21

Nagoya J. med. Sci. 39: 21-31,1976

BONE MARROW o-AMINOLEVULINIC ACID SYNTHETASEACTIVITY IN HEMATOLOGICAL DISORDERS
MASAO TANAKA

1st Department of Internal Medicine. Nagoya University School of Medicine(Director:[tsuro Sobue .}ABSTRACTAn improved radiochemical method to measure the o-aminoJevulinic acid (ALA) synthetaseactivity in human bone marrow erythroblasts by incubating bone marrow cell homogenate with
14C-succinate and succinyl CoA generating system has been reported. Studies were made to obtain the optimal conditions for the enzyme assay and to confirm the reliability of the assay procedures.
ALA-synthetase in erythroblasts was found to be decreased in all 3 cases of iron deficiency
anemia and 3 cases of l3-thalassemia minor. In 5 cases of idiopathic sideroblastic anemia, theenzyme activity was markedly decreased. A case of sideroblastic anemia, who restored normal
erythropoiesis following the administration of pyridoxal phosphate (PLP) showed low PLP level inserum & blood as well as in vivo evidence of PLP deficiency and an in vitro stimulation of ALAsynthetase activity by PLP. Erythroblasts of 4 patients with erythroleukemia showed diminishedbone marrow ALA-synthetase activity.

INTRODUCTION
The initial and rate-limiting step in the heme biosynthetic pathway is the enzymatic condensation of succinyl-CoA and glycine to form delta-aminolevulinic acid (ALA). 1)
This
condensation is mediated by the enzyme, deIta-aminolevulinic acid synthetase (succinylCoA: glycine succinyItransferase). This enzyme was shown to be inducible in high level in
Althe liver by a number of compounds which produced experimental porphyria. 2) - 3)though the normal level of ALA-synthetase in bone marrow erythroblasts is too low to bemeasured by the colorimetric method, the recent development of sufficiently sensitive
radiochemical assay method has made it possible to assay ALA-synthetase activity in humanbone marrow cells with limited number of erythroblasts. 4 ) - 10) The assay procedure adopted herein is the method developed by Evert et al and has the advantage of being both simple
and rapid.
Previous investigation on erythroleukemia, "refractory sideroblastic anemia", thalassemia
and iron deficiency anemia have suggested the presence of a disturbance in hemoglobin formation in these conditions. The author reports the results of experimental studies on assay
procedures of bone marrow ALA-synthetase activity in normal human subjects and patientswith sideroblastic anemia, iron deficiency anemia, beta thalassemia minor, anderythroleukemia in oder to elucidate the possible involvement of this enzyme in these disorders.
EB<:t-JiE~

Received for publication February 5, 1976
Present Address: Department ~f Medicine, Nagoya National Hospital, Nagoya, JapanM. TANAKA

22

METERIALS AND METHODSA.

Normal subjects and patients.

I.

Normal controls. Bone marrow ALA-synthetase activity was assayed in 6 healthy individuals with the age ranging from 19 to 24 years old.

2. Sideroblastic anemia. Bone marrow ALA-synthetase activity was assayed in 5 patientswith idiopathic sideroblastic anemia (lSA) at the time of diagnosis. In 2 cases, theenzyme was assayed for 2 to 3 times after the treatment with pyridoxal phosphate.
The diagnosis in each case was made on the basis of the following findings: moderate
to severe dimorphic anemia, reticulocytopenia, normal or increased serum iron concentration, erythroid hyperplasia and the presence of ringed sideroblasts in the bonemarrow aspirate, ineffective erythropoiesis by ferrokinetic studies, and the absence ofrecognizable underlying diseases.
3. Beta-thalassemia minor. The diagnosis in each case was established on the basis of
hypochromic anemia, normal or increased serum iron, increased Hb-A 2 content ranging
from 4.2 to 8.2% and family study revealing the presence of inheritance of A2 -typebeta-thalassemia.
4. Iron deficiency anemia. The three patients investigated had hypochromic anemia, low
serum iron and high unsaturated iron binding capacity of serum. The cause of iron deficiency was bleeding from gastrointestinal tract in all cases. The enzyme assay was
performed before any treatment in all cases.
5. Erythroleukemia. All three patients had very bizzare nucleated red blood cell morphology as well as numerous circulating nucleated red blood cells and erythroid hyperplasia of the bone marrow with megaloblastoid change. They were on no therapy at
the time of the study and were studied at their erythremic stage prior to their evolution to frank acute myelogenous leukemia.
B.

