Introduction

According to present knowledge, hyperhomocysteinemia (HHC) is one of the risk factors of cardio-vascular pathology [1, 2, 3]. Patients with chronic kidney disease (CKD) are known to develop HHC more often than those in general population [4, 5]. Today it is proved that in general population total homocysteine (HC) levels depend on age, sex, race and ethnic factors as well as on the rate of enzyme genetic polymorphism, involved in HC metabolism [6]. Equelly important cause of HHC development is the deficiency of vitamins В6, В9 and В12, involved in HC exchange, providing enzymes with cofactors involved in HC exchange and degradation [7, 8]. Taking into consideration rather high prevalance of vitamin deficiency even among people with rather high material status [9, 10], it is suggested to be one of the major reasons of HHC both in normal population and in the patients with chronic renal failure (CRF). It should be noted that in Ukraine neither the supply of those vitamins in the patients with CRF nor the relationship between their status and the level of HC in blood plasma have been previously studied.

The aim

In view of this, the aim of the study was to study homocystein contents in blood plasma of the patients with chronic kidney disease and to evaluate its association with vitamins В6, В9 та В12 levels.

Materials and Methods

148 in-patients and/or outpatients with CKD were examined at Vinnytsia Regional Pirogov Memorial Clinical Hospital, the clinical basis of Vinnytsia National Pirogov Memorial Medical University. The patients gave an informed consent to participate in the study. Chronic glomerulonephritis was diagnosed in 99 (66.9 %) and chronic pyelonephritis – in 49 patients (33.1 %). There were 77 females (52%) and 71 males (48%). All the patients were divided into two groups: the first one included 113 patients in whom underlying disease was accompanied by decreased glomerular filtration rate (GFR), and the second group consisted of 35 patients with no GFR impairement (CKD-I). In the group of patients with impaired GFR the following stages of CKD were diagnosed: stage II – in 31patients with impaired GFR, stage III – in 43, stage ІV – in 14, stage V (terminal) – in 25 patients. The age of patients in the group with decreased GFR ranged from 18 to 60 years, the average age being 39.6 ± 1.13 years. The average age of the patients was 40.3 ± 2.33 years in CKD-II group, 45.4 ± 2.0 years in CKD-III group, 45.5 ± 3.38 years in CKD-IVgroup and 37.8 ± 2.53 years in CKD-V group. Average age of the patients in CKD-I group was 36.9 ± 2.4 years. 30 apparently healthy individuals (14 men and 16 women) aged 21-57 years (average age 40.6 ± 2.38 years) served as representative control group.

The stage of CKD was determined according to the classification adopted by the Second Ukrainian Congress of Nephrologists (2005).

Total plasma HC level was determined by immunoenzyme method using “Axis-Shield” set (UK) on immunoenzyme analyzer “Santinaile”. Plasma level of folic acid (FA) was determined by microbiological method using folate-deficient strain Lactobacillus casei АТСС 7469. Cobalamin status was established by quantitation of urinary methyl-malonic acid excretion, vitamin В6 status – by pyridoxal-dependent enzymes ALAT and ASAT as well as by PLP-effect [11].

The study was carried out in compliance with the provisions of the Council of Europe Convention on Human Rights and Biomedicine, Declaration of Helsinki and recommendations of the Committee on Bioethics of the Presidium of National Academy of Medical Sciences of Ukraine.

Statistical analysis of obtained data was performed with application package STATISTICA (StatSoft, USA, v6.0). Nonparametric Mann-Whitney U-test was used to assess the difference between groups, Pearson correlation analysis – to determine the relationships between the indices, Fisher’s test – to compare the frequency of changes. P <0.05 was considered to be significant difference.

