Serum electrolyte levels in relation to macrovascular complications in Chinese patients with diabetes mellitus

Study population

A gender- and age- matched case–control analysis was performed based on data obtained from the follow-up study of the Shanghai Diabetes Study (SHDS) and SHDS II[15, 16]. As shown in Figure 1, this follow-up study was conducted from August 2010 to December 2011 in the Caoyang, Anting, and Huayang communities in Shanghai, China. Subjects enrolled in the analysis met the following inclusion criteria: aged 20 years or above and under 75 years, with complete serum electrolytes and fasting glucose data. Exclusion criteria included subjects once taking diuretics in the 2 weeks before being interviewed (n = 21), persistent diarrhea or vomiting (n = 0), severe renal insufficiency, which was determined using an estimated glomerular filtration rate (eGFR) <60 ml · min^-1 · 1.73 m^-2 (n = 34) and any self-report of type 1 diabetes mellitus (T1DM) or a history of diabetes of an unknown type (n = 39). In total, 2,723 subjects were enrolled in the study and were grouped according to their glucose tolerance. They were matched (the number ratio of subjects in each group nearly 3:1:1) according to their gender and age (5-year intervals). Then, a normal glucose regulation (NGR) group (n = 1,170), an impaired glucose regulation (IGR) group (n = 389) and a DM group (n = 343) were formed.

This study was approved by the institutional review board of Shanghai Jiao Tong University Affiliated Sixth People’s Hospital in accordance with the principle of the Helsinki Declaration II. Written informed consent was obtained from each subject before the survey.

Laboratory assessment

Subjects were invited to the local hospital between 6 and 8 am following an overnight 8–10 h fast. Venous blood samples were collected at 0 and 120 min following a 75-g oral glucose tolerance test (OGTT) for subjects without self-reported diabetes, or after the consumption of a steamed bun containing approximately 80 g of complex carbohydrates for subjects with self-reported histories of DM. Fasting blood samples were used to obtain serum electrolyte levels and other biochemical analyses. Plasma glucose, electrolytes, creatinine levels (SCr) and lipid profiles were determined with an autoanalyzer (Hitachi 7600 analyzer, Hitachi, Japan). The inter- and intra-assay coefficients of variation for Na⁺, K⁺, Ca²⁺, Mg²⁺ were 0.77% and 1.13%; 1.01% and 1.22%; 1.80% and 3.00%; and 1.15% and 1.92% respectively. Serum glycosylated hemoglobin (HbA1c) was measured using high-performance liquid chromatography. The eGFR was assessed using the following formula: eGFR (ml · min^-1 · 1.73 m^-2) = 186 × SCr (mg · dl^-1)^-1.154 × age^-0.203 (for women × 0.742)[17].

Interviews and measurements

Demographic and disease history information was collected again using a standardized questionnaire during this follow-up study. The anthropometric indices of height and weight were measured while subjects were barefoot and wearing light clothing. The waist circumference (WC) was measured at the point between the costal margin and iliac crests. Body mass index (BMI) was calculated as weight in kilograms divided by height in meters squared[15]. A blood pressure measurement was taken twice using a mercury sphygmomanometer and the results averaged[18]. A Doppler probe (Nicolet VersaLab SE) was used to calculate the ankle brachial index (ABI), which is equal to the highest measured arterial pressure in the ankle or foot divided the pressure in the arm on each side[19].

Definitions and diagnostic criteria

DM was defined as a fasting plasma glucose (FPG) ≥7.0 mmol/l and/or a 2-hour post-load plasma glucose (2hPG) ≥11.1 mmol/l, or on diabetic medication treatment for type 2 diabetes (T2DM). IGR was defined by an impaired fasting glucose (6.1 mmol/l < FPG <7.0 mmol/l and 2hPG <7.8 mmol/l), impaired glucose tolerance (FPG < 6.1 mmol/l and 7.8 mmol/l < 2hPG < 11.1 mmol/l), and impaired fasting glucose with impaired glucose tolerance (6.1 mmol/l < FPG <7.0 mmol/l and 7.8 mmol/l < 2hPG <11.1 mmol/l) in the follow-up[20]. Normal serum electrolyte reference ranges were used as follows: 137 mmol/l ≤ Na⁺ ≤ 145 mmol/l, 3.5 mmol/l ≤ K⁺ ≤ 5.1 mmol/l, 2.08 mmol/l ≤ Ca²⁺ ≤ 2.60 mmol/l, 0.65 mmol/l ≤ Mg²⁺ ≤ 1.05 mmol/l. Hypertension was defined as a blood pressure of >140/90 mmHg or the use of antihypertensive medications[21]. Dyslipidemia was defined as a serum triglyceride (TG) level ≥1.7 mmol/L or a high density lipoprotein cholesterol (HDL-C) level <0.9 mmol/L in men and <1.0 mmol/L in women[22].

