Relation between plasma homocysteine, folate, vitamin b12 levels with blood pressure, residual renal function, anemia, dialysis duration and serum albumin in maintenance hemodialysis

Journal of military pharmaco-medicine no5-2017

RELATION BETWEEN PLASMA HOMOCYSTEINE, FOLATE,
VITAMIN B12 LEVELS WITH BLOOD PRESSURE, RESIDUAL
RENAL FUNCTION, ANEMIA, DIALYSIS DURATION
AND SERUM ALBUMIN
IN MAINTENANCE HEMODIALYSIS PATIENTS
Diem Thi Van*; Hoang Trung Vinh*
Summary
Objectives: To analyze relation between levels of plasma homocysteine (Hcy), folate, vitamin
B12 with blood pressure (BP), residual renal function (RRF), anemia, dialysis duration and
serum albumin in hemodialysis (HD) patients Subjects and methods: 112 HD patients were
clinically and subclinically examined including measurements of plasma Hcy, folate and vitamin
B12 levels. Assessments of Hcy, folate and vitamin B12 levels in HD patients were based on
their levels in 56 healthy individuals. Results: Patients with unaccepted BP control had higher
level of plasma Hcy and rate of hyperhomocysteinemia but a lower rate of vitamin B12
deficiency than those with accepted BP control. Patients with RRF had significantly lower rate of
hyperhomocysteinemia and higher rate of vitamin B12 decrease than patients without RRF.
Levels of plasma Hcy, folate and vitamin B12 in HD patients were not significantly relative to
anemia. With dialysis duration, folate was negatively correlated (r = -0.47; p < 0.001) whereas
vitamin B12 was positively correlated (r = 0.26; p < 0.01). Hcy was positively correlated with
serum albumin (r = 0.27; p < 0.01). Conclusions: In HD patients, Hcy was significantly related to
BP control, RRF and serum albumin; folate was only related to dialysis duration; vitamin B12
had significant relation to BP control, RRF and dialysis duration.
* Key words: Maintenance hemodialysis; Homocysteine; Folate; Vitamin B12; Relation.

INTRODUCTION
Cardiovascular disease (CVD) is the
leading cause of death in HD patients,
accounting for 45 - 50% of causes of
death [5]. Chronic kidney disease (CKD)

patients, who have the mortality rate due
to CVD were 16 times higher than that
of population [5]. There are many risk
factors for CVD in HD patients. Apart
from traditional risk factors known such
as hypertension, age, gender, smoking,

diabetes, dyslipidemia, physical inactivity,
obesity. HD patients also have renal
disease-related risk factors such as chronic
volume overload, hyperhomocysteinemia,
anemia,
hyperparathyroidism,
hypoalbuminemia, oxidative stress and
chronic inflammation, among them,
hyperhomocystenemia and anemia play
an important role in appearance and
progress of CVD [5]. Hcy is an
independent risk factor for CVD. Hcy is
elevated in 85 - 100% of chronic kidney

* 103 Hospital
Corresponding author: Diem Thi Van ()

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Journal of military pharmaco-medicine no5-2017
disease (CKD) patients and negatively
correlated with glomerular filtration rate

(GFR) [8]. In cases of advanced CKD and
hemodialysis, Hcy level is 1.5 - 2 fold higher
than that of normal people [9]. Folate and
vitamin B12 are not only essential
cofactors in Hcy metabolism but also
important causes contributing to anemia
in hemodialysis patients [8]. Therefore,
we conducted this study for purpose: To
analyze relation between levels of plasma
Hcy, folate, vitamin B12 with BP, residual
renal function, anemia, dialysis duration
and serum albumin in HD patients.
SUBJECTS AND METHODS
1. Subjects.
168 participants were enrolled in our
study and divided into 2 groups: the study
group consisted of 112 hemodialysis
patients at the Department of Nephrology
and Hemodialysis, 103 Hospital, from
June to October, 2016. The control group
consisted of 56 healthy individuals who
age, gender matched with those of the
study group.
* Selected criteria for the study group:
- Patients with 3 hemodialysis sessions
per week.
- Using the same dialyzers with lowflux membranes.
- Using hypertensive drugs, erythropoietin,
tardyferol B9 in treatment.
- ESRD patients with different age and

gender and dialysis duration > 1 month.
* Excluded criteria for the study group:
210

