Elevated serum sialic acids, a potent biomarker of alloxan-induced type 1 diabetes in dogs by ethanolic extract of Anogeissus leiocarpus

Abstract

Objectives

This study investigated serum sialic acids for a predictive and diagnostic biomarker of diabetes mellitus (DM) in dogs and its prognostic value with ethanolic extract of Anogeissus leiocarpus.

Design

Four groups of 3 dogs were used; non-diabetic controls (ND), diabetic-untreated (DU), diabetic insulin-treated (DI) and diabetic extract-treated (DE). Free serum sialic acids (FSSA) and erythrocyte surface sialic acids (ESSA) were assayed in all groups, pre-and post-induction of hyperglycaemia and results were presented as means ± standard error of means (SEM) and subjected to ANOVA using Tukey’s post-hoc tests with GraphPad Prism® statistical package.

Results

FSSA increased in DU and plateaued at third week (61.8 ± 0.41 μg/ml), (P < 0.002) with additional 38.2 μg/ml (62%) generated, coinciding with hyperglycaemia. FSSA of DI increased but declined to 22.3 ± 1.55 μg/ml. Extract of Anogeissus leiocarpus effectively modulated FSSA in DE as increased value declined to 21.4 ± 0.78 μg/ml. Pre-induction DU ESSA (8.27 ± 0.11 μg/ml) significantly (P < 0.002) decreased by third week (2.33 ± 1.49 μg/ml), coinciding with hyperglycaemia. Strong negative correlation coefficient (r = −0.92) occurred between DU’s FSSA and ESSA and ND (P < 0.03). Sialic acid expression in dog’s insulin dependent diabetes mellitus (IDDM) is 18% lower than normal. Extract of A. leiocarpus restored ESSA completely. ESSA cleaved in DU, 5.94 μg/ml (72%), could not account for the extra FSSA (32.26 μg/ml); liver and kidneys are contributors.

Conclusion

FSSA predicts canine DM.

Introduction

Diabetes mellitus, (DM) a devastating non-communicable disease afflicts over 425 million people worldwide, with high economic cost to governments [1–4]. In particular, with the worldwide increases in type 2 diabetes mellitus (T2DM) without and with macrovascular and microvascular complications of retinopathy and nephropathy DM might become the 7th leading cause of death in 2030 as projected by World Health Organization (WHO) with other projections for 2035 [4]. Also, the increasing worldwide prevalence of canine and feline diabetes mellitus had been highlighted [5]. These imply a clamour for more clinical and experimental animal model research on diabetes mellitus; the reported clinical studies are examples [6, 7].

Sialic acid, a generic term for a family of acetylated derivative of neuraminic acid had been an essential component of glycoproteins, glycolipids, as a negatively charged monosaccharide expressed on erythrocyte membranes [8, 9]. Sialic acid, a cofactor of many cell surface receptors, for example insulin receptor, was positively associated with most of the serum acute phase reactants [10–14] and served as a marker of acute phase response [15].

Sialic acid derangement on red blood cell membranes of diabetic patients led to its release and elevation in urine and serum [16, 17]. Sialic acid as a risk factor, depicted acute inflammatory changes and damages in red cell membrane which led to ischaemia in the blood vessels, kidney, eyes and brain, such that increased levels of sialic acids indicated extensive damages in diabetes mellitus [18, 19].

Quantitative analysis of red cell membrane sialic acid became an important clinical parameter for diabetes mellitus [20–22]. Indeed, under expressed sialic acid on the erythrocyte membrane surface indicated diabetes mellitus; for instance, sialic acid expression in insulin-dependent diabetes mellitus (IDDM) patients was 38% less than that of healthy people [20].

Type 1 diabetes mellitus (TIDM) was successfully induced in Nigerian indigenous dogs using alloxan monohydrate and all haemato-biochemical aberrations, similarly produced by alloxan-induced diabetes in adult male Wistar albino rats [23] and mice [24] were corrected by ethanolic extract of Anogeissus leiocarpus.

The dynamics of FSSA and ESSA in relation to persistent hyperglycaemia in alloxan monohydrate-induced TIDM in Nigerian indigenous dogs were investigated for a predictive diagnostic biomarker and a modulating effect of A. leiocarpus.

Materials and methods

Plant collection, extraction, qualitative and quantitative phytochemical screenings

Collection

The stem bark of Anogeissus leiocarpus, was collected from Samaru, Zaria and environs and authenticated in the herbarium of the Department of Botany, Faculty of Life Science, Ahmadu Bello University, Zaria, with a voucher sample number 167. The stem bark, was air dried for about two weeks under shade at room temperature and pulverized with mortar and pestle.

