Type 2 Diabetes Mellitus (T2DM) is a primary public health concern globally, characterized by chronic hyperglycemia, insulin resistance, impaired beta-cell function and associated with significant metabolic disturbances, including leading to a spectrum of carbohydrate, lipid, and protein metabolic derangements that exacerbate the challenges of the pathophysiological condition [1][2]. A critical aspect of T2DM pathology involves vascular complications driven by endothelial dysfunction, which contributes to the high prevalence of cardiovascular disease (CVD) in diabetic populations [3].

In recent years, some progress has been made in discovering novel biomarkers associated with the pathogenesis of T2DM and improvements in procedures involved in diagnosing, monitoring, and therapeutic managing the disease [4]. Among these biomarkers, methylarginine has been of special interest due to its effects on endothelial function and nitric oxide (NO) availability [5]. NO signaling in maintaining endothelial health cannot be overstated [6]. NO, a critical vasodilator, is synthesized by nitric oxide synthase (NOS), which requires arginine as a substrate [7][8] (Fig. 1).

In T2DM, however, NO bioavailability is often compromised, and endothelial dysfunction becomes more pronounced, potentially increasing the risk of atherosclerosis and related CVD complications [9][10]. A significant factor contributing to impair NO production in T2DM is the presence of methylarginine derivatives, specifically asymmetric dimethylarginine (ADMA) and L-N Mono-methylarginine (L-NMMA) [11][12]. These Compounds act as endogenous inhibitors of NOS, reducing NO synthesis and thus limiting vasodilation, which in turn promotes endothelial dysfunction [13][14].

Research has established that concentrations of methylarginine and its derivatives are elevated in individuals with T2DM, and this elevation might relate to endothelial dysfunction [15][16], increased cardiovascular risk [17], and impaired metabolic parameters [18][19]. T2DM patients frequently experience dyslipidemia — elevated triglycerides, low HDL, and high LDL levels — raising their risk for atherosclerosis and cardiovascular complications [20].

Endothelium plays a crucial role in maintaining vascular homeostasis by regulating vascular tone, vascular smooth muscle cell proliferation, immune cell adhesion, and vascular inflammation through bioactive factors [21]. The molecular mechanisms underlying endothelial dysfunction are complex [10][22]. They are influenced by multiple pathologic stimuli, including NO, LDL, reactive oxygen species (ROS) and high glucose [23].

Endothelial dysfunction has been associated with diabetic animal models and human patients, and excessive ROS production plays a central role in early microvascular damage [24]. Mitochondrial and cytoplasmic oxidases, such as NADPH oxidase, are significant sources of ROS in endothelial cells [25]. High levels of glucose or fatty acids accelerate this process [26]. Shah et al. suggested hyperglycemia stimulates endothelial cells to overproduce ROS, causing DNA damage [27]. Given this information, it has become necessary to discuss the metabolic pathways of methylarginine within the biochemical cycle of associated molecules such as ADMA, L-NMMA, and symmetric dimethylarginine (SDMA).

This study examines serum levels of methylarginine derivatives (ADMA, SDMA, L-NMMA) and related metabolites (arginine, homoarginine, citrulline, ornithine) in T2DM patients with hyperlipidemia (DM-HL) and normolipidemia (DM-NL) to determine how lipid status may affect these biomarkers. Understanding these relationships is essential for clarifying the link between dyslipidemia and endothelial dysfunction and could enhance cardiovascular risk prediction in T2DM populations.

This cross-sectional, comparative study was conducted between July 2024 and September 2024, with patient recruitment carried out at the endocrinology outpatient clinics of Selçuk University Faculty of Medicine Hospital. A total of one hundred and twenty individuals over 18 years old participated in this cross-sectional, comparative study. 41 DM-HL aged (55.76±12.5), 42 with DM-NL aged (53.78±10.5) and 37 healthy controls (HC) aged (51.73±9.29).

