AN ABSTRACT OF THE THESIS OF
Xu Wang for the degree of Doctor of Philosophy in Nutrition
and Food Management presented on May 3. 1995.
Title: Effect of Controlled Vitamin B-6 Intake on in vitro
Lymphocyte Proliferation and Interleukin 2 Production in
Young Women
■__
Abstract approved:
;
Lorraine T. Miller
In two studies we tested the effect of vitamin B-6
(B-6) intake on in vitro lymphocyte, proliferation and IL-2
production in healthy young women.
In Study I, 6 women were
fed a constant diet containing 0.84 mg (4.96 jimols) of B-6
for 12 d, and 1.24 mg (7.33 jimols) and 2.44 mg (14.42 nmols)
of B-6 during two subsequent 10-d periods.
Lymphocyte
proliferation in response to the mitogens concanavalin A
(Con A) and pokeweed mitogen (PWM) was significantly higher
(p < 0.05) at 2.44 mg than at 0.84 mg intake and the prestudy value.
In Study II, 10 women who consumed their self-
selected diets were randomly divided into a PN and a placebo
group of five each.
Following a 5-d baseline period, the PN
group received a daily supplement of 1.5 mg (7.29 jimols) and
50 mg (243 nmols) pyridoxine.HC1 (PN.HC1) for 7 and 6 d,
respectively.
This was followed by a 28-d washout period
during which no supplement or placebo was administered.
After daily 1.5 mg supplementary PN.HC1 for seven days,
lymphocyte proliferation in response to phytohemagglutinin
(PHA), Con A and PWM, and IL-2 production were significantly
higher than the baseline (p < 0.05) and that of the placebo
group (p < 0.05).
The 50 mg PN.HC1 supplement increased
IL-2 production, but not lymphocyte proliferation as
compared with the 1.5 mg PN.HC1 supplement period.
Lymphocyte proliferation and IL-2 production were
significantly correlated with lymphocyte pyridoxal 5'phosphate concentrations (p < 0.01).
We conclude that a B-6
intake of 1.5 to 2 times the RDA improves lymphocyte
proliferation and IL-2 production in healthy young women and
this effect of B-6 on immunocompetence is transitory.
To
explore the basis for the effect of B-6 on immune function,
putrescine was added to the culture medium (2 - 200 pimol/L) .
In vitro lymphocyte proliferation and IL-2 production were
not changed by the addition of putrescine under our
experimental conditions.
Effect of Controlled Vitamin B-6 Intake
on in vitro Lymphocyte Proliferation and
Interleukin 2 Production
in Young Women
by
Xu Wang
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Doctor of Philosophy
Completed May 3, 1995
Commencement June, 1995
Doctor of Philosophy thesis of Xu Wang presented
on May 3. 1995
APPROVED:
Major Professor, representing
Nutrition and Food Management
Head of Department of Nutrition and Food Management
Dean of Gradiiate School
I understand that my thesis will become part of the
permanent collection of Oregon State University library.
My signature below authorizes release of my thesis to any
reader upon request.
Xu Wang, Author
TABLE OF CONTENTS
Page
INTRODUCTION
1
REVIEW OF LITERATURE
3
A. Vitamin B-6 Metabolism
3
B. The Safety of PN.HCl Supplementation
9
C. Recommended Dietary Allowance for Vitamin B-6
10
D. Assessment of Vitamin B-6 Status
11
E. Vitamin B-6 and Immunocompetence
15
1. Animal Studies
2. Human Studies
F. Proposed Roles of Vitamin B-6 in Immune Function
1.
2.
3.
4.
One-carbon Unit Metabolism
Modulation of Hormone Receptors
Polyamine Synthesis
Vitamin B-6 and Polyamines
MATERIALS AND METHODS
15
18
23
24
25
27
30
32
A. Subjects
32
B. Experimental Designs
33
C. Blood Drawing
38
D. Isolation of Mononuclear Cells
38
E. Cell Viability Tests and Counts
39
F. Lymphocyte Proliferation
39
G. IL-2 Production (Study II)
40
H. Effect of Putrescine on Lymphocyte Proliferation
and IL-2 Production (Study II)
41
I. PLP Determination
42
TABLE OF CONTENTS (Continued)
Page
J. Erythrocyte Aspartate Aminotransferase Activity
Coefficient (EAST AC) (Study II)
43
K. Collection of Dietary Information (Study II)
43
L. Statistics
44
RESULTS
A. Study I
1. Plasma PLP Concentration
2. Lymphocyte Proliferation
B. Study II
1. Subjects' Dietary Vitamin B-6 Intake
and Nutritional Status
2. Plasma and Lymphocyte PLP Concentrations
3. Erythrocyte Aspartate Aminotransferase
Activity Coefficient (EAST AC)
4. Lymphocyte Proliferation
5. IL-2 Production
6. Effect of Putrescine on Lymphocyte
Proliferation and IL-2 Production
45
45
45
45
49
49
52
57
58
66
69
DISCUSSION
75
SUMMARY AND CONCLUSIONS
92
BIBLIOGRAPHY
96
LIST OF FIGURES
Figure
1. Formulae of B-6 vitamers, metabolites
and PN-glucoside
Page
4
2. The experimental protocols
37
3. Vitamin B-6 intake and the changes of
lymphocyte proliferation in response
to Con A and PWM stimulation (Study I)
47
4. Changes of plasma and lymphocyte PLP
concentrations in the PN and placebo
groups (Study II)
56
5. Lymphocyte proliferation in response
to PHA (A), Con A (B) and PWM (C)
stimulation in the PN and placebo
groups (Study II)
62
6. Correlation between lymphocyte
proliferation, IL-2 production and
lymphocyte PLP concentrations (Study II)
64
7. IL-2 production in response to PHA
stimulation in the PN and placebo
groups (Study II)
68
8. Changes of lymphocyte proliferation
and IL-2 production in response to
putrescine concentrations in culture
medium (Study II)
74
LIST Of TABLES
Table
Page
1. Descriptive data on subjects in Study I
34
2. Descriptive data on subjects in Study II
36
3. Vitamin B-6 intake and plasma PLP
concentration in six healthy young
women consuming a constant diet with
controlled vitamin B-6 intake (Study I)
46
4. Vitamin B-6 intake and mitogen response,
in cpm, of lymphocytes in six healthy
young women consuming a constant diet
with controlled vitamin B-6 intake
(Study I)
48
5. Average daily dietary intakes of energy,
vitamin B-6 and protein by the PN and
placebo groups during baseline, supplement
and washout periods (Study II)
50
6. Average weekly hemoglobin (Hb)
concentration and hematocrit (Hct) value
of the PN and placebo groups (Study II)
51
7. Plasma PLP concentrations of the PN
and placebo groups (Study II)
54
8. Lymphocyte PLP concentrations of the PN
and placebo groups (Study II)
55
9. Erythrocyte aspartate aminotransferase
activity coefficient (EAST AC) of the PN
and placebo groups (Study II)
57
10. PN.HCl supplementation and mitogen response,
in cpm, of lymphocytes to PHA in the PN
and placebo groups (Study II)
59
11. PN.HCl supplementation and mitogen response,
in cpm, of lymphocytes to Con A in the PN
and placebo groups (Study II)
60
LIST OF TABLES (Continued)
Table
Page
12. PN.HCl supplementation and mitogen response,
in cpm, of lymphocytes to PWM in the PN
and placebo groups (Study II)
61
13. PN.HCl supplementation and IL-2
production (U/L) of lymphocytes in
the PN and placebo groups (Study II)
67
14. Effect of PN.HCl supplementation and
putrescine concentrations in the culture
medium on mitogen response, in cpm,
of lymphocytes to PHA in the PN and
placebo groups (Study II)
70
15. Effect of PN.HCl supplementation and
putrescine concentrations in the culture
medium on mitogen response, in cpm,
of lymphocytes to Con A in the PN and
placebo groups (Study II)
71
16. Effect of PN.HCl supplementation and
putrescine concentrations in the culture
medium on mitogen response, in cpm,
of lymphocytes to PWM in the PN and
placebo groups (Study II)
72
17. Effect of PN.HCl supplementation and
putrescine concentrations in the culture
medium on IL-2 production (U/L) of
lymphocytes in the PN and placebo groups
(Study II)
73
EFFECT OF CONTROLLED VITAMIN B-6 INTAKE ON IN VITRO
LYMPHOCYTE PROLIFERATION AND INTERLEUKIN 2
PRODUCTION IN YOUNG WOMEN
INTRODUCTION
Vitamin B-6 is necessary for the development and
maintenance of normal immune function (1).
Although the
effect of vitamin B-6 deficiency on immune function has been
examined widely in animals (2-5), relatively few studies
have been conducted in humans.
In a vitamin B-6 depletion-
repletion study in elderly people, vitamin B-6 deficiency
significantly decreased the percentage and total number of
lymphocytes, in vitro proliferation responses of peripheral
blood lymphocytes to T- and B-cell mitogens and interleukin
2 (IL-2) production (6).
These decreased parameters for
immune function were not restored completely by a vitamin B6 intake of 2.88 mg/d by men and 1.90 mg/d by women, which
is slightly higher than the current recommended dietary
allowance (RDA) for vitamin B-6.
In independently living
elderly having an adequate vitamin B-6 intake, a
pharmacological level of vitamin B-6 supplementation (50 mg
pyridoxine hydrochloride per day) significantly increased
immunocompetence as evaluated by lymphocyte subpopulation
analysis and in vitro lymphocyte proliferation in response
to mitogen stimulation (7).
Also in independently living
elderly having an adequate vitamin B-6 intake, a supplement
of 3 mg vitamin B-6 per day, together with other
micronutrients, for one year significantly increased IL-2
production, IL-2 receptor expression (8), delayed
hypersensitivity skin test (DHST)
(9) and the resistance to
infectious diseases (8) of the subjects.
On the other hand,
there has been reported that short-term marginal vitamin B-6
deficiency did not affect immune response in health young
men (10).
There have been no data to evaluate the influence
of vitamin B-6 intake on immune function in young women.
The research presented in this thesis was designed
to test the effect of different vitamin B-6 intakes on in
vitro lymphocyte proliferation and IL-2 production of
healthy young women in both controlled and self-selected
dietary conditions.
We determined the relationship between
vitamin B-6 intake, plasma and lymphocyte pyridoxal 5'phosphate concentrations and in vitro peripheral blood
lymphocyte proliferation in response to mitogen stimulation
and IL-2 production.
The duration of the effect of vitamin
B-6 supplement on lymphocyte proliferation and IL-2
production was also determined.
An attempt was made to
explore a possible mechanism of the effect of vitamin B-6 on
immune function.
REVIEW OF LITERATURE
A. Vitamin B-6 Metabolism
Vitamin B-6 was first defined and delineated as a
distinct entity in the vitamin B-2 complex by Gyorgy sixty
years ago (11).
Eight years later, Snell and his colleagues
(12) provided evidence that vitamin B-6 is a group of
related compounds.
Vitamin B-6 is recommended as the
generic descriptor for all 3-hydroxy-2-methylpyridine
derivatives exhibiting qualitatively the biological activity
of pyridoxine in rats (13).
The six forms of vitamin B-6
include the free forms pyridoxine (PN), pyridoxal (PL) and
pyridoxamine (PM); and their phosphorylated forms,
pyridoxine 5'-phosphate (PNP), pyridoxal 5'-phosphate (PLP)
and pyridoxamine 5'-phosphate (PMP)
(Fig. 1).
The
predominate urinary metabolite of vitamin B-6 is 4-pyridoxic
acid (4-PA)
(Fig. 1), which has no vitamin B-6 activity.
Vitamin B-6 occurs in a wide variety of foods.
In
general, PN and PNP are the predominant forms of vitamin B-6
in plant-derived foods, while PL and PM and their
phosphorylated forms are found chiefly in animal-derived
foods (14).
In addition, glycosylated pyridoxine
(pyridoxine 5'-fi-D-glucoside)
(Fig. 1) has been identified
and comprises from 5% to more than 80% of the total vitamin
B-6 in rice bran, wheat, orange juice, and other plant
CH, -R2
HoC
Rl
(PN)
Pyridoxine
(PM)
Pyridoxamine
Pyridoxal
(PL)
(PNP)
Pyridoxine 5'-phosphate
Pyridoxamine 5•-phosphate (PMP)
(PLP)
Pyridoxal 5'-phosphate
(4-PA)
4-pyridoxic acid
Pyridoxine 5'-B-D-glucosidee
(PN-glucoside)
-CH2OH
-CH2NH3+
-CHO
-CHjOH
-CH2NH3+
-CHO
-COO"
-CH2OH
R2
-OH
-OH
-OH
-OP03=
-OP03=
-OPO3-OH
-B-D-glucose
Fig. 1. Formulae of B-6 vitamers, metabolites and
PN-glucoside.
foods (15-17).
Glycosylated vitamin B-6 is less
bioavailable than the free and phosphorylated forms of
vitamin B-6.
Studies in human subjects using an orally
administered, purified deuterium-labeled PN-glucoside
indicated that the bioavailability of this form of vitamin
B-6 is 50-70% relative to PN (18).
In the intestinal lumen, PLP and PMP are
dephosphorylated by intestinal phosphatases.
The three free
forms are absorbed by the jejunum through a nonsaturable,
passive process (19).
The B-6 vitamers cross the
basolateral membrane to enter circulation, mainly in the
nonphosphorylated forms (19,20).
After transport to the
liver, the free forms of vitamin B-6 enter hepatocytes by
diffusion followed by metabolic trapping (21,22).
Liver is the primary organ responsible for vitamin B-6
metabolism.
In human hepatocytes, PL, PN and PM are
phosphorylated by a single kinase to their respective
phosphorylated forms.
The activity of pyridoxal kinase (EC
2.7.1.35) appears to be zinc- and ATP-dependent (23).
Since
the activity of pyridoxal kinase predominates over hepatic
phosphatase, phosphorylated forms of vitamin B-6 are the
major forms of the vitamin B-6 in liver.
PNP and PMP are
oxidized to PLP by the FMN-dependent oxidase, pyridoxine
(pyridoxamine) 5'-phosphate oxidase (EC 1.4.3.5)
(24).
PLP
is bound to apoenzymes and other proteins or released into
plasma attached to albumin.
When PLP is synthesized in
excess of the binding capacity of hepatic proteins, the
phosphate ester binding PLP is hydrolyzed by alkaline
phosphatases (25).
Finally, excess PL is oxidized by
aldehyde oxidase (EC 1.2.3.1) to 4-PA, the dead-end
catabolite and the major urinary vitamin B-6 excretory
product (23).
Plasma PLP bound to albumin is the major transport form
of vitamin B-6.
Protein binding protects PLP from
dephosphorylation by phosphatases.
cross cell membranes.
PLP does not readily
PL, formed when PLP is
dephosphorylated by a membrane phosphatase, is the primary
form crossing cell membranes.
Within the cells PL can be
rephosphorylated to PLP by the action of pyridoxal kinase,
which exists in most tissues (26) .
In contrast, the
distribution of PNP (PMP) oxidase has been identified only
in the liver (23) and erythrocytes in humans (27).