Assay Procedure.

1. Preparation of Succinyl-CoA Synthetase.
Crude succinyl CoA synthetase from Rhodepseudomonas spheroides ALA-requiringmutant H-5 (generously provided by Dr. June Lascelles of the University of California,
Los Angeles) was used. Cultures were grown at 30°C with aeration for 16 hrs. The
cells were then concentrated by centrifugation, washed in one half culture volume of
40 mH tris, pH 7.5 and sonicated. The cells were spun at 42,000 rpm for I hour andthe supernatant was assayed for succinyl CoA synthetase activity by the method of
Kaufman. 11)
The crude preparation contained 28.6 Ulml of succinyl CoA synthetase. One unit can catalize the formation of 1 micromole of succinyl CoA per hour.
2. Preparation of bone marrow lysate.
Cells from 3 to 4 ml aliquot of a heparinized bone marrow aspirate were dispersed
through a needle and washed twice with 0.15M NaC!. The total nucleated cell counto-AMINOLEVULINIC ACID SYNTHETASE

23

and the percent of erythroid cells were determined on the cell suspension prior to thesecond wash. The washed cells were exposed to 2 volumes of water for 4 minutes,
followed by addition of KCI to restore the isotonicity and then diluted to represent thedesired erythroid cell content. To completely disrupt the cell membrane, the preparation was homogenized prior to assay.

3. Enzyme assay procedure.
ALA-synthetase was measured by quantifying 14C-ALA formed after incubating thebone marrow lysate (final concentration ranging from 2.5 to 5% of the original cellvolume) in the presence of the following substances: 0.08M tris-HCI buffer, glycine,
sodium EDTA, sodium succinate containing a certain amount of 1-4 14C-succinate(specific activity 10.2 mCi/m mole), 10 mM MgCI 2 , I mM ATP, 0.1 mM CoA and succinyl CoA synthetase prepared from Rhodepseudomonas spheroides, which could catalize the formation of approximately 3.0 micromoles of succinyl CoA.
For assessment of ALA utilization during the incubation, duplicate samples wereincubated with 14 C-ALA instead of 14 C-succinate. The conditions of the incubation invarious experiments are indicated under Results and in the appropriate figures andtables. Incubations were terminated by addition of 0.5 ml of 25% trichloracetic acid.
14C-ALA was isolated from the trichloracetic acid supernatant as described by Evert etat. 5) with the following modifications: after the application of the sample to theresin, the column was washed successively with 20 ml of O.IM acetate buffer, pH 3.9,
20 ml of methanol: acetate buffer pH 3.9 (4: I, v/v) and 5 ml ofO.OIM HCI. 14C-ALA
was then eluted from the resin column with 3 ml of 0.1 M NaOH. One ml aliquot wasadded to Aquasol (New England Nuclear) and counted in a liquid scintilation spectrometer with the efficiency of greater than 90%.

RESULTS

1.

Experimental studies on ALA-synthetase assay procedures.

A.

