View of Association between Vitamin K Status and Ankle Brachial Index as a Marker of Atherosclerosis in Chronic Kidney Disease

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Association between Vitamin K Status and Ankle Brachial Index as a Marker of Atherosclerosis in Chronic Kidney Disease

Lalit Rhea Surana ¹, Leena Chand², Manikandan Ayyapasamy³, Anusha Raja Jagadeesan^*, Rajeev Roy Neelakanta⁴, Shalini Lakshmanan⁵

1 5^th Semester MBBS Student, Sri Ramachandra Institute of Higher Education and Research, Porur, Chennai, Tamilnadu

2 Assistant Professor, Department of Biochemistry, Sri Ramachandra Institute of Higher Education and Research, Porur, Chennai, Tamilnadu

3 Associate Professor, Department of Biochemistry, Sri Ramachandra Institute of Higher Education and Research, Porur, Chennai, Tamilnadu

* Associate Professor, Department of Biochemistry, Panimalar Medical College Hospital & Research Institute, Poonamallee, Chennai, Tamilnadu

4 Research Scholar (ICMR integrated PhD), Department of Community Medicine, Sri Ramachandra Institute of Higher Education and Research, Porur, Chennai, Tamilnadu

5Assistant Professor, Department of Biochemistry, Panimalar Medical College Hospital & Research Institute, Poonamallee, Chennai, Tamilnadu

*[email protected]

ABSTRACT

BACKGROUND: Patients suffering from CKD due to increased triglyceride plasma content and decreased HDL levels are at higher risk of atherosclerosis. CKD patients are known to be prone to vitamin K deficiency. Vitamin K by means of Matrix Gla Protein (MGP) helps prevent formation of arterial plaques. Thus, lower vitamin K levels may be reflected by the increase in plasma up-uc MGP thereby making it a potential indicator of Vitamin D deficiency. This research aims to prove that vitamin K deficiency in a CKD population increases the chances of cardiovascular problems by virtue of arterial plaque formation.

METHODS: This is an observational study done among patients presenting to the Nephrology Out Patient Department of a tertiary care medical college and hospital situated in South India. The collection of data involved food frequency questionnaires for dietary intake of vitamins K and D, Ankle Brachial Index (ABI) measurement using a handheld arterial Doppler and blood samples for analysis of plasma up-uc MGP. CONCLUSION: In this study 56 stage III-V CKD patients with a mean age of 56.04 ±6.23 years were studied of which 55.36% were male and 44.64% were female. The 56 patients were divided into two groups based on their glomerular filtration rate (GFR) of which 46.43% belonged to Group A and 53.57% belonged to Group B. With progression of CKD through the stages an increase in plasma up-uc MGP with a consequent decrease in plasma vitamin K concentration, and a slight increase in ABI was noticed.

Keywords

Matrix Gla Protein; Vitamin K; Ankle Brachial Index; Atherosclerosis

Introduction

The process of gamma-carboxylation of glutamic acid residues of certain proteins requires Vitamin K as a co-factor, enabling the conversion of inactive un-carboxylated forms into active carboxylated forms to confer functioning¹. Vitamin K-dependent carboxylase, converts specific glutamic acid residues of a small number of proteins to glutamic carboxyl

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(Gla) residues by the addition of a CO2. Vitamin K dependent proteins include blood coagulation factors (II, VII, IX, X), osteocalcin synthesized by osteoblasts in bone, Matrix Gla Protein (MGP) etc². MGP is a small extracellular matrix protein, synthesized by chondrocytes and vascular smooth muscle cells which binds to calcium ions in the vascular wall. The predominant function of MGP is that it acts as a potent inhibitor of vascular calcification³.