The
absence of PNP (PMP) oxidase in skeletal muscle is important
because this tissue represents the major storage site of
vitamin B-6 in the body.
with
In an isolated hind-limb perfused
3
H-PN, there was a large accumulation of labeled PNP,
but virtually no PLP (28) .
This means that the liver, via
plasma PLP, provides PL which other tissues take up from
circulation and convert to PLP for their own usage.
The erythrocytes also have an important role in
metabolizing and transporting vitamin B-6.
are rapidly taken up by red cells (29).
Both PL and PN
Since both
pyridoxal kinase and PNP (PMP) oxidase exist in human
erythrocytes (27,30), PL and PN are readily phosphorylated
and PNP is converted to PLP.
PLP, the biologically active form of vitamin B-6, is
utilized as a coenzyme by over 100 enzymes (31).
For
example, PLP (or PMP) plays an essential role as a coenzyme
for aminotransferases in the interconversion of amino acids
and their respective a-ketoacids (e.g., aspartateoxaloacetate aminotransferase, EC 2.6.1.1; alanine-pyruvate
aminotransferase, EC 2.6.1.2; etc.).
Transamination is
central to the biosynthesis and catabolism of essential and
non-essential amino acids.
In a number of cases,
transamination provides a simple link between amino acids
and intermediates of the glycolysis pathway and the
tricarboxylic acid cycle.
Reactions of PLP-dependent
decarboxylases, another example, include the synthesis of
various amines such as polyamines, serotonin, tyramine and
x-amino butyric acid by the decarboxylation of precursor
amino acids.
Withers et al.
(32) reported a structural role of PLP
in glycogen phosphorylase (EC 2.4.1.1) by demonstrating
conformational changes in the enzyme due to ionic
interactions of the phosphate group of PLP with the active
site in the enzyme.
Glycogen phosphorylase is the major
binding protein for PLP in muscle (33) and this enzyme
accounts for as much as 5% of the soluble protein in
skeletal muscle, which is approximately 40% of the lean body
mass in humans (34).
Krebs and Fisher (35) proposed that
muscle glycogen phosphorylase is a storage site for vitamin
B-6 in the body.
Black et al. (33) reported that high
intakes of vitamin B-6 increased muscle glycogen
phosphorylase activity, but it was the starvation rather
than a vitamin B-6 deficient diet that decreased the
activity of muscle glycogen phosphorylase in rats (36).
A
deficiency of vitamin B-6 in rats resulted in decreased
activity of liver glycogen phosphorylase (37).
Plasma PLP concentration increases during and
immediately following a strenuous exercise (38).
In a
recent study, Crozier et al. (39) reported that this rapid
increase in plasma PLP and 4-PA concentrations after the
onset of exercise was not affected by exercise intensity.
They proposed that the elevated plasma PLP concentration
immediately after exercise came from the redistribution of
PLP from extravascular spaces to the plasma pool along with
albumin and other proteins shifted from interstitial space
into blood during exercise.
This increased plasma PLP was
oxidized to 4-PA and was destined for urinary excretion
rather than for utilization by liver to facilitate
gluconeogenesis during exercise.
B. The Safety of PN Supplementation
The acute toxicity of pyridoxine is very low.
A single
dose in the range of 3 to 4 g per kg body weight produces
convulsions and death in animals (40).
In mice and rats,
the LD5Q of an oral dose is 4 - 6 g per kg body weight (41).
Such extremely large doses of vitamin B-6 intake does not
occur naturally at any place but toxicological laboratories.
Chronic toxicity of pyridoxine is also low.
Schaumburg
et al. (42) reported that in humans who had used PN for
self-medication, toxic effects occurred after 2 to 6 g of
pyridoxine intake daily for 2-40 months.
Clinical
manifestations of pyridoxine toxicity included peripheral
sensory neuropathy with atactic gait disorders, absence of
limb reflexes and impairment of the sensation of touch,
vibration, temperature, pinprick and the joint position.
Anatomically, the peripheral sensitive nerves undergo
nonspecific axonal degeneration of large and small
myelinated fibers (42).
Considerable and even complete
improvement occurred within 6 months of withdrawing
pyridoxine.
Rudman et al. (43) speculated that the toxic
side effects of pyridoxine were due to the basic pyridine
structure of vitamin B-6 (Fig. 1).
Rudman et al. also
suggested that high pyridoxine concentrations may
competitively inhibit the binding of PLP to apoenzymes,
leading to a deficiency of active enzymes in the peripheral
10
nervous system.
Possibly, toxic impurities in vitamin B-6
preparations may be significant in high-dose intakes.
Human data on the safety of pyridoxine have shown that
doses of PN less than 500 mg per day appear to produce no
detectable adverse effects for periods ranging from 6 months
to 6 years (44-46).
C. Recommended Dietary Allowance for Vitamin B-6
The crucial role of vitamin B-6 in metabolism clearly
points to the importance of an adequate intake of this
nutrient.
The 1989 edition of RDA sets the recommended
allowance of vitamin B-6 as 2 mg for adult men and 1.6 mg
for adult women per day (47).
Since the requirement for
vitamin B-6 is related to protein intake, this
recommendation is based on the ratio of 0.016 mg of vitamin
B-6 to one gram of protein intake.
It was calculated from
the reported average daily protein intakes of approximately
100 g by adult females and 125 g by adult males.
The
present RDA for vitamin B-6 is lower than the one in 19 86,
which set the recommendation for vitamin B-6 as 2.2 mg for
men and 2 mg for women per day (47).
These numbers were
based on the ratio of 0.02 mg of vitamin B-6 to one g of
protein intake.
Justification for reducing the RDA for
vitamin B-6 in 1989
included human studies and survey data of protein intake.
11
Studies showed acceptable biochemical measurements of
vitamin B-6 status in subjects who were receiving less than
the 1986 RDA for vitamin B-6.
Survey data also indicated
that the average daily protein intakes of women and men were
60 g and 100 g, respectively.
The use of dietary intake
data assumes that the survey sample represents all
populations of different age and socioeconomic groups, the
nutrient databases are accurate and the populations studied
are healthy.
Very few studies have aimed to assess human nutrient
requirements in terms of maximizing bodily functions.
Based
on the measurements of immunocompetence in the elderly
receiving controlled intakes of vitamin B-6, Meydani et al.
(6) suggested that the elderly need a higher intake of
vitamin B-6 than the present RDA to maintain normal immune
function.
D. Assessment of Vitamin B-6 Status
The indices for assessing vitamin B-6 nutriture can be
divided into three categories: direct and indirect
biochemical measures of vitamin B-6 status and dietary
vitamin B-6 intake.
Direct measurement of the concentrations of vitamin B-6
and its metabolites in blood and urine is widely used to
assess vitamin B-6 status.
Since PLP is the active form of
12
vitamin B-6 and the level of PLP in plasma reflects tissue
levels of vitamin B-6 (48), plasma PLP concentration is
considered a good indicator of vitamin B-6 status (49).
Plasma PLP levels respond to changes in vitamin B-6 intake
(50,51).
Leklem (52) recommended that the normal value for
plasma PLP concentration is > 30 nmol/L.
Protein (53,54)
and riboflavin (55,56) intake influence plasma PLP levels.
Other factors affecting plasma PLP concentration include
plasma volume and physical activity (37), smoking (57), age
(58), and use of oral contraceptive agents (59).
Plasma PL is also an important indicator of vitamin B-6
status.
Fifteen minutes after an orally administrated
physiological dose of
3
H-labeled pyridoxine (60) or
pyridoxamine (61) to mice, more than 50% of the
B-6 vitamers in plasma was PL.
Barnard et al.
3
H-labeled
(62) reported
that in pregnant women the plasma PLP concentration was 50%
lower than in nonpregnant controls, but plasma PLP and PL
together only showed a slightly lower value in pregnant than
in nonpregnant women.
Another direct measure of vitamin B-6 status is the
urinary excretion of 4-PA, the dead-end catabolite and the
major urinary excretory product of vitamin B-6 (54).
Urinary excretion of 4-PA normally accounts for about
40 - 60% of the dietary intake of vitamin B-6 (50,54).
Given individual doses of 100 mg PL, PM or PN, human
subjects excreted 90% of PL and PM, and 70% of PN in urine
13
as 4-PA within 3 6 hours (63).
A short-term indicator of
vitamin B-6 status, urinary 4-PA excretion parallels plasma
PLP concentrations in vitamin B-6 depletion-repletion
studies (64,65).
The liver is thought to be the major site
of 4-PA formation (23,66).
Indirect measurements of vitamin B-6 status include
activity of erythrocyte aminotransferases, tryptophan load
test and methionine load test.
A commonly used measure of
vitamin B-6 status is the activity of erythrocyte alanine
aminotransferase (EALT) and aspartate aminotransferase
(EAST).
For assessing vitamin B-6 status, the basal
activity and the activity of these enzymes stimulated with
exogenous PLP added in vitro are measured.
When vitamin B-6
status is inadequate, less endogenous PLP is available to
bind to the apoenzyme, resulting in lower basal activity and
higher PLP-stimulated activity.
The ratio of stimulated to
basal aminotransferase activity, known as the activity
coefficient (AC), indicates vitamin B-6 status (67).
Leklem
(52) suggests that an activity coefficient of < 1.25 and
< 1.80 for EALT and EAST, respectively, reflects adequate
vitamin B-6 intake.
Vitamin B-6 deficiency results in
higher activity coefficients for both aminotransferases.
Since erythrocyte has a long life span (120 days),
erythrocyte aminotransferase activity is a
long term index of vitamin B-6 status.
Tryptophan metabolism requires PLP for the conversion
14
of 3-hydroxykynurenine to 3-hydroxyanthranilic acid (68).
In vitamin B-6 deficiency, 3-hydroxykynurenine accumulates
and is converted to xanthurenic acid, which is another
PLP-dependent step.
Measurement of urinary tryptophan
metabolites, particularly xanthurenic acid, following an
oral load (2 - 5 g) of L-tryptophan was at one time used to
assess vitamin B-6 status in humans (69).
Activity of
enzymes in the tryptophan-niacin pathway is also affected by
dietary protein intake (53), specific hormones and use of
oral contraceptive agents (70).
These factors complicate
the interpretation of tryptophan load test as an index of
vitamin B-6 status.
In addition, in 19 89 Food and Drug
Administration (FDA) banned L-tryptophan from the market
following the determination that a contaminated lot of this
product had contributed to public health problems (71).
The methionine load test is also an indirect measure of
vitamin B-6 status (72).
In the metabolism of methionine,
conversion of cystathionine to a-ketobutyrate and cysteine
is PLP-dependent (73).
Following a 3-g oral load of
L-methionine, increased urinary excretion of cystathionine
indicates inadequate vitamin B-6 status.
Like tryptophan
load test, the level of protein influences methionine
metabolism (72).
Dietary vitamin B-6 intake alone (i.e., without using
biochemical test of vitamin B-6 status) is not sufficient to
assess vitamin B-6 status in humans.
The inherent problems
15
in collecting dietary intake data include difficulties of
obtaining accurate food intakes (74) and incomplete nutrient
databases used to determine vitamin B-6 content of foods
(75).
Additionally, nutrient databases do not include
information on the glycosylated form of this vitamin in
plant-derived foods which may affect bioavailability of
vitamin B-6 (76).
However, collecting dietary intake data
is relatively easy, cheap and quick (77).
The expected
improvement of nutrient databases and data collecting
methodology make dietary intake data still valuable in
estimating intake and identifying vulnerable populations who
may be susceptible to vitamin B-6 deficiency, especially
along with other biochemical or functional measurements of
vitamin B-6 status.
Several studies have shown that the
majority of those who had evidence of biochemical vitamin B6 deficiency also had low intakes of this vitamin (78-80).
E. Vitamin B-6 and Immunocompetence
l. Animal Studies
Since there are several recent reviews on the effect of
vitamin B-6 deficiency on immune function in animals
(81-83), this subject will be reviewed only briefly here.
Vitamin B-6 plays a critical role in the development
and maintenance of normal immune function in experimental
animals.
As early as in 1944, Stoerk and Zucher (84) showed
16
that vitamin B-6 deficiency produced a severe atrophy of
the thymus in rats.
Thymuses from vitamin B-6 deficient
rats were virtually depleted of lymphocytes and consisted
almost exclusively of epithelial cells and stroma.
Stoerk
and Zucher also observed atrophied lymph nodes and impaired
antibody responses in these vitamin B-6 deficient rats.
Splenic hypoplasia was reported in fetuses of pregnant
rats fed a vitamin B-6 deficient diet or given the vitamin
B-6 antagonist, deoxypyridoxine (85).
Pups of vitamin B-6
deficient dams had diminished numbers of circulating blood
lymphocytes, fewer splenic plaque-forming cells, and
diminished vitamin B-6 content of splenic and thymic tissues
(86) .
Vitamin B-6 deficiency produces profound effects on
cell-mediated immunity.
Cell-mediated immune function,
measured by delayed dermal hypersensitivity, allograft
response and mixed lymphocyte cultures, was decreased in the
vitamin B-6 deficient rats (87), mice (88), and guinea pigs
(89).
Thoracic duct lymphocytes from vitamin B-6 deficient
female rats and their three-month old offspring showed
decreased ability to respond in mixed lymphocyte cultures
and to incorporate tritiated uridine (90).
Axelrod et al.
(91) reported that vitamin B-6 deficient guinea pigs
inoculated with Mycobacterium tuberculosis had depressed
delayed type hypersensitivity skin response to this antigen.
Axelrod et al. (92) also reported that in rats immune
17
responses to antigens were inhibited and viability of skin
homografts was increased by vitamin B-6 deficiency.
al.
Ha et
(93) found that in mice challenged with Moloney sarcoma
virus, tumor incidence was higher and regression time was
longer in animals fed diets containing 0 or 0.1 mg of PN/kg
than in those fed 0.5 - 1.0 mg of PN/kg diet.
Gridley et al. (94) observed that the proliferation
response of peripheral blood and splenic lymphocytes to
T-cell mitogens was higher in mice fed diets containing 7.7
and 74.3 mg of PN/kg than in those fed 0.2 and 1.2 mg of
PN/kg diets.
Ha et al. (95) investigated different levels
of vitamin B-6 on cell-mediated immunity in mice.
In female
C57BL/6 mice, they observed that primary and secondary
splenic and peritoneal T-cell mediated cytotoxicity was
significantly reduced in animals receiving 10 and 0% of
their required vitamin B-6 compared with animals receiving
700 and 100% of their vitamin B-6 requirement.
Increasing
the vitamin B-6 intake to as much as seven times the
requirement did not further improve immunocompetence.
These
results suggest that high doses of vitamin B-6 may not
produce benefits beyond those observed with moderate
supplementation of the vitamin in mice.
Vitamin B-6 deficiency impairs humoral immunity.
Stoerk and coworkers (96) observed that the formation of
circulating antibodies to sheep red blood cells was
suppressed in rats fed a vitamin B-6 deficient diet.
18
Subsequent studies confirmed this original work and
demonstrated further that vitamin B-6 deficiency
significantly inhibited antibody production in response to
influenza virus (97), B. typhosus (98), Salmonella pullorum
(99), or a synthetic antigen poly Glu-Lys-Tyr (100).
Additionally, antibodies produced by vitamin B-6 deficient
animals have decreased in vitro binding affinity for
antigens (5).