Crude Ethanolic extraction of stem bark of A. leiocarpus

The cold maceration method with 95% v/v ethanol was applied to obtain the crude ethanolic extract of the plant. With a packed layer of cotton wool at the bottom of a separating funnel, 100 g of the powdered plant material were placed on it; five hundred (500) ml of 95% v/v ethanol was used to completely immerse the material for a 48 h extraction, at room temperature [25].

Qualitative phytochemical screening of crude ethanolic extracts of Anogeissus leiocarpus

Standard protocols [26, 27], were applied to detect the different phytochemical constituents in the crude ethanolic extract.

Quantitative phytochemical screening of crude ethanolic extracts of Anogeissus leiocarpus

Also using standard procedures [28–31], the yields of alkaloids, flavonoids, saponins, tannins and phenols in the crude extract were determined and expressed in percentages.

Experimental animals

Twelve apparently healthy adult (between 1 and 2 years) Nigerian indigenous dogs of both sexes, sourced around Zaria and environs, were subjected to pre-experimental screening in the arthropod-proof animal pen of the Department of Veterinary Parasitology and Entomology, Faculty of Veterinary Medicine, Ahmadu Bello University, Zaria, at ambient temperature and humidity. They were on standardized dog food of 60% carbohydrate, 25% protein, 15% fat and provided water ad libitum, weighed and colour-coded for identification. Screening included treatment against endo-and ectoparasites using Prazisam® plus (praziquantel 50 mg, pyrantel pamoate 144 mg and fenbendazole 500 mg Vetoquinol India Animal Health Pvt. Ltd. 801, Sigma, 8th Floor) at a dose of 1 tablet per 10 kg body weight administered orally based on manufacturer’s recommendation and Ivermectin subcutaneously at a dose of 100 μg/kg body weight. They were also vaccinated against rabies using dogs’ anti-rabies vaccine obtained from Nigeria Veterinary Research Institute, Vom, Plateau State, Nigeria. An acclimatization period of one month was allowed before commencement of the experiment.

The choice of dogs as experimental model was due to by the use of some identical drugs Veterinary practitioners and Human physicians for diseases of dogs and humans, respectively, for example, Ivermectin. Thus, results from dogs, in this study can readily be extrapolated for modification and used in humans.

Experimental dogs grouping and design

The dogs were assigned at random into 4 groups of three dogs each:

• Group 1 (ND): (Non-diabetic dogs). Diabetes was not induced and were each administered normal saline orally at a dose of 2 ml/kg body weight daily and served as normal control; Group 2 (DU): (Diabetic untreated dogs). Diabetes was induced and left untreated and served as negative control; Group 3 (DI): (Diabetic insulin-treated dogs). Diabetes was induced and the conventional drug, insulin at a dose of 0.5 IU/kg body weight was administered subcutaneously daily to each animal and served as positive control; Group 4 (DE): (Diabetic extract-treated dogs): Diabetes was induced and crude ethanolic extract of Anogeissus leiocarpus at a dose of 1000 mg/kg body weight was administered orally daily to each dog, using a tiny plastic tube attached to a syringe, such that the tube got into the esophagus. The dose of extract was selected based on previous study [25]. The crude ethanolic extracts were reconstituted by dissolving 1 g of extract in 2.5 ml distilled water to obtain a concentration of 400 mg/ml. All administrations were given at 9:00 am daily for a period of three weeks.

Induction of diabetes mellitus with alloxan monohydrate

Diabetes mellitus was induced using alloxan monohydrate (Sigma-Aldrich™) to produce type 1 diabetes mellitus. The dogs in groups 2, 3 and 4 were fasted overnight. Blood glucose level was measured at 0 min using a portable glucometer and test strip to establish baseline glucose level. (Accu-Chek® Active, Roche, Roche Diabetes care Middle East, FZCO) Alloxan monohydrate was reconstituted in cold normal saline to obtain a concentration of 200 mg/ml and administered rapidly through the saphenous vein at a dose rate of 100 mg/kg body weight in the range, as applied by Aluwong et al. [32]. The dogs were maintained on 10% dextrose to prevent fatal hypoglycaemia and monitored continuously for fatal clinical signs. Feeding and water supply ad. libitum continued. Fasting blood glucose (FBG) were tested 72 h, 7, 14 and 21 days after the alloxan monohydrate administration. Dogs having elevated glucose levels (6.3–8.3 mmol/L) were considered diabetic.