Determination of optimal condition for the enzyme assay.
The results of experiments designed to determine the optimal condition for themeasurement of ALA-synthetase activity are presented in figure 1, 2, 3, 4. The optimal pH was 7.5 in trisbuffer (fig. 1). Enzyme activity was maximal with 50 mM
glycine (fig. 2-a),l 50 j..lM 14 C-succinate (fig. 2-d) and I mM Na-EDTA (fig. 2-b) and was
not enhanced by PLP over a concentration range of 0.2 mM to 1.2 mM (fig. 2-c).
Under the optimal condition of the substrate and EDTA, ALA generation was nearly6linear for 30 minutes and was a direct function of enzyme concentration up to 4 x 10
erythroid cells per assay (fig. 3). Under the above conditions, 14 C-ALA utilization was
not completely inhibited by EDTA. 14 C-ALA loss was proportional to the concentration of erythroid precursor cells as well as to mature red cells. However, 14 C-ALA
utilization was linear with the concentration of ALA only at a final concentration oflysate of 10% or less (fig. 4). At greater lysate concentration, ALA utilization increased disproportionately with increasing ALA-concentration. For the assay of the
enzyme as reported below, lysate and erythroid cell concentration were kept below 5%
and 5 x 106 , respectively.

24

M. TANAKA

100

..e

o

7.0

7.4

7.8

pH
Fig. I
Fig. 1.

Effect of pH on ALA·synthetase activity.
Incubation conditions were: bone marrow lysate, 1.0 ml; tris buffer, 80 mM; glycine 100
mM; 14C·Na Succinate, 150 /lM; NaEDTA, 1 mM; pyridoxal phosphate, 0.2 mM; MgCI 2 ,10 mM;ATP, 1 mM;CoA,O.1 mM.
Three units of partially purified succinyl CoA synthetase was added to each assay. Specific activity of 1-4 14C-succinate was 5.5 Ci/mole. Incubation was done at 37°C for 30
minutes. Incubation volume was 2 ml.

100~

~ sol

I-

U

<l

T

~

.....

-I

•

:;E

x

~

?-

40

lL

o

25

Fig. 2.

50

75

o

1

10

GLYCINE (mM)

No - EDTA (m M )Fig. 2 (a)Fig. 2 (b)100

Effect of the concentration of glycine (a), NaEDTA (b), pyridoxal-phosphate (c), and Na·
succinated (d) on ALA·synthetase activity. Incubation condition was the same as in thelegend to fig. 1.

25

8-AMINOLEVULINIC ACID SYNTHETASE

12001 ----;;::::==::::::::;;;:::::::::::;:/<!

<i 800

6.0

'"

0

~I

X

"U

::a
0..

0

III
t!>

u

~400

3.0

o

0.4

0.8

i.2

0.05

0.10

0.15

020

PYRIDOXAL PHOSPHATE(mM)

No-SUCCINATE (mM )Fig. 2 (c)Fig. 2 (d)6.0.---------------,

6.0
M

S?
x

~3.0

4.0

0..
U

15

Fig. 3.

30

45

60

1

2

3

MINUTESERYTHROBLA STS X10 6

Fig. 3 (a)Fig. 3 (b)4Effect of time (a) and enzyme concentration (b) on ALA-synthetase activity. Incubation
condition was the same as in the legend to fig. 1. The vertical axis is the CPM of 14C_
ALA formed during incubation.

B.

Identification of ALA.
Column eluate (ALA-fraction) resulting from an incubation of normal bone marrow
with 14 C-succinate as well as 14 C-ALA added to a "killed" lysate were subjected to an
ascending paper chromatography. ALA in the eluate was first converted to pyrrole byboiling with acetylaceton; then concentrated to dryness and the residue was dissolvedin a small amount of methanol. This solution was subjected to paper chromatography.
Fig. 5 shows that the radioactivity of the eluate consisting mostly (above 90%) of 14 CALA-pyrrole.

2.

ALA-synthetase activity of marrow cells in normal subjects, idiopathic sideroblastic
anemia, iron deficiency anemia, beta-thalassemia minor and erythroleukemia patients.
Table I depicts the values of ALA-synthetase activity in bone marrow erythroblastsof 6 normal subjects and those of 5 patients with ISA. In all patients with ISA, the26

M. TANAKA- - 10 Of, LYSATE
e>--O
15 Of, LYSATE.-----.. 30'10 LYSATE

o

W
N

::i
i==

40

::J

«...I
«

~l

U

400

800

1200

C14_ ALA ADDED (p moles)

Fig. 4
Fig. 4.

