Inflammatory cytokine and plasma C-reactive protein response to ketoacidosis in adults with type 1 diabetes: Egyptian multicenter study

In adults with diabetic ketoacidosis (DKA), high plasma levels of C-reactive protein (CRP) and inflammatory cytokines such as interleukin-6 (IL-6) were reported to be sensitive markers of infection. However, elevation of both CRP and the pro-inflammatory cytokines which regulate CRP are probable without infection and may act as indicator of systemic inflammatory response (SIR). So, we wanted to determine if increased plasma CRP levels occur in young patients with severe DKA who do not have clinical evidence of infection and to relate observed changes in CRP level to the pro-inflammatory cytokine levels. Upon admission of 30 DKA patients with age ranging from 17 to 40 years, we found significant elevation of CRP (P = 0.001) and IL-6 (P = 0.001) in DKA patients than control group. After resolution of DKA, there was significant reduction in CRP and IL-6 compared to before DKA correction (P value = 0.054 and 0.001), respectively. There was a significant negative correlation between CRP after correction of DKA and RBCs (P = 0.042), and serum K (P = 0.021) and between CRP prior to DKA correction and PH (P = 0.025). Patients with DKA without infection had increased levels of CRP and inflammatory cytokines. Intensive insulin therapy has a powerful anti-inflammatory effect that, at the time of DKA improvement, can normalize levels of CRP and inflammatory cytokines, and this beneficial effect is greater for IL-6 than CRP.


Background
Diabetic ketoacidosis (DKA) is a life-threatening metabolic crisis that may be the first symptom of previously undiagnosed diabetes and occurs predominantly in those with type 1 diabetes or it can also occur in those with type 2 diabetes under certain conditions. DKA results from an absolute insulin deficiency, in response the body switches to burning fatty acids and producing acidic ketone bodies that cause most of the symptoms and complications [1].
C-reactive protein (CRP) is a type I acute phase response protein synthetized in the liver and regulated by the pro-inflammatory cytokines IL-6, IL-1, and TNF-α [2]. CRP has been established as a marker for adverse outcome in acute coronary syndrome, atherosclerosis [3], and ischemic cerebro-vascular accidents [4].
Experimental evidence indicates that CRP induces adhesion molecule expression in endothelial cells [5] and stimulates macrophage production of cytokines at sites of inflammation. Support for a direct role of CRP on endothelial function has been demonstrated by Verma et al. [6], who reported a quenching effect of CRP on nitric oxide production. The pleiotropic proinflammatory cytokines that regulate CRP synthesis can directly affect endothelial cell function by increasing capillary permeability [7].
Elevated plasma levels of CRP have been reported to be markers for endothelial cell dysfunction in uncomplicated, well-controlled, type 1 diabetes mellitus (TIDM), and in children with TIDM within the first year after diagnosis [8]. Positive association between CRP and HbA1c has recently been found in adults with diabetes mellitus [9]. CRP and IL-6 have also been reported to be early markers of infection in adults with diabetic ketoacidosis (DKA) and with diabetic hyperosmolar non-ketotic coma (DHNK) [10]. However, counter-regulatory hormones, which are elevated in DKA, have been reported to potentiate IL-6 in the in vitro production of CRP [11].
Our hypothesis was that both CRP and the proinflammatory cytokines, which regulate CRP, can be elevated without infection in young patients with severe DKA and may serve as a marker for systemic inflammatory response syndrome (SIRS).

Aim of the work
To determine if increased plasma CRP levels occur in young patients with severe DKA who do not have clinical evidence of infection and to relate observed changes in CRP level to the pro-inflammatory cytokine levels.

Methods
This prospective cohort study included 30 patients diagnosed as DKA for treatment and follow-up at Intermediate Care Units and 20 age-and sex-matched healthy control subjects.The patients' age range from 17 to 40 years. The included 30 patients fulfilling the American Diabetes Association criteria of DKA [1].
Mild: patient is alert, 7.30 ≥ pH ≤ 7.25 serum HCO3 − 15-18 mmol/l Moderate: mild disturbed conscious level 7.00 ≤ pH ≤ 7.25, HCO3 − 10-15, Severe: patient is with disturbed conscious level or pH is less than 7, HCO3 − is less than 10 These patients undergo measurement of CRP, IL6 prior to, and after correction of DKA after exclusion of acute infection based upon the medical histories and physical examinations.
All patients and controls included in this study are subjected to the following:

Data collection
Medical records of all patients were reviewed. The demographic, laboratory, and clinical data were collected.

Measurement of CRP, IL6
Blood samples were drawn at presentation before initial hydration and after resolution of DKA. Samples for CRP and interleukin-6 were withdrawn into chilled EDTA tubes and immediately centrifuged at 4°C at 2500 rpm for 20 min. The plasma was separated and kept at − 80°C until assayed. Interleukin-6 was detected by the Assay Max Human Interleukin-6 ELISA (enzyme-linked immunosorbent assay) kit. Plasma CRP was measured, by a high sensitivity ELISA assay. Other laboratory tests were performed using standard clinical chemistry and hematological procedures in our hospital's clinical laboratories.