2. Human Studies
In contrast to the numerous animal studies, studies
exploring the effect of vitamin B-6 status on immune
function in human beings are few.
An early study by Hodges et al. (101) investigated the
effect of vitamin B-6 deficiency on antibody production in
response to immunization with tetanus and typhoid antigens
in six healthy young men.
The subjects were made vitamin
B-6 deficient by feeding them a vitamin B-6 deficient diet
with and without deoxypyridoxine, a vitamin B-6 antagonist.
Compared with the controls who took 2 mg PN daily, the
vitamin B-6 deficient subjects had decreased antibody
responses to tetanus and typhoid antigens.
Hodges and
coworkers also observed that the combined deficiencies of
vitamin B-6 and pantothenic acid resulted in a further
decline of antibody titer in response to tetanus and typhoid
antigen immunization (102).
19
Van den Berg et al.
(10) investigated the effect of
marginal vitamin B-6 status on immunological indices in 24
young males.
During the first two weeks, all subjects
received the basal diet, which provided 0.68 mg of vitamin
B-6, plus a multinutrient supplement containing 4 mg of
vitamin B-6.
During the following 11 week period,
12
subjects were maintained on this regimen, while 12 other
subjects were given the same diet without vitamin B-6
supplementation.
At the end of the 11 weeks, the subjects
receiving no vitamin B-6 supplement were marginally depleted
as evaluated by decreased plasma PLP concentrations and
erythrocyte aminotransferase activities.
The percentage of
T-helper cells and the Ig D concentration were slightly
lower in the vitamin B-6 deficient group than in the vitamin
B-6 supplemented group.
Other indices (total lymphocytes,
total T-cells, total B-cells, IL-2 receptors, other
immunoglobulins and complement concentrations) were not
significantly different between the two groups.
The authors
concluded that short-term marginal vitamin B-6 deficiency
did not affect immune function in young men.
However, the
subjects in this study received twice the RDA for all other
vitamins and minerals.
Many of those vitamins and minerals,
such as vitamins A (103), C (104) and E (105), and minerals
Se (106) and Zn (107) may improve immune function of the
subjects.
Additionally, what the authors measured is
actually a static status of the immune cells rather than the
20
dynamic response of immune system to a stimulation.
It is
possible that the marginal vitamin B-6 nutriture could
maintain apparently normal cell number without challenge,
but the immune system may not have the optimal competence
upon a stimulation.
Talbott et al. (7) examined the effect of PN
supplementation on lymphocyte function in an independently
living elderly population.
The mean dietary vitamin B-6
intake in this study group was 1.51 mg/d or 0.022 mg vitamin
B-6/g protein, close to the current RDA.
Eleven subjects
received 50 mg PN.HC1 per day for 2 months and 4 subjects
received a placebo.
The results showed clearly that PN
supplementation significantly increased lymphocyte
proliferation response to both T- and B-cell mitogens in the
elderly.
The percentage of CD3+ and CD4+ cells increased
significantly in subjects receiving the PN supplement.
Moreover, when the PN-treated subjects were divided into two
groups according to their pre-study plasma PLP
concentrations, the effect of PN supplementation on
lymphocyte proliferation was much greater in the subjects
who had a low pre-study plasma PLP concentration ( z 30
nmol/L) than in those with normal level ( > 30 nmol/L).
More recently, Meydani et al.
(6) reported that in
elderly persons who had been depleted in vitamin B-6,
lymphocyte proliferation in response to mitogen stimulation
and IL-2 production increased with vitamin B-6 repletion.
21
The decline in IL-2 production and lymphocyte proliferation
induced by vitamin B-6 depletion, however, was not
completely restored to the baseline level by a vitamin B-6
intake of 2.88 mg/d by men and 1.9 0 mg/d by women.
On the
other hand, in response to 50 mg PN.HCl/day for 4 days,
lymphocyte proliferation and IL-2 production increased above
the baseline level.
Meydani et al. suggested that for
maintenance of normal immune function, older adults may
require more vitamin B-6 than the current RDA.
Vitamin B-6
status indicators (tryptophan load test, plasma PLP level,
urinary 4-PA excretion, and erythrocyte aspartate
aminotransferase activity coefficient) measured in the same
subjects during vitamin B-6 depletion and repletion also
supported this conclusion (108).
It is not certain,
however, whether this higher vitamin B-6 requirement for
maintaining optimal immune function is specific to the
elderly or is a general effect regardless of age.
From the studies summarized above, we see that an
intake of vitamin B-6 above the RDA improves in vitro immune
responses in the elderly.
Although less certain, the study
by Van den Berg et al. suggested that young subjects may
also have an increased immune response with the increased
intake of vitamin B-6.
However, in these studies the number
of subjects was small and the duration of vitamin B-6 was
relatively short and follow-up on the subjects' health was
not done.
22
There are only two relatively large scale field studies
of long duration on the effect of micronutrients including
vitamin B-6 supplementation on immune function.
In a
double-blind controlled investigation reported by Chandra
(8), 96 healthy Canadians over 65 years of age and in the
upper middle to high socioeconomic status were divided into
a supplemented group and a placebo group of 48 each.
Daily
for one year, the supplemented group received a multivitamin
and mineral supplement containing 3 mg of vitamin B-6 and
approximately 1-2 times of the RDA for other vitamins and
essential trace elements.
The percentage of the subjects
with plasma PLP concentrations lower than 50 nmol/L in
supplemented group was decreased from 16.7% at the beginning
of the study to 4.4% after taking the supplement for one
year.
This supplement of physiological amounts of vitamins
and minerals resulted in a significant improvement in immune
indices, including percentage of CD4+ cells, natural killer
(NK) cells, IL-2 production and IL-2 receptors, and in vitro
lymphocyte proliferation in response to PHA stimulation.
The supplemented group also had a significantly decreased
incidence of infectious diseases compared with the placebo
group.
Although this study did not clarify the role of
individual vitamins or minerals, it confirmed both the
effect of physiological amounts of supplementing vitamins
and minerals on laboratory tests of immune response and
improvement in health.
23
Recently, Bogden et al.
(9) administered daily for one
year an over the counter micronutrient supplement to 29
independently living healthy elderly subjects, aged 59 - 85
years, whose vitamin B-6 intake was adequate (1.55 - 2 mg of
vitamin B-6).
This over the counter supplement included 3
mg of vitamin B-6 and other vitamins and minerals in the
range of 20 - 450% of the RDA.
Daily intake of this
nutrient supplement for one year resulted in a significant
increase of DHST in response to a panel of seven recall
antigens.
The authors concluded that a modest daily
supplement of micronutrients enhanced cellular immunity, and
suggested that the current RDAs of micronutrients including
vitamin B-6 are too low to support optimal immunity in older
individuals.
Again, since the subjects had increased intake
of other vitamins and minerals, the role of individual
vitamin or mineral in immune response could not be clarified
in this investigation.
F. Proposed Roles of Vitamin B-6 in Immune Function
Although the importance of vitamin B-6 in developing
and maintaining normal immune function has been recognized
for a long time (5,80,101), the mechanism of the effect of
vitamin B-6 on immune system is still unclear.
Discussed
here are the three proposed mechanisms which include the
role of vitamin B-6 in one-carbon unit metabolism, hormone
24
receptor modulation and polyamine synthesis.
1. One-carbon Unit Metabolism
PLP is the coenzyme of serine hydroxymethyltransferase
(EC 2.1.2.1), which catalyzes a step in one-carbon unit
metabolism.
In 1964, Axelrod and Trakatellis (5) proposed
this as a role of vitamin B-6 in immune function.
This
enzyme catalyzes the transfer of a hydroxymethyl group from
serine to tetrahydrofolate to form N5,N10methylenetetrahydrofolate (109).
The methylene group from
N5,N10-methylenetetrahydrofolate participates in the
biosynthesis of thymidylic acid from deoxyuridine 5'phosphate and of purine bases from glycine amide ribotide.
DNA and RNA synthesis precedes all cell proliferation.
In
response to PHA stimulation, mRNA synthesis in cultured
human T-cells increased significantly within ten minutes
(110).
Trakatellis et al. reported that vitamin B-6
deficient rats had less DNA in splenic cells (111) and less
ribosomal RNA in liver and splenic cells (112) than the
corresponding control animals.
Axelrod and Trakatellis (5)
reported that vitamin B-6 deficiency resulted in a decreased
incorporation of C
rat tissue.
from serine-3-C14 into DNA and RNA in
Vitamin B-6 deficiency, therefore, may suppress
immune function by compromising DNA and RNA synthesis when
immune cells are activated.
To the knowledge of this reviewer, there is no recent
25
research on this role of vitamin B-6 on one-carbon unit
metabolism.
Many authors (4,6,82,83,113) cite this as the
role of vitamin B-6 in immune function.
2. Modulation of Hormone Receptors
An active biomolecule, PLP interacts with many proteins
forming a Schiff base with the e-amino group of the lysine
residue of the protein.
Nishigori et al.
(114) were the
first to report that PLP inhibits the binding of avian
progesterone receptors to ATP-sepharose.
After PLP
treatment, this inhibition could be made irreversible by
reduction with NaBH4 which fixes the formed Schiff base.
Following binding with specific receptors, transporting the
glucocorticoid-receptor complex from cytoplasm to the
nucleus and binding the complex to DNA is considered to be
the first step in glucocorticoid function in cells (115) .
Cidlowski et al. (116) demonstrated that PLP inhibited the
binding of the receptor-hormone complex to the nuclei of rat
thymic lymphocytes, which contain specific, high-affinity
glucocorticoid receptors.
Their study also showed that only
PLP effectively inhibited the binding of the receptorhormone complex to nuclei.
Neither PL, PN, PMP nor 5-
deoxypyridoxal (a nonphysiological analog of PL) fulfilled
this role.
In in vitro studies, PLP inhibited the binding
of estrogen (117) and androgen (118) receptors to DNA and
nuclei.
Compared with control animals, vitamin B-6
26
deficient ones accumulated more radioisotope-labeled
estradiol in uterine tissue (117), while vitamin B-6
sufficient animals exhibited a decreased nuclear
localization of uterine estrogen receptors (119).
Although the mechanism of the action is unclear, the
immunosuppressive effect of glucocorticoid hormones has been
recognized for a long time (120,121).
Allgood and Cidlowski
(122) proposed that the effect of vitamin B-6 on immune
function may be mediated by compromising the
immunosuppressive effect of glucocorticoid hormones.
In
human lymphocytes treated in vitro, glucocorticoids
inhibited production of a variety of cytokines (123) and the
expression of Fc receptors (12 0).
Glucocorticoid treatment
induced specific restriction endonuclease degradation of DNA
in rat thymocytes (124).
The morphologic characteristics of
apoptosis have been observed widely in glucocorticoidinduced atrophy of lymph tissues (125,126).
Recently, Reeve et al. (127) reported that vitamin B-6
intake equivalent to four times the RDA for mice protected
these animals from suppression of contact hypersensitivity
induced by ultraviolet B (UVB) radiation (280 - 320 nm) or
cis-urocanic acid.
Reeve et al. proposed that vitamin B-6
modulates the binding of cis-urocanic acid to H-2 histamine
receptors and compromises the immunosuppressive effect
induced by cis-urocanic acid or UVB radiation.
Trans-
urocanic acid, a metabolite of histidine, is a natural
27
epidermal constituent.
UVB radiation photoisomerizes trans -
urocanic acid to the cis-form, which suppresses immune
response in mice (128).
Cis-urocanic acid shares the
imidazole structure of histamine.
Both cis-urocanic acid
and histamine activate T-suppressor cell function by binding
to H-2 histamine receptors on surface of T-lymphocytes
(129) .
3. Polyamine Synthesis
Polyamines are aliphatic amines associated with nucleic
acids and are synthesized in all cells.
The most common
ones are putrescine, spermidine and spermine.
The first and
rate limiting step of polyamine biosynthesis is the
conversion of ornithine, an intermediate in the urea cycle,
to putrescine by ornithine decarboxylase (ODC, EC 4.1.1.17),
a PLP-dependent enzyme (130).
Putrescine is subsequently
converted to spermidine and spermine by the action of
spermidine synthase (EC 2.5.1.16) and spermine synthase (EC
2.5.1.22), respectively (131).
ODC, the key regulatory
enzyme in this pathway, has a short half-life (11 - 90
minutes depending on source of tissue and experimental
conditions)
(132).
Rapidly growing cells have higher ODC activities and
higher concentrations of polyamines than non-proliferating
cells (133).
Growth stimulation results in a parallel rise
in the levels of polyamines, DNA and RNA (134).
Although
28
the exact role of polyamines in cell growth and development
is not clear, polyamines are important in cellular
macromolecule (e.g., DNA, RNA and protein) synthesis.
Polyamines affect replication, transcription and translation
possibly by influencing the conformation of templates and
their products (135).
At a physiological pH, polyamines
function as polyvalent cations which interact with nucleic
acids and nucleic acid-containing structures such as
ribosomes (13 6).
Polyamines are required for isolated
nuclei from herpes simplex virus infected cells to
synthesize viral DNA (137).
Blocking polyamine synthesis by
adding the ODC inhibitor, difluoromethylornithine (DFMO),
resulted in loss of malarial DNA polymerase (EC 2.7.7.7)
activity.
The addition of putrescine to DFMO-treated
cultures led to increased DNA polymerase activity (13 8).
Physiological concentrations of polyamines stimulated
transcription of both bacterial and eukaryotic RNA
polymerases (EC 2.7.7.6) in vitro (136).
Polyamines affect
a number of steps of protein synthesis: low concentrations
of spermidine stimulate aminoacyl tRNA synthetase activity
(139); and polyamines facilitate the movement of the
ribosomes along mRNA, enabling them to surmount obstacles
(140).
Atkins et al. (141) demonstrated that the addition
of polyamines resulted in a qualitative difference in the
polypeptides synthesized in a cell-free system.
They showed
that when the system was supplemented with polyamines,
29
polypeptides synthesized in vitro were larger and resembled
the in vivo products more closely.
In absence of
polyamines, the cell-free polypeptide synthesis system
produced a greater amount of incomplete translation products
(142).
Abraham et al. (142) and Jelenc and Kurland (143)
suggested that polyamines contribute to the specificity of
the codon anti-codon interaction and that the high fidelity
of translation is partly due to the presence of polyamines.
It is well documented that the conversion of many types
of mammalian cells from resting to the rapidly growing and
dividing state is accompanied by a marked increase in
the ODC activity and subsequent polyamine content of cells
(130,144-146).
Upon antigenic or mitogenic stimulation,
lymphocytes are quickly transformed into a rapidly
proliferative state.
Fillingame et al.
methylglyoxal bis(guanylhydrazone)
(147) reported that
(MGBG), an anti-leukemic
agent and a potent inhibitor of polyamine synthesis,
inhibited DNA synthesis in bovine lymphocytes activated by
Con A by 60% compared with the control.
Addition of
exogenous spermidine or spermine 24 hours after Con A
stimulation produced complete reversal of inhibition of DNA
synthesis by MGBG.
Moreover, when MGBG was added more than
40 hours after Con A stimulation, the length of time
lymphocytes need to accumulate polyamines after stimulation,
no inhibition of DNA synthesis was observed.