Effect of ALA concentration on ALA-utilization.
Incubation condition was the same as in the legend to fig. I.

2

/\
1
(

I

I

I

I

I

I
I

I

I
I

I
I

I

5

10

------,,"
15

20

DISTANCE FROM ORIGIN

Fig. 5
Fig. 5.

Identification of 14 C_ALA in the eluate.
14C-succinate (l uCi, 0.045 J.l mole) was incubated with normal bone marrow lysate andthen chromatographed on column as described in materials and method. 14C_ALA in thepresence of trichloracetic acid -treated tissue homogeneite was not incubated, but was
treated similarly.
14C_ALA in the column eluate was converted to the pyrrole form, spotted on paper, andthen subjected to ascending chromatography, using n-butanol-acetic acid -water (4: I : 8).
Chromatographs were sprayed with ninhydrin to observe aminoacid contamination andwith Ehrlich reagent to localize the pyrrole spot. No ninhydrin-positive spot attributable
to amino-acid was seen.

27

o-AMINOLEVULINIC ACID SYNTHETASE
Table I. Bone marrow ALA-synthetase activity in normal subjects and patients with idiopathic
sideroblastic anemia.
p moles 14C-ALA formed/lO"
erythroblasts/30 minutes

Case

PLP** (-)

PLP** (+)

I. Normal
2. Normal
3. Normal
4. Normal
5. Normal
6. Normal
Normal Range

220
325
320
257
226
337
281 ±53

226
337
320
262
229
342
286 ±54

7.ISA*
8.ISA*
9.ISA*
10. ISA * - before PLP therapy

132
60.5,53.5
88
52
68
78
243
58
38
60

137.5
65.5,50
75
290
190
108
253
50
50
58

- after PLP therapy- 3 months after PLP therapy
11. ISA * - before PLP therapy- after PLP therapy*ISA = Idiopathic sideroblastic anemia**PLP = Pyridoxal phosphate

Table 2. Bone Marrow ALA synthetase activity in {3 thalassemia, iron deficiency anemia, anderythroleukemia.

casedisorderage

p moles of 14 C-ALA formed/10" erythroblasts/30 minutes *

l.

2.
3.

l3-thalassemia minor
l3-thalassemia minor
l3-thalassemia minor

23
26
18

103
112
104

4.
5.
6.

Iron deficiency anemia
Iron deficiency anemia
Iron deficiency anemia

42
28
61

121
71
82

7.
8.
9.
10.

Ery throleukemia
Erythroleukemia
Erythorleukemia
Erythroleukemia

61
64
42
38

177
80
56
41

* Assayed with PLP in the incubation mixture

ALA-synthetase activity was significantly diminished. In case 10, who was unresponsive to pyridoxine treatment, addition of pyridoxal phosphate (PLP) (0.2 mM) tothe enzyme assay restored the enzyme activity to normal. Because his blood PLP concentration was low, the patient was given parenteral PLP. A gradual and nearly normal
complete erythropoietic response followed this therapy. After the initial reticulocytosis, bone marrow ALA-synthetase activity remained low and was no longer
stimulated by PLP in vitro. After the full erythropoietic response, enzyme activity re-28

M. TANAKA
turned to nonnal. In the remaining 4 cases, PLP failed to improve the enzyme activity in vitro.
ALA-synthetase activity in iron deficiency anemia and beta-thalassemia minor are
shown in table 2. All 3 patients with iron deficiency anemia had a diminished activity
in bone marrow erythroblasts. The enzyme activity was also depressed in patients withbeta-thalassemia minor. Table 2 also shows the. enzyme activity in the bone marrow
erythroblasts of erythroleukemia. The enzyme activity in this disorder was also quitelow.
DISCUSSION