- Patients were treated by HDF online
method.
- Patients with currently serious
complications or malabsorption syndrome.
2. Methods.
* Study design: a cross-sectional
description, in comparison with control
group.
* Study contents:
- For the study group: We collected
history of disease, performed clinical
examinations and laboratory tests.
Patients were asked to provisionally stop
using folate and vitamin B12-containing
drugs at least one day before taking
plasma homocysteine, folate and vitamin
B12 tests. Blood sample was drawn under
fasting condition and before the first
dialysis session of the week. Plasma Hcy,
folate and vitamin B12 levels were
measured by microparticle chemiluminescent
immunoassay in the Department of
Biochemistry, 103 Military Hospital.
- For the control group: We also asked
for history of health and performed clinical
examinations of organs. After assessing

heatlthy individuals, they also measured
plasma Hcy, folate and vitamin B12 levels
by the same method as in the study
group.
* Statistical analysis: Stata 12.0 were
used to analyse data. Mean Hcy, folate
and vitamin B12 levels of the study group
were considered as increases when > X +
SD and decreases when < X - SD of the
control group.


Journal of military pharmaco-medicine no5-2017
RESULTS
Table 1: General characteristics of the study group (n = 112).
Mean age (year)
Uncontrolled BP
(n, %)
Urine conservation
(n, %)

49.93 ± 14.74

Mean dialysis duration (months)
Anemia

71 (63.4)

48.62 ± 47.26
106 (94.6)

(n, %)
Serum albumin < 35 g/L

27 (24.1)

28 (25.2)

(n, %)

Table 2: Features of Hcy, folate, vitamin B12 levels in HD patients.
Variable

X ± SD

Min

Max

Hcy (µmol/L)

25.43 ± 10.15

3.91

50

Folate (ng/mL)

12.61 ± 6.74

3.63

23.5

635.70 ± 297.52

235

1500

Vitamin B12 (pg/mL)

Table 3: Relation between Hcy, folate, vitamin B12, and blood pressure control (n = 112).
Unaccepted control

Accepted controlled

(n = 71)

(n = 41)

X ± SD

27.39 ± 9.75

22.04 ± 10.04

< 0.01

High n (%)

71 (100)

38 (92.7)

< 0.05

Folate

X ± SD

12.17 ± 6.44

13.36 ± 7.24

> 0.05

(ng/mL)

Low n (%)

20 (28.2)

12 (29.3)

> 0.05

Vit B12

X ± SD

668.77 ± 290.57

578.44 ± 304.28

> 0.05

(pg/mL)

Low n (%)

2 (2.8)

9 (22.0)

< 0.01

Variable

Hcy
(µmol/L)

p

+ Mean Hcy level and rate of hyperhomocysteinemia of group with unaccepted BP
control were significantly higher than those of the other group.
+ Unaccepted BP control group had a lower folate level and a higher rate of folate
deficiency than the other group, but these differences had no satistical significance.
+ There were no significant differences in mean vitamin B12 levels between two

groups (p > 0.05).
+ Rate of vitamin B12 deficiency in unaccepted BP control group was lower than
that in the other group (p < 0.01).
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Journal of military pharmaco-medicine no5-2017
Table 4: Relation between Hcy, folate, vitamin B12 and residual kidney function (n = 112).
Variable
Hcy (µmol/L)
Folate
(ng/mL)
Vit B12
(pg/mL)

Yes (n = 27)

No (n = 85)

p

X ± SD

22.89 ± 9.41

26.23 ± 10.29

> 0.05

High n (%)

24 (88.9)

85 (100)

< 0.05

X ± SD

14.85 ± 7.17

11.89 ± 6.48

> 0.05

Low n (%)

6 (22.2)

26 (30.6)

X ± SD

463.63 ± 171.12

690.36 ± 308.82

< 0.001

Low n (%)