More recently,
Singh et al. (148) tested the effect of DFMO and exogenous
30
putrescine on normal immune responses and malignant cell
growth.
Inhibition of ODC by DFMO suppressed lymphocyte
proliferation in response to Con A and lipopolysaccharide.
DFMO-mediated inhibition of cell proliferation correlated
with depletion of intracellular polyamines.
Repletion with
putrescine reversed the inhibitory effects of DFMO.
Scott
et al. (149) showed that ODC activity and polyamine
biosynthesis were rapidly increased by lectin or lymphokine
stimulated human T-lymphocytes.
Putrescine also reversed
the growth inhibitory effects of DFMO on four tumor cell
lines (148) .
After activation, lymphocyte proliferation is regulated
by the production of IL-2 by T-helper cells and the
induction of specific membrane receptors for IL-2 (150,151).
Responding to stimulation, T-helper cells also produce IL-3
(152).
IL-3, which is species-specific, stimulates stem
cell growth and fosters a dose-dependent rise in peripheral
blood lymphocyte counts (153).
Depletion of polyamines by
DFMO reduces both IL-2 and IL-3 dependent proliferation of
cell lines.
The addition of exogenous putrescine reversed
this decreased response to lymphokines (154).
Intracellular
polyamine biosynthesis is also required for IL-2
responsiveness during lymphocyte mitogenesis (155) .
4. Vitamin B-6 and Polyamines
Since PLP is the coenzyme of ODC, the relationship
31
between vitamin B-6 status and polyamine biosynthesis has
been explored.
Pegg (156) reported that polyamine
concentrations in the liver, kidney and brain of adult
vitamin B-6 deficient rats were significantly lower than in
the control group.
ODC activity (measured without adding
PLP in vitro) in hepatic extracts from the vitamin B-6deficient rats was significantly lower than that of the
controls.
After adding PLP to the hepatic extracts, ODC
activity increased markedly and was higher than that of the
controls.
Pegg suggested that more ODC apoenzyme had been
synthesized in the vitamin B-6 deficient rats and that the
reduced tissue polyamine concentration was due to the
decreased availability of PLP for ODC.
Recently, Sampson et al. (157) reported that ODC apoprotein, measured as total ODC activity (with PLP added in
vitro). was elevated 2.7-, 5.8-, and 8.5-fold, respectively,
in the kidney, liver, and intestine, respectively, in
vitamin B-6 deficient rats compared with control animals.
This is consistent with the results reported by Pegg (156) .
On the other hand, Sampson et al. found that ODC holoenzyme
activity (measured without adding PLP in vitro) also
increased in the kidney, liver and intestine of vitamin
B-6-deficient rats.
They did not measure tissue polyamine
concentrations and gave no explanation for the increased ODC
holoenzyme in vitamin B-6 deficient rats.
32
MATERIALS AND METHODS
Two studies were conducted in young women.
In Study I,
the effect of vitamin B-6 intake ranging from 0.84 mg to
2.44 mg vitamin B-6 on lymphocyte proliferation in vitro was
investigated.
This study was designed primarily to
determine the effect of varying physiological levels of
vitamin B-6 intake on the metabolism of this nutrient in
young women.
Results on vitamin B-6 metabolism from this
investigation will be reported elsewhere.
Study II was
designed to test the effect of physiological and
pharmacological doses of supplementary PN and its removal on
in vitro
lymphocyte proliferation and IL-2 production in
young women.
Unless mentioned otherwise, all reagents used in this
investigation were obtained from Sigma Chemical Company, St.
Louis, MO.
A. Subjects
The subjects were recruited by advertisements placed on
bulletin boards on Oregon State University (OSU) campus and
in the city of Corvallis, Oregon.
The volunteers completed
a questionnaire to provide information on their health,
menstrual cycle, eating habits, medical history, use of
drugs including alcoholic beverages and nutritional
33
supplements, and physical activity.
Written informed
consent was obtained from each subject.
These two
investigations were approved by the OSU Committee for
Protection of Human Subjects.
In both studies, all subjects were women between the
ages of 2 0 - 35 years, and, as determined by a
questionnaire, were in good health; had no acute or chronic
disease; were non-smokers; were not pregnant; were not
taking an oral contraceptive agent (except one subject in
Study II) or any other medications such as hydrazine
derivatives, cycloserine, L-Dopa, and penicillamine which
could affect the metabolism of vitamin B-6 (158); and were
not taking vitamin supplements.
In addition, the subjects
were not taking drugs that are known to affect immune
function, such as corticosteroids, cyclosporine,
azathioprine, and cyclophosphamide, or receiving any
immunostimulative agent such as lymphokines (159) .
B. Experimental Designs
Study I was a repeated measures design.
In this 32-day
investigation, the six subjects (Table 1) received a
constant basal diet which was adequate in all nutrients
(except vitamin B-6 during the first 12-day period) and was
composed of natural foods.
The basal diet contained 13.6 g
N (85 g crude protein) and 0.84 mg (4.96 jimols) of vitamin
34
B-6.
To lower the subjects' body stores of vitamin B-6, the
subjects received the basal diet without a PN supplement
during the first 12 days of the study.
This preliminary 12-
day period was followed by two consecutive 10-day periods.
The subjects' basal diet was supplemented daily with 0.4 mg
PN (as PN.HCl) during the first 10-day period, and with 1.6
mg PN (as PN.HCl) during the second 10-day period.
The
subjects' total daily vitamin B-6 intake was 0.84 mg (4.96
jimols) , 1.24 mg (7.33 ^mols) and 2.44 mg (14.42 nmols) ,
respectively, during the 3 consecutive periods (Fig. 2A).
The composition of the basal diet, as well as preparation
and administration of the PN supplements are reported
elsewhere.
Table l.
Descriptive data on subjects in Study I
Age
Height
(y)
(cm)
beginning
end of study
1
2
3
4
5
6
28
28
27
33
28
25
162
185
162
183
170
173
68.2
64.5
94.1
68.8
62.2
57.7
67.7
63.2
95.0
68.4
61.8
57.7
Mean
±SD
28.2
2.6
172.5
9.9
69.3
12.8
69.0
13.4
Subject
Weight (kg)
35
The experimental protocol for Study II is illustrated
in Fig. 2B.
This was a completely randomized design study.
Ten female subjects, described in Table 2, were randomly
assigned to two groups of five each: one group received a
PN.HCl supplement (PN group), the other received a placebo
(placebo group).
The subjects consumed self-selected diets
throughout the 46-day investigation.
Following a 5-day
preliminary period, the PN group received 1.5 mg of
supplementary PN.HCl (7.29 iimols) daily for 7 days, followed
by a supplement of 50 mg PN.HCl (243 umols) daily during the
subsequent 6-day period.
The control group received a
placebo during these 13 days.
The subjects were not aware
of which preparation they were receiving.
Compliance was
verified by providing the subjects their PN supplement or
placebo every morning during this period.
This "treatment"
period was followed by a 28-day "washout" period during
which the subjects received no PN supplement or placebo.
The subjects provided their 3-day dietary records between
days 1 and 5, 6 and 12, and 3 6 and 46 of the study.
36
Table 2.
Descriptive data on subjects in Study II
Sub- Group
ject
Age
Height
Weight (kg)
(y)
(cm)
34
36
20
24
25
25
34
22
24
27
164
160
175
160
162
179
168
171
164
161
62.2
53.1
68.1
56.3
53.3
72.2
61.3
63.1
52.7
54.9
61.7
53.1
68.1
55.4
52.7
72.6
59.9
64.0
52.9
52.9
Means
±SD
25. 8
6. 3
165.4
7.1
59.1
6.3
58.7
6.9
Placebo Means
±SD
28. 4
5. 1
167.4
6.8
60.3
8.0
60.0
8.1
1
2
3
4
5
6
7*
8
9
10
placebo
PN
PN
PN
placebo
Placebo
placebo
PN
placebo
PN
beginning
end of study
Group
PN
* Took an oral contraceptive agent during the experiment.
The vitamin B-6 supplement was prepared by mixing
PN.HCl with dextrose at ratios of 1:400 and 1:12 for the 1.5
mg PH.HC1 and 50 mg PN.HCl supplements, respectively.
Pyridoxine hydrochloride and dextrose were mixed thoroughly
and sieved 7 times.
Six hundred milligrams of this PN.HC1-
sugar mixture were placed in a gelatin capsule.
Six hundred
milligrams of dextrose were placed in a gelatin capsule for
the placebo.
The concentration of the PN.HCl in capsules
was verified spectrophotometrically in 0.1 N HC1 using an
extinction coefficient of 422 at 291 run (160).
37
(A)
blood draws
1
I
I
0.84mg B-6/day
1.24mg B-6/day 2.44mg B-6/day
day
12
1
22
32
(B)
3day dietary record
blood draws
I
+50mg
+1.5mg
washout
PN/day
(no PN or placebo)
PN/day
or placebo
day
12
15
18
22
26
36
46
Fig. 2. The experimental protocols.
(A). Study I. During
the first period, days 1 through 12, the subjects received
0.84 mg (4.96 jimols) of vitamin B-6 daily, and during days
13 through 22, and days 23 through 32, they received a total
of 1.24 mg (7.33 jimols) and 2.44 mg (14.42 jimols) of vitamin
B-6 daily, respectively. Blood was drawn from fasting
subjects in the morning of day 1, and in the morning
following days 12, 22, and 32.
(B). Study II. The subjects
consumed their self-selected diets throughout this
investigation. Following a 5-day preliminary period, the PN
group received a supplement of 1.5 mg PN.HC1 (7.29 jimols)
daily for seven days, followed by a supplement of 50 mg
PN.HC1 (243 nmols) daily during the subsequent six day. The
placebo group received a placebo during these 13 days. This
"treatment" period was followed by a 28-day "washout" period
of no supplement or placebo. The subjects provided their 3day dietary records between days 1 and 5, 6 and 12, and 3 6
and 46.
38
C. Blood Drawing
Between 7 and 9 am on specified days for both studies
(Fig. 2A and 2B), blood was obtained from the antecubital
vein of fasting subjects by a registered medical
technologist.
The blood was collected in evacuated tubes
containing heparin and processed immediately.
25 ml of blood were drawn each time.
Approximately
Of this volume,
approximately 8 ml of blood were used in both studies to
separate mononuclear cells for lymphocyte proliferation test
(both studies) and for measuring interleukin 2 production
and lymphocyte PLP concentration (Study II only).
The
plasma and erythrocytes (washed with normal saline) from the
remaining blood were stored at -20
0
C for measuring plasma
PLP concentration (both studies) and erythrocyte aspartate
aminotransferase activity later (Study II only).
D. Isolation of Mononuclear Cells
Immediately after it was obtained, whole blood was
diluted 1:1 with phosphate buffered saline.
Diluted blood
was carefully layered with a Pasteur pipette on top of 8 10 ml of lymphocyte separation reagent (Histopaque-1077) in
a 50 ml centrifuge tube.
The this tube was centrifuged at
400 x g for 3 0 minutes at room temperature.
Mononuclear
cells at the interface were removed with a Pasteur pipette,
39
placed in a 15 ml centrifuge tube, and washed twice with
RPMI 1640.
E. Lymphocyte Viability Tests and Counts
Lymphocyte viability was determined by trypan blue
exclusion.
The percentage of viable cells in the cell
suspension was greater than 95%.
For further experiments,
the cells were then resuspended in appropriate amount of
RPMI 1640 with 10% fetal bovine serum (FBS)
(10% RPMI) to
obtain a concentration of 1X109 viable cells/L.
F. Lymphocyte Proliferation
Lymphocyte proliferation was measured by 3H-thymidine
incorporation following culture with the T-cell mitogens,
PHA and Con A, and the T-cell-dependent B-cell mitogen, PWM.
Appropriate dilutions of each mitogen were made in 10%
RPMI.
PHA was diluted to concentrations of 10, 2.5, and
0.625 jig/ml;
Con A was diluted to 40, 10, and 2.5 ng/ml ;
and PWM was diluted to 4, l, and 0.25 ng/ml.
These
concentrations of each mitogen were plated in triplicate,
0.1 ml per well, into a 96-well flat-bottomed microtiter
plate.
One tenth milliliter of cell suspension of IxlO9
cells/L was plated into each well.
All plates were
incubated 96 hours at 370C in an atmosphere of 5% CO2 (Napco
40
CO2 incubator, Model 304; Napco Inc., Minneapolis, MN).
The
microtiter plates were wrapped with plastic film to prevent
water evaporation from the medium.
After 72 hours, 0.5 jiCi
3
H-thymidine (6.7 Ci/M; ICN Biomedical, Inc. Irvine, CA) was
added to each well in a volume of 20 jil.
At the end of 96
hours, the plates were frozen at -700C until harvested.
When thawed, the cells were harvested onto microfiber
paper using a semi-automatic cell harvest system (Type
11019, Skatron Instrument Inc., Sterling, VA).
Filter disks
were dried at 350C and placed in pre-labeled minivials.
After adding 2 ml of liquid scintillation fluid to each
vial, the vials were counted in a S-scintillation counter
(Beckman Model LS5000TD, Beckman Instruments, Inc.,
Fulleton, CA).
Data were recorded as counts per minute
(cpm) using a tritium channel.
The cpm of the unstimulated,
control culture was subtracted from each subject's mitogen
responses.
G. IL-2 Production (Study II)
Lymphocytes (IxlO9 cells/L) in 10% RPMI were cultured
in 24-well, flat-bottomed plates with PHA (2.5 mg/L) for 48
hours.
The cell-free supernatant was stored at -700C for
measuring IL-2.
IL-2 activity was measured by the
microassay method by Coligan et al. (161) using CTLL-2 cell
line (ATCC TIB 214).
Recombinant human IL-2 was used as the
41
standard.
IL-2 standard and samples were diluted with 10% RPMI.
One tenth milliliter of standard or sample was plated in
triplicate into a 96-well flat-bottomed microtiter plate.
CTLL-2 cells were counted and cell concentration was
adjusted to SxlO7 cells/L in 10% RPMI.
One tenth milliliter
of the cell suspension was added to each well of the
microtiter plates containing either an IL-2 standard or
sample.
A negative control with no standard or sample was
included in each assay.
The plates were wrapped with
plastic film and incubated for a total of 2 8 hours at 3 70C
and 5% CO2.
After 20 hours of incubation, 0.5 nCi
3
H-
thymidine was added to each well in a volume of 20 jil.
At
the end of 28 hours, the plates were frozen and stored at
-700C until harvested.
Cells were harvested and 3H-
thymidine incorporation was measured as described for the
lymphocyte proliferation assay given above.
IL-2 activity
was calculated using probit analysis (161).
H. Effect of Putrescine on Lymphocyte Proliferation and IL-2
Production (Study II)
To examine the effect of putrescine on the
proliferation of lymphocytes, putrescine was added in
culture medium at concentrations of 0, 2, 20 and 200 pimol/L
when testing the lymphocyte proliferation in response
42
to PHA, Con A and PWM stimulation.
putrescine on IL-2 production,
To test the effect of
of
0, 20 and 200 nmol/L
putrescine were added in the culture medium.
The procedures
for testing lymphocyte proliferation and IL-2 production
were then followed as described above.
