Blood, Vol. 91 No. 6 (March 15), 1998:
pp. 2169-2174
Delayed Onset of Hemolytic Anemia in
CBA-Pk-1slc/Pk-1slc Mice With a Point
Mutation of the Gene Encoding Red Blood Cell Type Pyruvate Kinase
By
Kumiko Tsujino,
Hitoshi Kanno,
Koji Hashimoto,
Hisaichi Fujii,
Tomoko Jippo,
Eiichi Morii,
Young-Mi Lee,
Hidekazu Asai,
Shiro Miwa, and
Yukihiko Kitamura
From the Department of Pathology, Osaka University Medical School,
Suita, Osaka; Okinaka Memorial Institute for Medical Research,
Minato-ku, Tokyo; the Department of Blood Transfusion Medicine, Tokyo
Women's Medical College, Shinjyuku-ku, Tokyo; and Japan SLC Co Ltd,
Hamamatsu, Shizuoka, Japan.
 |
ABSTRACT |
The Pk-1slc gene encodes a mutant red blood cell
(RBC) type pyruvate kinase (PK), and adult
CBA-Pk-1slc/Pk-1slc mice show a severe
nonspherocytic hemolytic anemia. However, the number of RBCs and the
proportion of reticulocytes were comparable between neonatal
CBA-Pk-1slc/Pk-1slc mice and control
-+/+ mice. Since the age-dependent increase of RBCs was much
greater in CBA-+/+ mice than in
CBA-Pk-1slc/Pk-1slc mice, significant
anemia was observed in the latter mice on day 14 after birth. The
increase of RBCs in CBA-+/+ mice was due to the prolongation of
their survival time. The half life of RBCs increased in CBA-+/+
mice with ages, but it decreased in
CBA-Pk-1slc/Pk-1slc mice. The
relatively longer half life of RBCs in neonatal
CBA-Pk-1slc/Pk-1slc mice appeared to be
due to the delayed switching from M2-type PK that are expressed by
undifferentiated erythroid precursor cells to RBC-type PK that are
expressed by mature RBCs.
| |
INTRODUCTION |
PYRUVATE KINASE (PK, EC 2.7.1.40)
catalyzes the conversion of phosphoenolpyruvate to pyruvate in the
glycolytic pathway. In humans, deficiency of PK activity is the most
common cause of hereditary anemias due to deficiency of glycolytic
enzymes.1-3 PK has four isozymes in mammals4;
the red blood cell (RBC) type PK (R-PK) is expressed almost exclusively
in mature RBCs.5 A unique structural profile of the R-PK is
the longer amino-terminal sequence as compared with that of liver-type
PK (L-PK), which is transcribed from alternative promoter of the L/R-PK
gene.6 In mice, L/R-PK is encoded by the Pk-1
gene.7 In contrast with mature RBCs, undifferentiated erythroid precursor cells express a different isozyme of PK
(M2-PK),8-10 which is encoded by the Pk-3 gene of
the mouse. The Pk-3 gene encodes both M1-PK and M2-PK; they are
produced by alternative RNA splicing,11 and the former was
expressed chiefly by muscle and the latter by fetus and most adult
tissues.12-15 The expression of the Pk-3 gene
switches to that of the Pk-1 gene during differentiation of
RBCs. The persistence of M2-PK in RBCs has been described in some
patients of hereditary PK deficiency.16,17
We recently found the mutant Pk-1slc gene in the
CBA/N strain of mice (hereafter called CBA mice).18,19 Mice
of CBA-Pk-1slc/Pk-1slc showed severe
nonspherocytic hemolytic anemia. The activity of PK in RBCs of the
adult mutants decreased to 16.2% of normal (+/+) adults. Because
CBA-Pk-1slc/Pk-1slc mice showed a
remarkable reticulocytosis (41.6%) and PK activity of reticulocytes is
much higher than that of mature RBCs, the PK activity in mature RBCs of
the CBA-Pk-1slc/Pk-1slc mice was
calculated to be 2.8% that of mature RBCs of CBA-+/+ mice.19
The abnormality of CBA-Pk-1slc/Pk-1slc
mice was characterized by comparing normal and mutant Pk-1
cDNAs. A missense mutation at nucleotide 1013 GGT 
GAT was
identified in the cDNA sequence of the mutant, causing a single amino
acid substitution of 338Gly to Asp. The missense mutant was also
confirmed in genomic sequence by the analysis using polymerase chain
reaction for restriction fragment length polymorphism.18
Although adult CBA-Pk-1slc/Pk-1slc mice
show a severe hemolytic anemia, neonatal
CBA-Pk-1slc/Pk-1slc mice are not pale.