Results

The first stage of the study was devoted to evaluation of HC level in the patients with CRF and comparison of the data received with those in apparently healthy persons (Table I). Only 6.7% of practically healthy persons appeared to have increased HC level (above 15 mcmol/l). Increase of HC level in blood plasma was proportional to the severity of CKD which, in its turn, lead to the increase of HHC cases with higher HC concentrations. Among the patients with CRF the following HC levels were determined: normal – in 26 patients (17.6%), subnormal – in 35 patients (23.6%), and HHC – in 87 persons (58.7%).

The level of vitamin B6 in the studied patients was assessed indirectly by PLP-effect value related to aspartat transaminase (ASAT) activity (Table II). Decreased level of ASAT in erythrocytes by 15% with simultaneous increase of PLP-effect (by 15%) was considered to be a significant sign of vitamin B6 deficiency as compared to the control group. As no distinct relationship between ASAT activity value and PLP-effect rate was detected in the patients with various degrees of CKD, Table II gives the data on vitamin B6 stores according to enzyme activation by pyridoxal phosphate. Average indices of ASAT activity as well as those of PLP-effect were found to be normal with no differences between them in all studied groups.

Vitamin B6 deficiency was found in only one patient with CKD-I, while marginal deficiency (PLP-effect – 70-80%) was observed in all studied groups with equal frequency. Overall 32 cases of vitamin B6 deficiency or marginal deficiency were diagnosed in the patients with CKD-I – CKD-IV (26%). The data obtained suggested that the level of vitamin B6 stores was equal in the studied healthy persons and in the patients with CKD and was not influenced by CKD degree.

Somewhat different data were obtained after evaluation of vitamin В9 status determining its serum level by direct (microbiologic) method. The test with high diagnostic value for detecting vitamin В9 deficiency proved to be serum folic acid contents and that for vitamin В12 deficiency – excretion of methyl-malonic acid [12]. The data received, presented in Table III, are indicative of the fact that folic acid level gradually decreased insignificantly in the patients with CKD proportionally to the degree of renal insufficiency.

Significant decrease of folic acid concertration was revealed in CKD-III and CKD-IV patients. Those patients had higher frequency of marginal folate deficiency – in 34.9% and 35.7% of cases, respectively (P <0.05 according to Chi-squre test when compared to groups 1 and 2). Folate deficiency was found only in one person of the control group. A total of 29 cases of folate deficiency or marginal deficiency (23.6%) were diagnosed in the patients of groups 2-5 (123 individuals).

Supply of vitamin В12 was determined by methyl-malonic acid (MMA), its urine excretetion increased in inverse proportion to concentration of that vitamin in blood. The MMA excretion indices were not found to vary among all the patients, in no occasion deficiency was revealed (Table IV). Higher proportion of persons with marginal deficiency was found only in CKD-IV patients. The total number of patients having marginal deficiency of vitamin В12 was 21 (17.1%).

Additional statistical analysis found no relationship between HC level in blood plasma and indices of vitamins B6 and B12 supply (Table V), unlike folate – its level appeared to be the least in the persons with higher HC level.

In addition, potential correlation relationship between three factors – plasma HC level (factor 1), vitamin status (separately by vitamins B6, B9 and B12 – factor 2), and degree of CKD (factor 3) – was studied in comparison with apparently healthy individuals. The data received are given in Table VI, they are indicative of the absence of correlation between all three factors concerning vitamins B6 and B12 (in all cases index t was less than 2.2, Р>0.05). But there was significant inverse relashionship between plasma levels of HC and folic acid even in healthy individuals. Similar relationship between those indices by its direction and intensity, was observed in CKD-І, CKD -ІІ and CKD –ІІІ groups of patients (t=2.38, 2.36, 2.95 and 2.56, respectively; Р<0.05 in all cases). Only in group 5 (CKD-IV) the tendency or weak correlation between them was revealed (r=0.4; t=1.51), possibly because of a small number of patients in the group (14 persons). Even in the same index r=0.4 the result would have been significant if the group had consisted of 28 persons (n=28, t=2.23, P<0.05).