Collection of blood sample for sialic acids assays

From the 5 ml of blood collected, 2 ml was dispensed into a vacutainer containing 0.3 ml of Acid Citrate Dextrose (ACD) as anticoagulant for the preparation of haemoglobin-free erythrocyte membranes (ghosts) for erythrocyte membrane sialic acid assay. Three (3) ml of blood was dispensed into anticoagulant free vacutainers, allowed to clot, removed and serum aspirated into serum tubes and stored at −20 °C until when needed for assay for free serum sialic acid.

Sialic acid assay

Haemoglobin-free erythrocyte membranes were prepared as outlined by Dodge et al. [33]. Both erythrocyte membrane sialic acid and free serum sialic acid were analyzed by the principles of Warren [34] and Aminoff [35] using the Quantichrome™ Sialic Acid Assay Kit (Bioassay Systems, 3191 Corporate Place, Hayward, CA 94545, USA) as described by the manufacturer.

Statistical analysis

Results were expressed as means ± SEM. The data were subjected to ANOVA with Tukey’s post-hoc tests using the GraphPad Prism® statistical package. Values of P ≤ 0.05 were considered significant.

Correlation coefficient analysis between FSSA and ESSA of all four groups was performed using standard statistical package.

Results

Intravenous glucose tolerance test (IVGTT) indicated no existing DM in the dogs prior to commencement of the study; by 1st week post-induction of DM blood glucose levels were DU, 25.0 ± 1.88 mmol/L; DI, 5.5 ± 0.20 mmol/L; DE, 5.5 ± 0.39 mmol/L against ND, 4.30 ± 0.11 mmol/L. Third week values were 25.0 ± 1.86; 5.2 ± 0.31. 4.1 ± 0.26 and 4.30 ± 0.06 mmol/L for DU, DI, DE and ND respectively; there was impaired glucose utilization in DU, from IVGTT and macrocytic normochromic anaemia also developed.

FSSA

The variations, dynamics and the effects of the administration of insulin and crude ethanolic stem bark extract of A. leiocarpus on FSSA concentrations in the Alloxan-induced diabetic dogs are presented in Fig. 1. There were increases in the FSSA concentration in DU, which occurred throughout the study when compared with ND, DI and DE groups (Table 1). FSSA increased in DU from a pre-induction value of 23.6 ± 0.92 μg/ml to 38.0 ± 0.55 μg/ml by first week and then 60.5 ± 0.69 μg/ml and plateaued with 61.8 ± 0.41 μg/ml by the second and third weeks post-induction (P < 0.002), respectively. Additional 38.20 μg/ml of FSSA was generated in DU by the third week. The increased FSSA coincided causally with persistent hyperglycaemia (25.0 ± 1.86 mmol/L) and macrocytic normochromic anaemia of the DU.

Fig. 1.

Effects of administration of insulin and crude ethanolic extract of A. leiocarpus on free serum sialic acid concentrations in alloxan-induced diabetic dogs

Table 1.

Mean (± SEM) Effects of the administration of insulin and crude ethanolic stem bark extract of A. leiocarpus on free serum sialic acid concentrations in Alloxan-induced diabetic dogs (μg/ml)

	ND	DU	DI	DE
Pre-Induction	26.9 ± 0.27	23.6 ± 0.92	18.4 ± 2.04	20.3 ± 1.11
Week 1	25.8 ± 0.82	38.0 ± 0.55	27.9 ± 2.02	39.2 ± 2.70
Week 2	26.0 ± 0.82	60.5 ± 0.69a	25.2 ± 0.52	34.9 ± 3.36
Week 3	26.7 ± 0.71	61.8 ± 0.41a	22.3 ± 1.55	21.4 ± 0.78

Approximately 38.2 μg/ml of FSSA was generated in the same DU dogs at the third week post-induction of diabetes mellitus

^a = P < 0.002 (Highly significant), ND Non diabetic, DU Diabetic untreated, DI Diabetic insulin treated, DE Diabetic extract treated

Dosages:

i Insulin; 0.5 IU per kg bd. wt

ii Ethanolic extract of A. leiocarpus; 1000 mg per kg.bd.wt

In DI, pre-induction value of 18.4 ± 2.04 μg/ml increased to 27.9 ± 2.02 μg/ml by first week but declined to 25.2 ± 0.52 μg/ml and 22.3 ± 1.55 μg/ml by second and third weeks post-induction, respectively. Ethanolic extract of Anogeissus leiocarpus effectively modulated the FSSA in DE as the pre-induction FSSA value of 20.3 ± 1.11 μg/ml which increased to 39.2 ± 2.70 μg/ml at first week declined to 34.9 ± 3.36 and 21.4 ± 0.78 μg/ml, at second and third weeks, post-induction, respectively.