Vascular calcification is an important factor which increases the risk of cardiovascular mortality and is seen with increasing frequency in patients with Chronic Kidney Disease (CKD).⁴ 25% of patients with CKD stage 3 and over 50% in patients on haemodialysis have been reported to present with vascular calcification.⁵ CKD patients suffer from subclinical vitamin K deficiency, caused by exhaustion of vitamin K due to the increased requirement of vitamin K-dependent proteins to inhibit calcification, thereby putting them at risk of consequences due to poor vitamin K status.⁶ However, dietary recommendations for CKD patients, such as diets low in potassium (fewer leafy green vegetables rich in vitamin K1) and low in phosphate (fewer dairy products rich in vitamin K2) could also contribute to this deficiency¹

Vitamin K deficiency results in the synthesis of under-carboxylated, biologically inactive Gla proteins which is a risk factor for vascular calcification and cardiovascular disease. Since vitamin K is essential for the activation of MGP, functional vitamin K deficiency may contribute to high vascular calcification burden among CKD patients which in turn directly predisposes to increased cardiovascular morbidity and mortality among dialysis patients.⁷ Several studies have shown that circulating dephosphorylated uncarboxylated MGP (dp-ucMGP) formed as a result of vitamin K deficiency in CKD, is associated with cardiovascular disease and has a profound impact on overall survival.^8-12

Previously, studies which had attempted to establish the relationship of vitamin K with cardiovascular disease relied on cumbersome measurements of dietary intake of vitamin K. Measurement of dephosphorylated uncarboxylated Matrix Glial Protein (dp-ucMGP) provides a technique for quantifying the vitamin K levels indirectly.^12,13 Warfarin which is commonly administered in CKD is known to influence vitamin K metabolism and function.

Warfarin by antagonizing vitamin K and by depressing the vitamin K-dependent carboxylation of MGP, leads to rapid arterial calcification. Evidence of this process comes from a study conducted in rat, wherein diets high in vitamin K have been shown to reverse aortic calcification and improve arterial elasticity in warfarin-treated rats suggesting that the calcification in response to warfarin treatment is due to the inhibition of the vitamin K- dependent gamma carboxylation of MGP ^14,15

Very limited studies are available which relate vitamin K deficiency to atherosclerosis by means of MGP protein analysis and those studies have shown that circulating dp-ucMGP levels a measure of vitamin K, is significantly and inversely correlated with the extent of coronary artery calcification among haemodialysis patients.^16,17,18 In this study, we aimed to determine the level of atherosclerosis by a non-invasive simple technique of measuring Ankle Brachial Index using a hand-held arterial Doppler. This method is less time consuming and the accuracy is similar to other methods of determination of atherosclerosis especially in case of the elderly population. This study hence aims at finding the association between vitamin K deficiency measured by dp-ucMGP levels and development of atherosclerosis among patients with various levels of chronic kidney disease.

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Methodology

This observational study was done at the Department of Nephrology of a tertiary care centre between May 2019 to October 2019. The study was conducted after obtaining clearance from Institutional Ethics Committee. 56 adult patients of age 18 or above who presented to the Nephrology OPD or the haemodialysis unit with CKD (determined by an elevated serum creatinine) and / or its complications, during the study period were included in the study. Exclusion criteria included a history of peripheral angioplasty and non- atherosclerotic cardiovascular diseases as these could make the measurements of ABI less accurate or unreliable especially in CKD patients.

After obtaining written informed consent, the study participants were subjected to ankle brachial index measurement for assessment of atherosclerosis and dp-ucMGP measurement as an index of Vitamin K status. Serum creatinine and Blood Urea Nitrogen levels were obtained from the study participants’ medical records with their consent. The value of serum creatinine was used to estimate the GFR by means of the CKD-EPI formula (a specialised formula used for estimation of GFR in CKD patients) approved by The National Kidney Foundation.

Ankle Brachial Index (ABI): ABI, a non-invasive vascular screening test to identify large vessel, peripheral arterial disease is performed using a continuous wave Doppler, a sphygmomanometer and pressure cuffs. An ABI of 1.4 or greater points to presence of vascular calcification.

