Type 2 diabetes mellitus (T2DM) is associated with accelerated vascular aging and a markedly increased burden of cardiovascular morbidity and mortality. Beyond overt atherosclerotic disease, abnormalities in large-artery function are increasingly recognized as important contributors to cardiovascular risk in diabetes, with arterial stiffness emerging as an early and clinically meaningful marker of vascular damage [1,2]. Among the available measures of vascular stiffness, carotid-femoral pulse wave velocity (cfPWV) is regarded as the reference standard for noninvasive assessment of aortic stiffness and has been recommended for clinical and research use because of its reproducibility and prognostic relevance [3].

In patients with T2DM, increased arterial stiffness reflects the combined effects of chronic hyperglycemia, endothelial dysfunction, low-grade inflammation, oxidative stress, and medial vascular calcification. These mechanisms contribute to loss of arterial elasticity, increased pulse wave transmission, and adverse ventricular-vascular coupling, thereby promoting hypertension, target-organ damage, and cardiovascular events [1,2]. Importantly, prospective studies in high-risk T2DM have shown that higher cfPWV predicts future cardiovascular and renal outcomes, supporting its value not only as a physiological marker but also as an indicator of long-term prognosis [2].

Vitamin K has attracted growing interest in this context because of its role in the activation of vitamin K-dependent proteins involved in vascular health. Matrix Gla protein (MGP), one of the most important of these proteins, acts as a potent inhibitor of vascular calcification, but requires vitamin K-dependent carboxylation for full biological activity [4,5]. In states of suboptimal vitamin K status, inactive forms of MGP accumulate, potentially facilitating arterial mineralization and stiffening [4]. This biological framework provides a plausible mechanistic link between vitamin K deficiency and increased arterial stiffness, particularly in populations already predisposed to vascular injury such as patients with T2DM.

Clinical studies have further supported this association. Sardana et al. demonstrated that higher levels of inactive matrix Gla protein were associated with greater arterial stiffness in patients with T2DM, suggesting that impaired vitamin K-dependent activation of MGP may contribute to large-artery dysfunction in diabetes [6]. Similarly, Liabeuf et al. reported that markers of vitamin K-dependent calcification inhibition were linked to vascular calcification in patients with T2DM, reinforcing the relevance of this pathway in diabetic vasculopathy [7]. At the population level, Pivin et al. also found that inactive MGP was independently associated with arterial stiffness, supporting the broader vascular significance of impaired vitamin K-dependent protection against calcification [8].

Despite these observations, data directly examining serum vitamin K levels in relation to arterial stiffness among patients with T2DM remain limited, especially in Indian clinical settings. Most available studies have focused on surrogate markers of vitamin K status, calcification-related proteins, or interventional supplementation outcomes rather than direct clinicobiochemical correlation. Therefore, the present study was undertaken to evaluate serum vitamin K levels and arterial stiffness in patients with T2DM and to examine the association between vitamin K status and carotid-femoral pulse wave velocity.

Study design and setting

This prospective observational study was conducted at K.M. Medical College and Hospital, Mathura, Uttar Pradesh, over a period of 1 year during Feb 2021– Jan 2022. The study was designed to evaluate the association between serum vitamin K levels and arterial stiffness in patients with type 2 diabetes mellitus (T2DM).

Study population

A total of 100 adult patients with established T2DM were included in the study. Eligible patients attending the hospital during the study period were enrolled consecutively after obtaining written informed consent.

Inclusion criteria

Patients were included if they fulfilled all of the following criteria:

1. Age ≥18 years
2. Diagnosed case of type 2 diabetes mellitus
3. Willing to participate and provide written informed consent

Exclusion criteria

Patients were excluded if they had any of the following:

1. Type 1 diabetes mellitus
2. Chronic liver disease
3. Chronic kidney disease stage 4 or 5
4. Current vitamin K supplementation
5. Use of warfarin or other vitamin K antagonists
6. Acute illness, sepsis, or recent hospitalization
7. Pregnancy
8. Known peripheral arterial disease or any condition interfering with assessment of arterial stiffness

Clinical and biochemical assessment

All patients underwent detailed history taking and clinical examination. Baseline demographic and clinical variables recorded included age, sex, duration of diabetes, smoking, alcohol use, hypertension, dyslipidemia, coronary artery disease, and current medications including metformin, sulfonylureas, insulin, statins, and ACE inhibitors/angiotensin receptor blockers.