The Inclusion criteria of this study are individuals with a confirmed diagnosis of T2DM, HbA1c levels above 6.5%, fasting blood glucose levels of more than 6.93 mmol/L, serum cholesterol of DM-HL was more than 5.17 mmol/L and triglyceride were higher than 1.69 mmol/L and stable health conditions with normal kidney and liver function tests. Healthy control subjects were age-sex matched to patient group. They had no history of chronic metabolic or cardiovascular diseases, and normal biochemical and hematological profiles were confirmed prior to inclusion.

We exclude some health conditions, including the presence of other types of DM like gestational and T1DM, Pregnancy or lactation, patients with a history of major surgery, Chronic liver or kidney disease, Substance abuse or alcoholism, and Severe CVD. This selection process was guided by strict ethical considerations, with the study receiving approval from the Selçuk University, Faculty of Medicine, Clinical Research Ethics Committee (Decision No: 2024/324).

Arginine (CAS Number: 202468-25-5), ADMA (CAS Number 220805-22-1), citrulline (CAS Number: 372-75-8), SDMA (CAS Number: 1266235-58-8), L-NMMA (CAS Number: 53308-83-1), ornithine (CAS Number: 3184-13-2), methanol (CAS Number: 67-56-1), HPLC grade water (CAS Number: 7732-18-5), n-butanol (CAS Number: 71-36-3), acetyl chloride (CAS Number: 75-36-5), formic acid (CAS Number: 64-18-6) and d7-ADMA (Catalog No: DLM-7476-5) were purchased from Sigma Aldrich and Cambridge Isotope Laboratories (St. et al., USA) respectively.

Shimadzu HPLC system (Kyoto, Japan) and Phenomenex C18 HPLC column (50 mm × 4.6 mm) were used for chromatographic separation. The detection was performed using a API 3200 triple quadrupole mass spectrometer equipped with an electrospray ionization interface that was used (Applied Biosystems/MDS Sciex) as detector.

Fasting venous blood samples were collected for analysis of serum methylarginine derivatives (ADMA, SDMA, L-NMMA) and related metabolites (arginine, homoarginine, citrulline, ornithine).

Serum methylarginine derivatives (ADMA, SDMA, L-NMMA) and related metabolites (arginine, homoarginine, citrulline, ornithine) levels were measured with a modification of the previously published method [28]. The sum of ADMA, SDMA and L-NMMA levels was calculated as a total methylated arginine load [29]. Global arginine bioavailability ratio (GABR) was calculated by the formula as [Arginine / (Citrulline + Ornithine)] [30].

The Statistical analyses were performed using IBM SPSS version 26.0. Data normality was assessed via the Shapiro-wilk test. We employed the Mann-Whitney U test for two group comparisons; for more than two group comparisons One-Way ANOVA test was used for normal distributed parameters while Kruskal-Wallis tests was used for abnormally distributed parameters. Spearman correlation was used. Statistical significance was established at p<0.05.

A total of 83 patients diagnosed with DM participated in the study; 55 were female, and 28 were male. The mean age was 56.22±11.4, 51.96±11.5 years respectively. Also 37 healthy controls, 25 were female and 12 were male included in the study. The mean age was 51.72±10.01, 51.75±8.01 respectively.

DM groups had higher fasting blood glucose (FBG), HbA1c, Insulin, HOMA-IR, Triglyceride (TG), Cholesterol (CHE), Low-density lipoprotein (LDL), White blood cell (WBC) and Platelets (PLT) levels than the Control group (p=0.000, p=0.000, p=0.000, p=0.000, p=0.000, p=0.000, p=0.000 p=0.000 and p=0.033 respectively), while High-density Lipoprotein (HDL), Hemoglobin (HGB), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) were higher in control than DM group (p=0.000, p=0.000, p=0.011, p=0.000 and p=0.000 respectively). The values are shown detailed in Table 1.

When we compared between three groups, ADMA (p=0.001), SDMA (p<0.001), Arginine (p=0.013), Citrulline (p<0.001), L-NMMA (p=0.001), Ornithine (p<0.001), Arginine/ADMA ratio (p=0.028), Homoarginine/ADMA ratio (p<0.001), Citrulline/ADMA ratio (p<0.001), GABR (p=0.002) and Total methylated Arginine load (p<0.001) were statistically significant. The results presented are detailed in Table 2.