Procedure

The participant lying supine, a pressure cuff was tied around their brachium and the brachial artery was palpated medial to the biceps brachii tendon at the cubital fossa. A small quantity of ultrasound gel was placed at the site and the probe of the Hi-Dop arterial Doppler was pressed against the region and manoeuvred till the arterial wave sounds could be heard. The pressure at the cuff is then increased till the arterial sounds cease and further increased by about 10-20 mm Hg after which the pressure is released slowly till the sounds can be heard again, which represents the brachial pressure. Similarly, the pressure is taken at the ankle by palpation of the dorsalis pedis artery at the proximal end of the first and second metacarpals or the posterior tibial artery postero-inferior to the medial malleolus. The ratio of the ankle and the brachial pressure is taken to be the ABI.

Measurement of dp-ucMGP:

5ml of random venous blood sample was collected from the study participant under aseptic precautions in red topped plain vacuum tubes without any anticoagulant. After 30 minutes of blood collection, the samples were then centrifuged at 3000rpm for 15 minutes. Serum was separated and stored at -80°C until analysis. The serum dp-ucMGP was quantitatively estimated by human dephospho-uncarboxylated Matrix Gla Protein (dp-ucMGP) ELISA kit [catalogue No：E13652559 (Type II), 96T/48T Enzyme Linked Immunosorbent Assay for Quantitative Detection].

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Results

The mean age of study participants was 56.045 ± 6.23 years with minimum age observed at 28 years and maximum age observed at 77 years. Of the 56 patients 31(55.36%) were male and 25 (44.64%) were female. The study participants were divided into two groups based on estimated Glomerular Filtration Rate (GFR):

 Group A included patients in CKD stages 3,4 with GFR between 30 and 60 ml/min/1.73m²

 Group B included patients in CKD stage 5 with GFR less than 30 ml/min/1.73m² All physical parameters were compared statistically by Student-t test analysis between the two groups shown in Table 1. There is no significant statistical difference between age and sex ratio between two groups. However, there is significant statistical difference between body mass index, systolic blood pressure and diastolic blood pressure in between the two groups with systolic blood pressure elevated in the CKD 5 group and diastolic blood pressure elevated in the CKD 3-4 group.

Table 1. Descriptive statistics of study groups

Parameter Group A (n=26) Group B (n=30) p value*

Age (years) 56.05 ± 4.53 56.06 ± 10.33 0.9964

BMI 25.68 ± 5.72 25.5 ± 4.53 0.8960

SBP (mmHg) 114.42 ± 17.23 138.7 ± 18.43 < 0.0001**

DBP (mmHg) 84 ± 8.2 76.92 ± 7.7 0.0016**

* Student t test, ** statistically significant

Table 2 shows the distribution of hypertension and diabetes mellitus among the study population with a higher prevalence of hypertension among Group A and diabetes among group B.

Table 2. Hypertension and Diabetes distribution among study participants Group A (n=26) Group B (n=30)

Hypertension 20 (76.9%) 16 (53.3%)

Diabetes mellitus 10 (38.46%) 13 (43.3%)

The comparison of biochemical parameters between two groups is shown in Table 3.

dp-ucMGP levels which depicts the vitamin K status showed a significant statistical difference between the two groups. Table 4 shows the correlation analysis between two study

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groups. Correlation analysis between the vitamin k status and ABI though positive among both groups, Group A has a marginally higher correlation coefficient than Group B.

Table 3. Ankle Brachial Index and Vitamin K values among study groups

Parameter Group A (n=26) Group B (n=30) p value*

ABI

1.132 ± 0.230 1.191 ± 0.227 0.3736 Vitamin K

3.835 ± 2.151 1.408 ± 0.714 < 0.0001**

*Student t test, ** statistically significant

Table 4. Correlation analysis between two study groups Parameter Group A Group B

dp-ucMGP r = 0.2062 r = 0.1743 Positive correlation seen in Group A was of a low degree and was

statistically significant

When stratified among Group B, mild correlation between dp-ucMGP level and ABI in stage 3-4 of CKD was seen (Figure 1), whereas there was no correlation between dp- ucMGP and ABI in stage 5 of CKD (Figure 2).