Statistical analysis
The collected data were analyzed by using the Statistical Package for Social Sciences (SPSS/PC/VER 17).

Results
This prospective cohort study included 30 patients diagnosed as DKA for treatment and follow-up at intermediate care units and 20 age-and sex-matched healthy control subjects. The patients' age range from 17 to 40 years (24.33 ± 6.12 years) (Fig. 1). There was a highly significant difference in WBCs count, random blood sugar, positive acetone detection in urine, arterial blood gases (ABG), and urea, (P < 0.001) and a significant difference in K + level (P < 0.015) between DKA patients and control (Figs. 2, 3, 4, 5, 6, 7 and 9, respectively).
There was a highly significant difference in CRP level between DKA patients before treatment and control (P < 0.001) represented in Table 1. Also, there was significant difference in CRP level before and after treatment (P < 0.054) as represented in Table 2.
There was a highly significant difference in IL6 level between DKA patients before treatment and control and before and after treatment of DKA (P < 0.001) as represented in Tables 3 and 4, respectively. There was a significant negative correlation between CRP after correction of DKA and RBCs, and serum K and between CRP before correction of DKA and PH P = 0.042*, 0.021*, 0.025*, respectively (Figs. 8, 9, and 10, respectively). As regards IL6, no correlation between IL6 before and after correction of DKA with laboratory data as represented in Table 5.

Discussion
This prospective cohort study investigated after exclusion of acute infection of the correlation between IL-6, CRP, and severity of DKA prior to and after correction of DKA. The study included 30 patients diagnosed as DKA for treatment and follow-up at intermediate care units 20 age-and sex-matched healthy control subjects.
The current study showed elevated levels of inflammatory markers in patients with DKA without infection at time of hospital admission while value of inflammatory markers decreased after medical treatment of DKA with insulin intensification. This result matched with Aljada et al. [12] who had suggested the beneficial role of insulin as anti-inflammatory drug in treatment of DKA. Rolla, 2004 [13] showed that persistent elevation of the blood glucose level is associated with impairment of the levels of inflammatory biomarkers as well as intermittent hyperglycemia. The possible mechanisms may be due to the effect of CRP and acute phase reaction proteins formed from different cytokines and counter-regulatory hormones released in critically ill patients [11] or may be due to the effect of elevated triglycerides levels during DKA that is through elevated levels of CRP, IL-6 via different complex mechanisms [14].
In the current study, there was a significant decrease of CRP levels in patients with DKA without infection after improvement of the Acute illness in comparison to its levels at time of admission (P = 0.054) was still higher than the CRP levels determined in control subjects with mean ± SD (17.13 ± 24.66, 2.02 ± 1.25) respectively matching with a study done by Dragana et al. 2019 [15].
Elevation of circulating proinflammatory cytokines are reduced to normal levels promptly in response to insulin therapy and normalization of blood glucose concentration. Of interest, similarly high levels of these markers occurred in patients with DKA and non-ketotic hyperglycemia indicating that hyperglycemia, independent of the presence of ketoacidosis, induces changes in proinflammatory cytokines [16].
On the other hand, the levels of IL-6 were significantly decreased at time of DKA improvement rather than at time of admission (P = 0.001) especially after the start and intensification of insulin therapy which has a potent   Mandrup [17,18]. Moreover, our results suggested that insulin therapy in these metabolic impairments has stronger beneficial effect and reduction of levels of IL-6 than on CRP with P value = 0.001, 0.054, respectively.
We had a limited resource; so, we recruited all limited number of patients and we used non-parametric value in spite of parametric one. Future direction in our investigation will be to include a larger number of patients in each group which will add a strongest value to the research  Authors' contributions GM gave the idea of the research and design of the work part of the research and follow-up of patients in Assiut University Hospital, statistical analysis, and data reviewing and interpretation. MA was responsible for the laboratory part of the research (CRP and IL-6) and contributed to writing and data interpretation. RB contributed to writing, shared in clinical part of the research in Specialized Medical Hospital in Mansoura University, statistical analysis, and data reviewing and interpretation. AA contributed to writing (main role), shared in clinical part and follow-up of patients in Specialized Medical Hospital in Mansoura University, responsible for statistical analysis, and data reviewing and interpretation. All authors read and approved the final manuscript.

Funding
This research is not funded.

Availability of data and materials
The data that support the findings of this study are available from AA (corresponding author).

Ethics approval and consent to participate
It is not applicable. Prof. GA who is the first author confirmed that there was no ethical committee available during the start of our study; on the other hand, our subject did not receive any interventions or exceptional treatment during their stay in hospital, and the blood sample was aspirated during their routine investigations in the hospital after written and informed consents from all patients were included in our study Prof. GA took an oral approval from the head of the internal medicine department at the time of start of our study which was the accepted method at that time. Consent was taken from all participants.

Consent for publication
Taken from participants. All authors read and approved the final manuscript.