Anthropometric measurements included body mass index (BMI). Blood pressure was measured using standard technique, and systolic blood pressure (SBP) and diastolic blood pressure (DBP) were recorded. Biochemical investigations included fasting blood sugar, postprandial blood sugar, glycated hemoglobin (HbA1c), total cholesterol, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, and triglycerides.

Measurement of serum vitamin K

Venous blood samples were collected under standard aseptic precautions for estimation of serum vitamin K levels. Serum vitamin K concentration was recorded in ng/mL. For descriptive and comparative analysis, patients were also categorized into low, intermediate, and adequate vitamin K groups according to serum vitamin K status.

Assessment of arterial stiffness

Arterial stiffness was assessed using carotid-femoral pulse wave velocity (cfPWV), expressed in m/s. Based on cfPWV findings, patients were categorized into three arterial stiffness groups: normal/mild stiffness, moderate stiffness, and high stiffness. These categories were used for comparison of clinical and metabolic variables across stiffness groups.

Data collection

Data were entered into a predesigned proforma and included:

1. Demographic profile
2. Clinical characteristics and comorbidities
3. Anthropometric measurements
4. Biochemical parameters
5. Serum vitamin K levels
6. Arterial stiffness measured by cfPWV
7. Vitamin K category
8. Arterial stiffness category

Outcome measures

The primary outcome measure was the association between serum vitamin K levels and arterial stiffness in patients with T2DM.

Secondary outcome measures included:

1. Distribution of serum vitamin K status in the study population
2. Distribution of arterial stiffness categories
3. Comparison of clinical and metabolic variables according to arterial stiffness category
4. Correlation of cfPWV with serum vitamin K and selected clinical and biochemical variables
5. Independent association of serum vitamin K with arterial stiffness after adjustment for relevant covariates

Statistical analysis

Data were entered into Microsoft Excel and analyzed using appropriate statistical software. Continuous variables were expressed as mean ± standard deviation, and categorical variables were expressed as number and percentage.

Comparisons between patients with normal/mild arterial stiffness and moderate arterial stiffness were performed using the independent samples t-test for continuous variables and the chi-square test or Fisher’s exact test for categorical variables, as appropriate. Correlation between cfPWV and serum vitamin K as well as other continuous clinical and biochemical variables was assessed using Pearson correlation analysis. The association between vitamin K category and arterial stiffness category was analyzed using the chi-square test.

To identify independent determinants of arterial stiffness, multivariable linear regression analysis was performed with cfPWV as the dependent variable. Serum vitamin K, age, duration of T2DM, BMI, SBP, and HbA1c were included as explanatory variables. Beta coefficients with 95% confidence intervals were reported. A p-value <0.05 was considered statistically significant.

Ethical considerations

The study was conducted after approval from the Institutional Ethics Committee. Written informed consent was obtained from all participants prior to inclusion in the study.

Baseline clinical and biochemical profile

A total of 100 patients with type 2 diabetes mellitus were included in the final analysis

The mean age of the study population was 51.92 ± 9.59 years, and 58.0% of participants were male. The mean duration of diabetes was 6.48 ± 4.63 years. Hypertension and dyslipidemia were present in 70.0% and 52.0% of patients, respectively. The overall mean serum vitamin K level was 0.88 ± 0.41 ng/mL, while the mean carotid–femoral pulse wave velocity (cfPWV) was 7.88 ± 1.15 m/s (Table 1).

Table 1. Baseline clinical and biochemical profile of the study population

Distribution of vitamin K levels and arterial stiffness categories

Low vitamin K levels were observed in 16 patients (16.0%), while 48.0% had intermediate levels and 36.0% had adequate levels. Most patients had normal or mildly increased arterial stiffness (80, 80.0%), whereas 20.0% showed moderate stiffness. No participant fell in the high arterial stiffness category (Table 2).