Diabetic macrovascular complications in this study included any patient with a history of CVD events or PAD. Subjects were defined as having had CVD events if a history of coronary heart disease (CHD) or stroke was evident. Various non-fatal CVDs were determined according to the self-reports of patients, which were confirmed by their medical records. The definition of CHD included a history of hospitalization for myocardial infarction, a surgical history of coronary balloon angioplasty, coronary stent implantation or coronary artery bypass. Stroke was defined as a history of language or physical dysfunction that continued for more than 24 h, and ischemic or hemorrhagic stroke was diagnosed using imaging examinations (computed tomography or magnetic resonance imaging)[23].

According to the current guidelines proposed by the American Heart Association (AHA), PAD was identified as an ABI <0.90 in either leg. Those with an ABI >1.3 were excluded from the analysis to avoid those with significant medial artery layer calcification, which is independent of atherosclerotic plaques[24]; other individuals were considered as non-PAD[19].

Statistical methods

Summarized data are shown as mean ± standard deviation or as frequencies (%). One-way analysis of variance (one-way ANOVA) was used to compare the mean differences and the Pearson’s chi-square test was used to compare the proportional differences in demographic characteristics and serum electrolyte levels among the NGR, IGR and DM groups, or among the mildly decreased eGFR, normal eGFR and elevated eGFR groups. The linear trends of electrolyte levels within the groups were tested. Multiple linear regression analyses were used to determine the relationships between electrolytes and glucose levels. Analysis of covariance (ANCOVA) was used to compare the mean differences in electrolyte levels after adjustment by possible confounding factors between DM subjects with macrovascular complications and those without macrovascular complications. SPSS for windows (version 17.0, SPSS, Chicago, IL, USA) was used to perform the statistical analyses. The significance level was P value <0.05, and the results were adjusted with the Bonferroni correction for multiple testing.

Characteristics of the study population

Serum electrolyte levels among the three groups

Among the NGR, IGR and DM groups, 9 (0.8%), 4 (1.0%) and 14 (4.1%) subjects had hyponatremia (P < 0.01), while 55 (4.7%), 17 (4.4%), and 10 (2.9%) subjects had hypernatremia (P = 0.36); 14 (1.2%), 11 (2.8%) and 2 (0.6%) had hypokalemia (P = 0.02), while 17 (1.5%), 3 (0.5%) and 4 (1.2%) subjects had hyperkalemia (P = 0.57); 6 (0.5%), 0 (0.0%) and 4 (1.2%) had hypocalcemia (P = 0.09), while 3 (0.3%), 0 (0.0%) and 3 (0.9%) subjects had hypercalcemia (P = 0.09); 0 (0.0%), 0 (0.0%) and 2 (0.6%) had hypomagnesemia (P = 0.01), while 26 (2.2%), 11 (2.8%) and 3 (0.9%) subjects had hypermagnesemia (P = 0.16).

Serum electrolyte levels in DM subjects with different eGFRs

Correlation of electrolytes and glucose levels in subjects with DM

Serum electrolyte levels in subjects with and without DM macrovascular complications

Alterations in serum electrolyte levels with elevated glucose levels

Significant reductions in serum Na⁺ and Mg²⁺ levels and an elevation in serum Ca²⁺ in subjects with DM were observed. These results were consistent with those reported by previous studies[6, 7, 26, 27]. Furthermore, levels of serum Na⁺ and Mg²⁺ were negatively correlated with plasma glucose levels, measured as FPG, 2hPG and HbA1c, while Ca²⁺ level was positively correlated with HbA1c in subjects with DM.

Previous studies have estimated that serum Na⁺ concentrations decrease from 0.24 mmol/l to 0.29 mmol/l for every 1.0 mmol/l decrease in glucose concentration[28, 29]. Our findings suggested there was a 0.32 mmol/l decrease of Na⁺ per 1.0 mmol/l fasting plasma glucose. Yajnik et al. observed that plasma Mg²⁺ concentrations were inversely related to plasma glucose levels in subjects with DM[30], and our results are in accordance with this.

In the present study, serum electrolyte levels of Na⁺ and Mg²⁺ decreased along with an elevation in the eGFR in subjects with DM. Under physiological conditions, most of the Na⁺ is reabsorbed in the proximal tubule of the kidney and Mg²⁺ is mostly reabsorbed in the loop of Henle[31]. Hyperglycemia-induced osmotic diuresis, which can increase excretion, is thought to be a primary mechanism underlying the decreased serum concentrations of Na⁺ and Mg²⁺ observed in response to elevated glucose levels. Additionally, overly aggressive volume re-expansion and glomerular hyperfiltration can induce renal Mg²⁺ wasting at the proximal tubule and the loop of Henle. Mg²⁺ reabsorption parallels Na⁺ reabsorption in the proximal tubules; therefore, volume expansion would further decrease the reabsorption of both Mg²⁺ and Na⁺[8, 32].

