1. No category

From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
1995 86: 4323-4330
Pyruvate kinase deficiency of mice associated with nonspherocytic
hemolytic anemia and cure of the anemia by marrow transplantation
without host irradiation
M Morimoto, H Kanno, H Asai, T Tsujimura, H Fujii, Y Moriyama, T Kasugai, A Hirono, Y Ohba
and S Miwa
Updated information and services can be found at:
http://www.bloodjournal.org/content/86/11/4323.full.html
Articles on similar topics can be found in the following Blood collections
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American
Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.
From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
Pyruvate Kinase Deficiency of Mice Associated With Nonspherocytic
Hemolytic Anemia and Cure of the Anemia by Marrow Transplantation
Without Host Irradiation
By Masahiro Morimoto, Hitoshi Kanno, Hidekazu Asai, Tohru Tsujimura, Hisaichi Fujii, Yasuhiro Moriyama,
Tsutomu Kasugai, Akira Hirono, Yuzou Ohba, Shiro Miwa, and Yukihiko Kitamura
Mutant mice with splenomegaly and nonspherocytic hemolytic anemia were found in an inbred colony of the CBA/N
(hereafter CBA) strain maintained in the Japan SLC Haruno
farm (Shuchi-gun, Shizuoka, Japan). The activity of pyruvate
kinase (PK) in red blood cells (RBCs) of the anemic mutants
decreased to 16.2% of normal (+/+l CBA mice. Because the
mutant CBA mice showed a remarkable reticulocytosis
(41.6Y0)and because the PK activity of reticulocytes is much
higher than that of mature RBCs, the PK activity in mature
RBCs of the mutant CBA mice was calculated to be 2.840
that of mature RBCsof CBA-+I+ mice. Because RBC type
PK is encoded by theP&-llocus of the mouse (chromosome
3). we designated the mutant locus as P&-l””. The anemia
P
(H.A.) found spontaneous development of splenomegaly in a litter
derived from the inbred colony of the CBA strain. All mice of the
second litter from the same parents exhibited splenomegaly, and the
offspring ofthe third litter from the parents was maintained by
brother-sister mating. All CBA mice belonging to this colony
showed significant splenomegaly from 3 weeks of age. Mice with
splenomegaly appeared to be healthy, and their life span was comparable to that of CBA-+/+ mice without splenomegaly. Recently, we
recognized that the splenomegaly was accompanied with a severe
anemia, andin the present study we show thatthe splenomegaly
and anemia resulted from the PK deficiency. Because the PK activity
inredblood cells (RBCs) is controlled by the Pk-l locus of the
mouse (chromosome 3),” we designated this mutation as Pk-l”‘.
Tissue sampling. Mice were weighed, anesthetized by ether, and
killed by exsanguination from the heart. Fresh blood was used for
determination of hematologic and biochemical parameters, enzyme
activities, glycolytic intermediates, adenine nucleotides, and reduced
glutathione. Blood films were prepared and stained with May-Griinwald-Giemsa. The spleen, liver, lung, heart, and kidney were removed and weighed. In some cases, the spleen was embedded in
paraffin; sections were stained with hematoxylin-eosin (H & E) or
with Berlin blue and nuclear fast red to show the deposition of iron.
Hematologic parameters. Three-month-old mice were used to
assess hematologic parameters. The number of RBCs, hematocrit,
hemoglobin level, and osmotic fragility of RBCs were measured
YRUVATE 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 nonspherocytic anemia
due to deficiency of glycolytic enzymes.’ PK deficiency was
first demonstrated in 1961 by Valentine et al.* Thereafter,
more than 300 cases with PK deficiency have been de~ c r i b e d In
. ~ Japan, Miwa and associates found 75 PK-deficient families (Miwa S , Fujii H, Hirono A, Kanno H, unpublished data, 1995). Because some PK-deficient patients show
severe sympt0ms,4,~therapy withnew techniques such as
transfer of normal PK genes into patients’ hematopoietic
stem cells are being considered.6Animal models of the target
diseases are useful to investigate new therapeutic methods.
As PK-deficient animal models, Basenji
and captured
wild mice’”,” have been reported, but both of them are not
easily available.
Approximately 6 years ago, one of us (H.A.) found hereditary splenomegaly in an inbred colony of the CBA/N (hereafter CBA) strain maintained in the Japan SLC Haruno farm
(Shuchi-gun, Shizuoka, Japan). The mutant CBA mice with
splenomegaly were segregated from normal (+/+) CBA
mice and have been kept by brother-sister mating without
further studies. Recently blood cell counts of the mutant
CBA mice with splenomegaly were done, and the presence
of a severe anemia was recognized in these mutant mice.
Because we characterized it as the deficiency ofPK with
hereditary nonspherocytic hemolytic anemia, we describe
here the spontaneously developed PK mutant mice as a potential animal model that may be useful to investigate the
pathophysiology and new therapeutic methods of PK deficiency. The anemia of the mutant was cured by bone marrow
transplantation (BMT) without the prior irradiation of the
hosts, suggesting that the homing of transplanted stem cells
and their differentiation to mature erythrocytes may be easy
in hematopoietic tissues of anemic hosts.
MATERIALSANDMETHODS
Mice. The CBA strain was originally obtained from the National
Institute of Health (Bethesda, MD) and has been maintained by
brother-sister mating at the Japan SLC Haruno farm (Shuchi-gun,
Shizuoka, Japan) since 1984. Approximately 6 years ago, one of US
Blood, Vol 86, No 11 (December l ) , 1995: pp 4323-4330
and PK deficiency of CBA-Pk-lhlPk-lh mice were cured by
bone marrow transplantation (BMT) from CBA-+I+ mice.
Prior irradiation was notnecessary for the curative BMT. On
the other hand, the BMT from CBA-Pk-lh/Pk-l” mice to
nonirradiated CBA-+/+ mice did not result in the decrease
of RBCs and the reduction of PK activity. The present results
indicate that CBA-Pk-ls’C/Pk-l*’cmice are a potentially useful
animal model for studying pathophysiology of PK deficiency
and for developing new therapeutic methods to correct PK
deficiency.