I. PLP Determination
Plasma PLP was determined by measuring the
14
C02
evolved during the decarboxylation of tyrosine-l-14C by
tyrosine decarboxylase apoenzyme.
The method used was
proposed by Chabner and Livingston (162) and modified by
Leklem (163).
A minimum of 1 x 105 lymphocytes were used to measure
PLP concentration in the cells.
The lymphocytes were broken
by freezing and thawing, followed by sonification (Sonifier
Model 200, Branson Sonic Power Company, Danbury, CT) for 30
seconds.
Complete breakage of cells was verified under a
light microscope.
Lymphocytes and FBS PLP concentrations
were measured the same as plasma as described above.
The coefficient of variation (CV) of a control plasma
sample analyzed with each assay was 4.2% (n = 11).
The
percent recovery of PLP added to the subjects' plasma
samples was 91.7% ± 5.7% (mean ± SD).
43
J. Erythrocyte Aspartate Aminotransferase Activity
Coefficient (EAST AC) (Study II)
The kinetic method described by Beutler (164) was used
to measure erythrocyte aspartate aminotransferase activity
with and without in vitro PLP stimulation.
Results are
expressed as erythrocyte aspartate aminotransferase activity
coefficient (activity with PLP stimulation/activity without
PLP stimulation).
K. Collection of Dietary Information (Study II)
Between the days 1 and 5, 6 and 12 and 3 6 and 46 of
Study II, each subject completed three three-day dietary
records.
Each three-day dietary record included two week
days and one weekend day.
Foods were coded for computer analysis for general
nutrient intake with special emphasis on vitamin B-6 and
protein intake.
The nutrient database used was The Food
Processor Plus (Nutrition & Fitness Software, Version 5.0,
ESHA Research, Salem, OR).
This nutrient database is based
on data from USDA and information from manufacturers for
over 5000 food items.
44
L. Statistics
The data were analyzed for statistical significance by
using the software packages Statgraphics (Statistical
Graphics System, Version 5.0, STSC, Inc., Rockville, MD) and
SAS (Version 6.04, SAS Institute Inc., Gary, NC).
Comparisons between means were made with ANOVA, multiple
range test and Student's t test.
Correlation coefficients
were calculated to determine relationships between immune
parameters and plasma PLP concentration and lymphocyte PLP
concentration.
p 4 0.05
Statistical significance was based on
45
RESULTS
A. Study I
1. Plasma PLP Concentration
The six subjects' plasma PLP concentrations are given
in Table 3.
Their initial (baseline) vitamin B-6 status was
adequate according to Leklem (52).
After a daily vitamin B-
6 intake of 0.84 mg for 12 days, the subjects' mean plasma
PLP concentration dropped more than 40 %, from 47.4 nmol/L
at baseline to 26.5 nmol/L.
An intake of 1.24 mg of vitamin
B-6 per day for 10 days did not restore the subjects' plasma
PLP to the baseline level.
After ten days of 2.44 mg
vitamin B-6 daily intake, the subjects' mean plasma PLP
level increased to 58.9 nmol/L, almost 25 % higher than the
baseline level.
2. Lymphocyte Proliferation
When the subjects received 0.84 mg of vitamin B-6, the
lymphocyte proliferation response to the T-cell mitogen, Con
A, decreased slightly, while the response to the T-dependent
B-cell mitogen, PWM, did not change from the baseline
response (Table 4 and Fig. 3).
An intake of 1.24 mg of
vitamin B-6 increased their lymphocyte proliferation
response to both mitogens, but only the response to Con A
was significantly higher (p < 0.05) than that at 0.84 mg of
46
vitamin B-6 intake.
Increasing the subjects' vitamin B-6
intake to 2.44 mg per day resulted in a significantly higher
(p < 0.05) lymphocyte response to both mitogens than at
baseline and when the subjects received 0.84 mg of vitamin
B-6.
Table 3.
Vitamin B-6 intake and plasma PLP concentrations in six
healthy young women consuming a constant diet with
controlled vitamin B-6 intake (Study I)*
B-6 intake (mg/d)
Baselinefl
0.84§
1.24§
2.44§
Plasma PLP (nmol/L)
47.4
26.5
29.4
58.9
±
±
±
±
17.9
12.4
12.5
22.3
* Mean ± SD; plasma PLP was measured by C. Hanson; details
are described elsewhere.
II First day of the study; reflects previous self-selected
diet.
§ Blood drawn on days 13, 23, and 33, after subjects had
consumed 0.84 mg, 1.24 mg, and 2.44 mg of vitamin B-6 per
day for 12, 10, and 10 days, respectively.
47
Con A
H PWM
150
120
E
Q.
O
9 ^
I2 3«=
I
2 o
gb
a
o
Baseline
0.84
1.24
B-6 intake (mg)
2.44
Fig. 3. Vitamin B-6 intake and lymphocyte proliferation in
response to Con A and PWM stimulation (Study I). Baseline
reflects the pre-study value (self-selected diet); 0.84,
1.24, and 2.44 mg B-6 indicate the subjects' vitamin B-6
intake.
(*) shows significant difference (p < 0.05)
compared with the baseline value and (#) significant
difference (p < 0.05) compared with the value during 0.84 mg
vitamin B-6 intake period.
y
48
Table 4.
Vitamin B-6 intake and mitogen response, in cpm, of
lymphocytes in six healthy young women consuming a constant
diet with controlled vitamin B-6 intake (Study I)*
Mitogens
B-6 intake
(mg/d)
Baselinefl
0.84§
1.24§
2.44§
Con A
73154
67306
91130
107447
± 11604
± 4425
± 10325B
± 5697^
PWM
24644
27058
35876
48792
± 5983
± 5458
± 7960
± 10704AB
* Mean ± SEM.
fl First day of the study; values reflect subjects' previous
self-selected diet.
§ Blood drawn on days 13, 23 and 33, after subjects consumed
0.84 mg, 1.24 mg and 2.44 mg of vitamin B-6 per day for
12, 10 and 10 days, respectively.
A Significant difference (p < 0.05) compared with baseline
value.
B Significant difference (p < 0.05) compared with 0.84 mg
vitamin B-6 intake period.
49
B. Study II
1. Subjects' Dietary Vitamin B-6 Intake and Nutritional
Status
The subjects' dietary intakes were adequate in protein,
vitamin B-6 and energy (Table 5) as well as other nutrients
(data not reported here).
There were no significant
differences between the PN and placebo groups in their
dietary intakes of vitamin B-6 and protein.
Their mean
vitamin B-6 intake and mean ratio of mg of vitamin B-6 to g
of protein were above the RDA for women (47).
There were no significant differences between the PN
and placebo groups in their hemoglobin concentrations and
hematocrit values (Table 6).
The hemoglobin and hematocrit
values of all 10 subjects were within the normal ranges for
women (165) .
50
Table 5.
Average daily dietary intakes of energy, vitamin B-6, and
protein by the PN and placebo groups during baseline,
supplement and washout periods (Study II)*1I
Periods
Baseline
Supplement!
Washout
1642 ± 136
1872 + 431
1694 ± 490
1822 ± 259
Vitamin B-6 (mg)
PN
1.75 ± 0.23
Placebo
1.75 ± 0.40
1.80 ± 0.23
1.81 ± 0.73
1.83 ± 0.37
2.33 ±- 0.80
Protein (g)
PN
Placebo
70.6 ± 16.0
68.9 ± 9.2
65.4 ± 27.5
65.5 ± 15.6
0.026 ± 0.007
0.027 ± 0.013
0.032 ± 0.020
0.035 ± 0.005
Energy (kcal)
PN
Placebo
1733 ± 286
1831 ± 668
66.0 ± 13.4
61.8 ± 19.6
B-6/Protein (mg/g)
PN
0.028 ± 0.009
Placebo
0.031 ± 0.011
* Mean ± SD; n = 5 per group. There were no significant
differences in dietary intakes between the PN and placebo
groups.
II The subjects consumed their self-selected diet throughout
this investigation. They recorded their three-day dietary
intakes between days 1 and 5, 6 and 12 and 36 and 46.
§ Vitamin B-6 intake of PN group during the days 6 to 12
does not include the 1.5 mg PN.HC1 supplement.
51
Table 6.
Average weekly hemoglobin (Hb) concentration and hematocrit
(Hct) value of the PN and placebo groups (Study II)*
Time
Baseline
PN
Placebo
Hb(g/L)
Hct(l)
127 ± 8
0.41 ± 0.01
128 ± 8
0.41 ± 0.01
Supplement period
+1.5mg PN.HCl/d
or placebo
day 7
Hb(g/L)
Hct(l)
128 ± 8
0.41 ± 0.02
132 ± 7
0.41 ± 0.01
+50mg PN.HCl/d
or placebo
day 6
Hb(g/L)
Hct(l)
133 ± 13
0.41 ± 0.03
134 ± 8
0.40 ± 0.01
Washout period
day 4
Hb(g/L)
Hct(l)
132 ±7
0.41 ± 0.01
133 ± 6
0.40 ± 0.01
day 8
Hb(g/L)
Hct(l)
128 ± 9
0.40 ± 0.02
131 ± 10
0.40 ± 0.02
day 18
Hb(g/L)
Hct(l)
131 ± 10
0.40 ± 0.02
127 ± 4
0.40 ± 0.01
day 28
Hb(g/L)
Hct(l)
131 ± 9
0.41 ± 0.02
130 ± 4
0.41 ± 0.02
* Mean ± SD; n = 5 per group. The subjects consumed their
self-selected diet throughout this investigation. There
were no significant differences in Hb and Hct between the
PN and placebo groups.
52
2. Plasma and Lymphocyte PLP concentrations
The mean plasma PLP concentrations at baseline of the
two groups were normal (Table 7, Fig. 4A)
(52).
Although
the range of plasma PLP concentrations within the placebo
group was large and the mean was higher than that of the PN
group, there was no significant difference between the PN
and placebo groups in their baseline plasma PLP
concentrations.
Following PN.HCl supplementation, the PN group had
higher plasma PLP concentration than the placebo group.
The PN-treated subjects' mean plasma PLP concentration
increased from 35.6 nmol/L at baseline to 54.3 nmol/L after
a daily 1.5 mg PN.HCl supplement for seven days.
After
having received 50 mg of PN.HCl for three days, mean plasma
PLP concentration increased to 248 nmol/L, seven times
higher than the baseline level.
The mean plasma PLP
concentration plateaued at approximately 250 nmol/L in
response to 50 mg of PN.HCl supplement.
During the washout
period, the plasma PLP concentrations in the PN group
dropped rapidly.
After 18 days of no PN.HCl
supplementation, the PN group's mean plasma PLP
concentration dropped to the baseline level.
The placebo
group's plasma PLP level remained relatively stable during
the 46 days of study.
In general, the changes in lymphocyte PLP concentration
53
in the PN group during the supplement and washout period
paralleled those in plasma PLP concentration (Table 8, Fig.
4B).
The initial mean lymphocyte PLP concentrations in two
groups were similar.
In the PN group, 1.5 mg PN.HC1
supplement increased mean lymphocyte PLP concentration from
0.27 to 0.55 pmol/10° cells.
After six days of supplement
with 50 mg PN.HC1 per day, this value increased to 1.68
pmol/106 cells.
During the washout period, the PN group's
lymphocyte PLP concentration dropped more slowly compared
with the decline in plasma PLP concentration.
After 18 days
of PN.HC1 withdrawal, their lymphocyte PLP concentration
dropped to the baseline level.
Lymphocyte PLP concentration
in the placebo group did not change during the 46 days of
this study.
Note in Table 8 that sufficient sample size was
not always available for the lymphocyte PLP measurement.
54
Table 7.
Plasma PLP concentrations
groups (Study II)*
(nmol/L)
of the PN and placebo
PN
Placebo
Baseline
35.6 ± 7.4
54.8 ± 38.8
Supplement period
+1.5mg PN.HCl/d
or placebo
day 7
54.3 ± 8.8J
44.3 ± 30.9
+50mg PN.HCl/d
or placebo
day 3
day 6
Washout period
day 4
day 8
day 18
day 28
248.0 ± 32.3 AB
254.8 ± 48.1 AB
100.6
70.2
47.1
39.7
±
±
±
±
17.1 AB
19.5^
6.9
7.0
60.8 ± 39.4
46.7 ± 37.2
48.2
42.7
50.5
54.6
±
±
±
±
30.3
33.6
28.7
43.1
* Mean ± SD; n = 5 per group.
The subjects consumed their
self-selected diet throughout this investigation.
A Significant difference (p < 0.05) compared with baseline
value.
B Significant difference (p < 0.05) compared with control
group during same period.
55
Table 8.
Lymphocyte PLP concentrations (pmol/106 cells) of the PN and
placebo groups (Study II)*
PN (n=5§)
Placebo (n§)
Baseline
0.27 ± 0.04
0.26 ± 0.04(4)
Supplement period
+1.5mg PN.HCl/d
or placebo
day 7
0.55 ± 0.30
0.32 ± 0.13(5)
1.28 ± 0-21^
1.68 + 0.45^
0.57 ± 0.25(3)
0.52 ± 0.33(2)
1.17
1.10
0.40
0.50
0.37
0.35
0.54
0.52
+5 0mg PN.HCl/d
or placebo
day 3
day 6
Washout period
day 4
day 8
day 18
day 2 8
± 0-35^
± 0>49AB
± 0.12
± 0.30
±
±
+
±
0.21(2)
0.26(2)
0.02(2)
0.33(3)
* Mean ± SD. The subjects consumed their self-selected diet
throughout this investigation.
§ Number of subjects. Sufficient blood was not always
available to measure lymphocyte PLP in all of the subjects
in the placebo group.
A Significant difference (p < 0.05) compared with baseline
value.
B Significant difference (p < 0.05) compared with control
group during same period.
56
-+-
PN
— O— Placebo
300i
200
a
E
V)
a
100:.
a.
Ilia
lllb
lllc
Hid
a.
-i
a.
u
nid
Fig. 4. Changes of plasma and lymphocyte PLP concentrations
in the PN and placebo groups (Study II). A: Plasma PLP
concentration; B: Lymphocyte PLP concentration. Periods
are: (B) baseline value; (I) a daily supplement of 1.5 mg
PN.HCl or placebo for 7 days; (Ila) a daily supplement of 50
mg PN.HCl or placebo for 3 days and (lib) for 6 days; (Ilia)
indicates discontinuation of PN supplement or placebo for 4
days, (lllb) for 8 days, (IIIc) for 18 days and (Hid) for
28 days.
(*) shows significant difference (p < 0.05)
compared with the baseline value and (#) significant
difference (p < 0.05) compared with the placebo group.
57
3. Erythrocyte Aspartate Aminotransferase Activity
Coefficient (EAST AC)
Although the PN group's EAST AC decreased slightly
during the supplement period, there were no significant
differences in EAST AC between the two groups throughout
this investigation (Table 9).
Table 9.