Serum ADMA (p=0.033), SDMA (p=0.001), Citrulline (p<0.001), L-NMMA (p<0.001), Citrulline/Arginine (p<0.001), Citrulline/ADMA (p<0.001), GABR (p=0.001) and Total methylated Arginine load (p=0.001) levels were found to be statistically significantly higher in DM-HL group compared to HC group, while serum Homoarginine (p<0.001), Ornithine (p<0.001), Arginine/ADMA (p=0.022) and Homoarginine/ADMA (p<0.001) were found to be statistically significantly lower in DM-HL group compared to HC group.

When DM-NL compared to HC group, serum Citrulline (p=0.002), SDMA/ADMA (p=0.031), Citrulline/Arginine (p<0.001), Citrulline/ADMA (p=0.001) levels were found to be statistically significantly higher, while Arginine (p=0.026), Homoarginine (p<0.001) and Homoarginine/ADMA (p=0.001) were found to be statistically significantly lower.

Moreover, DM group statistically compared according to lipemic profile and serum ADMA (p<0.001), SDMA (p<0.001), Arginine (p=0.005), Citrulline (p<0.001), L-NMMA (p=0.003), GABR (p=0.014) and Total methylated Arginine load (p<0.001) were found to be statistically significantly higher in lipemic group, while Arginine/ADMA (p=0.029) and Homoarginine/ADMA (p<0.001) were found to be statistically significantly lower. The laboratory parameters of DM and HC groups were described in Figure 2 and Table 3.

When we performed spearman correlation test on serum methylarginine derivatives and some biochemical parameters, the results showed that ADMA, SDMA, L-NMMA, homoarginine, ornithine, and their derivatives show correlations with glucose metabolism, insulin resistance, and inflammation, potentially linking them to metabolic syndrome and cardiovascular risks, the results presented in Table 4.

This study investigates the serum level of methylarginine derivatives (ADMA, SDMA, L-NMMA) and related metabolites (arginine, homoarginine, citrulline, ornithine), which are associated with NO metabolism in DM with and without hyperlipidemia. Unlike previous studies, our study uniquely investigates how hyperlipidemia modulates arginine metabolism and nitric oxide bioavailability in the diabetic state. It allows us to isolate the specific contribution of lipid profile abnormalities to endothelial dysfunction in T2DM patients.

NO is an unstable molecule with a very short half-life (2–6 s) and rapidly oxidizes to nitrite and nitrate. The unstable structure of NO complicates the direct measurement of NO levels [28]. Arginine divided through a reaction catalyzed by NOS to form citrulline and NO [31]. Therefore, in this study, we calculated the citrulline / Arginine ratio, which can be an indicator for NOS activity and NO levels. When we compared the 3 groups the citrulline/Arginine ration were significantly statistically higher in DM-HL than HC (p=0.000) and higher in DM-NL than HC (p=0.000). however, When the results between the two DM groups were examined, no statistical significance was found (p>0.05) (Table 2, Fig. 1).

Rainer H. Böger et al. investigated the levels of ADMA and arginine in hypercholesterolemic and normocholesterolemic participants. 49 hypercholesterolemic and 31 normocholesterolemic participants were included in the study. As a result of the study, plasma ADMA are statistically significantly elevated in hypercholesterolemic than in normocholesterolemic (P<0.05) [32].

Riccioni et al., investigated the level of ADMA and SDMA in asymptomatic carotid atherosclerosis. 180 subjects with carotid atherosclerosis included in this study. 14% had carotid intima-media thickening (CIMT) localized to the common carotid arteries (CCAs), 36% had atherosclerotic plaque in the CCAs, 44% had atherosclerotic plaque in the internal carotid arteries (ICAs) and 6% had atherosclerotic plaque in the external carotid arteries (ECAs). The study found that elderly subjects with asymptomatic carotid atherosclerosis, as measured by CIMT and plaque, had significantly higher plasma concentrations of ADMA and SDMA compared to those without carotid atherosclerosis (p<0.001) [33].