In addition, we found that serum Ca²⁺ level was positively correlated with glucose level. This observation was consistent with previous studies[27, 33, 34]. Ca²⁺ is mainly reabsorbed in the proximal tubule. Its reabsorption is coupled to Na⁺ absorption, and it appears to compete with Mg²⁺ for transport in the loop of Henle. In the distal convoluted tubule, Ca²⁺ absorption is regulated independently of Na⁺, where numerous factors, such as calcitonin, parathyroid hormone, and vitamin D, can have marked effects on Ca²⁺ reabsorption and secretion[31, 35]. Energy metabolism is associated with bone remodeling, and glucose levels also regulate parathyroid hormone and vitamin D levels[36]. This means that the excess excretion of Ca²⁺, induced by hyperglycemia, may be weakened by several pathways. Alternatively, the Resnick ionic hypothesis suggested that metabolic disorders, such as hypertension, metabolic syndrome, and diabetes, share a common, altered intracellular condition, characterized by decreased Mg²⁺ level and reciprocally elevated free intracellular Ca²⁺ level[37]. Intracellular Ca²⁺ plays a critical role in muscle contractions, insulin secretion, and glucose uptake after the binding of insulin to muscle cell membranes. The most common clinical manifestations of type 2 DM is insulin resistance, which mostly occurs in skeletal muscle, and the impairment of insulin secretion[38]. All these factors may account for the elevation of serum Ca²⁺ level in subjects with DM, and especially in patients whose eGFR levels are increased.

Furthermore, it has been observed that cellular membrane electrolyte transporter Na⁺-K⁺-ATPase dysfunction in diabetic subjects, can be secondary to hyperglycemia[39]. And the functions of Ca²⁺-Mg²⁺-ATPase, Na⁺/Ca²⁺ exchanger and Ca²⁺ pump, which are located in cell membrane, mitochondria or endoplasmic reticulum, have been shown to be impaired in diabetes[40, 41]. Electrolyte disturbances within cells can be induced by them above mentioned. The combination of intracellular and extracellular electrolyte disturbances may be implicated in the pathogenesis of neuropathy, nephropathy and vascular complications in diabetic patients.

Serum Mg²⁺ level is further decreased in subjects with diabetic macrovascular complications

We found that serum Mg²⁺ level was reduced in subjects with diabetic macrovascular complications. Ma et al. also demonstrated that, in African Americans and Caucasians, serum Mg²⁺ level was significantly lower in subjects with prevalent CVD and diabetes[42]. Recently, Larsson et al. performed a meta-analysis based on eight prospective studies and concluded that there was a significant inverse relationship between dietary Mg²⁺ intake and stroke risk[43]. It has also been observed that serum Mg²⁺ depletion is associated with some PAD symptoms, such as foot ulcers, in subjects with type 2 DM[44]. In addition, Resnick ionic hypothesis suggested that the intracellular Mg²⁺ level is generally suppressed, which summarizes the primacy of a common cellular defect in ion handling in various clinical manifestations of metabolic disorders[37]. Both decreased intracellular and extracellular Mg²⁺ concentrations may accelerate atherogenesis via the elevation of inflammatory cytokines and lipid oxidation, an increase in endothelial cell growth and the inhibition of cellular DNA repair[45–48].

Diabetic macrovascular complications, CVD in particular, are currently the leading cause of death for diabetic patients. It has been reported that even low Mg²⁺ levels that are still within the normal reference range are associated with all-cause mortality in patients with T2DM[10]. Our results would raise awareness of physicians, especially in China, and allow them to promptly correct mild electrolyte disturbances in diabetic patients. Even though many observational studies of electrolyte disturbances in patients with diabetes have consistently report that Mg²⁺ intervention may be extremely useful in the prevention and treatment of the cardiometabolic syndrome[49], some results of intervention studies, especially those involving daily electrolyte supplements for the prevention of DM or against diabetic complications, are controversial[50].

Strengths and limitations

The first strength was that our subjects were from a large population-based survey. The symptoms and complications of the diabetes group were relatively mild, and the effects of iatrogenic factors on electrolytes were less than in those patients who were administrated in hospital. Secondly, we matched subjects among the NGR, IGR and DM groups to reduce the significant differences in gender and age distribution. A main limitation of our study was that we only measured the total serum electrolytes levels, but not free ionized or intracellular electrolytes or levels in other biological samples from the subjects[51], which need some special apparatus to measure[52]. Although questionnaire data are of great value in collecting the information of the history of non-fatal CVDs, misclassification remains a possibility[53]. Moreover, even though the analyses were adjusted for the common risk factors of age, gender, obesity, hypertension and dyslipidemia, unmeasured factors, such as eating habits and other medications, may have indirect effects on the electrolyte regulation and interfere with the relationship between CVD and electrolyte disturbances. Our study also did not elucidate the causal relationship of serum electrolyte levels on the development of DM and diabetic macrovascular complications.