A CORRELATIVE STUDY OF SERUM INORGANIC PHOSPHATE CONCENTRATION AND FASTING BLOOD GLUCOSE IN TYPE 2 DIABETES MELLITUS – A HOSPITAL BASED STUDY

Dr. Girindra Kr. Bora 1 , Dr. Rashmi Rajkakati 2 and Dr Sushma Yadav 1 . 1. Demonstrator, Department of Biochemistry, Jorhat Medical College, Jorhat-785001, Assam, India. 2. Associate professor. Department of Biochemistry, Assam Medical College, Dibrugarh-786002, Assam, India. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History


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Diabetes is an "iceberg" disease. Although increase in the prevalence and incidence of type 2 diabetes have occurred globally, they have been especially dramatic in societies in economic transition, in newly industrialized countries and in developing countries. Currently the number of cases of diabetes worldwide is estimated to be around 347 million; of these more than 90 percent are type 2 diabetes 3 .
Diabetes is a major cause of mortality, but several studies indicate that diabetes is likely underreported as a cause of death. A recent estimate suggested that diabetes was the fifth leading cause of death worldwide and was responsible for almost 4 million deaths in 2010 (6.8% of deaths were attributed to diabetes worldwide) 4 .
Type 2 diabetes is a global public health crisis that threatens the economies of all nations, particularly developing countries. Fueled by rapid urbanization, nutrition transition, and increasingly sedentary lifestyles, the epidemic has grown in parallel with the worldwide rise in obesity. Asia's large population and rapid economic development have made it an epicenter of the epidemic 5 .
The recently published ICMR-INDIAB national study reported that there are 62.4 million people with type 2 diabetes (T2DM) and 77 million people with pre-diabetes in India. These numbers are projected to increase to 101 million by the year 2030. The complications related to diabetes pose a significant health care burden and a deterrent to overall quality of life. The Chennai Urban Population Study (CUPS) and Chennai Urban Rural Epidemiology Study (CURES) are one of the few population based studies on complications of diabetes in India and show that there is a huge burden due to diabetes related complications in India. The prevalence of diabetic retinopathy (DR) was 17.6%, microalbuminuria was 26.9%, neuropathy was 26.1%, coronary artery disease (CAD) was 21.4% and peripheral vascular disease (PVD) was 6.3%. The cost of treatment for diabetic complications adds to the health care costs. India thus faces a huge health care burden due to high prevalence of type 2 diabetes and its complications 6 .
An adult has about 600g or approximately 20 mol of phosphorus as inorganic and organic phosphates, of which about 85% is in the skeleton, and the rest is principally in soft tissue. Plasma contains both inorganic and organic phosphate, but only inorganic phosphate is measured. Inorganic phosphate is a major component of hydroxyapatite in bone; thus it plays an important role in the structural support of the body and provides phosphate for the extracellular and intracellular pool 7 .
Early in the progression of diabetes, a paradoxical metabolic imbalance in inorganic phosphate (Pi) occurs that may lead to reduced high energy phosphate and tissue hypoxia. These changes take place in the cells and tissues in which the entry of glucose is not controlled by insulin, particularly in poorly regulated diabetes patients in whom long-term vascular complications are more likely. Various conditions are involved in this disturbance in Pi. First, the homeostatic function of the kidneys is suboptimal in diabetes, because elevated blood glucose concentrations depolarize the brush border membrane for Pi reabsorption and lead to lack of intracellular phosphate and hyperphosphaturia. Second, during hyperglycemic-hyperinsulinemic intervals, high amounts of glucose enter muscle and fat tissues, which are insulin sensitive. Intracellular glucose is metabolized by phosphorylation, which leads to a reduction in plasma Pi, and subsequent deleterious effects on glucose metabolism in insulin insensitive tissues. While low and high uncontrolled blood sugars give rise to easily recognizable clinical symptoms, low and high plasma inorganic phosphate remains unrecognizable or presents vague and general symptoms. Hypophosphatemia is strongly related to a decrease in intracellular adenosine triphosphate (ATP) in the aging process and in uremia. Any interruption of optimal ATP production might lead to cell injury and possible cell death 8 .
In this background, it has become necessary to study the status of serum inorganic phosphate in type 2 diabetes mellitus and its association with fasting blood glucose in this region where no such study has been undertaken before. Therefore a humble effort is made to undertake this study with the following aims and objectives. Case-Control Study.

Selection of cases:
Patients were selected randomly on a weekly basis.
Inclusion criteria:-Newly diagnosed cases of type 2 diabetes mellitus who attended and/or was admitted in Department of Medicine, Assam Medical College and Hospital.
Patients were selected on the basis of clinical history, clinical examination and relevant clinical investigations. The individuals were selected irrespective of sex and socio-economic status.
Sex-Male and Female. Controls-age and sex matched healthy subjects. Criteria for diagnosis of diabetes mellitus:-Patients will be considered diabetic according to the criteria defined by American Diabetes Association 2011criteria 9 . For all parameters, a semi-autoanalyzer( MERCK Microlab 300) was used. Required reagents were used for estimation of parameters.
Test procedures:-Serum inorganic phosphate (molybdate u .v. method) Fasting blood glucose, postprandial blood glucose (GOD/POD Method) Statistical analysis:-Arithmetic mean and standard deviation were worked out to assess the levels of various parameters in both groups under study. Students 't' test was used for comparison of quantitative variables. Co-relation between serum inorganic phosphate and Fasting blood glucose in patients was evaluated using Pearson Co-relation Co-efficient. All tests were considered statistically significant if the p-value was <0.05. All statistical analysis were done in Microsoft Excel, Graphpad.