Table 2. Distribution of vitamin K status and arterial stiffness categories

Comparison according to arterial stiffness category

Patients with moderate arterial stiffness were older and had higher body mass index, blood pressure, fasting and postprandial blood glucose, and HbA1c values than those with normal or mild stiffness. The mean serum vitamin K level was significantly lower in patients with moderate stiffness (0.68 ± 0.31 ng/mL) than in those with normal or mild stiffness (0.93 ± 0.41 ng/mL; p = 0.005). Duration of diabetes showed a borderline association with stiffness category, while the proportions of male sex, smoking, dyslipidemia, and coronary artery disease did not differ significantly (Table 3).

Table 3. Comparison of clinical and metabolic variables by arterial stiffness category

Correlation between serum vitamin K and arterial stiffness

Serum vitamin K showed a significant inverse correlation with cfPWV (r = -0.364, p < 0.001), indicating higher arterial stiffness with lower vitamin K levels. cfPWV also correlated positively with age, duration of diabetes, body mass index, systolic and diastolic blood pressure, fasting and postprandial blood glucose, and HbA1c, while HDL cholesterol showed an inverse correlation (Table 4). The relationship between serum vitamin K and cfPWV is illustrated in Figure 1.

Table 4. Correlation of arterial stiffness (cfPWV) with vitamin K and selected clinical variables

 Vitamin K category and arterial stiffness

The proportion of patients with moderate arterial stiffness was highest in the low vitamin K group (43.8%), followed by the intermediate (18.8%) and adequate vitamin K groups (11.1%). This distribution was statistically significant (p = 0.024), suggesting that lower vitamin K status was associated with an unfavorable stiffness profile (Table 5). Mean cfPWV values also demonstrated a graded decline across low, intermediate, and adequate vitamin K categories (Figure 2).

Table 5. Association between vitamin K category and arterial stiffness category

Multivariable analysis

On multivariable linear regression, serum vitamin K remained independently associated with cfPWV after adjustment for age, duration of diabetes, body mass index, systolic blood pressure, and HbA1c. Lower vitamin K levels, older age, longer diabetes duration, higher body mass index, higher systolic blood pressure, and higher HbA1c were each associated with greater arterial stiffness (Table 6).

Table 6. Multivariable linear regression for determinants of arterial stiffness (cfPWV)

Abbreviations: ACEI/ARB, angiotensin-converting enzyme inhibitor/angiotensin receptor blocker; BMI, body mass index; cfPWV, carotid–femoral pulse wave velocity; HDL, high-density lipoprotein; LDL, low-density lipoprotein; PPBS, postprandial blood sugar; T2DM, type 2 diabetes mellitus.

This research proposes that reduced serum vitamin K levels relate to increased arterial stiffness in individuals with T2DM and that this association cannot be attributed to traditional vascular risk factors. Our cohort showed a significant negative relation between vitamin K and cfPWV (r = -0.364, p < 0.001), even after multivariate adjustment, which suggests that vitamin K could be capturing an age-independent, adiposity-independent, blood pressure-independent, and glycemic control-independent component of vascular risk. This explanation is backed by the previous diabetes-related studies on the stiffness. In a 2021 study conducted by Monteiro et al., the value of cfPWV of 7.9 m/s was revealed as a discriminative value of arterial stiffness in T2DM, and the stiffness was associated with hypertension, inadequate glycemic regulation, and dyslipidemia [9]. We have a mean cfPWV of 7.88/1.15 m/s which is disturbingly near that cutoff point, indicating that the vascular phenotype in our group is in a clinically significant range of early arterial stiffening as opposed to being merely advanced disease.