ESSA

The variations, dynamics and the effects of the administrations of insulin and crude ethanolic stem bark extract of A. leiocarpus on ESSA concentrations in the Alloxan-induced diabetic dogs are presented in Fig. 2. The ESSA concentration significantly decreased from a pre-induction value of 8.27 ± 0.11 μg/ml to 4.70 ± 1.35 μg/ml by week 1(P < 0.05) and 3.9 ± 0.46 by week 2 (P < 0.002) in the DU group and continued to decline throughout the study to 2.33 ± 1.49 μg/ml by the third week (P < 0.002) post-induction and these decreases causally coincided with the hyperglycaemia (25.0 ± 1.86 mmol/L) and macrocytic normochromic anaemia. The decreases were significant when compared with all the other groups. In the DI, there were fluctuations in ESSA concentrations but by the third week post-induction, 18% of ESSA had been cleaved, when compared with the pre-induction ESSA concentration (Table 2). Similarly, in the DE, there were very steady and constant ESSA values all through the study as observed in ND. The concentrations of ESSA in the DI and DE groups were not significantly (P > 0.05) different.

Fig. 2.

Effects of administration of insulin and crude ethanolic stem bark extract of A. leiocarpus on erythrocyte surface sialic acid concentrations in alloxan-induced diabetic dogs

Table 2.

Mean (± SEM) Effects of the administration of insulin and crude ethanolic stem bark extract of A. leiocarpus on erythrocyte surface sialic acid concentrations in Alloxan-induced diabetic dogs (μg/ml)

	ND	DU	DI	DE
Pre-Induction	8.63 ± 0.02	8.27 ± 0.11	9.18 ± 0.12	8.90 ± 0.04
Week 1	8.27 ± 0.82	4.70 ± 1.35a	7.93 ± 1.27	8.47 ± 0.97
Week 2	8.37 ± 0.83	3.9 ± 0.46b	9.73 ± 0.79	9.00 ± 0.98
Week 3	8.67 ± 0.91	2.33 ± 1.49b	7.53 ± 2.02	8.80 ± 0.67

In the DI group, ESSA dropped by approximately 18% by the third week post-induction. In the DU group, approximately 5.94 μg/ml of ESSA had been cleaved by the third week post-induction of diabetes mellitus

^a = P < 0.05, ^b = P < 0.002, ND Non diabetic, DU Diabetic untreated, DI Diabetic insulin treated, DE Diabetic extract treated

Dosages:

i Insulin; 0.5 IU per kg bd. wt

ii Ethanolic extract of A. leiocarpus; 1000 mg per kg.bd.wt

By assessing the diabetic untreated (DU) group of dogs, approximately 5.94 μg/ml of ESSA had been cleaved by the third week post-induction of diabetes mellitus. (Table 2); from the additionally generated 38.2 μg/ml FSSA by third week post-induction in DU (Table 1) a difference of 32.26 μg/ml was obtained.

Correlation coefficient analysis values between FSSA and ESSA of all four groups with highlights, are presented on Table 3. There is a very strong negative correlation coefficient (r = −0.92) between FSSA and ESSA in DU dogs.

Table 3.

Coefficient correlation analysis between FSSA and ESSA in controls alloxan-induced diabetes mellitus untreated, insulin- and A. leiocarpus ethanolic extract-treated dogs

Groups	Correlation Coefficient (r)	P value	Confidence interval
ND	0.97	0.03	0.14 to 0.99
DU	−0.93	0.08	−0.99 to 0.32
DI	−0.21	0.79	−0.97 to 0.94
DE	−0.47	0.53	−0.99 to 0.89

Highlights of the analysis: There are

1. ND: a strong positive correlation coefficient (r = 0.97) between controls’ FSSA and ESSA.

2. DU: a strong negative correlation coefficient (r = −0.92) between diabetic untreated FSSA and ESSA.

3. DI: a weak negative correlation coefficient (r = −0.21) between diabetic insulin-treated FSSA and ESSA

4. DE: a moderate negative correlation coefficient (r = −0.46) between diabetic extract-treated FSSA and ESSA.

ND compared with other groups is significant, P < 0.03

Discussion

The significantly increased FSSA in the diabetic untreated dogs remained persistently higher throughout the study. This finding agrees with the elevated serum sialic acid in human patients with TIDM [18, 36] and T2DM without vascular complications [37] including those with vascular complications that led to either retinopathy or nephropathy [17–19, 38–42].