We cannot distinguish neonatal
CBA-Pk-1slc/Pk-1slc mice from neonatal
CBA-+/+ mice by appearance. In the present study, we investigated when
the significant anemia developed in CBA-Pk-1slc/Pk-1slc mice. Moreover, we
compared the survival time of RBCs between CBA-Pk-1slc/Pk-1slc and -+/+ mice of
various ages.
| |
MATERIALS AND METHODS |
Mice.
CBA-Pk-1slc/Pk-1slc mice and CBA-+/+
mice were raised in the Japan SLC farm (Shuchi-gun, Shizuoka, Japan)
and sent to Osaka University. As described in a previous
report,19
CBA-Pk-1slc/Pk-1slc mutant strain was
found in the inbred CBA colony that was originally obtained from
National Institutes of Health (Bethesda, MD) and kept in the Japan SLC
farm. Although the CBA-Pk-1slc/Pk-1slc
mice were maintained by brother-sister mating for 7 years, no difference was detectable in 16 biochemical genetic markers between the
CBA-Pk-1slc/Pk-1slc and CBA-+/+
mice.19 Moreover, bone marrow transplantation from the
CBA-+/+ mice to CBA-Pk-1slc/Pk-1slc
mice cured the anemia.19
Female and male CBA-Pk-1slc/Pk-1slc
mice were mated together to obtain mice of
Pk-1slc/Pk-1slc genotypes; female and
male CBA-+/+ mice were mated together to obtain mice of +/+ genotypes.
On various days after birth, mice were weighed, anesthetized by ether,
and killed by exsanguination from the carotid artery. Blood was
collected using heparinized Pasteur pipette. The spleen was removed and
weighed. To obtain fetal livers, pregnant mice were killed on day 16 postcoitus.
Hematologic parameters.
The number of RBCs was measured with a hemocytometer. Reticulocytes
were counted on blood films stained with new methylene blue, and the
percentage of reticulocytes was determined by counting 1,000 RBCs. In
some cases, the blood films were post-stained with May-Grünwald-Giemsa to examine the contamination of nucleated erythroblasts.
Refinement of RBCs and PK activity.
Heparinized whole blood was diluted three times with ice-cold
physiologic saline and passed through a column of 
-cellulose (Sigma
Chemical Co, St Louis, MO) and Sigmacell (Sigma) to deplete leukocytes
and platelets.20 Refined RBCs were washed three times and
added to the same volume of preservative composed of 28% glycerin, 2.8% sorbitol, and 0.85% NaCl, and stored frozen at 
80°C. PK activity was measured spectrophotometrically by the consumption of NADH
in the coupled optical assay in which LDH is used as an auxiliary
enzyme as previously described.21
Survival of RBCs.
Blood was obtained from
CBA-Pk-1slc/Pk-1slc and -+/+ mice of 1, 7, 42, and 90 days of age. The blood was diluted three times with Hanks' balanced salt solution (HBSS), centrifuged at 1,500g
for 5 minutes, and the supernatant and buffy coat were removed. The remaining RBCs were diluted three times with HBSS and incubated for 1 hour at 37°C with 740 kBq/mL
Na251CrO4 (Amersham International,
Little Chalfont, Buckinghamshire, England; specific activity, 3.7 to
18.5 MBq/µg Cr).22 The labeled RBCs were washed twice and
diluted three times with HBSS, and injected into 90-day-old CBA-+/+
mice via the carotid vein in a volume of 0.5 mL. Blood samples of 0.1 mL were obtained from the tail tip on various times after the
injection, and the radioactivity was measured with a Minaxi 
5550
gamma counter (Packard, Meriden, CT). Blood samples that were obtained
2 hours after the infusion were kept as controls, and the correction
was made for radioactive decay. The radioactivity of each blood sample
was expressed as a percentage of the control, and the percentage was
plotted semi-logarithmically.
Polyacrylamide gel electrophoresis (PAGE).
Erythrocytes were lysed by sonication in the PK sample buffer
containing 10 mmol/L Tris/HCl, pH 7.5, 100 mmol/L KCl, 2 mmol/L 2-mercaptoethanol, 10 mmol/L 
-aminocaproic acid, and 10 mmol/L EDTA.
Protein extracts prepared from fetal liver were also homogenized in the
PK sample buffer. PK activity assay, PAGE, and staining for PK activity
were performed as described previously.23
Antibody neutralization of PK.