7 (25.9)

4 (4.7)

< 0.01

> 0.05

+ Mean Hcy level of patients with RRF was lower than that of patients without RRF
but had no statistical significance (p > 0.05).
+ Rate of hyperhomocysteinemia of RRF group was significantly higher than that of
the other group (p < 0.05).
+ Mean folate level and rate of folate deficiency were not different between the
two groups.
+ RRF group had a lower mean vitamin B12 level and a higher rate of vitamin B12
deficiency than the other group.
Table 5: Relation between Hcy, folate, vitamin B12 and anemia (n = 112).
Variable
Hcy
(µmol/L)
Folate
(ng/mL)
Vit B12
(pg/mL)

X ± SD

Anemia (n = 106)

Non-anemia (n = 6)

p

25.45 ± 9.98

24.98 ± 14.02

> 0.05

High n (%)

103 (97.2)

6 (100)

> 0.05

X ± SD

12.74 ± 6.72

10.18 ± 7.14

> 0.05

Low n (%)

29 (27.4)

3 (50.0)

> 0.05

X ± SD

628.41 ± 291.73

764.50 ± 396.10

> 0.05

Low n (%)

10 (9.4)

1 (16.7)

> 0.05

Mean Hcy, folate, vitamin B12 levels, rates of high Hcy, low folate and low vitamin
B12 were not different between two groups of anemia (p > 0.05).
Table 6: Correlation between Hcy, folate, vitamin B12 and dialysis duration, serum albumin.
Variable

Hcy (µmol/L)

Folate (ng/mL)

Vitamin B12 (pg/mL)

r

p

r

p

r

p

Dialysis duration (month) (n = 112)

0.11

> 0.05

-0.47

< 0.001

0.26

< 0.01

Serum albumin (g/L) (n = 111)

0.27

< 0.01

0.04

> 0.05

-0.06

> 0.05

+ With dialysis duration: folate had moderately negative correlation (r = -0.47, p < 0.001),
vitamin B12 had slightly positive correlation (r = 0.26, p < 0.01), Hcy had no correlation.
+ With serum albumin: plasma Hcy level had slightly positive correlation (r = 0.27;
p < 0.01), plasma folate and vitamin B12 levels had no correlation.
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Journal of military pharmaco-medicine no5-2017

DISCUSSION
1. Relation with blood pressure
control.
* Hcy: Lim and Cassano (2002) explored
the relationship between Hcy and blood
pressure by analyzing data in the NHANES
III study from 1988 to 1994. The results
indicated that the average diastolic and
systolic blood pressure measurements
increased by 3.7 and 9.3 mmHg,

respectively, from the lowest to the
highest quintile of Hcy, unadjusted for
age. An increase of 5 µmol/L in Hcy was
associated with increases in diastolic and
systolic blood pressure of 0.5 and 0.7
mmHg, respectively, in men and of 0.7
and 1.2 mmHg in women [6]. Mechanisms
that could explain the relationship between
homocysteine and blood pressure are
homocysteine-induced arteriolarconstriction,
renal dysfunction and increased sodium
reabsorption, and increased arterial stiffness
[10]. Because of relation between Hcy and
blood pressure, blood pressure control is
affected by plasma Hcy level. Our study
demonstrated that patients with unaccepted
BP control had higher level of plasma Hcy
and rate of hyperhomocysteinemia than
patients with accepted BP control (table 3).
In hemodialysis patients, Huynh Van
Nhuan (2009) also showed that plasma
Hcy level in patients without hypertension
was 18.04 ± 8.47 µmol/L, significantly lower
than that of patients with hypertension
(27.63 ± 12.70 µmol/L, p < 0.05) [2].
Ha Van Hung (2016) indicated that nonhypertensive patients had mean plasma
Hcy level of 23.76 ± 8.10 µmol/L and rate
of elevated homocystein of 18.1%, which

