We have studied a female mongrel dog found in Kanagawa Prefecture, Japan. This dog was selected and examined thoroughly because she naturally ...
Exp.
Anim. 37(2), 187-190,
1988
Note
A Dog Possessing with
High
an Increased
Glutathione
Na, K-ATPase
(GSH) and K Concentrations Activity
in its Erythrocytes
Eni OGAWA, Hiroshi FUJISE and Kosaku KOBAYASHI* Deparhnentof PathologyII and Internal Medicine II*, Schoolof Veterinary Medicine, AzabuUniversity, Feuchinobe, Saganiihara,Kanagawa229,Japan (Received3 April 1987/Accepted 8 December1987) We have studied a female mongrel dog found in Kanagawa Prefecture, Japan. This dog was selected and examined thoroughly because she naturally maintained a high glutathione (GSH) concentration in her erythrocytes and did not exhibit any clinical signs or hematologic disorders. Erythrocytes from this animal demonstrated high K and low Na concentrations, as well as accumulation of the amino acids, glutamic acid, aspartic acid and glutamine. The Na, K-ATPase activity was also markedly elevated and the osmotic fragility of the dog's erythrocytes was found to be significantly increased. Crossbreeding of our dog with a normal dog and also with a heterozygous carrier dog revealed that the genetic abnormality possessed by our dog is transmitted as an autosomal recessive trait. All of the clinical data obtained from studying this animal strongly suggest that it possesses a genetic trait similar to that of the HK dogs previously described by Maede.
During the process of selecting clinically and hematologically normal dogs, we discovered a female mongrel dog which showed an increased glutathione (GSH) concentration in her erythrocytes but did not exhibit clinical signs or hematologic disorders. GSH is known to be a reducing agent in erythrocytes, protecting the sulf hydryl groups in the hemoglobin and red cell membrane from oxidation. Previously, Maede found two families of dogs in Hokkaido, Japan, which also demonstrated increased GSH concentration in their erythrocytes and the genetic inheritance of this abnormality was determined to be by the recessive autosomal mode [5]. It has been documented that the accumulation of GSH was the result of an increased uptake of glutamic acid due to the high level of Na, K-ATPase
activity in the red cell membrane [4, 6, 7]. Moreover, owing to increased activity of the enzyme, these erythrocytes also had high K and low Na concentrations, which is the opposite of the condition encountered in normal canine erythrocytes which almost completely lack this enzyme [7]. Therefore, erythrocytes exhibiting high K, low Na and high GSH levels are called HK cells and dogs that possess these HK cells are referred to as HK dogs [8]. We conducted both hematological and biochemical studies on our subject dog and normal dog (control group) following the protocol developed by Maede in his earlier report on HK dogs [6, 7]. Our subject animal, the female mongrel dog from Kanagawa, Japan, was mature and weighed 7 kg when we began our study. We
188
were
unable
or
to
include
study
she
with
GSH
subject
and
dog
study and
(ED-1,
Sanwa
before
the
nations
were
methods. by
al.
the
[1].
osmotic
[10].
The
measured the
hemolyzed
the
the
a
year
examilaboratory deter-
by
Beutler
values
method
were
of
Parpart
ATPase of of
activity
Nakao
the cells
red
deproteinized
food
was
method
determination washed
and
for
standard
erythrocyte
by
[9]. For concentrations,
Ltd.)
fragility
to
in
dog
described
according
al.
housed
dry
concentration
procedure
The
calculated
by
inOur
hematological
performed GSH
were
were a
Co.
in dogs
group.
dogs
The
con-
dogs
normal
fed
our
camp
adult
control
Chemical
The
mined
a
age
in
unowned
and
study.
a
healthy
control
kennels
in
as
as
her
family
found
accepted
our
individual
her
Eight
levels
in
was
was
Japan.
cluded
determine
from
abandoned
Yokohama,
et
dogs
because
taining
et
accurately
other
by
et
amino [2]
the
al.
acid were
procedure Fig.
described
by
acids
were
acid
analyzer
single
Co.
Table
1
the
creased
packed
reticulocyte volume
strated
in
In Fig.
markedly dogs findings mongrel previously
within
the
control
count
the our
as
when
(control
group).
obtained were
reported
demon-
fragility
of
subject
dog
compared The
from
our
consistent for
with
is clearly
GSH
increased
dog
and
concentra-
osmotic high
in-
group
compared
addition, 1,
(retic.) were
control
decreased
from
the mean
were
(MCV)
the
count
the
hemoglobin
was
erythrocytes
data
with
corpuscular
normal
by
The
dogs.
female
(MCH)
cell
(MCHC)
and
defined
mean
normal cell
(PCV),
(Hb)
as
compared
blood
volume
dogs.
mean
logical
cell
bio-
examination with
red
the
was
the
fragility dog
normal
obtained dogs
with
curves the
dogs from (control
for
increased (• ).
group)
erythrocytes
GSH The
mean
the
level
normal
values and
(•¡) curve
from
four
the
bars
were
and
the
the
represent•}SD.