Rabbit antiserums against L-PK and M1-PK of the rat were kindly
provided by Dr Tamio Noguchi of Fukui Medical School. We used them to
characterize the isozyme expression in RBCs of
CBA-Pk-1slc/Pk-1slc and -+/+ mice of
various ages. RBC lysate was mixed with anti-L-PK or anti-M1-PK
antibody, followed by overnight incubation at 4°C. The mixture was
then centrifuged at 15,000g at 4°C for 15 minutes, and the PK
activity in the supernatant was measured.
| |
RESULTS |
Delayed onset of anemia.
Number of RBCs and proportion of reticulocytes were measured in
CBA-Pk-1slc/Pk-1slc and -+/+ mice on
days 1, 7, 14, 28, and 42 after birth. No significant differences were
detectable between CBA-Pk-1slc /Pk-1slc
and CBA-+/+ mice on day 1 (Fig 1). Although
the number of RBCs increased in both mutant and normal mice with ages,
the number was significantly greater in normal mice than in mutant mice
on day 14 and thereafter. As a result, significant anemia was
detectable in CBA-Pk-1slc/Pk-1slc mice
from day 14 after birth. The proportion of reticulocytes continuously
decreased after birth in CBA-+/+ mice. On the other hand, it increased
until day 14 and then retained the high value in
CBA-Pk-1slc/Pk-1slc mice (Fig 1). The
spleen weight was comparable between
CBA-Pk-1slc/Pk-1slc and -+/+ mice on
day 1 after birth, but a significant splenomegaly was observed in
CBA-Pk-1slc/Pk-1slc mice on day 42 (Table 1). In spite of the severe anemia of
CBA-Pk-1slc/Pk-1slc mice, body weight
of CBA-Pk-1slc/Pk-1slc mice were
comparable with that of CBA-+/+ mice throughout the observation period.
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| Fig 1.
Number of RBCs and proportion of reticulocytes in
CBA-+/+ ( ) and -Pk-1slc/Pk-1slc
( ) mice on various days after birth. Each point represents the mean
of ten mice. Bars are the standard error. In some points the standard
error was too small to be shown by bars.
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|
When RBCs of adult CBA-Pk-1slc/Pk-1slc
and -+/+ mice were labeled with 51Cr and injected into
adult CBA-+/+ mice, the disappearance of RBCs derived from
CBA-Pk-1slc/Pk-1slc mice was much
faster than that of RBCs from CBA-+/+ mice.19 Since the
number of RBCs of 1-day-old
CBA-Pk-1slc/Pk-1slc mice was comparable
with that of 1-day-old CBA-+/+ mice, we compared the survival time of
RBCs of CBA-Pk-1slc/Pk-1slc mice of
various ages to that of RBCs of CBA-+/+ mice of the same ages. RBCs of
1-day-old, 7-day-old, 42-day-old, and 90-day-old CBA-Pk-1slc/Pk-1slc and -+/+ mice were
labeled with 51Cr and injected into 90-day-old CBA-+/+
mice. Radioactivity retained in RBCs was measured at various times
after the injection. In contrast with the large difference between
90-day-old CBA-Pk-1slc/Pk-1slc and -+/+
mice, the difference between 1-day-old
CBA-Pk-1slc/Pk-1slc and -+/+ mice was
slight (Fig 2). When the radioactivity
retained in the RBCs was plotted semi-logarithmically, two-component
curves were observed after the injection of labeled RBCs obtained from 1-day-old and 7-day-old
CBA-Pk-1slc/Pk-1slc and -+/+ mice.
Two-component curves were also observed after the injection of labeled
RBCs from 42-day-old and 90-day-old
CBA-Pk-1slc/Pk-1slc mice, whereas
one-component curves were observed after the injection of labeled RBCs
from 42-day-old and 90-day-old CBA-+/+ mice (Fig 2). Since the
second-component line observed after the injection of labeled RBCs from
1-day-old and 7-day-old
CBA-Pk-1slc/Pk-1slc and -+/+ mice, and
42-day-old and 90-day-old
CBA-Pk-1slc/Pk-1slc mice were roughly
parallel to the line observed after the injection of labeled RBCs from
90-day-old CBA-+/+ mice, these lines appeared to represent the
destruction of the RBCs of 90-day-old CBA-+/+ hosts, which were
secondarily labeled by the 51Cr released from the injected
RBCs. The half life of RBCs decreased in
CBA-Pk-1slc/Pk-1slc mice but increased
in CBA-+/+ mice. The difference became greater in parallel with the
increase of age (Table 2).
