Relationship between serum insulin and point-of-admission blood glucose of ill neonates at a tertiary health facility in Nigeria

Introduction Optimal glucose metabolism is important in neonatal survival especially in the first days of life. Insulin play a significant role in maintaining blood glucose homeostasis. This study set out to determine the serum insulin levels of ill neonates as related to their point-of-admission blood glucose estimation at the Wesley Guild Hospital, Ilesa, Nigeria. Methods Three hundred babies took part in the study. Blood glucose and serum insulin levels were assayed at admission using Accu-Chek Active glucometer(R) and Accu-Ɔ-Bind ELISA Microwells(R) respectively. Hyperglycaemia was defined as blood glucose ≥7mmol/L and hypoglycaemia as blood glucose <2.2mmol/L. Results The median (IQR) age of the babies was 10.0 (0.5 - 70.0) hours with male to female ratio of 1.5:1. Seventy-four (24.7%) were preterms and 35 (11.7%) were small-for-gestational age. The mean (SD) blood glucose level of the babies was 4.1(2.1) mmol/L with a range of 0.6-13.4mmol/L. Hyperglycaemia and hypoglycaemia were observed in 18(6.0%) and 40(13.3%) babies respectively. The median (IQR) serum insulin level was 9.8(3.0-35.3) μIU/ml. There was weak positive correlation between serum insulin and blood glucose levels of the babies (r = 0.197, p = 0.001). Birth asphyxia was associated with lower serum insulin, while probable sepsis with relatively higher levels. Conclusion Serum insulin level increases with increasing blood glucose in ill Nigerian babies at presentation to the hospital. Babies with asphyxia and sepsis particularly tend to have abnormal serum insulin at admission. Hyperinsulinaemia in ill babies may connote a compensatory mechanism to normalise abnormal blood glucose rather than playing significant role in its aetio-pathogenesis.


Introduction
Blood glucose homeostasis is an important aspect of newborn physiology as abnormalities including hypoglycaemia and hyperglycaemia constitute significant risks to the newborn health [1,2]. Insulin is secreted by the beta cells of the islet of Langerhans of the pancreas [3]. It causes rapid uptake, storage, and use of glucose by the tissues of the body especially the muscles, adipose tissue, and liver [4]. The counter-regulatory hormones are hormones that oppose the action of insulin leading to increased blood glucose levels [4]. They include glucagon, cortisol, growth hormone, adrenaline and noradrenaline. The levels of the counter-regulatory hormones are increased during stressful clinical conditions [3].
Neonatal hyperglycaemia (NNH) and its relationship with serum insulin in ill neonates is largely understudied especially in the West African sub-region. Many studies have attributed increased level of stress hormones which are known to stimulate gluconeogenesis as being important in the pathogenesis of hyperglycaemia in ill neonates [1,5,6] but the importance of serum insulin in this respect needs to be further studied. There is paucity of studies relating blood glucose levels in ill newborns and the respective serum insulin levels at the point of admission especially in Nigeria. Knowing the serum insulin levels in ill babies with hyperglycaemia may justify insulin use or otherwise early in the management of NNH. The aim of the study was to determine the point-of-admission serum insulin and its relationship with the blood glucose levels in neonates admitted to a Nigerian tertiary health facility.

Methods
The study was a prospective cross-sectional study carried out at the Exclusion criteria included babies who had major congenital anomalies or parental refusal of consent. History obtained about the recruited babies included the age at presentation, parent's socioeconomic class (using Oyedeji's classification) [7], place of antenatal care, place of delivery, mode of delivery, gestational age (GA) and the mother's parity. These were documented in the proforma designed for the study. Physical examination of each baby was done and significant signs were documented in the proforma.
Babies were also classified as preterm (GA <37 weeks), term (GA between 37-42weeks), and postterm (GA >42 weeks). The babies were also classified into small-for-gestational age (SGA) i.e. weight <10 th percentile of the expected for the gestational age, appropriatefor-gestational age (AGA) when the weight is between the 10 th and 90 th percentile, and large-for-gestational age (LGA) when weight is >90 th percentile using an intrauterine growth chart [8]. Babies with sepsis were further classified as presumed sepsis, probable sepsis, and confirmed sepsis. Babies with confirmed sepsis not only had risk factors or clinical features of sepsis, but they also had bacteria growth on blood culture [9]. Presumed sepsis were babies with one or more risk factors to suggest sepsis including prolonged rupture of membrane, peripartum fever, or chorioamnionitis but yet to manifest clinical features of sepsis [9]. Babies with probable sepsis were babies with clinical features to suggest sepsis such as fever, hypothermia, poor suck, lethargy, respiratory distress etc. [9].
Blood glucose and serum insulin determination: peripheral venous blood was collected using aseptic procedure from each baby following venepuncture at the dorsum of the hand. A drop of venous blood was used for cot-side measurement of blood glucose using the Accu-Chek Active ® glucometer (Roche Diagnostics GmbH, Germany) while two millilitres of blood sample was collected in a plain bottle.
Blood glucose value >6.9mmol/L was taken as hyperglycaemia while blood glucose value <2.2mmol/L was taken as hypoglycaemia [2,5,10,11]. The two ml of blood sample that was collected in the plain bottle was centrifuged at 3000rpm for five minutes shortly after collection. The supernatant serum was then separated into another plain bottle and immediately stored inside the freezer in the hospital laboratory at temperature of -20° centigrade. and median with interquartile range (IQR) were used to summarize normally and non-normally distributed continous variables respectively, while proportions and percentages were used to summarize categorical variables such as sex, and age range.
Differences in serum insulin level of the various categories of babies which were not normally distributed were assessed using the Mann Whitney U test (U) or Kruskal Wallis test (k) as appropriate.
Spearman's correlation was used to determine the relationship between serum insulin level and the blood glucose of the babies.
Statistical significance was set at p value <0.05 at 95% confidence interval.

