Diabetes Mellitus (DM) is a major public health problem worldwide; amongst the most common chronic disorders of modern time. It remains unique because of its systemic micro and macrovascular complications. The complications of Diabetes Mellitus have far reaching effects on the lives of people, and have a big constraint on the budget for health services [1]. Saudi Arabia has the second highest rate of diabetes in the Middle East, and the seventh highest rate in the world [2].

With an estimated number of 3.8 million people diagnosed with the disease in KSA by 2014, this represents more than 25% of the population, and that figure is expected to exceed by more than double by 2030. Half of the population over 30 years of age is prone to diabetes [3].

Pre-diabetes is defined as the elevation of blood glucose and glycated Hb (HbA1c), above the normal range, but below levels of diabetes. Insulin resistance and b-cell dysfunction are characteristics of this disorder. Recently, pre-diabetes was found to be associated with disorders that were considered to be restricted to established diabetes. These disorders include micro vascular disease, metabolic syndrome, cardiovascular disease, cognitive dysfunction, blood pressure abnormalities, fatty liver disease, periodontal disease, obstructive sleep apnea, low testosterone, cancer [4], and birth defects [5].

In early pregnancy, HbA1c elevations close to the reference range may identify women with both underlying hyperglycemia, and association with adverse pregnancy outcomes [6].

Diabetic women with elevated blood glucose and glycosylated hemoglobin levels in the first trimester have been significantly associated with increased risk of adverse pregnancy outcomes (e.g., abortion, stillbirth, and congenital abnormalities) [7,8]. This is also true for those with insulin resistance, obesity, and even lesser degrees of hyperglycemia, which affect the intrauterine metabolic environment and have long-term implications on the future health of the offspring, so that achieving optimal outcomes in mothers can no longer be simply measured by whether or not maternal glucose values are at goal. Thus, the pregnant women with undiagnosed diabetes are a high-risk group that may benefit from early intervention [9].

The relation between zinc, diabetes and insulin was discovered in the 1930s, a decade following discovery of the hormone, when a study showed that crystallized insulin contains zinc and that zinc, along with other metal ions, could induce insulin crystallization [10].

Later on, zinc was found to prolong insulin action when co-injected with the hormone [11]. More recent findings show association between type 2 diabetes (T2D) risk, and the inheritance of gene variants encoding a critical β cell zinc transporter [12]. This documented the role of zinc in diabetes aetiology, and as a possible therapeutic target [13].

Globally, scientists observed beneficial outcomes in decreasing HbA1c levels with zinc supplementation. However, it is still unclear if dietary zinc supplementation would have an effect in preventing T2D [14,15].

Hypomagnesemia was found to be associated with the development of impaired glucose tolerance (IGT) and T2D [16].

Prospective studies have found that Mg intake can reduce the risk of developing pre-diabetes among those with normal glucose regulation [17], and can also improve glycemic status in adults with pre-diabetes [18] However, a cross-sectional study has failed to demonstrate a relationship between serum Mg and precursor states of diabetes, including impaired fasting glucose and IGT [19].

Selenium has contradictory effects in diabetes; the antioxidant property of selenium prevents the development of complications in diabetic patients. Some studies found that higher serum selenium concentrations were associated with a higher prevalence of diabetes [20]. More convincing data focuses on specific selenoproteins in glucose metabolism. Recently, a study showed that both selenoprotein deficiency and a high expression of selenoproteins cause glucose disturbances [21] Selenium has been shown to have insulin-like properties, [22] which qualify it as a potential anti-diabetic agent.

Subjects/Methods

This study was done to compare serum levels of zinc, magnesium and selenium between pregnant women with different glycemic status, (normal, pre-diabetic and diabetic).

The data was part of a large prospective cohort study with the aim of identifying some preventable risk factors that can cause congenital malformations among Saudi women.

A total of 1,180 healthy first trimesters pregnant were randomly selected from antenatal clinics in two hospitals, and 21 primary health care centres in Al-Madinah Al-Monawarah, KSA. Inclusion criteria included the following factors:

• Pregnant women in the first trimester

• Agreeability to participate until delivery

• Absence of systemic diseases that are known to cause birth defects such as diabetes (type 1 and 2) epilepsy, heart diseases etc.

Clinical history, examination, and lab analysis were done (serum levels of magnesium, zinc, selenium and HbA1c). Subjects were classified by their HbA1c levels as normal (HbA1c<5.7%), pre-diabetic (HbA1c 5.7<6.5%), and undiagnosed T2D (HbA1c ≥ 6.5%). Comparison in the lab results was done between the groups [23].

Laboratory methods: Blood samples

Fasting blood samples were taken after an overnight fast, placed into EDTA tubes, and centrifuged at 3000 x g for 10 minutes. The plasma obtained was frozen at -80°C until the time of analysis.