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| Fig 2.
The effect of ages of RBC donors on the elimination
of 51Cr-labeled RBCs. RBCs were collected from CBA-+/+
() and -Pk-1slc/Pk-1slc () mice
of various ages, labeled, and injected into 90-day-old CBA-+/+
mice. The radioactivity retained in RBCs was measured at various times
after the transfusion and the percentage was plotted
semi-logarithmically. Each point represents the mean of eight mice. In
all points the standard error was too small to be shown by bars.
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Table 2.
Half Life of RBCs Obtained From CBA-+/+ and
-Pk-1slc/Pk-1slc Mice of Various
Ages After Transfusion Into 90-Day-Old CBA-+/+
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|
Age-dependent changes of PK activity.
In either CBA-+/+ or
CBA-Pk-1slc/Pk-1slc mice, the PK
activity was much higher in neonatal mice than in 90-day-old mice
(Table 3). In spite of the high activity of
PK, examination of smears detected no nucleated erythroblasts. The PK
activity gradually decreased with ages in both CBA-+/+ and
cba-Pk-1slc/Pk-1slc mice. Although
age-dependent decrease in PK activity of RBCs was observed in both
CBA-+/+ and -Pk-1slc/Pk-1slc mice, the
absolute value of PK activity was much greater in CBA-+/+ mice than in
CBA-Pk-1slc/Pk-1slc mice. Moreover,
different isozyme pattern was observed by electrophoresis between
CBA-+/+ and -Pk-1slc/Pk-1slc mice of 1 and 7 days of age. Only R-PK was detectable in RBCs of 1-day-old and
7-day-old CBA-+/+ mice, whereas only M2-PK was detectable in RBCs of
1-day-old and 7-day-old
CBA-Pk-1slc/Pk-1slc mice (Fig
3). In the fetal liver of CBA-+/+ mice,
both R-PK and M2-PK were detectable, but only M2-PK was detectable in
the fetal liver of CBA-Pk-1slc/Pk-1slc
mice (Fig 3). Moreover, anti-L-PK antibody neutralized the PK activity
in the RBC lysate obtained from CBA-+/+ mice but did not neutralize the
PK activity in the RBC lysate of CBA-Pk-1slc
/Pk-1slc mice (Table 4). On the other hand, anti-M1-PK
antibody neutralized the PK activity in the RBC lysate of
CBA-Pk-1slc/Pk-1slc mice but did not
neutralize the PK activity in the RBC lysate of CBA-+/+ mice (Table
4).
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| Fig 3.
PAGE and staining for PK activity in extracts of fetal
livers or RBCs. Fetal livers were obtained from 16-day embryos of
CBA-+/+ or -Pk-1slc/Pk-1slc mice;
RBCs from 1-day-old and 7-day-old CBA-+/+ or
-Pk-1slc/Pk-1slc mice. M2, M2-PK; R/L,
R- or L-PK.
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|
| |
DISCUSSION |
Although PK encoded by the mutant Pk-1slc gene
hardly possesses the enzymatic activity,18,19 the numbers
of RBCs were comparable between 1-day-old CBA-+/+ and
-Pk-1slc/Pk-1slc mice. The comparable
RBC values in 1-day-old mice was chiefly attributable to the low RBC
counts of 1-day-old CBA-+/+ mice. The RBC number increased continuously
from day 1 to day 42 after birth in both CBA-+/+ and
-Pk-1slc/Pk-1slc mice, but the
magnitude of the increase was much greater in CBA-+/+ mice than in
CBA-Pk-1slc/Pk-1slc mice. The greater
increase in RBC number may be explained by the prolongation of the
survival time observed in CBA-+/+ mice from day 1 to day 42 after
birth.24 In contrast with CBA-+/+ mice, the survival time
decreased with ages in
CBA-Pk-1slc/Pk-1slc mice (Table 2).