Results
Over a six-month study period (January to June 2017), 300 babies were recruited into the study. Maternal age: the mean (SD) maternal age was 29.4 (6.2) years with a range of 17 to 51 years. The highest proportion (82.4%) of the mothers were in the age range 20-35 years.

Some obstetric characteristics and the babies' classification:
the obstetric characteristics of the mothers and the babies' classification are shown in Table 1.  Table 3. There was weak positive correlation between the random blood glucose and the serum insulin levels of the babies (r = 0.197, p = 0.001). Figure 1 shows a scatter plot of the relationship between the serum insulin levels and random blood glucose of the babies.

Discussion
The age of the babies in this study was significantly related to serum insulin levels as the babies that were older than seven days tend to have higher serum insulin levels. This is in agreement with the finding of Abdel-Azeem et al. [12] who also noted that serum insulin level tend to increase with the age of the babies. This is possibly because by seven days of life most mothers would have established breastfeeding. It is expected that with established or adequate feeding, the insulin secretion is increased to ensure adequate glucose uptake, use, and storage [3,4]. The present study showed that serum insulin level in the babies with probable sepsis was significantly higher than that of the other groups of babies. To the best of the investigator's knowledge, no study was found to relate neonatal sepsis with elevated serum insulin. However, it is possible that since this study showed that probable sepsis was a risk factor for neonatal hyperglycaemia, serum insulin was most likely elevated as a compensatory mechanism to correct hyperglycaemia in those babies.
In addition, serum insulin could possibly be elevated in babies with sepsis in reaction to the effect of stress and other counter-regulatory hormones like glucagon and cortisol [3][4][5].
These counter-regulatory hormones mobilise stored glucose from the liver and muscles which insulin tends to catabolise [3][4][5]. Could insulin be a biomarker of sepsis in newborn like C reactive protein and procalcitonin which are known biomarkers of sepsis [13]. This will be a good future research. Contrary to the investigator's expectation [14][15][16], there was no significant difference in the serum insulin levels of babies who were born low birth weight or macrosomic and those who had normal birth weight. Also, the serum insulin level was not significantly related to whether baby was small, appropriate or large for gestational age. These may be due to the small proportion of macrosomic, large for gestational age and small for gestational age babies compared to other groups of babies. Furthermore, in this study, the babies were recruited at different presenting ages when other factors such as inadequate lactation by mothers leading to reduced breastmilk intake must have set in which could have brought down the insulin level to normal, unlike the cord blood estimation of insulin level done in them at birth [14][15][16].
It was also shown in this study that the serum insulin level was not significantly related to the gestational age of the babies. This was against the finding of Wang et al. [17] in the United States of America who found that the serum insulin level was higher in preterm babies.
The reason for this difference was because the babies recruited into the present study were recruited at different ages unlike the study by Wang et al. [17] in which babies were recruited at birth. Also, the proportion of preterm babies in this study was fewer compared to those of term babies. This study equally showed that serum insulin level was significantly lower in babies who had birth asphyxia. This was contrary to previous studies, most of which implicated birth asphyxia as a cause of high serum insulin levels [18][19][20]. This was possibly because a major portion of the recruited babies for this study were not admitted immediately after birth even when they had birth asphyxia, hence, the serum insulin most likely had reduced after an initial rise. The explanation for this is the negative feedback mechanism which is normally stimulated in the body to stop insulin secretion when it is elevated [3,21]. Furthermore, when there is high level of insulin, it predisposes to hypoglycaemia which also turns off the insulin secretion thus reducing its level [3,18].
The current study showed that babies with hyperglycaemia have significantly higher level of serum insulin more than the babies with normoglycaemia (36.0 versus 8.6µIU/ml). This was also supported by Verhoeven et al. [22] who also found elevated serum insulin in hyperglycaemic babies compared to normoglycaemic babies (30.8 versus 8.3µIU/ml). This shows that insulin does not constitute an important factor in the pathogenesis of hyperglycaemia at point of admission in most neonates, but it may be elevated as a compensatory mechanism to reduce the blood glucose back to normal [3]. Also, because there is elevated counter-regulatory hormones which is thought to be responsible for stress hyperglycaemia, there is associated relative insulin resistance [23].

Conclusion
Serum insulin level increases with increasing blood glucose in ill Nigerian babies at presentation to the hospital. Babies with asphyxia and sepsis particularly tend to have abnormal serum insulin at admission. Hyperinsulinaemia in ill babies may connote a compensatory mechanism to normalise abnormal blood glucose rather than playing significant role in its aetio-pathogenesis.
What is known about this topic • The age of the babies was significantly related to serum insulin levels as the babies that were older than seven days tend to have higher serum insulin levels; • The babies with hyperglycaemia had significantly higher level of serum insulin more than the babies with normoglycaemia; • There was positive correlation between the blood glucose and serum insulin levels.

What this study adds
• Serum insulin level in the babies with probable sepsis was significantly higher than that of the other groups of babies; • This study equally showed that serum insulin level was significantly lower in babies who had birth asphyxia compared with babies without birth asphyxia; • Serum insulin may play more of a compensatory role in reaction to high blood glucose levels. review of manuscript. JBE was involved in data interpretation and critical review of manuscript. All authors read and approved the final version of the manuscript.

Acknowledgments
The authors hereby acknowledge the contributions of the clinicians and other health professionals who were involved in the management of the babies admitted into the Special Care Baby Unit, WGH, Ilesa.
We also acknowledge the babies and the parents/guardians who participated in the study. Table 1: obstetric characteristics and the babies' classification