Figure 1. ABI vs dp-ucMGP in patients with CKD grade 3 & 4

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Figure 2. ABI vs dp-ucMGP in patients with CKD grade 5

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Discussion

The high prevalence and associated risk of ESRD, cardiovascular disease (CVD) and premature death makes chronic kidney disease a major global health burden. Results from a study of 35 reports from 33 studies conducted in 32 countries, which represent 48.6% of the global population ≥20 years old, revealed the crude prevalence of CKD stages 1–5. Of them, 236 million had moderate or severe decreases in kidney function, ranging between CKD stages 3–5. ESRD has a severe impact on the patients by reducing life expectancy and leading to a poor quality of life among them.¹⁶

The age-standardized global prevalence of CKD stages 1–5 among adults aged ≥20 years in 2010 was 10.4% in men and 11.8% in women. The prevalence of CKD among men and women though were similar in younger age groups, it is seen to with higher frequencies among women in middle-age groups with the difference increasing as the age groups increases, especially for stages 3–5. The pattern of age-related increase in CKD prevalence in men and women is seen to be consistent in high-income and low- and middle-income countries.¹⁶ But contradictory to generalized statistics, in the current study 55.36% of the study participants were male and 44.64% were female.

In this study, hypertension was seen with increasing frequency among earlier stages of CKD while the frequency of diabetes was among those with CKD in late stages. Diabetes and hypertension are the leading causes of CKD in all high-income countries and most low- and middle-income countries. The global epidemic of diabetes and hypertension could lead to a worldwide increase in prevalence of persons with CKD and its complications lest some

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effective interventions are introduced. ¹⁹ CVD is the leading cause of death in the world, and ischemic heart disease and stroke collectively killed 12.9 million people in 2010, which represents one in four deaths worldwide.⁸

In India on an average, 40–60% cases of CKD patients present with either diabetes or hypertension as co-morbidities.²⁰ As per recent Indian Council of Medical Research data, diabetes prevalence in Indian adult population has risen to 7.1%, and is high especially among adults above 40 years living in urban population (28%)^15,16. Though only a minority of CKD patients reach ESKD (0.15–0.20%/year over next 10–25 years), this population is about 10–100 times vulnerable for cardiovascular (CV) events and the vulnerability increases further in the presence of comorbidities.¹⁹ It was also noted that with the progression through the different grades of CKD, the incidence of hypertension and diabetes also increases.

Similar results have been seen in other studies among CKD patients involving estimation of co-morbidities and their relationship with progression of CKD. A Canadian study that examined the burden of CKD stages 3-5 in adults in a primary care centre in terms of its prevalence, variation by sociodemographic and clinical characteristics also generated similar results.^21,22

In 2011 a study on relationship between distinct circulating inactive vitamin K- dependent proteins and cardiovascular mortality was explored, wherein Kaplan-Meier analysis showed that patients with low levels of dp-ucMGP had an increased risk for all- cause and cardiovascular mortality.²³ Furthermore, it was found that patients with higher vascular calcification scores showed lower levels of dp-ucMGP. The results obtained in the present study after statistical analysis by student–t test shows there was a highly significant difference observed for dp-ucMGP between two CKD groups with a highly significant probability thus pointing to the variation in plasma vitamin K in the different CKD groups.

Despite only the slight relation between ABI and dp-ucMGP found in this study we strongly believe that, vitamin K strongly influences vascular calcification in CKD patients.

Hence, large population studies and the use of other more accurate and novel methods of determination of atherosclerosis is required to generate better results.

CKD in its later stages leads to dependence on haemodialysis or need for kidney transplant and hence decreases one’s life quality. From this study it is also seen that with progression of CKD the incidence of hypertension, diabetes is also increased. Additional co- morbidities of CKD such as atherosclerosis which may manifest as CAD, stroke, PAD etc further reduce the life expectancy of the individual. Vitamin K deficiency coupled with increased susceptibility to atherosclerosis of CKD patients only aggravates the condition.

Hence early detection and vitamin K supplementation in these cases may help prevent further damage and avoid future cardiovascular events.

Acknowledgement

We sincerely thank ICMR for funding this project under ICMR – STS.

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