Results:-
Concentration of fasting, postprandial blood glucose was higher in cases than in controls (p<0.01). The mean ± SD of blood urea and serum creatinine was slightly higher in cases than control but statistically this difference was not significant (p>0.05). The mean serum inorganic phosphate concentration was found to be significantly lower in cases than controls (2.68±0.56 vs 3.64±0.42) and p< 0.01. Present study revealed a negative relationship between serum inorganic phosphate concentration and Fasting blood glucose in patients with type 2 diabetes mellitus with a pearson correlation coefficient , r = -0.67. It is observed from table 1 that the mean serum inorganic phosphate level in male cases had been 2.73 ± 0.58 mg/dL and that in female cases 2.6 ±.0.54 mg/dL. In the control group, the mean serum inorganic phosphate level in males had been 3.64 ± 0.46 mg/dL and that in female, 3.63 ± 0.36 mg/dL. The difference between the mean serum inorganic phosphate level between males and females in cases and in controls was very minimal and it was statistically not significant (p>0.05). Overall, the mean serum inorganic phosphate level in cases was 2.68 ± 0.56 mg/dL and that in controls is 3.64± 0.42 mg/dL. It was observed that the serum inorganic phosphate level in cases was lower than that in controls and it was statistically highly significant (p < 0.01). It is observed from Table 3 that the mean concentration of fasting blood glucose and post prandial blood glucose in cases were significantly higher than in controls.  It is seen from table 5 that serum inorganic phosphate levels showed a negative correlation with fasting blood glucose levels, that was statistically significant (p<0.01). The Pearson correlation coefficient "r" which was found to be -0.67 established the negative correlation between the two parameters.