0 1995 by The American Societyof Hematology.
Fromthe Department of Pathology. Osaka University Medical
School, Suita, Osaka; Okinaka Memorial Institute for Medical Research, Toranomon Hospital, Minato-ku, Tokyo; Japan SLC CO Ltd,
Hamamatsu, Shizuoka; the Department of Blood Transfusion Medicine, Tokyo Women’s Medical College, Shinjuku-ku, Tokyo; and the
Department of Laboratory Medicine, Yamaguchi University, Ube,
Yamaguchi, Japan.
Submitted February 17, 1995; accepted August 3, 1995.
Supported by grants from the Ministry of Education, Science and
Culture, and the Ministry of Health and Welfare.
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.
0 1995 by The American Society of Hematology.
0006-4971/95/8611-0037$3.00/0
4323
From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
4324
MORIMOTO ET AL
with standard techniq~es.'~
Reticulocytes were counted on blood
marked difference in hematologic values, no difference was
films stained with brilliant cresyl blue, and thepercentage of reticulodetectable in 16 biochemical genetic markers between anecytes was determined by counting 1,000 RBCs.
mic and nonanemic CBA mice (data not shown), suggesting
Analysis of hemoglobin. Blood samples were stored frozen as
that the splenomegaly and anemia were ascribed to a new
hemolysates. The heat denaturation test,I4isopropanol test," isoelecmutation in the original nonanemic CBA colony rather than
trofocusing,I6and anion exchange high-performance liquid chromato
the contamination of the colony by mice of other strains
tography on a DEAE-3SW column (Tosoh, Tokyo, Japan)" were
with a mutant gene. A11 F, hybrid mice obtained by the cross
performed to study the structure of hemoglobin.
of normal and anemic CBA mice were not anemic (Table
Enzyme activity and contents of reduced glutathione, glycolytic
1). When F, hybrids were mated together, the ratio of normal
intermediates and nucleotides. Three-month-old mice were used
to assess these biochemical parameters. Heparinized whole blood
to anemic offspring was approximately 3: 1 (Table 1). When
was washed three times with ice-cold saline andpassed through
F, hybrids were mated to anemic CBA mice, the ratio of
a column of a-cellulose and microcrystalline cellulose to deplete
normal to anemic offspring was approximately 1:1 (Table
leukocytes and platelets.'' Enzyme activities and contents of reduced
1). The crossing data suggested that the anemia was caused
glutathione in RBCs were measured by the methods recommended
by a mutation of an autosomal recessive gene.
by International Committee for Standardization in H a e m a t ~ l o g y . ' ~ ~ ~ ~
The number of RBCs, hematocrit, and concentration of
Contents of glycolytic intermediates and nucleotides were deterhemoglobin decreased in the anemic CBA mice (Table 2).
mined with the methods described by Minakami et
Reticulocyte counts in the anemic CBA mice were 41.6%,
Survival of RBCs. Packed RBCs obtained from CBA-+/+ or
whereas the values in normal CBA mice and the F, hybrids
CBA-Pk-1""/Pk-l'" mice were diluted three times with Hanks' Balwere 2.2% and 2.4%,respectively (Table 2). The mean celluanced Salt Solution (HBSS) and were incubated for l hour at 37°C
lar volume increased significantly in the anemic mice, and
with1,110 kBq/nL Na;'CrO,(DuPontlNJZN
Research Products,
Boston, MA; 0.071 pg/mL; specific activity, 15.6 MBq/yg).22The
this increase appeared to result from the increased proportion
labeled RBCs were washed twice with HBSS solution and injected
of reticulocytes. The increase of mean cellular hemoglobin
to CBA-+I+ mice via the lateral tail vein in a volume of 0.5 mL.
in the anemic CBA mice was attributed to the increase of
Blood samples of 0.1 mL were obtained from the tail tip on various
mean cellular volume, because no significant difference was
times after the injection, and the radioactivity was measured with a
detectable between anemic and nonanemic CBA mice in the
MINAXI y 5550 gamma counter (Packard, Meriden, CT). To correct
mean
cellular hemoglobin concentration (Table 2).
the spontaneous reduction of radioactivity, blood samples that were
Blood films from anemic CBA mice were compared with
obtained 2 hours after the infusion were kept and usedas the control.
those of nonanemic CBA mice under the microscope. No
The radioactivity of each blood sample was expressed as a percentabnormalities were detectable in the RBC morphology (data
age of the control.
not shown). When the osmotic fragility curve of RBCs from
Proportion of erythroblasts. BM cells and spleen cells were suspended in Eagle's medium (Nissui Pharmaceutical CO, Ltd, Tokyo,
anemic CBA mice was compared with that of RBCs from
Japan) as described previously?' Nucleated cells were counted with
nonanemic CBA mice, the former showed a slight shift toa hemocytometer. The proportion of erythroblasts was determined
ward lower NaCl concentrations (data not shown). Because
by classifying 1,000 nucleated cells in cytocentrifuge specimens
young RBCs from normalhumanRBC populations have
which were stained with May-Griinwald-Giemsa.
been reported to be more resistant to hypotonic salt solutions
Spleen colony-forming unit (CFU-S). Mice of CBA-+/+ were
than old RBCS,*~
the alteration of osmotic fragility curve in
used as recipients and subjected to lethal whole-body irradiation (9.0
anemic
mutants
appeared
rather to be a result of reticuloGy) with an RF-350 x-ray apparatus (Rigaku, Tokyo, Japan). Marcytosis than to be a direct consequence of the mutation.
row and spleen cells of CBA-Pk-1''/Pk-Ish and the control CBA-+/+
Structure of hemoglobin from the anemic CBA mice was
mice were suspended in Eagle's medium as mentioned above; marcompared with that of normal CBA mice by the heat denaturrow cells (5.0 X lo4) and spleen cells (5.0 X lo') were injected into
the lateral tail vein of a recipient within 3 hours after the irradiation.