The fact that our data has a graded relationship among vitamin K categories further supports the biological plausibility of the association. The proportion of patients with low vitamin K level (43.8%) exhibited moderate stiffness as opposed to 18.8% in intermediate and 11.1% in adequate group, which has a stepwise deterioration of the vascular profile with worse vitamin K status. Though, most of the past studies have involved indirect measures of vitamin K biology, and not direct serum vitamin K measurement, the findings of these studies have also directed at the same direction. Sardana et al. demonstrated that circulating inactive matrix Gla protein, which is an indicator of vascular protection impairment, was independently related to arterial stiffness in T2DM [6]. On the same note, Pivin et al. in a population based study found that inactive MGP was independently associated with arterial stiffness [8]. When these data are added together, they imply that our finding of a lower serum vitamin K, in combination with a higher cfPWV, is mechanistically consistent with the idea of impaired activation of calcification-inhibitory pathways rather than a nutritional epiphenomenon.

The other significant result of our investigation is that the vitamin Kstiffness correlation co-existed with the anticipated impacts of age, the duration of diabetes, blood pressure, and the metabolic control. The patients with moderate stiffness were almost 11 years older than the patients with normal/mild stiffness (60.60 ± 7.07 vs 49.75 ± 8.90 years), and the covariance of the PWV was strongly correlated with the age (r = 0.647, p = 0.001). This is very close to a cross-sectional study done by Gašparini et al. where 45.05% of T2DM patients with abnormally high cfPWV and elevated PWV was related to older age and longer diabetes time [10]. Similarly, Staef et al. examined 266 patients with early T2DM and found an average of PWV of 8.9 ± 1.8 m/s, with the stiffness of the arteries depending on systolic blood pressure, waist-related adiposity, LDL cholesterol, and the duration of diabetes [13]. Our data sets are no exception, finding moderate-stiffness patients had higher BMI, SBP, DBP, fasting glucose, postprandial glucose, and HbA1c and in multivariate analysis age, duration of diabetes, BMI, SBP and HbA1c were all significant with vitamin K. This is consistent with earlier cohorts which contend that the current results are not unique, but are within the existing hemodynamic and metabolic context of diabetic vascular stiffening.

HDL and cmPWV also have an inverse relationship that should be considered in our cohort. The moderate-stiffness group had lower HDL levels and an anticorrelation between HDL and cfPWV (r = -0.383, p < 0.001), indicating that a more atherogenic metabolic milieu can be associated with arterial stiffening. Monteiro et al. also highlighted dyslipidemia as one of the clinical domains linked to stiffness in T2DM [9], and Staef et al. suggested LDL-related parameters as one of the stiffness determinants in their cohort of early-diabetes individuals [13]. These comparisons indicate that vitamin K is not likely to act alone but it can be one component of a larger system of vascular calcification, endothelial dysfunction and metabolic injury. The key fact, though, is that even after these traditional factors were taken into consideration, the association with vitamin K remained.

The changing literature on the intervention should also be used to interpret our findings. There is an overall observational evidence of a relationship between worse vitamin K status and worse vascular health, but supplementation trials have reported more complex findings. Mansour et al. found that vitamin K2 supplementation in patients receiving renal transplantation was linked with the positive effect on subclinical vitamin K deficiency and arterial stiffness, and the vascular outcome was predicted by the decrease in the inactive MGP [12]. More recently, Eelderink et al. reported that vitamin K supplementation inhibited the progression of arterial stiffness, but made no significant changes to serum calcification propensity [15]. Meanwhile Vlasschaert et al., in a systematic review of controlled trials, found that there is an incongruent overall evidence regarding vitamin K supplementation on cardiovascular surrogate outcomes [14]. It is on that basis that our study will add clinically relevant observational data of a T2DM population by demonstrating lower serum vitamin K to be linked with higher cfPWV, less favourable stiffness-category distribution and an independent regression coefficient of -0.874 of cfPWV. Therefore, the primary implication is that it has not been shown to be causal, but that vitamin K status can be a biologically plausible and potentially modifiable predictor of vascular risk in T2DM.

Limitation

This was a single-center study with a relatively small sample size, which may limit the generalizability of the findings.

Lower serum vitamin K levels were significantly associated with greater arterial stiffness in patients with type 2 diabetes mellitus. The independent inverse association between vitamin K and cfPWV suggests that vitamin K status may be an important marker of vascular risk in this population.

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