The radiochemical assay methods of ALA-synthetase have been reported by several
authors in recent years. Irving and Elliot 4) described a radiochemical method employing
14 C-succinate as precursor. Evert 5) used 14 C-succinate and 14 Ca-ketoglutarate. Freshney
and Paul 6) used 14C-glycine as a precursor. Aoki et al. 9) developed an assay method
using 14 C-succinyl CoA in bone marrow erythroblasts. Strand et at. 8) and Ohashi et al.
10) established the method which used succinyl-CoA synthetase as the source of succinylCoA. The author modified Evert's method by employing succinyl-CoA generating system(succinic thiokinase from the ALA-synthetase lacking mutant of Rhodepseudomonas
spheroidesJ. The advantage of this method is that it uses 14 C-succinyl CoA as a direct
precursor of ALA-synthetase and that it does not employ the tedious procedure of partiallypurifying enzyme solution from bone marrow. In comparison to existing radiochemical
assay, the sensitivity of this present assay has been increased by an improved method
employing 14C-succinyl CoA generation and bone marrow lysate as enzyme solution without any loss of enzyme activity in the sonicated supernate. The assay method using succinyl
CoA generating system increased the sensitivity of assay 2.5 fold as compared with that of
the assay without succinyl CoA generating system (unpublished data). The result confirmsthat methods which do not employ a succinyl CoA generating system understimate total
ALA-synthetase activity.8)
The ALA-synthetase activity in the erythroblasts of normalbone marrow was 281 ±53 p moles/l0 6 erythroblasts/30 minutes. This value is quite close
to the value of 305 ± 17 p moles of ALA generated by partially purified enzyme solution
from normal bone marrow hemolysate mitochondria reported by Aoki. 9)This factsuggests that the assay of enzyme activity is not interfered by the low permeability ofthe substrate or inhibitor contained in the crude hemolysate.
The activity and the systhesis of ALA-synthetase is partially controlled by end productinhibition of heme. 12 ) - 13) Similar mechanism affecting heme synthetase may limit theformation of heme from protoporphyrin 1X. 14 ) This is the first report of diminished
ALA-synthetase activity in the bone marrow erythroblasts in (3-thalassemia. Although thetrue mechanism for the reduced ALA-synthetase in thalassemia is not clear, there is someevidence that heme synthesis is controlled to some extent by globin synthesis: when protein
synthesis in reticulocytes is inhibited by cyclohexamide, heme synthesis is also rapidly andreversibly reduced. Vavra and Poff 15) reported that heme synthesis is abnonnally reducedin thalassemia reticulocytes, presumably as a result of the primary decrease in globin synthesis in this disorder. The data of the present study and the Takaku's report 7) suggest thatthe reduced activity of ALA-synthetase is mediated by the feedback inhibition of free heme
present in the erythroblasts of thalassemic bone marrow.
Experimental results on the effect of iron on ALA-synthetase are controversial. Morrow