ation test, isopropanol test, isoelectrofocusing and anion exThe recipients were killed 8 days after the transplantation, and the
change high performance liquid chromatography on a
spleens were procured. Spleens were fixedin Bouin's fluidand
DEAE-3SW (Tosoh) column, but no apparent abnormalities
colonies measuring more than l-mm diameter were counted under
were detectable (data not shown).
the dissection micro~cope.~~
Enzyme activities in RBCs. The activities of RBC enBMT. Various doses of BM cells obtained from CBA-+/+ mice
zymes involved in the glycolytic pathway, pentose-phoswere injected into the tail vein of CBA-Pk-l""/Pk-I"" mice with or
phate shunt, glutathione metabolism, and nucleotide metabowithout theprior whole-body irradiation (6.0 Gy). In one experiment,
lism were assayed. The PK activity in the anemic CBA mice
IO' BM cells of CBA-Pk-IS''/Pk-I'L mice were injected into the
was 16.2% that of the normal CBA mice and the value of
tail vein of nonirradiated CBA-+I+ mice. Numbers of RBCs were
F, hybrids between anemic andnormalCBA
mice was
measured at various days after the transplantation. In some cases,
the activities of PK in RBCs of recipients were measured 15 weeks
3 1.2% (Table 3). The activities of hexokinase and glucoseafter the BMT.
6-phosphate dehydrogenase were significantly higher in the
RESULTS
Hematologic data. Two distinct inbred colonies of the
CBA strain have been maintained in the Japan SLC Haruno
farm; all mice of one colony showed apparent splenomegaly
and anemia, and all mice of the other colony had the spleen
of normal size and normal hematologic values. Despite the
anemic CBA mice than in the normal CBA mice (Table 3).
This may result from the reticulocytosis observed in the
anemic CBA mice. Because the PK activity was specifically
deficient in the anemic CBA mice and F, hybrid mice (Table
3) and because the PK activity of RBCs is controlled by the
Pk-l locus of the mouse (chromosome 3),12 we designated
the mutant locus as Pk-l"".
From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
MICE
PYRUVATE
OF
4325
Table 1. Segregation of Mutant Mica Wkh Splenomegaly and Anemia
No. of Mice With Each Phenotype (Genotype)'
~~
Parents and Cross
Presumed Genotype of Parents
Anemic x anemic
Normal x normal
Normal x anemic
F1 X FI
F1 x anemia
pk- ls'c/pk-Isic x pk- ISrc/pk-Is'c
+It
x +l+
+l
+ X Pk- I"'/PkPk- l'lcf + X Pk- 1"'lf
Pk- 7"f' + X Pk- Iaic/Pk-IS"
217
Nonanemic (Pk-lmrc/+or
Anemic
l'k/Pk-l*lc~
(Pk-
256
0
0
0
359
118
67
75
79
+/+l
Mice approximately 3 months old were used.
* Mice with RBC number <600 x lO'*/L were considered to be anemic, and mice with RBC number >g00 x 101zlLto be nonanemic. Male
and female mice were pooled because no significant difference was detectable between them.
Levels of glycolytic intermediates, reduced glutathione,
and nucleotides. The effect of PK deficiency on the energy
metabolism of RBCs was evaluated by measuring the concentrations of intermediate metabolites. The concentration
of pyruvate, the product of PK, decreased significantly in
RBCs of CBA-Pk-I"'/Pk-I"" mice, but the concentrations of
other glycolytic intermediates, which are proximal to pyruvate, increased significantly (Table 4).These findings were
compatible with the fact that the glycolysis was blocked at
the step catalyzed by PK. Reduced glutathione content was
increased in homozygotes, probably because of the young
mean RBC age. Despite severe blocking of glycolysis, the
adenosine triphosphate (ATP) content increased in RBCs of
CBA-Pk-I""/Pk-I"" mice. In severe PK deficiency, RBC
ATP is produced almost exclusively by the oxidative phosphorylation." Therefore, the increased ATP levels may be
explained by the predominance of reticulocytes in CBA-Pkls~c/Pk-ls~c
mice. Increased contents of the upstream glycolytic intermediates such as 2,3-diphosphoglycerate and 3phosphoglycerate were observed in CBA-Pk-I""/+ mice
(Table 4),suggesting that the extent of PK deficiency was
severe enough to impair the glycolysis in the heterozygotes.
In contrast to the increase of ATP contents in CBA-PkI""/Pk-l"[" RBCs, the ATP content of CBA-Pk-I""/+ RBCs
decreased significantly when compared with the value of
CBA-+/+ RBCs (Table 4).This is consistent with the result
reported in human cases; the ATP levels are lower in mild
PK deficiency than in severe PK deficiency." RBCs of heterozygous CBA-Pk-I""/+ mice, as those of patients with
mild PK deficiency, may survive long enough to show the
impaired production of ATP by glycolysis. On the other
hand, mature RBCs of CBA-Pk-I""/Pk-I"" mice maynot
survive by glycolysis as in the case of severe form of human
PK deficiency. Inaddition, the content of nicotinamide dinucleotide was significantly decreased in heterozygous CBAPk-l""l+ mice, as reported in human PK deficiency."
