Download Effect of Vitamin B12 and Folic Acid Deficiencies on Neutrophil

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BRIEF
REPORT
Effect
of Vitamin
Deficiencies
B12 and
on Neutrophil
By Sandra
S. Kaplan
and
Morphological
and
quantitative
neutrophil abnormalities
are common
in the
megaloblastic
anemias
of vitamin
B,2 and
folic acid deficiency.
Little is known,
however, about
the role of these vitamins
in
normal
leukocyte
function.
Seven patients
with
megaloblastic
bone
marrows,
four
with
vitamin
B,2 deficiency
and
three
with folic acid deficiency,
were studied
to
determine
the effect,
if any,
of these
deficiencies
on leukocyte
function.
Phagocytosis of staphylococci,
hexose monophosphate shunt activation
with
phagocytosis,
and microbicidal
capacity
against
Staphylococcus
aureus
were
determined
prior
to the institution
of specific
therapy.
In
two instances,
these studies were repeated
T
HE
MEGALOBLASTIC
ficiencies
developing
of
ficiencies
are
and
usually
Function
following
treatment.
There was no impairment of phagocytosis
per se, and resting
metabolism
was
not
significantly
decreased.
With
phagocytosis,
however,
metabolic
activation
was
decreased
to
35%-36%
of control values
in the leukocytes of patients
with
vitamin
B,2 deficiency but not in the leukocytes
of patients with
folic acid deficiency.
Bacterial
killing
was slightly
decreased
in vitamin
B,2 but not in folic acid deficiency.
These
abnormalities
of function
were
reversed
after specific therapy.
These findings
suggested a specific role for vitamin
B,2 in the
production
of intermediates
necessary
for normal cell function.
of
vitamin
with
qualitative
polymorphonuclear
associated
with
Acid
R. E. Basford
ANEMIAS
associated
mature
are
Folic
acid
de-
and
quantitative
abnormalities
(PMN)
leukocytes.
Such
de-
macrocytic
B,2
PMN
and
precursors
folic
and
reduced
numbers
of circulating
PM N leukocytes
with hypersegmented
nuclei.’
Relatively
little research
has been done
on the role ofvitamin
B,2
acid in leukocyte
hypersegmentation
to impaired
the production
states,
selves
metabolism
of PMN
DNA
of
leukocyte
suggest
ordered
synthesis.2
succinyl
the
has
Vitamin
B,2 coenzyme
CoA
from
methylmalonyl
propionate
that
leukocyte
morphology
impairment
qualitative
or function,
but
leukocytes
generally
oxidation
function
ofdeveloping
of leukocyte
platelet
defect
and
function
in
in vitamin
macrocytosis
been believed
also
folic
and
nuclear
to be related
serves
as a cofactor
for
CoA
and,
in deficiency
is reduced.3’4
These
may
be impaired.
mature
and
facts
do
However,
leukocytes,
as well
protein-calorie
B,2 deficiency7’8
malnutrition
provoked
not
the
as the
5,6
this
themdis-
known
and
the
investi-
gation.
The
four
cific
leukocyte
anemia.
associated
From
instances,
of Pathology
patients,
three
was evaluated
studies
were
with
folic
efficiency
of phagocytosis,
monophosphate
shunt
and Biochemistry.
acid
deficiency
prior
to the institution
repeated
after
reversal
the
(HMPS)
and
of speof the
phagocytosisand micro-
University
of
Pittsburgh
Medical
School.
University
of
Pittsburgh
Medical
School.
Pa.
Submitted
Blood,
several
the Departments
A ddress
Pittsburgh.
of seven
B,2 deficiency,
This evaluation
included
activation
of the hexose
Pittsburgh,
©
function
with vitamin
therapy.
In
for
August
21. 1975;
reprint
requests:
Pa.
accepted
Sandra
November
S. Kaplan.
27, 1975.
M.D..
/526/.
1976 by Grune
Vol. 47, No.
& Stratton.
5 (May).
Inc.
1976
801
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802
KAPLAN
1.
Table
Hexose
Monophosphate
Shunt
Resting
B12 deficiency
Vitamin
4.5
4.4
39
10
R.R.
0.6
125
45
136
9.1
p
26
12
±
37
<0.001
C.W.
6.6
295
161
EM.
6.5
302
170
N.J.
2.8
185
5.3 ±
p
1.25
249
±
23
pro duced
93
141 ±
>0.5
9.9 ± 1.3
n
<0.005
38.15
±
261
<0.3
values
14C02
57
102 ±
<0.2
Mean
*Nanomoles
36
108
4.6 ± 2
Normal
Staph
D.C.
E.H.
deficiency
Associated
Latex
Ci.
Mean
Folic acid
BASFORD
Activity*
Phagocytosis
Conditions
AND
<0.5
12.9
±
104
23
fro m 1- ‘4C glucose
per i07
leukocytes
24.34
8.4
18
per 30 mm; mean
± SE.
bicidal
capacity
against
Staphylococcus
aureus.