The significantly increased FSSA in the DU all through the study period coincided with the persistent hyperglycaemia (25.0 ± 1.86 mmol/L) observed in the DU during same period. This suggests a causal relationship between hyperglycaemia and increased FSSA. This causal relationship between hyperglycaemia and increased FSSA is supported by the reduction in FSSA in this study, as a result of decreased blood glucose levels (5.2 ± 0.31 mmol/L) following insulin treatment of the DI group of dogs. The oral administration of ethanolic extract of Anogeissus leiocarpus also which reduced blood glucose levels in alloxan-induced diabetic rats and mice [23, 24], produced a remarkable reduction in blood glucose concentrations (4.1 ± 0.26 mmol/L) in the DE dogs and also simultaneously reduced FSSA of DE dogs as shown in this study. Futher support comes from a study by Rahman et al. [43] that linked T2DM, hyperglycaemia with elevated plasma sialic acid levels in diabetic subjects, and a subsequent decline in sialic acid levels during treatment with metformin rosiglitazone.

The significantly increased FSSA concentrations in the DU group could have resulted from hyperglycaemia-induced injuries on glycoproteins, glycolipids on the tissues and surfaces of cells including red blood cells. Englyst et al. [16] reported that circulating plasma sialic acids could have resulted, partly, from cellular cleavage due to tissue inherent ability to synthesize sialic acids; cells expressed sialidases [16, 44] which cleaved sialic acids from the glycoconjugates of cell surfaces. Indeed, mammalian sialidase, neuraminidase induced insulin resistance through cellular signaling [45]. In a novel study, Olanzapine-induced Neu 3 sialidase activity down-regulated Neu 1, which eventually disrupted the signaling platform critical for the insulin receptor that led to insulin resistance and T2DM [45]. Any attempt for mammalian cell sialidase to cleave its membrane sialic acid is a ‘suicide’ mission, as such desialylated red blood cells will become senescent and age faster out of circulation, a situation that ‘resembles’ an auto-immune haemolytic anaemia. This may account for the anaemia associated with DM. Sialyltransferase that replaces membrane sialic acid may ameliorate and perform better in a diabetic diseased condition. The increased sialic acid concentrations in diabetes mellitus [16, 19, 43, 46–48] was attributed to increased output of acute phase proteins and diabetes-induced modulation of sialic acid metabolizing enzymes such as sialidase and sialyl transferase [46, 49]. From this study, it is being suggested that red blood cell surface sialic acids are involved in such diabetes-induced release into the plasma. In addition, local cytokine released from macrophages and endothelial cells, during diabetic vascular injury induced an acute phase response, release of acute phase glycoproteins with sialic acids from liver into peripheral circulation [50]. These acute phase glycoproteins include ceruloplasmin in α-globulins; haptoglobin, an α-2 glycoprotein and transferrin, a β-globulin glycoprotein [51].

The decrease and the steady decline of the ESSA from the first week, up to the third week post induction of DM in DU, also causally coincided with the severe hyperglycaemia (25.0 ± 1.86 mmol/L) observed in DU. Both the increased FSSA and the simultaneously decreased ESSA, causally coinciding with the persistent hyperglycaemia could have been due, in part, to cellular injury and cleavage of ESSA. In addition, rapid generation of reactive oxygen species (ROS) and subsequent apostosis, from alloxan administration [52, 53] might have caused cell surface injury.

The ESSA in DI and DE showed slight fluctuations in concentrations that were not significant throughout the study, a variation that was similar to FSSA of these same groups.

A decrease of ESSA in the DI from pre-induction value 9.18 ± 0.12 μg/ml to 7.53 ± 2.02 μg/ml by the third week post-induction of DM showed that 18% of ESSA was cleaved. This under expression of ESSA in the DI dogs, by the third week tacitly confirms the existence of DM in the dogs. It therefore suggests that ESSA expression in insulin-dependent diabetes mellitus (IDDM) in dogs is 18% less than ND control dogs, a value which is lower than the sialic acid expression in IDDM human patients, reported as 38% less than the healthy people [20] and the difference can be host or species related. Therefore, the dog may conveniently serve as experimental model for studies of DM, chronic hyperglycaemia, ESSA, FSSA and urinary sialic acids.