Erythrocyte aspartate aminotransferase activity coefficient
(EAST AC) of the PN and placebo groups (Study II)*
PN
Placebo
Baseline
1.67 ± 0.22
1.77 ± 0.18
Supplement period
+1.5mg PN.HCl/d
or placebo
day 7
1.55 ± 0.12
1.60 ± 0.18
+50mg PN.HCl/d
or placebo
day 3
day 6
1.43 ± 0.28
1.56 ± 0.23
1.60 ± 0.34
1.90 ± 0.24
1.56
1.59
1.47
1.59
1.78
1.64
1.76
1.57
Washout period
day 4
day 8
day 18
day 28
±
±
±
±
0.29
0.29
0.26
0.28
±
±
±
±
0.18
0.22
0.36
0.20
* Mean ± SD; n = 5 per group. The subjects consumed their
self-selected diet. There were no significant differences
at EAST AC between the PN and the placebo groups
throughout this investigation.
58
4. Lymphocyte Proliferation
The lymphocyte proliferation in response to PHA (Table
10, Fig. 5A), Con A (Table 11, Fig. 5B) and PWM (Table 12,
Fig. 5C), significantly increased following a daily
supplement of 1.5 mg PN.HC1 for 7 days.
Increasing the
PN.HC1 supplement to 50 mg, however, did not further
increase lymphocyte proliferation in response to PHA and PWM
stimulation and enhanced the lymphocyte proliferation
response to Con A only slightly.
The increased lymphocyte
proliferation in response to all three mitogens in PN group
during the supplement period were statistically significant
when compared with that in the placebo group.
During the washout period, the elevated lymphocyte
proliferation in response to three mitogens in the PN group
gradually decreased.
However, three weeks after removed the
PN.HC1 supplement, the lymphocyte proliferation in response
to all three mitogens remained elevated and significantly
higher than baseline and placebo group values.
By the end
of the fourth week, the proliferation response of
lymphocytes to all mitogens from the PN group dropped to the
pre-supplement level and were not significantly different
from the placebo group.
In the placebo group, the
lymphocyte proliferation in response to mitogen stimulation
showed only minor fluctuations throughout this 46-day study.
59
Table 10.
PN supplementation and mitogen response, in cpm, of
lymphocytes to PHA in the PN and placebo groups (Study II)*
Period
Baseline
PN
Placebo
84679 ± 3893
82117 ± 2838
Supplement period
+1.5mg PN.HCl/d
or placebo
day 7
106120 ± 5042 AB
91892 ± 2326
+50mg PN.HCl/d
or placebo
day 3
day 6
106983 ± 2443^
108262 ± 3690^
93069 ± 2812
84821 ± 3143
108329
107945
97654
85645
82249
85483
75681
86194
Washout period
day 4
day 8
day 18
day 28
±
±
±
±
4757^
6424^?
3017AB
5866
±
±
±
±
3449
6316
3150
3067
* Mean ± SEM; n = 5 per group. The subjects consumed their
self-selected diet throughout this investigation.
A Significant difference (p < 0.05) compared with baseline
value.
B Significant difference (p < 0.05) compared with control
group during same period.
60
Table 11.
PN supplementation and mitogen response, in cpm, of
lymphocytes to Con A in the PN and placebo groups
(Study II)*
Period
PN
Placebo
Baseline
69473 ± 1974
Supplement period
+1.5mg PN.HCl/d
or placebo
day 7
81287 ± 3031 AB
73073 ± 3588
+50mg PN.HCl/d
or placebo
day 3
day 6
92626 ± 2705^
83351 ± 941 AB
80555 ± 3255
69325 ± 5648
Washout period
day 4
day 8
day 18
day 28
88676
83546
79451
74239
±
±
±
±
1400 AB
2946"AB
5224^
4425
69813 ± 3928
68136
73925
67581
68544
±
±
±
±
6123
5598
4148
3418
* Mean ± SEM; n = 5 per group. The subjects consumed their
self-selected diet throughout this investigation.
A Significant difference (p < 0.05) compared with baseline
value.
B Significant difference (p < 0.05) compared with control
group during same period.
61
Table 12.
PN supplementation and mitogen response, in cpm, of
lymphocytes to PWM in the PN and placebo groups*
Period
PN
Placebo
Baseline
19335 ± 3007
Supplement period
+1.5mg PN.HCl/d
or placebo
day 7
36219 ± 5796AB
22273 ± 1084
+50mg PN.HCl/d
or placebo
day 3
day 6
38682 ± 4158^
34240 ± 5658^
27421 ± 5482
25110 ± 3665
43478
33077
37805
24011
23196 ± 5288
30492 ± 4737A
28595 ± 3224A
28203 ± 5945
Washout period
day 4
day 8
day 18
day 28
±
±
±
±
4156^
5947A
2366^
5839
19641 ± 2521
* Mean ± SEM; n = 5 per group. The subjects consumed their
self-selected diet throughout this investigation.
A Significant difference (p < 0.05) compared with baseline
value.
B Significant difference (p < 0.05) compared with control
group during same period.
62
PN
mM Placebo
ISO'
PHA
12<H
90-
60
30
I
B
lla
Mb
Ilia
lllb
lllc
Hid
ISO
120
B
I
lla
Mb
Ilia
lllb
lllc
Hid
150
PWM
Fig. 5. Lymphocyte proliferation in response to PHA (A), Con
A (B) and PWM (C) stimulation in the PN and placebo groups
(Study II). Periods, * and # are the same as in Fig. 4.
63
To explore further the relationship between lymphocyte
PLP concentration and the proliferation response of
lymphocytes to mitogen stimulation, correlation analyses
were conducted.
The lymphocyte proliferation in response to
each of the three mitogens was positively and significantly
(p < 0.01) correlated to lymphocyte PLP concentrations (Fig.
6A-C).
The correlation coefficient for lymphocyte
proliferation in response to PHA was 0.44 (p < 0.01), to Con
A was 0.47 (p < 0.01) , and to PWM was 0.45 (p < 0.01) .
The
best-fit correlation lines in Figure 6A-C could be roughly
separated into two parts at a PLP concentration of 1.0
pmol/10° cells.
Changes in proliferation response were
greater at the lower concentrations of lymphocyte PLP
( < 1.0 pmol/106 cells) than those at the higher lymphocyte
PLP concentrations ( ^ 1.0 pmol/106 cells).
64
Fig. 6. Correlation between lymphocyte proliferation, IL-2
production and lymphocyte PLP concentrations (Study II).
Data are from both PN and placebo groups. A: lymphocyte
proliferation in response to PHA stimulation; B: lymphocyte
proliferation in response to Con A stimulation;
C: lymphocyte proliferation in response to PWM stimulation;
D: IL-2 production. The correlations are positive and
statistically significant (p < 0.01).
65
(X 1000)
(X 1000)
Con fii
PHA
r=0.47,P<0.01
r=0.44,P<0.01
104
146
94
8
o
126
84
a
a
c
o
a.
n
e
o
a
74
106
L
C
a
a
o
64
86
54
44
66
J
1
1
1
1
'
0
0.4
0.8
1.2
1.6
2
2.4
J
'
'
0
0.4
0.8
L.
1.2
1
1
1.6
2
L
2.4
<X 10000)
PUfl
r = 0.45,P<0.01
IL-2
r=0.63,P<0.01
3
a
a
c
0
a.
m
a
L
C
0
a
o
J
L
0
0.4
0.8
J
1
1
L
1. 2
1.6
2
2.4
J
1
1
L
J
L
0
0.4
0.8
1.2
1.6
2
Lymphocyte PLP concentration (pmol/106 cells)
2.4
66
5. IL-2 Production
There was no significant difference in IL-2 production
between PN and placebo groups at baseline (Table 13, Fig.
7) .
In the PN group, a daily supplement of 1.5 mg PN.HC1
for 7 days increased the mean IL-2 production by more than
60%.
When the PN.HC1 supplement was increased to 50 mg per
day, mean 11-2 production in the PN group increased to 3470
U/L of culture medium after 3 days and 433 6 U/L of culture
medium after 6 days.
Withdrawing the PN.HC1 supplement
resulted in decreased IL-2 production, which was still
higher than the baseline level and the placebo group during
the first three weeks of the washout period.
At the end of
fourth week of the washout period, the IL-2 production in PN
group was still higher than the baseline level but showed no
significant difference between the PN and placebo groups.
In the placebo group, IL-2 production fluctuated slightly
from time to time during the 46-day study.
IL-2 production in the PN group was significantly (r =
0.63, p < 0.01) correlated to lymphocyte PLP concentration
(Fig. 7D).
67
Table 13
PN supplementation and IL-2 production (U/L) of lymphocytes
in the PN and placebo groups (Study II)*
Period
PN (n=5)
Baseline
988 ±
62
Placebo (n=3)
1137 ± 192
Supplement period
+1.5mg PN.HCl/d
or placebo
day 7
1602 ± 122^
1207 ± 212
+50mg PN.HCl/d
or placebo
day 3
day 6
3474 ± 866^
4336 ± 676AB
1880 ± 700
1710 ± 416
3622
3032
2032
1220
1300
1460
1390
1063
Washout period
day 4
day 8
day 18
day 28
±
±
±
±
740^
925^^
213^^
120A
± 72
± 292
± 10
± 144
* Mean ± SEM. The subjects consumed their self-selected
diet throughout this investigation.
A Significant difference (p < 0.05) comparing with baseline
value;
B Significant difference comparing with control group
during same period.
68
-+-
RN
-o-
Placebo
SOOOn
4000
c
o
o
3
•o
o
Q.
3000"
2000-
CVJ
I
_l
1000^ =
Hid
Fig. 7. IL-2 production in response to PHA stimulation in
the PN and placebo groups (Study II). Periods, * and # are
the same as in Fig. 4.
69
6. Effect of Putrescine on Lymphocyte Proliferation and
IL-2 Production
Effect of putrescine added in vitro on proliferation of
lymphocytes from subjects in the PN and placebo groups was
also tested in Study II (Table 14-16).
Lymphocyte
proliferation in response to each mitogen was at its peak
after the PN group subjects had taken the 50 mg PN.HC1
supplement for 3 days.
Figure 8A shows the effect of 0, 2,
20, and 200 nmol/L of putrescine concentrations on
lymphocyte proliferation at this peak response to mitogen
stimulation.
For all three mitogens, PHA, Con A and PWM,
the lymphocyte responses to different putrescine
concentrations in both PN and placebo groups were flat lines
(Fig. 8A), indicating that the putrescine concentrations
used did not affect the lymphocyte response to mitogen
stimulation under the experimental conditions used.
Putrescine was also added in culture medium at
concentrations of 0, 20, 200 nmol/L
to
test the effect of
this polyamine on IL-2 production by lymphocytes from the PN
and placebo groups (Table 17).
Figure 8B shows the IL-2
production at the different added putrescine concentrations
by the placebo group and PN group who had received 50 mg
PN.HC1 for 6 days.
The putrescine added to culture medium
did not significantly affect the IL-2 production in the
experiment.
70
Table 14.
Effect of PN supplementation and putrescine concentrations
in the culture medium on mitogen response, in cpm, of
lymphocytes to PHA in the PN and placebo groups (Study II)*
Putrescine
Cone. (iAmol/L)
PN
placebo
Baseline
0
2
20
200
84679
80882
82506
75976
±
±
±
±
3893
4388
5200
4037
82117
78918
78514
74994
±
±
±
±
2838
4046
2803
3814
Supplement period
+1.5mg PN.HCl/d
or placebo
day 7
0
2
20
200
106120
115314
114154
111747
±
±
±
±
5042
5033
5271
8565
91892
92357
86750
81596
±
±
±
±
2326
2685
1396
2798
+50mg PN. HCl/d
or placebo
day 3
0
2
20
200
106983
104838
102916
96315
±
±
±
±
2443
3193
4485
3554
93069
90340
91230
89535
±
±
±
±
2812
3290
3899
2940
0
2
20
200
108262
107006
105234
97484
±
±
±
±
3690
1968
2152
3903
84821
81700
81088
81687
±
±
±
±
3143
2655
2738
3984
0
2
20
200
108329
111065
111116
103560
±
±
±
±
4757
6704
6553
3850
82249
78000
77036
71675
±
±
±
±
3449
4328
4551
6812
day 8
0
2
20
200
107945
108677
110053
101969
±
±
±
±
6424
6023
4344
4710
85483
82662
85232
86172
± 6316
± 5964
± 6125
± 803
day 18
0
2
20
200
97654
97154
98069
88131
±
±
±
±
3017
3761
3390
6310
75681
71318
72045
71379
±
±
±
±
3150
2124
2024
2409
day 28
0
2
20
200
85645
91908
91426
86991
±
±
±
±
5866
5873
7073
5381
86194
81450
80993
78811
±
±
±
±
3067
4978
4490
3472
day 6
Washout period
day 4
* Mean ± SEM; n = 5 per group.
The subjects consumed their
self-selected diet throughout this investigation.
71
Table 15.
Effect of PN supplementation and putrescine concentrations
in the culture medium on mitogen response, in cpm, of
lymphocytes to Con A in PN and placebo groups (Study II)*
Putrescine
Cone. (jimol/L)
PN
placebo
Baseline
0
2
20
200
69473
66231
65376
65458
±
±
±
±
1974
2051
1814
2764
69813
63514
64984
61064
±
±
±
±
3928
3149
2313
3770
Supplement period
+1.5mg PN.HCl/d
or placebo
day 7
0
2
20
200
81287
73925
74014
70809
±
±
±
±
3031
2983
2559
3841
73073
71146
68801
72102
±
±
±
±
3588
3922
3459
3168
+50mg PN. HCl/d
or placebo
day 3
0
2
20
200
92626
89198
88305
89014
±
±
±
±
2705
2114
2220
4353
80555
78544
79949
79011
±
±
±
±
3255
2565
5365
4447
0
2
20
200
83351
77742
77010
77160
± 941
± 1796
± 1931
± 2378
69325
67832
65651
68134
±
±
±
±
5648
7423
7433
6522
0
2
20
200
88676
85820
85318
87581
±
±
±
±
1400
2777
2399
2291
68136
64603
67167
61749
±
±
±
±
6123
5925
5297
8717
day 8
0
2
20
200
83546
78335
79205
77648
±
±
±
±
2946
3656
3830
1641
73925
68585
68950
71144
±
±
±
±
5598
4132
5554
2987
day 18
0
2
20
200
79451
68480
67254
72983
±
±
±
±
5224
4605
3953
4806
67581
64984
65357
66149
±
±
±
±
4148
3953
4090
4013
day 2 8
0
2
20
200
74239
66954
64499
65219
±
±
±
±
4425
6882
7317
5640
68544
68061
67842
68499
±
±
±
±
3418
1906
1126
2709
day 6
Washout period
day 4
* Mean ± SEM; n = 5 per group.
The subjects consumed their
self-selected diet throughout this investigation.
72
Table 16.