Demonstration of RBC destruction. CBA-Pk-1"''/Pk-1""
mice exhibited hyperbilirubinemia because of the increase
of indirect bilirubin (Table 4). To show the accelerated destruction of RBCs in CBA-Pk-l"'c/Pk-l"'cmice, we labeled
RBCs obtained from either CBA-+l+ or CBA-Pk-I""/PkIs' mice with"Cr. The labeled RBCs were injected into
CBA-+'+ mice; the disappearance of RBCs derived from
CBA-Pk-I""/Pk-I"" mice was much faster than that of RBCs
from CBA-+/+ mice (Fig 1). In some cases, blood samples
were obtained immediately after the injection. The recovery
rate of labeled RBCs was calculated on the assumption that
total bloodvolume is 10% of bodyweight. Because the
Table 3. Activity of Various Enzymea in RBCs of CBA-+I+. -Pk-l*/
+, and -Pk-l*/Pk-lmkMice
Activity in Mice of Each Genotype
(univgram hemoglobin)*
Enzymes
Table 2. Hematologic Data in CBA-+/+, -Pk-l*'c/+
and
-Pk-lak/Pk-l*Mice
Values in Mice of Each Genotype*
Hematologic
Parameters
RBCs (x lO"/L)
Hematocrit (%)
Hemoglobin (g/dL)
Mean cellular
volume
(fL)
Mean cellular
hemoglobin (pg)
Mean cellular
hemoglobin
concentration
(gldL)
Reticulocytes (%)
l"'/Pk-
+I+ Pk-
Pk-
lek
1,020 f 64 (10)
48 f 1 (10)
15 f 1 (10)
999 f (18)
6
50 f l ( 1 8 )
16 f 1 (18)
536 f 11 (10)t
33 f l (10)t
10 f 1 (10)t
51 t 3 (10)
50 f 1 (18)
61 f 1 (10)t
16 f 1 (10)
16 2 l ( 1 8 )
19
31 2 l ( 1 0 )
2.2 f 0.2 (101
32 f l ( 1 8 )
2.4 t 0.3 (10)41.6
c 1 (10)t
31 f 1 1101
f 1.5 (10)t
Mice 3 months old were used.
Mean f SE. Number of mice is shown in parentheses,
t P < .01 when compared with values of CBA-+I+ mice by the t-test.
Hexokinase
Glucosephosphate isomerase
Phosphofructokinase
Aldolase
Triosephosphate isomerase
Glyceraldehyde-3-phosphate
dehydrogenase
Phosphoglycerate kinase
Monophosphoglyceromutase
Enolase
Pyruvate kinase
Lactate dehydrogenase
Glucose-&phosphate
dehydrogenase
BPhosphogluconic
dehydrogenase
Glutathione reductase
Glutathione peroxidase
Adenylate kinase
Adenosine deaminase
+I+
3.0 f 0.1
140 f 2
11 f 1
2.9 f 0.1
1,200 f 28
202 f 4
1 8 81f 8- 26 f 2
6.3 f 0.7
12 f 1
25.3f 0.4
271 f 2
P&- l*"/+
Pk- lak/Pk-l"
3.0 f 0.1
153 f 1
10 f 1
3.2 ? 0.1
1.400 f 22
8.5 f O.lt
176f 2t
lo? 1
6.5 f O.lt
1,984 f 53t
218 f 6
340 f 13t
223 2 4t
16.2 f 1.2t
26 f 2t
4.1 f O.lt
427 f l l t
8.0 c 0.4
12 f 1
7.9 f O.lt
303 f 5
24 f 1
29 t 1
47 f I t
7.2 f 0.1
10 f 1
381 f 1 1
5.7 f 0.2
2.9 ? 0.1
8.1 f 0.1
102 1
3 8 9 2 12
4.8 f 0.1
2.8 f 0.1
10.6 f 0.2t
18 f I t
441 + 3 7
17.8 f 0.8t
3.9 t 0.2t
Mice 3 months old were used.
Mean f SE of 5 mice.
t P < .01 when compared with values of CBA-+I+ mice by t-test.
From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
4326
MORIMOTO ET AL
Table 4. Levels of Glycolytic Intermediates, Nucleotides, Reduced Glutathione in RBCs and Bilirubin in Plasma
of CBA-+I+. -Pk-lWk/+, and -Pk-l"c/Pk-l*" Mice
Values in Mice of Each Genotype*
+/+
Biochemical Parameters
Glucoset
Pk-
9,380 t 338 (8)
79 t 5 (10)
46 t 5 (10)
17 2 2 (10)
34 2 5 (IO)
15 t 2 (10)
9,460 2 368 (10)
47 ? 4 (10)
17 t 1 (10)
22 +- 2 (10)
106 2 5 (10)
4,300 t 220 (7)
70 2 1 (5)
1,430 t 26 (10)
218 ? 49 (4)
255 2 8 (8)
0.45 ? 0.13 (IO)
0.32 t 0.02 (10)
0.13 i 0.04 (10)
Glucose-6-phosphate*
Fructose-6-phosphate*
Fructose-1.6-diphosphate*
Dihydroxyacetone phosphate*
Glyceraldehyde-3-phosphate*
2.3-DiphosphoglycerateS
3-Phosphoglycerate*
2-Phosphoglycerate*
Phosphoenolpyruvate*
Pyruvatet
Lactatet
Reduced glutathione*
Adenosine triphosphate*
Adenosine diphosphate*
Nicotinamide dinucleotide*
Total bilirubin§
Direct bilirubin§
Indirect bilirubin§
9,730 t 319 (9)
70 t 6 (9)
33 t 3 (9)1/
18 2 1 (6)
39 i 2 (9)
19 t 3 (9)
11,090 t 181 (9)Il
90 t 5 (9)lj
18 t 1 (9)
40 +- 4 (9)11
87 t 3 (9111
4,000 2 207 (9)
72 t 1 (91
950 t 22 (9)11
220 2 19 (9)
177 t 7 (9111
0.45 ? 0.08 (10)
0.35 2 0.03 (10)
0.11 2 0.08 (10)
Pk- l"'/Pk. l"'
10,130 2 349 (10)
212 t 7 (9)ll
116 i- 6 (10)11
112 t 9 (10)
185 ? 7 (lO)/l
69 i 11
12,490 t 576 (10)/1
504 -c 20 (10)/1
75 2 4 (10)/1
261 t 12(1O)ll
70 2 5 (1O)Il
4,060 i 276 (IO)
109 t 1 (5111
2,100 ? 32 (10)/1
449 t 25(10)1!
300 t 16 (10)11
0.92 -C 0.08 (10)/1
0.31 2 0.03 (10)
0.61 t 0.06(1O)Il
Mice 3 months old were used.
* Mean 2 SE, number of mice is shown in parentheses.
t Data expressed in nmol/mL whole blood.
