Vitamin K2 status in children with chronic kidney diseases

doi:10.21203/rs.3.rs-4295154/v1

Background: We aimed to investigate the relationship between vitamin K2 status, as measured by uOC, and bone health assessed through a bone turnover marker (bone alkaline phosphatase- BAP) in children with CKD taking in consideration the impact of eGFR.

Methods: We enrolled 75 patients classified into 3 groups: group A; CKD without KRT, group B; CKD on regular HD and group C; renal transplant recipients. Another 25 healthy individuals were involved as a control group. Under carboxylated osteocalcin (uOC) (as a sensitive indicator of vitamin K2 level) and BAP were measured in all patients. 24-hour dietary recall was used to assess vitamin k2, calcium, and phosphorus intake. Vitamin and mineral intake was calculated as a percent of target requirements of age and sex-matched healthy children.

Results: uOC was found significantly higher in the patient groups in comparison to the control group (p <0.001). The highest level of uOC was detected in the HD group. In all groups, robust negative correlations were observed between uOC and vitamin K intake (%) levels. There was a statistically significant difference in uOC (p < 0.001) between those with history of bone fractures (No.= 7) compared to those without fractures (No.= 93). By logistic regression analysis, patients with a history of fractures were more likely to have a higher uOC (p = 0.012).

Conclusion: elevated uOC levels were observed in children with CKD and demonstrated a correlation with eGFR. Additionally, they exhibited a notable association with heightened bone turnover status, as indicated by BAP levels.

Vitamin K2

osteocalcin

bone alkaline phosphatase

CKD

Vitamin K encompasses a set of fat-soluble compounds with differing side chain lengths and levels of oxygenation. This vitamin exists in two primary forms: phylloquinone, denoted as K1, and menaquinone, known as K2. The production of vitamin K2 occurs within the intestine through the activity of gut bacteria, while rich dietary sources of this vitamin include fermented items such as cheese and butter. Additionally, vitamin K2 can be found in egg yolks, poultry, and liver (1).

Serum osteocalcin (OC) is a small protein of 5.6 kDa, consisting of 49 amino acids, which is produced in bone by osteoblasts, featuring three gamma carboxyglutamic (Gla) residues. Vitamin K2 acts as a co-factor for OC. The activation of OC is facilitated by vitamin K2-dependent carboxylation of these Gla residues. The form without gamma carboxylation is referred to as uncarboxylated osteocalcin (uOC). Hydroxyapatite crystals are unable to attach to uOC, leading to alterations in the bone matrix (2).

Patients with chronic kidney disease (CKD) often exhibit inadequate levels of vitamin K2 (3). There are various factors contributing to this deficiency. Major iatrogenic causes of vitamin K2 deficiency in CKD patients include hemodialysis treatments and the use of vitamin K2 antagonists (4). Numerous studies have proposed that vitamin K2 may have a significant impact on pathogenesis of severe complications in CKD patients, particularly concerning bone fractures and vascular calcification (5).

In this context, our objective was to investigate the relationship between vitamin K2 status, as measured by uOC, and bone health assessed through a bone turnover marker (bone alkaline phosphatase- BAP) in children with CKD taking in consideration the impact of eGFR.

This cross-sectional study included 75 patients of both genders and different ages classified into 3 groups: group A (25 patients); CKD without KRT stages 3-5, group B (25 patients); CKD on regular HD for at least 3 months on regular dialysis prior to the study and group C (25 patients); renal transplant recipients for at least 3 months after transplantation. The patients were convenience samples recruited from the pediatric nephrology department and the out-patient clinic, Cairo University Children’s Hospital. The study included another 25 age and gender matched healthy group as a control group (group D) recruited from general pediatric out-patient clinics coming for routine check-up. The study was approved by local ethical committee and conducted according to Declaration of Helsinki. An informed written consent was obtained from patients’ caregivers.

The CKD diagnostic criteria based on the guidelines proposed by the Kidney Disease Outcomes Quality Initiative (K/DOQI) of the National Kidney Foundation, and the classiﬁcations were made according to estimated glomerular ﬁltration rate (e-GFR) [ml/min/1.73 m²] as follows: stage 3, e-GFR = 30–59, stage 4, e-GFR = 15–29 and stage 5, e-GFR <15. eGFR was assessed according to Schwartz formula (plasma creatinine was measured by Jaffe’s method) (6).

None of the enrolled patients in this study received vitamin K supplementation, warfarin, calcimimetics, probiotics or bisphosphonates. Vitamin D derivatives, calcium supplements and phosphate binders were given according to their needs. Patients that had metabolic bone diseases, acute significant illness, active infection requiring antibiotic therapy within 2 weeks prior to the study or malignancy were excluded from the study.