(Dainiseiko
compared The
range
normal
the
dog
hemoglobin
of
was
from
concentration
the
tion
concentrations
Osmotic
normal
citrate
hematologic
group).
normal
the
the
GSH
corpuscular
and
a
measuring
lithium
1. from and
using
71)
photometer
obtained
high
hemoglobin
group
amino
by
K
flame
lists
data
(control
the
automatic
graded
and a
amino
SAS-727).
chemical
(RBC),
a
Na
with
our
an
The
(ATTO-SC
with
Model
dogs
[7].
(ATTO-MLC203),
The
measured
al.
with
column
mm,
buffer.
et
analyzed
resin
4 •~ 250
of
Maede
with hematosubject
with HK
dog
the by
Maede [7]. The erythrocytes from our dog exhibited a greatly increased GSH level, and high K and low Na concentrations with elevated Na, K-ATPase activity, which were almost the same as the mean values obtained by Maede from his HK dogs [7]. The MgATPase activity and the plasma K and Na concentrations were similar to those from normal dogs and also to the levels found in the HK dogs. Glutathione, Na and K concentrations in the erythrocytes and hematologic values were occasionally determined in our subject dog and the control group, but they did not exhibit significant fluctuations during the past 2 years. Our analysis of the erythrocyte amino acids revealed a striking increase in glutamic acid, aspartic acid and glutamine levels which were nearly the same as the increases detected in the HK dogs . The other amino acid concentrations in the red cells were within the normal range and are not shown in Table 1. Crossbreeding our subject dog with a
189
Table
1. Hematologic both the elevated
and biochemical data GSH dog and normal
(control group). The data from the dogs are expressed as the mean+SD.
a.
Only one of control group of amino
from dogs
normal
eight normal clogs from the was used in the determination
acids.
normal LK male dog produced a litter of three puppies all of which possessed LK red cells. Then we attempted to crossbreed our dog with another LK dog, a heterozygous carrier of the HK gene, donated by Dr. Maede. However, the mating appeared to be unsuccessful. Then, our dog was artificially fertilized with the sperm of her son, also a carrier obtained in the previous crossbreeding, and consequently produced a litter containing two HK and one LK offspring. The two HK puppies displayed K concentrations of 128.5 and 132.6 mEq /l, Na concentrations of 5.8 and 5.2 mEq /l and GSH levels of 262 and 277 mg/dl RBC, respectively. The other littermate, the LK puppy, had a K concentration of 19.6 mEq/l and a Na concentration of 148.5 mEq/l. The above results clearly demonstrate that the genetic abnor-
mality in the erythrocytes of our dog is transmitted as an autosomal recessive trait. All of the evidence obtained from this study suggests that our subject dog is similar to the HK dogs reported by Maede. Since he found two HK families in 1976 [4], he had not encountered any HK dogs for ten years until he recently found one in Hokkaido. Considering that the HK dogs discovered so far are all mongrel dogs and that our subject dog is presumably unrelated to Maede's HK dogs from Hokkaido, the HK dogs may possibly exist elsewhere in Japan or in other parts of the world. Maede's earlier report describes two cases of acute hemolytic anemia which led him to the discovery of the new variant [4]. However, our subject dog has not displayed clinical evidence of any hemolytic disorder during the past two years while under observation in our laboratory. Only the evidence of the increased osmotic fragility and the large red cell volume, as indicated by the MCV and MCHC values, implies that our dog would be more susceptible to hemolytic anemia than normal dogs. How the increased Na, K-ATPase activity may influence the erythrocyte lesion is unknown at present. Na, KATPase is responsible for the maintenance of high K and low Na concentrations in the red cells by exchanging extracellular K for intracellular Na due to the hydrolysis of ATP [11, 13]. Humans, horses, pigs and rats have HK cells (high K and low Na concentrations in the cells) in relation to high Na, K-ATPase activity, while normal dogs and cats have LK cells (low K and high Na concentrations in the cells) because they lack the enzyme Na, K-ATPase in their erythrocytes. In contrast, ruminants have both HK and LK variant strains in each species because of the diversity of this enzyme's activity. In sheep, the allele for LK cells is dominant over the allele for HK cells and the genetic inheritance is characterized as autosomal recessive [15]. Such dimorphism of the cellular ion concentrations has been extensively used for studies of ion transport in cell membranes, although their clinical and physiological roles are not well understood [12, 14, 16]. Thus, the HK dogs are expected to provide new information to
190
researchers
in this
field of study. [4]
Acknowledgements
[5] [6]
The authors are grateful to Professor Y. Maede, Department of Veterinary Medicine, Hokkaido University, for proving us with a carrier dog heterozygous for the HK gene. We wish to thank Professor S. Ikemoto, Laboratory of Human Biology and Department of Legal Medicine, Jichi Medical School, for identification of parents and offspring in our dog family. We are also indebted to Drs. T. Samata and T. Iriki of our university for their technical assistance in analysis of amino acids.