Effect of PN supplementation and putrescine concentrations
in the culture medium on mitogen response, in cpm, of
lymphocytes to PWM in the PN and placebo groups (Study II)*
Putrescine
Cone.(nmol/L)
eline
PN
placebo
0
2
20
200
19335
19129
18343
15965
±
±
±
±
3007
2923
1531
2495
19641
19765
20953
16095
±
±
±
±
2521
3202
3523
2388
Supplement period
+1.5mg PN.HCl/d
or placebo
day 7
0
2
20
200
36219
35976
33444
23896
±
±
±
±
5796
5378
5580
5273
22273
23570
22051
25272
±
±
±
±
1084
1379
1648
6880
+50mg PN. HCl/d
or placebo
day 3
0
2
20
200
38682
39286
38882
33329
±
±
±
±
4158
3816
3802
4131
27421
27444
29325
24422
±
±
±
±
5482
6473
6429
6257
0
2
20
200
34240
35479
33460
28168
±
±
±
±
5658
5554
5329
6100
25110
28124
27662
21810
±
±
±
±
3665
5282
5079
4375
0
2
20
200
43478
44352
44530
38996
±
±
±
±
4156
3987
4156
6113
23196
24755
24830
21167
±
±
±
±
5288
6210
6162
6233
day 8
0
2
20
200
33077
32213
34059
28227
±
±
±
±
5947
5682
6171
7605
30492
28174
32726
29978
±
±
±
+
4737
2416
5640
5640
day 18
0
2
20
200
37805
35664
37130
32047
±
±
±
±
2366
2218
2115
4621
28595
31555
30671
26565
±
±
±
±
3224
2733
3217
3312
day 2 8
0
2
20
200
24011
25509
25181
21563
±
±
±
±
5839
6260
5736
5998
28203
31382
31063
24628
±
±
±
±
5945
5829
5538
4764
day 6
'ashout peiriod
day 4
* Mean ± SEM; n = 5 per group.
The subjects consumed their
self-selected diet throughout this investigation.
73
Table 17.
Effect of PN supplementation and putrescine concentrations
in the culture medium on IL-2 production (U/L) of
lymphocytes in the PN and placebo groups (Study II)*
Putrescine
Cone, (iimol)
Placebo (n=3i
62
75
44
1137 ± 192
1173 ± 186
1070 ± 55
1602 ± 122
1406 ± 137
1440 ± 346
1207 ± 213
1240 ± 171
1057 ± 243
0
20
200
3474 ± 866
4304 ± 813
2820 ± 160
1880 ± 700
1325 ± 345
1030 ± 90
0
20
200
4336 ± 676
4492 ± 598
3464 ± 937
1710 ± 416
1287 ± 272
1197 ± 200
0
20
200
3622 + 740
3634 ± 568
2512 ± 338
1300 ± 72
1267 ± 123
1093 ± 47
day 8
0
20
200
3032 ± 925
3210 ± 723
2608 ± 716
1460 ± 292
1380 ± 355
957 ± 109
day 18
0
20
200
2032 ± 213
2082 ± 232
1886 ± 48
1390 ± 10
1505 ± 25
1430 ± 330
day 28
0
20
200
1220 ± 120
1506 ± 112
1234 ± 131
1063 ± 144
1423 ± 160
1250 ± 190
Baseline
0
20
200
PN (n=5)
Supplement period 0
+1.5mg PN.HCl/d 20
200
or placebo
day 7
+50mg PN.HCl/d
or placebo
day 3
day 6
Washout pe:riod
day 4
988 ±
1116 ±
1020 ±
* Mean ± SEM. The subjects consumed their self-selected
diet throughout this investigation.
74
- + - pN
— O—
placebo
200
E
Q.
S 3
+—
100
S
O—
£ o
'—+
CO
-i
+putrescine
50001
1
2
1—
20
200
umol/L
B
4000
3000
a.
2000
1000-
+putr9»cino
h-- ~r20
-2
200
umol/L
Putroscino in medium
Fig. 8. Changes of lymphocyte proliferation and IL-2
production in response to putrescine concentrations in
culture medium (Study II). A: lymphocyte proliferation
after a daily supplement of 50 mg PN.HCl or placebo for 3
days; B: IL-2 production after a daily supplement of 50 mg
PN.HCl or placebo for 6 days.
75
DISCUSSION
While the damaging effect of vitamin B-6 deficiency on
cellular and humoral immunity in animals is well documented
(1,2,3,4,5), in limited human studies the effect of vitamin
B-6 on immune function is not as clear as shown in animal
studies.
In addition to the inherent limitations of human
studies, such as not existing in real life of single vitamin
B-6 difference and unethical to induce a severe B-6
deficient status in controlled studies, the different
experimental designs and the short experiment duration all
contribute to the uncertainty of the reported human study
results.
The two studies reported here show the effect of
controlled vitamin B-6 intake on in vitro lymphocyte
proliferation and IL-2 production in young women.
The
subjects who participated in these studies were apparently
healthy and free of any known disorder.
any illness throughout each study.
They were free of
The subjects also had
adequate intakes of protein, vitamin B-6 and energy (Table
5) as well as other nutrients.
After taking 2.44 mg vitamin B-6 per day (about 1.5
times of the RDA) for 10 days, the subjects in Study I had a
significantly increased lymphocyte proliferation in response
to T-cell mitogen, Con A, and T-dependent B-cell mitogen,
PWM, stimulation, compared with the baseline and the vitamin
76
B-6 intake of 0.84 mg per day (Table 4).
In Study II,
addition of daily supplement of 1.5 mg PN.HC1 to their selfselected diet (Table 5) brought the total vitamin B-6 intake
of the PN group to about 2 times of the RDA.
With this
intake of vitamin B-6, there was a significant (p < 0.05)
increase in the subjects' lymphocyte proliferation in
response to PHA, Con A and PWM, and IL-2 production compared
with the placebo group and the PN group's response at the
baseline.
The results of these two studies reported here
clearly show that a vitamin B-6 intake above the RDA
increased in vitro lymphocyte proliferation and IL-2
production in healthy young women as had been shown in
elderly (6,7).
Lymphocyte proliferation in response to mitogen
stimulation and IL-2 production were used to assess the
effect of vitamin B-6 intake on immunocompetence in healthy
young women in our studies.
In vitro proliferation of
peripheral blood lymphocytes in response to mitogen
stimulation is a widely used method for assessing
immunocompetence in humans (166) and is thought to mimic the
events occurring in vivo following challenge by specific
antigens.
Lymphocyte activation by either antigens or
mitogens results in similar intracellular changes and
subsequent proliferation.
The difference is that a specific
antigen stimulates a specific cell clone, while mitogens
(such as those used in this investigation) are multiclonal
77
stimulators (167).
IL-2, formerly called T-cell growth
factor, is one of the key factors in immune response.
Stimulated T-cells produce IL-2, which is essential to
initiate the proliferation of activated T-cells and NK cells
(168,169)
. High affinity receptors for IL-2, which are
absent on resting T-cells, appear within hours of
activation.
The binding of IL-2 to these receptors induces
the clonal expansion of the T-cells activated by the
specific antigen (168).
Activated B-cells also express
receptors for IL-2, which can act on those B-cells as a
potent growth factor (167).
Hodges et al. (101,102) found slightly impaired
antibody response to tetanus and typhoid antigens in young
men maintained on a vitamin B-6 deficient diet for five
weeks.
In an independently living elderly population with a
normal vitamin B-6 intake according to the current RDA
levels of B-6 intake and the ratio of the intakes of vitamin
B-6 and protein, a 50 mg PN.HC1 supplement per day for 2
months resulted in a significant increase of lymphocyte
proliferation response to both T- and B-cell mitogen
stimulation, compared with the pre-supplemental level and
the control group.
When the PN-treated subjects were
divided into two groups according to their pre-study plasma
PLP concentration, the effect of PN supplementation on
lymphocyte proliferation was much greater in the subjects
who had a low pre-study plasma PLP concentration (i 30
78
nmol/L) than in those with normal level (> 30 nmol/L).
Although the total lymphocyte count did not change with the
PN supplement, the number of CD3+ and CD4+ cells increased
significantly (7).
In a vitamin B-6 depletion-repletion
study, the depletion of vitamin B-6 from elderly people
resulted in a significant decrease of lymphocyte
proliferation response to both T- and B-cell mitogen
stimulation and a decline in IL-2 production.
An intake of
2.88 mg vitamin B-6 per day for elderly men and 1.90 mg per
day ^for elderly women, which was slightly higher than the
current vitamin B-6 RDA for men and women, respectively,
partly restored lymphocyte proliferation and IL-2
production.
A supplement of 50 mg PN.HC1 per day further
increased lymphocyte proliferation and IL-2 production in
these elderly subjects (6).
There are no reported studies
to evaluate the effect of vitamin B-6 intake on immune
function in young women.
It has been known that a decline in immunocompetence is
one of the general physiological changes associated with
aging (170-173).
It is possible that a high vitamin B-6
intake in the elderly specifically stimulated the decreased
immune function accompanying aging through a not yet clearly
defined nonnutritional effect.
It is important, therefore,
to study the effect of vitamin B-6 intake, especially in a
nutritional level, on immune function in young adults.
Based on a 13 weeks study of 24 young males, Van
79
den Berg et al. (10) concluded that short term vitamin B-6
marginal deficiency did not affect immune function as
evaluated by total lymphocytes, total T- and B-cells, IL-2
receptor expression, immunoglobulin and complement
concentrations.
However, these subjects, whether or not
they received vitamin B-6 supplement, received twice the RDA
for all other vitamins and minerals.
Many of these vitamins
and minerals, such as vitamins A (103), C (104) and E (105),
and minerals Se (106) and Zn (106) , may improve immune
function of the subjects.
Second, what the authors measured
is actually a static status of the immune cells, rather than
dynamic response of immune system to a stimulation.
It is
possible that the marginal vitamin B-6 status could maintain
apparently normal immune indices without challenge, but the
immune system may not have the optimal competence upon a
stimulation.
Additionally, sexual difference of the
subjects may also be a contributing factor in the
discrepancy between their conclusion and our results.
The American Medical Association (AMA) stated that, in
general, the nutritional supplementary intake of vitamins is
0.5 - 1.5 times that of the RDA, while 2 - 10 times RDA of
supplementary vitamins are therapeutic amounts for certain
nonnutritional or pharmacological purposes (174).
(The
amount suggested by the AMA is unrealistic in view of the
levels of vitamin B-6 administered in some studies (6,7,175)
and amount available over the counter.)
In the two studies
80
reported here, the vitamin B-6 intakes included both
nutritional and pharmacological levels.
The results of our
studies demonstrated that in young healthy women, a vitamin
B-6 intake 1.5-2 times of the current RDA increased the
lymphocyte function evaluated by in vitro proliferation in
response to mitogen stimulation and IL-2 production.
In
Study II, a daily supplement of 50 mg PN.HC1 in PN group
brought their total vitamin B-6 intake to about 25 times the
RDA of this vitamin for women.
This amount of vitamin B-6
intake did not further increase lymphocyte proliferation in
response to mitogen stimulation (Table 10-12, Fig. 5)
compared with the period of lower vitamin B-6 intake (2
times of the RDA).
IL-2 production was increased following
50 ml of PN.HC1 supplement (Table 13, Fig.7).
We speculate
that the effect of vitamin B-6 on immune function is by
virtue of providing PLP, the coenzyme for some enzymes which
in turn support the activation of immune cells.
Conceivably, there are apoenzymes in lymphocytes that are
inactive under normal conditions; in response to a higher
intake of vitamin B-6, these apoenzymes are activated by PLP
and support a rapid proliferation of lymphocytes in response
to mitogen stimulation.
In our study, lymphocyte
proliferation in response to mitogen stimulation could be
roughly separated into two phases (Fig. 6): changes in
proliferation were greater at the lower lymphocyte PLP
concentrations (roughly < 1.0 pmol/106 cells) than those at
81
the higher lymphocyte PLP concentrations {z. 1.0 pmol/106
cells).
Talbott et al. (7) also reported that the effect of
PN supplementation on lymphocyte proliferation was much
greater in the subjects who had low pre-study plasma PLP
concentrations U 30 nmol/L) than in those with normal
levels (> 30 nmol/L).
These results suggest that at lower
lymphocyte and plasma PLP concentrations, there are more
apoenzymes in lymphocytes that are activated by increased
PLP concentration and support lymphocyte proliferation.
It
makes sense that a high level of supplementary vitamin B-6
(i.e., 50 mg PN.HC1) would not have a proportionate effect
on immunocompetence, and that at higher lymphocyte and
plasma PLP levels the effect of PN supplementation on
lymphocyte proliferation was smaller than at lower PLP
levels, since the available amount of PLP-dependent enzymes
in the cells would be a limiting factor.
The results of
Talbott's and our studies support the view that the effect
of vitamin B-6 on immune function is by virtue of a
nutritional or physiological role rather than a
nonnutritional or pharmacological one.
To our knowledge, the persistence of improved
immunocompetence after withdrawing a micronutrient
supplement has not been reported elsewhere.
The study
reported here showed that when vitamin B-6 supplementation
ended, the increased lymphocyte proliferation in
response to mitogen stimulation and IL-2 production
82
gradually decreased, paralleling the decrease in plasma and
lymphocyte PLP concentrations (Fig. 4,5,7).
It is therefore
demonstrated that the effect of a high vitamin B-6 intake on
immunocompetence is transitory.
These results support our
speculation that the increased intake of vitamin B-6
provides the coenzyme for PLP-dependent enzymes of
lymphocytes, which, in turn, facilitate the rapid
proliferation in response to stimulation.
After cessation
of the higher intake of vitamin B-6, the plasma and
lymphocyte PLP concentration gradually decreased to a lower
level.
The improved laboratory values for immune function
during PN supplementation decreased since the higher plasma
and lymphocyte PLP concentrations to support the increased
lymphocyte proliferation and IL-2 production were no longer
available.
If a higher intake of vitamin B-6 improves
immunocompetence as indicated by in vitro lymphocyte
proliferation and IL-2 production, does this mean the
subjects have improved in vivo immune function?
Long-term
population studies are needed to clarify this question.
Two
studies which tested the effect of supplementary multiple
micronutrients including vitamin B-6 on immune function in
humans have been reported.
elderly populations.
Both studies were carried out in
Bogden et al. (9) gave 29
independently living, healthy elderly subjects a daily
multiple micronutrient supplement including 3 mg of vitamin
83
B-6 for one year.
At the end of one year, the subjects
receiving this daily supplement had a significantly
increased DHST in response to a panel of seven recall
antigens compared with the controls who took a placebo.
Bogden et al. concluded that a modest supplement of
micronutrients including vitamin B-6 improved cell-mediated
immunity in these elderly subjects.
Chandra (8) gave 4 8
independently living healthy elderly persons a daily
multinutrient supplement which also included 3 mg of vitamin
B-6 for one year.
At the end of the year, the subjects had
a significant improvement in immune indices including
percentage of CD4+ cells, NK cells, IL-2 production, IL-2
receptor expression and lymphocyte proliferation in response
to PHA stimulation.
Additionally, Chandra observed a
significantly (p < 0.05) decreased incidence of infectious
diseases among the subjects receiving the micronutrient
supplement compared with the control group who received a
placebo.
This in vivo experiment by Chandra suggests that
the elderly subjects benefitted from the improved
immunocompetence resulting from increased intake of
micronutrients including vitamin B-6 by having a lower
incidence of infectious diseases.
It is recognized that the
elderly subjects who took the supplement received vitamins
and minerals at the level of approximately 1-2 times of
the RDA.
Many of these micronutrients such as vitamins A
(103), C (104), E (105) and minerals Se (106), Zn (107) and
84
S-carotene (176) have been reported to improve laboratory
tests of immune function.
Therefore, the improvement of
immunocompetence reported in these investigations can not be
attributed to any individual nutrient or combination.