Demographic data were collected from the study cohort and the anthropometric measurements were plotted and calculated with the standard z-score using WHO Anthro software for WHO Growth Reference for children less than 5 years of age, while WHO AnthroPlus software was used for the global application of the WHO Growth Reference for 5-19 years (7). Kt/V was used to assess dialysis adequacy. The documentation of any instances of bone fractures, whether linked to minor injuries or occurring spontaneously, within the three months preceding the study was recorded.

Dietary assessment:

24-hour dietary recall was used to assess total caloric, vitamin K (with emphasis on dietary sources of vitamin K2, primarily found in dairy products, fermented foods, and animal products), calcium, and phosphorus intake according to the 2nd Edition of food composition tables for Egypt. Caloric, vitamin and mineral intake was calculated as a percent of target requirements of age and sex-matched healthy children (8). There were no pre-requisite dietary precautions before blood sampling.

Biochemical analysis:

Fasting blood samples (pre-dialysis for patients on hemodialysis (HD)) were taken for measurement of serum Ca, P, BUN, and creatinine. Serum samples are allowed to clot at room temperature then centrifuged at 3000xg for 10 minutes for assay of uOC and BAP by ELISA using Sun Red (Shanghai Sun Red Biological Technology Co., Ltd). Two ml of blood in a sterile ethylene diamine tetra acetate (EDTA) tube collected and plasma separated immediately for assay of intact PTH by chemiluminescence assay using IMMULITE 1000 system (Simens Healthineers, Germany).

The primary outcome was assessment of vitamin K2 status in children with CKD by measurement of uOC and its relation to bone turnover by measuring BAP. The secondary outcome was the relation between vitamin K intake and the level of uOC.

Statistical analysis:

Numerical data were presented as mean and standard deviation, while categorical data were presented as frequencies and percentages. The Independent T test and Mann-Whitney test were used to compare parametric and non-parametric variables, respectively. Kruskal-wallis test was used to analyze the difference between means of the different groups. The Pearson correlation coefficient was used to determine significant correlations between quantitative data. Logistic regression analysis was done to demonstrate the effects of different variables on the history of likelihood bone fractures. The significance level was set at p < 0.05. Statistical analysis was performed with SPSS 27.0 (statistical package for scientific studies) for Macintosh.

We enrolled 75 patients in the 3 groups with a mean age of 8.12 ± 3.91 years while it was 6.95 ± 2.51 years in the control group. Descriptive characteristics and biochemical analysis data of the different groups are provided in Table (1).

uOC was found significantly higher in the CKD without KRT and HD patients in comparison to transplant and control group (p <0.001). The highest level of uOC was detected in the HD group with a statistically significant difference in comparison to the other groups (Figure 1).

There were no notable discrepancy in phosphorus intake among the various groups under study (p= 0.249). Nevertheless, a substantial statistical variance was observed in terms of vitamin K, calcium and caloric intake relative to the Recommended Dietary Allowance (RDA) across the groups (p= <0.001, 0.012 and 0.047, respectively). The CKD on regular HD group displayed the highest caloric intake, whereas the lowest was recorded in the CKD without KRT group (Table2).

There was a statistically significant difference in uOC (p < 0.001) between those with history of bone fractures (No.= 7; 953.08 ± 494,4) compared to those without fractures (No.= 93; 370.52 ± 172.55) in all groups.

Except in the control group, there was a statistically significant negative correlation between uOC and eGFR (Figure 2). While in all groups, robust negative correlations were observed between uOC and vitamin K intake (%) levels (Figures 3). Conversely, in groups A and B, statistically significant positive correlations were detected between uOC and both serum PTH and BAP (Figure 4).

uOC did not show a notable correlation with serum calcium, phosphorus intake percentage, or BMI z-score across all the groups. Among transplant patients, there was a significant negative correlation between uOC and caloric intake. However, in the CKD group, a remarkable positive correlation was noted between uOC and dietary calcium intake percentage (p < 0.001), and between uOC and serum phosphorus in the CKD on regular HD group (p = 0.012).

A logistic regression was performed to ascertain the effects of uOC, serum Ca, P, BAP, PTH and dietary vitamin K (%) on the likelihood that participants had history of bone fractures. Patients with a history of fractures were more likely to have a higher uOC (p = 0.047, OR = 1.7). Serum Ca (p = 0.862), P (p = 0.454), BAP (p = 0.447), PTH (p = 0.384), BMI (z-score) (p = 0.583) and dietary vitamin K (%) (p = 0.091) were not significant.