* Data expressed in nmol/mL RBCs.
§
Data expressed in mg/dL plasma.
I/ P < .01 when compared with values of CBA-+/+ mice by t-test.
1000
Pk- 7'"/pk- 7'"
80-
60-
4020 0-
I
I
I
1
30
20
Days after RBC Transfusion
0
10
Fig 1. The fasterelimination of CBA-Pk-l&/Pk-F RBCI from the
circulationof CBA-+I+ mice.RBCsfrom CBA-P&-FP&-l" (01and CBA+I+ (0)
micewerelabewwith 6'Crandtrandud intoCBA-+/+ mice.
The radioactivity retained in RBCs was measured at verious times after
the transfusion. Each point
the mean of Gght mice.
arB
the standard error. In some points the standard error was too small to
be shown by bars.
recovery rate was approximately 90%, the RBC survival
curve shown in Fig 1 appeared to reflect the behavior of
the majority of labeled RBCs. When half-life of RBCs was
calculated according to the method recommended bythe
International Committee for Standardization in Haematology,22 the value for CBA-+I+RBCs was approximately
13.3 days, whereas that of CBA-Pk-I"'~/Pk-I""' RBCswas
approximately 2.2 days.
Reaction to hemolysis. CBA-Pk-I""/Pk-I"' micegained
body weight normally at least upto 10 weeks of age; no
significant difference was detectable in body weight between
CBA-Pk-l""/Pk-I"" and C B A - + / + mice of both sexes. The
spleen weight of male CBA-Pk-I""/Pk-I~""
mice was 4.6
times as great as that of male C B A - + / + mice and that of
female CBA-Pk-1""/Pk-1"'' mice was 6.4 times as great as
that of female C B A - + / + mice. Despite the significant enlargement of spleen in CBA-Pk-I"'/Pk-I"" mice, no significant change was observed in the weight of the liver, lung,
heart, and kidney of CBA-Pk-i""/Pk-I"' mice (data not
shown). When the enlarged spleen of CBA-Pk-l""/Pk-I""
mice was examined histologically, marked hemosiderosis
was observed in the red pulp (Fig 2, A and B ) . In addition
to hemosiderosis, erythropoiesis was remarkably enhanced
in the red pulp of CBA-Pk-1"'/Pk-I"'' mice (Fig 2, C through
E).
The total number of nucleated cells inthe femur and
spleen, the proportion of erythroblasts andthatof C W - S
were compared between C B A - + / + and CBA-Pk-I""/Pk-I"'
mice. The total number of nucleated cells and proportion Of
erythroblasts were significantly greater in CBA-Pk-I'"/Pk-
From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
PYRUVATEKINASEDEFICIENCYOFMICE
4327
Fig 2. Deposition of iron and enhancederythropoiesis in the spleen of the CBA-Pk-T*/Pk-T* mouse. (A) The spleen of a CBA-+I+ mouse
stained with Berlin blue and counterstained with nuclear fast red, original magnification (OM) x 80. (B) The spleen of a CBA-Pk-T*/Pk-pk
mouse stained with Berlin blue and counterstainedwith nuclear fast red, OM x 80. The deposition of iron isapparent. (C) The spleen of the
CBA-+I+ mouse stained with H & E, OM x 150. (D) The spleen of the CBA-Pk-pk/Pk-7* mouse stained with H & E, OM x 150. Enhanced
erythropoiesis in the red pulp is shown. (E) A higher magnifmtion of (D), showing increased erythroid cells in the red pulp, OM x 1250.
Asterisks in (A-D) indicate white pulps.
I"" mice than in CBA-+/+ mice (Table 5). The difference
in both parameters was much moreremarkable in the spleen
than in the femur. Although the proportion of CFU-Sin
the femur was comparable between CBA-Pk-I"'C/Pk-IS" and
CBA-+/+ mice, the total number of CFU-S was greater in
CBA-Pk-IsfC/Pk-Isfc
mice than in CBA-+/+ mice because of
the increase of the nucleated cells. Because both the number
of nucleated cells and the proportion of CFU-S increased in
the spleen of CBA-Pk-~sfc/Pk-ls'c
mice, the total number of
C m - S was much p a t e r in the spleen of CBA-Pk-Y'PkI"" mice than in the spleen of CBA-+/+ mice (Table 5).
Cure of anemia with BMT. Because severe hereditary
hemolytic anemia in Basenji dogs related to PK deficiency
is cured byBMT from nonanemic histocompatible lit-
Table 5. Numbers of Erythroblastsand CFU-S in the Femur and Spleen of CBA-+/+ and -Pk-T.k/Pk-l"
No. of
Nucleated Cells
Organ
Femur
Genotype
+l+
l'"/PkPk-
Spleen
+I+
l*"/PkPk-
* Mean 2
l'"
l*"
(X109*
15 2 1
23 2 l §
8
312t12
321 2 54
195
Proportion of
Erythroblasts
(%)*
No. of
Erythroblasts
(X109*
36 2 2
53 2
12.3
35
5.4
6.32 0.3
t 5.9
0.85
0.49
2.6 2 0.2
2172
35
t 101
Mice
Proportion of CFU-S
(per 5 x lo4 cells)t
1.89t 0.7
t
2.7
01
.6
0.302 0.05
1.78 t 0.100
11.43
SE of 5 mice.
t Mean t SE of 8 recipients.
*The value was calculated from the numberof nucleated cells and the proportion of CFU-S per 5.0 x
5 P .01 when compared with values of CBA-+I+ mice by t-test.
lo4 nucleated cells.