The PK activity of 1-day-old CBA-+/+ mice was over six times higher
than adults, and it decreased with ages (Table 3). Since the PK
activity in CBA-+/+ mice paralleled with the proportion of
reticulocytes, the high PK activity in neonatal CBA-+/+ mice was
attributable to the high proportion of reticulocytes. This is
consistent with the result reported by Chapman and
Schaumburg,25 Nathan et al,26 Paglia and
Valentine27 and Lakomek et al28 that PK
activity was higher in reticulocytes than in mature RBCs. Although the
PK activity was higher in 1-day-old and 7-day-old CBA-Pk-1slc/Pk-1slc mice than in the
older mice of the same genotype, the PK activity did not parallel with
the proportion of reticulocytes in mice of the
Pk-1slc/Pk-1slc genotype. In fact, the
PK activity significantly decreased on day 14 after birth whereas the
proportion of reticulocytes significantly increased on the same day
(Fig 1 and Table 3). Therefore, the relatively high PK activity in
neonatal CBA-Pk-1slc/Pk-1slc mice
cannot be explained by the high proportion of reticulocytes. When
isozyme pattern of PK was compared between 1-day-old and 7-day-old
CBA-+/+ mice and CBA-Pk-1slc/Pk-1slc
mice by zymograms as well as the antibody neutralization, R-PK was
exclusively observed in +/+ RBCs, whereas M2-PK in
Pk-1slc/Pk-1slc RBCs (Fig 3, Table 4).
Therefore, the relatively high PK activity in RBCs of 1-day-old and
7-day-old CBA-Pk-1slc/Pk-1slc mice may
be attributable to the delayed switching from M2-PK to R-PK.
Switching of isozymes from R-PK to M2-PK during erythroid
differentiation have been demonstrated by several investigators. Takegawa et al8 examined the transition of PK isozyme
expression by immunologic methods; the M2-PK predominantly expressed in
proerythroblasts, and R-PK expression became detectable at the stage of
basophilic erythroblasts. Nijhof et al9 demonstrated that
both R-PK and M2-PK activity was detected in murine erythroid precursor
cells (CFU-E) and that M2-PK activity rapidly disappeared during the erythropoietin-induced differentiation. Max-Audit et al10
reported that the synthetic rates of PK in human erythroid progenitors were highest in proerythroblasts. They also described that R-PK synthesis remained at the same level during differentiation and that
M2-PK activity decreased due to rapid decline of synthesis as well as
accelerated degradation.10
Present study showed that the half life of 51Cr-labeled
RBCs of 1-day-old and 7-day-old
CBA-Pk-1slc/Pk-1slc mice was longer
than the half life of 42-day-old and 90-day-old CBA-Pk-1slc/Pk-1slc mice. The
difference was slight but significant (Table 2). Since the PK activity
was greater in the former mice than in the latter mice and since the PK
activity in RBCs was attributed to M2-PK not only in suckling
CBA-Pk-1slc/Pk-1slc mice but also in
adult CBA-Pk-1slc/Pk-1slc mice, the
relatively large amount of M2-PK expressed by RBCs of the younger
CBA-Pk-1slc/Pk-1slc mice may play a
significant role for the survival of the RBCs. It is most likely that
the M2-PK activity beyond day 7 after birth may not be sufficient to
sustain survival of RBCs in
CBA-Pk-1slc/Pk-1slc mice.
There are numbers of globin gene abnormalities that influence switching
of active globin gene locus during hematopoietic development in humans.
Hereditary persistence of fetal hemoglobin (HPFH) is one of the genetic
abnormalities that increase hemoglobin F production in RBCs. Clinical
significance of HPFH has been recognized in its relationship to
thalassemia and sickle cell anemia. Phenotype of 
-thalassemia or the
sickle cell anemia was found to be alleviated or even asymptomatic with
HPFH.29-31 Beneficial effects on hemoglobinopathies would
be obtained if globin gene expression could be reactivated in adult
erythroid progenitors by pharmacologic means.32 Similarly, the persistent expression of M2-PK in mature RBCs has been reported in
severe PK deficiency of humans,4,16,17,33,34 and the M2-PK
activity seemed to compensate hemolysis to some extent in those cases.
Further investigations are required to understand PK isozyme switching
during erythroid differentiation and the significance of M2-PK
expression in severe PK deficiency. Intact M2-PK gene in erythroid
cells of mutants might become a novel therapeutic strategy to hemolytic
anemia due to PK deficiency.
| |
FOOTNOTES |
Submitted May 27, 1997;
accepted November 7, 1997.
Supported by grants from the Japanese Ministry of Education, Science,
Sports, and Culture.
Address reprint requests to Yukihiko Kitamura, MD, Department of
Pathology, Osaka University Medical School, Yamada-oka 2-2, Suita,
Osaka, 565, Japan.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank Professor Tamio Noguchi of Fukui Medical School for supplying
us with rabbit antibodies against rat L-PK and M1-PK.
| |
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Abstract
Introduction
Abstract