Significant inverse relashionship between plasma levels of HC and folic acid detected in studied patients made it possible to calculate the role of folate in HC concentration decrease. Actually, with augmentation of renal failure signs, plasma HC level in the patients with CKD increased in the group as a whole, but in some individuals it was partially dependent on folic acid level, and its high concentration inhibited the indicated action of CKD. After absolute difference between folate and HC concentrations had been determined in each group as compared to the next one with higher degree of CKD, relative percentage concentration of folic acid and its specific gravity were calculated. Hypohomocysteinemic contribution of folic acid was found to be 15.8%.

Because of direct assosiation of HC metabolism with folate status, Table VII presents the data on the level of that vitamin supply in dialysis patients.

Folate supply in dialysis patients appeared to be 22% lower than in the control group and 18% lower than in the patients with CKD-I. It is noteworthy that more than a half dialysis patients (56%) had marginal folate deficiency, while there were only 10% of such persons in the control group.

Discussion

Thus, the study of plasma HC concentration first conducted in Ukrainian population of patients with CKD revealed nomal HC level in 26 (17.6%), subnormal – in 35 (23.6%), and HHC – in 87 patients (58.7%). HHC was found to increase together with the increase of CKD stage. For example, an average HC contents in the patients with CKD-IV was significantly higher by 21.9% and 73.4% than in those with CKD-III and CKD-II, respectively, and the number of persons with HHC among the patients with CKD-IV significantly exceeded the proportion of those with HHC among the patients with CKD-II and CKD-I in 2.9 and 3.8 times, respectively.

According to the data from literature, homocystein level is closely correlated to the severity of kidney damage, and it is significantly higher in the patients with terminal than initial stage of renal failure. An important role of kidneys in elimination of HC from blood plasma has been shown in other studies as well [13].

The study of vitamins В6, В9, В12 status revealed unsatisfactory supply of those vitamins in both practically healthy individuals and in the patients with CKD. Deficiency, or marginal supply of vitamins В6, В9, В12 was found in 26.7% – 13.3% of apparently healthy individuals and in 26%, 23.5% and 17.1% of patients with CKD, respectively. The deficiency of those nutrients (except folate) proved to be independent of renal insufficiency stage. There were no significant differences in average indices of pyridoxin and cobalamin as well as in proportion of persons with marginal supply of those vitamins among the patients with various stages of renal failure. Only the level of folic acid significantly decreased proportionally to the increase of renal failure. While marginal folate supply occurred in one of ten patients among those with CKD-I, it was detected in one of two dialysis patients.

Literature data indicate the deficiency of B vitamins to be a common occurrence among the patients with CKD [14]. Glomerular filtration rate in the patients with CRF was shown to be assosiated with low folic acid level [15] while vitamin В12 supply is not assosiated with microalbuminuria [16]. It is noteworthy that some authors showed an additional administration of folate and vitamin B12 to prevent the progression of CKD in a high proportion of patients [17, 18].

At the second stage of the study we tried to find the relationship between the changes in vitamin B6, B9, B12 status and plasma homocystein level. Plasma homocystein level in CKD patients was proved to be closely assosiated with folic acid status but not with cobalamin and pirydoxin ones. Among the patients with optimal supply of vitamin B9 and B12 hyperhomocysteinemia occurred in 30.6% and 26.4% of patients, respectively, while in the patients with deficiency of one of the vitamins – in 63.6% and 75.8% of cases. Pathogenetic role of vitamin B9 in HHC formation among CKD patients was additionally confirmed by correlation analysis (r=-0.38 – -0.48). Therefore, a large proportion of studied patients with CRF was believed to have marginal, i.e. virtually moderate folate deficiency which can be partially the reason of HHC syndrome development.So, folate-independent and folate-dependent ways of HC metabolism were suggested in renal insufficiency.

Conclusions

1. In the patients with chronic renal failure normal homocysteine level was revealed in 26 patients (17.6%), subnormal – in 35 (23.6%), and hyperhomocysteinemia – in 87 persons (58.7%). Hyperhomocysteinemia was found to increase together with the increase of chronic kidney disease stage.