Vitamin K2 plays a crucial role in the formation of osteocalcin in bones, matrix GLa protein in cartilage, and the walls of blood vessels (9). This study revealed that the groups examined had lower vitamin K intake compared to the Recommended Dietary Allowance (RDA). Since vitamin K2, also known as menaquinone (MK), is primarily produced by the gut microbiota, relying solely on dietary assessments for vitamin K intake proves challenging. uOC serves as a sensitive indicator of the body's vitamin K status (10). We observed elevated levels of uOC in the studied groups compared to the control group, with a negative correlation to vitamin K intake (%). Furthermore, we noted that the HD group exhibited the highest level of uOC in comparison to the other groups. The precise reason for these observations in HD group requires further elucidation. Notably, it is worth mentioning that vitamin K2 is a is a lipophilic vitamin and it would not be anticipated that vitamin K2 could be eliminated through the dialysis process (11).

In a previous study, Nagata et al. observed high levels of uncarboxylated osteocalcin/intact osteocalcin (uOC/iOC) ratio in HD patients. They explained this observation as being linked to a heightened release of osteocalcin molecules from the bone into the bloodstream. This was supported by the finding of a distinct connection between initial levels of serum bone markers and the rise in serum ucOC/iOC ratio in HD patients during the inter-dialytic period (12).

Numerous prior studies, encompassing both adults and children, have established connections between biochemical indicators of vitamin K2 status and bone health (13) (14) (15). In our research, we observed significantly elevated levels of uOC in individuals with a history of prior fractures. A study conducted by Kohmeier et al. (16) yielded comparable findings. Their research delved into the function of uOC in bone metabolism, explored the correlation between uOC and vitamin K2, investigated the history of old fracture bones, and assessed the risk of bone fractures in patients with renal failure. The heightened bone turnover and reduced intake of vitamin K2 likely influence the bone's integrity, rendering it more susceptible to fractures (17). This is reinforced by two observations in our study: firstly, our discovery of positive correlations between uOC and BAP levels. Secondly, elevated uOC was found to be linked with a higher probability of experiencing bone fractures. These patterns were particularly evident in the CKD on regular HD and CKD groups without KRT. Following renal transplantation, an increase in eGFR is linked with improved metabolic bone health and dietary habits (as indicated by the decline in uOC levels).

In our research, we observed inverse relationships between uOC and eGFR, except within the control group. Various factors can impact vitamin K2 levels in CKD patients, with the primary culprits for its deficiency being food limitations, uremia-induced dysbiosis, and certain medications (18). Additionally, the necessity to restrict dietary intake due to the high potassium content in many vitamin K-rich green vegetables contributes to its insufficiency (19). In addition to dietary sources, vitamin K undergoes recycling through the "vitamin K cycle," which involves enzymes like vitamin K epoxide reductase, DT-diaphorase, and g-glutamylcarboxylase. In rats with CKD, a reduction in this recycling process, likely stemming from diminished g-glutamyl-carboxylase activity, was observed and akin to the mechanism seen with coumarins (20).

Kremer et al., in a separate study in transplant patients, discovered an adverse univariable linear relationship between uOC and eGFR in CKD patients (p = 0.049). They observed an even stronger association with both dephosphorylated uncarboxylated matrix Gla protein (dp-ucMGP) and dephosphorylated carboxylated matrix Gla protein (dp-cMGP) (p < 0.001). They suggested that the proportion of uOC could serve as a potential indicator of vitamin K2 status independent of kidney function (21). They attributed their findings to the negative charges of uOC which are less able to cross the negatively charged glomerular membrane (22).

While this study represents the initial attempt to evaluate vitamin K2 status in children with CKD using both nutritional assessment and the measurement of uOC as a sensitive indicator, it is important to consider some limitations in interpreting our findings. Firstly, the study was conducted with a relatively small number of patients. Secondly, we did not explore the potential influence of phosphate binders on vitamin K2 status due to presence of different types of phosphate binders used. Thirdly, we did not investigate the correlation between vitamin D levels and uOC due to variations in vitamin D therapy and the diverse forms of vitamin D administered to the patients.

Well-designed, randomized trial or prospective cohort study is needed to evaluate the potential causal relationships and the value of uOC as a biomarker for fracture risk in children with CKD.

In conclusion, elevated uOC levels were observed in children with CKD and demonstrated a correlation with eGFR. Additionally, they exhibited a notable association with heightened bone turnover status, as indicated by BAP levels.

Funding:

N/A

Conflict of interest statement:

The authors declare no competing interests

The authors declare that there is no conflict of interest.
An informed consent was obtained from patients’ caregivers
The research does not include animals
The research includes clinical trials on human subjects.