o-AMINOLEVULINIC ACID SYNTHETASE

29

et al. 16)
observed that ferrous iron depressed the activity of the enzyme in particles ofavian hemolysate. Aoki 9) reported an inhibitory effect of ferrous iron on partially purified ALA-synthetase from rabbit reticulocytes. However, in chicken reticulocytes
mitochondria, iron had an enhancing effect on ALA-synthetase activity, 17) and in rabbitbone marrow mitochondria, ferrous iron had no effect on this enzyme in concentration
ranging from 1.7 x 10-6 M to 1.7 x 10-3 M. 18 )Our present study indicated that ALA-synthetase activity is depressed in iron deficientbone marrow erythroblasts. This data is consistent with Brown's observation 19) that theformation of ALA from glycine is decreased in iron deficient duck erythrocytes. It is suggested that the decreased activity of ALA-synthetase in this condition is due to the feedback
inhibition of accumulated protoporphyrin in erythroblasts.
The present study demonstrates that the erythroblasts of patients with ISA had invariably
diminished ALA-synthetase activity. This data confirms previous observation of Tanaka et
al. 20)and Aoki et al. 9) that bone marrow ALA-synthetase is diminished in ISA. The
fact that 4 patients did not respond to PLP in vitro could be explained by the heterogeneityof ISA: pyridoxine responsive and non-responsive cases. The reported findings of PLP deficiency provide basis for the low ALA-synthetase activity in certain cases of this disorder.
Although the impaired heme synthesis in ISA has been postulated by the presence of
hypochromic anemia, the presence of ineffective erythropoiesis on ferro kinetic study andthe accumulation of iron in the mitochondria of erythroblasts, Tanaka et al. 20)
have
shown the decreased activity of ALA-synthetase in this condition. The decreased PLP concentration in blood and plasma was shown in experimental sideroblastic anemia in guinea
pig given INH and cycloserine. 21 )Complete understanding of the pathogensis of ISA
must take into acount other apparent abnormalities in heme biosynthesis, such as the accumulation of large amount of protoporphyrin and the possible disturbance of ferrochelataseand moreover the as yet unexplained mechanism for the massive accumulation of
mitochondrial iron in erythroblasts in this disorder. However, the alteration in the activityof ALA-synthetase can be a factor in the pathogenesis of anemia and mitochondrial change
in ISA, since this enzyme is one of the mitochondrial, rate limiting enzyme in heme biosynthesis.
Defective heme and globin synthesis have been reported in erythroleukemia. Steiner et
al. 22)and Necheles and Dameschek 23)
demonstrated lowered activities of ALA-dehydrase and heme synthetase in erythroleukemia. In the studies of globin synthesis inerythroleukemia, Pagnier et a124 ) found a very unusual large pool of {32 dimer, which is
not a precursor of normal hemoglobin. They reported that the presence of Hb-H was always
due to un balanced synthesis of 0' and {3 chains, large excess of {3 chain, which was caused
either by a defect in 0' chain synthesis or to an excess of (3 chain synthesis.
previously reported the decreased ALA-synthetase activity in bone
Tanaka et al. 25)
marrow erythroblasts in erythroleukemia. In that report ALA-synthetase activity was assayed by the method using 14 C-O'ketoglutarate, which did not use succinyl CoA generating
system.
The present report confirms our previous data by the assay method using succinyl CoA
generating system. If, as Damescheck 26) suggested, sideroblastic anemia is a preleukemic
condition finally ending with leukemia, decreased ALA-synthetase activity might be acommon pathologic phenomenon of erythroleukemia and sideroblastic anemia.
It is possible with the impaired heme synthesis does not represent the primary defect inerythroleukemia and sideroblastic anemia, but it would conceivably be a reflection of a

30

M. TANAKA

more basic underlying disturbances in proliferation and maturation of stem cells which issuggested by the presence of the chromosomal abnormalities in this disorder. However, thesimilarity of the biochemical, cytochemical and clinical manifestation between erythroleukemia and sideroblastic anemia suggests that they may constitute a group of relatedchanges due to an aquired and probably neoplastic disturbances in proliferation and differentiation of stem cells. The activity of ALA-synthetase was uniformly depressed in erythroleukemia, whereas in sideroblastic anemia, the depression of this enzyme activity was not
uniform. The reduction of bone marrow ALA-synthetase has thus been revealed in various
hematological disorders, although the mechanism of the reduction might be diverse.
SUMMARY

A sensitive and simple radiochemical assay method was established and employed for themeasurement of ALA-synthetase in human bone marrow erythroblasts. Low ALA-synthetase activity has been documented in the bone marrow of five patients with idiopathic
sideroblastic anemia. One patient showed an in vitro stimulation of bone marrow ALAsynthetase activity by PLP as well as in vivo response of the anemia by PLP treatment. In
iron deficiency anemia, (3-thalassemia minor and erythroleukemia, bone marrow ALAsynthetase activity was all depressed. Although the mechanism of the lowered enzyme activity was considered to be diverse in each disorder, the pathogenetic significance of thediminished enzyme activity in the development of anemia of these disorders were discussed.
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19) Brown, E. G. Evidence for the involvement of ferrous iron in the biosynthesis of aminolevulinate(ALA) synthetase in rabbit reticulocytes. Biochem. Biophys, Res. Comun., 42: 568.1958.
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