th ed. Grune & Stratton, New York and
London.
Castaldi G, Zavagli G , Ambroso G, Dallapiccola B &
Trotta F (1974) Anaemia in beta-thalassaemic car-
riers. Brit Med J 1, 518.
Cazzola M, Alessandrino P, Barosi G , Morandi S &
Stefanelli M (1979) Quantitative evaluation of the
mechanisms of anaemia in heterozygous p-thalassae-
mia. Scand J Haematol23, 107-14.
Chanarin I, Dacie J V & Mollin D L (1959) Folic-acid
deficiency in haemolytic anaemia. Br J Haematol 5 ,
245-56.
Committee on Iron-Deficiency (1968) Iron deficiency in
the United States. JAMA 203,407-12.
Delamore I W, Richmond J & Davies S H (1961)
Megaloblastic anaemia in congenital spherocytosis.
Br Med J 1,543-45.
Galanello R, Melis M A, Ruggeri R, Addis M, Scalas
M T, Maccioni L, Furbetta M, Angius A, Tuveri T &
Cao A (1979) v-thalassaemia trait in Sardinia.
Hemoglobin 3 , 3346.
Gyftaki H, Loukopoulos D, Kesse-Ehas M & Alevizou-
Terzaki V (1973) Estimation of serum folic acid with
competitive protein-binding method in beta thalas-
saemia. In Proc Intern Atom En A g , Istambul, 10-14
September 1973, vol 11, pp 201-04, IAEA, Vienna,
1974.
Herbert V & Zalusky R (1962) Interrelations of vitamin
Bl2 and folic acid metabolism: folic acid clearance
studies. J Clin Invest 41, 1263-76.
Jandl J H & Greenberg M S (1959) Bone marrow
failure due to relative nutritional deficiency in
Cooley’s hemolytic anemia. N Engl J Med 260, 461-
68.
Knox-MacAulay H H M, Weatherall D J, Clegg J B &
Pembrey M E (1973) Thalassaemia in the British. Br
Med J iii, 15C-55.
Komminos Z D, Minogue W F & Saravajic J (1965)
Folic acid deficiency in hereditary spherocytosis:
failure of response to massive parenteral doses of
cyanocobalamin. Arch Intern Med US,663-66.
Lindenbaum J & Klipstein F A (1963) Folic acid de-
ficiency in sickle cell anemia. N Engl J Med 269.
875-82.
Liu Y K (1975) Folic acid deficiency in sickle cell
anaemia. Scand J Haematoll4, 71-79.
Malacarne P, Zavagli G , Castaldi G & Piffanelli A
(1974) 2,4-diaminopyrimidines and heterozygous beta-
thalassaemia. Lancet ii, 904.
Mazza U, Saglio G , Caligaris Cappio F, Camaschella
C, Neretto G & Gallo E (1976) Clinical and haema-
tological data in 254 cases of beta-thalassaemia trait
in Italy. Br J Haematol33, 91-99.
Meital V, Izak G & Rachmilewitz M (1961) L’effet de
la grossesse sur la thalasskmie. Nouv Rev Fr Hematol
1, 389409.
 FOLIC ACID IN P-THALASSAEMIA TRAIT
Mincey E K, Wilcox E & Morrison R T (1973) Estima-
tion of serum and red cell folate by a simple radio-
metric technique. Clin Biochem 6 , 274-84.
Pavlic G J & Bouroncle B A (1965) Megaloblastic crisis
in paroxysmal nocturnal hemoglobinuria. N Engl J
Med 273,789-92.
Pearson H A & Cobb W T (1964) Folic acid studies in
sickle-cell anemia. J Lab Clin Med 64, 913-21.
Pootrakul P, Wasi P & Na-Nakorn S (1973) Haema-
tological data in 312 cases of beta-thalassaemia trait
in Thailand. Br J Haematol24, 703-12.
Robinson M G & Watson R J (1963) Megaloblastic
anemia complicating thalassemia major. Am J Dis
Child 105, 275-79.
Rothenberg S P, da Costa M & Rosenberg Z (1972) A
radioassay for serum folate: Use of two-phase se-
quential-incubation, ligand-binding system. N Engl J
Med 286, 1335-39.
Scand J Haematol(1983) 30
129
Rotoli B (1976) Thalassaemia in Italy: treatment of
Cooley disease and iron kinetics in heterozygotes.
Birth Defects l2,5561.
Silvestroni E & Bianco I (1975) Screening for micro-
cytemia in Italy: analysis of data collected in the past
30 years. Am J Hum Genet 27, 198-212.
Vatanavicharn S, Anuvatanakulchai M, Na-Nakorn S
& Wasi P (1979) Serum erythrocyte folate levels in
thalassaemic patients in Thailand. Scand J Haemarol
22,241-45.
Vigi V, Volpato S, Gaburro D, Conconi F, Bargellesi
A & Pontremoli S (1969) The correlation between
red cell survival and excess of a-globin synthesis in
fi-thalassaemia. Br J Haematof 16,25-30.
Weatherall D J (1965) The Thalassaemia Syndromes, p
215. Blackwell Sci Publ, Oxford.
9