The anaemia observed in these DU dogs was macrocytic normochromic, a regenerative (responding) anaemia. Therefore, with reticulocytosis and an influx of reticulocytes into the peripheral circulation, especially in dogs, an increased ESSA in DU dogs would be expected by the third week post-induction of DM; the influx of reticulocytes (young red blood cells) into peripheral circulation normally produced increased ESSA content and high negative charge [54, 55]. Any level of regenerative anaemia with an attendant reticulocytosis, young red cells with high sialic acid into circulation could not influence a return to normalcy of ESSA in DU dogs, even at the third week. This non-return to normal ESSA in DU dogs is probably due to the existence of hyperglycaemia (25.0 ± 1.86 mmol/L), the main and underlining contributor to the cell surface injury and the cleavage of ESSA.

However, in the DI and DE groups, insulin and the ethanolic extract of A. leiocarpus, respectively, restored the ESSA towards normalcy. The ethanolic extract of the stem bark of A. leiocarpus effectively modulated the FSSA and ESSA which can have prognostic values. By assessing the DU group of dogs, approximately 5.94 μg/ml of ESSA had been cleaved by the third week post-induction of DM (Table 2), whereas approximately 38.2 μg/ml of FSSA was generated in the same DU dogs at this time (Table 1). The difference of 32.26 μg/ml (38.2 μg/ml – 5.94 μg/ml) suggests the existence of other sources of FSSA in the alloxan-induced DM in these dogs. The liver and the kidneys are being implicated as contributors to the increased FSSA observed in this study, due to hyperglycaemia-induced injuries on cells of the liver and kidneys; this is supported by the increased ALT, ALP activities and the serum urea concentrations, respectively, observed in these DU dogs indicative of some level of hepatocellular and renal damage, supported by disorders of biochemical indices in alloxan-induced diabetic rats [23]. Additional support can be obtained from the study of Ibrahim et al. [48] in which sialic acid metabolism was investigated under insulin resistance or chronic hyperglycaemia among various body organs using the effects of high fructose- and glucose-induced hyperglycaemia in wistar rats, to identify the sources and level of the sialic acids in serum. The liver and kidney tended to stimulate sialic acid synthesis while the pancrease down regulated sialic acids synthesis and/or promoted sialic acid release from glycoconjugates, which made these organs contributors to high-serum sialic acid levels observed during type 2 diabetes mellitus [48]. This gives credence to the suggestion that hyperglycaemia-induced injuries in the liver and kidney implicated the organs as additional sources of the increased FSSA observed in DU in the present study. Sialic acids bound to plasma glycoproteins, as listed above are also exposed to hyperglycaemia-induced cleavage and release to impact on the increased FSSA of this study.

It is very noteworthy that the ethanolic extract of A. leiocarpus which ameliorated the hyperglycaemia, other biochemical indices relating to hepato-cellular damage [23, 24], macrocytic normochromic anaemia has effectively modulated FSSA and ESSA in alloxan-induced T1DM in dogs. This further supports additional research on A. leiocarpus for the search of a non-conventional treatment for DM.

Conclusion

Elevated FSSA occurred in alloxan monohydrate-induced T1DM in dogs. The elevated FSSA coincided and had a causal relationship with the persistent hyperglycaemia of the DM. Hyperglycaemia-induced cellular injuries cleaved and released erythrocytes surface sialic acids into plasma. Liver and kidney cells and plasma glycoproteins are implicated in the elevated FSSA due to the hyperglycaemia-induced injuries. The elevated FSSA can serve as a predictive and diagnostic biomarker in alloxan-induced T1DM with a co-existing impaired glucose homeostasis and hyperglycaemia [5, 24]. It also has a prognostic value and should be a routine laboratory analysis particularly for dogs presenting with clinical sign of obesity. Ethanolic extract of Anogeissus successfully modulated the FSSA and ESSA and requires more research for its use to treat DM.

Funding

This study was partly funded by Tertiary Education Trust Fund (TETFUND) of Nigeria.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All applicable international, national and institutional guidelines for the care and use of animals were followed. This article contains studies with animal subjects performed by the authors and ethical approval for this study was provided by Ahmadu Bello University Committee on Animal Use and Care (ABUCAUC) (Ethical clearance No ABUCAC/2019/16).