were significantly lower than those in

hypertensive patients (30.76 ± 7.36 µmol/L
and 81.9%, respectively) [1].
* Folate and vitamin B12: folate and
vitamin B12 are the main nutrition factors
affecting Hcy levels and have a protective
role against cardiovascular disease. Protective
effect of these B vitamins are not only due
to their ability of lowering Hcy level but
also due to the ability to act as an
antioxidant, to increase the concentration of
NO - a vasodilator of endothelial cells.
Many studies have shown that treatment
with B vitamins may reduce BP and
cardiovascular events. A study by Scazzone
(2014) in 116 hypertensive patients and
81 healthy individuals reported that average
level of folate in the hypertensive group
(6.7 ± 5.0 ng/mL) was significantly lower
than that of the group control (9.0 ±
4.4 ng/mL), while vitamin B12 level was
not different between the two groups (440
± 223 pg/mL in the hypertensive group
versus 491 ± 185 pg/mL in the control
group) [9]. Our study indicated that no
differences in folate and vitamin B12
levels were observed between the group
with accepted BP control and the group
with unaccepted BP control (table 3). We
believe that our results could not find out
the relation between folate, vitamin B12

and blood pressure because in HD
patients, blood pressure was influenced
by many factors, in which folate and
vitamin B12 play only a small role.
2. Relation with residual renal
function (or urine conservation).
Residual renal function is assessed by
volume of 24-hour urine while patients still
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Journal of military pharmaco-medicine no5-2017
remain dialysis. If 24-hour urine output
< 500 mL is considered to have no residual
renal function or no urine conservation. In
contrast, 24-hour urine output ≥ 500 mL is
considered as having residual renal
function or urine conservation. RRF plays
an important role for patients on dialysis,
especially peritoneal dialysis patients.
RRF contributes significantly to the overall
health and well-being of dialysis patients. It
does not only provide small solute
clearance but also plays an important role
in maintaining fluid balance, phosphorus
control, and removal of middle molecular
uremic toxins such as beta 2 microglobulin.
Decline of RRF also contributes significantly
to anemia, inflammation, and malnutrition
in patients on dialysis [12]. Plasma Hcy,

folate and vitamin B12 levels are all
influenced by residual renal function
because all of them are filtered through
the glomeruli. Therefore, if patients still
has residual renal function, levels of these
substances will be lower than those of
patients without RRF. Our results showed
that patients with RRF had lower rate of
hyperhomocysteinemia than patients
without RRF. We also found that patients
with RRF had lower vitamin B12 levels
and higher rate of vitamin B12 deficiency
than patients without RRF. But regarding
folate, our study revealed that no differences
in folate level and rate of folate deficiency
were observed between the two groups
(table 4). In our study, folate was not
related to RRF, which can be attributable
to folate level in hemodialysis patients
influenced by many factors such as diet,
using folate-containing drugs in treatment
before.
214

3. Relation to anemia.
* Hcy: Anemia is a common manifestation
in hemodialysis patients and has multifactorial
etiology, in which lack of materials for
hematopoiesis such as folic acid and
vitamin B12 also significantly contributed

to appearance and progression of anemia.
Hcy level is affected by folate and vitamin
B12 because they act as cofactors in the
process of Hcy re-methylation into
methionine. Thus between Hcy and
anemia may have a relation with each
other because they are all affected by the
status of folate, vitamin B12 in the body.
In our study, no significant differences
were observed in Hcy level and the rate of
hyperhomocysteinemia between anemia
patients and non-anemia patients (table 5).
Similar to our results, Ha Van Hung (2016)
reported that Hcy levels of patients with
anemia was 28.64 ± 7.65 µmol/L, which
was not different from that of patients
without anemia (34.02 ± 8.07 µmol/l)
(p > 0.05). There were no differences in
rate of elevated Hcy between group with
anemia and group without anaemia [1].
Tayebi (2016) showed that no significant
correlation was found between Hcy level
and hemoglobin [11].
* Folate and vitamin B12: Folate and
vitamin B12 are essential for the process
of hematopoiesis so that the lack of them
will cause macrocytic anemia. However,
in this study we did not observe any
significant relations between folate,
vitamin B12 levels and anemia (table 5).

This could be explained by anemia in HD
patients attributable to many causes such
as shortened red blood cell survival, blood
loss, inflammation and the deficiency of
EPO and vitamin.