Our
results
indicate
that,
although
leukocytes
appeared
to be quantitatively
and morphologically
similar
in vitamin
B,2 and folic acid deficiencies,
their
functional
integrity,
in terms
of
the parameters
studied,
is decidedly
dissimilar
with
impaired
function
in vitamm B,2 deficiency
and essentially
normal
function
in folic acid deficiency.
MATERIALS
Segmented
patients
done
neutrophils
were
from
counts
Wright’s
including
crest
were
folic
acid
red
deficiency
in two
lost.
All
B,2
had
and
of
had
tests.
value
of
57
basis
of
prior
was
pg/ml.
assessment
grown
Bacteria
for
activity
was
either
These
basis
was
of
not
patients,
resulting
in
had
who
Ime
lower
of
of
E.H.
the
at the
and
lack of
a markedly
posterior
superior
iliac
patients
with
three
initial
diagnosis.
and
limits
of
for
the
The
patients
B,2
values
R.R.,
were
acid
with
vitamin
B,2
One
Ci.
vitamin
abnormal
but
and
was
therapy
and
diagnosis,
leukocytes,
labora-
patient
folic
anemia.
vitamin
B,2 absorption.
deficient
diet while
the
third
to
were
polymorpho-
of normal
of his
All
showed
vitamin
time
were
blood
the
responded
serum
anemic
of
specimen
patients
low
Complete
the
polymorphonuclear
of phagocytosis
1- ‘4C
counts
the addition
in
two
and
trypticase
from
shunt
glucose
from
he had
vitamin
deficient
of
had
a
B,2
on
the
the patients,
characteristic
as
of a suitably
of bacteria.
The
were
and
heat
smears
rest
and
of
4C02
described.9
0.01-mI
cell
were
stained
at
evolution
previously
diluted
activity
washed
prepared
diluted
killed
at 60’C
prepared
30
from
to
an
for
30
and/or
an
18-hr
appropriate
mm.
120
Phago-
mm
after
per cell.
activity
the
broth,
studies
Wright’s
or ten staphylococci
determined
microbicidal
soy
use in metabolic
evaluated
monophosphate
particles was
of
(R.R.)
the
leukocytes.
marrow
original
improvement
the
these
diabetic
aureus
introducing
colony
of
surgery
below
levels.
the
vitamin-deficient
preparation
Each
bone
The
hypersegmented
S.
concentration.
ence
Two
bowel
were
ng/ml).
with
four
for
of
Hexose
marrow,
levels
B,2
on
at
from
WBC
from
patients.
anemia
the
PMN
aspirates
megaloblastic
(<2.5
reticulocytosis
of the
marrow
and
of
anemia.
Bacteria
culture
acid
diagnosed
One
bone
85%-90%
macrocytic
vitamin
bone
showed
a frankly
patients
normal
a juvenile
pernicious
cytic
had
controls
counts
vitamin-deficient
a marked
Folic
were
megaloblastic
the
normal
Differential
and
and
of
all
three
a characteristic
Schilling
after
the
three
deficiency
D.C.,
all
hypersegmentation.
tory
with
indices
in
METHODS
from
quantitated.
smears
cell
examined
leukocytes)
and
stained
thrombocytopenic,
nuclear
(PMN
prepared9
AND
ratio
aliquot
of these
during
uptake
after
30
Microbicidal
taken
at
of
mm
of
staphylococci
incubation
activity
time
leukocyte-bacteria
0 and
was
at
30,
suspensions
in
or
latex
the
pres-
determined
60,
and
was
1:2.
from
120
mm
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NEUTROPHIL
803
FUNCTION
l0
-0-
06
Fig. 1 .
The mean microbicidal
activity of 20
normal
controls
at 30, 60, and 120 mm is shown
by the solid line. The shaded area indicates
2 SD
above
and
below
the
mean.
The
microbicidal
activity
of leukocytes
from three of the four patients
with
vitamin
B,2 deficiency
are shown
by
dashed
killed
lines.
fewer
The mean
After
2 hr of incubation,
staphylococci
than
microbicidal
activity
three patients
by the dotted
of differences
acid
of leukocytes
between
deficiencies
had
expected.
with
folic acid deficiency
line. All were within
normal
Significance
B,2 and folic
all
normally
from
is shown
limits.
normal
30
120
MIN.
control
was determined
60
with
and
the
the Student’s
results
t
from
patients
with
vitamin
test.
RESULTS
The
effects
associated
1 and Fig.
lower than
of
vitamin
activation
I. Resting
normal.
B,2
and
folic
acid
deficiencies
on
the
phagocytosis-
ofthe
HMPS
and on bacterial
killing
are shown
in Table
metabolism
tended
to be low, but it was not significantly
With phagocytosis
either
of latex or of staphylococci,
how-
ever, there was a statistically
and 35% ofcontrol
in the
regardless
of the cause
of
significant
impairment
ofHMPS
activation
to 36%
leukocytes
of patients
with
vitamin
B,2 deficiency,
the deficiency.
Microbicidal
activity
at 2 hr after
inoculation
was reduced
by more
than 2 SD from
the mean
control
level
three of the four vitamin
B,2-deficient
patients
where
this was measured.
patient
with true pernicious
anemia
(C.J.)
showed
the greatest
impairment
in all
The
of
microbicidal
activity.