No. of CFU-S
( ~ 1 0 3 ~
t 0.21
2 0.275
t 0.04
2 0.650
From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
4328
MORIMOTO ET AL
term ate^:^^^' BMT was carried out from CBA-+/+ to CBAPk-I""/Pk-l"~'mice. Transplantation of 2 X lo7 BM cells
from CBA-+/+ mice normalized the number of RBCs in
sublethally irradiated (6.0 Gy) CBA-Pk-I"'"/Pk-I"'' mice (Fig
3). Even in genetically normal recipients, irradiation is not
prerequisite for the successful engraftment of hematopoietic
stem cells."~'* When huge numbers ofBM cells were injected to nonirradiated normal mice, homing and differentiation of the donor cells were observed. In CBA-Pk-I""/PkI"" mice, the spleen enlarged and many CFU-S were present
in the spleen. Because transplanted stem cells appeared to
settle more easily in the spleen than in the BM," we transplanted various doses of +/+ BM cells to nonirradiated
CBA-Pk-I"'c/Pk-I"'cmice. Normalization of RBCnumber
and PK activity was observed in nonirradiated CBA-Pk-l""/
Pk-l"" mice that received 5 X lo7 or lo8 BM cells from
CBA-+/+ mice (Fig 3, P < .01 when compared with nontreated CBA-Pk-I""/Pk-I"" mice). The BMT of the opposite
direction was also attempted. The transplantation ofBM
cells (lo8)from CBA-Pk-I"''/Pk-1"" mice to CBA-+/+ mice
did not result in the decrease of RBCs and the reduction of
PK activity in the recipients (Table 6).
DISCUSSION
The mutant CBA mice with remarkable splenomegaly
showed nonspherocytic hemolytic anemia. Iron deposition
and enhanced erythropoiesis were observed in the enlarged
spleen, suggesting that the cause of the anemia was hemolysis. RBCs of the anemic CBA mice showed an accelerated
121
I
7
25. 8 -
8
'c
6-
0
o 2x107 Cells with Rad
1x1 O8 Cells without Rad
m 5x107 Cells without Rad
A 2x1 O7 Cells without Rad
0
0
1
A
Control
t
-//m
0
1
2
5
10 20
50
Weeks after Transplantation
Fig 3. Cure of anemia in CBA-Pk-l.h/Pk-l"c mice after BMT from
CBA-+I+ mice. Various numbers of BM cells were injected into the
irradiated or nonirradiatedCBA-Pk-ldc/Pk-l"c mica. Each point represents the mean of eight mice. (0).
BM cells, 2.0 x lo', were injected
to sublethally (6.0 Gy) irradlated hosts; (01,1.0 x lb BM cells were
injected to nonirradiatedhosts; (m), 5.0 x IO' BM cells were injected
to nonirradiatedhosts; (A),2.0 x IO' BM cells were injectedto nonirradiated hosts; (A), control CBA-Pk-T*/Pk-7* mice without irradiation and BM cell transplantation. Bars are the standard error. In some
points the standard error was too smell to be shown by bars.
Table 6. Number of RBCs and PK Activity 15 Weeks Aftor BMT
Mice
Nontreated CBA-Pk-18'c/Pk-Is"
Nontreated CBA-+/+
CBA-Pk-I"'/Pk-7'" transplanted
from C B A - t / t
CBA-+I+ transplanted from
CBA- Pk- leIc/Pk-l'"
No. of RBCs'
( X 1O ' V U
PK Activity'
(unitJgram
hemoglobin)
554 f 15 (9)
1,025 f 28 (9)
4.7 f 0.1 (8)
25.2 t 0.5 (8)
1,016 f 16 ( 7 ) t
21.5 -+ 1.1 (6)t
1,031 f 14 (7)
25.1 f 1.9 (7)
BM cells (1.0x lo8)were transplanted without the prior irradiation
of hosts.
* Mean f SE. Number of mice is shown in parentheses.
t P i.01 when compared with values of nontreated mice of the
same genotype by f-test.
destruction even in the circulation of normalCBA mice,
indicating that the accelerated destruction of mutant RBCs
was not ascribed to an extrinsic but to an intrinsic defect.
RBCs of the mutant CBA mice showed neither the abnormal
RBC morphology nor the increase of osmotic fragility. No
abnormalities of hemoglobin were found in RBCs of the
mutant CBA mice either. Examination of RBC enzymes revealed that the PK activity of the homozygous mutants decreased to 16.2%and that of heterozygous mutants to 31.2%
that of normal CBA mice. The PK activity of reticulocytes
is 16.7 times as great as the PK activity of mature erythrocyte~.'~
Since
, ~ ~ proportion of reticulocytes was very high in
anemic CBA mice (41.6%),only 13.2%of the PK activity
was ascribed to that of mature RBCs in these mutant mice.
On the other hand, 74.3% of the PK activity was ascribed
to that of mature RBCs in normal CBA mice. As a result,
when the PK activity of mature RBCs was compared between anemic and normal CBA mice, the value in the former
mice wasonly 2.8% that of the latter mice. The content
of pyruvate inRBCs decreased inboth homozygous and
heterozygous mutants, but the content of glycolytic intermediates located to the upstream of PK increased. Taken together, the cause of the hemolytic anemia in the mutant CBA
mice was considered to be the PK deficiency. Because PK
of RBCs is encoded by the Pk-I locus of mice (chromosome
3),'* we designated the present mutant allele as Pk-l'".
The PK activity of the heterozygous Pk-l%/+ mutants
decreased to 31.2% that of normal CBA mice. Because the
Pk-I"" gene was a single loss-of-function mutation, the enzymatic activity might be 50% of normal. Because PK is a
tetrameric protein with allosteric domain interdependence,'.'
there is a possibility that the 30% activity may be related to
defective interactions of the protein. However, the Pk-l"" is
an active site mutation as shown by Kanno et
in the
accompanying report, suggesting that the tetrameric conformation might not be interfered severely by the mutation. We
speculate that the mutation diminishes the PK activityof
both the mutant homotetramers and heterotetramers in RBCs
of Pk-l""/+ mice and that only homotetramers of the normal
peptides show the normal activity.