Blood HbA1c levels were measured by using standard biochemical procedures, and the results were identified with the use of reference ranges in this laboratory. NycoCard HbA1c is a boronate affinity assay.

Zinc and magnesium were measured using Bioassay Systems' kits that measured zinc and magnesium directly in biological samples without any pre-treatment. The present method utilizes a chromogen that forms a colored complex. The intensity of the color measured at 425 nm, and 500 nm for zinc and magnesium respectively.

Statistical analysis

Descriptive analysis (mean, standard deviation, and Student's t test) was done using Statistical Package for Social Sciences (SPSS17). The comparative outcome of lab data was analyzed by using T-test for paired samples for continuous data. The minimal level of significance was set at P<0.05. Correlation coefficients were determined by linear regression analysis.

A total of 1,103 cases were enrolled in the study, and 77 dropped out for various reasons. The mean age of the participants was 27.9 ± 6.25 years.

About 78.9% (870 subjects) had normal HbA1c, 19.1% (211 subjects) had HbA1c in the range of pre-diabetes and 1.99% (22 subjects) had HbA1c>6.5% denoting undiagnosed type 2 diabetes.

Serum levels of zinc, magnesium and selenium were significantly lower among pre-diabetics compared to normal subjects. The same results were obtained when comparing both diabetics and prediabetics, and diabetics and normal subjects. There was a significant negative correlation between the levels of HbA1c and all the studied minerals.

The cause-and-effect relationship of the deficiency of certain nutrients with pre-diabetes and diabetes remains unclear. In our study, mineral levels in pregnant women at the pre-diabetes stage were significantly lower than that of women with normal glycemic control, but higher than those with diabetes. It has been shown that various trace elements such as Ca, P, Mg, Zn, Cu, Fe, etc. are metabolically interrelated, and there is an alteration in their concentration during pregnancy [24].

Several studies have linked the deficiency of certain nutrients with glycemic control and a possible interaction of multiple nutrients' deficiencies; each of which may have a contributing effect.

In the complex processes of development or progression of diabetes mellitus, micronutrients are involved in several different areas. Some are components of antioxidant enzymes (e.g., Cu, Zn and Mn in the case of the superoxide dismutases, and Selenium for GSHPx), and cofactors in a variety of enzymatic processes of importance in glucose and lipid metabolism (e.g., Zn, Mn, Cu). Evidence for changes in trace mineral and vitamin metabolism as a consequence of diabetes pathophysiology is proved especially among micronutrients which have a recognized antioxidant or pro-oxidant functions, with consideration of the relevance of these changes to current hypotheses regarding the onset of secondary complications in the progression of diabetes [25]. Hyperglycemia also results in overproduction of oxygen free radicals, which contributes to the progression of diabetes [26].

Zinc has been shown to have insulin-like action and anti-diabetic effects; it is required for insulin storage and secretion and it has antioxidant properties. Chronic low intake of zinc is associated with an increased risk of diabetes, and conversely, diabetes also impairs zinc metabolism [27] Comparable to our study, diabetic patients were found to have significantly lower serum zinc levels compared with healthy controls [28].

A recent study reported an improvement in glucose handling in subjects with pre-diabetes supplemented with zinc, and this could potentially be a safe way to prevent the progression of the pre-diabetes stage to diabetes [29]. Previous studies on dietary and supplemental zinc showed a potential protective effect on type 2 diabetes [30].

Results from a recent study suggested that a low magnesium level is an independent risk factor for the development of pre-diabetes and type 2 diabetes [31].

Magnesium is an essential ion involved in multiple levels in insulin's secretion, its binding, and its activity, and it is also a critical cofactor of many enzymes in carbohydrate metabolism. Magnesium plays an important role to improve insulin resistance [32].

The increased incidence of hypomagnesemia among patients with T2D is presumably multifactorial. Altered insulin metabolism, poor glycemic control, and osmotic diuresis may be contributory factors [33].

Plasma selenium concentrations corresponding to optimal health are highly dynamic and based on a combination of factors that need to be considered when assessing epidemiological data, such as the findings linking serum selenium and T2D [34].

Comparable with our study, Ruiz et al found that mean plasma selenium content in diabetic patients was significantly lower than the controls, and that there was a negative correlation between plasma contents of selenium and glycosylated hemoglobin [35].

Studies have shown that selenium can protect against oxidative damage attributable to unregulated blood sugar [36, 37].

Changes in serum levels of some lab parameters occur at the prediabetic stage and precedes the development of diabetes, and may be responsible for medical complications that are recently reported at this stage. Studies should be done for re-establishment of safe target levels of HbA1c.

Further researches should be done on the exact relation between those altered micronutrients in pre-diabetes, and the possible role of their correction on the development and progression of diabetes.

The investigators are deeply indebted and wish to express their sincere thanks to King Abdul Aziz City for Science and Technology (KACST) for the generous funding throughout this research. This project was made possible by Grant Number AT. 28-113 from KACST.