[7] [8] [9]
[10]
[11]
[12]
References [1] [2] [3]
Beutler, J. Lab. Dodge, (1963). Gupta,
E., Duron, O., and Kelly, B. M. (1963). Clin. Med., 61, 882-888. J. T., Mitchell, C., and Hanahan, D. J. Arch. Biochem. Biophys., 100, 119-130. J. D., Peterson, V. J., and Harley, J. D.
赤 血 球Na,K-ATPase活
[13] [14] [15] [16]
(1974). Comp. Biochem. Physiol., 47, 1123-1126. Inaba, M., and Maede, Y. (1984). J. Biol. Chem., 259, 312-317. Maede, Y. (1977). Jap. J. Vet. Sci., 39, 187-189. Maede, Y., Kasai, N., and Taniguchi, N. (1982). Blood, 59, 883-889. Maede, Y., Inaba, M., and Taniguchi, N. (1983). Blood, 61, 493-499. Maede, Y., and Inaba, M. (1985). J. Biol. Chem., 260, 3337-3343. Nakao, T., Nagano, K., Adachi, K., and Nakao, M. (1963). Biochem. Biophys Res. Comm., 13, 444-448. Parpart, A. K., Lorenz, P. B., Parpart, E. R., Gregg, J. R., and Chase, A. M. (1947). J. Clin. Invest., 26, 636-641. Post, R. L., Merrit, G. R., Kinsolving, C. R., and Albright, C. D. (1960). J. Biol. Chem., 235, 17961802. Rasmusen, B. A., Tucker, E. M., Ellory, J. C., and Spooner, R. L. (1974). Anim. Blood Grps. Biochem. Genet., 5, 95-104. Seou, J. C. (1965). Physiol. Rev., 45, 596-617. Tucker, E. M. (1971). Biol. Rev., 46, 341-386. Tucker, E. M., and Ellory, J. C. (1970). Anim. Blood Grps. Biochem. Gent., 1, 101-112. Tucker, E. M., Ellory, J. C., and Kilgour, L. (1979). Biochim. Biophys. Acta, 583, 388-393.
性 上 昇 に よ り高 い グ ル タ チ オ ン(GSH)
お よび カ リ ウ ム 濃 度 を 示 す 犬 に つ い て
小 川 絵 里 ・藤 瀬
浩 ・小 林 好 作*
麻布大学獣医学部病理学第2講 座 *麻布大学獣医学部内科学第2講 座
神 奈 川 県 で グ ル タ チ オ ン濃 度 の高 い 赤 血 球 を持 つ 雑 種
透 圧 脆 弱性 は亢 進 して い た。 交 配 に よ り,こ の よ うな 赤
メ ス犬 が発 見 され た。 この 犬 は何 ら臨床 症 状 を示 さず,
血 球 の異 常 は常 染 色 体 性 の劣 性 遺 伝 子 に よ って 支 配 され
血 液 学 的 異 常 も見 出 され な か った 。 赤 血 球 中 で は,Na,
て い る こ とが 強 く示 唆 され た。 以 上 の 結 果 か ら,こ の 犬
K-ATPase活
性 の 上 昇 に 伴 な って 高 カ リウ ム,低
リウ ム濃 度 が 認め られ,さ
ら に グル タ ミン酸,ア
ナ ト スパ ラ
ギ ン酸 お よび グル タ ミンの 蓄 積 が 生 じて い た 。 また,浸
は前 出 に よ り報 告 され たHK犬(1977) sci. (39, 187-189)と もの と考 え られ る。
Jap. J. Vet.
遺 伝 的 に 類 似 の 特 性 を 有 して い る