Symptoms of CBA-Pk-I"'/Pk-l"" mice are similar to those
of human PK defi~iency'~
and those of Basenji dogs with PK
defi~iency.~.~
In humans, PK deficiency is the most common
From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
PYRUVATE KINASE DEFICIENCY
4329
OF MICE
hemolytic anemia caused by the defect of glycolytic enzymes, and more than 300 cases have been reported. In the
most severe form, death might occur during perinatal and
neonatal periods.38Most of all CBA-Pk-ls'c/Pk-lsk mice appeared to survive to adulthood because segregation of homozygous mutants in adult population was consistent with the
Mendelian law. Adult CBA-Pk-Islc/Pk-Is'' mice appeared to
be as active as CBA-+/+ mice throughout the observation
period (up to 9 months after birth), probably because the
oxygen dissociation curve of hemoglobin was shifted to the
right bythe elevated 2,3-diphosphoglycerate level in the
affected -CS? The molecular characterization of the PkIStc mutant gene and the biochemical characterization of its
product are described by Kanno et
in the accompanying
report.
Because CBA-Pk-ISiC/Pk-IS"
mice developed as a mutant
in an inbred colony of the CBA strain, transplantation between anemic and nonanemic populations of CBA mice is
easy. In fact, BMT from the CBA-+/+ mice cured the anemia of irradiated CBA-Pk-I""/Pk-I"' mice. Moreover, transplantation of BM cells from CBA-+/+ mice cured the anemia even in nonirradiated CBA-Pk-I"''/Pk-I"'' mice. The PK
activity of RBCs increased to the level observed in CBA+/+ mice. On the other hand, the transplantation of BM
cells from CBA-Pk-Is''/Pk-IS" mice to CBA-+/+ mice did
not result in development of anemia; the PK activity of RBCs
did not decrease after the BMT. There are two possibilities
that may explain the different effect of BMT between CBAPk-ls'c/Pk-l''c and CBA-+/+ hosts. ( l ) The homing of transplanted stem cells to hematopoietic tissues may be easier
in CBA-Pk-Is'C/Pk-l"'cmice than in CBA-+/+ mice. The
increase of CFU-S pool in the spleen and BM of CBA-Pklslc/Pk-ls~c
mice may facilitate the homing of the transplanted
stem cells. (2) The efficiency of homing may be comparable
between CBA-Pk-I""/Pk-I"' and CBA-+/+ hosts. However,
the life span of CBA-+/+ RBCs is much longer than that
of CBA-Pk-I"c/Pk-I"'c RBCs. As a result, RBCs of CBA+/+ donor origin may accumulate inCBA-Pk-l''c/Pk-Is'c
hosts. On the other hand, RBCs produced by CBA-Pk-I"'/
Pk-I"' stem cells disappeared soon after the differentiation
and therefore the RBC number and the PK activity did not
decrease in CBA-+/+ hosts. These two possibilities are not
mutually exclusive, and bothmay occur after the BMT.
However, the second mechanism appears to be more probable than the first mechanism because the present data supported the shortened RBC survival and did not necessarily
support the facilitated engraftment.
The mechanism of the curative BMT in nonirradiated
CBA-Pk-I"'/Pk-1"" mice remains to be studied. Especially
gene marking studies of hematopoietic precursors could be
used to assess stability over time of the nucleated erythroid
lineage specific progenitors and could provide some answers.
Y chromosome-specific sequences may be used as a genetic
marker. Moreover, Kanno et
characterized the Pk-I""
mutation in the accompanying report. As a result of the
single nucleotide substitution at no. 1013, GGT to GAT,
- a
BstPI recognition sequence was lost in the Pk-I"" mutant
allele. We are now planning to use this as a genetic marker
of hematopoietic precursors. Taken all together, the CBA-
Pk-I"''/Pk-1'" mouse is a useful animal model for understanding the pathophysiology of PK deficiencyand for developing new therapeutic methods of severe PK deficiency.
REFERENCES
1. Miwa S, Kanno H. Fujii H: Pyruvate kinase deficiency: Historical perspective and recent progress of molecular genetics. Am J
Hematol 42:31, 1993
2. Valentine WN, Tanaka KR, Miwa S: A specific erythrocyte
glycolytic enzyme defect (pyruvate kinase) in three subjects with
congenital non-spherocytic hemolytic anemia. Trans Assoc Am Physicians 74:100, 1961
3. Tanaka KR, Paglia D E Pyruvate kinase and other enzymopathies of the erythrocyte, in Scriver CR. Beaudet AL, Sly WS, Valle
D (eds): The Metabolic and Molecular Bases of Inherited Disease
(ed 7). New York, NY, McGraw-Hill, 1995, p 3485
4. Tanaka KR, Valentine WN, Miwa S: Pyruvate kinase (PK)
deficiency hereditary non-spherocytic hemolytic anemia.Blood
19:267, 1962
5. Bowman HS, Procopio F: Hereditary non-spherocytic hemolytic anemia of the pyruvate kinase deficient type. Ann Intern Med
58567, 1963
6 . Tani K, Yoshikubo T, lkebuchi K, Takahashi K, Tsuchiya T,
Takahashi S, Shimane M, Ogura H, Tojo A, Ozawa K, Takahashi
Y, Nakauchi H, Markowitz D, Bank A, Asano S : Retrovirus-mediated gene transfer of human pyruvate kinase (PK) cDNA into murine
hematopoietic cells: Implications for gene therapy of humanPK
deficiency. Blood 83:2305, 1994
7. Tasker JB, Severin GA, Young S: Familial hemolytic anemia
in the Basenji dog. J Am Vet Med Assoc 154:158, 1969
8. Ewing GO: Familial nonspherocytic hemolytic anemia of Basenji dogs. J Am Vet Med Assoc 154503, 1969
9. Seacy GP, Miller DR, Tasker JB: Congenital hemolytic anemia
in the Basenji dog due to erythrocyte pyruvate kinase deficiency.