2. Supply of vitamins B6, B9, B12 was lower in the patients with chronic kidney disease than in apparently healthy individuals. Deficiency, or marginal supply of vitamins B6, B9, B12 was detected in 26.7% (В6) and13.3% (В9) of apparently healthy individuals and in 26%, 23.6% and 17.1%, respectively, of chronic kidney disease patients. At the same time only the level of folic acid significantly decreased proportionally to the increase of chronic kidney disease stage.

3. In the patients with chronic kidney disease homocystein level was found to be closely associated with folic acid status but it was independent of pyridoxin and cobalamin status.

Directions for future research involve the search for effective means of hypercysteinemia correction in the patients with chronic kidney disease by normalization of vitamin status in such patients.

The work was done as part of the research work of Scientific and Research Institute of Invalid Rehabilitation (Educational Scientific Treatment Complex) of National Pirogov Memorial Medical University: “To substantiate scientific approaches to definition of components of rehabilitation potential in patients with chronic kidney disease I-V D, Т”, state registration number 0116U00142. Financing – own funds.

Table І. Total plasma homocysteine level in the patients with different stages of chronic kidney disease and in healthy individuals (M±m, Р)

Studied individuals

Homocysteine

level, mcmol/l

M±m

Homocysteine level ranking , number of patients, %

Normal level

<10.0 mcmol/l

Subnormal level

10-15 mcmol/l

Hyperhomo-cysteinemia

≥15 mcmol/l

Practically healthy, n=30

9.35±0,57

24 (80%)

4 (13.3 %)

2 (6.7%)

CKD-І, n=35

10.7±0,72

17 (48,6%)*

10 (28.6%)

8 (22.8%)*

CKD-ІІ, n=31

15.4±1.45*#

6 (19,4)*#

16 (51.6%)*#

9 (29.0%)*

CKD-ІІІ, n=43

21.9±0.43*#$

3 (7%)*#

6 (14%)#$

34 (79.0%)*#$

CKD-ІV, n=14

26.7±2.1*#$

0

2 (14.3%)#$

12 (85.7%)*#$

CKD-V, n=25

27.3±1.83*#

0

1 (4%)#

24 (96.0%)*#$^

Notes: * – significance of differences with the group of healthy individuals; # – significance of differences with the group of patients with CKD-I; $ – significance of differences with the group of patients with CKD-II; ^ – significance of differences with the group of patients with CKD-III. The mark is indicated only in case of significant differences (P <0.05).

Table II. Vitamin B6 supply in the patients with various degrees of CKD and in healthy individuals (М±m)

GroupNo

Studied individuals

Erythrocytes ASAT, mcmol/h ml

PLP-effect, %

Pyridoxin supply

Normal

Marginal

Deficiency

< 70%

70-80%

>80%

1

Practically healthy, n=30

27.2±0.49

65.8±1.17

22

73.3%

8

26.7%

0

2

CKD-І, n=35

26.1±0.34

67.8±1.02

25

71.4%

9

25.7%

1

2.9%

3

CKD-ІІ, n=31

27.3±0.31

66.9± 1.0

23

74.2%

8

25.8%

0

4

CKD-ІІІ, n=43

24.8±0.30

67.3± 0.83

32

74.4%

11

25.6%

0

5

CKD-ІV, n=14

24.2±0.96

64.3± 2.77

11

78.6%

3

21.4%

0

Note: Р>0,05 in all cases.