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Table (1): Descriptive characteristics and biochemical analysis data of the different groups

Group A

(25 patients)

Group B

(25 patients)

Group C

(25 patients)

Group D

(25 patients)

p value

Age (years)

Gender

Female (%)

Male (%)

Original renal disease

Obstructive uropathy

Chronic GN

MAHA

Nephronophthisis

Others

Weight z-score*

Height z-score*

BMI z-score*

Previous fractures (%)

BUN (mg/dl)

Creatinine (mg/dl)

eGFR (ml/min/1.73m²)

Calcium (mg/dl)

Phosphorus (mg/dl)

PTH^* (pg/ml)

uOC^* (µmol/L)

BAP^* (IU/L)

6.32 ± 2.41

11 (44%)

14 (56%)

3 (12%)

6 (24%)

4 (16%)

3 (12%)

9 (36%)

-1.1 (-2.19 /-0.20)

-3.3 (-5.15 /-2.28)

1.4 (0.23 / 2.36)

3 (12%)

73.36 ± 27.24

1.62 ± 0.42

31.55 ± 9.82

8.25± 0.73

6.31 ± 1.09

149.12 ± 106.38

(83-176)

462.75 ± 166.33

286 (210-404)

7.12 ± 2.52

13 (52%)

12 (48%)

2 (8%)

6 (24%)

4 (16%)

7 (28%)

-2.0 (-2.77/-1.0)

-3.4 (-4.44/-2.5)

0.7 (-0.83 / 1.46)

3 (12%)

62.4 ± 15.3

3.39 ± 0.72

14.30 ± 4.46

7.5± 0.92

6.98 ± 1.14

149.36 ± 66.51

732.07 ± 810.76 (403.05-681.3)

319 (240-412.5)

8.22 ± 3.91

4 (16%)

21 (84%)

3 (12%)

5 (20%)

4 (16%)

10 (40%)

-3.2 (-4.49 / -1.15)

-3.8 (-4.22 / -3.30)

-0.6 (-1.01 / -0.05)

1 (4%)

73.88 ± 21.60

0.92 ± 0.1

66.58 ± 12.69

9.97 ± 1.22

5.03 ± 0.91

91.99 ± 23.83

282.82 ± 84.80

214 (161.5 – 250.5)

5.15 ± 2.72

7 (35%)

13(65%)

-

-0.3 (-1.26 /0.90)

-0.4 (-1.26 / 0.22)

-0.2 (-1.28 / 0.70)

-

21.24 ± 13.72

0.58± 0.22

90.95 ± 17.72

9.69± 0.7

4.32 ± 0.62

41.14 ± 27.54

(28.5-47)

287.58 ± 135.85

(193.75-360.85)

148 (113 -183.5)

0.142

0.054

0.28

<0.001

0.34

<0.001

0.385

0.004

<0.001

^*Median and interquartile range (for non-parametric data)

BAP= bone alkaline phosphatase; BMI = body mass index; BUN = blood urea nitrogen; GFR= glomerular filtration rate; MAHA = microangiopathic hemolytic anemia; PTH = parathormone

Table (2): Comparison between the different studied groups as regard dietary intake expressed as percentage achieved from the recommended intakes for age

Group A

(25 patients)

Group B

(25 patients)

Group C

(25 patients)

Group D

(25 patients)

p value

Calories (%)

Vitamin K (%)

Calcium (%)

Phosphorus (%)

72.75

(62.03-92.23)

24.3

(21.09 – 40.05)

48

(38.43 – 69.58)

54.83

(29.56 – 82.3)

80.61

(60.87-94.84)

27.36

(15.73 – 42.83)

29.71

(26.21 -44.37)

36.54

(25.68 – 59.69)

76.04

(53.46-94.61)

64.45

(43.79 -67.63)

33.25

(26.36 – 42.5)

26.75

(10.34 – 45.86)

79.52

(65.85-93.19)

79

(52 – 87.1)

59.55

(39.03-71.04)

84.33

(51.41- 89.08)

0.047

<0.001

0.012

0.249

median (IQR)

Table (3): Correlation of uOC with different parameters in the different studied groups

Group A

(25 patients)

Group B

(25 patients)

Group C

(25 patients)

Group D

(25 patients)

eGFR

Serum calcium

Serum phosphorus

BAP

PTH

Calories (%)

Dietary vitamin K (%)

Dietary calcium (%)

Dietary phosphorus (%)

BMI (z-score)

<0.001

0.004

0.085

0.049

0.014

0.887

0.002

<0.001

0.483

0.404

0.004

0.140

0.012

<0.001

0.038

0.948

0.011

0.484

0.192

0.072

<0.001

0.706

0.531

0.085

0.993

0.025

<0.001

0.392

0.381

0.119

0.724

0.840

0.569

0.709

0.603

0.492

0.002

0.185

0.534

0.590

PedNephGraphicalAbstractTemplate.pptx

Vitamin K2 status in children with chronic kidney diseases

Status:

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Abstract

Background

Methods

Results

Discussion

Declarations

References

Tables

Supplementary Files

Status:

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