Can J Comp Med 3567, 1971
10. Bulfield G, Moore K Screening and study of inherited enzyme deficiencies of erythrocyte glycolysis and associated pathways
in wild mice. Hereditas 89:140, 1978
11. Moore K, BulfieldG: An allele ( f k - l b ) from wild-caught
mice that affects &heactivity and kinetics of erythrocyte and liver
pyruvate kinase. Biochem Genet 19:771, 1981
12. Fitton LA, Bulfield G: The livederythrocyte pyruvate kinase
gene complex [ f k - I ] in the mouse: Structural gene mutations. Genet
Res 53:105, 1989
13. Rodak B F Routine laboratory evaluation of erythrocytes, in
Lotspeich-Steininger CA, Stiene-Martin EA, Koepke JA (eds): Clinical Hematology. Philadelphia, PA, Lippincott, 1992, p 107
14. Dacie JV, Grimes AJ, Meisler A, Steingold L, Hemsted EH,
Beaven GH, White JC: Hereditary Heinz body anemia: A report of
studies on 5 patients with mild anemia. Br J Haematol 10:388, 1964
15. Carrell R, Kay R: A simple method for the detection of unstable haemoglobin. Br J Haematol 23:615, 1972
16. Basset P, Beuzard Y, Garel MC, Rosa J: Isoelectric focusing
of human hemoglobin: Its application to screening, to characterizationof 70 variants, and to study of modified fractions of normal
hemoglobins. Blood 5 I :97 1, 1978
17. Ohba Y, Imai K, Kumada I, Osawa A, Miyaji T: Hb Moriguchi or a20297 (FG4)His Tyr. Substitution at the a102 interface.
Hemoglobin 13:367, 1989
18. Beutler E, West C, Blume KG: The removal of leukocytes
and platelets from whole blood. J Lab Clin Med 88:328, 1976
19. Beutler E, Blume KG, KaplanJC, Lohr GW, Ramot B, Valentine W: International Committee for Standardization in Haematol+
From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
4330
ogy:Recommendedmethodsfor
red cell enzymeanalysis.BrJ
Haematol 35:331, 1977
20. International Committee for Standardization in Haematology:
Recommended methods for an additional red cell enzyme (pyrimdine 5’-nucleotidase) assay and the determination of red cell adenosine-S’-triphosphate,2,3-diphosphoglycerateandreducedglutathione. Clin Lab Haematol 11:131, 1989
21. Minakami S, Suzuki C, Saito T, Yoshikawa
H: Studieson
erythrocyte glycolysis.1. Determinations of the glycolytic intermediates in human erythrocytes. J Biochem (Tokyo)
58543, 1965
red-cell
22. Glass HI:Recommendedmethodsforradioisotope
survival studies: A report by the ICSH panel on diagnostic applications of radioisotopes in haematology. Br J Haematol 21:241, 1971
23. Kitamura Y, Kawata T, Suda 0, Ezumi K: Changed differentiation pattern of parentalcolony-formingcells in F , hybridmice
sufferingfromgraft-versus-hostdisease.Transplantation
10:455.
1970
24. Tamai M, Kitamura Y: Hematologic changes during spleen
colony development in nonirradiated mice. Blood 52:1148, 1978
25. MarksPA,JohnsonAB:Relationship
between theage of
human erythrocytes and their osmotic resistance: A basis for separating young and old erythrocytes. J Clin Invest 37:1542, 1958
26. Mentzer WC, Baehner RL, Schmidt-Schonbein H, Robinson
SH,NathanDG:Selectivereticulocytedestruction
in erythrocyte
pyruvate kinase deficiency. J Clin Invest 50:688, 1971
27. Keitt AS: Pyruvate kinase deficiency and related disorders of
red cell glycolysis. Am J Med 41:762, 1966
28. Zerez CR, Tanaka KR: Impaired nicotinamide adenine dinucleotide synthesis in pyruvate kinase-deficient human erythrocytes:
A mechanism for decreased total NAD content and a possible secondary cause of hemolysis. Blood 69:999, 1987
29. WeidenPL,Storb
R, GrahamTC,Schroeder
ML: Severe
MORIMOTO ET AL
hereditaryhaemolyticanaemia
in dogs treated by muTow Iransplantation. Br J Haematol 33:357, 1976
30. Weiden PL. Hackman RC, Deeg J, Graham TC. Thomas ED,
Storb R: Long-termsurvivaland
reversal of iron overloadafter
marrow transplantation i n dogs with congenital hemolytic anemia.
Blood 57:66, 1981
31. Brecher G. Ansell JD, Micklem HS, Tjio JH, Cronkite EP:
Special proliferative sites are not needed for seeding and proliferation of transfused bone marrow cells in normal syngenic mice. Proc
Natl Acad Sci USA 79:5085, 1982
32. StewartFM.CrittendenRB,
Lowry PA,Pearson-White S,
Quesenberry PJ: Long-term engraftment of normal and post-5-fluorouracil murine marrow into normal nonmyeloablated mice. Blood
8 1 :2566, l993
33. Lahiri SK, Van PuttenLM:Distributionandmultiplication
of colony formit:? units from bone marrow andspleen after injection
i n irradiated 1nic.e. Cell Tissue Kinet 2:21, 1969
34. Lakomek M. Schroter W, De Maeyer G, Winkler H: On the
diagnosis of erythrocyteenzymedefects
in thepresenceofhigh
reticulocyte counts. Br J Haematol 72:445, 1988
35. Zimran A, Torem S, Beutler E: Thein-vivoageing of red
cell enzymes: Direct evidence of biphasic decay from polycythaemic
rabbits with reticulocytosis. Br J Haematol 69:67, 1988
36. Kanno H, Morimoto M, Fujii H, Tsujimura T, Asai H, Noguchi T, Kitamura Y, Miwa S: Primary structure of murine red celltype pyruvatekinase (PK) andmolecularcharacterization
ofPK
deficiency identified in the CBA strain. Blood 86:3205, 1995
37. Tanaka KR, Zerez CR: Red cell enzymopathies of the glycolytic pathway. Semin Hematol 27: 165, 1990
38. Bowman HS, McKusick VA, Dronamraju KR: Pyruvate
kinase deficienthemolyticanemia in anAmishisolate. Am J Hum
Genet 17: I , 196.5