Table ІІІ. Vitamin В9 supply in the patients with various degrees of chronic kidney disease and in healthy individuals (М±m)

GroupNo

Studied individuals

Serum folate level, mcg/l

Folate supply

Normal

Marginal

Deficiency

>6 mcg/l

3-6 mcg/l

<3 mcg/l

1

Practically healthy, n=30

7.67±0.25

26

86.7%

3

10%

1

3.33%

2

CKD-І, n=35

7.29±0.17

31

88.6%

4

11.4%

0

3

CKD-ІІ, n=31

6.88±0.17*

26

83.9%

5

16.1%

0

4

CKD-ІІІ, n=43

6.42±0.13*#

28

65.1%

15

34.9%*#

0

5

CKD- ІV, n=14

6.1±0.29*#

9

64.3%

5

35.7%*#

0

Notes: * – significance of differences with the group of healthy individuals; # – significance of differences with the group of patients with chronic glomerulo- and pyelonephritis without CKD. The mark is indicated only in case of significant differences (P <0.05).

Table ІV. Vitamin В12 supply in the patients with chronic renal failure and in healthy individuals by urine excretion of methyl-malonic acid (М±m)

GroupNo

Studied individuals

Creatinine ММA, mcg/g

Cobalamin supply

Normal

Marginal

Deficiency

<20 mcg/g

20-25 mcg/g

>25 mcg/g

1

Practically healthy, n=30

18.0±0.29

26

86.7%

4

13.3%

0

2

CKD-І, n=35

17.8±0.26

31

88.6%

4

11.4%

0

3

CKD-ІІ, n=31

18.2±0.36

25

80.6%

6

19.4%

0

4

CKD-ІІІ, n=43

18.3±0.25

36

83.7%

7

16.3%

0

5

CKD-ІV, n=14

18.6±0.51

10

71.4%

4

28.6%

0

Note: Р>0.05 in all cases.

Table V. Vitamin B6, B9, B12 supply depending on plasma homocysteine level in studied individuals (М ±m)

Groups

n

Indices of vitamins supply

PLP-effect, %

Serum folate,
mcg/l

ММA, urine excretion,
mcg/g of creatinine

1

Patients with HC<10
mcmol/l

50

68.0±0.96

7.82±0.12

18.1±0.20

2

Patients with HC 10-15 mcmol/l

38

67.6±1.22

7.06±0.13*

18.0±0.27

3

Patients with HC 15-25 mcmol/l

31

67.8±1.28

6.51±0.15*#

17.7±0.32

4

Patients with HC >25
mcmol/l

29

68.7±1.6

5.92± 0.08*#$

18.8±0.34

Notes: * – significance of differences with group 1, # – significance of differences with group 2, $ – significance of differences with group 3.

Table VI. Correlation relationship between total plasma homocysteine level and vitamins В6, В9, В12 status in the patients with chronic kidney disease of various degrees and in apparently healthy individuals of the control group (r)

Group No

HC level, mcmol/l

Vitamin supply indices

PLP-effect, %

Serum folate,
mcg/l

ММA, urine excretion,
mcg/g of creatinine

Apparently healthy individuals, n=30

1

9.35±0.57

0.23

-0.41*

-0.22

Patients with CKD-I, n=35

2

10.7±0.72

0.18

-0.38*

0.16

Patients with CKD -ІI, n=31

3

15.4±1.45

0.13

-0.48*

0.19

Patients with CKD -ІІI, n=43

4

21.9±0,43

-0.10

-0.43*

-0.02

Patients with CKD -ІV, n=14

5

26.7±2.1

0.05

-0.4*

0.12

Note: * – significance of correlation relationship.

Table VII. Vitamin В9 supply in dialysis patients, the control and in the patients with no signs of renal failure (M±m, Р)

Studied individuals

Serum folate level, mcg/l

Folate supply

Normal

Marginal

Deficiency

>6 mcg/l

3-6 mcg/l

<3 mcg/l

Practically healthy individuals, n=30

7.67±0.25

26

86.7%

3

10%

1

3.33%

CKD-I,

n=35

7.29±0.17

31

88.6%

4

11.4%

0

CKD-V,

n=25

6.0±0.24*#

11

44%*#

14

56%*#

0

Notes: * – significance of differences with the group of healthy individuals; # – significance of differences with the group of patients with chronic glomerulo- and pyelonephritis with no CKD.