In our study, elevated ADMA levels in the DM-HL group compared to both DM-NL and HC groups (p=0.000 and p=0.033, respectively) indicate that hyperlipidemia exacerbates endothelial dysfunction in diabetic patients. Conversely, the non-significant difference between DM-NL and HC (p=0.167) suggests that diabetes alone has a limited impact on ADMA levels. Elevated SDMA levels in DM-HL compared to DM-NL (p=0.000) and HC (p=0.001) highlight the potential role of SDMA as a marker of dyslipidemia-induced renal or vascular dysfunction. The absence of significant variation between DM-NL and HC (p=0.871) supports this interpretation.

Reduced arginine levels in both DM groups compared to HC suggest impaired nitric oxide synthesis, a result of diabetes-associated metabolic disturbances. The lack of a significant difference between the two DM groups (p=0.952) implies that hyperlipidemia does not further reduce arginine availability. Additionally, homoarginine levels are markedly lower in DM groups compared to HC (p=0.000 for both DM groups), likely reflecting altered urea cycle function or increased protein carbamylation in DM. The absence of significant differences between DM groups (p=0.566) suggests that hyperlipidemia does not independently influence homoarginine levels.

Citrulline levels were significantly lower in both DM groups compared to HC (p=0.000), suggesting dysregulation in nitric oxide or urea cycle pathways. In addition, the levels of Citrulline were significantly lower in DM-NL compared to DM-HL (p=0.000). Elevated L-NMMA levels in DM-HL compared to DM-NL (p=0.003) and HC (p=0.001) indicate heightened endogenous nitric oxide inhibition in DM-HL, further impairing vascular function. The lack of significant differences between DM-NL and HC (p=0.592) emphasizes the more pronounced role of hyperlipidemia in this dysfunction.

The levels of Ornithine showed no significant differences between the two DM groups (p=0.527) suggesting that Ornithine serum levels are not affected by dysregulated lipid profile. However, Ornithine levels were reduced in both DM groups compared to HC, highlighting disruptions in the urea cycle and potential shifts in arginine metabolism towards alternative pathways, such as polyamine synthesis or nitric oxide production. This reduction may reflect adaptive or maladaptive metabolic responses to diabetes-induced stress. Restoring metabolic balance through dietary arginine supplementation could be a potential therapeutic strategy to mitigate these disruptions.

Significantly elevated SDMA/ADMA ratios in DM groups, especially in DM-HL suggest an increase in nitric oxide inhibition, which is indicator for endothelial dysfunction. This ratio could be an important marker for identifying individuals at increased risk for cardiovascular complications. Also, the decrease in arginine/ADMA ratio across all DM groups indicates limited substrate availability for nitric oxide synthase due to high ADMA levels. The lack of significant differences between the DM-HL and DM-NL groups suggests that DM is responsible for these alterations rather than lipid status. Since, a low arginine/ADMA ratio strongly predicts CVD risk, the importance of early interventions to preserve endothelial function in diabetes management is mandatory.

The homoarginine/ADMA ratio, an alternative marker of nitric oxide production, was significantly lower in DM-HL. This compounded insufficiency in nitric oxide generation underscores the negative role of hyperlipidemia in vascular dysfunction. Monitoring homoarginine/ADMA ratio may provide valuable vision into the interplay between lipid status and vascular health in DM. Strategies to increase homoarginine levels, such as dietary supplementation, may hold promises for improving vascular outcomes in DM-HL patients.

The citrulline/arginine ratio, reflecting arginine recycling and utilization, was elevated in DM-HL suggesting altered metabolic flux due to increased arginine diversion toward other pathways (e.g., polyamine or proline synthesis). These disruptions contribute to endothelial dysfunction and impaired vascular homeostasis. Modulating arginine metabolism could restore nitric oxide synthesis efficiency and improve vascular function. The citrulline/ADMA ratio, integrating markers of nitric oxide recycling and inhibition, was significantly reduced across all DM groups, particularly in DM-HL. This finding highlights the additive effect of elevated ADMA levels and reduced substrate recycling in worsening endothelial dysfunction. Incorporating lipid-lowering strategies into comprehensive diabetes care is crucial to address these metabolic derangements.