The present study "A correlative study of Serum Inorganic Phosphate concentration and fasting blood glucose in type 2 Diabetes Mellitus -A hospital based study", was
undertaken in an attempt to understand the role of serum inorganic phosphate in diabetes mellitus as well as to understand the interplay between serum inorganic phosphate level and fasting blood glucose in the progression of type 2 diabetes mellitus.
In the present study it was observed that the mean serum inorganic phosphate level in male with type 2 diabetes was 2.73 ± 0.58 mg/dL and those in female cases, 2.6 ± 0.54 mg/dL. Here, the difference between male and female cases was very minimal and was statistically not significant (p > 0.05).
In control group also, the difference between male and female cases was very minimal and it was statistically not significant (p > 0.05). The above findings indicate the serum inorganic phosphate levels are independent of the gender of the study groups. Haap M et al 10 in 2006 showed that there was a significant association of low serum phosphate concentration with high 2-h blood glucose levels independent of anthropometric parameters like body fat, age and gender.
In the present study, the mean level of serum inorganic phosphate in diabetic cases was found to be 2.68 ± 0.56 mg/dL. On the contrary, in the control group the mean serum inorganic phosphate level was found to be 3.64 ± 0.42 mg/dL. The serum inorganic phosphate levels in diabetic cases was found to be lower than that in the control group and it was statistically highly significant (p < 0.01). The findings of the present study are consistent with findings of Ugwuja EI et al 11 in 2007. They had examined serum phosphate level along with other parameters in 60 Diabetic patients and compared with 60 apparently healthy, age and sex matched controls. They found that serum phosphate levels are significantly lower in diabetic patients than in controls (p < 0.05). In diabetes mellitus, increased urinary loss due to osmotic diuresis may be the common and most important cause of reduced phosphate, although intracellular shift may also be a factor.
Osmotic diuresis present in hyperglycemic and acidemic states may cause an increased phosphate excretion due to competition between phosphate and glucose in proximal tubular system 10 .
In Diabetes Mellitus, a major disturbance in phosphate handling occurs in the kidney tubules, where the excessive sodium-dependent glucose reabsorption depolarizes the electrochemical sodium gradient. Since inorganic phosphate use the same driving force, but have less binding to sodium than glucose, the inorganic phosphate reabsorption, particularly in poorly regulated patients, becomes impaired. This paradoxical phosphate imbalance may lead to affinity hypoxia and impaired formation of high energy phosphates. The lack of intracellular phosphate complementary to the increased intracellular glucose takes place in the insulin-insensitive cells and tissues, resulting in the possibility of late complications of diabetes mellitus 12 .
The findings of present study are also supported by study of Nagasaka S et al 13 in 1995. They showed that NIDDM patients had lower level of serum phosphate at the time of admission, and the values of serum phosphate significantly (p < 0.01) increases after hospitalization period, when glycemic control was markedly improved. They also showed the positive correlation between serum phosphate concentrations and renal threshold for phosphorus excretion on admission (r = 0.86, p < 0.01), and between urinary glucose and urinary phosphate on admission (r = 0.46, p < 0.05). These results indicate that hyperglycemia causes excess urinary phosphate excretion in patients with NIDDM 13 .
In NIDDM (Type 2 DM), abnormally high blood glucose is linked to non physiologic variations in plasma insulin content throughout a 24hr period. During hyperglycemic-hyperinsulinemic conditions, high amounts of glucose enter in to muscle and fat tissues which are insulin sensitive. Intra cellular glucose is metabolized by phosphorylation which leads to lower levels of plasma phosphate and consequent negative effects on glucose metabolism in the insulin-insensitive tissues 8 .
Ditzel et al 14 studied renal handling of Pi (inorganic phosphate) in 26 conventionally treated diabetic children and in 28 healthy children and found fasting urinary phosphate excretion 3 times higher in the former group despite a significantly lower fasting Pi. The maximal capacity of renal tubular reabsorption of phosphate per liter of filtrate (TmPO4/GFR) was significantly suppressed in the diabetic patients. The increased urinary phosphate excretion correlated positively with both urinary glucose excretion and blood glucose concentration (P <0.01). This finding was unrelated to serum PTH or to plasma growth hormone 14 .
Kalaitzidis R et al 15 in 2005 observed that patients with metabolic syndrome showed significantly lower phosphate and magnesium levels compared with controls. The findings in the present study were consistent with the reports of the above workers.
It is observed from present study that FBS ≥ 200 mg/dL group had lower phosphate (2.14 ± 0.25 mg/dL) than the group which had FBS < 200 mg/dL and it was statistically highly significant ( p< 0.01).
It was also observed in the present study that serum inorganic phosphate levels showed a negative correlation with fasting blood glucose levels, that was statistically significant (p<0.01). The Pearson correlation coefficient "r" which was found to be -0.67 established the negative correlation between the two parameters.
The findings of present study are almost consistent with findings of Nagasaka S et al 13 in 1995. They showed that in NIDDM patients at the time of admission in to hospital, HbA 1c was 11.1 ± 0.3% and the serum phosphate was 1.12 ± 0.03 mmol/l. The values of HbA 1c and serum phosphate became 9.3 ± 0.2% and 1.21 ± 0.03 mmol/l respectively at the time of discharge from the hospital when glycaemic control was markedly improved and the changes in the values were statistically significant (p<0.01). Thus, they showed that when there was significant reduction of HbA 1c % in NIDDM patients, the serum phosphate level rose significantly.
Raskin and Pak 16 studied 21 diabetic patients in whom treatment results ranged from "suboptimal" to "optimal" control and found that, as the mean plasma glucose decreased from 17.1 mmol/L to 5.2 mmol/L over 4 to 10days, serum phosphate level rose from 1.12 to 1.26 mmol/L (P < 0.001).
Gertner et al 17 studied mineral metabolism in 7 juvenile onset diabetic patients before and after achieving near normal glucose levels by 7 to 14 days treatment with a portable subcutaneous insulin infusion system. They found that as plasma glucose decreased from an average of 221 mg/dL to 95.9 mg/dL, serum inorganic phosphate rose from 4.09 to 5.01 mg/dL (P < 0.001) due to a 25% rise in renal tubular threshold for phosphate. No change was noted in immunoreactive parathyroid hormone (PTH) and in 1, 25hydroxy-vitamin D.
Haglin et al 18  The findings in the present study were supported by the reports of the above workers.

Conclusion:-
The present study was undertaken to determine the serum inorganic phosphate concentration and fasting blood glucose in newly diagnosed type 2 diabetes mellitus patients and also to determine the correlation of serum inorganic phosphate concentration with fasting blood glucose of the patients.
The study showed that the serum inorganic phosphate concentration was significantly decreased in the diagnosed type 2 diabetes mellitus patients in comparison to healthy individuals. Moreover, there was a significant reduction of serum inorganic phosphate concentration in cases whose glycaemic control was poor, which was reflected by increased fasting blood glucose. There was a statistically significant negative correlation between serum inorganic phosphate concentration and fasting blood glucose in patients of type 2 diabetes mellitus in the present study. Therefore, these findings indicate that there was a definite reduction of serum inorganic phosphate concentration in type 2 diabetes mellitus patients as long as glycaemic control was not achieved and that reduction of serum inorganic phosphate concentration may have a contributing role in the progression of the disease and development of complications of diabetes.
The major issue arises whether to estimate serum inorganic phosphate levels routinely in all type 2 diabetes patients and whether to set a cutoff value of serum inorganic phosphate for good glycaemic control remains to be seen.
Further studies with large sample size and longer duration of study with newer methods, which gives a more accurate picture of the actual serum inorganic phosphate and fasting blood glucose status of the body, are required which might shed greater light in this regard.