Population studies with long-term, low dose (i.e., 2-3 mg)
vitamin B-6 supplement only are needed to confirm the
beneficial effects, such as a lower incidence of infections
and/or a higher titer of the antibody in response to
immunizations, of a higher vitamin B-6 intake on immune
function in humans.
Nutrient requirements are currently defined as the
amounts of nutrients needed to maintain normal body
functions (177).
The indices of nutritional status,
therefore, are designed to measure if intake of a nutrient
equaled output, or if body functions dependent on that
nutrient remained "normal".
Along with our improved
understanding of nutrient functions, a new concept on
nutrient requirements has gradually formed: that it is not
only desirable to maintain "normal" function but also to
maximize body functions.
There is a shortage of
sophisticated methods to assess nutritional status in terms
of maximal function.
Potential measurements of maximal
function include reproduction, growth and development,
behavior and cognitive function and immunocompetence.
these measurements, immune function is the most viable
direction.
Among
85
The immune system is extremely important to
health and normal function of the whole body.
The immune
system is a large component of the body's nonmuscle lean
mass.
Lymphocytes alone account for approximately 2 per
cent of total body weight and 8 per cent of fat-free solids
(177).
The short life and active metabolism of most immune
cells make the immune system very sensitive to variations in
nutritional status.
The results of the two studies reported
here indicate that in healthy young women whose vitamin
B-6 intake is adequate according to the RDA and plasma PLP
concentration is normal (52), the addition of 1.5 - 2.5 mg
vitamin B-6 per day can significantly (p < 0.05) increase
the in vitro lymphocyte proliferation in response to mitogen
stimulation and IL-2 production, the same as had been
reported in elderly (6,7).
This suggests that the current
RDA for vitamin B-6 may not be the best level for maximizing
immune function.
Our results also suggest that the indices
of immunocompetence may be sensitive measurements of vitamin
B-6 and/or other nutrient status.
Since many factors affect
immune function (103-107,159,166-169), much more work needs
to be done before we can practically use immune parameters
as indices of nutritional status.
The results of our
studies and the investigations by Meydani et al.
Chandra (8), Bogden et al.
(9), Talbott et al.
(6),
(7) and
Miller and Kerkvliet (178) provided an encouraging
perspective for using immune function to assess nutritional
86
status.
The subjects' mean baseline lymphocyte PLP
concentration measured in Study II (Table 8, Fig. 4B) was
0.27 pmol/106 cells.
To our knowledge, there has been no
report on PLP concentrations in lymphocytes.
Early reports
revealed that the mean leukocyte PLP concentration in adults
(179), children (179) and mice (180) was 0.6, 1.0 and 1.2
pmol/10
cells, respectively, which is in the same order as
our results.
In response to supplementary PN, lymphocyte
PLP concentrations increased in a much flatter pattern than
did plasma PLP levels (Table 7,8, Fig. 4).
Since PLP can
not cross cell membranes directly, its transmembrane
movement is mediated through dephosphorylation by a
membrane-bound phosphatase followed by rephosphorylation by
a cellular pyridoxal kinase (181).
When a high intake of
vitamin B-6 dramatically increases plasma PLP concentration,
lymphocyte PLP concentrations are enhanced.
PLP must be in
lymphocytes to perform its physiological role in immune
response.
Lymphocyte PLP concentrations were well
correlated with the changes of lymphocyte proliferation in
response to mitogen stimulation (to PHA, r = 0.44, p <
0.01); to Con A, r = 0.47, p < 0.01; to PWM, r = 0.45, p <
0.01) and IL-2 production (r = 0.63, p < 0.01)
(Fig. 6).
Lymphocyte PLP, therefore, could be a good measure for
vitamin B-6 nutriture.
To measure the in vitro lymphocyte proliferation in
87
response to mitogen stimulation and IL-2 production, the
subjects' lymphocytes were put in the culture medium for 48
- 96 hours.
It is plausible that components in the culture
medium influence the response of lymphocytes to mitogen
stimulation.
In RPMI 1640, which was used in these
experiments, the form of vitamin B-6 is PN (182).
However,
PNP (PMP) oxidase, an enzyme converting PN to PLP, has been
identified only in liver and erythrocytes in humans (i.e.,
no one has measured this enzyme in lymphocytes)
(23,27).
If
there is no PNP (PMP) oxidase in lymphocytes, then they can
not directly use PN as a source of PLP without first
converting PN to PLP in the liver or erythrocytes.
In fact,
the omission of pyridoxine from the culture medium does not
significantly alter the response of lymphocytes in the
culture medium (183).
Ha et al. (93) also found that in a
culture medium containing the major part of vitamin B-6 as
PN and small amount of PLP, secondary T-cell responses
generated in vitro did not recover the impaired cytotoxic
response of the T-cells from vitamin B-6 deficient mice.
However, Sergeev et al. (184) reported that when PLP was
added to the medium, the impaired function of cytotoxic Tcells from vitamin B-6 deficient mice was partially
restored.
The results of these studies support the view
that lymphocytes can not convert PN into PLP in vitro.
Short-term mitogen activation and initial growth of
activated lymphocytes over a period of 5 days results in
88
about three cell divisions (185).
If the lymphocytes have a
reserve of PLP, a PLP-free culture medium may still support
optimal growth.
Therefore, the concentration of PLP in
lymphocytes when they are isolated from the subjects is an
important factor influencing lymphocyte proliferation in
response to mitogen stimulation.
A small amount of PLP in
our culture medium was from added 10% of FBS.
The FBS used
in our studies contained 4.8 nmol/L of PLP, which is very
low compared with the average PLP concentration in human
plasma (normally > 30 nmol/L).
It is unlikely that this
small amount of PLP in the culture medium could provide
enough PLP to affect the difference of PLP in lymphocytes
from the subjects of different vitamin B-6 status.
However,
a complete elimination of PLP and control of all components
in the culture medium is the best way of studying the effect
of vitamin B-6 on lymphocyte proliferation in in vitro
experiments.
Serum-free cell culture may provide an ideal
tool for further investigation in this area (186,187).
PLP is a highly active biomolecule.
In in vitro
studies, PLP, in concentrations of 0.2 - 0.5 mmol/L, which
is much higher than the physiological concentration of
plasma PLP, has been reported to inactivate human hepatitis
B virus and human immunodeficiency virus (HIV)
(189,190).
The maximum plasma PLP concentration that could be achieved
in response to different amounts of supplementary vitamin
B-6 in humans and the possible pharmacological effects of
89
elevated plasma PLP are interesting research topics and
worthy of further studies.
Talbott et al. (7) reported that
elderly people given 50 mg PN.HCl per day for one month, had
a plateau of plasma PLP concentration at around 200 nmol/L
as long as the subjects continued taking the PN supplement.
Speitling et al. (188) found that young healthy males given
daily supplement of 40 mg PN.HCl for one day increased their
mean plasma PLP concentration from 78 nmol/L to 298 nmol/L.
A steady state of about 500 nmol/L of plasma PLP occurred
after 4 days of receiving the daily supplement of 40 mg
PN.HCl.
Coburn et al. (175) reported that in healthy young
men a daily supplement of 200 mg PN.HCl for six weeks
increased the subjects' mean plasma PLP concentration to 455
nmol/L.
In our study, a daily supplement of 50 mg PN.HCl
for 3 days increased young women's mean plasma PLP
concentration to around 250 nmol/L and remained at this
level after 6 days of 50 mg PN.HCl supplement (Fig. 4A).
EAST AC did not change significantly in response to two
weeks' PN supplement in Study II.
EAST AC is considered an
indirect long-term indicator of vitamin B-6 nutriture
because of the long life span of erythrocyte.
Our samples
had been stored at -200C for 8 weeks and were thawed and
refrozen once due to a freezer malfunction.
The nature of
EAST AC as a long-term indicator of vitamin B-6 status, long
storage time and thawing and refreezing of our samples may
all have led to errors in measuring EAST AC in our study.
90
Westrick and Smolen (191) recently reported that EAST and
EALT activity in young erythrocytes separated by
fractionated centrifugation are better indicators of recent
vitamin B-6 status.
Polyamines are important for DNA, RNA, and protein
synthesis (144).
Activation of mammalian cells from a
resting to a rapidly dividing state is accompanied by a
marked increase in the polyamine content (135).
ODC, a PLP-
dependent enzyme, is the first and rate-limiting enzyme in
polyamine biosynthesis (131).
ODC (11 - 90 minutes)
The very short half-life of
(132) makes this enzyme sensitive to
regulatory factors and availability of its coenzyme, PLP.
Fillingame et al. observed that inhibiting ODC activity
suppressed lymphocyte proliferation in response to Con A and
lipopolysaccharide, and, furthermore, this inhibition was
correlated with the depletion of intracellular polyamines
(147).
Adding exogenous polyamines to the culture medium
reversed the inhibition of DNA synthesis in Con A stimulated
cells by the ODC inhibitor (147).
Vitamin B-6 deficient
rats had decreased tissue ODC activity and a lower tissue
polyamine concentration compared with normal vitamin B-6
status rats (156).
In a culture medium,
putrescine at
concentrations of 2 0 - 25 jimols/L had the strongest
stimulatory effect on human (192) and mouse (193)
lymphocytes.
Higher putrescine concentrations (100 - 200
nmol/L), however, inhibited proliferation of cultured
91
lymphocytes (154,192).
Based on these experimental results,
we hypothesized that a higher vitamin B-6 intake improves
immunocompetence by increasing intracellular PLP
concentration, which, in turn, increases ODC activity and
polyamine synthesis.
Increased concentration of polyamine
would optimize lymphocyte proliferation in response to
stimulations.
If this was true, exogenous polyamines added
to the culture medium would compromise the effect of
different vitamin B-6 status on the lymphocyte proliferation
in response to stimulation.
In our experiment, however, the
addition of polyamine to the culture medium (0 - 200 nmol/L)
had no effect on lymphocyte proliferation in response to
mitogen stimulation in both PN and placebo groups (Fig. 8).
Recently, Sampson et al. (157) reported that in vitamin B-6
deficient rats, ODC activity in liver, kidney and small
intestine tissues actually increased.
Further studies such
as measuring ODC activity and polyamine concentration in
lymphocytes with different B-6 status are needed to clarify
our hypothesis.
92
SUMMARY AND CONCLUSIONS
The effect of vitamin B-6 on in vitro lymphocyte
proliferation and IL-2 production in healthy women (20 - 35
years old) was examined in two studies.
The subjects of
both studies were healthy and in adequate nutritional status
as evaluated by dietary intake, health questionnaire,
hemoglobin and hematocrit values.
In Study I, a repeated measure design,
six subjects
received a constant diet supplying o.84 mg (4.96 jimols)
vitamin B-6 per day for 12 days, followed by 1.24 (7.33
nmols) and 2.44 mg (14.42 nmols) vitamin B-6 daily during
the following two 10-day periods, respectively.
Lymphocyte
proliferation in response to mitogens Con A and PWM was
higher when the subjects received 1.24 mg of vitamin B-6
than when their intake was 0.84 mg of vitamin B-6.
But only
the response to Con A increased significantly (p < 0.05) .
Increasing vitamin B-6 intake to 2.44 mg, which is about 50
% higher than the RDA, significantly (p < 0.05) increased
lymphocyte proliferation in response to both mitogens,
compared with the response at baseline and 0.84 mg vitamin
B-6 intake.
In study II, ten subjects, who consumed their selfselected diets throughout this 46-day investigation, were
randomly assigned to a PN group and a placebo group (5
subjects each).
Their daily mean vitamin B-6 intakes
93
calculated from dietary records were 1.80 mg (10.64 jimols)
and 1.96 mg (11.58 nmols) for the PN and placebo groups,
respectively.
After a 5-day baseline period (no treatment),
the PN group received a supplement of 1.5 mg PN.HCl (1.23 mg
PN) daily for 7 days, followed by a supplement of 50 mg
PN.HCl (41.12 mg PN) daily for the subsequent six days.
The
daily total mean vitamin B-6 intakes (supplement plus intake
from diets) for the PN group during the two time periods of
PN supplementation were 3.03 mg (17.91 jimols) and 42.92 mg
(253.60 jimols) , respectively.
The placebo group received a
capsule containing no PN during this period.
This
supplementary period was followed by a 28-day washout period
without a PN supplement or placebo.
In the PN group,
lymphocyte proliferation in response to mitogens PHA, Con A
and PWM, and IL-2 production significantly increased
following a daily 1.5 mg PN.HCl supplement compared with the
baseline (p < 0.05).
Increasing PN.HCl supplementation to
50 mg daily did not further enhance lymphocyte proliferation
in response to PHA and PWM.
However, when the PN group
received a daily supplementation of 50 mg PN.HCl, lymphocyte
response to Con A stimulation peaked after 3 days and IL-2
production maximized after 6 days.
During the PN
supplementation period, the PN group's lymphocyte
proliferation and IL-2 production were significantly higher
(p < 0.05) than placebo group's.
After withdrawing the PN
supplement, lymphocyte proliferation in response to mitogen
94
stimulation and IL-2 production gradually decreased.
At the
end of the 4-week washout period, lymphocyte proliferation
in PN group dropped to the baseline level and was not
significantly different from the placebo group.
IL-2
production in the PN group was still higher at the end of
the 28-day washout period compared with the baseline but not
significantly different from the placebo group.
The 1.5 mg and 50 mg supplement of PN.HC1 significantly
(p < 0.05) increased both plasma and lymphocyte PLP
concentrations in the PN group.
Lymphocyte PLP
concentration changed in a flatter pattern in response to
PN.HC1 supplement and subsequent withdrawal compared with
changes in plasma PLP.
Lymphocyte PLP concentrations were
significantly correlated with the lymphocyte proliferation
and IL-2 production (p < 0.01).
The changes of lymphocyte
proliferation and IL-2 production were greater at lower
lymphocyte PLP concentrations than at higher lymphocyte PLP
concentrations.
To explore a possible mechanism of the effect of
vitamin B-6 on immune function, 2 to 200 nmol/L of
putrescine were added to culture medium to assess its effect
on lymphocyte proliferation and IL-2 production.
However,
putrescine added in the culture medium in these
concentrations did not affect lymphocyte proliferation and
IL-2 production.
Further investigation is needed to
clarify the relationship between vitamin B-6 status,
95
polyamine metabolism and immune function.
The results of the studies reported here demonstrate
that a vitamin B-6 intake 1.5-2 times the RDA
significantly (p < 0.05) increased in vitro lymphocyte
proliferation in response to mitogen stimulation and IL-2
production in healthy young women as had been shown in
elderly.
The pharmacological level of vitamin B-6 intake
(i.e., 50 mg PN.HC1) did not improve these laboratory tests
of immune function proportionately.
Since lymphocyte PLP
concentrations were significantly (p < 0.01) correlated with
the lymphocyte proliferation and IL-2 production, this
suggests that lymphocyte PLP concentration may be a good
indicator of vitamin B-6 status.
During the washout period,
decreased lymphocyte proliferation in response to mitogen
stimulation and IL-2 production paralleled the drop of
plasma and lymphocyte PLP concentration.
The stimulatory
effect of an increased intke of vitamin B-6 on immune
function was transitory.
96
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