The GABR was significantly lower in both DM groups compared to the HC. GABR reflects the balance between circulating arginine and its primary inhibitors, such as ADMA, and is a critical indicator of nitric oxide bioavailability and vascular health. Both DM and hyperlipidemia are associated with increased oxidative stress and endothelial dysfunction, which reduce arginine availability for nitric oxide production. A reduced GABR in DM groups underscores diminished nitric oxide production capacity, which is pivotal for maintaining vascular tone and preventing atherogenesis. Interventions targeting arginine metabolisms, such as arginine supplementation or ADMA reduction, may improve endothelial function in these populations.

Finally, elevated total methylated arginine levels in DM-HL compared to both DM-NL (p=0.000) and HC (p=0.001) highlight the additive role of hyperlipidemia in endothelial dysfunction through elevated oxidative stress and impaired protein turnover. The lack of significant elevation in the DM-NL group suggests lipid status is critical in methylation-related dysfunction. Therapeutic strategies to reduce ADMA levels or enhance its clearance (e.g., through dimethylarginine dimethylaminohydrolase [DDAH] activity) could mitigate cardiovascular complications in hyperlipidemic diabetic patients.

This study highlights the complex impact of hyperlipidemia on endothelial dysfunction and metabolic disturbances in DM patients. The elevated levels of ADMA, SDMA, and total methylated arginine in the DM-HL group emphasize the additional role of lipid abnormalities in worsening nitric oxide inhibition, oxidative stress, and vascular dysfunction. The significant difference that observed in arginine, citrulline, and related metabolic ratios between patients and control further underline the dysregulation of nitric oxide synthesis and arginine metabolism associated with DM-HL.

These findings suggest that hyperlipidemia amplifies the harmful effects of DM on endothelial function, highlighting the importance of effective lipid management in diabetes care. Biomarkers, such as the SDMA/ADMA, arginine/ADMA, and homoarginine/ADMA ratios, provide valuable insights into the extent of endothelial dysfunction and cardiovascular risk in this population.

Therapeutic interventions targeting metabolic pathways — including arginine supplementation, reduction of ADMA levels, and lipid-lowering strategies — show promise for restoring endothelial function and reducing cardiovascular complications in DM-HL. Future studies should investigate these therapeutic approaches to develop comprehensive strategies for mitigating vascular dysfunction and improving outcomes in this high-risk population.

However, an important limitation of this study is the relatively small sample size, which may reduce the generalizability of the findings to larger populations. Furthermore, the study’s cross-sectional nature limits the ability to infer causality between hyperlipidemia, nitric oxide dysregulation, and cardiovascular outcomes. Future research should address these limitations through more extensive, longitudinal studies to confirm these findings and explore causal mechanisms.

Conflict of interest. The authors declare that they have no conflict of interest relevant to the content of this manuscript.

Funding. The authors did not receive support from any organization for the submitted work

Authorship contribution. Bik M.A. — conceptualization, methodology, supervision, project administration, verifying the results and ensuring accuracy, resources, writing (original draft, review & editing); Almaghrebi E. — writing (review & editing), methodology, validation, investigation and visualization; Akat F. — supervision, writing (review & editing), project administration, validation, supervision, resources, conceptualization; Gharab K. — methodology, supervision, project administration, writing (review & editing), data curation; Sak F. — resources, writing (original draft, review & editing, methodology, software, investigation; Karaman F. — data curation and visualization; Unlu A. — project administration, verifying the results and ensuring accuracy, writing (review & editing, original draft), software. All the authors approved the final version of the article before the publication and expressed their consent to be responsible for all aspects of the work, which implies proper investigation and resolving of issues related to the accuracy or integrity of any part of the work.