Two
of these
patients,
R.R.
and
E.H.,
were
restudied
after
receiving
vitamin
B,2. Of these,
E.H.
demonstrated
a marked
improvement of phagocytosis-associated
HMPS
activation
by the tenth
posttreatment
day.
Initially,
with
metabolism
the tenth
posttreatment
The other
patient,
leukocyte
function
The patients
with
HMPS
phagocytosis
of latex
and
staphylococci,
her leukocytes’
was 43% and 54% of the mean
control
value
(Table
I). At
day, this metabolic
activity
was 100%
of the control.
at 25 days
posttreatment,
also
showed
improvement
of
from approximately
40% of control
to about
60% of control.
folic
acid
deficiency
did not show
a decrease
either
of
phagocytosis-associated
HMPS
activation
or of microbicidal
activity.
DISCUSSION
The
primary
role
of
the
PMN
leukocyte
in
host
defense
against
bacterial
invaders
is well recognized.
It has been suggested
that deficiencies
of any one or
more of the B-complex
vitamins
may impair
the metabolic
activities
associated
with phagocytosis.’#{176}
Thiamin,
riboflavin,
and pyridoxine
deficiencies
all have
been
associated
with
decreased
activities
of enzymes
involved
in HMPS
activity.
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804
KAPLAN
In addition,
the leukocytes
have decreased
metabolic
of children
with
and microbicidal
severe
protein-calorie
activities,5
which
decreased
activity
of granule-bound
NADPH
oxidase.6
demonstrated
quantitatively
impaired
NBT
reduction
and
kinase
activity
with
normal
phagocytosis
in the leukocytes
refractory
Leukocyte
anemias
of unspecified
function,
in terms
types.’1
of metabolic
the course
of vitamin
B,2 and folic
investigated.
However,
two previous
mm B,2 deficiency
have shown
that
4 and
using
work
an
that the
newborn
increased
suspected
studies,
tabolism
deficiency.
decrease
susceptibility
with
caused
multiple
betic.
to fatal
malnutrition
be related
can
to
Another
study
has
decreased
pyruvate
of patients
with
microbicidal
activity
acid deficiencies,
has not been
studies
on leukocyte
metabolism
3-’4C
propionate
oxidation
to
of methylmalonyl
fed diets
deficient
BASFORD
during
specifically
in vitaCO2 is im-
CoA mutase
is decreased.3
Other
in folic acid has demonstrated
infections,’2
and
low
tissue
folate
levels
are
of being
a predisposing
factor
in the bacteriuria
of
‘#{176}
Our
however,
have
shown
no impairment
of phagocytosis-associated
meor microbicidal
activity
in the leukocytes
of patients
with
folic acid
Only
in the four patients
with vitamin
B,2 deficiency
did a marked
in the phagocytosis-associated
HMPS
activation
occur.
These
patients
also have
shown
bicidal
activity.
able
activity
animals
and
AND
a slight
Although
specific
therapy,
by vitamin
deficiencies
Only
one
to moderate
both
of these
it is not
impairment
defects
of
possible
B,2 deficiencies.
Two
due to malabsorption,
of the
patients
had
of
been
to
of
PMN
intraleukocytic
function
assume
that
they
are
microcorrect-
are
entirely
these
patients
may have
possessed
and one of the patients
also is dia-
vitamin
B,2 deficient
on
the
basis
of a
deficiency
of intrinsic
factor.
It is not unreasonable
to suppose
that,
in view of
the impaired
DNA
and protein
synthesis
in vitamin
B,2 deficiency,
one or more
of the enzymes
involved
in HMPS
activation
are reduced
in the cell.
Since
hexokinase
and NADP
are rate-limiting
for HMPS
activity,’3
quantitation
of
these factors
or the substances
necessary
for their
formation
would
seem
to be
the most
reduced
potentially
productive
PMN
reactivity
are
not
avenues
for future
investigation.
necessarily
incompatible
with
Our findings
the infrequency
of
oflife-threatening
infection
in vitamin
B,2 deficiency.
Metabolic
activity
is reduced
by approximately
40% and microbicidal
activity
only by 5%-lO%.
Much
more
profound
reductions
probably
are necessary
before
serious
compromise
of PMN
functional
integrity
occurs.
The fact that this defect
occurs
with
vitamin B,2 deficiency,
vitamin
B,2 in the
tabolism
and
but not with folic acid deficiency,
suggests
a specific
production
of intermediates
necessary
for normal
role for
cell me-
function.
ACKNOWLEDGMENT
We thank
tion
Michael
Nardi
of the manuscript.
for
We
expert
would
also
technical
like
assistance
to thank
Dr.
and
Mary
Ralph
Klayner
Schmeltz
for
for
help
in prepara-
allowing
us to study
in
Am
his patient.
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of vitamin
B,2
48:555-561,
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and
folate
man.
J Med
1970
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Giorgio
Al:
Leukocyte
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FUNCTION
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1976 47: 801-805
Effect of vitamin B12 and folic acid deficiencies on neutrophil function
SS Kaplan and RE Basford
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