Journal of military pharmaco-medicine no5-2017
4. Correlation with dialysis duration.
* Hcy: Hcy has a molecular weight of
135 dalton within range of glomerular
filtration. Absorption of Hcy in renal tubular
was clearly seen through kinetic studies in
rat renal cortex. Kidneys have an important
role in the metabolism of homocysteine.
Total homocysteine concentration increases
as impaired renal function and achieves
the highest level in end-stage renal disease.
Many studies have demonstrated that after
each hemodialysis session, Hcy levels
may decrease partly but then rise again
until the next session. Hence the longer
dialysis duration is, the more level of Hcy
accumulates. De Vecchi (2000) indicated
that plasma Hcy level was positively
correlated with dialysis duration (r = 0.32;
p < 0.0001) [4]. Moustapha A et al (1999)
reported that there was a positive correlation
between Hcy level and dialysis duration
(r = 0.15; p < 0.05) [7]. However, our study

demonstrated that plasma Hcy level was
not correlated with dialysis duration (table
6). We could not find out the correlation
between Hcy level and dialysis duration,
which could be explained by plasma Hcy
level influenced by many factors.
* Folate: Folate is a water-soluble vitamin,
small molecular weight of 441 dalton. It
exists in plasma mainly in the free form or
loosely bound with non-specific proteins,
so that folate may be lost through the
membrane. Hence, the longer dialysis
duration is, the higher the risk of folate
deficiency is. Our study showed that
plasma folate level was inversely correlated
with dialysis duration (table 6).
* Vitamin B12: Vitamin B12 has a greater
molecular weight than folate (1,355 dalton).
Moreover, in blood, vitamin B12 exists in

the form bound to the carrier proteins
(transcobalamin and haptocorrin), so vitamin
B12 is hardly lost through low-flux dialysis
membrane. Our research showed that a
positive correlation was observed between
vitamin B12 level and dialysis duration
(table 6), which may be firstly explained
by vitamin B12 level not affected by
hemodialysis; Moreover, patients with long
dialysis duration often have more nervous

complications than patients with short
dialysis duration, so they often use group
B vitamins including vitamin B12 more.
5. Correlation with level of serum
albumin.
Serum albumin is an important marker
of Hcy level because in plasma most
Hcy binds with protein (mainly albumin).
Meanwhile, only free form of Hcy is
filtered through the glomeruli. Thus, when
the level of serum albumin increases, the
rate of albumin-bound Hcy will increase,
leading to decreased elimination of Hcy
by kidneys, which results in elevated level
of plasma Hcy. Our study indicated that
there was a positive correlation between
Hcy level and serum albumin (table 6).
Similar to our result, many other researchers
also reported a positive correlation between
Hcy and serum albumin such as Huynh
Van Nhuan (2009) (r = 0.353; p < 0.01)
[2]; Arnadottir (1999) (r = 0.28; p < 0.05)
[3]. We did not find any correlation between
folate, vitamin B12 and serum albumin
(table 6), which may be also suitable
because folate in plasma exists mainly in
the free form and vitamin B12 is bound
with two transport proteins (not albumin)
(transcobalamin and haptocorrin), so that
the level of folate and vitamin B12 are not

affected by serum albumin.
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Journal of military pharmaco-medicine no5-2017
CONCLUSIONS
- Patients with unaccepted BP control
had higher level of plasma Hcy and rate
of hyperhomocysteinemia whereas a lower
rate of vitamin B12 decrease than those
with accepted BP control.
- Patients with RRF had significantly
lower rate of hyperhomocysteinemia and
higher rate of vitamin B12 decrease than
patients without RRF.
- Levels of plasma Hcy, folate and
vitamin B12 in HD patients were not
significantly relate to anemia.
- Folate was negatively correlated with
dialysis duration (r = -0.47; p < 0.001)
whereas vitamin B12 was positively
correlated with dialysis duration (r = 0.26;
p < 0.01).
- Hcy was positively correlated with
serum albumin (r = 0.27; p < 0.01).
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